Comparative analysis of BZR1/BES1 family transcription factors in Arabidopsis.

Brassinazole Resistant 1 (BZR1) and bri1 EMS Suppressor 1 (BES1) are key transcription factors that mediate brassinosteroid (BR)-responsive gene expression in Arabidopsis. The BZR1/BES1 family is composed of BZR1, BES1, and four BES1/BZR1 homologs (BEH1-BEH4). However, little is known about whether BEHs are regulated by BR signaling in the same way as BZR1 and BES1. We comparatively analyzed the functional characteristics of six BZR1/BES1 family members and their regulatory mechanisms in BR signaling using genetic and biochemical analyses. We also compared their subcellular localizations regulated by the phosphorylation status, interaction with GSK3-like kinases, and heterodimeric combination. We found that all BZR1/BES1 family members restored the phenotypic defects of bri1-5 by their overexpression. Unexpectedly, BEH2-overexpressing plants showed the most distinct phenotype with enhanced BR responses. RNA-Seq analysis indicated that overexpression of both BZR1 and BEH2 regulates BR-responsive gene expression, but BEH2 has a much greater proportion of BR-independent gene expression than BZR1. Unlike BZR1 and BES1, the BR-regulated subcellular translocation of the four BEHs was not tightly correlated with their phosphorylation status. Notably, BEH1 and BEH2 are predominantly localized in the nucleus, which induces the nuclear accumulation of other BZR1/BES1 family proteins through heterodimerization. Altogether, our comparative analyses suggest that BEH1 and BEH2 play an important role in the functional interaction between BZR1/BES1 family transcription factors.

TaSRO1 plays a dual role in suppressing TaSIP1 to fine tune mitochondrial retrograde signalling and enhance salinity stress tolerance.

Initially discovered in yeast, mitochondrial retrograde signalling has long been recognised as an essential in the perception of stress by eukaryotes. However, how to maintain the optimal amplitude and duration of its activation under natural stress conditions remains elusive in plants. Here, we show that TaSRO1, a major contributor to the agronomic performance of bread wheat plants exposed to salinity stress, interacted with a transmembrane domain-containing NAC transcription factor TaSIP1, which could translocate from the endoplasmic reticulum (ER) into the nucleus and activate some mitochondrial dysfunction stimulon (MDS) genes. Overexpression of TaSIP1 and TaSIP1-∆C (a form lacking the transmembrane domain) in wheat both compromised the plants' tolerance of salinity stress, highlighting the importance of precise regulation of this signal cascade during salinity stress. The interaction of TaSRO1/TaSIP1, in the cytoplasm, arrested more TaSIP1 on the membrane of ER, and in the nucleus, attenuated the trans-activation activity of TaSIP1, therefore reducing the TaSIP1-mediated activation of MDS genes. Moreover, the overexpression of TaSRO1 rescued the inferior phenotype induced by TaSIP1 overexpression. Our study provides an orchestrating mechanism executed by the TaSRO1-TaSIP1 module that balances the growth and stress response via fine tuning the level of mitochondria retrograde signalling.

Integrated regulation of periclinal cell division by transcriptional module of BZR1-SHR in Arabidopsis roots.

The timing and extent of cell division are crucial for the correct patterning of multicellular organism. In Arabidopsis, root ground tissue maturation involves the periclinal cell division of the endodermis to generate two cell layers: endodermis and middle cortex. However, the molecular mechanism underlying this pattern formation remains unclear. Here, we report that phytohormone brassinosteroid (BR) and redox signal hydrogen peroxide (H2 O2 ) interdependently promote periclinal division during root ground tissue maturation by regulating the activity of SHORT-ROOT (SHR), a master regulator of root growth and development. BR-activated transcription factor BRASSINAZOLE RESISTANT1 (BZR1) directly binds to the promoter of SHR to induce its expression, and physically interacts with SHR to increase the transcripts of RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs) and elevate the levels of H2 O2 , which feedback enhances the interaction between BZR1 and SHR. Additionally, genetic analysis shows that SHR is required for BZR1-promoted periclinal division, and BZR1 enhances the promoting effects of SHR on periclinal division. Together, our finding reveals that the transcriptional module of BZR1-SHR fine-tunes periclinal division during root ground tissue maturation in response to hormone and redox signals.

Interaction between BZR1 and EIN3 mediates signalling crosstalk between brassinosteroids and ethylene.

Plant growth and development are coordinated by multiple environmental and endogenous signals. Brassinosteroid (BR) and ethylene (ET) have overlapping functions in a wide range of developmental processes. However, the relationship between the BR and ET signalling pathways has remained unclear. Here, we show that BR and ET interdependently promote apical hook development and cell elongation through a direct interaction between BR-activated BRASSINOZALE-RESISTANT1 (BZR1) and ET-activated ETHYLENE INSENSITIVE3 (EIN3). Genetic analysis showed that BR signalling is required for ET promotion of apical hook development in the dark and cell elongation under light, and ET quantitatively enhances BR-potentiated growth. BZR1 interacts with EIN3 to co-operatively increase the expression of HOOKLESS1 and PACLOBUTRAZOL RESISTANCE FACTORs (PREs). Furthermore, we found that BR promotion of hook development requires gibberellin (GA), and GA restores the hookless phenotype of BR-deficient materials by activating EIN3/EIL1. Our findings shed light on the molecular mechanism underlying the regulation of plant development by BR, ET and GA signals through the direct interaction of master transcriptional regulators.

Phospho-Mutant Activity Assays Provide Evidence for the Negative Regulation of Transcriptional Regulator PRE1 by Phosphorylation.

The PACLOBUTRAZOL-RESISTANCE (PRE) gene family encodes a group of atypical helix-loop-helix (HLH) proteins that act as the major hub integrating a wide range of environmental and hormonal signals to regulate plant growth and development. PRE1, as a positive regulator of cell elongation, activates HBI1 DNA binding by sequestering its inhibitor IBH1. Furthermore, PRE1 can be phosphorylated at Ser-46 and Ser-67, but how this phosphorylation regulates the functions of PRE1 remains unclear. Here, we used a phospho-mutant activity assay to reveal that the phosphorylation at Ser-67 negatively regulates the functions of PRE1 on cell elongation. Both of mutations of serine 46, either to phospho-dead alanine or phospho-mimicking glutamic acid, had no significant effects on the functions of PRE1. However, the mutation of serine 67 to glutamic acid (PRE1S67E-Ox), but not alanine (PRE1S67A-Ox), significantly reduced the promoting effects of PRE1 on cell elongation. The mutation of Ser-67 to Glu-67 impaired the interaction of PRE1 with IBH1 and resulted in PRE1 failing to inhibit the interaction between IBH1 and HBI1, losing the ability to induce the expression of the subsequent cell elongation-related genes. Furthermore, we showed that PRE1-Ox and PRE1S67A-Ox both suppressed but PRE1S67E-Ox had no strong effects on the dwarf phenotypes of IBH1-Ox. Our study demonstrated that the PRE1 activity is negatively regulated by the phosphorylation at Ser-67.

KIN10 promotes stomatal development through stabilization of the SPEECHLESS transcription factor.

Stomata are epidermal structures that modulate gas exchanges between plants and the atmosphere. The formation of stomata is regulated by multiple developmental and environmental signals, but how these signals are coordinated to control this process remains unclear. Here, we showed that the conserved energy sensor kinase SnRK1 promotes stomatal development under short-day photoperiod or in liquid culture conditions. Mutation of KIN10, the catalytic α-subunit of SnRK1, results in the decreased stomatal index; while overexpression of KIN10 significantly induces stomatal development. KIN10 displays the cell-type-specific subcellular location pattern. The nuclear-localized KIN10 proteins are highly enriched in the stomatal lineage cells to phosphorylate and stabilize SPEECHLESS, a master regulator of stomatal formation, thereby promoting stomatal development. Our work identifies a module links connecting the energy signaling and stomatal development and reveals that multiple regulatory mechanisms are in place for SnRK1 to modulate stomatal development in response to changing environments.

The miR396-GRFs Module Mediates the Prevention of Photo-oxidative Damage by Brassinosteroids during Seedling De-Etiolation in Arabidopsis.

The switch from dark- to light-mediated development is critical for the survival and growth of seedlings, but the underlying regulatory mechanisms are incomplete. Here, we show that the steroids phytohormone brassinosteroids play crucial roles during this developmental transition by regulating chlorophyll biosynthesis to promote greening of etiolated seedlings upon light exposure. Etiolated seedlings of the brassinosteroids-deficient det2-1 (de-etiolated2) mutant accumulated excess protochlorophyllide, resulting in photo-oxidative damage upon exposure to light. Conversely, the gain-of-function mutant bzr1-1D (brassinazole-resistant 1-1D) suppressed the protochlorophyllide accumulation of det2-1, thereby promoting greening of etiolated seedlings. Genetic analysis indicated that phytochrome-interacting factors (PIFs) were required for BZR1-mediated seedling greening. Furthermore, we reveal that GROWTH REGULATING FACTOR 7 (GRF7) and GRF8 are induced by BZR1 and PIF4 to repress chlorophyll biosynthesis and promote seedling greening. Suppression of GRFs function by overexpressing microRNA396a caused an accumulation of protochlorophyllide in the dark and severe photobleaching upon light exposure. Additionally, BZR1, PIF4, and GRF7 interact with each other and precisely regulate the expression of chlorophyll biosynthetic genes. Our findings reveal an essential role for BRs in promoting seedling development and survival during the initial emergence of seedlings from subterranean darkness into sunlight.

Brassinosteroid and Hydrogen Peroxide Interdependently Induce Stomatal Opening by Promoting Guard Cell Starch Degradation.

Starch is the major storage carbohydrate in plants and functions in buffering carbon and energy availability for plant fitness with challenging environmental conditions. The timing and extent of starch degradation appear to be determined by diverse hormonal and environmental signals; however, our understanding of the regulation of starch metabolism is fragmentary. Here, we demonstrate that the phytohormone brassinosteroid (BR) and redox signal hydrogen peroxide (H2O2) induce the breakdown of starch in guard cells, which promotes stomatal opening. The BR-insensitive mutant bri1-116 accumulated high levels of starch in guard cells, impairing stomatal opening in response to light. The gain-of-function mutant bzr1-1D suppressed the starch excess phenotype of bri1-116, thereby promoting stomatal opening. BRASSINAZOLE-RESISTANT1 (BZR1) interacts with the basic leucine zipper transcription factor G-BOX BINDING FACTOR2 (GBF2) to promote the expression of ß-AMYLASE1 (BAM1), which is responsible for starch degradation in guard cells. H2O2 induces BZR1 oxidation, enhancing the interaction between BZR1 and GBF2 to increase BAM1 transcription. Mutations in BAM1 lead to starch accumulation and reduce the effects of BR and H2O2 on stomatal opening. Overall, this study uncovers the critical roles of BR and H2O2 in regulating guard cell starch metabolism and stomatal opening.

Hydrogen peroxide positively regulates brassinosteroid signaling through oxidation of the BRASSINAZOLE-RESISTANT1 transcription factor.

Hydrogen peroxide (H2O2) is an important signaling molecule in plant developmental processes and stress responses. However, whether H2O2-mediated signaling crosstalks with plant hormone signaling is largely unclear. Here, we show that H2O2 induces the oxidation of the BRASSINAZOLE-RESISTANT1 (BZR1) transcription factor, which functions as a master regulator of brassinosteroid (BR) signaling. Oxidative modification enhances BZR1 transcriptional activity by promoting its interaction with key regulators in the auxin-signaling and light-signaling pathways, including AUXIN RESPONSE FACTOR6 (ARF6) and PHYTOCHROME INTERACTING FACTOR4 (PIF4). Genome-wide analysis shows that H2O2-dependent regulation of BZR1 activity plays a major role in modifying gene expression related to several BR-mediated biological processes. Furthermore, we show that the thioredoxin TRXh5 can interact with BZR1 and catalyzes its reduction. We conclude that reversible oxidation of BZR1 connects H2O2-mediated and thioredoxin-mediated redox signaling to BR signaling to regulate plant development.

A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana.

Wheat is an important crop in the world. But most of the cultivars are salt sensitive, and often adversely affected by salt stress. WRKY transcription factors play a major role in plant responses to salt stress, but the effective salinity regulatory WRKYs identified in bread wheat are limited and the mechanism of salt stress tolerance is also not well explored. Here, we identified a salt (NaCl) induced class II WRKY transcription factor TaWRKY93. Its transcript level was strongly induced by salt (NaCl) and exogenous abscisic acid (ABA). Over-expression of TaWRKY93 in Arabidopsis thaliana enhanced salt (NaCl), drought, low temperature and osmotic (mannitol) stress tolerance, mainly demonstrated by transgenic plants forming longer primary roots or more lateral roots on MS plates supplemented with NaCl and mannitol individually, higher survival rate under drought and low temperature stress. Further, transgenic plants maintained a more proline content, higher relative water content and less electrolyte leakage than the wild type plants. The transcript abundance of a series of abiotic stress-related genes was up-regulated in the TaWRKY93 transgenic plants. In summary, TaWRKY93 is a new positive regulator of abiotic stress, it may increase salinity, drought and low temperature stress tolerance through enhancing osmotic adjustment, maintaining membrane stability and increasing transcription of stress related genes, and contribute to the superior agricultural traits of SR3 through promoting root development. It can be used as a candidate gene for wheat transgenic engineering breeding against abiotic stress.

Fine mapping a major QTL for kernel number per row under different phosphorus regimes in maize (Zea mays L.).

Phosphorus (P) is one of the essential macronutrients for plant growth and development. Grain yield is the primary trait of interest in maize breeding programs. Maize grain yield and yield-related traits are seriously affected by P deficiency. Kernel number per row (KN), as one of the major components of grain yield, has attracted the attention of more and more breeders. In our previous study, one major QTL (named qKN), controlling KN under different P regimes was mapped to the interval between molecular markers bnlg1360 and umc1645 on chromosome 10 using a F 2:3 population derived from the cross between maize inbreds 178 and 5,003 (107). In order to understand its genetic basis, we developed a population of near isogenic lines (NILs) and two P regimes were used to fine map and characterize qKN. The QTL qKN was finally localized in a region of ~480 kb. A single qKN allele of inbred 178 increased KN by 6.08-10.76 % in the 5,003 (107) background; qKN acted in a partially dominant manner. Our results will be instrumental for the future identification and isolation of the candidate gene underlying qKN. The tightly linked molecular markers that we developed for qKN will be useful in maize breeding programs for improving KN applying the marker-assisted selection.

Constitutive expression of a salinity-induced wheat WRKY transcription factor enhances salinity and ionic stress tolerance in transgenic Arabidopsis thaliana.

The isolation and characterization of TaWRKY79, a wheat class II WRKY transcription factor, is described. Its 1297 bp coding region includes a 987 bp long open reading frame. TaWRKY79 was induced by stressing seedlings with either NaCl or abscisic acid (ABA). When a fusion between an 843 bp segment upstream of the TaWRKY79 coding sequence and GUS was introduced into Arabidopsis thaliana, GUS staining indicated that this upstream segment captured the sequence(s) required to respond to ABA or NaCl treatment. When TaWRKY79 was constitutively expressed as a transgene in A. thaliana, the transgenic plants showed an improved capacity to extend their primary root in the presence of either 100 mM NaCl, 10 mM LiCl or 2 µM ABA. The inference was that TaWRKY79 enhanced the level of tolerance to both salinity and ionic stress, while reducing the level of sensitivity to ABA. The ABA-related genes ABA1, ABA2 ABI1 and ABI5 were all up-regulated in the TaWRKY79 transgenic plants, suggesting that the transcription factor operates in an ABA-dependent pathway.

Over-expression of TaMYB33 encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis.

Salt and drought stresses often adversely affect plant growth and productivity, MYB transcription factors have been shown to participate in the response to these stresses. Here we identified a new R2R3-type MYB transcription factor gene TaMYB33 from wheat (Triticum aestivum). TaMYB33 was induced by NaCl, PEG and ABA treatments, and its promoter sequence contains putative ABRE, MYB and other abiotic stress related cis-elements. Ectopic over-expression of TaMYB33 in Arabidopsis thaliana remarkably enhanced its tolerance to drought and NaCl stresses, but not to LiCl and KCl treatments. The expressions of AtP5CS and AtZAT12 which mirror the activities of proline and ascorbate peroxidase synthesis respectively were induced in TaMYB33 over-expression lines, indicating TaMYB33 promotes the ability for osmotic pressure balance-reconstruction and reactive oxidative species (ROS) scavenging. The up-regulation of AtAAO3 along with down-regulation of AtABF3, AtABI1 in TaMYB33 over-expression lines indicated that ABA synthesis was elevated while its signaling was restricted. These results suggest that TaMYB33 enhances salt and drought tolerance partially through superior ability for osmotic balance reconstruction and ROS detoxification.