Identification of NPF Family Genes in Brassica rapa Reveal Their Potential Functions in Pollen Development and Response to Low Nitrate Stress.

Nitrate Transporter 1/Peptide Transporter Family (NPF) genes encode membrane transporters involved in the transport of diverse substrates. However, little is known about the diversity and functions of NPFs in Brassica rapa. In this study, 85 NPFs were identified in B. rapa (BrNPFs) which comprised eight subfamilies. Gene structure and conserved motif analysis suggested that BrNFPs were conserved throughout the genus. Stress and hormone-responsive cis-acting elements and transcription factor binding sites were identified in BrNPF promoters. Syntenic analysis suggested that tandem duplication contributed to the expansion of BrNPFs in B. rapa. Transcriptomic profiling analysis indicated that BrNPF2.6, BrNPF2.15, BrNPF7.6, and BrNPF8.9 were expressed in fertile floral buds, suggesting important roles in pollen development. Thirty-nine BrNPFs were responsive to low nitrate availability in shoots or roots. BrNPF2.10, BrNPF2.19, BrNPF2.3, BrNPF5.12, BrNPF5.16, BrNPF5.8, and BrNPF6.3 were only up-regulated in roots under low nitrate conditions, indicating that they play positive roles in nitrate absorption. Furthermore, many genes were identified in contrasting genotypes that responded to vernalization and clubroot disease. Our results increase understanding of BrNPFs as candidate genes for genetic improvement studies of B. rapa to promote low nitrate availability tolerance and for generating sterile male lines based on gene editing methods.

Research Advances and Prospects of Orphan Genes in Plants.

Orphan genes (OGs) are defined as genes having no sequence similarity with genes present in other lineages. OGs have been regarded to play a key role in the development of lineage-specific adaptations and can also serve as a constant source of evolutionary novelty. These genes have often been found related to various stress responses, species-specific traits, special expression regulation, and also participate in primary substance metabolism. The advancement in sequencing tools and genome analysis methods has made the identification and characterization of OGs comparatively easier. In the study of OG functions in plants, significant progress has been made. We review recent advances in the fast evolving characteristics, expression modulation, and functional analysis of OGs with a focus on their role in plant biology. We also emphasize current challenges, adoptable strategies and discuss possible future directions of functional study of OGs.

Brassica rapa orphan genes largely affect soluble sugar metabolism.

Orphan genes (OGs), which are genes unique to a specific taxon, play a vital role in primary metabolism. However, little is known about the functional significance of Brassica rapa OGs (BrOGs) that were identified in our previous study. To study their biological functions, we developed a BrOG overexpression (BrOGOE) mutant library of 43 genes in Arabidopsis thaliana and assessed the phenotypic variation of the plants. We found that 19 of the 43 BrOGOE mutants displayed a mutant phenotype and 42 showed a variable soluble sugar content. One mutant, BrOG1OE, with significantly elevated fructose, glucose, and total sugar contents but a reduced sucrose content, was selected for in-depth analysis. BrOG1OE showed reduced expression and activity of the Arabidopsis sucrose synthase gene (AtSUS); however, the activity of invertase was unchanged. In contrast, silencing of two copies of BrOG1 in B. rapa, BraA08002322 (BrOG1A) and BraSca000221 (BrOG1B), by the use of an efficient CRISPR/Cas9 system of Chinese cabbage (B. rapa ssp. campestris) resulted in decreased fructose, glucose, and total soluble sugar contents because of the upregulation of BrSUS1b, BrSUS3, and, specifically, the BrSUS5 gene in the edited BrOG1 transgenic line. In addition, we observed increased sucrose content and SUS activity in the BrOG1 mutants, with the activity of invertase remaining unchanged. Thus, BrOG1 probably affected soluble sugar metabolism in a SUS-dependent manner. This is the first report investigating the function of BrOGs with respect to soluble sugar metabolism and reinforced the idea that OGs are a valuable resource for nutrient metabolism.

Synergistic effect of chlorogenic acid and levofloxacin against Klebsiella pneumonia infection in vitro and in vivo.

The study aimed to investigate the antibacterial effect and potential mechanisms of chlorogenic acid (CA) in Klebsiella pneumonia (KPN) induced infection in vitro and in vivo. 62 KPN strains were collected from the First People's Hospital of Yunnan Province. CA and CA combined Levofloxacin (LFX) were detected for KPN biofilm (BF) formation in vitro. The lung infection mice model were established by KPN. The effect of CA (500 mg/kg), LFX (50 mg/kg) and CA combined LFX (250 mg/kg + 25 mg/kg) were evaluated through the survival of mice, the changes of inflammation factors of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß and IL-6 in serum, the histopathological analysis of lung and the protein expression of NLRP3 signaling pathway in vivo. A total of 62 KPNs were isolated and identified, of which 13 (21%) strains were BF positive. 8 (13%) strains were extended spectrum ß-lactamase strains (ESBLs), and 20 (32%) strains are ESBLs biofilm positive. In vitro study, CA and LFX showed a synergistic effect on KPN biofilm formation. In vivo mice experiment, CA, especially CA + LFX treated group significantly decreased the serum levels of TNF-α, IL-1ß and IL-6, improved the survival ratio and lung pathology changes, and also reduced the protein expression of ASC, caspase 1 p20, IL-1ß and phosphor NF-κB p65. CA could effectively alleviate lung infection of KPN infected mice, and the antibacterial effection is strengthened by combined with LFX. The study provide a theroy basis for making rational and scientific antibacterial therapy strategy in clinic.

Molecular characterization of Arabidopsis thaliana LSH1 and LSH2 genes.

BACKGROUND: Arabidopsis thaliana genome encodes ten DUF640 (short for domain of unknown function 640)/ALOG (short for Arabidopsis LSH1 and Oryza G1) proteins, also known as light-dependent short hypocotyl (LSH) proteins. While some of the LSH genes regulate organ boundary determination and shade avoidance response, the function of most of these genes remains largely unknown. OBJECTIVE: In this study, we aimed to characterize the function of AtLSH1 and AtLSH2 in Arabidopsis. METHODS: We overexpressed AtLSH1 and AtLSH2 (with or without the FLAG tag) in Arabidopsis Col-0 plants under the control of the 35S promoter. We also generated knockout or knockdown lines of these genes by miRNA-induced gene silencing (MIGS). We conducted intensive phenotypic analysis of these transgenic lines, and finally performed RNA-seq analysis of two AtLSH2 overexpression (OX) lines. RESULTS: Although AtLSH1 and AtLSH2 amino acid sequences showed high similarly, AtLSH2-OX lines showed much higher levels of their transcripts than those of AtLSH1-OX lines. Additionally, overexpression of AtLSH1 and AtLSH2 greatly inhibited hypocotyl elongation in a light-independent manner, and reduced both vegetative and reproductive growth. However, knockout or knockdown of both these AtLSH genes did not affect plant phenotype. Gene Ontology (GO) analysis of differentially expressed genes (DEGs) identified by RNA-seq revealed enrichment of the GO term 'response to stimulus', included phytohormone-responsive genes; however, genes responsible for the abnormal phenotypes of AtLSH2-OX lines could not be identified. CONCLUSION: Although our data revealed no close association between light and phytohormone signaling components, overexpression of AtLSH1 and AtLSH2 greatly reduced vegetative and reproductive growth of Arabidopsis plants. This property could be used to generate new plants by regulating expression of AtLSH1 and AtLSH2.

Identification of source-sink tissues in the leaf of Chinese cabbage (Brassica rapa ssp. pekinensis) by carbohydrate content and transcriptomic analysis.

BACKGROUND: A leaf of Chinese cabbage (Brassica rapa ssp. pekinensis) is composed of a photosynthetic blade and a non-photosynthetic large midrib; thus each leaf contains both source and sink tissues. This structure suggests that, unlike in other plants, source-sink metabolism is present in a single leaf of Chinese cabbage. OBJECTIVE: This study was designed to identify the transport route of photosynthetic carbon and to determine whether both source and sink tissues were present in a leaf. METHODS: Plant samples were collected diurnally. Their carbohydrate contents were measured, and a genome-wide transcriptome analysis was performed using the Br300K microarray. Expression profiles of selected genes were validated using qRT-PCR analysis. RESULTS: The presence of two contrasting tissues (blade as source and midrib as sink) in a leaf was demonstrated by (1) diurnal distribution patterns of starch and sucrose content; (2) Gene Ontology (GO) enrichment analysis of microarray data; (3) expression profiles of photosynthetic and sucrose biosynthetic genes; and (4) expression patterns of a variety of sugar transporter genes. CONCLUSION: Source and sink tissues were both present in Chinese cabbage leaves, but the midrib functioned as a sink tissue as well as a site exporting to roots and other sink tissues. Function of most genes discriminating between source and sink tissue appeared to be regulated largely at the post-transcriptional level, not at the transcriptional level.

Evolutionary analysis of six chloroplast genomes from three Persea americana ecological races: Insights into sequence divergences and phylogenetic relationships.

Chloroplasts significantly influence species phylogenies because of their maternal inheritance and the moderate evolutionary rate of their genomes. Avocado, which is a member of the family Lauraceae, has received considerable attention from botanists, likely because of its position as a basal angiosperm. However, there is relatively little avocado genomic information currently available. In this study, six complete avocado chloroplast genomes from three ecological races were assembled to examine the sequence diversity among the three avocado ecological races. A comparative genomic analysis revealed that 515 simple sequence repeat loci and 176 repeats belonging to four other types were polymorphic across the six chloroplast genomes. Three highly variable regions (trnC-GCA-petN, petN-psbM, and petA-psbJ) were identified as highly informative markers. A phylogenetic analysis based on 79 common protein-coding genes indicated that the six examined avocado accessions from three ecological races form a monophyletic clade. The other three genera belonging to the Persea group clustered to form a sister clade with a high bootstrap value. These chloroplast genomes provide important genetic information for future attempts at identifying avocado races and for the related biological research.

Transcriptome Profiling Provides Insight into the Genes in Carotenoid Biosynthesis during the Mesocarp and Seed Developmental Stages of Avocado (Persea americana).

Avocado (Persea americana Mill.) is an economically important crop because of its high nutritional value. However, the absence of a sequenced avocado reference genome has hindered investigations of secondary metabolism. For next-generation high-throughput transcriptome sequencing, we obtained 365,615,152 and 348,623,402 clean reads as well as 109.13 and 104.10 Gb of sequencing data for avocado mesocarp and seed, respectively, during five developmental stages. High-quality reads were assembled into 100,837 unigenes with an average length of 847.40 bp (N50 = 1725 bp). Additionally, 16,903 differentially expressed genes (DEGs) were detected, 17 of which were related to carotenoid biosynthesis. The expression levels of most of these 17 DEGs were higher in the mesocarp than in the seed during five developmental stages. In this study, the avocado mesocarp and seed transcriptome were also sequenced using single-molecule long-read sequencing to acquired 25.79 and 17.67 Gb clean data, respectively. We identified 233,014 and 238,219 consensus isoforms in avocado mesocarp and seed, respectively. Furthermore, 104 and 59 isoforms were found to correspond to the putative 11 carotenoid biosynthetic-related genes in the avocado mesocarp and seed, respectively. The isoform numbers of 10 out of the putative 11 genes involved in the carotenoid biosynthetic pathway were higher in the mesocarp than those in the seed. Besides, alpha- and beta-carotene contents in the avocado mesocarp and seed during five developmental stages were also measured, and they were higher in the mesocarp than in the seed, which validated the results of transcriptome profiling. Gene expression changes and the associated variations in gene dosage could influence carotenoid biosynthesis. These results will help to further elucidate carotenoid biosynthesis in avocado.

Genome-Wide Analysis of Glycoside Hydrolase Family 1 ß-glucosidase Genes in Brassica rapa and Their Potential Role in Pollen Development.

Glycoside hydrolase family 1 (GH1) ß-glucosidases (BGLUs) are encoded by a large number of genes, and are involved in many developmental processes and stress responses in plants. Due to their importance in plant growth and development, genome-wide analyses have been conducted in model plants (Arabidopsis and rice) and maize, but not in Brassica species, which are important vegetable crops. In this study, we systematically analyzed B. rapa BGLUs (BrBGLUs), and demonstrated the involvement of several genes in pollen development. Sixty-four BrBGLUs were identified in Brassica databases, which were anchored onto 10 chromosomes, with 10 tandem duplications. Phylogenetic analysis revealed that 64 genes were classified into 10 subgroups, and each subgroup had relatively conserved intron/exon structures. Clustering with Arabidopsis BGLUs (AtBGLUs) facilitated the identification of several important subgroups for flavonoid metabolism, the production of glucosinolates, the regulation of abscisic acid (ABA) levels, and other defense-related compounds. At least six BrBGLUs might be involved in pollen development. The expression of BrBGLU10/AtBGLU20, the analysis of co-expressed genes, and the examination of knocked down Arabidopsis plants strongly suggests that BrBGLU10/AtBGLU20 has an indispensable function in pollen development. The results that are obtained from this study may provide valuable information for the further understanding of ß-glucosidase function and Brassica breeding, for nutraceuticals-rich Brassica crops.

Mining of Brassica-Specific Genes (BSGs) and Their Induction in Different Developmental Stages and under Plasmodiophora brassicae Stress in Brassica rapa.

Orphan genes, also called lineage-specific genes (LSGs), are important for responses to biotic and abiotic stresses, and are associated with lineage-specific structures and biological functions. To date, there have been no studies investigating gene number, gene features, or gene expression patterns of orphan genes in Brassica rapa. In this study, 1540 Brassica-specific genes (BSGs) and 1824 Cruciferae-specific genes (CSGs) were identified based on the genome of Brassica rapa. The genic features analysis indicated that BSGs and CSGs possessed a lower percentage of multi-exon genes, higher GC content, and shorter gene length than evolutionary-conserved genes (ECGs). In addition, five types of BSGs were obtained and 145 out of 529 real A subgenome-specific BSGs were verified by PCR in 51 species. In silico and semi-qPCR, gene expression analysis of BSGs suggested that BSGs are expressed in various tissue and can be induced by Plasmodiophora brassicae. Moreover, an A/C subgenome-specific BSG, BSGs1, was specifically expressed during the heading stage, indicating that the gene might be associated with leafy head formation. Our results provide valuable biological information for studying the molecular function of BSGs for Brassica-specific phenotypes and biotic stress in B. rapa.

Genome-Wide Identification and Characterization of Warming-Related Genes in Brassica rapa ssp. pekinensis.

For sustainable crop cultivation in the face of global warming, it is important to unravel the genetic mechanisms underlying plant adaptation to a warming climate and apply this information to breeding. Thermomorphogenesis and ambient temperature signaling pathways have been well studied in model plants, but little information is available for vegetable crops. Here, we investigated genes responsive to warming conditions from two Brassica rapa inbred lines with different geographic origins: subtropical (Kenshin) and temperate (Chiifu). Genes in Gene Ontology categories "response to heat", "heat acclimation", "response to light intensity", "response to oxidative stress", and "response to temperature stimulus" were upregulated under warming treatment in both lines, but genes involved in "response to auxin stimulus" were upregulated only in Kenshin under both warming and minor-warming conditions. We identified 16 putative high temperature (HT) adaptation-related genes, including 10 heat-shock response genes, 2 transcription factor genes, 1 splicing factor gene, and 3 others. BrPIF4, BrROF2, and BrMPSR1 are candidate genes that might function in HT adaptation. Auxin response, alternative splicing of BrHSFA2, and heat shock memory appear to be indispensable for HT adaptation in B. rapa. These results lay the foundation for molecular breeding and marker development to improve warming tolerance in B. rapa.

GDSL esterase/lipase genes in Brassica rapa L.: genome-wide identification and expression analysis.

GDSL esterase/lipase proteins (GELPs), a very large subfamily of lipolytic enzymes, have been identified in microbes and many plants, but only a few have been characterized with respect to their roles in growth, development, and stress responses. In Brassica crops, as in many other species, genome-wide systematic analysis and functional studies of these genes are still lacking. As a first step to study their function in B. rapa ssp. pekinensis (Chinese cabbage), we comprehensively identified all GELP genes in the genome. We found a total of 121 Brassica rapa GDSL esterase/lipase protein genes (BrGELPs), forming three clades in the phylogenetic analysis (two major and one minor), with an asymmetrical chromosomal distribution. Most BrGELPs possess four strictly conserved residues (Ser-Gly-Asn-His) in four separate conserved regions, along with short conserved and clade-specific blocks, suggesting functional diversification of these proteins. Detailed expression profiling revealed that BrGELPs were expressed in various tissues, including floral organs, implying that BrGELPs play diverse roles in various tissues and during development. Ten percent of BrGELPs were specifically expressed in fertile buds, rather than male-sterile buds, implying their involvement in pollen development. Analyses of EXL6 (extracellular lipase 6) expression and its co-expressed genes in both B. rapa and Arabidopsis, as well as knockdown of this gene in Arabidopsis, revealed that this gene plays an important role in pollen development in both species. The data described in this study will facilitate future investigations of other BrGELP functions.

Global Gene-Expression Analysis to Identify Differentially Expressed Genes Critical for the Heat Stress Response in Brassica rapa.

Genome-wide dissection of the heat stress response (HSR) is necessary to overcome problems in crop production caused by global warming. To identify HSR genes, we profiled gene expression in two Chinese cabbage inbred lines with different thermotolerances, Chiifu and Kenshin. Many genes exhibited >2-fold changes in expression upon exposure to 0.5- 4 h at 45°C (high temperature, HT): 5.2% (2,142 genes) in Chiifu and 3.7% (1,535 genes) in Kenshin. The most enriched GO (Gene Ontology) items included 'response to heat', 'response to reactive oxygen species (ROS)', 'response to temperature stimulus', 'response to abiotic stimulus', and 'MAPKKK cascade'. In both lines, the genes most highly induced by HT encoded small heat shock proteins (Hsps) and heat shock factor (Hsf)-like proteins such as HsfB2A (Bra029292), whereas high-molecular weight Hsps were constitutively expressed. Other upstream HSR components were also up-regulated: ROS-scavenging genes like glutathione peroxidase 2 (BrGPX2, Bra022853), protein kinases, and phosphatases. Among heat stress (HS) marker genes in Arabidopsis, only exportin 1A (XPO1A) (Bra008580, Bra006382) can be applied to B. rapa for basal thermotolerance (BT) and short-term acquired thermotolerance (SAT) gene. CYP707A3 (Bra025083, Bra021965), which is involved in the dehydration response in Arabidopsis, was associated with membrane leakage in both lines following HS. Although many transcription factors (TF) genes, including DREB2A (Bra005852), were involved in HS tolerance in both lines, Bra024224 (MYB41) and Bra021735 (a bZIP/AIR1 [Anthocyanin-Impaired-Response-1]) were specific to Kenshin. Several candidate TFs involved in thermotolerance were confirmed as HSR genes by real-time PCR, and these assignments were further supported by promoter analysis. Although some of our findings are similar to those obtained using other plant species, clear differences in Brassica rapa reveal a distinct HSR in this species. Our data could also provide a springboard for developing molecular markers of HS and for engineering HS tolerant B. rapa.

Suppression of ASKß (AtSK32), a Clade III Arabidopsis GSK3, Leads to the Pollen Defect during Late Pollen Development.

Arabidopsis Shaggy-like protein kinases (ASKs) are Arabidopsis thaliana homologs of glycogen synthase kinase 3/SHAGGY-like kinases (GSK3/SGG), which are comprised of 10 genes with diverse functions. To dissect the function of ASKß (AtSK32), ASKß antisense transgenic plants were generated, revealing the effects of ASKß down-regulation in Arabidopsis. Suppression of ASKß expression specifically interfered with pollen development and fertility without altering the plants' vegetative phenotypes, which differed from the phenotypes reported for Arabidopsis plants defective in other ASK members. The strength of these phenotypes showed an inverse correlation with the expression levels of ASKß and its co-expressed genes. In the aborted pollen of ASKß antisense plants, loss of nuclei and shrunken cytoplasm began to appear at the bicellular stage of microgametogenesis. The in silico analysis of promoter and the expression characteristics implicate ASKß is associated with the expression of genes known to be involved in sperm cell differentiation. We speculate that ASKß indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

Genome-wide transcriptome analysis of two contrasting Brassica rapa doubled haploid lines under cold-stresses using Br135K oligomeric chip.

Genome wide transcription analysis in response to stresses is important to provide a basis of effective engineering strategies to improve stress tolerance in crop plants. We assembled a Brassica rapa oligomeric microarray (Br135K microarray) using sequence information from 41,173 unigenes and analyzed the transcription profiles of two contrasting doubled haploid (DH) lines, Chiifu and Kenshin, under cold-treatments. The two DH lines showed great differences in electrolyte leakage below -4°C, but similar patterns from 4°C to -2°C. Cold-treatments induced 885 and 858 genes in Chiifu and Kenshin, respectively. Overall, 134, and 56 genes showed an intrinsic difference in expression in Chiifu and Kenshin, respectively. Among 5,349 genes that showed no hit found (NHF) in public databases, 61 and 24 were specifically expressed in Chiifu and Kenshin, respectively. Many transcription factor genes (TFs) also showed various characteristics of expression. BrMYB12, BrMYBL2, BrbHLHs, BrbHLH038, a C2H2, a WRKY, BrDREB19 and a integrase-type TF were induced in a Chiifu-specific fashion, while a bHLH (Bra001826/AT3G21330), bHLH, cycling Dof factor and two Dof type TFs were Kenshin specific. Similar to previous studies, a large number of genes were differently induced or regulated among the two genotypes, but many genes, including NHFs, were specifically or intrinsically expressed with genotype specificity. Expression patterns of known-cold responsive genes in plants resulted in discrepancy to membrane leakage in the two DH lines, indicating that timing of gene expression is more important to conferring freezing tolerance rather than expression levels. Otherwise, the tolerance will be related to the levels of transcripts before cold-treatment or regulated by other mechanisms. Overall, these results indicate common signaling pathways and various transcriptional regulatory mechanisms are working together during cold-treatment of B. rapa. Our newly developed Br135K oligomeric microarray will be useful for transcriptome profiling, and will deliver valuable insight into cold stresses in B. rapa.

Comprehensive analysis of genic male sterility-related genes in Brassica rapa using a newly developed Br300K oligomeric chip.

To identify genes associated with genic male sterility (GMS) that could be useful for hybrid breeding in Chinese cabbage (Brassicarapa ssp. pekinensis), floral bud transcriptome analysis was carried out using a B. rapa microarray with 300,000 probes (Br300K). Among 47,548 clones deposited on a Br300K microarray with seven probes of 60 nt length within the 3' 150 bp region, a total of 10,622 genes were differentially expressed between fertile and sterile floral buds; 4,774 and 5,848 genes were up-regulated over 2-fold in fertile and sterile buds, respectively. However, the expression of 1,413 and 199 genes showed fertile and sterile bud-specific features, respectively. Genes expressed specifically in fertile buds, possibly GMS-related genes, included homologs of several Arabidopsis male sterility-related genes, genes associated with the cell wall and synthesis of its surface proteins, pollen wall and coat components, signaling components, and nutrient supplies. However, most early genes for pollen development, genes for primexine and callose formation, and genes for pollen maturation and anther dehiscence showed no difference in expression between fertile and sterile buds. Some of the known genes associated with Arabidopsis pollen development showed similar expression patterns to those seen in this study, while others did not. BrbHLH89 and BrMYP99 are putative GMS genes. Additionally, 17 novel genes identified only in B. rapa were specifically and highly expressed only in fertile buds, implying the possible involvement in male fertility. All data suggest that Chinese cabbage GMS might be controlled by genes acting in post-meiotic tapetal development that are different from those known to be associated with Arabidopsis male sterility.

Ogura-CMS in Chinese cabbage (Brassica rapa ssp. pekinensis) causes delayed expression of many nuclear genes.

We investigated the mechanism regulating cytoplasmic male sterility (CMS) in Brassica rapa ssp. pekinensis using floral bud transcriptome analyses of Ogura-CMS Chinese cabbage and its maintainer line in B. rapa 300-K oligomeric probe (Br300K) microarrays. Ogura-CMS Chinese cabbage produced few and infertile pollen grains on indehiscent anthers. Compared to the maintainer line, CMS plants had shorter filaments and plant growth, and delayed flowering and pollen development. In microarray analysis, 4646 genes showed different expression, depending on floral bud size, between Ogura-CMS and its maintainer line. We found 108 and 62 genes specifically expressed in Ogura-CMS and its maintainer line, respectively. Ogura-CMS line-specific genes included stress-related, redox-related, and B. rapa novel genes. In the maintainer line, genes related to pollen coat and germination were specifically expressed in floral buds longer than 3mm, suggesting insufficient expression of these genes in Ogura-CMS is directly related to dysfunctional pollen. In addition, many nuclear genes associated with auxin response, ATP synthesis, pollen development and stress response had delayed expression in Ogura-CMS plants compared to the maintainer line, which is consistent with the delay in growth and development of Ogura-CMS plants. Delayed expression may reduce pollen grain production and/or cause sterility, implying that mitochondrial, retrograde signaling delays nuclear gene expression.