Genetic architecture of seed glycerolipids in Asian cultivated rice.

Glycerolipids are essential for rice development and grain quality but its genetic regulation remains unknown. Here we report its genetic base using metabolite-based genome-wide association study and metabolite-based quantitative traits locus (QTL) analyses based on lipidomic profiles of seeds from 587 Asian cultivated rice accessions and 103 chromosomal segment substitution lines, respectively. We found that two genes encoding phosphatidylcholine (PC):diacylglycerol cholinephosphotransferase (OsLP1) and granule-bound starch synthase I (Waxy) contribute to variations in saturated triacylglycerol (TAG) and lyso-PC contents, respectively. We demonstrated that allelic variation in OsLP1 sequence between indica and japonica results in different enzymatic preference for substrate PC-16:0/16:0 and different saturated TAG levels. Further evidence demonstrated that OsLP1 also affects heading date, and that co-selection of OsLP1 and a flooding-tolerant QTL in Aus results in the abundance of saturated TAGs associated with flooding tolerance. Moreover, we revealed that the sequence polymorphisms in Waxy has pleiotropic effects on lyso-PC and amylose content. We proposed that rice seed glycerolipids have been unintentionally shaped during natural and artificial selection for adaptive or import seed quality traits. Collectively, our findings provide valuable genetic resources for rice improvement and evolutionary insights into seed glycerolipid variations in rice.

Ectopic expression of OsJAZ6, which interacts with OsJAZ1, alters JA signaling and spikelet development in rice.

Jasmonates (JAs) are key phytohormones that regulate plant responses and development. JASMONATE-ZIM DOMAIN (JAZ) proteins safeguard JA signaling by repressing JA-responsive gene expression in the absence of JA. However, the interaction and cooperative roles of JAZ repressors remain unclear during plant development. Here, we found that OsJAZ6 interacts with OsJAZ1 depending on a single amino acid in the so-called ZIM domain of OsJAZ6 in rice JA signaling transduction and JA-regulated rice spikelet development. In vivo protein distribution analysis revealed that the OsJAZ6 content is efficiently regulated during spikelet development, and biochemical and genetic evidence showed that OsJAZ6 is more sensitive to JA-mediated degradation than OsJAZ1. Through over- and mis-expression experiments, we further showed that the protein stability and levels of OsJAZ6 orchestrate the output of JA signaling during rice spikelet development. A possible mechanism, which outlines how OsJAZ repressors interact and function synergistically in specifying JA signaling output through degradation titration, is also discussed.

SMALL REPRODUCTIVE ORGANS, a SUPERMAN-like transcription factor, regulates stamen and pistil growth in rice.

Organ size is determined mainly by cell division and cell expansion. Several genetic factors regulating development of plant lateral organs have been characterized, but those involved in determining reproductive organ size and separation in rice (Oryza sativa) remain unknown. We have isolated the rice gene SMALL REPRODUCTIVE ORGANS (SRO) encoding a nucleus-localized Cys2His2 (C2 H2 ) zinc finger protein orthologous to Arabidopsis transcription factor (TF) SUPERMAN (SUP). Combined developmental, genetic, histological and transcriptomic analyses were used to determine the function of SRO in regulating reproductive organ size. SRO affects genes involved in cell division, cell expansion and phytohormone signalling in the rice flower. SRO is specifically expressed in the first stages of stamen filament development to regulate their correct formation and separation. In addition, SRO noncell-autonomously regulates the size and functionality of male and female reproductive organs. The B-class MADS-box gene OsMADS16/SPW1 is epistatic to SRO, whereas SRO regulates reproductive organ specification and floral meristem determinacy synergistically with C-class genes OsMADS3 and OsMADS58. These findings provide insights into how an evolutionarily conserved TF has a pivotal role in reproductive organ development in core eudicots and monocots, through partially conserved expression, function and regulatory network.

Gibberellins orchestrate panicle architecture mediated by DELLA-KNOX signalling in rice.

Panicle architecture is a key determinant of grain yield in cereals, but the mechanisms governing panicle morphogenesis and organ development remain elusive. Here, we have identified a quantitative trait locus (qPA1) associated with panicle architecture using chromosome segment substitution lines from parents Nipponbare and 9311. The panicle length, branch number and grain number of Nipponbare were significantly higher than CSSL-9. Through map-based cloning and complementation tests, we confirmed that qPA1 was identical to SD1 (Semi Dwarf1), which encodes a gibberellin 20-oxidase enzyme participating in gibberellic acid (GA) biosynthesis. Transcript analysis revealed that SD1 was widely expressed during early panicle development. Analysis of sd1/osga20ox2 and gnp1/ osga20ox1 single and double mutants revealed that the two paralogous enzymes have non-redundant functions during panicle development, likely due to differences in spatiotemporal expression; GNP1 expression under control of the SD1 promoter could rescue the sd1 phenotype. The DELLA protein SLR1, a component of the GA signalling pathway, accumulated more highly in sd1 plants. We have demonstrated that SLR1 physically interacts with the meristem identity class I KNOTTED1-LIKE HOMEOBOX (KNOX) protein OSH1 to repress OSH1-mediated activation of downstream genes related to panicle development, providing a mechanistic link between gibberellin and panicle architecture morphogenesis.

PERSISTENT TAPETAL CELL2 Is Required for Normal Tapetal Programmed Cell Death and Pollen Wall Patterning.

The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. The mutant ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula, and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, Transposable Element Silencing Via AT-Hook, in Arabidopsis (Arabidopsis thaliana). Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches.

Rice transcription factor MADS32 regulates floral patterning through interactions with multiple floral homeotic genes.

Floral patterning is regulated by intricate networks of floral identity genes. The peculiar MADS32 subfamily genes, absent in eudicots but prevalent in monocots, control floral organ identity. However, how the MADS32 family genes interact with other floral homeotic genes during flower development is mostly unknown. We show here that the rice homeotic transcription factor OsMADS32 regulates floral patterning by interacting synergistically with E class protein OsMADS6 in a dosage-dependent manner. Furthermore, our results indicate important roles for OsMADS32 in defining stamen, pistil, and ovule development through physical and genetic interactions with OsMADS1, OsMADS58, and OsMADS13, and in specifying floral meristem identity with OsMADS6, OsMADS3, and OsMADS58, respectively. Our findings suggest that OsMADS32 is an important factor for floral meristem identity maintenance and that it integrates the action of other MADS-box homeotic proteins to sustain floral organ specification and development in rice. Given that OsMADS32 is an orphan gene and absent in eudicots, our data substantially expand our understanding of flower development in plants.

Two rice receptor-like kinases maintain male fertility under changing temperatures.

Plants employ dynamic molecular networks to control development in response to environmental changes, yet the underlying mechanisms are largely unknown. Here we report the identification of two rice leucine-rich repeat receptor-like kinases, Thermo-Sensitive Genic Male Sterile 10 (TMS10) and its close homolog TMS10-Like (TMS10L), which redundantly function in the maintenance of the tapetal cell layer and microspore/pollen viability under normal temperature conditions with TMS10 playing an essential role in higher temperatures (namely, 28 °C). tms10 displays male sterility under high temperatures but male fertility under low temperatures, and the tms10 tms10l double mutant shows complete male sterility under both high and low temperatures. Biochemical and genetic assays indicate that the kinase activity conferred by the intracellular domain of TMS10 is essential for tapetal degeneration and male fertility under high temperatures. Furthermore, indica or japonica rice varieties that contain mutations in TMS10, created by genetic crosses or genome editing, also exhibit thermo-sensitive genic male sterility. These findings demonstrate that TMS10 and TMS10L act as a key switch in postmeiotic tapetal development and pollen development by buffering environmental temperature changes, providing insights into the molecular mechanisms by which plants develop phenotypic plasticity via genotype-environment temperature interaction. TMS10 may be used as a genetic resource for the development of hybrid seed production systems in crops.

A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation.

In flowering plants, successful male reproduction requires the sophisticated interaction between somatic anther wall layers and reproductive cells. Timely degradation of the innermost tissue of the anther wall layer, the tapetal layer, is critical for pollen development. Ca2+ is a well-known stimulus for plant development, but whether it plays a role in affecting male reproduction remains elusive. Here we report a role of Defective in Exine Formation 1 (OsDEX1) in rice (Oryza sativa), a Ca2+ binding protein, in regulating rice tapetal cell degradation and pollen formation. In osdex1 anthers, tapetal cell degeneration is delayed and degradation of the callose wall surrounding the microspores is compromised, leading to aborted pollen formation and complete male sterility. OsDEX1 is expressed in tapetal cells and microspores during early anther development. Recombinant OsDEX1 is able to bind Ca2+ and regulate Ca2+ homeostasis in vitro, and osdex1 exhibited disturbed Ca2+ homeostasis in tapetal cells. Phylogenetic analysis suggested that OsDEX1 may have a conserved function in binding Ca2+ in flowering plants, and genetic complementation of pollen wall defects of an Arabidopsis (Arabidopsis thaliana) dex1 mutant confirmed its evolutionary conservation in pollen development. Collectively, these findings suggest that OsDEX1 plays a fundamental role in the development of tapetal cells and pollen formation, possibly via modulating the Ca2+ homeostasis during pollen development.

Interactions between FLORAL ORGAN NUMBER4 and floral homeotic genes in regulating rice flower development.

The floral meristem (FM) is self-maintaining at the early stages of flower development, but it is terminated when a fixed number of floral organs are produced. The FLORAL ORGAN NUMBER4 (FON4; also known as FON2) gene, an ortholog of Arabidopsis CLAVATA3 (CLV3), is required for regulating FM size and determinacy in rice. However, its interactions with floral homeotic genes remain unknown. Here, we report the genetic interactions between FON4 and floral homeotic genes OsMADS15 (an A-class gene), OsMADS16 (also called SUPERWOMAN1, SPW1, a B-class gene), OsMADS3 and OsMADS58 (C-class genes), OsMADS13 (a D-class gene), and OsMADS1 (an E-class gene) during flower development. We observed an additive phenotype in the fon4 double mutant with the OsMADS15 mutant allele dep (degenerative palea). The effect on the organ number of whorl 2 was enhanced in fon4 spw1. Double mutant combinations of fon4 with osmads3, osmads58, osmads13, and osmads1 displayed enhanced defects in FM determinacy and identity, respectively, indicating that FON4 and these genes synergistically control FM activity. In addition, the expression patterns of all the genes besides OsMADS13 had no obvious change in the fon4 mutant. This work reveals how the meristem maintenance gene FON4 genetically interacts with C, D, and E floral homeotic genes in specifying FM activity in monocot rice.

Glycerol-3-Phosphate Acyltransferase 3 (OsGPAT3) is required for anther development and male fertility in rice.

Lipid molecules are key structural components of plant male reproductive organs, such as the anther and pollen. Although advances have been made in the understanding of acyl lipids in plant reproduction, the metabolic pathways of other lipid compounds, particularly glycerolipids, are not fully understood. Here we report that an endoplasmic reticulum-localized enzyme, Glycerol-3-Phosphate Acyltransferase 3 (OsGPAT3), plays an indispensable role in anther development and pollen formation in rice. OsGPAT3 is preferentially expressed in the tapetum and microspores of the anther. Compared with wild-type plants, the osgpat3 mutant displays smaller, pale yellow anthers with defective anther cuticle, degenerated pollen with defective exine, and abnormal tapetum development and degeneration. Anthers of the osgpat3 mutant have dramatic reductions of all aliphatic lipid contents. The defective cuticle and pollen phenotype coincide well with the down-regulation of sets of genes involved in lipid metabolism and regulation of anther development. Taking these findings together, this work reveals the indispensable role of a monocot-specific glycerol-3-phosphate acyltransferase in male reproduction in rice.

Brassinosteroids promote development of rice pollen grains and seeds by triggering expression of Carbon Starved Anther, a MYB domain protein.

Transport of photoassimilates from leaf tissues (source regions) to the sink organs is essential for plant development. Here, we show that a phytohormone, the brassinosteroids (BRs) promotes pollen and seed development in rice by directly promoting expression of Carbon Starved Anther (CSA) which encodes a MYB domain protein. Over-expression of the BR-synthesis gene D11 or a BR-signaling factor OsBZR1 results in higher sugar accumulation in developing anthers and seeds, as well as higher grain yield compared with control non-transgenic plants. Conversely, knockdown of D11 or OsBZR1 expression causes defective pollen maturation and reduced seed size and weight, with less accumulation of starch in comparison with the control. Mechanically, OsBZR1 directly promotes CSA expression and CSA directly triggers expression of sugar partitioning and metabolic genes during pollen and seed development. These findings provide insight into how BRs enhance plant reproduction and grain yield in an important agricultural crop.

OsMADS32 interacts with PI-like proteins and regulates rice flower development.

OsMADS32 is a monocot specific MIKC(c) type MADS-box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clarified. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1-3 and identified its function in controlling rice flower development by bioinformatics and protein-protein interaction analysis. The cfo1-3 mutant produces defective flowers, including loss of lodicule identity, formation of ectopic lodicule or hull-like organs and decreased stamen number, mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis-splicing of OsMADS32 transcripts in the cfo1-3 mutant resulted in an extra eight amino acids in the K-domain of OsMADS32 protein. By yeast two hybrid and bimolecular fluorescence complementation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of OsMADS32 affects the interaction between OsMADS32 with PISTILLATA (PI)-like proteins OsMADS2 and OsMADS4. This work provides new insight into the mechanism by which OsMADS32 regulates rice lodicule and stamen identity, by interaction with two PI-like proteins via its K domain.

Rice CYP703A3, a cytochrome P450 hydroxylase, is essential for development of anther cuticle and pollen exine.

Anther cuticle and pollen exine act as protective envelopes for the male gametophyte or pollen grain, but the mechanism underlying the synthesis of these lipidic polymers remains unclear. Previously, a tapetum-expressed CYP703A3, a putative cytochrome P450 fatty acid hydroxylase, was shown to be essential for male fertility in rice (Oryza sativa L.). However, the biochemical and biological roles of CYP703A3 has not been characterized. Here, we observed that cyp703a3-2 caused by one base insertion in CYP703A3 displays defective pollen exine and anther epicuticular layer, which differs from Arabidopsis cyp703a2 in which only defective pollen exine occurs. Consistently, chemical composition assay showed that levels of cutin monomers and wax components were dramatically reduced in cyp703a3-2 anthers. Unlike the wide range of substrates of Arabidopsis CYP703A2, CYP703A3 functions as an in-chain hydroxylase only for a specific substrate, lauric acid, preferably generating 7-hydroxylated lauric acid. Moreover, chromatin immunoprecipitation and expression analyses revealed that the expression of CYP703A3 is directly regulated by Tapetum Degeneration Retardation, a known regulator of tapetum PCD and pollen exine formation. Collectively, our results suggest that CYP703A3 represents a conserved and diversified biochemical pathway for in-chain hydroxylation of lauric acid required for the development of male organ in higher plants.

[Design and application of an opening and breaking device of ampoule bottle].

Ampoule bottles are common clinical containers forliquid medicine. After use, ampoules occupy a lot of space, which will not only consume a large number of sharp boxes, but also lead to low work efficiency and easy to scratch the hands of medical staff by changing the sharp boxes frequently. In order to overcome the problems mentioned above, the neurosurgery medical staff of Yuechi County People's Hospital designed a new type of ampoule bottle opening and crushing device, and won the national utility model patent. The device includes a bottle-opening structure and a crushing structure, and has both ampoule opening and crushing function, the device is convenient to use, has high safety performance, and is suitable for clinical promotion.

Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding.

Although Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has been widely used for basic research in model plants, its application for applied breeding in crops has faced strong regulatory obstacles, due mainly to a poor understanding of the authentic output of this system, particularly in higher generations. In this study, different from any previous studies, we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1 (semi-dwarf 1) mutants from T2 to T4 generations, of which the selection of T1 and T2 was done only by visual phenotyping for semidwarf plants. Our data revealed not only on- and off-target mutations with small or lager indels but also exogenous elements in T2 plants. All indel mutants passed stably to T3 or T4 without additional modifications independent on the presence of Cas9, while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements. In addition, effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds. Taken together, our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis, but it may be not as precise as expected in rice, and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.

Development of a RAD-Seq Based DNA Polymorphism Identification Software, AgroMarker Finder, and Its Application in Rice Marker-Assisted Breeding.

Rapid and accurate genome-wide marker detection is essential to the marker-assisted breeding and functional genomics studies. In this work, we developed an integrated software, AgroMarker Finder (AMF: http://erp.novelbio.com/AMF), for providing graphical user interface (GUI) to facilitate the recently developed restriction-site associated DNA (RAD) sequencing data analysis in rice. By application of AMF, a total of 90,743 high-quality markers (82,878 SNPs and 7,865 InDels) were detected between rice varieties JP69 and Jiaoyuan5A. The density of the identified markers is 0.2 per Kb for SNP markers, and 0.02 per Kb for InDel markers. Sequencing validation revealed that the accuracy of genome-wide marker detection by AMF is 93%. In addition, a validated subset of 82 SNPs and 31 InDels were found to be closely linked to 117 important agronomic trait genes, providing a basis for subsequent marker-assisted selection (MAS) and variety identification. Furthermore, we selected 12 markers from 31 validated InDel markers to identify seed authenticity of variety Jiaoyuanyou69, and we also identified 10 markers closely linked to the fragrant gene BADH2 to minimize linkage drag for Wuxiang075 (BADH2 donor)/Jiachang1 recombinants selection. Therefore, this software provides an efficient approach for marker identification from RAD-seq data, and it would be a valuable tool for plant MAS and variety protection.

Interactions of OsMADS1 with Floral Homeotic Genes in Rice Flower Development.

During reproductive development, rice plants develop unique flower organs which determine the final grain yield. OsMADS1, one of SEPALLATA-like MADS-box genes, has been unraveled to play critical roles in rice floral organ identity specification and floral meristem determinacy. However, the molecular mechanisms underlying interactions of OsMADS1 with other floral homeotic genes in regulating flower development remains largely elusive. In this work, we studied the genetic interactions of OsMADS1 with B-, C-, and D-class genes along with physical interactions among their proteins. We show that the physical and genetic interactions between OsMADS1 and OsMADS3 are essential for floral meristem activity maintenance and organ identity specification; while OsMADS1 physically and genetically interacts with OsMADS58 in regulating floral meristem determinacy and suppressing spikelet meristem reversion. We provided important genetic evidence to support the neofunctionalization of two rice C-class genes (OsMADS3 and OsMADS58) during flower development. Gene expression profiling and quantitative RT-PCR analyses further revealed that OsMADS1 affects the expression of many genes involved in floral identity and hormone signaling, and chromatin immunoprecipitation (ChIP)-PCR assay further demonstrated that OsMADS17 is a direct target gene of OsMADS1. Taken together, these results reveal that OsMADS1 has diversified regulatory functions in specifying rice floral organ and meristem identity, probably through its genetic and physical interactions with different floral homeotic regulators.

Jasmonic acid regulates spikelet development in rice.

The spikelet is the basal unit of inflorescence in grasses, and its formation is crucial for reproductive success and cereal yield. Here, we report a previously unknown role of the plant hormone jasmonic acid (JA) in determining rice (Oryza sativa) spikelet morphogenesis. The extra glume 1 (eg1) and eg2 mutants exhibit altered spikelet morphology with changed floral organ identity and number, as well as defective floral meristem determinacy. We show that EG1 is a plastid-targeted lipase that participates in JA biosynthesis, and EG2/OsJAZ1 is a JA signalling repressor that interacts with a putative JA receptor, OsCOI1b, to trigger OsJAZ1's degradation during spikelet development. OsJAZ1 also interacts with OsMYC2, a transcription factor in the JA signalling pathway, and represses OsMYC2's role in activating OsMADS1, an E-class gene crucial to the spikelet development. This work discovers a key regulatory mechanism of grass spikelet development and suggests that the role of JA in reproduction has diversified during the flowering plant evolution.