A genome-wide association study identifies genes associated with cuticular wax metabolism in maize.

The plant cuticle is essential in plant defense against biotic and abiotic stresses. To systematically elucidate the genetic architecture of maize (Zea mays L.) cuticular wax metabolism, 2 cuticular wax-related traits, the chlorophyll extraction rate (CER) and water loss rate (WLR) of 389 maize inbred lines, were investigated and a genome-wide association study (GWAS) was performed using 1.25 million single nucleotide polymorphisms (SNPs). In total, 57 nonredundant quantitative trait loci (QTL) explaining 5.57% to 15.07% of the phenotypic variation for each QTL were identified. These QTLs contained 183 genes, among which 21 strong candidates were identified based on functional annotations and previous publications. Remarkably, 3 candidate genes that express differentially during cuticle development encode ß-ketoacyl-CoA synthase (KCS). While ZmKCS19 was known to be involved in cuticle wax metabolism, ZmKCS12 and ZmKCS3 functions were not reported. The association between ZmKCS12 and WLR was confirmed by resequencing 106 inbred lines, and the variation of WLR was significant between different haplotypes of ZmKCS12. In this study, the loss-of-function mutant of ZmKCS12 exhibited wrinkled leaf morphology, altered wax crystal morphology, and decreased C32 wax monomer levels, causing an increased WLR and sensitivity to drought. These results confirm that ZmKCS12 plays a vital role in maize C32 wax monomer synthesis and is critical for drought tolerance. In sum, through GWAS of 2 cuticular wax-associated traits, this study reveals comprehensively the genetic architecture in maize cuticular wax metabolism and provides a valuable reference for the genetic improvement of stress tolerance in maize.

Characterization and fine mapping of a maize lesion mimic mutant (Les8) with enhanced resistance to Curvularia leaf spot and southern leaf blight.

KEY MESSAGE: A novel light-dependent dominant lesion mimic mutant with enhanced multiple disease resistance was physiologically, biochemically, and genetically characterized; the causal gene was fine mapped to a 909 kb interval containing 38 genes. Identification of genes that confer multiple disease resistance (MDR) is crucial for the improvement of maize disease resistance. However, very limited genes are identified as MDR genes in maize. In this study, we characterized a dominant disease lesion mimics 8 (Les8) mutant that had chlorotic lesions on the leaves and showed enhanced resistance to both curvularia leaf spot and southern leaf blight. Major agronomic traits were not obviously altered, while decreased chlorophyll content was observed in the mutant, and the genetic effect of the Les8 mutation was stable in different genetic backgrounds. By BSR-seq analysis and map-based cloning, the LES8 gene was mapped into a 909 kb region containing 38 candidate genes on chromosome 9 wherein no lesion mimic or disease-resistance genes were previously reported. Using transcriptomics analysis, we found that genes involved in defense responses and secondary metabolite biosynthesis were enriched in the significantly up-regulated genes, while genes involved in photosynthesis and carbohydrate-related pathways were enriched in the significantly down-regulated genes in Les8. In addition, there was an overaccumulation of jasmonic acid and lignin but not salicylic acid in Les8. Taken together, this study revealed candidate genes and potential mechanism underlying Les8-conferred MDR in maize.

Efficient breeding of low glutelin content rice germplasm by simultaneous editing multiple glutelin genes via CRISPR/Cas9.

Chronic kidney disease (CKD) and phenylketonuria (PKU) patients need to eat rice with low glutelin content. Therefore, breeding low glutelin content rice varieties with high yield and delicious taste is one of the major goals of rice breeders due to the high demand for the product. In this study, we designed three sgRNAs targeting nine glutelin genes and generated nine T-DNA-free homozygous editing lines with reduced glutelin content compared with the wild-type due to simultaneous mutation(s) in 5-7 glutelin genes. The glutelin content of two lines is even significantly lower than that of the low glutelin content cultivar, LGC-1. Compared to the wild-type, these low glutelin lines showed similar agronomic traits, including yield components and viscosity properties, and can be used as new varieties or parental materials for further breeding.

Underlying mechanism of accelerated cell death and multiple disease resistance in a maize lethal leaf spot 1 allele.

Multiple disease resistance (MDR) in maize has attracted increasing attention. However, the interplay between cell death and metabolite changes and their contributions to MDR remains elusive in maize. In this study, we identified a mutant named as lesion mimic 30 (les30) that showed 'suicidal' lesion formation in the absence of disease and had enhanced resistance to the fungal pathogen Curvularia lunata. Using map-based cloning, we identified the causal gene encoding pheophorbide a oxidase (PAO), which is known to be involved in chlorophyll degradation and MDR, and is encoded by LETHAL LEAF SPOT1 (LLS1). LLS1 was found to be induced by both biotic and abiotic stresses. Transcriptomics analysis showed that genes involved in defense responses and secondary metabolite biosynthesis were mildly activated in leaves of the les30 mutant without lesions, whilst they were strongly activated in leaves with lesions. In addition, in les30 leaves with lesions, there was overaccumulation of defense-associated phytohormones including jasmonic acid and salicylic acid, and of phytoalexins including phenylpropanoids, lignin, and flavonoids, suggesting that their biosynthesis was activated in a lesion-dependent manner. Taken together, our study implies the existence of an interactive amplification loop of interrupted chlorophyll degradation, cell death, expression of defense-related genes, and metabolite changes that results in suicidal lesion formation and MDR, and this has the potential to be exploited by genetic manipulation to improve maize disease resistance.

Characterization and Functional Analysis of Pyrabactin Resistance-Like Abscisic Acid Receptor Family in Rice.

BACKGROUND: Abscisic acid (ABA) plays crucial roles in regulating plant growth and development, especially in responding to abiotic stress. The pyrabactin resistance-like (PYL) abscisic acid receptor family has been identified and widely characterized in Arabidopsis. However, PYL families in rice were largely unknown. In the present study, 10 out of 13 PYL orthologs in rice (OsPYL) were isolated and investigated. RESULTS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that expression of OsPYL genes is tissue-specific and display differential response to ABA treatment, implying their functional diversity. The interaction between 10 OsPYL members and 5 protein phosphatase 2C in rice (OsPP2C) members was investigated in yeast two-hybrid and tobacco transient expression assays, and an overall interaction map was generated, which was suggestive of the diversity and complexity of ABA-sensing signaling in rice. To study the biological function of OsPYLs, two OsPYL genes (OsPYL3 and OsPYL9) were overexpressed in rice. Phenotypic analysis of OsPYL3 and OsPYL9 transgenic rice showed that OsPYLs positively regulated the ABA response during the seed germination. More importantly, the overexpression of OsPYL3 and OsPYL9 substantially improved drought and cold stress tolerance in rice. CONCLUSION: Taken together, we comprehensively uncovered the properties of OsPYLs, which may be good candidates for the improvement of abiotic stress tolerance in rice.

Characterization and subcellular localization of two 14-3-3 genes and their response to abiotic stress in wheat.

In order to investigate biological functions of the 14-3-3 genes and their response to abiotic stress, two cDNAs (designated as Ta14R1 and Ta14R2) encoding putative 14-3-3 proteins were isolated from wheat by PCR and rapid amplification of cDNA end (RACE) technique. The cDNA of Ta14R1 is 999bp and encodes a protein of 262 amino acids, while the cDNA of Ta14R2 is 897bp in length and encodes a protein of 261 amino acids. Transient expression assays using Ta14R1/Ta14R2-GFP fusion constructs indicated that Ta14R1 and Ta14R2 were located in cytoplasm and cell membrane but not in chloroplasts. Real-time quantitative (RT-PCR) analysis revealed that Ta14R1 and Ta14R2 were differentially expressed in wheat tissues and significantly up-regulated in roots and shoots 1d after germination, indicating they may play a role in process of seed germination. The expression of the two genes in roots and leaves were significantly induced by plant hormone ABA, as well as heat, cold and drought treatments, suggesting that the two 14-3-3 genes in wheat may be involved in ABA dependent stress-responding pathway and response to heat, cold and drought stress.

The role of thioredoxin h in protein metabolism during wheat (Triticum aestivum L.) seed germination.

Thioredoxin h can regulate the redox environment in the cell and play an important role in the germination of cereals. In the present study, the thioredoxin s antisense transgenic wheat with down-regulation of thioredoxin h was used to study the role of thioredoxin h in protein metabolism during germination of wheat seeds, and to explore the mechanism of the thioredoxin s antisense transgenic wheat seeds having high resistance to pre-harvest sprouting. The qRT-PCR results showed that the expression of protein disulfide isomerase in the thioredoxin s antisense transgenic wheat was up-regulated, which induced easily forming glutenin macropolymers and the resistance of storage proteins to degradation. The expression of serine protease inhibitor was also up-regulated in transgenic wheat, which might be responsible for the decreased activity of thiocalsin during the germination. The expression of WRKY6 in transgenic wheat was down-regulated, which was consistent with the decreased activity of glutamine oxoglutarate aminotransferase. In transgenic wheat, the activities of glutamate dehydrogenase, glutamic pyruvic transaminase and glutamic oxaloacetic transaminase were down-regulated, indicating that the metabolism of amino acid was lower than that in wild-type wheat during seed germination. A putative model for the role of thioredoxin h in protein metabolism during wheat seed germination was proposed and discussed.

Identification of changes in wheat (Triticum aestivum L.) seeds proteome in response to anti-trx s gene.

BACKGROUND: Thioredoxin h (trx h) is closely related to germination of cereal seeds. The cDNA sequences of the thioredoxin s (trx s) gene from Phalaris coerulescens and the thioredoxin h (trx h) gene from wheat are highly homologous, and their expression products have similar biological functions. Transgenic wheat had been formed after the antisense trx s was transferred into wheat, and it had been certified that the expression of trx h decreased in transgenic wheat, and transgenic wheat has high resistance to pre-harvest sprouting. METHODOLOGY/PRINCIPAL FINDINGS: Through analyzing the differential proteome of wheat seeds between transgenic wheat and wild type wheat, the mechanism of transgenic wheat seeds having high resistance to pre-harvest sprouting was studied in the present work. There were 36 differential proteins which had been identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). All these differential proteins are involved in regulation of carbohydrates, esters, nucleic acid, proteins and energy metabolism, and biological stress. The quantitative real time PCR results of some differential proteins, such as trx h, heat shock protein 70, α-amylase, ß-amylase, glucose-6-phosphate isomerase, 14-3-3 protein, S3-RNase, glyceraldehyde-3-phosphate dehydrogenase, and WRKY transcription factor 6, represented good correlation between transcripts and proteins. The biological functions of many differential proteins are consistent with the proposed role of trx h in wheat seeds. CONCLUSIONS/SIGNIFICANCE: A possible model for the role of trx h in wheat seeds germination was proposed in this paper. These results will not only play an important role in clarifying the mechanism that transgenic wheat has high resistance to pre-harvest sprouting, but also provide further evidence for the role of trx h in germination of wheat seeds.

Transgenic barley with overexpressed PTrx increases aluminum resistance in roots during germination.

A transgenic barley line (LSY-11-1-1) with overexpressed Phalaris coerulescens thioredoxin gene (PTrx) was employed to measure the growth, protein oxidation, cell viability, and antioxidase activity in barley roots during germination on the presence of 2 mmol/L AlCl(3) on filter paper. The results show that (1) compared with the non-transgenic barley, LSY-11-1-1 had enhanced root growth, although both were seriously inhibited after AlCl(3) treatment; (2) the degree of protein oxidation and loss of cell viability in roots of LSY-11-1-1 were much less than those in roots of non-transgenic barley, as reflected by lower contents of protein carbonyl and Evans blue uptakes in LSY-11-1-1; (3) activities of catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR) in LSY-11-1-1 root tips were generally higher than those in non-transgenic barley root tips, although these antioxidase activities gave a rise to different degrees in both LSY-11-1-1 and non-transgenic barley under aluminum stress. These results indicate that overexpressing PTrx could efficiently protect barley roots from oxidative injury by increasing antioxidase activity, thereby quenching ROS caused by AlCl(3) during germination. These properties raise the possibility that transgenic barley with overexpressed PTrx may be used to reduce the aluminum toxicity in acid soils.

The level of expression of thioredoxin is linked to fundamental properties and applications of wheat seeds.

Work with cereals (barley and wheat) and a legume (Medicago truncatula) has established thioredoxin h (Trx h) as a central regulatory protein of seeds. Trx h acts by reducing disulfide (S-S) groups of diverse seed proteins (storage proteins, enzymes, and enzyme inhibitors), thereby facilitating germination. Early in vitro protein studies were complemented with experiments in which barley seeds with Trx h overexpressed in the endosperm showed accelerated germination and early or enhanced expression of associated enzymes (alpha-amylase and pullulanase). The current study extends the transgenic work to wheat. Two approaches were followed to alter the expression of Trx h genes in the endosperm: (1) a hordein promoter and its protein body targeting sequence led to overexpression of Trx h5, and (2) an antisense construct of Trx h9 resulted in cytosolic underexpression of that gene (Arabidopsis designation). Underexpression of Trx h9 led to effects opposite to those observed for overexpression Trx h5 in barley-retardation of germination and delayed or reduced expression of associated enzymes. Similar enzyme changes were observed in developing seeds. The wheat lines with underexpressed Trx showed delayed preharvest sprouting when grown in the greenhouse or field without a decrease in final yield. Wheat with overexpressed Trx h5 showed changes commensurate with earlier in vitro work: increased solubility of disulfide proteins and lower allergenicity of the gliadin fraction. The results are further evidence that the level of Trx h in cereal endosperm determines fundamental properties as well as potential applications of the seed.

Protective role of exogenous nitric oxide against oxidative-stress induced by salt stress in barley (Hordeum vulgare).

To probe into the potential of relieving the oxidative damage of salt stress, we investigated the protective role of nitric oxide on barley under salt stress. Salt stress resulted in increased ion leakage, lipid peroxidation and protein oxidation in barley leaves. Simultaneous treatments of barley leaves with 50 microM sodium nitroprusside, a nitric oxide donor, alleviated the damage of salt stress, reflected by decreased ion leakage, and malendialdehyde (MDA), carbonyl, and hydrogen peroxide content in barley leaves. The presence of the nitric oxide donor increased the activities of superoxide dismutases (SOD), ascorbate peroxidases (APX), and catalases (CAT). Meantime, sodium nitroprusside addition increased accumulation of ferritin at the protein level, indicating that nitric oxide directly regulated ferritin accumulation. These results suggested that nitric oxide can effectively protect seedlings from salt stress damage by enhancing activities of antioxidant enzymes to quench the excessive reactive oxygen species caused by salt stress and inducing the increase of ferritin accumulation to chelate larger number of ferrous ion. Information from this study can be used to improve soil management practices for sustainable use of salt-affected soils in the future.

Isolation and comparative expression analysis of six MBD genes in wheat.

The 5-methylcytosines (m5C) play critical roles in epigenetic control, often being recognized by proteins containing an MBD. In this study, we isolated six wheat cDNAs with open reading frame encoding putative methyl-binding domain proteins, which were designated as TaMBD1-TaMBD6, respectively. BLASTX searches and phylogenetic analysis suggested that the six TaMBD genes belonged to four (I, II, III and VIII) of the eight subclasses of MBD family. Genomic analysis showed that a 1386 bp intron was included in TaMBD1 and a 12-bp intron was found in TaMBD4. The expression profiles of the six TaMBDs were studied via Q-RT-PCR and the results indicated that the TaMBDs were differentially expressed in detected wheat tissues. It was interesting to note that 3 TaMBDs were highly expressed in dry seeds and endosperms. Moreover, the differential expression patterns of TaMBDs were observed in leaves and roots under water-stress. We concluded that multiple wheat MBD genes were present and they might play important roles in wheat growth and development, as well as in the water-stress response.

WITHDRAWN: Isolation and comparative expression analysis of six MBD genes in wheat.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Biochem. Biophys. Acta, doi:10.1016/j.bbagrm.2007.09.004. The duplicate article has therefore been withdrawn.

[Effects of antisense-thioredoxin s gene on expression of endogenous thioredoxin h gene in transgenic wheat seed].

To clarify the function mechanism of antisense-thioredoxin s (anti-trxs) gene in transgenic wheat, the expression pattern of endogenous trxh gene in transgenic line 01TY70-1-17-5 and non-transgenic cultivar 'Yumai 70' were detected by semi-quantitative RT-PCR using wheat actin gene as the endogenous control. The results of analysis of transgenic and non-transgenic seeds in different maturation periods, different tissues and different germinating processes indicated that the mRNA transcript amounts of trxh gene in transgenic line seed were lowered distinctly, though the trxh gene mRNA transcript level varied greatly in different developing and germination stages. The mRNA transcript amounts of trxh gene in transgenic line seed were significantly lower than the control seeds by 20.1% 15-30 d after anthesis. The lowest mRNA transcript amount of trxh gene appeared at 25 day after fluorescence and the difference was significant at the 0.05 level. The analysis of gene expression in different tissues also indicated that the transcript levels of trxh gene in transgenic seed were significantly lower than control seeds in 25 d and 30 d after anthesis. The lowest amounts of mRNA transcript of trxh gene was from the endosperm 25 d after anthesis followed by embryo and then by whole seed. During seed germination, the mRNA transcript amounts of trxh gene in transgenic seed were lower than control seed after imbibing 24 h, but the difference was not significant. The above result demonstrated that foreign antisense trxs gene directly interferes with the expression of the endogenous gene.