Seed coat pigmentation in transgenic soybean expressing the silencing suppressor 2b gene of Cucumber mosaic virus.

KEY MESSAGE: Soybean expressing the Cucumber mosaic virus 2b gene manifests seed coat pigmentation due to suppression of endogenous RNA silencing but no other morphological abnormality. This gene may help prevent transgene silencing. RNA silencing is an important mechanism for gene regulation and antiviral defense in plants. It is also responsible for transgene silencing, however, and thus hinders the establishment of transgenic plants. The 2b protein of Cucumber mosaic virus (CMV) functions as a suppressor of RNA silencing and therefore might prove beneficial for stabilization of transgene expression. We have now generated transgenic soybean that harbors the 2b gene of a CMV-soybean strain under the control of a constitutive promoter to investigate the effects of 2b expression. No growth abnormality was apparent in 2b transgenic plants, although the seed coat was pigmented in several of the transgenic lines. Genes for chalcone synthase (CHS), a key enzyme of the flavonoid pathway, are posttranscriptionally silenced by the inhibitor (I) locus in nonpigmented (yellow) soybean seeds. The levels of CHS mRNA and CHS small interfering RNA in strongly pigmented 2b transgenic seed coats were higher and lower, respectively, than those in the seed coat of a control transgenic line. The expression level of 2b also correlated with the extent of seed coat pigmentation. On the other hand, introduction of the 2b gene together with the DsRed2 gene into somatic embryos prevented the time-dependent decrease in transient DsRed2 expression. Our results indicate that the 2b gene alone is able to suppress RNA silencing of endogenous CHS genes regulated by the I locus, and that 2b is of potential utility for stabilization of transgene expression in soybean without detrimental effects other than seed coat pigmentation.

Suppressive mechanism of seed coat pigmentation in yellow soybean.

In soybean seeds, numerous variations in colors and pigmentation patterns exist, most of which are observed in the seed coat. Patterns of seed coat pigmentation are determined by four alleles (I, i(i), i(k) and i) of the classically defined I locus, which controls the spatial distribution of anthocyanins and proanthocyanidins in the seed coat. Most commercial soybean cultivars produce yellow seeds with yellow cotyledons and nonpigmented seed coats, which are important traits of high-quality seeds. Plants carrying the I or i(i) allele show complete inhibition of pigmentation in the seed coat or pigmentation only in the hilum, respectively, resulting in a yellow seed phenotype. Classical genetic analyses of the I locus were performed in the 1920s and 1930s but, until recently, the molecular mechanism by which the I locus regulated seed coat pigmentation remained unclear. In this review, we provide an overview of the molecular suppressive mechanism of seed coat pigmentation in yellow soybean, with the main focus on the effect of the I allele. In addition, we discuss seed coat pigmentation phenomena in yellow soybean and their relationship to inhibition of I allele action.

Accumulation of Potato spindle tuber viroid-specific small RNAs is accompanied by specific changes in gene expression in two tomato cultivars.

To better understand the biogenesis of viroid-specific small RNAs and their possible role in disease induction, we have examined the accumulation of these small RNAs in potato spindle tuber viroid (PSTVd)-infected tomato plants. Large-scale sequence analysis of viroid-specific small RNAs revealed active production from the upper portion of the pathogenicity and central domains, two regions previously thought to be underrepresented. Profiles of small RNA populations derived from PSTVd antigenomic RNA were more variable, with differences between infected Rutgers (severe symptoms) and Moneymaker (mild symptoms) plants pointing to possible cultivar-specific differences in small RNA synthesis and/or stability. Using microarray analysis, we monitored the effects of PSTVd infection on the expression levels of >100 tomato genes containing potential binding sites for PSTVd small RNAs. Of 18 such genes down-regulated early in infection, two genes involved in gibberellin or jasmonic acid biosynthesis contain binding sites for PSTVd small RNAs in their respective ORFs.

Endogenous RNA interference of chalcone synthase genes in soybean: formation of double-stranded RNA of GmIRCHS transcripts and structure of the 5' and 3' ends of short interfering RNAs.

In yellow soybean, seed coat pigmentation is inhibited via endogenous RNA interference (RNAi) of the chalcone synthase (CHS) genes. Genetic studies have shown that a single dominant gene, named the I gene, inhibits pigmentation over the entire seed coat in soybean. We previously isolated a candidate for the I gene from the yellow soybean genome with the I/I genotype, and designated it GmIRCHS. A structural feature of GmIRCHS is a perfect inverted repeat of the pseudoCHS gene lacking 5'-coding region. This suggests that the double-stranded RNA (dsRNA) structure of the pseudoCHS gene may be formed in the GmIRCHS transcript. RNAi is triggered by the dsRNA for a target gene, so the GmIRCHS transcript is likely to be a trigger for RNAi of CHS genes. In this study, we identified a 1087-bp dsRNA, including pseudoCHS region ranging from most of exon 2 to 3'-UTR, in the GmIRCHS transcript. Interestingly, this dsRNA was detected not only in the seed coat but also in the cotyledon and leaf tissues. Previously, CHS RNAi has been shown to be restricted to the seed coat, and we reported that endogenous short interfering RNAs of CHS genes (CHS siRNAs) are detected only in the seed coat and not in the cotyledon and leaf tissues. Taken together with these previous reports, our result suggests that seed-coat specificity of CHS RNAi may be determined in the amplification step of CHS siRNAs rather than dsRNA formation in the GmIRCHS transcript. Our studies further revealed that CHS siRNAs are modified at the 3' ends and bear 5' monophosphorylated ends, suggesting that CHS siRNA duplexes are generated by Dicer-like enzyme from CHS dsRNA and subsequently modified at the 3' ends for stabilizing CHS siRNAs.

Variation of GmIRCHS (Glycine max inverted-repeat CHS pseudogene) is related to tolerance of low temperature-induced seed coat discoloration in yellow soybean.

In yellow soybean, seed coat pigmentation is inhibited by post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. A CHS cluster named GmIRCHS (Glycine max inverted-repeat CHS pseudogene) is suggested to cause PTGS in yellow-hilum cultivars. Cold-induced seed coat discoloration (CD), a commercially serious deterioration of seed appearance, is caused by an inhibition of this PTGS upon exposure to low temperatures. In the highly CD-tolerant cultivar Toyoharuka, the GmIRCHS structure differs from that of other cultivars. The aim of this study was to determine whether the variation of GmIRCHS structure among cultivars is related to variations in CD tolerance. Using two sets of recombinant inbred lines between Toyoharuka and CD-susceptible cultivars, we compared the GmIRCHS genotype and CD tolerance phenotype during low temperature treatment. The GmIRCHS genotype was related to the phenotype of CD tolerance. A QTL analysis around GmIRCHS showed that GmIRCHS itself or a region located very close to it was responsible for CD tolerance. The variation in GmIRCHS can serve as a useful DNA marker for marker-assisted selection for breeding CD tolerance. In addition, QTL analysis of the whole genome revealed a minor QTL that also affected CD tolerance.

Molecular mechanism of seed coat discoloration induced by low temperature in yellow soybean.

Seed coat pigmentation is inhibited in yellow soybean. The I gene inhibits pigmentation over the entire seed coat. In yellow soybean, seed coat discoloration occurs when plants are exposed to low temperatures after the onset of flowering, a phenomenon named 'cold-induced discoloration (CD)'. Inhibition of seed coat pigmentation results from post-transcriptional gene silencing (PTGS) of the chalcone synthase (CHS) genes. PTGS is a sequence-specific RNA degradation mechanism in plants and occurs via short interfering RNAs (siRNAs). Similar post-transcriptional suppression is called RNAi (RNA interference) in animals. Recently, we identified a candidate of the I gene designated GmIRCHS. In this study, to elucidate the molecular mechanism of CD, CHS mRNA and siRNA levels in the seed coat were compared between CD-sensitive and CD-tolerant cultivars (Toyomusume and Toyoharuka, respectively). In Toyomusume, the CHS siRNA level was reduced markedly by low temperature treatment, and subsequently the CHS mRNA level increased rapidly after treatment. In contrast, low temperature treatment did not result in severe reduction of the CHS siRNA level in Toyoharuka, and the CHS mRNA level did not increase after the treatment. These results suggest that the rapid increase in CHS mRNA level after low temperature treatment may lead to enhanced pigmentation in some of the seed coat cells and finally in seed coat discoloration. Interestingly, we found a Toyoharuka-specific difference in the GmIRCHS region, which may be involved in CD tolerance.