However, as discussed above, BER and NCED are both induced by drought. Therefore, a new study was necessary to trigger NCED gene expression independent on inducing BER directly, such as during drought conditions. Based on those difficulties, different promoters could be used to achieve the best results. The ethanol-inducible system is derived from the fungus Aspergillus nidulans in which the alcR gene encodes transcription factor ALCR that controls the activation of several structural genes, such as alcA . In the absence of ethanol, the ALCR protein is inactive, but when ethanol is added, ALCR and ethanol interact to form an activated ALCR, which then binds to the promoter of the target gene such as alcA, inducing the expression of the gene . This system has been successfully used to control different genes in tobacco, Arabidopsis, potato, oilseed rape, tomato, and rice . As ethanol is less expensive, readily available, non-toxic in moderate amounts and can be easily supplied to the plants, this system is considered to have a great potential for field application . In this way, the manipulation of NCED gene expression using the alcohol-inducible promoter  could be used to stimulate ABA biosynthesis and responses in plants before drought stress occurs in a commercial setting. This approach would allow the control NCED expression and subsequent ABA generation during an effective timeframe, triggering preemptive xylem development and functionality, decreasing BER incidence. Therefore, growing raspberries in pots the objective of this study was to use ALC::NCED transgenic tomato plants to manipulate NCED expression and ABA biosynthesis in order to increase xylem functionality, fruit Ca2+ uptake, as well as improve plant WUE and diminish oxidative stress responses, reducing losses due to BER incidence.

Blossom-end rot incidence was markedly different between the wild type ‘New Yorker’ and the transgenic ALC::NCED lines 1 and 2 . Wild type plants had 52.6% of BER incidence , whereas both transgenic lines had less than 20% of BER incidence . NCED gene expression was higher in the transgenic line 1 and 2, after ethanol induction compared to NY . There was no difference in xylem functionality in the proximal fruit tissue among genotypes at 15 DAP . However, xylem functionality in the distal fruit tissue was different between wild type and transgenic lines at 15 DAP . Wild type fruit had a lower density of stained xylems compared to lines 1 and 2. At 30 DAP, line 1 presented a higher number of functional xylems compared to line 2 and NY in the proximal fruit tissue. On the other hand, in the distal fruit tissue lines 1 and 2 presented higher xylem functionality compared to the wild type NY . Fruit number, fresh and dry weights; diameter and length were higher in wild type plants compared to the transgenic lines . Wild type plants had higher water loss than transgenic lines, which resulted in severe water stress symptoms, reaching almost 90% of soil water lost among the 14 days of water monitoring . Both transgenic lines showed 60% of water loss during the evaluation period . Transgenic Line 1 plants had higher relative water content than wild type and transgenic line 2 plants . There were no differences in plant dry weight among genotypes . Transgenic line 1 plants had higher WUE than transgenic line 2 and wild type plants . Ca2+ concentration in the distal fruit tissue was higher in transgenic lines 1 and 2 than wild type . Transgenic lines 1 and 2 showed an average of 12% more Ca2+ concentration in the distal fruit tissue, compared to wild type . Chlorophyll A, chlorophyll B, and carotenoid contents were lower in wild type leaves compared to transgenic lines 1 and 2 leaves . Antioxidant capacity in leaves and distal fruit tissues were lower in wild type plants, compared to transgenic line 1 and 2 plants .Abscisic acid is known to be an important signaling molecule involved in many plant responses to stress conditions . ABA biosynthesis is mainly regulated by the increase in NCED expression that catalyzes a rate-limiting step in ABA production . Indeed, many studies have shown that NCED expression is highly correlated to ABA concentrations in petunia , rubber tree , tomato , Arabidopsis thaliana , and tobacco plants .

Our study shows a potential molecular approach to manipulate the internal plant NCED expression, increasing ABA biosynthesis and leading to higher fruit xylem functionality and Ca2+ concentration in distal tissues, as well as improving plant WUE and reducing fruit susceptibility to BER. The ALC::NCED transformed plants showed a great reduction in fruit susceptibility to BER, when NCED gene expression was stimulated by ethanol treatment, compared to wild type plants. Similar ABA responses on decreasing BER incidence have been reported in other studies with external ABA spray treatments . Treating tomato plants with ABA resulted in higher number of functional xylem vessels at later stages of fruit growth and development, favoring higher xylemic water and Ca2+ uptake into the fruit. Indeed, our study has also shown that triggering NCED expression, and possibly higher ABA biosynthesis in ALC::NCED transformed plants, resulted in a higher number of functional xylem vessels at earlier stage and later stages of growth and development . The higher number of functional xylems in ALC::NCED fruit resulted in higher fruit Ca2+ uptake and translocation into distal tissues, reducing fruit susceptibility to BER, as suggested in previous studies . Some studies have shown that ABA plays an important role on increasing primary and secondary vascular bundle development . Ramachandran et al. showed that xylem vessel differentiation is increased through protoxylem in response to VASCULAR-RELATEDNAC DOMAIN transcription factors expression that are activated by ABA treatment in Arabidopsis thaliana. Molecular and genetic analyses revealed that the two ABA-mediated xylem developmental changes are regulated by distinct members of this transcription factor family, with VND2 and VND3 promoting differentiation of metaxylem cells, while VND7 promotes the conversion of metaxylem into protoxylem . Moreover, higher NCED expression in transgenic lines also resulted in different plant and fruit growth and development, compared to the wild type plants. Transgenic plants and fruit grew slower than wild types. In addition, transgenic lines had fewer flowers and more abortion that resulted in lower number of fruit per plant, compared to wild type plants. The increase in NCED expression and ABA biosynthesis have also been suggested to result in higher expression of genes coding for DELLA proteins that inhibit plant responses to gibberellins. The ABA inhibition of gibberellin responses could explain the smaller fruit observed in the transgenic tomato lines, considering that gibberellins play an important role on triggering fruit growth . Indeed, mutant genotypes with repressed DELLA expression, such as PROCERA, have bigger plant phenotypes that produce larger fruit and leaves . In addition, previous studies have shown that gibberellins increase fruit susceptibility to BER . Therefore, triggering NCED expression in ALC::NCED transformed genotypes possibly decreasing BER incidence by inhibiting plant and/or fruit responses to gibberellins.In our study, triggering NCED expression in ALC::NCED transgenic plants resulted in a reduction of plant water loss, possibly due to reduced stomatal conductance, which is one of the main plant responses to ABA . ABA plays an important role in the plant response to drought by regulating stomatal conductance and root hydraulic conductivity . When applied exogenously, ABA causes rapid stomatal closure and reduces water loss through transpiration . However, CO2 diffusion into leaves becomes limiting to photosynthesis due to stomatal closure , and this also explains the smaller fruit growth, plant pot with drainage partially caused by DELLA repression, discussed above.Besides the fact that ABA can increase WUE by changing how plants can use water to produce assimilated carbon, a new study shows that ABA over expressing tomato lines can also increase xylem embolism under water stress conditions, in response to high ABA levels in plant tissue . However, the causes for the increase vascular embolism are still unclear. Thus, increases in WUE must be seek and analyzed case by case. Our results show that treating ALC::NCED transgenic lines with ethanol might have resulted in higher ABA biosynthesis in these plants, which was not observed in the wild type plants. In addition, triggering NCED expression in ALC::NCED transgenic plants also increased leaf chlorophyll and carotenoid contents , as well as leaf and fruit antioxidant capacity. Recent studies also show that ABA can play an important role on regulating antioxidant defenses during water stress conditions .

According to Jiang and Zhang , the accumulation of ABA induced by water stress causes an increase in the levels of ROS, triggering the antioxidant defense system in plants that leads to an increase in the levels of superoxide and hydrogen peroxide radicals, which was followed by an increase in the activity of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase.In our study, the antioxidant capacity was higher in leaf and fruit tissues of ALC::NCED transgenic plants treated with ethanol, compared to the wild type. Proteomic and transcriptomic analyses performed in several plant species subjected to different stress combinations have highlighted the importance of the antioxidant defense machinery to avoid cell death and plant tissue damage . Plants with higher antioxidant capacity and/or lower ROS levels have been shown to have higher tolerance to stress conditions . Indeed, our results show that higher antioxidant capacity was highly correlated with lower fruit susceptibility to BER in ALC::NCED transgenic plants. Calcium is a nutrient that acts as structural compound, binding to pectins and improving the structure and strength of the cell wall , but it is also an essential nutrient, involved in the proper membrane stability and functionality . The symptoms of BER have been suggested to develop in response to membrane damage caused by low apoplastic soluble calcium content and/or oxidative stresses , which also corroborate with our results showing that higher fruit antioxidant capacity due to higher NCED expression inhibited BER symptoms development possibly by inhibiting membrane damage caused by high ROS levels.Blossom-end rot is a physiological disorder believed to be regulated by the interaction between the genotype and environmental conditions. Under stress, such as drought, heat, salinity, and others, each genotype must have the capacity to trigger morphological, biochemical, physiological, and molecular changes that will maintain proper cell and plant tissue metabolism, avoiding cell death and tissue damage. According to our study, triggering NCED expression in ALC::NCED transgenic plants resulted in higher ABA biosynthesis that is supported by the observed lower plant water loss, leading to morphological changes such as lower fruit number, size, weight, and higher functional xylem vessels in the fruit, as well as biochemical changes such as increased chlorophyll and carotenoids contents and antioxidant capacity in leaves and fruit, and physiological changes such as reduced leaf transpiration and improved plant WUE, and possibly molecular changes related to the communication among ABA and other plant hormones that resulted in tomato plant with better performance under stress, using less water to produce fruit with less BER. The possibility to control plant ABA biosynthesis, using the ALC:: NCED approach, could enable the maintenance of higher plant ABA levels, promoting a reduction of cultivation costs in regions where irrigation water is limited, as well as turning well irrigated region in more efficient crop production sites. Besides that, this approach could help studies in which ABA is necessary only during certain parts of plant growth and development, in order to avoid negative effects of higher ABA levels, like short size, smaller fruits, floral abortion or even during a specific timing, when it is necessary to maintain the stomata close. In addition, the crosstalk between plant hormones could also be studied since ABA production can be easily manipulated. Therefore, this technique represents a powerful tool to improve plant WUE and allow for Ca2+ to reach the distal end of the fruit, decreasing BER incidence and reducing fruit losses. In addition, more studies must also be conducted, to check if ABA could lead to a higher xylem density and maintenance in fruit tissues, if the VND transcription factor family were highly expressed, and finally if genes related to GA-repressed growth were expressed in response to ABA treatment.

Unlike ACS and ACO1, SlCBF1 showed a clear spatial differentiation in both chilling and control conditions. Expression was higher in the pericarp compared to the columella , which suggests that the former might be more responsive to cold stress, possibly due to its external localization. Under chilling in both tissues, SlCBF1 expression peaked at 1h and was sustained for 24h in our study, which can be described as an early response , it then declined to the levels observed at the control temperature, after 3 weeks cold treatment. In other studies, cold storage induced expression of SlCBF1 for up to 8h; 8 days, and 14 days, however there was no induction at 6 °C in Micro-Tom fruit. Te upregulation of SlCBF1 may therefore be dependent on fruit developmental stage, the severity of cold stress, and genotype. After rewarming of the ‘control fruit’, SlCBF1 expression increased to levels higher than during chilling . This may be suggestive of two things: first, that SlCBF1 transcripts in chilled tissues were unable to reach the same levels as tissue held in control temperature, since they were developmentally repressed; and second, SlCBF1 is involved in ripening independent of ACO1 or ACS2, given the asynchrony of their expression . However, a correlation between endogenous ethylene production and SlCBF1 upregulation could partly explain this behavior and matches our observations . Dehydration stress response. Dehydrins , are protective proteins that accumulate in response to dehydration-associated stresses, including chilling. The expression of the clone FC11CA08-2, here named DHN, was analyzed in this study. DHN mRNA levels increased only after 24 h and 3w of post harvest chilling inthe pericarp, drainage collection pot or 3w in the columella with decreases in both tissues after rewarming. There were no detectable differences between tissues, however they responded differentially to temperature .

After rewarming, ‘control pericarp’ DHN expression was higher than that in the ‘chilled pericarp’, due to ripening taking place in the control. It appears that DHN transcript abundance in fruit increases as ripening progresses. The magnitude of changes were greater at 2.5 °C compared to 12.5 °C, consistent with a higher requirement for the molecular chaperones encoded under cold-stress. Oxidative damage. Prolonged or intense chilling stress induces ROS overproduction, which accelerates cell death. The alternative oxidase pathway is activated to minimize ROS levels, and in tomato fruit, AOX1a has been associated with enhanced PCI tolerance. Therefore, AOX1a was studied here. Lipoxygenases catalyze the peroxidation of polyunsaturated fatty acids and are associated with both ripening and redox balance, processes affected by PCI. The expression of the LoxB isoform has not been studied during fruit post harvest chilling storage, and was included. AOX1a expression levels in pericarp and columella were similar, but chilling induced a differential response over time. Transcript levels in the pericarp peaked at 24h and 3w, similar to that seen by Fung et al.. The ‘chilled pericarp’ had a reduced AOX1a expression after rewarming while the ‘chilled columella’ changed little even after rewarming. Under control conditions in both tissues, gradual increases were observed, but rewarming enhanced AOX1a expression , matching ethylene production rates , consistent with ethylene regulation of this gene. ACO1, ACS2 and AOX1a, were co-expressed, but chilling suppressed this correlation . PCI therefore contributes to the uncoupling of ripening-related ethylene biosynthesis, highlighted by the inability of chilled tomato to resume normal ripening after rewarming . LoxB expression displayed a mixed spatial-response that varied with temperature. Expression in both pericarp and columella was unchanged at 1h and 24h .

After 3 weeks, expression was down regulated, but in contrast, rewarming induced the upregulation of LoxB in both tissues. LoxB expression matched ethylene production, consistent with its regulation by this hormone. LoxB expression also paralleled MDA values after rewarming in the columella , in agreement with membrane alterations induced by PCI. The correlation of LoxB with ethylene production rates and ripening was in accordance with the strong correlation between LoxB and ACS2 at 12.5 °C . Interestingly, transcript levels in the ‘control pericarp’ plus rewarming were higher than those of rewarmed tissue after chilling, even though ethylene levels were 1.2-fold higher in the latter. In this case, ethylene production increased in response to chilling-induced stress and not due to ongoing ripening.Principal Component Analysis was performed to explore the structure of the gene expression data from a spatial perspective with respect to cold storage and rewarming of chilled tissue . The first and second principal components explained 75 and 15% of the variation present in the data, respectively. Data for the pericarp and columella portions under chilling for 3 weeks separated from the rewarmed tissues. More importantly, the data distinguished among tissues, with the pericarp and columella showing a clear separation even though gene expression differences between cold and rewarming were a greater determinant of the patterns seen on the PCA. Overall this analysis supports the hypothesis of a spatial and temporal differentiation in response to chilling stress at the gene expression level.Post harvest chilling injury is a complex multifactorial disorder with detrimental effects on tomato fruit quality and shelf-life. With the aim of representing the tomato fruit as a multilayered and integrated system of response to cold stress, we analyzed PCI impact on different fruit tissues and correlated it with known physiological parameters .

Overall, cold stress uncoupled key molecular, biochemical, and physiological processes occurring during the normal progression of storage and ripening. Increased water mobility and tissue liquefaction were also disrupted as evidenced by MRI-obtained D-values from the pericarp, columella and locular portions, and ion leakage obtained from the pericarp. MRI and color development confirmed three concepts: first, the system’s inability to restore or repair the chilling-affected mechanisms; second, that PCI is cumulative and progressive over time; and third, the need to examine each tissue to characterize PCI’s progression and symptomatology, since the most studied fraction, the pericarp, may not reflect processes occurring in other tissues. Reduced starch breakdown in columella and seed discoloration during cold storage reflect that, besides external changes, PCI extends to internal tissues. Tissues exhibited heterogeneous patterns of response to PCI at the biophysical, biochemical and molecular levels. D-values were intrinsically different in the three tissues under study, and their time evolution and temperature responses were also mixed. Responses to oxidative damage represented by the lipid peroxidation byproduct MDA varied in response to temperature but peaked after rewarming, which again highlights that after crossing a threshold of cumulative cold damage, rewarming aggravates PCI’s manifestation instead of alleviating it. Starch accumulation also showed significant spatial differences, suggesting that tissues may display a sharper specialization at the metabolite than at other levels. Responses to cold from the perspective of gene expression were highly dependent on the tissue-type, temperature and time of storage, but overall, they paralleled ethylene production trends via stress response or ripening. Some genes seemed to act concertedly across experimental conditions , others acted coordinately under either cold or control conditions or under apparently independent programs . Transcript accumulation was higher in the pericarp across conditions , square plastic pot equally expressed in both tissues , or, dependent on temperature and storage time . Taken together, this evidence reveals the dynamism of cold-stress in the tomato system and suggests that fruit may display specialized mechanisms to elaborate a response to this environmental challenge. It also unfolds numerous questions about the nature of such varied responses among fruit tissues: are they advantageous to the fruit under stress? What is the source of these differences? Would such relationships differ in the fruit from cold-tolerant tomato species? Exploring these questions in a comprehensive way may deepen our knowledge of this complex phenomenon to elaborate long-term, robust solutions.The efficacy and palatability of commercially available rodenticides can vary greatly, and bait effectiveness is often specific to particular pest species . Many rodenticides have been developed to control rodent populations , and several studies have assessed the materials’ ability to control rats and mice in natural areas . However, until now no peer-reviewed studies had tested the efficacy of rodenticides for roof rat control in nut or tree fruit crops, and few if any studies had been conducted on deer mice. We recently initiated an investigation into the efficacy of three rodenticide baits for control of roof rat and deer mouse activity in almond orchards and found that the 0.005% diphacinone oat bait, sold in many county Agricultural Commissioner’s offices, was highly effective . This study made use of elevated bait stations, which proved effective at supplying bait to target species while substantially limiting access to rodenticides for many non-target species. In this publication we provide information on how to identify damage from roof rats and deer mice in nut and tree fruit orchards, and how to effectively implement a baiting program to control these pests. This appears to be an efficacious, cost-effective, and safe baiting protocol for control of roof rats and deer mice in orchard crops, something that has thus far been unavailable to growers.Accurate identification of the species responsible for damage is essential to development of an effective pest management program. If your management plan focuses on the wrong species, it is likely to be ineffective and it may pose hazards to non-target species and even be an illegal misuse of the material, based on the rodenticide label information.

Fortunately, the presence of roof rats and deer mice can often be detected through indirect monitoring techniques. For example, roof rats often burrow at the bottom of trees, and these burrows are typically 2 to 3 inches in diameter . Burrows of the California ground squirrel are sometimes this same size, but usually they are a bit larger . Also, if ground squirrels are present, you will see them running around above ground and hanging out in burrows.Discarded almond shells at the entrance of a burrow can help you determine the depredating species , but distinguishing between damage from deer mice and roof rats can be difficult. Deer mouse burrow openings typically average around 1.5 inches in diameter. If burrow openings of this size are present, the depredating species may be the deer mouse. Vole and deer mouse burrow openings are similar in size, but voles are not typically found in almond orchards, so long as ground cover is limited. If burrow openings are larger , the roof rat is the likely culprit.he bait stations used in our field trials were tubular structures manufactured specifically for Orange County Vector Control . The bait station consisted of high-density polyethylene plastic tubes that were 13 in long and 3.94 in inside diameter . A steel end cap was fixed onto each end of the tube. Each end cap was penetrated with a 1.89-in opening, big enough to allow the roof rats and deer mice to enter the station and small enough to reduce or even eliminate any inadvertent loss of bait from the bait station. On the inside of the metal cap, under the opening, a 4.5-in long metal shelf is present. This also helps reduce bait loss. As of this writing, these bait stations are available for sale in a limited supply from the Los Angeles County Agricultural Commissioner’s office. We are exploring additional supply options.The current label for 0.005% diphacinone oat bait only allows baiting during the non-bearing season. This means that growers need to be proactive when dealing with rodent infestations. It is the responsibility of the grower to be aware of the presence of endangered species in orchards where they intend to implement a control program, since the bait may prove hazardous to non-target species. The killing of an endangered species may result in a fine and imprisonment under the Endangered Species Act 1973. The use of elevated bait stations will eliminate access to bait for many protected mammal species, such as kangaroo rats . Although other protected species, such as the Tulare grasshopper mouse , are not usually associated with climbing trees, growers must be vigilant in areas where these and other protected species are found. Growers can consult the California Department of Pesticide Regulations PRESCRIBE website for any endangered species restrictions associated with bait application. We recommend placing bait stations either 98 feet or 164 feet from each other, throughout the orchard.

However, some pathogens can exploit the SA/JA antagonism for their own benefit ; for example, B. cinerea produces an elicitor ofSA responses through the NPR1-dependent pathway, which leads to the inactivation of two JA-response genes, Proteinase I and II, that are required for resistance against necrotrophs . ET can counteract the negative effects of NPR1 on JA responses, but it also enhances the NPR1-dependent expression of SA defense genes . Leon-Reyes et al. proposed that the concurrent activation of ET and JA pathways promotes plant insensitivity to subsequent SA-mediated suppression of JAdependent defenses, which then favors effective resistance against pathogens of different lifestyles. Hence, localized synthesis and perception of JA, ET, and SA at the appropriate relative concentration and timing appear to be required for plant resistance. During infections of fruit, ET, SA, and JA networks might interact to stimulate defenses. Nonetheless, accumulation of susceptibility factors as a consequence of ET-triggered senescence/ripening and the antagonism between SA and JA responses may represent opposing influences in the fruit–pathogen interaction and, thus, lead to susceptibility.Increased expression of the tomato 9-cis-epoxycarotenoid dioxygenase 1 , a key ABA bio-synthetic gene, occurs during early infection of susceptible fruit , which suggests a link between ABA synthesis and fruit susceptibility. Several plant pathogens, including B. cinerea, generate ABA during infection or use effectors to induce its production by the host, round plastic plant pot facilitating senescence/ripening and subsequent colonization of the ripened tissue . ABA has been involved in fruit ripening of climacteric and non-climacteric fruit .

Exogenous treatments of ABA induce the expression of the ripening-associated ET biosynthetic genes LeACS2, LeACS4, and LeACO1, thereby, triggering ET production and ripening . In tomato fruit, expression of the 9-cis-epoxycarotenoid dioxygenase 1 increases at the onset of ripening prior to the ET climacteric rise . A slight induction of LeNCED1 was detected in infected MG fruit , which could have been prematurely induced to initiate climacteric ripening; however, a significant decrease in expression occurs at the late stage of ripening . The development and analysis of a genetic knock-out mutant line in LeNCED1 will be instrumental to understand the impact of ABA synthesis during the increase in ripe fruit susceptibility. The expression of FLACCA, a tomato molybdenum cofactor synthase that is involved in ABA biosynthesis, increases as consequence of ripening, but it is reduced in response to the B. cinerea infection . These observations indicate that the plant may reduce the expression of FLACCA in an effort to contain the rise in ABA production caused by the pathogen colonization; however, experimental evidence is needed to test this hypothesis. The interaction between tomato fruit and B. cinerea results in significant changes in the expression of 37% genes involved in the ABA signaling pathway . Alterations in regulators of ABA signaling/responses are detected as well as changes in membrane protein channels . In general, increased expression of the PYL/PYR/RCAR receptors was observed in RR fruit . The PYL/PYR/RCAR receptors are positive regulators of ABA response by blocking the PP2Cs inhibitors . In Arabidopsis, suppression of three PP2C clade A phosphatases results in constitutive activation of ABA signaling and increased susceptibility to fungal infection .

In agreement with these results, significant up-regulation of a RCAR1 homolog and down-regulation of a PP2C homolog in infected RR fruit at 1 and 3 dpi provides further support for a positive relationship between ABA responses and susceptibility . Enhanced expression of suppressor genes throughout the ABA hormonesignaling network is detected after inoculation with B. cinerea of resistant MG fruit . In contrast to the increased expression in MG fruit, the homolog RACK1_a is significantly down-regulated in RR fruit at 1 and 3 dpi . Previous studies have demonstrated a role for RACK1 in the activation of defense mechanisms in response to pathogens in rice. The rice RACK1 homolog triggers ROS production, defense gene expression, and disease resistance by interacting with OsRac1, a Rac/Rop small GTPase involved in basal immune responses . It is plausible that tomato homolog of RACK1 has a similar role in fruit by controlling infections in MG fruit. The contribution of ABA to the enhanced susceptibility of ripe fruit is supported by the disease development assays with the tomato sitiens mutant which fails to synthesize ABA . Inoculation of RR sitiens fruit with B. cinerea resulted in a significant decrease in disease incidence when compared to the infected wild-type RR fruit . Interestingly, about 40% of the inoculated sites in RR sitiens fruit displayed the typical localized necrotic response of wild-type MG green fruit . MG sitiens fruit are as resistant as MG wild-type fruit . The molecular mechanisms that mediate the reduction of susceptibility in RR sitiens fruit are not known; however, analysis of necrotrophic infections in leaves of sitiens plants suggest that a strong induction of defense-related genes , the oxidative burst, and an increase in cuticle permeability might be crucial for the resistant phenotype of this mutant .Plants modulate the ET, SA, JA, and ABA hormone networks to induce immune responses against the attacks by various classes of pathogens .

Recent studies indicate that other hormones such as auxin, gibberellins, cytokinins, cell wall oligogalacturonides, and brassinosteroids might also be implicated in responses to pathogens either directly or by interacting with other hormones . The interactions among hormones provide the plant with a powerful regulatory potential, but also give opportunities for pathogens tomanipulate the plant defense-signaling networks to their advantage . Plants in their natural environments infrequently interact with a single pathogen species, rather they are impacted by microbial communities, herbivores, and other plants, all of which could individually, collectively or cooperatively influence responses to contact with pathogens. This complexity should be taken into account when studying plant–pathogen associations. In fruit, high levels of ET and ABA, which stimulate senescence/ripening processes, may facilitate colonization by necrotrophs. The balance between SA and JA responses seems to be crucial for resistance in unripe fruit, while ABA production correlates with ripe fruit susceptibility. ET, at appropriate concentrations, also contributes to the resistance of fruit by activating JA and/or ET responses and possibly by blocking the antagonistic effect of SA on JA signaling. Hence, the role of plant hormones in promoting fruit resistance or susceptibility depends on the interaction of several factors, including: the concentration of the hormones, the timing of the synthesis and perception of the hormones, the competence of the host tissue to respond to active forms of the hormones, the localization of the plant’s response to the hormones, and the pathogen’s infection strategy, including its own production of hormones. The interaction between tomato fruit and B. cinerea causes transcriptional reprograming of multiple plant hormone networks simultaneously, and, depending on the developmental stage of the fruit contributes to either resistance or susceptibility outcomes. In Figure 6, we provide an overview of key expression changes of genes involved in biosynthesis, modification, signaling, and response pathways of the hormones that, based on our transcriptome profiling analysis and validation, we propose to be part of the regulation of the resistance-to-susceptibility transition associated with ripening and healthy fruit ripening. Analytical methods that allow the simultaneous profiling of multiple signaling molecules that are produced during fruit infections , will shed further light on the signaling networks that control fruit susceptibility in the context of ripening, but the challenge of identifying whether the hormones are synthesized by the host or by the pathogens will still be a limitation. New strategies to study complex gene networks involved in hormone signaling in fruit–pathogen interactions, including the analysis of natural or induced mutants in both plants and pathogens, 25 liter round pot the use of laser micro-dissection and cell-specific transcriptomics, and metabolomics can contribute novel important information to our understanding of the biological and ecological importance of plant development in modulating resistance and susceptibility. From an applied perspective, evaluating the specific hormonal events that promote fruit susceptibility may facilitate the development of commodities that ripen successfully and yet are less susceptible to pathogen infection.Diabetes is a multifaceted metabolic disorder affecting carbohydrate, fat, and protein metabolism. It is caused by increased levels of circulating blood sugar and insulin deficiency characterized by abnormal insulin secretion and insulin resistance in the body. Type-2diabetes affects 80% of those living with diabetes and is largely due to an unhealthy diet and a sedentary lifestyle, which might lead to persistent high glucose levels in the blood, oxidative stress, aging, and other metabolic imbalances. The use of the non-vertebrate organism Drosophila melanogaster as a model tool for research on various human diseases is important because this fly has biochemical features and characteristics similar to those of mammals; therefore, its use is increasing.

This model organism has been tested and has gained worldwide reputation for use in biomedical research, such as in diabetes studies and in other studies involving genetic manipulations. Certain reports identified that disease-causing genes in humans are conserved in Drosophila melanogaster, such as those associated with the insulin-like growth factor signaling pathway. About 70–75% of the brain cells in the fruit fly are insulin producing cells that are similar to the vertebrate pancreatic β cells and secrete seven insulin-like peptides. In addition to this, using the fruit fly for research studies is easy and cost-effective, since it is not expensive to maintain them. The indigenous eggplant belongs to the Solanaceae family and is locally called “Igba Yinrin” by the Yoruba people in South-West Nigeria. It is commonly known as a forest bitter berry and a non-tuberous and highly polymorphic indigenous medicinal fruit that is widely distributed in non-arid areas of Africa and has nutritional and therapeutic potential. A study reported that ripe and the unripe eggplants are used in folklore medicine. These eggplants is also used in Ghana and Cameroon cuisine as one of the ingredients of a dish called “Nkwi”. Despite the benefits of the unripe fruits being known, no study has reported the properties of the ripe fruit which is usually left to waste and rot in farms. The ripe eggplant fruits are usually discarded partly due to their low acceptability and the ignorance of their benefits and thus are among the post-harvest food crops lost after harvest in Nigeria, West Africa. Meanwhile, a study showed that ripening brings changes in the fruit content of phenolic compounds, organic acids, and carbohydrates, as well as in its color, texture, and flavor. Nigeria is facing an immense food insecurity challenge, a problem partly attributed to the increasing loss of food crops during post-harvest handling and distribution to retail markets. The reduction in post-harvest loss of this eggplant fruit and its use as a food ingredient for functional food development, such as in cookies and dumplings, could promote sustainable food security and a better health treatment/management of noncommunicable diseases and cardiovascular diseases , a task that requires an integrated approach . Therefore, this research was carried out on both ripe and unripe eggplants to compare their biological effects, with a focus on the anti-inflammatory, antioxidant, and anti-diabetic properties of a diet containing ripe Solanum anguivi lam fruits in diabetic Drosophila melanogaster .Ripe and unripe eggplant fruits were obtained from the botanical garden of the FUTA, Akure, as shown in Figure S1 . The identification and authentication of the fruits were performed at the FUTA Herbarium; and were recorded with number 0291a and 0291b. Common fruit processing methods were adopted, including cleaning, sorting, cutting, pulping, drying, and grinding the fruits as needed. The fruits were processed according to the method described by.Mineral elements were analyzed according to AOAC and Perkin-Elmer. The method described in was used to determine the total carotenoid content, while vitamin C determination was carried using a method reported in [16], vitamin A content was determined according to [17], and vitamin E content was obtained according to [18]. HPLC–UV [high-performance liquid chromatography with ultraviolet detection] was carried out on the samples as described in [19]. This is an advanced technique used for separating and quantifying bio-active compounds in a mixture. In this method, a liquid sample is loaded in a column, and its components are detected by their absorption of ultraviolet light, which provides quantitative and qualitative information. The standard compounds in Table 1 were investigated and identified according to a standard protocol.The eggplant fruits were washed, cut, and then blended into a puree. The puree was filtered through cheesecloth to collect the liquid, which was the eggplant extract and was freeze-dried.

As part of our horticultural program, we have crossed various cultivars of D. kaki and obtained their progenies segregating for fruit astringency. One of these crosses was made between two genetically distinct NA-types of D. kaki, cv. Luo Tian Tian Shi and cv. Taishu. Their F1 progenies were segregated into A- and NA-types, the latter of which appears to be determined by the presence of a dominant allele derived from cv. Luo Tian Tian Shi . In this report, we made use of these materials, attempting to elucidate the molecular mechanism of soluble PA accumulation in young persimmon fruits. We employed suppression subtractive hybridization to identify transcripts whose accumulation patterns were distinct between the segregated A- and NA-type fruits when their PA contents became distinct. Interestingly, only a few clones encoding Xavonoid biosynthetic enzymes were identified. Among cDNAs represented by multiple clones were those encoding a new member of the 1-Cys peroxiredoxin family and that of subgroup F of family 1 glycosyltransferases . A detailed sequence comparison and phylogenetic analysis revealed that the isolated 1-Cys Prx may be a novel type. In addition, UDP-galactose:anthocyanidin/Xavonol 3-O-galactosyltransferase activity of the GT homolog was conWrmed by using a bacterial expression system. These results may suggest complex mechanisms of PA accumulation in persimmon fruits.Generation of F1 progenies between the two distinct NAtype persimmon cultivars , their growth, and collection of their fruits were partly described by Ikegami et al. . Fruits usually become fully mature and ready to harvest for human consumption in the end of October to November. Immature green fruits used in this study were harvested considerably earlier, at three different dates , 25 liter plant pot during which PA accumulation started to decline in the NA-type but not in the A-type . The A- and NA-types used in this study were defined previously based on the size of PA-accumulating cells and the soluble tannin content of fully mature fruits . Fruit fresh was separated from seeds and peel, cut into small pieces , frozen with liquid nitrogen, and stored at ¡80°C before further analysis.

Total RNA was isolated from 5 g of the frozen sample using the hot borate method .Amino acid sequences were aligned with ClustalX . Phylogenetic analyses of the aligned amino acid sequences based on maximum parsimony were implemented in PAUP* with heuristic searches using the TBR branch-swapping algorithm 1,000 random taxon addition replicates and no limit on the number of trees saved. Relative support for clades was assessed using 1,000 bootstrap replicates with 10 random taxon addition replicates per bootstrap replicate. For the Prx sequences, 191 ambiguously aligned sites were excluded from the original alignment of 357 sites. A Wnal data set of 166 characters, of which 3 were constant, 6 were variable but parsimony-uninformative, and 157 were parsimony-informative, was subjected tophylogenetic analysis. The tree shown in Fig. 4a is one of four most parsimonious trees , arbitrarily rooted along a strongly supported internal branch. For the GT sequences, the complete alignment of 610 amino acid characters, of which 61 characters were constant, 62 were variable but parsimony-uninformative, and 487 were parsimony-informative, was used in phylogenetic analysis. The unrooted phylogram shown in Fig. 5a is one of three most parsimonious trees .The astringency of A-type fruits and that of NA-type fruits became distinct in those harvested in August. That is, the soluble PA content per dry weight of A-type fruits remained almost constant from June to August, whereas that of NA-type fruits dropped by more than 50% . By contrast, during this period, the concentrations of insoluble PAs remained largely unchanged and at comparable levels between the two fruit types ; and fresh weight of a fruit of both types increased similarly up to five times . These data suggest constant PA biosynthesis in A-type, but not in NA-type fruits.

It may also be possible that the level of Xavonoid oxidation, which has been shown to be negatively correlated with PA solubility , was lower in A-type than in NA-type. Hence, we decided to compare the transcript accumulation patterns in NA- and A-type fruits harvested in July and August. To identify differentially accumulating transcripts, we used suppression subtractive hybridization , which recently allowed us to isolate several transcripts including those encoding PA-biosynthetic enzymes whose accumulation levels were up- or down-regulated in persimmon fruits by ethanol treatment to remove astringency . We prepared RNAs from A- and NA-type fruits and generated reciprocal cDNA SSH libraries for samples of each data point. Among 4,800 recombinant clones that were randomly selected from these libraries, 37 clones showed significantly different accumulation patterns between the two fruit types by RNA dot blot assays, and their nucleotide sequences were determined . Based on the previous results , we had expected to find a number of PA-biosynthetic genes in A–NA libraries. Indeed, a total of nine independent clones involved in phenylpropanoid metabolism were isolated . However, none of them encodes an enzyme catalyzing one of the committed steps for PA biosynthesis, i.e., LAR or ANR. Some other clones identified in these libraries encode homologs to known proteins that do not play a direct role for PA biosynthesis . We also attempted to obtain full-length coding sequences of SSH clones isolated from A–NA libraries in order to facilitate further analysis. To this end, we screened a cDNA library from A-type persimmon fruits, and were able to obtain apparent full-length sequences for a subset of cDNAs . The presence of clones for the three Xavonoid biosynthetic enzymes, phenylalanine ammonia lyase , chalcone synthase , and dihydroXavonol 4-reductase , in A–NA libraries was consistent with the preliminary RNA-blotting data . Also identified in A-NA libraries were clones for two other Xavonoid biosynthetic enzymes, cinnamic acid 4-hydroxylase and Xavonoid 3 5 -hydroxylase .

Three truncated sequences may derive from nonoverlapping portions of a single mRNA encoding a C4H homolog . In addition, three SSH clones from the July sample were found to encode 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase , which is involved in the biosynthesis of aromatic amino acids to fuel the phenylpropanoid pathway. Finally, represented by the highest number of clones for both the July and August samples were the transcripts encoding a protein with high sequence identities to various plant Xavonoid GTs, such as UDPGlc:anthocyanidin 3-O-GlcT from Xower buds of Lobelia erinus L. , anthocyanin 3-O-GalT from cell suspension culture of Aralia cordata Thunb. , and kaempferol 3-O-GalT from pollen of Petunia x hybrida . The group of cDNAs not directly involved in phenylpropanoid metabolism consists of a total of eight independent clones. Three of them were found in A–NA libraries, encoding proteins similar to glucose acyltransferase , 1-Cysperoxiredoxin , and glutathione S-transferase , respectively. SCPL might be involved in PA accumulation, since correlation of its gene expression and accumulation of PAs has been reported at least twice, one in persimmon fruits and another in hairy roots of grape overproducing MYB transcription factors . The clone encoding a 1-Cys Prx homolog was represented by five SSH clones, whereas GST, which was shown to have multiple functions including conjugation to anthocyanidins in the cytoplasm for vacuole sorting , was represented by a single clone . Among the five cDNAs found in NA–A library, three clones may encode non-overlapping portions of a single protein with a high sequence similarity to LATE BLOOMER 1 from pea, which plays roles in photoperiodic Xowering, de-etiolation, and circadian regulation . The other two appear to encode distinct chitinases, among which is one for a class II chitinase-like protein, black plastic plant pots which was represented by Wve SSH clones.The citrus fruit, termed hesperidium, is a fleshy fruit which, like all berry-type fruit, is characterized by a thick and fleshy pericarp . The pericarp is usually divided into three tissues: the exocarp, which is the outer skin, the mesocarp, which usually refers to the major fleshy, edible interior, and the endocarp, an internal tissue composed of one or several cell layers. In true fruit, which develop from the ovary, these three tissues are part of the ovary wall. The exocarp of citrus fruit is the outer colored peel, often referred to as the flavedo . Proceeding inward is the albedo, the spongy white part of the peel. Most cell layers of the albedo are considered to be mesocarpal tissue, and the two or three innermost cell layers are referred to as endocarp . In mandarins, the albedo disintegrates during fruit maturation, leaving only the vascular system , which gives this group its name, Citrus reticulata. The pulp, the edible part of the fruit, is composed of juice sacs/vesicles that develop from the endocarp at an early stage of fruit development . Some authors refer to the juice sacs as endocarp, while others consider them to be a separate tissue. The juice sacs develop into the ovary locule, defined as the section in which the ovary wall that develops into fruit. The carpel and the juice sacs are covered by the same epidermal layer of segment epidermis . The juice sac is connected to the wall by a stalk, which joins the segment epidermis, so the latter provides one continuous layer covering both the segment and the juice sac.

Three major vascular bundles, a dorsal and two side bundles, are found in each section. Most juice sacs initiate from the dorsal wall, but some develop from the side wall, adjacent to the side vascular bundle . When present, seeds develop in the inner side of the fruit, where the carpels merge or along the ovary wall. Nutrition is supplied by a specific bundle, termed seed bundle, reaching from the fruit pedicle to the center of the fruit.The juice sac is a unique structure, found only in fruit of the genus Citrus and its close relatives. It is often referred to as a “sac of juice,” but this is misleading; the juice sac is composed of various layers of cells, each with distinct morphology . The vesicle primordia emerge from the endocarp soon after fertilization and fruit set. In a few cases, juice sac primordia are visible even before fertilization and fruit set, mainly when fertilization does not occur and parthenocarpic fruit develop . During fruit development, the vacuole of the juice sac cell becomes greatly enlarged, occupying over 90% of the total cell volume, and releases its content as juice. At fruit maturity, the vacuole contains about 100, 75, and 90% of the total cellular sucrose, hexose and citrate, respectively . The juice sac is considered the major fruit sink; however, it is disconnected from the vascular system, which ends in the albedo . This unique characteristic determines photo assimilate translocation rate into the sink cells and therefore, rate of fruit development, and the time required to reach maturity.In many citrus cultivars, the major external change that marks the conversion of the citrus ovary into a fruit let is usually petal fall . Fruit development is divided into three overlapping stages: cell division , cell expansion , and fruit maturation . During stage I, fruit growth is relatively moderate, and the peel, especially the albedo, thickens by cell division. During this stage, juice sacs grow out via cell division into the locule. Stage II is characterized by rapid fruit growth, mostly due to juice cell expansion. During stage III, the rate of fruit volume increase is greatly reduced. Externally, the major change is color break, and internally, sugar and acid levels reach the desired levels for harvesting and consumption, as discussed further by Spiegel-Roy and Goldschmidt . Changes in secondary metabolites give the fruit its unique aroma and flavor . As there is no respiration burst or autocatalytic ethylene production, the citrus fruit does not undergo the classical ripening process, typical of climacteric fruits. For a given citrus cultivar, the final flavor quality of the fruit has to be determined empirically and depends, largely, on consumer preference . The completion of fruit development is cultivar-dependent, with some cultivars, such as Satsuma mandarin , being ready for harvest 5–6 months after flowering, whereas others, such as Valencia orange , are harvested 12–14 months after flowering . In hot climates, fruit development is accelerated, potentially reducing the time needed for fruit maturation by ca. 50% .

This same spinach leaf antiserum has been shown to recognize AGPase protein purified from tomato leaf and fruit , and both of the antisera have been shown to recognize the small subunit of AGPase from a variety of other monocotyledonous and dicotyledonous species . The gels from which blots were prepared were loaded in two ways. First, lanes were loaded with samples of pellet, homogenate, and supernatant fractions, each of which contained the same activity of the plastidial marker enzyme, alkaline pyrophosphatase . If the AGPase protein is plastidial, the intensity of the AGPase band on the blot should be the same for these fractions. However, if the AGPase protein is wholly or substantially extraplastidial, the intensity of the band should be much greater in the homogenate and supernatant fractions than in the pellet fraction. Second, lanes were loaded with supernatant and pellet fractions that contained the same activity of the cytosolic marker enzyme, alcohol dehydrogenase . If the AGPase protein is cytosolic, the intensity of the AGPase band on the blot should be the same for the two fractions. However, if the AGPase protein is wholly or substantially plastidial, the intensity of the band should be much greater in the pellet fraction than in the supernatant fraction. In samples from pericarp and columella, both antisera strongly recognized only one band of an appropriate molecular mass to be a subunit of AGPase . Where lanes were loaded with equal activities of plastidial marker enzyme, the band was of approximately equal intensity in homogenate, pellet, and supernatant fractions . However, where lanes contained equal activities of cytosolic marker enzyme, growing strawberries vertically the band was visible in the pellet and not the supernantant fraction . These data indicate that AGPase protein is primarily or exclusively plastidial in the pericarp and the columella.

Our study provides strong evidence that AGPase activity and protein is mainly or exclusively plastidial in the pericarp and the columella of the developing tomato fruit. This conclusion is consistent with our observation that the ratio of ADP-Glc to UDP-Glc in developing fruit is very low . We suggest that ADP-Glc in tomato fruit is synthesized via a plastidial AGPase from Glc phosphate imported from the cytosol. Consistent with this idea, envelopes of plastids from tomato fruit are reported to have a hexose-phosphate-phosphate exchange transporter . The pathway we suggestfor tomato appears to occur in all other organs for which reliable information is available, including the embryos of oilseed rape and pea and the tubers of potato . Our results are at variance with those of Chen and colleagues, who reported that the stroma and the cytosol were labeled in sections of developing pericarp challenged with an antiserum to tomato AGPase. Chen et al. suggested that the cytosolic protein they detected might be an untransported precursor of the plastidial AGPase. It is likely that the antisera we used recognized primarily or exclusively the small subunit of the tomato enzyme. The amino acid sequences of small subunits are highly conserved between species, whereas those of large subunits are divergent . In studies with purified AGPase from tomato fruit, Chen and Janes found that the spinach antiserum recognized only the small subunit. It is possible, therefore, that the cytosolic protein detected by Chen and colleagues was an inactive form of the large subunit, which we did not detect. Regardless of the nature of the cytosolic antigen detected by Chen et al. , our results provide strong evidence that little or no active AGPase is present outside the plastid in developing fruit.Plant fruits protect developing seeds and aid in their dissemination .

They are also an important food source for humans and animals and are rich in nutrients such as carbohydrates, fats, proteins, vitamins, and trace elements . Fleshy fruit ripening and the generation of quality attributes occur towards the end of seed development and render fruit attractive to animal and human consumers, further aiding seed dispersal . Understanding fruit ripening provides an important theoretical and practical basis for manipulating the ripening process, improving fruit quality, and prolonging fruit shelf life . Tomato is a model plant for studying the ripening of climacteric fruit because of its simple diploid genetics, small genome size , short life cycle, ease of transient and stable transformation, distinct ripening phenotypes, and abundant bio-informatics resources . Molecular genetics studies have shown that tomato fruit ripening is governed by a transcription regulation network that is coordinated by a series of ripening-related transcription factors  and ethylene . Exploring the roles of these ripening-related TFs is an effective tool for understanding the mechanisms involved in fruit ripening. Tomato has abundant natural mutants , some of which have obvious ripening inhibited phenotypes, such as ripening-inhibitor , non-ripening , Colorless non-ripening , Never-ripe , and Green-ripe . Nr and Gr are related to ethylene signal transduction, while rin, Cnr, and nor TFs are involved in the transcription regulation network controlling the expression of tomato fruit ripening-related genes that determines quality attributes. However, several detailed studies of rin, Cnr, and nor mutants involving CRISPR/ Cas9 gene editing have caused the roles of these mutants to be re-evaluated . In rin mutant, almost all ripening-related phenotypes, including ethylene biosynthesis, carotenoid accumulation, fruit softening, and flavor synthesis, were significantly inhibited. In addition, the sepal size of rin mutant is increased, and the inflorescence is less ordered .

Studies have shown that rin is formed by the deletion of the 3′ end of the MADS-RIN gene and the 5′ end of the MADS-MC gene, resulting in the formation of a RIN–MC fusion gene. MADSRIN is considered to regulate tomato fruit ripening, while MADS-MC is considered to affect sepal development and inflorescence, and rin was considered a loss-of-function mutant of RIN . The phenotype of the rin mutant is a near complete inhibition of ripening, and based on this evidence, RIN was considered the core TF required for the tomato fruit ripening process, including ethylene biosynthesis and signal transduction, carotenoid synthesis, cell wall metabolism, aroma synthesis, sucrose metabolism, and other biological pathways . Recent studies, however, have shown that the fusion protein RIN–MC in the rin mutant retains biological functions, and the role of RIN has been re-evaluated in light of this evidence . The RIN protein segment of the RIN–MC fusion protein functions in binding DNA, while the adjacent MC region possesses a transcription repression function. This chimeric protein, RIN–MC, produced by the rin mutant is thus a gain-of-function mutant and active TF responsible for the inhibition of expression of ripening genes. It was concluded from this evidence that RIN was not required for the initiation of ripening but was essential for the completion of normal ripening . Compared with wild-type , the Cnr mutant has reduced ethylene synthesis, fruit softening, and carotenoid synthesis in pericarp tissue . Mapping and identification of Cnr by Manning et al. showed that SPL-CNR belongs to the SBP family of TFs. There was no alteration in the SPL-CNR DNA sequence, but its promoter region was hypermethylated, and the transcription of the SPLCNR gene was inhibited, giving rise to the Cnr ripening mutant phenotype . This was the first report of methylation affecting the expression of fruit ripening genes, but the exact cause of the methylation of SPL-CNR in the Cnr mutant remains unclear. Using CRISPR/Cas9 to edit SPLCNR in WT fruit, Gao et al. found that the ripening of CR-CNR fruits was similar to that of WT tomatoes, and CR-CNR fruits fail to show a Cnr mutant phenotype. Therefore, the mechanism of action of the Cnr mutant and the function of SPL-CNR requires explanation and further study. Studies on the nor mutant and the function of NAC-NOR have lagged behind those of RIN and SPL–CNR and there is little information available regarding the mechanism of action of nor and the function of NAC-NOR. The synthesis of ethylene and carotenoids in the fruit of the nor mutant is significantly inhibited, and the fruit does not ripen. Giovannoni et al. discovered by map-based cloning that the nor mutant was caused by the deletion of two adenines in the third exon of the NAC-NOR gene, which belongs to the NAC gene family. Due to this frameshift mutation, the NAC–NOR protein in the nor mutant encodes a truncated NOR protein of 186 amino acids , which disrupts the transcriptional activation region but preserves the complete DNA-binding region. Based on this evidence, the nor mutant phenotype was considered to be due to loss of function of the NAC-NOR gene, and NACNOR was considered to be a core TF regulating the initiation of tomato fruit ripening. Most NAC-NOR-related studies are based on the use of the nor mutant as experimental material. Yuan et al. compared the proteome differences between the nor mutant and WT tomato fruit by isobaric tags for relative and absolute quantification and found that the accumulation of many ripening-related and disease-resistance proteins was altered in the nor mutant. Additionally, the NACNOR mutation in Penjar tomato inhibited various metabolic processes and prolonged the shelf life of fruit , drainage planter pot whereas the over expression of NAC-NOR accelerated the senescence of tomato leaves . In addition to NAC-NOR, several other NAC TFs have been reported to be involved in regulating tomato fruit ripening. For example, the over expression of SlNAC1 in tomato resulted in a decrease in ethylene synthesis and the early softening of fruit, producing a yellow to orange phenotype . In addition, the silencing of SlNAC4 in tomato fruit resulted in a 2–3 d delay in fruit ripening and significantly inhibited ethylene biosynthesis, chlorophyll degradation, and carotenoid accumulation .

The ripening process in tomato fruit with CRISPR/Cas9 gene editing of NOR-like1 was significantly delayed for more than 2 weeks, and ethylene, carotenoid synthesis, and fruit softening were inhibited in CR-NOR-like1 fruit compared with WT . Surprisingly, however, we have recently been unable to obtain a nor mutant phenotype in NAC-NOR-edited fruit using CRISPR/Cas9 , which was published simultaneously by the de Maagd laboratory , who demonstrated Thus, the nor mutant may be a gain-of-function mutant, similar to rin, although the specific mechanism of action is unclear. If nor is a gain-of-function mutant, the role of NAC-NOR in the normal development and ripening of tomato and the function of the normal NAC-NOR gene in tomato fruit development and ripening need to be re-evaluated. In this study, we investigated the results of CR-NOR and OE-NOR at the physiological, cellular, and molecular levels. The results showed that the residual protein NOR186 of the nor mutant could not only enter the nucleus but also bind to the promoters of NAC-NOR target genes, but could not activate them. While mixing the WT NOR protein and the nor mutation NOR186 protein, the activation effect of NOR target promoters was inhibited compared with the WT NOR protein present alone. In addition, over expression of the NACNOR gene in the nor mutant did not restore the normal ripening phenotype of tomato, providing evidence for the gain-of-function of NOR186 in the nor mutant. Transcript accumulation studies indicate that NAC-NOR still plays an important role as a positive regulator in tomato fruit ripening. These results re-evaluated the role of NAC-NOR in tomato fruit ripening and help place it in the context of the transcriptional regulatory network regulating tomato fruit ripening.To investigate the activity of the NAC-NOR gene during fruit development, the accumulation of NAC-NOR transcripts in various WT tomato plant organs and during fruit development and ripening was measured by qRT-PCR. The results showed that the expression of the NAC-NOR gene in vegetative organs such as root, stem, and leaf of tomato was low, while it was high in reproductive organs such as flower and fruit , which suggested that it may play an important role in tomato fruit ripening. Ethylene is a key hormone in the ripening of climacteric tomato, and many ripening-related genes are induced by ethylene during fruit ripening . To study the relationship between NAC-NOR expression and ethylene, we used treatment with an ethylene-generating compound and an ethylene perception inhibitor to treat WT tomato fruits at mature green and breaker stages of fruit ripening, respectively. The results showed that the expression of the NAC-NOR gene in tomato fruit was induced by ethylene but inhibited by 1-MCP .

As overall invertases activities were considerably higher than SuSy activities, and assuming that the extractable activities of these enzymes in the total protein extracts paralleled their in vivo activities, our results would suggest that invertases provided the major route for Suc breakdown, as suggested in peach and tomato . Moreover, the reduction in invertase activities in both cultivars throughout the ripening-related stages, in agreement with the increase in transcript levels of INVINH was also observed in peach underlining the key role invertases play in determining overall fruit sugar composition . During ripening, Glu and Fru contents were higher in Santa Rosa fruits . Although VINV activity could be contributing to the higher hexose amounts in Santa Rosa fruits, the enhanced Sor breakdown in Santa Rosa fruits was a significant source of Glu and Fru in Santa Rosa. This conclusion is supported by the higher NAD+-SDH and SOX enzymatic activities and NAD+-SDH transcripts, and lower NAD+ levels in Santa Rosa fruits and by the report showing that NAD+-SDH is the key enzyme determining Fru concentrations in peach fruits . An additional observation supporting the above described scenario comes from both the HK transcript levels and HK enzyme activity. HK, which phosphorylates Glu into G6P, the precursor of Sor, displayed both lower activity and low HK transcript levels in Santa Rosa than in Sweet Miriam fruits . In apples, a decreased HK activity was observed throughout ripening, stacking flower pot tower suggesting a lower glucose metabolism similar to what was observed in Santa Rosa fruits.A noteworthy difference between Santa Rosa and Sweet Miriam fruits was their altered UDP-Gal metabolism.

These differences were indicated by the higher levels of the polyols Gol and Ino and the oligosaccharide Raf, and the lower contents of Gal in Sweet Miriam . UDP-Gal can be targeted towards cell wall biosynthesis through galactosyltransferases , can be used as a substrate for Suc synthesis through itsinterconversion to UDP-Glu , or can be used as a substrate for Raf biosynthesis via GolS . Our results suggested that in Santa Rosa, UDP-Gal was targeted towards Gal, while in Sweet Miriam it was targeted towards Raf . In Santa Rosa, these results were probably a consequence of the increased cleavage of galactosyl residues from cell wall polysaccharides through β-GAL, especially during the fruit softening stage , and increased cleavage of Raf into Gal and Suc through α-GAL . In Sweet Miriam fruits and leaves, UDP-Gal seemed to be targeted towards Raf biosynthesis via increased GolS and RS transcript levels, as overall Raf contents were higher in Sweet Miriam . In addition, Ino, a substrate for GolS and released by RS , provides a cycle which was overall increased in Sweet Miriam as compared with Santa Rosa . What might be the roles of the elevated contents of Gal in Santa Rosa and Gol, Raf, and Ino in Sweet Miriam? Free Gal, higher in Santa Rosa fruits, has been shown to increase ethylene production and induce earlier ripening in mature green tomatoes . This promotion of ripening due to the Gal-induced increase in ethylene production has been associated with the stimulation of 1-aminocyclopropane-1-carboxylic acid synthase activity, the rate-limiting step in ethylene biosynthesis, as well as to a transient increase in 1-aminocyclopropane-1-carboxylic acid . Thus, the higher levels of Gal in Santa Rosa  would suggest a link with the climacteric behavior of this cultivar, that could be further explored. Regarding Gol, Raf, and Ino, several reports have indicated that these metabolites are associated with protection against stresses due to their high antioxidant capacities . The fruit ripening process comprises a series of oxidative activities , specifically during cell wall breakdown and overall fruit softening .

Gol and Raf were dramatically elevated in peach fruits exposed to heat and cold stresses during post-harvest storage . Therefore, it is possible that the higher contents of Gol, Ino, and Raf in Sweet Miriam fruits improve their ability to cope with the oxidative processes occurring during ripening, as suggested by Aizat et al. in non-climacteric Capsicum. During ripening in post-harvest storage, these compounds were also higher in Sweet Miriam fruits, supporting their role as oxidative stress protectants . In addition to Gol, Ino, and Raf, Sweet Miriam fruits and leaves also displayed higher Tre contents, that were well correlated with lower TRE transcript levels . Tre has also been reported to play signaling/regulatory roles in plant stress responses , suggesting that the increase in Tre contents in Sweet Miriam could also contribute to cope with ripening-associated oxidative stress conditions. While in our previous publication we concentrated on characterizing sugar contents and some of their corresponding enzymes using two phenological stages and only in fruits, here we used a systems biology approach, combing gene expression, metabolomics, and biochemical analyses to show a reprograming of metabolism of major and minor sugars occurring in fruits and leaves of a non-climacteric bud mutant plum cultivar at four developmental stages. Non-climacteric plums accumulated higher amounts of Sor and lower amounts of Suc, Glu, and Fru than climacteric plums, and the higher amounts of Sor were a consequence of both increased synthesis, mediated by S6PDH, and decreased breakdown, mediated by NAD+-SDH and SOX. The non-climacteric behavior was also associated with a shift of UDP-Gal metabolism towards Raf and Gol, as well as the increase in Tre, probably playing a role in improving the overall ability of non-climacteric fruits to cope with oxidative processes associated with fruit ripening.

The lower Gal contents in Sweet Miriam could also play a role in its non-climacteric behavior due to the reported capacity of free Gal to induce ethylene production through stimulating ACS activity. Whether the differences in ethylene and ripening behavior between the two cultivars are also dependent on changes of other hormones is currently under investigation.Most flowering plants are perfect-flowered hermaphrodites , but hermaphrodites are not necessarily equisexual . Instead, pollen and ovule counts reveal continuous variation from predominantly male to predominantly female gamete production, both among plants in a population and among the flowers produced by an individual . This variation invites the question, why should one hermaphroditic individual be more or less female than another? Evolutionary stable strategy models indentify several factors that together determine a plant’s optimal sex allocation . These models find the sex allocation strategy that, if adopted by all members of the population, cannot be invaded by an alternative, initially rare, strategy. Factors affecting sex allocation optima include the shapes of male and female gain curves , the strength of competition among related pollen grains and related seeds, and the rate of self-fertilization . When the environment is assumed to be homogenous, ESS models find a single optimum for individuals in a population. If, however, the environment is heterogeneous, sex allocation optima may differ among plants for two reasons. First, environmental heterogeneity can alter the shape of gain curves. In wind-pollinated species, for example, the male gain curve may decelerate more rapidly in closed habitatsthan in open habitats . Second, environmental heterogeneity can induce variation in plant size, which may alter the shape of gain curves , and/or lead to variation in the size of reproductive investment made. Plants making a larger absolute investment are expected to allocate relatively more to the function whose gain curve decelerates slower . Optima can also differ among sequentially opening flowers on a plant. Like individuals, danish trolley flowers that differ in reproductive investment will vary in sex allocation optima if male and female gain curves are not identical . Variation in sex allocation optima also occurs when flowers vary in selfing rates: flowers with a higher selfing rate are expected to allocate more to female function than those with a lower selfing rate when inbreeding depression , 0.5; the prediction is reversed when d . 0.5 . The strongest driver of among-flower variation in allocation optima, however, is among-flower variation in pollen transfer probability. This can be caused by temporal shifts in the mating environment arising from flower dichogamy . Other causes of variation in pollen transfer probability include the tendency of pollinators to visit inflorescences from bottom-to-top , and varying pollinator attraction as display size changes . All else being equal, flowers with the greatest pollen transfer probability are expected to allocate relatively more to male function . Empirical study of sex allocation variation in hermaphroditic plants has focused largely on systems exhibiting dichogamy, or, to a lesserextent, pollinator directionality or variable selfing rates . Less attention has been paid to adichogamous, self-incompatible species lacking pollinator directionality, even though sex allocation variation can occur in such species if plants or flowers vary in reproductive investment .

Moreover, a particular type of variation in investment – declining fruit-set from first to last flowers on plants – might cause variation in the mating environment of adichogamous plants by altering the quality of siring opportunity over time. In their ESS model, Brunet & Charlesworth assumed a constant probability of fruit-set across all flowers, despite among-flower variation in reproductive investment. Fruit-set probability, however, frequently declines from first to last flowers on plants , and this decline is often attributed to resource pre-emption by first flowers . This post-fertilization decline in resources clearly lowers the expected female reproductive success of last flowers on plants. Less obvious are the effects on expected male mating opportunity. If first flowers are more likely to set fruit, then those flowers and plants that transfer pollen to the first flowers of others are more likely to fertilize ovules that successfully mature into seed. This siring advantage should fall to first flowers on plants, and to early-flowering plants in a population, because these are more likely to temporally coincide with the first flowers of others. Brunet recognized that declining fruit-set reduces the expected male success of last flowers on plants, and Weis and Kossler predicted that declining fruit-set leads to decreasing male success from early- to late- flowering plants . The consequences of this temporal shift in mate quality for relative mating success through the two genders have not, however, been fully examined. We asked the following questions. Does functional gender vary within plants when fruit-set declines? Does the predicted among-plant decline in male success cause appreciable variation in functional gender? Are within- and among-plant trends in expected functional gender matched by within- and among-plant trends in relative male and female investment? To answer these questions, we developed a numerical model examining effects of declining fruit-set probability on expected male success and functional gender. Informed by model results, we characterized within- and among-plant variation in pollen, ovule and fruit production in Brassica rapa . We contrast temporal trends in relative male and female investment in B. rapa to temporal trends in functional gender predicted by the model.The numerical model confirmed that declining fruit-set probability from first to last flowers on plants can, under a wide range of conditions, induce temporal variation in expected male success and functional femaleness . In most cases examined, average functional femaleness decreased sharply in the last flower class on plants. In contrast, at the among-plant level, functional femaleness increased substantially from early- to late-flowering plants in all cases where fruit-set declined . Temporal trends in phenotypic femaleness in B. rapa opposed expected trends in functional femaleness: phenotypic femaleness increased from first to last flowers on plants , and decreased from early- to late-flowering plants . Below, we discuss the numerical model in relation to previous ESS models, and examine the discrepancy between predicted trends in functional femaleness and observed trends in phenotypic femaleness.Variation in the mating environment of sequentially opening flowers on plants can lead to variation in their sex allocation optima . Effects of dichogamy are particularly well studied. Brunet and Charlesworth reasoned that dichogamy creates a temporal shift in the quantity of male- versus female-phase flowers , and therefore generates a temporal trend in total expected pollen transfer probability from first to last flowers on plants. This shift in the mating environment causes variation in sex allocation optima . This ESS model prediction is well supported by data from several dichogamous species . Dichogamy was not, however, the only scenario examined in this ESS model. Brunet and Charlesworth also reasoned that when resources decline from first to last flowers on plants, first flowers offer a higher quality siring opportunity than last flowers .

To our knowledge, no other study has tested whether naturally occurring soil biotas from multiple undisturbed habitats affect flowering phenology and selection on flowering time, nor has any study of the relationship between soil microbes and phenology explicitly accounted for genetic variation among undomesticated plant populations.Sterilized potting soil was saturated with inoculum and sterilized field soils were saturated with buffer for 10 days. Then, 48 accessions of Boechera stricta were transplanted as gnotobiotic seedlings into all treatments . All pots were randomized into blocks of 200 and maintained under controlled greenhouse conditions throughout the experiment, except for 7 weeks of 4°C vernalization in a growth chamber. We measured plant height and leaf number on the date of first flowering. Here we focus on phenology and fecundity of the 51% of plants that flowered successfully. Among those plants that flowered, the experimental design exhibited only modest imbalance, with substantial sample sizes remaining in every subspecies × treatment cell . Factors influencing probability of flowering and other fitness components are beyond the scope of this paper; we found no evidence that any experimental treatments affected flowering probability of surviving plants . Reproductive fitness was estimated as the number of fruits on each individual at 33 weeks of age; in B. stricta, fruit set is strongly and positively correlated with seed production in the greenhouse .Soils were collected from four natural Boechera stricta habitats in central Idaho, USA, separated by ~26 to ~92 km and differing in elevation, temperature, water availability, density and diversity of vegetation, and many soil properties .

Collection locations were named ‘Jackass Meadow’ , ‘Mahogany Valley’ , ‘Parker Meadow’ , and ‘Silver Creek’ . These remote sites have little history of disturbance by humans, are home to endogenous B. stricta populations, hydroponic net pots and function well as common gardens for B. stricta field experiments . Therefore, they are legitimate potential habitats that B. stricta likely encountered during its evolutionary history in this region. Each soil collection comprised five sub-samples from the four corners and approximate center of a ~150 m2 area, at a depth of ~10 to 30 cm. Subsamples were combined and mixed thoroughly, sieved through ~1.25 cm wire mesh to remove rocks and coarse detritus, shipped to Duke University, and stored in plastic bags at 4°C for ~3 months until use. We also collected seven ~1 mL soil vouchers from each site for microbial community analysis: three in August 2011, and at all sites except PAR, four in August 2012. Vouchers were frozen at -20°C until DNA extraction in late 2012. B. stricta seeds were collected from 48 natural populations, including four from the soil collection sites. Their sites of origin span over 1,000 m in elevation and are separated by between ~1 km and ~350 km, with the exception of the “SAD12” genotype from Colorado. Because B. stricta is naturally inbred and exhibits high FIS and FST , each population was represented by a distinct genotype. This diverse collection of genotypes included 24 from each of the ecologically divergent “east” and “west” subspecies . We used seeds from a single mother descended from the original field-collected accession, self-pollinated in the greenhouse for at least one generation, to minimize maternal effects; i.e. individuals within a genotype were self-full sibs .To create four soils that were identical except for their microbial communities, we extracted microbes from field soils into sterile buffer and soaked sterilized potting soil in the resulting suspensions.

We prepared inocula from 75 g sub-samples of each field-collected soil stirred into 1 L of 2.5 mM MES monohydrate in sterile diH2O . After settling for 30 min the suspensions were vacuum filtered to remove particulates. Filtrates were centrifuged 30 min at 3,000×G at room temperature to pellet microorganisms. To remove dissolved nutrients, we discarded the supernatants and re-suspended the microbe-enriched pellets in 1 L sterile 2.5 mM MES. This process mostly eliminated variation in chemical properties that differentiate the field soils . Each rack of 200 pots was bottom-saturated with 400 mL of one of the microbial suspensions, 6 g 20-10-20 fertilizer, and sterile diH2O for a total treatment volume of 4 L. The fertilizer was added to encourage seedling survival and to counteract possible soil impoverishment due to autoclaving . An additional 1 mL of undiluted microbial suspension was pipetted into each pot. Treatments derived by this process are termed “biotic” or “microbial treatments” throughout. It is possible that the filtration and re-colonization processes somewhat altered community structures; however, the differences between our experimental inocula—and their effects on the plants—originate from corresponding differences between real Boechera habitats.We sterilized soils from four natural habitats to create growth substrates with different physical and chemical properties, but without their natural microbiomes. After sub-sampling to extract microbial communities , we sterilized the four field-collected soils via autoclaving . These soils were loosely packed into clean pots and bottomsaturated with 400 mL sterile 2.5 mM MES, 6 g 20-10-20 fertilizer, and sterile diH2O to bring the treatment volume to 4 L.

An additional 1 mL sterile 2.5 mM MES was pipetted into each pot. Treatments derived by this process are termed “abiotic” or “sterilized field soils”. Although it is likely that autoclaving these soils changed their fertility, they appear to have retained at least some of their natural chemical variation .Surface-sterilized seeds of 48 genotypes were stratified on auto claved filter paper at 4°C in the dark for two weeks, then placed in a growth chamber to germinate for one week . Four germinated seedlings per genotype were transplanted into each of the eight experimental soils described above, one seedling per pot. Eight pots per treatment were left unplanted as controls. All pots were immediately rearranged into randomized blocks and maintained in controlled greenhouse conditions for the duration of the experiment. Plants were top-watered as needed with RO water, and received an additional 4 mL 20-10-20 fertilizer via pipet when one month old. Two-month-old plants were transferred to a 4°C vernalization treatment, where they remained for seven weeks. After vernalization, plants were returned to the greenhouse, checked three times weekly for flowers and allowed to set fruit. Flowering was defined as sufficient separation of the corollasuch that four distinct petals could be identified. The number of days between end of vernalization and first flowering is termed interchangeably “flowering time” and “flowering phenology” throughout. On the day of first flower for each plant we measured the individual’s height and number of leaves. The last census was done eight weeks post-vernalization; the 749 plants that had not flowered by this date were excluded from all future analyses. The experiment ended two months after the final flowering census, when almost all fruits had matured and dehisced. These cutoffs for flowering time and fruit production are realistic given the short growing season observed in the field. At this time we counted the number of fruits produced by each individual.Due to current methodological limitations, in this study we focus on the prokaryotic component of the soil microbiome . We extracted DNA from field collected soil vouchers using the MoBioTM PowerSoil DNA Isolation Kit and amplified variable region 4 of the bacterial 16S rRNA gene using established primer pairs 515F and 806R and PNA PCR clamps to reduce plastid and mitochondrial contamination . Paired-end 2×250bp sequencing of barcoded amplicons was performed on a MiSeq machine running v2 chemistry at the Joint Genome Institute . The primer sequences were trimmed from the paired-end sequences, blueberry grow pot which were then overlapped and merged using FLASH . Merged sequences were grouped into operational taxonomic units based on 97% sequence identity, and chimeric sequences were removed, using the UPARSE pipeline . Taxonomies were assigned as in Lundberg et al. . Unclassifiable OTUs at the kingdom level, OTUs matching Viridiplantae, mitochondrial, or plastid sequences were excluded by using BLAST to compare them to a custom database of contaminant sequences . Unclassifiable OTUs at the kingdom level and rare or non-reproducible OTUs were also excluded as in Lundberg et al. , resulting in 7,844 OTUs. To control for unequal sequencing effort, we normalized data by rarefaction to 40,000 reads/sample using QIIME-1.7.0 . Diversity analyses were performed after correcting data for 16S gene copy number variation using scripts provided in Kembel et al. ; OTUs without taxonomic information were assigned the mean copy number . Linear regressions were performed before this correction but the resulting parameter estimates were adjusted as necessary.To test the hypothesis that soil properties affect flowering time, we used restricted maximum likelihood linear mixed models with treatment, subspecies, and treatment × subspecies as fixed factors; block, genotype nested within subspecies, and treatment × genotype as random factors; and elongation rate , height at first flowering , and leaves per mm stem as covariates .

To test for treatment effects on overall plant size we used MANOVA with treatment, subspecies, and treatment × subspecies as fixed factors. We analyzed the parallel “biotic” and “abiotic” experiments separately, i.e. one model tested only for effects of soil microbiomes on flowering time, and another identical model tested only for effects of physical soil differences; we did not directly compare the effects of these two types of soil variation. These models were run using JMP® Pro version 10.0.0 . Statistical significance of random effects was determined by REML likelihood ratio test and results were graphed using ggplot2 in R version 3.0.2 . To test the hypothesis that soil properties alter selection on flowering time, we used a REML linear mixed model with the same terms as above, plus flowering time and flowering time × treatment as additional fixed effects. The response variable was number of fruits. Thus, the flowering time term describes the change in fecundity attributed to a change in flowering time, i.e. selection. We analyzed the parallel “biotic” and “abiotic” experiments separately as above. We performed these models both with and without including elongation rate, height at first flowering, and leaves per mm stem as covariates; the former model describes the selection gradient on flowering time , and the latter model describes the selection differential . Introducing a quadratic term did not improve fit, so our model considers only linear effects . Sample sizes were slightly smaller than for the flowering time models because 17 individuals were accidentally discarded after flowering. Main models were performed in JMP; selection differentials and selection gradients were calculated in R. For some models, non-uniformly distributed residuals might have influenced judgments about significance. In general, results with and without covariates were similar, suggesting that significance was robust to heteroscedasticity in the standard model. For the sake of caution, for all of our major results, we performed permutation tests to verify the results of the standard ANOVA. Rarefied microbial communities were analyzed in the R package ‘vegan’ . Principal coordinates of Bray-Curtis pairwise dissimilarities were identified using the vegan function ‘capscale’. Similarity of samples within vs. among sites was tested using the non-parametric permutation test ADONIS with 9,999 permutations constrained by collection year. To ask which components of microbial communities affect flowering phenology and selection, we regressed mean flowering time in each biotic Treatment onto the mean PCo score from the corresponding Site. To ask which OTUs underlie the observed phenotypic effect, we identified the ten OTUs most highly correlated with the PCo axes and regressed the same flowering time residuals on the OTUs’ mean abundances at each site. We used the Wilcoxon rank-sum test to compare relative abundances of common taxa between groups of samples associated with extreme phenotypes. P-values were adjusted using Benjamini-Hochberg false discovery rate. Our method of searching for microbial community members that underlie our phenotype of interest is described in more detail in Appendix S3.Selection on flowering time depended on soil microbiome. This result held both for selection gradients and selection differentials . The most extreme change was between the PAR and SIL soil biotas , with selection differentials of +0.034 and -0.043 fruits/day , respectively. The magnitude of this difference in selection is 1.2 times the selection differential measured in a nearby field site .

This finding is consistent with a report that both supine and standing HRs were significantly increased 1h and 3h after a 790 kcal meal in the morning after an overnight fast. However, the calorie content in mango and white bread in this current study was only 298 kcal. Studies regarding the consumption of fruits and postprandial BP and HR are scarce. Future research is encouraged to investigate whether fruit intake will induce similar hemodynamics as meals. In study I, the 2h change in blood glucose was not different between mango or no mango intake, despite the difference in sugar intake from the fruit. This observation was reinforced further in study II, where the blood glucose was significantly increased 1h after white bread intake but not after eating an isocalorically-matched amount of mango. The insulin level was also significantly increased 1h after white bread intake compared to 1h after no mango or mango intake. In addition, although the 2h change in blood glucose after eating white bread returned to a level similar to baseline values, the 2h change of insulin was still significantly elevated compared to the 2h value seen in the no mango group. These data are consistent with other reports regarding mango consumption and glucose regulation. For example, in obesity-prone mice fed a high-fat diet, dutch bucket hydroponic the fasting blood glucose, insulin, and homeostatic model assessment for insulin resistance score were significantly decreased after 10 weeks of mango fruit powder intake ateach of three levels .

In obese male C57BL/6J mice consuming a high-fat diet, daily supplementation freeze-dried mango at either 1% or 10% of the weight of the diet significantly reduced body fat compared to those consuming a non-supplemented control diet. Curiously, only the 1% mango group showed significantly decreased fasting blood glucose and postprandial blood glucose responses after tolerance tests, but no difference was noted for insulin or HOMA-IR, compared to those consuming the 10% supplementation or control diets.30 In overweight and obese humans, plasma insulin was significantly increased 45 min after consuming 100 kcal of mango , compared to their baseline levels, but did not increase as much as when the participants consumed an to isocaloric low-fat cookie. The same study also noted that capillary blood glucose levels were significantly elevated 30 min after mango intake compared to their baseline values, while returning to the baseline range at 60, 90, or 120 min after intake, whereas intake of the low-fat cookie showed significantly increased blood glucose at both 30 and 60 min, which is consistent with trends from our study. However, the above study measured insulin at baseline and 45 min after food intake, so the postprandial insulin levels cannot be compared directly with our study. Future research may consider assessing the association between postprandial BP, glucose, and insulin resistance at multiple time points. This study has several limitations. The Ataulfo mangos were not analyzed for nutrients or phenolic contents. Different mango cultivars vary in macronutrients, micronutrients, as well as phytonutrient content.

Among commonly consumed mango cultivars, Ataufo mango pulp contains the highest concentration of β-carotene, ascorbic acid, total phenolics, gallotannins, and mangiferin, in comparison to Haden, Keitt, Kent, and Tommy Atkins. The high concentrations were used in the selection of Ataulfo. The amount of white bread as an isocaloric control was calculated based on the USDA food database, which does not identify the cultivar or cultivars that were tested. Finally, the postprandial blood glucose and insulin responses in study II were not measured at 30 min, which may have missed the possible peak levels. Future studies may take the measurements at more frequent time points, as well as insulin resistance indicators, such as HOMA-IR, to better understand the role of mango in blood glucose management. In conclusion, two weeks of daily mango intake was associated with a decrease in SBP and PP. The glucose and insulin responses after mango intake were also moderated, compared to ingesting of an isocaloric amount of white bread. While the effects of mango intake on microvascular function were not as significant as the response from other whole foods, other measures of cardiovascular health, as well as glucoregulatory benefits, warrant further study.Epidemiological studies suggest that diets rich in carotenoids can be beneficial for vision, heart, bone health, cognitive performance, and cancer prevention. The current review focuses on the potential role of the xanthophyll carotenoids lutein and zeaxanthin in eye health, specifically their potential role in reducing risk of age-related macular degeneration .

We review the absorption, distribution, and metabolism of L and Z, and the current dietary recommendations for these carotenoids, then speculate about their putative role in maternal and infant health. Lastly, we discuss the potential value of goji berry within the diet as a food with the highest known amount of Z. Carotenoids contribute to the bright red, orange, and yellow color in plants. These fat-soluble phytochemicals are classified into two categories: carotenes, which include only hydrocarbons, and xanthophylls that also contain oxygen. While some dietary carotenoids serve as vitamin A precursors most of the approximately 100 carotenoids found in plants do not. Among the carotenoids devoid of vitamin A activity are L and Z, along with meso-zeaxanthin , a stereoisomeric metabolite of L. Absorption involves enterocyte uptake by CD36, scavenger receptor class B type I , and Niemann-Pick C1-like transporter 1 at the apical membrane. Xanthophylls are then secreted through the basolateral membrane of the enterocyte, mainly by ATP binding cassette A1 and carried by lipoproteins to target tissues. SR-B1, SR-B2, and CD36 transport L and Z into the tissues. Steroidogenic acute regulatory domain protein 3 has been identified as a binding protein for L in the retina, and glutathione S-transferase pi isoform for Z. Lutein, Z, and meso-Z impart a distinctive yellow color to the fovea of primates – the specialized central area of the macular region of the retina that is rich in cone photoreceptors and optimized for high-acuity central color vision. The compounds have a maximal absorbance at a wavelength near 460 nm and are most concentrated in the inner and outer plexiform layers, which consists primarily of axonal connections between the retinal layers. Their combined density is greatest in the center of the macula and decreases with increasing retinal eccentricity. In the central fovea, the concentration of Z and meso-Z is higher than L at a ratio of 2.4:1. Lutein is most abundant in the peripheral macula, with a Z + meso-Z to L ratio of 1:2 when measured by high performance liquid charomatography. However, a newer technique, confocal resonance Raman microscopy suggests that the Z + meso-Z to L ratio is as high as 9:1 at the central fovea.16 Protection from blue light is critical for eye health. Compared to longer wavelengths of visible light, short blue wavelengths are higher in energy and generate reactive oxygen species . Zeaxanthin can provide stronger oxidant defense than L during photooxidation, while lutein has a greater capacity to absorb short wavelength light irradiation in lipid membranes. Compared to other carotenoids , L and Z are more effective in scavenging ROS and can also reduce phospholipid peroxidation. The photoreceptor-retinal pigment epithelium complex in the outer retina is particularly susceptible to ROS damage due to its high polyunsaturated lipid content . Quenching of singlet oxygen appeared best when L, Z, and meso-Z were mixed in equal ratios rather than separately when assessed in an eye tissue model, suggesting some synergy between the these macular pigments in their antioxidant properties. The most common method to quantify xanthophylls in the retina is to assess macular pigment optical density . This parameter is measured through techniques such as heterochromatic flicker photometry , a non-invasive psychophysical technique, fundus reflectometry, resonance Raman spectroscopy, or autofluorescence imaging. The MPOD index is associated with plasma levels of L and Z, and has been used to assess the risk for AMD. However, some studies report no correlation between MPOD and risk of AMD,31 which suggests that other ocular measures may be useful to obtain a more complete profile of AMD risk. In human donor eyes, dutch buckets system the amount of L and Z was inversely associated with AMD. Supplementation of L, Z, and meso-Z have been shown to significantly increase MPOD in both healthy individuals and patients diagnosed with AMD. However, studies using foods rich in L and Z have produced inconsistent results, which may be due to the relatively modest amounts of these carotenoids in foods compared to supplements.

Importantly, the plasma concentration of L and Z has been more strongly associated with MPOD than the correlation between MPOD and dietary intake. Age-related macular degeneration is the third leading cause of blindness worldwide after uncorrected refractive errors and cataracts. An estimated 288 million people worldwide are projected to suffer from AMD by 2040. In the United States, the prevalence of early-stage AMD was 9.1 million in 2010, and this number is projected to increase to 17.8 million by 2050. AMD is characterized by a gradual loss of eyesight from the central visual field. Although the exact etiology of AMD is not clear, common pathologic progress includes oxidative stress, lipofuscin toxicity, lipid accumulation, immune dysregulation, and choroidal hyperperfusion. Age-related processes such as a decrease in retinal neuronal elements, alterations in the size and shape of RPE cells, and thickening of Bruch’s membrane also participate in the pathology of AMD. Damage to mitochondria in RPE cells has also been suggested to play a role. Dry AMD, also termed non-exudative AMD, involves the formation of drusen, which are mainly lipid and protein deposits that accumulate between the RPE and Bruch’s membrane in the macula. In contrast, wet AMD, also termed exudative or neovascular AMD, is a consequence of abnormal blood vessel formation arising from the choroid, known as choroidal neovascularization . Clinically, AMD is classified as early or intermediate stage based on the size and number of drusen, as well as presence of pigmentary changes. The AMD is considered late or advanced stage in the presence of CNV, where fluid accumulation may result in damage to the neurosensory retina and fibrous scarring, or geographic atrophy , where loss of the RPE result in damage to overlying photoreceptors and underlying choriocapillaris causing irreversible vision loss. The main risk factors for AMD are aging and smoking, although some studies have shown no difference in MPOD between healthy older individuals and healthy young. Other risk factors may include race, obesity, previous cataract surgery, presence of cardiovascular disease, and hypertension. According to the U.S. National Institutes of Health, the prevalence of AMD is highest among Caucasians as compared to other races, and higher in females than in males. Genetic factors are also associated with AMD, with several high-risk single-nucleotide polymorphisms identified from genome wide association studies. The strongest risk variants include the Y402H variant of complement factor H gene as well as those in the age-related maculopathy susceptibility 2 locus. Whether the color of the iris or sunlight exposure are related to the risk of AMD is still being explored. Dietary interventions using L- and Z-rich foods have generated inconsistent results regarding the risk of AMD. In a cohort study that assessed dietary carotenoid consumption among individuals without AMD at baseline over more than 20 years, increased predicted plasma carotenoid score of L, Z, β-carotene, α-carotene and β-cryptoxanthin were associated with a lower risk of advanced, but not early or intermediate AMD. Similarly, a meta-analysis of six longitudinal cohort studies found that the dietary intake of L and Z significantly reduced the risk of GA by 26% and CNV by 32%, with no apparent impact on early stages. Another metaanalysis concluded that supplementation with L, Z, and meso-Z significantly increased MPOD levels in both AMD patients and healthy individuals in a dose-response manner. However, whether the improvement in MPOD could be sustained after L and Z supplementation is discontinued remains unclear. The Age-Related Eye Disease Study was a multi-center study that assessed the efficacy of a dietary anti-oxidant supplements on subjects who are 50 to 80 years old, with and without AMD or cataracts, for more than seven years. The initial study used a formula containing 15 mg of β-carotene, 500 mg of vitamin C, 400 IU of vitamin E, with or without 80 mg of zinc and 2 mg of copper. Lutein and Z were not included because the scientific evidence to include these two carotenoids was not yet clear.

Climate change, including climate variability, must be considered as this may change extant regional dynamics of both coffee and CBB, and their interactions. Increased temperature may generate conditions favorable for coffee and CBB allowing range extensions to new areas, and changes in CBB damage levels in its current geographical distribution. Increased dry “El Niño” climatic events in some countries could increase CBB populations, while “La Niña” events with prolonged wet seasons would limit CBB populations. Te effects of such phenomena differ across geographical region, and the coffee/coffee berry borer system model provides a framework for analyzing the potential effect of variation in weather, climates and of climate change on coffee yield, and the dynamics of CBB across diverse bio-geographical zones. As an aside, the high pest status of this species in monocultures is a consequence of an evolutionary background, similar to what have been observed in other systems . From the prospective of the ecological theory, the large female bias appears to have had high adaptive value in the African tropical forest where it evolved so that large numbers of the small females with low searching rates could find scattered patches of suitable age berries. This adaptation would appear to occur at the expense of reduction in genetic variability caused by sib-mating and reported pseudo-arrhenotoky. As a final note, our C. arabica PBDM can easily be modified to include other species of coffee , strawberry gutter system and has transferability enabling its use in a bio-economic analysis on larger, albeit global scale, and in the face of climate change.Agriculture is a key human activity in terms of food production, economic importance and impact on the global carbon cycle.

As the human population heads toward 9 billion or beyond by 2050, there is an acute need to balance agricultural output with its impact on the environment, especially in terms of greenhouse gas production. An evolving set of tools, approaches and metrics are being employed under the term “climate smart agriculture” to help—from small and industrial scale growers to local and national policy setters—develop techniques at all levels and find solutions that strike that production-environment balance and promote various ecosystem services. California epitomizes the agriculture-climate challenge, as well as its opportunities. As the United States’ largest agricultural producing state agriculture also accounted for approximately 8% of California’s greenhouse gas emissions statewide for the period 2000–2013. At the same time, California is at the forefront of innovative approaches to CSA . Given the state’s Mediterranean climate, part of an integrated CSA strategy will likely include perennial crops, such as winegrapes, that have a high market value and store C long term in woody biomass. Economically, wine production and retail represents an important contribution to California’s economy, generating $61.5 billion in annual economic impact. In terms of land use, 230,000 ha in California are managed for wine production, with 4.2 million tons of winegrapes harvested annually with an approximate $3.2 billion farm gate value. This high level of production has come with some environmental costs, however, with degradation of native habitats, impacts to wildlife, and over abstraction of water resources. Although many economic and environmental impacts of wine production systems are actively being quantified, and while there is increasing scientific interest in the carbon footprint of vineyard management activities, efforts to quantify C capture and storage in annual and perennial biomass remain less well-examined.

Studies from Mediterranean climates have focused mostly on C cycle processes in annual agroecosystems or natural systems. Related studies have investigated sources of GHGs, on-site energy balance, water use and potential impacts of climate change on productivity and the distribution of grape production. The perennial nature and extent of vineyard agroecosystems have brought increasing interest from growers and the public sector to reduce the GHG footprint associated with wine production. The ongoing development of carbon accounting protocols within the international wine industry reflects the increased attention that industry and consumers are putting on GHG emissions and offsets. In principle, an easy-to-use, wine industry specific, GHG protocol would measure the carbon footprints of winery and vineyard operations of all sizes. However, such footprint assessment protocols remain poorly parameterized, especially those requiring time-consuming empirical methods. Data collected from the field, such as vine biomass, cover crop biomass, and soil carbon storage capacity are difficult to obtain and remain sparse, and thus limit the further development of carbon accounting in the wine sector. Simple yet accurate methods are needed to allow vineyard managers to measure C stocks in situ and thereby better parameterize carbon accounting protocols. Not only would removing this data bottleneck encourage broader participation in such activities, it would also provide a reliable means to reward climate smart agriculture.

Building on research that has used empirical data to compare soil and above ground C stocks in vineyards and adjacent oak woodlands in California, this study sought to estimate the C composition of a vine, including the relative contributions of its component parts . By identifying the allometric relationships among trunk diameter, plant height, and other vine dimensions, growers could utilize a reliable mechanism for translating vine architecture and biomass into C estimates. In both natural and agricultural ecosystems, several studies have been performed using allometric equations in order to estimate above ground biomass to assess potential for C sequestration. For example, functional relationships between the ground-measured Lorey’s height and above ground biomass were derived from allometric equations in forests throughout the tropics. Similarly, functional relationships have been found in tropical agriculture for above ground, below ground, and field margin biomass and C. In the vineyard setting, however, horticultural intervention and annual pruning constrain the size and shape of vines making existing allometric relationships less meaningful, though it is likely that simple physical measurements could readily estimate above ground biomass. To date, most studies on C sequestration in vineyards have been focused on soil C as sinks and some attempts to quantify biomass C stocks have been carried out in both agricultural and natural systems. In vineyards, studies in California in the late 1990s have reported net primary productivity or total biomass values between 550 g C m−2 and 1100 g C m−2. In terms of spatial distribution, some data of standing biomass collected by Kroodsma et al. from companies that remove trees and vines in California yielded values of 1.0–1.3 Mg C ha−1 year−1 woody C for nuts and stone fruit species, and 0.2–0.4 Mg C ha−1 year−1 for vineyards. It has been reported that mature California orchard crops allocate, on average, one third of their NPP to the harvested portion and mature vines 35–50% of the current year’s production to grape clusters. Pruning weight has also been quantified by two direct measurements which estimated 2.5 Mg of pruned biomass per ha for both almonds and vineyards. The incorporation of trees or shrubs in agroforestry systems can increase the amount of carbon sequestered compared to a monoculture field of crop plants or pasture. Additional forest planting would be needed to offset current net annual loss of above ground C, representing an opportunity for viticulture to incorporate the surrounding woodlands into the system. Astudy assessing C storage in California vineyards found that on average, grow strawberry in containers surrounding forested wildlands had 12 times more above ground woody C than vineyards and even the largest vines had only about one-fourth of the woody biomass per ha of the adjacent wooded wildlands.The objectives of this study were to: measure standing vine biomass and calculate C stocks in Cabernet Sauvignon vines by field sampling the major biomass fractions ; calculate C fractions in berry clusters to assess C mass that could be returned to the vineyard from the winery in the form of rachis and pomace; determine proportion of perennially sequestered and annually produced C stocks using easy to measure physical vine properties ; and develop allometric relationships to provide growers and land managers with a method to rapidly assess vineyard C stocks. Lastly, we validate block level estimates of C with volumetric measurements of vine biomass generated during vineyard removal.The study site is located in southern Sacramento County, California, USA , and the vineyard is part of a property annexed into a seasonal floodplain restoration program, which has since removed the levee preventing seasonal flooding. The ensuing vineyard removal allowed destructive sampling for biomass measurements and subsequent C quantification.

The vineyard is considered part of the Cosumnes River appellation within the Lodi American Viticultural Area, a region characterized by its Mediterranean climate— cool wet winters and warm dry summers—and by nearby Sacramento-San Joaquin Delta breezes that moderate peak summer temperatures compared to areas north and south of this location. The study site is characterized by a mean summer maximum air temperature of 32 °C, has an annual average precipitation of 90 mm, typically all received as rain from November to April. During summer time, the daily high air temperatures average 24 °C, and daily lows average 10 °C. Winter temperatures range from an average low 5 °C to average high 15 °C. Total heating degree days for the site are approximately 3420 and the frost-free season is approximately 360 days annually. Similar to other vineyards in the Lodi region, the site is situated on an extensive alluvial terrace landform formed by Sierra Nevada out wash with a San Joaquin Series soil . This soil-landform relationship is extensive, covering approximately 160,000 ha across the eastern Central Valley and it is used extensively for winegrape production. The dominant soil texture is clay loam with some sandy clay loam sectors; mean soil C content, based on three characteristic grab samples processed by the UC Davis Analytical Lab, in the upper 8 cm was 1.35% and in the lower 8–15 cm was 1.1% . The vineyard plot consisted of 7.5 ha of Cabernet Sauvignon vines, planted in 1996 at a density of 1631 plants ha−1 with flood irrigation during spring and summer seasons. The vines were trained using a quadrilateral trellis system with two parallel cordons and a modified Double Geneva Curtain structure attached to T-posts . Atypically, these vines were not grafted to rootstock, which is used often in the region to modify vigor or limit disease .In Sept.–Oct. of 2011, above ground biomass was measured from 72 vines. The vineyard was divided equally in twelve randomly assigned blocks, and six individual vines from each block were processed into major biomass categories of leaf, fruit, cane and trunk plus cordon . Grape berry clusters were collected in buckets, with fruit separated and weighed fresh in the field. Leaves and canes were collected separately in burlap sacks, and the trunks and cordons were tagged. Biomass was transported off site to partially air dry on wire racks and then fully dried in large ventilated ovens. Plant tissues were dried at 60 °C for 48 h and then ground to pass through a 250 μm mesh sieve using a Thomas Wiley® Mini-Mill . Total C in plant tissues was analyzed using a PDZ Europa ANCA-GSL elemental analyzer at the UC Davis Stable Isotope Facility. For cluster and berry C estimations, grape clusters were randomly selected from all repetitions. Berries were removed from cluster rachis. While the berries were frozen, the seeds and skins were separated from the fruit flesh or “pulp”, and combined with the juice . The rachis, skins and seeds were dried in oven and weighed. The pulp was separated from the juice + pulp with vacuum filtration using a pre-weighed Q2 filter paper . The largest portion of grape juice soluble solids are sugars. Sugars were measured at 25% using a Refractometer PAL-1 . The C content of sugar was calculated at 42% using the formula of sucrose. Below ground biomass was measured by pneumatically excavating the root system with compressed air applied at 0.7 Mpa for three of the 12 sampling blocks, exposing two vines each in 8 m3 pits. The soil was prewetted prior to excavation to facilitate removal and minimize root damage. A root restricting duripan, common in this soil, provided an effective rooting depth of about 40 cm at this site with only 5–10 fine and small roots able to penetrate below this depth in each plot. Roots were washed, cut into smaller segments and separated into four size classes , oven-dried at 60 °C for 48 h and weighed.

Nesting substrate, therefore, has a smaller or negligible effect on bacterial abundance compared to differences among states. Ceratina calcarata foraged from a slightly greater phylogenetic richness of floral genera in New Hampshire than in Georgia. The phylogenetic richness of floral genera in Missouri did not significantly differ from either Georgia or New Hampshire, perhaps because of its mid-lying geographic position and climate. As expected, our reanalysis of the New Hampshire data with 99% ESV matching recovered fewer genera than in McFrederick and Rehan, who found 110 genera compared to this study with 65. We identified the same five genera as being the most abundant , these genera accounting for 92% of the reads . Despite this more conserved estimate of genera, the floral resources used in New Hampshire are still rich compared to those utilized in Georgia. We also found that foraging females in Missouri foraged from more plants to form a single pollen provision mass than those in Georgia . This suggests that suitable floral resources at the time of brood provisioning may not be as diverse in Georgia as more northern areas of Ceratina’s range, or that they were simply not locally abundant in the area around the collected nests. Across its geographic range, C. calcarata encounters a broad variety of possible forage. Diets in Georgia, Missouri and New Hampshire were dominated by pollen from different plant genera . Out of the 96 floral genera found in provisions in this study, only Rubus was found in more than 1% of reads across all three states . All other genera, even if abundant in one or two states, were rare in provisions from the third. For example, grow bucket sumac was a key floral resource in New Hampshire but made up less than 10% of the reads in Georgia and was hardly utilized at all in Missouri .

It is important to note that while read counts have been correlated with microscopy pollen counts in many studies, factors such as pollen morphology can skew the abundance estimate obtained from DNA sequences. Our study uses the marker rbcl, which has shown strong correlation with pollen counts, outperforming trnL and ITS2. With this in mind, comparison of relative abundance between sites shows state-wise differences in diet. Many of these plant genera are common to all three states, so perhaps these dietary variations are due to differences in bee and floral phenologies, as well as possible microhabitat distinctions in floral assemblages in proximity to the bee nest. While we do not have data on floral distributions within each collecting site, our records of nest substrate allow us to determine that foraging was not skewed towards the host plants. Rubus was a common pollen source but even nests formed within Rubus plants did not show a bias in pollen collection. Different pollens vary in nutritional qualities, which may influence foraging decisions. Pollen can also have toxic constituents, and some generalist foragers appear to actively utilize a broad range of floral resources to alleviate the effects these may have on brood development. How these factors influence C. calcarata foraging is unknown but our results suggest that spatial orientation of floral resources alone does not determine foraging preferences. The presence of a consistent core microbial community despite the variation in pollen sources suggests that many of the most common bacterial genera do not have specific floral associations. We identified a number of tentative bacteria–plant correlations, but these were not consistent among states . In the overall analysis, the tupliptree genus Liriodendron was correlated with Lactobacillus, while the same plant genus was correlated with Sphingomonas in Georgia. In Missouri, Wolbachia was correlated with four plant genera: Brunia, Camptotheca, Rhus and Smilax but this bacterium was not correlated with plants in the other states or the overall analysis.

The correlations found in Georgia and Missouri also differ to those previously identified in New Hampshire, following the same methodology. These correlations broadly suggest that plants and bacteria are co-occurring but the variance in results between the overall dataset and the state-level analyses indicates these relationships are facultative or transient. Using read data to identify co-occurrence correlations is statistically challenging and further experiments sampling pollen bacterial communities with and without pollinator visitation, such as the study by McFrederick et al., are needed to directly test for plant–bacteria associations. Whether plants harbor certain microbes over others or not, there are many factors altering microbial floral communities. Long-term artificial warming of grassland plots was found to alter the microbial communities of plant leaves, including microbial groups common to bees. Aydogan et al. found Acinetobacter and Wolbachia increased in frequency, while Sphingomonas frequency decreased, these three bacterial genera being common to C. calcarata pollen provision and adult gut microbiomes. These temperature based microbial changes could translate into changes in insect microbiomes, and indeed climate has been correlated with changes in microbiome composition in some species such as the red palm weevil, the chestnut weevil and a spider mite. Flower visitation by bees can transfer microbes to flowers, but herbivorous insects, other pollinators including thrips and wind are thought to contribute to microbe dispersal as well. Similarly, the presence of potentially predatory or competitive species such as ants can reduce floral visitation and this in turn alters the microbes present on flowers. Any and all of these could be important factors influencing the observed microbiome variation in C. calcarata and are important considerations when concerned with wild bee health generally. Our study shows that the diet of C. calcarata varies widely with geography, with only Rubus found in more than 1% of reads at all three sites, indicating that this generalist bee species is able to utilize different resources as floral communities change. However, it seems that floral preference may not be simply determined by the proximity of the floral resource to the nest.

The same six bacterial genera consistently dominated provisions in all sites but the relative abundance of these fluctuated widely. There are still many unknowns regarding how microbes are acquired, both in the pollen provisions and subsequently the bees themselves. Flowers appear to be general points of bacterial transmission,but so far specific associations have not been identified. The current lack of knowledge on microbial associates is a major hindrance in our ability to maintain diverse wild bee populations.The fresh market berry industry in Santa Cruz and Monterey counties is an excellent example of transformation in the business of agriculture over the last 50 years. Located along the Central Coast of California, the two counties span the fertile Pajaro and Salinas valleys, and are well known for their amenable climate and production conditions, their diverse crop mix and grower demographics, and their developed agricultural infrastructure and support industries. The majority of the berry sector is comprised of strawberries , raspberries and blackberries , with blueberries and other miscellaneous berries produced on a much more limited basis. Substantial research-based literature and historical information is available for Central Coast strawberries; however, despite the area’s move towards greater production of raspberries and blackberries, less information exists for these crops. We seek here to provide a more complete portrayal and historical context for the berry industry in the Santa Cruz and Monterey area, which is the origin of the berry industry in California. While the berry industry has been very successful in recent decades, it now faces new challenges, such as invasive pests and the phaseout of the soil fumigant methyl bromide. This article draws on previous and more recent research to discuss some of the influences that have contributed to the berry industry’s dramatic expansion in Santa Cruz and Monterey counties, including selected innovations in agricultural practices and heightened consumer demand. Berry industry growth During the 1960s and 1970s, dutch bucket for tomatoes the number of acres planted to berries, tons produced and value of production fluctuated. The fluctuations can be partly explained by farm management: in the past growers often rotated berry and vegetable crops to assist with soil and pest management, thereby influencing these statistics. However, annual crop reports from the county agricultural commissioners show that since the 1980s, berries have become increasingly important to each county’s overall value of production, and by 2014 accounted for 64% and 17% of the total value of all agricultural products in Santa Cruz and Monterey counties, respectively . The industry’s growth can be explained by a shift of some acreage out of tree fruits and field crops , among others, into berries, and by additional acreage put into agricultural production.Strawberries are the undisputed leader in the berry sector and in 2014 represented 58% and 94% of the value of all berry production in Santa Cruz and Monterey counties, respectively , and 50% and 93% of all berry acreage . Table 2 documents the remarkable expansion of the strawberry industry over time in both counties with respect to acreage, tons produced and value of production. Between 1960 and 2014, acreage more than tripled and production increased tenfold. The value of production, in real dollars, increased by 424% in Monterey County and by 593% in Santa Cruz County, reaching an astonishing combined value of nearly $1 billion in both 2010 and 2014. The gains in all statistical categories in Monterey County were enabled in part by an expansion of production into the southern reaches of the county where more and larger blocks of farmland are available, and where land rents are lower than in Santa Cruz and northern Monterey counties.

However, from 2010 to 2014 Monterey County’s tonnage and production values declined, possibly because the area has recently experienced a shortage of labor to harvest fresh market crops. Tonnage was also lower in Santa Cruz County, but production values increased. This may be because of the county’s greater emphasis on local agriculture, organic production and direct market sales, which are often associated with higher crop values. For raspberries, the acreage, tons produced and value of production grew steadily and most strikingly in Santa Cruz County , where production conditions for caneberries are optimal. For example, caneberry fields in Santa Cruz County are situated in areas that have well-drained soils and are protected from damaging winds. Also, fields are planted to take advantage of the growth and yield gains associated with southern exposures. Moreover, field-to-cooler travel distances are shorter in Santa Cruz County, which is critical for safeguarding the quality and marketability of these highly perishable crops. By 2014, raspberries represented 33% of the county’s total value of production for all berries. In contrast, Monterey County raspberry production accounted for only 6% of the county’s total berry value. Blackberries have not been consistently reported as a separate category in archived statistical analyses, but instead were often included under the terms “bush- or miscellaneous berries”. Therefore, similar data for blackberry acreage and value of production cannotbe reported here. However, between 1990 and 2010, Santa Cruz County agricultural commissioner crop reports reported an upward trend for the broad category with respect to acreage planted and value of production . In 2010, blackberries were promoted to a position of prominence in the report and shown as a separate statistic; at the same time, the miscellaneous berry category was shown to be very small indeed. Between 2010 and 2014, however, blackberry acreage and value of production leveled off and have shown only modest gains . This may be because there has been less emphasis on production and market research and promotion for blackberries than for strawberries or raspberries. No comparable data are available for Monterey County. The two counties have contributed significantly to California’s total berry sector: in 2014, area strawberry acreage represented 35% of the statewide total, 37% of the total tons produced and 38% of the total value of production . Area raspberry acreage represented 43% of the statewide total, 42% of the total tons produced and 39% of the total value of production. Comparable statewide statistics are not available for blackberries. County agricultural commissioners’ reports show that the majority of all berries produced in the two counties — up to 98% — are sold as fresh market fruit . In years with adverse production conditions or low prices, a higher percentage of the crop may be diverted to the freezer or processed products market. Fresh market fruit is handled and sold primarily through local grower-shippers; a much smaller share is sold directly to consumers through farmers markets, community supported agriculture operations, farm stands and other direct and intermediated market channels such as restaurants, independent grocers and schools.