ABA-insensitive Mutants Research Articles

A novel ABA-insensitive mutant in Arabidopsis reveals molecular network of ABA-induced anthocyanin accumulation and abiotic stress tolerance

Abscisic acid (ABA) plays primary regulatory roles in abiotic stress tolerance and seed germination. Here, we report a unique novel Arabidopsis abscisic acid-insensitive mutant, abr (abscisic acid resistance), which was able to germinate in medium containing high ABA concentrations and tolerant to abiotic stress tolerance. We observed that abr mutant accumulated more anthocyanins by ABA treatment than did the wild type (WT). Dimethylthiourea (DMTU, an H2O2 scavenger) was effective in inhibiting ABA-induced anthocyanins accumulation. RNA-seq showed that the expression of anthocyanins synthesis, antioxidant enzyme and stress-related genes were specifically increased in ABA-treated abr seedlings, suggesting that the abr mutation affects stress response as well as ABA responses. Interestingly, seedlings accumulating anthocyanins exhibited more tolerance to mannitol and NaCl compared to wild type. We propose that ABA-induced H2O2 generation triggers the foliar anthocyanins accumulation, which, in turn, enhances the abiotic stress tolerance in abr mutant.

The involvement of abscisic acid-insensitive mutants in low phosphate stress responses during rhizosphere acidification, anthocyanin accumulation and Pi homeostasis in Arabidopsis

Phosphorus is an essential plant nutrient, used in the formation of macromolecules such as nucleic acids and phospholipids. Abscisic acid (ABA) may be involved in the process of low inorganic phosphate (Pi) responses. The phenotypes of ABA-insensitive Arabidopsis mutants (abi1/2/3/4/5) under low Pi stress were investigated to identify possible low Pi response mutant genes. The results showed enhanced rhizosphere acidification in the abi1–1/abi2–1/abi5–1 mutants under low Pi stress compared with wild-type (WT) seedlings. The abi1–1/abi2–1/ abi3–1/abi5–1 mutants accumulated less anthocyanin than the WT, while the abi4–1 mutant showed greater accumulation, implicating all the ABA-insensitive mutants in anthocyanin deposition under Pi deficiency. Alterations in the Pi contents of roots or shoots were also observed in the mutants in response to both Pi sufficiency and deficiency, indicating that the mutants were involved in Pi uptake or transportation. The primary root length and root-shoot ratio of abi3–1 and abi4–1 mutants decreased compared with WT seedlings under low Pi condition. Further research showed that ABI5 could regulate PHT1;5 and WRKY42 expression by combining with ACGT cis-acting elements of the PHT1;5 and WRKY42 promoters.

Stomata facilitate foliar sorption of silver nanoparticles by Arabidopsis thaliana

Application of nanopesticides may substantially increase surface attachment and internalization of engineered nanoparticles (ENPs) in food crops. This study investigated the role of stomata in the internalization of silver nanoparticles (Ag NPs) using abscisic acid (ABA)-responsive ecotypes (Ler and Col-7) and ABA-insensitive mutants (ost1-2 and scord7) of Arabidopsis thaliana in batch sorption experiments, in combination with microscopic visualization. Compared with those of the ABA-free control, stomatal apertures were significantly smaller for the Ler and Col-7 ecotypes (p ˂ 0.05) but remained unchanged for the ost1-2 and scord7 mutants, after exposure to 10 μM ABA for 1 h. Generally Ag NP sorption to the leaves of the Ler and Col-7 ecotypes treated with 10 μM ABA was lower than that in the ABA-free control, mainly due to ABA-induced stomatal closure. The difference in Ag NP sorption with and without ABA was less pronounced for Col-7 than for Ler, suggesting different sorption behaviors between these two ecotypes. In contrast, there was no significant difference in foliar sorption of Ag NPs by the ost1-2 and scord7 mutants with and without ABA treatment. Ag NPs were widely attached to the Arabidopsis leaf surface, and found at cell membrane, cytoplasm, and plasmodesmata, as revealed by scanning electron microscopy and transmission electron microscopy, respectively. These results highlight the important role of stomata in the internationalization of ENPs in plants and may have broad implications in foliar application of nanopesticides and minimizing contamination of food crops by ENPs.

Involvement of the miR156/SPL module in flooding response in Medicago sativa

The highly conserved plant microRNA, miR156, affects plant development, metabolite composition, and stress response. Our previous research revealed the role of miR156 in abiotic stress response in Medicago sativa exerted by downregulating SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE transcription factors. Here we investigated the involvement and possible mechanism of action of the miR156/SPL module in flooding tolerance in alfalfa. For that, we used miR156 overexpressing, SPL13RNAi, flood-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars exposed to flooding. We also used Arabidopsis ABA insensitive (abi1-2, abi5-8) mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) catalytic subunit of SnRK1 to investigate a possible role of ABA and SnRK1 in regulating miR156 expression under flooding. Physiological analysis, hormone profiling and global transcriptome changes revealed a role for miR156/SPL module in flooding tolerance. We also identified nine novel alfalfa SPLs (SPL1, SPL1a, SPL2a, SPL7, SPL7a, SPL8, SPL13a, SPL14, SPL16) responsive to flooding. Our results also showed a possible ABA-dependent SnRK1 upregulation to enhance miR156 expression, resulting in downregulation of SPL4, SPL7a, SPL8, SPL9, SPL13, and SPL13a. We conclude that these effects induce flooding adaptive responses in alfalfa and modulate stress physiology by affecting the transcriptome, ABA metabolites and secondary metabolism.

Abscisic acid-nitric oxide and auxin interaction modulates salt stress response in tomato roots

Integrated signaling network involving abscisic acid (ABA), nitric oxide (NO), and indole-acetic acid (IAA) controls root morphogenesis during salt stress by a mechanism still poorly understood. The present data unveiled an ABA-NO-IAA interaction underlying radicular morphological responses to salinity. Three Solanum lycopersicum genotypes were analyzed: wild type, ABA-insensitive mutant (sitiens), and auxin-responsive (DR5::GUS) plants. Nitric oxide fluorescence, nitrate reductase activity, auxin signaling, and some molecular analyses were performed. Pharmacological inhibitors and NO donor sodium nitroprusside were also used to evaluate NaCl-induced root morphological responses. Sodium nitroprusside inhibited primary root length, increased lateral root emergence, and rescued salt inhibited lateral root growth. The results showed that NO integrates the ABA-IAA signaling network of root system responses under salt stress, involving: (a) ABA and molybdenum-dependent enzymes as responsible for salt-induced NO production; (b) modulations of the plasma membrane H+-ATPase coupling and isoforms differential expression; and (c) ABA-mediated and NO-dependent antioxidative enzymes activities.

Bacillus subtilis and Pseudomonas fluorescens Trigger Common and Distinct Systemic Immune Responses in Arabidopsis thaliana Depending on the Pathogen Lifestyle.

Plants harbor various beneficial bacteria that modulate their innate immunity, resulting in induced systemic resistance (ISR) against various pathogens. However, the immune mechanisms underlying ISR triggered by Bacillus spp. and Pseudomonas spp. against pathogens with different lifestyles are not yet clearly elucidated. Here, we show that root drenching of Arabidopsis plants with Pseudomonas fluorescens PTA-CT2 and Bacillus subtilis PTA-271 can induce ISR against the necrotrophic fungus B. cinerea and the hemibiotrophic bacterium Pseudomonas syringae Pst DC3000. In the absence of pathogen infection, both beneficial bacteria do not induce any consistent change in systemic immune responses. However, ISR relies on priming faster and robust expression of marker genes for the salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) signaling pathways upon pathogen challenge. These responses are also associated with increased levels of SA, JA, and abscisic acid (ABA) in the leaves of bacterized plants after infection. The functional study also points at priming of the JA/ET and NPR1-dependent defenses as prioritized immune pathways in ISR induced by both beneficial bacteria against B. cinerea. However, B. subtilis-triggered ISR against Pst DC3000 is dependent on SA, JA/ET, and NPR1 pathways, whereas P. fluorescens-induced ISR requires JA/ET and NPR1 signaling pathways. The use of ABA-insensitive mutants also pointed out the crucial role of ABA signaling, but not ABA concentration, along with JA/ET signaling in primed systemic immunity by beneficial bacteria against Pst DC3000, but not against B. cinerea. These results clearly indicate that ISR is linked to priming plants for enhanced common and distinct immune pathways depending on the beneficial strain and the pathogen lifestyle.

Tartary Buckwheat Transcription Factor FtbZIP5, Regulated by FtSnRK2.6, Can Improve Salt/Drought Resistance in Transgenic Arabidopsis.

bZIP transcription factors have been reported to be involved in many different biological processes in plants. The ABA (abscisic acid)-dependent AREB/ABF-SnRK2 pathway has been shown to play a key role in the response to osmotic stress in model plants. In this study, a novel bZIP gene, FtbZIP5, was isolated from tartary buckwheat, and its role in the response to drought and salt stress was characterized by transgenic Arabidopsis. We found that FtbZIP5 has transcriptional activation activity, which is located in the nucleus and specifically binds to ABRE elements. It can be induced by exposure to PEG6000, salt and ABA in tartary buckwheat. The ectopic expression of FtbZIP5 reduced the sensitivity of transgenic plants to drought and high salt levels and reduced the oxidative damage in plants by regulating the antioxidant system at a physiological level. In addition, we found that, under drought and salt stress, the expression levels of several ABA-dependent stress response genes (RD29A, RD29B, RAB18, RD26, RD20 and COR15) in the transgenic plants increased significantly compared with their expression levels in the wild type plants. Ectopic expression of FtbZIP5 in Arabidopsis can partially complement the function of the ABA-insensitive mutant abi5-1 (abscisic acid-insensitive 5-1). Moreover, we screened FtSnRK2.6, which might phosphorylate FtbZIP5, in a yeast two-hybrid experiment. Taken together, these results suggest that FtbZIP5, as a positive regulator, mediates plant tolerance to salt and drought through ABA-dependent signaling pathways.

Elevated carbon dioxide decreases the adverse effects of higher temperature and drought stress by mitigating oxidative stress and improving water status in Arabidopsis thaliana.

This study revealed that elevated carbon dioxide increases Arabidopsis tolerance to higher temperature and drought stress by mitigating oxidative stress and improving water status of plants. Few studies have considered multiple aspects of plant responses to key components of global climate change, including higher temperature, elevated carbon dioxide (ECO2), and drought. Hence, their individual and combinatorial effects on plants need to be investigated in the context of understanding climate change impact on plant growth and development. We investigated the interactive effects of temperature, CO2, watering regime, and genotype on Arabidopsis thaliana (WT and ABA-insensitive mutant, abi1-1). Plants were grown in controlled-environment growth chambers under two temperature regimes (22/18°C and 28/24°C, 16h light/8h dark), two CO2 concentrations (400 and 700μmolmol-1), and two watering regimes (well-watered and water-stressed) for 18days. Plant growth, anatomical, physiological, molecular, and hormonal responses were determined. Our study provided valuable information about plant responses to the interactive effects of multiple environmental factors. We showed that drought and ECO2 had larger effects on plants than higher temperatures. ECO2 alleviated the detrimental effects of temperature and drought by mitigating oxidative stress and plant water status, and this positive effect was consistent across multiple response levels. The WT plants performed better than the abi1-1 plants; the former had higher rosette diameter, total dry mass, leaf and soil water potential, leaf moisture, proline, ethylene, trans-zeatin, isopentyladenine, and cis-zeatin riboside than the latter. The water-stressed plants of both genotypes accumulated more abscisic acid (ABA) than the well-watered plants; however, higher temperatures decreased the ability of WT plants to produce ABA in response to drought. We conclude that drought strongly, while higher temperature to a lesser extent, affects Arabidopsis seedlings, and ECO2 reduces the adverse effects of these stressors more efficiently in the WT plants than in the abi1-1 plants. Findings from this study can be extrapolated to other plant species that share similar characteristics and/or family with Arabidopsis.

Intact leaf gas exchange provides a robust method for measuring the kinetics of stomatal conductance responses to abscisic acid and other small molecules in Arabidopsis and grasses

BackgroundGuard cells perceive external and internal stimuli and regulate stomatal conductance in plants. With the use of gas exchange analyzers, time-resolved stomatal conductance responses to light intensity, [CO2] concentration and relative humidity changes can be measured. This is more difficult to achieve when measuring stomatal responses to small soluble molecules such as the plant hormone abscisic acid (ABA) or the bacterial peptide flagellin 22 (flg22), in particular when investigating mutants with response phenotypes.ResultsA method to evaluate the dynamic effects of small molecules on stomatal conductance in a time-resolved fashion using gas exchange analyzers is presented here. ABA-induced stomatal closure was investigated by adding ABA to the transpiration stream of intact leaves placed in a microcentrifuge tube containing water. Strong ABA responses were resolved in time- and in a dose-dependent manner in wild-type Arabidopsis leaves, whereas the same response was not observed in leaves of the ABA-insensitive mutant open stomata 1-3 (ost1-3). Moreover, when leaves of the Plasma membrane Intrinsic Protein (PIP) aquaporin quadruple mutant pip1;1 pip1;2 pip2;1 pip2;2 were tested, robust wild-type-like responses to ABA were observed. When the bacterial peptide flg22 was added to the transpiration stream of intact wild-type leaves, a strong flg22-induced stomatal closure effect was observed. Finally, the proposed technique was further developed and optimized for evaluation of stomatal conductance responses to small molecules in leaves of grasses using the reference plant Brachypodium distachyon.ConclusionsDue to the variable size of stomata in Arabidopsis and the limited dynamic response of stomata in isolated epidermal strips, evaluation of the effect of small molecules on stomatal physiology has been challenging and has led in some cases to inconsistent results. Moreover, potential signals from the mesophyll are missing when using epidermal peels to evaluate stomatal aperture responses. Here we propose a less invasive technique which allows for time-resolved measurements of stomatal conductance responses to small molecules optimized for both Arabidopsis and Brachypodium distachyon leaves.

Sulfate is Incorporated into Cysteine to Trigger ABA Production and Stomatal Closure.

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis (Arabidopsis thaliana). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3-1, and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2-1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18:GFP, and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response.

There is a direct link between allantoin concentration and cadmium tolerance in Arabidopsis

Allantoin, an important intermediate of ureide metabolism, has been the subject of investigation recently due to its dual function in nitrogen recycling and abiotic stress response in plants. Allantoin appears to be the dominant ureide accumulating in response to different abiotic stresses, and mutants containing elevated allantoin concentrations exhibit a stress-tolerant phenotype due to limited reactive oxygen species (ROS) generation. Here we describe the involvement of allantoin in stress response and attempt to explain the regulatory mechanism(s) underlying allantoin function in plants. Growth of wild type Col-0 seedlings in the presence of exogenous allantoin improved root elongation in response to Cd treatment. Allantoin treatment of Col-0 seeds increases superoxide dismutase activity causing an enhanced seed germination and seedling growth following Cd exposure. Additionally, allantoinase-overexpressed (ALNox) lines, with lower levels of allantoin, exhibited more susceptibility to Cd treatment than Col-0 Arabidopsis, implying that there is a positive correlation between allantoin concentration and Cd resistance in plants. Growing ABA-insensitive (abi) mutants on allantoin-containing media and comparison between abi mutants and their wild-type backgrounds demonstrated that the potential regulatory function of allantoin does not require ABA at germination but may be ABA-dependent at later stages of seedling growth, suggesting a potential crosstalk between allantoin-mediated stress response and ABA signalling pathway in plants.

Inoculation with Bacillus subtilis and Azospirillum brasilense Produces Abscisic Acid That Reduces Irt1-Mediated Cadmium Uptake of Roots.

Cadmium (Cd) contamination of agricultural soils represents a serious risk to crop safety. A new strategy using abscisic acid (ABA)-generating bacteria, Bacillus subtilis or Azospirillum brasilense, was developed to reduce the Cd accumulation in plants grown in Cd-contaminated soil. Inoculation with either bacterium resulted in a pronounced increase in the ABA level in wild-type Arabidopsis Col-0 plants, accompanied by a decrease in Cd levels in plant tissues, which mitigated the Cd toxicity. As a consequence, the growth of plants exposed to Cd was improved. Nevertheless, B. subtilis and A. brasilense inoculation had little effect on Cd levels and toxicity in the ABA-insensitive mutant snrk 2.2/2.3, indicating that the action of ABA is required for these bacteria to reduce Cd accumulation in plants. Furthermore, inoculation with either B. subtilis or A. brasilense downregulated the expression of IRT1 (iron-regulated transporter 1) in the roots of wild-type plants and had little effect on Cd levels in the IRT1-knockout mutants irt1-1 and irt1-2. In summary, we conclude that B. subtilis and A. brasilense can reduce Cd levels in plants via an IRT1-dependent ABA-mediated mechanism.

Regulation of stomatal movement by cortical microtubule organization in response to darkness and ABA signaling in Arabidopsis

Microtubule dynamics are essential for plant cell development and in producing responses to external stimuli. However, little is known about the regulation of microtubule dynamics or crosstalk between microtubule and stomatal movement. Here we identified microtubule reorganization as a crucial factor determining guard cell responses to dark and abscisic acid (ABA) signaling. As stomata opened, guard cells exhibited radially arranged cortical microtubules, which depolymerized into the cytosol when exposed to darkness and ABA. Suppression of microtubule disassembly by paclitaxel, a microtubule-stabilizing drug, significantly enhanced stomatal aperture under light, and partially blocked ABA- or darkness-induced stomatal closure. However, treatment with only the anti-microtubule drug, oryzalin, did not affect stomatal movement with or without external stimuli. Phosphatidic acid (PA) bound to a clade A type 2C protein phosphatase (PP2C), PP2CA, and deletion of PP2CA partially inhibited PA-induced microtubule depolymerization and stomatal closure. Moreover, microtubule reorganization was altered in the ABA-insensitive mutant pldα1, but not in the ABA-hypersensitive mutant pp2ca. We propose that a faithfully balanced reorganization of microtubules fulfills fundamental functions to enable the fast change of stomata in plant adaptive responses to developmental and environmental cues.

The role of Arabidopsis thaliana RASD1 gene in ABA-dependent abiotic stress response.

Abiotic stress is one of the key parameters affecting plant productivity. Drought and soil salinity, in particular, challenge plants to activate various response mechanisms to withstand these adverse growth conditions. While the molecular events that take place are complex and to a large extent unclear, the plant hormone abscisic acid (ABA) is considered a major player in mediating the adaptation of plants to stress. Here we report the identification of an ABA-insensitive mutant from Arabidopsis thaliana. A combination of molecular, genetic and physiology approaches were implemented, to characterise the AtRASD1 locus (RESPONSIVENESS TO ABA SALT AND DROUGHT 1) and to investigate its role in plant development. RASD1 is expressed predominantly in the vascular system of A.thaliana and encodes a peptide of unknown function with no similarity to any known sequence to date. The protein is localised in the nucleus and the cytoplasm, and RASD1-impaired plants are drought-intolerant and insensitive to exogenous ABA and NaCl during germination and root growth. Our data indicate that RASD1 is involved in ABA-dependent signal transduction pathways and therefore in enabling plants to activate response mechanisms related to seed germination and abiotic stress.

Stomatal VPD Response: There Is More to the Story Than ABA.

Guard cells shrink and close stomatal pores when air humidity decreases (i.e. when the difference between the vapor pressures of leaf and atmosphere [VPD] increases). The role of abscisic acid (ABA) in VPD-induced stomatal closure has been studied using ABA-related mutants that respond to VPD in some studies and not in others. The importance of ABA biosynthesis in guard cells versus vasculature for whole-plant stomatal regulation is unclear as well. Here, we show that Arabidopsis (Arabidopsis thaliana) lines carrying mutations in different steps of ABA biosynthesis as well as pea (Pisum sativum) wilty and tomato (Solanum lycopersicum) flacca ABA-deficient mutants had higher stomatal conductance compared with wild-type plants. To characterize the role of ABA production in different cells, we generated transgenic plants where ABA biosynthesis was rescued in guard cells or phloem companion cells of an ABA-deficient mutant. In both cases, the whole-plant stomatal conductance, stunted growth phenotype, and leaf ABA level were restored to wild-type values, pointing to the redundancy of ABA sources and to the effectiveness of leaf ABA transport. All ABA-deficient lines closed their stomata rapidly and extensively in response to high VPD, whereas plants with mutated protein kinase OST1 showed stunted VPD-induced responses. Another strongly ABA-insensitive mutant, defective in the six ABA PYR/RCAR receptors, responded to changes in VPD in both directions strongly and symmetrically, indicating that its VPD-induced closure could be passive hydraulic. We discuss that both the VPD-induced passive hydraulic stomatal closure and the stomatal VPD regulation of ABA-deficient mutants may be conditional on the initial pretreatment stomatal conductance.

Stress Tolerance in ABA-insensitive Mutant and Transgenic Lines of the Moss Physcomitrella patens

Stress Tolerance in ABA-insensitive Mutant and Transgenic Lines of the Moss Physcomitrella patens

Overexpression of OsDT11, which encodes a novel cysteine-rich peptide, enhances drought tolerance and increases ABA concentration in rice

Short-chain peptides play important roles in plant development and responses to abiotic and biotic stresses. Here, we characterized a gene of unknown function termed OsDT11, which encodes an 88 amino acid short-chain peptide and belongs to the cysteine-rich peptide family. It was found that the expression of OsDT11 can be activated by polyethylene glycol (PEG) treatment. Compared with wild-type lines, the OsDT11-overexpression lines displayed dramatically enhanced tolerance to drought and had reduced water loss, reduced stomatal density, and an increased the concentration of abscisic acid (ABA). The suppression of OsDT11 expression resulted in an increased sensitivity to drought compared to wild-type expression. Several drought-related genes, including genes encoding abscisic acid (ABA) signaling markers, were also strongly induced in the OsDT11-overexpressing lines. Moreover, the expression of OsDT11 was repressed in ABA-insensitive mutant Osbzip23 and Os2H16 RNAi lines. These results suggest that OsDT11-mediated drought tolerance may be dependent on the ABA signaling pathway.

The Chloroplast Protease AMOS1/EGY1 Affects Phosphate Homeostasis under Phosphate Stress.

Plastid intramembrane proteases in Arabidopsis (Arabidopsis thaliana) are involved in jasmonic acid biosynthesis, chloroplast development, and flower morphology. Here, we show that Ammonium-Overly-Sensitive1 (AMOS1), a member of the family of plastid intramembrane proteases, plays an important role in the maintenance of phosphate (P) homeostasis under P stress. Loss of function of AMOS1 revealed a striking resistance to P starvation. amos1 plants displayed retarded root growth and reduced P accumulation in the root compared to wild type (Col-0) under P-replete control conditions, but remained largely unaffected by P starvation, displaying comparable P accumulation and root and shoot growth under P-deficient conditions. Further analysis revealed that, under P-deficient conditions, the cell wall, especially the pectin fraction of amos1, released more P than that of wild type, accompanied by a reduction of the abscisic acid (ABA) level and an increase in ethylene production. By using an ABA-insensitive mutant, abi4, and applying ABA and ACC exogenously, we found that ABA inhibits cell wall P remobilization while ethylene facilitates P remobilization from the cell wall by increasing the pectin concentration, suggesting ABA can counteract the effect of ethylene. Furthermore, the elevated ABA level and the lower ethylene production also correlated well with the mimicked P deficiency in amos1 Thus, our study uncovers the role of AMOS1 in the maintenance of P homeostasis through ABA-antagonized ethylene signaling.

ABF2, ABF3, and ABF4 Promote ABA-Mediated Chlorophyll Degradation and Leaf Senescence by Transcriptional Activation of Chlorophyll Catabolic Genes and Senescence-Associated Genes in Arabidopsis

Chlorophyll (Chl) degradation is an integral process of leaf senescence, and NYE1/SGR1 has been demonstrated as a key regulator of Chl catabolism in diverse plant species. In this study, using yeast one-hybrid screening, we identified three abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors, ABF2 (AREB1), ABF3, and ABF4 (AREB2), as the putative binding proteins of the NYE1 promoter. Through the transactivation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrated that ABF2, ABF3, and ABF4 directly bound to and activated the NYE1 promoter in vitro and in vivo. ABA is a positive regulator of leaf senescence, and exogenously applied ABA can accelerate Chl degradation. The triple mutant of the ABFs, abf2abf3abf4, as well as two ABA-insensitive mutants, abi1-1 and snrk2.2/2.3/2.6, exhibited stay-green phenotypes after ABA treatment, along with decreased induction of NYE1 and NYE2 expression. In contrast, overexpression of ABF4 accelerated Chl degradation upon ABA treatment. Interestingly, ABF2/3/4 could also activate the expression of two Chl catabolic enzyme genes, PAO and NYC1, by directly binding to their promoters. In addition, abf2abf3abf4 exhibited a functional stay-green phenotype, and senescence-associated genes (SAGs), such as SAG29 (SWEET15), might be directly regulated by the ABFs. Taken together, our results suggest that ABF2, ABF3, and ABF4 likely act as key regulators in mediating ABA-triggered Chl degradation and leaf senescence in general in Arabidopsis.

Involvement of OST1 Protein Kinase and PYR/PYL/RCAR Receptors in Methyl Jasmonate-Induced Stomatal Closure in Arabidopsis Guard Cells.

Methyl jasmonate (MeJA) induces stomatal closure. It has been shown that stomata of many ABA-insensitive mutants are also insensitive to MeJA, and a low amount of ABA is a prerequisite for the MeJA response. However, the molecular mechanisms of the interaction between ABA and MeJA signaling remain to be elucidated. Here we studied the interplay of signaling of the two hormones in guard cells using the quadruple ABA receptor mutant pyr1 pyl1 pyl2 pyl4 and ABA-activated protein kinase mutants ost1-2 and srk2e. In the quadruple mutant, MeJA-induced stomatal closure, H2O2 production, nitric oxide (NO) production, cytosolic alkalization and plasma membrane Ca(2+)-permeable current (ICa) activation were not impaired. At the same time, the inactivation of the inward-rectifying K(+) current was impaired. In contrast to the quadruple mutant, MeJA-induced stomatal closure, H2O2 production, NO production and cytosolic alkalization were impaired in ost1-2 and srk2e as well as in aba2-2, the ABA-deficient mutant. The activation of ICa was also impaired in srk2e. Collectively, these results indicated that OST1 was essential for MeJA-induced stomatal closure, while PYR1, PYL1, PYL2 and PYL4 ABA receptors were not sufficient factors. MeJA did not appear to activate OST1 kinase activity. This implies that OST1 mediates MeJA signaling through an undetectable level of activity or a non-enzymatic action. MeJA induced the expression of an ABA synthesis gene, NCED3, and increased ABA contents only modestly. Taken together with previous reports, this study suggests that MeJA signaling in guard cells is primed by ABA and is not brought about through the pathway mediated by PYR1, PYL1 PYL2 and PYL4.