Jean-Francois Hausman

An improved protocol to study the plant cell wall proteome

Cell wall proteins were extracted from alfalfa stems according to a three-steps extraction procedure using sequentially CaCl2, EGTA, and LiCl-complemented buffers. The efficiency of this protocol for extracting cell wall proteins was compared with the two previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedure resulted in the identification of the highest number of cell wall proteins (242 NCBInr identifiers) and gave the lowest percentage of non-cell wall proteins (about 30%). However, the three protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol. This three-step protocol was therefore selected for a more detailed proteomic characterization using 2D-gel electrophoresis. With this technique, 75% of the identified proteins were shown to be fraction-specific and 72.7% were predicted as belonging to the cell wall compartment. Although, being less sensitive than LC-MS/MS approaches in detecting and identifying low-abundant proteins, gel-based approaches are valuable tools for the differentiation and relative quantification of protein isoforms and/or modified proteins. In particular isoforms, having variations in their amino-acid sequence and/or carrying different N-linked glycan chains were detected and characterized. This study highlights how the extracting protocols as well as the analytical techniques devoted to the study of the plant cell wall proteome are complementary and how they may be combined to elucidate the dynamism of the plant cell wall proteome in biological studies. Data are available via ProteomeXchange with identifier PXD001927.

Quantitative changes in protein expression of cadmium-exposed poplar plants

Cadmium (Cd) pollution is a worldwide major concern having, among others, deleterious effects on plants. In the present work, the effects of a 20 μM Cd exposure in hydroponics culture during 14 days were evaluated in young poplar leaves. Proteins were analysed by 2‐D DIGE, followed by MALDI‐TOF‐TOF identification. Additionally, growth and other physiological parameters were monitored during the experiment. Treated plants exhibited an inhibition of growth and visual symptoms appeared after 7 days. A significant accumulation of Cd in all organs was recorded by ICP‐MS analysis. A number of changes in the expression of proteins with various functions were identified; in particular a decreased abundance of oxidative stress regulating proteins, whereas pathogenesis‐related proteins showed a drastic increase in abundance. Furthermore, a large number of proteins involved in carbon metabolism showed a decrease in abundance, while proteins involved in remobilizing carbon from other energy sources were upregulated. In conclusion, the negative effect of Cd could be explained by a deleterious effect on protein expression from the primary carbon metabolism and from the oxidative stress response mechanism. Accumulation of Cd in stems of poplar, coupled with a low impact of Cd on physiological parameters, promotes the use of poplar trees for phytoremediation purposes.

Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach (Prunus persica L. Batsch)

In the temperate climate of the northern hemisphere, winter survival of woody plants is determined by the ability to acclimate to freezing temperatures and to undergo a period of dormancy. Cold acclimation in many woody plants is initially induced by short photoperiod and low, non-freezing temperatures. These two factors (5°C and short photoperiod) were used to study changes in the proteome of bark tissues of 1-year-old peach trees. Difference in-gel electrophoresis technology, a gel-based approach involving the labeling of proteins with different fluorescent dyes, was used to conduct a quantitative assessment of changes in the peach bark proteome during cold acclimation. Using this approach, we were able to identify differentially expressed proteins and to assign them to a class of either ‘temperature-responsive’ or ‘photoperiod-responsive’ proteins. The most significant factor affecting the proteome appeared to be low temperature, while the combination of low temperature and short photoperiod was shown to act either synergistically or additively on the expression of some proteins. Fifty-seven protein spots on gels were identified by mass spectrometry. They included proteins involved in carbohydrate metabolism (e.g., enolase, malate dehydrogenase, etc), defense or protective mechanisms (e.g., dehydrin, HSPs, and PR-proteins), energy production and electron transport (e.g., adenosine triphosphate synthases and lyases), and cytoskeleton organization (e.g., tubulins and actins). The information derived from the analysis of the proteome is discussed as a function of the two treatment factors: low temperature and short photoperiod.

Gene expression changes related to the production of phenolic compounds in potato tubers grown under drought stress.

Polyphenols represent a large family of plant secondary metabolites implicated in the prevention of various diseases such as cancers and cardiovascular diseases. The potato is a significant source of polyphenols in the human diet. In this study, we examined the expression of thirteen genes involved in the biosynthesis of polyphenols in potato tubers using real-time RT-PCR. A selection of five field grown native Andean cultivars, presenting contrasting polyphenol profiles, was used. Moreover, we investigated the expression of the genes after a drought exposure. We concluded that the diverse polyphenolic profiles are correlated to variations in gene expression profiles. The drought-induced variations of the gene expression was highly cultivar-specific. In the three anthocyanin-containing cultivars, gene expression was coordinated and reflected at the metabolite level supporting a hypothesis that regulation of gene expression plays an essential role in the potato polyphenol production. We proposed that the altered sucrose flux induced by the drought stress is partly responsible for the changes in gene expression. This study provides information on key polyphenol biosynthetic and regulatory genes, which could be useful in the development of potato varieties with enhanced health and nutritional benefits.

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.

Cannabis sativa: The Plant of the Thousand and One Molecules

Cannabis sativa L. is an important herbaceous species originating from Central Asia, which has been used in folk medicine and as a source of textile fiber since the dawn of times. This fast-growing plant has recently seen a resurgence of interest because of its multi-purpose applications: it is indeed a treasure trove of phytochemicals and a rich source of both cellulosic and woody fibers. Equally highly interested in this plant are the pharmaceutical and construction sectors, since its metabolites show potent bioactivities on human health and its outer and inner stem tissues can be used to make bioplastics and concrete-like material, respectively. In this review, the rich spectrum of hemp phytochemicals is discussed by putting a special emphasis on molecules of industrial interest, including cannabinoids, terpenes and phenolic compounds, and their biosynthetic routes. Cannabinoids represent the most studied group of compounds, mainly due to their wide range of pharmaceutical effects in humans, including psychotropic activities. The therapeutic and commercial interests of some terpenes and phenolic compounds, and in particular stilbenoids and lignans, are also highlighted in view of the most recent literature data. Biotechnological avenues to enhance the production and bioactivity of hemp secondary metabolites are proposed by discussing the power of plant genetic engineering and tissue culture. In particular two systems are reviewed, i.e., cell suspension and hairy root cultures. Additionally, an entire section is devoted to hemp trichomes, in the light of their importance as phytochemical factories. Ultimately, prospects on the benefits linked to the use of the -omics technologies, such as metabolomics and transcriptomics to speed up the identification and the large-scale production of lead agents from bioengineered Cannabis cell culture, are presented.

Lignocellulosic biomass: Biosynthesis, degradation, and industrial utilization

Lignocellulose biomass derived from plant cell walls is a rich source of biopolymers, chemicals, and sugars, besides being a sustainable alternative to petrochemicals. A natural armor protecting living protoplasts, the cell wall is currently the target of intense study because of its crucial importance in plant development, morphogenesis, and resistance to (a)biotic stresses. Beyond the intrinsic relevance related to the overall plant physiology, plant cell walls constitute an exquisite example of a natural composite material that is a constant source of inspiration for biotechnology, biofuel, and biomaterial industries. The aim of the present review is to provide the reader with an overview of the current knowledge concerning lignocellulosic biomass synthesis and degradation, by focusing on its three principal constituents, i.e. cellulose, hemicellulose (in particular xylan), and lignin. Furthermore, the current industrial exploitation of lignocellulose from fast growing fibre crops (such as hemp) is highlighted. We conclude this review by suggesting approaches for further research to fill gaps in our current knowledge and to highlight the potential of biotechnology and bioengineering in improving both biomass biosynthesis and degradation.

The impact of atmospheric composition on plants: a case study of ozone and poplar.

Tropospheric ozone is the main atmospheric pollutant that causes damages to trees. The estimation of the threshold for ozone risk assessment depends on the evaluation of the means that this pollutant impacts the plant and, especially, the foliar organs. The available results show that, before any visible symptom appears, carbon assimilation and the underlying metabolic processes are decreased under chronic ozone exposure. By contrast, the catabolic pathways are enhanced, and contribute to the supply of sufficient reducing power necessary to feed the detoxification processes. Reactive oxygen species delivered during ozone exposure serve as toxic compounds and messengers for the signaling system. In this review, we show that the contribution of genomic tools (transcriptomics, proteomics, and metabolomics) for a better understanding of the mechanistic cellular responses to ozone largely relies on spectrometric measurements.

Auxin polyamine interaction in the control of the rooting inductive phase of poplar shoots in vitro

Abstract Changes in endogenous free IAA and its conjugate IAAaspartate as well as in endogenous polyamines were analysed in in vitro poplar shoots during their inductive phase, under the effect of the rooting auxin NAA, in combination or not with polyamines or inhibitors of polyamine biosynthesis. In vitro raised poplar shoots rooted 100% when treated by NAA (0.3 mg/l) for 7 h, the previously determined duration of the inductive phase. Spermidine and aminoguanidine (AG, an inhibitor of diamine oxidase) alone were unable to promote rooting under these conditions, and counteracted the NAA rooting effect. Putrescine and cyclohexylamine (CHA, an inhibitor of spermidine synthase) on the contrary did not oppose the NAA effect and promoted up to 40% rooting when applied alone. The levels of free indoleacetic acid (IAA) and of its aspartate conjugate IAAsp elevated up to peaks situated at the 7th and 8th h, respectively in the basal parts of the NAA-treated shoots. Putrescine, when added toegether with NAA, did not affect the typical IAA and IAAsp increases (except a displacement of the peaks to the 8th and 10th h, respectively); when applied alone, it provoked elevation of their levels to limited extents. Quite similar results were recorded with CHA. Spermidine and AG counteracted the NAA induced elevations of IAA and IAAsp. The level of endogenous putrescine typically increased up to a peak at the 6th h in the basal part of the NAA-treated shoots only, was slightly affected by exogenous putrescine and CHA application but significantly reduced by spermidine and AG supply. The NAA inducing rooting treatment did not affect the variation of the levels of endogenous spermidine and spermine but putrescine treatment (also spermidine to a lesser extent) led to increases of their levels. The relationships between auxin and putrescine temporary accumulation are discussed as well as the involvement of the different polyamines in the rooting inductive process.

Silicon and the Plant Extracellular Matrix

Silicon (Si) is one of the most abundant elements on earth. Although not considered essential for the growth and development of higher plants, it is nonetheless known to increase vigor and to play protective roles. Its protective effects include for instance alleviation of (a)biotic stress damages and heavy metal toxicity. Si was shown to interact with several components of the plant cell walls in the form of silica (SiO2). In plants SiO2 promotes strengthening of the cell walls and provides increased mechanical support to the aerial parts. The relationship SiO2-plant cell wall has been well documented in monocots and pteridophytes, which are known Si accumulators, while much less is known on the interaction of Si with the cell walls of dicots. We here provide a concise up-to-date survey on the interaction between Si and plant cell wall components by focussing on cellulose, hemicelluloses, callose, pectins, lignin, and proteins. We also describe the effects of Si on cell wall-related processes by discussing the published results in both monocots and dicots. We conclude our survey with a description of the possible mechanisms by which Si exerts priming in plants.