Our study elucidates the differences in varieties’ sensitivities to climate and clusters the response by region of origin to explore the plastic phenotypic responses of present-day cultivars. Using published genetic data on thousands of varieties, we were able to incorporate geographic sub-regions of origin into our model comparison and produce predictions on timing for the three phenological stages with unique estimates for all 137 varieties. While geographic origins indicate trends in response to climate, more important are the outliers from the groups that are consistently late ripening with low sensitivity to changes in climate. Our approach to quantifying the responses of hundreds of varieties gives viticulturists insight to future alternative choices, such as the late ripening, heat tolerant Nebbiolo, with veraison predicted at 45-174 GDD later than the mean. Italian varieties also showed the lowest sensitivity to changes in climate, indicated by the relatively low CV compared to other regions. No other phenological studies in California test this many international varieties in the context of the hot central Northern Californian summer growing season with precise observations over years. Future research will target potential varieties for successful marketing in California under future climate conditions, round plastic plant pot and potentially elucidate physiological drivers of phenological variation that have been selected unintentionally through grapevine cultivation. These important phenological patterns and sensitivities could be useful for adapting plant material to new regions and new climates.
Drought is a threat to the quality and yield of grapevine in the world’s important wine grape growing regions . These regions are expected to have decreased precipitation with associated risks of developing soil water deficit in coming years . One adaptation strategy seen in plants to tolerate water limitation involves stomatal regulation of water loss . The opening of these pores controls leaf gas exchange and is regulated by changes in turgor pressure in the guard cells surrounding these pores. The two guard cells respond to a range of environmental signals, often in conflict with each other, and sometimes rapidly changing . In drought-stressed grapevine, stomatal closure is triggered by hydraulic signals and maintained by abscisic acid following re-watering . Genotypic variation for stomatal sensitivity to reduced water availability has been shown to exist in grapevine . Stomatal density and distribution in the epidermal tissue also plays a critical role in determining transpiration rate per unit of leaf area . Previous work focusing on natural variation for stomatal anatomical features provided evidence of a close negative relationship between plant water-use efficiency and stomatal density . Stomatal density and distribution are under the control of small cysteinerich peptides called epidermal patterning factors highly conserved in a wide range of higher plants . According to extensive studies carried out in Arabidopsis three members of this family play a key role in the formation of stomata: EPF1, EPF2 and EPFL9. EPF2 and EPF1 are expressed in the epidermis, in the earlier and later stages of leaf development, respectively. EPF2 inhibits the formation of cells considered the precursors of stomata guard cells, while EPF1 inhibits the subsequent differentiation of these same precursors and induces asymmetric cell division .
Epidermal Patterning Factor Like 9 , also known as STOMAGEN, plays an antagonist role with respect to EPF1 and EPF2 as it induces stomata formation . EPF-peptides interact with two transmembrane receptors of epidermal cells, ERECTA and Too Many Mouths . While EPF1 and EPF2 activate the receptor complex which in turn induces a MAPKs cascade leading to the destabilization of important transcription factors involved in the formation of stomata , STOMAGEN inactivates it. STOMAGEN is the only known positive regulator of stomata produced in mesophyll, and was confirmed to act independently of EPF1 and EPF2 . Its activity is antagonized by that of EPF2, however, it is not well understood if the antagonistic action is due to the sharing of an identical binding site in the common receptor or to other mechanisms . An evolutionary model suggests that EPFL9 may derive from the duplication of EPF1/2 with a subsequent alteration in the function . This is confirmed by the fact that EPF1/2 are more widespread in higher plants compared to EPFL9 . Moreover, it is known that cysteine-rich peptides are encoded by genes usually present in clusters, located in defined chromosomal regions, probably originating from gene duplication . Despite the different amino acid composition among the CRP different sub-classes and across species, the members of CRPs have in common a small size, a conserved N-terminal region that include an apoplast secretion signal and a functional Cterminal domain containing cysteine residues . Several functional genomics studies, based on the ectopic expression or silencing of EPF1, EPF2, or EPFL9, have recently demonstrated a highly conserved functional paradigm in Arabidopsis and cereals. In barley, Hughes et al. proved that HvEPF1 overexpression limits stomatal development. In a hexaploid bread wheat, Dunn et al. decreased stomatal density via the over expression of TaEPF1 and TaEPF2 orthologues and demonstrated improvements in water-use efficiency without affecting yield when SD reduction was moderate.
Similarly, in rice Caine et al. and Mohammed et al. elucidated the function of OsEPF1 adopting an over-expression approach. Adding to the studies on rice, Lu et al. confirmed the role of OsEPF1, OsEPF2 and OsEPF9 by a dual strategy, both over-expression and down-regulation via RNA interference. Yin and colleagues were the first to apply the genome editing technology in rice to disrupt OsEPFL9. Gene editing via the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 is to date the most powerful tool for functional genomics studies in plants . CRISPR/Cas9 system can efficiently produce nucleotide mutations into precise positions in the genome through the combined action of a specific guide RNA and the Cas9 nuclease which cleaves the DNA eliciting the non-homologous end-joining pathway for DNA repair . NHEJ may produce knock-out mutants with random insertion or deletion of variable lengths at the Cas9 cleavage site causing frameshift mutations or loss of amino acids in protein-coding sequences. These KO mutants are perfect systems to prove the function of a candidate gene . This technology is steadily progressing and, coupled with the advancements of in-vitro culture practices, represents a knowledge-based strategy for the genetic improvements of cultivated plants, with relevant advantages compared to traditional breeding . In grapevine, CRISPR/Cas9 technology has been successfully applied to evaluate the function of genes involved in susceptibility or tolerance to diseases, mainly caused by fungal pathogens or to enhance tolerance to cold stress . In this study, we inactivated VvEPFL9-1 in a grapevine table grape variety, ‘Sugraone’, adopting a genome editing approach based on CRISPR/Cas9 technology. Different edited lines with a significant reduction in stomatal density were produced and analyzed to investigate how reducing stomatal density affects grapevine physiological performance under different environmental conditions.Crops worldwide will experience warmer conditions in the next decades, followed by limited water availability and increasing atmospheric CO2 concentration, leading to possible detrimental effects on yield stability and food security . Genetic manipulation of stomatal density and stomatal size have been shown to be an effective approach to increase drought tolerance and reduce water loss in several species . Indeed, previous studies in Arabidopsis and grasses showed that water conservation, 25 liter round pot higher iWUE and enhanced tolerance to multiple stresses were achieved in lines over expressing EPF1/EPF2 or down regulating EPFL9, due to a reduction in stomatal density . In the grapevine genus we found two AtEPFL9 orthologs, we named VvEPFL9-1 and VvEPFL9-2, identical at 82% in the protein region corresponding to the functional peptide and respectively sharing 82% and 95% identity with the same region of AtEPFL9 peptide. So far, two EPFL9 paralogs have been found in maize and rice , showing respectively 84% and 73% and 82% and 73% identities to AtEPFL9 functional peptide. It has been suggested that EPFL9 paralogs in cereals might be functionally divergent but definitive evidence indicating a different function has never been produced. In the study of Lu et al. , the approach used to silence OsEPF9-1 was RNA interference with a 450 bp-long hairpin RNA, which hardly discriminated between the two variants. In our study, we focused on VvEPFL9-1, localized on chromosome 5 and selectively edited this paralog via CRISPR/Cas9 technology. According to our data, the knockout of VvEPFL9-1 can reduce stomatal density by up to 60%, leading to the hypothesis that VvEPFL9-1 and VvEPFL9-2 could be both involved in stomatal induction with a redundant function.
A similar approach using CRISPR/Cas9 technology to knock-out EPFL9 in rice achieved nearly 90% of stomatal density reduction compared to control by targeting a site on the first exon encoding for the signal peptide and thus not discriminating between OsEPFL9 paralogs . Our study also confirms the crucial role of cysteine residues in the C-terminal functional peptide. This is demonstrated by the lines S-epfl9KO5 and S-epfl9KO8 in which the loss of the second cysteine resulted in a stomatal density reduction similar to the one gained by a full frame-shift of the coding sequence. This is consistent with the finding of Ohki et al. who observed that impairing the formation of a disulphide bond prevented the correct protein folding and function. The design of a sgRNA that directed Cas9 cleavage next to the nucleotide triplet coding for the second cysteine proved to be a good choice for effective 3- and 6- bp deletions. Moreover, our data showed that the retention of almost 50% functional VvEPFL9-1 in some transgenic lines due to a partial editing of the target site, with a substantial maintenance of a WT peptide, still resulted in a significant decrease of SD, suggesting that a threshold amount of peptide may be required for EPFL9-1 to be functionally effective. Reduction in stomatal density following VvEPFL9-1 knock-out was significant, although partially compensated by an increase in stomatal size . The negative yet non-linear association between SD and SS has been frequently reported in many species and often linked to an improved economy of epidermal space allocation with the combination of low SD and high SS as a preferable strategy when low stomatal conductance is required . In our work, however, the increase in SS was only a partial compensation for the reduction in SD. Stomata are the main drivers of transpiration but at the same time are pivotal for CO2 uptake for mesophyll photosynthesis . For instance, in barley and wheat, a reduction in SD by 50% compared to WT led to a significant reduction in carbon assimilation and conductance and to an enhanced water use efficiency under optimal growth conditions . Similarly, in ‘Sugraone’ at well-watered conditions, we found that a 60% reduction in SD lead to a reduced Asat for the edited lines compared to the WT. Additionally, the reduction in gs was even greater, leading to a higher value of iWUE in edited versus WT lines. Moreover, the reduction in Asat was not concomitant to reductions in Rubisco velocity or to impairment in electron transport chain suggesting that the knock-out of VvEPFL9-1 did not affect the photosynthetic machinery, at least at the conditions applied in this work. Vitis vinifera genotypes with reduced SD and, in turn, limited Asat and greater iWUE, may be desirable to improve plant water conservation and to delay sugar accumulation under current and future climatic scenarios . Sugars and organic acids along with various secondary metabolites are determinants of grape berry quality and their accumulation during berry ripening is the result of the interaction between genotype and environment, a relationship made vulnerable by climate change . It is well known that grapevine physiology will be impacted by elevated carbon dioxide, increasing temperatures, and extreme heat events during the growing season . In particular, high temperature and increasing CO2 levels are already affecting viticulture with an evident shift towards an earlier onset of phenological stages and accelerated berry ripening . High temperatures and water stress slow down vine metabolism resulting in a lower accumulation of polyphenols and aromatic compounds in the berries . Thus, one of the consequences of a compressed phenology may be an earlier sugar accumulation in the berries that leads to anticipated harvest dates when the secondary metabolites content is sub-optimal .