PCR-based INDEL markers co-dominant between Oryza sativa, japonica cultivars and closely-related wild Oryza species.

Wild relatives genetically close to cultivars are precious genetic resources for plant breeding. Oryza rufipogon, O. barthii, O. glumaepatula, O. meridionalis and O. longistaminata are such wild species, and are also categorized as AA genome species based on their structural similarities. Chromosome segment substitution lines (CSSLs) are a powerful resource in breeding and genetics, and numerous rice CSSLs have been produced. This study aimed to develop DNA markers for evaluation of CSSLs directly by PCR and subsequent gel electrophoresis. We confirmed that up to 155 of 188 markers developed for detection of japonica-indica INDELs could also detect INDELs between rice cultivars and wild AA-species accessions. Percentages of applicable markers were higher in O. rufipogon accessions (61.7 to 85.6%), and lower in accessions of other four AA species (39.8 to 51.4%). These markers were distributed throughout the rice chromosomes, and will be useful for genotyping of CSSLs and other genetic resources derived from crosses between rice cultivars and closely related wild species.

Rice germline-specific Argonaute MEL1 protein binds to phasiRNAs generated from more than 700 lincRNAs.

Small RNAs that interact with Argonaute (AGO) proteins play central roles in RNA-mediated silencing. MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice AGO, has specific functions in the development of pre-meiotic germ cells and the progression of meiosis. Here, we show that MEL1, which is located mostly in the cytoplasm of germ cells, associates preferentially with 21-nucleotide phased small interfering RNAs (phasiRNAs) that bear a 5'-terminal cytosine. Most phasiRNAs are derived from 1171 intergenic clusters distributed on all rice chromosomes. From these clusters, over 700 large intergenic, non-coding RNAs (lincRNAs) that contain the consensus sequence complementary to miR2118 are transcribed specifically in inflorescences, and cleaved within the miR2118 site. Cleaved lincRNAs are processed via DICER-LIKE4 (DCL4) protein, resulting in production of phasiRNAs. This study provides the evidence that the miR2118-dependent and the DCL4-dependent pathways are both required for biogenesis of 21-nt phasiRNAs associated with germline-specific MEL1 AGO in rice, and over 700 lincRNAs are key factors for induction of this biogenesis during reproductive-specific stages.

Rice expression atlas in reproductive development.

Gene expression throughout the reproductive process in rice (Oryza sativa) beginning with primordia development through pollination/fertilization to zygote formation was analyzed. We analyzed 25 stages/organs of rice reproductive development including early microsporogenesis stages with 57,381 probe sets, and identified around 26,000 expressed probe sets in each stage. Fine dissection of 25 reproductive stages/organs combined with detailed microarray profiling revealed dramatic, coordinated and finely tuned changes in gene expression. A decrease in expressed genes in the pollen maturation process was observed in a similar way with Arabidopsis and maize. An almost equal number of ab initio predicted genes and cloned genes which appeared or disappeared coordinated with developmental stage progression. A large number of organ-/stage-specific genes were identified; notably 2,593 probe sets for developing anther, including 932 probe sets corresponding to ab initio predicted genes. Analysis of cell cycle-related genes revealed that several cyclin-dependent kinases (CDKs), cyclins and components of SCF E3 ubiquitin ligase complexes were expressed specifically in reproductive organs. Cell wall biosynthesis or degradation protein genes and transcription factor genes expressed specifically in reproductive stages were also newly identified. Rice genes homologous to reproduction-related genes in other plants showed expression profiles both consistent and inconsistent with their predicted functions. The rice reproductive expression atlas is likely to be the most extensive and most comprehensive data set available, indispensable for unraveling functions of many specific genes in plant reproductive processes that have not yet been thoroughly analyzed.

gorgon, a novel missense mutation in the SHOOT MERISTEMLESS gene, impairs shoot meristem homeostasis in Arabidopsis.

The shoot meristem is a group of self-perpetuating cells that ultimately gives rise to the aerial parts of plants. The Arabidopsis thaliana SHOOT MERISTEMLESS (STM) gene, which encodes a knotted1-like homeobox transcription factor, is required for shoot meristem formation and maintenance, and loss-of-function mutations in the gene result in complete loss or premature termination of the shoot meristem. Here, we report a novel missense allele of STM, gorgon (gor), which displays striking differences in shoot meristem defects compared with known stm alleles. The gor phenotype results from substitution of the highly conserved arginine at position 53 of the homeodomain, which is important for DNA binding in other homeodomain proteins. In gor, the shoot meristem enlarges continuously during post-embryonic development and the floral meristems frequently develop additional whorls. These phenotypes, together with enlarged expression domains of meristem markers, indicate that the mutation affects shoot meristem activity in the opposite direction to other loss-of-function alleles. However, detailed genetic analyses and overexpression studies indicate that gor represents a novel type of hypomorphic alleles rather than the hypermorph that is suggested by the phenotype. Consistently, the gor allele strictly requires the functional PENNYWISE (PNY) gene, which encodes a known binding partner of the STM protein, to maintain shoot meristem activity, whereas the wild-type allele efficiently maintains the meristem even in the absence of PNY. Our results suggest a critical role for Arg53 of the homeodomain in STM function and that the gor mutation at this residue impairs shoot meristem homeostasis.

Loss-of-function mutations of retromer large subunit genes suppress the phenotype of an Arabidopsis zig mutant that lacks Qb-SNARE VTI11.

Arabidopsis thaliana zigzag (zig) is a loss-of-function mutant of Qb-SNARE VTI11, which is involved in membrane trafficking between the trans-Golgi network and the vacuole. zig-1 exhibits abnormalities in shoot gravitropism and morphology. Here, we report that loss-of-function mutants of the retromer large subunit partially suppress the zig-1 phenotype. Moreover, we demonstrate that three paralogous VPS35 genes of Arabidopsis have partially overlapping but distinct genetic functions with respect to zig-1 suppression. Tissue-specific complementation experiments using an endodermis-specific SCR promoter show that expression of VPS35B or VPS35C cannot complement the function of VPS35A. The data suggest the existence of functionally specialized paralogous VPS35 genes that nevertheless share common functions.

ZIP genes encode proteins involved in membrane trafficking of the TGN-PVC/vacuoles.

The Arabidopsis zigzgag (zig) is a loss-of-function mutant of Qb-SNARE VTI11 which is involved in vesicle trafficking between the trans-Golgi network (TGN) and vacuoles. zig-1 exhibits abnormality in both shoot gravitropism and morphology. To elucidate the molecular network of the post-Golgi membrane trafficking in plant cells, we have isolated the suppressor mutants of zig. Here we report zig suppressor 2 (zip2) and zip4 that are recessive mutants and partially suppress abnormality in both gravitropism and morphology. ZIP2 encodes AtVPS41/AtVAM2 protein that is thought to be an Arabidopsis ortholog of yeast Vps41p/Vam2p, which is involved in protein sorting to vacuoles as a subunit of the tethering complex HOPS. Yeast Vps41p is also proposed to function in budding of adaptor protein (AP)-3-coated vesicles from the Golgi. The zip2 mutation is a missense mutation in a conserved amino acid of a putative clathrin heavy chain repeat (CHCR) domain. AtVPS41 is a single-copy gene in the Arabidopsis genome and the T-DNA insertion mutant appears to be lethal, whereas the zip2 single mutant showed no obvious phenotype. On the other hand, zip4 is a loss-of-function mutant of a putative ortholog of the yeast AP-3 mu subunit. In addition, loss-of-function mutants of other subunits of AP-3, ap-3beta and ap-3delta, also exhibit a suppressive effect on the zig-1 phenotype. Although these genes are also single-copy genes in the genome, the loss-of-function mutants of AP-3 grow normally. Our results suggest that AtVPS41 and AP-3 play roles in the proper function of the post-Golgi trafficking network and support membrane trafficking to vacuoles.

A SNARE complex unique to seed plants is required for protein storage vacuole biogenesis and seed development of Arabidopsis thaliana.

The SNARE complex is a key regulator of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. A functional SNARE complex consists of four coiled-coil helical bundles, three of which are supplied by Q-SNAREs and another from an R-SNARE. Arabidopsis thaliana VAMP727 is an R-SNARE, with homologs only in seed plants. We have found that VAMP727 colocalizes with SYP22/ VAM3, a Q-SNARE, on a subpopulation of prevacuolar compartments/endosomes closely associated with the vacuolar membrane. Genetic and biochemical analyses, including examination of a synergistic interaction of vamp727 and syp22 mutations, histological examination of protein localization, and coimmunoprecipitation from Arabidopsis lysates indicate that VAMP727 forms a complex with SYP22, VTI11, and SYP51 and that this complex plays a crucial role in vacuolar transport, seed maturation, and vacuole biogenesis. We suggest that the VAMP727 complex mediates the membrane fusion between the prevacuolar compartment and the vacuole and that this process has evolved as an essential step for seed development.

Conversion of functional specificity in Qb-SNARE VTI1 homologues of Arabidopsis.

In higher multicellular eukaryotes, highly specialized membrane structures or membrane trafficking events are required for supporting various physiological functions. SNAREs (soluble NSF attachment protein receptors) play an important role in specific membrane fusions. These protein receptors are assigned to subgroubs (Qa-, Qb-, Qc-, and R-SNARE) according to their specific SNARE structural motif. A specific set of Qa-, Qb-, and Qc-SNAREs, located on the target membrane, interact with R-SNARE on the vesicle to form a tight complex, leading to membrane fusion. The zig-1 mutant of Arabidopsis lacking Qb-SNARE VTI11 shows little shoot gravitropism and abnormal stem morphology. VTI11 and its homolog VTI12 exhibit partially overlapping but distinct intracellular localization and have different biological functions in plants. Little is known about how SNAREs are targeted to specific organelles, even though their functions and specific localization are closely linked. Here, we report that a novel mutation in VTI12 (zip1) was found as a dominant suppressor of zig-1. The zip1 mutation gave VTI12 the ability to function as VTI11 by changing both the specificity of SNARE complex formation and its intracellular localization. One amino acid substitution drastically altered VTI12, allowing it to suppress abnormalities of higher order physiological functions such as gravitropism and morphology. The zip1 mutation may be an indication of the flexibility in plant cell function afforded by gene duplication, particularly among the VTI11 genes and their recently diverged orthologs.

SGR2, a phospholipase-like protein, and ZIG/SGR4, a SNARE, are involved in the shoot gravitropism of Arabidopsis.

In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively. To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis. The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls. These genes probably are involved in the early step(s) of the gravitropic response. The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion. The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid-preferring phospholipase A1 containing a putative transmembrane domain. This gene family has been reported only in eukaryotes. The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport. Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism.