A transthyretin-like protein acts downstream of miR397 and LACCASE to regulate grain yield in rice.

Increasing grain yield is a major goal of breeders due to the rising global demand for food. We previously reported that the miR397-LACCASE (OsLAC) module regulates brassinosteroid (BR) signaling and grain yield in rice (Oryza sativa). However, the precise roles of laccase enzymes in the BR pathway remain unclear. Here, we report that OsLAC controls grain yield by preventing the turnover of TRANSTHYRETIN-LIKE (OsTTL), a negative regulator of BR signaling. Overexpressing OsTTL decreased BR sensitivity in rice, while loss-of-function of OsTTL led to enhanced BR signaling and increased grain yield. OsLAC directly binds to OsTTL and regulates its phosphorylation-mediated turnover. The phosphorylation site Ser226 of OsTTL is essential for its ubiquitination and degradation. Overexpressing the dephosphorylation-mimic form of OsTTL (OsTTLS226A) resulted in more severe defects than did overexpressing OsTTL. These findings provide insight into the role of an ancient laccase in BR signaling and suggest that the OsLAC-OsTTL module could serve as a target for improving grain yield.

MicroRNA397 promotes rice flowering by regulating the photorespiration pathway.

The precise timing of flowering plays a pivotal role in ensuring successful plant reproduction and seed production. This process is intricately governed by complex genetic networks that integrate internal and external signals. This study delved into the regulatory function of microRNA397 (miR397) and its target gene LACCASE-15 (OsLAC15) in modulating flowering traits in rice (Oryza sativa). Overexpression of miR397 led to earlier heading dates, decreased number of leaves on the main stem, and accelerated differentiation of the spikelet meristem. Conversely, overexpression of OsLAC15 resulted in delayed flowering and prolonged vegetative growth. Through biochemical and physiological assays, we uncovered that miR397-OsLAC15 had a profound impact on carbohydrate accumulation and photosynthetic assimilation, consequently enhancing the photosynthetic intensity in miR397-overexpressing rice plants. Notably, we identified that OsLAC15 is at least partially localized within the peroxisome organelle, where it regulates the photorespiration pathway. Moreover, we observed that a high CO2 concentration could rescue the late flowering phenotype in OsLAC15-overexpressing plants. These findings shed valuable insights into the regulatory mechanisms of miR397-OsLAC15 in rice flowering and provided potential strategies for developing crop varieties with early flowering and high-yield traits through genetic breeding.

Ubiquitin-dependent Argonauteprotein MEL1 degradation is essential for rice sporogenesis and phasiRNA target regulation.

MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.

OsmiR528 regulates rice-pollen intine formation by targeting an uclacyanin to influence flavonoid metabolism.

The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. The mir528 knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin gene OsUCL23 (encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition. OsUCL23 overexpression phenocopied the mir528 mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targeting OsUCL23 and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.

A Natural Variant of miR397 Mediates a Feedback Loop in Circadian Rhythm.

MicroRNAs (miRNAs) are small noncoding RNAs of ∼21 nt in length, which have regulatory roles in many biological processes. In animals, proper functioning of the circadian clock, which is closely linked to the fitness of almost all living organisms, is regulated by miRNAs. However, to date, there have been no reports of the roles of miRNA in regulation of the plant circadian rhythm. Here, we report a natural variant of miR397 that lengthens the circadian period and controls flowering time in Arabidopsis (Arabidopsis thaliana). Highly conserved among angiosperms, the miRNA miR397 has two members in Arabidopsis: miR397a and miR397b. However, only miR397b significantly delayed flowering. Our results suggest that miR397b controls flowering by targeting CASEIN KINASE II SUBUNIT BETA3 (CKB3), in turn modulating the circadian period of CIRCADIAN CLOCK ASSOCIATED1 (CCA1). We further demonstrated that CCA1 directly bound to the promoter of MIR397B and suppressed its expression, forming a miR397b-CKB3-CCA1 circadian regulation feedback circuit. Evolutionary analysis revealed that miR397b is a newly evolved genetic variant in Arabidopsis, and the miR397b targeting mode may have a role in enhancing plant fitness. Our results provide evidence for miRNA-mediated circadian regulation in plants and suggest the existence of a feedback loop to manipulate plant flowering through the regulation of circadian rhythm.

Genome-Wide Analysis Identified a Set of Conserved lncRNAs Associated with Domestication-Related Traits in Rice.

Crop domestication, which gives rise to a number of desirable agronomic traits, represents a typical model system of plant evolution. Numerous genomic evidence has proven that noncoding RNAs such as microRNAs and phasiRNAs, as well as protein-coding genes, are selected during crop domestication. However, limited data shows plant long noncoding RNAs (lncRNAs) are also involved in this biological process. In this study, we performed strand-specific RNA sequencing of cultivated rice Oryza sativa ssp. japonica and O. sativa ssp. indica, and their wild progenitor O. rufipogon. We identified a total of 8528 lncRNAs, including 4072 lncRNAs in O. rufipogon, 2091 lncRNAs in japonica rice, and 2365 lncRNAs in indica rice. The lncRNAs expressed in wild rice were revealed to be shorter in length and had fewer exon numbers when compared with lncRNAs from cultivated rice. We also identified a number of conserved lncRNAs in the wild and cultivated rice. The functional study demonstrated that several of these conserved lncRNAs are associated with domestication-related traits in rice. Our findings revealed the feature and conservation of lncRNAs during rice domestication and will further promote functional studies of lncRNAs in rice.

Transcriptional landscape of pathogen-responsive lncRNAs in rice unveils the role of ALEX1 in jasmonate pathway and disease resistance.

Plant defence is multilayered and is essential for surviving in a changing environment. The discovery of long noncoding RNAs (lncRNAs) has dramatically extended our understanding of post-transcriptional gene regulation in diverse biological processes. However, the expression profile and function of lncRNAs in disease resistance are still largely unknown, especially in monocots. Here, we performed strand-specific RNA sequencing of rice leaves infected by Xanthomonas oryzae pv. Oryzae (Xoo) in different time courses and systematically identified 567 disease-responsive rice lncRNAs. Target analyses of these lncRNAs showed that jasmonate (JA) pathway was significantly enriched. To reveal the interaction between lncRNAs and JA-related genes, we studied the coexpression of them and found 39 JA-related protein-coding genes to be interplayed with 73 lncRNAs, highlighting the potential modulation of lncRNAs in JA pathway. We subsequently identified an lncRNA, ALEX1, whose expression is highly induced by Xoo infection. A T-DNA insertion line constructed using enhancer trap system showed a higher expression of ALEX1 and exerted a significant resistance to rice bacterial blight. Functional study revealed that JA signalling is activated and the endogenous content of JA and JA-Ile is increased. Overexpressing ALEX1 in rice further confirmed the activation of JA pathway and resistance to bacterial blight. Our findings reveal the expression of pathogen-responsive lncRNAs in rice and provide novel insights into the connection between lncRNAs and JA pathway in the regulation of plant disease resistance.

Genome-wide analysis and functional annotation of chromatin-enriched noncoding RNAs in rice during somatic cell regeneration.

BACKGROUND: Plants have the remarkable ability to generate callus, a pluripotent cell mass that acquires competence for subsequent tissue regeneration. Global chromatin remodeling is required for this cell fate transition, but how the process is regulated is not fully understood. Chromatin-enriched noncoding RNAs (cheRNAs) are thought to play important roles in maintaining chromatin state. However, whether cheRNAs participate in somatic cell regeneration in plants has not yet been clarified. RESULTS: To uncover the characteristics and functions of cheRNAs during somatic cell reprogramming in plants, we systematically investigate cheRNAs during callus induction, proliferation and regeneration in rice. We identify 2284 cheRNAs, most of which are novel long non-coding RNAs or small nucleolar RNAs. These cheRNAs, which are highly conserved across plant species, shuttle between chromatin and the nucleoplasm during somatic cell regeneration. They positively regulate the expression of neighboring genes via specific RNA motifs, which may interact with DNA motifs around cheRNA loci. Large-scale mutant analysis shows that cheRNAs are associated with plant size and seed morphology. Further detailed functional investigation of two che-lncRNAs demonstrates that their loss of function impairs cell dedifferentiation and plant regeneration, highlighting the functions of cheRNAs in regulating the expression of neighboring genes via specific motifs. These findings support cis- regulatory roles of cheRNAs in influencing a variety of rice traits. CONCLUSIONS: cheRNAs are a distinct subclass of regulatory non-coding RNAs that are required for somatic cell regeneration and regulate rice traits. Targeting cheRNAs has great potential for crop trait improvement and breeding in future.

The parent-of-origin lncRNA MISSEN regulates rice endosperm development.

The cereal endosperm is a major factor determining seed size and shape. However, the molecular mechanisms of endosperm development are not fully understood. Long noncoding RNAs (lncRNAs) function in various biological processes. Here we show a lncRNA, MISSEN, that plays an essential role in early endosperm development in rice (Oryza sativa). MISSEN is a parent-of-origin lncRNA expressed in endosperm, and negatively regulates endosperm development, leading to a prominent dent and bulge in the seed. Mechanistically, MISSEN functions through hijacking a helicase family protein (HeFP) to regulate tubulin function during endosperm nucleus division and endosperm cellularization, resulting in abnormal cytoskeletal polymerization. Finally, we revealed that the expression of MISSEN is inhibited by histone H3 lysine 27 trimethylation (H3K27me3) modification after pollination. Therefore, MISSEN is the first lncRNA identified as a regulator in endosperm development, highlighting the potential applications in rice breeding.

Reproductive phasiRNAs regulate reprogramming of gene expression and meiotic progression in rice.

Plant spermatogenesis is a complex process that directly affects crop breeding. A rapid change in gene abundance occurs at early meiosis prophase, when gene regulation is selective. However, how these genes are regulated remains unknown. Here, we show that rice reproductive phasiRNAs are essential for the elimination of a specific set of RNAs during meiotic prophase I. These phasiRNAs cleave target mRNAs in a regulatory manner such that one phasiRNA can target more than one gene, and/or a single gene can be targeted by more than one phasiRNA to efficiently silence target genes. Our investigation of phasiRNA-knockdown and PHAS-edited transgenic plants demonstrates that phasiRNAs and their nucleotide variations are required for meiosis progression and fertility. This study highlights the importance of reproductive phasiRNAs for the reprogramming of gene expression during meiotic progression and establishes a basis for future studies on the roles of phasiRNAs with a goal of crop improvement.

[Mechanism of DATS Inhibiting Side Population Cell Proliferation in Multiple Myeloma].

OBJECTIVE: To study the effect of diallyl thiosulfinate (DATS) on the proliferation of side population (SP) cells in multiple myeloma (MM) and its mechanism. METHODS: RPMI-8226 and NCI-H929 cells were cultured, and the level of SP cells was detected by Hoechst33342 staining. The SP cells were cultured and treated with 10 µg/ml DATS, the CCK8 assay was carried out to examine the effect of DATS on the proliferation ability in SP cells, and plate colony-forming test was used to examine the colony-forming ability, the flow cytometry assay was carried out to examine the cell cycle, Western blot assay was used to examine the expression of cyclin D1, cyclin E, CDK2 and CDK4. RESULTS: SP cells were detected in RPMI-8226 and NCI-H929 cells with a proportion of 3.17±0.98 and 2.65±0.61, respectively. DATS treatment could significantly inhibit the SP cells survival in a time-dependent manner, and also could significantly inhibit the colony forming. In addition, DATS treatment could significantly induce the G1/S arrest and suppress the expression of cyclin D1, cyclin E, CDK2 and CDK4. CONCLUSION: DATS can inhibit the proliferation and colony-forming of SP cells in multiple myeloma, and induce the G1/S arrest that may be carried out via suppressing the expression of cyclin D1, cyclin E, CDK2 and CDK4.