Molecular mechanisms underlying low temperature inhibition of grain filling in maize (Zea mays L.): coordination of growth and cold responses.

Low temperature (LT) greatly restricts grain filling in maize (Zea mays L.), but the relevant molecular mechanisms are not fully understood. To better understand the effect of LT on grain development, 17 hybrids were subjected to LT stress in field trials over 3 years, and two hybrids of them with contrasting LT responses were exposed to 30/20°C and 20/10°C for 7 days during grain filling in a greenhouse. At LT, thousand-kernel weight declined, especially in LT-sensitive hybrid FM985, while grain-filling rate was on average about 48% higher in LT-tolerant hybrid DK159 than FM985. LT reduced starch synthesis in kernel mainly by suppression of transcript levels and enzyme activities for sucrose synthase and hexokinase. Brassinolide (BR) was abundant in DK159 kernel, and genes involved in BR and cytokinin signals were inducible by stress. LT downregulated the genes in light-harvesting complex and photosystem I/II subunits, accompanied by reduced photosynthetic rate and Fv/Fm in ear leaf. The LT-tolerant hybrid could maintain a high soluble sugar content and fast interconversion between sucrose and hexose in the stem internode and cob, improving assimilate allocation to kernel at LT stress and paving the way for simultaneous growth and LT stress responses.

The Mechanisms Underlying Salt Resistance Mediated by Exogenous Application of 24-Epibrassinolide in Peanut.

Peanut is one of the most important oil crops in the world, the growth and productivity of which are severely affected by salt stress. 24-epibrassinolide (EBL) plays an important role in stress resistances. However, the roles of exogenous EBL on the salt tolerance of peanut remain unclear. In this study, peanut seedlings treated with 150 mM NaCl and with or without EBL spray were performed to investigate the roles of EBL on salt resistance. Under 150 mM NaCl conditions, foliar application of 0.1 µM EBL increased the activity of catalase and thereby could eliminate reactive oxygen species (ROS). Similarly, EBL application promoted the accumulation of proline and soluble sugar, thus maintaining osmotic balance. Furthermore, foliar EBL spray enhanced the total chlorophyll content and high photosynthesis capacity. Transcriptome analysis showed that under NaCl stress, EBL treatment up-regulated expression levels of genes encoding peroxisomal nicotinamide adenine dinucleotide carrier (PMP34), probable sucrose-phosphate synthase 2 (SPS2) beta-fructofuranosidase (BFRUCT1) and Na+/H+ antiporters (NHX7 and NHX8), while down-regulated proline dehydrogenase 2 (PRODH). These findings provide valuable resources for salt resistance study in peanut and lay the foundation for using BR to enhance salt tolerance during peanut production.

A mutation in the promoter of the arabinogalactan protein 7-like gene PcAGP7-1 affects cell morphogenesis and brassinolide content in pear (Pyrus communis L.) stems.

Dwarfing rootstocks and dwarf cultivars are urgently needed for modern pear cultivation. However, germplasm resources for dwarfing pear are limited, and the underlying mechanisms remain unclear. We previously showed that dwarfism in pear is controlled by the single dominant gene PcDw (Dwarf). We report here that the expression of PcAGP7-1 (ARABINOGALACTAN PROTEIN 7-1), a key candidate gene for PcDw, is significantly higher in dwarf-type pear plants because of a mutation in an E-box in the promoter. Electrophoretic mobility shift assays and transient infiltration showed that the transcription factors PcBZR1 and PcBZR2 could directly bind to the E-box of the PcAGP7-1 promoter and repress transcription. Moreover, transgenic pear lines overexpressing PcAGP7-1 exhibited obvious dwarf phenotypes, whereas RNA interference pear lines for PcAGP7-1 were taller than controls. PcAGP7-1 overexpression also enhanced cell wall thickness, affected cell morphogenesis, and reduced brassinolide (BL) content, which inhibited BR signaling via a negative feedback loop, resulting in further dwarfing. Overall, we identified a dwarfing mechanism in perennial woody plants involving the BL-BZR/BES-AGP-BL regulatory module. Our findings provide insight into the molecular mechanism of plant dwarfism and suggest strategies for the molecular breeding of dwarf pear cultivars.

Optimal Brassinosteroid Levels Are Required for Soybean Growth and Mineral Nutrient Homeostasis.

Brassinosteroids (BRs) are steroid phytohormones that are known to regulate plant growth and nutrient uptake and distribution. However, how BRs regulate nutrient uptake and balance in legume species is not fully understood. Here, we show that optimal BR levels are required for soybean (Glycine max L.) seedling growth, as treatments with both 24-epicastasterone (24-epiCS) and the BR biosynthesis inhibitor propiconazole (PPZ) inhibit root growth, including primary root elongation and lateral root formation and elongation. Specifically, 24-epiCS and PPZ reduced the total phosphorus and potassium levels in the shoot and affected several minor nutrients, such as magnesium, iron, manganese, and molybdenum. A genome-wide transcriptome analysis identified 3774 and 4273 differentially expressed genes in the root tip after brassinolide and PPZ treatments, respectively. The gene ontology (GO) analysis suggested that genes related to "DNA-replication", "microtubule-based movement", and "plant-type cell wall organization" were highly responsive to the brassinolide and PPZ treatments. Furthermore, consistent with the effects on the nutrient concentrations, corresponding mineral transporters were found to be regulated by BR levels, including the GmPHT1s, GmKTs, GmVIT2, GmZIPs, and GmMOT1 genes. Our study demonstrates that optimal BR levels are important for growth and mineral nutrient homeostasis in soybean seedlings.

Amelioration of Chlorpyrifos-Induced Toxicity in Brassica juncea L. by Combination of 24-Epibrassinolide and Plant-Growth-Promoting Rhizobacteria.

Pervasive use of chlorpyrifos (CP), an organophosphorus pesticide, has been proven to be fatal for plant growth, especially at higher concentrations. CP poisoning leads to growth inhibition, chlorosis, browning of roots and lipid and protein degradation, along with membrane dysfunction and nuclear damage. Plants form a linking bridge between the underground and above-ground communities to escape from the unfavourable conditions. Association with beneficial rhizobacteria promotes the growth and development of the plants. Plant hormones are crucial regulators of basically every aspect of plant development. The growing significance of plant hormones in mediating plant-microbe interactions in stress recovery in plants has been extensively highlighted. Hence, the goal of the current study was to investigate the effect of 24-epibrassinolide (EBL) and PGPRs (Pseudomonas aeruginosa (Ma), Burkholderia gladioli (Mb)) on growth and the antioxidative defence system of CP-stressed Brassica juncea L. seedlings. CP toxicity reduced the germination potential, hypocotyl and radicle development and vigour index, which was maximally recuperated after priming with EBL and Mb. CP-exposed seedlings showed higher levels of superoxide anion (O2-), hydrogen peroxide (H2O2), lipid peroxidation and electrolyte leakage (EL) and a lower level of nitric oxide (NO). In-vivo visualisation of CP-stressed seedlings using a light and fluorescent microscope also revealed the increase in O2-, H2O2 and lipid peroxidation, and decreased NO levels. The combination of EBL and PGPRs reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) contents and improved the NO level. In CP-stressed seedlings, increased gene expression of defence enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APOX), glutathione peroxidase (GPOX), dehydroascorbate reductase (DHAR) and glutathione reductase (GPOX) was seen, with the exception of catalase (CAT) on supplementation with EBL and PGPRs. The activity of nitrate reductase (NR) was likewise shown to increase after treatment with EBL and PGPRs. The results obtained from the present study substantiate sufficient evidence regarding the positive association of EBL and PGPRs in amelioration of CP-induced oxidative stress in Brassica juncea seedlings by strengthening the antioxidative defence machinery.

A novel W/O/W double emulsion co-delivering brassinolide and cinnamon essential oil delayed the senescence of broccoli via regulating chlorophyll degradation and energy metabolism.

The postharvest senescence accompanied by yellowing limited the shelf-life of broccoli. In this study, we developed a novel W/O/W double emulsion co-delivering brassinolide and cinnamon essential oil and applied it to broccoli for preservation. Results showed that double emulsion prepared by whey protein concentrate-high methoxyl pectin (1:3) exhibited best storage stability with largest particle size (581.30 nm), lowest PDI (0.23) and zeta potential (-40.31 mV). This double emulsion also exhibited highest encapsulation efficiency of brassinolide (92%) and cinnamon essential oil (88%). The broccoli coated with double emulsion maintained higher chlorophyll contents and activities of chlorophyllase and magnesium-dechelatase were reduced by 9% and 24%, respectively. The energy metabolic enzymes (SDH, CCO, H+-ATPase, Ca2+-ATPase) were also activated, inducing higher level of ATP and energy charge. These results demonstrated W/O/W double emulsion co-delivering brassinolide and cinnamon essential delayed the senescence of broccoli via regulating chlorophyll degradation and energy metabolism.

Protein farnesylation negatively regulates brassinosteroid signaling via reducing BES1 stability in Arabidopsis thaliana.

Brassinosteroids (BRs) are a group of steroidal phytohormones, playing critical roles in almost all physiological aspects during the life span of a plant. In Arabidopsis, BRs are perceived at the cell surface, triggering a reversible phosphorylation-based signaling cascade that leads to the activation and nuclear accumulation of a family of transcription factors, represented by BES1 and BZR1. Protein farnesylation is a type of post-translational modification, functioning in many important cellular processes. Previous studies demonstrated a role of farnesylation in BR biosynthesis via regulating the endoplasmic reticulum localization of a key bassinolide (BL) biosynthetic enzyme BR6ox2. Whether such a process is also involved in BR signaling is not understood. Here, we demonstrate that protein farnesylation is involved in mediating BR signaling in Arabidopsis. A loss-of-function mutant of ENHANCED RESPONSE TO ABA 1 (ERA1), encoding a ß subunit of the protein farnesyl transferase holoenzyme, can alter the BL sensitivity of bak1-4 from a reduced to a hypersensitive level. era1 can partially rescue the BR defective phenotype of a heterozygous mutant of bin2-1, a gain-of-function mutant of BIN2 which encodes a negative regulator in the BR signaling. Our genetic and biochemical analyses revealed that ERA1 plays a significant role in regulating the protein stability of BES1.

The acyltransferase PMAT1 malonylates brassinolide glucoside.

Brassinosteroids (BRs) are steroid hormones of plants that coordinate fundamental growth and development processes. Their homeostasis is controlled by diverse means, including glucosylation of the bioactive BR brassinolide (BL), which is catalyzed by the UDP-glycosyltransferases (UGTs) UGT73C5 and UGT73C6 and occurs mainly at the C-23 position. Additional evidence had suggested that the resultant BL-23-O-glucoside (BL-23-O-Glc) can be malonylated, but the physiological significance of and enzyme required for this reaction had remained unknown. Here, we show that in Arabidopsis thaliana malonylation of BL-23-O-Glc is catalyzed by the acyltransferase phenolic glucoside malonyl-transferase 1 (PMAT1), which is also known to malonylate phenolic glucosides and lipid amides. Loss of PMAT1 abolished BL-23-O-malonylglucoside formation and enriched BL-23-O-Glc, showing that the enzyme acts on the glucoside. An overexpression of PMAT1 in plants where UGT73C6 was also overexpressed, and thus, BL-23-O-Glc formation was promoted, enhanced the symptoms of BR-deficiency of UGT73C6oe plants, providing evidence that PMAT1 contributes to BL inactivation. Based on these results, a model is proposed in which PMAT1 acts in the conversion of both endogenous and xenobiotic glucosides to adjust metabolic homeostasis in spatial and temporal modes.

Transcriptomic analyses show that 24-epibrassinolide (EBR) promotes cold tolerance in cotton seedlings.

In plants, brassinosteroids (BRs) are a class of steroidal hormones that are involved in numerous physiological responses. However, the function of BRs in cold tolerance in cotton has not been explored. In this study, cotton seedlings were treated with five concentrations (0, 0.05, 0.1, 0.2, 0.5 and 1.0 mg/L) of 24-Epibrassinolide (EBR) at 4°C. We measured the electrolyte leakage, malondialdehyde (MDA) content, proline content, and net photosynthesis rate (Pn) of the seedlings, which showed that EBR treatment increased cold tolerance in cotton in a dose-dependent manner, and that 0.2 mg/L is an optimum concentration for enhancing cold tolerance. The function of EBR in cotton cotyledons was investigated in the control 0 mg/L (Cold+water) and 0.2 mg/L (Cold+EBR) treatments using RNA-Seq. A total of 4,001 differentially expressed genes (DEGs), including 2,591 up-regulated genes and 1,409 down-regulated genes were identified. Gene Ontology (GO) and biochemical pathway enrichment analyses showed that EBR is involved in the genetic information process, secondary metabolism, and also inhibits abscisic acid (ABA) and ethylene (ETH) signal transduction. In this study, physiological experiments showed that EBR can increase cold tolerance in cotton seedlings, and the comprehensive RNA-seq data shed light on the mechanisms through which EBR increases cold tolerance in cotton seedlings.

Brassinosteroids Influence Arabidopsis Hypocotyl Graviresponses through Changes in Mannans and Cellulose.

The force of gravity is a constant environmental factor. Plant shoots respond to gravity through negative gravitropism and gravity resistance. These responses are essential for plants to direct the growth of aerial organs away from the soil surface after germination and to keep an upright posture above ground. We took advantage of the effect of brassinosteroids (BRs) on the two types of graviresponses in Arabidopsis thaliana hypocotyls to disentangle functions of cell wall polymers during etiolated shoot growth. The ability of etiolated Arabidopsis seedlings to grow upward was suppressed in the presence of 24-epibrassinolide (EBL) but enhanced in the presence of brassinazole (BRZ), an inhibitor of BR biosynthesis. These effects were accompanied by changes in cell wall mechanics and composition. Cell wall biochemical analyses, confocal microscopy of the cellulose-specific pontamine S4B dye and cellular growth analyses revealed that the EBL and BRZ treatments correlated with changes in cellulose fibre organization, cell expansion at the hypocotyl base and mannan content. Indeed, a longitudinal reorientation of cellulose fibres and growth inhibition at the base of hypocotyls supported their upright posture whereas the presence of mannans reduced gravitropic bending. The negative effect of mannans on gravitropism is a new function for this class of hemicelluloses. We also found that EBL interferes with upright growth of hypocotyls through their uneven thickening at the base.

A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae).

Feeding and oviposition deterrents help phytophagous insects to identify host plants. The taste organs of phytophagous insects contain bitter gustatory receptors (GRs). To explore their function, the GRs in Plutella xylostella were analyzed. Through RNA sequencing and qPCR, we detected abundant PxylGr34 transcripts in the larval head and adult antennae. Functional analyses using the Xenopus oocyte expression system and 24 diverse phytochemicals showed that PxylGr34 is tuned to the canonical plant hormones brassinolide (BL) and 24-epibrassinolide (EBL). Electrophysiological analyses revealed that the medial sensilla styloconica of 4th instar larvae are responsive to BL and EBL. Dual-choice bioassays demonstrated that BL inhibits larval feeding and female oviposition. Knock-down of PxylGr34 by RNAi attenuates the taste responses to BL, and abolishes BL-induced feeding inhibition. These results increase our understanding of how herbivorous insects detect compounds that deter feeding and oviposition, and may be useful for designing plant hormone-based pest management strategies.

Plant-eating insects use their sense of taste to decide where to feed and where to lay their eggs. They do this using taste sensors called gustatory receptors which reside in the antennae and legs of adults, and in the mouthparts of larvae. Some of these sensors detect sugars which signal to the insect that the plant is a nutritious source of food. While others detect bitter compounds, such as poisons released by plants in self-defense. One of the most widespread plant-eating insects is the diamondback moth, which feeds and lays its eggs on cruciferous vegetable crops, like cabbage, oilseed rape and broccoli. Before laying its eggs, female diamondback moths pat the vegetable's leaves with their antennae, tasting for the presence of chemicals. But little was known about the identity of these chemicals. Cabbages produce large amounts of a hormone called brassinolide, which is known to play a role in plant growth. To find out whether diamondback moths can taste this hormone, Yang et al. examined all their known gustatory receptors. This revealed that the adult antennae and larval mouthparts of these moths make high levels of a receptor called PxylGr34. To investigate the role of PxylGr34, Yang et al. genetically modified frog eggs to produce this receptor. Various tests on these receptors, as well as receptors in the mouthparts of diamondback larvae, showed that PxylGr34 is able to sense the hormone brassinolide. To find out how this affects the behavior of the moths, Yang et al. investigated how adults and larvae responded to different levels of the hormone. This revealed that the presence of brassinolide significantly decreased both larval feeding and the amount of eggs laid by adult moths. Farmers already use brassinolide to enhance plant growth and protect crops from stress. These results suggest that the hormone might also help to shield plants from insect damage. However, more research is needed to understand how this hormone acts as a deterrent. Further studies could improve understanding of insect behavior and potentially identify more chemicals that can be used for pest control.

Foliar application of 24-epibrassinolide improves Solanum nigrum L. tolerance to high levels of Zn without affecting its remediation potential.

Although Solanum nigrum L. is a phytoremediator for different metals, its growth and physiology are still compromised by toxic levels of zinc (Zn). Thus, the development of eco-friendly strategies to enhance its tolerance, maintaining remediation potential is of special interest. This study evaluated the potential of 24-epibrassinolide (24-EBL) to boost S. nigrum defence against Zn towards a better growth rate and remediation potential. After 24 days of exposure, the results revealed that Zn-mediated inhibitory effects on biomass and biometry were efficiently mitigated upon application of 24-EBL, without affecting Zn accumulation. The evaluation of oxidative stress markers reported that Zn excess stimulated the accumulation of superoxide anion (O2.-), but reduced hydrogen peroxide (H2O2) levels, while not altering lipid peroxidation (LP). This was accompanied by an up-regulation of the antioxidant system, especially proline, superoxide dismutase (SOD) and ascorbate peroxidase (APX) in both organs, and ascorbate in roots of Zn-exposed plants. Foliar application of 24-EBL, however, induced distinctive effects, lowering proline levels in both organs, as well as APX activity in shoots and SOD in roots, whilst stimulating GSH and total thiols in both organs, as well as SOD and APX activity, in shoots and in roots, respectively. Probably due to a better antioxidant efficiency, levels of O2.- and H2O2 in pre-treated plants remained identical to the control, while LP further decreased in shoots. Overall, our results indicate a protective effect of 24-EBL on S. nigrum response to excess Zn, contributing for a better tolerance and growth rate, without disturbing its phytoremediation potential.

Interactive role of exogenous 24 Epibrassinolide and endogenous NO in Brassica juncea L. under salinity stress: Evidence for NR-dependent NO biosynthesis.

The present study unravels origin of nitric oxide (NO) and the interaction between 24-Epibrassinolide (EBL) and nitrate reductase (NR) for NO production in Indian mustard (Brassica juncea L.) under salinity stress. Two independent experiments were performed to check whether (i) Nitrate reductase or Nitric oxide synthase takes part in the biosynthesis of endogenous NO and (ii) EBL has any regulatory effect on NR-dependent NO biosynthesis in the alleviation of salinity stress. Results revealed that NR-inhibitor tungstate significantly (P ≤ 0.05) decreased the NR activity and endogenous NO content, while NOS inhibitor l-NAME did not influence NO biosynthesis and plant growth. Under salinity stress, inhibition in NR activity decreased the activities of antioxidant enzymes, increased H2O2, MDA, protein carbonyl content and caused DNA damage, implying that antioxidant defense might be related to NO signal. EBL supplementation enhanced the NR activity but did not influence NOS activity, suggesting that NR was involved in endogenous NO production. EBL supplementation alleviated the inhibitory effects of salinity stress and improved the plant growth by enhancing nutrients, photosynthetic pigments, compatible osmolytes, and performance of AsA-GSH cycle. It also decreased the superoxide ion accumulation, leaf epidermal damages, cell death, DNA damage, and ABA content. Comet assay revealed significant (P ≤ 0.05) enhancement in tail length and olive tail moment, while flow cytometry did not showed any significant (P ≤ 0.05) changes in genome size and ploidy level under salinity stress. Moreover, EBL supplementation increased the G6PDH activity and S-nitrosothiol content which further boosted the antioxidant responses under salinity stress. Taken together, these results suggested that NO production in mustard occurred in NR-dependent manner and EBL in association with endogenous NO activates the antioxidant system to counter salinity stress.

Anabolic/anticatabolic and adaptogenic effects of 24-epibrassinolide on Lupinus angustifolius: Causes and consequences.

Lupinus angustifolius L. is a legume culture known as a source of valuable feed protein and the N2-fixator for improving soil fertility. However, its low ecological resistance does not allow for a stable yield of the crop. Earlier, we have shown that steroid phytohormone 24-epibrassinolide (EBR) increases the tolerance of lupine to chlorine ions by activating the protective proteins in ripening seeds (such as proteinase inhibitors that prevent protein breakdown) and lectins. Here we investigated the effect of EBR on the functional status of the N2-fixing system in root nodules, protein synthesis in ripening seeds and the resistance of lupine plants to various pathogens. It was found that EBR enhanced the nodulation process, N2-fixing activity of nitrogenase and the accumulation of poly-ß-hydroxybutirate in the bacteroides, increased the leghemoglobin content in the nodules as well as the metabolic activity of bacteroides. According to data on the inclusion of 14C-leucine in maturing seed proteins, EBR increased the accumulation of protein in them against the background of a short-term decrease in protein synthesis and its subsequent regeneration to the control level. Gradual inhibition of protein synthesis, characteristic of other legumes, was observed in control variants of lupine. EBR increased lupine resistance to phytopathogenic fungi of Colletotrichum genus and insects of Noctuidae and Scarabaeidae families. We concluded that a more complete implementation of the potential productivity and sustainability of lupine under the action of EBR was achieved due to the anabolic/anti-catabolic effect on the N2 fixation system in root nodules, as well as on protein synthesis in ripening seeds.

Molecular Mechanism of Brassinosteroid Perception by the Plant Growth Receptor BRI1.

Brassinosteroids (BRs) are essential phytohormones, which bind to the plant receptor, BRI1, to regulate various physiological processes. The molecular mechanism of the perception of BRs by the ectodomain of BRI1 remains not fully understood. It also remains elusive why a substantial difference in biological activity exists between the BRs. In this work, we study the binding mechanisms of the two most bioactive BRs, brassinolide (BLD) and castasterone (CAT), using molecular dynamics simulations. We report free-energy landscapes of the binding processes of both ligands, as well as detailed ligand binding pathways. Our results suggest that CAT has a lower binding affinity compared to BLD due to its inability to form hydrogen-bonding interactions with a tyrosine residue in the island domain of BRI1. We uncover a conserved nonproductive binding state for both BLD and CAT, which is more stable for CAT and may further contribute to the bioactivity difference. Finally, we validate past observations about the conformational restructuring and ordering of the island domain upon BLD binding. Overall, this study provides new insights into the fundamental mechanism of the perception of the two most bioactive BRs, which may create new avenues for genetic and agrochemical control of their signaling cascade.

A novel single-base mutation in CaBRI1 confers dwarf phenotype and brassinosteroid accumulation in pepper.

Dwarfing is the development trend of pepper breeding. It is of great practical and scientific value to generate new dwarf germplasms, and identify new genes or alleles conferring dwarf traits in pepper. In our previous study, a weakly BR-insensitive dwarf mutant, E29, was obtained by EMS mutagenesis of the pepper inbred line 6421. It can be used as a good parent material for breeding new dwarf varieties. Here, we found that this dwarf phenotype was controlled by a single recessive gene. Whole-genome resequencing, dCAPs analysis, and VIGs validation revealed that this mutation was caused by a nonsynonymous single-nucleotide mutation (C to T) in CaBRI1. An enzyme activity assay, transcriptome sequencing, and BL content determination further revealed that an amino-acid change (Pro1157Ser) in the serine/threonine protein kinase and catalytic (S_TKc) domain of CaBRI1 impaired its kinase activity and caused the transcript levels of two important genes (CaDWF4 and CaROT3) participating in BR biosynthesis to increase dramatically in the E29 mutant, accompanied by significantly increased accumulation of brassinolide (BL). Therefore, we concluded that the novel single-base mutation in CaBRI1 conferred the dwarf phenotype and resulted in brassinosteroid (BR) accumulation in pepper. This study provides a new allelic variant of the height-regulating gene CaBRI1 that has theoretical and practical values for the breeding of the plants suitable for the facility cultivation and mechanized harvesting of pepper varieties.

Brassinosteroid signaling delimits root gravitropism via sorting of the Arabidopsis PIN2 auxin transporter.

Arabidopsis PIN2 protein directs transport of the phytohormone auxin from the root tip into the root elongation zone. Variation in hormone transport, which depends on a delicate interplay between PIN2 sorting to and from polar plasma membrane domains, determines root growth. By employing a constitutively degraded version of PIN2, we identify brassinolides as antagonists of PIN2 endocytosis. This response does not require de novo protein synthesis, but involves early events in canonical brassinolide signaling. Brassinolide-controlled adjustments in PIN2 sorting and intracellular distribution governs formation of a lateral PIN2 gradient in gravistimulated roots, coinciding with adjustments in auxin signaling and directional root growth. Strikingly, simulations indicate that PIN2 gradient formation is no prerequisite for root bending but rather dampens asymmetric auxin flow and signaling. Crosstalk between brassinolide signaling and endocytic PIN2 sorting, thus, appears essential for determining the rate of gravity-induced root curvature via attenuation of differential cell elongation.

GSK3-like kinase BIN2 phosphorylates RD26 to potentiate drought signaling in Arabidopsis.

Plant steroid hormones brassinosteroids (BRs) regulate plant growth and development at many different levels. Recent research has revealed that stress-responsive NAC (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) transcription factor RD26 is regulated by BR signaling and antagonizes BES1 in the interaction between growth and drought stress signaling. However, the upstream signaling transduction components that activate RD26 during drought are still unknown. Here, we demonstrate that the function of RD26 is modulated by GSK3-like kinase BIN2 and protein phosphatase 2C ABI1. We show that ABI1, a negative regulator in abscisic acid (ABA) signaling, dephosphorylates and destabilizes BIN2 to inhibit BIN2 kinase activity. RD26 protein is stabilized by ABA and dehydration in a BIN2-dependent manner. BIN2 directly interacts and phosphorylates RD26 in vitro and in vivo. BIN2 phosphorylation of RD26 is required for RD26 transcriptional activation on drought-responsive genes. RD26 overexpression suppressed the brassinazole (BRZ)  insensitivity of BIN2 triple mutant bin2 bil1 bil2, and BIN2 function is required for the drought tolerance of RD26 overexpression plants. Taken together, our data suggest a drought signaling mechanism in which drought stress relieves ABI1 inhibition of BIN2, allowing BIN2 activation. Sequentially, BIN2 phosphorylates and stabilizes RD26 to promote drought stress response.

Sumoylation of BRI1-EMS-SUPPRESSOR 1 (BES1) by the SUMO E3 Ligase SIZ1 Negatively Regulates Brassinosteroids Signaling in Arabidopsis thaliana.

Brassinosteroids (BRs), a group of plant steroid hormones, participate in the regulation of plant growth and developmental processes. BR functions through the BES1/BZR1 family of transcription factors, however, the regulation of the BES1 activity by post-translational modifications remains largely unknown. Here, we present evidence that the SUMO E3 ligase SIZ1 negatively regulates BR signaling pathway. T-DNA insertion mutant siz1-2 shows BL (Brassinolide, the most active BR) hypersensitivity and BRZ (Brassinazole, a BR biosynthesis inhibitor) insensitivity during hypocotyl elongation. In addition, expression of BES1-dependent BR-response genes is hyper-regulated in siz1-2 seedlings. The siz1-2bes1-D double mutant exhibits longer hypocotyl than bes1-D. Moreover, SIZ1 physically interacts with BES1 in vivo and in vitro and mediates the sumoylation of BES1. A K302R substitution in BES1 blocks its sumoylation mediated by SIZ1 in plants, indicating that K302 is the principal site for SUMO conjugation. Consistently, we find that sumoylation inhibits BES1 protein stability and activity. Taken together, our data show that the sumoylation of BES1 via SIZ1 negatively regulates the BR signaling pathway.

24-Epibrassinolide promotes NO3- and NH4+ ion flux rate and NRT1 gene expression in cucumber under suboptimal root zone temperature.

BACKGROUND: Suboptimal root zone temperature (RZT) causes a remarkable reduction in growth of horticultural crops during winter cultivation under greenhouse production. However, limited information is available on the effects of suboptimal RZT on nitrogen (N) metabolism in cucumber seedlings. The aim of this study is to investigate the effects of 24-Epibrassinolide (EBR) on nitrate and ammonium flux rate, N metabolism, and transcript levels of NRT1 family genes under suboptimal RZT in cucumber seedlings. RESULTS: Suboptimal RZT (LT) negatively affected on cucumber growth and proportionately decreased EBR contents, bleeding rate, root activity, enzyme activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT), nitrate (NO3-) influx rate, ammonium (NH4+) efflux rate, and transcript levels of nitrate transporter (NRT1) encoding genes. However, exogenous EBR reduced the harmful effects of suboptimal RZT and increased endogenous EBR contents, bleeding rate, root activity, enzyme activities of NR, NiR, GS, and GOGAT, NH4+ and NO3- flux rates and contents, and N accumulation. EBR-treated seedlings also upregulated the transcript levels of nitrate transporters CsNRT1.1, CsNRT1.2A, CsNRT1.2B, CsNRT1.2C, CsNRT1.3, CsNRT1.4A, CsNRT1.5B, CsNRT1.5C, CsNRT1.9, and CsNRT1.10, and downregulated CsNRT1.5A and CsNRT1.8. LT treatment upregulated the expression level of CsNRT1.5A, while exogenous BZR application downregulated the expression level of NRT1 genes. CONCLUSION: These results indicate that exogenous application of EBR alleviated the harmful effects of suboptimal RZT through changes in N metabolism, NH4+ and NO3- flux rates, and NRT1 gene expression, leading to improved cucumber seedlings growth. Our study provides the first evidence of the role of EBR in the response to suboptimal RZT in cucumber, and can be used to improve vegetable production.