Populus euphratica plant cadmium resistance 2 mediates Cd tolerance by root efflux of Cd ions in poplar.

KEY MESSAGE: Populus euphratica PePCR2 increases Cd resistance by functioning as a Cd extrusion pump and by mediating the expression of genes encoding other transporters. Cadmium (Cd) is a non-essential, toxic metal that negatively affects plant growth. Plant cadmium resistance (PCR) proteins play key roles in the response to heavy metal stress. In this study, we isolated the gene PePCR2 encoding a plant PCR from Populus euphratica. PePCR2 gene transcription was induced by Cd, and its transcript level peaked at 24 h after exposure, at a level approximately 18-fold higher than that at 0 h. The PePCR2 protein was localized to the plasma membrane. Compared with yeast cells harboring the empty vector, yeast cells expressing PePCR2 showed enhanced Cd tolerance and a lower Cd content. Compared with wild-type (WT) plants, poplar overexpressing PePCR2 showed higher Cd resistance. Net Cd2+ efflux measurements showed that Cd2+ efflux from the roots was 1.5 times higher in the PePCR2-overexpressing plants than in WT plants. Furthermore, compared with WT plants, the PePCR2-overexpressing plants showed increased transcript levels of ABCG29, HMA5, PDR2, YSL7, and ZIP1 and decreased transcript levels of NRAMP6, YSL3, and ZIP11 upon exposure to Cd. These data show that PePCR2 increased Cd resistance by acting as a Cd extrusion pump and/or by regulating other Cd2+ transporters to decrease Cd toxicity in the cytosol. The results of this study identify a novel plant gene with potential applications in Cd removal, and provide a theoretical basis for reducing Cd toxicity and protecting food safety.

Populus euphratica plant cadmium tolerance PePCR3 improves cadmium tolerance.

Contamination of soils with toxic heavy metals is a major environmental problem. Growing crop plants that can promote the efflux of heavy metals is an effective strategy in contaminated soils. The plant cadmium resistance (PCR) protein is involved in the translocation of heavy metals, specifically zinc and cadmium (Cd). In this study, yeast expressing Populus euphratica PCR3 (PePCR3) showed enhanced Cd tolerance and decreased Cd accumulation under Cd treatment. Real-time quantitative PCR analyses revealed up-regulation of PePCR3 in poplar seedlings under Cd stress. Localization analysis revealed that PePCR3 localizes at the plasma membrane. The plant growth and biomass were greater in PePCR3-overexpressing (OE) transgenic hybrid poplar lines than in wild type (WT). Physiological parameters analyses indicated that, compared with WT, PePCR3-OE transgenic lines were more tolerant to Cd. In addition, more Cd was excreted in the roots of the PePCR3-OE transgenic lines than in those of WT, but the remaining Cd in transgenic lines was more translocated into the stems and leaves. Eight genes encoding transporters showed increased transcript levels in PePCR3-OE transgenic lines under Cd treatment, implying that PePCR3 interacts with other transporters to translocate Cd. Thus, PePCR3 may be an important genetic resource for generating new lines that can enhance Cd translocation to phytoremediation in contaminated soils.

Plant cadmium resistance 6 from Salix linearistipularis (SlPCR6) affects cadmium and copper uptake in roots of transgenic Populus.

Phytoextraction in phytoremediation is one of the environmentally friendly methods used for restoring soils contaminated by heavy metals (HMs). The screening and identification of HM-resistant plants and their regulatory genes associated with HM ion transport are the key research aims in this field. In this study, a plant cadmium (Cd) resistance (PCR) gene family member, SlPCR6, was identified in roots of Salix linearistipularis, which exhibits strong HM resistance. The results revealed that SlPCR6 expression was induced in S. linearistipularis roots in response to Cd stress. Furthermore, SlPCR6 was mainly localized on the plasma membrane. Compared with the wild type, SlPCR6 overexpression reduced the Cd and copper (Cu) contents in the transgenic poplar (84 K) and increased its Cd and Cu resistance. The roots of transgenic poplar seedlings had lower net Cd and Cu uptake rates than wild type roots. Further investigation revealed that the transcript levels of multiple HM ion transporters were not significantly different between the roots of the wild type and those of the transgenic poplar. These results suggest that SlPCR6 is directly involved in Cd and Cu transport in S. linearistipularis roots. Therefore, SlPCR6 can serve as a candidate gene to improve the phytoextraction of the HMs Cd and Cu through genetic engineering.

Study on a new type of environment-friendly polymer and its preliminary application as soil consolidation agent during tree transplanting.

Transplanting trees with rhizospheric soil is an important way to facilitate tree survival in the process of landscaping and reforestation. Traditional way to prevent looseness of rhizospheric soil is forming soil balls around the roots with bags, boxes or rope wrapping, which is cumbersome, laborious and easy to break. This study is aimed to develop a new type of degradable environment-friendly polymer as soil consolidation agent to facilitate tree transplanting. In this paper, the KGM/CA/PVA ternary blending soil consolidation agent was prepared by using Konjac glucomannan (KGM), chitosan (CA) and polyvinyl alcohol (PVA) as raw materials. Through the verification and evaluation, the clay and sandy soil can be consolidated and formed into soil balls by the ternary blend adhesive, which was convenient for transportation. The preliminary application of the ternary blend adhesive in the transplanting process of sierra salvia, Japanese Spindle (Euonymus japonicus) and Juniperus sabina 'Tamaricifolia' confirmed that the application of soil consolidation agent can effectively solve the problem that the root ball of seedling is easily broken in the process of transplant. And the application of soil consolidation agent has no adverse effect on the growth of transplanted seedlings. The research and development of ternary blending soil consolidation agent and its preliminary application in seedling transplanting will provide a new solution to solve the problem of soil ball breakage in the process of seedling transplanting. This is an important stage in the development of new seedling transplanting technology. Therefore, the research and development of soil consolidation agent is of great significance.

The Osmotin-Like Protein Gene PdOLP1 Is Involved in Secondary Cell Wall Biosynthesis during Wood Formation in Poplar.

Osmotin-like proteins (OLPs) mediate defenses against abiotic and biotic stresses and fungal pathogens in plants. However, no OLPs have been functionally elucidated in poplar. Here, we report an osmotin-like protein designated PdOLP1 from Populus deltoides (Marsh.). Expression analysis showed that PdOLP1 transcripts were mainly present in immature xylem and immature phloem during vascular tissue development in P. deltoides. We conducted phenotypic, anatomical, and molecular analyses of PdOLP1-overexpressing lines and the PdOLP1-downregulated hybrid poplar 84K (Populus alba × Populus glandulosa) (Hybrid poplar 84K PagOLP1, PagOLP2, PagOLP3 and PagOLP4 are highly homologous to PdOLP1, and are downregulated in PdOLP1-downregulated hybrid poplar 84K). The overexpression of PdOLP1 led to a reduction in the radial width and cell layer number in the xylem and phloem zones, in expression of genes involved in lignin biosynthesis, and in the fibers and vessels of xylem cell walls in the overexpressing lines. Additionally, the xylem vessels and fibers of PdOLP1-downregulated poplar exhibited increased secondary cell wall thickness. Elevated expression of secondary wall biosynthetic genes was accompanied by increases in lignin content, dry weight biomass, and carbon storage in PdOLP1-downregulated lines. A PdOLP1 coexpression network was constructed and showed that PdOLP1 was coexpressed with a large number of genes involved in secondary cell wall biosynthesis and wood development in poplar. Moreover, based on transcriptional activation assays, PtobZIP5 and PtobHLH7 activated the PdOLP1 promoter, whereas PtoBLH8 and PtoWRKY40 repressed it. A yeast one-hybrid (Y1H) assay confirmed interaction of PtoBLH8, PtoMYB3, and PtoWRKY40 with the PdOLP1 promoter in vivo. Together, our results suggest that PdOLP1 is a negative regulator of secondary wall biosynthesis and may be valuable for manipulating secondary cell wall deposition to improve carbon fixation efficiency in tree species.

Comparative Transcriptome Analysis Reveals Stem Secondary Growth of Grafted Rosa rugosa 'Rosea' Scion and R. multiflora 'Innermis' Rootstock.

Grafted plant is a chimeric organism formed by the connection of scion and rootstock through stems, so stem growth and development become one of the important factors to affect grafted plant state. However, information regarding the molecular responses of stems secondary growth after grafting is limited. A grafted Rosa plant, with R. rugosa 'Rosea' as the scion (Rr_scion) grafted onto R. multiflora 'Innermis' as the stock (Rm_stock), has been shown to significantly improve stem thickness. To elucidate the molecular mechanisms of stem secondary growth in grafted plant, a genome-wide transcription analysis was performed using an RNA sequence (RNA-seq) method between the scion and rootstock. Comparing ungrafted R. rugosa 'Rosea' (Rr) and R. multiflora 'Innermis' (Rm) plants, there were much more differentially expressed genes (DEGs) identified in Rr_scion (6887) than Rm_stock (229). Functional annotations revealed that DEGs in Rr_scion are involved in two Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways: the phenylpropanoid biosynthesis metabolism and plant hormone signal transduction, whereas DEGs in Rm_stock were associated with starch and sucrose metabolism pathway. Moreover, different kinds of signal transduction-related DEGs, e.g., receptor-like serine/threonine protein kinases (RLKs), transcription factor (TF), and transporters, were identified and could affect the stem secondary growth of both the scion and rootstock. This work provided new information regarding the underlying molecular mechanism between scion and rootstock after grafting.

Proline-rich protein gene PdPRP regulates secondary wall formation in poplar.

Proline-rich protein (PRP) is a plant cell wall associated protein. Its distinct patterns of regulation and localization studied in a number of plants indicate that it may play important roles in growth and development. However, the mechanism of how these genes control secondary cell wall development in tree species is largely unknown. Here, we report that a Populus deltoides (Marsh.) proline-rich protein gene PdPRP was preferentially expressed in immature/mature phloem and immature xylem in P. deltoides. PdPRP overexpression increased poplar plant height and diameter as well as the radial width of the phloem and xylem regions, facilitated secondary wall deposition, and induced expression of genes related to microfibril angle (MFA) and secondary wall biosynthesis. Downregulation of PdPRP retarded poplar growth, decreased the radial width of the secondary phloem and secondary xylem regions, reduced secondary wall thickening in fibers and vessels, and decreased the expression of genes related to MFA and secondary wall biosynthesis. These results suggest that PdPRP might positively regulate secondary cell wall formation by promoting secondary wall thickening and expansion in poplar. PdPRP-overexpressing poplar had a lower MFA, indicating that PdPRP may be useful for improving wood stiffness and properties in plants. Together, our results demonstrate that PdPRP is a proline-rich protein associated with cell wall development, playing a critical role in regulating secondary cell wall formation in poplar.

Expression profiling and functional characterization of a CBL-interacting protein kinase gene from Populus euphratica.

KEY MESSAGE: This is the first report on the function of a member of the CIPK family in Populus euphratica. The Ca(2+)-dependent salt overly sensitive (SOS) pathway has been shown to play an essential role in maintaining ion homeostasis and conferring salt tolerance. One component of the SOS pathway, SOS1, was identified in the salt-resistant tree P. euphratica. In this study, we identified and functionally characterized another component of the SOS pathway in this tree called PeSOS2 or PeCIPK26. On the basis of protein sequence similarity and complementation studies in Arabidopsis, PeCIPK26 was concluded to be the functional homolog of Arabidopsis AtSOS2. Yeast two-hybrid assays revealed that PeCIPK26 can interact with four calcineurin B-like (CBL) genes, i.e., PeCBL1, PeCBL4/PeSOS3, PeCBL9 and PeCBL10. Autophosphorylation assays showed that PeCIPK26 is an active protein kinase. Expression profile analysis demonstrated that PeCIPK26 is expressed in root, stem and leaf, and throughout the cell including cell membrane, cytoplasm and nucleus; in addition, it can be induced under salt-stress treatment. Functions of PeCIPK26 in salt tolerance were evaluated by gene overexpression in Arabidopsis cipk24 mutants. The better salt tolerance of transgenic plants relative to mutants was shown by their higher germination rate, lower Na(+) accumulation and higher capacity to discharge Na(+) when grown with NaCl. These results suggest the involvement of PeCIPK26 in the salt-stress response of P. euphratica.

The calcium sensor PeCBL1, interacting with PeCIPK24/25 and PeCIPK26, regulates Na(+)/K (+) homeostasis in Populus euphratica.

KEY MESSAGE: This paper is the first to directly link two types of ion channel regulation pathway into an emerging and complex CBL-CIPK signal system in wooden plant. In Arabidopsis thaliana, the calcineurin b-like (CBL) 1 gene has been shown to be necessary in response to abiotic stresses. In this study, we identified CBL1 in the woody plant Populus euphratica, designated as PeCBL1. Heterologous expression of PeCBL1 could build the resistance of sensitive phenotypes to low K(+) stress in the corresponding Arabidopsis cbl1/cbl9 mutant, and display a salt-sensitive phenotype compared with the mutant. Protein interaction analysis showed that PeCBL1 can interact with PeCIPK24, 25 and 26, and form different complexes of PeCBL-PeCIPK. To further investigate the mechanism of PeCBL1, we analyzed the fluxes of K(+) and Na(+) in roots of the wild-type Arabidopsis, cbl1/9 mutant, and PeCBL1 transgenic plants under low K(+) stress and high Na(+) stress. These analyses revealed that, compared to the cbl1/9 mutant, the PeCBL1 transgenic plant roots exhibited a higher capacity to absorb K(+) after exposure to low K(+) stress, and a lower capacity to discharge Na(+) after exposure to salt stress. The results suggest that CBL1 interacts with CIPK24, CIPK25 and CIPK26 to regulate Na(+)/K(+) homeostasis in Populus euphratica.