Yulong Ren

D14–SCF D3 -dependent degradation of D53 regulates strigolactone signalling

Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.

Ehd4 Encodes a Novel and Oryza-Genus-Specific Regulator of Photoperiodic Flowering in Rice

Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (Oryza sativa L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the CO-FT module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (Hd1-Hd3a in rice). However, in rice, there is a unique Ehd1-dependent flowering pathway that is Hd1-independent. Here, we report isolation and characterization of a positive regulator of Ehd1, Early heading date 4 (Ehd4). ehd4 mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that Ehd4 encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids in vitro and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. Ehd4 expression is most active in young leaves with a diurnal expression pattern similar to that of Ehd1 under both short-day and long-day conditions. We show that Ehd4 up-regulates the expression of the “florigen” genes Hd3a and RFT1 through Ehd1, but it acts independently of other known Ehd1 regulators. Strikingly, Ehd4 is highly conserved in the Oryza genus including wild and cultivated rice, but has no homologs in other species, suggesting that Ehd4 is originated along with the diversification of the Oryza genus from the grass family during evolution. We conclude that Ehd4 is a novel Oryza-genus-specific regulator of Ehd1, and it plays an essential role in photoperiodic control of flowering time in rice.

A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice

The brown planthopper (BPH) is the most destructive pest of rice (Oryza sativa) and a substantial threat to rice production, causing losses of billions of dollars annually. Breeding of resistant cultivars is currently hampered by the rapid breakdown of BPH resistance. Thus, there is an urgent need to identify more effective BPH-resistance genes. Here, we report molecular cloning and characterization of Bph3, a locus in rice identified more than 30 years ago that confers resistance to BPH. We show that Bph3 is a cluster of three genes encoding plasma membrane–localized lectin receptor kinases (OsLecRK1-OsLecRK3). Introgression of Bph3 into susceptible rice varieties by transgenic or marker-assisted selection strategies significantly enhanced resistance to both the BPH and the white back planthopper. Our results suggest that these lectin receptor kinase genes function together to confer broad-spectrum and durable insect resistance and provide a resource for molecular breeding of insect-resistant rice cultivars.

Pollen Semi-Sterility1 Encodes a Kinesin-1–Like Protein Important for Male Meiosis, Anther Dehiscence, and Fertility in Rice

This work describes Pollen Semi-Sterility1, a kinesin-1 like protein that is involved in regulating male meiosis in rice; in pss1 mutants, chromosome segregation is perturbed, causing reduced pollen viability and partial sterility. In flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1–like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1–green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice.

The vacuolar processing enzyme OsVPE1 is required for efficient glutelin processing in rice

Rice (Oryza sativa L.) accumulates prolamines and glutelins as its major storage proteins. Glutelins are synthesized on rough endoplasmic reticulum as 57-kDa precursors; they are then sorted into protein storage vacuoles where they are processed into acidic and basic subunits. We report a novel rice glutelin mutant, W379, which accumulates higher levels of the 57-kDa glutelin precursor. Genetic analysis revealed that the W379 phenotype is controlled by a single recessive nuclear gene. Using a map-based cloning strategy, we identified this gene, OsVPE1, which is a homolog of the Arabidopsis betaVPE gene. OsVPE1 encodes a 497-amino-acid polypeptide. Nucleotide sequence analysis revealed a missense mutation in W379 that changes Cys269 to Gly. Like the wild-type protein, the mutant protein is sorted into vacuoles; however, the enzymatic activity of the mutant OsVPE1 is almost completely eliminated. Further, we show that OsVPE1 is incorrectly cleaved, resulting in a mature protein that is smaller than the wild-type mature protein. Taken together, these results demonstrate that OsVPE1 is a cysteine protease that plays a crucial role in the maturation of rice glutelins. Further, OsVPE1 Cys269 is a key residue for maintaining the Asn-specific cleavage activity of OsVPE1.

Wax crystal-sparse leaf2, a rice homologue of WAX2/GL1, is involved in synthesis of leaf cuticular wax

Epicuticular wax in plants limits non-stomatal water loss, inhibits postgenital organ fusion, protects plants against damage from UV radiation and imposes a physical barrier against pathogen infection. Here, we give a detailed description of the genetic, physiological and morphological consequences of a mutation in the rice gene WSL2, based on a comparison between the wild-type and an EMS mutant. The mutant’s leaf cuticle membrane is thicker and less organized than that of the wild type, and its total wax content is diminished by ~80%. The mutant is also more sensitive to drought stress. WSL2 was isolated by positional cloning, and was shown to encode a homologue of the Arabidopsis thaliana genes CER3/WAX2/YRE/FLP1 and the maize gene GL1. It is expressed throughout the plant, except in the root. A transient assay carried out in both A. thaliana and rice protoplasts showed that the gene product is deposited in the endoplasmic reticulum. An analysis of the overall composition of the wax revealed that the mutant produces a substantially reduced quantity of C22–C32 fatty acids, which suggests that the function of WSL2 is associated with the elongation of very long-chain fatty acids.

OsRab5a regulates endomembrane organization and storage protein trafficking in rice endosperm cells.

Rice glutelins are synthesized at the endoplasmic reticulum (ER) as precursors (pro-glutelins), and are transported to protein storage vacuoles, where they are processed into mature proteins. The molecular basis of this process is largely unknown. Here, we report the isolation of a rice mutant, gpa1, that accumulates 57 kDa pro-glutelins in seeds and whose endosperm has a floury appearance. Transmission electron microscopy analysis showed that the gpa1 endosperm cells have an enlarged ER lumen and a smaller protein body II (PBII), and accumulated three types of newly generated subcellular structures. Moreover, a proportion of glutelins in the gpa1 endosperm cells were not delivered to PBII, and instead were mis-targeted to two of the newly generated structures or secreted. The gene corresponding to the gpa1 mutation was found to be OsRab5a, which encodes a small GTPase. In Arabidopsis protoplasts, OsRab5a protein was found to co-localize predominantly with AtVSR2, a molecular marker for the pre-vacuolar compartments (PVC). We conclude that OsRab5a plays an essential role in trafficking of storage protein to PBII, possibly as part of its function in organizing the endomembrane system in developing endosperm cells of rice.

A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants

Disruption of the OsClpP6 gene causes a virescent-yellow leaf phenotype, demonstrating an important role of caseinolytic proteases in regulating chloroplast biogenesis and leaf development in rice. The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS). The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana). In this work, we isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period. The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.

FLOURY ENDOSPERM6 encodes a CBM48 domain‐containing protein involved in compound granule formation and starch synthesis in rice endosperm

Starch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and related factors have been identified for starch biosynthesis, unknown players remain to be identified, given that it is a complicated and sophisticated process. The endosperm of rice (Oryza sativa) has been used for the study of starch synthesis. Here, we report the cloning and characterization of the FLOURY ENDOSPERM6 (FLO6) gene in rice. In the flo6 mutant, the starch content is decreased and the normal physicochemical features of starch are changed. Significantly, flo6 mutant endosperm cells show obvious defects in compound granule formation. Map-based cloning showed that FLO6 encodes a protein of unknown function. It harbors an N-terminal transit peptide that ensures its correct localization and functions in the plastid, and a C-terminal carbohydrate-binding module 48 (CBM48) domain that binds to starch. Furthermore, FLO6 can interact with isoamylase1 (ISA1) both in vitro and in vivo, whereas ISA1 does not bind to starch directly. We thus propose that FLO6 may act as a starch-binding protein involved in starch synthesis and compound granule formation through a direct interaction with ISA1 in developing rice seeds. Our data provide a novel insight into the role of proteins with the CBM48 domain in plant species.

Young Leaf Chlorosis 1, a chloroplast-localized gene required for chlorophyll and lutein accumulation during early leaf development in rice

Chlorophyll (Chl) and lutein are the two most abundant and essential components in photosynthetic apparatus, and play critical roles in plant development. In this study, we characterized a rice mutant named young leaf chlorosis 1 (ylc1) from a 60Co-irradiated population. Young leaves of the ylc1 mutant showed decreased levels of Chl and lutein compared to those of wild type, and transmission electron microscopy analysis revealed that the thylakoid lamellar structures were obviously loosely arranged. Whereas, the mutant turns green gradually and approaches normal green at the maximum tillering stage. The Young Leaf Chlorosis 1 (YLC1) gene was isolated via map-based cloning and identified to encode a protein of unknown function belonging to the DUF3353 superfamily. Complementation and RNA-interference tests confirmed the role of the YLC1 gene, which expressed in all tested rice tissues, especially in the leaves. Real-time PCR analyses showed that the expression levels of the genes associated with Chl biosynthesis and photosynthesis were affected in ylc1 mutant at different temperatures. In rice protoplasts, the YLC1 protein displayed a typical chloroplast location pattern. The N-terminal 50 amino acid residues were confirmed to be necessary and sufficient for chloroplast targeting. These data suggested that the YLC1 protein may be involved in Chl and lutein accumulation and chloroplast development at early leaf development in rice.