Yuan Hu Xuan

Rice Indeterminate 1 (OsId1) is necessary for the expression of Ehd1 (Early heading date 1) regardless of photoperiod.

Indeterminate 1 (Id1), a classical flowering gene first reported in 1946, is one of the earliest genes whose expression in leaf tissues affects the floral transition in the shoot meristem. How Id1 is integrated into the flowering process is largely unknown. In this study, we examined the genetic action of the rice (Oryza sativa) ortholog OsId1. In rice, OsId1 is preferentially expressed in young leaves, but the overall expression pattern is broader than that in maize (Zea mays). OsId1 is able to activate transcription in yeast. RNAi mutants show a delay in flowering under both short-day (SD) and long-day (LD) conditions. OsId1 regulates the expression of Ehd1 (Early heading date 1) and its downstream genes, including Hd3a (a rice ortholog of FT) and RFT1 (Rice Flowering Locus T1), under both SD and LD conditions. In rice, the expression of Ehd1 is also controlled by the photoperiodic flowering genes OsGI (a rice ortholog of GI) and OsMADS51. However, the expression of OsId1 is independent of OsGI, OsMADS51, and OsMADS50 (a rice SOC1 ortholog). This study demonstrates that the activation of Ehd1 by OsId1 is required for the promotion of flowering.

Functional role of oligomerization for bacterial and plant SWEET sugar transporter family

Significance SWEET sugar transporter homologs from bacteria were identified and named SemiSWEETs. They are small proteins with only three transmembrane domains (TMs); they are too small to create pores by themselves, but likely, they assemble multiple 3-TMs into a complex. SemiSWEETs are related to SWEETs, which play important roles in intercellular and interorgan sugar translocation in plants, and they are found in animals. SWEETs have fused two 3-TM units through a linker. However, SWEETs seem to be too small to transport sugars on their own. Here, we show that SWEET function requires assembly into oligomers, indicating that a pore requires at least an SWEET dimer. Eukaryotic sugar transporters of the MFS and SWEET superfamilies consist of 12 and 7 α-helical transmembrane domains (TMs), respectively. Structural analyses indicate that MFS transporters evolved from a series of tandem duplications of an ancestral 3-TM unit. SWEETs are heptahelical proteins carrying a tandem repeat of 3-TM separated by a single TM. Here, we show that prokaryotes have ancestral SWEET homologs with only 3-TM and that the Bradyrhizobium japonicum SemiSWEET1, like Arabidopsis SWEET11, mediates sucrose transport. Eukaryotic SWEETs most likely evolved by internal duplication of the 3-TM, suggesting that SemiSWEETs form oligomers to create a functional pore. However, it remains elusive whether the 7-TM SWEETs are the functional unit or require oligomerization to form a pore sufficiently large to allow for sucrose passage. Split ubiquitin yeast two-hybrid and split GFP assays indicate that Arabidopsis SWEETs homo- and heterooligomerize. We examined mutant SWEET variants for negative dominance to test if oligomerization is necessary for function. Mutation of the conserved Y57 or G58 in SWEET1 led to loss of activity. Coexpression of the defective mutants with functional A. thaliana SWEET1 inhibited glucose transport, indicating that homooligomerization is necessary for function. Collectively, these data imply that the basic unit of SWEETs, similar to MFS sugar transporters, is a 3-TM unit and that a functional transporter contains at least four such domains. We hypothesize that the functional unit of the SWEET family of transporters possesses a structure resembling the 12-TM MFS structure, however, with a parallel orientation of the 3-TM unit.

Border control--a membrane-linked interactome of Arabidopsis.

Degrees of Separation Proteins embedded in membranes represent an interesting point of communication between the cell and its environment, but their localization to membranes can make them difficult to study. Jones et al. (p. 711) found an approach to catalog thousands of interactions involving membrane proteins and membrane-associated signaling machinery—including many previously uncharacterized proteins. With a focus on the model plant Arabidopsis, several of the identified interactions fill gaps in important signal transduction chains, while others point to functions for enigmatic unknown proteins. Amembrane and signaling protein interaction network for gene discovery and hypothesis generation is identified in Arabidopsis. Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 106 pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split–green fluorescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

Allosteric Regulation of Transport Activity by Heterotrimerization of Arabidopsis Ammonium Transporter Complexes in Vivo

In plants, AMT-type ammonium transporters are posttranslationally regulated by phosphorylation. This study provides evidence that allosteric regulation is functional in planta and that C-terminal phosphorylation mediates trans-inactivation also in heteromeric AMT complexes containing different AMT isoforms. Ammonium acquisition by plant roots is mediated by AMMONIUM TRANSPORTERs (AMTs), ubiquitous membrane proteins with essential roles in nitrogen nutrition in all organisms. In microbial and plant cells, ammonium transport activity is controlled by ammonium-triggered feedback inhibition to prevent cellular ammonium toxicity. Data from heterologous expression in yeast indicate that oligomerization of plant AMTs is critical for allosteric regulation of transport activity, in which the conserved cytosolic C terminus functions as a trans-activator. Employing the coexpressed transporters AMT1;1 and AMT1;3 from Arabidopsis thaliana as a model, we show here that these two isoforms form functional homo- and heterotrimers in yeast and plant roots and that AMT1;3 carrying a phosphomimic residue in its C terminus regulates both homo- and heterotrimers in a dominant-negative fashion in vivo. 15NH4+ influx studies further indicate that allosteric inhibition represses ammonium transport activity in roots of transgenic Arabidopsis expressing a phosphomimic mutant together with functional AMT1;3 or AMT1;1. Our study demonstrates in planta a regulatory role in transport activity of heterooligomerization of transporter isoforms, which may enhance their versatility for signal exchange in response to environmental triggers.

RAV-Like1 Maintains Brassinosteroid Homeostasis via the Coordinated Activation of BRI1 and Biosynthetic Genes in Rice

Brassinosteroid (BR) homeostasis is established by the regulatory circuit between receptor BRI1-mediated signaling and BR synthesis. RAVL1 modulates the strength of the circuit by activating expression of both BRI1 and synthetic genes and is necessary for feedback responses to BR levels. Temporal and spatial variation in the levels of and sensitivity to hormones are essential for the development of higher organisms. Traditionally, end-product feedback regulation has been considered as the key mechanism for the achievement of cellular homeostasis. Brassinosteroids (BRs) are plant steroid hormones that are perceived by the cell surface receptor kinase Brassinosteroid Insensitive1. Binding of these hormones to the receptor activates BR signaling and eventually suppresses BR synthesis. This report shows that RAVL1 regulates the expression of the BR receptor. Furthermore, RAVL1 is also required for the expression of the BR biosynthetic genes D2, D11, and BRD1 that are subject to BR negative feedback. Activation by RAVL1 was coordinated via E-box cis-elements in the promoters of the receptor and biosynthetic genes. Also, RAVL1 is necessary for the response of these genes to changes in cellular BR homeostasis. Genetic evidence is presented to strengthen the observation that the primary action of RAVL1 mediates the expression of genes involved in BR signaling and biosynthesis. This study thus describes a regulatory circuit modulating the homeostasis of BR in which RAVL1 ensures the basal activity of both the signaling and the biosynthetic pathways.

OsCIPK31, a CBL-Interacting Protein Kinase Is Involved in Germination and Seedling Growth under Abiotic Stress Conditions in Rice Plants

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. Extensive studies using Arabidopsis plants have demonstrated that many calcium signatures that activate CIPKs originate from abiotic stresses. However, there are few reports on the functional demonstration of CIPKs in other plants, especially in grasses. In this study, we used a loss-of-function mutation to characterize the function of the rice CIPK gene OsCIPK31. Exposure to high concentrations of NaCl or mannitol effected a rapid and transient enhancement of OsCIPK31 expression. These findings were observed only in the light. However, longer exposure to most stresses resulted in downregulation of OsCIPK31 expression in both the presence and absence of light. To determine the physiological roles of OsCIPK31 in rice plants, the sensitivity of oscipk31::Ds, which is a transposon Ds insertion mutant, to abiotic stresses was examined during germination and seedling stages. oscipk31::Ds mutants exhibited hypersensitive phenotypes to ABA, salt, mannitol, and glucose. Compared with wild-type rice plants, mutants exhibited retarded germination and slow seedling growth. In addition, oscipk31::Ds seedlings exhibited enhanced expression of several stress-responsive genes after exposure to these abiotic stresses. However, the expression of ABA metabolic genes and the endogenous levels of ABA were not altered significantly in the oscipk31::Ds mutant. This study demonstrated that rice plants use OsCIPK31 to modulate responses to abiotic stresses during the seed germination and seedling stages and to modulate the expression of stress-responsive genes.

High-Glucose Inhibits Human Fibroblast Cell Migration in Wound Healing via Repression of bFGF-Regulating JNK Phosphorylation

One of the major symptoms of diabetes mellitus (DM) is delayed wound healing, which affects large populations of patients worldwide. However, the underlying mechanism behind this illness remains elusive. Skin wound healing requires a series of coordinated processes, including fibroblast cell proliferation and migration. Here, we simulate DM by application of high glucose (HG) in human foreskin primary fibroblast cells to analyze the molecular mechanism of DM effects on wound healing. The results indicate that HG, at a concentration of 30 mM, delay cell migration, but not cell proliferation. bFGF is known to promote cell migration that partially rescues HG effects on cell migration. Molecular and cell biology studies demonstrated that HG enhanced ROS production and repressed JNK phosphorylation, but did not affect Rac1 activity. JNK and Rac1 activation were known to be important for bFGF regulated cell migration. To further confirm DM effects on skin repair, a type 1 diabetic rat model was established, and we observed the efficacy of bFGF on both normal and diabetic rat skin repair. Furthermore, proteomic studies identified an increase of Annexin A2 protein nitration in HG-stressed fibroblasts and the nitration was protected by activation of bFGF signaling. Treatment with FGFR1 and JNK inhibitors delayed cell migration and increased Annexin A2 nitration levels, indicating that Annexin A2 nitration is modulated by bFGF signaling via activation of JNK. Together with these results, our data suggests that the HG-mediated delay of cell migration is linked to the inhibition of bFGF signaling, specifically through JNK suppression.

Analysis of gene-trap Ds rice populations in Korea

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes.

OsSNDP1, a Sec14-nodulin domain-containing protein, plays a critical role in root hair elongation in rice

Rice is cultivated in water-logged paddy lands. Thus, rice root hairs on the epidermal layers are exposed to a different redox status of nitrogen species, organic acids, and metal ions than root hairs growing in drained soil. To identify genes that play an important role in root hair growth, a forward genetics approach was used to screen for short-root-hair mutants. A short-root-hair mutant was identified and isolated by using map-based cloning and sequencing. The mutation arose from a single amino acid substitution of OsSNDP1 (Oryza sativa Sec14-nodulin domain protein), which shows high sequence homology with Arabidopsis COW1/AtSFH1 and encodes a phosphatidylinositol transfer protein (PITP). By performing complementation assays with Atsfh1 mutants, we demonstrated that OsSNDP1 is involved in growth of root hairs. Cryo-scanning electron microscopy was utilized to further characterize the effect of the Ossndp1 mutation on root hair morphology. Aberrant morphogenesis was detected in root hair elongation and maturation zones. Many root hairs were branched and showed irregular shapes due to bulged nodes. Many epidermal cells also produced dome-shaped root hairs, which indicated that root hair elongation ceased at an early stage. These studies showed that PITP-mediated phospholipid signaling and metabolism is critical for root hair elongation in rice.

Indeterminate domain 10 regulates ammonium‐mediated gene expression in rice roots

Indeterminate domain (IDD) genes are a family of plant transcriptional regulators that function in the control of development and metabolism during growth. Here, the function of Oryza sativa indeterminate domain 10 (OsIDD10) has been explored in rice plants. Compared with wild-type roots, idd10 mutant roots are hypersensitive to exogenous ammonium. This work aims to define the action of IDD10 on gene expression involved in ammonium uptake and nitrogen (N) metabolism. The ammonium induction of key ammonium uptake and assimilation genes was examined in the roots of idd10 mutants and IDD10 overexpressors. Molecular studies and transcriptome analysis were performed to identify target genes and IDD10 binding cis-elements. IDD10 activates the transcription of AMT1;2 and GDH2 by binding to a cis-element motif present in the promoter region of AMT1;2 and in the fifth intron of GDH2. IDD10 contributes significantly to the induction of several genes involved in N-linked metabolic and cellular responses, including genes encoding glutamine synthetase 2, nitrite reductases and trehalose-6-phosphate synthase. Furthermore, the possibility that IDD10 might influence the N-mediated feedback regulation of target genes was examined. This study demonstrates that IDD10 is involved in regulatory circuits that determine N-mediated gene expression in plant roots.