In planta evidence that the HAK transporter OsHAK2 is involved in Na+ transport in rice.

HAK family transporters primarily function as K+ transporters and play major roles in K+ uptake and translocation in plants, whereas several HAK transporters exhibit Na+ transport activity. OsHAK2, a rice HAK transporter, was shown to mediate Na+ transport in Escherichia coli in a previous study. In this study, we investigated whether OsHAK2 is involved in Na+ transport in the rice plant. Overexpression of OsHAK2 increased Na+ translocation from the roots to the shoots of transgenic rice. It also increased both root and whole-plant Na+ content, and enhanced shoot length under low Na+ and K+ conditions. Meanwhile, OsHAK2 overexpression increased salt sensitivity under a long-term salt stress condition, indicating that OsHAK2 is not involved in salt tolerance, unlike in the case of ZmHAK4 in maize. These results suggest that OsHAK2 is permeable to Na+ and contributes to shoot growth in rice plants under low Na+ and K+ conditions.

Molecular characterization of gliadins of Chinese Spring wheat in relation to celiac disease elicitors.

The wheat seed storage proteins gliadin and glutenin are encoded by multigenes. Gliadins are further classified into α-, γ-, δ- and ω-gliadins. Genes encoding α-gliadins belong to a large multigene family, whose members are located on the homoeologous group 6 chromosomes at the Gli-2 loci. Genes encoding other gliadins are located on the homoeologous group 1 chromosomes at the Gli-1 loci. Two-dimensional polyacrylamide gel electrophoresis (2-DE) was used to characterize and profile the gliadins. The gliadins in aneuploid Chinese Spring wheat lines were then compared in this study. Gliadin proteins separated into 70 spots after 2-DE and a total of 10, 10 and 16 spots were encoded on chromosomes 6A, 6B and 6D, respectively, which suggested that they were α-gliadins. Similarly, six, three and seven spots were encoded on chromosomes 1A, 1B and 1D, respectively, which indicated that they were γ-gliadins. Spots that could not be assigned to chromosomes were N-terminally sequenced and were all determined to be α-gliadins or γ-gliadins. The 2-DE profiles showed that specific α-gliadin spots assigned to chromosome 6D were lost in tetrasomic chromosome 2A lines. Furthermore, western blotting against the Glia-α9 peptide, an epitope for celiac disease (CD), suggested that α-gliadins harboring the CD epitope on chromosome 6D were absent in the tetrasomic chromosome 2A lines. Systematic analysis of α-gliadins using 2-DE, quantitative RT-PCR and genomic PCR revealed that tetrasomic 2A lines carry deletion of a chromosome segment at the Gli-D2 locus. This structural alteration at the Gli-D2 locus may provide a genetic resource in breeding programs for the reduction of CD immunotoxicity.

Erratum: Acetate-mediated novel survival strategy against drought in plants.

This corrects the article DOI: 10.1038/nplants.2017.97.

Acetate-mediated novel survival strategy against drought in plants.

Water deficit caused by global climate changes seriously endangers the survival of organisms and crop productivity, and increases environmental deterioration1,2. Plants' resistance to drought involves global reprogramming of transcription, cellular metabolism, hormone signalling and chromatin modification3-8. However, how these regulatory responses are coordinated via the various pathways, and the underlying mechanisms, are largely unknown. Herein, we report an essential drought-responsive network in which plants trigger a dynamic metabolic flux conversion from glycolysis into acetate synthesis to stimulate the jasmonate (JA) signalling pathway to confer drought tolerance. In Arabidopsis, the ON/OFF switching of this whole network is directly dependent on histone deacetylase HDA6. In addition, exogenous acetic acid promotes de novo JA synthesis and enrichment of histone H4 acetylation, which influences the priming of the JA signalling pathway for plant drought tolerance. This novel acetate function is evolutionarily conserved as a survival strategy against environmental changes in plants. Furthermore, the external application of acetic acid successfully enhanced the drought tolerance in Arabidopsis, rapeseed, maize, rice and wheat plants. Our findings highlight a radically new survival strategy that exploits an epigenetic switch of metabolic flux conversion and hormone signalling by which plants adapt to drought.

A Causal Gene for Seed Dormancy on Wheat Chromosome 4A Encodes a MAP Kinase Kinase.

Seed germination under the appropriate environmental conditions is important both for plant species survival and for successful agriculture. Seed dormancy, which controls germination time, is one of the adaptation mechanisms and domestication traits [1]. Seed dormancy is generally defined as the absence of germination of a viable seed under conditions that are favorable for germination [2]. The seed dormancy of cultivated plants has generally been reduced during domestication [3]. Bread wheat (Triticum aestivum L.) is one of the most widely grown crops in the world. Weak dormancy may be an advantage for the productivity due to uniform emergence and a disadvantage for the risks of pre-harvest sprouting (PHS), which decreases grain quality and yield [4]. A number of quantitative trait loci (QTLs) controlling natural variation of seed dormancy have been identified on various chromosomes [5]. A major QTL for seed dormancy has been consistently detected on chromosome 4A [6-13]. The QTL was designated as a major gene, Phs1, which could be precisely mapped within a 2.6 cM region [14]. Here, we identified a mitogen-activated protein kinase kinase 3 (MKK3) gene (designated TaMKK3-A) by a map-based approach as a candidate gene for the seed dormancy locus Phs1 on chromosome 4A in bread wheat. Complementation analysis showed that transformation of a dormant wheat cultivar with the TaMKK3-A allele from a nondormant cultivar clearly reduced seed dormancy. Cultivars differing in dormancy had a single nonsynonymous amino acid substitution in the kinase domain of the predicted MKK3 protein sequence, which may be associated with the length of seed dormancy.

Comprehensive molecular characterization of the α/ß-gliadin multigene family in hexaploid wheat.

To characterize the structure and expression of a large multigene family of α/ß-gliadin genes, 90 individual α/ß-gliadin genes harboring a promoter region were identified in the wheat cultivar Chinese Spring. These genes were classified into eleven groups by phylogenetic analysis, and the chromosomes they were derived from were determined. Of these genes, 50 had the basic α/ß-gliadin domains and six conserved cysteine residues and 16, 16 and 18 of them were, respectively, located on chromosome 6A, 6B and 6D. Six genes had an additional cysteine residue, suggesting that these α/ß-gliadins acquired the property of binding other proteins through intermolecular disulphide bands. Expression of α/ß-gliadin genes in developing seeds was measured by quantitative RT-PCR using group-specific primers over 3 years. Expression patterns of these genes on the basis of accumulated temperature were similar among gene groups, whereas expression levels differed for the 3 years. The expression of most α/ß-gliadin and other prolamin genes was correlated with the sunshine duration. On the other hand, although all α/ß-gliadin genes had a common E-box within the -300 promoter region, some genes showed a particular expression pattern with respect to the sunshine duration, similarly to gene encoding high-molecular weight glutenin subunits and endosperm enzymes. These observations suggested that expression of each α/ß-gliadin gene is differentially regulated by multiple regulatory factors.

Comparison of genome-wide gene expression patterns in the seedlings of nascent allohexaploid wheats produced by two combinations of hybrids.

Allopolyploidization in plants is an important event that enhances heterosis and environmental adaptation. Common wheat, Triticum aestivum (AABBDD), which is an allohexaploid that evolved from an allopolyploidization event between T. turgidum (AABB) and Aegilops tauschii (DD), shows more growth vigor and wider adaptation than tetraploid wheats. To better understand the molecular basis for the heterosis of hexaploid wheat, we systematically analyzed the genome-wide gene expression patterns of two combinations of newly hybridized triploids (ABD), their chromosome-doubled hexaploids (AABBDD), stable synthetic hexaploids (AABBDD) and natural hexaploids, in addition to their parents, T. turgidum (AABB) and Ae. tauschii (DD), using a microarray to reconstruct the events of allopolyploidization and genome stabilization. Overall comparisons of gene expression profiles showed that the newly generated hexaploids exhibited gene expression patterns similar to those of their maternal tetraploids, irrespective of hybrid combination. With successive generations, the gene expression profiles of nascent hexaploids became less similar to the maternal profiles, and belonged to a separate cluster from the natural hexaploids. Triploids revealed characteristic expression patterns, suggesting endosperm effects. In the newly hybridized triploids (ABD) of two independent synthetic lines, approximately one-fifth of expressed genes displayed non-additive expression; the number of these genes decreased with polyploidization and genome stabilization. Approximately 20% of the non-additively expressed genes were transmitted across generations throughout allopolyploidization and successive self-pollinations, and 43 genes overlapped between the two combinations, indicating that shared gene expression patterns can be seen during allohexaploidization. Furthermore, four of these 43 genes were involved in starch and sucrose metabolism, suggesting that these metabolic events play key roles in the hybrid vigor of hexaploid wheat.

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B.

BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Expression of a rice glutaredoxin in aleurone layers of developing and mature seeds: subcellular localization and possible functions in antioxidant defense.

MAIN CONCLUSION: A rice glutaredoxin isoform (OsGrxC2;2) with antioxidant capacity is expressed abundantly in seed tissues and is localized to storage vacuoles in aleurone layers in developing and mature seeds. Seed tissues undergo drastic water loss at the late stage of seed development, and thus need to tolerate oxidative injuries associated with desiccation. We previously found a rice glutaredoxin isoform, OsGrxC2;2, as a gene expressed abundantly in developing seeds. Since glutaredoxin is involved in antioxidant defense, in the present study we investigated the subcellular localization and expression profile of OsGrxC2;2 and whether OsGrxC2;2 has a role in the defense against reactive oxygen species. Western blotting and immunohistochemistry revealed that the OsGrxC2;2 protein accumulated at a high level in the embryo and aleurone layers of developing and mature seeds. The OsGrxC2;2 in developing seeds was particularly localized to aleurone grains, which are storage organelles derived from vacuoles. Overexpression of OsGrxC2;2 resulted in an enhanced tolerance to menadione in yeast and methyl viologen in green leaves of transgenic rice plants. These results suggest that OsGrxC2;2 participates in the defense against oxidative stress in developing and mature seeds.

QTL analysis of genetic loci affecting domestication-related spike characters in common wheat.

Domestication-related changes that govern a spike morphology suitable for seed harvesting in cereals have resulted from mutation and selection of the genes. A synthetic hexaploid wheat (S-6214, genome AABBDD) produced by a cross between durum wheat (AABB) and wild goat grass (DD) showed partial non-domestication-related phenotypes due to genetic effects of the wild goat grass genome. Quantitative trait loci (QTLs) affecting wheat domestication-related spike characters including spike threshability, rachis fragility and spike compactness were investigated in F2 progeny of a cross between Chinese Spring (CS) wheat (AABBDD) and S-6214. Of 15 relevant QTLs identified, eight seemed to be consistent with peaks previously reported in wheat, while four QTL regions were novel. Four QTLs that affected spike threshability were localized to chromosomes 2BS, 2DS, 4D and 5DS. The QTL on 2DS probably represents the tenacious glume gene, Tg-D1. Based on its map position, the QTL located on 2BS coincides with Ppd-B1 and seems to be a homoeolocus of the soft glume gene. Two novel QTLs were detected on 4D and 5DS, and their goat grass alleles increased glume tenacity. Three novel QTLs located on 2DL, 3DL and 4D for rachis fragility were found. Based on the map position, the QTL on 3DL seems different from Br1 and Br2 loci and its CS allele appears to promote the generation of barrel-type diaspores. Three disarticulation types of spikelets were found in F2 individuals: wedge-type, barrel-type and both types. Among eight QTL peaks that governed spike morphology, six, located on 2AS, 2BS, 2DS, 4AL and 5AL, coincided with ones previously reported. A QTL for spike compactness on 5AL was distinct from the Q gene. A novel QTL that controls spike length was detected on 5DL. Complex genetic interactions between genetic background and the action of each gene were suggested.

Changes in genome-wide gene expression during allopolyploidization and genome stabilization in hexaploid wheat.

Allopolyploidization is an important evolutionary event in plants, but its genome-wide effects are not fully understood. Common wheat, Triticum aestivum (AABBDD), evolved through amphidiploidization between T. turgidum (AABB) and Aegilops tauschii (DD). Here, global gene expression patterns in the seedlings of a synthetic triploid wheat line (ABD), its chromosome-doubled hexaploid (AABBDD) and stable synthetic hexaploid (AABBDD), and the parental lines T. turgidum (AABB) and Ae. tauschii (DD) were compared using an oligo-DNA microarray to identify metabolic pathways affected by the genome conflict that occurs during allopolyploidization and genome stabilization. Characteristic gene expression patterns of non-additively expressed genes were detected in the newly synthesized triploid and hexaploid, and in the stable synthetic hexaploid. Hierarchical clustering of all differentially expressed and non-additively expressed genes revealed that the gene expression patterns of the triploid (ABD) were similar to those of the maternal parent (AABB), and that expression patterns in successive generations arising from self-pollination became closer to that of the pollen parent (DD). The non-additive gene expression profiles markedly differed between the triploid (ABD) and chromosome-doubled hexaploid (AABBDD), as supported by Gene Ontology (GOSlim) analysis. Four hundred and nineteen non-additively expressed genes were commonly detected in all three generations. GOSlim analysis indicated that these non-additively expressed genes were predominantly involved in "biological pathways". Notably, four of 11 genes related to sugar metabolism displayed elevated expression throughout allopolyploidization. These may be useful candidates for promoting heterosis and adaptation in plants.

Next-generation survey sequencing and the molecular organization of wheat chromosome 6B.

Common wheat (Triticum aestivum L.) is one of the most important cereals in the world. To improve wheat quality and productivity, the genomic sequence of wheat must be determined. The large genome size (∼17 Gb/1 C) and the hexaploid status of wheat have hampered the genome sequencing of wheat. However, flow sorting of individual chromosomes has allowed us to purify and separately shotgun-sequence a pair of telocentric chromosomes. Here, we describe a result from the survey sequencing of wheat chromosome 6B (914 Mb/1 C) using massively parallel 454 pyrosequencing. From the 4.94 and 5.51 Gb shotgun sequence data from the two chromosome arms of 6BS and 6BL, 235 and 273 Mb sequences were assembled to cover ∼55.6 and 54.9% of the total genomic regions, respectively. Repetitive sequences composed 77 and 86% of the assembled sequences on 6BS and 6BL, respectively. Within the assembled sequences, we predicted a total of 4798 non-repetitive gene loci with the evidence of expression from the wheat transcriptome data. The numbers and chromosomal distribution patterns of the genes for tRNAs and microRNAs in wheat 6B were investigated, and the results suggested a significant involvement of DNA transposon diffusion in the evolution of these non-protein-coding RNA genes. A comparative analysis of the genomic sequences of wheat 6B and monocot plants clearly indicated the evolutionary conservation of gene contents.

Effects of barley chromosome addition to wheat on behavior and development of Locusta migratoria nymphs.

Locusta migratoria feeds on various Poaceae plants but barley. Barley genes related to feeding deterrence may be useful for developing novel resistant crops. We investigated the effects of barley cultivar Betzes, wheat cultivar Chinese Spring (CS), and six barley chromosome disomic addition lines of wheat (2H-7H) on locomotor activity, feeding behavior, survival and development of L. migratoria nymphs. Locomotor activity was similar in nymphs kept with wheat and 2H-7H in an actograph, whereas it was generally high in those kept with barely. No-choice and choice feeding tests suggested that barley genes related to inhibition of feeding by L. migratoria are located on barley chromosomes 5H and 6H and those related to the palatability of plants on chromosomes 2H, 5H and 6H. Rearing experiments suggested the presence of barley genes negatively affecting the survival and growth of locust nymphs on chromosomes 5H and 2H, respectively, and the effects are phase-dependent.

Identification of a novel homolog for a calmodulin-binding protein that is upregulated in alloplasmic wheat showing pistillody.

Intracellular signaling pathways between the mitochondria and the nucleus are important in both normal and abnormal development in plants. The homeotic transformation of stamens into pistil-like structures (a phenomenon termed pistillody) in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) has been suggested to be induced by mitochondrial retrograde signaling, one of the forms of intracellular communication. We showed previously that the mitochondrial gene orf260 could alter the expression of nuclear class B MADS-box genes to induce pistillody. To elucidate the interactions between orf260 and nuclear homeotic genes, we performed a microarray analysis to compare gene expression patterns in the young spikes of a pistillody line and a normal line. We identified five genes that showed higher expression levels in the pistillody line. Quantitative expression analysis using real-time PCR indicated that among these five genes, Wheat Calmodulin-Binding Protein 1 (WCBP1) was significantly upregulated in young spikes of the pistillody line. The amino acid sequence of WCBP1 was predicted from the full-length cDNA sequence and found to encode a novel plant calmodulin-binding protein. RT-PCR analysis indicated that WCBP1 was preferentially expressed in young spikes at an early stage and decreased during spike maturation, indicating that it was associated with spikelet/floret development. Furthermore, in situ hybridization analysis suggested that WCBP1 was highly expressed in the pistil-like stamens at early to late developmental stages. These results indicate that WCBP1 plays a role in formation and development of pistil-like stamens induced by mitochondrial retrograde signaling.

Comprehensive functional analyses of expressed sequence tags in common wheat (Triticum aestivum).

About 1 million expressed sequence tag (EST) sequences comprising 125.3 Mb nucleotides were accreted from 51 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including abiotic stresses and pathogen challenges in common wheat (Triticum aestivum). Expressed sequence tags were assembled with stringent parameters after processing with inbuild scripts, resulting in 37,138 contigs and 215,199 singlets. In the assembled sequences, 10.6% presented no matches with existing sequences in public databases. Functional characterization of wheat unigenes by gene ontology annotation, mining transcription factors, full-length cDNA, and miRNA targeting sites were carried out. A bioinformatics strategy was developed to discover single-nucleotide polymorphisms (SNPs) within our large EST resource and reported the SNPs between and within (homoeologous) cultivars. Digital gene expression was performed to find the tissue-specific gene expression, and correspondence analysis was executed to identify common and specific gene expression by selecting four biotic stress-related libraries. The assembly and associated information cater a framework for future investigation in functional genomics.

A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination.

Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.

Effect of barley chromosome addition on the susceptibility of wheat to feeding by a gall-inducing leafhopper.

The maize orange leafhopper Cicadulina bipunctata is distributed widely in tropical and subtropical regions of the Old World and feeds on various Poaceae. The leafhopper is recognized as an important pest of maize in several countries. Adults as well as nymphs of C. bipunctata induce growth stunting and galls characterized by the severe swelling of leaf veins on many cereal crops including wheat, rice, and maize, but do not on barley. To clarify the mechanism of growth stunting and gall induction by C. bipunctata, we used six barley chromosome disomic addition lines of wheat (2H-7H) and investigated the effect of barley (cv. Betzes) chromosome addition on the susceptibility of wheat (cv. Chinese Spring) to feeding by the leafhopper. Feeding by C. bipunctata significantly stunted the growth in 2H, 3H, 4H, and 5H, but did not in 6H and 7H. The degree of gall induction was significantly weaker and severer in 3H and 5H than in Chinese Spring, respectively. These results suggest that barley genes resistant to growth stunting and gall induction exist in 6H and 7H, and 3H, respectively. 5H is considered to be useful for future assays investigating the mechanism of gall induction by this leafhopper because of the high susceptibility to the feeding by C. bipunctata. Significant correlation between the degrees of growth stunting and gall induction was not detected in the six chromosome addition lines and Chinese spring. This implies that these two symptoms are independent phenomena although both are initiated by the feeding of C. bipunctata.

A new insight into application for barley chromosome addition lines of common wheat: achievement of stigmasterol accumulation.

Barley (Hordeum vulgare) has a much higher content of bioactive substances than wheat (Triticum aestivum). In order to investigate additive and/or synergistic effect(s) on the phytosterol content of barley chromosomes, we used a series of barley chromosome addition lines of common wheat that were produced by normal crossing. In determining the plant sterol levels in 2-week-old seedlings and dry seeds, we found that the level of stigmasterol in the barley chromosome 3 addition (3H) line in the seedlings was 1.5-fold higher than that in the original wheat line and in the other barley chromosome addition lines, but not in the seeds. Simultaneously, we determined the overall expression pattern of genes related to plant sterol biosynthesis in the seedlings of wheat and each addition line to assess the relative expression of each gene in the sterol pathway. Since we elucidated the CYP710A8 (cytochrome P450 subfamily)-encoding sterol C-22 desaturase as a key characteristic for the higher level of stigmasterol, full-length cDNAs of wheat and barley CYP710A8 genes were isolated. These CYP710A8 genes were mapped on chromosome 3 in barley (3H) and wheat (3A, 3B, and 3D), and the expression of CYP710A8 genes increased in the 3H addition line, indicating that it is responsible for stigmasterol accumulation. Overexpression of the CYP710A8 genes in Arabidopsis increased the stigmasterol content but did not alter the total sterol level. Our results provide new insight into the accumulation of bioactive compounds in common wheat and a new approach for assessing plant metabolism profiles.

Heteroplasmy and expression of mitochondrial genes in alloplasmic and euplasmic wheat.

The plant chondriome confers a complex nature. The atp4 gene (formerly called orf25) of Aegilops crassa (CR) harbors the promoter sequence of the rps7 gene from common wheat (Triticum aestivum cv. Chinese Spring, CS). The rps7 gene of CR has the promoter sequence of CS atp6. The atp6 gene of CR contains an unknown sequence inside of its coding region. Since repeat sequences have been found around the breaking points, these structural alterations are most likely generated through homologous recombination. In this study, PCR analysis was performed to detect structural alterations in each of three lines: euplasmic lines of Ae. crassa, Chinese Spring, and alloplasmic Chinese Spring wheat with the cytoplasm of Ae. crassa ((cr)-CS). We found that each of these lines contained both genotypes, although mitochondrial genotypes of CR in Chinese Spring wheat and CS genotypes in Ae. crassa were still retained as minor fractions (less than 10%). On the other hand, CS mitochondrial gene frequencies in ((cr)-CS) were shown to be ca. 30%. SNP analysis after DNA sequencing of these genes indicated that minor types of all three mitochondrial genes in alloplasmic wheat contained the mitochondrial gene types from pollens. Since the frequencies of paternal mitochondrial gene types in F(1) were about 20%, successive backcrossing increased the frequencies of paternal mitochondrial gene types to around 30% in alloplasmic wheat. Expression profiles of these mitochondrial genes were quantitatively analyzed by RT-PCR. Transcripts of paternal mitochondrial gene types were scarcely found. This suggests that minor fractions including paternal mitochondrial gene types are maintained and silenced in the descendants.

Comparative gene expression analysis of susceptible and resistant near-isogenic lines in common wheat infected by Puccinia triticina.

Gene expression after leaf rust infection was compared in near-isogenic wheat lines differing in the Lr10 leaf rust resistance gene. RNA from susceptible and resistant plants was used for cDNA library construction. In total, 55 008 ESTs were sequenced from the two libraries, then combined and assembled into 14 268 unigenes for further analysis. Of these ESTs, 89% encoded proteins similar to (E value of < or =10(-5)) characterized or annotated proteins from the NCBI non-redundant database representing diverse molecular functions, cellular localization and biological processes based on gene ontology classification. Further, the unigenes were classified into susceptible and resistant classes based on the EST members assembled from the respective libraries. Several genes from the resistant sample (14-3-3 protein, wali5 protein, actin-depolymerization factor and ADP-ribosylation factor) and the susceptible sample (brown plant hopper resistance protein, caffeic acid O-methyltransferase, pathogenesis-related protein and senescence-associated protein) were selected and their differential expression in the resistant and susceptible samples collected at different time points after leaf rust infection was confirmed by RT-PCR analysis. The molecular pathogenicity of leaf rust in wheat was studied and the EST data generated made a foundation for future studies.