Environmental Stress and Plants 2.0.

Following the success of our previous edition [...].

Biochemical and cytological interactions between callose synthase and microtubules in the tobacco pollen tube.

KEY MESSAGE: The article concerns the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in the pollen tube. Results confirmed this association and immunogold labeling showed a colocalization. Callose is a cell wall polysaccharide involved in fundamental biological processes, from plant development to the response to abiotic and biotic stress. To gain insight into the deposition pattern of callose, it is important to know how the enzyme callose synthase is regulated through the interaction with the vesicle-cytoskeletal system. Actin filaments likely determine the long-range distribution of callose synthase through transport vesicles but the spatial/biochemical relationships between callose synthase and microtubules are poorly understood, although experimental evidence supports the association between callose synthase and tubulin. In this manuscript, we further investigated the association between callose synthase and microtubules through biochemical and ultrastructural analyses in the pollen tube model system, where callose is an essential component of the cell wall. Results by native 2-D electrophoresis, isolation of callose synthase complex and far-western blot confirmed that callose synthase is associated with tubulin and can therefore interface with cortical microtubules. In contrast, actin and sucrose synthase were not permanently associated with callose synthase. Immunogold labeling showed colocalization between the enzyme and microtubules, occasionally mediated by vesicles. Overall, the data indicate that pollen tube callose synthase exerts its activity in cooperation with the microtubular cytoskeleton.

Environmental Stress and Plants.

Land plants are constantly subjected to multiple unfavorable or even adverse environmental conditions. Among them, abiotic stresses (such as salt, drought, heat, cold, heavy metals, ozone, UV radiation, and nutrient deficiencies) have detrimental effects on plant growth and productivity and are increasingly important considering the direct or indirect effects of climate change. Plants respond in many ways to abiotic stresses, from gene expression to physiology, from plant architecture to primary, and secondary metabolism. These complex changes allow plants to tolerate and/or adapt to adverse conditions. The complexity of plant response can be further influenced by the duration and intensity of stress, the plant genotype, the combination of different stresses, the exposed tissue and cell type, and the developmental stage at which plants perceive the stress. It is therefore important to understand more about how plants perceive stress conditions and how they respond and adapt (both in natural and anthropogenic environments). These concepts were the basis of the Special Issue that International Journal of Molecular Sciences expressly addressed to the relationship between environmental stresses and plants and that resulted in the publication of 5 reviews and 38 original research articles. The large participation of several authors and the good number of contributions testifies to the considerable interest that the topic currently receives in the plant science community, especially in the light of the foreseeable climate changes. Here, we briefly summarize the contributions included in the Special Issue, both original articles categorized by stress type and reviews that discuss more comprehensive responses to various stresses.

Expression of Clementine Asp-Rich Proteins (CcASP-RICH) in Tobacco Plants Interferes with the Mechanism of Pollen Tube Growth.

Low-molecular-weight, aspartic-acid-rich proteins (ASP-RICH) have been assumed to be involved in the self-incompatibility process of clementine. The role of ASP-RICH is not known, but hypothetically they could sequester calcium ions (Ca2+) and affect Ca2+-dependent mechanisms. In this article, we analyzed the effects induced by clementine ASP-RICH proteins (CcASP-RICH) when expressed in the tobacco heterologous system, focusing on the male gametophyte. The aim was to gain insight into the mechanism of action of ASP-RICH in a well-known cellular system, i.e., the pollen tube. Pollen tubes of tobacco transgenic lines expressing CcASP-RICH were analyzed for Ca2+ distribution, ROS, proton gradient, as well as cytoskeleton and cell wall. CcASP-RICH modulated Ca2+ content and consequently affected cytoskeleton organization and the deposition of cell wall components. In turn, this affected the growth pattern of pollen tubes. Although the expression of CcASP-RICH did not exert a remarkable effect on the growth rate of pollen tubes, effects at the level of growth pattern suggest that the expression of ASP-RICH may exert a regulatory action on the mechanism of plant cell growth.

Purification and Biochemical Characterization of Sucrose synthase from the Stem of Nettle (Urtica dioica L.).

Sucrose synthase is a key enzyme in sucrose metabolism as it saves an important part of sucrose energy in the uridine-5'-diphosphate glucose (UDP-glucose) molecule. As such it is also involved in the synthesis of fundamental molecules such as callose and cellulose, the latter being present in all cell walls of plant cells and therefore also in the gelatinous cell walls of sclerenchyma cells such as bast fibers. Given the importance of these cells in plants of economic interest such as hemp, flax and nettle, in this work we have studied the occurrence of Sucrose synthase in nettle stems by analyzing its distribution between the cytosol, membranes and cell wall. We have therefore developed a purification protocol that can allow the analysis of various characteristics of the enzyme. In nettle, Sucrose synthase is encoded by different genes and each form of the enzyme could be subjected to different post-translational modifications. Therefore, by two-dimensional electrophoresis analysis, we have also traced the phosphorylation profile of Sucrose synthase isoforms in the various cell compartments. This information paves the way for further investigation of Sucrose synthase in plants such as nettle, which is both economically important, but also difficult to study.

Insights into the Mechanisms of Heat Priming and Thermotolerance in Tobacco Pollen.

Global warming leads to a progressive rise in environmental temperature. Plants, as sessile organisms, are threatened by these changes; the male gametophyte is extremely sensitive to high temperature and its ability to preserve its physiological status under heat stress is known as acquired thermotolerance. This latter can be achieved by exposing plant to a sub-lethal temperature (priming) or to a progressive increase in temperature. The present research aims to investigate the effects of heat priming on the functioning of tobacco pollen grains. In addition to evaluating basic physiological parameters (e.g., pollen viability, germination and pollen tube length), several aspects related to a correct pollen functioning were considered. Calcium (Ca2+) level, reactive oxygen species (ROS) and related antioxidant systems were investigated, also to the organization of actin filaments and cytoskeletal protein such as tubulin (including tyrosinated and acetylated isoforms) and actin. We also focused on sucrose synthase (Sus), a key metabolic enzyme and on the content of main soluble sugars, including UDP-glucose. Results here obtained showed that a pre-exposure to sub-lethal temperatures can positively enhance pollen performance by altering its metabolism. This can have a considerable impact, especially from the point of view of breeding strategies aimed at improving crop species.

Immunohistochemical analyses on two distinct internodes of stinging nettle show different distribution of polysaccharides and proteins in the cell walls of bast fibers.

Stinging nettle is a perennial herbaceous species holding value as a multi-purpose plant. Indeed, its leaves and roots are phytofactories providing functional ingredients of medicinal interest and its stems produce silky and resistant extraxylary fibers (a.k.a. bast fibers) valued in the biocomposite sector. Similarly to what is reported in other fiber crops, the stem of nettle contains both lignified and hypolignified fibers in the core and cortex, respectively, and it is therefore a useful model for cell wall research. Indeed, data on nettle stem tissues can be compared to those obtained in other models, such as hemp and flax, to support hypotheses on the differentiation and development of bast fibers. The suitability of the nettle stem as model for cell wall-related research was already validated using a transcriptomics and biochemical approach focused on internodes at different developmental stages sampled at the top, middle, and bottom of the stem. We here sought to complement and enrich these data by providing immunohistochemical and ultrastructural details on young and older stem internodes. Antibodies recognizing non-cellulosic polysaccharides (galactans, arabinans, rhamnogalacturonans) and arabinogalactan proteins were here investigated with the goal of understanding whether their distribution changes in the stem tissues in relation to the bast fiber and vascular tissue development. The results obtained indicate that the occurrence and distribution of cell wall polysaccharides and proteins differ between young and older internodes and that these changes are particularly evident in the bast fibers.

Influence of CNV on transcript levels of HvCBF genes at Fr-H2 locus revealed by resequencing in resistant barley cv. 'Nure' and expression analysis.

Resequencing in resistant cultivar 'Nure' and structural comparison with the same region of susceptible 'Morex' was performed in order to gain a better insight into barley Frost-resistance-H2 locus. Accurate annotation showed copy number variation (CNV) in the proximal part of the locus. In 'Nure', two exact copies of the HvCBF4-HvCBF2A region and one of the HvCBF4-HvCBF2B segment were observed, while in 'Morex' the corresponding region harboured a single HvCBF4-HvCBF2A (22 kb) segment. Abundance and diversity of repetitive element classes, gene function gain/losses, regulatory motifs and SNPs in gene sequences were identified. An expression study of key HvCBFs with/without CNV on selected genotypes contrasting for frost resistance and estimated HvCBF4-HvCBF2B copy number (2-10 copies) was also performed. Under light stimulus at warm temperature (23 °C), CNV of HvCBF2A and HvCBF4 correlated with their expression levels and reported frost resistance of genotypes; moreover, expression levels of HvCBF2A and HvCBF14 were strongly correlated (r = 0.908, p < 0.01). On the other hand, frost resistance correlated to HvCBF14 expression (r = 0.871, p < 0.01) only after cold induction (6°C) in the dark. A complex interplay of HvCBFs expression levels under different light/temperature stimuli is discussed in light of CNV and presence/number of regulatory elements that integrate different signal transduction pathways.

Temperature-Dependent Compatible and Incompatible Pollen-Style Interactions in Citrus clementina Hort. ex Tan. Show Different Transglutaminase Features and Polyamine Pattern.

In clementine, failure of fertilization can result in parthenocarpic fruit development, which has several advantages, such as seedless fruit, longer shelf-life, and greater consumer appeal. Recently, S-RNases have been identified in Citrus grandis, thus revealing that the self-incompatibility (SI) reaction relies on the S-RNase gametophytic mechanism. The fundamental role of environmental factors, mostly temperature, in determining the numbers of pollen tubes reaching the ovary is also well established in Citrus. In the present work, temperature-dependent pollen-pistil interactions in C. clementina were analyzed, focusing on several morphological aspects, as well as on polyamine (PA) content and the activity and distribution of transglutaminase (TGase), both reported to be involved in the SI response in pear and in pummelo. Results clearly indicate that temperature contributed to a different activation of the SI response, which occurs at optimal temperature of 25°C but was by-passed at 15°C. TGase activity was stimulated during the SI response, and it localized differently in the compatible and incompatible interaction: in compatible pollinated styles, TGase localized inside the style canal, while it was detected all around it in incompatible crosses. TGase localization and activity were congruent with the levels of soluble and insoluble conjugated PAs and with morphological evidences, which highlighted cell wall modification occurring as a result of SI.

Drought Stress Affects the Response of Italian Local Tomato (Solanum lycopersicum L.) Varieties in a Genotype-Dependent Manner.

Drought stress is one of the most severe conditions for plants, especially in the face of the emerging problem of global warming. This issue is important when considering economically relevant crops, including the tomato. For these plants, a promising solution is the valorization of local agrobiodiversity as a source of genetic variability. In this paper we investigated how six Italian tomato varieties react to a prolonged period of water depletion. We used a multidisciplinary approach, from genetics to plant physiology and cytology, to provide a detailed overview of the response of plants to stress. The varieties analyzed, each characterized by a specific genetic profile, showed a genotype-specific response with the variety 'Fragola' being the most resistant and the variety 'Pisanello' the most susceptible. For all the parameters evaluated, 'Fragola' performed in a manner comparable to that of control plants. On the contrary, 'Pisanello' appeared to be more affected and showed an increase in the number of stomata and a drastic increase in antioxidants, a symptom of acute oxidative stress. Our work suggests the existence of a valuable reservoir of genetic biodiversity with more drought-tolerant tomato genotypes opening the way to further exploitation and use of local germplasm in breeding programs.