Iain W. Wilson

Expression Profile Analysis of the Low-Oxygen Response in Arabidopsis Root Cultures

We used DNA microarray technology to identify genes involved in the low-oxygen response of Arabidopsis root cultures. A microarray containing 3500 cDNA clones was screened with cDNA samples taken at various times (0.5, 2, 4, and 20 h) after transfer to low-oxygen conditions. A package of statistical tools identified 210 differentially expressed genes over the four time points. Principal component analysis showed the 0.5-h response to contain a substantially different set of genes from those regulated differentially at the other three time points. The differentially expressed genes included the known anaerobic proteins as well as transcription factors, signal transduction components, and genes that encode enzymes of pathways not known previously to be involved in low-oxygen metabolism. We found that the regulatory regions of genes with a similar expression profile contained similar sequence motifs, suggesting the coordinated transcriptional control of groups of genes by common sets of regulatory factors.

Global Changes in Gene Expression in Response to High Light in Arabidopsis

A range of environmental conditions can lead to oxidative stress; thus, a prompt and effective response to oxidative stress is crucial for the survival of plants. Microarray and northern-blot analyses were performed toward the identification of the factors and signaling pathways that enable plants to limit oxidative damage caused by exposure to high light (HL). Arabidopsis plants grown under moderate light (100 μmol m−2 s−1) were exposed to HL (1,000 μmol m−2 s−1) for 1 h. The microarray analyses revealed that exposure of Arabidopsis to HL caused an increase in known antioxidant genes, as well as several unknown genes. Some of these unknown genes had homologies to possible regulatory genes and metabolic enzymes. Furthermore, it was found that a range of chaperones were up-regulated in the HL treatment and that this induction was specifically due to the HL stress. The temporal expression under HL and different oxidative stress conditions of a subset of HL-responsive genes was confirmed via northern-blot analysis. Results from the arrays were also compared with publicly available microarray data sets from a range of different stress conditions at the Arabidopsis Functional Genomics Consortium. This cross comparison enabled the identification of genes that may be induced by changes in redox poise. Finally, to determine if the genes that were differentially expressed by HL stress were under similar transcriptional control, we analyzed the promoter sequences for the presence of common motifs.

Arabidopsis RAP2.2: An Ethylene Response Transcription Factor That Is Important for Hypoxia Survival

Arabidopsis (Arabidopsis thaliana) RAP2.2 (At3g14230) is an APETALA2/ethylene response factor-type transcription factor that belongs to the same subfamily as the rice (Oryza sativa) submergence tolerance gene SUB1A. RAP2.2 is expressed at constitutively high levels in the roots and at lower levels in the shoots, where it is induced by darkness. Effector studies and analysis of ethylene signal transduction mutants indicate that RAP2.2 is induced in shoots by ethylene and functions in an ethylene-controlled signal transduction pathway. Overexpression of RAP2.2 resulted in improved plant survival under hypoxia (low-oxygen) stress, whereas lines containing T-DNA knockouts of the gene had poorer survival rates than the wild type. This indicates that RAP2.2 is important in a plants ability to resist hypoxia stress. Observation of the expression pattern of 32 low-oxygen and ethylene-associated genes showed that RAP2.2 affects only part of the low-oxygen response, particularly the induction of genes encoding sugar metabolism and fermentation pathway enzymes, as well as ethylene biosynthesis genes. Our results provide a new insight on the regulation of gene expression under low-oxygen conditions. Lighting plays an important regulatory role and is intertwined with hypoxia conditions; both stimuli may act collaboratively to regulate the hypoxic response.

Systemic Gene Expression in Arabidopsis during an Incompatible Interaction with Alternaria brassicicola

Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of “high alert,” otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the β-oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks.

Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f. sp. vasinfectum.

Microarray analysis of large-scale temporal and tissue-specific plant gene expression changes occurring during a susceptible plant-pathogen interaction revealed different gene expression profile changes in cotton root and hypocotyl tissues. In hypocotyl tissues infected with Fusarium oxysporum f. sp. vasinfectum, increased expression of defense-related genes was observed, whereas few changes in the expression levels of defense-related genes were found in infected root tissues. In infected roots, more plant genes were repressed than were induced, especially at the earlier stages of infection. Although many known cotton defense responses were identified, including induction of pathogenesis-related genes and gossypol biosynthesis genes, potential new defense responses also were identified, such as the biosynthesis of lignans. Many of the stress-related gene responses were common to both tissues. The repression of drought-responsive proteins such as aquaporins in both roots and hypocotyls represents a previously unreported response of a host to pathogen attack that may be specific to vascular wilt diseases. Gene expression results implicated the phytohormones ethylene and auxin in the disease process. Biochemical analysis of hormone level changes supported this observation.

The Arabidopsis AMP1 Gene Encodes a Putative Glutamate Carboxypeptidase

Arabidopsis amp1 mutants show pleiotropic phenotypes, including altered shoot apical meristems, increased cell proliferation, polycotyly, constitutive photomorphogenesis, early flowering time, increased levels of endogenous cytokinin, and increased cyclin cycD3 expression. We have isolated the AMP1 gene by map-based cloning. The AMP1 cDNA encodes a 706;–amino acid polypeptide with significant similarity to glutamate carboxypeptidases. The AMP1 mRNA was expressed in all tissues examined, with higher expression in roots, stems, inflorescences, and siliques. Microarray analysis identified four mRNA species with altered expression in two alleles of amp1, including upregulation of CYP78A5, which has been shown to mark the shoot apical meristem boundary. The similarity of the AMP1 protein to glutamate carboxypeptidases, and in particular to N-acetyl α-linked acidic dipeptidases, suggests that the AMP1 gene product modulates the level of a small signaling molecule that acts to regulate a number of aspects of plant development, in particular the size of the apical meristem.

Hypoxia-responsive microRNAs and trans-acting small interfering RNAs in Arabidopsis

Low-oxygen (hypoxia) stress associated with natural phenomena such as waterlogging, results in widespread transcriptome changes and a metabolic switch from aerobic respiration to anaerobic fermentation. High-throughput sequencing of small RNA libraries obtained from hypoxia-treated and control root tissue identified a total of 65 unique microRNA (miRNA) sequences from 46 families, and 14 trans-acting small interfering RNA (tasiRNA) from three families. Hypoxia resulted in changes to the abundance of 46 miRNAs from 19 families, and all three tasiRNA families. Chemical inhibition of mitochondrial respiration caused similar changes in expression in a majority of the hypoxia-responsive small RNAs analysed. Our data indicate that miRNAs and tasiRNAs play a role in gene regulation and possibly developmental responses to hypoxia, and that a major signal for these responses is likely to be dependent on mitochondrial function.

Global Gene Expression Responses to Waterlogging in Roots and Leaves of Cotton (Gossypium hirsutum L.)

Waterlogging is a serious impediment to crop productivity worldwide which acts to reduce oxygen levels in the rhizosphere due to the low diffusion rate of molecular oxygen in water. Plants respond to low oxygen through rapid and specific changes at both the transcriptional and translational levels. Transcriptional changes to low-oxygen (hypoxia) stress have been studied in a number of plant species using whole genome microarrays. Using transcriptome data from root tissue from early time points (4-5 h) from cotton (Gossypium hirsutum), Arabidopsis and gray poplar (Populus x canescens), we have identified a core set of orthologous genes that responded to hypoxia in similar ways between species, and others that showed species specific responses . Responses to hypoxia were most similar between Arabidopsis and cotton, while the waterlogging tolerant poplar species exhibited some significant differences.Waterlogging stress causes yield reduction in cotton (Gossypium hirsutum L.). A major component of waterlogging stress is the lack of oxygen available to submerged tissues. While changes in expressed protein, gene transcription and metabolite levels have been studied in response to low oxygen stress, little research has been done on molecular responses to waterlogging in cotton. We assessed cotton growth responses to waterlogging and assayed global gene transcription responses in root and leaf cotton tissues of partially submerged plants. Waterlogging caused significant reductions in stem elongation, shoot mass, root mass and leaf number, and altered the expression of 1,012 genes (4% of genes assayed) in root tissue as early as 4 h after flooding. Many of these genes were associated with cell wall modification and growth pathways, glycolysis, fermentation, mitochondrial electron transport and nitrogen metabolism. Waterlogging of plant roots also altered global gene expression in leaf tissues, significantly changing the expression of 1,305 genes (5% of genes assayed) after 24 h of flooding. Genes affected were associated with cell wall growth and modification, tetrapyrrole synthesis, hormone response, starch metabolism and nitrogen metabolism The implications of these results for the development of waterlogging-tolerant cotton are discussed.

Spatial and temporal analysis of the local response to wounding in Arabidopsis leaves

We studied the local response to wounding in Arabidopsis thaliana leaves using a two-step microarray analysis. A microarray containing 3500 cDNA clones was first screened to enrich for genes affected by wounding in the immediate vicinity of the wound (4 h post wounding). 359 non-redundant putative wound responsive genes were then spotted on a smaller wound-response array for detailed analysis of spatial expression (local, adjacent and systemic), timing of expression (0.5, 4, 8, 17 h), and effect of hormone treatments (methyl jasmonate, ethylene and abscisic acid). Our results show that genes that respond early at the site of the wound also respond throughout the plant, with similar kinetics. Early-induced genes which respond systemically encode predominantly signal transduction and regulatory factors (36%), and the expression of many of them is also controlled by methyl jasmonate (about 35% of the 36%). Genes specific to the wound site and the wounded leaf have a slower response to wounding and are mainly metabolic genes. At the wound, many genes of the lignin biosynthesis pathway were induced. In silico analysis of the 5′ promoter regions of genes affected by wounding revealed G-box-related motifs in a significant proportion of the promoters. These results show that the establishment of a systemic response to wounding is a priority for the plant, and that the local response at the wound site is established later. Ethylene and abscisic acid are involved in the local response, regulating repression of photosynthetic genes and expression of drought responsive genes respectively.

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis.

BackgroundSiraitia grosvenorii (Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway.ResultsIn this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450 s and five UDPG s were selected as the candidates most likely to be involved in mogrosides biosynthesis.ConclusionA combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven CYP450 s and five UDPG s were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from S. grosvenorii.