SummaryIn cultivated rice two wild-type alleles, Wx a and Wx b , predominate at the waxy locus, which encodes granulebound starch synthase. The activity of Wx a is 10-fold higher than that of Wx b at the level of both protein and mRNA.Wx b has a ϩ1G to T mutation at the 5Ј splice site of the first intron. Sequence analysis of Wx b transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5Ј splice sites of both Wx a and Wx b , fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wx a reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wx b restored GUS activity and the level of spliced RNA to that of wild-type Wx a . These results demonstrated that the low level expression of Wx b results from a single base mutation at the 5Ј splice site of the first intron. It is of interest that the Wx b allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.

The quantitative trait locus controlling the number of primary rachis branches (PRBs) in rice was identified using backcrossed inbred lines of Sasanishiki/Habataki//Sasanishiki///Sasanishiki. The resultant gene was ABERRANT PANICLE ORGANIZATION 1 (APO1). Habataki-genotype segregated reciprocal recombinant lines for the APO1 locus increased both the number of PRB (12-13%) and the number of grains per panicle (9-12%), which increased the grain yield per plant (5-7%). Further recombination dividing this region revealed that different alleles regulated the number of PRB and the number of grains per panicle. The PRB1 allele, which includes the APO1 open reading frame (ORF) and the proximal promoter region, controlled only the number of PRB but not the number of grains per panicle. In contrast, the HI1 allele, which includes only the distal promoter region, increased the grain yield and harvest index in Habataki-genotype plants, nevertheless, the ORF expressed was Sasanishiki type. It also increased the number of large vascular bundles in the peduncle. APO1 expression occurred not only in developing panicles but also in the developing vascular bundle systems. In addition, Habataki plants displayed increased APO1 expression in comparison to Sasanishiki plants. It suggests that APO1 enhances the formation of vascular bundle systems which, consequently, promote carbohydrate translocation to panicles. The HI1 allele is suggested to regulate the amount of APO1 expression, and thereby control the development of vascular bundle systems. These findings may be useful to improve grain yield as well as quality through the improvement of translocation efficiency.

SummaryIn rice, silencing of the aleurone-specific Ltp2-gus transgene, causing easily detectable staining patterns on the grain surface, offers a convenient tool to study quantitative aspects of gene silencing in monocots. In this paper we analyzed phenotypes, occurrence, inheritance and environmental effects on the silencing. We also report on the cloning of transgenes, determination of their structure and analysis of transcripts from the transgene loci. The results show that various patterns of silencing appeared in the R2 generation at which most of the transgenes became homozygous and that they were inherited for five generations. In addition, silencing independently occurred in three generations and reversion to full expression was also found. Cloning of transgenes from a silenced L3.3 line demonstrated that this line carried two transgene loci: one carried an intact Ltp2-gus gene and the other carried a rearranged transgene in which part of the gus gene was in the antisense orientation. Analysis of gus transcripts indicated that partial antisense RNA derived from the rearranged transgene was present in silenced lines and was polyadenylated but that it was absent in non-silenced lines. RNA analyses suggested that the Ltp2-gus silencing in the aleurone layer was post-transcriptional and that it may be caused by interaction of partial antisense gus transcripts with normal sense transcripts. Possible involvement of antisense transcripts in post-transcriptional silencing is discussed.

An Ltp2 promoter was isolated from barley as an aleurone-specific promoter, and its tissue specificity was maintained in transgenic rice. Expression of the Ltp2-gus gene in rice could be detected by X-gluc staining of the seeds. Previously, we reported the isolation of silenced plants in the R2 generation and the involvement of antisense gus transcripts in aleurone-specific Ltp2-gus gene silencing in transgenic rice, the L3.3 line. In the current study, we analyzed the L0.1 line, a sibling of the L3.3 line, and the partially revertant line from the L0.1 line accompanied by a transgene structural change. Strong silencing of the Ltp2-gus gene was detected over five generations in the L0.1 line. DNA and RNA analysis revealed that there were incomplete transgenes and that several aberrant RNAs that carried an antisense gus region were expressed in the L0.1 line. Determination of the transgene structure in the L0.1 line revealed that the partial antisense gus gene and the partial Ltp2 promoter region fused to the Ltp2 promoter were lacking in the revertants. RNA analysis demonstrated that the antisense gus and the promoter transcripts were produced and found in the poly(A)- fraction. Results of these analyses suggested that the observed aberrant transcripts, including antisense and promoter transcripts, were associated with Ltp2-gus silencing in the L0.1 line.

Prohydrojasmon (PDJ) is a synthetic derivative of the plant hormone jasmonic acid (JA), which is used in apples, grapes, and mandarin oranges as a plant growth regulator. Here, we show that irrigation of PDJ could prevent spindly growth of rice seedlings in both indica and japonica cultivars. PDJ also inhibited root elongation in hydroponically growing seedlings in various cultivars. The higher dry root weight following PDJ application than the controls was observed in two japonica cultivars. Furthermore, its reduction was slight in comparison with the inhibition of root elongation. Starch degradation in the endosperm of seedlings grown in PDJ solution was slower than those grown in the control. The inhibition of root elongation could be mediated by the combined application of PDJ and appropriate concentrations of a synthetic auxin, NAA (1-Naphthaleneacetic acid). Additionally, we generated transgenic rice carrying sGFP driven by the PBZ1 promoter, a marker gene for abiotic and biotic stress responses. The expression of PBZ1p:: sGFP was induced by PDJ treatment in the roots and seeds. PDJ could be used as a plant growth regulator to prevent spindly growth and to induce stress responses in rice seedlings.

BackgroundReactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants.MethodsWe used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2).ResultsWe identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells.ConclusionsThe findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-017-0115-3) contains supplementary material, which is available to authorized users.

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Prohydrojasmon (PDJ), a synthetic derivative of the plant hormone jasmonic acid, has been identified as a plant growth regulator used in various fruit and rice seedlings. In this study, we demonstrate the inhibition of transpiration by PDJ in the indica rice cultivar cv. Takanari compared with commercially available microcrystalline and paraffin wax agents in a greenhouse. Microcrystalline and paraffin wax agents inhibited approximately 30%-50% transpiration 3 h after application, and the inhibition lasted for seven days after treatment, depending on the concentration and the agent used. In contrast, PDJ inhibited approximately 40%-80% transpiration compared with control 0.5 h after treatment, and the effect lasted for one day. However, the effect almost disappeared 7 days after treatment. Thus, PDJ affects transpiration differently than commercially available products and could be used to lower excess transpiration in rice, specifically when an early, transient, and reversible inhibition is needed.