Motoki Kanekatsu

Proteomic Analysis of Embryonic Proteins Synthesized from Long-Lived mRNAs During Germination of Rice Seeds

Dry seeds contain translatable, long-lived mRNAs that are stored during seed maturation. Early studies using transcriptional inhibitors supported the view that protein synthesis during the initial phase of germination occurs on long-lived mRNA templates. Rice seeds were treated with the transcriptional inhibitor actinomycin D (Act D), and the embryonic proteins translated from long-lived mRNAs during germination were identified using a proteomic analysis. De novo transcription was not required for germination of rice seeds, since >80% of seeds germinated when transcription was prevented by treatment with Act D. In contrast, germination was completely inhibited in the presence of cycloheximide, an inhibitor of translation. Thus, de novo protein synthesis is necessary for germination of rice seeds. The proteomic analysis revealed that 20 proteins are up-regulated during germination, even after Act D treatment. Many of the up-regulated proteins are involved in carbohydrate metabolism and cytoskeleton formation. These results indicate that some of the germination-specific proteins involved in energy production and maintenance of cell structure in rice seeds are synthesized from long-lived mRNAs. The timing of translation of eight up-regulated proteins was clearly later than that of the other up-regulated proteins under conditions in which transcription was inhibited by Act D, suggesting that translation of long-lived mRNAs in rice seeds is regulated according to the germination phase.

The β subunit of chloroplast ATP synthase (CF0CF1-ATPase) is phosphorylated by casein kinase II

We studied a phosphate acceptor for casein kinase II (CK‐II) in chloroplasts, and found a 56 kDa protein (p56) as an acceptor, which was partially purified from the stroma of spinach chloroplasts. The N‐terminal amino acid sequence of p56 was identical with that of the β subunit of chloroplast ATP synthase (CF0CF1‐ATPase). In addition, the recombinant β subunit of CF1 was phosphorylated when the subunit was incubated with CK‐II. These results suggest that the β subunit of CF1 is a substrate protein of CK‐II in the chloroplast.

Homologs of genes associated with programmed cell death in animal cells are differentially expressed during senescence of Ipomoea nil petals

In senescent petals of Ipomoea nil, we investigated the expression of genes showing homology to genes involved in animal programmed cell death (PCD). Three encoded proteins were homologous to apoptotic proteins in animals: Bax inhibitor-1 (BI-1), a vacuolar processing enzyme (VPE; homologous to caspases) and a monodehydroascorbate reductase [MDAR; homologous to apoptosis-inducing factor (AIF)]. AIFs harbor an oxidoreductase domain and an apoptotic domain. MDARs exhibit homology to the AIF oxidoreductase domain, not to the apoptotic domain. The three other genes studied relate to autophagy. They encode homologs to vacuolar protein sorting 34 (VPS34) and to the Arabidopsis autophagy-related proteins 4b and 8a (ATG4b and ATG8a). The transcript abundance of MDAR decreased continuously, whereas that of the other genes studies exhibited a transient increase, except ATG4b whose abundance stayed high after an increase. Treatment with ethylene advanced the time to visible petal senescence, and hastened the changes in expression of each of the genes studied. In order to assess the role of VPS34 in petal senescence, we studied the effect of its inhibitor 3-methyladenine (3-MA). 3-MA reduced the time to visible petal senescence, and also accelerated the time to DNA degradation. Remarkably, 3-MA increased the time to nuclear fragmentation, indicating that the time to visible petal senescence was independent of nuclear fragmentation. The data on 3-MA might suggest the idea that autophagy is not a cause of PCD, but part of the remobilization process.

Increased lodging resistance in long-culm, low-lignin gh2 rice for improved feed and bioenergy production.

Lignin modification has been a breeding target for the improvements of forage digestibility and energy yields in forage and bioenergy crops, but decreased lignin levels are often accompanied by reduced lodging resistance. The rice mutant gold hull and internode2 (gh2) has been identified to be lignin deficient. GH2 has been mapped to the short arm of chromosome 2 and encodes cinnamyl-alcohol dehydrogenase (CAD). We developed a long-culm variety, ‘Leaf Star’, with superior lodging resistance and a gh phenotype similar to one of its parents, ‘Chugoku 117’. The gh loci in Leaf Star and Chugoku 117 were localized to the same region of chromosome 2 as the gh2 mutant. Leaf Star had culms with low lignin concentrations due to a natural mutation in OsCAD2 that was not present in Chugoku 117. However, this variety had high culm strength due to its strong, thick culms. Additionally, this variety had a thick layer of cortical fiber tissue with well-developed secondary cell walls. Our results suggest that rice can be improved for forage and bioenergy production by combining superior lodging resistance, which can be obtained by introducing thick and stiff culm traits, with low lignin concentrations, which can be obtained using the gh2 variety.

Proteomic analysis of RNA-binding proteins in dry seeds of rice after fractionation by ssDNA affinity column chromatography

In proteomic analysis, one of the major limitations is the detection of low-abundance proteins. To detect low-abundance RNA-binding proteins in mature dry seeds of rice, fractionation by single stranded DNA (ssDNA) affinity column chromatography was carried out before analysis by two-dimensional gel electrophoresis (2-DE). Proteomic analysis of the ssDNA-binding fraction revealed the existence of three types of RNA-binding proteins, including a K homology (KH) domain containing protein, a putative RNA-binding protein and a glycine-rich RNA-binding protein, in mature seeds. In addition, decreases in the putative RNA-binding protein and glycine-rich RNA-binding protein after absorbing water in seeds appear to be associated with seed germination.

Casein kinase 2 during early embryonic development in silkworm Bombyx mori: cDNA sequence, gene expression, and enzyme activity

To clarify the property of casein kinase 2 (CK2) during early embryonic development in the silkworm, we compared the phosphorylation activities of CK2 in non-diapause and diapause eggs until 60 h after oviposition. In nondiapause eggs, the phosphorylated signals were found at each stage and became progressively stronger through each stage. On the other hand, in diapause eggs, the strongest phosphorylated signals were found at approximately 12 to 24 h after oviposition and became progressively weaker through each stage. To clarify the control mechanism of these enzyme activities, we tried to clone cDNAs encoding α- and β-subunit of CK2 and analyze the gene expressions. The deduced amino acid sequence of the isolated cDNAs comprised 342 and 220 residues, and these sequences showed 85–90% identities to the α- and β-subunit of CK2 in Spodoptera frugiperda. RT-PCR indicated that these genes were expressed in nondiapause and diapause eggs. However, these genes expressions were not parallel with the changes in CK2 activity. These results suggest that the changes in CK2 activity are regulated mainly at the level of post-transcription during embryonic development in Bombyx mori.

Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice

Highlight Long-lived mRNAs stored in dry seed are translated after imbibition for germination. We report accumulation of long-lived mRNAs in developing rice embryos and candidates of the mRNAs required for germination.

Application of Digital Image Analysis System for Fine Evaluation of Varietal Differences and the Role of Ethylene in Visible Petal Senescence of Morning Glory

We detected differences in both onset and progression of visible petal senescence among morning glory cultivars by application of a digital image analysis system. The system is based on semiautomated time-lapse measurement of corolla areas. The system could also be applied to evaluate the effects of ethylene and its inhibitor on visible petal senescence. Both onset and progression of visible petal senescence were accelerated by ethylene treatment in all six cultivars tested. Treatment with aminooxyacetic acid (AOA), an ethylene biosynthesis inhibitor, prolonged time to onset of visible petal senescence in three of the six tested cultivars. In contrast, AOA treatment had no effect on duration of visible petal senescence in any tested cultivars. These data suggested differences among morning glory cultivars in the role of endogenous ethylene in controlling onset of visible petal senescence. In addition, we propose a new application of image analysis to fine quantification of time-lapse changes in the shape of plant organs.

Removal of redundant contigs from de novo RNA-Seq assemblies via homology search improves accurate detection of differentially expressed genes

BackgroundFor plant species with unsequenced genomes, cDNA contigs created by de novo assembly of RNA-Seq reads are used as reference sequences for comparative analysis of RNA-Seq datasets and the detection of differentially expressed genes (DEGs). Redundancies in such contigs are evident in previous RNA-Seq studies, and such redundancies can lead to difficulties in subsequent analysis. Nevertheless, the effects of removing redundancy from contig assemblies on comparative RNA-Seq analysis have not been evaluated.ResultsHere we describe a method for removing redundancy from raw contigs that were primarily created by de novo assembly of Arabidopsis thaliana RNA-Seq reads. Specifically, the contigs with the highest bit scores were selected from raw contigs by a homology search against the gene dataset in the TAIR10 database. The two existing methods for removal of redundancy based on contig length or clustering analysis used to eliminate redundancies from raw contigs. Contig number was reduced most effectively with the method based on homology search. In a comparative analysis of RNA-Seq datasets, DEGs detected in contigs that underwent redundancy removal via the homology search method showed the highest identity to the DEGs detected when the TAIR10 gene dataset was used as an exact reference. Redundancy in raw contigs could also be removed by a homology search against integrated protein datasets from several plant species other than A. thaliana. DEGs detected using contigs that underwent such redundancy-removed also showed high homology to DEGs detected using the TAIR10 gene dataset.ConclusionHere we describe a method for removing redundant contigs within raw contigs; this method involves a homology search against a gene or protein database. In principal, this method can be used with unsequenced plant genomes that lack a well-developed gene database. Redundant contigs were not removed adequately via either of two existing methods, but our method allowed for removal of all redundant contigs. To our knowledge, this is the first reported improvement in accurate detection of DEGs via comparative RNA-Seq analysis that involved preparation of a non-redundant reference sequence. This method could be used to rapidly and cost-effectively detect useful genes in unsequenced plants.

Expression of an AtNAP gene homolog in senescing morning glory (Ipomoea nil) petals of two cultivars with a different flower life span.

AtNAP, a NAC family transcription factor, has been shown to promote leaf senescence in Arabidopsis. We isolated an AtNAP homolog in morning glory (Ipomoea nil), designated InNAP, and investigated its expression during petal senescence. We used two cultivars, one showing a normal short flower life span (cv. Peking Tendan) and another a longer life span (cv. Violet). InNAP was highly expressed in both cultivars. Expression was high before that of the senescence marker gene InSAG12. InNAP and InSAG12 expression was high in cv. Peking Tendan before cv. Violet. The expression of both genes was therefore temporally related to the onset of the visible senescence symptoms. An inhibitor of ethylene action (silver thiosulphate, STS) delayed petal senescence in cv. Peking Tendan but had no effect in cv. Violet. STS treatment had no clear effect on the InNAP expression in petals of both cultivars, suggesting that endogenous ethylene may not be necessary for its induction. These data suggest the hypothesis that InNAP plays a role in petal senescence, independent of the role of endogenous ethylene.