Rice leaf inclination2, a VIN3-1ike protein, regulates leaf angle through modulating cell division of the collar

As an important agronomic trait, inclination of leaves is crucial Ior crop architecture and grain yields. 10 understand the molecular mechanism controlling rice leaf angles, one rice leaf inclination2 （1c2, three alleles） mutant was identified and functionally characterized. Compared to wild-type plants, lc2 mutants have enlarged leaf angles due to increased cell division in the adaxial epidermis of lamina joint. The LC2 gene was isolated through positional cloning, and encodes a vernalization insensitive 3-like protein. Complementary expression of LC2 reversed the enlarged leaf angles of lc2 plants, confirming its role in controlling leaf inclination. LC2 is mainly expressed in the lamina joint during leaf development, and particularly, is induced by the phytohormones abscisic acid, gibberellic acid, auxin, and brassinosteroids. LC2 is localized in the nucleus and defects of LC2 result in altered expression of cell division and hormone-responsive genes, indicating an important role of LC2 in regulating leaf inclination and mediating hormone effects.

Genome-wide analysis of the phospholipase D family in Oryza sativa and functional characterization of PLDβ1 in seed germination

Phospholipase D （PLD） plays a critical role in plant growth and development, as well as in hormone and stress responses. PLD encoding genes constitute a large gene family that are present in higher plants. There are 12 members of the PLD family in Arabidopsis thaliana and several of them have been functionally characterized; however, the members of the PLD family in Oryza sativa remain to be fully described. Through genome-wide analysis, 17 PLD members found in different chromosomes have been identified in rice. Protein domain structural analysis reveals a novel subfamily, besides the C2-PLDs and PXPH-PLDs, that is present in rice - the SP-PLD. SP-PLD harbors a signal peptide instead of the C2 or PXPH domains at the N-terminus. Expression pattern analysis indicates that most PLD-encoding genes are differentially expressed in various tissues, or are induced by hormones or stress conditions, suggesting the involvement of PLD in multiple developmental processes. Transgenic studies have shown that the suppressed expression office PLDβ1 results in reduced sensitivity to exogenous ABA during seed germination. Further analysis of the expression of ABA signaling-related genes has revealed that PLDβ1 stimulates ABA signaling by activating SAPK, thus repressing GAmyb exoression and inhibiting seed germination.

Functional conservation of the meiotic genes SDS and RCK in male meiosis in the monocot rice

The Arabidopsis SDS （SOLO DANCERS） and RCK （ROCK-N-ROLLERS） genes are important for male meiosis, but it is still unknown whether they represent conserved functions in plants. We have performed phylogenetic analyses of SDS and RCK and their respective homologs, and identified their putative orthologs in poplar and rice. Quantitative real-time RT-PCR analysis indicated that rice SDS and RCK are expressed preferentially in young flowers, and transgenic RNAi rice lines with reduced expression of these genes exhibited normal vegetative development, but showed significantly reduced fertility with partially sterile flowers and defective pollens. SDS deficiency also caused a decrease in pollen amounts. Further cytological examination of male meiocytes revealed that the SDS deficiency led to defects in homolog interaction and bivalent formation in meiotic prophase I, and RCK deficiency resulted in defective meiotic crossover formation. These results indicate that rice SDS and RCK genes have similar functions to their Arabidopsis orthologs. Because rice and Arabidopsis, respectively, are members of monocots and eudicots, two largest groups of flowering plants, our results suggest that the functions of SDS and RCK are likely conserved in flowering plants.

Vacuolar H^(+)-ATPase and BZR1 form a feedback loop to regulate the homeostasis of BR signaling inArabidopsis

Brassinosteroid(BR)is a vital plant hormone that regulates plant growth anddevelopment.BRASSINAZOLE RESISTANT1(BZR1)is a key transcription factor in BR signaling,and its nucleocytoplasmic localization is crucial for BR signaling.However,the mechanisms that regulate BzR1 nucleocytoplasmic distribution and thus the homeostasis of BR signaling remain largely unclear.The vacuole is the largest organelle in mature plantcells and plays a key role in maintenance of cell ular pH,storage of intracellular substances,and transport ofions.In this study,weuncovered anovel mechanismof BR signaling homeostasis regulatedbythe vacuolar H+-ATPase(V-ATPase)and BZR1 feedback loop.Our results revealed that the vha-a2 vha-a3 mutant(vha2,lacking V-ATPase activity)exhibits enhanced BR signaling with increased total amount of BZR1,nuclearlocalized BZR1,and the ratio of BZR1/phosphorylated BZR1 in the nucleus.Further biochemical assays revealed that VHA-a2 and VHA-a3 of V-ATPase interact with the BZR1 protein through a domain that is conserved across multiple species.VHA-a2 and VHA-a3 negatively regulate BR signaling by interacting with BzR1 and promoting its retention in the tonoplast.Interestingly,a series of molecular analyses demonstrated that nuclear-localized BZR1 could bind directlyto specific motifs in the promoters of VHA-a2 andVHAa3topromote their expression.Taken together,these results suggest that V-ATPase and BzR1 mayforma feedback regulatory loop to maintain thehomeostasis of BR signaling in Arabidopsis,providing new insights into vacuole-mediated regulation of hormone signaling.

A Novel QTL qTGW3 Encodes the GSK3/ SHAGGY-Like Kinase OsGSK5/OsSK41 that Interacts with OsARF4 to Negatively Regulate Grain Size and Weight in Rice

Grain size and shape are important determinants of grain weight and yield in rice. Here, we report a new major quantitative trait locus （QTL）, qTGW3, that controls grain size and weight in rice. This locus, qTGW3, encodes OsSK41 （also known as OsGSK5）, a member of the GLYCOGEN SYNTHASE KINASE 3/SHAGGY-like family. Rice near-isogenic lines carrying the loss-of-function allele of OsSK41 have increased grain length and weight. We demonstrate that OsSK41 interacts with and phosphorylates AUXIN RESPONSE FACTOR 4 （OsARF4）. Co-expression of OsSK41 with OsARF4 increases the accumulation of OsARF4 in rice protoplasts. Loss of function of OsARF4 results in larger rice grains. RNA-sequencing analysis suggests that OsARF4 and OsSK41 repress the expression of a common set of downstream genes, including some auxin-responsive genes, during rice grain development. The loss-of-function form of OsSK41 at qTGW3 represents a rare allele that has not been extensively utilized in rice breeding. Suppression of OsSK41 function by either targeted gene editing or QTL pyramiding enhances rice grain size and weight. Thus, our study reveals the important role of OsSK41 in rice grain development and provides new candidate genes for genetic improvement of grain yield in rice and perhaps in other cereal crops.

EL1-like Casein Kinases Suppress ABA Signaling and Responses by Phosphorylating and Destabilizing the ABA Receptors PYR/PYLs in Arabidopsis

Unveiling the signal transduction of phytohormone abscisic acid （ABA） and its regulatory mechanisms is critical for developing the strategies toward improving plant responses to stressful environments. ABA signaling is perceived and mediated by multiple PYR/PYL receptors, whose post-translational modifications, especially phosphorylation, remain largely unknown. In this study, we demonstrate thatArabidopsis ELl-like （AEL） protein, a casein kinase that regulates various physiological processes, phosphorylate PYR/PYLs to promote their ubiquitination and degradation, resulting in suppressed ABA responses. Arabidopsis ael triple mutants display hypersensitive responses to ABA treatment, which is consistent with the suppressed degradation of PYR/PYL proteins. PYR/PYLs are phosphorylated in vivo and mutation of the conserved AEL phosphorylation sites results in reduced phosphorylation, ubiquitination, and degradation of PYR/PYLs, and hence enhanced ABA responses. Taken together, these results demonstrate that AEL-mediated phosphorylation plays crucial roles in regulating the stability and function of PYR/ PYLs, providing significant insights into the post-translational regulation of PYR/PYL receptors and ABA signaling.

OsLEC1/OsHAP3E Participates in the Determination of Meristem Identity in Both Vegetative and Reproductive Developments of Rice

In the vegetative phase of plant development, the shoot apical meristem （SAM） produces leaf primordia in regular phyllotaxy, and transforms to the inflorescence meristem when the plant enters reproductive growth, which will undergo a series of identity differentiations and will finally form a complete and fertile panicle. Our previous studies indicated a tissue-specific expression pattern of the OsLEC1 （leafy cotyledon） gene, which is homologous to the Arabidopsis AtLEC1 gene and belongs to the CCAAT-binding protein HAP3 subfamily, during embryo development. Expression of additional OsLEC1 genomic sequences resulted in abnormalities in the development of leaves, panicles and spikelets. The spikelets in particular presented abnormities, including panicle and spikelet-like structures that occurred reiteratively inside prior spikelets, and the occasional spikelet structures that completely transformed into plantlets （a reproductive habit alteration from sexual to asexual called ＂pseudovivipary＂）. Analysis showed that OsLEC1 interacts with several SEPALLATA-like MADS transcription factors, suggesting that increased levels of the OsLEC1 protein might interfere with the normal interaction network of these MADS proteins and lead to defective spikelet development. The expression of OsMADS1 was dramatically reduced, and the DNA methylation level of cytosine in certain regions of the OsMADS1 promoter was increased under OsLEC1 overexpression. These results indicate that OsLEC1 affects the development of leaves, panicles and spikelets, and is a key regulator of meristem identity determination in both rice （Oryza sativa） vegetative and reproductive development.

Global Analysis of Expression Profiles of Rice Receptor-Like Kinase Genes

The receptor-like kinases （RLKs） play critical roles in plant development and response to stress stimuli. By perceiving or sensing the extracellular signals, RLK activates the downstream signaling pathway through phosphorylating the specific targets. Up to now, only a few RLKs have been functionally identified, which are even fewer in rice （Oryza sativa L.）. We here report the systemic analysis of the expression profiles of rice RLK coding genes in different tissues, with the emphasis on seed development and in response to both abiotic stress and plant hormones. The results showed that most rice RLK genes are expressed in two or more tissues, of which the RLCK-RLKs have a higher, while WAK- and SD-RLKs have a lower, expression level in the vegetative tissues than other subfamily members. Interestingly, the constitutively highly expressed RLKs in rice and Arabidopsis are conserved, which is consistent with the previous hypothesis that RLKs existed before the differentiation of monocotyledon and dicotyledon plants. Nearly one-third of the detected rice RLKs are expressed during seed development, and the RLCK-RLK members possess a higher percentage during the endosperm de- velopment, suggesting a novel function of RLCK-RLK members in endosperm development. Further analysis revealed that many RLK genes expressed during seed development are also regulated by abiotic stresses （cold, salt, or drought） or hor- mones, indicating that RLKs may take part in the stress-related signaling pathways such as dehydration of endosperm. These results provide informative insights into the RLK studies and will be helpful to reveal the global regulatory network controlling rice seed development.

CRISPR Primer Designer:Design primers for knockout and chromosome imaging CRISPR-Cas system

The clustered regularly interspaced short palin- dromic repeats （CRiSPR）-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user- friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present researchers to design primers CRISPR Primer Designer for for CRISPR applications. Theprogram has a user-frfendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRiSPR-Cas system-based chromosome imaging system.

Rice miR394 suppresses leaf inclination through targeting an F-box gene, LEAF INCLINATION 4

Rice leaf inclination is an important agronomic trait, closely related to plant architecture and yield.Identification of genes controlling leaf inclination would assist in crop improvement. Although various factors,including the plant hormones auxin and brassinosteroids,have been shown to regulate lamina joint development,the role of microRNAs in regulating leaf inclination remains largely unknown. Here, we functionally characterize the role of rice miR394 and its target, LEAF INCLINCATION 4(LC4), which encodes an F-box protein, in the regulation of leaf inclination. We show that miR394 and LC4 work,antagonistically, to regulate leaf lamina joint development and rice architecture, by modulating expansion and elongation of adaxial parenchyma cells. Suppressed expression of miR394, or enhanced expression of LC4,results in enlarged leaf angles, whereas reducing LC4 expression by CRISPR/Cas9 leads to reduced leaf inclination, suggesting LC4 as candidate for use in rice architecture improvement. LC4 interacts with SKP1, a component of the SCF E3 ubiquitin ligase complex, and transcription of both miR394 and LC4 are regulated by auxin. Rice plants with altered expression of miR394 or LC4 have altered auxin responses, indicating that the miR394-LC4 module mediates auxin effects important for determining rice leaf inclination and architecture.

Cis-12-Oxo-Phytodienoic Acid Stimulates Rice Defense Response to a Piercing-Sucking Insect

The brown planthopper （BPH, Nilaparvata lugens） is a destructive, monophagous, piercing-sucking insect pest of rice. Previous studies indicated that jasmonic acid （JA） positively regulates rice defense against chewing insect pests but negatively regulates it against the piercing-sucking insect of BPH. We here demonstrated that overexpression of allene oxide cyclase （AOC） but not OPR3 （cis-12-oxo-phytodienoic acid （OPDA） reductase 3, an enzyme adjacent to AOC in the JA synthetic pathway） significantly increased rice resistance to BPH, mainly by reducing the feeding activity and survival rate. Further anal- ysis revealed that plant response to BPH under AOC overexpression was independent of the JA pathway and that significantly higher OPDA levels stimulated rice resistance to BPH. Microarray analysis identified multiple candidate resistance-related genes underAOCoverexpression. OPDA treatment stimulated the resistance of radish seedlings to green peach aphid Myzus persicae, another piercing-sucking insect. These results imply that rice resistance to chewing insects and to sucking insects can be enhanced simultaneously through AOC-mediated increases of JA and OPDA and provide direct evidence of the potential application of OPDA in stimulating plant defense responses to piercing-sucking insect pests in agriculture.

Arabidopsis AUTOPHAGY-RELATED3(ATG3)facilitates the liquid-liquid phase separation of ATG8e to promote autophagy

In each cell,various components/organelles(including membranous and non-membranous organelles)assemble at the proper time and space to function.How organelles lacking a membrane form and the physicochemical natures of such organelles remain unknown.Recent studies have shown that membrane-free structures can assemble by liquid-liquid phase separation(LLPS),an essential process in which protein molecules condense into a closed liquid compartment with other proteins or RNAs after they reach a critical threshold concentration[1].These components show highly sensitive fluidity and dynamic exchange with their surrounding liquid environments.LLPS plays crucial roles in various physiological and signaling processes by creating a relatively independent spatial domain to selectively enrich molecules and form distinct structures.Phase separation of SORTING OF cpTat SUBSTRATES TO THYLAKOID MEMBRANES1(STT1)and STT2,key protein transport sorting factors to the chloroplast stroma,drives protein sorting in chloroplasts[2].Arabidopsis thaliana EARLY FLOWERING3(ELF3)senses changes in ambient temperature through prion-like domain(PrD)-mediated phase transition[3].Two members of the plant guanylate-binding protein-like GTPase(GBPL)superfamily,GBPL1 and GBPL3,mobilize the host immune response to pathogen infection through phase transition,thereby enabling host defense[4].The prion-like protein FLOE1(a name inspired by ice floes,which are sheets of floating ice and,thus,phase-separated bodies of water)regulates seed germination by sensing changes in environmental moisture through phase separation[5].

Global Analysis of Gene Expression Profiles in Brassica napus Developing Seeds Reveals a Conserved Lipid Metabolism Regulation with Arabidopsis thaliana

In order to study Brassica napus fatty acid （FA） metabolism and relevant regulatory networks, a systematic identification of fatty acid （FA） biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization （〉8000 EST clones from seed）. The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17-21 d after flowering （DAF）, whereas FA biosynthesis-related genes were highly expressed pri- marily at 21 DAF. Hormone （auxin and jasmonate） signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.

LC2 and OsVIL2 Promote Rice Flowering by Photoperoid-lnduced Epigenetic Silencing of OsLF

Proper flowering time is essential for plant reproduction. Winter annual Arabidopsis thaliana needs ver-nalization before flowering, during which AtVILs （VIN3 and VRNS, components of PRC2 complex） mediate the H3K27 tri- methylation at the FLC locus （a floral repressor） to repress the FLC expression and hence to induce flowering. However, how VILs （VIL, VERNALIZATION INSENSITIVE 3-LIKE） function in rice is unknown. Here we demonstrated that rice LC2 （OsVIL3） and OsVIL2 （two OsVILs, possible components of PRC2 complex） promote rice flowering. Our results showed that expressions of LC2 and OsVIL2 are induced by SD （short-day） conditions and both Ic2 mutant and OsVIL2-RNAi lines display delayed heading date, consistent with the reduced expression levels of Hdl and Hd3a. Interestingly, LC2 binds to the promoter region of a floral repressor OsLF and represses the OsLF expression via H3K27 tri-methylation modification. In addition, OsLF directly regulates the Hdl expression through binding to Hdl promoter. These results first demonstrated that the putative PRC2 in rice is involved in photoperiod flowering regulation, which is different from that of Arabidopsis, and revealed that LC2 binds the promoter region of target gene, presenting a possible mechanism of the recruitment pro-cess of PRC2 complex to its target genes. The studies provide informative clues on the epigenetic control of rice flowering.

Rice SPL12 coevolved with GW5 to determine grain shape

Asian cultivated rice(Oryza sativa L.)is a staple food crop in China.Accordingly,many studies have focused on grain size and weight as a means to in crease rice yield.Two main subgroups of cultivated rice,Indica and Japonica,have evolved independently under natural and human selection,and a key morphological difference between these subgroups is grain shape:slender in Indica rice and ovate in Japonica.Elucidating the genetic basis of grain shape will provide insights into rice evolution and open new avenues for molecular breeding of improved grain shape.

Phosphatidic acid plays key roles regulating plant development and stress responses

Phospholipids, including phosphatidic acid （PA）, phosphatidylcholine （PC）, phosphatidylethanolamine （PE）, phosphatidylglycerol （PC）, phosphatidylserine （PS） and phosphoinositides, have emerged as an important class of cellular messenger molecules in various cellular and physiological processes, of which PA attracts much attention of researchers. In addition to its effect on stimulating vesicle trafficking, many studies have demonstrated that PA plays a crucial role in various signaling pathways by binding target proteins and regulating their activity and subcellular localization. Here, we summarize the functional mechanisms and target proteins underlying PA-mediated regulation of cellular signaling, development, hormonal responses, and stress responses in plants.

The rice PLATZ protein SHORT GRAIN6 determines grain size by regulating spikelet hull cell division

Grain size is a major determinant of cereal grain yields;however,the relevant regulatory mechanisms controlling this trait have not been fully elucidated.The rice(Oryza sativa)mutant short grain6(sg6)was identified based on its reduced grain length and weight.Here,we functionally characterized the role of SG6 in determining grain size through the regulation of spikelet hull cell division.SG6 encodes a previously uncharacterized plant ATrich sequence and zinc-binding(PLATZ)protein that is ubiquitously localized throughout the cell and is preferentially expressed in the early developing panicles but not in the endosperm.The overexpression of SG6 resulted in significantly larger and heavier grains,as well as increased plant heights,which is consistent with its elevated spikelet hull cell division rate.Yeast two-hybrid analyses revealed that SG6 interacts with the core cell cycle machinery DP protein and several other putative cell division regulators,consistent with our transcriptomic analysis,which showed that SG6 activates the expression of many DNA replication and cell-cycle-related genes.These results confirm the crucial role of SG6 in determining grain size by regulating spikelet hull cell division and provide clues for understanding the functions of PLATZ family proteins and the network regulating cereal grain size.

Phytohormone dynamics in developing endosperm influence rice grain shape and quality

Hormones are important signaling molecules regulating developmental processes and responses to environmental stimuli in higher plants.Rice endosperm,the portion of the seed surrounding the embryo,is the main determinant of rice grain shape and yield;how-ever,the dynamics and exact functions of phyto-hormones in developing endosperm remain elusive.Through a systemic study including transcriptome analysis,hormone measurement,and transgene-based endosperm-specific expression of phytohormone bio-synthetic enzymes,we demonstrated that dynamic phytohormone levels play crucial roles in the developing rice endosperm,particularly in regard to grain shape and quality.We detected diverse,differential,and dra-matically changing expression patterns of genes related to hormone biosynthesis and signaling during endosperm development,especially at early devel-opmental stages.Liquid chromatography measure-ments confirmed the dynamic accumulation of hor-mones in developing endosperm.Further transgenic analysis performed on plants expressing hormone bio-synthesis genes driven by an endosperm-specific pro-moter revealed differential effects of the hormones,especially auxin and brassinosteroids,in regulating grain shape and quality.Our studies help elucidate the dis-tinct roles of hormones in developing endosperm and provide novel and useful tools for influencing crop seed shape and yield.

Interaction of brassinosteroid and cytokinin promotes ovule initiation and increases seed number per silique in Arabidopsis

Ovule initiation is a key step that strongly influences ovule number and seed yield.Notably,mutants with enhanced brassinosteroid(BR)and cytokinin(CK)signaling produce more ovules and have a higher seed number per silique(SNS)than wild-type plants.Here,we crossed BR-and CKrelated mutants to test whether these phytohormones function together in ovule initiation.We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately,and BR and CK enhanced each other.Further,the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1(ARR1).Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation.Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants,suggesting that BR regulates ovule initiation and SNS through CK-mediated and-independent pathways.Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number,providing important clues for increasing the seed yield of dicot crops.

Pollen and Plant Reproduction Biology： Blooming from East to West

The idea of assembling a Special Issue on Pollen Tube and Reproduction in Molecular Plant was conceived during the organization of the 2012 Pollen Biology International Symposium and Workshop held at the Shanghai Institute of Plant Physiology and Ecology （SIPPE）, Chinese Academy of Science （CAS）, 27-31 October 2012. Wei-Hua Tang of the SIPPE led the organization, with the support of Hong-Wei Xue, Director of SIPPE acting as Chair of the Organization Committee. The 2-d symposium was attended by about 100 international participants and focused on reports and discus- sions on the most recent research activities in the field. The 2-d workshop that followed provided a forum for in-depth introduction on various aspects of pollen and plant reproduc- tion biology to about 200 graduate students, postdoctoral fellows, and new investigators to the field.