Original research
Characterization and Fine Mapping of a Necrotic Leaf Mutant in Maize (Zea mays L.)

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Abstract

Maize (Zea mays L.) is a commercially important crop. Its yield can be reduced by mutations in biosynthetic and degradative pathways that cause death. In this paper, we describe the necrotic leaf (nec-t) mutant, which was obtained from an inbred line, 81647. The nec-t mutant plants had yellow leaves with necrotic spots, reduced chlorophyll content, and the etiolated seedlings died under normal growth conditions. Transmission electron microscopy revealed scattered thylakoids, and reduced numbers of grana lamellae and chloroplasts per cell. Histochemical staining suggested that spot formation of nec-t leaves might be due to cell death. Genetic analysis showed that necrosis was caused by the mutation of a recessive locus. Using simple sequence repeat markers, the Nec-t gene was mapped between mmc0111 and bnlg2277 on the short arm of chromosome 2. A total of 1287 individuals with the mutant phenotype from a F2 population were used for physical mapping. The Nec-t gene was located between markers T31 and H8 within a physical region of 131.7 kb.

Introduction

A plant is affected by many kinds of stresses in its life, such as water, temperature, light, soil, and microbes. These stresses can result in various types of physiological damage. Plants resist such stresses by making metabolic and structural adjustments. If a defect exists in such processes, lesions and necrosis or even programmed cell death (PCD) may result. PCD has been defined as a series of events that lead to the death of cells, tissues, or whole organs (Lockshin and Zakeri, 2004). The hypersensitive response (HR) is recognized as rapid cell death in a host plant infected by a pathogen and is considered a type of PCD (Heath, 2000).

Many dominant and recessive necrotic or lesion mimic mutations have been identified in studies of the biological mechanism of cell death, such as in Arabidopsis (reviewed by Lorrain et al., 2003), barley (reviewed by Lundqvist et al., 1997, and Rostoks et al., 2003), rice (reviewed by Kinoshita, 1998), and maize (reviewed by Johal et al., 1995). The Arabidopsis lsd1 mutant showed necrotic lesions on leaves that occurred in the absence of a pathogen (Dietrich et al., 1994). LSD1 is a zinc finger protein that regulates transcription via suppression of a pro-death pathway that stems from cells suffering hypersensitive cell death (Dietrich et al., 1997). The necrotic mutant genes ACD2 and HLM1 were cloned from Arabidopsis, and encode an enzyme involved in the breakdown of chlorophyll (Mach et al., 2001) and a cyclic nucleotide-gated ion channel (CNGC) 4, a downstream component of HR/pathogen response signaling (Balague et al., 2003), respectively. The mlo mutant of barley causes leaf lesions and elevates resistance to fungal pathogens (Büschges et al., 1997). Mlo is predicted to be a negative regulatory protein in leaf cell death (Von et al., 1998; Devoto et al., 1999). There are many Spl (spotted leaf) genes in rice, one of which, Spl7, encodes a heat stress transcription factor (HSF) protein (Yamanouchi et al., 2002). Although spl mutants including spl11, OsPti1a, and OsACRD1 show a similar lesion phenotype in the absence of stresses, these genes have different functions during pathogen infection (Wang et al., 2005; Takahashi et al., 2007; Kim et al., 2009).

In maize, a large number of lesion mimic mutations were observed with phenotypes ranging from small HR spots to gray, tan or necrotic lesions (Walbot et al., 1983). Examples include Les22, which encodes an uroporphyrinogen decarboxylase (UROD), and Lls1, which encodes an inhibitor protein with two conserved motifs of aromatic ring-hydroxylating dioxygenases. In plants, the Les22 protein plays a key role in the biosynthetic pathway for heme and chlorophyll (Hu et al., 1998), while Lls1 functions in accelerating PCD through the degradation of a toxic phenolic compound (Gray et al., 1997). Another maize mutant, Ragged leaves 1 (Rg1), shows lesions on the leaves and leaf sheaths and was mapped within a 17-kb region on chromosome 3 based on the B73 genome (Guan et al., 2012). In addition, genes that affect signal transduction or interrupt metabolic processes can also induce cell death in plants (Gray et al., 2002).

In this paper, we identified a necrotic mutant named nec-t that has a yellow and necrotic leaf phenotype caused by cell death in leaves, reduced chlorophyll content, and abnormal chloroplasts. We mapped the mutant gene to the short arm of chromosome 2 within a physical region of 131.7 kb. Here, we describe the results of phenotypic characterization, histochemical detection, and fine mapping of the mutant gene.

Section snippets

Characterization and genetic analysis of nec-t mutant

Seedlings of necrotic maize mutant nec-t were isolated as described in the Materials and methods. At the seedling stage, the leaves of nec-t were yellow and could be distinguished from the wild type (WT) at the one-leaf stage. Necrotic spots were visible on the leaves of the nec-t mutant at the two-leaf stage (Fig. 1A). The necrosis was initially confined to a small region and gradually expanded until the leaf blades presented patches (Fig. 1B and C). Eventually, the necrotic regions spread

Discussion

Many lesion mimic (necrotic) mutants have been identified in higher plants with abnormal leaf variegation or morphology. Most lesion mimic genes are associated with signal pathways for plant defense, chlorophyll metabolism or responses to abiotic stresses (Jabs et al., 1996; Zhang et al., 2003). The maize camouflage1 (cf1) mutant presented a novel pattern of cell death in leaves and the Cf1 gene was found to be involved in the production of heme and chlorophyll (Huang et al., 2009). There have

Plant materials and growth conditions

Plant materials were obtained from crosses involving the nec-t mutant of maize. The nec-t mutant was isolated from the self-pollinated progeny of an inbred line, 81647, which was heterozygous at the Nec-t locus. The homozygous siblings of 81647 were used as the wild type (WT) for phenotypic observation. The 81647 line was crossed with B73 to obtain F1 plants. All of the F1 plants, approximately 50% of which had the Nec-t allele, showed a normal green phenotype and were self-crossed to produce

Acknowledgements

This work was financially supported by the grants from the Agriculture Thoroughbred Industrialization of Shandong Province (No. 2011-7), the Ministry of Agriculture of China (Nos. 2008ZX08003-003 and 2009ZX08003-023B) and the National High-tech ‘863’ Program of China (No. 2012AA101104).

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  • 1

    These authors contributed equally to this work.

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