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Summary Ethylene Response Factor 1 (ERF1) plays a crucial role in biotic and abiotic stress responses. Previous studies have shown that ERF1 regulates stress-responsive gene expression by binding to different cis-acting elements in response to various stress signals. ERF1 was also reported to be unstable in the dark, and it regulates hypocotyl elongation. Here, we elucidated the mechanism underlying degradation of ERF1. Yeast two-hybrid screening showed that UBIQUITIN-CONJUGATING ENZYME 18 (UBC18) interacted with ERF1. The interaction between ERF1 and UBC18 was verified using pull-down assays and coimmunoprecipitation analyses. We then compared the ERF1 protein abundance in the UBC18 mutant and overexpression plants. Based on the results of protein degradation and in vivo ubiquitination assays, we proposed that UBC18 mediates ERF1 ubiquitination and degradation. ERF1 was more stable in UBC18 mutants and less stable in UBC18 overexpression lines compared with that in wild-type plants. ERF1 was degraded by the 26S proteasome system via regulation of UBC18 and promotes dark-repression of downstream genes and proline accumulation. UBC18 negatively regulated drought and salt stress responses by altering the abundance of ERF1 and the expression of genes downstream of ERF1.

ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) of Arabidopsis thaliana is an AP2/ERF domain transcription factor that regulates jasmonate (JA) biosynthesis and is induced by methyl JA treatment. The regulatory mechanism of ORA47 remains unclear. ORA47 is shown to bind to the cis-element (NC/GT)CGNCCA, which is referred to as the O-box, in the promoter of ABI2. We proposed that ORA47 acts as a connection between ABA INSENSITIVE1 (ABI1) and ABI2 and mediates an ABI1-ORA47-ABI2 positive feedback loop. PORA47:ORA47-GFP transgenic plants were used in a chromatin immunoprecipitation (ChIP) assay to show that ORA47 participates in the biosynthesis and/or signaling pathways of nine phytohormones. Specifically, many abscisic acid (ABA) and JA biosynthesis and signaling genes were direct targets of ORA47 under stress conditions. The JA content of the P35S:ORA47-GR lines was highly induced under wounding and moderately induced under water stress relative to that of the wild-type plants. The wounding treatment moderately increased ABA accumulation in the transgenic lines, whereas the water stress treatment repressed the ABA content. ORA47 is proposed to play a role in the biosynthesis of JA and ABA and in regulating the biosynthesis and/or signaling of a suite of phytohormone genes when plants are subjected to wounding and water stress.

SUMMARY Although glutathione is well known for its reactive oxygen species (ROS) scavenging function and plays a protective role in biotic stress, its regulatory function in abiotic stress still remains to be elucidated. Our previous study showed that exogenously applied reduced glutathione (GSH) could improve abiotic stress tolerance in Arabidopsis. Here, we report that endogenously increased GSH also conferred tolerance to drought and salt stress in Arabidopsis. Moreover, both exogenous and endogenous GSH delayed senescence and flowering time. Polysomal profiling results showed that global translation was enhanced after GSH treatment and by the induced increase of GSH level by salt stress. By performing transcriptomic analyses of steady-state and polysome-bound mRNAs in GSH-treated plants, we reveal that GSH has a substantial impact on translation. Translational changes induced by GSH treatment target numerous hormones and stress signaling molecules, which might contribute to the enhanced stress tolerance in GSH-treated plants. Our translatome analysis also revealed that abscisic acid (ABA), auxin and jasmonic acid (JA) biosynthesis, as well as signaling genes, were activated during GSH treatment, which has not been reported in previously published transcriptomic data. Together, our data suggest that the increased glutathione level results in stress tolerance and global translational changes.

ETHYLENE RESPONSE FACTOR1 (ERF1) is an upstream component in both jasmonate (JA) and ethylene (ET) signaling and is involved in pathogen resistance. Accumulating evidence suggests that ERF1 might be related to the salt stress response through ethylene signaling. However, the specific role of ERF1 in abiotic stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. Here, we report that ERF1 was highly induced by high salinity and drought stress in Arabidopsis (Arabidopsis thaliana). The salt stress induction required both JA and ET signaling but was inhibited by abscisic acid. ERF1-overexpressing lines (35S:ERF1) were more tolerant to drought and salt stress. They also displayed constitutively smaller stomatal aperture and less transpirational water loss. Surprisingly, 35S:ERF1 also showed enhanced heat tolerance and up-regulation of heat tolerance genes compared with the wild type. Several suites of genes activated by JA, drought, salt, and heat were found in microarray analysis of 35S:ERF1. Chromatin immunoprecipitation assays found that ERF1 up-regulates specific suites of genes in response to different abiotic stresses by stress-specific binding to GCC or DRE/CRT. In response to biotic stress, ERF1 bound to GCC boxes but not DRE elements; conversely, under abiotic stress, we observed specific binding of ERF1 to DRE elements. Furthermore, ERF1 bound preferentially to only one among several GCC box or DRE/CRT elements in the promoter region of its target genes. ERF1 plays a positive role in salt, drought, and heat stress tolerance by stress-specific gene regulation, which integrates JA, ET, and abscisic acid signals.

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.

Transcriptional activities of plants play important roles in responses to environmental stresses. ETHYLENE RESPONSE FACTOR53 (AtERF53) is a drought-induced transcription factor that belongs to the AP2/ERF superfamily and has a highly conserved AP2 domain. It can regulate drought-responsive gene expression by binding to the GCC box and/or the dehydration-responsive element in the promoter of downstream genes. Overexpression of AtERF53 driven by the cauliflower mosaic virus 35S promoter resulted in an unstable drought-tolerant phenotype in T2 transgenic Arabidopsis (Arabidopsis thaliana) plants. Using a yeast two-hybrid screen, we identified a RING domain ubiquitin E3 ligase, RGLG2, which interacts with AtERF53 in the nucleus. The copine domain of RGLG2 exhibited the strongest interacting activity. We also demonstrated that RGLG2 could move from the plasma membrane to the nucleus under stress treatment. Using an in vitro ubiquitination assay, RGLG2 and its closest sequelog, RGLG1, were shown to have E3 ligase activity and mediated AtERF53 ubiquitination for proteasome degradation. The rglg1rglg2 double mutant but not the rglg2 or rglg1 single mutant exhibited a drought-tolerant phenotype when compared with wild-type plants. AtERF53-green fluorescent proteins expressed in the rglg1rglg2 double mutants were stable. The 35S:AtERF53-green fluorescent protein/rglg1rglg2 showed enhanced AtERF53-regulated gene expression and had greater tolerance to drought stress than the rglg1rglg2 double mutant. In conclusion, RGLG2 negatively regulates the drought stress response by mediating AtERF53 transcriptional activity in Arabidopsis.

The membrane protein AtTLP18.3 of Arabidopsis (Arabidopsis thaliana) contains a domain of unknown function, DUF477; it forms a polysome with photosynthetic apparatuses in the thylakoid lumen. To explore the molecular function of AtTLP18.3, we resolved its crystal structures with residues 83 to 260, the DUF477 only, and performed a series of biochemical analyses to discover its function. The gene expression of AtTLP18.3 followed a circadian rhythm. X-ray crystallography revealed the folding of AtTLP18.3 as a three-layer sandwich with three α-helices in the upper layer, four β-sheets in the middle layer, and two α-helices in the lower layer, which resembles a Rossmann fold. Structural comparison suggested that AtTLP18.3 might be a phosphatase. The enzymatic activity of AtTLP18.3 was further confirmed by phosphatase assay with various substrates (e.g. p-nitrophenyl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, O-phospho-l-serine, and several synthetic phosphopeptides). Furthermore, we obtained the structure of AtTLP18.3 in complex with O-phospho-l-serine to identify the binding site of AtTLP18.3. Our structural and biochemical studies revealed that AtTLP18.3 has the molecular function of a novel acid phosphatase in the thylakoid lumen. DUF477 is accordingly renamed the thylakoid acid phosphatase domain.

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.

This study examined the spatial pattern of chloroplast DNA (cpDNA) variation in Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. (Fagaceae) in 140 trees from Taiwan (25 populations), Japan (three), Ryukyus (two), Hong Kong (one) and Mainland China (one). By sequencing three cpDNA intergenic spacer fragments using universal primers (trnT-trnL, trnV-trnM, including the trnV intron, and petG-trnP), we found a total of 1,980 bp and 15 polymorphic sites. Among them, 12 sites were caused by point mutation, and three resulted from insertion. This gives rise to a total of 13 cpDNA haplotypes. The level of differentiation among the populations studied is relatively high (GST = 0.612). Two ancestral haplotypes (A and B) are distributed widely in East Asia. Interestingly, all the derived cpDNA variations are found only in Taiwan but not in other areas. The Central Mountain Ridge (CMR) of Taiwan creates an unsurpassed barrier to the east-west gene flow of C. glauca. Among the populations on the west of CMR, only three separated populations, Yangmingshan, Wushe and Chinshuiying, have high haplotype diversity, each consisting of sister haplotypes all mutated from the same ancestral haplotype. Thus, they have probably originated from de novo mutation after the last glaciation. This inference agrees with the observation that no spatial autocorrelation existed on the west side. Two unrelated dominant lineages on the east of the CMR (haplotypes D and F) showed significant spatial genetic structure. Estimate of NST - GST was -0.090 and differed significantly from zero. Thus at the local scale, the phylogeographical component of the genetic structure is significant on the east of the CMR. Accompanied by published palynological records of the last glaciation, this study suggests the possibility that these two types were colonized northward from the southeastern part of Taiwan. 'Star-like' genealogy is characterized, with all the haplotypes coalescing rapidly and as a general outcome of population expansion (Page & Holmes 1998). A neutrality test also suggested a demographic expansion recovered from a bottleneck. We therefore inferred that the southeastern part of Taiwan might be a potential refugium for C. glauca.

Rice blast disease resistance to the fungal pathogen Magnaporthe grisea is triggered by a physical interaction between the protein products of the host R (resistance) gene, Pi-ta, and the pathogen Avr (avirulence) gene, AVR-pita. The genotype variation and resistant/susceptible phenotype at the Pi-ta locus of wild rice (Oryza rufipogon), the ancestor of cultivated rice (O. sativa), was surveyed in 36 locations worldwide to study the molecular evolution and functional adaptation of the Pi-ta gene. The low nucleotide polymorphism of the Pi-ta gene of O. rufipogon was similar to that of O. sativa, but greatly differed from what has been reported for other O. rufipogon genes. The haplotypes can be subdivided into two divergent haplogroups named H1 and H2. H1 is derived from H2, with nearly no variation and at a low frequency. H2 is common and is the ancestral form. The leucine-rich repeat (LRR) domain has a high πnon/πsyn ratio, and the low polymorphism of the Pi-ta gene might have primarily been caused by recurrent selective sweep and constraint by other putative physiological functions. Meanwhile, we provide data to show that the amino acid Ala-918 of H1 in the LRR domain has a close relationship with the resistant phenotype. H1 might have recently arisen during rice domestication and may be associated with the scenario of a blast pathogen–host shift from Italian millet to rice.

† Background and Aims Evolutionary and ecological roles of the chloroplast small heat shock protein (CPsHSP) have been emphasized based on variations in protein contents; however, DNA sequence variations related to the evolutionary and ecological roles of this gene have not been investigated. In the present study, a basal angiosperm, Machilus, together with the eudicot Rhododendron were used to illustrate the evolutionary dynamics of gene divergence in CPsHSPs. † Methods Degenerate primers were used to amplify CPsHSP-related sequences from 16 Rhododendron and eight Machilus species that occur in Taiwan. Manual DNA sequence alignment was carried out according to the deduced amino acid sequence alignment performed by CLUSTAL X. A neighbour-joining tree was generated in MEGA using conceptual translated amino acid sequences from consensus sequences of cloned CPsHSP genes from eight Machilus and 16 Rhododendron species as well as amino acid sequences of CPsHSPs from five monocots and seven other eudicots acquired from GenBank. CPsHSP amino acid sequences of Funaria hygrometrica were used as the outgroups. The aligned DNA and amino acid sequences were used to estimate several parameters of sequence divergence using the MEGA program. Separate Bayesian inference of DNA sequences of Rhododendron and Machilus species was analysed and the resulting gene trees were used for detection of putative positively selected amino acid sites by the Codeml program implemented in the PAML package. Mean hydrophobicity profile analysis was performed with representative amino acid sequences for both Rhododendron and Machilus species by the Bioedit program. The computer program SplitTester was used to examine whether CPsHSPs of Rhododendron lineages and duplicate copies of the Machilus CPsHSPs have evolved functional divergence based on the hydrophobicity distance matrix. † Key Results Only one copy of the CPsHSP was found in Rhododendron. However, a higher evolutionary rate of amino acid substitutions in the Hymenanthes lineage of Rhododendron was inferred. Two positively selected amino acid sites may have resulted in higher hydrophobicity in the region of the a-crystallin domain (ACD) of the CPsHSP. By contrast, the basal angiosperm, Machilus, possessed duplicate copies of the CPsHSP, which also differed in their evolutionary rates of amino acid substitutions. However, no apparent relationship of ecological relevance toward the positively selected amino acid sites was found in Machilus. † Conclusions Divergent evolution was found for both Rhododendron lineages and the paralogues of CPsHSP in Machilus that were directed to the shift in hydrophobicity in the ACD and/or methionine-rich region, which might have played important roles in molecular chaperone activity.

Quercus glauca (Thunb. ex Murray) Oerst (Fagaceae) has a wide distributional range in Taiwan. In this study, the evolutionary history and the most genetically divergent sites of Q. glauca were studied using a nuclear gene marker, glyceraldehyde‐3‐phosphate dehydrogenase. Also, the consistency of the results obtained from nuclear gene and cytoplasmic loci was investigated. Using a genealogical approach (TCS software), we determined haplotypes and their relationships to one another. We used the level of divergence for each population from the remaining populations (calculated as mean values of pairwise population differentiation, FST, for each population) to locate the most genetically divergent areas in Taiwan. According to the average FST of each population in comparison with the remaining ones, a peak was found in the northern part of central Taiwan, and another was found in the southeastern region. The peak profiles of the mean FST values for all three DNA data sets (nDNA, cpDNA, and mtDNA) showed simil...

Background and Aims The genetic variation and divergence estimated by allozyme analysis were used to reveal the evolutionary history of Castanopsis carlesii in Taiwan. Two major questions were discussed concerning evolutionary issues: where are the diversity centres, and where are the most genetically divergent sites in Taiwan? Methods Twenty-two populations of C. carlesii were sampled throughout Taiwan. Starch gel electrophoresis was used to assay allozyme variation. Genetic parameters and mean FST values of each population were analysed using the BIOSYS-2 program. Mean FST values of each population against the remaining populations, considered as genetic divergence, were estimated using the FSTAT program. Key Results Average values of genetic parameters describing the within-population variation, the average number of alleles per locus (A = 25), the effective number of alleles per locus (Ae = 138), the allelic richness (Ar = 238), the percentage of polymorphic loci (P = 69%), and the expected heterozygosity (He = 0270) were estimated. High levels of genetic diversity were found for C. carlesii compared with other local plant species. Genetic differentiation between populations was generally low. Conclusions From the data of expected heterozygosity, one major diversity centre was situated in central Taiwan corroborating previous reports for other plant species. According to the mean FST value of each population, the most divergent populations were situated in two places. One includes populations located in north central Taiwan between 2480N and 2420N. The other is located in south-eastern Taiwan between 2240N and 2310N. These two regions are approximately convergent with the most divergent locations determined for several other plant species using chloroplast DNA markers published previously. An important finding obtained from this study is that unordered markers like allozymes can be used to infer past population histories as well as chloroplast DNA markers do.

Aims To unravel isolation and differentiation of the genetic structure of the Euphrasia transmorrisonensis complex, a showy herb, among alpine regions of mountain peaks in subtropical Taiwan and to infer its evolutionary history. Location Alpine ecosystems of high-montane regions of Taiwan. Methods Phylogenetic analyses of the trnL intron and the trnL–trnF intergenic spacer of chloroplast (cp) DNA, and the intertranscribed spacer (ITS) of nuclear ribosomal (nr) DNA between 18S and 26S were carried out on 18 populations of the E. transmorrisonensis complex in Taiwan. Results In total, 10 haplotypes for cpDNA and 14 haplotypes for nrDNA were detected. Three population groups located in the northern, north-eastern, and south-central regions of the Central Mountain Range (CMR) were revealed according to the frequencies of haplotypes and haplotype lineages of nrDNA. Balancing selection might have played a role in the evolution of Euphrasia in Taiwan. Main conclusions By integrating the spatial-genetic patterns of cpDNA and nrDNA, two possible evolutionary histories of Euphrasia in Taiwan were inferred. The favourable hypotheses for interpreting the data suggest at least three origins of the E. transmorrisonensis complex in Taiwan, corresponding to each nuclear lineage in the northern (II), northern/north-eastern (I), and central/southern regions (III) with subsequent hybridization between lineages I and II and lineages II and III. These lineage boundaries are strengthened by the finding that haplotypes of C derived from cpDNA were found in the geographical region of lineage II of nrDNA, while haplotypes of A derived from cpDNA were found in the region of lineage III of nrDNA. Thus, the origin of chloroplasts exclusive to lineages II and III supports their long-term isolation from one another.

In this study, we examined spatial patterns of chloroplast DNA (cpDNA) variation in a total of 30 populations of Castanopsis carlesii Hayata (Fagaceae), a subtropical and temperate tree species, including 201 individuals sampled throughout Taiwan. By sequencing two cpDNA fragments using universal primers (the trnL intron and the trnV-trnM intergenic spacer), we found a total of 1663 bp and 21 polymorphic sites. These gave rise to a total of 28 cpDNA haplotypes. The level of differentiation among the populations studied was relatively high (GST = 0.723). Two ancestral haplotypes are widely distributed. The Central Mountain Ridge (CMR) of Taiwan represents an insurmountable barrier to the east-west gene flow of C. carlesii. Among the populations studied, three separated populations, at Lienhuachih, Fushan and Lichia, have high nucleotide diversity. Estimates of NST-GST for populations on both sides of the CMR indicate that no phylogeographical structure exists. According to the genealogical tree, number of rare haplotype and population genetic divergence, this study suggests that two potential refugia existed during the last glaciation: the first refugium was located in a region to the north of Hsuehshan Range (HR) and west of the CMR; the second refugium was located in south, especially southeastern Taiwan. In fact, the second refugium happens to be the same as that reported for Quercus glauca. A 'star-like' genealogy is characteristic when all haplotypes rapidly coalesce and is a general outcome of population expansion. The neutrality test and mismatch distribution also suggest demographic expansion recovering from a bottleneck.

Luanta-fir (Cunninghamia konishii), an endemic to Taiwan, is an outcrossing, long-lived conifer. Populations of C. konishii are generally fragmented due to a once high intensity of timber exploitation. C. konishii and Cunninghamia lanceolata are two sibling taxa constituting derivative-progenitor species relationship. The amount of genetic variations within and between 11 and 10 populations of C. konishii and C. lanceolata, respectively, were assessed using amplified fragment length polymorphism (AFLP) markers in this report. Three AFLP primer pairs generated a total of 357 and 226 markers for C. konishii and C. lanceolata samples, of which 56.1 and 65.3% are polymorphic, respectively. Analysis of molecular variance indicates a 4.78% variation between C. konishii and C. lanceolata. A relatively high value of genetic variation (24.60%) was apportioned between the populations of C. konishii. In contrast, a lower divergence value (12.21%) between populations was found for C. lanceolata. The population with the highest genetic diversity was found in Nantou County, which concurred with the results of many other tree species investigated in Taiwan. The estimates of the number of migrants between populations (Nm), obtained from population pair-wise ΦST, suggest that gene flow in C. konishii is efficient in some adjacent populations but is restricted in the rest. Individual UPGMA tree, generated based on AFLP markers, suggests six evolutionary lineages for C. konishii. All evolutionary lineages of C. konishii were derived from C. lanceolata. In conclusion, the migration patterns of Cunninghamia from mainland China may have been established following multiple sources, migrant-pools, long-distance dispersal events, and via different directions.

Aim This paper described current phylogeographical patterns of chloroplastic DNA variation of Trochodendron aralioides, a temperate tree species, and inferred its possible refugium in Taiwan. This information was compared with the known phylogeographical pattern of subtropical tree species. Location A total of 24 populations were sampled including 20 from Taiwan, two each from the Ryukyus and Japan. Methods A haplotype network was constructed by computer program TCS, various parameters of genetic diversity were calculated and neutrality was tested by computer program DnaSP. To examine the similarity of genetic structure among populations, a maximum parsimony tree was reconstructed by computer program PAUP*. The results of isozyme of T. aralioides from a previous publication were incorporated into this study to infer the phylogeographical history. Results Nine haplotypes according to six substitutions, two indels and one inversion of the two cpDNA intergenic spacer fragments (petG-trnP and petA-psbJ) of T. aralioides were recognized. Genetic structure of the population of Japan is totally different from those of Taiwan and the Ryukyus. In Taiwan, the genetic structure was differentiated among populations revealed by Gst = 0.700 and Nst = 0.542, and the population genetics was clearly spatially structured. Two population groups were recognized. The first group was distributed islandwide and extended to the Ryukyus. The second group contained five of the seven known haplotypes, and was restricted to the area between latitude 24°46′ and 24°06′ N. Conclusions In Taiwan, north-central area between latitude 24°46′ and 24°06′ N is potentially a refugium during the last glaciations. This finding is contradicted to subtropical species as Cyclobalanopsis glauca.

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.

The relationship between sugar content and loss of desiccation tolerance of hydrated crop seeds (tomato, okra, snow pea, mung bean, and cucumber) was evaluated by imbibing seeds with or without ABA, followed by dehydration and germination. During the process of hydration, but before the seeds lost desiccation tolerance, monosaccharide content increased only slightly, sucrose increased in snow pea, mung bean and cucumber, but maintained its original level in other species and the oligosaccharides declined dramatically. At the time of losing desiccation tolerance, the sucrose content of imbibed seeds was 2-3 times higher than the original level in most species. Positive significant correlation coefficients (r) were found in many, but not all crop seeds between desiccation tolerance and the oligosaccharide mass, or oligo/sucrose ratio. The ratio of oligo/sucrose in intact seeds at the time of losing desiccation tolerance, however, was not a fixed value and varied among species. Oligosaccharides declined significantly in different seed parts of imbibed cucumber seeds while sucrose increased to a higher level in the radicle than in the hypocotyl. Radicles were far more sensitive to desiccation than hypocotyls. The same observation was found for cucumber seeds imbibed in 100 μM ABA, yet desiccation tolerance was largely maintained in hypocotyls and cotyledons. It is concluded that sucrose and oligosaccharides are not the determinants of the loss of desiccation tolerance in hydrated seeds. Imbibed seeds did not show any differences between seed parts in their ability to resynthesize sugars during the process of slow dehydration. Differences in sensitivity to desiccation among seed parts were not due to differences in the initial water content or to the rate of water content increase among seed parts. Physiological regulation of the loss of desiccation tolerance in crop seeds during hydration is discussed.

Seeds of Taxus mairei are known for their deep dormancy which can only be broken by a procedure involving warm stratification followed by cold stratification. Treatments with alternating temperatures of 25}15 or 23}11 ∞C (12 h light) for 6 months followed by 5 ∞C for 3 months were successful in overcoming seed dormancy. After 6 months of warm stratification, cytological changes observed included: enlargement of the embryo; a decrease in the number of lipid bodies; appearance of ER; and increases in mitochondria, plastids, dictyosomes, vacuoles and microbodies in the shoot apical meristem. Cold stratification following the warm treatment induced cell division, and one or two distinct nucleoli in the shoot apical meristem cells were observed. Both warm and cold stratification reduced endogenous ABA concentrations from the original 8888 pg per freshly harvested seed to 392 and 536 pg, respectively. Treatment with exogenous gibberellins after seeds had been warm-stratified showed that GA % and GA ( were eective at promoting seed germination, but GA $ was not. These results suggest that the strong seed dormancy of T. mairei could be caused by a high ABA content and underdevelopment of the embryos in freshly shed seeds. We conclude that warm stratification with alternating temperatures increases the growth of embryos by cell expansion and enlargement and decreases ABA content, but seeds still remain ungerminated. Cold stratification may induce the response to GAs and initiate cell division resulting in release from physiological dormancy and subsequent germination of T. mairei seeds. # 1998 Annals of Botany Company

The once dominant endemic broadleaf tree species In Taiwan, CInnamomum kanehlrae Hay., has become a rare and endangered species due to overcutting. A done bank, consisting of at least 220 clones collected istandwide, was established at the Uukuel station, Taiwan Forestry Research Institute. Genetic diversity wtthln and genetic differentiation among four geographic areas of C. kanehlrae were Investigated using 164 clones collected from the clone bank. Seven out of the 11 loci examined were polymorphic. The average proportion of polymorphic loci per area was 51% at the 99% criterion for polymorphism. Mean expected heterozygosity ranged from 0.188-0.261 In the different areas, and 0.238 at the species level. On average the percent heterozygous loci per individual ranged from 13.9-21.6%, the number of alleies per locus ranged from 1.7-1.9, and the effective number of alleies per locus ranged from 1.34-1.54 at the area level. The much higher expected heterozygosity and higher number of aJtofes per locus compared to other local woody species investigated may support the hypothesis that Taiwan was a distributional center of CInnamomum. Partitioning the genetic variability Into withinand among-geographlc area components with F statistics and gene diversity analysis led to an estimate of wtthin-area variation amounting to 88% of the total variation. A slight barrier to gene flow among areas could be due to the large seed size and to Insect pollination. The limitation of seed dispersion, erratic flowering times, and several subpopulations wtthln the same geographic areas may contribute to the lower levels of observed heterozygosity than expected heterozygosity among the seven polymorphic loci, indicating some degree of Inbreeding and the Wahlund effect and thus a slight deficiency of heterozygotes (Fto = 0.157). Area 4, located in southeastern Taiwan has a much lower elevational distribution and showed a higher proportion of polymorphic loci, implying that genetic variation of area 4 may have developed under different climate conditions.

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

Biochemical properties, i.e. endogenous abscisic acid, proline, sugars, respiration, adenosine phosphates and adenylate energy charge, and growth and moisture content were measured during the development of seeds of Machilus thunbergii. As dry matter accumulated in the embryo during development, moisture content, ABA, proline, respiration and sugars all declined. At maturity, the dry mass of the seeds failed to attain a plateau before the period of natural seed shedding; the axis and cotyledons reached moisture contents of 58 and 45%, respectively. Drying of immature seeds at 73% relative humidity and 25 °C for 30 d resulted in a complete loss of viability at all developmental stages tested with the exception of mature seeds that were able to tolerate a 5% decrease in moisture content before germination declined. ABA was detected in all embryos tested, with a maximum value 16.·16 μg g -1 d. wt about midway through development. Although the presence of ABA induced no tolerance to desiccation of mature seeds, it did coincide with decreased content of water in the developing seeds and decreased respiration. Desiccation damage of M. thunbergii seeds occurred when moisture content was still high (45%) and this damage was not related to the absence of oligosaccharides in the mature seeds. We conclude that developing embryos and mature seeds of M. thunbergii have properties common to many recalcitrant seeds, with seeds being sensitive to desiccation at all stages, having a prominent ABA peak, little proline, lacking oligosaccharides, and specifically, little dormancy and a moderate rate of respiration of mature seeds (0·9 μmol O 2 min -1 g -1 f. wt). Adenosine triphosphate content and energy charge decreased from stage four to stage eight of seed development, then increased again to 103 nmol g -1 d. wt and 0·73, respectively, in mature seeds. The moderate energy charge observed in mature seeds indicates that continuous metabolism is also a characteristic of recalcitrant seeds.

Genetic diversity within and genetic differentiation among three populations of Chamaecyparisformosensis Matsum. and two populations of Chamaecyparistaiwanensis Masam. & Suzuki were investigated using one-year-old seedlings collected from central and northern Taiwan. For C. formosensis 330 seedlings from 33 seed trees were used, while for C. taiwanensis 260 seedlings from 26 seed trees were used. Eleven enzyme systems were investigated. In C. formosensis, 5 of the 21 loci examined were polymorphic. The average percentage of polymorphic loci per population was 20.6% at the 99% criterion for polymorphism. Mean expected heterozygosity ranged from 0.079 to 0.100 in the different populations. On average, there were 6.6 to 9.2% heterozygous loci per individual and 1.24 to 1.29 alleles per locus; the effective number of alleles per locus ranged from 1.09 to 1.11. In C. taiwanensis, 7 of the 20 loci examined were polymorphic and the average percentage of polymorphic loci per population was 22.5%. Mean expected heterozygosity ranged from 0.044 to 0.060. On average there were 4.5 to 5.6% heterozygous loci per individual and 1.45 alleles per locus; the effective number of alleles per locus ranged from 1.05 to 1.08. The surprisingly low expected heterozygosity and percentage of polymorphic loci compared with other conifer probably reflects the insular nature of these species. Partitioning the genetic variability into within- and among-population components with F-statistics led to an estimate of within-population variation of 95% of the total variation in both C. formosensis and C. taiwanensis. Chamaecyparisformosensis had a positive fixation index (0.109) that was significantly different from zero at the 5% level, indicating that most loci have slightly higher frequencies of homozygotes. Chamaecyparistaiwanensis, however, had a fixation index close to zero (0.036), which suggests that most loci are in Hardy–Weinberg equilibrium. The genetic distance between C. formosensis and C. taiwanensis was 0.70, which clearly separates these two species.

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.

Fresh mature tree seeds of 16 species plus soybean were used for the analysis of soluble neutral sugar content, as well as for the determination of longevity in terms of the time required for the seeds to decrease to 50% of the original germination under 5 o C and 80-91% relative humidity. Oligosaccharides were sometimes found in species of recalcitrant; although the mass ratio of oligosaccharide/total sugar or mole ratio of stachyose and raffinose/sucrose was less than 0.05 or 0.044, respectively. The time required for seeds to decrease to 50% germination varied from a few days to 8.3 months for the seeds of desiccation-sensitive species. The low ratio of oligosaccharide to sucrose is, however, most unlikely to be a cause of short life-span in recalcitrant seeds

Although glutathione S-transferases (GSTs) are thought to play major roles in oxidative stress metabolism, little is known about the regulatory functions of GSTs. We have reported that Arabidopsis (Arabidopsis thaliana) GLUTATHIONE S-TRANSFERASE U17 (AtGSTU17; At1g10370) participates in light signaling and might modulate various aspects of development by affecting glutathione (GSH) pools via a coordinated regulation with phytochrome A. Here, we provide further evidence to support a negative role of AtGSTU17 in drought and salt stress tolerance. When AtGSTU17 was mutated, plants were more tolerant to drought and salt stresses compared with wild-type plants. In addition, atgstu17 accumulated higher levels of GSH and abscisic acid (ABA) and exhibited hyposensitivity to ABA during seed germination, smaller stomatal apertures, a lower transpiration rate, better development of primary and lateral root systems, and longer vegetative growth. To explore how atgstu17 accumulated higher ABA content, we grew wild-type plants in the solution containing GSH and found that they accumulated ABA to a higher extent than plants grown in the absence of GSH, and they also exhibited the atgstu17 phenotypes. Wild-type plants treated with GSH also demonstrated more tolerance to drought and salt stresses. Furthermore, the effect of GSH on root patterning and drought tolerance was confirmed by growing the atgstu17 in solution containing l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH biosynthesis. In conclusion, the atgstu17 phenotype can be explained by the combined effect of GSH and ABA. We propose a role of AtGSTU17 in adaptive responses to drought and salt stresses by functioning as a negative component of stress-mediated signal transduction pathways.

Based on fossil pollen, the distribution range of Taiwan fir [Abies kawakamii (Hay.) Ito] (Pinaceae) is smaller than it was 50 000 years ago. To characterize the present refuge populations of A. kawakamii, which survive only in subalpine forests in Taiwan, we surveyed nuclear genes and chloroplast intergenic spacers to assess the genetic diversity of Taiwan fir. Populations maintain high genetic diversity and contain similar numbers of haplotypes for the GapC (cytosolic glyceraldehyde 3-phosphate dehydrogenase) fragment. Haplotypes for GapC are generally widespread, and population-specific haplotypes accounted for 2.5% of the total. Differentiation among populations is very low (G(ST) = 0.01). Only three haplotypes were detected for the cpDNA marker, and every population had one or two haplotypes. In a neutrality test, the variation in nucleotides did not deviate from that expected with neutral evolution for either marker. A retreat route to higher elevations was not evident from either the GapC or cpDNA markers. Hsuehshan was the site of the most divergent population in Taiwan. We concluded that uniform genetic diversity, low differentiation, low numbers of population-specific haplotypes, and neutral evolution characterize contemporary refuge populations of Taiwan fir.

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.

†Background and Aims Positive selection in the a-crystallin domain (ACD) of the chloroplast small heat shock protein (CPsHSP) gene was found in a previous study and was suggested to be related to the ecological adaptation of Rhododendron species in the subgenus Hymenanthes. Consequently, it was of interest to examine whether gene duplication and subsequent divergence have occurred in other sHSP genes, for example class I cytosolic sHSP genes (CT1sHSPs )i nRhododendron in Taiwan, where many endemic species have evolved as a result of habitat differentiation. †Methods A phylogeny of CT1sHSP amino acid sequences was built from Rhododendron, Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Vitis vinifera and other species for elucidation of the phylogenetic relationships among CT1sHSPs. Phylogenies of Rhododendron CT1sHSP nucleotide and amino acid sequences were generated for positive selection and functional divergence analysis, respectively. Positively selected sites and amino acid differences between types of Rhododendron CT1sHSPs were mapped onto the wheat sHSP16.9 protein structure. Average genetic distance (Dxy) and dN/dS ratios between types of Rhododendron CT1sHSP genes were analysed using sliding window analysis. Gene conversion was also assessed between types of Rhododendron CT1sHSPs. †Key Results Two types of Rhododendron CT1sHSP were identified. A high level of genetic similarity and diversity within and flanking the ACD, respectively, between types of Rhododendron CT1sHSP were found. Main differences between the two types of Rhododendron CT1sHSPs were: (1) increased hydrophobicity by two positively selected amino acid sites and a seven-amino-acid insertion in the N-terminal arm; and (2) increased structural flexibility and solubility by a seven-amino-acid insertion in the N-terminal arm and one positively selected amino acid site in the C-terminal extension. †Conclusions Functional conservation of the ACD of Rhododendron CT1sHSP genes was inferred because of strong purifying selection. However, sequence variations flanking the ACD inRhododendronCT1sHSP gene duplicates may have resulted in functional divergence and played important roles in chaperon function enhancement.

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.

Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes alpha-amylase, beta-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutation at a locus designated sex1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.

Selaginella tamariscina, one of the most primitive vascular plants, can remain alive in a desiccated state and resurrect when water becomes available. To evaluate the nature of desiccation tolerance in this plant, we compared the composition of soluble sugars and saturation ratios of phospholipids (PLs) between hydrated and desiccated tissues of S. tamariscina using gas chromatography. In this study, differences in gene expression and ABA contents were also analyzed during dehydration. The results revealed that trehalose (at >130 mg g(-1) DW) was the major soluble sugar, and low saturated fatty acid content in PLs (0.31) was maintained in both hydrated and desiccated tissues. In addition, the ABA content of S. tamariscina increased 3-fold, and genes involved in ABA signaling and cellular protection were up-regulated while photosystem-related genes were down-regulated during dehydration. The biochemical and molecular findings suggest that both constitutive and inducible protective molecules contribute to desiccation tolerance of S. tamariscina.

In this work, we attempted to study genetic differentiation between populations of Quercus glauca in Taiwan using nuclear microsatellite markers to infer the potential refugium in the last glaciation stage. Four microsatellite loci for 20 individuals each in 10 populations of Taiwan were analyzed. We found that Q. glauca has relatively high within-population diversity (H(E) = 0.741) and low population differentiation (F(ST) = 0.042) but shows isolation by distance. The most divergent populations, according to the average F(ST) for individual populations in comparison with every other population, were found in populations Cy, Sa, and Hy in southern Taiwan and Pa in north-central Taiwan. Moreover, populations Cy, Sa, and Pa were recognized as being the source populations for gene recolonization after the last glaciation stage. In addition, the three sites of Wu, Ym, and Cy exhibited the highest gene diversities that coincided with populations with the highest chloroplast DNA variations. This may have resulted from an admixture of colonization routes. In conclusion, observations of the most divergent populations and source populations suggest that southern and probably north-central Taiwan may have potentially been refugia for Q. glauca in the last glaciation. This agrees with the possible refugium in southern Taiwan revealed by a previous study using chloroplast DNA markers.

This study infers a single origin and a once-widespread distribution of the Rhododendron pseudochrysanthum species complex in Taiwan based on chloroplast DNA (cpDNA) variation. In total, 124 individuals from five endemic Rhododendron species were used for amplifications of two chloroplast intergenic spacers: trnL-trnF and atpB-rbcL. The haplotype and nucleotide diversities were much lower for the R. pseudochrysanthum complex, comprised of the species R. pseudochrysanthum, R. morii, Rhododendron rubropunctatum, and Rhododendron hyperythrum, than for Rhododendron formosanum. Two measures of pairwise population differentiation, N(ST) and F(ST), consistently revealed mostly non-significant levels of genetic divergence between populations of the R. pseudochrysanthum complex. No genetic difference was found among the four species of the R. pseudochrysanthum complex by analysis of molecular variance (AMOVA), which is concordant with the parsimonious topology of cpDNA haplotypes for the complex. Nested clade analysis (NCA) of the cpDNA haplotypes indicated that restricted gene flow with isolation-by-distance characterized the recolonization pattern of the R. pseudochrysanthum complex. In contrast, the NCA analysis indicated a contiguous range expansion for cpDNA haplotypes of R. formosanum. This research suggests a once-widespread distribution of the R. pseudochrysanthum complex probably via north-to-south colonization of mid-elevations during low-temperature periods of the Pleistocene. Population fragmentation followed the warmer climate which began in the Holocene and resulted in the present-day range contraction into high elevations.

Phylogeographical and mismatch analysis of chloroplast DNA (cpDNA) variation were used to infer the temporal dynamics of distributional and demographic history of Taiwan fir (Cunninghamia konishii). We examined 64 and 52 trees from 17 populations of C. konishii and 14 provenances of C. lanceolata, respectively, by sequencing three intergenic spacers and one intron using cpDNA universal primers. Of the aligned 1888 base pairs (bp) sequence, 30 varied among 28 haplotypes, which consisted of three transitions, 14 transversions and 13 indels. One ancestral haplotype was found in 86 individuals across the surveyed range of both species, C. konishii and C. lanceolata, which was distributed in all populations and provenances. The 28 haplotypes also included 15 C. konishii specific and 12 C. lanceolata-specific haplotypes. Ancestral haplotype was found fixed in five populations of C. konishii and five provenances of C. lanceolata. Other haplotypes occurred mainly as singletons. The levels of population differentiation studied are relatively low in both Cunninghamia species. The nucleotide diversity (theta) of chloroplast DNA sequences within C. konishii was slightly higher than that of C. lanceolata. Excess in singletons as well as star-like phylogeny of haplotypes suggested no clearcut migration patterns of C. konishii after glacial maximum. One probable demographic history of C. konishii is the postglacial population growth of C. konishii after a glacial bottleneck event. This inference is supported by the combined results of fossil pollen record, low nucleotide diversity, significant Tajima's d-value, phylogeographical analysis and unimodal mismatch distribution. Similarities and discrepancies between our results and those of Lu et al. (2001) are discussed.

Variations in mitochondrial DNA in Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. were studied in 140 trees from 32 populations collected from within the tree's natural range. By sequencing two mitochondrial DNA intron fragments (nad4/3-nad4/4r and nad7/2-nad7/3r), we revealed a total of 1788 bp and five polymorphic sites which allowed us to distinguish six mitotypes. The mitochondrial DNA markers provided replicated data to support population phylogeographical scenarios suggested previously using chloroplastic DNA markers. The gene genealogical tree of mitochondrial DNA was partially congruent with the chloroplastic DNA tree owing to the slower mutation rate and different mutational direction. Significant linkage disequilibrium existed between the two organellar genomes. Further paring analyses between fragments synthesized using different primers, accompanied by exclusion of polymorphic sites, showed that the random association could be attributed specifically to one of the polymorphic sites of the petG-trnP fragment of the chloroplastic genome, and the three polymorphic sites of the nad4/3-nad4/4r fragment of the mitochondrial genome. The former was inferred to derive from paternal leakage, and the latter from recurrent mutation. These polymorphic sites were also responsible for uncoupling of the combined gene tree of mitotype and chlorotype. In conclusion, specific fragments found in this study contribute to the incomplete congruence of the two organellar lineages that otherwise associate well phylogeographically.

A new cyclitol which is abundant in the late developmental stages of leucaena (Leucaena leucocephala (Lam.) de Wit) seeds was identified by HPLC, NMR, and GCMS as O-a-D-gaIactopyranosyl-(l —*• l)-3-O-methyl-D-cA/roinositol, a new galactopinitol. This galactopinitol was initially detected midway through seed development and increased to 10.2 mg (g DW)~\ but decreased in mature seeds to its about a half. Stachyose content increased greatly and remained the most abundant of the soluble sugars in mature seeds (25.6 mg (g DW)' 1 ). Artificial drying at 73% relative humidity of 70 DPA immature seeds induced the accumulation of raffinose, stachyose, galactopinitol and galactinol, but the total amounts of these sugars were only about half of those found in mature seeds. Seed germination decreased following an initial increase after 8 d artificial drying to a moisture content of 24%, and this dehydration damage probably is because of underdevelopm ent of seed tissue. Galactopinitol changes in a similar fashion to the oligosaccharides during the late developmental stage and dehydration experiment, implying that galactopinitol may play a role in desiccation tolerance of leucaena seeds.

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.

Induction of pectinmethylesterase (PME) activity in the pericarp of achenes can be achieved by opening the syconium of the jelly-fig (Ficus awkeotsang Makino) and exposing the achenes to air. Among several factors examined, exposure to the atmosphere is the critical factor for the induction of the synthesis of PME in achenes obtained from green, pollinated syconia at different developmental stages. (...)

A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F1 progeny of a cross between the mutant and wild type had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus.

A starch deficient mutant of Arabidopsis thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild type. A single, nuclear mutation at a previously undescribed locus designated adg2 is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild type, it accumulated 40% as much starch when grown in a 12 hour photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates its steady state accumulation. Immunological analysis of leaf extracts using antibodies against the spinach 54 and 51 kilodalton (kD) ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54 kD polypeptide and has only about 4% as much as the wild type of a cross-reactive 51 kD polypeptide. This result and genetic studies suggested that adg2 is a structural gene which codes for the 54 kD polypeptide, and provides the first functional evidence that the 54 kD polypeptide is a required component of the native ADPglucose pyrophosphorylase enzyme.

Pectinmethylesterase from the pericarp of jelly fig (Ficus awkeotsang) achenes was extracted and purified to a specific activity of 289 micromole proton produced per minute per milligram protein. Pectinmethylesterase, a major protein with high specific activity in the crude extract, was monomeric with a molecular weight of 38,000. The enzyme preparation was stable in distilled water at 4 degrees C for at least 6 months, and at 60 degrees C for at least 10 minutes. This enzyme functioned optimally at pH 6.5 to 7.5 when the assay mixture contained no NaCl or at low NaCl concentration. The pH optimum shifted to lower pH as the NaCl concentration was increased. The K(m) value for pectin was 0.75 milligram per milliliter pectin, corresponding to a V(max) value of 310 micromoles per minute per milligram protein. Inhibition studies with antibodies indicated that jelly fig achene pectinmethylesterase and the two other pectinmethylesterases from orange and tomato were similar in their active site conformation; however, the surface determinants may be very different because no precipitation between anti-jelly fig pectinmethylesterase immune serum and the pectin methylesterase from orange and tomato could be observed in the double immunodiffusion analysis. Specific antisera raised against jelly fig achene pectinmethylesterase in a Western blot experiment also showed low similarity between jelly fig pectinmethylesterase with that from orange and tomato. This observation was also supported by the very low isoelectric point (pH 3.5) of jelly fig pectinmethylesterase, compared with high isoelectric points reported for most of the pectinmethylesterases. Amino acid composition and N-terminal sequence have been obtained. High homology of the N-terminal amino acid residues between jelly fig and tomato pectinmethylesterase (O Markovic, H Jornvall [1986] Eur J Biochem 158: 455-462) was observed. Pectinmethylesterase activity causes the release of protons from the deesterification of pectin such that a low pH environment is created, and this may be related to the cell growth. Pectinmethylesterase is not needed for jelly fig seed germination, however the gel formed from pectin and pectinmethylesterase may insure a water source for the germinating jelly fig seeds.

Three classes of mutants of Arabidopsis thaliana (L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants was due to an up to 40-fold increase in the activity of an extrachloroplast β-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of β-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that β-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.

A major triticale (X Triticosecale Wittmack) endosperm acid phosphatase (EC 3.1.2.2) (APase) from sib-lines producing plump and shriveled seed was purified 140- and 230-fold to a specific activity of 94 and 153 micromoles per minute per milligram protein respectively, by ammonium sulfate fractionation, ion-exchange chromatography, chromatofocusing, affinity column chromatography, and gel filtration. The purified enzyme from both materials is a monomeric glycoprotein with an apparent molecular weight of 45,700 +/- 500 containing 12% carbohydrate and an apparent isoelectric point of pH 5.9. It hydrolyzes tri- and di-phosphate of nucleosides as well as phosphate esters and exhibits characteristics of ATP-hydrolase and phosphatase. About 2-fold more of the APase was isolated from shriveled seeds, and the purified enzyme exhibited 3- and 5-fold higher V(max) for p-nitrophenyl phosphate and ATP, respectively, than that of plump seed. The I(50) for Pi concentration was 5.5-fold higher in APase of shriveled seed than the plump one. These varied quantitative and kinetic properties substantiate the role of APase in lines with shriveled seeds being reduction of starch accumulation by depleting substrates and energy supply in the cytosol.

Amylolytic enzymes of Arabidopsis leaf tissue were partially purified and characterized. Endoamylase, starch phosphorylase, d-enzyme (transglycosylase), and possibly exoamylase were found in the chloroplasts. Endoamylase, fraction A2, found only in the chloroplast, was resolved from the exoamylases by chromatography on a Mono Q column and migrated with an R(F) of 0.44 on 7% polyacrylamide gel electrophoresis. Exoamylase fraction, A1, has an R(F) of 0.23 on the polyacrylamide gel. Viscometric analysis showed that A1 has a slope of 0.013, which is same as that of A3, the extrachloroplastic amylase. A1, however, can be distinguished from A3 by having much higher amylolytic activity in succinate buffer than acetate buffer, and having much less reactivity with amylose. A1 probably is also localized in the chloroplast, and contributes to the 30 to 40% higher amylolytic activity of the chloroplast preparation in succinate than acetate buffer at pH 6.0. The high activity of d-enzyme compared to the amylolytic activity in the chloroplast suggests that transglycosylation probably has an important role during starch degradation in Arabidopsis leaf. Extrachloroplastic amylase, A3, has an R(F) of 0.55 on 7% electrophoretic gel and constitutes 80% of the total leaf amylolytic activity. The results of substrate specificity studies, action pattern and viscometric analyses indicate that the extrachloroplastic amylases are exolytic.

A certain amount of the carbon dioxide that is converted to 3-P-glycerate in plants during photosynthesis is utilized for formation of the reserve polysaccharides present in the leaf starch granule. However, the fraction of carbon accumulated as starch in different plants varies. Species such as barley, spinach and wheat accumulate proportionately more sucrose than starch whereas the leaves of peanuts, soybean and tobacco accumulate more starch than sucrose. It is believed that an important factor in the partitioning of carbon between sucrose and starch is the level of sucrose-P synthase (1,2). The levels of sucrose-P synthase in the leaf cell correlates fairly well with the amount of 14CO2 incorporated into sucrose and inversely correlates with the amount of 14CO2 fixed into the starch fraction.