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.

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.

Sterols and triterpenoids from Ganoderma lucidum and their reversal activities of tumor multidrug resistance.

Two sterols and seven triterpenoids were isolated and identified from Ganoderma lucidum by silica gel column chromatography, preparative high-performance liquid chromatography and spectra analysis. Then, the multidrug resistance reversal activities of these compounds were assessed using MTT assay. Among these compounds, ganoderol B (3), ganoderone A (4), ganodermanondiol (6) and ganoderiol F (8) were shown to reverse the resistance of human oral epidermoid carcinoma cell line KBv200 to doxorubicin, and the reversal folds were 6.59, 4.70, 4.01 and 7.09, respectively. Ganoderiol F could increase the intracellular accumulation of doxorubicin in KBv200 cells through inhibiting P-glycoprotein transport function. Further mechanistic investigation found that ganoderiol F did not alter P-glycoprotein expression. In conclusion, ganoderiol F has potent effect in reversing P-glycoprotein mediated tumor multidrug resistance. Potential reversal agents against multidrug resistance in tumor may be found in triterpenoids from Ganoderma lucidum.[Formula: see text].

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.

Identification and Characterization of Plant Circular RNAs.

Recent studies have reported that circular RNAs (circRNAs) are a newly discovered type of ubiquitous, abundant and stable noncoding RNAs (ncRNAs) that play important roles in various biological processes in eukaryotic organisms. However, the biological functions of circRNAs in plants remain largely unknown and need further studies. Identification of plant circRNAs from plant circRNA database or sequencing analysis is a first step to investigate their functions. Here, we provide a series of protocols for circRNA identification including circular forms, composition features and location even in plant tissues which are rich in polysaccharides and polyphenols and difficult to extract RNAs.

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.

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.

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.

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.

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.

circMYBL2, a circRNA from MYBL2, regulates FLT3 translation by recruiting PTBP1 to promote FLT3-ITD AML progression.

Internal tandem duplication (ITD) mutations within FMS-like tyrosine kinase-3 (FLT3) occur in up to 30% of acute myeloid leukemia (AML) patients and confer a very poor prognosis. The oncogenic form of FLT3 is an important therapeutic target, and inhibitors specifically targeting FLT3 kinase can induce complete remission; however, relapse after remission has been observed due to acquired resistance with secondary mutations in FLT3, highlighting the need for new strategies to target FLT3-ITD mutations. Recent studies have reported that the aberrant formations of circular RNAs (circRNAs) are biological tumorigenesis-relevant mechanisms and potential therapeutic targets. Herein, we discovered a circRNA, circMYBL2, derived from the cell-cycle checkpoint gene MYBL2. circMYBL2 is more highly expressed in AML patients with FLT3-ITD mutations than in those without the FLT3-ITD mutation. We found that circMYBL2 knockdown specifically inhibits proliferation and promotes the differentiation of FLT3-ITD AML cells in vitro and in vivo. Interestingly, we found that circMYBL2 significantly influences the protein level of mutant FLT3 kinase, which contributes to the activation of FLT3-ITD-dependent signaling pathways. Mechanistically, circMYBL2 enhanced the translational efficiency of FLT3 kinase by increasing the binding of polypyrimidine tract-binding protein 1 (PTBP1) to FLT3 messenger RNA. Moreover, circMYBL2 knockdown impaired the cytoactivity of inhibitor-resistant FLT3-ITD+ cells, with a significant decrease in FLT3 kinase expression, followed by the inactivation of its downstream pathways. In summary, we are the first to reveal a circRNA that specifically influences FLT3-ITD AML and regulates FLT3 kinase levels through translational regulation, suggesting that circMYBL2 may be a potential therapeutic target for FLT3-ITD AML.

The subunit of RNA N6-methyladenosine methyltransferase OsFIP regulates early degeneration of microspores in rice.

N6-Methyladenosine (m6A) RNA methylation plays important roles during development in different species. However, knowledge of m6A RNA methylation in monocots remains limited. In this study, we reported that OsFIP and OsMTA2 are the components of m6A RNA methyltransferase complex in rice and uncovered a previously unknown function of m6A RNA methylation in regulation of plant sporogenesis. Importantly, OsFIP is essential for rice male gametogenesis. Knocking out of OsFIP results in early degeneration of microspores at the vacuolated pollen stage and simultaneously causes abnormal meiosis in prophase I. We further analyzed the profile of rice m6A modification during sporogenesis in both WT and OsFIP loss-of-function plants, and identified a rice panicle specific m6A modification motif "UGWAMH". Interestingly, we found that OsFIP directly mediates the m6A methylation of a set of threonine protease and NTPase mRNAs and is essential for their expression and/or splicing, which in turn regulates the progress of sporogenesis. Our findings revealed for the first time that OsFIP plays an indispensable role in plant early sporogenesis. This study also provides evidence for the different functions of the m6A RNA methyltransferase complex between rice and Arabidopsis.

[Protective effects and action mechanism of extract from Tibetan medicine Yajima(Chrysosplenium nudicaule) on mice with intrahepatic cholestasis induced by ANIT].

Chrysosplenium nudicaule,Tibetan name " Yajima",is recorded as an effective medicine for the treatment of liver and gallbladder diseases by Tibetan Pharmacopoeia published in the past dynasties,but its traditional efficacy has not yet been investigated by means of modern pharmacological research methods. In this paper,the protective effect of extract of C. nudicaule(ECN) on liver injury in mice was observed by using the mice model of intrahepatic cholestasis(IC) induced by α-naphthyl isothiocyanate(ANIT) and the possible mechanism by which ECN work as the therapeutic agent was discussed. The results showed that the serum levels of AST,ALT,ALP,DBIL,TBIL and TBA of the model mice were notably reduced in dose-dependent manner(P<0. 01,P<0. 05). The activity of SOD and GSH-Px in the liver homogenate of mice was increased,while the content of MDA was decreased(P<0. 01,P<0. 05).Pathological examination of liver in mice showed that ECN could improve the pathological changes of liver tissue in mice. The mRNA expression level of genes related to bile acid metabolism were detected by RT-PCR and the results suggested that ECN could significantly increase the expression of genes such as BSEP,FXR and MRP2(P<0. 01,P<0. 05),meanwhile significantly reduce the expression of CYP7 A1(P<0. 01,P<0. 05). These results confirmed the protective effect of ECN on intrahepatic cholestasis-induced liver injury in mice,and indicated that the mechanism may be related to activating FXR and its target genes,reducing bile acid synthesis and increasing bile acid excretion. This study provides a modern pharmacological basis for the clinical application of Yajima in Tibetan medicine.

Rice UCL8, a plantacyanin gene targeted by miR408, regulates fertility by controlling pollen tube germination and growth.

BACKGROUND: Pollen tube formation and growth are crucial steps that lead to seed production. Despite the importance of pollen tube growth, the molecular mechanisms implicated in its spatial and temporal control are not fully known. In this study, we found an uclacyanin gene, OsUCL8, that regulates pollen intine deposition and pollen tube growth. FINDINGS: The overexpression of OsUCL8 led to a striking irregularity in pollen tube growth and pollination and thus affected the seed setting rate in rice; many pollen tubes appeared to lose the ability to grow directly into the style. Conversely, plants with OsUCL8 knocked out and plants overexpressing miR408, a negative regulator of OsUCL8, had vigorous pollens with a higher germination rate. We further demonstrated that OsUCL8 mainly affects pollen intine formation. The addition of Vitamin B1 (VB1) significantly contributed to the germination of OXUCL8 pollen grains, suggesting that OsUCL8 could be associated with VB1 production. Using a yeast two-hybrid system, we revealed that OsUCL8 interacts with the protein OsPKIWI, a homolog of the Arabidopsis FNRL protein. We thus hypothesized that OsUCL8 might regulate the production of VB components by interacting with OsPKIWI. This study revealed a novel molecular mechanism of pollen tube growth regulation. CONCLUSIONS: The rice plantacyanin family member OsUCL8 plays an important role in pollen tube formation and growth and, in turn, regulates fertility and the seed setting rate.

Mitofusin-2 mediated mitochondrial Ca2+ uptake 1/2 induced liver injury in rat remote ischemic perconditioning liver transplantation and alpha mouse liver-12 hypoxia cell line models.

AIM: To investigate the protective mechanism of mitofusin-2 (Mfn2) in rat remote ischemic perconditioning (RIC) models and revalidate it in alpha mouse liver-12 (AML-12) hypoxia cell lines. METHODS: Sprague-Dawley rats were divided into three groups (n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting (WB) and quantitative real-time (qRT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture (hypoxia) and anoxic incubator tank culture with Mfn2 knockdown (hypoxia + Si), and data of qRT-PCR, WB, mitochondrial membrane potential (ΔΨm), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected. RESULTS: Both sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group (P < 0.05). qRT-PCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2 (MICUs) axis was changed (P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of ΔΨm and apoptosis (P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group (P < 0.005). Finally, qRT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups (P < 0.005). CONCLUSION: Mfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.

MiR408 Regulates Grain Yield and Photosynthesis via a Phytocyanin Protein.

Increasing grain yield is the most important object of crop breeding. Here, we report that the elevated expression of a conserved microRNA, OsmiR408, could positively regulate grain yield in rice (Oryza sativa) by increasing panicle branches and grain number. We further showed that OsmiR408 regulates grain yield by down-regulating its downstream target, OsUCL8, which is an uclacyanin (UCL) gene of the phytocyanin family. The knock down or knock out of OsUCL8 also increases grain yield, while the overexpression of OsUCL8 results in an opposite phenotype. Spatial and temporal expression analyses showed that OsUCL8 was highly expressed in pistils, young panicles, developing seeds, and inflorescence meristem and was nearly complementary to that of OsmiR408. Interestingly, the OsUCL8 protein was localized to the cytoplasm, distinct from a majority of phytocyanins, which localize to the plasma membrane. Further studies revealed that the cleavage of OsUCL8 by miR408 affects copper homeostasis in the plant cell, which, in turn, affects the abundance of plastocyanin proteins and photosynthesis in rice. To our knowledge, this is the first report of the effects of miR408-OsUCL8 in regulating rice photosynthesis and grain yield. Our study further broadens the perspective of microRNAs and UCLs and provides important information for breeding high-yielding crops through genetic engineering.

Laccase-13 Regulates Seed Setting Rate by Affecting Hydrogen Peroxide Dynamics and Mitochondrial Integrity in Rice.

Seed setting rate is one of the most important components of rice grain yield. To date, only several genes regulating setting rate have been identified in plant. In this study, we showed that laccase-13 (OsLAC13), a member of laccase family genes which are known for their roles in modulating phenylpropanoid pathway and secondary lignification in cell wall, exerts a regulatory function in rice seed setting rate. OsLAC13 expressed in anthers and promotes hydrogen peroxide production both in vitro and in the filaments and anther connectives. Knock-out of OsLAC13 showed significantly increased seed setting rate, while overexpression of this gene exhibited induced mitochondrial damage and suppressed sugar transportation in anthers, which in turn affected seed setting rate. OsLAC13 also induced H2O2 production and mitochondrial damage in the root tip cells which caused the lethal phenotype. We also showed that high abundant of OsmiR397, the suppressor of OsLAC13 mRNA, increased the seed setting rate of rice plants, and restrains H2O2 accumulation in roots during oxidative stress. Our results suggested a novel regulatory role of OsLAC13 gene in regulating seed setting rate by affecting H2O2 dynamics and mitochondrial integrity in rice.