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.

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.

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.

Depletion of sucrose induces changes in the tip growth mechanism of tobacco pollen tubes.

Background and Aims: Pollen tubes are rapidly growing, photosynthetically inactive cells that need high rates of energy to support growth. Energy can derive from internal and external storage sources. The lack of carbon sources can cause various problems during pollen tube growth, which in turn could affect the reproduction of plants. Methods: We analysed the effects of energy deficiency on the development of Nicotiana tabacum pollen tubes by replacing sucrose with glycerol in the growth medium. We focused on cell growth and related processes, such as metabolite composition and cell wall synthesis. Key Results: We found that the lack of sucrose affects pollen germination and pollen tube length during a specific growth period. Both sugar metabolism and ATP concentration were affected by sucrose shortage when pollen tubes were grown in glycerol-based media; this was related to decreases in the concentrations of glucose, fructose and UDP-glucose. The intracellular pH and ROS levels also showed a different distribution in pollen tubes grown in sucrose-depleted media. Changes were also observed at the cell wall level, particularly in the content and distribution of two enzymes related to cell wall synthesis (sucrose synthase and callose synthase). Furthermore, both callose and newly secreted cell wall material (mainly pectins) showed an altered distribution corresponding to the lack of oscillatory growth in pollen tubes. Growth in glycerol-based media also temporarily affected the movement of generative cells and, in parallel, the deposition of callose plugs. Conclusion: Pollen tubes represent an ideal model system for studying metabolic pathways during the growth of plant cells. In our study, we found evidence that glycerol, a less energetic source for cell growth than sucrose, causes critical changes in cell wall deposition. The evidence that different aspects of pollen tube growth are affected is an indication that pollen tubes adapt to metabolic stress.

New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts.

Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the highest TPC and AA under non-saline conditions. Salinity increased TPC in all three landraces, with the strongest increase occurring in R49, and enhanced radical scavenging capacity in R49 and VR. Overall, results show that salinity deeply altered the seed proteome and amino acid profiles and, in general, increased the concentration of bioactive molecules and AA of protein extracts in a genotype-dependent manner.

Salares versus coastal ecotypes of quinoa: Salinity responses in Chilean landraces from contrasting habitats.

Quinoa (Chenopodium quinoa Willd.) is a highly salt-tolerant species subdivided into five ecotypes and exhibiting broad intra-specific differences in tolerance levels. In a greenhouse study, Chilean landraces belonging either to the salares (R49) or coastal lowlands (VI-1, Villarrica) ecotype with contrasting agro-ecological origins were investigated for their responses to high salinity. The effects of two levels of salinity, 100 (T1) and 300 (T2) mM NaCl, on plant growth and on some physiological parameters were measured. Leaf and root Na(+) accumulation differed among landraces. T2 reduced growth and seed yield in all landraces with maximum inhibition relative to controls in R49. Salinity negatively affected chlorophyll and total polyphenol content (TPC) in VI-1 and Villarrica but not R49. Germination on saline or control media of seeds harvested from plants treated or not with NaCl was sometimes different; the best performing landrace was R49 insofar as 45-65% of seeds germinated on 500 mM NaCl-containing medium. In all landraces, average seedling root length declined strongly with increasing NaCl concentration, but roots of R49 were significantly longer than those of VI-1 and Villarrica up to 300 mM NaCl. Salt caused increases in seed TPC relative to controls, but radical scavenging capacity was higher only in seeds from T2 plants of R49. Total SDS-extractable seed proteins were resolved into distinct bands (10-70 kDa) with some evident differences between landraces. Salt-induced changes in protein patterns were landrace-specific. The responses to salinity of the salares landrace are discussed in relation to its better adaptation to an extreme environment.

Spermine either delays or promotes cell death in Nicotiana tabacum L. corolla depending on the floral developmental stage and affects the distribution of transglutaminase.

The role of spermine (SM) was studied to verify if SM supplied to Nicotiana tabacum flower can modulate programmed cell death (PCD) of the corolla. SM has strong effects on the development and senescence of excised flowers despite its low physiological levels. The timing and duration of SM treatment is a key factor; SM counteracts PCD (verified by morphological observations, pigment contents and DNA laddering) only in the narrow developmental window of corolla expansion. Before and after, SM promotes PCD. SM exerts its pro-survival role by delaying fresh weight loss, by inhibiting reduction of pigments and finally by preventing DNA degradation. Moreover, SM deeply alters the distribution of the PA-conjugating enzyme transglutaminase (TGase). TGase is present in the epidermis during development, but it sprays also in the cell walls of inner parenchyma at senescence. After SM treatment, parenchyma cells accumulate TGase, increase in size and their cell walls do not undergo stiffening contrarily to control cells. The subcellular localization of TGase has been validated by biolistic-transformation of onion epidermal cells. Results indicated that SM is a critical factor in the senescence of N. tabacum corolla by controlling biochemical and morphological parameters; the lasts are probably interconnected with the action of TGase.

Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments.

TGases (transglutaminases) are a class of calcium-dependent enzymes that catalyse the interactions between acyl acceptor glutamyl residues and amine donors, potentially making cross-links between proteins. To assess the activity of apple (Malus domestica) pollen TGase on the functional properties of actin and tubulin, TGase was prepared from apple pollen by hydrophobic- interaction chromatography and assayed on actin and tubulin purified from the same cell type. The enzyme catalysed the incorporation of putrescine into the cytoskeleton monomers. When tested on actin filaments, pollen TGase induced the formation of high-molecular-mass aggregates of actin. Use of fluorescein-cadaverine showed that the labelled polyamine was incorporated into actin by pollen TGase, similar to with guinea pig liver TGase. The pollen TGase also reduced the enzyme activity and the binding of myosin to TGase-treated actin filaments. Polymerization of tubulin in the presence of pollen TGase also yielded the formation of high-molecular-mass aggregates. Furthermore, the pollen TGase also affected the binding of kinesin to microtubules and reduced the motility of microtubules along kinesin-coated slides. These results indicate that the pollen TGase can control different properties of the pollen tube cytoskeleton (including the ability of actin and tubulin to assemble and their interaction with motor proteins) and consequently regulate the development of pollen tubes.

The acropetal wave of developmental cell death of tobacco corolla is preceded by activation of transglutaminase in different cell compartments.

The activity of transglutaminase (TGase), an enzyme responsible for polyamine conjugation to proteins, was analyzed in relationship to developmental cell death (DCD) during the flower life span stages of the tobacco (Nicotiana tabacum) corolla. As the DCD exhibits an acropetal gradient, TGase was studied in corolla proximal, medial, and distal parts. TGase was immunorecognized by three TGase antibodies; the main 58-kD band decreased during corolla life, whereas a 38-kD band localized progressively from basal to distal parts. The former was present in the soluble, microsomal, plastidial (together with the 38-kD band), and cell wall fractions. The endogenous TGase activity increased during DCD reaching a maximum soon after the corolla opening. The activity maximum shifted from proximal to distal part, preceding the DCD acropetal pattern. A similar activity increase was observed by the exogenous TGase substrate (histidine(6)-Xpr-green fluorescent protein). Subcellular activities were detected in (1) the microsomes, where TGase activity is in general higher in the proximal part, peaking at the corolla opening; (2) the soluble fraction, where it is present only in the proximal part at senescence; (3) the plastids, where it shows an increasing trend; and (4) cell walls, prevailing in the distal part and progressively increasing. These data suggest a relationship between DCD and TGase; the latter, possibly released in the cell wall through the Golgi vesicles, could cooperate to cell wall strengthening, especially at the abscission zone and possibly during corolla shape change. The plastid TGase, stabilizing the photosystems, could sustain the energy requirements for the senescence progression.

Transglutaminase activity changes during the hypersensitive reaction, a typical defense response of tobacco NN plants to TMV.

The occurrence of glutamyl polyamines (PAs) and changes in activity and levels of transglutaminase (TGase, EC 2.3.2.13), the enzyme responsible for their synthesis, are reported during the progression of the hypersensitive reaction (HR) of resistant NN tobacco plants (Nicotiana tabacum L. cv. Samsun) to tobacco mosaic virus (TMV). Mature leaves of tobacco were collected over 0-72 h after inoculation with TMV or phosphate buffer (mock). In vivo synthesis of polyamine glutamyl derivatives (glutamyl PAs), catalyzed by TGase activity, was evaluated after supplying labeled putrescine (Pu, a physiological substrate of TGase) to leaves. Results show that, starting from 24 h, mono-(gamma-glutamyl)-Pu and bis-(gamma-glutamyl)-Sd were recovered in TMV-inoculated samples but not in mock-inoculated ones; 2 days later, in the former, the amount of glutamyl derivatives further increased. An in vitro radiometric assay showed that, in TMV-inoculated leaves, TGase activity increased from 24 h onwards relative to mock controls. An immunoblot analysis with AtPng1p polyclonal antibody detected a 72-kDa protein whose amount increased at 72 h in TMV-inoculated leaves and in the lesion-enriched areas. A biotin-labeled cadaverine incorporation assay showed that TGase activity occurred in S1 (containing soluble proteins), S2 (proteins released by both cell walls and membranes) and S3 (membrane intrinsic proteins) fractions. In S3 fraction, where changes were the most relevant, TGase activity was enhanced in both mock-inoculated and TMV-inoculated samples, but the stimulation persisted only in the latter case. These data are discussed in the light of a possible role of TGase activity and glutamyl PAs in the defense against a viral plant pathogen.