The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

Expression of the tetrahydrofolate‐dependent nitric oxide synthase from the green alga Ostreococcus tauri increases tolerance to abiotic stresses and influences stomatal development in Arabidopsis

Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants. NO plays a crucial role in growth and development, from germination to senescence, and is also involved in plant responses to biotic and abiotic stresses. In animals, NO is synthesized by well‐described nitric oxide synthase (NOS) enzymes. NOS activity has also been detected in higher plants, but no gene encoding an NOS protein, or the enzymes required for synthesis of tetrahydrobiopterin, an essential cofactor of mammalian NOS activity, have been identified so far. Recently, an NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) has been discovered and characterized. Arabidopsis thaliana plants were transformed with OtNOS under the control of the inducible short promoter fragment (SPF) of the sunflower (Helianthus annuus) Hahb‐4 gene, which responds to abiotic stresses and abscisic acid. Transgenic plants expressing OtNOS accumulated higher NO concentrations compared with siblings transformed with the empty vector, and displayed enhanced salt, drought and oxidative stress tolerance. Moreover, transgenic OtNOS lines exhibited increased stomatal development compared with plants transformed with the empty vector. Both in vitro and in vivo experiments indicate that OtNOS, unlike mammalian NOS, efficiently uses tetrahydrofolate as a cofactor in Arabidopsis plants. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool to improve plant fitness under adverse growth conditions.     

The ANGULATA7 gene encodes a DnaJ‐like zinc finger‐domain protein involved in chloroplast function and leaf development in Arabidopsis

The characterization of mutants with altered leaf shape and pigmentation has previously allowed the identification of nuclear genes that encode plastid‐localized proteins that perform essential functions in leaf growth and development. A large‐scale screen previously allowed us to isolate ethyl methanesulfonate‐induced mutants with small rosettes and pale green leaves with prominent marginal teeth, which were assigned to a phenotypic class that we dubbed Angulata. The molecular characterization of the 12 genes assigned to this phenotypic class should help us to advance our understanding of the still poorly understood relationship between chloroplast biogenesis and leaf morphogenesis. In this article, we report the phenotypic and molecular characterization of the angulata7‐1 (anu7‐1) mutant of Arabidopsis thaliana, which we found to be a hypomorphic allele of the EMB2737 gene, which was previously known only for its embryonic‐lethal mutations. ANU7 encodes a plant‐specific protein that contains a domain similar to the central cysteine‐rich domain of DnaJ proteins. The observed genetic interaction of anu7‐1 with a loss‐of‐function allele of GENOMES UNCOUPLED1 suggests that the anu7‐1 mutation triggers a retrograde signal that leads to changes in the expression of many genes that normally function in the chloroplasts. Many such genes are expressed at higher levels in anu7‐1 rosettes, with a significant overrepresentation of those required for the expression of plastid genome genes. Like in other mutants with altered expression of plastid‐encoded genes, we found that anu7‐1 exhibits defects in the arrangement of thylakoidal membranes, which appear locally unappressed.     

The wheat TaGBF1 gene is involved in the blue‐light response and salt tolerance

Light and abiotic stress both strongly modulate plant growth and development. However, the effect of light‐responsive factors on growth and abiotic stress responses in wheat (Triticum aestivum) is unknown. G–box binding factors (GBFs) are blue light‐specific components, but their function in abiotic stress responses has not been studied. Here we identified a wheat GBF1 gene that mediated both the blue light‐ and abiotic stress‐responsive signaling pathways. TaGBF1 was inducible by blue light, salt and exposure to abscisic acid (ABA). TaGBF1 interacted with a G–box light‐responsive element in vitro and promoted a blue‐light response in wheat and Aradidopsis thaliana. Both TaGBF1 over‐expression in wheat and its heterologous expression in A. thaliana heighten sensitivity to salinity and ABA, but its knockdown in wheat conferred resistance to high salinity and ABA. The expression of AtABI5, a key component of the ABA signaling pathway in A. thaliana, and its homolog Wabi5 in wheat was increased by transgenic expression of TaGBF1. The hypersensitivity to salt and ABA caused by TaGBF1 was not observed in the abi5 mutant background, showing that ABI5 is the mediator in TaGBF1‐induced abiotic stress responses. However, the hypersensitivity to salt conferred by TaGBF1 is not dependent on light. This suggests that TaGBF1 is a common component of blue light‐ and abiotic stress‐responsive signaling pathways.     

Arabidopsis PLC2 is involved in auxin‐modulated reproductive development

Phospholipase C (PLC) is an enzyme that plays crucial roles in various signal transduction pathways in mammalian cells. However, the role of PLC in plant development is poorly understood. Here we report involvement of PLC2 in auxin‐mediated reproductive development in Arabidopsis. Disruption of PLC2 led to sterility, indicating a significant role for PLC2 in reproductive development. Development of both male and female gametophytes was severely perturbed in plc2 mutants. Moreover, elevated auxin levels were observed in plc2 floral tissues, suggesting that the infertility of plc2 plants may be associated with increased auxin concentrations in the reproductive organs. We show that expression levels of the auxin reporters DR5:GUS and DR5:GFP were elevated in plc2 anthers and ovules. In addition, we found that expression of the auxin biosynthetic YUCCA genes was increased in plc2 plants. We conclude that PLC2 is involved in auxin biosynthesis and signaling, thus modulating development of both male and female gametophytes in Arabidopsis.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.     

A dominant‐negative form of Arabidopsis AP‐3 β‐adaptin improves intracellular pH homeostasis

Intracellular pH (pH_i) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pH_i regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid‐tolerant 1‐1D (wat1‐1D) are due to the expression of a truncated form of AP‐3 β‐adaptin (encoded by the PAT2 gene) that behaves as a as dominant‐negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pH_i and a more depolarized plasma membrane electrical potential than wild‐type cells. Additional phenotypes of wat1‐1D roots include increased rates of acetate efflux, K⁺ uptake and H⁺ efflux, the latter reflecting the in vivo activity of the plasma membrane H⁺‐ATPase. The in vitro activity of the enzyme was not increased but, as the H⁺‐ATPase is electrogenic, the increased ion permeability would allow a higher rate of H⁺ efflux. The AP‐3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1‐1D mutant can be explained if loss of function of the AP‐3 β‐adaptin causes activation of channels or transporters for organic anions (acetate) and for K⁺ at the plasma membrane, perhaps through miss‐localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.     

Cadmium‐induced and trans‐generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana

Trans‐generational adaptation is important to respond rapidly to environmental challenges and increase overall plant fitness. Besides well‐known mechanisms such as epigenetic modifications, vertically transmitted endophytic bacteria might contribute to this process. The cultivable and total endophytic communities of several generations of Arabidopsis thaliana seeds harvested from plants exposed to cadmium (Cd) or not exposed were investigated. The diversity and richness of the seed endophytic community decreased with an increasing number of generations. Aeromicrobium and Pseudonocardia were identified as indicator species in seeds from Cd‐exposed plants, while Rhizobium was abundantly present in both seed types. Remarkably, Rhizobium was the only genus that was consistently detected in seeds of all generations, which suggests that the phenotypic characteristics were more important as selection criteria for which bacteria are transferred to the next plant generation than the actual genera. Production of IAA was an important trait for endophytes from both seed types, while ACC deaminase activity and Cd tolerance were mainly associated with seed endophytes from Cd‐exposed plants. Understanding how different factors influence the seed endophytic community can help us to improve seed quality and plant growth through different biotechnological applications.     

Subcellular dynamics of proteins and metabolites under abiotic stress reveal deferred response of the Arabidopsis thaliana hexokinase‐1 mutant gin2‐1 to high light

Stress responses in plants imply spatio‐temporal changes in enzymes and metabolites, including subcellular compartment‐specific re‐allocation processes triggered by sudden changes in environmental parameters. To investigate interactions of primary metabolism with abiotic stress, the gin2‐1 mutant, defective in the sugar sensor hexokinase 1 (HXK1) was compared with its wildtype Landsberg erecta (Ler) based on time resolved, compartment‐specific metabolome and proteome data obtained over a full diurnal cycle. The high light sensitive gin2‐1 mutant was substantially delayed in subcellular re‐distribution of metabolites upon stress, and this correlated with a massive reduction in proteins belonging to the ATP producing electron transport chain under high light, while fewer changes occurred in the cold. In the wildtype, compounds specifically protecting individual compartments could be identified, e.g., maltose and raffinose in plastids, myo‐inositol in mitochondria, but gin2‐1 failed to recruit these substances to the respective compartments, or responded only slowly to high irradiance. No such delay was obtained in the cold. At the whole cell level, concentrations of the amino acids, glycine and serine, provided strong evidence for an important role of the photorespiratory pathway during stress exposure, and different subcellular allocation of serine may contribute to the slow growth of the gin2‐1 mutant under high irradiance.     

ATR and MKP1 play distinct roles in response to UV‐B stress in Arabidopsis

Ultraviolet‐B (UV‐B) stress activates MAP kinases (MAPKs) MPK3 and MPK6 in Arabidopsis. MAPK activity must be tightly controlled in order to ensure an appropriate cellular outcome. MAPK phosphatases (MKPs) effectively control MAPKs by dephosphorylation of phosphothreonine and phosphotyrosine in their activation loops. Arabidopsis MKP1 is an important regulator of MPK3 and MPK6, and mkp1 knockout mutants are hypersensitive to UV‐B stress, which is associated with reduced inactivation of MPK3 and MPK6. Here, we demonstrate that MPK3 and MPK6 are hyperactivated in response to UV‐B in plants that are deficient in photorepair, suggesting that UV‐damaged DNA is a trigger of MAPK signaling. This is not due to a block in replication, as, in contrast to atr, the mkp1 mutant is not hypersensitive to the replication‐inhibiting drug hydroxyurea, hydroxyurea does not activate MPK3 and MPK6, and atr is not impaired in MPK3 and MPK6 activation in response to UV‐B. We further show that mkp1 leaves and roots are UV‐B hypersensitive, whereas atr is mainly affected at the root level. Tolerance to UV‐B stress has been previously associated with stem cell removal and CYCB1;1 accumulation. Although UV‐B‐induced stem cell death and CYCB1;1 expression are not altered in mkp1 roots, CYCB1;1 expression is reduced in mkp1 leaves. We conclude that the MKP1 and ATR pathways operate in parallel, with primary roles for ATR in roots and MKP1 in leaves.     

Autophagy mediates caloric restriction‐induced lifespan extension in Arabidopsis

Caloric restriction (CR) extends lifespan in various heterotrophic organisms ranging from yeasts to mammals, but whether a similar phenomenon occurs in plants remains unknown. Plants are autotrophs and use their photosynthetic machinery to convert light energy into the chemical energy of glucose and other organic compounds. As the rate of photosynthesis is proportional to the level of photosynthetically active radiation, the CR in plants can be modeled by lowering light intensity. Here, we report that low light intensity extends the lifespan in Arabidopsis through the mechanisms triggering autophagy, the major catabolic process that recycles damaged and potentially harmful cellular material. Knockout of autophagy‐related genes results in the short lifespan and suppression of the lifespan‐extending effect of the CR. Our data demonstrate that the autophagy‐dependent mechanism of CR‐induced lifespan extension is conserved between autotrophs and heterotrophs.     

Arabidopsis RABA1 GTPases are involved in transport between the trans‐Golgi network and the plasma membrane,and are required for salinity stress tolerance

RAB GTPases are key regulators of membrane traffic. Among them, RAB11, a widely conserved sub‐group, has evolved in a unique way in plants; plant RAB11 members show notable diversity, whereas yeast and animals have only a few RAB11 members. Fifty‐seven RAB GTPases are encoded in the Arabidopsis thaliana genome, 26 of which are classified in the RAB11 group (further divided into RABA1–RABA6 sub‐groups). Although several plant RAB11 members have been shown to play pivotal roles in plant‐unique developmental processes, including cytokinesis and tip growth, molecular and physiological functions of the majority of RAB11 members remain unknown. To reveal precise functions of plant RAB11, we investigated the subcellular localization and dynamics of the largest sub‐group of Arabidopsis RAB11, RABA1, which has nine members. RABA1 members reside on mobile punctate structures adjacent to the trans‐Golgi network and co‐localized with VAMP721/722, R‐SNARE proteins that operate in the secretory pathway. In addition, the constitutive‐active mutant of RABA1b, RABA1b^Q72L , was present on the plasma membrane. The RABA1b ‐containing membrane structures showed actin‐dependent dynamic motion . Vesicles labeled by GFP–RABA1b moved dynamically, forming queues along actin filaments. Interestingly, Arabidopsis plants whose four major RABA1 members were knocked out, and those expressing the dominant‐negative mutant of RABA1B, exhibited hypersensitivity to salinity stress. Altogether, these results indicate that RABA1 members mediate transport between the trans‐Golgi network and the plasma membrane, and are required for salinity stress tolerance.