Transcriptomic analysis of developing seeds in a wheat (Triticum aestivum L.) mutant RSD32 with reduced seed dormancy.

Seed dormancy, a major factor regulating pre-harvest sprouting, can severely hinder wheat cultivation. Reduced Seed Dormancy 32 (RSD32), a wheat (Triticum aestivum L.) mutant with reduced seed dormancy, is derived from the pre-harvest sprouting tolerant cultivar, 'Norin61'. RSD32 is regulated by a single recessive gene and mutant phenotype expressed in a seed-specific manner. Gene expressions in embryos of 'Norin61' and RSD32 were compared using RNA sequencing (RNA-seq) analysis at different developmental stages of 20, 30, and 40 days after pollination (DAP). Numbers of up-regulated genes in RSD32 are equivalent in all developmental stages. However, down-regulated genes in RSD32 are more numerous on DAP20 and DAP30 than on DAP40. In central components affecting the circadian clock, homologues to the morning-expressed genes are expressed at lower levels in RSD32. However, higher expressions of homologues acting as evening-expressed genes are observed in RSD32. Homologues of Ca2+ signaling pathway related genes are specifically expressed on DAP20 in 'Norin61'. Lower expression is shown in RSD32. These results suggest that RSD32 mutation expresses on DAP20 and earlier seed developmental stages and suggest that circadian clock regulation and Ca2+ signaling pathway are involved in the regulation of wheat seed dormancy.

LARGE GRAIN Encodes a Putative RNA-Binding Protein that Regulates Spikelet Hull Length in Rice.

Grain size is a key determiner of grain weight, one of the yield components in rice (Oryza sativa). Therefore, to increase grain yield, it is important to elucidate the detailed mechanisms regulating grain size. The Large grain (Lgg) mutant, found in the nonautonomous DNA-based active rice transposon1 (nDart1)-tagged lines of Koshihikari, is caused by a truncated nDart1-3 and 355 bp deletion in the 5' untranslated region of LGG, which encodes a putative RNA-binding protein, through transposon display and cosegregation analysis between grain length and LGG genotype in F2 and F3. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9-mediated knockout and overexpression of LGG led to longer and shorter grains than wild type, respectively, showing that LGG regulates spikelet hull length. Expression of LGG was highest in the 0.6-mm-long young panicle and gradually decreased as the panicle elongated. LGG was also expressed in roots and leaves. These results show that LGG functions at the very early stage of panicle development. Longitudinal cell numbers of spikelet hulls of Lgg, knockout and overexpressed plants were significantly different from those of the wild type, suggesting that LGG might regulate longitudinal cell proliferation in the spikelet hull. RNA-Seq analysis of 1-mm-long young panicles from LGG knockout and overexpressing plants revealed that the expressions of many cell cycle-related genes were reduced in knockout plants relative to LGG-overexpressing plants and wild type, whereas some genes for cell proliferation were highly expressed in knockout plants. Taken together, these results suggest that LGG might be a regulator of cell cycle and cell division in the rice spikelet hull.

Transgenerational activation of an autonomous DNA transposon, Dart1-24, by 5-azaC treatment in rice.

KEY MESSAGE: Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.

Establishment of nDart1-tagged lines of Koshihikari, an elite variety of rice in Japan.

To utilize a transposon-tagged mutant as a breeding material in rice, an endogenous DNA transposon, nDart1-0, was introduced into Koshihikari by successive backcrossing together with aDart1-27, an active autonomous element. The founder line for nDart1-tagged lines of Koshihikari carried nDart1-0 on chromosome 9 and transposed nDart1-12s on chromosomes 1 and 8 and nDart1-3 on chromosome 11. In nDart1-tagged lines, there were the most abnormal phenotypic mutants and many aberrant chlorophyll mutants at seedling stage. At mature stage, many semi-sterile mutants were observed. Dwarf, reduced culm number and lesion mimic mutants were also found. In total, 43.2% of the lines segregated some phenotypic mutants. Thus, the nDart1-tagged lines of Koshihikari are expected to be potentially useful for screening stress-tolerant mutants under abiotic or biotic stress conditions.

Crystal Structure and Band-Gap Engineering of a Semiconducting Coordination Polymer Consisting of Copper(I) Bromide and a Bridging Acceptor Ligand.

A new semiconducting 3D coordination polymer, [Cu2Br2(ttz)] n (1), with an acceptor bridging ligand, 1,2,4,5-tetrazine (ttz), was synthesized. The complex shows large absorption bands extending to the near-IR region, indicating a small band gap in the coordination polymer. This complex shows higher conductivity than those of [CuBr(pyz)] n (2), including pyrazine (pyz) with a higher lowest unoccupied molecular orbital level. We performed density functional theory band calculations using the VASP program to understand the electronic states and conducting paths of the coordination polymer.

Easy sectioning of whole grain of rice using cryomicrotome.

To obtain a clear intact section of a ripened rice grain, which is suitable for biochemical and histological analysis, the Kawamoto method using a specific adhesive film was applied using a cryomicrotome. The longitudinal and sagittal sections were easily obtained together with the cross-section, and cell characteristics were clearly discerned in the ripened grain. It was demonstrated that the Kawamoto method is readily applicable for intact sectioning of hard tissue, including ripened grain. Intact section sampling may be useful for enzymatic analysis and transcriptomic analysis of plant tissue.

Amyloplast Membrane Protein SUBSTANDARD STARCH GRAIN6 Controls Starch Grain Size in Rice Endosperm.

Starch is a biologically and commercially important polymer of glucose. Starch is organized into starch grains (SGs) inside amyloplasts. The SG size differs depending on the plant species and is one of the most important factors for industrial applications of starch. There is limited information on genetic factors regulating SG sizes. In this study, we report the rice (Oryza sativa) mutant substandard starch grain6 (ssg6), which develops enlarged SGs in endosperm. Enlarged SGs are observed starting at 3 d after flowering. During endosperm development, a number of smaller SGs appear and coexist with enlarged SGs in the same cells. The ssg6 mutation also affects SG morphologies in pollen. The SSG6 gene was identified by map-based cloning and microarray analysis. SSG6 encodes a protein homologous to aminotransferase. SSG6 differs from other rice homologs in that it has a transmembrane domain. SSG6-green fluorescent protein is localized in the amyloplast membrane surrounding SGs in rice endosperm, pollen, and pericarp. The results of this study suggest that SSG6 is a novel protein that controls SG size. SSG6 will be a useful molecular tool for future starch breeding and applications.

SAD1, an RNA polymerase I subunit A34.5 of rice, interacts with Mediator and controls various aspects of plant development.

The DWARF14 (D14) gene of rice functions within the signaling pathway of strigolactones, a group of plant hormones that inhibits shoot branching. We isolated a recessive mutant named super apical dormant (sad1-1) from a suppressor screen of d14-1. The growth of tillers (vegetative shoot branches) is suppressed in both the d14-1 sad1-1 double mutant and the sad1-1 single mutant. In addition, the sad1-1 mutant shows pleiotropic defects throughout development. SAD1 encodes an ortholog of RPA34.5, a subunit of RNA polymerase I (Pol I). Consequently, the level of ribosomal RNA (rRNA) is severely reduced in the sad1-1 mutant. These results indicate that proper ribosome function is a prerequisite for normal development in plants. The Arabidopsis ortholog of SAD1 was previously isolated as a Mediator-interacting protein. Here we show that SAD1 interacts physically with the Mediator complex through direct binding with OsMED4, a component of the middle module of the Mediator complex in rice. It is known that Mediator interacts with Pol II, which transcribes mRNAs and functions as a central regulator of transcription. This study indicates a novel aspect of Mediator function in Pol I-controlled rRNA transcription. TFIIF2 and RPC53 are the counterparts of RPA34.5 in Pol II and Pol III, respectively. We demonstrate that the rice orthologs of these proteins also interact with OsMED4. Our results suggest that interaction with MED4 in the Mediator complex is a common feature of the three types of RNA polymerases.

Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes.

We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.

TAWAWA1, a regulator of rice inflorescence architecture, functions through the suppression of meristem phase transition.

Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant tawawa1-D, the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in TAWAWA1 (TAW1) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences. TAW1 encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs. TAW1 expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the SHORT VEGETATIVE PHASE subfamily of MADS-box genes function downstream of TAW1. We thus propose that TAW1 is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity.

Identification of QTLs for yield-related traits in RILs derived from the cross between pLIA-1 carrying Oryza longistaminata chromosome segments and Norin 18 in rice.

To improve rice yield, a wide genetic pool is necessary. It is therefore important to explore wild rice relatives. Oryza longistaminata is a distantly related wild rice relative that carries the AA genome. Its potential for improving agronomic traits is not well studied. Introgression line (pLIA-1) that carries Oryza longistaminata's chromosome segments, showed high performance in yield-related traits under non-fertilized conditions. Therefore, to illustrate Oryza longistaminata's potential for improving yield-related traits, RILs from the F1 of a cross between pLIA-1 and Norin 18 were developed and QTL analysis was done using the RAD-Seq method. In total, 36 QTLs for yield-related traits were identified on chromosomes 1, 2, 3, 5, 6, 7, 8, 10, and 11. Clusters of QTLs for strongly correlated traits were also identified on chromosomes 1, 3, 6, and 8. Phenotypic data from recombinant plants for chromosomes 1 and 8 QTL clusters revealed that the pLIA-1 genotype on chromosome 1 region was more important for panicle-related traits and a combination of pLIA-1 genotypes on chromosomes 1 and 8 showed a favorable phenotype under non-fertilized conditions. These results suggest that Oryza longistaminata's chromosome segments carry important alleles that can be used to improve yield-related traits of rice.

Geometrical Formation of Compound Starch Grains in Rice Implements Voronoi Diagram.

Starch forms transparent grains, called starch grains (SGs), in amyloplasts. One of the major morphological SG forms in Poaceae, called a compound SG, is formed by assemblies of small starch granules in an amyloplast. Starch granules assemble as a well-ordered structure; however, the mechanism that regulates this organization has not been identified. In this study, we examined how starch granules grow and converge into the final SG morphology. First, we found that the number of starch granules in an amyloplast is almost constant from the early developmental stage until endosperm maturity. Next, we quantitatively evaluated the geometrical similarities between starch granules and a Voronoi diagram, which is a mathematical tessellation of space based on the distance to a specific set of points in the space. The in silico growth simulation showed that the geometrical patterns of compound SGs resembling a Voronoi diagram is determined by physical interactions among the free-growing starch granules and the amyloplast envelope membrane. The geometrical similarity between compound SGs and a Voronoi diagram is likely a result of maximum loading and storage of starch in the amyloplast. The simulation described in this study provides a greater understanding of how compound SGs are formed and also has the potential to explain morphological variations of SGs.

Control of the Water Transport Activity of Barley HvTIP3;1 Specifically Expressed in Seeds.

Tonoplast intrinsic proteins (TIPs) are involved in the transport and storage of water, and control intracellular osmotic pressure by transporting material related to the water potential of cells. In the present study, we focused on HvTIP3;1 during the periods of seed development and desiccation in barley. HvTIP3;1 was specifically expressed in seeds. An immunochemical analysis showed that HvTIP3;1 strongly accumulated in the aleurone layers and outer layers of barley seeds. The water transport activities of HvTIP3;1 and HvTIP1;2, which also accumulated in seeds, were measured in the heterologous expression system of Xenopus oocytes. When they were expressed individually, HvTIP1;2 transported water, whereas HvTIP3;1 did not. However, HvTIP3;1 exhibited water transport activity when co-expressed with HvTIP1;2 in oocytes, and this activity was higher than when HvTIP1;2 was expressed alone. This is the first report to demonstrate that the water permeability of a TIP aquaporin was activated when co-expressed with another TIP. The split-yellow fluorescent protein (YFP) system in onion cells revealed that HvTIP3;1 interacted with HvTIP1;2 to form a heterotetramer in plants. These results suggest that HvTIP3;1 functions as an active water channel to regulate water movement through tissues during the periods of seed development and desiccation.

A Mutation in GIANT CHLOROPLAST Encoding a PARC6 Homolog Affects Spikelet Fertility in Rice.

Chloroplasts are not generated de novo but proliferate from a pre-existing population of plastids present in meristematic cells. Chloroplast division is executed by the co-ordinated action of at least two molecular machineries: internal machinery located on the stromal side of the inner envelope membrane and external machinery located on the cytosolic side of the outer envelope membrane. To date, molecular studies of chloroplast division in higher plants have been limited to several species such as Arabidopsis. To elucidate chloroplast division in rice, we performed forward genetics and isolated a mutant displaying large chloroplasts among an ethyl methanesulfonate (EMS)-mutagenized Oryza sativa spp japonica Nipponbare population. Using a map-based approach, this mutation, termed giant chloroplast (gic), was allocated in a gene that encodes a protein that is homologous to Paralog of ARC6 (PARC6), which is known to play a role in chloroplast division. GIC is unique in that it has a long C-terminal extension that is not present in other PARC6 homologs. Characterization of gic phenotypes in a rice field showed that gic exhibited defective growth in seed setting, suggesting that the gic mutant negatively affects the reproductive stage. This report is the first describing a chloroplast division mutant in monocotyledons and its effect on plant development.

Amyloplast-localized SUBSTANDARD STARCH GRAIN4 protein influences the size of starch grains in rice endosperm.

Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology.

Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

BACKGROUND: High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. RESULTS: High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. CONCLUSION: Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis.

A mutable albino allele in rice reveals that formation of thylakoid membranes requires the SNOW-WHITE LEAF1 gene.

Active DNA transposons are important tools for gene functional analysis. The endogenous non-autonomous transposon, nDart1-0, in rice (Oryza sativa L.) is expected to generate various transposon-insertion mutants because nDart1-0 elements tend to insert into genic regions under natural growth conditions. We have developed a specific method (nDart1-0-iPCR) for efficient detection of nDart1-0 insertions and successfully identified the SNOW-WHITE LEAF1 (SWL1) gene in a variegated albino (swl1-v) mutant obtained from the nDart1-promoted rice tagging line. The variegated albino phenotype was caused by insertion and excision of nDart1-0 in the 5'-untranslated region of the SWL1 gene predicted to encode an unknown protein with the N-terminal chloroplast transit peptide. SWL1 expression was detected in various rice tissues at different developmental stages. However, immunoblot analysis indicated that SWL1 protein accumulation was strictly regulated in a tissue-specific manner. In the swl1 mutant, formations of grana and stroma thylakoids and prolamellar bodies were inhibited. This study revealed that SWL1 is essential for the beginning of thylakoid membrane organization during chloroplast development. Furthermore, we provide a developmental perspective on the nDart1-promoted tagging line to characterize unidentified gene functions in rice.

A copper(I) thiocyanate-based photoresponsive semiconducting 2D coordination polymer.

A coordination polymer, [Cu(SCN)(iqi)]n (iqi = isoquinoline), containing copper(I) thiocyanate and a nitrogen-containing π-conjugated ligand, iqi, has been synthesized and its physical properties were evaluated. This coordination polymer has a two-dimensional (2D) sheet structure consisting of copper(I) thiocyanate and shows photoluminescence derived from 3MLCT and photoconductive properties.

ABERRANT PANICLE ORGANIZATION 2/RFL, the rice ortholog of Arabidopsis LEAFY, suppresses the transition from inflorescence meristem to floral meristem through interaction with APO1.

The temporal and spatial control of meristem identity is a key element in plant development. To better understand the molecular mechanisms that regulate inflorescence and flower architecture, we characterized the rice aberrant panicle organization 2 (apo2) mutant which exhibits small panicles with reduced number of primary branches due to the precocious formation of spikelet meristems. The apo2 mutants also display a shortened plastochron in the vegetative phase, late flowering, aberrant floral organ identities and loss of floral meristem determinacy. Map-based cloning revealed that APO2 is identical to previously reported RFL gene, the rice ortholog of the Arabidopsis LEAFY (LFY) gene. Further analysis indicated that APO2/RFL and APO1, the rice ortholog of Arabidopsis UNUSUAL FLORAL ORGANS, act cooperatively to control inflorescence and flower development. The present study revealed functional differences between APO2/RFL and LFY. In particular, APO2/RFL and LFY act oppositely on inflorescence development. Therefore, the genetic mechanisms for controlling inflorescence architecture have evolutionarily diverged between rice (monocots) and Arabidopsis (eudicots).

Two WUSCHEL-related homeobox genes, narrow leaf2 and narrow leaf3, control leaf width in rice.

Leaf shape is one of the key determinants of plant architecture. Leaf shape also affects the amount of sunlight captured and influences photosynthetic efficiency; thus, it is an important agronomic trait in crop plants. Understanding the molecular mechanisms governing leaf shape is a central issue of plant developmental biology and agrobiotechnology. Here, we characterized the narrow-leaf phenotype of FL90, a linkage tester line of rice (Oryza sativa). Light and scanning electron microscopic analyses of FL90 leaves revealed defects in the development of marginal regions and a reduction in the number of longitudinal veins. The narrow-leaf phenotype of FL90 shows a two-factor recessive inheritance and is caused by the loss of function of two WUSCHEL-related homeobox genes, NAL2 and NAL3 (NAL2/3), which are duplicate genes orthologous to maize NS1 and NS2 and to Arabidopsis PRS. The overexpression of NAL2/3 in transgenic rice plants results in wider leaves containing increased numbers of veins, suggesting that NAL2/3 expression regulates leaf width. Thus, NAL2/3 can be used to modulate leaf shape and improve agronomic yield in crop plants.