Search

Phosphorus (P) is essential for cellular processes like respiration, photosynthesis, biosynthesis of membrane phospholipids, etc. To cope with P deficiency stress, plants adopt reprograming of the expression of genes involved in different metabolic/signaling pathways for survival, growth, and development. Plants use transcriptional, post-transcriptional, and/or post-translational machinery to achieve P homeostasis. Several transcription factors (TFs), miRNAs, and P transporters play important roles in P deficiency tolerance; however, the underlying mechanisms responsible for P deficiency tolerance remain poorly understood. Studies on P starvation/deficiency responses in plants at early (seedling) stage of growth have been reported but only a few of them focused on molecular responses of the plant at advanced (tillering or reproductive) stage of growth. To decipher the strategies adopted by rice at tillering stage under P deficiency stress, a pair of contrasting genotypes [Pusa-44 (a high-yielding, P deficiency sensitive cultivar) and its near-isogenic line (NIL-23, P deficiency tolerant) for Pup1 QTL] was used for morphophysiological, biochemical, and molecular analyses. Comparative analyses of shoot and root tissues from 45-day-old plants grown hydroponically under P sufficient (16 ppm) or P deficient (4 ppm) medium confirmed some of the known morphophysiological responses. Moreover, RNA-seq analysis revealed the important roles of phosphate transporters, TFs, auxin-responsive proteins, modulation in the cell wall, fatty acid metabolism, and chromatin architecture/epigenetic modifications in providing P deficiency tolerance to NIL-23, which were brought in due to the introgression of the Pup1 QTL in Pusa-44. This study provides insights into the molecular functions of Pup1 for P deficiency tolerance, which might be utilized to improve P-use efficiency of rice for better productivity in P deficient soils. KEY MESSAGE: Introgression of Pup1 QTL in high-yielding rice cultivar modulates mainly phosphate transporters, TFs, auxin-responsive proteins, cell wall structure, fatty acid metabolism, and chromatin architecture/epigenetic modifications at tillering stage of growth under phosphorus deficiency stress.

Flag leaf heterosis of inter-subspecific hybrid rice is suggested to be related to leaf area, gene expression pattern and allele-specific expression, putatively related to DNA methylation differences between the hybrid and its parents. Inter-subspecific hybrid rice combinations of indica × japonica have great potential to broaden genetic diversity and enhance the heterosis. The genetic and epigenetic molecular mechanism of its heterosis is not completely understood. Here, the dissection of gene expression and epigenetic regulation of an elite inter-subspecific hybrid rice were reported. In the hybrid, plant height, flag leaf area and Pn showed significant heterosis at the heading stage. Chloroplast-related differentially expressed genes (DEGs) and 530 allele-specific expression genes in hybrid were identified. Analysis of the genome-wide distribution of DNA methylation (5-methylcytosine, 5mC) and its association with transcription showed that there were variant DNA methylation maps and that the regulation of gene expression levels was negatively regulated by DNA methylation in the inter-subspecific hybrid rice. Differentially methylated DEGs were significantly enriched in photosynthetic functions. Moreover, distinct 5mC sequence contexts and distinct functional elements (promoter/gene body) may have different influences on heterosis related genes. The data identified heterosis related molecular mechanisms in inter-subspecific hybrid rice and suggested that epigenetic changes could extensively influence the flag leaf gene expression and heterosis.

Key Message Endophytes are crucial for the promotion of rice growth and stress tolerance and can be used to increase rice crop yield. Endophytes can thus be exploited in biotechnology and genetic engineering as eco-friendly and cost-effective means for the development of high-yielding and stress-tolerant rice plants. Abstract Rice (Oryza sativa) crop is continuously subjected to biotic and abiotic stresses, compromising growth and consequently yield. The situation is exacerbated by climate change impacting on ecosystems and biodiversity. Genetic engineering has been used to develop stress-tolerant rice, alongside physical and chemical methods to mitigate the effect of these stresses. However, the success of these strategies has been hindered by short-lived field success and public concern on adverse effects associated. The limited success in the field of stress-tolerant cultivars developed through breeding or transgenic approaches is due to the complex nature of stress tolerance as well as to the resistance breakdown caused by accelerated evolution of pathogens. It is therefore necessary to develop novel and acceptable strategies to enhance rice stress tolerance and durable resistance and consequently improve yield. In the last decade, plant growth promoting (PGP) microbes, especially endophytes, have drawn the attention of agricultural scientists worldwide, due to their ability to mitigate environmental stresses in crops, without causing adverse effects. Increasing evidence indicates that endophytes effectively confer fitness benefits also to rice under biotic and abiotic stress conditions. Endophyte-produced metabolites can control the expression of stress-responsive genes and improve the physiological performance and growth of rice plants. This review highlights the current evidence available for PGP microbe-promoted tolerance of rice to abiotic stresses such as salinity and drought and to biotic ones, with special emphasis on endophytes. Associated molecular mechanisms are illustrated, and prospects for sustainable rice production also in the light of the impending climate change, discussed.

Key message This study showed the systematic identification of long non-coding RNAs (lncRNAs) involving in flag leaf senescence of rice, providing the possible lncRNA-mRNA regulatory relationships and lncRNA-miRNA-mRNA ceRNA networks during leaf senescence. Abstract LncRNAs have been reported to play crucial roles in diverse biological processes. However, no systematic identification of lncRNAs associated with leaf senescence in plants has been studied. In this study, a genome-wide high throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. A total of 3953 lncRNAs and 38757 mRNAs were identified, of which 343 lncRNAs and 9412 mRNAs were differentially expressed. Through weighted gene co-expression network analysis (WGCNA), 22 continuously down-expressed lncRNAs targeting 812 co-expressed mRNAs and 48 continuously up-expressed lncRNAs targeting 1209 co-expressed mRNAs were considered to be significantly associated with flag leaf senescence. Gene Ontology results suggested that the senescence-associated lncRNAs targeted mRNAs involving in many biological processes, including transcription, hormone response, oxidation–reduction process and substance metabolism. Additionally, 43 senescence-associated lncRNAs were predicted to target 111 co-expressed transcription factors. Interestingly, 8 down-expressed lncRNAs and 29 up-expressed lncRNAs were found to separately target 12 and 20 well-studied senescence-associated genes (SAGs). Furthermore, analysis on the competing endogenous RNA (CeRNA) network revealed that 6 down-expressed lncRNAs possibly regulated 51 co-expressed mRNAs through 15 miRNAs, and 14 up-expressed lncRNAs possibly regulated 117 co-expressed mRNAs through 21 miRNAs. Importantly, by expression validation, a conserved miR164-NAC regulatory pathway was found to be possibly involved in leaf senescence, where lncRNA MSTRG.62092.1 may serve as a ceRNA binding with miR164a and miR164e to regulate three transcription factors. And two key lncRNAs MSTRG.31014.21 and MSTRG.31014.36 also could regulate the abscisic-acid biosynthetic gene BGIOSGA025169 (OsNCED4) and BGIOSGA016313 (NAC family) through osa-miR5809. The possible regulation networks of lncRNAs involving in leaf senescence were discussed, and several candidate lncRNAs were recommended for prior transgenic analysis. These findings will extend the understanding on the regulatory roles of lncRNAs in leaf senescence, and lay a foundation for functional research on candidate lncRNAs.

Two OsDGAT1 genes showed the ability to restore TAG and LB synthesis in yeast H1246. Alterations in the N-terminal region of OsDGAT1-1 gene revealed its regulatory role in gene function. Accumulation of triacylglycerol (TAG) or oil in vegetative tissues has emerged as a promising approach to meet the global needs of food, feed, and fuel. Rice (Oryza sativa) has been recognized as an important cereal crop containing nutritional rice bran oil with high economic value for renewable energy production. To identify the key component involved in storage lipid biosynthesis, two type-1 diacylglycerol acyltransferases (DGAT1) from rice were characterized for its in vivo function in the H1246 (dga1, lro1, are1 and are2) yeast quadruple mutant. The ectopic expression of rice DGAT1 (designated as OsDGAT1-1 and OsDGAT1-2) genes restored the capability of TAG synthesis and lipid body (LB) formation in H1246. OsDGAT1-1 showed nearly equal substrate preferences to C16:0-CoA and 18:1-CoA whereas OsDGAT1-2 displayed substrate selectivity for C16:0-CoA over 18:1-CoA, indicating that these enzymes have contrasting substrate specificities. In parallel, we have identified the intrinsically disordered region (IDR) at the N-terminal domains of OsDGAT1 proteins. The regulatory role of the N-terminal domain was dissected. Single point mutations at the phosphorylation sites and truncations of the N-terminal region highlighted reduced lipid accumulation capabilities among different OsDGAT1-1 variants.

OsSIRP4 is an E3 ligase that acts as a negative regulator in the plant response to salt stress via the 26S proteasomal system regulation of substrate proteins, OsPEX11-1, which it provides important information for adaptation and regulation in rice. Plants are sessile organisms that can be exposed to environmental stress. Plants alter their cellular processes to survive under potentially unfavorable conditions. Protein ubiquitination is an important post-translational modification that has a crucial role in various cellular signaling processes in abiotic stress response. In this study, we characterized Oryza sativa salt-induced RING finger protein 4, OsSIRP4, a membrane and cytosol-localized RING E3 ligase in rice. OsSIRP4 transcripts were highly induced under salt stress in rice. We found that OsSIRP4 possesses E3 ligase activity; however, no E3 ligase activity was observed with a single amino acid substitution (OsSIRP4C269A). The results of the yeast two hybrid system, in vitro pull-down assay, BiFC analysis, in vitro ubiquitination assay, and in vitro degradation assay indicate that OsSIRP4 regulates degradation of a substrate protein, OsPEX11-1 (Oryza sativa peroxisomal biogenesis factor 11-1) via the 26S proteasomal system. Phenotypic analysis of OsSIRP4-overexpressing plants demonstrated hypersensitivity to salt response compared to that of the wild type and mutated OsSIRP4C269A plants. In addition, OsSIRP4-overexpressing plants exhibited significant low enzyme activities of superoxide dismutase, catalase, and peroxidase, and accumulation of proline and soluble sugar, but a high level of H2O2. Furthermore, qRT data on transgenic plants suggest that OsSIRP4 acted as a negative regulator of salt response by diminishing the expression of genes related to Na+/K+ homeostasis (AtSOS1, AtAKT1, AtNHX1, and AtHKT1;1) in transgenic plants under salt stress. These results suggest that OsSIRP4 plays a negative regulatory role in response to salt stress by modulating the target protein levels.

Role of Rubisco Activase in imparting thermotolerance to the photosynthetic apparatus under high temperature. Thus, to improve the grain filling, we need to fine tune these crucial enzymes and their regulation, which directly or indirectly affect spike photosynthesis. CO2 fixation in cereals crops like bread wheat (Triticum aestivum L.) is also contributed by ear photosynthesis beside the other organs like leaves or the flag leaf. 1000-grain weight of three Indian wheat cultivars (cvs.) PBW343, K7903, and HD2329 were calculated under three treatments until maturity stage (i.e. removal of flag leaf, removal of awns and shaded spikes). We observed that awn removal showed a significant decrease in 1000-grain weight in all cultivars. To delve deeper into the biological and molecular pathways taking place underlying the awn physiology, we conducted the awn transcriptome analysis of thermosusceptible Indian wheat cv. PBW343 under heat stress (HS) at 42 °C for 2 h using RNA-sequencing (RNA-seq). Differential expression analysis revealed, 160 transcripts, out of these, 143 transcripts were significantly upregulated and 17 transcripts were repressed under HS conditions. Of these Rca1β was selected for characterization and overexpression studies. Ectopic expression of TaRca1β in rice transgenics indicate a direct correlation with tolerance under HS conditions. TaRca1β provides a better photosynthate energy partitioning under HS with a significant reduction in the non-photochemical fluorescence quenching of the photosynthetic machinery.

OsHOX24 mediates regulation of desiccation stress response via complex regulatory network as indicated by its binding to several target genes including transcription factors in rice. HD-ZIP I subfamily of homeobox transcription factors (TFs) are involved in abiotic stress responses and plant development. Previously, we demonstrated the role of OsHOX24, a member of HD-ZIP I subfamily, in abiotic stress responses. In this study, we identified downstream targets of OsHOX24 under control and desiccation stress conditions via chromatin immunoprecipitation-sequencing (ChIP-seq) approach in wild-type and OsHOX24 over-expression transgenic in rice. OsHOX24-binding sites in each sample and differential binding sites between the samples were detected at various genomic locations, including genic and intergenic regions. Gene ontology enrichment analysis revealed that OsHOX24 direct target genes were involved in several biological processes, including plant development, ABA-mediated signalling pathway, ubiquitin-dependent protein catabolic process, ion transport, abiotic and biotic stress responses besides transcriptional and translational regulation. The enrichment of several cis-regulatory motifs representing binding sites of other TFs, such as ABFs, ERF1, MYB1, LTREs and SORLIP2, suggested the involvement of OsHOX24 in a complex regulatory network. These findings indicate that OsHOX24-mediated desiccation stress regulation involves modulation of a plethora of target genes, which participate in diverse pathways in rice.

Identification of infection process and defense response during M. oryzae infecting Acuce. Magnaporthe oryzae is a destructive rice pathogen. Recent studies have focused on the initial infectious stage, with a few studies conducted to elucidate the characteristics of the late infectious stages. This study aims to decipher the characteristics at different stages (biotrophic, biotrophy-necrotrophy switch (BNS), and necrotrophic) between the interaction of two M. oryzae-rice combinations and investigate the resistance mechanisms of rice to M. oryzae using cytological and molecular methods. The biotrophic phase of M. oryzae-LTH compatible interaction was found to be longer than that of M. oryzae-Acuce incompatible interaction. We also found that jasmonic acid (JA) signaling plays an important role in defense by regulating antimicrobial compound accumulation in infected Acuce via a synergistic interaction of JA-salicylic acid (SA) and JA-ethylene (ET). In infected LTH, JA-ET/JA-SA showed antagonistic interaction. Ibuprofen (IBU) is a JA inhibitor. Despite the above findings, we found that exogenous JA-Ile and IBU significantly alleviated blast symptoms in infected LTH at 36 hpi (biotrophic) and 72 hpi (BNS), indicating these two-time points may be critical for managing blast disease in the compatible interaction. Conversely, IBU significantly increased blast symptoms on the infected Acuce at 36 hpi, confirming that the JA signal plays a central role in the defense response in infected Acuce. According to transcriptional analysis, the number of genes enriched in the plant hormone signal pathway was significantly higher than in other pathways. Our findings suggested that JA-mediated defense mechanism is essential in regulating Acuce resistance, particularly during the biotrophic and BNS phases.

An R2R3-MYB transcription factor FOUR LIPS associated with B-type Cyclin-Dependent Kinase 1;1 confers salt tolerance in rice. The Arabidopsis FOUR LIPS (AtFLP), an R2R3 MYB transcription factor, acts as an important stomatal development regulator. Only one orthologue protein of AtFLP, Oryza sativa FLP (OsFLP), was identified in rice. However, the function of OsFLP is largely unknown. In this study, we conducted RNA-seq and ChIP-seq to investigate the potential role of OsFLP in rice. Our results reveal that OsFLP is probably a multiple functional regulator involved in many biological processes in growth development and stress responses in rice. However, we mainly focus on the role of OsFLP in salt stress response. Consistently, phenotypic analysis under salt stress conditions showed that osflp exhibited significant sensitivity to salt stress, while OsFLP over-expression lines displayed obvious salt tolerance. Additionally, Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that OsFLP directly bound to the promoter region of Oryza sativa B-type Cyclin-Dependent Kinase 1;1 (OsCDKB1;1), and the expression of OsCDKB1;1 was repressed in osflp. Disturbing the expression of OsCDKB1;1 remarkably enhanced the tolerance to salt stress. Taken together, our findings reveal a crucial function of OsFLP regulating OsCDKB1;1 in salt tolerance and largely extend the knowledge about the role of OsFLP in rice.

TaPSK5 is a less conserved target of miR164 in wheat encoding a positive regulator of root growth and yield traits that could be used for crop improvement. MicroRNAs (miRNAs) play key roles in regulating plant growth and development by targeting the mRNAs of conserved genes. However, little is known about the roles of less conserved miRNA-targeted genes in plants. In the current study, we identified TaPSK5, encoding a phytosulfokine precursor, as a novel target of miR164. Compared with miR164-targeted NAC transcription factor genes, TaPSK5 is less conserved between monocots and dicots. Expression analysis indicated that TaPSK5 homoeologs were constitutively expressed in wheat tissues, especially young spikes. Overexpression of TaPSK5-D and miR164-resistant TaPSK5-D (r-TaPSK5-D) led to increased primary root growth and grain yield in rice, with the latter having more significant effects. Comparison of the transcriptome between wild-type and r-TaPSK5-D overexpression plants revealed multiple differentially expressed genes involved in hormone signaling, transcription regulation, and reactive oxygen species (ROS) homeostasis. Moreover, we identified three TaPSK5-A haplotypes (TaPSK5-A-Hap1/2/3) and two TaPSK5-B haplotypes (TaPSK5-B-Hap1/2) in core collections of Chinese wheat. Both TaPSK5-A-Hap1 and TaPSK5-B-Hap2 are favorable haplotypes associated with superior yield traits that were under positive selection during wheat breeding. Together, our findings identify miR164-targeted TaPSK5 as a regulator of root growth and yield traits in common wheat with potential applications for the genetic improvement of crops.

This manuscript describes the functions of an Argonaute protein named AGO17 in rice. AGO17 is required for the development of rice reproductive tissues. Argonaute (AGO) proteins are a well-conserved multigene family of regulators mediating gene silencing across eukaryotes. Monocot plants have additional members of AGO, the functions of which are poorly understood. Among the non-dicot AGO1 clade members in monocots, AGO17 expresses highly in reproductive tissues. Here we show that overexpression of Oryza sativa indica AGO17 in rice resulted in robust growth and increased yield, whereas its silencing resulted in reduced panicle length, less fertility, and poor growth. Small (s)RNA transcriptome analysis revealed misregulation of several miRNAs and other categories of sRNAs in silenced and overexpression lines, in agreement with its likely competition with other AGO1 clade members. Targets of differentially expressed miRNAs included previously unreported target RNAs coding for proteins involved in development, phase transition, and transport. Our results indicate a distinctive role for OsAGO17 in rice reproductive development that could be harnessed to improve yield.

Key message We applied the phylogenomics to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity. Abstract Rice (genus Oryza) is one of the most important crops in the world, supporting half of the world’s population. Breeding of high-yielding and quality cultivars relies on genetic resources from both cultivated and wild species, which are collected and maintained in seed banks. Unfortunately, numerous seeds are mislabeled due to taxonomic issues or misidentifications. Here, we applied the phylogenomics of 58 complete chloroplast genomes and two hypervariable nuclear genes to determine species identity in rice seeds. Twenty-one Oryza species were identified. Conspecific relationships were determined between O. glaberrima and O. barthii, O. glumipatula and O. longistaminata, O. grandiglumis and O. alta, O. meyeriana and O. granulata, O. minuta and O. malampuzhaensis, O. nivara and O. sativa subsp. indica, and O. sativa subsp. japonica and O. rufipogon.D and L genome types were not found and the H genome type was extinct. Importantly, we evaluated the performance of four conventional plant DNA barcodes (matK, rbcL, psbA-trnH, and ITS), six rice-specific chloroplast DNA barcodes (psaJ-rpl33, trnC-rpoB, rps16-trnQ, rpl22-rps19, trnK-matK, and ndhC-trnV), two rice-specific nuclear DNA barcodes (NP78 and R22), and a chloroplast genome super DNA barcode. The latter was the most reliable marker. The six rice-specific chloroplast barcodes revealed that 17% of the 53 seed accessions from rice seed banks or field collections were mislabeled. These results are expected to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.

OsGTγ-2, a trihelix transcription factor, is a positive regulator of rice responses to salt stress by regulating the expression of ion transporters. Salinity stress seriously restricts rice growth and yield. Trihelix transcription factors (GT factors) specifically bind to GT elements and play a diverse role in plant morphological development and responses to abiotic stresses. In our previous study, we found that the GT-1 element (GAAAAA) is a key element in the salinity-induced OsRAV2 promoter. Here, we identified a rice OsGTγ family member, OsGTγ-2, which directly interacted with the GT-1 element in the OsRAV2 promoter. OsGTγ-2 specifically targeted the nucleus, was mainly expressed in roots, sheathes, stems and seeds, and was induced by salinity, osmotic and oxidative stresses and abscisic acid (ABA). The seed germination rate, seedling growth and survival rate under salinity stress was improved in OsGTγ-2 overexpressing lines (PZmUbi::OsGTγ-2). In contrast, CRISPR/Cas9-mediated OsGTγ-2 knockout lines (osgtγ-2) showed salt-hypersensitive phenotypes. In response to salt stress, different Na+ and K+ acclamation patterns were observed in PZmUbi::OsGTγ-2 lines and osgtγ-2 plants were observed. The molecular mechanism of OsGTγ-2 in rice salt adaptation was also investigated. Several major genes responsible for ion transporting, such as the OsHKT2; 1, OsHKT1; 3 and OsNHX1 were transcriptionally regulated by OsGTγ-2. A subsequent yeast one-hybrid assay and EMSA indicated that OsGTγ-2 directly interacted with the promoters of OsHKT2; 1, OsNHX1 and OsHKT1; 3. Taken together, these results suggest that OsGTγ-2 is an important positive regulator involved in rice responses to salt stress and suggest a potential role for OsGTγ-2 in regulating salinity adaptation in rice.

CHR721 functions as a chromatin remodeler and interacts with a known single-stranded binding protein, OsRPA1a, to regulate both male and female reproductive development in rice. Reproductive development and fertility are important for seed production in rice. Here, we identified a sterile rice mutant, chr721, that exhibited defects in both male and female reproductive development. Approximately 5% of the observed defects in chr721, such as asynchronous dyad division, occurred during anaphase II of meiosis. During the mitotic stage, approximately 80% of uninucleate microspores failed to develop into tricellular pollen, leading to abnormal development. In addition, defects in megaspore development were detected after functional megaspore formation. CHR721, which encodes a nuclear protein belonging to the SNF2 subfamily SMARCAL1, was identified by map-based cloning. CHR721 was expressed in various tissues, especially in spikelets. CHR721 was found to interact with replication protein A (OsRPA1a), which is involved in DNA repair. The expressions of genes involved in DNA repair and cell-cycle checkpoints were consistently upregulated in chr721. Although numerous genes involved in male and female development have been identified, the mode of participation of chromatin-remodeling factors in reproductive development is still not well understood. Our results suggest that CHR721, a novel gene cloned from rice, plays a vital role in both male and female reproductive development.

Leaf angle is a key factor in plant architecture and crop yield. Brassinosteroids (BRs) regulate many developmental processes, especially the leaf angle in monocots. However, the BR signalling pathway is complex and includes many unknown members. Here, we propose that Oryza sativa BRASSINOSTEROID-RESPONSIVE LEAF ANGLE REGULATOR 1 (OsBLR1) encodes a bHLH transcription factor, and positively regulates BR signalling to increase the leaf angle and grain length in rice (Oryza sativa L.). Lines overexpressing OsBLR1 (blr1-D and BLR1-OE-1/2/3) had similar traits, with increased leaf angle and grain length. Conversely, OsBLR1-knockout mutants (blr1-1/2/3) had erect leaves and shorter grains. Lamina joint inclination, coleoptile elongation, and root elongation assay results indicated that these overexpression lines were more sensitive to BR, while the knockout mutants were less sensitive. There was no significant difference in the endogenous BR contents of blr1-1/2 and wild-type plants. These results suggest that OsBLR1 is involved in BR signal transduction. The blr1-D mutant, with increased cell growth in the lamina joint and smaller leaf midrib, showed significant changes in gene expression related to the cell wall and leaf development compared with wild-type plants; furthermore, the cellulose and protopectin contents in blr1-D were reduced, which resulted in the increased leaf angle and bent leaves. As the potential downstream target gene of OsBLR1, the REGULATOR OF LEAF INCLINATION1 (OsRLI1) gene expression was up-regulated in OsBLR1-overexpression lines and down-regulated in OsBLR1-knockout mutants. Moreover, we screened OsRACK1A as an interaction protein of OsBLR1 using a yeast two-hybrid assay and glutathione-S-transferase pull-down.

Key message Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Abstract Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRÖSCHEN, DORNRÖSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity.

OsPUB67, a U-box E3 ubiquitin ligase, may interact with two drought tolerance negative regulators (OsRZFP34 and OsDIS1) and improve drought tolerance by enhancing the reactive oxygen scavenging ability and stomatal closure. E3 ubiquitin ligases are major components of the ubiquitination cascade and contribute to the biotic and abiotic stress response in plants. In the present study, we show that a rice drought responsive gene, OsPUB67, encoding the U-box E3 ubiquitin ligase was significantly induced by drought, salt, cold, JA, and ABA, and was expressed in nuclei, cytoplasm, and membrane systems. This distribution of expression suggests a significant role for OsPUB67 in a wide range of biological processes and abiotic stress response. Over-expression of OsPUB67 improved drought stress tolerance by enhancing the reactive oxygen scavenging ability and stomatal closure. Bimolecular fluorescence complementation assays revealed that a few E2s interacted with OsPUB67 with unique functional implications in different cell components. Further evidence showed that several E3 ubiquitin ligases interacted with OsPUB67, especially OsRZFP34 and OsDIS1, which are negative regulators of drought tolerance. This interaction on the stomata implied OsPUB67 might function as a heterodimeric ubiquitination complex in response to drought stress. Comprehensive transcriptome analysis revealed OsPUB67 participated in regulating genes involved in the abiotic stress response and transcriptional regulation in an ABA-dependent manner. Our findings revealed OsPUB67 mediated a multilayered complex drought stress tolerance mechanism.

Mutation of the BEIIb gene in an isa1 mutant background mitigates the negative effect of the ISA1 mutation on grain filling, and facilitates recovery of amyloplast formation in rice endosperm. In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the BEIIb and ISA1 genes and showed no BEIIb and ISA1 activity, implying that both lines are beIIb isa1 double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to beIIb mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to isa1 mutants. Furthermore, beIIb isa1 double mutant F

High levels of two major starch synthases, SSIIa and GBSSI, in ss3a ss4b double mutant rice alter the starch structure but fail to recover the polygonal starch granule morphology. The endosperm starch granule is polygonal in wild-type rice but spherical in double mutant japonica rice lacking genes encoding two of the five major Starch synthase (SS) isozymes expressed in endosperm, SSIIIa and SSIVb. Japonica rice naturally has low levels of SSIIa and Granule-bound SSI (GBSSI). Therefore, introduction of active SSIIa allele and/or high-expressing GBSSI allele from indica rice into the japonica rice mutant lacking SS isozymes can help elucidate the compensatory roles of SS isozymes in starch biosynthesis. In this study, we crossed the ss3a ss4a double mutant japonica rice with the indica rice to generate three new rice lines with high and/or low SSIIa and GBSSI levels, and examined their starch structure, physicochemical properties, and levels of other starch biosynthetic enzymes. Lines with high SSIIa levels showed more SSI and SSIIa bound to starch granule, reduced levels of short amylopectin chains (7 ≤ DP ≤ 12), increased levels of amylopectin chains with DP > 13, and consequently higher gelatinization temperature. Lines with high GBSSI levels showed an increase in amylose content. The ADP-glucose content of the crude extract was high in lines with low or high SSIIa and low GBSSI levels, but was low in lines with high GBSSI. Addition of high SSIIa and GBSSI altered the starch structure and physicochemical properties but did not affect the starch granule morphology, confirming that SSIIIa and SSIVb are key enzymes affecting starch granule morphology in rice. The relationship among SS isozymes and its effect on the amount of substrate (ADP-glucose) is discussed.

BEIIb plays a specific role in determining the structure of amylopectin in rice endosperm, whereas BEIIa plays the similar role in the culm where BEIIb is absent. Cereals have three types of starch branching enzymes (BEs), BEI, BEIIa, and BEIIb. It is widely known that BEIIb is specifically expressed in the endosperm and plays a distinct role in the structure of amylopectin because in its absence the amylopectin type changes to the amylose-extender-type (ae-type) or B-type from the wild-type or A-type and this causes the starch crystalline allomorph to the B-type from the wild-type A-type. This study aimed to clarify the role of BEIIa in the culm where BEIIb is not expressed, by using a be2a mutant in comparison with results with be2b and be1 mutants. The results showed that the amylopectin structure exhibited the B-type in the be2a culm compared with the A-type in the wild-type culm. The starch granules from the be2a culm also showed the B-type like allomorph when examined by X-ray diffraction analysis and optical sum frequency generation spectroscopy. Both amylopectin chain-length profile and starch crystalline properties were found to be the A-type at the very early stage of endosperm development at 4-6 days after pollination (DAP) even in the be2b mutant. All these results support a view that in the culm as well as in the endosperm at 4-6 DAP, BEIIa can play the role of BEIIb which has been well documented in maturing endosperm. The possible mechanism as to how BEIIa can play its role is discussed.

KEY MESSAGE We show that in rice, the amino acid-conjugates of JA precursor, OPDA, may function as a non-canonical signal for the production of phytoalexins in coordination with the innate chitin signaling. The core oxylipins, jasmonic acid (JA) and JA-Ile, are well-known as potent regulators of plant defense against necrotrophic pathogens and/or herbivores. However, recent studies also suggest that other oxylipins, including 12-oxo-phytodienoic acid (OPDA), may contribute to plant defense. Here, we used a previously characterized metabolic defense marker, p-coumaroylputrescine (CoP), and fungal elicitor, chitooligosaccharide, to specifically test defense role of various oxylipins in rice (Oryza sativa). While fungal elicitor triggered a rapid production of JA, JA-Ile, and their precursor OPDA, rice cells exogenously treated with the compounds revealed that OPDA, rather than JA-Ile, can stimulate the CoP production. Next, reverse genetic approach and oxylipin-deficient rice mutant (hebiba) were used to uncouple oxylipins from other elicitor-triggered signals. It appeared that, without oxylipins, residual elicitor signaling had only a minimal effect but, in synergy with OPDA, exerted a strong stimulatory activity towards CoP production. Furthermore, as CoP levels were compromised in the OPDA-treated Osjar1 mutant cells impaired in the oxylipin-amino acid conjugation, putative OPDA-amino acid conjugates emerged as hypothetical regulators of CoP biosynthesis. Accordingly, we found several OPDA-amino acid conjugates in rice cells treated with exogenous OPDA, and OPDA-Asp was detected, although in small amounts, in the chitooligosaccharide-treated rice. However, as synthetic OPDA-Asp and OPDA-Ile, so far, failed to induce CoP in cells, it suggests that yet another presumed OPDA-amino acid form(s) could be acting as novel regulator(s) of phytoalexins in rice.

Fungal endophytes establish symbiotic relationships with host plants, which results in a mutual growth benefit. However, little is known about the plant genetic response underpinning endophyte colonization. Phomopsis liquidambaris usually lives as an endophyte in a wide range of asymptomatic hosts and promotes biotic and abiotic stress resistance. In this study, we show that under low nitrogen conditions P. liquidambaris promotes rice growth in a hydroponic system, which is free of other microorganisms. In order to gain insights into the mechanisms of plant colonization by P. liquidambaris under low nitrogen conditions, we compared root and shoot transcriptome profiles of root-inoculated rice at different colonization stages. We determined that genes related to plant growth promotion, such as gibberellin and auxin related genes, were up-regulated at all developmental stages both locally and systemically. The largest group of up-regulated genes (in both roots and shoots) were related to flavonoid biosynthesis, which is involved in plant growth as well as antimicrobial compounds. Furthermore, genes encoding plant defense-related endopeptidase inhibitors were strongly up-regulated at the early stage of colonization. Together, these results provide new insights into the molecular mechanisms of plant-microbe mutualism and the promotion of plant growth by a fungal endophyte under nitrogen-deficient conditions.

Key message A wild relative of rice from the Australian savannah was compared with cultivated rice, revealing thermotolerance in growth and photosynthetic processes and a more robust carbon economy in extreme heat. Abstract Above ~ 32 °C, impaired photosynthesis compromises the productivity of rice. We compared leaf tissues from heat-tolerant wild rice (Oryza australiensis) with temperate-adapted O. sativa after sustained exposure to heat, as well as diurnal heat shock. Leaf elongation and shoot biomass in O. australiensis were unimpaired at 45 °C, and soluble sugar concentrations trebled during 10 h of a 45 °C shock treatment. By contrast, 45 °C slowed growth strongly in O. sativa. Chloroplastic CO2 concentrations eliminated CO2 supply to chloroplasts as the basis of differential heat tolerance. This directed our attention to carboxylation and the abundance of the heat-sensitive chaperone Rubisco activase (Rca) in each species. Surprisingly, O. australiensis leaves at 45 °C had 50% less Rca per unit Rubisco, even though CO2 assimilation was faster than at 30 °C. By contrast, Rca per unit Rubisco doubled in O. sativa at 45 °C while CO2 assimilation was slower, reflecting its inferior Rca thermostability. Plants grown at 45 °C were simultaneously exposed to 700 ppm CO2 to enhance the CO2 supply to Rubisco. Growth at 45 °C responded to CO2 enrichment in O. australiensis but not O. sativa, reflecting more robust carboxylation capacity and thermal tolerance in the wild rice relative.

MoSDT1, a rice blast fungus transcription factor, is as an inducer to activate defense response through mainly mediating phosphorylated proteins in rice. Pathogen effector proteins play a dual role in infecting the host or triggering a defense response. Our previous research found a Magnaporthe oryzae effector, MoSDT1, which could activate the rice defense response when it was overexpressed in rice. However, we still know little about the mechanisms on how MoSDT1 in vivo or in vitro influences the resistance ability of rice. Our results showed that decreased ROS and increased lignin contents appeared along with significant upregulation of defense-related genes, raffinose synthesis gene, and phenylalanine ammonialyase gene. Moreover, we revealed that the contents of lignin were increased, which was in accordance with the upregulation of its precursor phenylalanine gene despite the fact that the glutamate-/thiamine-responsive genes were inhibited in MoSDT1 transgenic rice, and these indicated that MoSDT1 triggered the defense system of rice in vivo. Interestingly, in vitro studies, we further found that MoSDT1 induced the defense system by ROS synthesis, callose deposition, PR gene expression and SA/JA synthesis/signal genes using the purified prokaryotic expression system in rice plants. In addition, this defense response was confirmed to be activated by the zinc finger domain of MoSDT1 via prokaryotic expression of MoSDT1 truncated mutants in rice plants. To elucidate the regulative effects of MoSDT1 on protein phosphorylation in rice, phosphoproteome analysis was performed in both MoSDT1-transgenic and wild type rice. We found that MoSDT1 specifically up-regulated the expression levels of a few phosphorylated proteins, which were involved in multiple functions, such as biotic/abiotic stress and growth. In addition, the motifs in these specific proteins ranked the top among the top-five conserved motifs in the MoSDT1-transgenic rice. MoSDT1 played a crucial role in enhancing rice resistance by modulating several genes and signaling pathways.

This study describes biological functions of the bHLH transcription factor RERJ1 involved in the jasmonate response and the related defense-associated metabolic pathways in rice, with particular focus on deciphering the regulatory mechanisms underlying stress-induced volatile emission and herbivory resistance. RERJ1 is rapidly and drastically induced by wounding and jasmonate treatment but its biological function remains unknown as yet. Here we provide evidence of the biological function of RERJ1 in plant defense, specifically in response to herbivory and pathogen attack, and offer insights into the RERJ1-mediated regulation of metabolic pathways of specialized defense compounds, such as monoterpene linalool, in possible collaboration with OsMYC2—a well-known master regulator in jasmonate signaling. In rice (Oryza sativa L.), the basic helix–loop–helix (bHLH) family transcription factor RERJ1 is induced under environmental stresses, such as wounding and drought, which are closely linked to jasmonate (JA) accumulation. Here, we investigated the biological function of RERJ1 in response to biotic stresses, such as herbivory and pathogen infection, using an RERJ1-defective mutant. Transcriptome analysis of the rerj1-Tos17 mutant revealed that RERJ1 regulated the expression of a typical family of conserved JA-responsive genes (e.g., terpene synthases, proteinase inhibitors, and jasmonate ZIM domain proteins). Upon exposure to armyworm attack, the rerj1-Tos17 mutant exhibited more severe damage than the wildtype, and significant weight gain of the larvae fed on the mutant was observed. Upon Xanthomonas oryzae infection, the rerj1-Tos17 mutant developed more severe symptoms than the wildtype. Among RERJ1-regulated terpene synthases, linalool synthase expression was markedly disrupted and linalool emission after wounding was significantly decreased in the rerj1-Tos17 mutant. RERJ1 appears to interact with OsMYC2—a master regulator of JA signaling—and many OsJAZ proteins, although no obvious epistatic interaction was detected between them at the transcriptional level. These results indicate that RERJ1 is involved in the transcriptional induction of JA-mediated stress-responsive genes via physical association with OsMYC2 and mediates defense against herbivory and bacterial infection through JA signaling.

Down-regulation of starch branching enzymes alters fine structure and starch properties, especially the B-type crystalline pattern and extremely high amylose content identified in the BEIIb-deficiency mutant in the indica rice. The relative importance of the starch branching enzymes in determining the molecular fine structure and starch functional properties were uncovered in this study. An indica rice, Guangluai 4 with high amylose content (AC) and high gelatinization temperature (GT) was used to generate the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein-9 (Cas9) knockout lines. Five mutant lines were identified including be1-1, be1-2, be2a-1, be2a-2 and be2b-1, and analysis of western blot showed the CRISPR/Cas9 system was successful in inducing mutations in the targeted genes. AC of be2b-1 (34.1%) was greater than that of wild type (WT) (27.4%) and other mutants. Mutations of either BEI or BEIIa did not alter the starch crystallite pattern (A-type). The BEIIb deficiency caused an opaque endosperm phenotype, changed the crystallite pattern from A- to B-type, and dramatically increased the degree of ordered structure, the relative proportion of amylose chains and intermediate to long amylopectin chains, average chain length of amylopectin molecules as well as GT. The BEIIa deficiency had no effect on the proportion of amylose chains, the length of amylopectin intermediate-long chains, conclusion temperature and enthalpy of gelatinization. Down-regulation of BEI increased the proportion of shortest amylopectin chains (fa) but decreased the proportion of long amylopectin chains (fb2 and fb3), leading to a lower GT. It is concluded that the relative importance in determining starch fine structures and functionality was in the order of BEIIb > BEI > BEIIa. Our results provide new information for utilizations of BE-deficient mutants in rice quality breeding.

Jasmonate-induced accumulation of anti-herbivore compounds mediates rice resistance to the leaf folder Cnaphalocrocis medinalis. The rice leaf folder (LF), Cnaphalocrocis medinalis, is one of the most destructive insect pests in the paddy field. LF larvae induces leaf folding and scrapes the upper epidermis and mesophyll tissues reducing photosynthesis and yield in rice. Identifying plant defense pathways and genes involved in LF resistance is essential to understand better this plant–insect interaction and develop new control strategies for this pest. Jasmonate (JA) signaling controls a plethora of plant defenses against herbivores. Using RNA-seq time series analysis, we characterized changes in the transcriptome of wild-type (WT) leaves in response to LF damage and measured the dynamics of accumulation of JA phytohormone pools in time-course experiments. Genes related to JA signaling and responses, known to mediate resistance responses to herbivores, were induced by LF and were accompanied by an increment in the levels of JA pools in damaged leaves. The accumulation of defense compounds such as phenolamides and trypsin proteinase inhibitor (TPI) also increased after LF infestation in WT but not in JA mutant plants impaired in JA biosynthesis (aoc-2) and signaling (myc2-5). Consistent with all these responses, we found that LF larvae performed better in the JA mutant backgrounds than in the WT plants. Our results show that JA signaling regulates LF-induced accumulation of TPI and phenolamides and that these compounds are likely an essential part of the defense arsenal of rice plants against this insect pest.

Coordinated regulation of amylose and amylopectin synthesis via manipulation of SSII-2, SSII-3 and Wx expression in endosperm can improve rice eating and cooking quality. With increasing rice consumption worldwide, many researchers are working to increase the yield and improve grain quality, especially eating and cooking quality (ECQ). The rice ECQ is mainly controlled by the expression of starch synthesis-related genes (SSRGs) in endosperm. Although the Wx and SSII-3/SSIIa/ALK genes, two major SSRGs, have been manipulated to improve rice ECQ via various breeding approaches, new methods to further improve ECQ are desired. In our previous study, we enhanced rice ECQ by knocking down SSII-2 expression in the japonica Nipponbare cultivar (carrying the Wxb allele) via RNA interference. Herein, the SSII-2 RNAi was introduced into two Nipponbare-derived near-isogenic lines (NILs), Nip(Wxa) and Nip(wx), carrying Wxa and wx alleles respond for high and no amylose levels, respectively. Analysis of physicochemical properties revealed that the improved grain quality of SSII-2 RNAi transgenic lines was achieved by coordinated downregulating the expression of SSII-2, SSII-3 and Wx. To further confirm this conclusion, we generated ssii-2, ssii-3 and ssii-2ssii-3 mutants via CRISPR/Cas9 technique. The amylopectin structure of the resulting ssii-2sii-3 mutants was similar to that in SSII-2 RNAi transgenic lines, and the absence of SSII-2 decreased the amylose content, gelatinisation temperature and rapid visco-analyser profile, indicating essential roles for SSII-2 in the regulation of amylopectin biosynthesis and amylose content in rice endosperm. The effect of SSII-2 was seen only when the activity of SSII-3 was very low or lacking. Our study provides novel approaches and valuable germplasm resources for improving ECQ via plant breeding.

Key message Subgroup IVc basic helix-loop-helix transcription factors OsbHLH058 and OsbHLH059 positively regulate major iron deficiency responses in rice in a similar but distinct manner, putatively under partial control by OsHRZs. Abstract Under low iron availability, plants transcriptionally induce the expression of genes involved in iron uptake and translocation. OsHRZ1 and OsHRZ2 ubiquitin ligases negatively regulate this iron deficiency response in rice. The basic helix-loop-helix (bHLH) transcription factor OsbHLH060 interacts with OsHRZ1, and positively regulates iron deficiency-inducible genes. However, the functions of three other subgroup IVc bHLH transcription factors in rice, OsbHLH057, OsbHLH058, and OsbHLH059, have not yet been characterized. In the present study, we investigated the functions of OsbHLH058 and OsbHLH059 related to iron deficiency response. OsbHLH058 expression was repressed under iron deficiency, whereas the expression of OsbHLH057 and OsbHLH060 was moderately induced. Yeast two-hybrid analysis indicated that OsbHLH058 interacts with OsHRZ1 and OsHRZ2 more strongly than OsbHLH060, whereas OsbHLH059 showed no interaction. An in vitro ubiquitination assay detected no OsbHLH058 and OsbHLH060 ubiquitination by OsHRZ1 and OsHRZ2. Transgenic rice lines overexpressing OsbHLH058 showed tolerance for iron deficiency and higher iron concentration in seeds. These lines also showed enhanced expression of many iron deficiency-inducible genes involved in iron uptake and translocation under iron-sufficient conditions. Conversely, OsbHLH058 knockdown lines showed susceptibility to iron deficiency and reduced expression of many iron deficiency-inducible genes. OsbHLH059 knockdown lines were also susceptible to iron deficiency, and formed characteristic brownish regions in iron-deficient new leaves. OsbHLH059 knockdown lines also showed reduced expression of many iron deficiency-inducible genes. These results indicate that OsbHLH058 and OsbHLH059 positively regulate major iron deficiency responses in a similar but distinct manner, and that this function may be partially controlled by OsHRZs.

Regulation of abscisic acid (ABA) signaling is crucial in balancing responses to abiotic stresses and retaining growth in planta. An ABA receptor (PYL/RCAR) and a protein phosphatase (PP2C), a co-receptor, form a complex upon binding to ABA. Previously we reported that the second and fourth positions in the VxGΦL motif of PP2Cs from Oryza sativa are critical in the interaction of PP2Cs with PYL/RCARs. Considering substantial effects of the VxGΦL motif on ABA signaling outputs, further comprehensive characterization of residues in the second and fourth positions are required. Here we surveyed the second and fourth positions of the VxGΦL motif by combination of biochemical, structural and physiological analyses. We found that the fourth position of the VxGΦL motif, highly conserved to small hydrophobic residues, was a key determinant of the OsPP2C50:OsPYL/RCAR interactions across subfamilies. Large hydrophobic or any hydrophilic residues in the fourth position abrogated ABA responsiveness. Analysis of crystal structures of OsPP2C50 mutants, S265L/I267V ("LV"), I267L ("SL") and I267W ("SW"), in complex with ABA and OsPYL/RCAR3, along with energy calculation of the complexes, uncovered that a bulky hydrophobic residue in the fourth position of the VxGΦL motif pushed away side chains of nearby residues, conferring side-chain rotameric energy stress. Hydrophilic residues in this position imposed solvation energy stress to the PP2C:PYL/RCAR complex. Germination and gene expression analyses corroborated that OsPP2C50 AS and AK mutants modulated ABA responsiveness in Arabidopsis. Our results suggest that ABA responsiveness could be fine-tuned by the fourth position of the VxGΦL motif on PP2Cs. KEY MESSAGE: We comprehensively surveyed the VxGΦL motif to find that the fourth position, highly conserved to small hydrophobic residues, was critical in regulating ABA responsiveness.

Anther dehiscence, one of the essential steps in pollination and double fertilization, is regulated by a complex signaling pathway encompassing hormones and environmental factors. However, key components underlying the signaling pathway that regulate anther dehiscence remain largely elusive. Here, we isolated a rice mutant anther dehiscence defected 1 (Osadd1) that exhibited defects in anther dehiscence and glume open. Map-based cloning revealed that OsADD1 encoded a GARP (Golden2, ARR-B and Psr1) transcription factor. Sequence analysis showed that a single base deletion in Osadd1 mutant resulted in pre-termination of the GARP domain. OsADD1 was constitutively expressed in various tissues, with more abundance in the panicles. The major genes associated with anther dehiscence were affected in the Osadd1 mutant, and the expression level of the cellulose synthase-like D sub-family 4 (OsCSLD4) was significantly decreased. We demonstrate that OsADD1 regulated the expression of OsCSLD4 by binding to its promoter, and affects rice anther dehiscence.

Several classes of transcription factors are involved in the activation of defensins. A new type of the transcription factor responsible for the regulation of wheat grain specific defensins was characterised in this work. HD-Zip class IV transcription factors constitute a family of multidomain proteins. A full-length cDNA of HD-Zip IV, designated TaGL7 was isolated from the developing grain of bread wheat, using a specific DNA sequence as bait in the Y1H screen. 3D models of TaGL7 HD complexed with DNA cis-elements rationalised differences that underlined accommodations of binding and non-binding DNA, while the START-like domain model predicted binding of lipidic molecules inside a concave hydrophobic cavity. The 3'-untranslated region of TaGL7 was used as a probe to isolate the genomic clone of TdGL7 from a BAC library prepared from durum wheat. The spatial and temporal activity of the TdGL7 promoter was tested in transgenic wheat, barley and rice. TdGL7 was expressed mostly in ovary at fertilisation and its promoter was active in a liquid endosperm during cellularisation and later in the endosperm transfer cells, aleurone, and starchy endosperm. The pattern of TdGL7 expression resembled that of genes that encode grain-specific lipid transfer proteins, particularly defensins. In addition, GL7 expression was upregulated by mechanical wounding, similarly to defensin genes. Co-bombardment of cultured wheat cells with TdGL7 driven by constitutive promoter and seven grain or root specific defensin promoters fused to GUS gene, revealed activation of four promoters. The data confirmed the previously proposed role of HD-Zip IV transcription factors in the regulation of genes that encode lipid transfer proteins involved in lipid transport and defence. The TdGL7 promoter could be used to engineer cereal grains with enhanced resistance to insects and fungal infections.

Rice WSL6 is involved in chloroplast ribosome biogenesis and is essential for early chloroplast development. Construction of the genetic translation system is a prerequisite for chloroplast development in plants. However, the molecular mechanism underlying this process is largely unknown. Here, we isolated a white stripe leaf6 (wsl6) mutant in rice. The mutant seedlings displayed white-striped leaves that were more severe under low-temperature conditions. Transmission electron microscopy analysis showed that the wsl6 mutant was defective in early chloroplast development. Map-based cloning revealed that WSL6 encodes an Era-type guanosine-5'-triphosphate (GTP)-binding protein located in chloroplasts. Immunoblotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses demonstrated an absence of 70S ribosomes in wsl6 chloroplasts. Further research showed that WSL6 binds to the 16S ribosomal RNA (rRNA) subunit of chloroplast ribosome 30S. In summary, these results show that WSL6 is essential for chloroplast ribosome biogenesis during early chloroplast development in rice.

CYP71Z18 exhibited plastic substrate specificity to catalyze oxidation of multiple rice diterpenes and elevated chemical defense against the blast fungus in transgenic rice. Diversified plant specialized metabolism relies on corresponding biosynthetic enzymes with differential substrate specificity. CYP71Z18 catalyzed formation of maize phytoalexins including zealexin A1, the sesquiterpenoid phytoalexin, and diterpenoid phytoalexin dolabralexin, indicating catalytic promiscuity on different terpene substrates. Here substrate specificity of CYP71Z18 was further explored through microbial metabolic engineering and it was identified to accept multiple rice diterpenes as substrates for oxidation. One CYP71Z18 enzymatic product derived from syn-pimaradiene was identified as 15,16-epoxy-syn-pimaradiene by NMR analysis, which was further elaborated by CYP99A3 to generate C19 hydroxylated product. 15,16-epoxy-syn-pimaradien-19-ol exhibited inhibitory effect on spore germination and appressorium formation of the blast pathogen Magnaporthe oryzae. Overexpression of CYP71Z18 in rice resulted in accumulation of several new diterpenoids, indicating promiscuous activity in planta. Transgenic rice also showed stronger resistance against M. oryzae infection, suggesting elevated chemical defense through changed diterpenoid metabolism by CYP71Z18 overexpression. This investigation sheds light on plant metabolic engineering using plastic substrate specificity of P450s to strengthen disease resistance and potentially provide abundant lead compounds.

Key message We have isolated several Osiaa23 rice mutants with different knockout genotypes, resulting in different phenotypes, which suggested that different genetic backgrounds or mutation types influence gene function. Abstract The Auxin/Indole-3-Acetic Acid (Aux/IAA) gene family performs critical roles in auxin signal transduction in plants. In rice, the gene OsIAA23 (Os06t0597000) is known to affect development of roots and shoots, but previous knockouts in OsIAA23 have been sterile and difficult for research continuously. Here, we isolate new Osiaa23 mutants using the CRISPR/Cas9 system in japonica (Wuyunjing24) and indica (Kasalath) rice, with extensive genome re-sequencing to confirm the absence of off-target effects. In Kasalath, mutants with a 13-amino acid deletion showed profoundly greater dwarfing, lateral root developmental disorder, and fertility deficiency, relative to mutants with a single amino acid deletion, demonstrating that those 13 amino acids in Kasalath are essential to gene function. In Wuyunjing24, we predicted that mutants with a single base-pair frameshift insertion would experience premature termination and strong phenotypic defects, but instead these lines exhibited negligible phenotypic difference and normal fertility. Through RNA-seq, we show here that new mosaic transcripts of OsIAA23 were produced de novo, which circumvented the premature termination and thereby preserved the wild-type phenotype. This finding is a notable demonstration in plants that mutants can mask loss of function CRISPR/Cas9 editing of the target gene through de novo changes in alternative splicing. Electronic supplementary material The online version of this article (10.1007/s11103-019-00871-5) contains supplementary material, which is available to authorized users.

Key message We demonstrate that the C-terminus of OsCDC48 is essential for maintaining its full ATPase activity and OsCDC48/48E interaction is required to modulate cellular processes and plant survival in rice. Abstract Cell division cycle 48 (CDC48) belongs to the superfamily protein of ATPases associated with diverse cellular activities (AAA). We previously isolated a rice CDC48 mutant (psd128) displaying premature senescence and death phenotype. Here, we showed that OsCDC48 (Os03g0151800) interacted with OsCDC48E (Os10g0442600), a homologue of OsCDC48, to control plant survival in rice. OsCDC48E knockout plants exhibited similar behavior to psd128 with premature senescence and plant death. Removal of the C-terminus of OsCDC48 caused altered expression of cell cycle-related genes, changed the percentage of cells in G1 and G2/M phases, and abolished the interaction between OsCDC48 itself and between OsCDC48 and OsCDC48E, respectively. Furthermore, the truncated OsCDC48–PSD128 protein lacking the C-terminal 27 amino acid residues showed a decreased level of ATPase activity. Overexpression of OsCDC48–psd128 resulted in differential expression of AAA-ATPase associated genes leading to increased total ATPase activity, accumulation of reactive oxygen species and decreased plant tiller numbers while overexpression of OsCDC48 also resulted in differential expression of AAA-ATPase associated genes leading to increased total ATPase activity, but increased plant tiller numbers and grain yield, indicating its potential utilization for yield improvement. Our results demonstrated that the C-terminal region of OsCDC48 was essential for maintaining the full ATPase activity and OsCDC48/48E complex might function in form of heteromultimers to modulate cellular processes and plant survival in rice. Electronic supplementary material The online version of this article (10.1007/s11103-019-00851-9) contains supplementary material, which is available to authorized users.

Key message GluA and GluB-4/5 glutelin subfamilies are mainly localized to outer region of the endosperm, particularly in its ventral side, in rice grain, but GluC is localized to throughout the endosperm. Abstract The major seed storage protein in rice (Oryza sativa) is glutelin, which forms a vacuole-derived protein body type-II. Glutelins are encoded by multiple genes, and generally comprise four protein subfamilies, namely, GluA, GluB, GluC, and GluD: however, the localization pattern of glutelin in rice grains remains obscure. In this study, we investigated the localization pattern of five subtypes of the glutelin protein in rice grains using glutelin-subtype specific antibodies. Immunoblot analysis against sequentially polished rice flour fractions from three crop years and seven japonica rice varieties revealed that GluA was strongly localized in the outer region of the endosperm, including the subaleurone layer, whereas GluC was distributed throughout the endosperm. Among the glutelin subtypes, GluA was mostly found in the outer region of the rice grain, followed by GluB-4/5, GluB-1, GluD, and GluC. Immunofluorescence labeling microscopy analysis using immature rice seeds clearly revealed that the localization pattern of GluC and GluD was completely different from that of GluA and GluB. Expression levels of all glutelins, particularly GluA, GluB-1, and GluB-4/5, were stronger on the ventral than dorsal side in rice grains. These results provide strong and consistent evidence that glutelins localize to the rice grain in a subfamily-dependent manner. Electronic supplementary material The online version of this article (10.1007/s11103-019-00855-5) contains supplementary material, which is available to authorized users.

The OsPLS2 locus was isolated and cloned by map-based cloning that encodes a Upf1-like helicase. Disruption of OsPLS2 accelerated light-dependent leaf senescence in the rice mutant of ospls2. Leaf senescence is a very complex physiological process controlled by both genetic and environmental factors, however its underlying molecular mechanisms remain elusive. In this study, we report a novel Oryza sativa premature leaf senescence mutant (ospls2). Through map-based cloning, a G-to-A substitution was determined at the 1st nucleotide of the 13th intron in the OsPLS2 gene that encodes a Upf1-like helicase. This mutation prompts aberrant splicing of OsPLS2 messenger and consequent disruption of its full-length protein translation, suggesting a negative role of OsPLS2 in regulating leaf senescence. Wild-type rice accordingly displayed a progressive drop of OsPSL2 protein levels with age-dependent leaf senescence. Shading and light filtration studies showed that the ospls2 phenotype, which was characteristic of photo-oxidative stress and reactive oxygen species (ROS) accumulation, was an effect of irritation by light. When continuously exposed to far-red light, exogenous H2O2 and/or abscisic acid (ABA), the ospls2 mutant sustained hypersensitive leaf senescence. In consistence, light and ROS signal pathways in ospls2 were activated by down-regulation of phytochrome genes, and up-regulation of PHYTOCHROME-INTERACTING FACTORS (PIFs) and WRKY genes, all promoting leaf senescence. Together, these data indicated that OsPLS2 played an essential role in leaf senescence and its disruption triggered light-dependent leaf senescence in rice.

Grain amyloplast and leaf chloroplast DNA sequences are identical in rice plants but are differentially methylated. The leaf chloroplast DNA becomes more methylated as the rice plant ages. Rice is an important crop worldwide. Chloroplasts and amyloplasts are critical organelles but the amyloplast genome is poorly studied. We have characterised the sequence and methylation of grain amyloplast DNA and leaf chloroplast DNA in rice. We have also analysed the changes in methylation patterns in the chloroplast DNA as the rice plant ages. Total genomic DNA from grain, old leaf and young leaf tissues were extracted from the Oryza sativa ssp. indica cv. MR219 and sequenced using Illumina Miseq. Sequence variant analysis revealed that the amyloplast and chloroplast DNA of MR219 were identical to each other. However, comparison of CpG and CHG methylation between the identical amyloplast and chloroplast DNA sequences indicated that the chloroplast DNA from rice leaves collected at early ripening stage was more methylated than the amyloplast DNA from the grains of the same plant. The chloroplast DNA became more methylated as the plant ages so that chloroplast DNA from young leaves was less methylated overall than amyloplast DNA. These differential methylation patterns were primarily observed in organelle-encoded genes related to photosynthesis followed by those involved in transcription and translation.

OsHIRP1 is an E3 ligase that acts as a positive regulator in the plant response to heat stress, thus providing important information relating to adaptation and regulation under heat stress in plant. Extreme temperature adversely affects plant growth, development, and productivity. Here, we report the molecular functions of Oryza sativa heat-induced RING finger protein 1 (OsHIRP1), which might play an important role in the response to heat. Transcription of the OsHIRP1 was upregulated in response to heat and drought treatment. We found that the OsHIRP1-EYFP fusion protein was localized to the nucleus after heat treatment (45 °C). Two interacting partners, OsARK4 and OsHRK1, were identified via yeast-two-hybrid screening, which were mainly targeted to the nucleus (OsARK4) and cytosol (OsHRK1), and their interactions with OsHIRP1 were confirmed by biomolecular fluorescence complementation (BiFC). An in vitro ubiquitination assay showed that OsHIRP1 E3 ligase directly ubiquitinates its interacting proteins, OsAKR4 and OsHRK1, as substrates. Using an in vitro cell-free degradation assay, we observed a clear reduction in the levels of the two proteins under high temperature (45 °C), but not under low temperature conditions (4 °C and 30 °C). Seeds of OsHIRP1-overexpressing plants exhibited high germination rates compared with the control under heat stress. The OsHIRP1-overexpressing plants presented high survival rates of approximately 62-68%, whereas control plants displayed a low recovery rate of 34% under condition of acquired thermo-tolerance. Some heat stress-inducible genes (HsfA3, HSP17.3, HSP18.2 and HSP20) were up-regulated in OsHIRP1-overexpressing Arabidopsis than control plants under heat stress conditions. Collectively, these results suggest that OsHIRP1, an E3 ligase, positively regulates plant response to heat stress.

We reported that knockdown of OsDCL3b decreased grain yield but increased grain quality in rice, which is helpful for molecular breeding in crops. Multiple DICER-LIKE (DCL) genes usually exist and show diverse biochemical and phenotypic functions in land plants. In rice, the biochemical function of OsDCL3b is known to process 24-nucleotide panicle phased small RNAs, however, its phenotypic functions are unclear. Here we reported that knockdown of OsDCL3b led to reduced pollen fertility, seed setting rate, and decreased grain yield but increased grain quality in rice. To reveal the molecular mechanism of the above phenomena, extracted RNAs from rice panicles of the wild type (WT) and OsDCL3b-RNAi line S6-1 were analyzed by deep sequencing. It showed that knockdown of OsDCL3b affected the biogenesis of both 21- and 24-nucleotide small RNAs including miRNAs and phased small RNAs. Using RNA-seq, 644 up- and 530 down-regulated mRNA genes were identified in panicles of line S6-1, and 550 and 273 differentially spliced genes with various alternative splicing (AS) events were observed in panicles of line S6-1 and WT, respectively, suggesting that OsDCL3b involved in influencing the transcript levels of mRNA genes and the AS events in rice panicles. Thus, our results show that knockdown of OsDCL3b will affect the biogenesis of small RNAs, which is involved in regulating the transcription of mRNA genes, and consequently influence the grain yield and quality in rice.