Many flowering plants have adopted self-incompatibility mechanisms to prevent inbreeding and promote out-crosses. In the Solanaceae, Rosaceae and Scrophulariaceae, two separate genes at the highly polymorphic S-locus control self-incompatibility interactions: the S-RNase gene encodes the pistil determinant and the previously unidentified S-gene encodes the pollen determinant. S-RNases interact with pollen S-allele products to inhibit the growth of self-pollen tubes in the style. Pollen-expressed F-box genes showing allelic sequence polymorphism have recently been identified near to the S-RNase gene in members of the Rosaceae and Scrophulariaceae; but until now have not been directly shown to encode the pollen determinant. Here we report the identification and characterization of PiSLF, an S-locus F-box gene of Petunia inflata (Solanaceae). We show that transformation of S1S1, S1S2 and S2S3 plants with the S2-allele of PiSLF causes breakdown of their pollen function in self-incompatibility. This breakdown of pollen function is consistent with 'competitive interaction', in which pollen carrying two different pollen S-alleles fails to function in self-incompatibility. We conclude that PiSLF encodes the pollen self-incompatibility determinant.

Flowering plants have evolved various stratagems to prevent inbreeding and promote outcrosses. One such mechanism, gametophytic self-incompatibility, provides a genetic barrier to self-fertilization, and in the simplest cases is controlled by the highly polymorphic S locus. Growth of a pollen tube in the style is arrested when the S allele carried by the pollen matches one of the two S alleles carried by the pistil. Putative S allele proteins of the pistil have been identified in several solanaceous species based on their co-segregation with S alleles, and they have been shown to be ribonucleases. So far, there has been only correlative or indirect evidence for the claim that these S allele-associated proteins (S proteins) are involved in recognition and rejection of self pollen. Here we show that inhibition of synthesis of S3 and S2 proteins in Petunia inflata plants of S2S3 genotype by the antisense S3 gene resulted in failure of the transgenic plants to reject S3 and S2 pollen. We further show that expression of the transgene encoding S3 protein in P. inflata plants of S1S2 genotype confers on the transgenic plants the ability to reject S3 pollen. The self-incompatibility behaviour of the pollen was not affected by the transgene in either set of experiments. Taken together, these findings provide direct in vivo evidence that S proteins control the self-incompatibility behaviour of the pistil.

In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C 19 -GAs. All bioactive GAs are C 19 -GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA 1 , GA 9 , and GA 20 were detected in seedlings of the transgenic line examined. GA 4 , which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GAoverproduction phenotype.

The major maize seed storage proteins, zeins, are deficient in lysine and tryptophan content, which contribute to the poor nutritional quality of corn. Whether through the identification of mutations or genetic engineering, kernels with reduced levels of zein proteins have been shown to have increased levels of lysine and tryptophan. It has been hypothesized that these increases are due to the reduction of lysine-poor zeins and a pleiotropic increase in the lysine-rich non-zein proteins. By transforming maize with constructs expressing chimeric double-stranded RNA, kernels derived from stable transgenic plants displayed significant declines in the accumulation of both 19- and 22-kD alpha-zeins, which resulted in higher lysine and tryptophan content than previously reported for kernels with reduced zein levels. The observation that lysine and tryptophan content is correlated with the protein levels measured in transgenic maize kernels is consistent with the hypothesis that a pleiotropic increase in non-zein proteins is contributing to an improved amino acid balance. In addition, a large increase in accumulation of free amino acids, consisting predominantly of asparagine, aspartate and glutamate, was observed in the zein reduction kernels.

SummaryBecause of the limited lysine content in corn grain, synthetic lysine supplements are added to corn meal-based rations for animal feed. The development of biotechnology, combined with the understanding of plant lysine metabolism, provides an alternative solution for increasing corn lysine content through genetic engineering. Here, we report that by suppressing lysine catabolism, transgenic maize kernels accumulated a significant amount of lysine. This was achieved by RNA interference (RNAi) through the endosperm-specific expression of an inverted-repeat (IR) sequence targeting the maize bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharopine dehydrogenase (ZLKR/SDH). Although plant-short interfering RNA (siRNA) were reported to lack tissue specificity due to systemic spreading, we confirmed that the suppression of ZLKR/SDH in developing transgenic kernels was restricted to endosperm tissue. Furthermore, results from our cloning and sequencing of siRNA suggested the absence of transitive RNAi.These results support the practical use of RNAi for plant genetic engineering to specifically target gene suppression in desired tissues without eliciting systemic spreading and the transitive nature of plant RNAi silencing.

SummaryCorn is one of the major crops in the world, but its low lysine content is often problematic for animal consumption. While exogenous lysine supplementation is still the most common solution for today's feed corn, high-lysine corn has been developed through genetic research and biotechnology. Reducing the lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme, CordapA, has shown increased total lysine or free lysine content in the grains of modified corn plants, respectively. Here, by combining these two approaches through genetic crosses, the total lysine content has more than doubled in F1 progeny. We also observe a synergy between the transgenic zein reduction and the enhanced lysine biosynthesis by CordapA expression. The zein reduction plants are found to accumulate higher levels of aspartate, asparagine and glutamate, and therefore, provide excess precursors for the enhanced lysine biosynthesis.

S proteins, pistil-specific ribonucleases that cosegregate with S alleles, have previously been shown to control rejection of self-pollen in Petunia inflata and Nicotiana alata, two solanaceous species that display gametophytic self-incompatibility. The ribonuclease activity of S proteins was thought to degrade RNA of self-pollen tubes, resulting in the arrest of their growth in the style. However, to date no direct evidence has been obtained. Here, the ribonuclease activity of S3 protein of P. inflata was abolished, and the effect on the pistil's ability to reject S3 pollen was examined. The S3 gene was mutagenized by replacing the codon for His-93, which has been implicated in ribonuclease activity, with a codon for asparagine, and the mutant S3 gene was introduced into P. inflata plants of S1S2 genotype. Two transgenic plants produced a level of mutant S3 protein comparable to that of the S3 protein produced in self-incompatible S1S3 and S2S3 plants, yet they failed to reject S3 pollen. The mutant S3 protein produced in these two transgenic plants did not exhibit any detectable ribonuclease activity. We have previously shown that transgenic plants (S1S2 plants transformed with the wild-type S3 gene) producing a normal level of wild-type S3 protein acquired the ability to reject S3 pollen completely. Thus, the results reported here provide direct evidence that the biochemical mechanism of gametophytic self-incompatibility in P. inflata involves the ribonuclease activity of S proteins.

SummaryAlthough it is one of the major crops in the world, corn has poor nutritional quality for human and animal consumption due to its low lysine content. Here, we report a method