Agnès Guilliot

Putative Role of Aquaporins in Variable Hydraulic Conductance of Leaves in Response to Light

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 (Kleaf) to irradiance. The effects of light and temperature on Kleaf are described. Under dark conditions, Kleaf was low, but increased by 400% upon exposure to light. In contrast to dark conditions, Kleaf values of light-exposed leaves responded to temperature and 0.1 mm cycloheximide treatments. Furthermore, Kleaf was not related to stomatal aperture. Data of real-time reverse transcription-polymerase chain reaction showed that Kleaf dynamics were tightly correlated with the transcript abundance of two walnut aquaporins (JrPIP2,1 and JrPIP2,2). Low Kleaf in the dark was associated with down-regulation, whereas high Kleaf in the light was associated with up-regulation of JrPIP2. Light responses of Kleaf 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.

Plasma Membrane Aquaporins Are Involved in Winter Embolism Recovery in Walnut Tree

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.

Carbohydrate storage in wood and bark of rubber trees submitted to different level of C demand induced by latex tapping

When the current level of carbohydrates produced by photosynthesis is not enough to meet the C demand for maintenance, growth or metabolism, trees use stored carbohydrates. In rubber trees (Hevea brasiliensis Muell. Arg.), however, a previous study (Silpi U., A. Lacointe, P. Kasemsap, S. Thanisawanyangkura, P. Chantuma, E. Gohet, N. Musigamart, A. Clement, T. Améglio and P. Thaler. 2007. Carbohydrate reserves as a competing sink: evidence from tapping the rubber tree. Tree Physiol. 27:881-889) showed that the additional sink created by latex tapping results not in a decrease, but in an increase in the non-structural carbohydrate (NSC) storage in trunk wood. In this study, the response of NSC storage to latex tapping was further investigated to better understand the trade-off between latex regeneration, biomass and storage. Three tapping systems were compared to the untapped Control for 2 years. Soluble sugars and starch were analyzed in bark and wood on both sides of the trunk, from 50 to 200 cm from the ground. The results confirmed over the 2 years that tapped trees stored more NSC, mainly starch, than untapped Control. Moreover, a double cut alternative tapping system, which produced a higher latex yield than conventional systems, led to even higher NSC concentrations. In all tapped trees, the increase in storage occurred together with a reduction in trunk radial growth. This was interpreted as a shift in carbon allocation toward the creation of reserves, at the expense of growth, to cover the increased risk induced by tapping (repeated wounding and loss of C in latex). Starch was lower in bark than in wood, whereas it was the contrary for soluble sugars. The resulting NSC was twice as low and less variable in bark than in wood. Although latex regeneration occurs in the bark, changes related to latex tapping were more marked in wood than in bark. From seasonal dynamics and differences between the two sides of the trunk in response to tapping, we concluded that starch in wood behaved as the long-term reserve compartment at the whole trunk level, whereas starch in bark was a local buffer. Soluble sugars behaved like an intermediate, ready-to-use compartment in both wood and bark. Finally, the dynamics of carbohydrate reserves appears a relevant parameter to assess the long-term performance of latex tapping systems.

Differential expression of four members of the H+-ATPase gene family during dormancy of vegetative buds of peach trees

Abstract. Vegetative-bud dormancy in peach (Prunus persica L. Batsch) trees is known to be correlated, at least partially, with properties of the underlying bud tissues during winter. Variations in the activity and amount of plasma-membrane H+-ATPase were observed. A full-length cDNA, PPA2 (Prunus persica H+-ATPase 2) and three partial cDNAs (PPA1, PPA3 and PPA4) for the plasma-membrane H+-ATPase from peach trees were isolated by reverse transcription (RT)-coupled rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR). The accumulation of plasma membrane H+-ATPase transcripts was then studied in vegetative buds during dormancy and breaking of dormancy. Competitive RT-PCR analysis revealed that, during dormancy, the plasma membrane H+-ATPase transcripts were higher in the tissues underlying the buds than in the buds themselves. After dormancy release, the level of PPA1, 2, 3 mRNA increased, whereas the level of PPA4 decreased in the buds. When trees were kept in a greenhouse (i.e. sheltered from chilling), no accumulation of PPA mRNA could be detected. These results suggest that there is a differential accumulation of H+-ATPase mRNA between the bud and the underlying bud tissues during dormancy, and that chilling could act as a decisive factor.

Walnut enzyme activity related with carbohydrate mobilization during dormancy period.

Juglans regia L. sprout depends on the carbohydrate mobilization from the parenchyma cells into the buds located in the upper portion of the twigs. The objective of this work was to contribute to the walnut tree sprout acrotony mechanism, in temperate climate, through the activity of alpha-amylase (EC 3.2.1.1), and Sucrose Phosphate Synthase (SPS - EC 2.4.1.14) mensuration, related to carbohydrate mobilization, during the dormancy period. From September to March, five one-year-old twigs were monthly sampled around. In April, close to budbreak, three samples were accomplished. The twigs were divided in apical, sub-apical, and base, and separated in bark, buds and wood (xylem). The average time of bud break in controlled conditions (25oC), water content, and activity of the above mentioned enzymes were determined. The results showed that the sprout gradient develops itself during ecodormancy period, and it is related with the alpha-amylase activity; the relationship of the SPS activity with this gradient is not evident.