Correlation between mtDNA complexity and mtDNA replication mode in developing cotyledon mitochondria during mung bean seed germination.

The currently accepted model of recombination-dependent replication (RDR) in plant mitochondrial DNA (mtDNA) does not clearly explain how RDR progresses and how highly complex mtDNA develops. This study aimed to investigate the correlation between RDR and mtDNA complexity during mitochondrial development in mung bean (Vigna radiata) seed, and the initiation and processing of RDR in plant mitochondria. Flow cytometry, pulsed-field gel electrophoresis, electron microscopy, real-time PCR and biochemical studies were used in this study. The highly dynamic changes in mtDNA complexity correspond to mtDNA RDR activity throughout mitochondrial development. With in vitro freeze-thaw treatment or prolonged in vivo cold incubation, the mtDNA rosette core disappeared and the rosette structure converted to a much longer linear DNA structure. D-loops, Holliday junctions and putative RDR forks often appeared near the rosette cores. We hypothesize that the rosette core may consist of condensed mtDNA and a replication starting sequence, and play an initial and central role in RDR. The satellite cores in the rosette structure may represent the re-initiation sites of mtDNA RDR in the same parental molecule, thereby forming highly complex and giant mitochondrial molecules, representing the RDR intermediates, in vivo.

Historical spatial range expansion and a very recent bottleneck of Cinnamomum kanehirae Hay. (Lauraceae) in Taiwan inferred from nuclear genes.

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.

Functional phosphoproteomic profiling of phosphorylation sites in membrane fractions of salt-stressed Arabidopsis thaliana.

BACKGROUND: Under conditions of salt stress, plants respond by initiating phosphorylation cascades. Many key phosphorylation events occur at the membrane. However, to date only limited sites have been identified that are phosphorylated in response to salt stress in plants. RESULTS: Membrane fractions from three-day and 200 mM salt-treated Arabidopsis suspension plants were isolated, followed by protease shaving and enrichment using Zirconium ion-charged magnetic beads, and tandem mass spectrometry analyses. From this isolation, 18 phosphorylation sites from 15 Arabidopsis proteins were identified. A unique phosphorylation site in 14-3-3-interacting protein AHA1 was predominately identified in 200 mM salt-treated plants. We also identified some phosphorylation sites in aquaporins. A doubly phosphorylated peptide of PIP2;1 as well as a phosphopeptide containing a single phosphorylation site (Ser-283) and a phosphopeptide containing another site (Ser-286) of aquaporin PIP2;4 were identified respectively. These two sites appeared to be novel of which were not reported before. In addition, quantitative analyses of protein phosphorylation with either label-free or stable-isotope labeling were also employed in this study. The results indicated that level of phosphopeptides on five membrane proteins such as AHA1, STP1, Patellin-2, probable inactive receptor kinase (At3g02880), and probable purine permease 18 showed at least two-fold increase in comparison to control in response to 200 mM salt-stress. CONCLUSION: In this study, we successfully identified novel salt stress-responsive protein phosphorylation sites from membrane isolates of abiotic-stressed plants by membrane shaving followed by Zr4+-IMAC enrichment. The identified phosphorylation sites can be important in the salt stress response in plants.

Isolation and characterization of the ATP-binding cassette (ABC) transporter system genes from loofah witches' broom phytoplasma.

A clone containing a 3903 bp EcoRI-restriction fragment was obtained from a lambda(ZAP) genomic library of loofah witches' broom (LfWB) phytoplasma by plaque hybridization using a PCR fragment as a probe. Sequence analysis revealed that this fragment contained three open reading frames (ORFs). The deduced amino acid sequences of ORF 1 and ORF 2 showed a high homology with the ATP-binding proteins of the ABC transporter system genes of prokaryotes and eukaryotes, and encoded proteins with a molecular mass of 36 and 30 kDa, respectively. Based on amino acid sequence similarity, secondary structure, hydrophilicity and a signal peptide sequence at the N-terminus, we predicted that ORF 3 might encode a specific solute-binding prolipoprotein of the ABC transporter system with a molecular mass of 62 kDa. The cleavage site of this prolipoprotein signal peptide was similar to those of gram-positive bacteria. In addition to nutrient uptake, ABC transporter systems of bacteria also play a role in signal transduction, drug-resistance and perhaps virulence. The possible implications of the system to the survival and the pathogenesis of phytoplasma were discussed.

Source populations of Quercus glauca in the last glacial age in Taiwan revealed by nuclear microsatellite markers.

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.