Frequent ploidy changes in Salicaceae indicates widespread sharing of the salicoid whole genome duplication by the relatives of Populus L. and Salix L.

BACKGROUNDS: Populus and Salix belong to Salicaceae and are used as models to investigate woody plant physiology. The variation of karyotype and nuclear DNA content can partly reflect the evolutionary history of the whole genome, and can provide critical information for understanding, predicting, and potentially ameliorating the woody plant traits. Therefore, it is essential to study the chromosome number (CN) and genome size in detail to provide information for revealing the evolutionary process of Salicaceae. RESULTS: In this study, we report the somatic CNs of seventeen species from eight genera in Salicaceae. Of these, CNs for twelve species and for five genera are reported for the first time. Among the three subfamilies of Salicaceae, the available data indicate CN in Samydoideae is n = 21, 22, 42. The only two genera, Dianyuea and Scyphostegia, in Scyphostegioideae respectively have n = 9 and 18. In Salicoideae, Populus, Salix and five genera closely related to them (Bennettiodendron, Idesia, Carrierea, Poliothyrsis, Itoa) are based on relatively high CNs from n = 19, 20, 21, 22 to n = 95 in Salix. However, the other genera of Salicoideae are mainly based on relatively low CNs of n = 9, 10, 11. The genome sizes of 35 taxa belonging to 14 genera of Salicaceae were estimated. Of these, the genome sizes of 12 genera and all taxa except Populus euphratica are first reported. Except for Dianyuea, Idesia and Bennettiodendron, all examined species have relatively small genome sizes of less than 1 pg, although polyploidization exists. CONCLUSIONS: The variation of CN and genome size across Salicaceae indicates frequent ploidy changes and a widespread sharing of the salicoid whole genome duplication (WGD) by the relatives of Populus and Salix. The shrinkage of genome size after WGD indicates massive loss of genomic components. The phylogenetic asymmetry in clade of Populus, Salix, and their close relatives suggests that there is a lag-time for the subsequent radiations after the salicoid WGD event. Our results provide useful data for studying the evolutionary events of Salicaceae.

NRAMP1 promotes iron uptake at the late stage of iron deficiency in poplars.

Iron (Fe) is an essential micronutrient for plant survival and proliferation. Plants have evolved complex mechanisms to maintain Fe homeostasis in response to Fe deficiency. In this study, we evaluated the physiological, biochemical and transcriptomic differences between poplars grown under Fe-sufficient and Fe-deficient conditions to elucidate the mechanistic responses of poplars to Fe deficiency. Our results revealed that chlorophyll synthesis and photosynthesis were inhibited under Fe-deficient conditions. The inhibition of these pathways caused chlorosis and reduced shoot growth. Although both photosynthetic systems (PSI and PSII) were inhibited under Fe limitation, PSI was affected more severely and earlier than PSII. Fe deficiency also promoted root growth and increased the accumulation of divalent metal ions in roots. IRT1 and NRAMP1 are both Fe2+ transporters for iron uptake in Arabidopsis. In this study, however, only NRAMP1 was induced to promote Fe2+ uptake in roots at the late stage of Fe deficiency response. It indicated that NRAMP1, rather than the more well-known IRT1, might be a major Fe2+ transporter at the late stage of Fe-deficiency in poplars.

Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia.

Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp.

Divergence in Enzymatic Activities in the Soybean GST Supergene Family Provides New Insight into the Evolutionary Dynamics of Whole-Genome Duplicates.

Whole-genome duplication (WGD), or polyploidy, is a major force in plant genome evolution. A duplicate of all genes is present in the genome immediately following a WGD event. However, the evolutionary mechanisms responsible for the loss of, or retention and subsequent functional divergence of polyploidy-derived duplicates remain largely unknown. In this study we reconstructed the evolutionary history of the glutathione S-transferase (GST) gene family from the soybean genome, and identified 72 GST duplicated gene pairs formed by a recent Glycine-specific WGD event occurring approximately 13 Ma. We found that 72% of duplicated GST gene pairs experienced gene losses or pseudogenization, whereas 28% of GST gene pairs have been retained in the soybean genome. The GST pseudogenes were under relaxed selective constraints, whereas functional GSTs were subject to strong purifying selection. Plant GST genes play important roles in stress tolerance and detoxification metabolism. By examining the gene expression responses to abiotic stresses and enzymatic properties of the ancestral and current proteins, we found that polyploidy-derived GST duplicates show the divergence in enzymatic activities. Through site-directed mutagenesis of ancestral proteins, this study revealed that nonsynonymous substitutions of key amino acid sites play an important role in the divergence of enzymatic functions of polyploidy-derived GST duplicates. These findings provide new insights into the evolutionary and functional dynamics of polyploidy-derived duplicate genes.

Molecular characterization of a dehydroascorbate reductase from Pinus bungeana.

Abstract Dehydroascorbate reductase (DHAR) plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR (PbDHAR) from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcription-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coli following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity (19.84 micromol/min per mg) and high affinity (a K(m) of 0.08 mM) towards the substrates dehydroascorbate (DHA). Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55 degrees C. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.

Catalytic properties of glutathione-binding residues in a tau class glutathione transferase (PtGSTU1) from Pinus tabulaeformis.

Glutathione transferases (GSTs) play important roles in stress tolerance and detoxification in plants. However, there is extremely little information on the molecular characteristics of GSTs in gymnosperms. In a previous study, we cloned a tau class GST (PtGSTU1) from a gymnosperm (Pinus tabulaeformis) for the first time. Based on the N-terminal amino acid sequence identity to the available crystal structures of plant tau GSTs, Ser13, Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 were proposed as glutathione-binding (G-site) residues. The importance of Ser13 as a G-site residue was investigated previously. The functions of Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 are examined in this study through site-directed mutagenesis. Enzyme assays and thermal stability measurements on the purified recombinant PtGSTU1 showed that substitution at each of these sites significantly affects the enzyme's substrate specificity and affinity for GSH, and these residues are essential for maintaining the stability of PtGSTU1. The results of protein expression and refolding analyses suggest that Ile54 is involved in the protein folding process. The findings demonstrate that the aforementioned residues are critical components of active sites that contribute to the enzyme's catalytic activity and structural stability.

Molecular characterization of a glutathione transferase from Pinus tabulaeformis (Pinaceae).

Glutathione transferases (GSTs) play important roles in stress tolerance and detoxification metabolism in plants. To date, studies on GSTs in higher plants have focused largely on agricultural plants. In contrast, there is virtually no information on the molecular characteristics of GSTs in gymnosperms. The present study reports for the first time the cloning, expression and characteristics of a GST gene (PtGSTU1) from a pine, Pinus tabulaeformis, which is widely distributed from northern to central China covering cold temperate and drought regions. The PtGSTU1 gene encodes a protein of 228 amino acid residues with a calculated molecular mass of 26.37 kDa. Reverse transcription PCR revealed that PtGSTU1 was expressed in different tissues, both above and below ground, of P. tabulaeformis. The over-expressed recombinant PtGSTU1 showed high activity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a Km of 0.47 mM and Vmax of 169.1 micromol/min per mg of protein. The recombinant PtGSTU1 retained more than 60% of its maximum enzymatic activity from 15 degrees C to 45 degrees C with a broad optimum Tm range of 25 degrees C - 35 degrees C. The enzyme had a maximum activity at approximately pH 8.5 - 9.0. Site-directed mutagenesis revealed that Ser13 in the N-terminal domain is a critical catalytic residue, responsible for stabilisation of the thiolate anion of enzyme-bound glutathione. Based on comparative analyses of its amino acid sequence, phylogeny and predicted three-dimensional structure, the PtGSTU1 should be classified as a tau class GST.