Mapping and Functional Analysis of LE Gene in a Lethal Etiolated Rice Mutant at Seedling Stage

An EMS(ethy methanesulfonate)-induced lethal etiolated(le)mutant obtained from the rice variety Zhongjian 100 was characterized by lethal etiolated phenotypes,with significantly reduced levels of chlorophyll a,chlorophyll b,total chlorophyll,and carotenoids.Additionally,the mutant displayed a significantly decreased number of chloroplast grana,along with irregular and less-stacked grana lamellae.The le mutant showed markedly diminished root length,root surface area,and root volume compared with the wild type.It also exhibited significantly lower catalase activity and total protein content,while peroxidase activity was significantly higher.Using the map-based cloning method,we successfully mapped the LE gene to a 48-kb interval between markers RM16107 and RM16110 on rice chromosome 3.A mutation(from T to C)was identified at nucleotide position 692 bp of LOC_Os03g59640(ChlD),resulting in a change from leucine to proline.By crossing HM133(a pale green mutant with a single-base substitution of A for G in exon 10 of ChlD subunit)with a heterozygous line of le(LEle),we obtained two plant lines heterozygous at both the LE and HM133 loci.Among 15 transgenic plants,3 complementation lines displayed normal leaf color with significantly higher total chlorophyll,chlorophyll a,and chlorophyll b contents.The mutation in le led to a lethal etiolated phenotype,which has not been observed in other ChlD mutants.The mutation in the AAA+domain of ChlD disrupted the interaction of ChlDle with ChlI as demonstrated by a yeast two-hybrid assay,leading to the loss of ChlD function and hindering chlorophyll synthesis and chloroplast development.Consequently,this disruption is responsible for the lethal etiolated phenotype in the mutant.

C-terminal frameshift mutations generate viable knockout mutants with developmental defects for three essential protein kinases

Loss-of-function mutants are fundamental resources for gene function studies.However,it is difficult to generate viable and heritable knockout mutants for essential genes.Here,we show that targeted editing of the C-terminal sequence of the embryo lethal gene MITOGEN-ACTIVATED PROTEIN KINASES 1(OsMPK1)results in weak mutants.This C-terminal-edited osmpk1 mutants displayed severe developmental defects and altered disease resistance but generated tens of viable seeds that inherited the mutations.Using the same C-terminal editing approach,we also obtained viable mutants for a wallassociated protein kinase(Os07g0493200)and a leucine-rich repeat receptor-like protein kinase(Os01g0239700),while the null mutations of these genes were lethal.These data suggest that protein kinase activity could be reduced by introducing frameshift mutations adjacent to the C-terminus,which could generate valuable resources for gene function studies and tune protein kinase activity for signaling pathway engineering.

Characterization and Fine Mapping of a Novel Vegetative Senescence Lethal Mutant Locus in Rice

Mutants showing spontaneous cell death in the absence of pathogen attack are called lesion mimic mutants （lmm） （Lorrain et al., 2003）. These mutants usually exhibit typical hypersensitive responses （HRs） within or around the lesion spots, which are frequently observed in plants challenged with avirulent pathogens （Lorrain et al., 2003）. A number of these mutants have been characterized in rice （Zeng et al., 2004）, Arabidopsis （Guo et al., 2013）, maize （Wang et al., 2013） and barley （Persson et al., 2008）. Most lmm show enhanced resistance to various pathogens （Huang et al., 2011）, because HR is usually accompanied with enhanced defense responses, such as reactive oxygen species （ROS） activation （Qiao et al., 2010） and increased expression of pathogenesis-related genes （Lorrain et al., 2003）. Additionally, most lmm exhibit defects in growth and development due to the disordered physiolog- ical and molecular processes caused by the lesion spots. Thus, lmm are powerful tools for the study of the molecular mech- anisms of cell death, plant development and disease resistance （Lorrain et al., 2003; Babu et al., 2011）.