Higher Abscisic Acid Research Articles

Impact of in vitro hormone treatments on the bibenzyl production of Radula complanata

Bibenzyls are a specialized metabolite class found throughout the plant kingdom. One of the most prolific producers of bibenzyls are liverworts, specifically plants of the Radula genera. These plants possess an incredible diversity of bibenzyls, prenylated bibenzyls, and a few (bis)bibenzyls, several of which have medicinal properties, including perrottetinene, an analog of tetrahydrocannabinol from cannabis. To provide insight into the bibenzyls’ biosynthesis in planta, exogenous phytohormones were applied to in vitro grown Radula complanata and bibenzyl metabolite production was monitored with targeted and untargeted metabolomics. The targeted metabolomic analysis of six prenylated bibenzyls revealed that production of these metabolites was largely reduced when plants were treated with abscisic acid (AA), salicylic acid (SA), 1-naphthaleneacetic acid (NAA), or 6-benzylaminopurine (BAP). The reduction of these metabolites in the BAP and NAA treatment suggests that prenylated bibenzyl production is negatively correlated with vegetative plant growth. The reduction of bibenzyls at low AA and SA concentrations and mild increase at higher AA and SA concentrations suggest that their production is regulated by these stress hormones. In addition, six other bibenzyl metabolites were tentatively identified from the untargeted analysis. These results provide insight into the influence of phytohormones on the bioactive bibenzyl content of R. complanata.

Abiotic Stress Induces Morphological, Physiological, and Genetic Changes in Orthosiphon stamineus Benth. In Vitro Cultures

Orthosiphon stamineus is a well-known medicinal herb that has long been used as a traditional treatment for a variety of ailments and disorders. In this study, in vitro grown O. stamineus plantlets were subjected to various stress factors to elucidate the effects of these stresses on the occurrence of somaclonal variation in this species. Murashige and Skoog (MS) media supplemented with 2.0 mg/L kinetin plus 0.5 mg/L IAA was identified as the optimum in vitro regeneration media (OM). The data analysis confirmed the clonal nature of the resulting plantlets. The plantlets were also exposed to various stress factors such as high salinity, abscisic acid (ABA), and high cytokinin concentrations, by subculturing the plantlets on OM added with various concentrations of NaCl, ABA, and high kinetin levels. Exposure to the various stress factors was observed to result in the occurrence of somaclonal variation in O. stamineus, where the incidence of hyperhydricity was found to be the most prominent. An ISSR analysis revealed 70.12% polymorphism among the variants. The results showed that the chlorophyll content and photosynthetic ability of plantlets were reduced when the abiotic stress levels increased.

Elevated CO2 and high endogenous ABA level alleviate PEG-induced short-term osmotic stress in tomato plants

Elevated CO 2 concentration ( e [CO 2 ]) alleviates the impact of drought stress on plants where abscisic acid (ABA) is involved. To explore the mechanisms by which tomato plants respond to short-term osmotic stress, Solanum lycopersicum cv. Ailsa Craig (AC), a transgenic line overproducing ABA (sp5), and an ABA-deficient mutant ( flacca ) were hydroponically grown under ambient CO 2 (400 ppm) and e [CO 2 ] (800 ppm) and then exposed to 10% or 15% (w/v) polyethylene glycol (PEG) 6000 for 24 h before transferring to PEG-free nutrient solution for 24 h. Under non-stress condition, e [CO 2 ] decreased root hydraulic conductance ( K root ), which was overridden by high endogenous ABA in sp5 through increasing specific leaf area and root branching intensity. Basically, e [CO 2 ] improved stress resistance through enhanced water status. PEG stress decreased stomatal conductance and osmotic potential in AC but these effects were less pronounced in sp5, which exhibited a stronger osmotic adjustment (OA) and improved plant fitness. A greater flexibility of hydraulic system and a reduced sensitivity of K root to ABA might confer sp5 a great ability to recover from PEG stress. On the contrary, high stomatal density, size and pore aperture of flacca rendered plants suffering severe stress. Moreover, the premise that PEG stress could mimic soil water deficit was the sufficient achievement of OA. Our results indicate that e [CO 2 ] and high endogenous ABA level could improve osmotic stress resistance in tomato plants via osmotic and hydraulic adjustments. • High endogenous ABA increased specific leaf area and root branching intensity. • High endogenous ABA overrode the effects of e [CO 2 ] on root hydraulic conductance. • Sufficient osmotic adjustment improved drought resistance. • PEG stress could not mimic soil drought unless it triggered fast osmotic adjustment.

Fluoride tolerance in rice is negatively regulated by the 'stress-phytohormone' abscisic acid (ABA), but promoted by ABA-antagonist growth regulators, melatonin, and gibberellic acid.

The present manuscript aimed at investigating whether abscisic acid (ABA) promotes fluoride tolerance, similar to inciting salt adaptation in rice. Seeds of three salt-tolerant rice genotypes were maintained at 32°C under 16/8h light/dark photoperiodic cycle with 700μmol photons m-2s-1 intensity and 50% relative humidity in a plant growth chamber for 20days. Suppressed ABA biosynthesis, and downregulated expression of ABA-inducible genes like Rab16A, Osem, and TRAB1 triggered NaCl-induced growth inhibition and physiological injuries like chlorophyll degradation, electrolyte leakage, formation of H2O2, malondialdehyde, and methylglyoxal in Matla. Reduced ABA accumulation increased the levels of melatonin and gibberellic acid in NaF (50mgL-1)-stressed Nonabokra and Matla, which altogether promoted fluoride tolerance. Higher ABA content in NaF-stressed Jarava stimulated fluoride uptake via chloride channels, thus exhibiting severe fluoride susceptibility, in spite of higher production of ABA-associated osmolytes like proline, glycine-betaine and polyamines via the concerted action of genes like PDH, ADC, ODC, SAMDC, SPDS, SPMS, DAO, and PAO. Increased accumulation of compatible solutes in presence of high endogenous ABA promoted salt tolerance in Jarava; the same was insufficient to ameliorate fluoride-induced injuries in this cultivar. Treatment with ABA biosynthetic inhibitor, Na2WO4 promoted fluoride tolerance in Jarava, whereas further supplementation with exogenous ABA resulted in reversion back to fluoride-susceptible phenotype. Our work clearly established that ABA cannot always be considered as a 'universal' stress hormone as known in literature, since it acts as a negative regulator of fluoride tolerance which is more tightly regulated in rice via melatonin- and gibberellic acid-dependent pathways in ABA-independent manner.

Parental drought priming enhances tolerance to low temperature in wheat (Triticum aestivum) offspring.

Low temperature is one of the major environmental stresses that limit crop growth and grain yield in wheat (Triticum aestivum L.). Drought priming at the vegetative stage could enhance wheat tolerance to later cold stress; however, the transgenerational effects of drought priming on wheat offspring's cold stress tolerance remains unclear. Here, the low temperature responses of offspring were tested after the parental drought priming treatment at grain filling stage. The offspring plants from parental drought priming treatment had a higher abscisic acid (ABA) level and lower osmotic potential (Ψo) than the control plants under cold conditions. Moreover, parental drought priming increased the antioxidant enzyme activities and decreased hydrogen peroxide (H2 O2 ) accumulation in offspring. In comparison to control plants, parental drought priming plants had a higher ATP concentration and higher activities of ATPase and the enzymes involved in sucrose biosynthesis and starch metabolism. The results indicated that parental drought priming induced low temperature tolerance in offspring by regulating endogenous ABA levels and maintaining the redox homeostasis and the balance of carbohydrate metabolism, which provided a potential approach for cold resistant cultivation in wheat.

Slsnrk2.3 Interacts with Slsui1 to Modulate High Temperature Tolerance Via Abscisic Acid (Aba) Controlling Stomatal Movement in Tomato

Slsnrk2.3 Interacts with Slsui1 to Modulate High Temperature Tolerance Via Abscisic Acid (Aba) Controlling Stomatal Movement in Tomato

Exogenous Abscisic Acid Priming Modulates Water Relation Responses of Two Tomato Genotypes With Contrasting Endogenous Abscisic Acid Levels to Progressive Soil Drying Under Elevated CO2.

Plants have evolved multiple strategies to survive and adapt when confronting the changing climate, including elevated CO2 concentration (e[CO2]) and intensified drought stress. To explore the role of abscisic acid (ABA) in modulating the response of plant water relation characteristics to progressive drought under ambient (a[CO2], 400 ppm) and e[CO2] (800 ppm) growth environments, two tomato (Solanum lycopersicum) genotypes, Ailsa Craig (AC) and its ABA-deficient mutant (flacca), were grown in pots, treated with or without exogenous ABA, and exposed to progressive soil drying until all plant available water in the pot was depleted. The results showed that exogenous ABA application improved leaf water potential, osmotic potential, and leaf turgor and increased leaf ABA concentrations ([ABA]leaf) in AC and flacca. In both genotypes, exogenous ABA application decreased stomatal pore aperture and stomatal conductance (gs), though these effects were less pronounced in e[CO2]-grown AC and gs of ABA-treated flacca was gradually increased until a soil water threshold after which gs started to decline. In addition, ABA-treated flacca showed a partly restored stomatal drought response even when the accumulation of [ABA]leaf was vanished, implying [ABA]leaf might be not directly responsible for the decreased gs. During soil drying, [ABA]leaf remained higher in e[CO2]-grown plants compared with those under a[CO2], and a high xylem sap ABA concentration was also noticed in the ABA-treated flacca especially under e[CO2], suggesting that e[CO2] might exert an effect on ABA degradation and/or redistribution. Collectively, a fine-tune ABA homeostasis under combined e[CO2] and drought stress allowed plants to optimize leaf gas exchange and plant water relations, yet more detailed research regarding ABA metabolism is still needed to fully explore the role of ABA in mediating plant physiological response to future drier and CO2-enriched climate.

Do the ends justify the means? Impact of drought progression rate on stress response and recovery in Vitis vinifera.

Plants are frequently exposed to prolonged and intense drought events. To survive, species must implement strategies to overcome progressive drought while maintaining sufficient resources to sustain the recovery of functions. Our objective was to understand how stress rate development modulates energy reserves and affects the recovery process. Grenache Vitis vinifera cultivar was exposed to either fast‐developing drought (within few days; FDD), typical of pot experiments, or slow‐developing drought (few weeks, SDD), more typical for natural conditions. FDD was characterized by fast (2–3 days) stomatal closure in response to increased stress level, high abscisic acid (ABA) accumulation in xylem sap (>400 μg L−1) without the substantial changes associated with stem priming for recovery (no accumulation of sugar or drop in xylem sap pH). In contrast, SDD was characterized by gradual stomatal closure, low ABA accumulation (<100 μg L−1) and changes that primed the stem for recovery (xylem sap acidification from 6 to 5.5 pH and sugar accumulation from 1 to 3 g L−1). Despite FDD and SDD demonstrating similar trends over time in the recovery of stomatal conductance, they differed in their sensitivity to xylem ABA. Grenache showed near‐isohydric and near‐anisohydric behavior depending on the rate of drought progression, gauging the risk between hydraulic integrity and photosynthetic gain. The isohydry observed during FDD could potentially provide protection from large sudden swings in tension, while transitioning to anisohydry during SDD could prioritize the maintenance of photosynthetic activity over hydraulic security.

Paclobutrazol Modulates Endogenous Level of Phytohormones in Inducing Early Flowering in Camellia tamdaoensis Hakoda et Ninh, a Golden Camellia Species

Golden camellia flowers are treasured for their unique yellow color and bioactive chemical compounds. Because of its high market demand, there is strong interest in inducing early flowering in golden camellias for earlier harvest. Previously, we have successfully induced flowering in Camelia chrysantha (Hu) Tuyama juvenile grafted plants and seedlings with paclobutrazol (PBZ). During this study, we investigated the efficacy of PBZ on C. tamdaoensis juvenile rooted cuttings. C. tamdaoensis is a yellow-flowering camellia species that is native to Vietnam and valued by the local population. It was found that applications of 100 and 200 ppm PBZ generated an average of 13 and 30 flowers per 5-year-old plant, respectively. None of the control plants flowered. The average flower diameter was 17.2 cm for 100-ppm-induced flowers and 26.0 cm for 200-ppm-induced flowers. The dynamics of various phytohormones (indoleacetic acid, abscisic acid, salicylic acid, and jasmonic acid) were altered by PBZ treatment. It is suggested that low indoleacetic acid, high abscisic acid, and jasmonic acid and a gradual increase in salicylic acid benefit floral initiation of golden camellias. The study provided the first insight regarding the action mechanism of PBZ for the initiation of camellia flowering.

Physiological and Molecular Responses of Six Apple Rootstocks to Osmotic Stress.

The growth and productivity of several apple rootstocks have been evaluated in various previous studies. However, limited information is available on their tolerance to osmotic stress. In the present study, the physiological and molecular responses as well as abscisic acid (ABA) levels were assessed in six apple rootstocks (M26, V3, G41, G935, B9 and B118) osmotically stressed with polyethylene glycol (PEG, 30%) application under greenhouse conditions. Our results showed that V3, G41, G935 and B9 had higher relative water content (RWC), and lower electrolyte leakage (EL) under stress conditions compared to M26 and B118. Additionally, water use efficiency (WUE) was higher in V3, G41 and B9 than M26, which might be partially due to the lower transpiration rate in these tolerant rootstocks. V3, G41 and B9 rootstocks also displayed high endogenous ABA levels which was combined with a reduction in stomatal conductance and decreased water loss. At the transcriptional level, genes involved in ABA-dependent and ABA-independent pathways, e.g., SnRK, DREB, ERD and MYC2, showed higher expression in V3, G41, G935 and B9 rootstocks compared to M26 in response to stress. In contrast, WRKY29 was down-regulated in response to stress in the tolerant rootstocks, and its expression was negatively correlated with ABA content and stomatal closure. Overall, the findings of this study showed that B9, V3 and G41 displayed better osmotic stress tolerance followed by G935 then M26 and B118 rootstocks.

Comparative transcriptomic and physiological analyses of weedy rice and cultivated rice to identify vital differentially expressed genes and pathways regulating the ABA response

Weedy rice is a valuable germplasm resource characterized by its high tolerance to both abiotic and biotic stresses. Abscisic acid (ABA) serves as a regulatory signal in plant cells as part of their adaptive response to stress. However, a global understanding of the response of weedy rice to ABA remains to be elucidated. In the present study, the sensitivity to ABA of weedy rice (WR04-6) was compared with that of temperate japonica Shennong9816 (SN9816) in terms of seed germination and post-germination growth via the application of exogenous ABA and diniconazole, an inhibitor of ABA catabolism. Physiological analysis and a transcriptomic comparison allowed elucidation of the molecular and physiological mechanisms associated with continuous ABA and diniconazole treatment. WR04-6 was found to display higher ABA sensitivity than SN9816, resulting in the rapid promotion of antioxidant enzyme activity. Comparative transcriptomic analyses indicated that the number of differentially expressed genes (DEGs) in WR04-6 seedlings treated with 2 μM ABA or 10 μM diniconazole was greater than that in SN9816 seedlings. Genes involved in stress defense, hormone signal transduction, and glycolytic and citrate cycle pathways were highly expressed in WR04-6 in response to ABA and diniconazole. These findings provide new insight into key processes mediating the ABA response between weedy and cultivated rice.

Overexpression of ZmDHN11 could enhance transgenic yeast and tobacco tolerance to osmotic stress.

Maize group II LEA protein ZmDHN11 could protect protein activity and confer resistance to osmotic stress on transgenic yeast and tobacco. Late embryogenesis abundant (LEA) proteins are widely assumed to play crucial roles in environmental stress tolerance, but their function has remained obscure. Dehydrins are group II LEA proteins, which are highly hydrophilic plant stress proteins. In the present study, a novel group II LEA protein, ZmDHN11, was cloned and identified from maize. The expression of ZmDHN11 was induced by high osmotic stress, low temperature, salinity, and ABA (abscisic acid). The ZmDHN11 protein specifically accumulated in the nuclei and cytosol. Further study indicated that ZmDHN11 is phosphorylated by the casein kinase CKII. ZmDHN11 protected the activity of LDH under water-deficit stress. The overexpression of ZmDHN11 endows transgenic yeast and tobacco with tolerance to osmotic stress.

The Resistance of Oilseed Rape Microspore-Derived Embryos to Osmotic Stress Is Associated With the Accumulation of Energy Metabolism Proteins, Redox Homeostasis, Higher Abscisic Acid, and Cytokinin Contents.

The present study aims to investigate the response of rapeseed microspore-derived embryos (MDE) to osmotic stress at the proteome level. The PEG-induced osmotic stress was studied in the cotyledonary stage of MDE of two genotypes: Cadeli (D) and Viking (V), previously reported to exhibit contrasting leaf proteome responses under drought. Two-dimensional difference gel electrophoresis (2D-DIGE) revealed 156 representative protein spots that have been selected for MALDI-TOF/TOF analysis. Sixty-three proteins have been successfully identified and divided into eight functional groups. Data are available via ProteomeXchange with identifier PXD024552. Eight selected protein accumulation trends were compared with real-time quantitative PCR (RT-qPCR). Biomass accumulation in treated D was significantly higher (3-fold) than in V, which indicates D is resistant to osmotic stress. Cultivar D displayed resistance strategy by the accumulation of proteins in energy metabolism, redox homeostasis, protein destination, and signaling functional groups, high ABA, and active cytokinins (CKs) contents. In contrast, the V protein profile displayed high requirements of energy and nutrients with a significant number of stress-related proteins and cell structure changes accompanied by quick downregulation of active CKs, as well as salicylic and jasmonic acids. Genes that were suitable for gene-targeting showed significantly higher expression in treated samples and were identified as phospholipase D alpha, peroxiredoxin antioxidant, and lactoylglutathione lyase. The MDE proteome profile has been compared with the leaf proteome evaluated in our previous study. Different mechanisms to cope with osmotic stress were revealed between the genotypes studied. This proteomic study is the first step to validate MDE as a suitable model for follow-up research on the characterization of new crossings and can be used for preselection of resistant genotypes.

RNA N6-Methyladenosine Responds to Low-Temperature Stress in Tomato Anthers.

Cold stress is a serious threat to subtropical crop pollen development and induces yield decline. N6-methyladenosine (m6A) is the most frequent mRNA modification and plays multiple physiological functions in plant development. However, whether m6A regulates pollen development is unclear, and its putative role in cold stress response remains unknown. Here, we observed that moderate low-temperature (MLT) stress induced pollen abortion in tomato. This phenotype was caused by disruption of tapetum development and pollen exine formation, accompanied by reduced m6A levels in tomato anther. Analysis of m6A-seq data revealed 1,805 transcripts displayed reduced m6A levels and 978 transcripts showed elevated m6A levels in MLT-stressed anthers compared with those in anthers under normal temperature. These differentially m6A enriched transcripts under MLT stress were mainly related to lipid metabolism, adenosine triphosphatase (ATPase) activity, and ATP-binding pathways. An ATP-binding transcript, SlABCG31, had significantly upregulated m6A modification levels, which was inversely correlated to the dramatically downregulated expression level. These changes correlated with higher abscisic acid (ABA) levels in anthers and disrupted pollen wall formation under low-temperature stress. Our findings characterized m6A as a novel layer of complexity in gene expression regulation and established a molecular link between m6A methylation and tomato anther development under low-temperature conditions.

Distinctive in-planta acclimation responses to basal growth and acute heat stress were induced in\xa0Arabidopsis by cattle manure biochar

In-planta mechanisms of biochar (BC)-mediated improved growth were evaluated by examining oxidative stress, metabolic, and hormonal changes of Arabidopsis wild-type plants under basal or acute heat stress (–HS/ + HS) conditions with or without BC (+ BC/–BC). The oxidative stress was evaluated by using Arabidopsis expressing redox-sensitive green fluorescent protein in the plastids (pla-roGFP2). Fresh biomass and inflorescence height were greater in + BC(‒HS) plants than in the –BC(‒HS) plants, despite similar leaf nutrient levels, photosystem II (PSII) maximal efficiencies and similar oxidative poise. Endogenous levels of jasmonic and abscisic acids were higher in the + BC(‒HS) treatment, suggesting their role in growth improvement. HS in ‒BC plants caused reductions in inflorescence height and PSII maximum quantum yield, as well as significant oxidative stress symptoms manifested by increased lipid peroxidation, greater chloroplast redox poise (oxidized form of roGFP), increased expression of DNAJ heat shock proteins and Zn-finger genes, and reduced expression of glutathione-S-transferase gene in addition to higher abscisic acid and salicylic acid levels. Oxidative stress symptoms were significantly reduced by BC. Results suggest that growth improvements by BC occurring under basal and HS conditions are induced by acclimation mechanisms to ‘microstresses’ associated with basal growth and to oxidative stress of HS, respectively.

Different abscisic acid‐deficient mutants show unique morphological and hydraulic responses to high air humidity

High relative humidity (RH) perturbs plant growth, stomatal functioning and abscisic acid (ABA) homeostasis, but the role of ABA in this physiological regulation is equivocal. To determine the role(s) of ABA in plant responses to high RH, wild-type (WT) tomato and barley plants and their respective ABA-deficient mutants flacca and Az34 (which are mutated in the same locus of the ABA biosynthesis pathway) were grown in contrasting RHs (60% and 90%) to measure biomass partitioning, stomatal traits and water relations. Surprisingly, growth RH did not affect foliar ABA levels in either species. While Az34 showed similar stomatal size and density as WT plants, flacca had larger and more abundant stomata. High RH increased stomatal size in tomato, but decreased it in barley, and decreased stomatal density in tomato, but not in barley. Altered stomatal responses in ABA-deficient plants to high RH had little effect on tomato photosynthesis, but Az34 barley showed lower photosynthesis. ABA deficiency decreased relative shoot growth rate (RGRSHOOT ) in both species, yet this was counteracted by high RH increasing leaf water status in tomato, but not in barley. High RH increased RGRSHOOT in flacca, but not in WT tomatoes, while having no effect on RGRSHOOT in barley, but affecting barley net assimilation rate, leaf area ratio (LAR) and specific leaf area in an ABA-dependent manner. ABA-RH interaction affected leaf development in tomato only. Thus, different crop species show variable responses to both high RH and ABA deficiency, making it difficult to generalise on the role of ABA in growth regulation at contrasting RHs.

Anatomical physiological and biochemical processes involved in grapevine rootstock drought tolerance

In order to explore the drought resistance mechanism of grape rootstocks, two grape rootstock species, '1103P' (a drought-tolerant rootstock) and '101-14M' (drought-sensitive), were treated with moderate water deficit (field capacity of 45-50 %). Throughout the experimental period, the leaves of '1103P' showed a higher stomatal conductance (gs), relative water content and photosynthetic rate (Pn) than '101-14M', indicating '1103P' was more resistant to tolerant than '101-14M'. We propose that '1103P' could prevent water loss from leaves under drought conditions based on the discoveries that '1103P' had higher leaf phytohormone abscisic acid (ABA) content and leaf cuticular wax content, and smaller stomata aperture than those of '101-14M'. Additionally, the activities of H2O2-scavenging enzymes in leaves of '1103P' were higher than those of '101-14M' under drought conditions, indicating the lipid peroxidation induced by H2O2 of '1103P' was less serious than that of '101-14M'. Therefore, better water-saving and higher reactive oxygen species (ROS) scavenging abilities contributed together to stronger drought resistance of '1103P' than '101-14M'.

Overexpression of isochorismate synthase enhances drought tolerance in barley

Isochorismate synthase (ICS) is a key enzyme for the synthesis of salicylic acid (SA) in plants. SA mediates plant responses to both biotic and abiotic stresses. In previous studies, we found that overexpression of ICS (ICSOE) or suppression of ICS (ICSRNAi) affected the host response to Fusarium graminearum in barley. However, whether the barley ICS gene plays a role in adapting to abiotic stresses remains to be determined. In the present study, expression of the ICS gene was upregulated when treated with 20 % PEG6000, and ICSOE lines were more drought tolerant than wild type (WT) and ICSRNAi. In addition, the abscisic acid (ABA) levels in the ICSOE lines were higher than those in the WT and ICSRNAi lines under drought stress. High ABA levels significantly reduced Gs and E, which may impact water retention under drought stress. Under drought conditions, the activity of antioxidant enzymes was significantly higher in the ICSOE lines, correlating with a lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Enhanced antioxidant competence also contributed to drought tolerance in ICSOE lines. These findings help elucidate the abiotic stress resistance of the ICS pathway in barley.

Regulation of the stability and ABA import activity of NRT1.2/NPF4.6 by CEPR2-mediated phosphorylation in Arabidopsis

Abscisic acid (ABA) transport plays an important role in systemic plant responses to environmental factors. However, it remains largely unclear about the precise regulation of ABA transporters in plants. In this study, we show that the C-terminally encoded peptide receptor 2 (CEPR2) directly interacts with the ABA transporter NRT1.2/NPF4.6. Genetic and phenotypic analyses revealed that NRT1.2/NPF4.6 positively regulates ABA response and that NRT1.2/NPF4.6 is epistatically and negatively regulated by CEPR2. Further biochemical assays demonstrated that CEPR2 phosphorylates NRT1.2/NPF4.6 at serine 292 to promote its degradation under normal conditions. However, ABA treatment and non-phosphorylation at serine 292 prevented the degradation of NRT1.2/NPF4.6, indicating that ABA inhibits the phosphorylation of this residue. Transport assays in yeast and Xenopus oocytes revealed that non-phosphorylated NRT1.2/NPF4.6 had high levels of ABA import activity, whereas phosphorylated NRT1.2/NPF4.6 did not import ABA. Analyses of complemented nrt1.2 mutants that mimicked non-phosphorylated and phosphorylated NRT1.2/NPF4.6 confirmed that non-phosphorylated NRT1.2S292A had high stability and ABA import activity in planta. Additional experiments showed that NRT1.2/NPF4.6 was degraded via the 26S proteasome and vacuolar degradation pathways. Furthermore, we found that three E2 ubiquitin-conjugating enzymes, UBC32, UBC33, and UBC34, interact with NRT1.2/NPF4.6 in the endoplasmic reticulum and mediate its ubiquitination. NRT1.2/NPF4.6 is epistatically and negatively regulated by UBC32, UBC33, and UBC34 in planta. Taken together, these results suggest that the stability and ABA import activity of NRT1.2/NPF4.6 are precisely regulated by its phosphorylation and degradation in response to environmental stress.

Maize microRNA166 Inactivation Confers Plant Development and Abiotic Stress Resistance.

MicroRNAs are important regulators in plant developmental processes and stress responses. In this study, we generated a series of maize STTM166 transgenic plants. Knock-down of miR166 resulted in various morphological changes, including rolled leaves, enhanced abiotic stress resistance, inferior yield-related traits, vascular pattern and epidermis structures, tassel architecture, as well as abscisic acid (ABA) level elevation and indole acetic acid (IAA) level reduction in maize. To profile miR166 regulated genes, we performed RNA-seq and qRT-PCR analysis. A total of 178 differentially expressed genes (DEGs) were identified, including 118 up-regulated and 60 down-regulated genes. These DEGs were strongly enriched in cell and intercellular components, cell membrane system components, oxidoreductase activity, single organism metabolic process, carbohydrate metabolic process, and oxidation reduction process. These results indicated that miR166 plays important roles in auxin and ABA interaction in monocots, yet the specific mechanism may differ from dicots. The enhanced abiotic stress resistance is partly caused via rolling leaves, high ABA content, modulated vascular structure, and the potential changes of cell membrane structure. The inferior yield-related traits and late flowering are partly controlled by the decreased IAA content, the interplay of miR166 with other miRNAs and AGOs. Taken together, the present study uncovered novel functions of miR166 in maize, and provide insights on applying short tandem target mimics (STTM) technology in plant breeding.