Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] functions as a site-specific signal on membranes to promote cytoskeletal reorganization and membrane trafficking. Localization of PtdIns(4,5)P 2 to apices of growing root hairs and pollen tubes suggests that it plays an important role in tip growth. However, its regulation and mode of action remain unclear. We found that Arabidopsis thaliana PIP5K3 (for Phosphatidylinositol Phosphate 5-Kinase 3) encodes a phosphatidylinositol 4-phosphate 5-kinase, a key enzyme producing PtdIns(4,5)P 2 , that is preferentially expressed in growing root hairs. T-DNA insertion mutations that substantially reduced the expression of PIP5K3 caused significantly shorter root hairs than in the wild type. By contrast, overexpression caused longer root hairs and multiple protruding sites on a single trichoblast. A yellow fluorescent protein (YFP) fusion of PIP5K3, driven by the PIP5K3 promoter, complemented the short-root-hair phenotype. PIP5K3-YFP localized to the plasma membrane and cytoplasmic space of elongating root hair apices, to growing root hair bulges, and, notably, to sites about to form root hair bulges. The signal was greatest in rapidly growing root hairs and quickly disappeared when elongation ceased. These results provide evidence that PIP5K3 is involved in localizing PtdIns(4,5)P 2 to the elongating root hair apex and is a key regulator of the machinery that initiates and promotes root hair tip growth.

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

SummaryLaser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.

CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5′-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.

We characterized a rice monoculm mutant moc2, which showed significantly reduced tiller numbers, pale-green leaves, a reduced growth rate, and a consequent dwarf phenotype. The monoculm feature was attributed to a deficiency in the efficient outgrowth of tiller buds, although the moc2 mutant produced tiller buds. Inconsistent change was observed in the expression of genes involved in tiller bud outgrowth, suggesting that the moc2 mutant has a defective function necessary for the tiller bud outgrowth. The gene responsible for the moc2 mutant was mapped to a locus encoding cytosolic fructose-1,6-bisphosphatase 1 (FBP1), in which a Tos17 retrotransposon was inserted in exon 4. Reverse-transcription PCR for the FBP1 gene amplified a shorter transcript from the moc2 mutant than from the wild-type plant. The sequence of the shorter transcript revealed a deletion of exon 4 by abnormal splicing, and the resulting frameshift generated a new translation termination signal. The moc2 mutant showed a very low level of FBPase activity, suggesting that it involves a loss-of-function mutation of FBP1. Cytosolic FBPase is considered a key enzyme in the sucrose biosynthesis pathway. Defective FBPase activity is anticipated to lead a shortage of sucrose supply, which probably causes the inhibition of tiller bud outgrowth in the moc2 mutant. The monoculm phenotype of the moc2 mutant supports the idea that sucrose supply may be an important cue to outgrow tiller buds.

Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.

Dihydrosphingosine C4 hydroxylase is a key enzyme in the biosynthesis of phytosphingosine, a major constituent of sphingolipids in plants and yeasts. The rice genome contains five homologue genes for dihydrosphingosine C4 hydroxylase, DSH1-DSH5, whose gene products show high degrees of homology to the yeast counterpart, SUR2. Among them, expression of DSH1, DSH2 and DSH4 was detected, and DSH1 and DSH4 complement the yeast sur2 mutation. The DSH1 gene was specifically and abundantly expressed in vascular bundles and apical meristems. In particular, very strong expression was detected in the stigmas of flowers. Repression of DSH1 expression by the antisense gene or RNA interference (RNAi) resulted in a severe reduction of fertility. In the transformants in which DSH1 expression was suppressed, significantly increased expression of DSH2 was found in leaves but not in pistils, suggesting that there was tissue-specific correlation between DSH1 and DSH2 expression. Our results indicate that the product of DSH1 may be involved in plant viability or reproductive processes, and that the phenotype of sterility is apparently caused by loss of function of DSH1 in the stigma. It is also suggested that there is a complex mechanism controlling the tissue-specific expression of the DSH1 gene.

Thanatin is an antimicrobial peptide with a strong and wide-ranging antimicrobial spectrum, including certain species of fungi and Gram-negative and -positive bacteria. To evaluate the application of thanatin to the generation of disease-resistant plants, we introduced a synthetic thanatin gene into rice. Several transformants that expressed the introduced gene showed significant level of antimicrobial activity. The substances showing antimicrobial activity were partially purified from these transformants and their properties were determined. The molecule with characteristics similar to those of native thanatin on the elution pattern in HPLC analysis had an identical molecular mass to that of native molecule. It should also be noted that the transformant acquired a sufficient level of resistance to the rice blast fungus, Magnaporthe oryzae, presumably due to the repressive activity of thanatin to its initial stage of infection. This result demonstrates that thanatin has antifungal activity for M. oryzae and that the introduction of the thanatin gene into rice is effective in generating a plant resistant to rice blast disease.