Project description:The interplay between plant chemistry and architecture was investigated using a pharmacological approach combined with mutant analysis. Due to the high abundance and essential role of cellulose in plant development and function, it was hypothesized that perturbation of cellulose biosynthesis would have far-reaching effects on plant chemistry and resource allocation. The impact of cellulose disruption was studied through comprehensive multiphase-NMR (CMP-NMR) using the Arabidopsis thaliana cellulose synthase mutant ectopic lignification1 (eli1). CMP-NMR confirmed several known metabolic impacts of cellulose disruption including increased lignification, increased starch production, and a shift from crystalline to amorphous cellulose. It also revealed unexpected metabolic impacts such as increased methanol production, increased seed-derived lipid content, and the presence of the peptide pentaglycine, which had not been previously observed in plants. It was further hypothesized that the metabolic impacts of cellulose disruption are mediated by one or more signal molecules that would be elevated in cellulose synthase mutants such as eli1. The presence of one or more signal molecules in eli1 that induce a lignification response was confirmed by exposing wild-type seedlings to ground tissue from eli1. Treatment of wild-type seedlings with pentaglycine suggested that the peptide may be at least partially responsible for mediating this response. A case is made for glycine rich proteins (GRPs) as the source of pentaglycine in vivo, and for wall associated kinases (WAKs) as receptors that trigger either developmental or defence responses through differential binding of intact GRPs and oligoglycine peptides.
Project description:Transcriptional profiling after inhibition of cellulose synthesis by thaxtomin A and isoxaben in Arabidopsis thaliana suspension cells Perturbations in the cellulose content of the plant cell wall lead to global modifications in cellular homeostasis, as seen in cellulose synthase mutants or after inhibiting cellulose synthesis. In particular, application of inhibitors of cellulose synthesis such as thaxtomin A (TA) and isoxaben (IXB) initiates a programmed cell death (PCD) in Arabidopsis thaliana suspension cells that is dependent on de novo gene transcription. To further understand how TA and IXB activate PCD, a whole genome microarray analysis was performed on mRNA isolated from Arabidopsis suspension cells exposed to TA and IXB. More than 75% of the genes upregulated by TA were also upregulated by IXB, including genes encoding cell wall-related and calcium-binding proteins, defence/stress-related transcription factors, signalling components and cell death-related proteins. Comparisons with published transcriptional analyses revealed an important subset of genes generally induced in response to various biotic and abiotic stress.
Project description:Transcriptional profiling after inhibition of cellulose synthesis by thaxtomin A and isoxaben in Arabidopsis thaliana suspension cells; Perturbations in the cellulose content of the plant cell wall lead to global modifications in cellular homeostasis, as seen in cellulose synthase mutants or after inhibiting cellulose synthesis. In particular, application of inhibitors of cellulose synthesis such as thaxtomin A (TA) and isoxaben (IXB) initiates a programmed cell death (PCD) in Arabidopsis thaliana suspension cells that is dependent on de novo gene transcription. To further understand how TA and IXB activate PCD, a whole genome microarray analysis was performed on mRNA isolated from Arabidopsis suspension cells exposed to TA and IXB. More than 75% of the genes upregulated by TA were also upregulated by IXB, including genes encoding cell wall-related and calcium-binding proteins, defence/stress-related transcription factors, signalling components and cell death-related proteins. Comparisons with published transcriptional analyses revealed an important subset of genes generally induced in response to various biotic and abiotic stress. Experiment Overall Design: TA, IXB and methanol (control) were added to Arabidopsis thaliana suspension cells three days after sub-culture. Cells were harvested for RNA isolation and frozen in liquid nitrogen after 6 hours of contact with the inhibitors. Samples consisted of four replicates for each condition. A total of 12 Affymetrix GeneChips® were used in this study, which correspond to 12 RNA samples from the four biological replicates for each of the TA, IXB or methanol addition.
Project description:We compared the transcriptome of homozygous mutants for AtCesA4 and AtCesA6 (cellulose synthase genes) to their heterozygous counterparts that have a wild type phenotype. All plants were 4-weeks-old and grown under short day conditions.
Project description:The barley brittle stem mutants, fs2, designated X054 and M245, have reduced levels of cellulose compared with their isogenic parents Ohichi and Shiroseto. A custom-designed microarray, based on Agilent technology and including genes involved in cell wall metabolism, was used to compare transcript levels in the mutant and parental lines. For both mutants, the microarray revealed a marked decrease in mRNA for the HvCesA4 cellulose synthase gene in specific zones of stem internodes, and this was confirmed by quantitative PCR.
Project description:Background: Cell walls (CWs) are protein-rich polysaccharide matrices essential for plant growth and environmental acclimationadaptation. The CW constitutes the first physical barrier as well as a primary source of sugars for plant microbes, such as the vascular pathogen Fusarium oxysporum (Fo). Fo colonizes roots, advancing through the plant primary CWs towards the vasculature, where it grows causing devastation in many crops. The pathogenicity of Fo and other vascular microbes relies on their capacity to reach and colonize the xylem. However, little is known about the root-microbe interaction before the pathogen reaches the vasculature and the role of the plant CW during this process. Results: Using the pathosystem Arabidopsis-Fo5176, we show dynamic transcriptional changes in both fungus and root during their interaction. One of the earliest plant responses to Fo5176 was the downregulation of primary CW synthesis genes. We observed enhanced resistance to Fo5176 in Arabidopsis mutants impaired in primary CW cellulose synthesis. Previous studies showed an induction of ectopic lignification, accumulation of defense-related phytohormones, and dwarfism in primary CW cellulose synthesis deficient plants, potentially explaining their resistance to Fo5176. We confirmed that Arabidopsis roots deposit lignin in response to Fo5176 infection but we show that lignin-deficient mutants were as susceptible as wildtype plants to Fo5176. Genetic impairment of jasmonic acid biosynthesis and signaling did not alter Arabidopsis response to Fo5176, whereas impairment of ethylene signaling did increase vasculature colonization by Fo5176. AbolishingThis ethylene signaling interruption attenuated the observed resistance while maintaining the dwarfism observed in primary CW cellulose-deficient mutants. Conclusions: Our study provides significant insights on the dynamic root-vascular pathogen interaction at the transcriptome level and the vital role of primary CW cellulose during defense response to these pathogens. These findings represent an essential resource for the generation of plant resistance to Fo that can be transferred to other vascular pathosystems.
Project description:In bacteria, the biosynthesis of cysteine is accomplished by two enzymes that are encoged by the cysK and cysM genes. CysM is also able to incorporate thiosulfate to produce S-sulfocysteine. In plant cells, the biosynthesis of cysteine occurs in the cytosol, mitochondria and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of cs26 suppressed the S-sulfocysteine synthase activity that was detected in wild type; furthermore, the mutants exhibited a growth phenotype, but penetrance depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants), as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify ROS, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our finding reveals that S-sulfocysteine and the activity of S-sulfocysteine synthase play an important role in chloroplast function and are essential for light-dependent redox regulation within the chloroplast.
Project description:Background: Cell walls (CWs) are protein-rich polysaccharide matrices essential for plant growth and environmental acclimationadaptation. The CW constitutes the first physical barrier as well as a primary source of sugars for plant microbes, such as the vascular pathogen Fusarium oxysporum (Fo). Fo colonizes roots, advancing through the plant primary CWs towards the vasculature, where it grows causing devastation in many crops. The pathogenicity of Fo and other vascular microbes relies on their capacity to reach and colonize the xylem. However, little is known about the root-microbe interaction before the pathogen reaches the vasculature and the role of the plant CW during this process. Results: Using the pathosystem Arabidopsis-Fo5176, we show dynamic transcriptional changes in both fungus and root during their interaction. One of the earliest plant responses to Fo5176 was the downregulation of primary CW synthesis genes. We observed enhanced resistance to Fo5176 in Arabidopsis mutants impaired in primary CW cellulose synthesis. Previous studies showed an induction of ectopic lignification, accumulation of defense-related phytohormones, and dwarfism in primary CW cellulose synthesis deficient plants, potentially explaining their resistance to Fo5176. We confirmed that Arabidopsis roots deposit lignin in response to Fo5176 infection but we show that lignin-deficient mutants were as susceptible as wildtype plants to Fo5176. Genetic impairment of jasmonic acid biosynthesis and signaling did not alter Arabidopsis response to Fo5176, whereas impairment of ethylene signaling did increase vasculature colonization by Fo5176. AbolishingThis ethylene signaling interruption attenuated the observed resistance while maintaining the dwarfism observed in primary CW cellulose-deficient mutants. Conclusions: Our study provides significant insights on the dynamic root-vascular pathogen interaction at the transcriptome level and the vital role of primary CW cellulose during defense response to these pathogens. These findings represent an essential resource for the generation of plant resistance to Fo that can be transferred to other vascular pathosystems.