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.

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.

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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.