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.