Evaluation of Tunisian wheat endophytes as plant growth promoting bacteria and biological control agents against Fusarium culmorum.

Plant growth-promoting rhizobacteria (PGPR) applications have emerged as an ideal substitute for synthetic chemicals by their ability to improve plant nutrition and resistance against pathogens. In this study, we isolated fourteen root endophytes from healthy wheat roots cultivated in Tunisia. The isolates were identified based from their 16S rRNA gene sequences. They belonged to Bacillota and Pseudomonadota taxa. Fourteen strains were tested for their growth-promoting and defense-eliciting potentials on durum wheat under greenhouse conditions, and for their in vitro biocontrol power against Fusarium culmorum, an ascomycete responsible for seedling blight, foot and root rot, and head blight diseases of wheat. We found that all the strains improved shoot and/or root biomass accumulation, with Bacillus mojavensis, Paenibacillus peoriae and Variovorax paradoxus showing the strongest promoting effects. These physiological effects were correlated with the plant growth-promoting traits of the bacterial endophytes, which produced indole-related compounds, ammonia, and hydrogen cyanide (HCN), and solubilized phosphate and zinc. Likewise, plant defense accumulations were modulated lastingly and systematically in roots and leaves by all the strains. Testing in vitro antagonism against F. culmorum revealed an inhibition activity exceeding 40% for five strains: Bacillus cereus, Paenibacillus peoriae, Paenibacillus polymyxa, Pantoae agglomerans, and Pseudomonas aeruginosa. These strains exhibited significant inhibitory effects on F. culmorum mycelia growth, sporulation, and/or macroconidia germination. P. peoriae performed best, with total inhibition of sporulation and macroconidia germination. These finding highlight the effectiveness of root bacterial endophytes in promoting plant growth and resistance, and in controlling phytopathogens such as F. culmorum. This is the first report identifying 14 bacterial candidates as potential agents for the control of F. culmorum, of which Paenibacillus peoriae and/or its intracellular metabolites have potential for development as biopesticides.

Assessment of Tunisian Trichoderma Isolates on Wheat Seed Germination, Seedling Growth and Fusarium Seedling Blight Suppression.

Beneficial microorganisms, including members of the Trichoderma genus, are known for their ability to promote plant growth and disease resistance, as well as being alternatives to synthetic inputs in agriculture. In this study, 111 Trichoderma strains were isolated from the rhizospheric soil of Florence Aurore, an ancient wheat variety that was cultivated in an organic farming system in Tunisia. A preliminary ITS analysis allowed us to cluster these 111 isolates into three main groups, T. harzianum (74 isolates), T. lixii (16 isolates) and T. sp. (21 isolates), represented by six different species. Their multi-locus analysis (tef1, translation elongation factor 1; rpb2, RNA polymerase B) identified three T. afroharzianum, one T. lixii, one T. atrobrunneum and one T. lentinulae species. These six new strains were selected to determine their suitability as plant growth promoters (PGP) and biocontrol agents (BCA) against Fusarium seedling blight disease (FSB) in wheat caused by Fusarium culmorum. All of the strains exhibited PGP abilities correlated to ammonia and indole-like compound production. In terms of biocontrol activity, all of the strains inhibited the development of F. culmorum in vitro, which is linked to the production of lytic enzymes, as well as diffusible and volatile organic compounds. An in planta assay was carried out on the seeds of a Tunisian modern wheat variety (Khiar) by coating them with Trichoderma. A significant increase in biomass was observed, which is associated with increased chlorophyll and nitrogen. An FSB bioprotective effect was confirmed for all strains (with Th01 being the most effective) by suppressing morbid symptoms in germinated seeds and seedlings, as well as by limiting F. culmorum aggressiveness on overall plant growth. Plant transcriptome analysis revealed that the isolates triggered several SA- and JA-dependent defense-encoding genes involved in F. culmorum resistance in the roots and leaves of three-week-old seedlings. This finding makes these strains very promising in promoting growth and controlling FSB disease in modern wheat varieties.

Genome-Wide Identification, Structure Characterization, and Expression Pattern Profiling of the Aquaporin Gene Family in Betula pendula.

Aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life. They play key roles in the flux of water and many solutes across the membranes. The AQP diversity, protein features, and biological functions of silver birch are still unknown. A genome analysis of Betula pendula identified 33 putative genes encoding full-length AQP sequences (BpeAQPs). They are grouped into five subfamilies, representing ten plasma membrane intrinsic proteins (PIPs), eight tonoplast intrinsic proteins (TIPs), eight NOD26-like intrinsic proteins (NIPs), four X intrinsic proteins (XIPs), and three small basic intrinsic proteins (SIPs). The BpeAQP gene structure is conserved within each subfamily, with exon numbers ranging from one to five. The predictions of the aromatic/arginine selectivity filter (ar/R), Froger's positions, specificity-determining positions, and 2D and 3D biochemical properties indicate noticeable transport specificities to various non-aqueous substrates between members and/or subfamilies. Nevertheless, overall, the BpePIPs display mostly hydrophilic ar/R selective filter and lining-pore residues, whereas the BpeTIP, BpeNIP, BpeSIP, and BpeXIP subfamilies mostly contain hydrophobic permeation signatures. Transcriptional expression analyses indicate that 23 BpeAQP genes are transcribed, including five organ-related expressions. Surprisingly, no significant transcriptional expression is monitored in leaves in response to cold stress (6 °C), although interesting trends can be distinguished and will be discussed, notably in relation to the plasticity of this pioneer species, B. pendula. The current study presents the first detailed genome-wide analysis of the AQP gene family in a Betulaceae species, and our results lay a foundation for a better understanding of the specific functions of the BpeAQP genes in the responses of the silver birch trees to cold stress.

Fungal X-Intrinsic Protein Aquaporin from Trichoderma atroviride: Structural and Functional Considerations.

The major intrinsic protein (MIP) superfamily is a key part of the fungal transmembrane transport network. It facilitates the transport of water and low molecular weight solutes across biomembranes. The fungal uncharacterized X-Intrinsic Protein (XIP) subfamily includes the full protein diversity of MIP. Their biological functions still remain fully hypothetical. The aim of this study is still to deepen the diversity and the structure of the XIP subfamily in light of the MIP counterparts-the aquaporins (AQPs) and aquaglyceroporins (AQGPs)-and to describe for the first time their function in the development, biomass accumulation, and mycoparasitic aptitudes of the fungal bioagent Trichoderma atroviride. The fungus-XIP clade, with one member (TriatXIP), is one of the three clades of MIPs that make up the diversity of T. atroviride MIPs, along with the AQPs (three members) and the AQGPs (three members). TriatXIP resembles those of strict aquaporins, predicting water diffusion and possibly other small polar solutes due to particularly wider ar/R constriction with a Lysine substitution at the LE2 position. The XIP loss of function in ∆TriatXIP mutants slightly delays biomass accumulation but does not impact mycoparasitic activities. ∆TriatMIP forms colonies similar to wild type; however, the hyphae are slightly thinner and colonies produce rare chlamydospores in PDA and specific media, most of which are relatively small and exhibit abnormal morphologies. To better understand the molecular causes of these deviant phenotypes, a wide-metabolic survey of the ∆TriatXIPs demonstrates that the delayed growth kinetic, correlated to a decrease in respiration rate, is caused by perturbations in the pentose phosphate pathway. Furthermore, the null expression of the XIP gene strongly impacts the expression of four expressed MIP-encoding genes of T. atroviride, a plausible compensating effect which safeguards the physiological integrity and life cycle of the fungus. This paper offers an overview of the fungal XIP family in the biocontrol agent T. atroviride which will be useful for further functional analysis of this particular MIP subfamily in vegetative growth and the environmental stress response in fungi. Ultimately, these findings have implications for the ecophysiology of Trichoderma spp. in natural, agronomic, and industrial systems.

Early transcriptional response to gravistimulation in poplar without phototropic confounding factors.

In response to gravistimulation under anisotropic light, tree stems showing an active cambium produce reaction wood that redirects the axis of the trees. Several studies have described transcriptomic or proteomic models of reaction wood relative to the opposite wood. However, the mechanisms leading to the formation of reaction wood are difficult to decipher because so many environmental factors can induce various signalling pathways leading to this developmental reprogramming. Using an innovative isotropic device where the phototropic response does not interfere with gravistimulation we characterized the early molecular responses occurring in the stem of poplar after gravistimulation in an isotropic environment, and without deformation of the stem. After 30 min tilting at 35° under anisotropic light, we collected the upper and lower xylems from the inclined stems. Controls were collected from vertical stems. We used a microarray approach to identify differentially expressed transcripts. High-throughput real-time PCR allowed a kinetic experiment at 0, 30, 120 and 180 min after tilting at 35°, with candidate genes. We identified 668 differentially expressed transcripts, from which we selected 153 candidates for additional Fluidigm qPCR assessment. Five candidate co-expression gene clusters have been identified after the kinetic monitoring of the expression of candidate genes. Gene ontology analyses indicate that molecular reprogramming of processes such as 'wood cell expansion', 'cell wall reorganization' and 'programmed cell death' occur as early as 30 min after gravistimulation. Of note is that the change in the expression of different genes involves a fine regulation of gibberellin and brassinosteroid pathways as well as flavonoid and phosphoinositide pathways. Our experimental set-up allowed the identification of genes regulated in early gravitropic response without the bias introduced by phototropic and stem bending responses.

Voltage-gating of aquaporins, a putative conserved safety mechanism during ionic stresses.

Aquaporins are transmembrane water channels found in almost every living organism. Numerous studies have brought a good understanding of both water transport through their pores and the regulations taking place at the molecular level, but subtleties remain to be clarified. Recently, a voltage-related gating mechanism involving the conserved arginine of the channel's main constriction was captured for human aquaporins through molecular dynamics studies. With a similar approach, we show that this voltage-gating could be conserved among this family and that the underlying mechanism could explain part of plant AQPs diversity when contextualized to high ionic concentrations provoked by drought. Finally, we identified residues as adaptive traits which constitute good targets for drought resistance plant breeding research.

Hydrogen peroxide generated by over-expression of cytosolic superoxide dismutase in transgenic plums enhances bacterial canker resistance and modulates plant defence responses.

H2O2 generated during the oxidative burst, plays important roles in plant defenses responses against pathogens. In this study we examined the role of H2O2 on bacterial canker resistance in transgenic plums over-expressing cytosolic superoxide dismutase. Three transgenic lines (C64, C66 and F12) with elevated levels of H2O2 accumulation showed enhanced resistance against bacterial canker disease caused by Pseudomonas syringae pv. syringae, when compared to the non-transformed control. Analysis of the expression of several genes involved in the plant-pathogen interaction showed that the expression of those involved in SA pathway (pr1 and npr1) and JA (lox3) were activated earlier and transiently in transgenic lines C66 and F12 when compared to the wild type. However, the expression of genes involved in anthocyanin synthesis (chi, chs, f3h, dfr, atcs, myb10) and ethylene (acs) was induced at very low levels whereas it was activated by the pathogen at exaggerated levels in the non-transformed line. These results suggest that resistance observed in transgenic lines over-producing H2O2 is correlated with an early and transient induction of defense genes associated with the SA and JA pathways and inhibition of gene expression associated with ethylene and anthocyanin biosynthesis.

Genome Wild Analysis and Molecular Understanding of the Aquaporin Diversity in Olive Trees (Olea Europaea L.).

Cellular aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life, playing important roles in the uptake of water and many solutes across the membranes. In olive trees, AQP diversity, protein features and their biological functions are still largely unknown. This study focuses on the structure and functional and evolution diversity of AQP subfamilies in two olive trees, the wild species Olea europaea var. sylvestris (OeuAQPs) and the domesticated species Olea europaea cv. Picual (OleurAQPs), and describes their involvement in different physiological processes of early plantlet development and in biotic and abiotic stress tolerance in the domesticated species. A scan of genomes from the wild and domesticated olive species revealed the presence of 52 and 79 genes encoding full-length AQP sequences, respectively. Cross-genera phylogenetic analysis with orthologous clustered OleaAQPs into five established subfamilies: PIP, TIP, NIP, SIP, and XIP. Subsequently, gene structures, protein motifs, substrate specificities and cellular localizations of the full length OleaAQPs were predicted. Functional prediction based on the NPA motif, ar/R selectivity filter, Froger's and specificity-determining positions suggested differences in substrate specificities of Olea AQPs. Expression analysis of the OleurAQP genes indicates that some genes are tissue-specific, whereas few others show differential expressions at different developmental stages and in response to various biotic and abiotic stresses. The current study presents the first detailed genome-wide analysis of the AQP gene family in olive trees and it provides valuable information for further functional analysis to infer the role of AQP in the adaptation of olive trees in diverse environmental conditions in order to help the genetic improvement of domesticated olive trees.

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

MIP diversity from Trichoderma: Structural considerations and transcriptional modulation during mycoparasitic association with Fusarium solani olive trees.

Major intrinsic proteins (MIP) are characterized by a transmembrane pore-type architecture that facilitates transport across biomembranes of water and a variety of low molecular weight solutes. They are found in all parts of life, with remarkable protein diversity. Very little is known about MIP from fungi. And yet, it can legitimately be stated that MIP are pivotal molecular components in the privileged relationships fungi enjoy with plants or soil fauna in various environments. To date, MIP have never been studied in a mycoparasitism situation. In this study, the diversity, expression and functional prediction of MIP from the genus Trichoderma were investigated. Trichoderma spp. genomes have at least seven aquaporin genes. Based on a phylogenetic analysis of the translated sequences, members were assigned to the AQP, AQGP and XIP subfamilies. In in vitro and in planta assays with T. harzianum strain Ths97, expression analyses showed that four genes were constitutively expressed. In a mycoparasitic context with Fusarium solani, the causative agent of fusarium dieback on olive tree roots, these genes were up-regulated. This response is of particular interest in analyzing the MIP promoter cis-regulatory motifs, most of which are involved in various carbon and nitrogen metabolisms. Structural analyses provide new insights into the possible role of structural checkpoints by which these members transport water, H2O2, glycerol and, more generally, linear polyols across the membranes. Taken together, these results provide the first evidence that MIP may play a key role in Trichoderma mycoparasitism lifestyle.

Integrated drought responses of black poplar: how important is phenotypic plasticity?

Climate change is expected to increase drought frequency and intensity which will threaten plant growth and survival. In such fluctuating environments, perennial plants respond with hydraulic and biomass adjustments, resulting in either tolerant or avoidant strategies. Plants' response to stress relies on their phenotypic plasticity. The goal of this study was to explore physiology of young Populus nigra in the context of a time-limited and progressive water deficit in regard to their growth and stress response strategies. Fourteen French 1-year-old black poplar genotypes, geographically contrasted, were subjected to withholding water during 8 days until severe water stress. Water fluxes (i.e. leaf water potentials and stomatal conductance) were analyzed together with growth (i.e. radial and longitudinal branch growth, leaf senescence and leaf production). Phenotypic plasticity was calculated for each trait and response strategies to drought were deciphered for each genotype. Black poplar genotypes permanently were dealing with a continuum of adjusted water fluxes and growth between two extreme strategies, tolerance and avoidance. Branch growth, leaf number and leaf hydraulic potential traits had contrasted plasticities, allowing genotype characterization. The most tolerant genotype to water deficit, which maintained growth, had the lowest global phenotypic plasticity. Conversely, the most sensitive and avoidant genotype ceased growth until the season's end, had the highest plasticity level. All the remaining black poplar genotypes were close to avoidance with average levels of traits plasticity. These results underpinned the role of plasticity in black poplar response to drought and calls for its wider use into research on plants' responses to stress.

Effect of acibenzolar-S-methyl phototransformation on its elicitation activity in tobacco cells.

The plant activator acibenzolar-S-methyl (BTH) undergoes phototransformation when exposed to solar radiation. Here we investigated the changes in its elicitation properties on BY-2 tobacco cells at different stages of the photochemical reaction. Both pure BTH and formulated BTH were irradiated in controlled conditions to achieve different extents of conversion. Both pure BTH (900 µM) and Bion® (0.4 g.L-1) induced BY-2 cell death, but BTH photoconverted to an extent of 25 ± 3% lowered the cell death rate. A kinetic study of ß-1,3-glucanase and chitinase activities was conducted on BY-2 extracellular medium. Exposure of tobacco cells to either pure BTH or Bion® resulted in a significant increase in the activities of both defense enzymes, which peaked 48 h after the treatment. The pathogenesis-related (PR) protein activities were quantified 48 h after elicitation for a range of phototransformed BTH solutions. The enzyme activities were reduced when BY-2 cells were treated with solutions in which BTH conversion was 22 ± 3%, 42 ± 3% and 100 ± 3%, but were not affected by the solution in which BTH was phototransformed at 60%, suggesting that some of the secondary photoproducts also exhibit eliciting properties. Solar irradiation of BTH thus impairs its elicitation properties, but this impairment depends strongly on the extent of phototransformation.

The Hevea brasiliensis XIP aquaporin subfamily: genomic, structural and functional characterizations with relevance to intensive latex harvesting.

X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.

Gravity sensing, a largely misunderstood trigger of plant orientated growth.

Gravity is a crucial environmental factor regulating plant growth and development. Plants have the ability to sense a change in the direction of gravity, which leads to the re-orientation of their growth direction, so-called gravitropism. In general, plant stems grow upward (negative gravitropism), whereas roots grow downward (positive gravitropism). Models describing the gravitropic response following the tilting of plants are presented and highlight that gravitropic curvature involves both gravisensing and mechanosensing, thus allowing to revisit experimental data. We also discuss the challenge to set up experimental designs for discriminating between gravisensing and mechanosensing. We then present the cellular events and the molecular actors known to be specifically involved in gravity sensing.

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.

Modulation of bud survival in Populus nigra sprouts in response to water stress-induced embolism.

Understanding drought tolerance mechanisms requires knowledge about the induced weakness that leads to tree death. Bud survival is vital to sustain tree growth across seasons. We hypothesized that the hydraulic connection of the bud to stem xylem structures was critical for its survival. During an artificial drastic water stress, we carried out a census of bud metabolic activity of young Populus nigra L. trees by microcalorimetry. We monitored transcript expression of aquaporins (AQPs; plasma membrane intrinsic proteins (PIPs), X intrinsic proteins (XIPs) and tonoplast membrane intrinsic proteins (TIPs)) and measured local water status within the bud and tissues in the bearer shoot node by nuclear magnetic resonance (NMR) imaging. We found that the bud respiration rate was closely correlated with its water content and decreased concomitantly in buds and their surrounding bearer tissues. At the molecular level, we observed a modulation of AQP pattern expressions (PIP, TIP and XIP subfamilies) linked to water movements in living cells. However, AQP functions remain to be investigated. Both the bud and tree died beyond a threshold water content and respiration rate. Nuclear magnetic resonance images provided relevant local information about the various water reservoirs of the stem, their dynamics and their interconnections. Comparison of pith, xylem and cambium tissues revealed that the hydraulic connection between the bud and saturated parenchyma cells around the pith allowed bud desiccation to be delayed. At the tree death date, NMR images showed that the cambium tissues remained largely hydrated. Overall, the respiration rate (Rco2) and a few AQP isoforms were found to be two suitable, complementary criteria to assess the bud metabolic activity and the ability to survive a severe drought spell. Bud moisture content could be a key factor in determining the capacity of poplar to recover from water stress.

Light-mediated K(leaf) induction and contribution of both the PIP1s and PIP2s aquaporins in five tree species: walnut (Juglans regia) case study.

Understanding the response of leaf hydraulic conductance (K(leaf)) to light is a challenge in elucidating plant-water relationships. Recent data have shown that the effect of light on K(leaf) is not systematically related to aquaporin regulation, leading to conflicting conclusions. Here we investigated the relationship between light, K(leaf), and aquaporin transcript levels in five tree species (Juglans regia L., Fagus sylvatica L., Quercus robur L., Salix alba L. and Populus tremula L.) grown in the same environmental conditions, but differing in their K(leaf) responses to light. Moreover, the K(leaf) was measured by two independent methods (high-pressure flow metre (HPFM) and evaporative flux method (EFM)) in the most (J. regia) and least (S. alba) responsive species and the transcript levels of aquaporins were analyzed in perfused and unperfused leaves. Here, we found that the light-induced K(leaf) value was closely related to stronger expression of both the PIP1 and PIP2 aquaporin genes in walnut (J. regia), but to stimulation of PIP1 aquaporins alone in F. sylvatica and Q. robur. In walnut, all newly identified aquaporins were found to be upregulated in the light and downregulated in the dark, further supporting the relationship between the light-mediated induction of K(leaf) and aquaporin expression in walnut. We also demonstrated that the K(leaf) response to light was quality-dependent, K(leaf) being 60% lower in the absence of blue light. This decrease in K(leaf) was correlated with strong downregulation of three PIP2 aquaporins and of all the PIP1 aquaporins tested. These data support a relationship between light-mediated K(leaf) regulation and the abundance of aquaporin transcripts in the walnut tree.

Development of a Real-Time PCR assay for quantitative assessment of uncultured freshwater zoosporic fungi.

Recently, molecular environmental surveys of the eukaryotic microbial community in lakes have revealed a high diversity of sequences belonging to uncultured zoosporic fungi. Although they are known as saprobes and algal parasites in freshwater systems, zoosporic fungi have been neglected in microbial food web studies. Recently, it has been suggested that zoosporic fungi, via the consumption of their zoospores by zooplankters, could transfer energy from large inedible algae and particulate organic material to higher trophic levels. However, because of their small size and their lack of distinctive morphological features, traditional microscopy does not allow the detection of fungal zoospores in the field. Hence, quantitative data on fungal zoospores in natural environments is missing. We have developed a quantitative PCR (qPCR) assay for the quantification of fungal zoospores in lakes. Specific primers were designed and qPCR conditions were optimized using a range of target and non-target plasmids obtained from previous freshwater environmental 18S rDNA surveys. When optimal DNA extraction protocol and qPCR conditions were applied, the qPCR assay developed in this study demonstrated high specificity and sensitivity, with as low as 100 18S rDNA copies per reaction detected. Although the present work focuses on the design and optimization of a new qPCR assay, its application to natural samples indicated that qPCR offers a promising tool for quantitative assessment of fungal zoospores in natural environments. We conclude that this will contribute to a better understanding of the ecological significance of zoosporic fungi in microbial food webs of pelagic ecosystems.

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 (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.

Expression of viral EPS-depolymerase reduces fire blight susceptibility in transgenic pear.

Erwinia amylovora is the causal agent of fire blight of Maloideae. One of the main pathogenicity factors of this bacterium is the exopolysaccharide (EPS) of its capsule. In this paper, we used genetic transformation tools to constitutively express an EPS-depolymerase transgene in the pear (Pyrus communis L.) cv. Passe Crassane with the aim of decreasing its high susceptibility to fire blight. Expression of the depolymerase gene in 15 independent transgenic clones led, on average, to low depolymerase activity, although relatively high expression was observed at the transcriptional and translational levels. Only two of the transgenic clones (9X and 10M) consistently showed a decrease in fire blight susceptibility in vitro and in the greenhouse. These clones were also among the highest expressers of depolymerase at the RNA and enzyme activity levels. The correlation observed among all transgenic clones between depolymerase expression and fire blight resistance suggested the potential of this strategy.