Understanding the role of stomatal traits and tissue-tolerance mechanisms in salinity stress responses in quinoa and wild barley

Agricultural production needs to be doubled by year 2050; this task is complicated by various abiotic stresses severely affecting crop production. Salinity stress is among major environmental stresses that influences crop production globally and it is estimated that around 950 million hectares of arable land is affected by this environmental stress. Considering this fact, it is necessary to introduce new approaches to manage this main challenge. One of them is breeding for enhanced salinity tolerance. An alternative solution may be the use of halophyte relatives. Despite having high tolerance to salt stress, halophytic plants have not been extensively used to study salinity tolerance mechanisms. Also, studies investigating the salinity tolerance mechanism in halophytes have concentrated on physiological or anatomical aspects with relatively little focus being given to the omics-based studies such as metabolomics and transcriptome analysis. Epidermal bladder cells (EBCs) are specialized structures in some of halophytic plants that provide external store for toxic ions such as Na\\(^+\\) and Cl\\(_-\\), and hence understanding the function of EBCs may eventually play an important role in transferring this ability to crop plants. Stomata also being another focus of this study. Although there have been significant advances of understanding mechanisms that controlling stomatal development and also the signalling pathways that regulate guard cells function in glycophytes, much less is known about stomata development and operation in halophytes. In light with this fact a question on how environmental variables and in particular salinity stress change the basal stomatal development pathway requires more studies. Given the fact that osmotic stress and toxic Na\\(^+\\) level negatively affect stomatal parameters under saline conditions the question is that why are halophytes capable to optimise their stomata performance? Do halophytic plants possess unique stomata operation mechanisms? How does salinity stress affect epidermal cell differentiation which leads to either an increase or decrease in stomatal density? Hence, the major aim of this PhD project was to fill some of above discussed gaps in our knowledge by addressing the following specific objectives: (i) investigate the role of EBC in salinity tolerance in quinoa; (ii) identifying key genes related to salt sequestration into EBCs by transcriptome analysis of EBC through comparing bladder-bearing quinoa plants with those that EBCs were mechanically removed; (iii) evaluate the effects of salinity on EBC patterning in quinoa and correlate the extent of variability in this trait with the genetic variation in salinity stress tolerance; (iv) investigate stomata patterning and development and associate the extent of variability in stomata characteristics with genetic variation in salinity stress tolerance; (v) comparing stomatal traits as a component of the tolerant mechanism between halophytic crops and their wild relatives (using cultivated and wild barley as a case study). To provide direct supporting evidence for the role of EBCs that have been postulated to assist halophytes to cope with saline environment, Chenopodium quinoa plants were grown under saline conditions for 5 weeks. One day prior to commencement of salinity stress EBC from all leaves and petioles were gently removed using soft cosmetic brush. Physiological, ionic and metabolic changes in brushed and non-brushed leaves were compared. Gentle removal of EBC did neither initiate wound metabolism nor affected physiology and biochemistry of control-grown plants but had a pronounced effect on salt-grown plants resulting in a salt-sensitive phenotype. Of 91 detected metabolites, more than half (50) were significantly affected by salinity. Removal of EBC has dramatically modified these metabolic changes, with the biggest differences reported for gamma-aminobutyric acid (GABA), proline, sucrose and inositol, affecting ion transport across cellular membranes (as shown in electrophysiological experiments). This work provides the first direct evidence for the role of EBC in salt tolerance in halophytes and attributes this role to (1) key role of EBC as a salt dumper to externally sequester salt load; (2) improved K\\(^+\\) retention in leaf mesophyll and (3) storage space for several metabolites known to modulate plant ionic relations. To identify key genes related to salt sequestration abilities in EBCs, a transcriptome study was conducted with bladder-bearing and bladderless plants similar to above experiment. Comparing differently expressed genes (DEGs) of brushed and non-brushed leaves grown under 400 mM NaCl using a p-value < 0.05 and fold change > 2 as the significance cut-offs, indicated that 2015 genes were differently expressed where 1399 genes were up-regulated and 616 genes were down-regulated in bladder-bearing leaves. Significant alterations of genes related to ion transport, DNA replication, and genes related to stress signalling in response to salinity stress were determined. Altogether, the finding that the transcriptome of bladder-bearing leaves differed from those of bladderless leaves suggests that EBCs do not function as a passive external store place for salt as it was perceived before but play active metabolic role in quinoa plant. Varietal differences in salinity tolerance of quinoa was explored by evaluation of 114 accessions grown under control and 400 mM NaCl conditions, and different physiological and anatomical characteristics were measured. Accessions were grouped to sensitive, intermediate and tolerant classes based on relative dry weight defined as salinity tolerance index (STI). Results showed a large variability for fresh and dry weights indicating a strong genetic variation for salinity tolerance in quinoa. Bladder density increased in majority of accessions under saline condition while bladder diameter remained unchanged; this resulted in a large variability in a bladder volume as a dependant variable. Stomata density remained unchanged between saline and non-saline conditions while stomata length declined between 3% to 43% among accessions. Correlation analysis indicated a significant positive association between EBC diameter and STI on one hand and EBC volume and STI on the other hand, in a salt-tolerant group. A negative association between STI and stomata length was also found in a salt-tolerant group, suggesting that these plants were able to efficiently regulate stomatal patterning to efficiently balance water loss and CO\\(_2\\) assimilation under saline condition. Both salt-sensitive and salt-tolerant groups had the same Na\\(^+\\) content under saline condition; however, a negative association between leaf Na\\(^+\\) concentration and STI in salt-sensitive plants indicated an efficient Na\\(^+\\) sequestration into the EBCs in salt-tolerant plants. While sequestration of toxic ions into EBCs is an efficient mechanism contributing to salinity tolerance in quinoa, many halophytes do not utilize EBCs to modulate their tissue ion concentrations but still possess superior salinity tolerance ability. To elucidate possible compensation mechanism(s) underlying superior salinity tolerance in the absence of external salt storage capacity, we have selected four accessions from our previous experiment to address this issue. Whole-plant physiological and electrophysiological characteristics were assessed after 2 days and 3 weeks of 400 mM NaCl stress. The results showed that accession Q21 that had low EBC volume had superior photosynthetic rate and stomatal conductance at both 2 days and 3 weeks of salt stress than the counterpart Q68 with high EBC volume. Both accessions with low EBC volume (Q21 and Q30) utilised Na\\(^+\\) exclusion at the root level and were capable to maintain low Na\\(^+\\)concentration in leaves, to compensate for inability to sequester Na\\(^+\\) load in EBC. These conclusions were further confirmed by electrophysiological experiments showing higher Na\\(^+\\) efflux from Q21 and Q30 roots as compared with 195 and Q68 as accessions with high EBC volume. Furthermore, accessions with low EBC volume had significantly higher K\\(^+\\) concentration in their leaves at long-term salinity stress compared to plants with high EBC sequestration ability suggesting that the ability to maintain high K\\(^+\\) content in mesophyll was as another key compensation mechanism. In the light of importance of stomatal traits as a determinant of salinity tolerance in quinoa, we have extrapolated this work to cereal plants, comparing cultivated (CB; Hordeum vulgare) and wild (WB; Hordeum spontaneum ) barley. Twenty-six genotypes of WB and CB were grown under control and saline conditions and stomatal characteristics, leaf ion content and epidermal strips response to Na\\(^+\\) and K\\(^+\\) were measured. WB had higher relative biomass than CB when exposed to salinity stress. Under saline conditions, WB plants were able to keep constant stomata density (SD) while SD significantly decreased in CB. The higher SD in WB also resulted in higher stomatal conductance (gs) under saline conditions, with gs reduction being 51% and 72% in WB and CB, respectively. Furthermore, WB showed faster stomatal response to light, indicating their better ability to adapt to changing environmental conditions. Experiments with isolated epidermal strips indicated that CB genotypes have the higher stomatal aperture when incubated in 80 mM KCl solution, and its aperture declined when KCl was substituted by NaCl, indicating strong preference to KCl for stomatal operation in CB. On the contrary, WB genotype had the highest stomata aperture being exposed to 80 mM NaCl suggesting that WB plants may use Na\\(^+\\) instead of K\\(^+\\) for stomata movements. Our data suggest that CB employ a stress-escaping strategy by reducing stomata density, in an attempt to conserve water when grown under salinity conditi...