Sang-Gu Kim

SUPPRESSOR OF FRIGIDA3 Encodes a Nuclear ACTIN-RELATED PROTEIN6 Required for Floral Repression in Arabidopsis

Flowering traits in winter annual Arabidopsis thaliana are conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FLC acts as a flowering repressor and is regulated by multiple flowering pathways. We isolated an early-flowering mutant, suppressor of FRIGIDA3 (suf3), which also shows leaf serration, weak apical dominance, and infrequent conversion of the inflorescence shoot to a terminal flower. The suf3 mutation caused a decrease in the transcript level of FLC in both a FRI-containing line and autonomous pathway mutants. However, suf3 showed only a partial reduction of FLC transcript level, although it largely suppressed the late-flowering phenotype. In addition, the suf3 mutation caused acceleration of flowering in both 35S-FLC and a flc null mutant, indicating that SUF3 regulates additional factor(s) for the repression of flowering. SUF3 is highly expressed in the shoot apex, but the expression is not regulated by FRI, autonomous pathway genes, or vernalization. SUF3 encodes the nuclear ACTIN-RELATED PROTEIN6 (ARP6), the homolog of which in yeast is a component of an ATP-dependent chromatin-remodeling SWR1 complex. Our analyses showed that SUF3 regulates FLC expression independent of vernalization, FRI, and an autonomous pathway gene, all of which affect the histone modification of FLC chromatin. Subcellular localization using a green fluorescent protein fusion showed that Arabidopsis ARP6 is located at distinct regions of the nuclear periphery.

Distinct Roles of the First Introns on the Expression of Arabidopsis Profilin Gene Family Members

Profilin is a small actin-binding protein that regulates cellular dynamics of the actin cytoskeleton. In Arabidopsis (Arabidopsis thaliana), five profilins were identified. The vegetative class profilins, PRF1, PRF2, and PRF3, are expressed in vegetative organs. The reproductive class profilins, PRF4 and PRF5, are mainly expressed in pollen. In this study, we examined the role of the first intron in the expression of the Arabidopsis profilin gene family using transgenic plants and a transient expression system. In transgenic plants, we examined PRF2 and PRF5, which represent vegetative and reproductive profilins. The expression of the PRF2 promoter fused with the β-glucuronidase (GUS) gene was observed in the vascular bundles, but transgenic plants carrying the PRF2 promoter-GUS with its first intron showed constitutive expression throughout the vegetative tissues. However, the first intron of PRF5 had little effect on the reporter gene expression pattern. Transgenic plants containing PRF5 promoter-GUS fusion with or without its first intron showed reproductive tissue-specific expression. To further investigate the different roles of the first two introns on gene expression, the first introns were exchanged between PRF2 and PRF5. The first intron of PRF5 had no apparent effect on the expression pattern of the PRF2 promoter. But, unlike the intron of PRF5, the first intron of PRF2 greatly affected the reproductive tissue-specific expression of the PRF5 promoter, confirming a different role for these introns. The results of a transient expression assay indicated that the first intron of PRF1 and PRF2 enhances gene expression, whereas PRF4 and PRF5 do not. These results suggest that the first introns of profilin genes are functionally distinctive and the first introns are required for the strong and constitutive gene expression of PRF1 and PRF2 in vegetative tissues.

Brassinosteroid Signals Control Expression of the AXR3/IAA17 Gene in the Cross-Talk Point with Auxin in Root Development

Transgenic plants overexpressing AXR3/IAA17 were impaired in root growth. Specifically, they exhibited severe defects in lateral root and root hair development similar to the root phenotypes of epi-brassinolide (epiBL)-treated wild-type plants. Here, we investigated the involvement of AXR3/IAA17 gene expression in brassinosteroid (BR)-regulated root development. Exogenous epiBL application significantly induced expression of the AXR3/IAA17 gene as well as several Aux/IAA genes, such as AXR2/IAA7, SLR/IAA14, and IAA28. We analyzed the transcription levels of several Aux/IAA genes related to root development in the BR signaling mutant bri1 and the BR biosynthesis mutant det2. AXR3/IAA17 gene expression was significantly decreased in bri1 plants. In det2 plants, expression of AXR3/IAA17 slightly decreased. This in turn suggests that epiBL induced these Aux/IAA genes, and that these induced gene products might function as factors in root development. Furthermore, AXR3/IAA17 might be involved in the BR signaling pathway, suggesting an intersection node of BR-auxin signaling in root development.

Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron.

Actin-depolymerizing factor (ADF) is one of the actin cytoskeleton-modulating proteins. We have characterized the accumulation pattern of petunia ADF proteins. PhADF proteins are accumulated in every petunia organ and their accumulation is differentially regulated by developmental signals. Their cellular localization is vascular tissue-preferential in vegetative organs, whereas somewhat different in reproductive organs. In reproductive organs, PhADFs are present in outer integument, endocarp of ovary wall, transmitting tissue of style, and epidermis and endothecium of young anther. From a petunia genomic library, we have isolated a genomic clone encoding PhADF1. Comparison to complementary DNA sequence revealed that the coding region of PhADF1 gene consists of three exons and two introns. Analysis of chimeric gene expression using beta-glucuronidase as a reporter gene in transgenic Arabidopsis revealed that PhADF1 was strongly expressed in every vegetative tissue except petal. In addition, expression of the gene was highly enhanced by its first intron. These results suggest that PhADF1 gene of petunia is mainly expressed in vascular tissues and its expression is regulated by intron-mediated enhancement mechanism.

Molecular cloning and characterization of the gene encoding osmotin protein in Petunia hybrida

Abstract A cDNA clone encoding osmotin, PhOSM , was isolated from a cDNA library constructed from petal protoplast cultures of Petunia hybrida . PhOSM cDNA was composed of an open reading frame corresponding to protein of 246 amino acids and had a calculated molecular weight of 26.7 kDa. Database comparisons of the PhOSM protein sequences revealed high identity (89%) with the tobacco osmotin and tobacco OLP (Osmotin-Like-Protein). The increased accumulation of PhOSM mRNA in petal protoplast cultures was completely inhibited by treatment with Dhp (3,4-dehydro- l -proline), which is a inhibitor of peptydyl proline hydroxylation in cell wall regeneration. RNA blot analyses revealed that PhOSM was expressed primarily in roots and slightly in the pistil, 3 days after flowering. PhOSM expression was strongly induced in leaves that were exposed to Penicillium funiculosum , Erwinia stewartii and Pseudomonas syringae but not to Aspergillus nidulans . Upon wounding, PhOSM transcripts were induced in the directly damaged leaf but not systemically. Moreover, PhOSM transcript levels increased in response to octadecanoid pathway intermediates and treatments with aspirin and salicylic acid. Our results indicate that PhOSM is developmentally regulated as well as involved in wound-stress signal transduction.

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.

Two closely related cDNAs encoding actin-depolymerizing factors of petunia are mainly expressed in vegetative tissues.

Actin-depolymerizing factor (ADF) is one of the small actin-binding proteins that regulate actin dynamics in cells. We have isolated two cDNA clones, PhADF1 and PhADF2, encoding ADF from cDNA libraries constructed from petal protoplast cultures and flowers of Petunia hybrida. PhADF1 and PhADF2 encode polypeptides of 139 and 143 amino acids with a calculated molecular mass of 16.04 and 16.51kDa, respectively. Co-sedimentation assay showed that the recombinant PhADF1 protein produced in Escherichia coli binds to F-actin at pH7. 0 and preferentially depolymerizes it at pH8.0. Gene tree analysis indicates that the plant ADF family can be grouped into four classes, and PhADFs are included in class I. Southern blot analyses revealed that one or two copies of PhADF genes are present in petunia genome, and several other related isoforms also exist. Northern blot analyses indicated that PhADF1 and PhADF2 are closely related and abundantly expressed in every plant organ except pollen. In addition, they are highly accumulated in mature vegetative tissue (petal, leaf, and stem). Our results indicate that the transcription of petunia ADF genes is differentially regulated by developmental signals.

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.

Identification of Tyrosyl-DNA Phosphodiesterase as a Novel DNA Damage Repair Enzyme in Arabidopsis

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a key enzyme that hydrolyzes the phosphodiester bond between tyrosine of topoisomerase and 3′-phosphate of DNA and repairs topoisomerase-mediated DNA damage during chromosome metabolism. However, functional Tdp1 has only been described in yeast and human to date. In human, mutations of the Tdp1 gene are involved in the disease spinocerebellar ataxia with axonal neuropathy. In plants, we have identified the functional nuclear protein AtTDP, homolog to human Tdp1 from Arabidopsis (Arabidopsis thaliana). The recombinant AtTDP protein certainly hydrolyzes the 3′-phosphotyrosyl DNA substrates related to repairing in vivo topoisomerase I-DNA-induced damage. The loss-of-function AtTDP mutation displays developmental defects and dwarf phenotype in Arabidopsis. This phenotype is substantially caused by decreased cell numbers without any change of individual cell sizes. The tdp plants exhibit hypersensitivities to camptothecin, a potent topoisomerase I inhibitor, and show rigorous cell death in cotyledons and rosette leaves, suggesting the failure of DNA damage repair in tdp mutants. These results indicate that AtTDP plays a clear role in the repair of topoisomerase I-DNA complexes in Arabidopsis.

Cortical tissue-specific accumulation of the root-specific ns-LTP transcripts in the bean (Phaseolus vulgaris) seedlings

The characterization of a cDNA clone encoding non-specific lipid transfer protein (PvLTP, formerly named PVR3) in the roots of bean seedlings has been previously reported. In this study, we examined the temporal and spatial accumulation of PvLTP mRNA and the effect of the auxin naphthaleneacetic acid (NAA) on the accumulation of PvLTP mRNA during root development. In situ hybridization showed that accumulation of PvLTP mRNA is highly tissue-specific. Accumulation was detected in the cortical tissue, but not in other tissues of root, including the quiescent center and root cap. Within the cortical tissue, accumulation of PvLTP mRNA was developmentally regulated; accumulation of PvLTP mRNA was high in the cortical tissue of the proximal and ground meristem and declined as cortical tissue developed further. Since the appropriate distribution of auxin is an important factor responsible for the maintenance of root meristem organization. We examined effect of auxin on the accumulation of PvLTP mRNA in relation to the development of cortical tissue. In bean seedlings grown on medium supplemented with 5 μM NAA, morphological alternations, including radial root expansion and abnormal tissue organization in the root apical meristem, were observed. Only faint accumulation signals of PvLTP mRNA were observed in the cortical tissue of proximal meristem region, indicating that cortical tissue development was repressed by exogenous NAA. However, our results suggest that the change in accumulation of PvLTP mRNA is not direct regulatory effect but reflective effect of altered development of cortical tissue that was induced by exogenous NAA. The temporal and spatial accumulation of PvLTP mRNA indicates that PvLTP is a useful marker for the development of cortical tissue in the root tip in bean seedlings.