Genome-Wide Analysis of the Auxin/Indoleacetic Acid (Aux/IAA) Gene Family in Autopolyploid Sugarcane (Saccharum spontaneum).

The auxin/indoleacetic acid (Aux/IAA) family plays a central role in regulating gene expression during auxin signal transduction. Nonetheless, there is limited knowledge regarding this gene family in sugarcane. In this study, 92 members of the IAA family were identified in Saccharum spontaneum, distributed on 32 chromosomes, and classified into three clusters based on phylogeny and motif compositions. Segmental duplication and recombination events contributed largely to the expansion of this superfamily. Additionally, cis-acting elements in the promoters of SsIAAs involved in plant hormone regulation and stress responsiveness were predicted. Transcriptomics data revealed that most SsIAA expressions were significantly higher in stems and basal parts of leaves, and at nighttime, suggesting that these genes might be involved in sugar transport. QRT-PCR assays confirmed that cold and salt stress significantly induced four and five SsIAAs, respectively. GFP-subcellular localization showed that SsIAA23 and SsIAA12a were localized in the nucleus, consistent with the results of bioinformatics analysis. In conclusion, to a certain extent, the functional redundancy of family members caused by the expansion of the sugarcane IAA gene family is related to stress resistance and regeneration of sugarcane as a perennial crop. This study reveals the gene evolution and function of the SsIAA gene family in sugarcane, laying the foundation for further research on its mode of action.

RLB (RICE LATERAL BRANCH) recruits PRC2-mediated H3K27 tri-methylation on OsCKX4 to regulate lateral branching.

Lateral branches such as shoot and panicle are determining factors and target traits for rice (Oryza sativa L.) yield improvement. Cytokinin promotes rice lateral branching; however, the mechanism underlying the fine-tuning of cytokinin homeostasis in rice branching remains largely unknown. Here, we report the map-based cloning of RICE LATERAL BRANCH (RLB) encoding a nuclear-localized, KNOX-type homeobox protein from a rice cytokinin-deficient mutant showing more tillers, sparser panicles, defected floret morphology as well as attenuated shoot regeneration from callus. RLB directly binds to the promoter and represses the transcription of OsCKX4, a cytokinin oxidase gene with high abundance in panicle branch meristem. OsCKX4 over-expression lines phenocopied rlb, which showed upregulated OsCKX4 levels. Meanwhile, RLB physically binds to Polycomb repressive complex 2 (PRC2) components OsEMF2b and co-localized with H3K27me3, a suppressing histone modification mediated by PRC2, in the OsCKX4 promoter. We proposed that RLB recruits PRC2 to the OsCKX4 promoter to epigenetically repress its transcription, which suppresses the catabolism of cytokinin, thereby promoting rice lateral branching. Moreover, antisense inhibition of OsCKX4 under the LOG promoter successfully increased panicle size and spikelet number per plant without affecting other major agronomic traits. This study provides insight into cytokinin homeostasis, lateral branching in plants, and also promising target genes for rice genetic improvement.

ABF1 Positively Regulates Rice Chilling Tolerance via Inducing Trehalose Biosynthesis.

Chilling stress seriously limits grain yield and quality worldwide. However, the genes and the underlying mechanisms that respond to chilling stress remain elusive. This study identified ABF1, a cold-induced transcription factor of the bZIP family. Disruption of ABF1 impaired chilling tolerance with increased ion leakage and reduced proline contents, while ABF1 over-expression lines exhibited the opposite tendency, suggesting that ABF1 positively regulated chilling tolerance in rice. Moreover, SnRK2 protein kinase SAPK10 could phosphorylate ABF1, and strengthen the DNA-binding ability of ABF1 to the G-box cis-element of the promoter of TPS2, a positive regulator of trehalose biosynthesis, consequently elevating the TPS2 transcription and the endogenous trehalose contents. Meanwhile, applying exogenous trehalose enhanced the chilling tolerance of abf1 mutant lines. In summary, this study provides a novel pathway 'SAPK10-ABF1-TPS2' involved in rice chilling tolerance through regulating trehalose homeostasis.

RMS2 Encoding a GDSL Lipase Mediates Lipid Homeostasis in Anthers to Determine Rice Male Fertility.

Plant male gametogenesis is a coordinated effort involving both reproductive tissues and sporophytic tissues, in which lipid metabolism plays an essential role. Although GDSL esterases/lipases have been well known as key enzymes for many plant developmental processes and stress responses, their functions in reproductive development remain unclear. Here, we report the identification of a rice male sterile2 (rms2) mutant in rice (Oryza sativa), which is completely male sterile due to the defects in tapetum degradation, cuticle formation in sporophytic tissues, and impaired exine and central vacuole development in pollen grains. RMS2 was map-based cloned as an endoplasmic reticulum-localized GDSL lipase gene, which is predominantly transcribed during early anther development. In rms2, a three-nucleotide deletion and one base substitution (TTGT to A) occurred within the GDSL domain, which reduced the lipid hydrolase activity of the resulting protein and led to significant changes in the content of 16 lipid components and numerous other metabolites, as revealed by a comparative metabolic analysis. Furthermore, RMS2 is directly targeted by the male fertility regulators Undeveloped Tapetum1 and Persistent Tapetal Cell1 both in vitro and in vivo, suggesting that RMS2 may serve as a key node in the rice male fertility regulatory network. These findings shed light on the function of GDSLs in reproductive development and provide a promising gene resource for hybrid rice breeding.

OsABF1 Represses Gibberellin Biosynthesis to Regulate Plant Height and Seed Germination in Rice (Oryza sativa L.).

Gibberellins (GAs) are diterpenoid phytohormones regulating various aspects of plant growth and development, such as internode elongation and seed germination. Although the GA biosynthesis pathways have been identified, the transcriptional regulatory network of GA homeostasis still remains elusive. Here, we report the functional characterization of a GA-inducible OsABF1 in GA biosynthesis underpinning plant height and seed germination. Overexpression of OsABF1 produced a typical GA-deficient phenotype with semi-dwarf and retarded seed germination. Meanwhile, the phenotypes could be rescued by exogenous GA3, suggesting that OsABF1 is a key regulator of GA homeostasis. OsABF1 could directly suppress the transcription of green revolution gene SD1, thus reducing the endogenous GA level in rice. Moreover, OsABF1 interacts with and transcriptionally antagonizes to the polycomb repression complex component OsEMF2b, whose mutant showed as similar but more severe phenotype to OsABF1 overexpression lines. It is suggested that OsABF1 recruits RRC2-mediated H3K27me3 deposition on the SD1 promoter, thus epigenetically silencing SD1 to maintain the GA homeostasis for growth and seed germination. These findings shed new insight into the functions of OsABF1 and regulatory mechanism underlying GA homeostasis in rice.

Cyclin-Dependent Kinase Inhibitors KRP1 and KRP2 Are Involved in Grain Filling and Seed Germination in Rice (Oryza sativa L.).

Cyclin-dependent kinase inhibitors known as KRPs (kip-related proteins) control the progression of plant cell cycles and modulate various plant developmental processes. However, the function of KRPs in rice remains largely unknown. In this study, two rice KRPs members, KRP1 and KRP2, were found to be predominantly expressed in developing seeds and were significantly induced by exogenous abscisic acid (ABA) and Brassinosteroid (BR) applications. Sub-cellular localization experiments showed that KRP1 was mainly localized in the nucleus of rice protoplasts. KRP1 overexpression transgenic lines (OxKRP1), krp2 single mutant (crkrp2), and krp1/krp2 double mutant (crkrp1/krp2) all exhibited significantly smaller seed width, seed length, and reduced grain weight, with impaired seed germination and retarded early seedling growth, suggesting that disturbing the normal steady state of KRP1 or KRP2 blocks seed development partly through inhibiting cell proliferation and enlargement during grain filling and seed germination. Furthermore, two cyclin-dependent protein kinases, CDKC;2 and CDKF;3, could interact with KRP1 in a yeast-two-hybrid system, indicating that KRP1 might regulate the mitosis cell cycle and endoreduplication through the two targets. In a word, this study shed novel insights into the regulatory roles of KRPs in rice seed maturation and germination.

Exogenous abscisic acid represses rice flowering via SAPK8-ABF1-Ehd1/Ehd2 pathway.

INTRODUCTION: Rice flowering is a major agronomic trait, determining yield and ecological adaptability in particular regions. ABA plays an essential role in rice flowering, but the underlying molecular mechanism remains largely elusive. OBJECTIVES: In this study, we demonstrated a "SAPK8-ABF1-Ehd1/Ehd2" pathway, through which exogenous ABA represses rice flowering in a photoperiod-independent manner. METHODS: We generated abf1 and sapk8 mutants using the CRISPR-Cas9 method. Using yeast two-hybrid, Pull down, BiFC and kinase assays, SAPK8 interacted and phosphorylated ABF1. ABF1 directly bound to the promoters of Ehd1 and Ehd2 using ChIP-qPCR, EMSA, and LUC transient transcriptional activity assay, and suppressed the transcription of these genes. RESULTS: Under both long day and short day conditions, simultaneous knock-out of ABF1 and its homolog bZIP40 accelerated flowering, while SAPK8 and ABF1 over-expression lines exhibited delayed flowering and hypersensitivity to ABA-mediated flowering repression. After perceiving the ABA signal, SAPK8 physically binds to and phosphorylates ABF1 to enhance its binding to the promoters of master positive flowering regulators Ehd1 and Ehd2. Upon interacting with FIE2, ABF1 recruited PRC2 complex to deposit H3K27me3 suppressive histone modification on Ehd1 and Ehd2 to suppress these genes transcription, thereby leading to later flowering. CONCLUSION: Our work highlighted the biological functions of SAPK8 and ABF1 in ABA signaling, flowering control and the involvement of a PRC2-mediated epigenetic repression mechanism in the transcription regulation governed by ABF1 on ABA-mediated rice flowering repression.

The OsNAC23-Tre6P-SnRK1a feed-forward loop regulates sugar homeostasis and grain yield in rice.

Tre6P (trehalose-6-phosphate) mediates sensing of carbon availability to maintain sugar homeostasis in plants, which underpins crop yield and resilience. However, how Tre6P responds to fluctuations in sugar levels and regulates the utilization of sugars for growth remains to be addressed. Here, we report that the sugar-inducible rice NAC transcription factor OsNAC23 directly represses the transcription of the Tre6P phosphatase gene TPP1 to simultaneously elevate Tre6P and repress trehalose levels, thus facilitating carbon partitioning from source to sink organs. Meanwhile, OsNAC23 and Tre6P suppress the transcription and enzyme activity of SnRK1a, a low-carbon sensor and antagonist of OsNAC23, to prevent the SnRK1a-mediated phosphorylation and degradation of OsNAC23. Thus, OsNAC23, Tre6P, and SnRK1a form a feed-forward loop to sense sugar and maintain sugar homeostasis by transporting sugars to sink organs. Importantly, plants over-expressing OsNAC23 exhibited an elevated photosynthetic rate, sugar transport, and sink organ size, which consistently increased rice yields by 13%-17% in three elite-variety backgrounds and two locations, suggesting that manipulation of OsNAC23 expression has great potential for rice improvement. Collectively, these findings enhance our understanding of Tre6P-mediated sugar signaling and homeostasis, and provide a new strategy for genetic improvement of rice and possibly also other crops.

MoWhi2 regulates appressorium formation and pathogenicity via the MoTor signalling pathway in Magnaporthe oryzae.

Magnaporthe oryzae causes rice blast disease, which seriously threatens the safety of food production. Understanding the mechanism of appressorium formation, which is one of the key steps for successful infection by M. oryzae, is helpful to formulate effective control strategies of rice blast. In this study, we identified MoWhi2, the homolog of Saccharomyces cerevisiae Whi2 (Whisky2), as an important regulator that controls appressorium formation in M. oryzae. When MoWHI2 was disrupted, multiple appressoria were formed by one conidium and pathogenicity was significantly reduced. A putative phosphatase, MoPsr1, was identified to interact with MoWhi2 using a yeast two-hybridization screening assay. The knockout mutant ΔMopsr1 displayed similar phenotypes to the ΔMowhi2 strain. Both the ΔMowhi2 and ΔMopsr1 mutants could form appressoria on a hydrophilic surface with cAMP levels increasing in comparison with the wild type (WT). The conidia of ΔMowhi2 and ΔMopsr1 formed a single appressorium per conidium, similar to WT, when the target of rapamycin (TOR) inhibitor rapamycin was present. In addition, compared with WT, the expression levels of MoTOR and the MoTor signalling activation marker gene MoRS3 were increased, suggesting that inappropriate activation of the MoTor signalling pathway is one of the important reasons for the defects in appressorium formation in the ΔMowhi2 and ΔMopsr1 strains. Our results provide insights into MoWhi2 and MoPsr1-mediated appressorium development and pathogenicity by regulating cAMP levels and the activation of MoTor signalling in M. oryzae.

Genome-Wide Identification of lncRNAs During Rice Seed Development.

Rice seed is a pivotal reproductive organ that directly determines yield and quality. Long non-coding RNAs (lncRNAs) have been recognized as key regulators in plant development, but the roles of lncRNAs in rice seed development remain unclear. In this study, we performed a paired-end RNA sequencing in samples of rice pistils and seeds at three and seven days after pollination (DAP) respectively. A total of 540 lncRNAs were obtained, among which 482 lncRNAs had significantly different expression patterns during seed development. Results from semi-qPCR conducted on 15 randomly selected differentially expressed lncRNAs suggested high reliability of the transcriptomic data. RNA interference of TCONS_00023703, which is predominantly transcribed in developing seeds, significantly reduced grain length and thousand-grain weight. These results expanded the dataset of lncRNA in rice and enhanced our understanding of the biological functions of lncRNAs in rice seed development.

Mitochondrial dynamics and mitophagy are necessary for proper invasive growth in rice blast.

Magnaporthe oryzae causes blast disease, which is one of the most devastating infections in rice and several important cereal crops. Magnaporthe oryzae needs to coordinate gene regulation, morphological changes, nutrient acquisition and host evasion in order to invade and proliferate within the plant tissues. Thus far, the molecular mechanisms underlying the regulation of invasive growth in planta have remained largely unknown. We identified a precise filamentous-punctate-filamentous cycle in mitochondrial morphology during Magnaporthe-rice interaction. Interestingly, disruption of such mitochondrial dynamics by deletion of genes regulating either the mitochondrial fusion (MoFzo1) or fission (MoDnm1) machinery, or inhibition of mitochondrial fission using Mdivi-1 caused significant reduction in M. oryzae pathogenicity. Furthermore, exogenous carbon source(s) but not antioxidant treatment delayed such mitochondrial dynamics/transition during invasive growth. In contrast, carbon starvation induced the breakdown of the mitochondrial network and led to more punctate mitochondria in vitro. Such nutrient-based regulation of organellar dynamics preceded MoAtg24-mediated mitophagy, which was found to be essential for proper biotrophic development and invasive growth in planta. We propose that precise mitochondrial dynamics and mitophagy occur during the transition from biotrophy to necrotrophy and are required for proper induction and establishment of the blast disease in rice.