The arabinose kinase,ARA1, gene of Arabidopsis is a novel member of the galactose kinase gene family

The arabinose-sensitive ara1-1 mutant of Arabidopsis is deficient in arabinose kinase activity. A candidate for the ARA1 gene, ISA1, has been previously identified through the Arabidopsis genome sequencing initiative. Here we demonstrate that (1) the ARA1 gene coincides with ISA1 in a positional cloning strategy; (2) there are mutations in the ISA1 gene in both the ara1-1 mutant and an intragenic suppressor mutant; and (3) the ara1-1 and suppressor mutant phenotypes can be complemented by the expression of the ISA1 cDNA in transgenic plants. Together these observations confirm that ISA1 is the ARA1 gene. ARA1 is a member of the galactose kinase family of genes and represents a new substrate specificity among this and other families of sugar kinases. A second gene with similarities to members of the galactose kinase gene family has been identified in the EST database. A 1.8 kb cDNA contained an open reading-frame predicted to encode a 496 amino acid polypeptide. The GAL1 cDNA was expressed in a galK mutant of Escherichia coli and in vitro assays of extracts of the strain expressing GAL1 confirmed that the cDNA encodes a galactose kinase activity. Both GAL1 and ARA1 cross-hybridise at low stringency to other sequences suggesting the presence of additional members of the galactose kinase gene family.     

EMBRYONIC FACTOR 31 encodes a tyrosyl-tRNA synthetase that is essential for seed development

Aminoacyl-tRNA synthetases (AARSs) involve the process of catalyzing the ligation of specific amino acids to their cognate tRNAs. Here we identified an Arabidopsis mutant embryonic factor 31 (fac31), its embryos arrested at development from one cell to globular stage. The FAC31 gene was identified by positional cloning and confirmed by a genetic complementation test with two independent T-DNA insertion lines and transgenic rescue with full-length genomic DNA. FAC31 encodes a Tyrosyl-tRNA synthetase and localize to mitochondria and cytoplasm. Fac31 mutants contain a point mutation from CAA to a stop codon TAA which may lead to a truncated protein. The phenotype of fac31 mutants are very similar to the T-DNA insertion lines Salk_016722 and Salk_045570 displayed smaller embryo sac contains only less number of endosperm nucleolus. Genetic analysis showed that the FAC31 gene had no parental effects through the transmission of mutated FAC31 gene by gametes. FAC31 is a high-conserved protein among animals and plants. RT-PCR analysis and promoter-GUS expression showed that it is expressed in nearly all tissues tested, strongly expressed in meristem of seedlings, the primordium of lateral root, young inflorescences, mature pollen, germinated pollen tubes and embryo sacs before heart stage. Our findings suggest that FAC31 is essential for the seed development through regulation the expanding of embryo sac and proliferation of endosperm nucleolus.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

An Auxin-Inducible F-Box Protein CEGENDUO Negatively Regulates Auxin-Mediated Lateral Root Formation in Arabidopsis

Previously, we characterized 92 Arabidopsis genes (AtSFLs) similar to the S-locus F-box genes involved in S-RNase-based self-incompatibility and found that they likely play diverse roles in Arabidopsis. In this study, we investigated the role of one of these genes, CEGENDUO (CEG, AtSFL61), in the lateral root formation. A T-DNA insertion in CEG led to an increased lateral root production, which was complemented by transformation of the wild-type gene. Its downregulation by RNAi also produced more lateral roots in transformed Arabidopsis plants whereas its overexpression generated less lateral roots compared to wild-type, indicating that CEG acts as a negative regulator for the lateral root formation. It was found that CEG was expressed abundantly in vascular tissues of the primary root, but not in newly formed lateral root primordia and the root meristem, and induced by exogenous auxin NAA (α-naphthalene acetic acid). In addition, the ceg mutant was hyposensitive to NAA, IAA (indole-3-acetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), as well as the auxin transport inhibitor TIBA (3,3,5-triiodobenzoic acid), showing that CEG is an auxin-inducible gene. Taken together, our results show that CEG is a novel F-box protein negatively regulating the auxin-mediated lateral root formation in Arabidopsis. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

The axr4 auxin-resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation

To understand the molecular mechanism of auxin action, mutants of Arabidopsis thaliana with altered responses to auxin have been identified and characterized. Here the isolation of two auxin-resistant mutants that define a new locus involved in auxin response, named AXR4, is reported. The axr4 mutations are recessive and map near the ch1 mutation on chromosome 1. Mutant plants are specifically resistant to auxin and defective in root gravitropism. Double mutants between axr4 and the recessive auxin-resistant mutants axr1-3 and aux1-7 were characterized to ascertain possible genetic interactions between the mutations. The roots of the axr4 axr1-3 double mutant plants are less sensitive to auxin, respond more slowly to gravity, and form fewer lateral roots than either parental single mutant. These results suggest that the two mutations have additive or even synergistic effects. The AXR1 and AXR4 gene products may therefore act in separate pathways of auxin response or perhaps perform partially redundant functions in a single pathway. The axr4 aux1-7 double mutant has the same sensitivity to auxin as the aux1-7 mutant but forms far fewer lateral roots than either parental single mutant. The aux1-7 mutation thus appears to be epistatic to axr4 with respect to auxin-resistant root elongation, whereas in lateral root formation, the effects of the two mutations are additive. The complexity of the genetic interactions indicated by these results may reflect differences in the mechanism of auxin action during root elongation and the formation of lateral roots. The AXR4 gene product, along with those of the AXR1 and AUX1 genes, is important for normal auxin sensitivity, gravitropic response in roots and lateral root formation.     

ydk1-D, an auxin-responsive GH3 mutant that is involved in hypocotyl and root elongation

To study the GH3 gene family of Arabidopsis, we investigated a flanking sequence database of Arabidopsis activation-tagged lines. We found a dwarf mutant, named yadokari 1-D (ydk1-D), that had a T-DNA insertion proximal to a GH3 gene. ydk1-D is dominant and has a short hypocotyl not only in light but also in darkness. Moreover, ydk1-D has a short primary root, a reduced lateral root number, and reduced apical dominance. A GH3 gene, named YDK1, was upregulated in ydk1-D, and YDK1 transgenic plants showed the ydk1-D phenotype. YDK1 gene expression was induced by exogenously applied auxin and regulated by auxin-response factor (ARF)7. In addition, YDK1 gene expression was downregulated by blue and far-red (FR) lights. Strong promoter activity of YDK1 was observed in roots and flowers. These results suggest that YDK1 may function as a negative component in auxin signaling by regulating auxin activity.     

Arabidopsis ASA1 Is Important for Jasmonate-Mediated Regulation of Auxin Biosynthesis and Transport during Lateral Root Formation

Plant roots show an impressive degree of plasticity in adapting their branching patterns to ever-changing growth conditions. An important mechanism underlying this adaptation ability is the interaction between hormonal and developmental signals. Here, we analyze the interaction of jasmonate with auxin to regulate lateral root (LR) formation through characterization of an Arabidopsis thaliana mutant, jasmonate-induced defective lateral root1 (jdl1/asa1-1). We demonstrate that, whereas exogenous jasmonate promotes LR formation in wild-type plants, it represses LR formation in jdl1/asa1-1. JDL1 encodes the auxin biosynthetic gene ANTHRANILATE SYNTHASE α1 (ASA1), which is required for jasmonate-induced auxin biosynthesis. Jasmonate elevates local auxin accumulation in the basal meristem of wild-type roots but reduces local auxin accumulation in the basal meristem of mutant roots, suggesting that, in addition to activating ASA1-dependent auxin biosynthesis, jasmonate also affects auxin transport. Indeed, jasmonate modifies the expression of auxin transport genes in an ASA1-dependent manner. We further provide evidence showing that the action mechanism of jasmonate to regulate LR formation through ASA1 differs from that of ethylene. Our results highlight the importance of ASA1 in jasmonate-induced auxin biosynthesis and reveal a role for jasmonate in the attenuation of auxin transport in the root and the fine-tuning of local auxin distribution in the root basal meristem.     

OsRPK1, a novel leucine-rich repeat receptor-like kinase,negatively regulates polar auxin transport and root development in rice

Background

Leucine-rich-repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of putative RLKs in plants. Although several members in this subfamily have been identified, the studies about the relationships between LRR-RLKs and root development are still few. We previously identified a novel LRR-RLK in rice roots, and named it OsRPK1.

Methods

In this study, we first detected OsRPK1 kinase activity in vitro, and assessed its expression profile. We then investigated its biological function using transgenic rice plants over- and under-expressing OsRPK1.

Results

The OsRPK1 gene, which encodes a Ca^2 +-independent Ser/Thr kinase, was predominantly expressed in root tips, leaf blades, and undifferentiated suspension cells, and was markedly induced by treatment with auxin or ABA. Knockdown of OsRPK1 promoted the growth of transgenic rice plants, and increased plant height and tiller numbers. In contrast, over-expressing plants showed undeveloped adventitious roots, lateral roots, and a reduced root apical meristem. OsRPK1 over-expression also inhibited the expression of most auxin efflux carrier OsPIN genes, which was accompanied by changes in PAT and endogenous free IAA distribution in the leaves and roots.

Conclusions

The data indicated that OsRPK1, a novel leucine-rich-repeat receptor-like kinase, affects the root system architecture by negatively regulating polar auxin transport in rice.

General significance

This study demonstrated a common regulatory pathway of root system development in higher plants, which might be initiated by external stimuli via upstream receptor-like kinases and downstream carriers for polar auxin transport.     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

VLN2 Regulates Plant Architecture by Affecting Microfilament Dynamics and Polar Auxin Transport in Rice

As a fundamental and dynamic cytoskeleton network, microfilaments (MFs) are regulated by diverse actin binding proteins (ABPs). Villins are one type of ABPs belonging to the villin/gelsolin superfamily, and their function is poorly understood in monocotyledonous plants. Here, we report the isolation and characterization of a rice (Oryza sativa) mutant defective in VILLIN2 (VLN2), which exhibits malformed organs, including twisted roots and shoots at the seedling stage. Cellular examination revealed that the twisted phenotype of the vln2 mutant is mainly caused by asymmetrical expansion of cells on the opposite sides of an organ. VLN2 is preferentially expressed in growing tissues, consistent with a role in regulating cell expansion in developing organs. Biochemically, VLN2 exhibits conserved actin filament bundling, severing and capping activities in vitro, with bundling and stabilizing activity being confirmed in vivo. In line with these findings, the vln2 mutant plants exhibit a more dynamic actin cytoskeleton network than the wild type. We show that vln2 mutant plants exhibit a hypersensitive gravitropic response, faster recycling of PIN2 (an auxin efflux carrier), and altered auxin distribution. Together, our results demonstrate that VLN2 plays an important role in regulating plant architecture by modulating MF dynamics, recycling of PIN2, and polar auxin transport.     

Ribosome biogenesis factor OLI2 and its interactor BRX1-2 are associated with morphogenesis and lifespan extension in Arabidopsis thaliana

Mutations that reduce the expression of ribosomal proteins (RPs) or limit the activity of ribosome biogenesis-related factors frequently cause physiological and morphological changes in Arabidopsis. Arabidopsis OLI2/NOP2A, a homolog of yeast Nop2, encodes a nucleolar methyltransferase that is required for the maturation of the 25S ribosomal RNA of the 60S large ribosomal subunit. Mutant oli2 plants exhibit pointed leaves and shortened primary roots. In this study, detailed phenotypic analysis of oli2 mutant and OLI2 overexpressor lines revealed a range of phenotypes. Seeds produced by oli2 mutant and OLI2 overexpressor plants were lighter and heavier than wild-type seeds, respectively. Seeds of the oli2 mutant also showed delayed germination, whereas seeds from the OLI2 overexpressor lines germinated earlier than the wild type. The oli2 mutant also had fewer and shorter lateral roots than the wild type. The lateral root development phenotype in the oli2 mutant was similar to that of auxin-related mutants, but was not enhanced by exogenously supplied auxin. Furthermore, the oli2 mutant and OLI2 overexpressor lines were hypersensitive and less sensitive to high concentrations of sugar, respectively. Split-GFP-based bimolecular fluorescence complementation analysis revealed that OLI2 interacted with a nucleolar protein, BRX1-2, which is involved in rRNA processing for the large ribosomal subunit. Moreover, overexpression of OLI2 and BRX1-2 caused similar morphological changes, including extension of plant lifespans. These results suggest that the functions of OLI2 and its interactor BRX1-2 are intimately associated with a range of developmental events in Arabidopsis.     

Disturbed Local Auxin Homeostasis Enhances Cellular Anisotropy and Reveals Alternative Wiring of Auxin-ethylene Crosstalk in Brachypodium distachyon Seminal Roots

Observations gained from model organisms are essential, yet it remains unclear to which degree they are applicable to distant relatives. For example, in the dicotyledon Arabidopsis thaliana (Arabidopsis), auxin biosynthesis via indole-3-pyruvic acid (IPA) is essential for root development and requires redundant TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and TAA1-RELATED (TAR) genes. A promoter T-DNA insertion in the monocotyledon Brachypodium distachyon (Brachypodium) TAR2-LIKE gene (BdTAR2L) severely down-regulates expression, suggesting reduced tryptophan aminotransferase activity in this mutant, which thus represents a hypomorphic Bdtar2l allele (Bdtar2l^hypo). Counterintuitive however, Bdtar2l^hypo mutants display dramatically elongated seminal roots because of enhanced cell elongation. This phenotype is also observed in another, stronger Bdtar2l allele and can be mimicked by treating wild type with L-kynerunine, a specific TAA1/TAR inhibitor. Surprisingly, L-kynerunine-treated as well as Bdtar2l roots display elevated rather than reduced auxin levels. This does not appear to result from compensation by alternative auxin biosynthesis pathways. Rather, expression of YUCCA genes, which are rate-limiting for conversion of IPA to auxin, is increased in Bdtar2l mutants. Consistent with suppression of Bdtar2l^hypo root phenotypes upon application of the ethylene precursor 1-aminocyclopropane-1-carboxylic-acid (ACC), BdYUCCA genes are down-regulated upon ACC treatment. Moreover, they are up-regulated in a downstream ethylene-signaling component homolog mutant, Bd ethylene insensitive 2-like 1, which also displays a Bdtar2l root phenotype. In summary, Bdtar2l phenotypes contrast with gradually reduced root growth and auxin levels described for Arabidopsis taa1/tar mutants. This could be explained if in Brachypodium, ethylene inhibits the rate-limiting step of auxin biosynthesis in an IPA-dependent manner to confer auxin levels that are sub-optimal for root cell elongation, as suggested by our observations. Thus, our results reveal a delicate homeostasis of local auxin and ethylene activity to control cell elongation in Brachypodium roots and suggest alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis.