Moon-Soo Soh

REP1, a basic helix-loop-helix protein, is required for a branch pathway of phytochrome A signaling in Arabidopsis.

Phytochromes are primary photoreceptors mediating diverse responses ranging from induction of germination to floral induction in higher plants. We have isolated novel recessive rep1 (reduced phytochrome signaling 1) mutants, which exhibit a long-hypocotyl phenotype only under far-red light but not under red light. Physiological characterization showed that rep1 mutations greatly reduced a subset of phytochrome A–regulated responses, including the inhibition of hypocotyl elongation, cotyledon expansion, modulation of gravitropic growth of hypocotyl, and induction of the CAB (encoding chlorophyll a/b binding protein) gene, without affecting the accumulation of anthocyanin, far-red-preconditioned blocking of greening, induction of germination, and induction of CHS (encoding chalcone synthase) and FNR (encoding ferredoxin-NADP+ oxidoreductase) genes. These results suggest that REP1 is a positive signaling component, functioning in a branch of the phytochrome A signaling pathway. Molecular cloning and characterization of the REP1 gene revealed that it encodes a light-inducible, putative transcription factor containing the basic helix-loop-helix motif.

Overexpression of a mutant basic helix-loop-helix protein HFR1, HFR1-ΔN105, activates a branch pathway of light signaling in arabidopsis

The HFR1, a basic helix-loop-helix protein, is required for a subset of phytochrome A-mediated photoresponses in Arabidopsis. Here, we show that overexpression of the HFR1-ΔN105 mutant, which lacks the N-terminal 105 amino acids, confers exaggerated photoresponses even in darkness. Physiological analysis implied that overexpression of HFR1-ΔN105 activated constitutively a branch pathway of light signaling that mediates a subset of photomorphogenic responses, including germination, de-etiolation, gravitropic hypocotyl growth, blocking of greening, and expression of some light-regulated genes such as CAB, DRT112, PSAE, PSBL, PORA, and XTR7, without affecting the light-responsiveness of anthocyanin accumulation and expression of other light-regulated genes such as CHS and PSBS. Although the end-of-day far-red light response and petiole elongation were suppressed in the HFR1-ΔN105-overexpressing plants, flowering time was not affected by HFR1-ΔN105. In addition, the HFR1-ΔN105-overexpressing plants showed hypersensitive photoresponses in the inhibition of hypocotyl elongation, dependently on phytochrome A, FHY1, and FHY3 under FR light or phyB under R light, respectively. Moreover, our double mutant analysis suggested that the hypersensitive photoresponse is due to functional cooperation between HFR1-ΔN105 and other light-signaling components including HY5, a basic leucine zipper protein. Taken together, our results of gain-of-function approach with HFR1-ΔN105 suggest the existence of a complex and important basic helix-loop-helix protein-mediated transcriptional network controlling a branch pathway of light signaling and provide a useful framework for further genetic dissection of light-signaling network in Arabidopsis.

Genetic identification of ACC-RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana.

1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana.

Evaluation of rice promoters conferring pollen-specific expression in a heterologous system, Arabidopsis

Promoters can direct gene expression specifically to targeted tissues or cells. Effective with both crop species and model plant systems, these tools can help researchers overcome the practical obstacles associated with transgenic protocols. Here, we identified promoters that allow one to target the manipulation of gene expression during pollen development. Utilizing published transcriptomic databases for rice, we investigated the promoter activity of selected genes in Arabidopsis. From various microarray datasets, including those for anthers and pollen grains at different developmental stages, we selected nine candidate genes that showed high levels of expression in the late stages of rice pollen development. We named these Oryza sativa late pollen-specific genes. Their promoter regions contained various cis-acting elements that could be responsible for anther-/pollen-specific expression. Promoter::GUS–GFP reporters were constructed and introduced into Arabidopsis plants. Histochemical GUS staining revealed that six of the nine rice promoters conferred strong GUS expression that was restricted to the anthers in Arabidopsis. Further analysis showed that although the GUS signals were not detected at the unicellular stage, they strengthened in the bicellular or tricellular stages, peaking at the mature pollen stage. This paralleled their transcriptomic profiles in rice. Based on our results, we proposed that these six rice promoters, which are active in the late stages of pollen formation in the dicot Arabidopsis, can aid molecular breeders in generating new varieties of a monocot plant, rice.

Genetic Identification of a Second Site Modifier of ctr1-1 that Controls Ethylene-Responsive and Gravitropic Root Growth in Arabidopsis thaliana

Ethylene controls myriad aspects of plant growth throughout developmental stages in higher plants. It has been well established that ethylene-responsive growth entails extensive crosstalk with other plant hormones, particularly auxin. Here, we report a genetic mutation, named 1-aminocyclopropane carboxylic acid (ACC) resistant root1-1 (are1-1) in Arabidopsis thaliana (L.) Heynh. The CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) encodes a Raf-related protein, functioning as an upstream negative regulator of ethylene signaling in Arabidopsis thaliana. We found that the ctr1-1, a kinase-inactive allele exhibited slightly, but significantly, longer root length, compared to ACC-treated wild-type or ctr1-3, a null allele. Our genetic studies unveiled the existence of are1-1 mutation in the ctr1-1 mutant, as a second-site modifier which confers root-specific ethylene-resistance. Based on well-characterized crosstalk between ethylene and auxin during ethylene-responsive root growth, we performed various physiological analyses. Whereas are1-1 displayed normal sensitivity to synthetic auxins, it showed modest resistance to an auxin transport inhibitor, 1-Nnaphthylphthalamic acid. In addition, are1-1 mutant exhibited ectopically altered DR5:GUS activity upon ethylenetreatment. The results implicated the involvement of are1-1 in auxin-distribution, but not in auxin-biosynthesis, -uptake, or -sensitivity. In agreement, are1-1 mutant exhibited reduced gravitropic root growth and defective redistribution of DR5:GUS activity upon gravi-stimulation. Taken together with genetic and molecular analysis, our results suggest that ARE1 defines a novel locus to control ethylene-responsive root growth as well as gravitropic root growth presumably through auxin distribution in Arabidopsis thaliana.

Isolation and characterization of a novel mutation that confers gibberellin-sensitive dwarfism inArabidopsis thaliana

Gibberellins (GAs) regulate diverse aspects of plant growth and development. Despite extensive analysis of the GA-metabolic pathway, only a few genes have been identified as regulatory components of GA metabolism. In searching for those genes, we screened and isolated a novel dominant mutant,GA-sensitive dwarf1-1D (gsd1-1D), fromArabidopsis thaliana. This mutant exhibited the characteristic phenotypes of GA-deficient mutants, including semi-dwarfism, dark-green leaves, late-flowering, and reduced fertility. Exogenously applied GA rescued thegsd1-1D mutant phenotypes, implying that this phenomenon was likely due to a reduced level of GA. Likewise, transcripts of GA-responsive genes were affected by thisgsd1-1D mutation, which genetic analysis showed to be semi-dominant and monogenic. Chromosomal mapping of theGSD1 locus indicated that it resides on the middle of Chromosome 3, where no loci related to GA metabolism exist. These results suggest that theGSD1 locus encodes a novel regulatory component controlling the bioactive GA level inA. thaliana.

Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage

Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

Bioengineering of Male Sterility in Rice (Oryza sativa L.)

Male sterility is an important trait for crop breeding program based on heterosis. Recent advances in molecular researches have led to the identification of genes involved in plant reproductive development and understanding the molecular functions of rice male gametophyte including roles of phytohormones in reproduction process. Here, we review the genes required for key aspects of anther/pollen development and conventional methods for the production of hybrid seeds in rice. Finally, we discuss the molecular approaches for the generation of male-sterile lines through the regulation of phytohormonal biosynthesis in reproductive organs.

How plants make and sense changes in their levels of Gibberellin

To cope with constantly changing environments, plants employ versatile mechanisms. Gibberellins (GAs) are a class of well-characterized plant hormones that enable plastic growth and developments in higher plants throughout their life cycles. Several key components of GA metabolism and signaling have now been revealed through elegant molecular genetics analyses powered by genomics information fromArabidopsis and rice. Here, we highlight recent findings concerning the molecular mechanisms by which plants control their bioactive GA levels and sense/respond to changes in gibberellin concentrations.

GA-sensitive dwarf1-1D (gsd1-1D) Defines a New Mutation that Controls Endogenous GA Levels in Arabidopsis

Gibberellin (GA) regulates diverse plant growth and developmental processes. A number of GA inactivation mechanisms and the associated enzyme-encoding genes and transcriptional regulators have been identified in plants. Identification of a novel dominant GA-sensitive mutant of Arabidopsis, GA-sensitive dwarf1-1D (gsd1-1D), exhibiting phenotypes typical of GA-deficient mutants, including semidwarfism and smaller and dark green leaves, has been reported previously. Unlike the severe GA-deficient mutants such as ga1-3, gsd1-1D seeds displayed a normal GA-sensitive germination phenotype. To gain insights into how the gsd1-1D mutation affects GA metabolism, we performed dose-response studies with several GA biosynthetic intermediates. The results implied that the gsd1-1D mutation preferentially affects the activity of the non-13-hydroxylated bioactive GA, GA4. Our gene expression analysis indicated upregulation of GA2ox1 in the gsd1-1D mutant relative to that in the wild-type and GA-deficient ga3ox1/ga3ox2 mutant; however, this does not appear to be the major cause for the phenotype exhibited by gsd1-1D as the basal level of GA2ox1 expression is low. Measurement of endogenous levels of GAs showed a substantial decrease in the amounts of non-13-hydroxylated GA precursors and GA4 and their respective catabolites in gsd1-1D. Although to a lesser extent, the levels of some 13-hydroxylated GA precursors and bioactive GA1 were also reduced by the gsd1-1D mutation. Taken together, our results suggest that GSD1 is involved in the GA inactivation process and thereby regulates the levels of bioactive GAs in Arabidopsis.