Hur-Song Chang

Transcriptional Profiling Reveals Novel Interactions between Wounding, Pathogen, Abiotic Stress, and Hormonal Responses in Arabidopsis

Mechanical wounding not only damages plant tissues, but also provides pathways for pathogen invasion. To understand plant responses to wounding at a genomic level, we have surveyed the transcriptional response of 8,200 genes in Arabidopsis plants. Approximately 8% of these genes were altered by wounding at steady-state mRNA levels. Studies of expression patterns of these genes provide new information on the interactions between wounding and other signals, including pathogen attack, abiotic stress factors, and plant hormones. For example, a number of wound-responsive genes encode proteins involved in pathogen response. These include signaling molecules for the pathogen resistance pathway and enzymes required for cell wall modification and secondary metabolism. Many osmotic stress- and heat shock-regulated genes were highly responsive to wounding. Although a number of genes involved in ethylene, jasmonic acid, and abscisic acid pathways were activated, many in auxin responses were suppressed by wounding. These results further dissected the nature of mechanical wounding as a stress signal and identified new genes that may play a role in wounding and other signal transduction pathways.

Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization

Glomalean fungi induce and colonize symbiotic tissue called arbuscular mycorrhiza on the roots of most land plants. Other fungi also colonize plants but cause disease not symbiosis. Whole-transcriptome analysis using a custom-designed Affymetrix Gene-Chip and confirmation with real-time RT-PCR revealed 224 genes affected during arbuscular mycorrhizal symbiosis. We compared these transcription profiles with those from rice roots that were colonized by pathogens (Magnaporthe grisea and Fusarium moniliforme). Over 40% of genes showed differential regulation caused by both the symbiotic and at least one of the pathogenic interactions. A set of genes was similarly expressed in all three associations, revealing a conserved response to fungal colonization. The responses that were shared between pathogen and symbiont infection may play a role in compatibility. Likewise, the responses that are different may cause disease. Some of the genes that respond to mycorrhizal colonization may be involved in the uptake of phosphate. Indeed, phosphate addition mimicked the effect of mycorrhiza on 8% of the tested genes. We found that 34% of the mycorrhiza-associated rice genes were also associated with mycorrhiza in dicots, revealing a conserved pattern of response between the two angiosperm classes.

Expression profiling reveals COI1 to be a key regulator of genes involved in wound- and methyl jasmonate-induced secondary metabolism, defence, and hormone interactions

The Arabidopsis gene COI1 is required for jasmonic acid (JA)-induced growth inhibition, resistance to insect herbivory, and resistance to pathogens. In addition, COI1 is also required for transcription of several genes induced by wounding or by JA. Here, we use microarray gene transcription profiling of wild type and coi1 mutant plants to examine the extent of the requirement of COI1 for JA-induced and wound-induced gene transcription. We show that COI1 is required for expression of approximately 84% of 212 genes induced by JA, and for expression of approximately 44% of 153 genes induced by wounding. Surprisingly, COI1 was also required for repression of 53% of 104 genes whose expression was suppressed by JA, and for repression of approximately 46% of 83 genes whose expression was suppressed by wounding. These results indicate that COI1 plays a pivotal role in wound- and JA signalling.

Gene Expression Phenotypes of Arabidopsis Associated with Sensitivity to Low Temperatures

Chilling is a common abiotic stress that leads to economic losses in agriculture. By comparing the transcriptome of Arabidopsis under normal (22°C) and chilling (13°C) conditions, we have surveyed the molecular responses of a chilling-resistant plant to acclimate to a moderate reduction in temperature. The mRNA accumulation of approximately 20% of the approximately 8,000 genes analyzed was affected by chilling. In particular, a highly significant number of genes involved in protein biosynthesis displayed an increase in transcript abundance. We have analyzed the molecular phenotypes of 12 chilling-sensitive mutants exposed to 13°C before any visible phenotype could be detected. The number and pattern of expression of chilling-responsive genes in the mutants were consistent with their final degree of chilling injury. The mRNA accumulation profiles for the chilling-lethal mutants chs1, chs2, and chs3 were highly similar and included extensive chilling-induced and mutant-specific alterations in gene expression. The expression pattern of the mutants upon chilling suggests that the normal function of the mutated loci prevents a damaging widespread effect of chilling on transcriptional regulation. In addition, we have identified 634 chilling-responsive genes with aberrant expression in all of the chilling-lethal mutants. This reference gene list, including genes related to lipid metabolism, chloroplast function, carbohydrate metabolism and free radical detoxification, represents a potential source for genes with a critical role in plant acclimation to suboptimal temperatures. The comparison of transcriptome profiles after transfer of Arabidopsis plants from 22°C to 13°C versus transfer to 4°C suggests that quantitative and temporal differences exist between these molecular responses.

Toward elucidating the global gene expression patternsof developing Arabidopsis: Parallel analysis of 8 300 genesby a high-density oligonucleotide probe array

Arabidopsis thaliana has been widely used as a model system, in various aspects of biological studies, such as genomics, genetics, cellular, developmental and molecular biology. In order to reveal the molecular events and regulatory networks controlling Arabidopsis development and responses to genetic and environmental changes, we designed and used a high-density oligonucleotide probe array (GeneChip) to profile global gene expression patterns. The Arabidopsis oligonucleotide probe array consists of probes from 8 300 unique Arabidopsis genes, which covers approximately one-third of the genome. Global transcription profiles of A. thaliana in various developmental stages, and their responses to different environments were generated using this microarray, and archived. Here, we analyze data sets derived from nineteen independent experiments. Constitutively and differentially expressed genes in seedlings, roots, leaves, inflorescences, flowers and siliques at different developmental stages were identified. Functions of these genes based on homologs were determined and categorized. Our results provide insight into the coordinated transcriptional regulation of the genes during plant growth and development.

EMF Genes Maintain Vegetative Development by Repressing the Flower Program in Arabidopsis

The EMBRYONIC FLOWER (EMF) genes EMF1 and EMF2 are required to maintain vegetative development and repress flower development. EMF1 encodes a putative transcriptional regulator, and EMF2 encodes a Polycomb group protein homolog. We examined expression profiles of emf mutants using GeneChip technology. The high degree of overlap in expression changes from the wild type among the emf1 and emf2 mutants was consistent with the functional similarity between the two genes. Expression profiles of emf seedlings before flower development were similar to that of Arabidopsis flowers, indicating the commitment of germinating emf seedlings to the reproductive fate. The germinating emf seedlings ectopically expressed flower organ genes, suggesting that vegetative development in wild-type plants results from EMF repression of the flower program, directly or indirectly. In addition, the seed development program is derepressed in the emf1 mutants. Gene expression analysis showed no clear regulation of CONSTANS (CO), FLOWERING LOCUS T (FT), LEAFY (LFY), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 by EMF1. Consistent with epistasis results that co, lfy, or ft cannot rescue rosette development in emf mutants, these data show that the mechanism of EMF-mediated repression of flower organ genes is independent of these flowering genes. Based on these findings, a new mechanism of EMF-mediated floral repression is proposed.

Expression profiling of rice segregating for drought tolerance QTLs using a rice genome array

Plants alter their gene expression patterns in response to drought. Sometimes these transcriptional changes are successful adaptations leading to tolerance, while in other instances the plant ultimately fails to adapt to the stress and is labeled as sensitive to that condition. We measured the expression of approximately half of the genes in rice (∼21,000) in phenotypically divergent accessions and their transgressive segregants to associate stress-regulated gene expression changes with quantitative trait loci (QTLs) for osmotic adjustment (OA, a trait associated with drought tolerance). Among the parental lines, a total of 662 transcripts were differentially expressed. Only 12 genes were induced in the low OA parent, CT9993, at moderate dehydration stress levels while over 200 genes were induced in the high OA parent, IR62266. The high and low OA parents had almost entirely different transcriptional responses to dehydration stress suggesting a complete absence of an appropriate response rather than a slower response in CT9993. Sixty-nine genes were up-regulated in all the high OA lines and nine of those genes were not induced in any of the low OA lines. The annotation of four of those genes, sucrose synthase, a pore protein, a heat shock and an LEA protein, suggests a role in maintaining high OA and membrane stability. Of the 3,954-probe sets that correspond to the QTL intervals, very few had a differential expression pattern between the high OA and low OA lines that suggest a role leading to the phenotypic variation. However, several promising candidates were identified for each of the five QTL including a snRNP auxiliary factor, a LEA protein, a protein phosphatase 2C and a Sar1 homolog.

A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function.

Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations, yet only a few calcium-binding proteins have been identified in plants. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer. A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA.

Gene Expression Microarrays: Improvements and Applications towards Agricultural Gene Discovery

In agriculture, the associations between genes and resulting traits revealed by high throughput approaches such as transcription profiling could be used to select more environmentally friendly chemicals for plant protection and to develop plants with increased grain yields and better nutrition value, with more resistance to diseases and tolerance to abiotic stress. However, one of the major challenges to apply such approaches is the limited genomic information for most of the very diversified crop species. We developed multiple strategies and platform technologies to address this issue. Here we report our improvements of these technologies towards large-scale transcription profiling and their applications in agricultural gene discovery.