A natural gene drive system confers reproductive isolation in rice.

Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.

The transcriptional hub SHORT INTERNODES1 integrates hormone signals to orchestrate rice growth and development.

Plants have evolved sophisticated mechanisms to coordinate their growth and stress responses via integrating various phytohormone signaling pathways. However, the precise molecular mechanisms orchestrating integration of the phytohormone signaling pathways remain largely obscure. In this study, we found that the rice (Oryza sativa) short internodes1 (shi1) mutant exhibits typical auxin-deficient root development and gravitropic response, brassinosteroid (BR)-deficient plant architecture and grain size as well as enhanced abscisic acid (ABA)-mediated drought tolerance. Additionally, we found that the shi1 mutant is also hyposensitive to auxin and BR treatment but hypersensitive to ABA. Further, we showed that OsSHI1 promotes the biosynthesis of auxin and BR by activating the expression of OsYUCCAs and D11, meanwhile dampens ABA signaling by inducing the expression of OsNAC2, which encodes a repressor of ABA signaling. Furthermore, we demonstrated that 3 classes of transcription factors, AUXIN RESPONSE FACTOR 19 (OsARF19), LEAF AND TILLER ANGLE INCREASED CONTROLLER (LIC), and OsZIP26 and OsZIP86, directly bind to the promoter of OsSHI1 and regulate its expression in response to auxin, BR, and ABA, respectively. Collectively, our results unravel an OsSHI1-centered transcriptional regulatory hub that orchestrates the integration and self-feedback regulation of multiple phytohormone signaling pathways to coordinate plant growth and stress adaptation.

A CYP78As-small grain4-coat protein complex â ¡ pathway promotes grain size in rice.

CYP78A, a cytochrome P450 subfamily that includes rice (Oryza sativa L.) BIG GRAIN2 (BG2, CYP78A13) and Arabidopsis thaliana KLUH (KLU, CYP78A5), generate an unknown mobile growth signal (referred to as a CYP78A-derived signal) that increases grain (seed) size. However, the mechanism by which the CYP78A pathway increases grain size remains elusive. Here, we characterized a rice small grain mutant, small grain4 (smg4), with smaller grains than its wild type due to restricted cell expansion and cell proliferation in spikelet hulls. SMG4 encodes a multidrug and toxic compound extrusion (MATE) transporter. Loss of function of SMG4 causes smaller grains while overexpressing SMG4 results in larger grains. SMG4 is mainly localized to endoplasmic reticulum (ER) exit sites (ERESs) and partially localized to the ER and Golgi. Biochemically, SMG4 interacts with coat protein complex Ⅱ (COPⅡ) components (Sar1, Sec23, and Sec24) and CYP78As (BG2, GRAIN LENGTH 3.2 [GL3.2], and BG2-LIKE 1 [BG2L1]). Genetically, SMG4 acts, at least in part, in a common pathway with Sar1 and CYP78As to regulate grain size. In summary, our findings reveal a CYP78As-SMG4-COPⅡ regulatory pathway for grain size in rice, thus providing new insights into the molecular and genetic regulatory mechanism of grain size.

Dwarf and High Tillering1 represses rice tillering through mediating the splicing of D14 pre-mRNA.

Strigolactones (SLs) constitute a class of plant hormones that regulate many aspects of plant development, including repressing tillering in rice (Oryza sativa). However, how SL pathways are regulated is still poorly understood. Here, we describe a rice mutant dwarf and high tillering1 (dht1), which exhibits pleiotropic phenotypes (such as dwarfism and increased tiller numbers) similar to those of mutants defective in SL signaling. We show that DHT1 encodes a monocotyledon-specific hnRNP-like protein that acts as a previously unrecognized intron splicing factor for many precursor mRNAs (pre-mRNAs), including for the SL receptor gene D14. We find that the dht1 (DHT1I232F) mutant protein is impaired in its stability and RNA binding activity, causing defective splicing of D14 pre-mRNA and reduced D14 expression, and consequently leading to the SL signaling-defective phenotypes. Overall, our findings deepen our understanding of the functional diversification of hnRNP-like proteins and establish a connection between posttranscriptional splicing and SL signaling in the regulation of plant development.

SUBSTANDARD STARCH GRAIN7 regulates starch grain size and endosperm development in rice.

Starch is synthesized as insoluble, semicrystalline particles within plant chloroplast and amyloplast, which are referred to as starch grains (SGs). The size and morphology of SGs in the cereal endosperm are diverse and species-specific, representing a key determinant of the suitability of starch for industrial applications. However, the molecular mechanisms modulating SG size in cereal endosperm remain elusive. Here, we functionally characterized the rice (Oryza sativa) mutant substandard starch grain7 (ssg7), which exhibits enlarged SGs and defective endosperm development. SSG7 encodes a plant-specific DUF1001 domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) CRUMPLED LEAF (AtCRL). SSG7 localizes to the amyloplast membrane in developing endosperm. Several lines of evidence suggest that SSG7 functions together with SSG4 and SSG6, known as two regulators essential for SG development, to control SG size, by interacting with translocon-associated components, which unveils a molecular link between SG development and protein import. Genetically, SSG7 acts synergistically with SSG4 and appears to be functional redundancy with SSG6 in modulating SG size and endosperm development. Collectively, our findings uncover a multimeric functional protein complex involved in SG development in rice. SSG7 represents a promising target gene for the biotechnological modification of SG size, particularly for breeding programs aimed at improving starch quality.

A regulatory loop establishes the link between the circadian clock and abscisic acid signaling in rice.

There is a close regulatory relationship between the circadian clock and the abscisic acid (ABA) signaling pathway in regulating many developmental processes and stress responses. However, the exact feedback regulation mechanism between them is still poorly understood. Here, we identified the rice (Oryza sativa) clock component PSEUDO-RESPONSE REGULATOR 95 (OsPRR95) as a transcriptional regulator that accelerates seed germination and seedling growth by inhibiting ABA signaling. We also found that OsPRR95 binds to the ABA receptor gene REGULATORY COMPONENTS OF ABA RECEPTORS10 (OsRCAR10) DNA and inhibits its expression. Genetic analysis showed OsRCAR10 acts downstream of OsPRR95 in mediating ABA responses. In addition, the induction of OsPRR95 by ABA partly required a functional OsRCAR10, and the ABA-responsive element-binding factor ABSCISIC ACID INSENSITIVE5 (OsABI5) bound directly to the promoter of OsPRR95 and activated its expression, thus establishing a regulatory feedback loop between OsPRR95, OsRCAR10, and OsABI5. Taken together, our results demonstrated that the OsRCAR10-OsABI5-OsPRR95 feedback loop modulates ABA signaling to fine-tune seed germination and seedling growth, thus establishing the molecular link between ABA signaling and the circadian clock.

Rice STOMATAL CYTOKINESIS DEFECTIVE2 regulates cell expansion by affecting vesicular trafficking in rice.

Vesicular trafficking plays critical roles in cell expansion in yeast and mammals, but information linking vesicular trafficking and cell expansion in plants is limited. Here, we isolated and characterized a rice (Oryza sativa) mutant, decreased plant height 1-1 (dph1-1), which exhibited a wide spectrum of developmental phenotypes, including reduced plant height and smaller panicles and grains. Cytological analysis revealed that limited cell expansion was responsible for the dph1-1 mutant phenotype compared to the wild-type. Map-based cloning revealed that DPH1 encodes a plant-specific protein, OsSCD2, which is homologous to Arabidopsis (Arabidopsis thaliana) STOMATAL CYTOKINESIS DEFECTIVE2 (SCD2). Subcellular localization revealed that OsSCD2 is associated with clathrin. Confocal microscopy showed that the dph1-1 mutant has defective endocytosis and post-Golgi trafficking. Biochemical and confocal data indicated that OsSCD2 physically interacts with OsSCD1 and that they are associated with intracellular structures that colocalize with microtubules. Furthermore, we found that cellulose synthesis was affected in the dph1-1 mutant, evidenced by reduced cellulose synthase gene accumulation at the transcript and protein levels, most likely resulting from an impaired localization pattern. Our results suggest that OsSCD2 is involved in clathrin-related vesicular trafficking with an important role in maintaining plant growth in rice.

The APC/CTE E3 Ubiquitin Ligase Complex Mediates the Antagonistic Regulation of Root Growth and Tillering by ABA and GA.

The antagonistic regulation of seed germination by the phytohormones abscisic acid (ABA) and gibberellic acid (GA) has been well-established. However, how these phytohormones antagonistically regulate root growth and branching (tillering in rice, Oryza sativa) remains obscure. Rice TILLER ENHANCER (TE) encodes an activator of the APC/CTE E3 ubiquitin ligase complex that represses tillering but promotes seed germination. In this study, we identified a dual role of GA and APC/CTE in regulating root growth. High GA levels can activate APC/CTE to promote the degradation of rice SHORT-ROOT1 (OsSHR1, a key factor promoting root growth) in the root meristem (RM) or MONOCULM1 (MOC1, a key factor promoting tillering) in the axillary meristem (AM), leading to restricted root growth and tillering, while low GA levels can activate the role of APC/CTE in stimulating RM cell division to promote root growth. In addition, moderate enhancement of ABA signaling helps maintain the RM and AM size, sustaining root growth and tillering by antagonizing the GA-promoted degradation of OsSHR1 and MOC1 through the SnRK2-APC/CTE regulatory module. We conclude that APC/CTE plays a key role in regulating plant architecture by mediating the crosstalk between ABA and GA signaling pathways.

GPA5 Encodes a Rab5a Effector Required for Post-Golgi Trafficking of Rice Storage Proteins.

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice (Oryza sativa) glutelin precursor accumulation5 (gpa5) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant-specific VAMP727-containing R-soluble N-ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm.

Young Leaf White Stripe encodes a P-type PPR protein required for chloroplast development.

Pentatricopeptide repeat (PPR) proteins function in post-transcriptional regulation of organellar gene expression. Although several PPR proteins are known to function in chloroplast development in rice (Oryza sativa), the detailed molecular functions of many PPR proteins remain unclear. Here, we characterized a rice young leaf white stripe (ylws) mutant, which has defective chloroplast development during early seedling growth. Map-based cloning revealed that YLWS encodes a novel P-type chloroplast-targeted PPR protein with 11 PPR motifs. Further expression analyses showed that many nuclear- and plastid-encoded genes in the ylws mutant were significantly changed at the RNA and protein levels. The ylws mutant was impaired in chloroplast ribosome biogenesis and chloroplast development under low-temperature conditions. The ylws mutation causes defects in the splicing of atpF, ndhA, rpl2, and rps12, and editing of ndhA, ndhB, and rps14 transcripts. YLWS directly binds to specific sites in the atpF, ndhA, and rpl2 pre-mRNAs. Our results suggest that YLWS participates in chloroplast RNA group II intron splicing and plays an important role in chloroplast development during early leaf development.

ESCRT-III component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice.

The endosomal sorting complex required for transport (ESCRT) is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes. Although ESCRT components affect a variety of plant growth and development processes, their impact on leaf development is rarely reported. Here, we found that OsSNF7.2, an ESCRT-III component, controls leaf rolling in rice (Oryza sativa). The Ossnf7.2 mutant rolled leaf 17 (rl17) has adaxially rolled leaves due to the decreased number and size of the bulliform cells. OsSNF7.2 is expressed ubiquitously in all tissues, and its protein is localized in the endosomal compartments. OsSNF7.2 homologs, including OsSNF7, OsSNF7.3, and OsSNF7.4, can physically interact with OsSNF7.2, but their single mutation did not result in leaf rolling. Other ESCRT complex subunits, namely OsVPS20, OsVPS24, and OsBRO1, also interact with OsSNF7.2. Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation. Both Osyuc8 and rl17 Osyuc8 showed rolled leaves, indicating that OsYUC8 and OsSNF7.2 function in the same pathway, conferring leaf development. This study reveals a new biological function for the ESCRT-III components, and provides new insights into the molecular mechanisms underlying leaf rolling.

OsSHI1 Regulates Plant Architecture Through Modulating the Transcriptional Activity of IPA1 in Rice.

Tillering and panicle branching are important determinants of plant architecture and yield potential in rice (Oryza sativa). IDEAL PLANT ARCHITECTURE1 (IPA1) encodesSQUAMOSA PROMOTER BINDING PROTEIN-LIKE14, which acts as a key transcription factor regulating tiller outgrowth and panicle branching by directly activating the expression of O. sativa TEOSINTE BRANCHED1 (OsTB1) and O. sativa DENSE AND ERECT PANICLE1 (OsDEP1), thereby influencing grain yield in rice. Here, we report the identification of a rice mutant named shi1 that is characterized by dramatically reduced tiller number, enhanced culm strength, and increased panicle branch number. Map-based cloning revealed that O. sativa SHORT INTERNODES1 (OsSHI1) encodes a plant-specific transcription factor of the SHI family with a characteristic family-specific IGGH domain and a conserved zinc-finger DNA binding domain. Consistent with the mutant phenotype, OsSHI1 is predominantly expressed in axillary buds and young panicle, and its encoded protein is exclusively targeted to the nucleus. We show that OsSHI1 physically interacts with IPA1 both in vitro and in vivo. Moreover, OsSHI1 could bind directly to the promoter regions of both OsTB1 and OsDEP1 through a previously unrecognized cis-element (T/GCTCTAC motif). OsSHI1 repressed the transcriptional activation activity of IPA1 by affecting its DNA binding activity toward the promoters of both OsTB1 and OsDEP1, resulting in increased tiller number and diminished panicle size. Taken together, our results demonstrate that OsSHI1 regulates plant architecture through modulating the transcriptional activity of IPA1 and provide insight into the establishment of plant architecture in rice.

Genetic characterization and fine mapping of qHMS4 responsible for pollen sterility in hybrids between Oryza sativa L. and Oryza glaberrima Steud.

African cultivated rice (Oryza glaberrima Steud) contains many favorable genes for tolerance to biotic and abiotic stresses and F1 hybrids between Asian cultivated rice (Oryza sativa L.) show strong heterosis. However, the hybrids of two species often exhibit hybrid sterility. Here, we identified a male sterility locus qHMS4 on chromosome 4 (Chr.4), which induces pollen semi-sterility in F1 hybrids of japonica rice variety Dianjingyou1 (DJY1) and a near-isogenic line (NIL) carrying a Chr.4 segment from Oryza glaberrima accession IRGC101854. Cytological observations indicated that non-functional pollen grains produced by the hybrids and lacking starch accumulation abort at the late bicellular stage. Molecular genetic analysis revealed distorted segregation in male gametogenesis carrying qHMS4 allele from DJY1. Fine-mapping of qHMS4 using an F2 population of 22,500 plants delimited qHMS4 to a region of 110-kb on the short arm of Chr.4. Sequence analysis showed that the corresponding sequence region in DJY1 and Oryza glaberrima were 114-kb and 323-kb, respectively, and that the sequence homology was very poor. Gene prediction analysis identified 16 and 46 open reading frames (ORFs) based on the sequences of DJY1 and O. glaberrima, respectively, among which 3 ORFs were shared by both. Future map-based cloning of qHMS4 will help to understand the underlying molecular mechanism of hybrid sterility between the two cultivated rice species. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01306-8.

OsRE1 interacts with OsRIP1 to regulate rice heading date by finely modulating Ehd1 expression.

Heading date is a key agronomic trait affecting crop yield. In rice, Early heading date 1 (Ehd1) is an important B-type response regulator in determination of heading date. Although many regulatory factors of Ehd1 expression have been functionally characterized, the direct regulators of Ehd1 largely remain to be identified. Here, we identified a new regulator of Ehd1, OsRE1, that directly binds to the A-box motif in the Ehd1 promoter. Osre1 confers an early heading phenotype due to elevated expression levels of Ehd1. OsRE1 is a nucleus-localized bZIP transcription factor with a diurnal rhythmic expression pattern. Furthermore, we identified an OsRE1-interacting protein, OsRIP1, and demonstrated that OsRIP1 can repress the transcript expression of Ehd1 in an OsRE1-dependent manner. Our genetic data showed that OsRE1 and OsRIP1 may function upstream of Ehd1 in regulating heading date. Together, our results suggest that OsRE1 functions cooperatively with OsRIP1 to regulate heading date through finely modulating the expression of Ehd1. In addition, OsRE1 and OsRIP1 are two minor heading date regulators, which are more desirable for fine-tuning heading date to improve rice regional adaptability.

Small grain and semi-dwarf 3, a WRKY transcription factor, negatively regulates plant height and grain size by stabilizing SLR1 expression in rice.

KEY MESSAGE: OsWRKY36 represses plant height and grain size by inhibiting gibberellin signaling. Plant height and grain size are important agronomic traits affecting yield in cereals, including rice. Gibberellins (GAs) are plant hormones that promote plant growth and developmental processions such as stem elongation and grain size. WRKYs are transcription factors that regulate stress tolerance and plant development including height and grain size. However, the relationship between GA signaling and WRKY genes is still poorly understood. Here, we characterized a small grain and semi-dwarf 3 (sgsd3) mutant in rice cv. Hwayoung (WT). A T-DNA insertion in the 5'-UTR of OsWRKY36 induced overexpression of OsWRKY36 in the sgsd3 mutant, likely leading to the mutant phenotype. This was confirmed by the finding that overexpression of OsWRKY36 caused a similar small grain and semi-dwarf phenotype to the sgsd3 mutant whereas knock down and knock out caused larger grain phenotypes. The sgsd3 mutant was also hyposensitive to GA and accumulated higher mRNA and protein levels of SLR1 (a GA signaling DELLA-like inhibitor) compared with the WT. Further assays showed that OsWRKY36 enhanced SLR1 transcription by directly binding to its promoter. In addition, we found that OsWRKY36 can protect SLR1 from GA-mediated degradation. We thus identified a new GA signaling repressor OsWRKY36 that represses GA signaling through stabilizing the expression of SLR1.

Ubiquitin Specific Protease 15 Has an Important Role in Regulating Grain Width and Size in Rice.

Ubiquitination and deubiquitination are reversible processes that play crucial roles in regulating organ size in plants. However, information linking deubiquitination and seed size in rice (Oryza sativa) is limited. Here, we characterized a dominant large-grain mutant, large grain1-D (lg1-D), with a 30.8% increase in seed width and a 34.5% increase in 1,000-grain weight relative to the wild type. The lg1-D mutant had more cells oriented in the lateral direction of the spikelet hull compared with the wild type. Map-based cloning showed that LG1 encodes a constitutively expressed ubiquitin-specific protease15 (OsUBP15) that possesses deubiquitination activity in vitro. Loss-of-function and down-regulated expression of OsUBP15 produced narrower and smaller grains than the control. A set of in vivo experiments indicated that the mutant Osubp15 had enhanced protein stability relative to wild-type OsUBP15. Further experiments verified that OsDA1 directly interacted with OsUBP15. Genetic data indicated that OsUBP15 and GRAIN WIDTH 2 (GW2) were not independent in regulating grain width and size. In summary, we identified OsUBP15 as a positive regulator of grain width and size in rice and provide a promising strategy for improvement of grain yield by pyramiding OsUBP15 and gw2.

OsCNGC13 promotes seed-setting rate by facilitating pollen tube growth in stylar tissues.

Seed-setting rate is a critical determinant of grain yield in rice (Oryza sativa L.). Rapid and healthy pollen tube growth in the style is required for high seed-setting rate. The molecular mechanisms governing this process remain largely unknown. In this study, we isolate a dominant low seed-setting rate rice mutant, sss1-D. Cellular examination results show that pollen tube growth is blocked in about half of the mutant styles. Molecular cloning and functional assays reveals that SSS1-D encodes OsCNGC13, a member of the cyclic nucleotide-gated channel family. OsCNGC13 is preferentially expressed in the pistils and its expression is dramatically reduced in the heterozygous plant, suggesting a haploinsufficiency nature for the dominant mutant phenotype. We show that OsCNGC13 is permeable to Ca2+. Consistent with this, accumulation of cytoplasmic calcium concentration ([Ca2+]cyt) is defective in the sss1-D mutant style after pollination. Further, the sss1-D mutant has altered extracellular matrix (ECM) components and delayed cell death in the style transmission tract (STT). Based on these results, we propose that OsCNGC13 acts as a novel maternal sporophytic factor required for stylar [Ca2+]cyt accumulation, ECM components modification and STT cell death, thus facilitating the penetration of pollen tube in the style for successful double fertilization and seed-setting in rice.

Disruption of gene SPL35, encoding a novel CUE domain-containing protein, leads to cell death and enhanced disease response in rice.

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)-like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL-PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H2 O2 , up-regulated expression of defence-related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain-containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta-COP1 and Delta-COP2 through the CUE domain, and down-regulation of these interacting proteins also cause development of HR-like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.

Rice FLOURY ENDOSPERM10 encodes a pentatricopeptide repeat protein that is essential for the trans-splicing of mitochondrial nad1 intron 1 and endosperm development.

Endosperm, the major storage organ in cereal grains, determines grain yield and quality. Despite the fact that a role for P-type pentatricopeptide repeat (PPR) proteins in the regulation of endosperm development has emerged, molecular functions of many P-type PPR proteins remain obscure. Here, we report a rice endosperm defective mutant, floury endosperm10 (flo10), which developed smaller starch grains in starchy endosperm and abnormal cells in the aleurone layer. Map-based cloning and rescued experiments showed that FLO10 encodes a P-type PPR protein with 26 PPR motifs, which is localized to mitochondria. Loss of function of FLO10 affected the trans-splicing of the mitochondrial nad1 intron 1, which was accompanied by the increased accumulation of the nad1 exon 1 and exons 2-5 precursors. The failed formation of mature nad1 led to a dramatically decreased assembly and activity of complex I, reduced ATP production, and changed mitochondrial morphology. In addition, loss of function of FLO10 significantly induced an alternative respiratory pathway involving alternative oxidase. These results reveal that FLO10 plays an important role in the maintenance of mitochondrial function and endosperm development through its effect on the trans-splicing of the mitochondrial nad1 intron 1 in rice.

OsPEX5 regulates rice spikelet development through modulating jasmonic acid biosynthesis.

Spikelet is the primary reproductive structure and a critical determinant of grain yield in rice. The molecular mechanisms regulating rice spikelet development still remain largely unclear. Here, we report that mutations in OsPEX5, which encodes a peroxisomal targeting sequence 1 (PTS1) receptor protein, cause abnormal spikelet morphology. We show that OsPEX5 can physically interact with OsOPR7, an enzyme involved in jasmonic acid (JA) biosynthesis and is required for its import into peroxisome. Similar to Ospex5 mutant, the knockout mutant of OsOPR7 generated via CRISPR-Cas9 technology has reduced levels of endogenous JA and also displays an abnormal spikelet phenotype. Application of exogenous JA can partially rescue the abnormal spikelet phenotype of Ospex5 and Osopr7. Furthermore, we show that OsMYC2 directly binds to the promoters of OsMADS1, OsMADS7 and OsMADS14 to activate their expression, and subsequently regulate spikelet development. Our results suggest that OsPEX5 plays a critical role in regulating spikelet development through mediating peroxisomal import of OsOPR7, therefore providing new insights into regulation of JA biosynthesis in plants and expanding our understanding of the biological role of JA in regulating rice reproduction.