Pentatricopeptide Repeat Genes Research Articles

Small is big in Arabidopsis mitochondrial ribosome.

Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.

Tissue-specific alternative splicing of pentatricopeptide repeat (PPR) family genes in Arabidopsis thaliana.

Alternative splicing is a post- and co-transcriptional regulatory mechanism of gene expression. Pentatricopeptide repeat (PPR) family proteins were recently found to be involved in RNA editing in plants. The aim of this study was to investigate the tissue-specific expression and alternative splicing of PPR family genes and their effects on protein structure and functionality. Of the 27 PPR genes in Arabidopsis thaliana, we selected six PPR genes of the P subfamily that are likely alternatively spliced, which were confirmed by sequencing. Four of these genes show intron retention, and the two remaining genes have 3' alternative-splicing sites. Alternative-splicing events occurred in the coding regions of three genes and in the 3' UTRs of the three remaining genes. We also identified five previously unannotated alternatively spliced isoforms of these PPR genes, which were confirmed by PCR and sequencing. Among these, three contain 3' alternative-splicing sites, one contains a 5' alternative-splicing site, and the remaining gene contains a 3'-5' alternative-splicing site. The new isoforms of two genes affect protein structure, and three other alternative-splicing sites are located in 3' UTRs. These findings suggest that tissue-specific expression of different alternatively spliced transcripts occurs in Arabidopsis, even at different developmental stages.

Genome-wide analysis of the pentatricopeptide repeat gene family in different maize genomes and its important role in kernel development

BackgroundThe pentatricopeptide repeat (PPR) gene family is one of the largest gene families in land plants (450 PPR genes in Arabidopsis, 477 PPR genes in rice and 486 PPR genes in foxtail millet) and is important for plant development and growth. Most PPR genes are encoded by plastid and mitochondrial genomes, and the gene products regulate the expression of the related genes in higher plants. However, the functions remain largely unknown, and systematic analysis and comparison of the PPR gene family in different maize genomes have not been performed.ResultsIn this study, systematic identification and comparison of PPR genes from two elite maize inbred lines, B73 and PH207, were performed. A total of 491 and 456 PPR genes were identified in the B73 and PH207 genomes, respectively. Basic bioinformatics analyses, including of the classification, gene structure, chromosomal location and conserved motifs, were conducted. Examination of PPR gene duplication showed that 12 and 15 segmental duplication gene pairs exist in the B73 and PH207 genomes, respectively, with eight duplication events being shared between the two genomes. Expression analysis suggested that 53 PPR genes exhibit qualitative variations in the different genetic backgrounds. Based on analysis of the correlation between PPR gene expression in kernels and kernel-related traits, four PPR genes are significantly negatively correlated with hundred kernel weight, 12 are significantly negatively correlated with kernel width, and eight are significantly correlated with kernel number. Eight of the 24 PPR genes are also located in metaQTL regions associated with yield and kernel-related traits in maize. Two important PPR genes (GRMZM2G353195 and GRMZM2G141202) might be regarded as important candidate genes associated with maize kernel-related traits.ConclusionsOur results provide a more comprehensive understanding of PPR genes in different maize inbred lines and identify important candidate genes related to kernel development for subsequent functional validation in maize.

High intraspecific diversity of Restorer‐of‐fertility‐like genes in barley

SUMMARY Nuclear restorer of fertility (Rf) genes suppress the effects of mitochondrial genes causing cytoplasmic male sterility (CMS), a condition in which plants fail to produce viable pollen. Rf genes, many of which encode RNA‐binding pentatricopeptide repeat (PPR) proteins, are applied in hybrid breeding to overcome CMS used to block self‐pollination of the seed parent. Here, we characterise the repertoire of restorer‐of‐fertility‐like (RFL) PPR genes in barley (Hordeum vulgare). We found 26 RFL genes in the reference genome (‘Morex’) and an additional 51 putative orthogroups (POGs) in a re‐sequencing data set from 262 barley genotypes and landraces. Whereas the sequences of some POGs are highly conserved across hundreds of barley accessions, the sequences of others are much more variable. High sequence variation strongly correlates with genomic location – the most variable genes are found in a cluster on chromosome 1H. A much higher likelihood of diversifying selection was found for genes within this cluster than for genes present as singlets. This work includes a comprehensive analysis of the patterns of intraspecific variation of RFL genes. The RFL sequences characterised in this study will be useful for the development of new markers for fertility restoration loci.

Genomewide identification of PPR gene family and prediction analysis on restorer gene in Gossypium

Pentatricopeptide repeat (PPR) gene family plays an essential role in the regulation of plant growth and organelle gene expression. Some PPR genes are related to fertility restoration in plant, but there is no detailed information in Gossypium. In the present study, we identified 482 and 433 PPR homologues in Gossypium raimondii (D5) and G. arboreum (A2) genomes, respectively. Most PPR homologues showed an even distribution on the whole chromosomes. Given an evolutionary analysis to PPR genes from G. raimondii (D5), G. arboreum (A2) and G. hirsutum genomes, eight PPR genes were clustered together with restoring genes of other species. Most cotton PPR genes were qualified with no intron, high proportion of α-helix and classical tertiary structure of PPR protein. Based on bioinformatics analyses, eight PPR genes were targeted in mitochondrion, encoding typical P subfamily protein with protein binding activity and organelle RNA metabolism in function. Further verified by RNA-seq and quantitative real-time PCR (qRT-PCR) analyses, two PPR candidate genes, Gorai.005G0470 (D5) and Cotton_A_08373 (A2), were upregulated in fertile line than sterile line. These results reveal new insights into PPR gene evolution in Gossypium.

Genome-wide generation and genotyping of informative SNPs to scan molecular signatures for seed yield in chickpea

We discovered 2150 desi and 2199 kabuli accessions-derived SNPs by cultivar-wise individual assembling of sequence-reads generated through genotyping-by-sequencing of 92 chickpea accessions. Subsequent large-scale validation and genotyping of these SNPs discovered 619 desi accessions-derived (DAD) SNPs, 531 kabuli accessions-derived (KAD) SNPs, 884 multiple accessions-derived (MAD) SNPs and 1083 two accessions (desi ICC 4958 and kabuli CDC Frontier)-derived (TAD) SNPs that were mapped on eight chromosomes. These informative SNPs were annotated in coding/non-coding regulatory sequence components of genes. The MAD-SNPs were efficient to detect high intra-specific polymorphic potential and wide natural allelic diversity level including high-resolution admixed-population genetic structure and precise phylogenetic relationship among 291 desi and kabuli accessions. This signifies their effectiveness in introgression breeding and varietal improvement studies targeting useful agronomic traits of chickpea. Six trait-associated genes with SNPs including quantitative trait nucleotides (QTNs) in combination explained 27.5% phenotypic variation for seed yield per plant (SYP). A pentatricopeptide repeat (PPR) gene with a synonymous-coding SNP/QTN significantly associated with SYP trait was found most-promising in chickpea. The essential information delineated can be of immense utility in genomics-assisted breeding applications to develop high-yielding chickpea cultivars.

QTL mapping and validation of fertility restoration in West African sorghum A1 cytoplasm and identification of a potential causative mutation for Rf2

Key messageMajor A1cytoplasm fertility restoration loci,Rf2andRf5, were found in the West African sorghum. A potential causative mutation forRf2was identified. KASP markers were validated on independent material.To accelerate the identification and development of hybrid parental lines in West African (WA) sorghum, this study aimed to understand the genetics underlying the fertility restoration (Rf) in WA A1 cytoplasmic male sterility system and to develop markers for a routine use in WA breeding programs. We genotyped by sequencing three F2 populations to map the Rf quantitative trait loci (QTL), validated the molecular KASP markers developed from those QTL in two F2:3 populations, and assessed the most promising markers on a set of 95 R- and B-lines from WA breeding programs. Seven QTL were found across the three F2 populations. On chromosome SBI-05, we found a major fertility restorer locus (Rf5) for two populations with the same male parent, explaining 19 and 14% of the phenotypic variation in either population. Minor QTL were detected in these two populations on chromosomes SBI-02, SBI-03, SBI-04 and SBI-10. In the third population, we identified one major fertility restorer locus on chromosome SBI-02, Rf2, explaining 31% of the phenotypic variation. Pentatricopeptide repeat genes in the Rf2 QTL region were sequenced, and we detected in Sobic.002G057050 a missense mutation in the first exon, explaining 81% of the phenotypic variation in a F2:3 population and clearly separating B- from R-lines. The KASP marker developed from this mutation stands as a promising tool for routine use in WA breeding programs.

Three new pentatricopeptide repeat proteins facilitate the splicing of mitochondrial transcripts and complex I biogenesis in Arabidopsis.

Group II introns are common features of most angiosperm mitochondrial genomes. Intron splicing is thus essential for the expression of mitochondrial genes and is facilitated by numerous nuclear-encoded proteins. However, the molecular mechanism and the protein cofactors involved in this complex process have not been fully elucidated. In this study, we characterized three new pentatricopeptide repeat (PPR) genes, called MISF26, MISF68, and MISF74, of Arabidopsis and showed they all function in group II intron splicing and plant development. The three PPR genes encode P-type PPR proteins that localize in the mitochondrion. Transcript analysis revealed that the splicing of a single intron is altered in misf26 mutants, while several mitochondrial intron splicing defects were detected in misf68 and misf74 mutants. To our knowledge, MISF68 and MISF74 are the first two PPR proteins implicated in the splicing of more than one intron in plant mitochondria, suggesting that they may facilitate splicing differently from other previously identified PPR splicing factors. The splicing defects in the misf mutants induce a significant decrease in complex I assembly and activity, and an overexpression of mRNAs of the alternative respiratory pathway. These results therefore reveal that nuclear encoded proteins MISF26, MISF68, and MISF74 are involved in splicing of a cohort of mitochondrial group II introns and thereby required for complex I biogenesis.

The Pentatricopeptide Repeat Gene Family in Salvia miltiorrhiza: Genome-Wide Characterization and Expression Analysis.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants and plays important roles in posttranscriptional regulation. In this study, we combined whole genome sequencing and transcriptomes to systematically investigate PPRs in Salvia miltiorrhiza, which is a well-known material of traditional Chinese medicine and an emerging model system for medicinal plant studies. Among 562 identified SmPPRs, 299 belong to the P subfamily while the others belong to the PLS subfamily. The majority of SmPPRs have only one exon and are localized in the mitochondrion or chloroplast. As many as 546 SmPPRs were expressed in at least one tissue and exhibited differential expression patterns, which indicates they likely play a variety of functions in S. miltiorrhiza. Up to 349 SmPPRs were salicylic acid-responsive and 183 SmPPRs were yeast extract and Ag+-responsive, which indicates these genes might be involved in S. miltiorrhiza defense stresses and secondary metabolism. Furthermore, 23 salicylic acid-responsive SmPPRs were co-expressed with phenolic acid biosynthetic enzyme genes only while 16 yeast extract and Ag+-responsive SmPPRs were co-expressed with tanshinone biosynthetic enzyme genes only. Two SmPPRs were co-expressed with both phenolic acid and tanshinone biosynthetic enzyme genes. The results provide a useful platform for further investigating the roles of PPRs in S. miltiorrhiza.

Genome Analysis of the Ancient Tracheophyte Selaginella tamariscina Reveals Evolutionary Features Relevant to the Acquisition of Desiccation Tolerance

Resurrection plants, which are the “gifts” of natural evolution, are ideal models for studying the genetic basis of plant desiccation tolerance. Here, we report a high-quality genome assembly of 301 Mb for the diploid spike moss Selaginella tamariscina, a primitive vascular resurrection plant. We predicated 27 761 protein-coding genes from the assembled S. tamariscina genome, 11.38% (2363) of which showed significant expression changes in response to desiccation. Approximately 60.58% of the S. tamariscina genome was annotated as repetitive DNA, which is an almost 2-fold increase of that in the genome of desiccation-sensitive Selaginella moellendorffii. Genomic and transcriptomic analyses highlight the unique evolution and complex regulations of the desiccation response in S. tamariscina, including species-specific expansion of the oleosin and pentatricopeptide repeat gene families, unique genes and pathways for reactive oxygen species generation and scavenging, and enhanced abscisic acid (ABA) biosynthesis and potentially distinct regulation of ABA signaling and response. Comparative analysis of chloroplast genomes of several Selaginella species revealed a unique structural rearrangement and the complete loss of chloroplast NAD(P)H dehydrogenase (NDH) genes in S. tamariscina, suggesting a link between the absence of the NDH complex and desiccation tolerance. Taken together, our comparative genomic and transcriptomic analyses reveal common and species-specific desiccation tolerance strategies in S. tamariscina, providing significant insights into the desiccation tolerance mechanism and the evolution of resurrection plants.

Genome-wide investigation of pentatricopeptide repeat gene family in poplar and their expression analysis in response to biotic and abiotic stresses

Pentatricopeptide repeat (PPR) proteins, which are characterized by tandem 30–40 amino acid sequence motifs, constitute of a large gene family in plants. Some PPR proteins have been identified to play important roles in organellar RNA metabolism and organ development in Arabidopsis and rice. However, functions of PPR genes in woody species remain largely unknown. Here, we identified and characterized a total of 626 PPR genes containing PPR motifs in the Populus trichocarpa genome. A comprehensive genome-wide analysis of the poplar PPR gene family was performed, including chromosomal location, phylogenetic relationships and gene duplication. Genome-wide transcriptomic analysis showed that 154 of the PtrPPR genes were induced by biotic and abiotic treatments, including Marssonina brunnea, salicylic acid (SA), methyl jasmonate (MeJA), mechanical wounding, cold and salinity stress. Quantitative RT-PCR analysis further investigated the expression profiles of 11 PtrPPR genes under different stresses. Our results contribute to a comprehensive understanding the roles of PPR proteins and provided an insight for improving the stress tolerance in poplar.

A donor-specific QTL, exhibiting allelic variation for leaf sheath hairiness in a nested association mapping population, is located on barley chromosome 4H.

Leaf sheath hairiness is a morphological trait associated with various advantages, including tolerance to both abiotic and biotic stresses, thereby increasing yield. Understanding the genetic basis of this trait in barley can therefore improve the agronomic performance of this economically important crop. We scored leaf sheath hairiness in a two-year field trial in 1,420 BC1S3 lines from the wild barley nested association mapping (NAM) population HEB-25. Leaf sheath hairiness segregated in six out of 25 families with the reference parent Barke being glabrous. We detected the major hairy leaf sheath locus Hs (syn. Hsh) on chromosome 4H (111.3 cM) with high precision. The effects of the locus varied across the six different wild barley donors, with donor of HEB family 11 conferring the highest score of leaf sheath hairiness. Due to the high mapping resolution present in HEB-25, we were able to discuss physically linked pentatricopeptide repeat genes and subtilisin-like proteases as potential candidate genes underlying this locus. In this study, we proved that HEB-25 provides an appropriate tool to further understand the genetic control of leaf sheath hairiness in barley. Furthermore, our work represents a perfect starting position to clone the gene responsible for the 4H locus observed.

RNA Editing Responses to Oxidative Stress between a Wild Abortive Type Male-Sterile Line and Its Maintainer Line.

RNA editing of mitochondrial gene transcripts plays a central role during plant development and evolutionary adaptation. RNA editing has previously been reported to differ between the rice cytoplasmic male sterile (CMS) line and its maintainer line, which has been suggested as a cause for their different performances under environmental stress. To specifically test this hypothesis, a wild abortive (WA) CMS line (Huhan-1A) and its maintainer line (Huhan-1B) were utilized to investigate performances in response to oxidative stress, as well as RNA editing efficiencies on transcripts of six selected mitochondrial genes. Compared to the maintainer line, Huhan-1A represented both lower plant height and total antioxidant capacity, possessed higher total soluble protein and chlorophyll contents, accumulated less H2O2 content on the 3rd day after treatment (DAT), and exhibited higher survival ratio after re-watering. Furthermore, a total of 90 editing sites were detected on transcripts of six mitochondrial genes (atp9, nad2, nad7, nad9, ccmB, and ccmC) in both Huhan-1A and Huhan-1B on the 0, 1st, and 3rd DAT. Forty-eight sites were furthermore determined as stress-responsive sites (SRS). Generally, in response to oxidative stress, SRS in Huhan-1A increased the resulting editing efficiencies, while SRS in Huhan-1B decreased the resulting editing efficiencies. In addition, 33 and 22 sites at ccmB and ccmC were differentially edited between Huhan-1A and Huhan-1B, respectively, on the 0, 1st, and 3rd DAT. Editing efficiencies of ccmB and ccmC were generally lower in Huhan-1A (ccmB, 37.3–47.8%; ccmC, 41.2–52.3%) than those in Huhan-1B (ccmB, 82.6–86.5%; ccmC, 81.0–82.9%). Deficiencies of RNA editing in Huhan-1A at ccmB and ccmC could lead to the loss of transmembrane domains in their protein structures. Consequently, differences in RNA editing at ccmB and ccmC between the WA-CMS line and its maintainer line partially explained their different performances under stress. Moreover, we detected differences in expressions of pentatricopeptide repeat (PPR) genes between both lines, as well as significant correlations with RNA editing. Our study indicated potential associations of RNA editing and PPR genes in rice tolerance to abiotic stresses. However, the underlying molecular mechanisms of stress-adaptation, which are attributed to RNA editing on transcripts of mitochondrial genes, require further investigation.

Map-based cloning of the fertility restoration locus Rfm1 in cultivated barley (Hordeum vulgare)

Hybridization technology has proven valuable in enhancing yields in many crops, but was only recently adopted in the small grain cereals. Hybrid varieties in barley (Hordeum vulgare) rely on the cytoplasmic male sterility (CMS) system msm1 derived from Hordeum vulgare ssp. spontaneum. The major restorer gene described for the msm1 system is known as Rfm1 and maps to the top of chromosome 6H. To gain further insight into mechanisms underlying male fertility restoration in barley, we used a map-based cloning approach to identify the nuclear gene involved in the restoration mechanism of this hybridization system. Taking advantage of the available genomic resources in barley in combination with a custom-made non-gridded BAC library developed from a restorer line, we cloned and sequenced the Rfm1 restorer locus. The characterization and annotation of the nucleotide sequence for the Rfm1 restorer allele allowed for the identification of the candidate gene for Rfm1. The Rfm1 locus carries a tandem repeat of a gene encoding a pentatricopeptide repeat (PPR) protein. Surprisingly, Rfm1 belongs to the PLS-DYW subfamily of PPR genes known for their involvement in RNA editing in plants organelles, but that to date have not been identified as restorer genes.

Primers for Castilleja and their utility across Orobanchaceae: II. Single-copy nuclear loci.

Premise of the study:We developed primers targeting nuclear loci in Castilleja with the goal of reconstructing the evolutionary history of this challenging clade. These primers were tested across other major clades in Orobanchaceae to assess their broader utility.Methods and Results:We assembled low-coverage genomes for three taxa in Castilleja and developed primer combinations for the single-copy conserved ortholog set (COSII) and the pentatricopeptide repeat (PPR) gene family. These primer combinations were designed to take advantage of the Fluidigm microfluidic PCR platform and are well suited for high-throughput sequencing applications. Eighty-seven primers were designed for Castilleja, and 27 were found to have broader utility in Orobanchaceae.Conclusions:These results demonstrate the utility of these primers, not only across Castilleja, but for other lineages within Orobanchaceae as well. This expanded molecular toolkit will be an asset to future phylogenetic studies in Castilleja and throughout Orobanchaceae.

Transcriptomic analysis of salt stress responsive genes in Rhazya stricta.

Rhazya stricta is an evergreen shrub that is widely distributed across Western and South Asia, and like many other members of the Apocynaceae produces monoterpene indole alkaloids that have anti-cancer properties. This species is adapted to very harsh desert conditions making it an excellent system for studying tolerance to high temperatures and salinity. RNA-Seq analysis was performed on R. stricta exposed to severe salt stress (500 mM NaCl) across four time intervals (0, 2, 12 and 24 h) to examine mechanisms of salt tolerance. A large number of transcripts including genes encoding tetrapyrroles and pentatricopeptide repeat (PPR) proteins were regulated only after 12 h of stress of seedlings grown in controlled greenhouse conditions. Mechanisms of salt tolerance in R. stricta may involve the upregulation of genes encoding chaperone protein Dnaj6, UDP-glucosyl transferase 85a2, protein transparent testa 12 and respiratory burst oxidase homolog protein b. Many of the highly-expressed genes act on protecting protein folding during salt stress and the production of flavonoids, key secondary metabolites in stress tolerance. Other regulated genes encode enzymes in the porphyrin and chlorophyll metabolic pathway with important roles during plant growth, photosynthesis, hormone signaling and abiotic responses. Heme biosynthesis in R. stricta leaves might add to the level of salt stress tolerance by maintaining appropriate levels of photosynthesis and normal plant growth as well as by the participation in reactive oxygen species (ROS) production under stress. We speculate that the high expression levels of PPR genes may be dependent on expression levels of their targeted editing genes. Although the results of PPR gene family indicated regulation of a large number of transcripts under salt stress, PPR actions were independent of the salt stress because their RNA editing patterns were unchanged.

A genome-wide identification and analysis of the DYW-deaminase genes in the pentatricopeptide repeat gene family in cotton (Gossypium spp.).

The RNA editing occurring in plant organellar genomes mainly involves the change of cytidine to uridine. This process involves a deamination reaction, with cytidine deaminase as the catalyst. Pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain are reportedly associated with cytidine deamination, similar to members of the deaminase superfamily. PPR genes are involved in many cellular functions and biological processes including fertility restoration to cytoplasmic male sterility (CMS) in plants. In this study, we identified 227 and 211 DYW deaminase-coding PPR genes for the cultivated tetraploid cotton species G. hirsutum and G. barbadense (2n = 4x = 52), respectively, as well as 126 and 97 DYW deaminase-coding PPR genes in the ancestral diploid species G. raimondii and G. arboreum (2n = 26), respectively. The 227 G. hirsutum PPR genes were predicted to encode 52–2016 amino acids, 203 of which were mapped onto 26 chromosomes. Most DYW deaminase genes lacked introns, and their proteins were predicted to target the mitochondria or chloroplasts. Additionally, the DYW domain differed from the complete DYW deaminase domain, which contained part of the E domain and the entire E+ domain. The types and number of DYW tripeptides may have been influenced by evolutionary processes, with some tripeptides being lost. Furthermore, a gene ontology analysis revealed that DYW deaminase functions were mainly related to binding as well as hydrolase and transferase activities. The G. hirsutum DYW deaminase expression profiles varied among different cotton tissues and developmental stages, and no differentially expressed DYW deaminase-coding PPRs were directly associated with the male sterility and restoration in the CMS-D2 system. Our current study provides an important piece of information regarding the structural and evolutionary characteristics of Gossypium DYW-containing PPR genes coding for deaminases and will be useful for characterizing the DYW deaminase gene family in cotton biology and breeding.

Response of microRNAs to cold treatment in the young spikes of common wheat

BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in biotic and abiotic stresses by regulating their target genes. For common wheat, spring frost damage frequently occurs, especially when low temperature coincides with plants at early floral organ differentiation, which may result in significant yield loss. Up to date, the role of miRNAs in wheat response to frost stress is not well understood.ResultsWe report here the sequencing of small RNA transcriptomes from the young spikes that were treated with cold stress and the comparative analysis with those of the control. A total of 192 conserved miRNAs from 105 families and nine novel miRNAs were identified. Among them, 34 conserved and five novel miRNAs were differentially expressed between the cold-stressed samples and the controls. The expression patterns of 18 miRNAs were further validated by quantitative real time polymerase chain reaction (qRT-PCR). Moreover, nearly half of the miRNAs were cross inducible by biotic and abiotic stresses when compared with previously published work. Target genes were predicted and validated by degradome sequencing. Gene Ontology (GO) enrichment analysis showed that the target genes of differentially expressed miRNAs were enriched for response to the stimulus, regulation of transcription, and ion transport functions. Since many targets of differentially expressed miRNAs were transcription factors that are associated with floral development such as ARF, SPB (Squamosa Promoter Binding like protein), MADS-box (MCM1, AG, DEFA and SRF), MYB, SPX (SYG1, Pho81 and XPR1), TCP (TEOSINTE BRANCHED, Cycloidea and PCF), and PPR (PentatricoPeptide Repeat) genes, cold-altered miRNA expression may cause abnormal reproductive organ development.ConclusionAnalysis of small RNA transcriptomes and their target genes provide new insight into miRNA regulation in developing wheat inflorescences under cold stress. MiRNAs provide another layer of gene regulation in cold stress response that can be genetically manipulated to reduce yield loss in wheat.

The Rice Pentatricopeptide Repeat Gene TCD10 is Needed for Chloroplast Development under Cold Stress.

BackgroundChloroplast plays a vital role in plant development and growth. The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants. In addition, cold stress affects a broad spectrum of cellular components, e.g. chloroplast, and metabolism in plants. However, the regulatory mechanism for rice PPR genes on chloroplast development still remains elusive under cold stress.ResultIn this paper, we characterized a new rice PPR gene mutant tcd10 (thermo-sensitive chlorophyll-deficient mutant 10) that exhibits the albino phenotype, malformed chloroplast and could not survive after the 5-leaf stage when grown at 20 °C, but does the normal phenotype at 32 °C. Map-based cloning, followed by RNA interference and CRISPR/Cas9 genome editing techniques, revealed that TCD10 encoding a novel PPR protein, mainly localized to the chloroplasts, with 27 PPR motifs, is responsible for the mutant phenotype. In addition, TCD10 is specific expression in tissues. The disruption of TCD10 resulted in an evidently reduced expression of chloroplast-associated genes under cold stress (20 °C), whereas they did recovered to normal levels at high temperature (32 °C). These results showed an important role of TCD10 for chloroplast development under cold stress.ConclusionsThe TCD10 encodes a novel rice PPR protein, mainly located in chloroplasts, which is important for chloroplast development, growth and the maintenance of photosynthetic electron transport and its disorder would lead to an aberrant chloroplast and abnormal expressions in these genes for chloroplast development and photosynthesis in rice under cold stress.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-016-0134-1) contains supplementary material, which is available to authorized users.

Discovery, utilization and perspective of Honglian cytoplasmic male sterile rice

Hybrid rice has been researched and utilized in China for 50 years. Three mainly cytoplasmic male sterile rice are wild abortive (WA), boro-II (BT), and Honglian (HL). Honglian (HL) type hybrid rice has made important contributions to the development of hybrid rice by combining scientific research, application research and industrial utilization. We identified Honglian cytoplasmic male sterile (HL-CMS) gene, orfH79 , which located downstream of atp6 in mitochondrial genome. CMS RNA, atp6-orfH79 , were translated into a cytotoxic protein, ORFH79. Data showed ORFH79 interacts with P61, which is a subunit of complex III of mitochondrial electron transfer chain. This interaction may impair energy production and leads to the accumulation of reactive oxygen species (ROS) in mitochondria, consequently resulting in abnormal pollen development. Furthermore, we cloned two restorer of fertility ( Rf ) genes, Rf5 and Rf6 , both of which are PPR genes. Neither RF5 nor RF6 binds to CMS RNA, atp6-orfH79 , directly. RF5 interacts with glycine rich protein 162 (GRP162) and RFC3 (restoration of fertility complex subunit 3) thus forming a restoration of fertility complex (RFC) to process CMS RNA. GRP162 can form homodimer and bind to CMS RNA with two binding sites directly. RCF3 is transported into the mitochondria as a membrane protein and functions as a scaffold protein for the assembly of the RFC complex. The RFC complex was detected with a size ranging from 400 to 500 kD. Moreover, Rf6 encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3 to 5. Results show RF6 is also targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS RNA, atp6-orfH79 , to restore the fertility of CMS line. To create more available germplasm in hybrid rice, we developed a molecular marker of orfH79 to screen wild rice and bred some novel CMS lines. We also developed molecular markers of Rf5 and Rf6 to breed novel stronger restore lines which contain two Rf genes. Consequently, we bred several combinations such as Honglian you 6, Yueyou 938, Luoyou 8 and Luoyou 9348 with high yield, good quality, multiple resistance and wide adaptation. HL hybrid rice has been widely planted in Yangzte river areas and South China with the yield of 10 to 12 tons per hectare. HL hybrid rice combinations also have been planted in Southeast Asian countries, such as Vietnam, Indonesia, Malaysia, Bangladesh and Phillippine. Most combinations exhibited 140% or more of yield compared with the local rice cultivars in Southeast Asian countries. HL hybrid rice even more is becoming the leading hybrid rice and priority choice in most of Asian countries and other Africa countries.