Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (Populus trichocarpa) and birch (Betula pendula). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (P. tremula ؋ tremuloides) to express a cytokinin catabolic gene, Arabidopsis CYTOKININ OXI-DASE 2, under the promoter of a birch CYTOKININ RECEPTOR 1 gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development.cambial activity ͉ cambium ͉ secondary development ͉ Populus ͉ CYTOKININ OXIDASE

In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.Winter embolism, the generation of air bubbles in xylem vessels induced by freeze-thaw cycles, often leads to a loss of hydraulic conductivity of the vessels (Cochard and Tyree, 1990; Améglio et al., 2001; Ewers et al., 2001). Vulnerability to winter embolism is related to the anatomy and vessel diameter of woody plants (Cochard and Tyree, 1990) and affects the ability of plants to survive cold climates and the geographic distribution of species (Tyree and Cochard, 1996; Pockman and Sperry, 1997; Lemoine et al., 1999).Detailed physiological studies of the responses of temperate woody plants to winter embolism have been made. Plants minimize the impact of winter embolism by replacing embolized vessels by new functional vessels every year and/or by refilling embolized vessels by generating positive xylem pressures (Holbrook and Zwieniecki, 1999; Tyree et al., 1999; Améglio et al., 2002). Although making new vessels is common to all the plants that exhibit secondary growth, the generation of xylem pressures has only been reported in a few species such as maple (Acer pseudoplatanus; O'Malley and Milburn, 1983; Tyree, 1983; Sperry et al., 1987 Sperry et al., , 1994, grapevine (Vitis vinifera; Sperry et al., 1987), birch (Betula alleghaniensis) (Sperry et al., 1994; Zhu et al., 2000), and walnut (Juglans regia; Améglio et al., 1995Améglio et al., , 2001 Ewers et al., 2001).In walnut trees, depending on the temperature, two types of positive xylem pressures have been ...

Bud break pattern is a key determinant of tree architecture. The mechanisms leading to the precedence of certain buds over the others are not yet fully explained, but the availability of soluble sugars may play a significant role, especially those in the xylem sap at the onset of the growing period. Here, we measured carbon availability in the different tissues (bud, xylem and bark). To assess the capacity of buds to use the xylem sap carbohydrates, the fluxes between xylem vessels and parenchyma cells, bark and buds of walnut (Juglans regia cv 'Franquette') were measured during the rest period until bud break. This uptake capacity varies according to the temperature, the sugar and the position on the branch of the fragment studied. Between December and March, in xylem tissues, the active component of sucrose uptake was predominant compared with diffusion (90% of the total uptake), whereas the active component accounted for more moderate amounts in buds (50% of the uptake). The active uptake of hexoses took place belatedly (April) in xylem. The flow rates between xylem vessels and buds increased 1 month before bud break and reached 2000 microg sucrose h(-)(1) g DW(-)(1). Fluxes seemed to depend on bud position on the branch. However, this study strongly suggests that they were mainly dependent on the sink strength of the buds and on the sink competition between bud, xylem parenchyma and bark.

Mechanical signals are important factors that control plant growth and development. External mechanical loadings lead to a decrease in elongation and a stimulation of diameter growth, a syndrome known as thigmomorphogenesis. A previous study has demonstrated that plants perceive the strains they are subjected to and not forces or stresses. On this basis, an integrative biomechanical model of mechanosensing was established ("sum-of-strains model") and tested on tomato (Solanum lycopersicum) elongation but not for local responses such as diameter growth or gene expression. The first aim of this interdisciplinary work was to provide a quantitative study of the effect of a single transitory bending on poplar (Populus tremula 3 alba) diameter growth and on the expression level of a primary mechanosensitive transcription factor gene, PtaZFP2. The second aim of this work was to assess the sum-of-strains model of mechanosensing on these local responses. An original bending device was built to study stem responses according to a controlled range of strains. A single bending modified plant diameter growth and increased the relative abundance of PtaZFP2 transcripts. Integrals of longitudinal strains induced by bending on the responding tissues were highly correlated to local plant responses. The sum-of-strains model of mechanosensing established for stem elongation was thus applicable for local responses at two scales: diameter growth and gene expression.