Abstract
In plants, lesion mimic mutants (LMMs) reveal spontaneous disease-like lesions in the absence of pathogen that constitutes powerful genetic material to unravel genes underlying programmed cell death (PCD), particularly the hypersensitive response (HR). However, only a few LMMs are reported in soybean, and no related gene has been cloned until now. In the present study, we isolated a new LMM named spotted leaf-1 (spl-1) from NN1138-2 cultivar through ethyl methanesulfonate (EMS) treatment. The present study revealed that lesion formation might result from PCD and excessive reactive oxygen species (ROS) accumulation. The chlorophyll content was significantly reduced but antioxidant activities, viz., superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the malondialdehyde (MDA) contents, were detected higher in spl-1 than in the wild-type. According to segregation analysis of mutant phenotype in two genetic populations, viz., W82×spl-1 and PI378692×spl-1, the spotted leaf phenotype of spl-1 is controlled by a single recessive gene named lm1. The lm1 locus governing mutant phenotype of spl-1 was first identified in 3.15 Mb genomic region on chromosome 04 through MutMap analysis, which was further verified and fine mapped by simple sequence repeat (SSR) marker-based genetic mapping. Genetic linkage analysis narrowed the genomic region (lm1 locus) for mutant phenotype to a physical distance of ~76.23 kb. By searching against the Phytozome database, eight annotated candidate genes were found within the lm1 region. qRT-PCR expression analysis revealed that, among these eight genes, only Glyma.04g242300 showed highly significant expression levels in wild-type relative to the spl-1 mutant. However, sequencing data of the CDS region showed no nucleotide difference between spl-1 and its wild type within the coding regions of these genes but might be in the non-coding regions such as 5′ or 3′ UTR. Hence, the data of the present study are in favor of Glyma.04g242300 being the possible candidate genes regulating the mutant phenotype of spl-1. However, further validation is needed to prove this function of the gene as well as its role in PCD, which in turn would be helpful to understand the mechanism and pathways involved in HR disease resistance of soybean.
Highlights
Plants have evolved complicated signaling pathways and defense system for protecting themselves against pathogen attack
Our results show that activities of superoxide dismutase (SOD), POD, and CAT were significantly higher in the spl-1 mutant than in wild-type plants for both High-lesion mimic (HLM) and low-lesion mimic (LLM), except for CAT that exhibited significantly lower activity at LLM in the spl-1 mutant (Figure 5C)
This can be explained because lesions are present throughout the HLM leaf from bottom to top and are at final stage of development, whereas in the case of LLM leaf lesion mimic mutant phenotype is in its initial stage, i.e., yet to be developing, and lesions are very less in number usually at the bottom of leaf as well as small in size
Summary
Plants have evolved complicated signaling pathways and defense system for protecting themselves against pathogen attack. Hypersensitive response (HR) is the most efficient and prominent response characterized by the rapid death of plants cells that come in direct contact or are close to a pathogen. Extensive efforts have been made to identify the signaling pathway as well as to identify candidate genes involved in the control and execution of the hypersensitive cell death [1,2,3,4]. Isolation and identification of mutants in which cell death is misregulated are one of the approaches used for this study. These mutants are named as lesion mimic mutants (LMMs) showing either unregulated or constitutive cell death formation that mimic the pathogen-inducible, HR cell death [5]. LMMs are very promising genetic materials for exploring the regulatory mechanisms of plant growth and defense response
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