EGR1 and EGR2 positively regulate plant ABA signaling by modulating the phosphorylation of SnRK2.2.

During abscisic acid (ABA) signaling, reversible phosphorylation controls the activity and accumulation of class III SNF1-RELATED PROTEIN KINASE 2s (SnRK2s). While protein phosphatases that negatively regulate SnRK2s have been identified, those that positively regulate ABA signaling through SnRK2s are less understood. In this study, Arabidopsis thaliana mutants of Clade E Growth-Regulating 1 and 2 (EGR1/2), which belong to the protein phosphatase 2C family, exhibited reduced ABA sensitivity in terms of seed germination, cotyledon greening, and ABI5 accumulation. Conversely, overexpression increased these ABA-induced responses. Transcriptomic data revealed that most ABA-regulated genes in egr1 egr2 plants were expressed at reduced levels compared with those in Col-0 after ABA treatment. Abscisic acid up-regulated EGR1/2, which interact directly with SnRK2.2 through its C-terminal domain I. Genetic analysis demonstrated that EGR1/2 function through SnRK2.2 during ABA response. Furthermore, SnRK2.2 de-phosphorylation by EGR1/2 was identified at serine 31 within the ATP-binding pocket. A phospho-mimic mutation confirmed that phosphorylation at serine 31 inhibited SnRK2.2 activity and reduced ABA responsiveness in plants. Our findings highlight the positive role of EGR1/2 in regulating ABA signaling, they reveal a new mechanism for modulating SnRK2.2 activity, and provide novel insight into how plants fine-tune their responses to ABA.

S-nitrosylation of RABG3E positively regulates vesicle trafficking to promote salt tolerance.

Nitric oxide (NO) is a key signaling molecule affecting the response of plants to salt stress; however, the underlying molecular mechanism is poorly understood. In this study, we conducted a phenotype analysis and found that the small GTPase RABG3E (RAB7) promotes salt tolerance in Arabidopsis thaliana. NO promotes the S-nitrosylation of RAB7 at Cys-171, which in turn helps maintain the ion balance in salt-stressed plants. Furthermore, the S-nitrosylation of RAB7 at Cys-171 enhances the enzyme's GTPase activity, thereby promoting vesicle trafficking and increasing its interaction with phosphatidylinositol phosphates-especially phosphatidylinositol-4-phosphate (PI4P). Exogenously applied PI4P increases vesicle trafficking and promotes salt tolerance depending on the S-nitrosylation of RAB7 at Cys-171. These findings illustrate a unique mechanism in salt tolerance, by which NO regulates vesicle trafficking and ion homeostasis through the S-nitrosylation of RAB7 and its interaction with PI4P.

The Role of Nitric Oxide in Plant Responses to Salt Stress.

The gas nitric oxide (NO) plays an important role in several biological processes in plants, including growth, development, and biotic/abiotic stress responses. Salinity has received increasing attention from scientists as an abiotic stressor that can seriously harm plant growth and crop yields. Under saline conditions, plants produce NO, which can alleviate salt-induced damage. Here, we summarize NO synthesis during salt stress and describe how NO is involved in alleviating salt stress effects through different strategies, including interactions with various other signaling molecules and plant hormones. Finally, future directions for research on the role of NO in plant salt tolerance are discussed. This summary will serve as a reference for researchers studying NO in plants.

AtPFA-DSP5 interacts with MPK3/MPK6 and negatively regulates plant salt responses.

Protein tyrosine phosphatases play essential roles in plant growth and development and in plant responses to biotic or abiotic stresses. We recently demonstrated that an atypical dual-specificity protein tyrosine phosphatase in plants, AtPFA-DSP3 (DSP3), negatively regulates plant salt tolerance. Here, we report that a homolog of DSP3, AtPFA-DSP5 (DSP5), affects the response of plants to high-salt conditions. A loss-of-function mutant of DSP5 showed reduced sensitivity to salt treatment at the seed germination and vegetative stages of development while a gain-of-function mutant of DSP5 showed increased sensitivity to salt stress. The salt responses of dsp3dsp5 double-mutant plants were similar to those of dsp3 and dsp5 single-mutant plants. Gel overlay and firefly luciferase complementation assays showed that DSP5 interacts with MPK3 and MPK6 in vitro and in vivo. These results indicate that DSP5 is a novel negative regulator of salt responses in Arabidopsis that interacts directly with MPK3 and MPK6.

OsWAK112, A Wall-Associated Kinase, Negatively Regulates Salt Stress Responses by Inhibiting Ethylene Production.

The wall-associated kinase (WAK) multigene family plays critical roles in various cellular processes and stress responses in plants, however, whether WAKs are involved in salt tolerance is obscure. Herein, we report the functional characterization of a rice WAK, WAK112, whose expression is suppressed by salt. Overexpression of OsWAK112 in rice and heterologous expression of OsWAK112 in Arabidopsis significantly decreased plant survival under conditions of salt stress, while knocking down the OsWAK112 in rice increased plant survival under salt stress. OsWAK112 is universally expressed in plant and associated with cell wall. Meanwhile, in vitro kinase assays and salt tolerance analyses showed that OsWAK112 possesses kinase activity and that it plays a negative role in the response of plants to salt stress. In addition, OsWAK112 interacts with S-adenosyl-L-methionine synthetase (SAMS) 1/2/3, which catalyzes SAM synthesis from ATP and L-methionine, and promotes OsSAMS1 degradation under salt stress. Furthermore, in OsWAK112-overexpressing plants, there is a decreased SAMS content and a decreased ethylene content under salt stress. These results indicate that OsWAK112 negatively regulates plant salt responses by inhibiting ethylene production, possibly via direct binding with OsSAMS1/2/3.

Plastid-Localized EMB2726 Is Involved in Chloroplast Biogenesis and Early Embryo Development in Arabidopsis.

Embryogenesis is a critical developmental process that establishes the body organization of higher plants. During this process, the biogenesis of chloroplasts from proplastids is essential. A failure in chloroplast development during embryogenesis can cause morphologically abnormal embryos or embryonic lethality. In this study, we isolated a T-DNA insertion mutant of the Arabidopsis gene EMBRYO DEFECTIVE 2726 (EMB2726). Heterozygous emb2726 seedlings produced about 25% albino seeds with embryos that displayed defects at the 32-cell stage and that arrested development at the late globular stage. EMB2726 protein was localized in chloroplasts and was expressed at all stages of development, such as embryogenesis. Moreover, the two translation elongation factor Ts domains within the protein were critical for its function. Transmission electron microscopy revealed that the cells in emb2726 embryos contained undifferentiated proplastids and that the expression of plastid genome-encoded photosynthesis-related genes was dramatically reduced. Expression studies of DR5:GFP, pDRN:DRN-GFP, and pPIN1:PIN1-GFP reporter lines indicated normal auxin biosynthesis but altered polar auxin transport. The expression of pSHR:SHR-GFP and pSCR:SCR-GFP confirmed that procambium and ground tissue precursors were lacking in emb2726 embryos. The results suggest that EMB2726 plays a critical role during Arabidopsis embryogenesis by affecting chloroplast development, possibly by affecting the translation process in plastids.

AtPFA-DSP3, an atypical dual-specificity protein tyrosine phosphatase, affects salt stress response by modulating MPK3 and MPK6 activity.

Protein phosphorylation, especially serine/threonine and tyrosine phosphorylation, plays significant roles in signalling during plant growth and development as well as plant responses to biotic or abiotic stresses. Dual-specificity protein tyrosine phosphatases dephosphorylate components of these signalling pathways. Here, we report that an atypical dual-specificity protein tyrosine phosphatase, AtPFA-DSP3 (DSP3), negatively affects the response of plants to high-salt conditions. A DSP3 loss-of-function mutant showed reduced sensitivity to salt treatment. DSP3 was primarily localized in nuclei and was degraded during salt treatment. Compared to wild type, the level of ROS was lower in the dsp3 mutant and higher in plants ectopically expressing DSP3, indicating that higher DSP3 level was associated with increased ROS production. DSP3 interacted with and dephosphorylated MPK3 and MPK6. Genetic analyses of a dsp3mpk3 double mutant revealed that DSP3's effect on salt stress depends on MPK3. Moreover, the phosphatase activity of DSP3 was required for its role in salt signalling. These results indicate that DSP3 is a negative regulator of salt responses in Arabidopsis by directly modulating the accumulation of phosphorylated MPK3 and MPK6.

Identification of BZR1-interacting proteins as potential components of the brassinosteroid signaling pathway in Arabidopsis through tandem affinity purification.

Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response.

Brassinosteroid, gibberellin and phytochrome impinge on a common transcription module in Arabidopsis.

Brassinosteroid and gibberellin promote many similar developmental responses in plants; however, their relationship remains unclear. Here we show that BR and GA act interdependently through a direct interaction between the BR-activated BZR1 and GA-inactivated DELLA transcription regulators. GA promotion of cell elongation required BR signalling, whereas BR or active BZR1 suppressed the GA-deficient dwarf phenotype. DELLAs directly interacted with BZR1 and inhibited BZR1-DNA binding both in vitro and in vivo. Genome-wide analysis defined a BZR1-dependent GA-regulated transcriptome, which is enriched with light-regulated genes and genes involved in cell wall synthesis and photosynthesis/chloroplast function. GA promotion of hypocotyl elongation requires both BZR1 and the phytochrome-interacting factors (PIFs), as well as their common downstream targets encoding the PRE-family helix-loop-helix factors. The results demonstrate that GA releases DELLA-mediated inhibition of BZR1, and that the DELLA-BZR1-PIF4 interaction defines a core transcription module that mediates coordinated growth regulation by GA, BR and light signals.

Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors.

Brassinosteroid (BR) regulates gene expression and plant development through a receptor kinase-mediated signal transduction pathway. Despite the identification of many components of this pathway, it remains unclear how the BR signal is transduced from the cell surface to the nucleus. Here we describe a complete BR signalling pathway by elucidating key missing steps. We show that phosphorylation of BSK1 (BR-signalling kinase 1) by the BR receptor kinase BRI1 (BR-insensitive 1) promotes BSK1 binding to the BSU1 (BRI1 suppressor 1) phosphatase, and BSU1 inactivates the GSK3-like kinase BIN2 (BR-insensitive 2) by dephosphorylating a conserved phospho-tyrosine residue (pTyr 200). Mutations that affect phosphorylation/dephosphorylation of BIN2 pTyr200 (bin2-1, bin2-Y200F and quadruple loss-of-function of BSU1-related phosphatases) support an essential role for BSU1-mediated BIN2 dephosphorylation in BR-dependent plant growth. These results demonstrate direct sequential BR activation of BRI1, BSK1 and BSU1, and inactivation of BIN2, leading to accumulation of unphosphorylated BZR (brassinazole resistant) transcription factors in the nucleus. This study establishes a fully connected BR signalling pathway and provides new insights into the mechanism of GSK3 regulation.

Effect of erythropoietin on hepcidin, DMT1 with IRE, and hephaestin gene expression in duodenum of rats.

BACKGROUND: Erythropoietin (Epo) is the central regulator of red blood cell production and can stimulate proliferation and differentiation of erythroid progenitor cells. Now, recombinant human erythropoietin (rHuEpo) is widely used in patients with renal disease, chronic anemia, and iron deficiency of early childhood. It has been reported that the enhanced erythropoiesis associated with erythropoietin therapy increases intestinal iron absorption, but the molecular mechanisms underlying are unknown. Therefore, we have investigated the effect of rHuEpo on duodenal iron transport protein synthesis in rats. METHODS: Male Sprague-Dawley rats weighing 250 g were randomly divided into two groups: (1) rHuEpo injection group (rHuEpo, 500 IU/day, s.c.), and (2) control group (injection of the same volume of saline). After 3 days injection, blood parameters, serum iron status, and non-heme iron concentrations in the liver and duodenum were examined at the fifth day. The mRNA levels and protein synthesis of duodenal divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hephaestin (Hp) were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Hepatic hepcidin mRNA expression was analyzed by RT-PCR. RESULTS: rHuEpo injection significantly stimulated erythropoiesis and decreased serum iron status, non-heme iron concentrations in the liver and duodenum. DMT1 (+IRE) and Hp expression in duodenum were increased significantly. However, DMT1 (-IRE) and FPN1 expression had no apparent change. Hepatic hepcidin mRNA expression was decreased dramatically, reaching an almost undetectable level in rHuEpo-treated rats. CONCLUSIONS: rHuEpo administration improved the duodenal iron absorption by increasing the expression of DMT1 (+IRE) and Hp.