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.

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.

Stomatal CO2 responses at sub- and above-ambient CO2 levels employ different pathways in Arabidopsis.

Stomatal pores that control plant CO2 uptake and water loss affect global carbon and water cycles. In the era of increasing atmospheric CO2 levels and vapor pressure deficit (VPD), it is essential to understand how these stimuli affect stomatal behavior. Whether stomatal responses to sub-ambient and above-ambient CO2 levels are governed by the same regulators and depend on VPD remains unknown. We studied stomatal conductance responses in Arabidopsis (Arabidopsis thaliana) stomatal signaling mutants under conditions where CO2 levels were either increased from sub-ambient to ambient (400 ppm) or from ambient to above-ambient levels under normal or elevated VPD. We found that guard cell signaling components involved in CO2-induced stomatal closure have different roles in the sub-ambient and above-ambient CO2 levels. The CO2-specific regulators prominently affected sub-ambient CO2 responses, whereas the lack of guard cell slow-type anion channel SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) more strongly affected the speed of above-ambient CO2-induced stomatal closure. Elevated VPD caused lower stomatal conductance in all studied genotypes and CO2 transitions, as well as faster CO2-responsiveness in some studied genotypes and CO2 transitions. Our results highlight the importance of experimental setups in interpreting stomatal CO2-responsiveness, as stomatal movements under different CO2 concentration ranges are controlled by distinct mechanisms. Elevated CO2 and VPD responses may also interact. Hence, multi-factor treatments are needed to understand how plants integrate different environmental signals and translate them into stomatal responses.

Impacts of methyl jasmonate on Selaginella martensii: volatiles, transcriptomics, phytohormones, and gas exchange.

Methyl jasmonate (MeJA) induces various defence responses in seed plants, but for early plant lineages, information on the potential of jasmonates to elicit stress signalling and trigger physiological modifications is limited. The spikemoss Selaginella martensii was exposed to a range of MeJA concentrations (0, 10, 25, and 50 mM), and biogenic volatile organic compound (BVOC) emissions, photosynthetic rate (A), and stomatal conductance (gs) were continuously measured. In addition, changes in phytohormone concentrations and gene expression were studied. Enhancement of methanol, lipoxygenase pathway volatiles and linalool emissions, and reductions in A and gs, were MeJA dose-dependent. Before MeJA treatment, the concentration of 12-oxo-phytodienoic acid (OPDA) was 7-fold higher than jasmonic acid (JA). MeJA treatment rapidly increased OPDA and JA concentrations (within 30 min), with the latter more responsive. Some genes involved in BVOC biosynthesis and OPDA-specific response were up-regulated at 30 min after MeJA spraying, whereas those in the JA signalling pathway were not affected. Although JA was synthesized in S. martensii, OPDA was prioritized as a signalling molecule upon MeJA application. MeJA inhibited primary and enhanced secondary metabolism; we propose that fast-emitted linalool could serve as a marker of elicitation of stress-induced metabolism in lycophytes.

Leaf temperature responses to ABA and dead bacteria in wheat and Arabidopsis.

Stomatal densities, aperture openness and their responsiveness to environmental change determine plant water loss and regulate entry of pathogens. Stomatal responsiveness is usually assessed on restricted areas of leaves or isolated epidermal peels floated in solution. Analyzing these responses in the whole plant context could give valuable additional information, for example on the role of mesophyll in stomatal responses. We analyzed stomatal responses to the phytohormone abscisic acid (ABA) and pathogenic elicitors in intact plants by dynamic measurement of leaf temperature. We tested whether ABA-induced stomatal closure in wheat requires external nitrate and whether bacterial elicitor-induced stomatal closure can be detected by dynamic thermal imaging in intact Arabidopsis. We found that wheat was hypersensitive to all applied treatments, as even mock-treated leaves showed a strong increase in leaf temperature. Nevertheless, ABA activated stomatal closure in wheat independent of exogenous nitrate. Pathogenic elicitors triggered a fast and transient increase in leaf temperature in intact Arabidopsis, indicating short-term stomatal closure. The data suggest that the dynamics of pathogen-induced stomatal closure is different in whole plants compared to epidermal peels, where elicitor-induced stomatal closure persists longer. We propose that dynamic thermal imaging could be applied to address the effect of pathogenic elicitors on stomatal behavior in whole plants to complement detached sample assays and gain a better understanding of stomatal immunity.

Dynamic thermal imaging confirms local but not fast systemic ABA responses.

Abscisic acid (ABA) signals regulating stomatal aperture and water loss are usually studied in detached leaves or isolated epidermal peels and at infrequent timepoints. Measuring stomatal ABA responses in attached leaves across a time course enables the study of stomatal behaviour in the physiological context of the plant. Infrared thermal imaging is often used to characterize steady-state stomatal conductance via comparisons of leaf surface temperature but is rarely used to capture stomatal responses over time or across different leaf surfaces. We used dynamic thermal imaging as a robust, but sensitive, tool to observe stomatal ABA responses in a whole plant context. We detected stomatal responses to low levels of ABA in both monocots and dicots and identified differences between the responses of different leaves. Using whole plant thermal imaging, stomata did not always behave as described previously for detached samples: in Arabidopsis, we found no evidence for fast systemic ABA-induced stomatal closure, and in barley, we observed no requirement for exogenous nitrate during ABA-induced stomatal closure. Thus, we recommend dynamic thermal imaging as a useful approach to complement detached sample assays for the study of local and systemic stomatal responses and molecular mechanisms underlying stomatal responses to ABA in the whole plant context.