Most commercial Glycine max (soybean) varieties have yellow seeds because of loss of pigmentation in the seed coat. It has been suggested that inhibition of seed coat pigmentation in yellow G. max may be controlled by homology-dependent silencing of chalcone synthase (CHS) genes. Our analysis of CHS mRNA and short-interfering RNAs provide clear evidence that the inhibition of seed coat pigmentation in yellow G. max results from posttranscriptional rather than transcriptional silencing of the CHS genes. Furthermore, we show that mottling symptoms present on the seed coat of G. max plants infected with some viruses can be caused by suppression of CHS posttranscriptional gene silencing (PTGS) by a viral silencing suppressor protein. These results demonstrate that naturally occurring PTGS plays a key role in expression of a distinctive phenotype in plants and present a simple clear example of the elucidation of the molecular mechanism for viral symptom induction.

SummaryVirus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various

An allele of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (Md-ACS1), the transcript and translated product of which have been identified in ripening apples (Malus domestica), was isolated from a genomic library of the apple cultivar, Golden Delicious. The predicted coding region of this allele (ACS1-2) showed that seven nucleotide substitutions in the corresponding region of ACS1-1 resulted in just one amino acid transition. A 162-bp sequence characterized as a short interspersed repetitive element retrotransposon was inserted in the 5 -flanking region of ACS1-2 corresponding to position ؊781 in ACS1-1. The XhoI site located near the 3 end of the predicted coding region of ACS1-2 was absent from the reverse transcriptase-polymerase chain reaction product, revealing that exclusive transcription from ACS1-1 occurs during ripening of cv Golden Delicious fruit. DNA gel-blot and polymerase chain reaction analyses of genomic DNAs showed clearly that apple cultivars were either heterozygous for ACS1-1 and ACS1-2 or homozygous for each type. RNA gel-blot analysis of the ACS1-2 homozygous Fuji apple, which produces little ethylene and has a long storage life, demonstrated that the level of transcription from ACS1-2 during the ripening stage was very low.

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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.

Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The I gene inhibits pigmentation over the entire seed coat, resulting in a uniform yellow color of mature harvested seeds. We previously demonstrated that the inhibition of seed coat pigmentation by the I gene results from post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. Little is known about the structure of the I gene and the mechanism by which it induces PTGS of CHS genes. Here, we report a candidate of the I gene, GmIRCHS, which consists of a 5'-portion of a DnaJ-like gene containing a promoter region and a perfect inverted repeat (IR) of 1.1-kb truncated CHS3 sequences (5'-DeltaCHS3 and 3'-DeltaCHS3). RT-PCRs and RNase protection assay indicated the existence of the read-through product from 5'-DeltaCHS3 to 3'-DeltaCHS3 and the dsRNA region of DeltaCHS3, suggesting that dsRNA of DeltaCHS3 could be transcribed from GmIRCHS and could induce PTGS of CHS genes. Moreover, the IR structure of DeltaCHS3 in GmIRCHS was lost in the soybean mutants in which I was changed to i, supporting the conclusion that GmIRCHS is the I gene.

The dose-dependent hypocholesterolemic and antiatherogenic effects of dietary apple polyphenol (AP) from unripe apple, which contains approximately 85% catechin oligomers (procyanidins), were examined in male Sprague-Dawley rats (4 wk of age) given a purified diet containing 0.5% cholesterol. Dietary AP at 0.5 and 1.0% levels significantly decreased the liver cholesterol level compared with that in the control (AP-free diet-fed) group. Dietary AP also significantly lowered the serum cholesterol level compared with that in the control group. However, the HDL cholesterol level was significantly higher in the 1.0% AP-fed group than in the control group. Accordingly, the ratio of HDL-cholesterol/total cholesterol was significantly higher in the 0.5% AP-fed group and 1.0% AP-fed group than in the control group. Moreover, the atherogenic indices in the 0.5 and 1.0% AP-fed groups were significantly lower than those in the control group. The activity of hepatic cholesterol 7alpha-hydroxylase tended to be increased by dietary AP in a dose-dependent manner. In accord with this observation, dietary AP increased the excretion of acidic steroids in feces. Dietary AP also significantly promoted the fecal excretion of neutral steroids in a dose-dependent manner. These observations suggest that dietary AP at a 0.5 or 1.0% level exerts hypocholesterolemic and antiatherogenic effects through the promotion of cholesterol catabolism and inhibition of intestinal absorption of cholesterol.

Low temperature is among the critical environmental factors that limit soybean production. To elucidate the genetic basis for chilling tolerance and identify useful markers, we conducted quantitative trait loci (QTL) analysis of seed-yielding ability at low temperature in soybean (Glycine max), using artificial climatic environments at usual and low temperatures and recombinant inbred lines derived from a cross between two contrasting cultivars in terms of chilling tolerance. We identified a QTL of a large effect (LOD > 15, r (2) > 0.3) associated with seed-yielding ability only at low temperature. The QTL was mapped near marker Sat_162 on linkage group A2, where no QTL for chilling tolerance has previously been identified. The tolerant genotype did not increase the pod number but maintained the seed number per pod and single seed weight, namely, the efficiency of seed development at low temperature. The effect of the QTL was confirmed in a segregating population of heterogeneous inbred families, which provided near-isogenic lines. The genomic region containing the QTL also influenced the node and pod numbers regardless of temperature condition, although this effect was not primarily associated with chilling tolerance. These results suggest the presence of a new major genetic factor that controls seed development specifically at low temperature. The findings will be useful for marker-assisted selection as well as for understanding of the mechanism underlying chilling tolerance in reproductive organs.