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AP2/ERF proteins play crucial roles in plant growth and development and in responses to biotic and abiotic stresses. ETHYLENE RESPONSE FACTOR 53 (AtERF53) belongs to group 1 in the ERF family and is induced in the early hours of dehydration and salt treatment. The functional study of AtERF53 is hampered because its protein expression in Arabidopsis is vulnerable to degradation in overexpressed transgenic lines. Taking advantage of the RING domain ligase1/RING domain ligase2 (rglg1rglg2) double mutant in which the AtERF53 can express stably, we investigate the physiological function of AtERF53. In this study, we demonstrate that expression of AtERF53 in wild-type Arabidopsis was responsive to heat and abscisic acid (ABA) treatment. From results of the cotransfection experiment, we concluded that AtERF53 has positive transactivation activity. Overexpression of AtERF53 in the rglg1rglg2 double mutant conferred better heat-stress tolerance and had resulted in higher endogenous ABA and proline levels compared to rglg1rglg2 double mutants. AtERF53 also has a function to regulate guard-cell movement because the stomatal aperture of AtERF53 overexpressed in rglg1rglg2 double mutant was smaller than that in the rglg1rglg2 double mutant under ABA treatment. In a global gene expression study, we found higher expressions of many stress-related genes, such as DREB1A, COR15A, COR15B, PLC, P5CS1, cpHSC70 s and proline and ABA metabolic-related genes. Furthermore, we identified several downstream target genes of AtERF53 by chromatin immunoprecipitation assay. In conclusion, the genetic, molecular and biochemical result might explain how AtERF53 serving as a transcription factor contributes to abiotic stress tolerance in Arabidopsis.

Cinnamomum kanehirae Hayata (Lauraceae), the most valuable subtropical and temperate broadleaf timber tree in Taiwan, is rapidly disappearing from the wild. Taking advantage of a scion garden established by the Taiwan Forestry Research Institute, we examined patterns of chloroplast DNA (cpDNA) variations in 19 populations including 94 individuals. By sequencing two cpDNA fragments using universal primers (the trnL-trnF and petG-trnP intergenic spacers), we found eight polymorphic sites, six haplotypes, and extremely low nucleotide diversity (π = 0.00016) from 792 bp aligned sequences. The ancestral haplotype is widely distributed. Among the populations studied, three separated populations, at Yungfeng, Fuli, and Tahu have high nucleotide diversity. No phylogeographical structures of haplotypes were revealed because the tests of NST−GST for populations did not differ from zero in any situations; a ‘star-like’ genealogy is characteristic when all haplotypes rapidly coalesce and is a general outcome of population expansion. The neutrality test also suggested demographic expansion. The genetic divergence and diversity analyses suggested that two potential refugia existed during the last glaciation with a major one located in southeastern Taiwan and a minor one located in Tahu in north-central Taiwan in the Hsuehshan Range, west of the Central Mountain Range.

The genus Chamaecyparis comprises five species and one variety native to Taiwan, Japan, Canada, and USA, which demonstrates a classical eastern Asian, western North American, and eastern North American disjunct distributional pattern. The phylogenetic relationships of the species of Chamaecyparis were inferred by comparing 1130 bp of the combined data set of chloroplast trnV intron and petG-trnP intergenic spacer. The phylogenetic tree shows that Chamaecyparis nootkatensis (Cupressus nootkatensis or Xanthocyparis nootkatensis) is clearly diverged from other Chamaecyparis species. For Chamaecyparis species, C. thyoides is sister to C. formosensis and C. pisifera and these together form a monophyletic group. C. lawsoniana is sister to C. obtusa and C. taiwanensis; and these form another monophyletic group. Homogeneity in evolutionary rates was found among species in these two monophyletic groups. Results indicate the divergent evolution of C. taiwanensis and C. formosensis and molecular evidence in this investigation supports C. taiwanensis as a variety of C. obtusa. Utility of cpDNA intergenic spacer petG-trnP in Chamaecyparis is also discussed. Several biogeographical implications were inferred: (1) at least two divergence events have produced the eastern Asian, and both western and eastern North American disjunct distribution in Chamaecyparis; (2) intercontinental sister species pairs are found in Chamaecyparis; (3) cpDNA divergence between two intercontinental sister pairs of C. thyoides and C. pisifera, and C. lawsoniana and C. obtusa is 2.8% and 1.1%, which suggest an estimated divergence time of 14 and 5.5 million years ago during middle and late Miocene, respectively; (4) cpDNA divergence of two Asian Chamaecyparis groups between C. obtusa and C. taiwanensis, and between C. pisifera and C. formosensis is 0.25% and 0.57%, which suggest an estimated divergence time of 1.3 and 2.9 million years ago during Pleistocene and late Pliocene, respectively; these estimated divergence times suggest a relatively recent migration of Chamaecyparis to Taiwan from the Japanese Archipelago; (5) that climatic deterioration caused the disappearance of Chamaecyparis in continental Asia is probable.

Michelia formosana is distributed islandwide in Taiwan and also occurs in the Ryukyu Archipelago, Japan. Allozyme genetic variability in M. formosana (Magnoliaceae) was investigated using five polymorphic loci (Pgm-1, Idh-1, Mr-1, Skdh-1, and Ppo-3) from five enzyme systems. The average value of expected heterozygosity (He) describing within-population variation was 0.241. The overall Fis (0.0736) indicates a significant deficiency of heterozygotes at the population level. This positive value of Fis is mainly contributed from populations found at Pinglin, Chienshi, Wufeng, and the Ryukyus, all located at northern latitudes, and is also caused by locus Ppo-3. Among-population variation, Fst, accounted for 10.6% of the total heterozygosity and deviates significantly from zero. This populational differentiation agrees well with that of general tropical woody species outcrossed by animals. The population on Lanyu (Orchid Island) has some morphological differences from the plants native to Taiwan, but this was not reflected in the cluster analysis using the unweighted pair group method. Pgm-1d, however, is the diagnostic characteristic for the plants growing on Lanyu as it is entirely absent from all other populations of M. formosana. About 18% of the alleles of this study show clinal geographical variation and were found to be significantly related to latitudinal gradients throughout the species range. This, however, is not a linear relation but a curve with a peak form which is exactly the same as the relationship between expected heterozygosities of populations and latitude. This observation suggests that Nantou County, in central Taiwan could be a glacial refugium for genetic diversity of M. formosana. This inference is further supported by analysis of published studies on seven widely spread plant species in Taiwan. Location of this glacial refugium is probably related to the first emergence of the Central Mountain Ridge millions of years ago that naturally becamed the major window to receive genetic diversity from the Asian mainland.

Low genetic variation in Amentotaxus formosana Li revealed by isozyme analysis and random amplified polymorphic DNA markers

Genomic DNA of Paulownia fortunei, P. kawakamii and P. taiwaniana were amplified with 10-base primers of arbitrary sequences using the polymerase chain reaction (PCR). A total of 351 DNA fragments were amplified from 23 primers and of these 265 fragments (75.5%) were polymorphic. Almost all of the PCR-amplified products of P. taiwaniana were shared by either P. fortunei or P. kawakamii, or both, and the number of polymorphic fragments shared by P. taiwaniana and P. fortunei was about equivalent to those shared by P. taiwaniana and P. kawakamii. Restriction fragments of chloroplast DNA (cpDNA) purified from Paulownia species and from reciprocal crosses between P. fortunei and P. kawakamii were analyzed. Restriction enzyme SalI-digested cpDNA showed an identical pattern in both P. kawakamii and P. taiwaniana. These results further support the hypothesis that P. taiwaniana is the natural hybrid between P. fortunei and P. kawakamii and that the maternal parent of P. taiwaniana is P. kawakamii.

Background Species in the varied geographic topology of Taiwan underwent obvious demographic changes during glacial periods. Cinnamomum kanehirae has been exploited for timber and to obtain medicinal fungi for the past 100 years. Understanding anthropogenic factors influencing the demography of this species after the last glacial maximum (LGM) is critically important for the conservation of this species. Results Populations of C. kanehirae were classified into four geographic regions: northwestern (NW), west-central (WC), southwestern (SW), and southeastern (SE). In total, 113 individuals from 19 localities were sampled, and variations in the chalcone synthase gene (Chs) intron and leafy (Lfy) intron-2 sequences of nuclear DNA were examined in order to assess phylogeographic patterns, the timescales of demographic and evolutionary events, and recent anthropogenic effects. In total, 210 Chs and 170 Lfy sequences, which respectively constituted 36 and 35 haplotypes, were used for the analyses. Estimates of the migration rate (M) through time revealed a pattern of frequent gene flow during previous and the present interglacials. The isolation-by-distance test showed that there generally was no significant correlation between genetic and geographic distances. The level of among-region genetic differentiation was significant when comparing eastern to western populations. However, no significant among-region genetic differentiation was found in comparisons among the four geographic regions. Moreover, essentially no genetic structuring was found for the three regions west of the CMR. A fit of spatial range expansion was found for pooled and regional samples according to the non-significant values of the sum of squared deviations. Using the Bayesian skyline plot (BSP) method, a recent bottleneck after the LGM expansion was detected in both regional and pooled samples. Conclusions Common haplotype distributions among geographic regions and the relatively shallow genetic structuring displayed are the result of historical gene flows. Southward dispersals in an earlier time frame from the NW region and in a later time frame from the SE region were inferred. The BSP analysis suggested a postglacial expansion event. Recent trends, however, refer to a bottleneck due to human interventions observed for both pooled and regional C. kanehirae samples.

Paulownia taiwaniana, the widely cultivated, commercially important tree, has been suspected of being of hybrid origin at least since its original publication in 1975. Evidence in support of this thesis, derived from a number of different investigations, is presented in this paper.—Strong evidence comes from a controlled pollination study of the two supposed parental species,P. kawakamii andP. fortunei. F1 seedlings, derived from reciprocal crosses between the suspected parents, exhibited identical banding patterns for a number of enzymes (such as SKDH, GOT, and IDH) withP. taiwaniana, when separated by electrophoresis. Furthermore, comparative morphological studies of trichomes and wood parenchyma patterns between the purported parents andP. taiwaniana reveal that this latter qualitatively exhibits characteristics that combine features of both of the suspected parental types. Biochemically, eight enzyme systems were compared in the three species here under discussion, and, without exception, the electrophoretic banding patterns exhibited byP. taiwaniana represented a combination of the alleles of the other two species. Perhaps the most convincing evidence comes from a genetic analysis of the progeny obtained by selfingP. taiwaniana. Genotypic segregation of the offspring based on a single locus each of SKDH and PGI fit the 1:2:1 hypothesis. Genotypic segregation of the offspring based on two loci each of SP and GOT fit the ratio of 3:6:3:1:2:1. This, taken in conjunction with the other data presented, clearly suggests thatP. taiwaniana is a hybrid involvingP. kawakamii andP. fortunei.

Background CBF/DREB duplicate genes are widely distributed in higher plants and encode transcriptional factors, or CBFs, which bind a DNA regulatory element and impart responsiveness to low temperatures and dehydration. Results We explored patterns of genetic variations of CBF1, -2, and -3 from 34 accessions of Arabidopsis thaliana. Molecular population genetic analyses of these genes indicated that CBF2 has much reduced nucleotide diversity in the transcriptional unit and promoter, suggesting that CBF2 has been subjected to a recent adaptive sweep, which agrees with reports of a regulatory protein of CBF2. Investigating the ratios of Ka/Ks between all paired CBF paralogus genes, high conservation of the AP2 domain was observed, and the major divergence of proteins was the result of relaxation in two regions within the transcriptional activation domain which was under positive selection after CBF duplication. With respect to the level of CBF gene expression, several mutated nucleotides in the promoters of CBF3 and -1 of specific ecotypes might be responsible for its consistently low expression. Conclusion We concluded from our data that important evolutionary changes in CBF1, -2, and -3 may have primarily occurred at the level of gene regulation as well as in protein function.