Kotaro T. Yamamoto

The NPH4 Locus Encodes the Auxin Response Factor ARF7, a Conditional Regulator of Differential Growth in Aerial Arabidopsis Tissue

Organ bending through differential growth represents a major mechanism by which plants are able to adaptively alter their morphology in response to local changes in the environment. Two plant hormones, auxin and ethylene, have been implicated as regulators of differential growth responses; however, the mechanisms by which they elicit their effects remain largely unknown. Here, we describe isolation of the NPH4 gene of Arabidopsis, which is conditionally required for differential growth responses of aerial tissues, and we report that NPH4 encodes the auxin-regulated transcriptional activator ARF7. The phenotypes of nph4 mutants, which include multiple differential growth defects associated with reduced auxin responsiveness, including impaired auxin-induced gene expression, are consistent with the predicted loss of function of a transcriptional activator, and these phenotypes indicate that auxin-dependent changes in gene transcription are prerequisite for proper organ bending responses. Although NPH4/ARF7 appears to be a major regulator of differential growth, it is not the sole regulator because phenotypes of nph4 null mutants were suppressed by application of ethylene. This latter finding illustrates the intimate connection between auxin and ethylene in the control of growth in higher plants.

Characterization of the Class IV Homeodomain-Leucine Zipper Gene Family in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome contains 16 genes belonging to the class IV homeodomain-Leucine zipper gene family. These include GLABRA2, ANTHOCYANINLESS2, FWA, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), and PROTODERMAL FACTOR2 (PDF2). Our previous study revealed that atml1 pdf2 double mutants have severe defects in the shoot epidermal cell differentiation. Here, we have characterized additional members of this gene family, which we designated HOMEODOMAIN GLABROUS1 (HDG1) through HDG12. Analyses of transgenic Arabidopsis plants carrying the gene-specific promoter fused to the bacterial β-glucuronidase reporter gene revealed that some of the promoters have high activities in the epidermal layer of the shoot apical meristem and developing shoot organs, while others are temporarily active during reproductive organ development. Expression profiles of highly conserved paralogous gene pairs within the family were found to be not necessarily overlapping. Analyses of T-DNA insertion mutants of these HDG genes revealed that all mutants except hdg11 alleles exhibit no abnormal phenotypes. hdg11 mutants show excess branching of the trichome. This phenotype is enhanced in hdg11 hdg12 double mutants. Double mutants were constructed for other paralogous gene pairs and genes within the same subfamily. However, novel phenotypes were observed only for hdg3 atml1 and hdg3 pdf2 mutants that both exhibited defects in cotyledon development. These observations suggest that some of the class IV homeodomain-Leucine zipper members act redundantly with other members of the family during various aspects of cell differentiation. DNA-binding sites were determined for two of the family members using polymerase chain reaction-assisted DNA selection from random oligonucleotides with their recombinant proteins. The binding sites were found to be similar to those previously identified for ATML1 and PDF2, which correspond to the pseudopalindromic sequence 5′-GCATTAAATGC-3′ as the preferential binding site.

A methyl viologen-resistant mutant of Arabidopsis, which is allelic to ozone-sensitive rcd1, is tolerant to supplemental ultraviolet-B irradiation.

To better understand the role of active oxygen species (AOS) in acquired resistance to increased levels of ultraviolet (UV)-B irradiation in plants, we isolated an Arabidopsis mutant that is resistant to methyl viologen, and its sensitivity to UV-B was investigated. A complementation test revealed that the obtained mutant was allelic to the ozone-sensitive radical-induced cell death1-1 (rcd1-1). Therefore, this mutant was named rcd1-2. rcd1-2 was recessive and nearly 4-fold more resistant to methyl viologen than wild type. It exhibited a higher tolerance to short-term UV-B supplementation treatments than the wild type: UV-B-induced formation of cyclobutane pyrimidine dimers was reduced by one-half after 24 h of exposure; the decrease in quantum yield of photosystem II was also diminished by 40% after 12 h of treatment. Furthermore, rcd1-2 was tolerant to freezing. Steady-state mRNA levels of plastidic Cu/Zn superoxide dismutase and stromal ascorbate peroxidase were higher in rcd1-2 than in wild type, and the mRNA level of the latter enzyme was enhanced by UV-B exposure more effectively in rcd1-2. UV-B-absorbing compounds were more accumulated in rcd1-2 than in wild type after UV-B exposure for 24 h. These findings suggest that rcd1-2 methyl viologen resistance is due to the enhanced activities of the AOS-scavenging enzymes in chloroplasts and that the acquired tolerance to the short-term UV-B exposure results from a higher accumulation of sunscreen pigments. rcd1 appears to be a mutant that constitutively shows stress responses, leading to accumulation of more pigments and AOS-scavenging enzymes without any stresses.

The Arabidopsis STV1 Protein, Responsible for Translation Reinitiation, Is Required for Auxin-Mediated Gynoecium Patterning

Ribosomal protein L24 (RPL24) is implicated in translation reinitiation of polycistronic genes. A newly isolated Arabidopsis thaliana short valve1 (stv1) mutant, in which one of the RPL24-encoding genes, RPL24B, is deleted, shows specific defects in the apical-basal patterning of the gynoecium, in addition to phenotypes induced by ribosome deficiency. A similar gynoecium phenotype is caused by mutations in the auxin response factor (ARF) genes ETTIN (ETT) and MONOPTEROS (MP), which have upstream open reading frames (uORFs) in their 5′-transcript leader sequences. Gynoecia of a double mutant of stv1 and a weak ett mutant allele are similar to those of a strong ett allele, and transformation with a uORF-eliminated ETT construct partially suppressed the stv1 gynoecium phenotype, implying that STV1 could influence ETT translation through its uORFs. Analyses of 5′-leader-reporter gene fusions showed that the uORFs of ETT and MP negatively regulate the translation of the downstream major ORFs, indicating that translation reinitiation is an important step for the expression of these proteins. Taken together, we propose that perturbation of translation reinitiation of the ARF transcripts causes the defects in gynoecium patterning observed in the stv1 mutant.

Spermine is not essential for survival of Arabidopsis

Spermine is the final product of the polyamine biosynthetic pathway and is ubiquitously present in most organisms. The genome of Arabidopsis thaliana has two genes encoding spermine synthase: ACAULIS5 (ACL5), whose loss‐of‐function mutants show a severe defect in stem elongation, and SPMS. In order to elucidate the function of spermine in plants, we isolated a T‐DNA insertion mutant of the SPMS gene. Free and conjugated spermine levels in the mutant, designated spms‐1, were significantly decreased compared with those in the wild‐type, but no obvious morphological phenotype was observed in spms‐1 plants. We further confirmed that acl5‐1 spms‐1 double mutants contained no spermine. Surprisingly, acl5‐1 spms‐1 was fully as viable as the wild‐type and showed no phenotype except for the reduced stem growth due to acl5‐1. These results indicate that spermine is not essential for survival of Arabidopsis, at least under normal growth conditions.

The dwarf phenotype of the Arabidopsis acl5 mutant is suppressed by a mutation in an upstream ORF of a bHLH gene

Loss-of-function mutants of the Arabidopsis thaliana ACAULIS 5 (ACL5) gene, which encodes spermine synthase, exhibit a severe dwarf phenotype. To elucidate the ACL5-mediated regulatory pathways of stem internode elongation, we isolated four suppressor of acaulis (sac) mutants that reverse the acl5 dwarf phenotype. Because these mutants do not rescue the dwarfism of known phytohormone-related mutants, the SAC genes appear to act specifically on the ACL5 pathways. We identify the gene responsible for the dominant sac51-d mutant, which almost completely suppresses the acl5 phenotype. sac51-d disrupts a short upstream open reading frame (uORF) of SAC51, which encodes a bHLH-type transcription factor. Our results indicate that premature termination of the uORF in sac51-d results in an increase in its own transcript level, probably as a result of an increased translation of the main ORF. We suggest a model in which ACL5 plays a role in the translational activation of SAC51, which may lead to the expression of a subset of genes required for stem elongation.

Arabidopsis AUXIN RESPONSE FACTOR6 and 8 Regulate Jasmonic Acid Biosynthesis and Floral Organ Development via Repression of Class 1 KNOX Genes

Two mutations in Arabidopsis thaliana, auxin response factor6 (arf6) and arf8, concomitantly delayed the elongation of floral organs and subsequently delayed the opening of flower buds. This phenotype is shared with the jasmonic acid (JA)-deficient mutant dad1, and, indeed, the JA level of arf6 arf8 flower buds was decreased. Among JA biosynthetic genes, the expression level of DAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) was markedly decreased in the double mutant, suggesting that ARF6 and ARF8 are required for activation of DAD1 expression. The double mutant arf6 arf8 also showed other developmental defects in flowers, such as aberrant vascular patterning and lack of epidermal cell differentiation in petals. We found that class 1 KNOX genes were expressed ectopically in the developing floral organs of arf6 arf8, and mutations in any of the class 1 KNOX genes (knat2, knat6, bp and hemizygous stm) partially suppressed the defects in the double mutant. Furthermore, ectopic expression of the STM gene caused a phenotype similar to that of arf6 arf8, including the down-regulation of DAD1 expression. These results suggested that most defects in arf6 arf8 are attributable to abnormal expression of class 1 KNOX genes. The expression of AS1 and AS2 was not affected in arf6 arf8 flowers, and as1 and arf6 arf8 additively increased the expression of class 1 KNOX genes. We concluded that ARF6 and ARF8, in parallel with AS1 and AS2, repress the class 1 KNOX genes in developing floral organs to allow progression of the development of these organs.

The massugu1 Mutation of Arabidopsis Identified with Failure of Auxin-Induced Growth Curvature of Hypocotyl Confers Auxin Insensitivity to Hypocotyl and Leaf

Unilateral application of indole-3-acetic acid (IAA) in a lanolin base to hypocotyls of partially etiolated seedlings of wild-type Arabidopsis thaliana induced growth curvature in a dose-dependent manner. The effects of IAA in concentrations from 1 to 1000 [mu]M were studied, with maximum IAA-induced curvature at 100 [mu]M. Three IAA-insensitive mutants were isolated and are all in the same locus, massugu1 (msg1). They did not undergo hypocotyl growth curvature at any of the IAA concentrations tested. msg1 is recessive and is located on chromosome 5. msg1 hypocotyl growth is resistant to 2,4-dichlorophenoxyacetic acid (2,4-D), but the roots are as sensitive to 2,4-D as the wild type. Growth of the hypocotyl was inhibited to essentially the same extent as the wild type by 6-benzylaminopurine, abscisic acid, and l-aminocyclopropane-1-carboxylate, an ethylene precursor. The msg1 leaves were also resistant to 2,4-D-induced chlorosis. The gravitropic response of the msg1 hypocotyl takes much more time to initiate and achieve the wild-type degree of curvature, whereas the msg1 roots responded normally to gravity. The mature plants and the etiolated seedlings of msg1 were generally wild type in appearance, except that their rosette leaves were either epinastic or hyponastic. msg1 is the first auxin-insensitive mutant in which its effects are mostly restricted to the hypocotyl and leaf, and msg1 also appears to be auxin specific.

Specificity and Similarity of Functions of the Aux/IAA Genes in Auxin Signaling of Arabidopsis Revealed by Promoter-Exchange Experiments among MSG2/IAA19, AXR2/IAA7, and SLR/IAA14

As indicated by various and some overlapped phenotypes of the dominant mutants, the Aux/IAA genes of Arabidopsis (Arabidopsis thaliana) concomitantly exhibit a functional similarity and differentiation. To evaluate the contributions of their expression patterns determined by promoter activity and molecular properties of their gene products to Aux/IAA function, we examined phenotypes of transgenic plants expressing the green fluorescent protein (GFP)-tagged msg2-1/iaa19, axr2-1/iaa7, or slr-1/iaa14 cDNA by the MSG2 or AXR2 promoter. When driven by the MSG2 promoter (pMSG2), each GFP-tagged cDNA caused the msg2-1 phenotype, that is, the wild-type stature in the mature-plant stage, long and straight hypocotyls in the dark, reduced lateral root formation, relatively mild agravitropic traits in hypocotyls, and a normal gravitropic response in roots. However, development of one or two cotyledonary primordia was often arrested in embryogenesis of the pMSG2∷axr2-1∷GFP and pMSG2∷slr-1∷GFP plants, resulting in monocotyledonary or no cotyledonary seedlings. Such defects in embryogenesis were never seen in pMSG2∷msg2-1∷GFP or the msg2-1, axr2-1, or slr-1 mutant. The MSG2 promoter-GUS staining showed that expression of MSG2 started specifically in cotyledonary primordia of the triangular-stage embryos. When driven by the AXR2 promoter (pAXR2), each GFP-tagged mutant cDNA caused, in principle, aberrant aboveground phenotypes of the corresponding dominant mutant. However, either the axr2-1∷GFP or slr-1∷GFP cDNA brought about dwarf, agravitropic stems almost identical to those of axr2-1, and the pAXR2∷msg2-1∷GFP and pAXR2∷slr-1∷GFP hypocotyls exhibited complete loss of gravitropism as did axr2-1. These results showed functional differences among the msg2-1, axr2-1, and slr-1 proteins, though some phenotypes were determined by the promoter activity.

Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis

Degradation of Aux/IAA proteins which are triggered by the ubiquitin ligase complex containing the auxin F-box receptors (AFBs), is thought to be the primary reaction of auxin signaling. Upon auxin perception, AFBs bind domain II of Aux/IAA proteins that is conserved in most of the 29 family members in Arabidopsis. However, IAA20 and IAA30 lack domain II. Furthermore, IAA31, which forms a single clade with IAA20 and IAA30 in Aux/IAA protein family, has a partially conserved domain II, which contains an amino acid substitution that would cause a dominant mutation of Aux/IAA genes. It has been shown that the half-lives of these proteins are much longer than those of the canonical Aux/IAA proteins. We generated overexpression lines (OXs) of IAA20, IAA30 and IAA31 by the use of cauliflower mosaic virus 35S promoter to better understand the molecular function of atypical Aux/IAA proteins in Arabidopsis. OXs of the three genes exhibited similar auxin-related aberrant phenotypes, with IAA20 OX showing the most severe defects: Some of them showed a semi-dwarf phenotype; gravitropic growth orientation was often affected in hypocotyl and root; vasculature of cotyledons was malformed; the primary root stopped growing soon after germination because of collapse of root apical meristem. IAA 20 and IAA30 were early auxin inducible, but IAA31 was not. These results showed that the wild-type genes of the three Aux/IAAs could disturb auxin physiology when ectopically overexpressed.