Pan-transcriptomic analysis reveals alternative splicing control of cold tolerance in rice.

Plants have evolved complex mechanisms to adapt to harsh environmental conditions. Rice (Oryza sativa) is a staple food crop that is sensitive to low temperatures. However, its cold stress responses remain poorly understood, thus limiting possibilities for crop engineering to achieve greater cold tolerance. In this study, we constructed a rice pan-transcriptome and characterized its transcriptional regulatory landscape in response to cold stress. We performed Iso-Seq and RNA-Seq of 11 rice cultivars subjected to a time-course cold treatment. Our analyses revealed that alternative splicing-regulated gene expression plays a significant role in the cold stress response. Moreover, we identified CATALASE C (OsCATC) and Os03g0701200 as candidate genes for engineering enhanced cold tolerance. Importantly, we uncovered central roles for the 2 serine-arginine-rich proteins OsRS33 and OsRS2Z38 in cold tolerance. Our analysis of cold tolerance and resequencing data from a diverse collection of 165 rice cultivars suggested that OsRS2Z38 may be a key selection gene in japonica domestication for cold adaptation, associated with the adaptive evolution of rice. This study systematically investigated the distribution, dynamic changes, and regulatory mechanisms of alternative splicing in rice under cold stress. Overall, our work generates a rich resource with broad implications for understanding the genetic basis of cold response mechanisms in plants.

Identification of hub genes involved in GA-regulated coleoptile elongation under submerged germinations in rice.

Submergence stress hinders the direct seeding in rice cultivation. Rapid elongation of rice seed coleoptiles to reach the water surface enables rice to survive submergence stress. Gibberellin (GA) positively influences rice growth. However, the molecular mechanisms underlying GA-regulated coleoptile elongation under submergence conditions remain unclear. Here, we performed a WGCNA analysis to preliminarily investigate the mechanisms. Our results identify four key modules with a high correlation to the GA regulation of rice submergence tolerance. The genes within these modules are mainly involved in Golgi apparatus and carbohydrate metabolic pathways, suggesting involvement of these biological processes in enhancing rice submergence tolerance. Moreover, natural variation analysis reveals that the hub genes, specifically, Os03g0337900, Os03g0355600, and Os07g0638400, exhibited a strong correlation with the subspecies divergence of the coleoptile elongation phenotype. Mutation of Os07g0638400 results in a lower germination potential and a stronger inhibition of coleoptile elongation under submergence conditions in rice, indicating the reliability of the analyses. The hub genes identified in this study provide deep insights into understanding the molecular mechanisms underlying GA-dependent submergence stress tolerance in rice and provide a theoretical basis for innovating rice germplasm for direct seeding application.

Identification of hub genes that variate the qCSS12-mediated cold tolerance between indica and japonica rice using WGCNA.

KEY MESSAGE: Cold-tolerant QTL qCSS12-regulated 14 hub genes are involved in the chloroplastic biological processes and in the protein synthesis and degradation processes in japonica rice. Low temperature is a main constraint factor for rice growth and production. To better understand the regulatory mechanisms underlying the cold tolerance phenotype in rice, here, we selected a cold-sensitive nearly isogenic line (NIL) NIL(qcss12) as materials to identify hub genes that are mediated by the cold-tolerant locus qCSS12 through weighted gene co-expression network analysis (WGCNA). Fourteen cold-responsive genes were identified, of which, 6 are involved in regulating biological processes in chloroplasts, including the reported EF-Tu, Prk, and ChlD, and 8 are involved in the protein synthesis and degradation processes. Differential expression of these genes between NIL(qcss12) and its controls under cold stress may be responsible for qCSS12-mediated cold tolerance in japonica rice. Moreover, natural variations in 12 of these hub genes are highly correlated with the cold tolerance divergence in two rice subspecies. The results provide deep insights into a better understanding of the molecular basis of cold adaptation in rice and provide a theoretical basis for molecular breeding.

Weighted gene coexpression network analysis-based identification of key modules and hub genes associated with drought sensitivity in rice.

BACKGROUND: Drought stress is an adverse factor with deleterious effects on several aspects of rice growth. However, the mechanism underlying drought resistance in rice remains unclear. To understand the molecular mechanism of the drought response in rice, drought-sensitive CSSL (Chromosome Single-substitution Segment Line) PY6 was used to map QTLs of sensitive phenotypes and to reveal the impact of the QTLs on transcriptional profiling. RESULTS: The QTL dss-1 was mapped onto the short arm of chromosome 1 of rice. According to transcriptomic analysis, the identified differentially expressed genes (DEGs) exhibited a downregulated pattern and were mainly enriched in photosynthesis-related GO terms, indicating that photosynthesis was greatly inhibited under drought. Further, according to weighted gene coexpression network analysis (WGCNA), specific gene modules (designating a group of genes with a similar expression pattern) were strongly correlated with H2O2 (4 modules) and MDA (3 modules), respectively. Likewise, GO analysis revealed that the photosynthesis-related GO terms were consistently overrepresented in H2O2-correlated modules. Functional annotation of the differentially expressed hub genes (DEHGs) in the H2O2 and MDA-correlated modules revealed cross-talk between abiotic and biotic stress responses for these genes, which were annotated as encoding WRKYs and PR family proteins, were notably differentially expressed between PY6 and PR403. CONCLUSIONS: We speculated that drought-induced photosynthetic inhibition leads to H2O2 and MDA accumulation, which can then trigger the reprogramming of the rice transcriptome, including the hub genes involved in ROS scavenging, to prevent oxidative stress damage. Our results shed light on and provide deep insight into the drought resistance mechanism in rice.

Correction to: Weighted gene coexpression network analysis-based identification of key modules and hub genes associated with drought sensitivity in rice.

An amendment to this paper has been published and can be accessed via the original article.

The rice pds1 locus genetically interacts with partner to cause panicle exsertion defects and ectopic tillers in spikelets.

BACKGROUND: Rice (Oryza sativa L.) is a staple food crop worldwide. Its yield and quality are affected by its tillering pattern and spikelet development. Although many genes involved in the vegetative and reproductive development of rice have been characterized in previous studies, the genetic mechanisms that control axillary tillering, spikelet development, and panicle exsertion remain incompletely understood. RESULTS: Here, we characterized a novel rice recombinant inbred line (RIL), panicle exsertion defect and aberrant spikelet (pds). It was derived from a cross between two indica varieties, S142 and 430. Intriguingly, no abnormal phenotypes were observed in the parents of pds. This RIL exhibited sheathed panicles at heading stage. Still, a small number of tillers in pds plants were fully exserted from the flag leaves. Elongated sterile lemmas and rudimentary glumes (occurred occasionally) were observed in the spikelets of the exserted panicles and were transformed into palea/lemma-like structures. Furthermore, more interestingly, tillers occasionally grew from the axils of the elongated rudimentary glumes. Via genetic linkage analysis, we found that the abnormal phenotype of pds manifesting as genetic incompatibility or hybrid weakness was caused by genetic interaction between a recessive locus, pds1, which was derived from S142 and mapped to chromosome 8, and a locus pds2, which not yet mapped from 430. We fine-mapped pds1 to an approximately 55-kb interval delimited by the markers pds-4 and 8 M3.51. Six RGAP-annotated ORFs were included in this genomic region. qPCR analysis revealed that Loc_Os080595 might be the target of pds1 locus, and G1 gene might be involved in the genetic mechanism underlying the pds phenotype. CONCLUSIONS: In this study, histological and genetic analyses revealed that the pyramided pds loci resulted in genetic incompatibility or hybrid weakness in rice might be caused by a genetic interaction between pds loci derived from different rice varieties. Further isolation of pds1 and its interactor pds2, would provide new insight into the molecular regulation of grass inflorescence development and exsertion, and the evolution history of the extant rice.

Chloroplasts- Beyond Energy Capture and Carbon Fixation: Tuning of Photosynthesis in Response to Chilling Stress.

As organelles for photosynthesis in green plants, chloroplasts play a vital role in solar energy capture and carbon fixation. The maintenance of normal chloroplast physiological functions is essential for plant growth and development. Low temperature is an adverse environmental stress that affects crop productivity. Low temperature severely affects the growth and development of plants, especially photosynthesis. To date, many studies have reported that chloroplasts are not only just organelles of photosynthesis. Chloroplasts can also perceive chilling stress signals via membranes and photoreceptors, and they maintain their homeostasis and promote photosynthesis by regulating the state of lipid membranes, the abundance of photosynthesis-related proteins, the activity of enzymes, the redox state, and the balance of hormones and by releasing retrograde signals, thus improving plant resistance to low temperatures. This review focused on the potential functions of chloroplasts in fine tuning photosynthesis processes under low-temperature stress by perceiving stress signals, modulating the expression of photosynthesis-related genes, and scavenging excess reactive oxygen species (ROS) in chloroplasts to survive the adverse environment.

Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach.

Ubiquitination modulates nearly all aspects of plant life. Here, we reconstituted the Arabidopsis thaliana ubiquitination cascade in Escherichia coli using a synthetic biology approach. In this system, plant proteins are expressed and then immediately participate in ubiquitination reactions within E. coli cells. Additionally, the purification of individual ubiquitination components prior to setting up the ubiquitination reactions is omitted. To establish the reconstituted system, we co-expressed Arabidopsis ubiquitin (Ub) and ubiquitination substrates with E1, E2 and E3 enzymes in E. coli using the Duet expression vectors. The functionality of the system was evaluated by examining the auto-ubiquitination of a RING (really interesting new gene)-type E3 ligase AIP2 and the ubiquitination of its substrate ABI3. Our results demonstrated the fidelity and specificity of this system. In addition, we applied this system to assess a subset of Arabidopsis E2s in Ub chain formation using E2 conjugation assays. Affinity-tagged Ub allowed efficient purification of Ub conjugates in milligram quantities. Consistent with previous reports, distinct roles of various E2s in Ub chain assembly were also observed in this bacterial system. Therefore, this reconstituted system has multiple advantages, and it can be used to screen for targets of E3 ligases or to study plant ubiquitination in detail.

Comparative proteomic analysis of QTL CTS-12 derived from wild rice (Oryza rufipogon Griff.), in the regulation of cold acclimation and de-acclimation of rice (Oryza sativa L.) in response to severe chilling stress.

BACKGROUND: Rice (Oryza sativa L.) is a thermophilic crop vulnerable to chilling stress. However, common wild rice (Oryza rufipogon Griff.) in Guangxi (China) has the ability to tolerate chilling stress. To better understand the molecular mechanisms underlying chilling tolerance in wild rice, iTRAQ-based proteomic analysis was performed to examine CTS-12, a major chilling tolerance QTL derived from common wild rice, mediated chilling and recovery-induced differentially expressed proteins (DEPs) between the chilling-tolerant rice line DC90 and the chilling-sensitive 9311. RESULTS: Comparative analysis identified 206 and 155 DEPs in 9311 and DC90, respectively, in response to the whole period of chilling and recovery. These DEPs were clustered into 6 functional groups in 9311 and 4 in DC90. The majority were enriched in the 'structural constituent of ribosome', 'protein-chromophore linkage', and 'photosynthesis and light harvesting' categories. Short Time-series Expression Miner (STEM) analysis revealed distinct dynamic responses of both chloroplast photosynthetic and ribosomal proteins between 9311 and DC90. CONCLUSION: CTS-12 might mediate the dynamic response of chloroplast photosynthetic and ribosomal proteins in DC90 under chilling (cold acclimation) and recovery (de-acclimation) and thereby enhancing the chilling stress tolerance of this rice line. The identified DEPs and the involvement of CTS-12 in mediating the dynamic response of DC90 at the proteomic level illuminate and deepen the understanding of the mechanisms that underlie chilling stress tolerance in wild rice.

Functional analysis of host factors that mediate the intracellular lifestyle of Cryptococcus neoformans.

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.

An Integration of MicroRNA and Transcriptome Sequencing Analysis Reveal Regulatory Roles of miRNAs in Response to Chilling Stress in Wild Rice.

A chromosome single segment substitution line (CSSL) DC90, which was generated by introgressing CTS-12, a locus derived from common wild rice (Oryza rufipogon Griff.), into the 9311 (Oryza sativa L. ssp. indica) background, exhibits a chilling tolerance phenotype under chilling stress. Here, an integration of microRNA (miRNA) deep sequencing and transcriptomic sequencing analysis was performed to explore the expression profiles of miRNAs and their target genes mediated by CTS-12 under chilling stress, and to reveal the possible regulatory mechanisms of miRNAs that are involved in chilling tolerance. Integration analysis revealed that a number of differentially expressed miRNAs (DEMs) and putative target genes with different expression patterns and levels were identified in 9311 and DC90 under chilling stress. KEGG enrichment analysis revealed that the target genes that are regulated by chilling-induced miRNAs are involved in the regulation of various biological processes/pathways, including protein biosynthesis, redox process, photosynthetic process, and chloroplast development in two genotypes. CRISPR/Cas9 editing of the target genes of the key DEMs in a chilling tolerant rice variety Zhonghua 11 (ZH11) found that LOC_Os11g48020 (OsGL1-11), one of the putative target genes of osa-miR1846a/b-5p and encoding a wax synthesis protein, is correlated with a chilling stress tolerance phenotype, implying osa-miR1846a/b-5p/OsGL1-11 plays an important role in CTS-12-mediated chilling stress tolerance regulatory pathway(s). Therefore, we speculate that the CTS-12 may regulate the key miRNA target genes in response to chilling stress by differential regulation of miRNAs in wild rice, thereby resulting in the variation of chilling tolerance phenotype between 9311 and DC90.

The Molecular Regulatory Pathways and Metabolic Adaptation in the Seed Germination and Early Seedling Growth of Rice in Response to Low O2 Stress.

As sessile organisms, flooding/submergence is one of the major abiotic stresses for higher plants, with deleterious effects on their growth and survival. Therefore, flooding/submergence is a large challenge for agriculture in lowland areas worldwide. Long-term flooding/submergence can cause severe hypoxia stress to crop plants and can result in substantial yield loss. Rice has evolved distinct adaptive strategies in response to low oxygen (O2) stress caused by flooding/submergence circumstances. Recently, direct seeding practice has been increasing in popularity due to its advantages of reducing cultivation cost and labor. However, establishment and growth of the seedlings from seed germination under the submergence condition are large obstacles for rice in direct seeding practice. The physiological and molecular regulatory mechanisms underlying tolerant and sensitive phenotypes in rice have been extensively investigated. Here, this review focuses on the progress of recent advances in the studies of the molecular mechanisms and metabolic adaptions underlying anaerobic germination (AG) and coleoptile elongation. Further, we highlight the prospect of introducing quantitative trait loci (QTL) for AG into rice mega varieties to ensure the compatibility of flooding/submergence tolerance traits and yield stability, thereby advancing the direct seeding practice and facilitating future breeding improvement.

The Wild Rice Locus CTS-12 Mediates ABA-Dependent Stomatal Opening Modulation to Limit Water Loss Under Severe Chilling Stress.

A near-isogenic line (NIL) DC90 which was generated by introgressing a wild rice (Oryza rufipogon Griff.) locus CTS-12 into the 9311(Oryza sativa L. ssp. indica) background confers chilling tolerance phenotype. Here, our pilot trials showed that chilling tolerance was positively correlated with abscisic acid (ABA) biosynthesis. To understand how CTS-12 mediated the ABA-dependent multi-levels of regulation, the integration of transcriptomic and metabolomic profiling using the two-way orthogonal projections to latent structures (O2PLS) and discriminant analysis (OPLS-DA) modeling was performed to investigate the mechanisms underlying chilling tolerance. Our results revealed that metabolic shifts, including the activation of stachyose biosynthesis, amino acid metabolism pathways, phenylpropanoid/flavonoid biosynthesis, ABA biosynthesis, and perturbation of glycolysis, occurred under chilling treatment; in the recovery period, glutamate-related pathways, ß-alanine biosynthesis and degradation, and serotonin biosynthesis pathways were differentiated between 9311 and DC90. Particularly, the differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs), including galactinol, ß-alanine, glutamate, naringenin, serotonin, ABA, and LOC_Os03g44380 (9-cis-epoxycarotenoid dioxygenase 3, OsNCED3), might be involved in the chilling tolerance variation of 9311 and DC90. CRISPR/Cas9-edited OsNCED3 resulted in chilling sensitive of japonica rice ZH11, demonstrating the involvement of ABA pathway in chilling stress response. In addition, chilling tolerance of rice was associated with the balance of water uptake and loss that was modulated by stomatal movement under chilling stress. Therefore, we speculated that the CTS-12-mediated ABA signaling pathway leads to transcriptional regulation of chilling-responsive genes and, in turn, triggers metabolic shifts to coordinately regulate the stomatal movement of guard cells. The results of this study improve our understanding of the multilevel regulation of wild rice in response to chilling stress.

Genetic analysis and fine mapping of a qualitative trait locus wpb1 for albino panicle branches in rice.

Chloroplast plays an important role in the plant life cycle. However, the details of its development remain elusive in rice. In this study, we report the fine-mapping of a novel rice gene wpb1 (white panicle branch 1), which affects chloroplast biogenesis, from a tropical japonica variety that results in an albino panicle branches at and after the heading stage. The wpb1 variety was crossed with Nipponbare to generate the F2 and BC1F2 populations. Green and white panicle branch phenotypes with a 3:1 segregation ratio was observed in the F2 population. Bulked segregant analysis (BSA) based on whole genome resequencing was conducted to determine the wpb1 locus. A candidate interval spanning from 11.35 to 23.79M (physical position) on chromosome 1 was identified. The results of BSA analysis were verified by a 40K rice SNP-array using the BC1F2 population. A large-scale F2 population was used to pinpoint wpb1, and the locus was further narrowed down to a 95-kb interval. Furthermore, our results showed that the expression levels of the majority of the genes involved in Chl biosynthesis, photosynthesis and chloroplast development were remarkably affected in wpb1 variety and in F2 plants with a white panicle branch phenotype. In line with the results mentioned above, anatomical structural examination and chlorophyll (Chl) content measurement suggested that wpb1 might play an important role in the regulation of chloroplast development. Further cloning and functional characterization of the wpb1 gene will shed light on the molecular mechanism underlying chloroplast development in rice.

Heterotrimeric G proteins regulate nitrogen-use efficiency in rice.

The drive toward more sustainable agriculture has raised the profile of crop plant nutrient-use efficiency. Here we show that a major rice nitrogen-use efficiency quantitative trait locus (qNGR9) is synonymous with the previously identified gene DEP1 (DENSE AND ERECT PANICLES 1). The different DEP1 alleles confer different nitrogen responses, and genetic diversity analysis suggests that DEP1 has been subjected to artificial selection during Oryza sativa spp. japonica rice domestication. The plants carrying the dominant dep1-1 allele exhibit nitrogen-insensitive vegetative growth coupled with increased nitrogen uptake and assimilation, resulting in improved harvest index and grain yield at moderate levels of nitrogen fertilization. The DEP1 protein interacts in vivo with both the Gα (RGA1) and Gß (RGB1) subunits, and reduced RGA1 or enhanced RGB1 activity inhibits nitrogen responses. We conclude that the plant G protein complex regulates nitrogen signaling and modulation of heterotrimeric G protein activity provides a strategy for environmentally sustainable increases in rice grain yield.

Polo-like kinase 1, on the rise from cell cycle regulation to prostate cancer development.

Polo-like kinase 1 (Plk1), a well-characterized member of serine/threonine kinases Plk family, has been shown to play pivotal roles in mitosis and cytokinesis in eukaryotic cells. Recent studies suggest that Plk1 not only controls the process of mitosis and cytokinesis, but also, going beyond those previously described functions, plays critical roles in DNA replication and Pten null prostate cancer initiation. In this review, we briefly summarize the functions of Plk1 in mitosis and cytokinesis, and then mainly focus on newly discovered functions of Plk1 in DNA replication and in Pten-null prostate cancer initiation. Furthermore, we briefly introduce the architectures of human and mouse prostate glands and the possible roles of Plk1 in human prostate cancer development. And finally, the newly chemotherapeutic development of small-molecule Plk1 inhibitors to target Plk1 in cancer treatment and their translational studies are also briefly reviewed.

Fine mapping of Spr3, a locus for spreading panicle from African cultivated rice (Oryza glaberrima Steud.).

A CSSL (chromosome segment substitution line), SG-64, carrying a segment of chromosome 4 from African cultivated rice (CG-14) in the genetic background of var. Wuyujing-7 (japonica), showed a spreading panicle, which was different significantly from that of Wuyujing-7 with an erect compact panicle. The gene controlling a spreading panicle is referred to as Spr3, and is mapped on chromosome 4. To uncover the genetic basis of Spr3, a large F(2) population derived from cross between SG-64 and Wuyujing-7 was constructed for fine mapping of the Spr3 locus. The high-resolution linkage analysis revealed that the Spr3 locus was narrowed down to a 4.6-kb region. The delimited genomic DNA regions of Wuyujing-7 and CG-14 were sequenced and compared. Sequence mutations between Wuyujing-7 and CG-14 were evident and the candidate genes for the locus were predicted. Publicly available databases were searched for homologous cDNA sequences. However, any coding regions or other meaningful sequences for the Spr3 locus were not found within this delimited region. This result suggested that Spr3 is an unknown genetic factor in controlling the outspreading of the primary branches in rice inflorescence. In addition, NIL(Spr3) exhibited seed shattering. The formation of spreading panicle was accompanied by a few undesirable traits and the spreading panicle links with seed shattering suggest that the spreading panicle was likely lost during the domestication and selection for high seed productivity of cultivated rice.