Zhangliang Chen

An Indole-3-Acetic Acid Carboxyl Methyltransferase Regulates Arabidopsis Leaf Development

Auxin is central to many aspects of plant development; accordingly, plants have evolved several mechanisms to regulate auxin levels, including de novo auxin biosynthesis, degradation, and conjugation to sugars and amino acids. Here, we report the characterization of an Arabidopsis thaliana mutant, IAA carboxyl methyltransferase1-dominant (iamt1-D), which displayed dramatic hyponastic leaf phenotypes caused by increased expression levels of the IAMT1 gene. IAMT1 encodes an indole-3-acetic acid (IAA) carboxyl methyltransferase that converts IAA to methyl-IAA ester (MeIAA) in vitro, suggesting that methylation of IAA plays an important role in regulating plant development and auxin homeostasis. Whereas both exogenous IAA and MeIAA inhibited primary root and hypocotyl elongation, MeIAA was much more potent than IAA in a hypocotyl elongation assay, indicating that IAA activities could be effectively regulated by methylation. IAMT1 was spatially and temporally regulated during the development of both rosette and cauline leaves. Changing expression patterns and/or levels of IAMT1 often led to dramatic leaf curvature phenotypes. In iamt1-D, the decreased expression levels of TCP genes, which are known to regulate leaf curvature, may partially account for the curly leaf phenotype. The identification of IAMT1 and the elucidation of its role in Arabidopsis leaf development have broad implications for auxin-regulated developmental process.

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis.

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene (PDS3) encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarf phenotypes of the pds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in the pds3 mutant. Further analysis showed that high level of phytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype of the pds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.

Genomic Evidence for COP1 as a Repressor of Light-Regulated Gene Expression and Development in Arabidopsis

Microarray gene expression profiling was used to examine the role of COP1 in the light control of Arabidopsis genome expression. Qualitatively similar gene expression profiles were observed between wild-type seedlings grown in white light and multiple cop1 mutant alleles grown in the dark. Furthermore, overexpression of the dominant-negative-acting N terminus of COP1 (N282) in darkness produced a genome expression profile similar to those produced by white light and the cop1 mutations. Different cop1 mutant alleles, N282, and light treatment also resulted in distinct expression profiles in a small fraction of the genes examined. In the light, the genome expression of cop1 mutations displayed an exaggerated light response. COP1-regulated genes in the dark were estimated to account for >20% of the genome. Analysis of these COP1-regulated genes revealed that >28 cellular pathways are coordinately but antagonistically regulated by light and COP1. Interestingly, the gene expression regulation attributable to HY5 in the light is included largely within those genes regulated by COP1 in the dark. Thus, this genomic study supports the hypothesis that COP1 acts as a repressor of photomorphogenesis, possibly by controlling the degradation of transcription factors and their target gene expression. The majority of light-controlled genome expression could be accounted for by the negative regulation of COP1 activity.

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem–loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.

A genome-wide analysis of blue-light regulation of Arabidopsis transcription factor gene expression during seedling development

A microarray based on PCR amplicons of 1,864 confirmed and predicted Arabidopsis transcription factor genes was produced and used to profile the global expression pattern in seedlings, specifically their light regulation. We detected expression of 1,371 and 1,241 genes in white-light- and dark-grown 6-d-old seedlings, respectively. Together they account for 84% of the transcription factor genes examined. This array was further used to study the kinetics of transcription factor gene expression change of dark-grown seedlings in response to blue light and the role of specific photoreceptors in this blue-light regulation. The expression of about 20% of those transcription factor genes are responsive to blue-light exposure, with 249 and 115 genes up or down-regulated, respectively. A large portion of blue-light-responsive transcription factor genes exhibited very rapid expression changes in response to blue light, earlier than the bulk of blue-light-regulated genes. This result suggests the involvement of transcription cascades in blue-light control of genome expression. Comparative analysis of the expression profiles of wild type and various photoreceptor mutants demonstrated that during early seedling development cryptochromes are the major photoreceptors for blue-light control of transcription factor gene expression, whereas phytochrome A and phototropins play rather limited roles.

Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants

Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene. Transformants were directly selected and rooted on medium containing cadmium and kanamycin. A total of 49 individual transgenic tobacco plants were regenerated. Among them 20% showed a very high expression level and their growth was unaffected by up to 200 μM cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 μM cadmium. The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting. In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.

Molecular Cloning and Functional Analysis of a Novel Type of Bowman-Birk Inhibitor Gene Family in Rice

Bowman-Birk inhibitor (BBI) genes encode serine protease inhibitors that have repetitive cysteine-rich domains with reactive sites for the trypsin or chymotrypsin family. We have identified seven BBI genes from japonica rice (Oryza sativa subsp. japonica var Teqing). All of the genes identified were found in a single cluster on the southern end of the long arm of rice chromosome 1. Four of the seven BBI genes have two repetitive cysteine-rich domains, whereas one has a truncated domain with only one reactive site. We have also identified three novel BBI genes, each of which possesses three repetitive domains instead of two. In situ hybridization analyses indicated that the accumulation of rice BBI transcripts was differentially regulated in germinating embryos and also in the leaves, roots, and flower organs at later developmental stages. Different members of the rice BBI gene family displayed different expression patterns during rice seed germination, and wounding induced the expression of rice BBI transcripts. The three-domain BBIs had higher expression levels than the two-domain BBIs. It was also found that the mRNA of rice BBI genes was present in abundant amounts in scutellar epithelium and aleurone layer cells. RBBI3-1, one of the three-domain RBBI, exhibited in vitro trypsin-inhibiting activity but no chymotrypsin-inhibiting activity. Overexpression of RBBI2-3 in transgenic rice plants resulted in resistance to the fungal pathogen Pyricularia oryzae, indicating that proteinase inhibitors confer resistance against the fungal pathogen in vivo and that they might play a role in the defense system of the rice plant.

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development.

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcl1 is male sterile as a result of the failure of pollen germination. We show that the bcl1 mutant allele cannot be transmitted by male gametophytes and no homozygous bcl1 mutants were obtained. Analysis of pollen developmental stages indicates that the bcl1 mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting (VPS) in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.

Obtaining and analysis of flanking sequences from T-DNA transformants of Arabidopsis

Abstract Large collections of insertional Arabidopsis lines are valuable for research on functional genomics. Using the activation tagging vector pSKI015, more than 45 000 T-DNA insertion lines were generated by Agrobacterium -mediated floral-dip transformation protocol. 2304 insertion lines were analyzed and 1502 items of plant sequences flanking the T-DNA insertion sites were obtained by a modified thermal asymmetric interlaced PCR (TAIL-PCR) protocol. These sequences were searched against Genebank database using blast and 1194 insertion sites were determined according to the sequences matching to the Arabidopsis genome sequence. The insertion sites were distributed on all the five Arabidopsis chromosomes and the interfered genes were classified into 14 function categories. Analysis on 1194 items of 100-bp sequences surrounding T-DNA insertion sites showed that 27 and 31% GC contents were likely to favor the T-DNA integration. Sixty-eight items of these 100-bp sequences having more than two insertions were chosen to look for motifs in favor of T-DNA integration. The results showed that “ATNTT” (N represents A/T/C/G) and the polyT and polyA motifs probably play a role in the T-DNA integration event.

Purification and characterization of a novel anti-fungal protein from Gastrodia elata

Abstract An anti-fungal protein GAFP-1 ( Gastrodia anti-fungal protein, also called gastrodianin) was purified from Gastrodia elata B1. f. flavida S. Chow (Orchidaceae), a parasitic plant on the fungus Armillaria mellea . It can inhibit the hyphal growth of some phytopathogenic fungi such as Valsa ambiens, Rhizoctonia solani, Gibberella zeae, Ganoderma lucidum and Botrytis cinerea in vitro. GAFP-1 is a monomer with a molecular mass of 10 kDa and a pI of 8.45. The optimum pH for its inhibitory activity is 5.0 ∼ 6.0. GAFP-1 is insensitive to high temperatures. It can preserve 75% inhibitory activity after 30 min at 60°C. Amino acid composition analysis revealed that GAFP-1 is rich in Asp (22.1%), Gly (10.0 %) and Leu (9.4 %), and does not contain any Pro. The amino acid sequence of the N-terminal was determined and found to share high homology with those of other lectins from orchids such as Listera ovata and Epipactis helleborine . GAFP-1 could not agglutinate trypsin-treated rabbit erythrocytes. It could bind to chitin, immobilized mannose and N-acetylglucosamine in 50mM sodium acetate buffer (pH 5.0) with 2 M ammonium sulfate. These data suggest that GAFP-1 could be a lectin-like protein with strong inhibitory activity against certain fungal pathogens.