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Identification of stably expressed reference genes for expression studies in Arabidopsis thaliana using mass spectrometry-based label-free quantification

ABSTRACT: Arabidopsis thaliana has been used regularly as a model plant in gene expression studies on transcriptional reprogramming upon pathogen infection, such as that by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), or when subjected to stress hormone treatments including jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). RT-qPCR has been extensively employed to quantitate these gene expression changes. However, the accuracy of the quantitation is largely dependent on the stability of the expressions of reference genes used for normalization. Recently, RNA-seq has been widely used to mine stably expressed genes for use as references in RT-qPCR. However, the amplification step in RNA-seq creates an intrinsic bias against those genes with relatively low expression levels, and therefore does not provide an accurate quantification of all expressed genes. In this study, we employed mass spectrometry-based label-free quantification (LFQ) in proteomic analyses to identify those proteins with abundances unaffected by Pst DC3000 infection. We verified, using RT-qPCR, that the levels of their corresponding mRNAs were also unaffected by Pst DC3000 infection. In addition, using RT-qPCR, we verified that the mRNAs were stably expressed upon stress hormone treatments including JA, SA, and ABA. Results indicated that the candidate genes identified here had stable expressions upon these stresses and are suitable to be used as reference genes for RT-qPCR. Among the 18 candidate reference genes reported in this study, many of them had greater expression stability than the commonly used reference genes, such as ACT7, in previous studies. Here, besides proposing more appropriate reference genes for Arabidopsis expression studies, we also demonstrated the capacity of mass spectrometry-based LFQ to quantify protein abundance and the possibility to extend protein expression studies to the transcript level.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Aerial Part

SUBMITTER: Sau Shan Susan

LAB HEAD: Hon-Ming Lam

PROVIDER: PXD035677 | Pride | 2022-09-05

REPOSITORIES: Pride

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Project description:Plant 9-lipoxygenases (9-LOX) and α-dioxygenases (α-DOX) initiate the synthesis of oxylipins after bacterial infection. Here, the role of these enzymes in plant’s defense was investigated using individual Arabidopsis thaliana lox1 and dox1 mutants and a double lox1 dox1 mutant. Studies with Pseudomonas syringae pv tomato (Pst) revealed the enhanced susceptibility of lox1 to the virulent strain Pst DC3000 and the partial impairment of lox1 and dox1 mutants to activate systemic acquired resistance. Notably, both defects were enhanced in the lox1 dox1 plants as compared with individual mutants. We found that pre-treatment with 9-LOX- and -DOX-generated oxylipins protected plant tissues against bacterial infection. The strongest effect in this respect was exerted by 9-ketooctadecatrienoic acid (9-KOT), which is produced from linolenic acid by 9-LOX. Quantification of 9-KOT revealed its accumulation after bacterial infection. The levels were reduced in lox1 and lox1 dox1 plants but strongly increased in the dox1 mutant due to metabolic interaction of the two pathways. Transcriptional analyses indicated that 9-KOT pre-treatment modifies hormone homeostasis during bacterial infection. The nature of the changes detected suggested that 9-KOT interfers the hormonal changes caused by bacterial effectors. This notion was substantiated by the finding that 9-KOT failed to reduce the growth of PstDC3000hrpA, a mutant compromised in effector secretion, and of the avirulent strain Pst DC3000 avrRpm1. Further support to the action of the 9-LOX- and -DOX-oxylipin pathways as modulators of hormone homeostasis was the observation that lox1 dox1 seedlings are hypersensitive to the growth-inhibitory effect of ABA and showed enhanced activation of ABA-inducible marker genes. Two experiments, Col-0 treated with 9-KOT vs. Col-0 treated with water and Col-0 treated with 9-KOT vs. Col-0 treated with water after treatment with Pst DC3000. Biological replicates: 4 control replicates treated , 4 9-KOT treated replicates; 4 control treated and Pst DC3000 replicates and 4 9-KOT treated and Pst DC3000 replicates

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Identification of stably expressed reference genes for expression studies in Arabidopsis thaliana using mass spectrometry-based label-free quantification

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