Low relative air humidity and increased stomatal density independently hamper growth in young Arabidopsis.

Stomatal pores in plant leaves mediate CO2 uptake for photosynthesis and water loss via transpiration. Altered stomatal density can affect plant photosynthetic capacity, water use efficiency, and growth, potentially providing either benefits or drawbacks depending on the environment. Here we explore, at different air humidity regimes, gas exchange, stomatal anatomy, and growth of Arabidopsis lines designed to combine increased stomatal density (epf1, epf2) with high stomatal sensitivity (ht1-2, cyp707a1/a3). We show that the stomatal density and sensitivity traits combine as expected: higher stomatal density increases stomatal conductance, whereas the effect is smaller in the high stomatal sensitivity mutant backgrounds than in the epf1epf2 double mutant. Growth under low air humidity increases plant stomatal ratio with relatively more stomata allocated to the adaxial epidermis. Low relative air humidity and high stomatal density both independently impair plant growth. Higher evaporative demand did not punish increased stomatal density, nor did inherently low stomatal conductance provide any protection against low relative humidity. We propose that the detrimental effects of high stomatal density on plant growth at a young age are related to the cost of producing stomata; future experiments need to test if high stomatal densities might pay off in later life stages.

Responses of leaf gas exchange and metabolites to drought stress in different organs of sugarcane and its closely related species Erianthus arundinaceus.

MAIN CONCLUSION: The high intrinsic water-use efficiency of Erianthus may be due to the low abaxial stomatal density and the accumulation of leaf metabolites such as betaine and gamma-aminobutyric acid. Sugarcane is an important crop that is widely cultivated in tropical and subtropical regions of the world. Because drought is among the main impediments limiting sugarcane production in these regions, breeding of drought-tolerant sugarcane varieties is important for sustainable production. Erianthus arundinaceus, a species closely related to sugarcane, exhibits high intrinsic water-use efficiency (iWUE), the underlying mechanisms for which remain unknown. To improve the genetic base for conferring drought tolerance in sugarcane, in the present study, we performed a comprehensive comparative analysis of leaf gas exchange and metabolites in different organs of sugarcane and Erianthus under wet and dry soil-moisture conditions. Erianthus exhibited lower stomatal conductance under both conditions, which resulted in a higher iWUE than in sugarcane. Organ-specific metabolites showed gradations between continuous parts and organs, suggesting linkages between them. Cluster analysis of organ-specific metabolites revealed the effects of the species and treatments in the leaves. Principal component analysis of leaf metabolites confirmed a rough ordering of the factors affecting their accumulations. Compared to sugarcane leaf, Erianthus leaf accumulated more raffinose, betaine, glutamine, gamma-aminobutyric acid, and S-adenosylmethionine, which function as osmolytes and stress-response compounds, under both the conditions. Our extensive analyses reveal that the high iWUE of Erianthus may be due to the specific accumulation of such metabolites in the leaves, in addition to the low stomatal density on the abaxial side of leaves. The identification of drought-tolerance traits of Erianthus will benefit the generation of sugarcane varieties capable of withstanding drought stress.

MYC2 regulates stomatal density and water use efficiency via targeting EPF2/EPFL4/EPFL9 in poplar.

Although polyploid plants have lower stomatal density than their diploid counterparts, the molecular mechanisms underlying this difference remain elusive. Here, we constructed a network based on the triploid poplar transcriptome data and triple-gene mutual interaction algorithm and found that PpnMYC2 was related to stomatal development-related genes PpnEPF2, PpnEPFL4, and PpnEPFL9. The interactions between PpnMYC2 and PagJAZs were experimentally validated. PpnMYC2-overexpressing poplar and Arabidopsis thaliana had reduced stomatal density. Poplar overexpressing PpnMYC2 had higher water use efficiency and drought resistance. RNA-sequencing data of poplars overexpressing PpnMYC2 showed that PpnMYC2 promotes the expression of stomatal density inhibitors PagEPF2 and PagEPFL4 and inhibits the expression of the stomatal density-positive regulator PagEPFL9. Yeast one-hybrid system, electrophoretic mobility shift assay, ChIP-qPCR, and dual-luciferase assay were employed to substantiate that PpnMYC2 directly regulated PagEPF2, PagEPFL4, and PagEPFL9. PpnMYC2, PpnEPF2, and PpnEPFL4 were significantly upregulated, whereas PpnEPFL9 was downregulated during stomatal formation in triploid poplar. Our results are of great significance for revealing the regulation mechanism of plant stomatal occurrence and polyploid stomatal density, as well as reducing stomatal density and improving plant water use efficiency by overexpressing MYC2.

Nutrient and Water Availability Influence Rice Physiology, Root Architecture and Ionomic Balance via Auxin Signalling.

Water and soil nutrients are the vital ingredients of crop production, and their efficient uptake is essentially dependent on root development, majorly regulated by auxin. For a water-loving crop like rice, how water availability regulates nutrient acquisition, additionally, how ambient nutrient level modulates water uptake, and the role of auxin therein is not well studied. While investigating the cross-talks among these components, we found water to be essential for auxin re-distribution in roots and shaping the root architecture. We also found that supplementing rice seedlings with moderate concentrations of mineral nutrients facilitated faster water uptake and greater nutrient enrichment in leaves compared to adequate nutrient supplementation. Additionally, moderate nutrient availability favoured greater stomatal density, stomatal conductance, photosynthesis, transpiration rate and water use efficiency when water was not limiting. Further, auxin supplementation enhanced root formation in rice, while affecting their water uptake ability, photosynthesis and transpiration causing differential mineral-specific uptake trends. The present study uncovers the existence of an intricate crosstalk among water, nutrients and auxin signalling the knowledge of which will enable optimizing the growth conditions for speed breeding of rice and harnessing the components of auxin signalling to improve water and nutrient use efficiency of rice.

Greater aperture counteracts effects of reduced stomatal density on WUE: a case study on sugarcane and meta-analysis.

Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (gsw). However, the quantitative relationship between reduced SD, gsw, and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEFP2) in sugarcane reduced SD by 26-38% but did not affect gsw compared to wildtype. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C3 dicots, C3 monocots, and C4 monocots revealed engineered reductions in SD are strongly correlated with lower gsw (r2=0.60-0.98), but this response is damped relative to the change in anatomy.

Stomatal patterning is differently regulated in adaxial and abaxial epidermis in Arabidopsis.

Stomatal pores in leaves mediate CO2 uptake into the plant and water loss via transpiration. Most plants are hypostomatous with stomata present only in the lower leaf surface (abaxial epidermis). Many herbs, including the model plant Arabidopsis thaliana, have substantial numbers of stomata also on the upper (adaxial) leaf surface. Studies of stomatal development have mostly focused on abaxial stomata and very little is known of adaxial stomatal formation. We addressed the role of leaf number in determination of stomatal density and stomatal ratio, and studied adaxial and abaxial stomatal patterns in mutants deficient in known abaxial stomatal development regulators. We found that stomatal density in some genetic backgrounds varies between different fully expanded leaves and recommend using defined leaves for analyses of stomatal patterning. Our results indicate that stomatal development is at least partly independently regulated in adaxial and abaxial epidermis, as i) plants deficient in ABA biosynthesis and perception have increased stomatal ratios, ii) the epf1epf2, tmm and sdd1 mutants have reduced stomatal ratios, iii) erl2 mutants have increased adaxial but not abaxial stomatal index, and iv) stomatal precursors preferentially occur in abaxial epidermis. Further studies of adaxial stomata can reveal new insights into stomatal form and function.

Natural genetic variation in dynamic photosynthesis is correlated with stomatal anatomical traits in diverse tomato species across geographical habitats.

Plants grown under field conditions experience fluctuating light. Understanding the natural genetic variations for a similarly dynamic photosynthetic response among untapped germplasm resources, as well as the underlying mechanisms, may offer breeding strategies to improve production using molecular approaches. Here, we measured gas exchange under fluctuating light, along with stomatal density and size, in eight wild tomato species and two tomato cultivars. The photosynthetic induction response showed significant diversity, with some wild species having faster induction rates than the two cultivars. Species with faster photosynthetic induction rates had higher daily integrated photosynthesis, but lower average water use efficiency because of high stomatal conductance under natural fluctuating light. The variation in photosynthetic induction was closely associated with the speed of stomatal responses, highlighting its critical role in maximizing photosynthesis under fluctuating light conditions. Moreover, stomatal size was negatively correlated with stomatal density within a species, and plants with smaller stomata at a higher density had a quicker photosynthetic response than those with larger stomata at lower density. Our findings show that the response of stomatal conductance plays a pivotal role in photosynthetic induction, with smaller stomata at higher density proving advantageous for photosynthesis under fluctuating light in tomato species. The interspecific variation in the rate of stomatal responses could offer an untapped resource for optimizing dynamic photosynthetic responses under field conditions.

A continental-scale analysis reveals the latitudinal gradient of stomatal density across amphistomatous species: Evolutionary history vs. present-day environment.

BACKGROUND AND AIMS: Amphistomy is a potential method for increasing photosynthetic rate; however, the latitudinal gradients of stomatal density across amphistomatous species and their drivers remain unknown. METHODS: Here, the adaxial stomatal density (SDad) and abaxial stomatal density (SDab) of 486 amphistomatous species-site combinations, belonging to 32 plant families, were collected from China, and their total stomatal density (SDtotal) and stomatal ratio (SR) were calculated. KEY RESULTS: Overall, these four stomatal traits did not show significant phylogenetic signals. There were no significant differences in SDab and SDtotal between woody and herbaceous species, but SDad and SR were higher in woody species than in herbaceous species. Besides, a significantly positive relationship between SDab and SDad was observed. We also found that stomatal density (including SDab, SDad, and SDtotal) decreased with latitude while SR increased with latitude, and temperature seasonality was the most important environmental factor driving it. Besides, evolutionary history (represented by both phylogeny and species) explained about 10-22 fold more of the variation in stomatal traits than the present-day environment (65.2%-71.1% vs. 2.9%-6.8%). CONCLUSIONS: Our study extended our knowledge of trait-environment relationships and highlighted the importance of evolutionary history in driving stomatal trait variability.

Growing at the arid edge: Anatomical variations in leaves are more extensive than in stems of five Mediterranean species across contrasting moisture regimes.

PREMISE: Increasing aridity in the Mediterranean region affects ecosystems and plant life. Various anatomical changes in plants help them cope with dry conditions. This study focused on anatomical differences in leaves and xylem of five co-occurring Mediterranean plant species namely Quercus calliprinos, Pistacia palaestina, Pistacia lentiscus, Rhamnus lycioides, and Phillyrea latifolia in wet and dry sites. METHODS: Stomatal density, stomatal length, leaf mass area, lamina composition, percentage of intercellular air spaces, and mesophyll cell area in leaves of plants in wet and dry sites were analyzed. Xylem anatomy was assessed through vessel length and area in branches. RESULTS: In the dry site, three species had increased stomatal density and decreased stomatal length. Four species had increased palisade mesophyll and reduced air space volume. In contrast, phenotypic changes in the xylem were less pronounced; vessel length was unaffected by site conditions, but vessel diameter decreased in two species. Intercellular air spaces proved to be the most dynamic anatomical feature. Quercus calliprinos had the most extensive anatomical changes; Rhamnus lycioides had only minor changes. All these changes were observed in comparison to the species in the wet site. CONCLUSIONS: This study elucidated variations in anatomical responses in leaves among co-occurring Mediterranean plant species and identified the most dynamic traits. Understanding these adaptations provides valuable insights into the ability of plants to thrive under changing climate conditions.

Coordination of pinna, petiole, and root anatomical traits in 24 tropical-subtropical fern species.

Ferns are primitive vascular plants with diverse morphologies and structures. Plant anatomical traits and their linkages can reflect adaptation to the environment; however, these remain are still poorly understood in ferns. The main objective of this study was to explore whether there was structural coordination among and within organs in fern species. We measured 16 hydraulically related anatomical traits of pinnae, petioles, and roots of 24 representative fern species from the tropical and subtropical forest understory and analyzed trait correlation networks. In addition, we examined phylogenetic signals for the anatomical traits and analyzed co-evolutionary relationships. These results indicated that stomatal density and all petiole anatomical traits exhibited significant phylogenetic signals. Evolutionary correlations were observed between the tracheid diameter and wall thickness of the petiole and between the water transport capacity of the petiole and stomatal density. Conversely, anatomical traits of roots (e.g., root diameter) showed no phylogenetic signals and were not significantly correlated with those of the pinnae and petioles, indicating a lack of structural coordination between the below- and above-ground organs. Unlike angiosperms, vein density is unrelated to stomatal density or pinna thickness in ferns. As root diameter decreased, the cortex-to-stele diameter ratio decreased significantly (enhanced water absorption) in angiosperms but remained unchanged in ferns. These differences lead to different responses of ferns to climate change and improve our knowledge of the water adaptation strategies of ferns.

Altering Stomatal Density for Manipulating Transpiration and Photosynthetic Traits in Rice through CRISPR/Cas9 Mutagenesis.

Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0 progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0 mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1 generation. Evaluation of T1 progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1 plants showed 54-95% increased stomatal density. The homozygous T1 lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.

Molecular Evolution of Plant 14-3-3 Proteins and Function of Hv14-3-3A in Stomatal Regulation and Drought Tolerance.

Drought significantly affects stomatal regulation, leading to the reduced growth and productivity of plants. Plant 14-3-3 proteins were reported to participate in drought response by regulating the activities of a wide array of target proteins. However, the molecular evolution, expression pattern and physiological functions of 14-3-3s under drought stress remain unclear. In this study, a comparative genomic analysis and the tissue-specific expression of 14-3-3s revealed the highly conserved and early evolution of 14-3-3s in green plants and duplication and expansion of the 14-3-3s family members in angiosperms. Using barley (Hordeum vulgare) for the functional characterization of 14-3-3 proteins, the transcripts of five members out of six Hv14-3-3s were highly induced by drought in the drought-tolerant line, XZ141. Suppression of the expression of Hv14-3-3A through barley stripe mosaic virus-virus induced gene silencing resulted in significantly increased drought sensitivity and stomatal density as well as significantly reduced net CO2 assimilation (A) and stomatal conductance (gs) in barley. Moreover, we showed the functional interactions between Hv14-3-3s and key proteins in drought and stomatal responses in plants-such as Open Stomata 1 (HvOST1), Slow Anion Channel 1 (HvSLAC1), three Heat Shock Proteins (HvHSP90-1/2/5) and Dehydration-Responsive Element-Binding 3 (HvDREB3). Taken together, we propose that 14-3-3s are highly evolutionarily conserved proteins and that Hv14-3-3s represent a group of the core regulatory components for the rapid stomatal response to drought in barley. This study will provide important evolutionary and molecular evidence for future applications of 14-3-3 proteins in breeding drought-tolerant crops in a changing global climate.

Increased stomatal conductance and leaf biochemical capacity, not mesophyll conductance, contributing to the enhanced photosynthesis in Oryza plants during domestication.

MAIN CONCLUSION: Leaf biochemical capacity and the ratio of leaf biochemical capacity to stomatal conductance are promising to enhance leaf photosynthetic rate and water use efficiency in rice plants, respectively. Domestication may have great impact on crop photosynthetic rate, which has not been fully understood, especially from the perspective of stomatal conductance, mesophyll conductance, and leaf biochemical capacity simultaneously. In this study, we constructed a database consisting of 141 and 92 sets of data from wild and cultivated rice, respectively, including leaf gas exchange parameters, hydraulic conductance, structural traits, and nitrogen content. We found that, compared to wild rice, enhanced leaf photosynthetic rate in cultivated rice was mainly resulted by the increased stomatal conductance and leaf biochemical capacity, rather than mesophyll conductance. The unchanged mesophyll conductance observed during domestication suggested that it might have been optimized during plant evolution in wild rice. Additionally, the positive relationship between stomatal density and stomatal conductance disappeared in Oryza plants during domestication, suggesting that stomatal conductance in cultivated rice is less restricted by stomatal density, compared to wild rice. Moreover, in both wild and cultivated rice, leaf photosynthetic rate was mainly determined by leaf biochemical capacity, rather than stomatal conductance and mesophyll conductance. This study highlighted the important role of stomatal conductance and leaf biochemical capacity in improvement of leaf photosynthetic rate in rice during domestication. Leaf biochemical capacity and the ratio of leaf biochemical capacity to stomatal conductance should be further investigated to enhance leaf photosynthetic rate and water use efficiency in rice plants, respectively.

A reduced role for water transport during the Cenozoic evolution of epiphytic Eupolypod ferns.

The Cretaceous-Cenozoic expansion of tropical forests created canopy space that was subsequently occupied by diverse epiphytic communities including Eupolypod ferns. Eupolypods proliferated in this more stressful niche, where lower competition enabled the adaptive radiation of thousands of species. Here, we examine whether xylem traits helped shape the Cenozoic radiation of Eupolypod ferns. We characterized the petiole xylem anatomy of 39 species belonging to the Eupolypod I and Eupolypod II clades occupying the epiphytic, hemiepiphytic, and terrestrial niche, and we assessed vulnerability to embolism in a subset of species. The transition to the canopy was associated with reduced xylem content and smaller tracheid diameters, but no differences were found in species vulnerability to embolism and pit membrane thickness. Phylogenetic analyses support selection for traits associated with reduced water transport in Eupolypod 1 species. We posit that in Eupolypod epiphytes, selection favored water retention via thicker leaves and lower stomatal density over higher rates of water transport. Consequently, lower leaf water loss was coupled with smaller quantities of xylem and narrower tracheid diameters. Traits associated with water conservation were evident in terrestrial Eupolypod 1 ferns and may have predisposed this clade toward radiation in the canopy.

Photosynthesis - beyond the leaf.

Although leaves are considered the main site for photosynthesis, other green nonfoliar tissues can carry out considerable amounts of photosynthetic carbon assimilation. With photosynthesis, a potential target for improving crop productivity, physiology and contribution of nonfoliar tissues to overall plant carbon acquisition is gaining increasing attention. This review will provide an overview of nonfoliar photosynthesis, the role of stomata in these tissues and methodologies for quantification and the contribution to overall carbon gain.

Domestication has reduced leaf water use efficiency associated with the anatomy of abaxial stomata in cotton.

Crop domestication for increasing growth rates and yields appears to have altered the features of adaxial and abaxial stomata, but its effect on leaf water use efficiency (WUE) have not been experimentally verified. In this study, we characterized stomatal anatomy and carbon isotope discrimination (δ13C) in 32 wild and 36 domesticated genotypes of cotton grown under agricultural field conditions. The results showed that domesticated genotypes possessed lower WUE, as indicated by low or more negative δ13C compared with wild genotypes. Higher theoretical maximum stomatal conductance (gsmax) after domestication was accounted for by more stomata rather than significantly enlarged stomata. Specifically, abaxial stomatal density was higher whilst there was no change in the adaxial density. The size of both adaxial and abaxial stomata was greater due to larger guard cells but without there being any increase in pore size. However, there was a negative relationship between δ13C and stomatal size across wild and domesticated genotypes, especially on the abaxial leaf surface, because bigger stomata resulted in a lower maximum stomatal response rate to fluctuating canopy light, resulting in increased water loss. Overall, our results indicate that cotton domestication has resulted in substantial variation in stomatal anatomy, and that WUE and drought tolerance can potentially be improved in future breeding by decreasing the size of abaxial stomata to produce a faster stomatal response and hence a reduction in unnecessary water loss.

The impact of growth at elevated [CO2] on stomatal anatomy and behavior differs between wheat species and cultivars.

The ability of plants to respond to changes in the environment is crucial to their survival and reproductive success. The impact of increasing the atmospheric CO2 concentration (a[CO2]), mediated by behavioral and developmental responses of stomata, on crop performance remains a concern under all climate change scenarios, with potential impacts on future food security. To identify possible beneficial traits that could be exploited for future breeding, phenotypic variation in morphological traits including stomatal size and density, as well as physiological responses and, critically, the effect of growth [CO2] on these traits, was assessed in six wheat relative accessions (including Aegilops tauschii, Triticum turgidum ssp. Dicoccoides, and T. turgidum ssp. dicoccon) and five elite bread wheat T. aestivum cultivars. Exploiting a range of different species and ploidy, we identified key differences in photosynthetic capacity between elite hexaploid wheat and wheat relatives. We also report differences in the speed of stomatal responses which were found to be faster in wheat relatives than in elite cultivars, a trait that could be useful for enhanced photosynthetic carbon gain and water use efficiency. Furthermore, these traits do not all appear to be influenced by elevated [CO2], and determining the underlying genetics will be critical for future breeding programmes.

Diverse mangroves deviate from other angiosperms in their genome size, leaf cell size and cell packing density relationships.

BACKGROUND AND AIMS: While genome size limits the minimum sizes and maximum numbers of cells that can be packed into a given leaf volume, mature cell sizes can be substantially larger than their meristematic precursors and vary in response to abiotic conditions. Mangroves are iconic examples of how abiotic conditions can influence the evolution of plant phenotypes. METHODS: Here, we examined the coordination between genome size, leaf cell sizes, cell packing densities and leaf size in 13 mangrove species across four sites in China. Four of these species occurred at more than one site, allowing us to test the effect of climate on leaf anatomy. RESULTS: We found that genome sizes of mangroves were very small compared to other angiosperms, but, like other angiosperms, mangrove cells were always larger than the minimum size defined by genome size. Increasing mean annual temperature of a growth site led to higher packing densities of veins (Dv) and stomata (Ds) and smaller epidermal cells but had no effect on stomatal size. In contrast to other angiosperms, mangroves exhibited (1) a negative relationship between guard cell size and genome size; (2) epidermal cells that were smaller than stomata; and (3) coordination between Dv and Ds that was not mediated by epidermal cell size. Furthermore, mangrove epidermal cell sizes and packing densities covaried with leaf size. CONCLUSIONS: While mangroves exhibited coordination between veins and stomata and attained a maximum theoretical stomatal conductance similar to that of other angiosperms, the tissue-level tradeoffs underlying these similar relationships across species and environments were markedly different, perhaps indicative of the unique structural and physiological adaptations of mangroves to their stressful environments.

Overexpression of Lolium multiflorum LmMYB1 Enhances Drought Tolerance in Transgenic Arabidopsis.

Lolium multiflorum is one of the world-famous forage grasses with rich biomass, fast growth rate and good nutritional quality. However, its growth and forage yield are often affected by drought, which is a major natural disaster all over the world. MYB transcription factors have some specific roles in response to drought stress, such as regulation of stomatal development and density, control of cell wall and root development. However, the biological function of MYB in L. multiflorum remains unclear. Previously, we elucidated the role of LmMYB1 in enhancing osmotic stress resistance in Saccharomyces cerevisiae. Here, this study elucidates the biological function of LmMYB1 in enhancing plant drought tolerance through an ABA-dependent pathway involving the regulation of cell wall development and stomatal density. After drought stress and ABA stress, the expression of LmMYB1 in L. multiflorum was significantly increased. Overexpression of LmMYB1 increased the survival rate of Arabidopsis thaliana under drought stress. Under drought conditions, expression levels of drought-responsive genes such as AtRD22, AtRAB and AtAREB were up-regulated in OE compared with those in WT. Further observation showed that the stomatal density of OE was reduced, which was associated with the up-regulated expression of cell wall-related pathway genes in the RNA-Seq results. In conclusion, this study confirmed the biological function of LmMYB1 in improving drought tolerance by mediating cell wall development through the ABA-dependent pathway and thereby affecting stomatal density.

Bna.EPF2 Enhances Drought Tolerance by Regulating Stomatal Development and Stomatal Size in Brassica napus.

Drought stress severely affects global plant growth and production. The enhancement of plant water-use efficiency (WUE) and drought tolerance by the manipulation of the stomata is an effective strategy to deal with water shortage. However, increasing the WUE and drought tolerance by manipulation on the stomata has rarely been tested in Brassica napus. Here, we isolated Bna.EPF2, an epidermal patterning factor (EPF) in Brassica napus (ecotype Westar), and identified its role in drought performance. Bna.EPF2 overexpression lines had a reduction average of 19.02% in abaxial stomatal density and smaller stomatal pore size, leading to approximately 25% lower transpiration, which finally resulted in greater instantaneous WUE and enhanced drought tolerance. Interestingly, the reduction in stomatal density did not affect the CO2 assimilation or yield-related agronomic traits in Bna.EPF2 overexpression plants. Together with the complementation of Bna.EPF2 significantly decreasing the stomatal density of Arabidopsis epf2, and Bna.EPF2 being expressed in mature guard cells, these results suggest that Bna.EPF2 not only functions in stomatal density development, but also in stomatal dimension in Brassicas. Taken together, our results suggest that Bna.EPF2 improves WUE and drought tolerance by the regulation of stomatal density and stomatal size in Brassica without growth and yield penalty, and provide insight into the manipulation of this gene in the breeding of drought tolerant plants with increased production under water deficit conditions.