Arabidopsis SMALL AUXIN UP RNA63 promotes hypocotyl and stamen filament elongation

Auxin regulates plant growth and development in part by activating gene expression. Arabidopsis thaliana SMALL AUXIN UP RNAs (SAURs) are a family of early auxin-responsive genes with unknown functionality. Here, we show that transgenic plant lines expressing artificial microRNA constructs (aMIR-SAUR-A or -B) that target a SAUR subfamily (SAUR61-SAUR68 and SAUR75) had slightly reduced hypocotyl and stamen filament elongation. In contrast, transgenic plants expressing SAUR63:GFP or SAUR63:GUS fusions had long hypocotyls, petals and stamen filaments, suggesting that these protein fusions caused a gain of function. SAUR63:GFP and SAUR63:GUS seedlings also accumulated a higher level of basipetally transported auxin in the hypocotyl than did wild-type seedlings, and had wavy hypocotyls and twisted inflorescence stems. Mutations in auxin efflux carriers could partially suppress some SAUR63:GUS phenotypes. In contrast, SAUR63:HA plants had wild-type elongation and auxin transport. SAUR63:GFP protein had a longer half-life than SAUR63:HA. Fluorescence imaging and microsomal fractionation studies revealed that SAUR63:GFP was localized mainly in the plasma membrane, whereas SAUR63:HA was present in both soluble and membrane fractions. Low light conditions increased SAUR63:HA protein turnover rate. These results indicate that membrane-associated Arabidopsis SAUR63 promotes auxin-stimulated organ elongation.     

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H⁺-ATPases (PM H⁺-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H⁺-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H⁺-ATPase function. We find that SAUR19 stimulates PM H⁺-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H⁺-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H⁺-ATPases, and negatively regulate PM H⁺-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion.     

Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

INTRODUCTIONAuxin plays an important role in regu1ating celldivision, e1ongation and differentiatiou, vascular tis-sue fOrmation[1], pollen deve1opment[2] and 1eafyhead fOrmation[3]. Adrin polar transport is be-1ieved to invohe in a variety of important growthand developmenial processes, including the patternfOrmation of eInbryO, leaf morphogenesis and theroot gravity response[4--8]. Auxin po1ar transportinhibitor has been proved essential illterference ofataln transport leading to patte…     

CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants

Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) mediate plant responses to a variety of external stresses. Here we report that Arabidopsis CIPK6 is also required for the growth and development of plants. Phenotype of tobacco plants ectopically expressing a homologous gene ( CaCIPK6 ) from the leguminous plant chickpea ( Cicer arietinum ) indicated its functional conservation. A lesion in AtCIPK6 significantly reduced shoot-to-root and root basipetal auxin transport, and the plants exhibited developmental defects such as fused cotyledons, swollen hypocotyls and compromised lateral root formation, in conjunction with reduced expression of a number of genes involved in auxin transport and abiotic stress response. The Arabidopsis mutant was more sensitive to salt stress compared to wild-type, while overexpression of a constitutively active mutant of CaCIPK6 promoted salt tolerance in transgenic tobacco. Furthermore, tobacco seedlings expressing the constitutively active mutant of CaCIPK6 showed a developed root system, increased basipetal auxin transport and hypersensitivity to auxin. Our results provide evidence for involvement of a CIPK in auxin transport and consequently in root development, as well as in the salt-stress response, by regulating the expression of genes.     

Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport.     

Functional expression and characterization of Arabidopsis ABCB,AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe

Heterologous expression systems based on tobacco BY‐2 cells, Arabidopsis cell cultures, Xenopus oocytes, Saccharomyces cerevisiae, and human HeLa cells have been used to express and characterize PIN, ABCB (PGP), and AUX/LAX auxin transporters from Arabidopsis. However, no single system has been identified that can be used for effective comparative analyses of these proteins. We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway^®‐compatible expression vectors for functional analysis and subcellular localization of recombinant proteins. We expressed Arabidopsis ABCB1 and ABCB19 in mam1pdr1 host lines under the inducible nmt41 promoter. ABCB19 showed a higher ³H‐IAA export activity than ABCB1. Arabidopsis PIN proteins were expressed in a mutant lacking the auxin effluxer like 1 (AEL1) gene. PIN1 showed higher activity than PIN2 with similar protein expression levels. Expression of AUX1 in a permease‐deficient vat3 mutant resulted in increased net auxin uptake activity. Finally, ABCB4 expressed in mam1pdr1 displayed a concentration‐dependent reversal of ³H‐IAA transport that is consistent with its observed activity in planta. Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19, thus providing a rationale for the observed substrate activation. Taken together, these results suggest that the S. pombe system described here can be employed for comparative analyses and subsequent structural characterizations of plant transport proteins.     

Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein

Since auxin was first isolated and characterized as a plant hormone, the underlying molecular mechanism of auxin signaling has been elucidated primarily in dicot plants represented by Arabidopsis. In monocot plants, the molecular mechanism of auxin signaling has remained unclear, despite various physiological experiments. To understand the function and mechanism of auxin signaling in rice ( Oryza sativa ), we focused on the IAA gene, a well-studied gene in Arabidopsis that serves as a negative regulator of auxin signaling. We found 24 IAA gene family members in the rice genome. OsIAA3 is one of these family members whose expression is rapidly increased in response to auxin. We produced transgenic rice harboring m OsIAA3 - GR , which can overproduce mutant OsIAA3 protein containing an amino acid change in domain II to cause a gain-of-function phenotype, by treatment with dexamethasone. The transgenic rice was insensitive to auxin and gravitropic stimuli, and exhibited short leaf blades, reduced crown root formation, and abnormal leaf formation. These results suggest that , in rice, auxin is important for development and its signaling is mediated by IAA genes.     

SAUR proteins and PP2C.D phosphatases regulate H+-ATPases and K+ channels to control stomatal movements

Activation of plasma membrane (PM) H⁺-ATPase activity is crucial in guard cells to promote light-stimulated stomatal opening, and in growing organs to promote cell expansion. In growing organs, SMALL AUXIN UP RNA (SAUR) proteins inhibit the PP2C.D2, PP2C.D5, and PP2C.D6 (PP2C.D2/5/6) phosphatases, thereby preventing dephosphorylation of the penultimate phosphothreonine of PM H⁺-ATPases and trapping them in the activated state to promote cell expansion. To elucidate whether SAUR–PP2C.D regulatory modules also affect reversible cell expansion, we examined stomatal apertures and conductances of Arabidopsis thaliana plants with altered SAUR or PP2C.D activity. Here, we report that the pp2c.d2/5/6 triple knockout mutant plants and plant lines overexpressing SAUR fusion proteins exhibit enhanced stomatal apertures and conductances. Reciprocally, saur56 saur60 double mutants, lacking two SAUR genes normally expressed in guard cells, displayed reduced apertures and conductances, as did plants overexpressing PP2C.D5. Although altered PM H⁺-ATPase activity contributes to these stomatal phenotypes, voltage clamp analysis showed significant changes also in K⁺ channel gating in lines with altered SAUR and PP2C.D function. Together, our findings demonstrate that SAUR and PP2C.D proteins act antagonistically to facilitate stomatal movements through a concerted targeting of both ATP-dependent H⁺ pumping and channel-mediated K⁺ transport.

SMALL AUXIN UP RNA (SAUR) proteins and PP2C.D phosphatases antagonistically regulate stomatal aperture in Arabidopsis by modulating the activities of plasma membrane H⁺-ATPases and K⁺ channels.     

Growth promotion of Chinese cabbage and Arabidopsis by Piriformospora indica is not stimulated by mycelium-synthesized auxin

Piriformospora indica, an endophytic fungus of the order Sebacinales, interacts with the roots of a large variety of plant species. We compared the interaction of this fungus with Chinese cabbage (Brassica campestris subsp. chinensis) and Arabidopsis seedlings. The development of shoots and roots of Chinese cabbage seedlings was strongly promoted by P. indica and the fresh weight of the seedlings increased approximately twofold. The strong stimulation of root hair development resulted in a bushy root phenotype. The auxin level in the infected Chinese cabbage roots was twofold higher compared with the uncolonized controls. Three classes of auxin-related genes, which were upregulated by P. indica in Chinese cabbage roots, were isolated from a double-subtractive expressed sequence tag library: genes for proteins related to cell wall acidification, intercellular auxin transport carrier proteins such as AUX1, and auxin signal proteins. Overexpression of B. campestris BcAUX1 in Arabidopsis strongly promoted growth and biomass production of Arabidopsis seedlings and plants; the roots were highly branched but not bushy when compared with colonized Chinese cabbage roots. This suggests that BcAUX1 is a target of P. indica in Chinese cabbage. P. indica also promoted growth of Arabidopsis seedlings but the auxin levels were not higher and auxin genes were not upregulated, implying that auxin signaling is a more important target of P. indica in Chinese cabbage than in Arabidopsis. The fungus also stimulated growth of Arabidopsis aux1 and aux1/axr4 and rhd6 seedlings. Furthermore, a component in an exudate fraction from P. indica but not auxin stimulated growth of Chinese cabbage and Arabidopsis seedlings. We propose that activation of auxin biosynthesis and signaling in the roots might be the cause for the P. indica-mediated growth phenotype in Chinese cabbage.     

葡萄SAUR基因家族鉴定与生物信息学分析

为了研究葡萄早期应答生长素基因SAUR(Small auxin-up RNA)家族,本研究利用全基因组信息鉴定了葡萄64个SAUR家族成员,并对SAUR家族成员的基因结构、氨基酸特性、染色体定位、基因进化、基因功能以及组织表达进行分析。结果表明,葡萄全基因组上64个SAUR家族成员在19条染色体中的8条染色体上呈现簇状分布,主要分布在3、4号染色体上,其中3号染色体上数量最多为37个;葡萄SAUR家族基因长度较短,有59个基因是无内含子基因;蛋白理化特征分析显示,多数SAUR蛋白呈碱性,结构稳定性较差,蛋白脂溶指数高,呈亲水性;基因功能预测结果表明,葡萄SAUR基因主要担当生长因子、结构蛋白、转录、转录调控以及响应胁迫应答和免疫应答6种功能,其中更多参与生长调节功能;根据系统进化分析将其分为10个分支,另外不同组织表达谱的分析结果表明SAUR基因家族成员具有不同的组织表达模式,对于非生物胁迫具有一定的调节作用。这些信息为葡萄SAUR基因家族功能分析奠定了一定的工作基础。     

Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP-glucose:thiohydroximate S-glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low Km values for phenylacetothiohydroximic acid (approximately 6 microm) and UDP-glucose (approximately 50 microm) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss-of-function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light-grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole-3-acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.     

Ethylene directs auxin to control root cell expansion

Root morphogenesis is controlled by the regulation of cell division and expansion. We isolated an allele of the eto1 ethylene overproducer as a suppressor of the auxin-resistant mutant ibr5, prompting an examination of crosstalk between the phytohormones auxin and ethylene in control of root epidermal cell elongation and root hair elongation. We examined the interaction of eto1 with mutants that have reduced auxin response or transport and found that ethylene overproduction partially restored auxin responsiveness to these mutants. In addition, we found that the effects of endogenous ethylene on root cell expansion in eto1 seedlings were partially impeded by dampening auxin signaling, and were fully suppressed by blocking auxin influx. These data provide insight into the interaction between these two key plant hormones, and suggest that endogenous ethylene directs auxin to control root cell expansion.