The cis-acting region of the rd29A promoter contains three dehydration-responsive elements and one abscisic acid responsive element.We hypothesized that under water stress conditions, the over expression of LeNCED1 using the stress-inducible promoter rd29A would increase the ABA and proline concentration in leaves, leading to stomatal closure, reducing the osmotic potential and enhancing drought resistance in petunia, while under non-stress conditions, there would be no negative effects on plant growth and development.Our data strongly support the hypothesis that over expression of LeNCED1 using a stress-specific promoter would enhance drought resistance in petunia without negative effects on plant growth and development under non-stress conditions. Previous researchers overexpressed NCED using a constitutive promoter such as CaMV- 35S. Although they found increased ABA accumulation, faster stomatal closure and enhanced drought resistance in several plants including tomato,Arabidopsis,Nicotiana plumbaginifolia,and tobacco;the transgenic plants showed defective growth and development, increased seed dormancy and delayed germination.In the CaMV-35S:LeNCED1 petunia lines that we generated, similar effects,stacking pots including a marked photobleaching phenotype in leaves of mature plants, were observed . To avoid these pleiotropic effects,strawberry gutter system we generated petunia plants over expressing the LeNCED1 gene under the control of the stress responsive rd29A promoter.

Inducible promoters are a very powerful tool used in plant genetic engineering to regulate the expression of genes in a particular tissue or at specific stages of development.In normal growth conditions, the transgenic plants were indistinguishable from the control plants , indicating that the transgene was tightly controlled. Even in line 7D, whose stomatal conductance was significantly reduced under non-stress conditions , suggesting some ‘leakiness,’ there were only minor negative effects on plant growth and development. Seed germination rate , flower longevity , and stomatal density of the transgenic plants grown under non-stressed conditions were also similar to those of control plants. The photo bleaching symptom observed in the mature leaves of the CaMV-35S:LeNCED1 plants might be attributed to reduced production of photo protective carotenoids resulting from diversion of precursors to ABA synthesis. Since there was no photo bleaching in the rd29A:LeNCED1 plants, photo bleaching in the constitutive NCED plants cannot be explained in this way, and must instead be the result of other correlated effects, such as stomatal closure and limited CO2 fixation under non-stressed conditions. One of the more remarkable responses to drought stress of the rd29A:LeNCED1 transgenic plants was their ability to recover from severe water stress. After 14 days without water, most of the transgenic plants recovered, whereas almost all of the control plants died when re-watered. This difference may simply reflect the reduction in water loss and accompanying physiological stresses in the transgenic plants, but it may also be the result of the greatly increased concentration of proline and perhaps other osmoregulatory compounds in the cells of the stressed transgenic plants. We found that the relatively low concentration of ABA in the leaves of constitutive CaMV-35S:LeNCED1 plants under non-stressed conditions had about the same effect on gas exchange as the much higher levels in the drought-stressed rd29A:LeNCED1 transgenic plants.

This suggests that mechanisms other than stomatal closure are key to the improved stress resistance resulting from stress induced over expression of NCED. In response to drought stress, many plants reduce their water potential to maintain turgor by accumulating compatible solutes such as proline, mannitol, or trehalose in the cytoplasm.Proline, one of the most commonly reported compatible solutes, is important not only in osmoregulation,3but also in protecting cells by enhancing the stability of proteins and membranes.The increased transcript abundance of P5CS, considered to be the rate limiting enzyme in proline biosynthesis,was correlated with a significantly higher accumulation of free proline in rd29A:LeNCED1 transgenic plants during drought stress . Other researchers have shown correlations between endogenous increase and exogenous application of ABA and proline concentrations in drought-stressed plants, but the relationship between proline and ABA in stress survival requires further examination. Our results demonstrated that over expression of a target gene related to ABA biosynthesis pathway such as NCED using the inducible promoter rd29A is an effective strategy for improving the drought resistance and avoiding the negative effects resulting from a constitutive promoter. Drought is a complex stress. Drought resistance can vary widely among crops; accurately assessing the drought resistance of different crops and varieties requires extensive field trials over a number of growing seasons in varied environments. Whether the approach described in this study would be widely applicable to other plants or agricultural field crops or the mechanism based on the conditionally enhanced ABA biosynthesis would be useful under field conditions requires further study. In addition, it would be informative to examine whether this approach could enhance water use efficiency and affect biomass production in the future.Major progress has been made in recent years in identifying the master regulators of sex determination in plants, but less is known about the transcriptional networks that account for sex dimorphism.

Transcription factors, small RNAs, and cytokinin-response regulators have all been identified as mechanisms of sex determination in angiosperms. Development of separate sexes, however, requires that the genes controlling sex determination regulate the transcription of many genes and metabolic pathways in order to coordinate development of sex-specific characteristics, such as gametes and floral morphology. With the notable exception of Diospyros, little is known about the metabolic pathways that are regulated by these sex-determination genes and the resulting transcriptome-wide expression differences. Evidence from multiple angiosperm taxa, as well as leading sex determination models, suggests that in plants, sex is controlled by one or two regulatory factors, termed “sex determination” or “master regulator” genes. These factors in turn lead to primary sex dimorphisms as well as secondary sex dimorphisms, such as floral volatile profiles, pigmentation, floral phenology, and organ morphology, and often involve both sex-linked and autosomal genes. While primary sex dimorphisms are under direct control of the sex determination gene, secondary sex dimorphisms may be either under the control of sex-linked genes, or under direct control of the sex-determining genes themselves.Because their expression in the opposite sex may be deleterious, linkage between sex determination genes and genes controlling secondary sexual dimorphisms may be favored by natural selection. Such genes are termed “sexually antagonistic” . As a result, it can be challenging to discriminate among sex determination genes and other sex linked genes that influence sex dimorphisms. In angiosperms,hydroponic fodder system the maintenance of separate sexes typically involves factors controlling sex determination residing alongside linked sexually antagonistic genes controlling sex dimorphisms on heterogametic sex chromosomes, where typically one sex is heterogametic and the other homogametic. Two heterogametic systems, XY and ZW, have been observed in angiosperms. The XY system, where the male is the heterogametic sex, tends to be more prevalent, and is found in 84.7% of diecious angiosperm species, including Asparagus officinalis, Carica papaya, Diospyros, and Phoenix dactylifera. In the less common ZW system, females are heterogametic, as in Fragaria and Silene otitis. The Salicaceae family is of particular interest for use as a model in understanding the evolution of sex chromosomes and sex determination in plants, because the family is almost exclusively diecious, yet the sex determination region has been remarkably dynamic. The Salicaceae family exhibits both ZW and XY heterogametic systems and SDRs that are localized in different chromosomes across species. The SDR in S. purpurea has been localized to a 6.73-Mb pericentromeric region on chr15W, which includes approximately 2 Mb of sequence that is not present in the corresponding region of chr15Z. Other Salix spp. in the section Vetrix also have a chr15 ZW SDR, including S. viminalis and S. suchowensis, whereas S. nigra in the section Salix has a chr07 XY system. In contrast, most Populus species have chr19 SDRs, including XY in P. trichocarpa and ZW in P. alba but, exceptionally, P. euphratica exhibits a chr14 XY SDR. This indicates that sex determination has a complex evolutionary history in the Salicaceae, and that the SDR has shifted chromosomes as well as heterogametic systems, possibly through translocation, the rise of an entirely new locus becoming sexually antagonistic, or independent origins of diecy. Differing sex determination systems in closely related taxa have been observed across both plants and animals. For instance, both XY and ZW systems are represented in Silene.

In cichilid fish, multiple sex determination loci and systems have been identified in specific populations. Further, examples of a sex determination region evolving independently in related taxa include: sticklebacks, medaka fishes, and true frogs. Thus, even closely related species with similar genomic regions involved in sex determination, as in the Salicaceae, may have evolved independently and can involve unique mechanisms. The Salicaceae family contains many species of economic importance in the genera Populus and Salix. Shrub willows in particular, are grown throughout North America and Eurasia for bioenergy and bioproducts. Despite its commonality across the Salicaceae family, diecy presents a challenge for breeding efforts and the cultivation of shrub willow, with sex showing linkage to biomass-related traits, such as leaf area and catkins showing distinct phenology and secondary metabolite profiles between sexes, affecting pollinator and pest attraction. There is a strong interest in understanding the genetic mechanisms controlling sex determination in Salix, along with the gene pathways involved in sex dimorphism, in order to advance current breeding efforts and genetic studies to improve Salix as a bioenergy crop. Nevertheless, there is still relatively little data characterizing sex-determination genes and sex dimorphism pathways in Salix, despite substantial research and identifcation of putative master regulators of sex in the related genus Populus. Moreover, willows are typically insect pollinated, whereas members of the genus Populus are wind pollinated and show little evidence of sex dimorphism in vegetative traits that may point to unique pathways and genes involved in sex dimorphism and underscore the need for sex determination research that is specific to Salix. A previous study of transcriptomic data in Salix identified differentially expressed genes associated with sex in shoot tips containing floral primordia. It is hypothesized that sexually dimorphic genes are regulated through complex pathways that are ultimately controlled by one or more elements in the SDR, most likely via transcription factors, plant hormones, and/or small RNAs. This would be consistent with characterized sex determination systems in plants. Elements in the SDR controlling sex determination are termed “master regulator genes” and likely control several top-level regulatory genes that may or may not be located in the SDR, which in turn regulate both primary and secondary sex characteristics through intermediate metabolic pathways, as described by Feng et al., 2020. Unfortunately, identification of master regulator genes in Salix purpurea is complicated by an SDR that comprises nearly 40% of chr15W and contains 488 linked genes, including repetitive regions, and tandem duplications, requiring the use of transcriptomic data and coexpression analyses to characterize candidate genes. This study captures the transcriptome-wide primary and secondary sex dimorphism profile in emerging inflorescences, which contain hundreds of achlamydeous flowers across a range of developmental time points, in addition to exhibiting distinct terpenoid and phenylpropanoid profiles leading to pigmentation and volatile organic compound emissiondimorpism. Using eQTL analysis, we associated the expression levels of differentially expressed genes in catkins with genomic loci. We identified multiple genes associated in trans with the SDR that could serve as top level regulators of primary and secondary floral sex dimorphisms under direct control by master regulator genes, as well as enriched pathways predicted to serve as intermediate pathways involved in sex dimorphism. Furthermore, based on these multi-omics results, six gene families are presented as candidates for the master regulators of sex: homologs of Arabidopsis GATA15, ARR17, AGO4, and DRB1, three genes coding hypothetical proteins, and a CCHC zinc finger. Characterizing the master regulator genes and the mechanisms of sex determination in willow provides insight into the complex evolution of diecy in the Salicaceae. These data represent one of the most comprehensive studies of sex dimorphism expression in a diecious crop plant, incorporating RNA expression, genotyping-by-sequencing , differential methylation, and small RNA expression, and provide a valuable addition to the nascent body of knowledge on sex-determination mechanisms in crop plants.Principal component analysis showed a clear separation of male and female genotypes based on transcriptome-wide expression . A total of 36,518 gene models, including alternative transcripts, accounting for 63.6% of all genomewide protein-coding transcripts, were expressed in floral tissue across the 159 samples, with 24,074 showing significant differential expression between males and females.