Glutathione imparts stress tolerance against <i>Alternaria brassicicola</i> infection via miRNA mediated gene regulation

Glutathione (GSH) is well known to play a crucial role in imparting resistance against various pathogen invasions. Nevertheless, the role of GSH in regulating miRNA-mediated defense response is yet to be explored. To decipher the GSH-mediated regulation of miRNA expression during necrotrophic infection in Arabidopsis thaliana, wild-type Col-0 and AtECS1, the transgenic line exhibiting enhanced GSH content, were infected with necrotrophic pathogen Alternaria brassicicola. AtECS1 plants exhibited enhanced resistance as compared to wild-type. MiRNA next-generation sequencing (NGS) was performed to compare the miRNA expression in Col-0 and AtECS1 leaves. Under control condition, differentially expressed 96 known miRNAs and 17 novel miRNAs viz. ath-miR8167f, ath-miR1886.3, ath-miR3932b-5p, etc. were identified. However, under infected condition, 73 known and 43 novel differentially expressed miRNAs viz. ath-miR5652, ath-miR160b, ath-miR865-5p, etc. were identified. Functional annotation and enrichment analysis revealed that several miRNAs that target defense-related genes like leucine-rich repeat protein kinase, MYB transcription factors, TCP8, etc. were down regulated in the AtECS1 line, which, in turn, relieves the repression of their target gene expression, leading to resistance against infection. Together, the present investigation suggests that GSH plays a decisive role in modulating the miRNA-mediated regulation of defense-related genes during pathogen invasion.</p

Heat map with hierarchical cluster tree for differentially expressed genes.

<p>Tree classified on the basis of gene expression. Over-expressed (log2 fold change > = 1) and under-expressed (log2 fold change < = -1) genes are shown by red and green colour respectively.</p

DAVID functional annotation categories of differentially expressed genes.

<p>Significantly enriched categories for (A) up-regulated genes and (B) down-regulated genes in <i>pad</i>2.1.</p

Functionally categorized (A) down-accumulated and (B) up-accumulated proteins in response to combined stress treatment in <i>pad</i>2.1.

<p>Functionally categorized (A) down-accumulated and (B) up-accumulated proteins in response to combined stress treatment in <i>pad</i>2.1.</p

MapMan analysis of different (A) transcription factors (B) cellular redox maintaining genes.

<p>Up-regulated genes in blue and down-regulated genes in red colour respectively.</p

Transcriptomic Profiling of <i>Arabidopsis thaliana</i> Mutant <i>pad</i>2.1 in Response to Combined Cold and Osmotic Stress

<div><p>The contribution of glutathione (GSH) in stress tolerance, defense response and antioxidant signaling is an established fact. In this study transcriptome analysis of <i>pad</i>2.1, an <i>Arabidopsis thaliana</i> mutant, after combined osmotic and cold stress treatment has been performed to explore the intricate position of GSH in the stress and defense signaling network <i>in planta</i>. Microarray data revealed the differential regulation of about 1674 genes in <i>pad</i>2.1 amongst which 973 and 701 were significantly up- and down-regulated respectively. Gene enrichment, functional pathway analysis by DAVID and MapMan analysis identified various stress and defense related genes viz. members of heat shock protein family, <i>peptidyl prolyl isomerase (PPIase)</i>, <i>thioredoxin peroxidase</i> (<i>TPX</i>2), <i>glutathione-S-transferase</i> (<i>GST</i>), <i>NBS-LRR</i> type resistance protein etc. as down-regulated. The expression pattern of the above mentioned stress and defense related genes and <i>APETALA</i> were also validated by comparative proteomic analysis of combined stress treated Col-0 and <i>pad</i>2.1. Functional annotation noted down-regulation of <i>UDP-glycosyl transferase</i>, <i>4-coumarate CoA ligase</i> 8, <i>cinnamyl alcohol dehydrogenase</i> 4 <i>(CAD</i>4<i>)</i>, <i>ACC synthase</i> and <i>ACC oxidase</i> which are the important enzymes of phenylpropanoid, lignin and ethylene (ET) biosynthetic pathway respectively. Since the only difference between Col-0 (Wild type) and <i>pad</i>2.1 is the content of GSH, so, this study suggested that in addition to its association with specific stress responsive genes and proteins, GSH provides tolerance to plants by its involvement with phenylpropanoid, lignin and ET biosynthesis under stress conditions.</p></div

Effect of stress treatment on (A) GSH content and (B) GSH:GSSG ratio in Col-0 and <i>pad</i>2.1.

<p>Data are presented as mean ± SE (n = 3). Lower case letters indicate significant difference from Col-0 at ^<i>a</i><i>P</i><0.05, ^<i>b</i><i>P</i><0.01 and ^<i>c</i><i>P</i><0.001 (Student-Newman-Keuls multiple comparison test).</p

In response to combined stress treatment, (A) Functional annotation of the differentially expressed gene in Col-0 and <i>pad</i>2.1 through MapMan.

<p>Differentially expressed genes related to (B) hormones, (C) metabolic pathways and (D) secondary metabolites in Col-0 and <i>pad</i>2.1.</p

Identified genes related to (A) phenylpropanoid and lignin (B) ET biosynthetic pathways by MapMan analysis.

<p>Up-regulated genes in red and down-regulated genes in blue colour respectively.</p

Stress response overview of transcriptome altered in response to combined stress treatment in <i>pad</i>2.1 as visualized by MapMan analysis.

<p>Stress response overview of transcriptome altered in response to combined stress treatment in <i>pad</i>2.1 as visualized by MapMan analysis.</p