Polyphenol-enriched spelt husk extracts improve growth and stress-related biochemical parameters under moderate salt stress in maize plants.

Biostimulants improve yield, quality, and stress acclimation in crops. In this work, we tested the possibility of using phenolics-rich extracts from spelt (Triticum dicoccum L.) husks to attenuate the effects of salt stress (100-200 mM NaCl) in maize. Two methanolic extracts were prepared from the soluble-conjugated (SC), and the insoluble-bound (IB) phenolic acid fractions of the spelt husk, and their effects were investigated on several stress-associated biochemical parameters, such as proline, lipid peroxidation, H2O2, GSH levels, and ion content. Results show that SC and IB fractions of husk extracts behaved very differently, no doubt due to their greatly divergent chemical composition, as revealed by both GC-MS and HPLC analyses. The efficacy of treatments in mitigating salt stress was also dose- and timing-dependent. IB, even at the lower concentration tested, was able to recover the performance of stressed plants in terms of growth, photosynthetic pigments content, and levels of salt stress markers. Recovery of shoot growth to control levels and reduction of stress-induced proline accumulation occurred regardless of whether plants were pre-treated or post-treated with IB, whereas only pre-treatment with the higher dose of IB was effective in mitigating oxidative stress. Although in some cases SC and even methanol alone exerted some positive effects, they could also be deleterious whereas IB never was. Overall, results indicate that a polyphenol-containing extract obtained from spelt by-products can behave as biostimulant in maize plants and can mitigate their response to salt stress, by acting on different biochemical targets.

Proteasome-mediated remodeling of the proteome and phosphoproteome during kiwifruit pollen germination.

Proteasome activity is essential for pollen tube emergence and growth; nevertheless, little is known about proteasome function at the molecular level. The objective of this study was to identify molecular targets and pathways which are directly/indirectly controlled by the proteasome during pollen germination. To this aim, changes in the proteome and phosphoproteome of Actinidia pollen, germinated in the presence of the proteasome inhibitor MG132, were investigated. Phosphoproteins were enriched by metal oxide/hydroxide affinity chromatography and phosphopeptides were further isolated using titanium ion (Ti4+) functional magnetic microparticles prior to liquid chromatography-tandem mass spectrometry analysis. Our results show that proteasome inhibition affects the phosphoproteome more profoundly than the proteome. Accordingly, the steady-state abundance of some kinases and phosphatases was changed and/or their phosphorylation status altered. Notably, affected proteins are involved in processes that are fundamental to pollen germination such as cytoskeletal organization, vesicular transport, cell wall synthesis and remodeling, protein synthesis, folding and degradation as well as energetic metabolism. Our data provide a molecular framework for the structural alterations observed when the proteasome is inhibited, contribute to the understanding of how proteasome activity regulates pollen germination, show the cross-talk between phosphorylation and proteasomal degradation and are a resource for further functional analyses. SIGNIFICANCE: Pollen germination and tube growth are fundamental to successful fertilization in seed plants. These events are based on dramatic remodeling and the dismantling of existing programs, which are replaced by new ones. Degradation plays a prominent role in reshaping the protein repertoire, also cross talking with the bulk of post-translational modifications. At present, phosphorylation is the only modification studied in germinating pollen on a large scale. The proteasome has been universally recognized as one of the most important sites for protein degradation and its function has been shown to be essential for pollen tube emergence and elongation. Upon proteasome inhibition structural alterations and dysregulation of pivotal processes governing pollen germination have been described; however, a mechanistic framework for the proteasome function at the molecular level is still lacking. In this investigation we provide the very first view of the global impact of the proteasome in remodeling the proteome and phosphoproteome during germination and tube growth. Our results show how proteasome inhibition alters the levels, and profoundly affects the phosphorylation status of many proteins involved, controlling energetic and synthetic pathways and signaling cascades.

Oxidative stress and antioxidant responses to increasing concentrations of trivalent chromium in the Andean crop species Chenopodium quinoa Willd.

Quinoa (Chenopodium quinoa Willd), an ancient Andean seed crop, exhibits exceptional nutritional properties and resistance to abiotic stress. The species' tolerance to heavy metals has, however, not yet been investigated nor its ability to take up and translocate chromium (Cr). This study aimed to investigate the metabolic adjustments occurring upon exposure of quinoa to several concentrations (0.01-5mM) of CrCl3. Young hydroponically grown plants were used to evaluate Cr uptake, growth, oxidative stress, and other biochemical parameters three and/or seven days after treatment. Leaves accumulated the lowest amounts of Cr, while roots and stems accumulated the most at low and at high metal concentrations, respectively. Fresh weight and photosynthetic pigments were reduced only by the higher Cr(III) doses. Substantially increased lipid peroxidation, hydrogen peroxide, and proline levels were observed only with 5mM Cr(III). Except for a significant decrease at day 7 with 5mM Cr(III), total polyphenols and flavonoids maintained control levels in Cr(III)-treated plants, whereas antioxidant activity increased in a dose-dependent manner. Maximum polyamine accumulation was observed in 1mM CrCl3-treated plants. Even though α- and γ-tocopherols also showed enhanced levels only with the 1mM concentration, tyrosine aminotransferase (TAT, EC 2.6.1.5) activity increased under Cr(III) treatment in a dose- and time-dependent manner. Taken together, results suggest that polyamines, tocopherols, and TAT activity could contribute to tolerance to 1mM Cr(III), but not to the highest concentration that, instead, generated oxidative stress.

Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

Role of the ubiquitin-proteasome pathway and some peptidases during seed germination and copper stress in bean cotyledons.

The role of the ubiquitin (Ub)-proteasome pathway and some endo- and aminopeptidases (EPs and APs, respectively) was studied in cotyledons of germinating bean seeds (Phaseolus vulgaris L.). The Ub system appeared to be important both in the early (3 days) and late (9 days) phases of germination. In the presence of copper, an increase in protein carbonylation and a decrease in reduced -SH pool occurred, indicating protein damage. This was associated with an enhancement in accumulation of malondialdehyde, a major product of lipid peroxidation, and an increase in content of hydrogen peroxide (H2O2), showing oxidative stress generation. Moreover, copper induced inactivation of the Ub-proteasome (EC 3.4.25) pathway and inhibition of leucine and proline aminopeptidase activities (EC 3.4.11.1 and EC 3.4.11.5, respectively), thus limiting their role in modulating essential metabolic processes, such as the removal of regulatory and oxidatively-damaged proteins. By contrast, total trypsin and chymotrypsin-like activities (EC 3.4.21.4 and EC 3.4.21.1, respectively) increased after copper exposure, in parallel with a decrease in their inhibitor capacities (i.e. trypsin inhibitor and chymotrypsin inhibitor activity), suggesting that these endoproteases are part of the protective mechanisms against copper stress.

Over-accumulation of putrescine induced by cyclohexylamine interferes with chromium accumulation and partially restores pollen tube growth in Actinidia deliciosa.

Both trivalent and hexavalent chromium, i.e., Cr(III) and Cr(VI), respectively, were previously demonstrated to affect in vitro germination and ultrastructure of kiwifruit (Actinidia deliciosa) pollen. In the present work, the response to chromium in germinating pollen was evaluated in terms of changes in the polyamine profile. Slight, though significant, increases in free spermidine and spermine occurred after exposure to Cr(III), while the levels remained almost unchanged after Cr(VI) treatment. The spermidine synthase inhibitor cyclohexylamine (CHA) caused a dramatic increase in free putrescine in both chromium-treated and untreated samples, while spermidine content was not affected. Interestingly, CHA positively affected the performance of chromium-treated pollen by partially, though significantly, restoring pollen tube growth. The major growth recovery was registered with 1 mM CHA in the presence of Cr(VI), concomitant with a considerable reduction in uptake of the metal. Conversely, endogenous calcium levels were more heavily affected in Cr(III)-treated pollen. The effect of CHA on production of reactive oxygen species also varied depending on the chromium species. The response of pollen to the CHA-induced putrescine excess was compared with that exerted by an exogenous supply of the same diamine. Results show that in Cr(III)-treated pollen, putrescine over-accumulation induced by CHA exerted similar effects as exogenous putrescine, while this was not true in the Cr(VI) treatment. It appears that the diamine was able to improve pollen tolerance to metal stress through different mechanisms, mostly depending upon the chromium species, namely via reduced metal uptake or by substituting for calcium.

In vitro toxicity of silver nanoparticles to kiwifruit pollen exhibits peculiar traits beyond the cause of silver ion release.

The vast use of silver nanoparticles (AgNPs) mandates thorough investigation of their impact on biosystems at various levels. The cytotoxicity of PVP coated-AgNPs to pollen, the aploid male gametophyte of higher plants, has been assessed here for the first time. The negative effects of AgNPs include substantial decreases in pollen viability and performance, specific ultrastructural alterations, early changes in calcium content, and unbalance of redox status. Ag⁺ released from AgNPs damaged pollen membranes and inhibited germination to a greater extent than the AgNPs themselves. By contrast, the AgNPs were more potent at disrupting the tube elongation process. ROS deficiency and overproduction were registered in the Ag⁺- and AgNP-treatment, respectively. The peculiar features of AgNP toxicity reflected their specific modes of interaction with pollen surface and membranes, and the dynamic exchange between coating (PVP) and culture medium. In contrast, the effects of Ag⁺ were most likely induced through chemical/physicochemical interactions.

New insights into an old story: pollen ROS also play a role in hay fever.

Reactive oxygen species (ROS) can exhibit negative and benign traits. In plants, ROS levels increase markedly during periods of environmental stress, and defense against pathogen attack. ROS form naturally as a by-product of normal oxygen metabolism, and evenly play an essential role in cell growth. The short ROS lifespan makes them ideal molecules to act in cell signaling, a role they share in both plants and animals. A particular plant organism, the pollen grain, may closely interact with human mucosa and an allergic inflammatory response often results. Pollen grain ROS represent a first, crucial signal which primes and magnifies a cascade of events in the allergic response.

Pd-nanoparticles cause increased toxicity to kiwifruit pollen compared to soluble Pd(II).

In the present study, endpoints including in vitro pollen performance (i.e., germination and tube growth) and lethality were used as assessments of nanotoxicity. Pollen was treated with 5-10 nm-sized Pd particles, similar to those released into the environment by catalytic car exhaust converters. Results showed Pd-nanoparticles altered kiwifruit pollen morphology and entered the grains more rapidly and to a greater extent than soluble Pd(II). At particulate Pd concentrations well below those of soluble Pd(II), pollen grains experienced rapid losses in endogenous calcium and pollen plasma membrane damage was induced. This resulted in severe inhibition and subsequent cessation of pollen tube emergence and elongation at particulate Pd concentrations as low as 0.4 mg L(-1). Particulate Pd emissions related to automobile traffic have been increasing and are accumulating in the environment. This could seriously jeopardize in vivo pollen function, with impacts at an ecosystem level.

Species-dependent chromium accumulation, lipid peroxidation, and glutathione levels in germinating kiwifruit pollen under Cr(III) and Cr(VI) stress.

The accumulation of chromium by germinating kiwifruit pollen appears to be significantly affected by Cr species, Cr concentration and calcium availability. Cr(III) accumulation always occurred in a linear manner while Cr(VI) uptake followed a logarithmic model. In the absence of exogenous calcium, Cr(III) accumulation was much higher than that of Cr(VI). It was observed that, as the Cr(III) concentration increased, there was a significant decrease in the endogenous calcium content of pollen, ultimately leading to complete calcium depletion after 90 min of incubation at 150 microM Cr(III). This loss of calcium could be responsible for the strong inhibition of tube emergence and growth following exposure of pollen to Cr(III). Indeed, when exogenous calcium was added to the kiwifruit pollen culture medium, significant growth recovery and reduced Cr(III) uptake occurred; the opposite was true in Cr(VI)-treatments. A significant rise in lipid peroxide production occurs in the presence of both Cr species; the effect was more pronounced following Cr(VI) exposure. Finally, glutathione pool dynamics appears to be differentially affected by chromium species and concentrations. In conclusion, results of the present study have provided important information regarding the different activity profiles of Cr(III) and Cr(VI) in relation to kiwifruit pollen performance, and have also demonstrated differences in some biochemical responses of pollen to metal stress.

Both trivalent and hexavalent chromium strongly alter in vitro germination and ultrastructure of kiwifruit pollen.

Due to its widespread industrial use, chromium is considered a dangerous environmental pollutant. It is known to inhibit plant growth and development. The present study provides the first evidence of the toxicity of this metal on the male haploid generation of a higher plant. Both Cr(III) and Cr(VI) species, supplied as CrCl(3) and CrO(3), respectively, exerted a strong dose-dependent inhibitory effect on kiwifruit pollen tube emergence and growth. Cr(III) resulted more effective than Cr(VI) in the 16-75microM interval; moreover, complete inhibition of germination was attained at much lower doses than Cr(VI). Also tube morphology was affected. While the plasma membrane was still undamaged in the large majority of the treated pollen grains, dramatic ultrastructural alterations were induced by chromium including chromatin condensation, swelling of mitochondria, cytoplasmic vacuolization, and perturbed arrangement of endoplasmic reticulum cisternae. Thus, it seems that the impact of the two chromium species on kiwifruit pollen may result in severe compromission of both essential structures and functions of the male gametophyte.

Uptake and toxicity of Cr(III) in celery seedlings.

The present study shows that in celery Cr(III) induces deleterious effects on seedling development and morphology, and a number of metabolic responses related to stress. Exogenous CrCl3 from 0.01 to 1 mM increasingly inhibited seed germination and hypocotyl elongation, or completely blocked it (10 mM), while the root apparatus was dramatically damaged even at the lowest dose. Seedlings took up exogenous Cr(III) in a dose-dependent manner, roots being the site of major metal accumulation; translocation towards the hypocotyl and cotyledonary leaves was also detected. Either total or chlorophyll a content was significantly reduced by chromium as low as 0.01 mM. A large accumulation of free and, to a lesser extent, conjugated polyamines occurred in all segments of treated plants. A dose-dependent relationship linking actual amounts of Cr(III) recovered in the entire seedling or organ and the respective polyamine titre was evidenced. Free putrescine, in particular, was the polyamine exhibiting the highest rate of increase, and cotyledonary leaves the organ where the major response occurred. A marked increase in ubiquitin-protein conjugates after Cr(III) treatment was also observed, particularly in roots. Thus, the study suggests for the first time a possible relationship between ubiquitination and Cr(III)-stress. The putative function of polyamines as a stress response, and the recruitment of the ubiquitin pathway to remove damaged or aberrant proteins which might have been produced in metal-treated seedlings are discussed.