Growth kinetics, spatialization and quality of potato tubers monitored in situ by MRI - long-term effects of water stress.

Understanding the potato tuber development and effects of drought at key stages of sensitivity on yield is crucial, particularly when considering the increasing incidence of drought due to climate change. So far, few studies addressed the time course of tuber growth in soil, mainly due to difficulties in accessing underground plant organs in a non-destructive manner. This study aims to understand the tuber growth and quality and the complex long-term effects of realistic water stress on potato tuber yield. MRI was used to monitor the growth kinetics and spatialization of individual tubers in situ and the evolution of internal defects throughout the development period. The intermittent drought applied to plants reduced tuber yield by reducing tuber growth and increasing the number of aborted tubers. The reduction in the size of tubers depended on the vertical position of the tubers in the soil, indicating water exchanges between tubers and the mother plant during leaf dehydration events. The final size of tubers was linked with the growth rate at specific developmental periods. For plants experiencing stress, this corresponded to the days following rewatering, suggesting tuber growth plasticity. All internal defects occurred in large tubers and within a short time span immediately following a period of rapid growth of perimedullary tissues, probably due to high nutrient requirements. To conclude, the non-destructive 3D imaging by MRI allowed us to quantify and better understand the kinetics and spatialization of tuber growth and the appearance of internal defects under different soil water conditions.

Plant growth regulator extracts from seaweeds promote plant growth and confer drought tolerance in canola (Brassica napus).

Brassica napus, commonly known as canola, is an important oilseed crop in Canada contributing over $29.9 billion CAD to the Canadian economy annually. A major challenge facing Canadian canola is drought, which has become increasingly prevalent in recent years due to the changing climate. Research investigating novel agronomic techniques in mitigating drought is key to securing yields and sustainability in canola and other crops. One such technique is the use of bio-stimulant sprays to help offset biotic and abiotic stresses in plants through promoting stand establishment. Previous studies have shown that the application of seaweed extracts as bio-stimulant sprays to Brassicaceae has been successful in improving plant growth and development along with stress tolerance. However, this method has yet to be tested on canola. The organic nutrients that are waste products from processed seaweed help stimulate plant growth, yielding higher quality plants as a result. In association with Le Groupe Roullier, this study demonstrates that the Roullier extracts (RE) help increase plant growth characteristics and drought tolerance in canola when sprayed 3 times over a 3-week period. A high yielding but drought sensitive mutant of canola, d14 (developed through gene editing) was used for drought assays after 8 weeks of growth and where water was withheld for 6 days. Application of the REs prior to drought resulted in plants having enhanced survival rate and improved biomass retention indicating high drought tolerance. Subsequent RNA sequencing and gene ontological term analysis performed using RE treated plants in triplicates, revealed substantial levels of differential expression of growth-related genes along with stress-related genes. These REs elicited responses in plants that had previously only been achieved through gene editing and transgenic methodologies. Using bio-stimulant sprays provides a novel platform to promote beneficial agronomic traits, independent of genetic manipulation.

Nutritional and tissue-specific regulation of cytochrome P450 CYP711A MAX1 homologues and strigolactone biosynthesis in wheat.

Strigolactones (SLs) are a class of phytohormones regulating branching/tillering, and their biosynthesis has been associated with nutritional signals and plant adaptation to nutrient-limiting conditions. The enzymes in the SL biosynthetic pathway downstream of carlactone are of interest as they are responsible for structural diversity in SLs, particularly cytochrome P450 CYP711A subfamily members, such as MORE AXILLARY GROWTH1 (MAX1) in Arabidopsis. We identified 13 MAX1 homologues in wheat, clustering in four clades and five homoeologous subgroups. The utilization of RNA-sequencing data revealed a distinct expression pattern of MAX1 homologues in above- and below-ground tissues, providing insights into the distinct roles of MAX1 homologues in wheat. In addition, a transcriptional analysis showed that SL biosynthetic genes were systematically regulated by nitrogen supply. Nitrogen limitation led to larger transcriptional changes in the basal nodes than phosphorus limitation, which was consistent with the observed tillering suppression, as wheat showed higher sensitivity to nitrogen. The opposite was observed in roots, with phosphorus limitation leading to stronger induction of most SL biosynthetic genes compared with nitrogen limitation. The observed tissue-specific regulation of SL biosynthetic genes in response to nutritional signals is likely to reflect the dual role of SLs as rhizosphere signals and branching inhibitors.

Two Carbohydrate-Based Natural Extracts Stimulate in vitro Pollen Germination and Pollen Tube Growth of Tomato Under Cold Temperatures.

To date, it is widely accepted by the scientific community that many agricultural regions will experience more extreme temperature fluctuations. These stresses will undoubtedly impact crop production, particularly fruit and seed yields. In fact, pollination is considered as one of the most temperature-sensitive phases of plant development and until now, except for the time-consuming and costly processes of genetic breeding, there is no immediate alternative to address this issue. In this work, we used a multidisciplinary approach using physiological, biochemical, and molecular techniques for studying the effects of two carbohydrate-based natural activators on in vitro tomato pollen germination and pollen tube growth cultured in vitro under cold conditions. Under mild and strong cold temperatures, these two carbohydrate-based compounds significantly enhanced pollen germination and pollen tube growth. The two biostimulants did not induce significant changes in the classical molecular markers implicated in pollen tube growth. Neither the number of callose plugs nor the CALLOSE SYNTHASE genes expression were significantly different between the control and the biostimulated pollen tubes when pollens were cultivated under cold conditions. PECTIN METHYLESTERASE (PME) activities were also similar but a basic PME isoform was not produced or inactive in pollen grown at 8°C. Nevertheless, NADPH oxidase (RBOH) gene expression was correlated with a higher number of viable pollen tubes in biostimulated pollen tubes compared to the control. Our results showed that the two carbohydrate-based products were able to reduce in vitro the effect of cold temperatures on tomato pollen tube growth and at least for one of them to modulate reactive oxygen species production.

Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds.

In temperate trees, optimal timing and quality of flowering directly depend on adequate winter dormancy progression, regulated by a combination of chilling and warm temperatures. Physiological, genetic and functional genomic studies have shown that hormones play a key role in bud dormancy establishment, maintenance and release. We combined physiological and transcriptional analyses, quantification of abscisic acid (ABA) and gibberellins (GAs), and modeling to further investigate how these signaling pathways are associated with dormancy progression in the flower buds of two sweet cherry cultivars. Our results demonstrated that GA-associated pathways have distinct functions and may be differentially related with dormancy. In addition, ABA levels rise at the onset of dormancy, associated with enhanced expression of ABA biosynthesis PavNCED genes, and decreased prior to dormancy release. Following the observations that ABA levels are correlated with dormancy depth, we identified PavUG71B6, a sweet cherry UDP-GLYCOSYLTRANSFERASE gene that up-regulates active catabolism of ABA to ABA glucosyl ester (ABA-GE) and may be associated with low ABA content in the early cultivar. Subsequently, we modeled ABA content and dormancy behavior in three cultivars based on the expression of a small set of genes regulating ABA levels. These results strongly suggest the central role of ABA pathway in the control of dormancy progression and open up new perspectives for the development of molecular-based phenological modeling.

Simultaneous untargeted and targeted metabolomics profiling of underivatized primary metabolites in sulfur-deficient barley by ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry.

BACKGROUND: Metabolomics based on mass spectrometry analysis are increasingly applied in diverse scientific domains, notably agronomy and plant biology, in order to understand plants' behaviors under different stress conditions. In fact, these stress conditions are able to disrupt many biosynthetic pathways that include mainly primary metabolites. Profiling and quantifying primary metabolites remain a challenging task because they are poorly retained in reverse phase columns, due to their high polarity and acid-base properties. The aim of this work is to develop a simultaneous untargeted/targeted profiling of amino acids, organic acids, sulfur metabolites, and other several metabolites. This method will be applied on sulfur depleted barley, in order to study this type of stress, which is difficult to detect at early stage. Also, this method aims to explore the impact of this stress on barley's metabolome. RESULTS: Ultra-high performance liquid chromatography-high resolution mass spectrometry-based method was successfully applied to real samples allowing to discriminate, detect, and quantify primary metabolites in short-runs without any additional sampling step such as derivatization or ion pairing. The retention of polar metabolites was successfully achieved using modified C18 columns with high reproducibility (relative standard deviation below 10%). The quantification method showed a high sensitivity and robustness. Furthermore, high resolution mass spectrometry detection provided reliable quantification based on exact mass, eliminating potential interferences, and allowing the simultaneous untargeted metabolomics analysis. The untargeted data analysis was conducted using Progenesis QI software, performing alignment, peak picking, normalization and multivariate analysis. The simultaneous analysis provided cumulative information allowing to discriminate between two plant batches. Thus, discriminant biomarkers were identified and validated. Simultaneously, quantification confirmed coherently the relative abundance of these biomarkers. CONCLUSIONS: A fast and innovated simultaneous untargeted/targeted method has successfully been developed and applied to sulfur deficiency on barley. This work opens interesting perspectives in both fundamental and applied research. Biomarker discovery give precious indication to understand plant behavior during a nutritional deficiency. Thus, direct or indirect measurement of these compounds allows a real time fertilization management and encounter the challenges of sustainable agriculture.

Induction of Barley Silicon Transporter HvLsi1 and HvLsi2, increased silicon concentration in the shoot and regulated Starch and ABA Homeostasis under Osmotic stress and Concomitant Potassium Deficiency.

Drought is one of the major stress factors reducing cereal production worldwide. There is ample evidence that the mineral nutrient status of plants plays a critical role in increasing plant tolerance to different biotic and abiotic stresses. In this regard, the important role of various nutrients e.g., potassium (K) or silicon (Si) in the mitigation of different stress factors, such as drought, heat or frost has been well documented. Si application has been reported to ameliorate plant nutrient deficiency. Here, we used K and Si either solely or in combination to investigate whether an additive positive effect on barley growth can be achieved under osmotic stress and which mechanisms contribute to a better tolerance to osmotic stress. To achieve this goal, barley plants were subjected to polyethylene glycol (PEG)-induced osmotic stress under low or high K supply and two Si regimes. The results showed that barley silicon transporters HvLsi1 and HvLsi2 regulate the accumulation of Si in the shoot only when plant suffered from K deficiency. Si, in turn, increased the starch level under both osmotic stress and K deficiency and modulated the glycolytic and TCA pathways. Hormone profiling revealed that the beneficial effect of Si is most likely mediated also by ABA homeostasis and active cytokinin isopentenyl adenine (iP). We conclude that Si may effectively improve stress tolerance under K deficient condition in particular when additional stress like osmotic stress interferes.

An ethoxylated surfactant enhances the penetration of the sulfated laminarin through leaf cuticle and stomata, leading to increased induced resistance against grapevine downy mildew.

Some ß-1,3-glucans and particularly sulfated laminarin (PS3) are known as resistance inducers (RIs) in grapevine against the downy mildew. However, their efficacy in vineyard is still often too low, which might be caused by a limited penetration through the leaf cuticle following spray application. We used (14) C-sucrose uptake experiments with grapevine leaves in order to select a surfactant as saccharide penetration enhancer. Our results showed that although sucrose foliar uptake was low, it was strongly enhanced by Dehscofix CO125 (DE), a highly ethoxylated surfactant. Fluorescent saccharides were then produced and laser scanning microscopy was used to analyze their foliar diffusion pattern in Arabidopsis thaliana and grapevine. Interestingly, sucrose and PS3 were seemingly able to penetrate the leaf cuticle only when formulated with DE. Diffusion could preferentially occur via stomata, anticlinal cell walls and trichomes. In grapevine, PS3 penetration rate was much higher on the stomateous abaxial surface of the leaf than on the adaxial surface. Finally, using DE allowed a higher level of downy mildew control by PS3, which corroborated diffusion observations. Our results have practical consequences for the improvement of treatments with saccharidic inducers on grape. That is, formulation of such RIs plays a critical role for their cuticular diffusion and consequently their efficacy. Also, spray application should preferentially target the abaxial surface of the leaves in order to maximize their penetration.

Effect of sulphur deprivation on osmotic potential components and nitrogen metabolism in oilseed rape leaves: identification of a new early indicator.

Identification of early sulphur (S) deficiency indicators is important for species such as Brassica napus, an S-demanding crop in which yield and the nutritional quality of seeds are negatively affected by S deficiency. Because S is mostly stored as SO4 (2-) in leaf cell vacuoles and can be mobilized during S deficiency, this study investigated the impact of S deprivation on leaf osmotic potential in order to identify compensation processes. Plants were exposed for 28 days to S or to chlorine deprivation in order to differentiate osmotic and metabolic responses. While chlorine deprivation had no significant effects on growth, osmotic potential and nitrogen metabolism, Brassica napus revealed two response periods to S deprivation. The first one occurred during the first 13 days during which plant growth was maintained as a result of vacuolar SO4 (2-) mobilization. In the meantime, leaf osmotic potential of S-deprived plants remained similar to control plants despite a reduction in the SO4 (2-) osmotic contribution, which was fully compensated by an increase in NO3 (-), PO4 (3-) and Cl(-) accumulation. The second response occurred after 13 days of S deprivation with a significant reduction in growth, leaf osmotic potential, NO3 (-) uptake and NO3 (-) reductase activity, whereas amino acids and NO3 (-) were accumulated. This kinetic analysis of S deprivation suggested that a ([Cl(-)]+[NO3 (-)]+[PO4 (3-)]):[SO4 (2-)] ratio could provide a relevant indicator of S deficiency, modified nearly as early as the over-expression of genes encoding SO4 (2-) tonoplastic or plasmalemmal transporters, with the added advantage that it can be easily quantified under field conditions.

Sesquiterpene volatile organic compounds (VOCs) are markers of elicitation by sulfated laminarine in grapevine.

Inducing resistance in plants by the application of elicitors of defense reactions is an attractive plant protection strategy, particularly for grapevine (Vitis vinifera), which is susceptible to severe fungal diseases. Although induced resistance (IR) can be successful under controlled conditions, in most cases, IR is not sufficiently effective for practical disease control under outdoor conditions. Progress in the application of IR requires a better understanding of grapevine defense mechanisms and the ability to monitor defense markers to identify factors, such as physiological and environmental factors, that can impact IR in the vineyard. Volatile organic compounds (VOCs) are well-known plant defense compounds that have received little or no attention to date in the case of grape-pathogen interactions. This prompted us to investigate whether an elicitor, the sulfated laminarin (PS3), actually induces the production of VOCs in grapevine. An online analysis (proton-transfer-reaction quadrupole mass spectrometry) of VOC emissions in dynamic cuvettes and passive sampling in gas-tight bags with solid-phase microextraction-GC-MS under greenhouse conditions showed that PS3 elicited the emission of VOCs. Some of them, such as (E,E)-α-farnesene, may be good candidates as biomarkers of elicitor-IR, whereas methyl salicylate appears to be a biomarker of downy mildew infection. A negative correlation between VOC emission and disease severity suggests a positive role of VOCs in grape defense against diseases.

ß-(1→3)-Glucan-mannitol conjugates: scope and amazing results.

It is well known that ß-(1→3)-Glucans present high applicative potential in human health as immunostimulating agents. Numerous studies have highlighted this, but mainly used native polysaccharides extracted from various natural sources. These compounds are therefore inevitably polydisperse but also present structures that are not homogeneous, in an analytical point of view. This is the reason why we have achieved the chemical synthesis of small glucan-mannitol derivatives especially found in brown seaweeds. The targets differ from each other by the nature of the conjunction between the laminaribiose and the mannose or mannitol, i.e., (1→6) or (1→3). We established that (I) these molecules were efficiently obtained from glucose, laminaribiose and/or mannose derivatives; (II) the synthetic plan has to be adapted to the first connection between a glucosyl entity and the mannosyl residue; and (III) resulting pure compounds may be used as the standard for analytical purposes.

New 4-deoxy-(1→3)-ß-D-glucan-based oligosaccharides and their immunostimulating potential.

(1→3)-ß-D-Glucans are well-established natural biological immunomodulators. However, problems inherited with the natural origin of these polysaccharides bring about significant setbacks, including batch-to-batch heterogeneity and significant differences based on the source and isolation techniques. In this study, we tried to overcome these problems by preparation of a quantitatively new set of oligo-(1→3)-ß-D-glucan-based synthetic immunomodulators. Some of these non-natural oligosaccharides showed biological activities, such as stimulation of phagocytosis, modulation of gene expression, and anti-cancer activity, which were superior to natural glucans.

Biological properties of (1 → 3)-ß-D-glucan-based synthetic oligosaccharides.

Despite the fact that ß-glucans are well-established immunomodulators, the problems with batch-to-batch heterogeneity remains problematic. The aim of this study was to prepare and evaluate new type of synthetic oligosaccharides. A new family of oligo-(1 → 3)-ß-d-glucans modified on the reducing end was synthesized using a controlled and specific inversion of configuration at C-2 starting from already formed oligo-(1 → 3)-ß-d-glucans. The designed glycosides are characterized by the presence of four or five glucopyranose entities and a mannose residue at the reducing end. To study of the impact of well-defined structural modulations, we used murine and human models to evaluate their immunostimulating potential. These novel oligosaccharides showed strong and long-lasting stimulation of phagocytosis and significant potentiation of synthesis and/or secretion of interleukin (IL-2, IL-4, IL-5, IL-6), tumor necrosis factor-α, and vascular endothelial growth factor. In addition, the oligosaccharides tested showed significant effects on expression of several genes in human fibroblasts and breast cancer cells. From our results it is clear that these synthetic oligosaccharides represent a better alternative to natural ß-glucans.

Double diastereoselection explains limitations in synthesizing mannose-containing beta-(1,3)-glucans.

It is known that 3-O-glycosylation of glucosidic acceptors bearing acyl groups in the 4 and 6 positions instead of a 4,6-O-benzylidene ring mainly affords alpha-glycosides. Described here is an unexpected stereochemical outcome for elongation at glucose O-3 of a beta-d-Glcp-(1-->3)-alpha-d-Manp disaccharide using peracetylated ethyl thioglucoside as a donor. This unexpected reaction was correlated with match-mismatch effects, as shown by efficient coupling of the same acceptor by a donor of l-configuration.

New oligo-beta-(1,3)-glucan derivatives as immunostimulating agents.

Oligo-beta-(1,3)-glucans were chemically modified in order to introduce a structural variation specifically on the reducing end of the oligomers. The impact of well defined structural modulations was further studied on cancer cells and murin models to evaluate their cytotoxicity and immunostimulating potential.

Recent progress in the field of beta-(1,3)-glucans and new applications.

Beta-(1,3)-glucans are widely distributed within microorganisms or seaweeds in which they act as membrane components or for energy storage, respectively. Since these glucans are not biosynthesized by mammals, they are likely to activate the immune system of their host. Since the discovery of their positive involvement as immunomodulator agents, numerous studies were published all around the glycosciences. These works deal with purification procedures, analytical chemistry, synthetic processes, chemical modification of the natural polysaccharides, determination of their physicochemical properties, and assessment of their biological and medicinal effects through in vitro and in vivo studies. This article aims at presenting some recent results linked to beta-(1,3)-glucans through two closely connected points of view, i.e. biology and chemistry. Biological aspects will be focused more particularly on discovery of some receptors present on immunocompetent cells and scope and limitations of chemical synthesis and/or modifications will be described. Moreover, this paper will also introduce some new chemo-enzymatic synthetic methods using wild-type or mutant glycosidases and will be extended to novel opportunities of applications of beta-(1,3)-glucans in nanotechnology resulting from a better understanding of their self-assembling propensity in aqueous media.

Glucan-like synthetic oligosaccharides: iterative synthesis of linear oligo-beta-(1,3)-glucans and immunostimulatory effects.

Small reducing and linear oligo-beta-(1,3)-glucans, which are able to act as phytoallexin elicitors or as immunostimulating agents in anticancer therapy, were synthesized according to an iterative strategy that involved a unique key monosaccharidic donor. To avoid anomeric mixtures, the reducing entity of the target oligomers was first locked with benzyl alcohol and further selective deprotection of the 3-OH with DDQ afforded the desired building block as an acceptor. The latter was then used in a second cycle of glycosylation/deprotection to afford the desired disaccharide, and successive reiterations of this process provided the desired oligomers. Unusual conformational behaviors were observed by standard NMR sequences and supported by NOESY studies. Finally, removal of protecting groups afforded free tri-, tetra-, and pentaglucosides in good overall yields. Two oligosaccharides representing linear laminaritetraose and laminaripentaose were compared to the recently described beta-(1,3)-glucan phycarine. Following an intraperitoneal injection, the influx of monocytes and granulocytes into the blood and macrophages into the peritoneal cavity was comparable to that caused by phycarine. Similarly, both oligosaccharides stimulated phagocytic activity of granulocytes and macrophages. Using ELISA, we also demonstrated a significant stimulation of secretion of IL-1beta. Together these results suggest that the synthetic oligosaccharides have similar stimulatory effects as natural beta-(1,3)-glucans.

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis.

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.