EMS-mutagenized Population Research Articles

The Role of BrKS in Leafy Head Formation Was Confirmed by Two Allelic Mutants of Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Considering that the presence of a leafy head is a key morphological characteristic that determines the yield and quality of Chinese cabbage, identifying the major genes regulating the formation of a leafy head is crucial for variety improvement. A heading-related gene, BrKS, was previously predicted from a non-heading mutant, nhm1, derived from a heading variety, which encodes a key enzyme involved in gibberellin biosynthesis. Here, another mutant, nhm2, was identified from the same EMS-mutagenized population, and the phenotype of nhm2 was consistent with that of nhm1. We crossed mutants nhm1 and nhm2, and their F1 plants exhibited the mutant phenotype, which indicated that their mutant genes were allelic. A single non-synonymous mutation in the fourth exon of BrKS in mutant nhm1 and another single non-synonymous mutation in the tenth exon of BrKS in mutant nhm2. The same gene mutation in mutants nhm1 and nhm2 produced a similar non-heading phenotype, which confirmed the role of BrKS in the leafy head formation of Chinese cabbage. RNA-Seq analysis indicated that a transcription factor gene, BrERF1A, which is associated with leaf development, significantly down-regulated expression in mutant nhm1, and after the mutant was treated with GA3, the expression level of BrERF1A was recovered, which indicated that BrKS might be involved in leafy head formation through regulating the expression level of BrERF1A. Our findings provide important clues for revealing the molecular mechanism of leafy head formation in Chinese cabbage.

Cloning and functional verification of the male sterile gene BrQRT3 in Chinese cabbage

Chinese cabbage is a cross-pollinated crop with significant heterosis, and male sterile lines are an important way to produce hybrid seeds. In this study, a male sterile mutant msm0795 was identified in an EMS-mutagenized population of Chinese cabbage. Cytological observations revealed that the microspores failed to separate after the tetrad stage, and thus developed into abnormal pollen grains, resulting in anther abortion. MutMap combined with Kompetitive Allele Specific PCR genotyping showed that BraA01g011280.3.5 C was identified as the candidate gene, which encodes polygalacturonase QRT3 and plays a direct role in the degradation of pollen mother cell wall during microspore development, named BrQRT3. Subcellular localization and expression analyses demonstrated that BrQRT3 was localized in the cell membrane and was ubiquitously expressed in roots, stems, leaves, flower buds, and flowers, but the expression of BrQRT3 was gradually suppressed with the anther development. Ectopic expression confirmed that over-expression of BrQRT3 in qrt3 background Arabidopsis mutant can rescue the pollen defects caused by loss of AtQRT3 function. It is the first time to achieve a male sterile mutant caused by the mutation of BrQRT3 in Chinese cabbage. These findings contribute to elucidate the mechanism of BrQRT3 in regulating stamen development of Chinese cabbage.

Identification and characterization of CsERECTA, a major gene controlling stem elongation through regulating GA biosynthesis in cucumber.

Dwarfing is an ideal agronomic trait in crop breeding, which can improve lodging resistance and increase crop productivity. In this study, we identified a dwarf mutant cp-3 from an EMS-mutagenized population, which had extremely short internodes, and the cell length and number of internodes were significantly reduced. Meanwhile, exogenous GA3 treatment partially rescued the plant height of the cp-3. Inheritance analysis showed that the cp-3 mutant was regulated via a recessive nuclear locus. A candidate gene, CsERECTA, encoding an LRR receptor-like serine/threonine-protein kinase, was cloned through a map-based cloning strategy. Sequence analysis showed that a nucleotide mutation (C ~ T) in exon 26 of CsERECTA led to premature termination of the protein. Subsequently, two transgenic lines were generated using the CRISPR/Cas9 system, and they showed plant dwarfing. Plant endogenous hormones quantitative and RNA-sequencing analysis revealed that GA3 content and the expression levels of genes related to GA biosynthesis were significantly reduced in Cser knockout mutants. Meanwhile, exogenous GA3 treatment partially rescued the dwarf phenotype of Cser knockout mutants. These findings revealed that CsERECTA controls stem elongation by regulating GA biosynthesis in cucumber.

Development and characterization of an EMS-mutagenized population of wheat (Triticum aestivum L.) for agronomic trait variation and increased micronutrients content

Development and characterization of an EMS-mutagenized population of wheat (Triticum aestivum L.) for agronomic trait variation and increased micronutrients content

Screening and Resistance Locus Identification of the Mutant fcrZ22 Resistant to Crown Rot Caused by Fusarium pseudograminearum.

Crown rot caused by Fusarium pseudograminearum is a devastating wheat disease worldwide. In addition to yield losses, the fungi causing Fusarium crown rot (FCR) also deteriorate the quality and safety of food because of the production of mycotoxins. Planting resistant cultivars is an effective way to control FCR. However, most wheat cultivars are susceptible to FCR. Therefore, development of new sources and detection of loci for FCR resistance are necessary. In the present study, a resistant mutant, fcrZ22, was identified from an ethyl methane sulfonate (EMS)-mutagenized population of the cultivar Zhoumai 22, and then fcrZ22 was crossed with the wild type to produce an F2 population. Genetic analysis of the F2 population was carried out by the mixed inheritance model of major genes plus polygenes, and 20 resistant and 20 susceptible plants were selected to assemble mixed pools. Combining 660K SNP arrays, the resistance loci were detected by bulked segregant analysis. The resistance to FCR caused by F. pseudograminearum in the F2 population was in accordance with the "mixed model with two major genes of additive-epistasis effect + additive-dominant polygenes," and the heritability of the major gene was 0.92. Twenty-one loci were detected, which were located on 10 chromosomes, namely, 1B (1), 1D (1), 2A (3), 1B (1), 3A (3), 3B (3), 4A (2), 5A (2), 7A (3), and 7B (2). Among the 21 loci, eight were new loci for FCR resistance. This is the first report of detecting loci for FCR resistance from a mutant. The results of the present study provided excellent germplasm resources for breeding wheat cultivars with FCR resistance and laid the foundation for fine mapping of FCR resistance loci.

LESION MIMIC MUTANT 1 confers basal resistance to Sclerotinia sclerotiorum in rapeseed via a salicylic acid-dependent pathway.

Rapeseed (Brassica napus) is a major edible oilseed crop consumed worldwide. However, its yield is seriously affected by infection from the broad-spectrum non-obligate pathogen Sclerotinia sclerotiorum due to a lack of highly resistant germplasm. Here, we identified a Sclerotinia-resistant and light-dependent lesion mimic mutant from an ethyl methanesulfonate-mutagenized population of the rapeseed inbred Zhongshuang 11 (ZS11) named lesion mimic mutant 1 (lmm1). The phenotype of lmm1 is controlled by a single recessive gene, named LESION MIMIC MUTANT 1 (LMM1), which mapped onto chromosome C04 by bulked segregant analysis within a 2.71-Mb interval. Histochemical analysis indicated that H2O2 strongly accumulated and cell death occurred around the lesion mimic spots. Among 877 differentially expressed genes (DEGs) between ZS11 and lmm1 leaves, 188 DEGs were enriched in the defense response, including 95 DEGs involved in systemic acquired resistance, which is consistent with the higher salicylic acid levels in lmm1. Combining bulked segregant analysis and transcriptome analysis, we identified a significantly up-regulated gene, BnaC4.PR2, which encodes β-1,3-glucanase, as the candidate gene for LMM1. Overexpression of BnaC4.PR2 may induce a reactive oxygen species burst to trigger partial cell death and systemic acquired resistance. Our study provides a new genetic resource for S. sclerotiorum resistance as well as new insights into disease resistance breeding in B. napus.

AGRONOMIC CHARACTERIZATION OF AN EMS-MUTAGENIZED POPULATION FOR SELECTING HIGH-YIELDING AND GLUTENENRICHED INDUSTRIAL WHEAT

A mutant population was generated using a popular wheat cultivar of Bangladesh; BARI GOM-28, with ethyl methanesulfonate (EMS) to create diversity in terms of different agronomic characteristics and grain quality parameters. An EMS concentration ranging from 0.2% to 1.2% was used and the optimum was found to be 0.8%. This study was initiated with 16,000 seeds, where 1,581 lines survived under greenhouse conditions up to M4 generation. Through 3 subsequent field trials, 3 promising lines, namely, 0037/17, 0020/17 and 0023/17 were selected with enhanced spike length, number of spikes per plant, number of spikelets per spike, number of grains per spike, grains weight per plant and 1000-grain weight. Such improved agronomic traits contributed towards a greater yield potential of 0037/17 (5.94-6.10 t ha-1), 0020/17 (5.47-5.54 t ha-1) and 0023/17 (4.97-5.20 t ha-1) than BARI GOM-28 (3.63-3.69 t ha-1) in the multi-location trial. Improvement in certain bread-making qualities like wet gluten content (>28%) and total protein content (~13%) was also observed and compared to BARI GOM-28 which had 22% wet gluten and 11% total protein. Therefore, these mutant lines could be used as a valuable resource for genetic studies to dissect the function of the genes controlling such desired parameters as well as superior breeding lines.

The reverse mutation of CsMLO8 results in susceptibility to powdery mildew via inhibiting cell wall apposition formation and cell death in cucumber (Cucumis sativus L.)

Powdery mildew (PM) is an obligate biotrophic pathogen that requires living cells to grow and reproduce, causing huge losses in cucumbers. However, the resistance mechanism against PM in cucumber remains poorly understood. In this study, a susceptible mutant m6326 was found from the EMS-mutagenized population of No.26. Genetic analysis of the F2 population and sequence analysis showed that No.26 was a natural mlo mutant, and m6326 was a CsMLO8 revertant. The phytopathological investigation and transcriptome analysis of the resistant line (No.26) and susceptible revertant (m6326) after PM infection were carried out. The higher rate of spore penetration, longer hypha and more conidia in m6326 were observed than that in No.26. Histological analysis showed that the formation of cell wall apposition and cell death in No.26 leaves was faster and more than that in m6326 after powdery mildew infection. Transcriptome analysis identified differentially expressed genes between the resistant and susceptible lines. The phenylpropanoid biosynthesis pathway and ROS scavenging genes were significantly up-regulated in No.26 compared with that in m6326. Our findings suggested that the reverse mutation of CsMLO8 results in susceptibility to powdery mildew, which was related to cell wall apposition formation and cell death.

A Tomato EMS-Mutagenized Population Provides New Valuable Resources for Gene Discovery and Breeding of Developmental Traits.

Tomato (Solanum lycopersicum L.) is a major horticultural crop and a model species among eudicots, especially for traits related to reproductive development. Although considerable progress has been made since the tomato genome sequence project was completed, most of the genes identified remain predictions with an unknown or hypothetical function. This lack of functional characterization hampers the use of the huge amount of genomic information available to improve the quality and productivity of this crop. Reverse genetics strategies such as artificial mutagenesis and next-generation sequencing approaches build the perfect tandem for increasing knowledge on functional annotation of tomato genes. This work reports the phenotypic characterization of a tomato mutant collection generated from an EMS chemical mutagenesis program aimed to identify interesting agronomic mutants and novel gene functions. Tomato mutants were grouped into fourteen phenotypic classes, including vegetative and reproductive development traits, and the inheritance pattern of the identified mutations was studied. In addition, causal mutation of a selected mutant line was isolated through a mapping-by-sequencing approach as a proof of concept of this strategy’s successful implementation. Results support tomato mutagenesis as an essential tool for functional genomics in this fleshy-fruited model species and a highly valuable resource for future breeding programs of this crop species aimed at the development of more productive and resilient new varieties under challenging climatic and production scenarios.

A putative SUBTILISIN-LIKE SERINE PROTEASE 1 (SUBSrP1) regulates anther cuticle biosynthesis and panicle development in rice

IntroductionPanicle abortion is a severe physiological defect and causes a reduction in grain yield. ObjectivesIn this study, we aim to provide the characterization and functional analysis of a mutant apa1331 (apical panicle abortion1331). MethodsThe isolated mutant from an EMS-mutagenized population was subjected to SSR analysis and Mutmap assay for candidate gene mapping. We performed phenotypic analysis, anthers cross-sections morphology, wax and cutin profiling, biochemical assays and phylogenetic analysis for characterization and evaluation of apa1331. We used CRISPR/Cas9 disruption for functional validation of its candidate gene. Furthermore, comparative RNA-seq and relative expression analysis were performed to get further insights into mechanistic role of the candidate gene. ResultsThe anthers from the apical spikelets of apa1331 were degenerated, pollen-less and showed defects in cuticle formation. Transverse sections of apa1331 anthers showed defects in post-meiotic microspore development at stage 8–9. Gas Chromatography showed a significant reduction of wax and cutin in anthers of apa1331 compared to Wildtype (WT). Quantification of H2O2 and MDA has indicated the excessive ROS (reactive oxygen species) in apa1331. Trypan blue staining and TUNEL assay revealed cell death and excessive DNA fragmentation in apa1331. Map-based cloning and Mutmap analysis revealed that LOC_Os04g40720, encoding a putative SUBTILISIN-LIKE SERINE PROTEASE (OsSUBSrP1), harbored an SNP (A > G) in apa1331. Phenotypic defects were only seen in apical spikelets due to highest expression of OsSUBSrP1 in upper panicle portion. CRISPR-mediated knock-out lines of OsSUBSrP1 displayed spikelet abortion comparable to apa1331. Global gene expression analysis revealed a significant downregulation of wax and cutin biosynthesis genes. ConclusionsOur study reports the novel role of SUBSrP1 in anther cuticle biosynthesis by ROS-mediated programmed cell death in rice.

GWC1 is essential for high grain quality in rice

Appearance quality is an important determinant of rice quality. Many genes that affect grain appearance quality have been identified, but the regulatory mechanisms that contribute to this trait remain unclear. Here, two grains with chalkiness (gwc1) mutants, gwc1−1 and gwc1−2, were identified from an EMS-mutagenized population of indica rice cultivar Shuhui498 (R498). The gwc1 mutants had poor grain appearance quality consistent with the measured values for the percentage of grains with chalkiness, square of chalky endosperm, the total starch, amylose and sucrose contents. Milling quality and grain size were also affected in the gwc1 mutants. The gwc1−1 and gwc1−2 were found to be loss-of-function allelic mutants. GWC1 was mapped to the long arm of rice chromosome 8 using the MutMap strategy and incorrectly annotated in the reference genome for Nipponbare (MSU). The GWC1 gene corresponds to the WTG1/OsOTUB1 gene, which encodes an otubain-like protease with deubiquitinating activity that is homologous to human OTUB1. GWC1 transcripts accumulated to high levels in early endosperm after fertilization and developing inflorescences, and GWC1−green fluorescent protein (GFP) signal was detected in the nucleus and cytoplasm. GWC1 is likely to regulate grain appearance quality through genes involved in sucrose metabolism and starch biosynthesis. Overall, the present findings reveal that GWC1 is important for grain quality and yield due to its effects on grain chalkiness and size.

A single-base change at a splice site in Wx-A1 caused incorrect RNA splicing and gene inactivation in a wheat EMS mutant line.

An EMS-induced single-base mutation at a splice site caused abnormal RNA splicing and resulted in the gene inactivation and the lack of Wx-A1 protein in a wheat EMS mutant line. An EMS-mutagenized population was generated using common wheat cv. SM126 consisting of 10,600 M2 plants. One Wx-A1 null mutant was identified through analyses of 390 grains produced from 130 M2 plants using electrophoresis analyses. The Wx-A1 sequences of parental line SM126 and M2-31 mutant were determined as 2781bp, and there was only one SNP mutation between them. The SNP was a mutation from G to A at nucleotide sequence position 2168bp (G2168A) downstream of the start codon which was located at the splicing site within the eighth intron. All 52 cDNA transcripts were found to be incorrectly spliced and can be summarized as five types of variations. The deletion of the exon and the exclusion of intron were structural features in abnormal splicing RNA. Together with the prediction of potential splice regulatory motifs, the mutation G2168A happened within the 5' splice site of the eighth intron and destroyed the splice donor site from GU to AU, which may have brought about a barrier against correct RNA splice, and generated abnormal mRNA, which was the mechanism of the inactivation of Wx-A1 in M2-31. The lack of Wx-A1 has resulted in changes in starch properties in the M2-31 mutant, with the reduction in amylose and starch contents. The increased grains hardness was observed in M2-31, which may be related to the lower expression level of Pinb-D1 gene. As the waxy wheat foods have a lot of advantages, the null waxy genes will be widely applied in breeding waxy wheat for varied amylose contents.

YO-09 - Genetic screen of Arabidopsis cat2 suppressor mutants to understand the signaling pathways leading to photorespiratory H2O2-induced cell death

Abiotic stresses, such as heat, drought, flooding, high salinity and high light, contribute to the reduction of plant yield and are going to occur more and more often due to climate change. In order to prevent the yield loss it is therefore crucial to understand the mechanisms of plant stress signaling. In response to adverse environmental conditions that limit CO2 availability photorespiration in plant tissues increases, which leads to enhanced H2O2 production in peroxisomes. Arabidopsis thaliana has three catalase isoforms that scavenge peroxisomal H2O2, with catalase 2 reaching highest transcript and protein levels. Upon transfer to photorespiratory conditions cat2 mutants acumulate H2O2 and eventually die. To understand the mechanisms of ROS-induced cell death, we have conducted a forward genetic screen of an EMS-mutagenized population of cat2 and isolated 21 suppressor mutants that revert the photorespiratory phenotype. The causative mutations were revealed by Next Generation Sequencing followed by SHORE/SHOREmap analysis. To further validate the mutations we described the stress responses, performed metabolomic and transcriptomic profiling of double mutant lines lacking cat2 and the gene of interest. So far we have published our findings on the loss of-function mutations in GLYCOLATE OXIDASE 1 (GOX1) and SHORT ROOT (SHR) genes, with several others currently in progress.

Development and Genetic Characterization of A Novel Herbicide (Imazethapyr) Tolerant Mutant in Rice (Oryza sativa L.)

BackgroundIncreased water and labour scarcity in major rice growing areas warrants a shift towards direct seeded rice cultivation under which management of weeds is a major issue. Use of broad spectrum non-selective herbicides is an efficient means to manage weeds. Availability of rice genotypes with complete tolerance against broad-spectrum non-selective herbicides is a pre-requisite for advocating use of such herbicides. In the present study, we developed an EMS induced rice mutant, ‘HTM-N22‘, exhibiting tolerance to a broad spectrum herbicide, ‘Imazethapyr‘, and identified the mutations imparting tolerance to the herbicide.ResultsWe identified a stable and true breeding rice mutant, HTM-N22 (HTM), tolerant to herbicide, Imazethapyr, from an EMS-mutagenized population of approximately 100,000 M2 plants of an upland rice variety, Nagina 22 (N22). Analysis of inheritance of herbicide tolerance in a cross between Pusa 1656-10-61/HTM showed that this trait is governed by a single dominant gene. To identify the causal gene for Imazethapyr tolerance, bulked segregant analysis (BSA) was followed using microsatellite markers flanking the three putative candidate genes viz., an Acetolactate Synthase (ALS) on chromosome 6 and two Acetohydroxy Acid Synthase (AHAS) genes, one on chromosomes 2 and another on chromosome 4. RM 6844 on chromosome 2 located 0.16 Mbp upstream of AHAS (LOC_Os02g30630) was found to co-segregate with herbicide tolerance. Cloning and sequencing of AHAS (LOC_Os02g30630) from the wild type, N22 and the mutant HTM and their comparison with reference Nipponbare sequence revealed several Single Nucleotide Polymorphisms (SNPs) in the mutant, of which eight resulted in non-synonymous mutations. Three of the eight amino acid substitutions were identical to Nipponbare and hence were not considered as causal changes. Of the five putative candidate SNPs, four were novel (at positions 30, 50, 81 and 152) while the remaining one, S627D was a previously reported mutant, known to result in Imidazolinone tolerance in rice. Of the novel ones, G152E was found to alter the hydrophobicty and abolish an N myristoylation site in the HTM compared to the WT, from reference based modeling and motif prediction studies.ConclusionsA novel mutant tolerant to the herbicide “Imazethapyr” was developed and characterized for genetic, sequence and protein level variations. This is a HTM in rice without any IPR (Intellectual Property Rights) infringements and hence can be used in rice breeding as a novel genetic stock by the public funded organizations in the country and elsewhere.

A new temperature-insensitive allele of the Arabidopsis AXR6/CUL1 locus derived from a missense mutation in the C-terminal RBX1 binding region

We isolated a new recessive allele at the AUXIN RESISTANT6/CULLIN1 (AXR6/CUL1) locus, axr6–101, from an EMS-mutagenized population of Arabidopsis thaliana, the Landsberg erecta ecotype. axr6–101 is auxin resistant and semi-dwarf similar to the other recessive axr6 mutants. The axr6–101 phenotype is caused by the E716K substitution of the CUL1 protein, which is likely to affect its ability to bind to the C-terminal RING domain of RING-box 1 (RBX1). The previously reported allele of AXR6, cul1–7, is caused by a substitution at T510 that binds to the N-terminal β-strand of RBX1. Although cul1–7 shows temperature-sensitive phenotype, the axr6–101 phenotype is largely unaffected by temperature. axr6–101 may provide an important genetic resource for study of the structure−function relationship of the CUL1 protein.

A Method for Screening Sensors of Calcium Signaling in Rice

Although rice is an important staple food crop of nearly half of the world population, the discoveries in calcium ion signal were mainly focused on the model plant Arabidopsis and researches in rice are very superficial and no calcium ion sensors have been found yet. Recently, the recombinant aequorin was introduced into rice, and the Ca2+ responses to NaCl and H2O2 in rice roots were characterized. This system can also be used to identify novel Ca2+-coupled stress sensors or pathways after the establishment of an EMS-mutagenized population. In this paper, we present the screening design of the specific program in details.

Characterization of novel Sorghum brown midrib mutants from an EMS-mutagenized population.

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles.

Characterization of fs10.1, a major QTL controlling fruit elongation in Capsicum

We previously identified fs10.1 as a major QTL controlling fruit shape (index of length to width) in an interspecific F(2) cross of Capsicum annuum (round fruit)×C. chinense (elongated fruit) in pepper. To more precisely map and characterize the QTL, we constructed near-isogenic lines for fs10.1 and mapped it in a BC(4)F(2) population. In this population, fs10.1 segregated as a Mendelian locus and mapped 0.3cM away from the closest molecular marker. We further verified the effect of fs10.1 in an F(2) population from an independent cross between elongated- and conical-fruited parents. To identify additional allelic variation at fruit shape loci, we screened an EMS-mutagenized population of the blocky-fruited cv. Maor and identified the mutant E-1654 with elongated fruit. This fruit shape mutation was mapped to the fs10.1 region and was determined to be allelic to the QTL. By measuring fruit shape of near-isogenic lines for fs10.1 during fruit development, we found that the shape of the fruit is determined primarily in the first 2weeks after anthesis. Histological measurements of cell size and cell shape in pericarp sections of fruits of the isogenic lines throughout fruit development indicated that the shape of the fruit is determined primarily by cell shape and that the development of fruit shape is correlated with cell shape.

Genetic Analysis and Gene Mapping of Short Root Mutant Rice ksr1

A short root mutant ksr1 with the Kasalath background was isolated from an EMS-mutagenized population in rice. The root length of 6-day-old ksr1 seedlings was only about 20% of the wild type. Genetic analysis indicated that the short root phenotype of ksr1 was controlled by a recessive mutation in a single nuclear-encoded gene. To map the ksr1 mutation, an F 2 population was generated by crossing the ksr1 mutant with Nipponbare. The KSR1 locus was linked to the SSR marker RM1223 on rice chromosome 4. Eight new SSR markers and two InDel markers were developed around this marker. KSR1 gene was further mapped to a 155 kb region, flanked by the InDel marker 4-24725K and the SSR marker RM17182.

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.