Stress and innate immune responses in plants

Reactive Oxygen Species in Plant Stress Responses

Successful defense against abiotic stress and pathogens requires perception of the environment followed by the execution of a preplanned defense posture. There are commonalties in the way plants react to stress and pathogens among them the production of reactive oxygen species (ROS). These are versatile molecules that play an indispensable role in mediating a diversity of cellular responses in plant cells, including necrotic reactions, programmed cell death (PCD), development, gravitropism, and hormonal signaling. The membrane-bound NADPH oxidase or respiratory burst oxidase homologues (Rboh) are key enzymes that generate superoxide radicals in plant cells and act as signal transponders. Our work shows their localization to plasma membrane fractions and regulation by Ca2+ ions during defense responses to wounding. During Phytophthora cryptogea interaction with tobacco a small fungal effector molecule called cryptogein that can serve as a carrier of sterols, elicits a plant hypersensitive response. This response is crucial for plant defense. When cryptogein is applied to tobacco BY-2 suspension cells, one of the early events is cryptogein-induced ROS production. These show temporal ROS accumulation in endomembrane, cytoplasmic and nuclear compartments. How these events are controlled and how signal specificity to cell death is achieved are our research goals

Cryptogein-induced ROS produced by NADPH oxidases causes cell death and impacts on cytoplasmic and cellular redox.
Ahstamker et al (2007)

“Reactive Oxygen Species Control of the Wound Response” Sagi et al (2004)

ROS production by NADPH oxidases controls the response to wounding. Left: Shown are wild type (WT) and antisense plants (M3) without (control) or with wounding. The brown precipitate (DAB) reveals H2O2 that accumulates in normal systemic leaves 5 h after wounding but not in M3 mutant leaves. Right: Transcriptome analysis of tomato gene expression that reveals the extent of control ROS plays in transducing the wound response. MC, mutant control leaf; MS, mutant systemic leaf; WC, wild type control leaf; WS, wild type systemic leaf. The arrows and numbers (brackets) indicate groups of genes with similar behavior. Sagi et al (2004)

Splicing Dynamics in the Plant Transcriptome

Alternative splicing (AS) can add significantly to genome complexity. Plants are thought to exhibit less alternative splicing then animals. We have previously shown that the major form of plant AS is intron retention. An algorithm, based on EST pairs gapped alignment (EPGA), was developed that takes advantage of the relatively small intron and exon size in plants and directly compares pairs of ESTs to search for alternative splicing. EPGA was tested in Arabidopsis and rice for which annotated genome sequence is available and was shown to accurately predict splicing events. We show that direct transcript expression analysis using high-density oligonucleotide-based whole-genome microarrays (WGAs) is particularly amenable for assessing global intron retention in Arabidopsis. This rate of detection predicts an overall total AS rate of 20% for Arabidopsis compared to 10-22% based on EST/cDNA-based analysis. Our research interests are in examining dynamic aspects of alternative splicing and its control.

Intron retention is present in transcripts that are ribosome-associated. Sub cellular fractionation with or without puromycin (+/- Pu) treatment. The RNA was extracted from the resultant pellet (p) and supernatant (s). The RT-PCR results of At1g79090 show a retained intron that is part of the UTR. The results show release of retained introns (I) from ribosome pellets. Ner-Gaon et al (2004)

Analysis of whole genome microarrays enables unbiased evaluation of gene activity. The distribution of the mean hybridization values of the probes in exons and introns of all Arabidopsis transcripts. Probes were divided into exons and introns. The mean hybridization value of all the exons in a transcript, as well as the mean hybridization value of all its introns were calculated to evaluate genome-wide alternative splicing. Ner Gaon et al (2006)

“Intron retention is the major form of alternative splicing in plants” Ner-Gaon et al( 2004,6,7)