Jean-Louis Julien

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.

Insights into Populus XIP aquaporins: evolutionary expansion, protein functionality, and environmental regulation

A novel category of major intrinsic proteins which share weak similarities with previously identified aquaporin subfamilies was recently identified in land plants, and named X (for unrecognized) intrinsic proteins (XIPs). Because XIPs are still ranked as uncharacterized proteins, their further molecular characterization is required. Herein, a systematic fine-scale analysis of XIP sequences found in flowering plant databases revealed that XIPs are found in at least five groups. The phylogenetic relationship of these five groups with the phylogenetic organization of angiosperms revealed an original pattern of evolution for the XIP subfamily through distinct angiosperm taxon-specific clades. Of all flowering plant having XIPs, the genus Populus encompasses the broadest panel and the highest polymorphism of XIP isoforms, with nine PtXIP sequences distributed within three XIP groups. Comprehensive PtXIP gene expression patterns showed that only two isoforms (PtXIP2;1 and PtXIP3;2) were transcribed in vegetative tissues. However, their patterns are contrasted, PtXIP2;1 was ubiquitously accumulated whereas PtXIP3;2 was predominantly detected in wood and to a lesser extent in roots. Furthermore, only PtXIP2;1 exhibited a differential expression in leaves and stems of drought-, salicylic acid-, or wounding-challenged plants. Unexpectedly, the PtXIPs displayed different abilities to alter water transport upon expression in Xenopus laevis oocytes. PtXIP2;1 and PtXIP3;3 transported water while other PtXIPs did not.

Is membrane potential involved in calmodulin gene expression after external stimulation in plants

In Bidens pilosa (cv. radiata), a non‐injurious stimulus induces a local and transient change in membrane potential, and an injurious stimulus induces a transmitted electrical signal described as the combination of an action potential and a slow wave. We have studied calmodulin gene expression after these stimuli. When the stimulus is non‐injurious, calmodulin mRNA accumulation is only increased in the stimulated region. In contrast, when the stimulus is injurious, mRNA accumulation takes place in both wounded and distant, unwounded tissue. We propose that the slow wave plays a role in the long‐distance transmission of a wound‐induced information in plants.

Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production.

BACKGROUND AND AIMS The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described. METHODS Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel. KEY RESULTS Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application. CONCLUSION The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.

Characterization and expression analysis under bending and other abiotic factors of PtaZFP2, a poplar gene encoding a Cys2/His2 zinc finger protein

In plants, mechanoperception and transduction of mechanical signals have been studied essentially in Arabidopsis thaliana L. and Lycopersicon esculentum L. plants, i.e., in nonwoody plants. Here, we have described the isolation of both the full-length cDNA and the regulatory region of PtaZFP2, encoding a member of Cys2/His2 zinc finger protein (ZFP) family in Populus tremula L. x Populus alba L. Time course analysis of expression demonstrated that PtaZFP2 mRNA accumulated as early as 5 min in response to a controlled stem bending and is restricted to the organ where the mechanical stimulus is applied. The real-time quantitative Reverse Transcriptase Polymerase Chain Reaction experiments showed that PtaZFP2 was also rapidly up-regulated in poplar stems in response to gravitropism suggesting that PtaZFP2 is induced by different mechanical signals. Abundance of PtaZFP2 transcripts also increased highly in response to wounding and to a weaker extent to salt treatment and cold, which is consistent with the numerous putative cis-elements found in its regulatory region. As in other species, these data suggest that Cys2/His2 ZFPs could function in poplar as key transcriptional regulators in the acclimation response to different environmental factors.

Plasma membrane H+-ATPase, succinate and isocitrate dehydrogenases activities of vessel-associated cells in walnut trees

Summary In winter and spring, walnut trees exhibit variations of sugar content in the vascular sap. According to their location, the vessel-associated cells (VACs, also called contact cells) could control nutrient exchanges between the storage parenchyma and the xylem vessels. According to the literature, the recovery of sap (influx) occurs at the VAC/vessel interface via an H+/sugar symport that depends on the transmembrane pH gradient generated by the plasma membrane H+-ATPase. The aim of this study was to investigate the ATPase activity, using a perfusion technique that allows the use of several effectors: carbonyl cyanide m-chlorophenylhydrazone (CCCP) and fusicoccin (FC). During winter, the uncoupler CCCP revealed a low pH gradient between the xylem vessels and the vessel-associated cells. Under these conditions, FC, an activator of the H+-ATPase, had no effect on the pH of the perfusion solution, suggesting that the enzyme could be lightly active. In contrast, close to bud break, a high pH gradient was revealed by the use of CCCP, and an acidification of the perfusion solution was observed in the presence of FC. Moreover, cytochemical investigation showed high activity of two respiratory enzymes located in mitochondria: NAD-dependent isocitrate dehydrogenase and succinate dehydrogenase. The hypothesis is that in spring this high respiratory activity of VACs provides a consequent increase in available ATP that can be utilized by the plasma membrane H+-ATPase.

Integrative Mechanobiology of Growth and Architectural Development in Changing Mechanical Environments

Mechanosensitive control of plant growth is a major process shaping how terrestrial plants acclimate to the mechanical challenges set by wind, self-weight, and autostresses. Loads acting on the plant are distributed down to the tissues, following continuum mechanics. Mechanosensing, though, occurs within the cell, building up into integrated signals; yet the reviews on mechanosensing tend to address macroscopic and molecular responses, ignoring the biomechanical aspects of load distribution to tissues and reducing biological signal integration to a “mean plant cell.” In this chapter, load distribution and biological signal integration are analyzed directly. The Sum of Strain Sensing model S 3 m is then discussed as a synthesis of the state of the art in quantitative deterministic knowledge and as a template for the development of an integrative and system mechanobiology.

Phylogenetic Study of Plant Q-type C2H2 Zinc Finger Proteins and Expression Analysis of Poplar Genes in Response to Osmotic, Cold and Mechanical Stresses

Plant Q-type C2H2 zinc finger transcription factors play an important role in plant tolerance to various environmental stresses such as drought, cold, osmotic stress, wounding and mechanical loading. To carry out an improved analysis of the specific role of each member of this subfamily in response to mechanical loading in poplar, we identified 16 two-fingered Q-type C2H2-predicted proteins from the poplar Phytozome database and compared their phylogenetic relationships with 152 two-fingered Q-type C2H2 protein sequences belonging to more than 50 species isolated from the NR protein database of NCBI. Phylogenetic analyses of these Q-type C2H2 proteins sequences classified them into two groups G1 and G2, and conserved motif distributions of interest were established. These two groups differed essentially in their signatures at the C-terminus of their two QALGGH DNA-binding domains. Two additional conserved motifs, MALEAL and LVDCHY, were found only in sequences from Group G1 or from Group G2, respectively. Functional significance of these phylogenetic divergences was assessed by studying transcript accumulation of six poplar C2H2 Q-type genes in responses to abiotic stresses; but no group specificity was found in any organ. Further expression analyses focused on PtaZFP1 and PtaZFP2, the two genes strongly induced by mechanical loading in poplars. The results revealed that these two genes were regulated by several signalling molecules including hydrogen peroxide and the phytohormone jasmonate.

Mechanosensitive control of plant growth: bearing the load, sensing, transducing, and responding

As land plants grow and develop, they encounter complex mechanical challenges, especially from winds and turgor pressure. Mechanosensitive control over growth and morphogenesis is an adaptive trait, reducing the risks of breakage or explosion. This control has been mostly studied through experiments with artificial mechanical loads, often focusing on cellular or molecular mechanotransduction pathway. However, some important aspects of mechanosensing are often neglected. (i) What are the mechanical characteristics of different loads and how are loads distributed within different organs? (ii) What is the relevant mechanical stimulus in the cell? Is it stress, strain, or energy? (iii) How do mechanosensing cells signal to meristematic cells? Without answers to these questions we cannot make progress analyzing the mechanobiological effects of plant size, plant shape, tissue distribution and stiffness, or the magnitude of stimuli. This situation is rapidly changing however, as systems mechanobiology is being developed, using specific biomechanical and/or mechanobiological models. These models are instrumental in comparing loads and responses between experiments and make it possible to quantitatively test biological hypotheses describing the mechanotransduction networks. This review is designed for a general plant science audience and aims to help biologists master the models they need for mechanobiological studies. Analysis and modeling is broken down into four steps looking at how the structure bears the load, how the distributed load is sensed, how the mechanical signal is transduced, and then how the plant responds through growth. Throughout, two examples of adaptive responses are used to illustrate this approach: the thigmorphogenetic syndrome of plant shoots bending and the mechanosensitive control of shoot apical meristem (SAM) morphogenesis. Overall this should provide a generic understanding of systems mechanobiology at work.

Aquaporins and Leaf Hydraulics: Poplar Sheds New Light

To help understand leaf hydraulic conductance (Kleaf) modulation under high irradiance, well-watered poplars (Populus trichocarpa Torr. & Gray ex Hook and Populus nigra L.) were studied diurnally at molecular and ecophysiological scales. Transcriptional and translational modulations of plasma membrane intrinsic protein (PIP) aquaporins were evaluated in leaf samples during diurnal time courses. Among the 15 poplar PIP genes, a subset of two PIP1s and seven PIP2s are precociously induced within the first hour of the photoperiod concomitantly with a Kleaf increase. Since expression patterns were cyclic and reproducible over several days, we hypothesized that endogenous signals could be involved in PIP transcriptional regulation. To address this question, plants were submitted to forced darkness during their subjective photoperiod and compared with their control counterparts, which showed that some PIP1s and PIP2s have circadian regulation while others did not. Promoter analysis revealed that a large number of hormone, light, stress response and circadian elements are present. Finally, involvement of aquaporins is supported by the reduction of Kleaf by HgCl2 treatment.