Stomata operation in halophytes

The ability of plants to grow under adverse environmental conditions implies optimising the water and gas exchange by controlling operation of stomata, microscopic valves at the leaf surface. Stomata represent approximately 0.5 to 5% of total leaf surface but play a vital role in gas exchange where they are responsible for over 95% of all the water lost by the leaf. Soil salinity is one of the major environmental hurdles and affects stomata operation by imposing osmotic, oxidative and ionic stresses onto stomata operation. This results in reduced growth and yield and associated economic penalties in crop production systems. While significant advances have been made in our understanding of stomatal function in glycophyte plants, much less is known about stomata operation in halophytes, naturally salt-loving species. Therefore, a comparative analysis of the effects of salinity stress on stomatal operation in both glycophytes and halophytes may be an important step in improving salt stress tolerance in traditional crops. In light of the above, the major aim of this PhD study was to address the following gaps in our knowledge: (i) investigate stomata patterning and operation in various plant species contrasting in their salinity stress tolerance; (ii) investigate the difference in protein profile of stomata in closely related halophytic (quinoa) and glycophytic (spinach) plant species that might assist plant adaptation and stomata operation under hyperosmotic saline conditions; (iii) to conduct comparative RNA-sequencing analysis of GCs of halophytic quinoa plants under control and saline conditions with the aim of understanding salt-induced transcriptome changes in GC and leaf lamina; and (iv) reveal the molecular identity of proteins and ion transporters that likely are involved in salt stress responses and stomatal movement. Understanding the adaptive strategies of halophytes to maintain their productivity under saline conditions in comparison to traditional plants is essential for plant breeding programs. Working along these lines, three halophytic species [quinoa (Chenopodium quinoa Wild.), sea barley (Hordeum marinum), and sea beet (Beta maritima)] and their glycophytic relatives [Chenopodium album, cultivated barley (Hordeum vulgare) and sugar beet (Beta vulgaris)] were grown under control and saline (100, 200, 300, 400 and 500 mM NaCl) conditions. Plant agronomical, ionic, and photosynthetic traits were then determined. Reduction in stomatal conductance decreased in a dose-dependent manner with increasing NaCl concentrations. However, net CO\\(_2\\) assimilation rates remained constant or displayed higher values at the medium level of salinity in halophytes. Stomatal densities were lower in halophytic species and they increased with increasing salinity levels in the studied species except for C. album and quinoa. Stomatal size was reduced with salinity in all studies species. The speed of stomatal response to light and dark was species-specific, with the highest values in the cultivated barley and sea barley and the lowest ones in sugar beet and sea beet. Halophytes exhibited a higher speed of stomatal opening and closure in response to light/dark fluctuations. Fast stomatal responses to internal signals and environmental factors may be essential for synchronization of photosynthetic and stomatal conductance responses, thus optimizing water use efficiency under changing conditions. To be able to apply transcriptomics and proteomics approaches in stomatal function studies, we then developed and validated protocols for mechanical isolation of GC-enriched epidermal peels for omics studies. Accordingly, we develop and optimised the method for GC isolation from the leaf epidermis of spinach, sugar beet and quinoa under control and salt-stress conditions. The RNA integrity, gene expression and 1D SDS-PAGE tests were performed to validate the suitability of this technique for omics studies. The results confirmed the appropriateness of this method for proteomics analysis as well as the suitability of RNA integrity for gene expression analysis. Transcriptional levels of several GCspecific genes such as MYB60, FAMA, ICE1, SLAC1 and PP2CA were quantified in both leaf and GC samples. A high abundance of transcripts of these genes was observed in GC compared with those in the leaf. Isolated GCs were enriched in GC-specific genes suggesting suitability of this isolation approach for molecular studies. By successfully validating the aforementioned GC isolation method and in order to better understand the molecular mechanisms underlying stomatal function under saline conditions, we used a proteomics approach to study isolated GCs from the salt-tolerant sugar beet species. Of the 2088 proteins identified in sugar beet GCs, 82 were differentially regulated by salt treatment. According to bioinformatics analysis (GO enrichment analysis and protein classification), these proteins were involved in lipid metabolism, cell wall modification, ATP biosynthesis, and signalling. Among the significant differentially expressed proteins several proteins classified as stress proteins‚ÄövÑvp were upregulated, including non-specific lipid transfer proteins, chaperone proteins, heat shock proteins, and inorganic pyrophosphatase 2, responsible for the vacuole membrane for ion transportation. Moreover, several antioxidant enzymes (peroxide, superoxidase dismutase) were highly upregulated. Furthermore, cell wall proteins detected in GCs provided some evidence that GC walls were more flexible in response to salt stress. Proteins such as L-ascorbate oxidase that were constitutively high under both control and high salinity conditions may contribute to the ability of sugar beet GCs to adapt to salinity by mitigating salinity-induced oxidative stress. We then applied proteomics analyses of GCs in halophyte quinoa under salinity. In total, 2147 proteins were identified, of which 36% were differentially expressed in response to salinity stress in GCs. Up and downregulated proteins included signaling molecules, enzyme modulators, transcription factors and oxidoreductases. Several proteins involved in stress in general and osmotic/salt stresses in particular, were found to be highly abundant in GCs following salinity treatment, including desiccation-responsive protein 29B (50-fold), osmotin-like protein OSML13 (13-fold), PLAT domain-containing protein 3-like (8-fold), and dehydrin ERD14 (8-fold). Ten proteins related to the gene ontology term response to ABA‚ÄövÑvp were upregulated in quinoa GC such as aspartic protease in guard cell-1, phospholipase D and plastid-lipid-associated protein. In addition, seven proteins in the sucrose-starch pathway were upregulated in the GC in response to salinity stress. Furthermore, the accumulation of two enzymes involved in the amino acid biosynthesis (tryptophan synthase and L-methionine synthase) was observed in the GC under salt stress. Exogenous application of sucrose and amino acids on stomatal conductance showed that tryptophan, L-methionine and sucrose were associated with less stomatal aperture and conductance, which could be advantageous for plants under salt stress. Given the lack of comparative transcriptomics analysis of GC, we further isolated GC for RNA-sequencing. We performed comparative transcriptome analysis of halophyte (Chenopodium quinoa Wild.) and glycophyte (Spinacia oleracea) guard cells in response to salt stress. The RNA-sequencing analysis of mechanically prepared guard cell-enriched epidermal fragments of quinoa and spinach demonstrated that these two plant species had similar responses where in both plants salt-responsive genes were mainly related to biological processes such as protein metabolism, secondary metabolites, signal transduction, and transportation system. On the other hand, genes related to ABA signalling and ABA biosynthesis were strongly induced in quinoa GCs. Furthermore, GCs in quinoa as a halophytic plant showed higher expression levels of amino acids, proline, sugars, sucrose and potassium transporters under saline condition. This study also shown that one of the main transcriptomic differences between quinoa and spinach was occurred at the cell wall where spinach as a traditional crop plant developed more rigid guard cell wall while quinoa as a halophyte had flexible cell wall upon exposure to salt stress. These differences resulted in higher stomatal movements in response to light and dark in quinoa. Furthermore, genes involving in inhibition of stomata development and differentiation were highly expressed by salt in quinoa but not in spinach. This resulted in lesser stomatal density and index in quinoa leaf while salinity did not alter stomatal formation in the epidermal tissue in spinach. Overall, this comparative transcriptomic analysis revealed that quinoa GCs had better saltadaptability responses compared to spinach. Altogether, the results of this project revealed that at the whole plant level, halophytes were superior to glycophytes under saline conditions through higher velocity of carboxylation, higher K\\(^+\\)/Na\\(^+\\) selectivity and faster stomatal responses to environmental factors. Also, the results of guard cell transcriptomics and proteomics studies demonstrated that many proteins and genes are either expressed constitutively or induced by salt stress which may contribute to the ability of halophytes to adapt to saline conditions.