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Bitter gourd is an economically important horticultural crop for its edible and medicinal value. However, the regulatory mechanisms of bitter gourd in response to cold stress are still poorly elucidated. In this study, phytohormone determination and comparative transcriptome analyses in XY (cold-tolerant) and QF (cold-sensitive) after low temperature treatment were conducted. Under cold stress, the endogenous contents of abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) in XY were significantly increased at 24 h after treatment (HAT), indicating that ABA, JA and SA might function in regulating cold resistance. RNA-seq results revealed that more differentially expressed genes were identified at 6 HAT in QF and 24 HAT in XY, respectively. KEGG analysis suggested that the plant hormone signal transduction pathway was significantly enriched in both genotypes at all the time points. In addition, transcription factors showing different expression patterns between XY and QF were identified, including CBF3, ERF2, NAC90, WRKY51 and WRKY70. Weighted gene co-expression network analysis suggested MARK1, ERF17, UGT74E2, GH3.1 and PPR as hub genes. These results will deepen the understanding of molecular mechanism of bitter gourd in response to cold stress and the identified genes may help to facilitate the genetic improvement of cold-resistant cultivars. [ABSTRACT FROM AUTHOR]

For soybean, novel single dominant Resistance to Phytophthora sojae (Rps) genes are sought to manage Phytophthora root and stem rot. In this study, resistance to P. sojae was mapped individually in four recombinant inbred line (RIL) populations derived from crosses of the susceptible cultivar Williams with PI 407985, PI 408029, PI 408097, and PI424477 previously identified as putative novel sources of disease resistance. Each population was screened for resistance with five to seven isolates of P. sojae separately over multiple F7-F10 generations. Additionally, three of the populations were screened with inoculum from the combination of three P. sojae isolates (PPR), which comprised virulence to 14 Rps genes. Over 2,300 single-nucleotide polymorphism markers were used to construct genetic maps in each population to identify chromosomal regions associated with resistance to P. sojae. Resistance segregated as one or two genes to the individual isolates and one gene toward PPR in each population and mapped to chromosomes 3, 13, or 18 in one or more of the four RIL populations. Resistance to five isolates mapped to the same chromosome 3 region are as follows: OH7 (PI 424477 and PI408029), OH12168, OH7/8, PPR (PI 407985), and 1.S.1.1 (PI408029). The resistance regions on chromosome 13 also overlapped for OH1, OH25, OH-MIA (PI424477), PPR (PI 424477, PI 407985, and PI 408097), PPR and OH0217 (PI 408097), and OH4 (PI 408029), but were distinct for each population suggesting multiple genes confer resistance. Two regions were identified on chromosome 18 but all appear to map to known loci; notably, resistance to the combined inoculum (PPR) did not map at this locus. However, there are putative new alleles in three of four populations, three on chromosome 3 and two on chromosome 13 based on mapping location but also known virulence in the isolate used. This characterization of all the Rps genes segregating in these populations to these isolates will be informative for breeding, but the combined inoculum was able to map a novel loci. Furthermore, within each of these P. sojae isolates, there was virulence to more than the described Rps genes, and the effectiveness of the novel genes requires testing in larger populations. [ABSTRACT FROM AUTHOR]

In plants, small RNAs (sRNAs), mainly microRNAs (miRNAs) and small interfering RNAs (siRNAs), have been described as key regulators of plant development, growth, and abiotic and biotic responses. Despite reports indicating the involvement of certain sRNAs in regulating the interaction between Botrytis cinerea (a major necrotrophic fungal phytopathogen) and host plants, there remains a lack of analysis regarding the potential regulatory roles of plant sRNAs during early stages of the interaction despite early immune responses observed then during infection. We present the first transcriptome-wide analysis of small RNA expression on the early interaction between the necrotrophic fungus Botrytis cinerea and the model plant Arabidopsis thaliana. We found that evolutionary conserved A. thaliana miRNAs were the sRNAs that accumulated the most in the presence of B. cinerea. The upregulation of miR167, miR159 and miR319 was of particular interest because these, together with their target transcripts, are involved in the fine regulation of the plant hormone signaling pathways. We also describe that miR173, which triggers the production of secondary siRNAs from TAS1 and TAS2 loci, as well as secondary siRNAs derived from these loci, is upregulated in response to B. cinerea. Thus, at an early stage of the interaction there are transcriptional changes of sRNA-guided silencing pathway genes and of a subset of sRNAs that targeted genes from the PPR gene superfamily, and these may be important mechanisms regulating the interaction between A. thaliana and B. cinerea. This work provides the basis for a better understanding of the regulation mediated by sRNAs during early B. cinerea-plant interaction and may help in the development of more effective strategies for its control. [ABSTRACT FROM AUTHOR]

Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX), the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene (LRPPRC). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions, tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study, we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC, homozygous for the LRPPRC*354V allele, and from a control, homozygous for the LRPPRC*A354 allele. Specifically, for both of these fibroblast lines we generated hiPSC, hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines, as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells, with a reduction ranging from ~70% in hiPSC-CMs to undetectable levels in hiPSC-HLC, reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts, this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC. [ABSTRACT FROM AUTHOR]

In citrus breeding programs, male sterility is an important trait for developing seedless varieties. Sterility associated with the male sterile cytoplasm of Kishu mandarin (Kishu-cytoplasm) has been proposed to fit the cytoplasmic male sterility (CMS) model. However, it remains undetermined whether CMS in citrus is controlled by interactions between sterile cytoplasm and nuclear restorer-offertility (Rf) genes. Accordingly, mechanisms underlying the control of the wide phenotypic variation in pollen number for breeding germplasm should be elucidated. This study aimed to identify complete linkage DNA markers responsible for male sterility at the MS-P1 region based on fine mapping. Two P-class pentatricopeptide repeat (PPR) family genes were identified as candidates for Rf based on predicted mitochondrial localization and higher expression in a male fertile variety/selected strain than in a male sterile variety. Eleven haplotypes (HT1-HT11) at the MS-P1 region were defined based on genotyping of DNA markers. Association analysis of diplotypes at the MS-P1 region and the number of pollen grains per anther (NPG) in breeding germplasms harboring Kishu-cytoplasm revealed that the diplotypes in this region influenced NPG. Among these haplotypes, HT1 is a non-functional restorer-of-fertility (rf) haplotype; HT2, a less-functional Rf; HT3-HT5 are semi-functional Rfs; and HT6 and HT7 are functional Rfs. However, the rare haplotypes HT8-HT11 could not be characterized. Therefore, P-class PPR family genes in the MS-P1 region may constitute the nuclear Rf genes within the CMS model, and a combination of the seven haplotypes could contribute to phenotypic variation in the NPG of breeding germplasms. These findings reveal the genomic mechanisms of CMS in citrus and will contribute to seedless citrus breeding programs by selecting candidate seedless seedlings using the DNA markers at the MS-P1 region. [ABSTRACT FROM AUTHOR]

ChinaMu is the largest sequence‐indexed Mutator (Mu) transposon insertional library in maize (Zea mays). In this study, we made significant improvements to the size and quality of the ChinaMu library. We developed a new Mu‐tag isolation method Mu‐Tn5‐seq (MuT‐seq). Compared to the previous method used by ChinaMu, MuT‐seq recovered 1/3 more germinal insertions, while requiring only about 1/14 of the sequencing volume and 1/5 of the experimental time. Using MuT‐seq, we identified 113,879 germinal insertions from 3,168 Mu‐active F1 families. We also assembled a high‐quality genome for the Mu‐active line Mu‐starter, which harbors the initial active MuDR element and was used as the pollen donor for the mutation population. Using the Mu‐starter genome, we recovered 33,662 (15.6%) additional germinal insertions in 3,244 (7.4%) genes in the Mu‐starter line. The Mu‐starter genome also improved the assignment of 117,689 (54.5%) germinal insertions. The newly upgraded ChinaMu dataset currently contains 215,889 high‐quality germinal insertions. These insertions cover 32,224 pan‐genes in the Mu‐starter and B73Ref5 genomes, including 23,006 (80.4%) core genes shared by the two genomes. As a test model, we investigated Mu insertions in the pentatricopeptide repeat (PPR) superfamily, discovering insertions for 92% (449/487) of PPR genes in ChinaMu, demonstrating the usefulness of ChinaMu as a functional genomics resource for maize. [ABSTRACT FROM AUTHOR]

Pentatricopeptide repeat (PPR) proteins, with tandem 30–40 amino acids, were characterized as one kind of nucleus coding protein. They have been demonstrated to play important roles in RNA editing, plant growth and development, and plant immunity. Although the PPR gene family has been characterized in some plant species, less is known about this family in peanut, especially their functions in response to Ralstonia solanacearum. In this study, we performed a genome-wide analysis to identify PPR genes and their functions in resistance to R. solanacearum. Here, 389, 481, and 1079 PPR genes were identified from Arachis duranensis, Arachis ipaensis, and Arachis hypogaea, respectively. Allopolyploidization was the main reason for the increased number of the AhPPR members. Gene duplication brought about 367 pairs of homologous genes of PPRs in A. hypogaea. Whole-genome replication, tandem repeats, scattered repeats, and unconnected repeats constituted the replication types. The substitution rates of nonsynonymous (Ka) versus synonymous (Ks) of all homologous pairs were less than 1.0, suggesting that the homologous AhPPRs underwent intense purifying selection pressure and remained conserved in both structure and function. RNA-seq and RT-qPCR analyses showed that AhPPR598 gene was highly expressed in the aerial part of peanut and involved in response to R. solanacearum. The transient expression of AhPPR598 in Nicotiana benthamiana induced the HR-mediated cell death, up-regulated expression of resistant marker genes, and enhanced the resistance to R. solanacearum, suggesting AhPPR598 was a positive regulator of immunity by regulating the JA and SA pathways. These results provide a new understanding of the origin, distribution, and evolution of the AhPPR gene family and potential gene resources for peanut-resistant breeding. [ABSTRACT FROM AUTHOR]

This article, published in the Journal of Integrative Plant Biology, discusses the molecular bases for the three-line hybrid rice breeding system. The authors focus on the fertility restoration gene Rf4 and its impact on the Wild Abortive (WA) cytoplasmic male sterility type three-line system. They identify a two-copy haplotype of Rf4 (H1) as the most valuable for future hybrid rice breeding. The study provides insights into the evolutionary trajectory of Rf genes and the origin of cytoplasmic male sterility and fertility restoration systems in rice breeding. The findings contribute to our understanding of the CMS/Rf system and offer guidance for the molecular design breeding of three-line hybrid rice. [Extracted from the article]

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in land plants. However, current knowledge about the evolution of the PPR gene family remains largely limited. In this study, we performed a comparative genomic analysis of the PPR gene family in O. sativa and its wild progenitor, O. rufipogon, and outlined a comprehensive landscape of gene duplications. Our findings suggest that the majority of PPR genes originated from dispersed duplications. Although segmental duplications have only expanded approximately 11.30% and 13.57% of the PPR gene families in the O. sativa and O. rufipogon genomes, we interestingly obtained evidence that segmental duplication promotes the structural diversity of PPR genes through incomplete gene duplications. In the O. sativa and O. rufipogon genomes, 10 (~33.33%) and 22 pairs of gene duplications (~45.83%) had non-PPR paralogous genes through incomplete gene duplication. Segmental duplications leading to incomplete gene duplications might result in the acquisition of domains, thus promoting functional innovation and structural diversification of PPR genes. This study offers a unique perspective on the evolution of PPR gene structures and underscores the potential role of segmental duplications in PPR gene structural diversity. [ABSTRACT FROM AUTHOR]

With a diversity of approximately 22,000 species, bryophytes (hornworts, liverworts, and mosses) represent a major and diverse lineage of land plants. Bryophytes can thrive in many extreme environments as they can endure the stresses of drought, heat, and cold. The moss Niphotrichum japonicum (Grimmiaceae, Grimmiales) can subsist for extended periods under heat and drought conditions, providing a good candidate for studying the genetic basis underlying such high resilience. Here, we de novo assembled the genome of N. japonicum using Nanopore long reads combined with Hi-C scaffolding technology to anchor the 191.61 Mb assembly into 14 pseudochromosomes. The genome structure of N. japonicum's autosomes is mostly conserved and highly syntenic, in contrast to the sparse and disordered genes present in its sex chromosome. Comparative genomic analysis revealed the presence of 10,019 genes exclusively in N. japonicum. These genes may contribute to the speciesspecific resilience, as demonstrated by the gene ontology (GO) enrichment. Transcriptome analysis showed that 37.44% (including 3,107 unique genes) of the total annotated genes (26,898) exhibited differential expression as a result of heat-induced stress, and the mechanisms that respond to heat stress are generally conserved across plants. These include the upregulation of HSPs, LEAs, and reactive oxygen species (ROS) scavenging genes, and the downregulation of PPR genes. N. japonicum also appears to have distinctive thermal mechanisms, including species-specific expansion and upregulation of the Self-incomp_S1 gene family, functional divergence of duplicated genes, structural clusters of upregulated genes, and expression piggybacking of hub genes. Overall, our study highlights both shared and species-specific heat tolerance strategies in N. japonicum, providing valuable insights into the heat tolerance mechanism and the evolution of resilient plants. [ABSTRACT FROM AUTHOR]

Summary: Restorer‐of‐fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer‐of‐fertility C‐terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target.A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility.We show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2‐nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria.We provide a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins and the evolution of fertility restoration in flowering plants. [ABSTRACT FROM AUTHOR]

Although several pentatricopeptide repeat (PPR) proteins are involved in post-transcriptional processing of mitochondrial RNA, it is unclear which specific protein is involved in the RNA editing of ccmC in maize (Zea mays). Here we report the identification of the maize empty pericarp 601 (emp601) mutant and the map-based cloning of the Emp601 gene, which encodes an E2-type PPR protein that is targeted to mitochondria. A single-nucleotide deletion in the emp601 mutant caused a frameshift and introduced a premature stop codon into the predicted EMP601. This mutation was associated with reduced accumulation of mitochondrial complex III as well as with inhibition of growth and differentiation of basal endosperm transfer layer cells, leading to final degeneration of the embryo and endosperm. We determine that loss of EMP601 function prevents the C-to-U RNA editing of the mitochondrial transcript ccmC at position 358. EMP601 binds to the ccmC transcript and directly interacts with Multiple organellar RNA editing factor 8 and may be a component of the plant mitochondrial editosome. We conclude that EMP601 functions in RNA editing of mitochondrial ccmC transcripts and influences mitochondrial function and seed development. [ABSTRACT FROM AUTHOR]

In higher plants, the chloroplast is the most important organelle for photosynthesis and for numerous essential metabolic processes in the cell. Although many genes involved in chloroplast development have been identified, the mechanisms underlying such development are not fully understood. In this study, a rice (Oryza sativa) mutant exhibiting pale green color and seedling lethality was isolated from a mutant library. The mutated gene was identified as an ortholog of THA8 (thylakoid assembly 8) in Arabidopsis and maize. This gene is designated as OsTHA8 hereafter. OsTHA8 showed a typical pentatricopeptide repeat (PPR) characteristic of only four PPR motifs. Inactivation of OsTHA8 led to a deficiency in chloroplast development in the rice seedling stage. OsTHA8 was expressed mainly in young leaves and leaf sheaths. The OsTHA8 protein was localized to the chloroplast. Loss of function of OsTHA8 weakened the editing efficiency of ndhB-611/737 and rps8-182 transcripts under normal conditions. Y2H and BiFC indicated that OsTHA8 facilitates RNA editing by forming an editosome with multiple organellar RNA editing factor (OsMORF8) and thioredoxin z (OsTRXz), which function in RNA editing in rice chloroplasts. Defective OsTHA8 impaired chloroplast ribosome assembly and resulted in reduced expression of PEP-dependent genes and photosynthesis-related genes. Abnormal splicing of the chloroplast gene ycf3 was detected in ostha8. These findings reveal a synergistic regulatory mechanism of chloroplast biogenesis mediated by RNA, broaden the function of the PPR family, and shed light on the RNA editing complex in rice. [ABSTRACT FROM AUTHOR]

Kiwifruit is a perennial fruit tree with high nutritional and economic value; however, various pathogen stresses have resulted in reductions in its yield and quality. Pentatricopeptide repeat proteins (PPRs), characterized by tandem repetitions of 35 amino acid motifs, play roles in RNA editing, mRNA stability, and splicing. They may also regulate plant development and growth. Nevertheless, the roles of PPRs in plant development and disease resistance remain unclear. In this study, we focused on the roles of PPRs in the fruit development and pathogen stress of kiwifruit and conducted a series of analyses of the PPR gene family in two representative kiwifruit species (Actinidia chinensis (Ach) and Actinidia eriantha (Ace)) with markedly different degrees of disease resistance. A total of 497 and 499 PPRs were identified in Ach and Ace, respectively. All the kiwifruit PPRs could be phylogenetically divided into four subfamilies. There were about 40.68% PPRs predicted to be localized to mitochondria or chloroplasts. A synteny analysis showed that the expansion of the kiwifruit PPRs mainly originated from segmental duplication. Based on RNA-seq data from the fruit over 12 periods of development and maturity, a weighted correlation network analysis suggested that two PPRs, Actinidia20495.t1 and Actinidia15159.t1, may be involved in fruit development and maturation. In addition, we observed different responses with respect to the expression of PPRs and RNA editing between resistant and susceptible kiwifruits following infection with pathogenic bacteria, indicating the regulatory role of PPRs in the stress response via the modulation of RNA editing. The differentially expressed upstream transcription factors of the PPRs were further identified; they may regulate resistance adaption by modulating the expression of the PPRs. Collectively, these results suggest that PPRs play roles in the development and disease resistance of kiwifruit and provide candidate genes for further clarifying the resistance mechanisms in kiwifruits. [ABSTRACT FROM AUTHOR]

Peanut (Arachis hypogaea L.) is an important oilseed crop worldwide. Improving its yield is crucial for sustainable peanut production to meet increasing food and industrial requirements. Deciphering the genetic control underlying peanut kernel weight and size, which are essential components of peanut yield, would facilitate high-yield breeding. A high-density single nucleotide polymorphism (SNP)-based linkage map was constructed using a recombinant inbred lines (RIL) population derived from a cross between the variety Yuanza9102 and a germplasm accession wt09-0023. Kernel weight and size quantitative trait loci (QTLs) were co-localized to a 0.16 Mb interval on Arahy07 using inclusive composite interval mapping (ICIM). Analysis of SNP, and Insertion or Deletion (INDEL) markers in the QTL interval revealed a gene encoding a pentatricopeptide repeat (PPR) superfamily protein as a candidate closely linked with kernel weight and size in cultivated peanut. Examination of the PPR gene family indicated a high degree of collinearity of PPR genes between A. hypogaea and its diploid progenitors, Arachis duranensis and Arachis ipaensis. The candidate PPR gene, Arahy.JX1V6X, displayed a constitutive expression pattern in developing seeds. These findings lay a foundation for further fine mapping of QTLs related to kernel weight and size, as well as validation of candidate genes in cultivated peanut. [ABSTRACT FROM AUTHOR]

In citrus breeding programs, male sterility is an important trait for developing seedless varieties. Sterility associated with the male sterile cytoplasm of Kishu mandarin (Kishu-cytoplasm) has been proposed to fit the cytoplasmic male sterility (CMS) model. However, it remains undetermined whether CMS in citrus is controlled by interactions between sterile cytoplasm and nuclear restorer-offertility (Rf) genes. Accordingly, mechanisms underlying the control of the wide phenotypic variation in pollen number for breeding germplasm should be elucidated. This study aimed to identify complete linkage DNA markers responsible for male sterility at the MS-P1 region based on fine mapping. Two P-class pentatricopeptide repeat (PPR) family genes were identified as candidates for Rf based on predicted mitochondrial localization and higher expression in a male fertile variety/selected strain than in a male sterile variety. Eleven haplotypes (HT1–HT11) at the MS-P1 region were defined based on genotyping of DNA markers. Association analysis of diplotypes at the MS-P1 region and the number of pollen grains per anther (NPG) in breeding germplasms harboring Kishu-cytoplasm revealed that the diplotypes in this region influenced NPG. Among these haplotypes, HT1 is a non-functional restorer-of-fertility (rf) haplotype; HT2, a less-functional Rf; HT3–HT5 are semi-functional Rfs; and HT6 and HT7 are functional Rfs. However, the rare haplotypes HT8–HT11 could not be characterized. Therefore, P-class PPR family genes in the MS-P1 region may constitute the nuclear Rf genes within the CMS model, and a combination of the seven haplotypes could contribute to phenotypic variation in the NPG of breeding germplasms. These findings reveal the genomic mechanisms of CMS in citrus and will contribute to seedless citrus breeding programs by selecting candidate seedless seedlings using the DNA markers at the MS-P1 region. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) serve as covalently closed single-stranded RNAs and have been proposed to influence plant development and stress resistance. Grapevine is one of the most economically valuable fruit crops cultivated worldwide and is threatened by various abiotic stresses. Herein, we reported that a circRNA (Vv-circPTCD1) processed from the second exon of the pentatricopeptide repeat family gene PTCD1 was preferentially expressed in leaves and responded to salt and drought but not heat stress in grapevine. Additionally, the second exon sequence of PTCD1 was highly conserved, but the biogenesis of Vv-circPTCD1 is species-dependent in plants. It was further found that the overexpressed Vv-circPTCD1 can slightly decrease the abundance of the cognate host gene, and the neighboring genes are barely affected in the grapevine callus. Furthermore, we also successfully overexpressed the Vv-circPTCD1 and found that the Vv-circPTCD1 deteriorated the growth during heat, salt, and drought stresses in Arabidopsis. However, the biological effects on grapevine callus were not always consistent with those of Arabidopsis. Interestingly, we found that the transgenic plants of linear counterpart sequence also conferred the same phenotypes as those of circRNA during the three stress conditions, no matter what species it is. Those results imply that although the sequences are conserved, the biogenesis and functions of Vv-circPTCD1 are species-dependent. Our results indicate that the plant circRNA function investigation should be conducted in homologous species, which supports a valuable reference for further plant circRNA studies. [ABSTRACT FROM AUTHOR]

The Pentatricopeptide repeat (PPR) superfamily is a large gene family in plants that regulates organelle RNA metabolism, which is important for plant growth and development. However, a genome-wide analysis of the PPR gene family and its response to abiotic stress has not been reported for the relict woody plant Liriodendron chinense. In this paper, we identified 650 PPR genes from the L. chinense genome. A phylogenetic analysis showed that the LcPPR genes could roughly be divided into the P and PLS subfamilies. We found that 598 LcPPR genes were widely distributed across 19 chromosomes. An intraspecies synteny analysis indicated that duplicated genes from segmental duplication contributed to the expansion of the LcPPR gene family in the L. chinense genome. In addition, we verified the relative expression of Lchi03277, Lchi06624, Lchi18566, and Lchi23489 in the roots, stems, and leaves and found that all four genes had the highest expression in the leaves. By simulating a drought treatment and quantitative reverse transcription PCR (qRT-PCR) analysis, we confirmed the drought-responsive transcriptional changes in four LcPPR genes, two of which responded to drought stress independent of endogenous ABA biosynthesis. Thus, our study provides a comprehensive analysis of the L. chinense PPR gene family. It contributes to research into their roles in this valuable tree species' growth, development, and stress resistance. [ABSTRACT FROM AUTHOR]

Soybean is the most important oilseed in the tropical and subtropical parts of the world unfortunately has poor competence for seed longevity. It's strongly affected by environment (Temperature and RH) during seed formation, harvest and storage. Tagging of seed longevity associated markers in the crosses (Birsa Soya1 × DS-228) which are good and poor storer genotypes resp. by using 26 SSR primers in genotypes contrasting for these traits in soybean. The polymorphism was observed between genotypes differing for seed longevity trait for six primer pairs, viz; Sat_162, Satt632, Sat_202, Satt523, Satt184 and Satt460, among them seed coat hardiness QTL, SCH 1-2 linked Satt632 amplified distinct markers. Bulked segregant analysis was carried out by using SSR markers in segregating 93 F2 populations. The Satt632-237 bp marker was further validated in individual F2 plant population. The genotype, Birsa Soya1 and it segregants Satt632-237 bp marker was found to be closely associated with high seed longevity in soybean. Hence, the primer Satt632 is impending marker for marker assisted breeding for high seed longevity marker to be linked to seed coat hardiness loci qSCH1-2 like gene present on chromosome A2. Seed longevity trait is governed by one or few gene with observed segregation ratio of 3.65:1 as against expected segregated ratio 3:1. Candidate gene analysis at qSCH1-2 loci, indicated for role of either a brassinosteriod regulated protein or pentatricopeptide repeat gene to regulate seed longevity. [ABSTRACT FROM AUTHOR]

Cold acclimation refers to a phenomenon in which plants become more tolerant to freezing after exposure to non-lethal low temperatures. Aulacomnium turgidum (Wahlenb.) Schwaegr is a moss found in the Arctic that can be used to study the freezing tolerance of bryophytes. To improve our understanding of the cold acclimation effect on the freezing tolerance of A. turgidum, we compared the electrolyte leakage of protonema grown at 25 °C (non-acclimation; NA) and at 4 °C (cold acclimation; CA). Freezing damage was significantly lower in CA plants frozen at −12 °C (CA-12) than in NA plants frozen at −12 °C (NA-12). During recovery at 25 °C, CA-12 demonstrated a more rapid and greater level of the maximum photochemical efficiency of photosystem II than NA-12, indicating a greater recovery capacity for CA-12 compared to NA-12. For the comparative analysis of the transcriptome between NA-12 and CA-12, six cDNA libraries were constructed in triplicate, and RNA-seq reads were assembled into 45,796 unigenes. The differential gene expression analysis showed that a significant number of AP2 transcription factor genes and pentatricopeptide repeat protein-coding genes related to abiotic stress and the sugar metabolism pathway were upregulated in CA-12. Furthermore, starch and maltose concentrations increased in CA-12, suggesting that cold acclimation increases freezing tolerance and protects photosynthetic efficiency through the accumulation of starch and maltose in A. turgidum. A de novo assembled transcriptome can be used to explore genetic sources in non-model organisms. [ABSTRACT FROM AUTHOR]

Background: Starch are the main nutritional components of maize (Zea mays L.), and starch pasting properties are widely used as essential indicators for quality estimation. Based on the previous studies, various genes related to pasting properties have been identified in maize. However, the loci underlying variations in starch pasting properties in maize inbred lines remain to be identified. Results: To investigate the genetic architecture of these traits, the starch pasting properties were examined based on 292 maize inbred lines, which were genotyped with the MaizeSNP50 BeadChip composed of 55,126 evenly spaced, random SNPs. A genome-wide association study (GWAS) implemented in the software package FarmCPU was employed to identify genomic loci for the starch pasting properties. 48 SNPs were found to be associated with pasting properties. Moreover, 37 candidate genes were correlated with pasting properties. Among the candidate genes, GRMZM2G143646 and GRMZM2G166407 were associated with breakdown and final viscosity significantly, and both genes encode PPR (Pentatricopeptide repeat) protein. We used GWAS to explore candidate genes of maize starch pasting properties in this study. The identified candidate genes will be useful for further understanding of the genetic architecture of starch pasting properties in maize. Conclusion: This study showed a complex regulation network about maize quality trait and starch pasting properties. It may provide some useful markers for marker assisted selection and a basis for cloning the genes behind these SNPs. [ABSTRACT FROM AUTHOR]

SUMMARY: The Arabidopsis thaliana genome harbors more than 450 nuclear genes encoding pentatricopeptide repeat (PPR) proteins that operate in the RNA metabolism of mitochondria and/or plastids. To date, the molecular function of many PPR proteins is still unknown. Here we analyzed the nucleus‐encoded gene At4g19440 coding for a P‐type PPR protein. Knockout of this gene interferes with normal embryo development and seed maturation. Two experimental approaches were applied to overcome lethality and to investigate the outcome of At4g19440 knockout in adult plants. These studies revealed changes in the abundance of several mitochondria‐encoded transcripts. In particular, steady‐state levels of dicistronic rpl5‐cob RNAs were markedly reduced, whereas levels of mature ccmC and rpl2‐mttB transcripts were clearly increased. Predictions according to the one repeat to one nucleotide code for PPR proteins indicate binding of the At4g19440 protein to a previously detected small RNA at the 3′ termini of the dicistronic rpl5‐cob transcripts. This potential interaction indicates a function of this protein in 3′ end formation and stabilization of these RNA species, whereas the increase in the levels of the ccmC mRNA along with other mitochondria‐encoded RNAs seems to be a secondary effect of At4g19440 knockout. Since the inactivation of At4g19440 influences the stability of several mitochondrial RNAs we call this gene MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4). This factor will be an interesting subject to study opposing effects of a single nucleus‐encoded protein on mitochondrial transcript levels. Significance Statement: The nucleus‐encoded gene MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 is essential for accumulation of rpl5‐cob dicistronic transcripts in mitochondria of Arabidopsis thaliana. Knockout of this nuclear gene interferes with the formation of viable seeds and leads to a severe reduction in rpl5‐cob mRNAs but concomitantly induces an increase in other mitochondrial RNAs. [ABSTRACT FROM AUTHOR]

This document is a corrigendum for an article titled "Allelic haplotype combinations at the MS-P1 region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus." The corrigendum addresses two errors in Table 2 of the published article, correcting the names of the founders from which the haplotypes were derived. The authors apologize for the errors and state that they do not affect the scientific conclusions of the article. The original article has been updated. [Extracted from the article]

Allopolyploids are believed to inherit the genetic characteristics of its progenitors and exhibit stronger adaptability and vigor. The allotetraploid Isoetes sinensis was formed by the natural hybridization and polyploidization of two diploid progenitors, Isoetes taiwanensis and Isoetes yunguiensis, and was believed to have the potential to adapt to plateau environments. To explore the expression pattern of homoeologous genes and their contributions to altitude adaptation, we transplanted natural allotetraploid I. sinensis (TnTnYnYn) along the altitude gradient for a long‐term, and harvested them in summer and winter, respectively. One year after transplanting, it still lived well, even in the extreme environment of the Qinghai‐Tibet Plateau. Then, we performed high‐throughput RNA sequencing to measure their gene expression level. A total of 7801 homoeologous genes were expressed, among which 5786 were identified as shared expression in different altitudes and seasons. We further found that altitude variations could change the subgenome bias trend of I. sinensis, but season could not. Moreover, the functions of uniquely expressed genes indicated that temperature might be an important restrictive factor during the adaptation process. Through the analysis of DEGs and uniquely expressed genes, we found that Y subgenome provided more contributions to high altitude adaptation than T subgenome. These adaptive traits to high altitude may be inherited from its plateau progenitor I. yunguiensis. Through weighted gene co‐expression network analysis, pentatricopeptide repeats gene family and glycerophospholipid metabolism pathway were considered to play important roles in high‐altitude adaptation. Totally, this study will enrich our understanding of allopolyploid in environmental adaptation. [ABSTRACT FROM AUTHOR]

Photosynthesis, a fundamental process for plant growth and development, is dependent on chloroplast formation and chlorophyll synthesis. Severe disruption of chloroplast structure results in albinism of higher plants. In the present study, we report a cucumber albino alc mutant that presented white cotyledons under normal light conditions and was unable to produce first true leaf. Meanwhile, alc mutant could grow creamy green cotyledons under dim light conditions but died after exposure to normal light irradiation. No chlorophyll and carotenoid were detected in the alc mutant grown under normal light conditions. Using transmission electron microscopy, impaired chloroplasts were observed in this mutant. The genetic analysis indicated that the albino phenotype was recessively controlled by a single locus. Comparative transcriptomic analysis between the alc mutant and wild type revealed that genes involved in chlorophyll metabolism and the methylerythritol 4-phosphate pathway were affected in the alc mutant. In addition, three genes involved in chloroplast development, including two FtsH genes and one PPR gene, were found to have negligible expression in this mutant. The quality of RNA sequencing results was further confirmed by real-time quantitative PCR analysis. We also examined 12 homologous genes from alc mutant in other plant species, but no genetic variation in the coding sequences of these genes was found between alc mutant and wild type. Taken together, we characterized a cucumber albino mutant with albinism phenotype caused by chloroplast development deficiency and this mutant can pave way for future studies on plastid development. [ABSTRACT FROM AUTHOR]

Oryza longistaminata (OL), an AA-genome African wild rice which can propagate clonally via rhizome, is an important germplasm for improvement of Asian cultivated rice, however recessive lethal alleles can hitchhike clonal propagation in heterozygous state. Selfing of OL is difficult due to its self-incompatibility, but simple selfing of hybrid progeny between OL and O. sativa is effective to disclose and eliminate recessive lethal alleles. Here, we identified an exhibited albino-lethal phenotype mutant, from an F 2 population between OL and O. sativa, named it albino seedling-lethal (asl). The leaves of asl mutant showed abnormal chloroplast development. The albino characteristics of asl were determined to be governed by a set of recessive nuclear genes through genetic analysis. Map-based cloning experiments found that a single nucleotide variation (G to A) was detected in the exon of OsASL in OL , which causes a premature stop codon. OsASL encodes a PLS-type PPR protein with 12 pentratricopeptide repeat domains, and is translocalized to chloroplasts. Complementation and knockout transgenic experiments further confirmed that OsASL is responsible for the albino-lethal phenotype. Loss-of-function OsASL (i.e. osasl) resulted in devoid of intron splicing of chloroplast RNA atpF , ndhA , rpl2 and rps12 , and also RNA editing of ndhB , but facilitates the RNA editing of rpl2 in the plastid. Transcriptome sequencing showed that OsASL was mainly involved in chlorophyll synthesis pathway. The expression of Chlorophyll-associated genes were significantly decreased in asl plants, especially PEP (plastid-encoded RNA polymerase)-mediated genes. Our results suggest that OsASL is crucial for RNA editing, RNA splicing of chloroplast RNA group II genes, and plays an essential role in chloroplast development during early leaf development in rice. • We identified a lethal albino phenotype mutant asl) from the progeny of the Oryza longistaminata and O. sativa cross. • Fine mapping, complementation and knockout experiments confirmed that the gene OsASL is responsible for the phenotype of asl. • OsASL gene encodes a PPR protein located in the chloroplast, its mutation affects splicing and editing of chloroplast RNAs. [ABSTRACT FROM AUTHOR]

Pentatricopeptide repeat (PPR) proteins are one type of helical repeat protein that are widespread in eukaryotes. In particular, there are several hundred PPR members in flowering plants. The majority of PPR proteins are localized in the plastids and mitochondria, where they play a crucial role in various aspects of RNA metabolism at the post-transcriptional and translational steps during gene expression. Among the early land plants, the moss Physcomitrium (formerly Physcomitrella) patens has at least 107 PPR protein-encoding genes, but most of their functions remain unclear. To elucidate the functions of PPR proteins, a reverse-genetics approach has been applied to P. patens. To date, the molecular functions of 22 PPR proteins were identified as essential factors required for either mRNA processing and stabilization, RNA splicing, or RNA editing. This review examines the P. patens PPR gene family and their current functional characterization. Similarities and a diversity of functions of PPR proteins between P. patens and flowering plants and their roles in the post-transcriptional regulation of organellar gene expression are discussed. [ABSTRACT FROM AUTHOR]

Background: Pentatricopeptide repeat (PPR) proteins play an essential role in the post-transcriptional regulation of genes in plastid genomes. Although important advances have been made in understanding the functions of these genes, there is little information available on chloroplastic PPR genes in non-model plants and less in plants without chloroplasts. In the present study, a comprehensive and multifactorial bioinformatic strategy was applied to search for putative PPR genes in the foliar and meristematic tissues of green and albino plantlets of the non-model plant Agave angustifolia Haw. Results: A total of 1581 PPR transcripts were identified, of which 282 were chloroplastic. Leaf tissue in the albino plantlets showed the highest levels of expression of chloroplastic PPRs. The search for hypothetical targets of 12 PPR sequences in the chloroplast genes of A. angustifolia revealed their action on transcripts related to ribosomes and translation, photosystems, ATP synthase, plastid-encoded RNA polymerase and RuBisCO. Conclusions: Our results suggest that the expression of PPR genes depends on the state of cell differentiation and plastid development. In the case of the albino leaf tissue, which lacks functional chloroplasts, it is possible that anterograde and retrograde signaling networks are severely compromised, leading to a compensatory anterograde response characterized by an increase in the expression of PPR genes. [ABSTRACT FROM AUTHOR]

Ferroptosis is a form of iron- and lipid peroxidation-mediated programmed cell death that occurs widely in mammalian cells. However, this phenomenon is rarely reported in unicellular eukaryotes. Here, we address whether ferroptosis occurs in the model unicellular eukaryote Schizosaccharomyces pombe (S. pombe). Deletion of the pentatricopeptide repeat (PPR) gene ppr2 encoding as a general mitochondrial translation factor required for mitochondrial translation disrupts iron homeostasis and induces oxidative stress, resulting in loss of cell viability. The small-molecular ferroptosis inhibitors deferoxamine (DFO) and ferrostatin-1 (Fer-1) partially rescued the ppr2 deletion-induced cell death. The amount of malondialdehyde, a lipid peroxidation marker, in Δppr2 cells was higher than that in wild type. Using C11-BODIPY 581/591, an oxidation-sensitive fluorescent lipid peroxidation probe, we showed that Δppr2 cells have a large amount of lipid peroxidation compared to wild-type cells. Deletion of ferric reductase transmembrane component 1 (frp1) encoding S. pombe ferric reductase, which is required for ferric iron uptake, partially rescued the cell death of Δppr2 cells. Our results suggest that ppr2 deletion causes an imbalance in iron homeostasis and redox, leading to ferroptosis-like cell death in S. pombe. [ABSTRACT FROM AUTHOR]

The North Eastern Karnataka, which consists of seven districts viz., Bidar, Kalaburgi (Earlier Gulbarga), Yadgir, Raichur, Koppal, Bellary and part of Bijapur (Sindagi and Indi talukas) with sizable sheep and goat population experiences the outbreaks of PPR inspite of regular vaccination but the attempts to isolate and characterize the prevailing PPR virus is limited. Therefore, the goal of the current study was to detect PPR virus isolates circulating in North Eastern Karnataka using microscopic (TEM) and molecular (RT-PCR and sequencing) approaches. Various clinical samples viz., occular, oral, nasal and rectal swabs including blood from ailing kids and adult goats were taken from a total of 15 suspected PPR outbreaks. The samples were first examined for the presence of the PPR virus by observing the infected Vero-cells for development of characteristic CPE. The isolates showing CPE were further confirmed for the virus by RT-PCR and sequencing using M (MF-Morb/ MRPPR3) gene of PPR virus.All the six isolates obtained by isolation yielded desired amplicon of 191 bp size which were specific to M region of PPR virus genome. The PCR confirmed isolates were further subjected to transmission electron microscopy (TEM) and sequencing. The TEM images showed pleomorphic virion particles with varying sizes indicative of Morbilli-virus. The sequenced data of all isolates confirmed them as PPR virus by NCBI blast. These findings provide the first scientific documentation of PPR outbreak confirmation in the region by microscopic and molecular characterization using M gene. [ABSTRACT FROM AUTHOR]

Ribonucleic acid editing (RE) is a post-transcriptional process that altered the genetics of RNA which provide the extra level of gene expression through insertion, deletions, and substitutions. In animals, it converts nucleotide residues C-U. Similarly in plants, the role of RNA editing sites (RES) in rice under alkaline stress is not fully studied. Rice is a staple food for most of the world population. Alkaline stress cause reduction in yield. Here, we explored the effect of alkaline stress on RES in the whole mRNA from rice chloroplast and mitochondria. Ribonucleic acid editing sites in both genomes (3336 RESs) including chloroplast (345 RESs) and mitochondria (2991 RESs) with average RES efficiency ∼55% were predicted. Our findings showed that majority of editing events found in non-synonymous codon changes and change trend in amino acids was hydrophobic. Four types of RNA editing A-G (A-I), C-T (C-U), G-A, and T-C were identified in treated and untreated samples. Overall, RNA editing efficiency was increased in the treated samples. Analysis of Gene Ontology revealed that mapped genes were engaged in many biological functions and molecular processes. We also checked the expression of pentatricopeptide repeat (PPR), organelle zinc-finger (OZI), and multiple organellar RNA editing factors/RNA editing factor interacting proteins genes in control and treatment, results revealed upregulation of PPR and OZ1 genes in treated samples. This induction showed the role of these genes in RNA editing. The current findings report that RNA editing increased under alkaline stress which may contribute in adaptation for rice by changing amino acids in edited genes (88 genes). These findings will provide basis for identification of RES in other crops and also will be useful in alkaline tolerance development in rice. [ABSTRACT FROM AUTHOR]

Key Message: Unstable Restorer-of-fertility (Rfu), conferring unstable fertility restoration in the pepper CGMS system, was delimited to a genomic region near Rf and is syntenic to the PPR-like gene-rich region in tomato. The use of cytoplasmic-genic male sterility (CGMS) systems greatly increases the efficiency of hybrid seed production. Although marker development and candidate gene isolation have been performed for the Restorer-of-fertility (Rf) gene in pepper (Capsicum annuum L.), the broad use of CGMS systems has been hampered by the instability of fertility restoration among pepper accessions, especially sweet peppers, due to the widespread presence of the Unstable Restorer-of-fertility (Rfu) locus. Therefore, to investigate the genetic factors controlling unstable fertility restoration in sweet peppers, we developed a segregation population (BC4F5) from crosses using a male-sterile line and an Rfu-containing line. Segregation did not significantly deviate from a 3:1 ratio for unstable fertility restoration to sterility, indicating single dominant locus control for unstable fertility restoration in this population. Genetic mapping delimited the Rfu locus to a 398 kb genomic region on chromosome 6, which is close to but different from the previously identified Rf-containing region. The Rfu-containing region harbors a pentatricopeptide repeat (PPR) gene, along with 10 other candidate genes. In addition, this region is syntenic to the genomic region containing the largest number of Rf-like PPR genes in tomato. Therefore, the dynamic evolution of PPR genes might be responsible for both the restoration and instability of fertility in pepper. During genetic mapping, we developed various molecular markers, including one that co-segregated with Rfu. These markers showed higher accuracy for genotyping than previously developed markers, pointing to their possible use in marker-assisted breeding of sweet peppers. [ABSTRACT FROM AUTHOR]

Wheat is an important cereal and half of the world population consumed it. Wheat faces environmental stresses and different techniques (CRISPR, gene silencing, GWAS, etc.) were used to enhance its production but RNA editing (RESs) is not fully explored in wheat. RNA editing has a special role in controlling environmental stresses. The genome-wide identification and functional characterization of RESs in different types of wheat genotypes was done. We employed six wheat genotypes by RNA-seq analyses to achieve RESs. The findings revealed that RNA editing events occurred on all chromosomes equally. RNA editing sites were distributed randomly and 10–12 types of RESs were detected in wheat genotypes. Higher number of RESs were detected in drought-tolerant genotypes. A-to-I RNA editing (2952, 2977, 1916, 2576, 3422, and 3459) sites were also identified in six wheat genotypes. Most of the genes were found to be engaged in molecular processes after a Gene Ontology analysis. PPR (pentatricopeptide repeat), OZ1 (organelle zinc-finger), and MORF/RIP gene expression levels in wheat were also examined. Normal growth conditions diverge gene expression of these three different gene families, implying that normal growth conditions for various genotypes can modify RNA editing events and have an impact on gene expression levels. While the expression of PPR genes was not change. We used Variant Effect Predictor (VEP) to annotate RNA editing sites, and Local White had the highest RESs in the CDS region of the protein. These findings will be useful for prediction of RESs in other crops and will be helpful in drought tolerance development in wheat. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility (CMS) is widely used to control pollination in the production of commercial F1 hybrid seed in sorghum. So far, 6 major fertility restorer genes, Rf1 to Rf6, have been reported in sorghum. Here, we fine-mapped the Rf5 locus on sorghum chromosome 5 using descendant populations of a ‘Nakei MS-3A’ × ‘JN43’ cross. The Rf5 locus was narrowed to a 140-kb region in BTx623 genome (161-kb in JN43) with 16 predicted genes, including 6 homologous to the rice fertility restorer Rf1 (PPR.1 to PPR.6). These 6 homologs have tandem pentatricopeptide repeat (PPR) motifs. Many Rf genes encode PPR proteins, which bind RNA transcripts and modulate gene expression at the RNA level. No PPR genes were detected at the Rf5 locus on the corresponding homologous chromosome of rice, foxtail millet, or maize, so this gene cluster may have originated by chromosome translocation and duplication after the divergence of sorghum from these species. Comparison of the sequences of these genes between fertile and CMS lines identified PPR.4 as the most plausible candidate gene for Rf5. [ABSTRACT FROM AUTHOR]

Alsophila spinulosa , a relict tree fern, is a valuable plant for investigating environmental adaptations. Its genetic resources, however, are scarce. We used the PacBio and Illumina platforms to sequence the polyadenylated RNA of A. spinulosa root, rachis, and pinna, yielding 125,758, 89,107, and 89,332 unigenes, respectively. Combining the unigenes from three organs yielded a non-redundant reference transcriptome with 278,357 unigenes and N50 of 4141 bp, which were further reconstructed into 38,470 UniTransModels. According to functional annotation, pentatricopeptide repeat genes and retrotransposon-encoded polyprotein genes are the most abundant unigenes. Clean reads mapping to the full-length transcriptome is used to assess the expression of unigenes. The stress-induced ASR genes are highly expressed in all three organs, which is validated by qRT-PCR. The organ-specific upregulated genes are enriched for pathways involved in stress response, secondary metabolites, and photosynthesis. Genes for five types of photoreceptors, CRY signaling pathway, ABA biosynthesis and transduction pathway, and stomatal movement-related ion channel/transporter are profiled using the high-quality unigenes. The gene expression pattern coincides with the previously identified stomatal characteristics of fern. This study is the first multi-organ full-length transcriptome report of a tree fern species, the abundant genetic resources and comprehensive analysis of A. spinulosa , which provides the groundwork for future tree fern research. [ABSTRACT FROM AUTHOR]

Key message: Rf candidate genes were related to the super D05_PPR-cluster and verified to be individually nonfunctional. Restorer of fertility (Rf) genes of cytoplasmic male sterility (CMS) is commonly found to be PPR (pentatricopeptide repeat) genes, which are mostly located in a cluster of PPR genes with high similarity. Here, Homocap-seq was applied to analyze PPR clusters in 'three lines,' and we found broad variations within the D05_PPR-cluster in a restorer line and deduced that the D05_PPR-cluster was associated with fertility restoration. Genetic mapping of Rf and Homocap-seq analysis of three genotypes in the F2 population validated that the D05_PPR-cluster was the origin of Rf. Three Rf candidates were cloned that were the most actively expressed genes in the D05_PPR-cluster in the restorer line as revealed by their high-depth amplicons. However, further transgenic experiments showed that none of the candidates could restore fertility of the CMS line independently. Then, the members of the brand-new super D05_PPR-cluster in the restorer line, containing 14 full-length PPRs and at least 13 PPR homologous sequences, were identified by long-read resequencing, which validated the effectiveness of variation and expression prediction of Homocap-seq. Additionally, we found that several PPR duplications, including 2 of the 3 Rf candidates, had undergone site-specific selection as potentially important anther development-associated genes. Finally, we proposed that multiple PPRs were coordinately responsible for the fertility restoration of the CMS line. [ABSTRACT FROM AUTHOR]

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method, we show that synthetic S-type PPR proteins are easy to design and bind with high affinity and specificity and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools. [ABSTRACT FROM AUTHOR]

Summary: Lesion mimic mutants are used to elucidate mechanisms controlling plant responses to pathogen attacks and environmental stresses. Although dozens of genes had been functionally demonstrated to be involved in lesion mimic phenotype in several plant species, the molecular mechanisms underlying the hypersensitive response are largely unknown. Here, a rice (Oryza sativa) lesion mimic mutant natural blight leaf 3 (nbl3) was identified from T‐DNA insertion lines. The causative gene, OsNBL3, encodes a mitochondrion‐localized pentatricopeptide repeat (PPR) protein. The nbl3 mutant exhibited spontaneous cell death response and H2O2 accumulation, and displayed enhanced resistance to the fungal and bacterial pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. This resistance was consistent with the up‐regulation of several defence‐related genes; thus, defence responses were induced in nbl3. RNA interference lines of OsNBL3 exhibited enhanced disease resistance similar to that of nbl3, while the disease resistance in overexpression lines did not differ from that of the wild type. In addition, nbl3 displayed improved tolerance to salt, accompanied by up‐regulation of several salt‐associated marker genes. OsNBL3 was found to mainly participate in the splicing of mitochondrial gene nad5 intron 4. Disruption of OsNBL3 leads to the reduction in complex I activity, the elevation of alternative respiratory pathways and the destruction of mitochondrial morphology. Overall, the results demonstrated that the PPR protein‐coding gene OsNBL3 is essential for mitochondrial development and functions, and its disruption causes the lesion mimic phenotype and enhances disease resistance and tolerance to salt in rice. [ABSTRACT FROM AUTHOR]

Cytidine-to-uridine (C-to-U) RNA editing has been generally observed in land plants; however, reverse (U-to-C) RNA editing is a rare phenomenon. In this study, we investigated the U-to-C RNA editing-related genes in Arabidopsis tissues and the effects on mRNA stability, with a special focus on PPR proteins. A previous study showed the extensive occurrence of U-to-C RNA editing in 12-day and 20-dayold Arabidopsis seedlings. Here, we have demonstrated the effects of this "reverse" RNA editing on the mRNA stability for all seven edited genes. We also identified U-to-C RNA editing in the nuclear PPR gene (AT2G19280) in 12-day-old seedlings of Arabidopsis thaliana. The U-to-C RNA editing sites were found in the untranslated region (3' UTR) of the mature mRNA and may affect its secondary structure. We also examined the correlation between U-to-C RNA editing-related genes and their mRNA abundance. Furthermore, we investigated the effects of U-to-C RNA editing in Arabidopsis using the transcription inhibitor actinomycin D (Act D). The addition of Act D to the seedlings of transgenic Arabidopsis generated by Agrobacterium -mediated transformation showed that single nucleotide base conversion adversely affected the mRNA secondary structure and stability. • The U-to-C RNA editing-related genes in Arabidopsis tissues and the effects on mRNA stability. • U-to-C RNA editing in the nuclear PPR gene (AT2G19280) in 12-day-old seedlings of Arabidopsis thaliana. • Transgenic Arabidopsis that single nucleotide base conversion adversely affected the mRNA secondary structure and stability. [ABSTRACT FROM AUTHOR]

Pentatricopeptide repeat (PPR) proteins play important roles in plant growth and development. However, little is known about their functions in the leaf morphogenesis of Jingxiu grape (Vitis vinifera L.). Here, we explored the function of VvPPR1 , which encodes a DYW-type PPR protein in grape. We showed that VvPPR1 is involved in the regulation of leaf rolling, anthocyanin accumulation, and trichome formation in Arabidopsis thaliana. Analysis of structural characteristics showed that VvPPR1 is a DYW-type PPR gene in the PLS subfamily consisting of 15 PPR motifs. The N-terminal had a targeted chloroplast site, and the C-terminal had a DYW domain. Quantitative PCR analysis revealed that the expression level of VvPPR1 was highest in grape leaves. Subcellular localization revealed that VvPPR1 is localized in the cytoplasm and chloroplast. VvPPR1 -overexpressing plants had rolled leaves, high degrees of anthocyanin accumulation, and longer trichomes. The expression levels of genes related to these phenotypes were either significantly up-regulated or down-regulated. These results demonstrate that VvPPR1 is involved in leaf rolling, anthocyanin accumulation, and trichome formation in Arabidopsis; more generally, our findings indicate that VvPPR1 could be a target for improving the cultivation of horticultural crops. • VvPPR1 belongs to DYW class in the PLS subfamily of the PPR gene family. • VvPPR1 is mainly expressed in leaves, especially young leaves. • VvPPR1 localized in the cytoplasm and chloroplast. • Overexpressing VvPPR1 lead to the results that leaf rolling, anthocyanin accumulation and trichome longer. [ABSTRACT FROM AUTHOR]

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins. RNA editing in plants is carried out by pentatricopeptide repeat (PPR) proteins. Royan et al construct an editing factor from synthetic PPR motifs and show that it can be expressed in plants and bacteria and edits the chosen target RNA with high specificity, demonstrating the potential of PPR proteins as programmable RNA editing factors. [ABSTRACT FROM AUTHOR]

Pentatricopeptide repeat (PPR) proteins play an important role in post-transcriptional regulation of mitochondrial gene expression. Functions of many PPR proteins and their roles in plant growth and development remain unknown. Through characterization of an empty pericarp32 (emp32) mutant, we identified the function of Emp32 in mitochondrial intron splicing and seed development in maize. The loss-of-function mutant emp32 shows embryo lethality with severely arrested embryo and endosperm development, and over-expression of Emp32 rescues the embryo-lethality. EMP32 is a P-type PPR protein targeted to mitochondria. Loss of function in Emp32 dramatically decreases the splicing efficiency of nad7 intron 2, while complementation of Emp32 restores the splicing efficiency. Although nad7 intron 2 is partially spliced in the wild type, over-expression of Emp32 does not increase the splicing efficiency. The splicing deficiency of nad7 intron 2 blocks the assembly of mitochondrial complex I and dramatically reduces its activity, which may explain the embryo-lethality in emp32. In addition to the one copy of nad7 in the maize mitochondrial genome, we identified one to six copies of nad7 in the nuclear genomes in different maize inbred lines. These copies appear not to be expressed. Together, our results revealed that the P-type PPR protein EMP32 is required for the cis-splicing of nad7 intron 2 and seed development in maize. [ABSTRACT FROM AUTHOR]

All paired sensory organs arise from a common precursor domain called the pre-placodal region (PPR). In Xenopus, Zic1 non-cell autonomously regulates PPR formation by activating retinoic acid (RA) production. Here, we have identified two Zic1 targets, the RA catabolizing enzyme Cyp26c1 and the transcription factor Pitx2c, expressed in the vicinity of the PPR as being crucially required for maintaining low RA levels in a spatially restricted, PPR-adjacent domain.Morpholino- or CRISPR/Cas9-mediated Cyp26c1 knockdown abrogated PPR gene expression, yielding defective cranial placodes. Direct measurement of RA levels revealed that this is mediated by a mechanism involving excess RA accumulation. Furthermore, we show that pitx2c is activated by RA and required for Cyp26c1 expression in a domain-specific manner through induction of FGF8. We propose that Zic1 anteriorly establishes a programofRAcontainment and regulation through activation of Cyp26c1 and Pitx2c that cooperates to promote PPR specification in a spatially restricted domain. [ABSTRACT FROM AUTHOR]

Keywords: oomycete; fungi; genomics; effector; small RNAs; plant-pathogen interactions EN oomycete fungi genomics effector small RNAs plant-pathogen interactions N.PAG N.PAG 4 02/05/21 20210203 NES 210203 Fungi and oomycetes are filamentous microorganisms that include a diversity of highly developed pathogens of plants. Notably, PsGH7a protein triggers hypersensitive response in diverse plants, and it is highly conserved in oomycetes. de Vries et al. performed comparative genomics to understand how the repertoire of the carbohydrate esterase (CE)1 and CE10-encoding gene families is shaped across oomycete evolution. Oomycete, fungi, genomics, effector, small RNAs, plant-pathogen interactions. [Extracted from the article]

Identifying whether a pentatricopeptide repeat (PPR) exists in an amino acid is a significant task in the field of bioinformatics. To address this problem, an identification method that combines an optimal feature set selection framework and machine learning algorithms is proposed to recognize the PPR coding genes and proteins in the sequence of amino acid. The original 188-dimensional (D) features are obtained using a feature extraction method, which is successively optimised through a covariance analysis, max-relevant-max-distance processing, and principal component analysis to reduce it to an optimal feature set that has fewer but more expressive features. Four machine learning methods are then used to serve as the classifiers for the identification task. The final number of feature data dimensions is reduced from 188 to only 10, and according to the experimental results from support vector machine methods, the loss of the AUC and the F 1 values are only 3.26% and 10.1%, respectively. Moreover, after applying the J48, random forest, and naïve Bayes methods as classifiers, it was also found that the optimal feature set with 10 dimensions has an almost equivalent performance for a 10-fold validation test. Identifying whether a pentatricopeptide repeat (PPR) exists in an amino acid is a significant task in the field of bioinformatics. To address this problem, an identification method that combines an optimal feature set selection framework and machine learning algorithms is proposed to recognise the PPR coding genes and proteins in the sequence of an amino acid. The original 188-dimensional (D) features are obtained using a feature extraction method, which is successively optimised through a covariance analysis, max-relevant-max-distance processing, and a principal component analysis to reduce it to an optimal feature set that has fewer but more expressive features. Four machine learning methods are then used to serve as the classifiers for the identification task. The final number of feature data dimensions is reduced from 188 to only 10, and according to the experimental results from support vector machine methods, the loss of the AUC and F 1 the value are only 3.26% and 10.1%, respectively. Moreover, after applying the J48, random forest, and naïve Bayes methods as classifiers, it was also found that the optimal feature set with 10 dimensions has an almost equivalent performance for a 10-fold validation test Image, graphical abstract [ABSTRACT FROM AUTHOR]

Fusarium crown rot (FCR) and Fusarium head blight (FHB) are caused by Fusarium pseudograminearum and are newly emerging diseases of wheat in China. In this study, we characterized FpPPR1 , a gene that encodes a protein with 12 pentatricopeptide repeat (PPR) motifs. The radial growth rate of the Δ Fpppr1 deletion mutant was significantly slower than the wild type strain WZ-8A on potato dextrose agar plates and exhibited significantly smaller colonies with sector mutations. The aerial mycelium of the mutant was almost absent in culture tubes. The Δ Fpppr1 mutant was able to produce spores, but spores of abnormal size and altered conidium septum shape were produced with a significant reduction in sporulation compared to wild type. Δ Fpppr1 failed to cause disease on wheat coleoptiles and barley leaves using mycelia plugs or spore suspensions. The mutant phenotypes were successfully restored to the wild type levels in complemented strains. FpPpr1-GFP signals in spores and mycelia predominantly overlapped with Mito-tracker signals, which substantiated the mitochondria targeting signal prediction of FpPpr1. RNAseq revealed significant transcriptional changes in the Δ Fpppr1 mutant with 1,367 genes down-regulated and 1,333 genes up-regulated. NAD-binding proteins, thioredoxin, 2Fe-2S iron-sulfur cluster binding domain proteins, and cytochrome P450 genes were significantly down-regulated in Δ Fpppr1 , implying the dysfunction of mitochondria-mediated reductase redox stress in the mutant. The mating type idiomorphic alleles MAT1-1-1, MAT1-1-2 , and MAT1-1-3 in F. pseudograminearum were also down-regulated after deletion of FpPPR1 and validated by real-time quantitative PCR. Additionally, 21 genes encoding putative heterokaryon incompatibility proteins were down-regulated. The yellow pigmentation of the mutant was correlated with reduced expression of PKS12 cluster genes. Taken together, our findings on FpPpr1 indicate that this PPR protein has multiple functions in fungal asexual development, regulation of heterokaryon formation, mating-type, and pathogenesis in F. pseudograminearum. [ABSTRACT FROM AUTHOR]

Background: Cytoplasmic male sterile (CMS) with cytoplasm from Gossypium Trilobum (D8) fails to produce functional pollen. It is useful for commercial hybrid cotton seed production. The restore line of CMS-D8 containing Rf2 gene can restore the fertility of the corresponding sterile line. This study combined the whole genome resequencing bulked segregant analysis (BSA) with high-throughput SNP genotyping to accelerate the physical mapping of Rf2 locus in CMS-D8 cotton. Methods: The fertility of backcross population ((sterile line×restorer line)×maintainer line) comprising of 1623 individuals was investigated in the field. The fertile pool (100 plants with fertile phenotypes, F-pool) and the sterile pool (100 plants with sterile phenotypes, S-pool) were constructed for BSA resequencing. The selection of 24 single nucleotide polymorphisms (SNP) through high-throughput genotyping and the development insertion and deletion (InDel) markers were conducted to narrow down the candidate interval. The pentapeptide repeat (PPR) family genes and upregulated genes in restore line in the candidate interval were analysed by qRT-PCR. Results: The fertility investigation results showed that fertile and sterile separation ratio was consistent with 1:1. BSA resequencing technology, high-throughput SNP genotyping, and InDel markers were used to identify Rf2 locus on candidate interval of 1.48 Mb on chromosome D05. Furthermore, it was quantified in this experiment that InDel markers co-segregated with Rf2 enhanced the selection of the restorer line. The qRT-PCR analysis revealed PPR family gene Gh_D05G3391 located in candidate interval had significantly lower expression than sterile and maintainer lines. In addition, utilization of anther RNA-Seq data of CMS-D8 identified that the expression level of Gh_D05G3374 encoding NB-ARC domain-containing disease resistance protein in restorer lines was significantly higher than that in sterile and maintainer lines. Conclusions: This study not only enabled us to precisely locate the restore gene Rf2 but also evaluated the utilization of InDel markers for marker assisted selection in the CMS-D8 Rf2 cotton breeding line. The results of this study provide an important foundation for further studies on the mapping and cloning of restorer genes. [ABSTRACT FROM AUTHOR]

Summary: Pentatricopeptide repeat (PPR) proteins form a large family of proteins targeted to organelles, where they post‐transcriptionally modulate gene expression through binding to specific RNA sequences. Among them, the mitochondria‐targeted restorer‐of‐fertility (Rf) PPRs inhibit peculiar mitochondrial genes that are detrimental to male gametes and cause cytoplasmic male sterility (CMS). Here, we revealed three nuclear loci involved in CMS in a cross between two distant Arabidopsis thaliana strains, Sha and Cvi‐0. We identified the causal gene at one of these loci as RFL24, a conserved gene encoding a PPR protein related to known Rf PPRs. By analysing fertile revertants obtained in a male sterile background, we demonstrate that RFL24 promotes pollen abortion, in contrast with the previously described Rf PPRs, which allow pollen to survive in the presence of a sterilizing cytoplasm. We show that the sterility caused by the RFL24 Cvi‐0 allele results from higher expression of the gene during early pollen development. Finally, we predict a binding site for RFL24 upstream of two mitochondrial genes, the CMS gene and the important gene cob. These results suggest that the conservation of RFL24 is linked to a primary role of ensuring a proper functioning of mitochondria, and that it was subsequently diverted by the CMS gene to its benefit. Significance Statement: In cytoplasmic male sterility (CMS), pollen abortion is caused by mitochondrial genes, and fertility is restored by nuclear‐encoded proteins. Most identified restorers of fertility are pentatricopeptide repeat (PPR) proteins that suppress the mitochondrial sterilizing factors. Here, on the contrary, we report a restorer‐of‐fertility‐like PPR protein whose expression induces pollen death, revealing an unexpected role of PPRs in CMS. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility (CMS) is peculiar to all higher plants; it occurs in natural populations or arises under distant (sexual or somatic) hybridization. CMS and fertility restoration are important speciation characters and also the most valuable for breeding. The main features of structural and functional organization of CMS-associated mitochondrial genes in forms with different sterilizing cytoplasms are considered. The origin of nuclear PPR genes as the most probable candidates associated with pollen fertility restoration trait is discussed; examples of "atypical" Rf genes of some species are given. On the basis of data on studies of diverse CMS-Rf genetic systems, the possibility of parallelism in the variability pattern of genes responsible for sterility and male fertility restoration in plants is shown. [ABSTRACT FROM AUTHOR]