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 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.

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.

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.

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.

MERISTEM-DEFECTIVE, an RS domain protein, is required for the correct meristem patterning and function in Arabidopsis.

Plant growth and development is dependent on the specification and maintenance of pools of stem cells found in the meristems. Mutations in the Arabidopsis MERISTEM-DEFECTIVE (MDF) gene lead to a loss of stem cell and meristematic activity in the root and vegetative shoot. MDF encodes a putative RS domain protein with a predicted role in transcription or RNA processing control. mdf mutants exhibit decreased levels of PINFORMED2 (PIN2) and PIN4 mRNAs, which is associated with a reduction in PIN:GFP levels, and with a defective auxin maximum in the basal region of the developing mdf embryo and seedling root meristem. Seedling roots also exhibit reduced PLETHORA (PLT), SCARECROW and SHORTROOT gene expression, a loss of stem cell activity, terminal differentiation of the root meristem and defective cell patterning. MDF expression is not defective in the bodenlos, pin1 or eir1/pin2 auxin mutants, and is not modulated by exogenous auxin. plt1 plt2 double mutants have unaffected levels of MDF RNA, indicating that MDF acts upstream of PIN and PLT gene expression. Differentiation of the shoot stem cell pool also occurs in mdf mutants, associated with a reduced WUSCHEL (WUS) expression domain and expanded CLAVATA3 (CLV3) domain. Overexpression of MDF leads to the activation of markers of embryonic identity and ectopic meristem activity in vegetative tissues. These results demonstrate a requirement for the MDF-dependent pathway in regulating PIN/PLT- and WUS/CLV-mediated meristem activity.

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.

Laser-capture microdissection to study global transcriptional changes during plant embryogenesis.

A key objective in the study of plant embryogenesis is to identify genes expressed in temporal and spatial patterns during development, in order to understand transcriptional control mechanisms regulating pattern formation, differentiation and morphogenesis. Mutagenic approaches have proved powerful to identify essential genes, but global, transcriptome-wide analysis of mRNA profiles in cells at different stages of differentiation would allow the identification of changes in the abundance of major classes of transcripts expressed from genes that are known to respond to regulatory signals, such as hormones. Particular classes of transcription factors or other genes might also be discovered to be associated with particular aspects of cell differentiation. This information would allow the construction of models to describe how signalling pathways might modulate transcriptional changes associated with cell differentiation. Previous limitations in tissue accessibility for RNA isolation have been overcome through the use of laser-capture microdissection, which allows cells from different embryonic tissues to be isolated, for RNA isolation, amplification and analysis by either polymerase chain reaction or DNA microarray techniques.

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.

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.

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].

Environmental regulation of stomatal development.

Stomata are microscopic structures in the epidermis of the aerial parts of flowering plants formed by two specialized guard cells flanking a central pore. The role of stomata is to optimize gas exchange (the uptake of carbon dioxide and the loss of water vapor) to suit the prevailing environmental conditions. To do this plants open and close the stomatal pores and regulates the number of stomata that develop on the epidermes. Both these responses are controlled by integrating information from environmental cues and hormonal signals. Recent work has resulted in significant advances in our understanding of the underlying pathway controlling stomatal development. Here we shall discuss how environmental cues might modulate this pathway such that gas exchange is optimized to suit the prevailing environmental conditions.

phytochrome B and PIF4 regulate stomatal development in response to light quantity.

Stomata are pores on the surfaces of leaves that regulate gas exchange between the plant interior and the atmosphere [1]. Plants adapt to changing environmental conditions in the short term by adjusting the aperture of the stomatal pores, whereas longer-term changes are accomplished by altering the proportion of stomata that develop on the leaf surface [2, 3]. Although recent work has identified genes involved in the control of stomatal development [4], we know very little about how stomatal development is modulated by environmental signals, such as light. Here, we show that mature leaves of Arabidopsis grown at higher photon irradiances show significant increases in stomatal index (S.I.) [5] compared to those grown at lower photon irradiances. Light quantity-mediated changes in S.I. occur in red light, suggesting that phytochrome photoreceptors [6] are involved. By using a genetic approach, we demonstrate that this response is dominated by phytochrome B and also identify a role for the transcription factor, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) [7]. In sum, we identify a photoreceptor and downstream signaling protein involved in light-mediated control of stomatal development, thereby establishing a tractable system for investigating how an environmental signal modulates stomatal development.

Transcriptional profiling of the Arabidopsis embryo.

We have used laser-capture microdissection to isolate RNA from discrete tissues of globular, heart, and torpedo stage embryos of Arabidopsis (Arabidopsis thaliana). This was amplified and analyzed by DNA microarray using the Affymetrix ATH1 GeneChip, representing approximately 22,800 Arabidopsis genes. Cluster analysis showed that spatial differences in gene expression were less significant than temporal differences. Time course analysis reveals the dynamics and complexity of gene expression in both apical and basal domains of the developing embryo, with several classes of synexpressed genes identifiable. The transition from globular to heart stage is associated in particular with an up-regulation of genes involved in cell cycle control, transcriptional regulation, and energetics and metabolism. The transition from heart to torpedo stage is associated with a repression of cell cycle genes and an up-regulation of genes encoding storage proteins, and pathways of cell growth, energy, and metabolism. The torpedo stage embryo shows strong functional differentiation in the root and cotyledon, as inferred from the classes of genes expressed in these tissues. The time course of expression of the essential EMBRYO-DEFECTIVE genes shows that most are expressed at unchanging levels across all stages of embryogenesis. We show how identified genes can be used to generate cell type-specific markers and promoter activities for future application in cell biology.

The turnip mutant of Arabidopsis reveals that LEAFY COTYLEDON1 expression mediates the effects of auxin and sugars to promote embryonic cell identity.

The transition from embryonic to vegetative growth marks an important developmental stage in the plant life cycle. The turnip (tnp) mutant was identified in a screen for modifiers of POLARIS expression, a gene required for normal root growth. Mapping and molecular characterization of tnp shows that it represents a gain-of-function mutant of LEAFY COTYLEDON1 (LEC1), due to a promoter mutation. This results in the ectopic expression of LEC1, but not of other LEC genes, in vegetative tissues. The LEC class of genes are known regulators of embryogenesis, involved in the control of embryonic cell identity by currently unknown mechanisms. Activation of the LEC-dependent pathway in tnp leads to the loss of hypocotyl epidermal cell marker expression and loss of SCARECROW expression in the endodermis, the ectopic accumulation of starch and lipids, and the up-regulation of early and late embryonic genes. tnp also shows partial deetiolation during dark growth. Penetrance of the mutant phenotype is strongly enhanced in the presence of exogenous auxin and sugars, but not by gibberellin or abscisic acid, and is antagonized by cytokinin. We propose that the role of LEC1 in embryonic cell fate control requires auxin and sucrose to promote cell division and embryonic differentiation.

The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling.

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.

Characterization of a proteinase inhibitor from Brachypodium distachyon suggests the conservation of defence signalling pathways between dicotyledonous plants and grasses.

SUMMARY Proteinase inhibitors (PIs) are established markers for wound- and especially jasmonate-mediated signalling in dicot species such as tomato and potato. Differential screening of a cDNA library constructed from RNA isolated from wounded leaves of the grass Brachypodium distachyon led to the identification of a proteinase inhibitor gene (Bdpin1). Bdpin1 exhibited the highest homology to the subtilisin/chymotrypsin-inhibiting subgroup of the pin1 class of plant PIs. Northern analyses indicated that Bdpin1 was induced within 6 h at the site of wounding and systemically, by 24 h, thereby providing evidence for long-distance signalling in grasses. Bdpin1 also proved to be more rapidly induced in susceptible than in resistant ecotypes of B. distachyon following challenge with the Rice blast pathogen, Magnaporthe grisea. Screening with chemical signals indicated that Bdpin1 could be induced with MeJA but not with the putative mimic of salicylic acid, benzothiadiazole. Genomic Southern hybridization was consistent with Bdpin1 existing at a single locus, which was isolated following screening of a genomic cosmid library. DNA upstream of the Bdpin1 coding sequence was characterized via fusion to a GUS reporter and was found to confer wound-responsive transcription in B. distachyon and other cereals following biolistic bombardment. Both wound- and TMV-activated Bdpin1-GUS activity was detected in transgenic tobacco. Given that B. distachyon represents an ancestral grass species, our data suggest that there is considerable conservation in defence-associated signalling between dicots and grasses.

The POLARIS gene of Arabidopsis encodes a predicted peptide required for correct root growth and leaf vascular patterning.

The POLARIS (PLS) gene of Arabidopsis was identified as a promoter trap transgenic line, showing beta-glucuronidase fusion gene expression predominantly in the embryonic and seedling root, with low expression in aerial parts. Cloning of the PLS locus revealed that the promoter trap T-DNA had inserted into a short open reading frame (ORF). Rapid amplification of cDNA ends PCR, RNA gel blot analysis, and RNase protection assays showed that the PLS ORF is located within a short ( approximately 500 nucleotides) auxin-inducible transcript and encodes a predicted polypeptide of 36 amino acid residues. pls mutants exhibit a short-root phenotype and reduced vascularization of leaves. pls roots are hyperresponsive to exogenous cytokinins and show increased expression of the cytokinin-inducible gene ARR5/IBC6 compared with the wild type. pls seedlings also are less responsive to the growth-inhibitory effects of exogenous auxin and show reduced expression of the auxin-inducible gene IAA1 compared with the wild type. The PLS peptide-encoding region of the cDNA partially complements the pls mutation and requires the PLS ORF ATG for activity, demonstrating the functionality of the peptide-encoding ORF. Ectopic expression of the PLS ORF reduces root growth inhibition by exogenous cytokinins and increases leaf vascularization. We propose that PLS is required for correct auxin-cytokinin homeostasis to modulate root growth and leaf vascular patterning.