Discovery of Functional Genes for Systemic Acquired Resistance in Arabidopsis Thaliana through Integrated Data Mining

Various data mining techniques combined with sequence motif information in the promoter region of genes were applied to discover functional genes that are involved in the defense mechanism of systemic acquired resistance (SAR) in Arabidopsis thaliana. A series of K-Means clustering with difference-in-shape as distance measure was initially applied. A stability measure was used to validate this clustering process. A decision tree algorithm with the discover-and-mask technique was used to identify a group of most informative genes. Appearance and abundance of various transcription factor binding sites in the promoter region of the genes were studied. Through the combination of these techniques, we were able to identify 24 candidate genes involved in the SAR defense mechanism. The candidate genes fell into 2 highly resolved categories, each category showing significantly unique profiles of regulatory elements in their promoter regions. This study demonstrates the strength of such integration methods and suggests a broader application of this approach.Diff\ue9rentes techniques d'exploration de donn\ue9es, combin\ue9es \ue0 de l'information sur le motif de s\ue9quence dans la r\ue9gion promotrice de g\ue8nes, ont \ue9t\ue9 appliqu\ue9es pour d\ue9couvrir les g\ue8nes fonctionnels qui interviennent dans le m\ue9canisme de d\ue9fense de la r\ue9sistance syst\ue9mique acquise (RSA ou SAR) chez Arabidopsis thaliana. On a initialement utilis\ue9 une s\ue9rie de classifications par les K moyennes et la diff\ue9rence de forme comme mesure de distance. On a utilis\ue9 une mesure de stabilit\ue9 pour valider ce processus de classification, et un algorithme d'arbre de d\ue9cision ainsi que la technique de d\ue9couverte et de masquage pour identifier un groupe de g\ue8nes sup\ue9rieurement informatifs. On a \ue9tudi\ue9 l'apparence et l'abondance de diff\ue9rents sites de liaison de facteurs de transcription dans la r\ue9gion promotrice des g\ue8nes. En combinant ces techniques, nous avons pu identifier 24 g\ue8nes candidats intervenant dans le m\ue9canisme de d\ue9fense de la RSA. Ces g\ue8nes candidats se classaient dans deux cat\ue9gories hautement r\ue9solues, chacune pr\ue9sentant des profils v\ue9ritablement uniques d'\ue9l\ue9ments r\ue9gulateurs dans leurs r\ue9gions promotrices. Cette \ue9tude d\ue9montre le potentiel de pareilles m\ue9thodes d'int\ue9gration et laisse entrevoir une plus vaste application de cette approche.Peer reviewed: YesNRC publication: Ye

Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts

<p>Abstract</p> <p>Background</p> <p>Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of <it>DGAT1 </it>results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating <it>DGAT1 </it>levels during seed development, a concerted transcriptome and metabolome analysis of transgenic <it>B. napus </it>prototypes was performed.</p> <p>Results</p> <p>Using a targeted <it>Brassica </it>cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24–33% of the targets showing significant changes have no matching gene in <it>Arabidopsis </it>although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 <it>Arabidopsis </it>genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development.</p> <p>Conclusion</p> <p>Our results indicate that the increased TAG accumulation observed in transgenic <it>DGAT1 </it>plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in <it>Arabidopsis </it>compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.</p

Role of transcription factors in storage lipid accumulation in plants

With few exceptions, seeds oil accumulates as triacylglycerols; three fatty acid molecules esterified to a glycerol backbone. The synthesis of normal fatty acids and unusual fatty acids, their assembly into triacylglycerols and subsequent packaging to form oil bodies have been reviewed elsewhere, and appear as separate contributions to the AOCS Lipid Library by Harwood, Hildebrand, Weselake et al., and Huang, respectively. These processes are highly regulated, involving spatial compartmentalization between different organelles and the exquisite control of several biosynthetic steps by one or more of a variety of biochemical mechanisms. The important role of transcriptional regulation towards seed oil accumulation has recently emerged. Not only are mRNA levels of many genes involved in seed oil biosynthesis coordinated during seed development, but altering the levels of certain transcription factors can affect the expression of multiple lipid biosynthetic genes, resulting in altered seed oil content. This article reviews salient features of plant transcription factors that have been implicated in the control of seed oil deposition. To set the stage, fundamental concepts in transcription factor biology are first summarized. Subsequently, the role of key transcription factors is highlighted by describing seed oil phenotypes associated with altering their expression levels by mutagenesis or transgenic means, and providing information on the putative target genes and pathways affected. The location of the transcription factors in hierarchical cascades and the modes of transcriptional regulation are also discussed, when this information is known. A summary of the information presented is depicted in Figure 1.NRC publication: Ye

<i>Arabidopsis</i> Clade I TGA Factors Regulate Apoplastic Defences against the Bacterial Pathogen <i>Pseudomonas syringae</i> through Endoplasmic Reticulum-Based Processes

<div><p>During the plant immune response, large-scale transcriptional reprogramming is modulated by numerous transcription (co) factors. The <i>Arabidopsis</i> basic leucine zipper transcription factors TGA1 and TGA4, which comprise the clade I TGA factors, have been shown to positively contribute to disease resistance against virulent strains of the bacterial pathogen <i>Pseudomonas syringae</i>. Despite physically interacting with the key immune regulator, NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), following elicitation with salicylic acid (SA), clade I function was shown to be largely independent of NPR1. Unlike mutants in NPR1, <i>tga1-1 tga4-1</i> plants do not display reductions in steady-state levels of SA-pathway marker genes following treatment with this phenolic signaling metabolite or after challenge with virulent or avirulent <i>P. syringae</i>. By exploiting bacterial strains that have limited capacity to suppress <i>Arabidopsis</i> defence responses, the present study demonstrates that <i>tga1-1 tga4-1</i> plants are compromised in basal resistance and defective in several apoplastic defence responses, including the oxidative burst of reactive oxygen species, callose deposition, as well as total and apoplastic PATHOGENESIS-RELATED 1 (PR-1) protein accumulation. Furthermore, analysis of <i>npr1-1</i> and the <i>tga1-1 tga4-1 npr1-1</i> triple mutant indicates that clade I TGA factors act substantially independent of NPR1 in mediating disease resistance against these strains of <i>P. syringae</i>. Increased sensitivity to the <i>N</i>-glycosylation inhibitor tunicamycin and elevated levels of endoplasmic reticulum (ER) stress marker genes encoding ER-resident chaperones in mutant seedlings suggest that loss of apoplastic defence responses is associated with aberrant protein secretion and implicate clade I TGA factors as positive regulators of one or more ER-related secretion pathways.</p> </div

Defence-related gene expression in Col-0 and <i>tga1-1 tga4-1.</i>

<p>Four-week-old leaves were syringe-infiltrated with 10⁸ cfu ml^-1 of <i>Pst </i><i>hrcC</i>^<i>-</i> or <i>Psp</i> 1448a. Leaf tissues from three plants were collected and pooled as one sample for RNA isolation. Values were normalized to the expression of <i>UBIQUITIN5</i>. An asterisk indicates a statistically significant difference compared with Col-0 at the same time point (p<0.05, Student’s <i>t</i>-test), and two asterisks indicate p<0.01. The error bars represent the standard deviation of three biological samples.</p

Growth of <i>Pst hrcC</i>^<i>-</i> (A) and <i>Psp</i> 1448a (B) in Col-0, <i>tga1-1 tga4-1, npr1-1 and tga1-1 tga4-1 npr1-1</i> plants.

<p>Four-week old leaves were syringe-infiltrated with a bacterial suspension of <i>Pst </i><i>hrcC</i>^<i>-</i> at 10⁵ colony forming units (cfu) ml^-1 or <i>Psp</i> 1448a at 10⁶ cfu ml^-1. The error bars represent the standard deviation of six replicates, each containing 8 leaf discs from one plant. An ANOVA of the log-transformed data was performed at α = 0.05; treatments with common letters over bars are not significantly different from each other. Post-hoc tests are presented in Table S2. Each experiment was repeated twice with similar results.</p

MAMPs-induced oxidative burst in Col-0 and <i>tga1-1tga4-1</i> plants.

<p>Four-week-old leaf discs (3 per each sample) were treated with or without 2 µM flg22 in the presence of luminol, and the H₂O₂ generated was measured every 30 sec after treatment for 20 min. The error bars represent the standard deviation of six replicates. The experiment was repeated five times with similar results.</p

PR-1 protein accumulation in Col-0 and <i>tga1-1 tga4-1</i> after pathogen inoculation.

<p>Four-week-old leaves were syringe-infiltrated with 10⁸ cfu ml^-1 of <i>Pst </i><i>hrcC</i>^<i>-</i> or <i>Psp</i> 1448a. Intercellular washing fluids (IWFs) and total protein were collected at 0, 1 and 2 dpi, separated on 16% Tricine-SDS-polyacrylamide gels and blotted with a PR-1 antibody. The <i>Arabidopsis</i> PR-1 protein has apredicted molecular weight of 16 kilodaltons (kDa) [74]. Arrows indicate the position of a 17-kDa molecular weight masker. The same gels were stained with Coomassie Brilliant Blue R250 (Sigma) as a loading control. These experiments were repeated three times with similar results.</p