SummaryGateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available 'entry vector' and are then recombined into various 'destination vectors' for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of 'pEarleyGate' plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.

Although the evolutionary success of polyploidy in higher plants has been widely recognized, there is virtually no information on how polyploid genomes have evolved after their formation. In this report, we used synthetic polyploids of Brassica as a model system to study genome evolution in the early generations after polyploidization. The initial polyploids we developed were completely homozygous, and thus, no nuclear genome changes were expected in selffertilized progenies. (14) and the linkage orders of RFLP loci (15). However, these and other studies on polyploid evolution (5, 16) have compared natural polyploids, which are usually hundreds or thousands of years old, to present forms of hypothesized progenitors. Thus, it was not possible to distinguish between genome change after formation of the polyploid and genome divergence within the diploid progenitor species or to determine how quickly newly formed polyploid genomes evolve. Synthetic polyploids provide a model system to study early events in the evolution of polyploid genomes. Because the exact progenitors for a synthetic polyploid are known, we can determine precisely whether extensive genome changes occur after synthesis of polyploids and if so, the timing and processes of genome changes. We recently developed a series of synthetic Brassica polyploids by reciprocal interspecific hybridizations between the diploid species, followed by chromosome doubling of the F1 hybrids (17). We now report direct evidence for nuclear genome changes in these synthetic polyploids on the basis of comparing RFLP patterns of synthetic polyploids and their self-pollinated progenies by using a large number of cloned DNA probes.Polyploidy is one of the most distinctive and widespread modes of speciation in higher plants. Thirty to 70% of angiosperms, including many important crop plants, are estimated to have polyploidy in their lineages (1-6). The success ofpolyploid species has been attributed to their ability to colonize a wider range of habitats and to survive better in unstable climates compared with their diploid progenitors (7-10), presumably due to increased heterozygosity and flexibility provided by the presence of additional alleles (11-13). Genome multiplicity also provides a genetic buffer against the effects of individual alleles; and thus, new mutations are expected to contribute less to the evolution of polyploids compared to diploids (6). However, this hypothesis assumes that diploids and polyploids have equal mutation rates. It is possible that genome change is greatly accelerated in new polyploids derived from interspecies hybrids, due to greater instabilities created by the interaction of diverse genomes. Such changes could result in rapid genetic divergence of newly formed polyploids and might have contributed to the evolutionary success of many polyploid lineages.The potential contribution of genome change to the evolution of polyploids has been overlooked, mainly due to lack of information on how polyploid genomes have evolved after their for...

We summarize our recent studies showing that angiosperm mitochondrial (mt) genomes have experienced remarkably high rates of gene loss and concomitant transfer to the nucleus and of intron acquisition by horizontal transfer. Moreover, we find substantial lineage-specific variation in rates of these structural mutations and also point mutations. These findings mostly arise from a Southern blot survey of gene and intron distribution in 281 diverse angiosperms. These blots reveal numerous losses of mt ribosomal protein genes but, with one exception, only rare loss of respiratory genes. Some lineages of angiosperms have kept all of their mt ribosomal protein genes whereas others have lost most of them. These many losses appear to reflect remarkably high (and variable) rates of functional transfer of mt ribosomal protein genes to the nucleus in angiosperms. The recent transfer of cox2 to the nucleus in legumes provides both an example of interorganellar gene transfer in action and a starting point for discussion of the roles of mechanistic and selective forces in determining the distribution of genetic labor between organellar and nuclear genomes. Plant mt genomes also acquire sequences by horizontal transfer. A striking example of this is a homing group I intron in the mt cox1 gene. This extraordinarily invasive mobile element has probably been acquired over 1,000 times separately during angiosperm evolution via a recent wave of cross-species horizontal transfers. Finally, whereas all previously examined angiosperm mtDNAs have low rates of synonymous substitutions, mtDNAs of two distantly related angiosperms have highly accelerated substitution rates.

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