Several studies have compared hydrolyzable tannin profiles in developing pomegranate fruits. However, the fruit developmental stages were defined by different standards, such as days after fruit set/full bloom, physico-chemical properties, or physiological attributes of the fruit . Developing fruits of two cultivars, “Wonderful” and “Rosh Hapered,” grown in Israel were collected during a span of 8 or 10 weeks . Three major hydrolyzable tannins, gallagic acid, punicalin isomers , and punicalagins, were quantified in water extracts of the developing fruits. Fruits of “Ruby” grown in South Africa were harvested at five stages according to days after full bloom . Total hydrolyzable tannins in aril juice declined during the progression of fruit maturation, and were accompanied by decreases in ellagic acid and gallic acid . Relative amounts of hydrolyzable tannins in fruit peel, aril juice, and seed of developing pomegranate fruits were also investigated. Fruits of the Chinese cultivar “Taishanhong” were harvested at 10-day intervals for nine collections. Unicalagins, ellagic acid, and gallic acid were higher in fruit peel than aril juice and in seed; all three metabolites showed decreased accumulation in the three tissues during fruit development . When quantified by absorption of the methanolic extracts at 550 nm, total hydrolyzable tannins gradually decreased in fruit peels at low, low-medium, medium, plant pots with drainage and medium-high stages of the Spanish cultivar “Mollar de Elche.” In contrast, they were not detectable in aril juice at all stages.
In seeds total hydrolizable tannins increased at medium and then decreased at medium-high stages . Overall, despite the differences in the genetic background, growth conditions, harvesting scheme, and extraction and quantification methods, there is a consistent trend of decreasing hydrolyzable tannin accumulation in fruit peels, aril juice, and seed through pomegranate fruit development.To understand the impact of growth environment on hydrolyzable tannin profiles, fruit peel and aril juice hydrolyzable tannins were compared for 11 accessions grown in the Mediterranean or desert climate in Israel . Mediterranean climate promoted high levels of hydrolyzable tannins in aril juice in most of the accessions evaluated; in contrast, desert climate had a positive impact on hydrolyzable tannins in fruit peels . It was reported that the sweet/sour phenotype and environment interactions had the most influence on total tannin variations in aril juice of 10 commercial cultivars grown in four different regions in China, followed by the growth environment . There were negative correlations of overall average temperature with total polyphenol, total tannin, and punicalagin concentrations. The sweet/sour phenotype only accounted for 0.06% of the variations in tannins among different cultivars . The quality of aril juice under deficit irrigation was investigated in Spain . Three water regimes were applied to pomegranate trees at 75% evapotranspiration , 43% ETo , and 12% ETo . Water stress drastically decreased punicalagins, causing 30 and 70% reduction in moderate and sever stresses, respectively, in aril juices of fruits harvested from the corresponding trees . This study provided valuable information on the implications of water stress on the hydrolyzable tannin metabolism and the nutritional value of aril juice.Plants have developed diverse mechanisms to regulate their biological and metabolic processes via transcription factor regulatory networks .
Among the TF families, the basic leucine zipper family is present in all eukaryotes and is one of the largest and most diverse TF groups in higher plants. There are about four times more bZIP genes in the Arabidopsis genome than in the genomes of other model organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster . Large numbers of bZIP TF family members have been found in many plant species including rice , maize , tomato , common wheat , sorghum , soybean , banana , cassava , grape , peach , strawberry , apple , rapeseed , radish , cucumber , tea plant , sweet potato, watermelon/melon , Chinese jujube , pepper , Chinese pear , poplar , quinoa and plum . The bZIP family is phylogenetically categorized into different groups, with different species having various members of homologs. For example, the Arabidopsis AtbZIP family members were systematically classified into 10 groups based on conserved motifs . Subsequently, a more complete classification was expanded into 13 groups, designated as A-J, M, and S . The tomato SlbZIPs were classified as nine clades . The cucumber CsbZIPs and sorghum SbbZIPs were separately categorized into six and seven groups . The bZIP family in both rice and maize has 11 groups which are the same as castor bean . The plum PmbZIP proteins were divided into 12 groups . Chinese pear PbbZIPs were categorized into 13 groups . Several interspecies clustering studies indicate that the S group found in Arabidopsis has especially high homology across different species , although some clades might be specific to Arabidopsis compared to peach, strawberry, and apple . These classifications, phylogeny, and homology analyses define the possible biological roles of bZIPs in green plant evolution .
Basic leucine zipper TFs orchestrate a diverse array of functions in multiple biological processes including flower development and pollen development , seed maturation , senescence , light signaling , anthocyanin and chlorophyll biosynthesis , nutrient signaling , hormone signaling such as salicylic acid, ABA, ethylene, auxin, and cytokinin , sugar signaling , and abiotic/biotic stress signaling in plants. Group S is the largest bZIP subgroup in several species such as Arabidopsis and safflower and comprises three to four even smaller subgroups. In this review, we focus on the well-studied S1-bZIP subgroup, whose members contain unique conserved upstream open reading frames in the 50 region of their transcripts and play important regulatory roles in many metabolic processes relating to fruit quality and stress responses. Our review aims to provide perspectives for further surveying the biological function, exploring regulatory mechanisms, and genome engineering the S1-bZIPs to obtain desirable traits for quality improvement in horticultural plants.Of the AtbZIPs, the 17 members of the S group are further separated into three subgroups based on homology: S1, S2, and S3 . The S1 subgroup in Arabidopsis contains five members: AtbZIP1, −2, −11, −44, and −53. Recent studies indicate that other species, including many horticultural plants, also have multiple members of the S1- bZIP subgroup . Like other bZIP members, those in the S1 subgroup are characterized by a conserved bZIP domain, composed of two functionally distinct motifs located on a contiguous α-helix. The basic region of −18 amino acids contains, sequentially, a nuclear localization signal and an invariant N-x7-R/K-x9 motif for DNA binding. This motif preferentially binds to the A-box, C-box, and G-box of target promoters which contain DNA sequences with an ACGT core . The leucine zipper comprises a heptad repeat of leucines or other numerous hydrophobic amino acids . Compared to other groups, members of the S group include the extraordinarily high number of eight hydrophobic amino acid repeats . The two subunits form a zipper structure that binds DNA to form dimers through interactions with the hydrophobic sides of the helices . Of three S subgroups, only members of the S1 subgroup show specific heterodimerization with C group bZIP proteins , whereas weak homodimerization within members of the S1 subgroup is detected . Phylogenetic analysis between S1 and C group bZIPs from angiosperms, gymnosperms, mosses, and algae suggests that the S1 and C groups evolved from a proto-S/C bZIP in algae species that homodimerized, which has since diverged into heterodimerizing pairs prior to the evolution of seeds plants .Besides their common structural features, S1-bZIPs are unique in that they have an unusually long 50 -leader sequence in the upstream region of the main open reading frame of the mRNA. This leader sequence contains several upstream open reading frames that encode small peptides . Among those, the second uORF is conserved and encodes a Sucrose Control peptide of 28 residues, plastic plants pots which regulates the translation of the mORF and reduces protein expression through a mechanism known as Sucrose-Induced Repression of Translation , which contributes to sucrosehomeostasis in the cells . Here, we summarize uORFs of the S1-bZIP subgroup from different horticultural plants, including banana , grape , apple , peach , cucumber , strawberry , petunia, and white pear . The regulation of gene expression involves different layers, including transcriptional and translational controls . Compared with transcriptional regulation, translational control allows more immediate responses to adjust protein expression and reprogram metabolism upon cellular signals or environmental stimuli . The translation process of mRNA includes four major steps: initiation, elongation, termination, and ribosome re-initiation . Translation initiation is the major step that determines the rate of protein biosynthesis and is regulated by multiple mechanisms . uORFs have been suggested to play a critical role in regulating the translation of the mORF . uORFs of S1-bZIPs are involved in the translational regulation in a SIRT manner .
The SC-peptide encoded by the uORF in the 50 leader region of AtbZIP11 is capable of repressing translation of the subsequent mORF in the presence of sucrose . High sucrose levels enhance ribosome stalling on the uORF, which results in poor translation of the mORF . The members of the Arabidopsis S1-bZIP subfamily show similar responses to sucrose. Translation of AtbZIP1, AtbZIP2, AtbZIP11, AtbZIP44 and AtbZIP53 is down regulated by sucrose . Transgenic seedlings with 35S:bZIP11 50 leader::LUC show significantly reduced luciferase activities when treated with sucrose while those incubated in media lacking sucrose show two- to three-fold higher luciferase activities . SIRT-mediating S1-bZIP orthologs exist in all seed plants .Previous research showed that amino acids such as serine, leucine, and tyrosine in the conserved peptide of uORF are essential for SIRT . However, it has been shown that expressing the gymnosperm 50 uORF sequence, which only contains the conserved leucine and tyrosine in Arabidopsis cells efficiently mediates the translational repression of the LUC reporter gene in response to sucrose . This study suggests that the SIRT mechanism most likely depends on structural conformation, but not on recognition of specific sequence motifs . Recently, interesting research conducted using gene-editing technology in strawberry demonstrated that uORFs are involved in regulating protein translation efficiency and sucrose content . In the study, to manipulate the SC-uORF of FvebZIPs1.1, the start codons of the uORF and the codons encoding a conserved pair of amino acid arginine within the SCpeptide were edited using the CRISPR/Cas9 system. Mutations in the start codons and the conserved C-terminal region of the SC-peptide significantly reduced translation of the SC-uORF. This consequently enhanced the translation efficiency of the downstream mORF. Seven novel alleles with C-to-T substitutions and small deletions within the uORF were identified. To test if phenotypic effects were additive in heterozygous and biallelic plants, 4000 T1 seedlings were generated by crossing the biallelic and homozygous T0 mutants to each other and to wild type. 35 novel genotypes were obtained in T1 and inherited in T2 generation. In comparison with wild-type fruits, the mutants had significantly higher levels of fructose, glucose, and total sugar contents, demonstrating that engineering the conserved SCuORF of FvebZIPs1.1 can increase the sugar content in strawberry . In addition, the citric acid content was slightly lower in the homozygous mutants than that in wild type. A continuum of gradual increase of sugar contents was generated in T1 by combining heterozygous, homozygous, and biallelic mutants, and inherited in T2 generation by propagating stolons of these T1 mutants, therefore confirming the transmissibility of novel genotypes and phenotypes from T1 to T2 by asexual propagation . Given that sugars can modulate multiple growth and development processes, the agricultural traits including leaf shapes, leaf areas, plant height, growth rates, pollination, fruit size and fruit weight were further evaluated in FvebZIPs1.1 uORF mutants. Remarkably, editing SC-uORF does not severely impair plant growth. The agricultural traits in FvebZIPs1.1 uORF mutants were similar to wild-type , whereas impaired phenotypes and retarded growth are observed in transgenic lines with the overexpression of AtbZIP11, tbz17, and FvbZIP11 mORF . Taken together, this suggests a broad application of editing uORFs of S1-bZIPs for quality improvement in horticultural plants.Amino acids are not only involved in plant response to stress but also influence fruit flavor . For example, asparagine is present in almost all fruits and determines fruit flavor and quality in a concentration dependent manner . Glutamate is responsible for “umami” or savory taste . Glycine, alanine, serine, threonine, proline, glutamine, and lysine are highly correlated with sweetness , while phenylalanine and tyrosine are bitter .