STN7 is not essential for developmental acclimation of Arabidopsis to light intensity.

Plants respond to changing light intensity in the short term through regulation of light harvesting, electron transfer, and metabolism to mitigate redox stress. A sustained shift in light intensity leads to a long-term acclimation response (LTR). This involves adjustment in the stoichiometry of photosynthetic complexes through de novo synthesis and degradation of specific proteins associated with the thylakoid membrane. The light-harvesting complex II (LHCII) serine/threonine kinase STN7 plays a key role in short-term light harvesting regulation and was also suggested to be crucial to the LTR. Arabidopsis plants lacking STN7 (stn7) shifted to low light experience higher photosystem II (PSII) redox pressure than the wild type or those lacking the cognate phosphatase TAP38 (tap38), while the reverse is true at high light, where tap38 suffers more. In principle, the LTR should allow optimisation of the stoichiometry of photosynthetic complexes to mitigate these effects. We used quantitative label-free proteomics to assess how the relative abundance of photosynthetic proteins varied with growth light intensity in wild-type, stn7, and tap38 plants. All plants were able to adjust photosystem I, LHCII, cytochrome b6 f, and ATP synthase abundance with changing white light intensity, demonstrating neither STN7 nor TAP38 is crucial to the LTR per se. However, stn7 plants grown for several weeks at low light (LL) or moderate light (ML) still showed high PSII redox pressure and correspondingly lower PSII efficiency, CO2 assimilation, and leaf area compared to wild-type and tap38 plants, hence the LTR is unable to fully ameliorate these symptoms. In contrast, under high light growth conditions the mutants and wild type behaved similarly. These data are consistent with the paramount role of STN7-dependent LHCII phosphorylation in tuning PSII redox state for optimal growth in LL and ML conditions.

High cyclic electron transfer via the PGR5 pathway in the absence of photosynthetic control.

The light reactions of photosynthesis couple electron and proton transfers across the thylakoid membrane, generating NADPH, and proton motive force (pmf) that powers the endergonic synthesis of ATP by ATP synthase. ATP and NADPH are required for CO2 fixation into carbohydrates by the Calvin-Benson-Bassham cycle. The dominant ΔpH component of the pmf also plays a photoprotective role in regulating photosystem II light harvesting efficiency through nonphotochemical quenching (NPQ) and photosynthetic control via electron transfer from cytochrome b6f (cytb6f) to photosystem I. ΔpH can be adjusted by increasing the proton influx into the thylakoid lumen via upregulation of cyclic electron transfer (CET) or decreasing proton efflux via downregulation of ATP synthase conductivity (gH+). The interplay and relative contributions of these two elements of ΔpH control to photoprotection are not well understood. Here, we showed that an Arabidopsis (Arabidopsis thaliana) ATP synthase mutant hunger for oxygen in photosynthetic transfer reaction 2 (hope2) with 40% higher proton efflux has supercharged CET. Double crosses of hope2 with the CET-deficient proton gradient regulation 5 and ndh-like photosynthetic complex I lines revealed that PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET is the major pathway contributing to higher proton influx. PGR5-dependent CET allowed hope2 to maintain wild-type levels of ΔpH, CO2 fixation and NPQ, however photosynthetic control remained absent and PSI was prone to photoinhibition. Therefore, high CET in the absence of ATP synthase regulation is insufficient for PSI photoprotection.

The origin and evolution of stomata.

The acquisition of stomata is one of the key innovations that led to the colonisation of the terrestrial environment by the earliest land plants. However, our understanding of the origin, evolution and the ancestral function of stomata is incomplete. Phylogenomic analyses indicate that, firstly, stomata are ancient structures, present in the common ancestor of land plants, prior to the divergence of bryophytes and tracheophytes and, secondly, there has been reductive stomatal evolution, especially in the bryophytes (with complete loss in the liverworts). From a review of the evidence, we conclude that the capacity of stomata to open and close in response to signals such as ABA, CO2 and light (hydroactive movement) is an ancestral state, is present in all lineages and likely predates the divergence of the bryophytes and tracheophytes. We reject the hypothesis that hydroactive movement was acquired with the emergence of the gymnosperms. We also conclude that the role of stomata in the earliest land plants was to optimise carbon gain per unit water loss. There remain many other unanswered questions concerning the evolution and especially the origin of stomata. To address these questions, it will be necessary to: find more fossils representing the earliest land plants, revisit the existing early land plant fossil record in the light of novel phylogenomic hypotheses and carry out more functional studies that include both tracheophytes and bryophytes.

Physio-biochemical responses and expressional profiling analysis of drought tolerant genes in new promising rice genotype.

Rice cultivation in Egypt is limited by the scarcity of water resources. The main strategy of rice breeders to overcome this problem is to develop new high-yielding varieties that are tolerant to drought stress. In this study, an drought-tolerant (IR60080-46A) variety was crossed with commercial Egyptian varieties using the back-cross method and marker-assisted selection (MAS) approach. The advanced lines of these crosses were selected under drought stress conditions. The best-performing candidate line, RBL-112, and its parental genotypes, were evaluated under drought stress and control conditions. The RBL-112 line showed superior its root system, which in turn produced higher grain yield under drought-stress conditions than its parental and check genotypes. Furthermore, physiological and biochemical studies showed that the RBL-112 line maintained higher relative water content (RWC), maximum quantum efficiency of photosystem II (Fv/Fm) values, proline content, superoxide dismutase (SOD) activity, and lower malondialdehyde (MDA) content compared to its parents and the check. The functional expression profiles of 22 drought tolerance-related genes were studied, out of which the genes OsAHL1, OsLEA3, OsCATA, OsP5CS, OsSNAC1, Os1g64660, OsRab21, OsAPX2, OsDREB2A, OsSKIPa, and OsLG3 were strongly induced in the newly developed RBL-112 line under drought-stress conditions. It could be concluded that the new line has a higher capacity to modulate physiological activities and expression levels of several drought-induced genes to withstand drought stress with high yielding ability. This finding suggests that the RBL-112 line presents a promising new addition to enable sustainable rice cultivation under water-limited conditions, and confirms the efficiency of the approach implemented in the current study.

Inhibition of Arabidopsis stomatal development by plastoquinone oxidation.

Stomata are the pores in the epidermal surface of plant leaves that regulate the exchange of water and CO2 with the environment thus controlling leaf gas exchange.1 In the model dicot plant Arabidopsis thaliana, the transcription factors SPEECHLESS (SPCH) and MUTE sequentially control formative divisions in the stomatal lineage by forming heterodimers with ICE1.2 SPCH regulates entry into the stomatal lineage and its stability or activity is regulated by a mitogen-activated protein kinase (MAPK) signaling cascade, mediated by its interaction with ICE1.3-6 This MAPK pathway is regulated by extracellular epidermal patterning factor (EPFs) peptides, which bind a transmembrane receptor complex to inhibit (EPF1 and EPF2) or promote (STOMAGEN/EPFL9) stomatal development.7-9 MUTE controls the transition to guard mother cell identity and is regulated by the HD-ZIP transcription factor HDG2, which is expressed exclusively in stomatal lineage cells.10,11 Light signals acting through phytochrome and cryptochrome photoreceptors positively regulate stomatal development in response to increased irradiance.12,13 Here we report that stomatal development is also regulated by the redox state of the photosynthetic electron transport chain (PETC). Oxidation of the plastoquinone (PQ) pool inhibits stomatal development by negatively regulating SPCH and MUTE expression. This mechanism is dependent on MPK6 and forms part of the response to lowering irradiance, which is distinct to the photoreceptor dependent response to increasing irradiance. Our results show that environmental signals can act through the PETC, demonstrating that photosynthetic signals regulate the development of the pores through which CO2 enters the leaf.

Stomatal responses to carbon dioxide and light require abscisic acid catabolism in Arabidopsis.

In plants, stomata control water loss and CO2 uptake. The aperture and density of stomatal pores, and hence the exchange of gases between the plant and the atmosphere, are controlled by internal factors such as the plant hormone abscisic acid (ABA) and external signals including light and CO2. In this study, we examine the importance of ABA catabolism in the stomatal responses to CO2 and light. By using the ABA 8'-hydroxylase-deficient Arabidopsis thaliana double mutant cyp707a1 cyp707a3, which is unable to break down and instead accumulates high levels of ABA, we reveal the importance of the control of ABA concentration in mediating stomatal responses to CO2 and light. Intriguingly, our experiments suggest that endogenously produced ABA is unable to close stomata in the absence of CO2. Furthermore, we show that when plants are grown in short day conditions ABA breakdown is required for the modulation of both elevated [CO2]-induced stomatal closure and elevated [CO2]-induced reductions in leaf stomatal density. ABA catabolism is also required for the stomatal density response to light intensity, and for the full range of light-induced stomatal opening, suggesting that ABA catabolism is critical for the integration of stomatal responses to a range of environmental stimuli.

The Arabidopsis R-SNARE VAMP714 is essential for polarisation of PIN proteins and auxin responses.

The plant hormone auxin and its directional intercellular transport play a major role in diverse aspects of plant growth and development. The establishment of auxin gradients requires the asymmetric distribution of members of the auxin efflux carrier PIN-FORMED (PIN) protein family to the plasma membrane. An endocytic pathway regulates the recycling of PIN proteins between the plasma membrane and endosomes, providing a mechanism for dynamic localisation. N-Ethylmaleimide-sensitive factor adaptor protein receptors (SNAP receptors, SNAREs) mediate fusion between vesicles and target membranes and are classed as Q- or R-SNAREs based on their sequence. We analysed gain- and loss-of-function mutants, dominant-negative transgenics and localisation of the Arabidopsis R-SNARE VAMP714 protein to understand its function. We demonstrate that VAMP714 is essential for the insertion of PINs into the plasma membrane, for polar auxin transport, root gravitropism and morphogenesis. VAMP714 gene expression is upregulated by auxin, and the VAMP714 protein co-localises with endoplasmic reticulum and Golgi vesicles and with PIN proteins at the plasma membrane. It is proposed that VAMP714 mediates the delivery of PIN-carrying vesicles to the plasma membrane, and that this forms part of a positive regulatory loop in which auxin activates a VAMP714-dependent PIN/auxin transport system to control development.

Dynamic thylakoid stacking and state transitions work synergistically to avoid acceptor-side limitation of photosystem I.

TAP38/STN7-dependent (de)phosphorylation of light-harvesting complex II (LHCII) regulates the relative excitation rates of photosystems I and II (PSI, PSII) (state transitions) and the size of the thylakoid grana stacks (dynamic thylakoid stacking). Yet, it remains unclear how changing grana size benefits photosynthesis and whether these two regulatory mechanisms function independently. Here, by comparing Arabidopsis wild-type, stn7 and tap38 plants with the psal mutant, which undergoes dynamic thylakoid stacking but lacks state transitions, we explain their distinct roles. Under low light, smaller grana increase the rate of PSI reduction and photosynthesis by reducing the diffusion distance for plastoquinol; however, this beneficial effect is only apparent when PSI/PSII excitation balance is maintained by state transitions or far-red light. Under high light, the larger grana slow plastoquinol diffusion and lower the equilibrium constant between plastocyanin and PSI, maximizing photosynthesis by avoiding PSI photoinhibition. Loss of state transitions in low light or maintenance of smaller grana in high light also both bring about a decrease in cyclic electron transfer and over-reduction of the PSI acceptor side. These results demonstrate that state transitions and dynamic thylakoid stacking work synergistically to regulate photosynthesis in variable light.

HY5 is not integral to light mediated stomatal development in Arabidopsis.

Light is a crucial signal that regulates many aspects of plant physiology and growth including the development of stomata, the pores in the epidermal surface of the leaf. Light signals positively regulate stomatal development leading to changes in stomatal density and stomatal index (SI; the proportion of cells in the epidermis that are stomata). Both phytochrome and cryptochrome photoreceptors are required to regulate stomatal development in response to light. The transcription factor ELONGATED HYPOCOTYL 5 (HY5) is a key regulator of light signalling, acting downstream of photoreceptors. We hypothesised that HY5 could regulate stomatal development in response to light signals due to the putative presence of HY5 binding sites in the promoter of the STOMAGEN (STOM) gene, which encodes a peptide regulator of stomatal development. Our analysis shows that HY5 does have the potential to regulate the STOM promoter in vitro and that HY5 is expressed in both the epidermis and mesophyll. However, analysis of hy5 and hy5 hyh double mutants (HYH; HY5-HOMOLOG), found that they had normal stomatal development under different light conditions and the expression of stomatal developmental genes was not perturbed following light shift experiments. Analysis of stable lines overexpressing HY5 also showed no change in stomatal development or the expression of stomatal developmental genes. We therefore conclude that whilst HY5 has the potential to regulate the expression of STOM, it does not have a major role in regulating stomatal development in response to light signals.

Molecular control of stomatal development.

Plants have evolved developmental plasticity which allows the up- or down-regulation of photosynthetic and water loss capacities as new leaves emerge. This developmental plasticity enables plants to maximise fitness and to survive under differing environments. Stomata play a pivotal role in this adaptive process. These microscopic pores in the epidermis of leaves control gas exchange between the plant and its surrounding environment. Stomatal development involves regulated cell fate decisions that ensure optimal stomatal density and spacing, enabling efficient gas exchange. The cellular patterning process is regulated by a complex signalling pathway involving extracellular ligand-receptor interactions, which, in turn, modulate the activity of three master transcription factors essential for the formation of stomata. Here, we review the current understanding of the biochemical interactions between the epidermal patterning factor ligands and the ERECTA family of leucine-rich repeat receptor kinases. We discuss how this leads to activation of a kinase cascade, regulation of the bHLH transcription factor SPEECHLESS and its relatives, and ultimately alters stomatal production.

Origin and function of stomata in the moss Physcomitrella patens.

Stomata are microscopic valves on plant surfaces that originated over 400 million years (Myr) ago and facilitated the greening of Earth's continents by permitting efficient shoot-atmosphere gas exchange and plant hydration1. However, the core genetic machinery regulating stomatal development in non-vascular land plants is poorly understood2-4 and their function has remained a matter of debate for a century5. Here, we show that genes encoding the two basic helix-loop-helix proteins PpSMF1 (SPEECH, MUTE and FAMA-like) and PpSCREAM1 (SCRM1) in the moss Physcomitrella patens are orthologous to transcriptional regulators of stomatal development in the flowering plant Arabidopsis thaliana and essential for stomata formation in moss. Targeted P. patens knockout mutants lacking either PpSMF1 or PpSCRM1 develop gametophytes indistinguishable from wild-type plants but mutant sporophytes lack stomata. Protein-protein interaction assays reveal heterodimerization between PpSMF1 and PpSCRM1, which, together with moss-angiosperm gene complementations6, suggests deep functional conservation of the heterodimeric SMF1 and SCRM1 unit is required to activate transcription for moss stomatal development, as in A. thaliana7. Moreover, stomata-less sporophytes of ΔPpSMF1 and ΔPpSCRM1 mutants exhibited delayed dehiscence, implying stomata might have promoted dehiscence in the first complex land-plant sporophytes.

Plant Development: Suppression the Key to Asymmetric Cell Fate.

A new study shows that SPEECHLESS determines cell fate in the stomatal lineage but is inherited equally by daughter cells following an asymmetric cell division. The polarity determinant BASL acts as a MAPK scaffold, targeting SPEECHLESS for degradation in the larger daughter cell.

Elevated CO2-Induced Responses in Stomata Require ABA and ABA Signaling.

An integral part of global environment change is an increase in the atmospheric concentration of CO2 ([CO2]) [1]. Increased [CO2] reduces leaf stomatal apertures and density of stomata that plays out as reductions in evapotranspiration [2-4]. Surprisingly, given the importance of transpiration to the control of terrestrial water fluxes [5] and plant nutrient acquisition [6], we know comparatively little about the molecular components involved in the intracellular signaling pathways by which [CO2] controls stomatal development and function [7]. Here, we report that elevated [CO2]-induced closure and reductions in stomatal density require the generation of reactive oxygen species (ROS), thereby adding a new common element to these signaling pathways. We also show that the PYR/RCAR family of ABA receptors [8, 9] and ABA itself are required in both responses. Using genetic approaches, we show that ABA in guard cells or their precursors is sufficient to mediate the [CO2]-induced stomatal density response. Taken together, our results suggest that stomatal responses to increased [CO2] operate through the intermediacy of ABA. In the case of [CO2]-induced reductions in stomatal aperture, this occurs by accessing the guard cell ABA signaling pathway. In both [CO2]-mediated responses, our data are consistent with a mechanism in which ABA increases the sensitivity of the system to [CO2] but could also be explained by requirement for a CO2-induced increase in ABA biosynthesis specifically in the guard cell lineage. Furthermore, the dependency of stomatal [CO2] signaling on ABA suggests that the ABA pathway is, in evolutionary terms, likely to be ancestral.

phytochrome B Is required for light-mediated systemic control of stomatal development.

Stomata are pores found on the surfaces of leaves, and they regulate gas exchange between the plant and the environment [1]. Stomatal development is highly plastic and is influenced by environmental signals [2]. Light stimulates stomatal development, and this response is mediated by plant photoreceptors [3-5], with the red-light photoreceptor phytochrome B (phyB) having a dominant role in white light [3]. Light also regulates stomatal development systemically, with the irradiance perceived by mature leaves modulating stomatal development in young leaves [6, 7]. Here, we show that phyB is required for this systemic response. Using a combination of tissue-specific expression and an inducible expression system in the loss-of-function phyB-9 mutant [8], we show that phyB expression in the stomatal lineage, mesophyll, and phloem is sufficient to restore wild-type stomatal development. Induction of PHYB in mature leaves also rescues stomatal development in young untreated leaves, whereas phyB mutants are defective in the systemic regulation of stomatal development. Our data show that phyB acts systemically to regulate cell fate decisions in the leaf epidermis.

Putting the brakes on: abscisic acid as a central environmental regulator of stomatal development.

Stomata are produced by a controlled series of epidermal cell divisions. The molecular underpinnings of this process are becoming well understood, but mechanisms that determine plasticity of stomatal patterning to many exogenous and environmental cues remain less clear. Light quantity and quality, vapour pressure deficit, soil water content, and CO2 concentration are detected by the plant, and new leaves adapt their stomatal densities accordingly. Mature leaves detect these environmental signals and relay messages to immature leaves to tell them how to adapt and grow. Stomata on mature leaves may act as stress signal-sensing and transduction centres, locally by aperture adjustment, and at long distance by optimizing stomatal density to maximize future carbon gain while minimizing water loss. Although mechanisms of stomatal aperture responses are well characterized, the pathways by which mature stomata integrate environmental signals to control immature epidermal cell fate, and ultimately stomatal density, are not. Here we evaluate current understanding of the latter through the influence of the former. We argue that mature stomata, as key portals by which plants coordinate their carbon and water relations, are controlled by abscisic acid (ABA), both metabolically and hydraulically, and that ABA is also a core regulator of environmentally determined stomatal development.

Developmental priming of stomatal sensitivity to abscisic acid by leaf microclimate.

Plant water loss and CO2 uptake are controlled by valve-like structures on the leaf surface known as stomata. Stomatal aperture is regulated by hormonal and environmental signals. We show here that stomatal sensitivity to the drought hormone abscisic acid (ABA) is acquired during leaf development by exposure to an increasingly dryer atmosphere in the rosette plant Arabidopsis. Young leaves, which develop in the center of the rosette, do not close in response to ABA. As the leaves increase in size, they are naturally exposed to increasingly dry air as a consequence of the spatial arrangement of the leaves, and this triggers the acquisition of ABA sensitivity. Interestingly, stomatal ABA sensitivity in young leaves is rapidly restored upon water stress. These findings shed new light on how plant architecture and stomatal physiology have coevolved to optimize carbon gain against water loss in stressing environments.

GSK3-like kinases integrate brassinosteroid signaling and stomatal development.

Developmental pathways are often regulated by multiple signals, and a major challenge is to understand how the different signaling pathways triggered by these signals interact to modulate a specific process. Brassinosteroids (BRs) are plant hormones that regulate cell expansion, cell division, and photomorphogenesis. A key regulator in BR signaling, the GSK3- and SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE2, regulates two distinct steps in the stomatal development signaling pathway to either enhance or inhibit this process.

Land plants acquired active stomatal control early in their evolutionary history.

Stomata are pores that regulate plant gas exchange [1]. They evolved more than 400 million years ago [2, 3], but the origin of their active physiological responses to endogenous and environmental cues is unclear [2-6]. Recent research suggests that the stomata of lycophytes and ferns lack pore closure responses to abscisic acid (ABA) and CO(2). This evidence led to the hypothesis that a fundamental transition from passive to active control of plant water balance occurred after the divergence of ferns 360 million years ago [7, 8]. Here we show that stomatal responses of the lycophyte Selaginella [9] to ABA and CO(2) are directly comparable to those of the flowering plant Arabidopsis [10]. Furthermore, we show that the underlying intracellular signaling pathways responsible for stomatal aperture control are similar in both basal and modern vascular plant lineages. Our evidence challenges the hypothesis that acquisition of active stomatal control of plant carbon and water balance represents a critical turning point in land plant evolution [7, 8]. Instead, we suggest that the critical evolutionary development is represented by the innovation of stomata themselves and that physiologically active stomatal control originated at least as far back as the emergence of the lycophytes (circa 420 million years ago) [11].