Yoshibumi Komeda

Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis.

In higher plants, the outermost cell layer (L1) of the shoot apex gives rise to the epidermis of shoot organs. Our previous study demonstrated that an 8-bp motif named the L1 box functions as a cis-regulatory element for L1-specific gene expression in the shoot system of Arabidopsis. We show here that PROTODERMAL FACTOR2 (PDF2), a member of the HD-GL2 class of homeobox genes, is expressed exclusively in the L1 of shoot meristems and that recombinant PDF2 protein specifically binds to the L1 box in vitro. Although knockout mutants of PDF2 and ATML1, another L1-specific HD-GL2 class gene sharing the highest homology with PDF2, display normal shoot development, the double mutant results in severe defects in shoot epidermal cell differentiation. This suggests that PDF2 and ATML1 are functionally interchangeable and play a critical role in maintaining the identity of L1 cells, possibly by interacting with their L1 box and those of downstream target-gene promoters.

Spermidine Synthase Genes Are Essential for Survival of Arabidopsis

The cellular polyamines putrescine, spermidine, and spermine are ubiquitous in nature and have been implicated in a wide range of growth and developmental processes. There is little information, however, on mutant plants or animals defective in the synthesis of polyamines. The Arabidopsis genome has two genes encoding spermidine synthase, SPDS1 and SPDS2. In this paper, we describe T-DNA insertion mutants of both of these genes. While each mutant allele shows normal growth, spds1-1 spds2-1 double-mutant seeds are abnormally shrunken and they have embryos that are arrested morphologically at the heart-torpedo transition stage. These seeds contain significantly reduced levels of spermidine and high levels of its precursor, putrescine. The embryo lethal phenotype of spds1-1 spds2-1 is complemented by the wild-type SPDS1 gene. In addition, we observed a nearly identical seed phenotype among an F2 seed population from the cross between the spds2-1 allele and SPDS1 RNA interference transgenic lines. These data provide the first genetic evidence indicating a critical role of the spermidine synthase in plant embryo development.

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.

Isolation of an Arabidopsis thaliana Mutant, mto1, That Overaccumulates Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation).

We isolated Arabidopsis thaliana mutants that are resistant to ethionine, a toxic analog of methionine (Met). One of the mutants was analyzed further, and it accumulated 10- to 40-fold more soluble Met than the wild type in the aerial parts during the vegetative growth period. When the mutant plants started to flower, however, the soluble Met content in the rosette region decreased to the wild-type level, whereas that in the inflorescence apex region and in immature fruits was 5- to 8-fold higher than the wild type. These results indicate that the concentration of soluble Met is temporally and spatially regulated and suggest that soluble Met is translocated to sink organs after the onset of reproductive growth. The causal mutation, designated mto1, was a single, nuclear, semidominant mutation and mapped to chromosome 3. Accumulation profiles of soluble amino acids suggested that the mutation affects a later step(s) in the Met biosynthesis pathway. Ethylene production of the mutants was only 40% higher than the wild-type plants, indicating that ethylene production is tightly regulated at a step after Met synthesis. This mutant will be useful in studying the translocation of amino acids, as well as regulation of Met biosynthesis and other metabolic pathways related to Met.

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.

Characterization of two genes encoding small heat-shock proteins in Arabidopsis thaliana

SummaryUsing the technique of differential hybridization screening, we have isolated the cDNAs for two low-molecular-mass heat-shock proteins and their corresponding genes, HSP17.4 and HSP18.2, from Arabidopsis thaliana. These two genes encode polypeptides that are 79.2% identical to each other with respect to amino acid sequence, and contain several overlapping sequences that are similar to the consensus sequences for the heat-shock elements (HSE) in Drosophila in the regions upstream from the promoters. The 5′ region of the HSP18.2 gene has been fused, in frame, to the uidA gene from Escherichia coli which encodes β-glucuronidase (GUS), and the product has been introduced into petunia by Agrobacterium-mediated transformation. We have demonstrated that the GUS activity in transformed petunia plants is enhanced by heat shock.

Characterization of the spermidine synthase-related gene family in Arabidopsis thaliana

The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase‐deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells.

Expression of a Soybean (Glycine max [L.] Merr.) Seed Storage Protein Gene in Transgenic Arabidopsis thaliana and Its Response to Nutritional Stress and to Abscisic Acid Mutations

Among the three subunits of [beta]-conglycinin, the 7S seed storage protein of soybean (Glycine max [L.] Merr.), expression of the [beta] subunit gene is unique. Accumulation of the [beta] subunit is enhanced in sulfate-deficient soybean plants, and its mRNA levels increase when abscisic acid (ABA) is added to the in vitro cotyledon culture medium. Transgenic Arabidopsis thaliana lines carrying a gene encoding the [beta] subunit was constructed and grown under sulfate deficiency. Accumulation of both [beta] subunit mRNA and protein were enhanced in developing A. thaliana seeds. Accumulation of one of the A. thaliana seed storage protein mRNAs was also enhanced by sulfate deficiency, although the response was weaker than that observed for the soybean [beta] subunit mRNA. When the aba1–1 or abi3–1 mutations were crossed into the transgenic A. thaliana line, accumulation of the [beta] subunit was significantly reduced, whereas accumulation of the A. thaliana seed storage protein was not greatly affected. These results indicate that soybean and A. thaliana share a common mechanism for response to sulfate deficiency and to ABA, although the sensitivity is different between the species. The transgenic A. thaliana carrying the [beta] subunit gene of [beta]-conglycinin will be a good system to analyze these responses.

The role of cAMP in flagellation of Salmonella typhimurium.

SummaryA mutational alteration either in adenylate cyclase (cya-) or in cyclic-3′5′-AMP (cAMP) receptor protein (crp-) rendered Salmonella typhimurium incapable of producing flagella. The amount of mRNA specific for flagellin in these mutants was almost negligible when assayed in an in vitro protein synthesizing system. A secondary mutation, cfs, partially suppressing the cya- mutation, was identified among the revertants of cya-. A mutation in the same cistron as cfs resulted in a non-flagellate phenotype either by itself or in combination with cfs. The cistron, which was given the gene symbol flaT, was located between flaE and flaL. It was suggested that cAMP receptor protein together with cAMP modulates the gene flaT, which in turn acts as a positive effector on the synthesis of active mRNA specific for flagellin.

The Arabidopsis OBERON1 and OBERON2 genes encode plant homeodomain finger proteins and are required for apical meristem maintenance.

Maintenance of the stem cell population located at the apical meristems is essential for repetitive organ initiation during the development of higher plants. Here, we have characterized the roles of OBERON1 (OBE1) and its paralog OBERON2 (OBE2), which encode plant homeodomain finger proteins, in the maintenance and/or establishment of the meristems in Arabidopsis. Although the obe1 and obe2 single mutants were indistinguishable from wild-type plants, the obe1 obe2 double mutant displayed premature termination of the shoot meristem, suggesting that OBE1 and OBE2 function redundantly. Further analyses revealed that OBE1 and OBE2 allow the plant cells to acquire meristematic activity via the WUSCHEL-CLAVATA pathway, which is required for the maintenance of the stem cell population, and they function parallel to the SHOOT MERISTEMLESS gene, which is required for preventing cell differentiation in the shoot meristem. In addition, obe1 obe2 mutants failed to establish the root apical meristem, lacking both the initial cells and the quiescent center. In situ hybridization revealed that expression of PLETHORA and SCARECROW, which are required for stem cell specification and maintenance in the root meristem, was lost from obe1 obe2 mutant embryos. Taken together, these data suggest that the OBE1 and OBE2 genes are functionally redundant and crucial for the maintenance and/or establishment of both the shoot and root meristems.