Picea wilsonii NAC31 and DREB2A Cooperatively Activate ERD1 to Modulate Drought Resistance in Transgenic Arabidopsis.

The NAC family of transcription factors (TFs) regulate plant development and abiotic stress. However, the specific function and response mechanism of NAC TFs that increase drought resistance in Picea wilsonii remain largely unknown. In this study, we functionally characterized a member of the PwNAC family known as PwNAC31. PwNAC31 is a nuclear-localized protein with transcriptional activation activity and contains an NAC domain that shows extensive homology with ANAC072 in Arabidopsis. The expression level of PwNAC31 is significantly upregulated under drought and ABA treatments. The heterologous expression of PwNAC31 in atnac072 Arabidopsis mutants enhances the seed vigor and germination rates and restores the hypersensitive phenotype of atnac072 under drought stress, accompanied by the up-regulated expression of drought-responsive genes such as DREB2A (DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A) and ERD1 (EARLY RESPONSIVE TO DEHYDRATION STRESS 1). Yeast two-hybrid and bimolecular fluorescence complementation assays confirmed that PwNAC31 interacts with DREB2A and ABF3 (ABSCISIC ACID-RESPONSIVE ELEMENT-BINDING FACTOR 3). Yeast one-hybrid and dual-luciferase assays showed that PwNAC31, together with its interaction protein DREB2A, directly regulated the expression of ERD1 by binding to the DRE element of the ERD1 promoter. Collectively, our study provides evidence that PwNAC31 activates ERD1 by interacting with DREB2A to enhance drought tolerance in transgenic Arabidopsis.

Interspecific Gene Flow and Selective Sweeps in Picea wilsonii, P. neoveitchii and P. likiangensis.

Genome-wide single nucleotide polymorphism (SNP) markers were obtained by genotyping-by-sequencing (GBS) technology to study the genetic relationships, population structure, gene flow and selective sweeps during species differentiation of Picea wilsonii, P. neoveitchii and P. likiangensis from a genome-wide perspective. We used P. jezoensis and P. pungens as outgroups, and three evolutionary branches were obtained: P. likiangensis was located on one branch, two P. wilsonii populations were grouped onto a second branch, and two P. neoveitchii populations were grouped onto a third branch. The relationship of P. wilsonii with P. likiangensis was closer than that with P. neoveitchii. ABBA-BABA analysis revealed that the gene flow between P. neoveitchii and P. wilsonii was greater than that between P. neoveitchii and P. likiangensis. Compared with the background population of P. neoveitchii, the genes that were selected in the P. wilsonii population were mainly related to plant stress resistance, stomatal regulation, plant morphology and flowering. The genes selected in the P. likiangensis population were mainly related to plant stress resistance, leaf morphology and flowering. Selective sweeps were beneficial for improving the adaptability of spruce species to different habitats as well as to accelerate species differentiation. The frequent gene flow between spruce species makes their evolutionary relationships complicated. Insight into gene flow and selection pressure in spruce species will help us further understand their phylogenetic relationships and provide a scientific basis for their introduction, domestication and genetic improvement.

NAC Transcription Factor PwNAC11 Activates ERD1 by Interaction with ABF3 and DREB2A to Enhance Drought Tolerance in Transgenic Arabidopsis.

NAC (NAM, ATAF1/2, and CUC2) transcription factors are ubiquitously distributed in eukaryotes and play significant roles in stress response. However, the functional verifications of NACs in Picea (P.) wilsonii remain largely uncharacterized. Here, we identified the NAC transcription factor PwNAC11 as a mediator of drought stress, which was significantly upregulated in P. wilsonii under drought and abscisic acid (ABA) treatments. Yeast two-hybrid assays showed that both the full length and C-terminal of PwNAC11 had transcriptional activation activity and PwNAC11 protein cannot form a homodimer by itself. Subcellular observation demonstrated that PwNAC11 protein was located in nucleus. The overexpression of PwNAC11 in Arabidopsis obviously improved the tolerance to drought stress but delayed flowering time under nonstress conditions. The steady-state level of antioxidant enzymes' activities and light energy conversion efficiency were significantly increased in PwNAC11 transgenic lines under dehydration compared to wild plants. PwNAC11 transgenic lines showed hypersensitivity to ABA and PwNAC11 activated the expression of the downstream gene ERD1 by binding to ABA-responsive elements (ABREs) instead of drought-responsive elements (DREs). Genetic evidence demonstrated that PwNAC11 physically interacted with an ABA-induced protein-ABRE Binding Factor3 (ABF3)-and promoted the activation of ERD1 promoter, which implied an ABA-dependent signaling cascade controlled by PwNAC11. In addition, qRT-PCR and yeast assays showed that an ABA-independent gene-DREB2A-was also probably involved in PwNAC11-mediated drought stress response. Taken together, our results provide the evidence that PwNAC11 plays a dominant role in plants positively responding to early drought stress and ABF3 and DREB2A synergistically regulate the expression of ERD1.

Picea wilsonii transcription factor NAC2 enhanced plant tolerance to abiotic stress and participated in RFCP1-regulated flowering time.

KEY MESSAGE: Picea wilsonii transcription factor PwNAC2 enhanced plant tolerance to salt and drought stress through multiple signaling pathway and interacted with PwRFCP1 to participate in flowering regulation. NAC is one of the largest transcription factor families in plants, however, its role is not yet fully understood. Here, we identified a transcription factor PwNAC2 in Picea wilsonii, which localized in nucleus with transcriptional activity in C-terminal region and can form homodimer by itself. Expression analysis by real-time PCR showed that PwNAC2 was induced by multiple abiotic stresses and phytohormones stimuli. PwRFCP1 (Resemble-FCA-contain-PAT1 domain), an interaction protein of PwNAC2 was screened via yeast two hybrid. Luciferase complementation assay confirmed the interaction in vivo and bimolecular fluorescence complementation assay showed the interaction in nucleus. PwNAC2 overexpression retarded Arabidopsis hypocotyls growth which is closely related to light, whereas promotion of hypocotyls growth by PwRFCP1 is independent on light. Under drought or salt treatment, overexpression of PwNAC2 in Arabidopsis showed more vigorous seed germination and significant tolerance for seedlings by ROS scavenging, reducing of membrane damage, slower water loss and increased stomatal closure. ABA or CBF-pathway marker genes were substantially higher in PwNAC2 transgenic Arabidopsis. Overexpression of PwRFCP1 promotes flowering in transgenic Arabidopsis, whereas PwNAC2 delayed flowering by altering the expression of FT, SOC1 and FLC. In addtioin, PwRFCP1 overexpression plants showed no higher tolerance to stress treatment than Col-0. Collectively, our results indicate that PwNAC2 enhanced plant tolerance to abiotic stress through multiple signaling pathways and participated in PwRFCP1-regulated flowering time.

PicW2 from Picea wilsonii: preparation, purification, crystallization and X-ray diffraction analysis.

Low temperature is a major limiting factor for plant growth and development. Dehydrin proteins are generally induced in response to low-temperature stress. In previous research, a full-length dehydrin gene, PicW2, was isolated from Picea wilsonii and its expression was associated with hardiness to cold. In order to gain insight into the mechanism of low-temperature tolerance by studying its three-dimensional crystal structure, prokaryotically expressed PicW2 dehydrin protein was purified using chitosan-affinity chromatography and gel filtration, and crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.1 M HEPES pH 8.0, 25%(w/v) PEG 3350 using 4 mg ml-1 protein solution at 289 K. X-ray diffraction data were collected from a crystal at 100 K to 2.82 Šresolution. The crystal belonged to space group C121, with unit-cell parameters a = 121.55, b = 33.26, c = 73.39 Å, α = γ = 90.00, ß = 109.01°. The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.87 Å3 Da-1 and a solvent content of 57.20%. Owing to a lack of structures of homologous dehydrin proteins, molecular-replacement trials failed. Data collection for selenium derivatization of PicW2 and crystal structure determination is currently in progress.

Overexpression of a NF-YB3 transcription factor from Picea wilsonii confers tolerance to salinity and drought stress in transformed Arabidopsis thaliana.

Nuclear factor Y (NF-Y) is a highly conserved transcription factor comprising NF-YA, NF-YB and NF-YC subunits. To date, the roles of NF-Y subunit in plant still remain elusive. In this study, a subunit NF-YB (PwNF-YB3), was isolated from Picea wilsonii Mast. and its role was studied. PwNF-YB3 transcript was detected in all vegetative and reproductive tissues with higher levels in stem and root and was greatly induced by salinity, heat and PEG but not by cold and ABA treatment. Over-expression of PwNF-YB3 in Arabidopsis showed a significant acceleration in the onset of flowering and resulted in more vigorous seed germination and significant tolerance for seedlings under salinity, drought and osmotic stress compared with wild type plants. Transcription levels of salinity-responsive gene (SOS3) and drought-induced gene (CDPK1) were substantially higher in transgenic Arabidopsis than in wild-type plants. Importantly, CBF pathway markers (COR15B, KIN1, LEA76), but not ABA pathway markers CBF4, were greatly induced under condition of drought. The nuclear localization showed that NF-YB3 acted as a transcription factor. Taken together, the data provide evidence that PwNF-YB3 positively confers significant tolerance to salt, osmotic and drought stress in transformed Arabidopsis plants probably through modulating gene regulation in CBF-dependent pathway.

Different heavy metals have various effects on Picea wilsonii pollen germination and tube growth.

Heavy metal pollution has became one of the realistic matters of globality. Previous reports indicated that heavy metals could significantly inhibit pollen germination and tube growth. In the present study, comparative studies on the effects of different heavy metals (As, Hg, Cd, Cr and Cu) on in-vitro picea wilsonii pollen germination and tube growth were carried out. Microscopic evaluation revealed that different heavy metals had various degree of toxicity on P. wilsonii pollen tube development. As showed the most toxic effects on pollen germination, which was followed by Hg and Cd, while Cr and Cu showed relatively lower toxicity. Besides, pollen tubes showed varying shapes in response to different heavy metal stress. Pollen tubes treated with Cd, Hg and As were usually characterized by irregularly increasing diameters and swelling tips with distinct cytoplasimic vacuolation. On the other hand, except for the slightly increased diameters, no obvious abnormal shape were observed in tubes treated with Cr or Cu. Lyso-Tracker Green staining indicated that only Cd-treated pollen tubes showed numerous vacuole-like acidic organelles, though cytoplasmic vacuolization were also observed in pollen tubes treated with Hg and A. In brief, our data indicated that different heavy metals have various effects on Picea wilsonii pollen germination and tube growth, and that in-vitro pollen culture might be used as a competent system for biomonitoring of air pollution.

Population genetic evidence for speciation pattern and gene flow between Picea wilsonii, P. morrisonicola and P. neoveitchii.

BACKGROUND AND AIMS: Genetic drift due to geographical isolation, gene flow and mutation rates together make it difficult to determine the evolutionary relationships of present-day species. In this study, population genetic data were used to model and decipher interspecific relationships, speciation patterns and gene flow between three species of spruce with similar morphology, Picea wilsonii, P. neoveitchii and P. morrisonicola. Picea wilsonii and P. neoveitchii occur from central to north-west China, where they have overlapping distributions. Picea morrisonicola, however, is restricted solely to the island of Taiwan and is isolated from the other two species by a long distance. METHODS: Sequence variations were examined in 18 DNA fragments for 22 populations, including three fragments from the chloroplast (cp) genome, two from the mitochondrial (mt) genome and 13 from the nuclear genome. KEY RESULTS: In both the cpDNA and the mtDNA, P. morrisonicola accumulated more species-specific mutations than the other two species. However, most nuclear haplotypes of P. morrisonicola were shared by P. wilsonii, or derived from the dominant haplotypes found in that species. Modelling of population genetic data supported the hypothesis that P. morrisonicola derived from P. wilsonii within the more recent past, most probably indicating progenitor-derivative speciation with a distinct bottleneck, although further gene flow from the progenitor to the derivative continued. In addition, the occurrence was detected of an obvious mtDNA introgression from P. neoveitchii to P. wilsonii despite their early divergence. CONCLUSIONS: The extent of mutation, introgression and lineage sorting taking place during interspecific divergence and demographic changes in the three species had varied greatly between the three genomes. The findings highlight the complex evolutionary histories of these three Asian spruce species.