Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.     

Acute metal stress in Populus tremula×P. alba (717‐1B4 genotype): Leaf and cambial proteome changes induced by cadmium2+

The comprehension of metal homeostasis in plants requires the identification of molecular markers linked to stress tolerance. Proteomic changes in leaves and cambial zone of Populus tremula×P. alba (717‐1B4 genotype) were analyzed after 61 days of exposure to cadmium (Cd) 360 mg/kg soil dry weight in pot‐soil cultures. The treatment led to an acute Cd stress with a reduction of growth and photosynthesis. Cd stress induced changes in the display of 120 spots for leaf tissue and 153 spots for the cambial zone. It involved a reduced photosynthesis, resulting in a profound reorganisation of carbon and carbohydrate metabolisms in both tissues. Cambial cells underwent stress from the Cd actually present inside the tissue but also a deprivation of photosynthates caused by leaf stress. An important tissue specificity of the response was observed, according to the differences in cell structures and functions.     

Poplar under drought: comparison of leaf and cambial proteomic responses

The forest ecosystem is of particular importance from an economic and ecological perspective. However, the stress physiology of trees, perennial and woody plants, is far from being fully understood. For that purpose, poplar plants were exposed to drought; the plants exhibited commonly reported drought stress traits in the different plant tissues. Leafy rooted cuttings of poplar were investigated through a proteomic approach in order to compare the water constraint response of two plant tissues, namely leaf and cambium. Sampling was realized during the drought period at 2 time points with increased drought intensity and 7 days after rewatering. Our data show that there is a difference in the moment of response to the water constraint between the two tissues, cambium being affected later than leaves. In leaves, drought induced a decrease in rubisco content, and an increase in the abundance of light harvesting complex proteins as well as changes in membrane-related proteins. In the cambial tissue, the salient proteome pattern change was the decrease of multiple proteins identified as bark storage proteins. After rewatering, almost all changes in cambial proteome disappeared whereas a significant number of leaf proteins appeared to be differentially regulated only during the recovery from drought.     

Osmotic stress sensing in Populus: components identification of a phosphorelay system

To study the Populus response to an osmotic stress, we have isolated one cDNA encoding a histidine-aspartate kinase (HK1) and four cDNAs encoding histidine-containing phosphotransfer proteins (HPts), HPt1-4. The predicted HK1 protein shares a typical structure with ATHK1 and SLN1 osmosensors. The 4 HPTs are characterized by the histidine phosphotransfer domain. We have shown that HK1 is upregulated during an osmotic stress in hydroponic culture. We have detected an interaction between HK1 and HPt2, using the yeast two-hybrid system. These results suggest the existence of a multi-step phosphorelay pathway probably involved in osmotic stress sensing in Populus.     

Characterization of histidine‐aspartate kinase HK1 and identification of histidine phosphotransfer proteins as potential partners in a Populus multistep phosphorelay

In poplar, we identified proteins homologous to yeast proteins involved in osmosensing multistep phosphorelay Sln1p‐Ypd1p‐Ssk1p. This finding led us to speculate that Populus cells could sense osmotic stress by a similar mechanism. This study focuses on first and second protagonists of this possible pathway: a histidine‐aspartate kinase (HK1), putative osmosensor and histidine phosphotransfer proteins (HPt1 to 10), potential partners of this HK. Characterization of HK1 showed its ability to homodimerize in two‐hybrid tests and to act as an osmosensor with a kinase activity in yeast, by functional complementation of sln1Δ sho1Δ strain. Moreover, in plant cells, plasma membrane localization of HK1 is shown. Further analysis on HPts allowed us to isolate seven new cDNAs, leading to a total of 10 different HPts identified in poplar. Interaction tests showed that almost all HPts can interact with HK1, but two of them exhibit stronger interactions, suggesting a preferential partnership in poplar. The importance of the phosphorylation status in these interactions has been investigated with two‐hybrid tests carried out with mutated HK1 forms. Finally, in planta co‐expression analysis of genes encoding these potential partners revealed that only three HPts are co‐expressed with HK1 in different poplar organs. This result reinforces the hypothesis of a partnership between HK1 and these three preferential HPts in planta. Taken together, these results shed some light on proteins partnerships that could be involved in the osmosensing pathway in Populus.     

Cadmium and Zinc are differentially distributed in Populus tremula x P. alba exposed to metal excess

Abstract

Poplar plants were exposed during 61 days to a soil added with heavy metals so as to contain 300 mg Zn²⁺.kg^?1 soil dry weight (SDW) (Zinc) or 50 mg Cd²⁺.kg^?1 SDW (Cadmium). The Cd treatment induced a delayed growth of poplar, whereas Zn induced no change in physiological parameters. Both treatments resulted in a significant metal accumulation in plants. Zn²⁺ and Cd²⁺ exhibited contrasting distribution within tissues, indicating dissimilar handling by the plant. The main difference was the efficient compartmentalisation of Zn²⁺ in specific organ parts: old leaves and bark, while Cd²⁺ did not exhibit such a compartmentalisation. Results were also compared with a previous work where plants were exposed to 360 mg Cd²⁺.kg^?1 SDW.     

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.     

Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor

Background

Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca²⁺-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca²⁺ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca²⁺ influx mediated by mechanosensitive Ca²⁺ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.

Results

We here identified and investigated OsMCA1, the sole rice homolog of putative Ca²⁺-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca²⁺ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca²⁺ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca²⁺-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.

Conclusions

We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca²⁺-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca²⁺ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.