Molecular and physiological studies in walnut (Juglans regia) are combined to establish the putative role of leaf plasma membrane aquaporins in the response of leaf hydraulic conductance (K leaf ) to irradiance. The effects of light and temperature on K leaf are described. Under dark conditions, K leaf was low, but increased by 400% upon exposure to light. In contrast to dark conditions, K leaf values of light-exposed leaves responded to temperature and 0.1 mM cycloheximide treatments. Furthermore, K leaf was not related to stomatal aperture. Data of real-time reverse transcription-polymerase chain reaction showed that K leaf dynamics were tightly correlated with the transcript abundance of two walnut aquaporins (JrPIP2,1 and JrPIP2,2). Low K leaf in the dark was associated with down-regulation, whereas high K leaf in the light was associated with up-regulation of JrPIP2. Light responses of K leaf and aquaporin transcripts were reversible and inhibited by cycloheximide, indicating the importance of de novo protein biosynthesis in this process. Our results indicate that walnut leaves can rapidly change their hydraulic conductance and suggest that these changes can be explained by regulation of plasma membrane aquaporins. Model simulation suggests that variable leaf hydraulic conductance in walnut might enhance leaf gas exchanges while buffering leaf water status in response to ambient light fluctuations.

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.

Vegetative buds of peach (Prunus persica L. Batsch.) trees act as strong sinks and their bud break capacity can be profoundly affected by carbohydrate availability during the rest period (November-February). Analysis of xylem sap revealed seasonal changes in concentrations of sorbitol and hexoses (glucose and fructose). Sorbitol concentrations decreased and hexose concentrations increased with increasing bud break capacity. Sucrose concentration in xylem sap increased significantly but remained low. To clarify their respective roles in the early events of bud break, carbohydrate concentrations and uptake rates, and activities of NAD-dependent sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX) and cell wall invertase (CWI) were determined in meristematic tissues, cushion tissues and stem segments. Only CWI activity increased in meristematic tissues shortly before bud break. In buds displaying high bud break capacity (during January and February), concentrations of sorbitol and sucrose in meristematic tissues were almost unchanged, paralleling their low rates of uptake and utilization by meristematic tissues, and indicating that sorbitol and sucrose play a negligible role in the bud break process. Hexose concentrations in meristematic tissues and glucose imported by meristematic tissues correlated positively with bud break capacity, suggesting that hexoses are involved in the early events of bud break. These findings were confirmed by data for buds that were unable to break because they had been collected from trees deprived of cold. We therefore conclude that hexoses are of greater importance than sorbitol or sucrose in the early events of bud break in peach trees.