Hormonal response to recurrent seasonal stress in coastal and mountain scabiouses growing in their natural habitat: linking ABA and jasmonates with photoprotection.

Plant species distribution across ecosystems is influenced by multiple environmental factors, and recurrent seasonal stress events can act as natural selection agents for specific plant traits and limit species distribution. For that, studies aiming at understanding how environmental constraints affect adaptive mechanisms of taxonomically closely related species are of great interest. We chose two Scabiosa species inhabiting contrasting environments: the coastal scabious S. atropurpurea, typically coping with hot-dry summers in a Mediterranean climate, and the mountain scabious S. columbaria facing cold winters in an oceanic climate. A set of functional traits was examined to assess plant performance in these congeneric species from contrasting natural habitats. Both S. atropurpurea and S. columbaria appeared to be perfectly adapted to their environment in terms of adjustments in stomatal closure, CO2 assimilation rate and water use efficiency over the seasons. However, an unexpected dry period during winter followed by the typical Mediterranean hot-dry summer forced S. atropurpurea plants to deploy a set of photoprotective responses during summer. Aside from reductions in leaf water content and Fv/Fm, photoprotective molecules (carotenoids, α-tocopherol and anthocyanins) per unit of chlorophyll increased, mostly as a consequence of a severe chlorophyll loss. The profiling of stress-related hormones (ABA, salicylic acid and jasmonates) revealed associations between ABA and the bioactive jasmonoyl-isoleucine with the underlying photoprotective response to recurrent seasonal stress in S. atropurpurea. We conclude that jasmonates may be used together with ABA as a functional trait that may, at least in part, help understand plant responses to recurrent seasonal stress in the current frame of global climate change.

Malondialdehyde Assays in Higher Plants.

Malondialdehyde is a three-carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation widely used as a marker of the extent of lipid peroxidation in plants. There are several methodological approaches to quantify malondialdehyde contents in higher plants, ranging from the simplest, cheapest, and quickest spectrophotometric approaches to the more complex ones using tandem mass spectrometry. This chapter summarizes the advantages and limitations of approaches followed and provides brief protocols with some tips to facilitate the selection of the best method for each experimental condition and application.

Desiccation tolerance in the resurrection plant Barbacenia graminifolia involves changes in redox metabolism and carotenoid oxidation.

Desiccation tolerance in vegetative tissues enables resurrection plants to remain quiescent under severe drought and rapidly recover full metabolism once water becomes available. Barbacenia graminifolia is a resurrection plant that occurs at high altitudes, typically growing on rock slits, exposed to high irradiance and limited water availability. We analyzed the levels of reactive oxygen species (ROS) and antioxidants, carotenoids and its cleavage products, and stress-related phytohormones in fully hydrated, dehydrated, and rehydrated leaves of B. graminifolia. This species exhibited a precise adjustment of its antioxidant metabolism to desiccation. Our results indicate that this adjustment is associated with enhanced carotenoid and apocarotenoids, α-tocopherol and compounds of ascorbate-glutathione cycle. While α-carotene and lutein increased in dried-leaves suggesting effective protection of the light-harvesting complexes, the decrease in ß-carotene was accompanied of 10.2-fold increase in the content of ß-cyclocitral, an apocarotenoid implicated in the regulation of abiotic stresses, compared to hydrated plants. The principal component analysis showed that dehydrated plants at 30 days formed a separate cluster from both hydrated and dehydrated plants for up to 15 days. This regulation might be part of the protective metabolic strategies employed by this resurrection plant to survive water scarcity in its inhospitable habitat.

Abscisic acid triggers vitamin E accumulation by transient transcript activation of VTE5 and VTE6 in sweet cherry fruits.

Tocopherols are lipophilic antioxidants known as vitamin E and synthesized from the condensation of two metabolic pathways leading to the formation of homogentisate and phytyl diphosphate. While homogentisate is derived from tyrosine metabolism, phytyl diphosphate may be formed from geranylgeranyl diphosphate or phytol recycling from chlorophyll degradation. Here, we hypothesized that abscisic acid (ABA) could induce tocopherol biosynthesis in sweet cherries by modifying the expression of genes involved in vitamin E biosynthesis, including those from the phytol recycling pathway. Hence, the expression of key tocopherol biosynthesis genes was determined together with vitamin E and chlorophyll contents during the natural development of sweet cherries on the tree. Moreover, the effects of exogenously applied ABA on the expression of key tocopherol biosynthesis genes were also investigated during on-tree fruit development, and tocopherols and chlorophylls contents were analyzed. Results showed that the expression of tocopherol biosynthesis genes, including VTE5, VTE6, HPPD and HPT showed contrasting patterns of variation, but in all cases, increased by 2- and 3-fold over time during fruit de-greening. This was not the case for GGDR and VTE4, the first showing constitutive expression during fruit development and the second with marked down-regulation at ripening onset. Furthermore, exogenous ABA stimulated the production of both α- and γ-tocopherols by 60% and 30%, respectively, promoted chlorophyll degradation and significantly enhanced VTE5 and VTE6 expression, and also that of HPPD and VTE4, altogether increasing total tocopherol accumulation. In conclusion, ABA increases promote the transcription of phytol recycling enzymes, which may contribute to vitamin E biosynthesis during fruit development in stone fruits like sweet cherries.

Ancient trees are essential elements for high-mountain forest conservation: Linking the longevity of trees to their ecological function.

Mature forests and their extremely old trees are rare and threatened ancient vestiges in remote European high-mountain regions. Here, we analyze the role that extremely long-living trees have in mature forests biodiversity in relation to their singular traits underlying longevity. Tree size and age determine relative growth rates, bud abortion, and the water status of long-living trees. The oldest trees suffer indefectible age-related constraints but possess singular evolutionary traits defined by fitness adaptation, modular autonomy, and a resilient metabolism that allow them to have irreplaceable roles in the ecosystem as biodiversity anchors of vulnerable lichen species like Letharia vulpina. We suggest that the role of ancient trees as unique biodiversity reservoirs is linked to their singular physiological traits associated with longevity. The set of evolutionarily plastic tools that can only be provided by centuries or millennia of longevity helps the oldest trees of mature forests drive singular ecological relationships that are irreplaceable and necessary for ecosystem dynamics.

Fruit quality in organic and conventional farming: advantages and limitations.

Fruit quality is essential for nutrition and human health and needs urgent attention in current agricultural practices. Organic farming is not as productive as conventional agriculture, but it can provide higher quality in some fruit crops, thanks to the absence of synthetic fertilizers and pesticides, enhanced pollination, and the reduction of protection treatments, hence boosting antioxidant compound production. Although organic farming does not always provide healthier food than conventional farming, some lessons from organic farming can be extrapolated to new sustainable production models. Exploiting natural resources and an adequate knowledge transfer will undoubtedly help improve the quality of climacteric and nonclimacteric fruits in new agricultural systems.

Mother trees, altruistic fungi, and the perils of plant personification.

There are growing doubts about the true role of the common mycorrhizal networks (CMN or wood wide web) connecting the roots of trees in forests. We question the claims of a substantial carbon transfer from 'mother trees' to their offspring and nearby seedlings through the CMN. Recent reviews show that evidence for the 'mother tree concept' is inconclusive or absent. The origin of this concept seems to stem from a desire to humanize plant life but can lead to misunderstandings and false interpretations and may eventually harm rather than help the commendable cause of preserving forests. Two recent books serve as examples: The Hidden Life of Trees and Finding the Mother Tree.

Achieving the impossible: prevention and eradication of invasive plants in Mediterranean-type ecosystems.

An in-depth analysis of the mechanistic processes underlying the evolution and ecophysiology of typical invasive plants such as Carpobrotus spp., Acacia spp., Agave spp., and Opuntia spp. in Mediterranean-type ecosystems shows very sophisticated, complex, and efficient strategies for invasion success, particularly in fragmented habitats. Propagule pressure at both geographical and temporal scales and the establishment of long-term seed banks are determinant for invasion success. A two-sword strategy based on long-term prevention and eradication is proposed for the management of Mediterranean-type ecosystems. Eradication of invasive plants in Mediterranean-type ecosystems appears to be extremely difficult nowadays and, at least for some invaders like Carpobrotus spp., long-term approaches that ultimately culminate in the elimination of seed banks is the only path for success.

Hormonal involvement in boosting the vegetative vigour underlying caffeine-related improvements in lentil production.

Previous studies have shown that caffeine (1,3,7-trimethylxanthine) has some potential for its use as a biostimulant ingredient for boosting lentil production at suboptimal temperatures. However, some limitations to its use include its potential side effects as an emerging contaminant and the current lack of knowledge of its mechanism of action. Here, we aimed to study the mechanisms underlying improved lentil production upon caffeine application. Greenhouse-grown plants treated with caffeine (at 10-5 M, 10-4 M, and 10-3 M) were compared to an untreated, control treatment, and both reproductive and vegetative vigour were evaluated in parallel with endogenous foliar concentrations of phytohormones, including both stress and growth-related hormones. Results showed an enhanced lentil production at the highest caffeine concentration (10-3 M) which might be attributed, at least in part, to a greater vegetative vigour. The hormonal profiling revealed a dual effect. Firstly, there was a specific increase in jasmonoyl-isoleucine (JA-Ile) in the short term, which may provide a priming effect. Secondly, abscisic acid (ABA) content kept at low levels and the active cytokinin (CK) isopentenyl adenine (2-iP) increased and persisted at high levels throughout the reproductive stage. Cytokinin-mediated effects on growth, and more specifically the high CK/ABA ratios in leaves, appeared to mediate caffeine-related effects in boosting vegetative vigour. In conclusion, caffeine emerges as a compelling alkaloid for integration into biostimulant formulations due to its favorable effect in boosting lentil production through an improvement of vegetative vigour. These outcomes appear to be modulated by phytohormones, most notably jasmonates, priming plants for improved performance under suboptimal temperatures, and cytokinins, alongside ABA and its associated ratios, collectively enhancing plant growth and reproductive vigour in challenging conditions.

Parasitic plant-host interaction between the holoparasite Cytinus hypocistis and the shrub Cistus albidus in their natural Mediterranean habitat: local and systemic hormonal effects.

Mediterranean-type ecosystems provide a unique opportunity to study parasitic plant-host interactions, such as the relationship between the dominant shrub Cistus albidus L. and the root holoparasitic plant Cytinus hypocistis L. We examined this interaction (i) locally, by measuring the hormonal profiling of the interaction zone between the holoparasitic plant and the host, and (ii) systemically, by examining the hormonal profiling and physiological status of leaves from infested and uninfested plants. Furthermore, we explored how temporal variation (seasonal effects) and geographical location influenced the systemic hormonal and physiological response of leaves. Results shed light on tissue-related variations in hormones, suggesting the parasite exerted a sink effect, mainly influenced by cytokinins. Jasmonates triggered a defense response in leaves, far from the infestation point, and both jasmonates and abscisic acid (ABA) appeared to be involved in the tolerance to holoparasitism when plants were simultaneously challenged with summer drought. Parasitism did not have any major negative impact on the host, as indicated by physiological stress markers in leaves, thus indicating a high tolerance of the shrub C. albidus to the root holoparasitic plant C. hypocistis. Rather, parasitism seemed to exert a priming-like effect and some compensatory effects were observed (increased chlorophyll contents) in the host under mild climatic conditions. We conclude that (i) cytokinins, jasmonates and ABA play a role at the local and systemic levels in the response of C. albidus to the biotic stress caused by C. hypocistis, and that (ii) seasonal changes in environmental conditions and geographical location may impact holoparasitic plant-host interactions in the field, modulating the physiological response.

Organ-specific responses during acclimation of mycorrhizal and non-mycorrhizal tomato plants to a mild water stress reveal differential local and systemic hormonal and nutritional adjustments.

MAIN CONCLUSION: Tomato plant acclimation to a mild water stress implied tissue-specific hormonal and nutrient adjustments, being the root one of the main modulators of this response. Phytohormones are key regulators of plant acclimation to water stress. However, it is not yet clear if these hormonal responses follow specific patterns depending on the plant tissue. In this study, we evaluated the organ-specific physiological and hormonal responses to a 14 day-long mild water stress in tomato plants (Solanum lycopersicum cv. Moneymaker) in the presence or absence of the arbuscular mycorrhizal fungus Rhizoglomus irregulare, a frequently used microorganism in agriculture. Several physiological, production, and nutritional parameters were evaluated throughout the experiments. Additionally, endogenous hormone levels in roots, leaves, and fruits at different developmental stages were quantified by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Water deficit drastically reduced shoot growth, while it did not affect fruit production. In contrast, fruit production was enhanced by mycorrhization regardless of the water treatment. The main tissue affected by water stress was the root system, where huge rearrangements in different nutrients and stress-related and growth hormones took place. Abscisic acid content increased in every tissue and fruit developmental stage, suggesting a systemic response to drought. On the other hand, jasmonate and cytokinin levels were generally reduced upon water stress, although this response was dependent on the tissue and the hormonal form. Finally, mycorrhization improved plant nutritional status content of certain macro and microelements, specially at the roots and ripe fruits, while it affected jasmonate response in the roots. Altogether, our results suggest a complex response to drought that consists in systemic and local combined hormonal and nutrient responses.

A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance.

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood. Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.

Acclimation to a combination of water deficit and nutrient deprivation through simultaneous increases in abscisic acid and bioactive jasmonates in the succulent plant Sempervivum tectorum L.

Activation of hormonal responses defines the drought acclimation ability of plants and may condition their survival. However, aside ABA, little is known about the possible contribution of other phytohormones, such as jasmonates and salicylates, in the response of CAM plants to water deficit. Here, we aimed to study the physiological mechanisms underlying the stress tolerance of house leek (Sempervivum tectorum L.), a CAM plant adapted to survive harsh environments, to a combination of water deficit and nutrient deprivation. We exposed plants to the combination of these two abiotic stresses by withholding nutrient solution for 10 weeks and monitored their physiological response every two weeks by measuring various stress makers together with the accumulation of stress-related phytohormones and photoprotective molecules, such as tocopherols (vitamin E). Results showed that ABA content increased by 4.2-fold after four weeks of water deficit to keep later constant up to 10 weeks of stress, variations that occurred concomitantly with reductions in the relative leaf water content, which decreased by up to 20% only. The bioactive jasmonate, jasmonoyl-isoleucine was the other stress-related phytohormone that simultaneously increased under stress together with ABA. While contents of salicylic acid and the jasmonoyl-isoleucine precursors, 12-oxo-phytodienoic acid and jasmonic acid decreased with water deficit, those of jasmonoyl-isoleucine increased 3.6-fold at four weeks of stress. The contents of ABA and jasmonoyl-isoleucine correlated positively between them and with the content of α-tocopherol per unit of chlorophyll, thus suggesting a photoprotective activation role. It is concluded that S. tectorum not only withstands a combination of water deficit and nutrient deprivation for 10 weeks without any symptom of damage but also activates effective defense strategies through the simultaneous accumulation of ABA and the bioactive jasmonate form, jasmonoyl-isoleucine.

α-Tocopherol in chloroplasts: Nothing more than an antioxidant?

Among the eight forms of vitamin E, only tocopherols are essential compounds that are distributed throughout the entire plant kingdom, with α-tocopherol being the most predominant form in photosynthetic tissues. At the cellular level, α-tocopherol is of special relevance inside the chloroplast, where it eliminates singlet oxygen and modulates lipid peroxidation. This is of utmost relevance since tocopherols are the only antioxidants that counteract lipid peroxidation. Moreover, at the whole-plant level, α-tocopherol appears to modulate several physiological processes from germination to senescence. The antioxidant role of α-tocopherol at the cellular level can have profound effects at the whole-plant level, including the modulation of physiological processes that are apparently not related to redox processes and could be considered non-antioxidant functions. Here, we discuss whether non-antioxidant functions of α-tocopherol at the whole-plant level are mediated by its antioxidant role in chloroplasts and the regulation of redox processes at the cellular level.

Improved production and quality of peppers irrigated with regenerated water by the application of 24-epibrassinolide.

Water shortage for crop irrigation is reducing agricultural production worldwide and the use of sewage treatment plant (STP) water to irrigate horticultural fields is a solution to avoid the use of drinkable water in agriculture. In this study, two different genotypes of pepper (Red Cherry Small and Italian green) were irrigated with STP water, as an alternative to potable water. Moreover, the foliar application of a molecule with biostimulant properties (24-epibrassinolide; EBR) was tested as a strategy to ameliorate the production and quality of fruits. Both genotypes differed on their tolerance to the suffered oxidative stress due to their different salinity tolerance, but fruit commercial weight was reduced by 49% on the salt sensitive and by 37% on the salt tolerant. Moreover, ascorbic acid was also decreased by 37% after STP water irrigation in the Red Cherry Small peppers. However, EBR applications alleviated STP watering stress effects improving pepper plants fruit production and quality parameters, such as ascorbic acid and capsaicinoids. These results have important economic and environmental relevance to overcome present and future water deficiencies in the agricultural sector derived from climate change, guaranteeing the maintenance of production in peppers irrigated with STP water for a more sustainable agriculture following relevant circular economy actions.

Abscisic Acid Synthesis and Signaling during the Ripening of Raspberry (Rubus idaeus 'Heritage') Fruit.

The raspberry (Rubus idaeus L.) fruit is characterized by its richness in functional molecules and high nutritional value, but the high rate of fruit softening limits its quality during postharvest. Raspberry drupelets have a particular ripening regulation, depending partially on the effect of ethylene produced from the receptacle. However, the possible role of abscisic acid (ABA) in the modulation of quality parameters during the ripening of raspberry is unclear. This study characterized the fruit quality-associated parameters and hormonal contents during fruit development in two seasons. The quality parameters showed typical changes during ripening: a drastic loss of firmness, increase in soluble solids content, loss of acidity, and turning to a red color from the large green stage to fully ripe fruit in both seasons. A significant increase in the ABA content was observed during the ripening of drupelets and receptacles, with the higher content in the receptacle of ripe and overripe stages compared to the large green stage. Moreover, identification of ABA biosynthesis-(9-cis-epoxycarotenoid dioxygenase/NCED) and ABA receptor-related genes (PYRs-like receptors) showed three genes encoding RiNCEDs and nine genes for RiPYLs. The expression level of these genes increased from the large green stage to the full-ripe stage, specifically characterized by a higher expression of RiNCED1 in the receptacle tissue. This study reports a consistent concomitant increase in the ABA content and the expression of RiNCED1, RiPYL1, and RiPYL8 during the ripening of the raspberry fruit, thus supporting the role for ABA signaling in drupelets.

Photo-oxidative stress leading to oxylipin accumulation may trigger drought-induced leaf abscission in olive trees.

Olive trees shed their leaves under severe drought as a defence mechanism. Foliar drought-induced abscission is a programmed process occurring in a differentiated cell layer at the base of the petiole. Considering the antioxidant properties of vitamin E and its interplay with lipid peroxidation-derived jasmonates in abiotic stress responses, we hypothesized about their possible role in abscission signaling by forming a jasmonates-increasing basipetal gradient along the leaf up to the abscission zone. We exposed young olives trees to water withdrawal for 21 days, after which five leaf sections, from the apex to the petiole, were sampled on both attached and detached leaves of irrigated and water-stressed trees. We found that prolonged drought stress resulted in a sharp reduction in the photosystem II efficiency, chlorophyll and vitamin E contents in leaves, leading to photo-oxidative stress, reflected by the increase in lipid peroxidation. In addition, the content of chloroplast-derived oxylipins and phytohormones, such as jasmonoyl-isoleucine and salicylic acid, increased. At the same time, α-tocopherol decreased in the petiole of water-stressed attached leaves, suggesting a conditioning for the abscission process to occur. Although no differences were observed in petioles from attached and detached leaves, the dropped ones showed higher oxidative stress in the leaf blade. It is concluded that redox signaling through oxylipins accumulation may trigger leaf abscission in drought-stressed olive trees. Mechanical stress is, however, additionally needed to execute leaf abscission once the abscission zone is properly conditioned.

Differential tissue-specific accumulation and function of tocochromanols in grape berries.

Grape berries have been extensively studied in terms of antioxidant characterization, specifically in anthocyanin, total phenol, and tannin accumulation. However, very little is known about vitamin E composition and contents in this fruit. Aiming to examine the function of vitamin E during grape berries ripening, tocochromanol contents and composition were evaluated in berries and leaves of grapevines (Vitis vinifera L. cv. Merlot), from just before veraison to commercial harvest. We also determined the time-course evolution of tocochromanol accumulation in various fruit tissues, including the skin, pulp, and seeds, and measured the extent of primary and secondary lipid peroxidation, as well as fruit technological maturity parameters. Vitamin E accumulated at higher levels in leaves than in fruits, although the tissue-specific evaluation of tocochromanol contents revealed that berry skin is also rich in α-tocopherol whereas tocotrienols were present in seeds only. α-Tocopherol content decreased during ripening, more specifically in the skin, and it was accompanied by an increase in the extent of lipid peroxidation. Contents and variations in the levels of α-tocopherol, but not those of the other tocochromanols, were inversely related to changes in lipid peroxidation during fruit ripening, as indicated by tissue-specific variations in malondialdehyde contents. In conclusion, α-tocopherol is more abundant in leaves than fruit, yet it apears to exert a role in the modulation of the extent of lipid peroxidation in grape berries, more specifically in the skin, where α-tocopherol depletion and malondialdehyde accumulation may be related to an adequate progression of fruit ripening.

Experimental approaches in studying active biomolecules modulating fruit ripening: Melatonin as a case study.

Phytohormones are naturally occurring small organic molecules found at low concentrations in plants. They perform essential functions in growth and developmental processes, from organ initiation to senescence, including fruit ripening. These regulatory molecules are studied using different experimental approaches, such as performing exogenous applications, evaluating endogenous levels, and/or obtaining genetically modified lines. Here, we discuss the advantages and limitations of current experimental approaches used to study active biomolecules modulating fruit ripening, focusing on melatonin. Although melatonin has been implicated in fruit ripening in several model fruit crops, current knowledge is affected by the different experimental approaches used, which have given different and sometimes even contradictory results. The methods of application and the doses used have produced different results in studies based on exogenous applications, while different measurement methods and ways of expressing results explain most of the variability in studies using correlative analyses. Furthermore, studies on genetically modified crops have focused on tomato (Solanum lycopersicum L.) plants only. However, TILLING and CRISPR methodologies are becoming essential tools to complement the results from the experimental approaches described above. This will not only help the scientific community better understand the role of melatonin in modulating fruit ripening, but it will also help develop technological advances to improve fruit yield and quality in major crops. The combination of various experimental approaches will undoubtedly lead to a complete understanding of the function of melatonin in fruit ripening in the near future, so that this knowledge can be effectively transferred to the field.