Drought Protective Effects of Exogenous ABA and Kinetin on Lettuce: Sugar Content, Antioxidant Enzyme Activity, and Productivity.

Drought is an environmental stressor that significantly impacts plant growth and development. Comprehending the complexity of drought stress and water utilization in the context of plant growth and development holds significant importance for sustainable agriculture. The aim of this study was to evaluate the effect of exogenously applied phytohormones on lettuce (Lactuca sativa L.) sugar content profiles and antioxidant enzyme activity and productivity. Lettuce plants were grown under normal and drought conditions in a growth chamber with a photoperiod of 14/10 h (day/night). Kinetin and abscisic acid were applied separately and in combinations when the second leaf was fully expanded. The results showed that sugar accumulation and productivity of the pretreated plants under drought were significantly higher than the controls. The perspective offered by this work showed that growth-related and stress-related phytohormones significantly influenced plant sugar metabolism, metabolic profiles, and productivity, thus enabling the control of yield and quality.

Resurrection Plants-A Valuable Source of Natural Bioactive Compounds: From Word-of-Mouth to Scientifically Proven Sustainable Use.

Resurrection plant species are a group of higher plants whose vegetative tissues are able to withstand long periods of almost full desiccation and recover quickly upon rewatering. Apart from being a model system for studying desiccation tolerance, resurrection plant species appear to be a valuable source of metabolites, with various areas of application. A significant number of papers have been published in recent years with respect to the extraction and application of bioactive compounds from higher resurrection plant species in various test systems. Promising results have been obtained with respect to antioxidative and antiaging effects in various test systems, particularly regarding valuable anticancer effects in human cell lines. Here, we review the latest advances in the field and propose potential mechanisms of action of myconoside-a predominant secondary compound in the European members of the Gesneriaceae family. In addition, we shed light on the possibilities for the sustainable use of natural products derived from resurrection plants.

Antioxidative Defense, Suppressed Nitric Oxide Accumulation, and Synthesis of Protective Proteins in Roots and Leaves Contribute to the Desiccation Tolerance of the Resurrection Plant Haberlea rhodopensis.

The desiccation tolerance of plants relies on defense mechanisms that enable the protection of macromolecules, biological structures, and metabolism. Although the defense of leaf tissues exposed to solar irradiation is challenging, mechanisms that protect the viability of the roots, yet largely unexplored, are equally important for survival. Although the photosynthetic apparatus in leaves contributes to the generation of oxidative stress under drought stress, we hypothesized that oxidative stress and thus antioxidative defense is also predominant in the roots. Thus, we aimed for a comparative analysis of the protective mechanisms in leaves and roots during the desiccation of Haberlea rhodopensis. Consequently, a high content of non-enzymatic antioxidants and high activity of antioxidant enzymes together with the activation of specific isoenzymes were found in both leaves and roots during the final stages of desiccation of H. rhodopensis. Among others, catalase and glutathione reductase activity showed a similar tendency of changes in roots and leaves, whereas, unlike that in the leaves, superoxide dismutase activity was enhanced under severe but not under medium desiccation in roots. Nitric oxide accumulation in the root tips was found to be sensitive to water restriction but suppressed under severe desiccation. In addition to the antioxidative defense, desiccation induced an enhanced abundance of dehydrins, ELIPs, and sHSP 17.7 in leaves, but this was significantly better in roots. In contrast to leaf cells, starch remained in the cells of the central cylinder of desiccated roots. Taken together, protective compounds and antioxidative defense mechanisms are equally important in protecting the roots to survive desiccation. Since drought-induced damage to the root system fundamentally affects the survival of plants, a better understanding of root desiccation tolerance mechanisms is essential to compensate for the challenges of prolonged dry periods.

Reactivation of the Photosynthetic Apparatus of Resurrection Plant Haberlea rhodopensis during the Early Phase of Recovery from Drought- and Freezing-Induced Desiccation.

Haberlea rhodopensis is a unique desiccation-tolerant angiosperm that also survives winter frost. As, upon freezing temperatures, H. rhodopensis desiccates, the taxon is proposed to survive low temperature stress using its desiccation tolerance mechanisms. To reveal the validity of this hypothesis, we analyzed the structural alterations and organization of photosynthetic apparatus during the first hours of recovery after drought- and freezing-induced desiccation. The dynamics of the ultrastructure remodeling in the mesophyll cells and the restoration of the thylakoid membranes shared similarities independent of the reason for desiccation. Among the most obvious changes in thylakoid complexes, the proportion of the PSI-LHCII complex strongly increased around 70% relative water content (RWC), whereas the proportion of Lhc monomers decreased from the beginning of rehydration. We identified enhanced levels of cyt b6f complex proteins that contributed to the enhanced electron flow. The high abundance of proteins related to excitation energy dissipation, PsbS, Lhcb5, Lhcb6 and ELIPs, together with the increased content of dehydrins contributed to the preservation of cellular integrity. ELIP expression was maintained at high levels up to 9 h into recovery. Although the recovery processes from drought- and freezing-induced desiccation were found to be similar in progress and time scale, slight variations indicate that they are not identical.

Antioxidant Defense during Recovery of Resurrection Plant Haberlea rhodopensis from Drought- and Freezing-Induced Desiccation.

In this study, the contribution of nonenzymatic (ascorbate, glutathione) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase) in the first hours of recovery of the resurrection plant Haberlea rhodopensis from drought- and freezing-induced desiccation was assessed. The initial stage of recovery after desiccation is critical for plants, but less investigated. To better understand the alterations in the activity of antioxidant enzymes, their isoenzyme patterns were determined. Our results showed that ascorbate content remained high during the first 9 h of rehydration of desiccated plants and declined when the leaves' water content significantly increased. The glutathione content remained high at the first hour of rehydration and then strongly decreased. The changes in ascorbate and glutathione content during recovery from drought- and freezing-induced desiccation showed great similarity. At the beginning of rehydration (1-5 h), the activities of antioxidant enzymes were significantly increased or remained as in dry plants. During 7-24 h of rehydration, certain differences in the enzymatic responses between the two plant groups were registered. The maintenance of a high antioxidant activity and upregulation of individual enzyme isoforms indicated their essential role in protecting plants from oxidative damage during the onset of recovery.

Ethylene signaling in salt-stressed Arabidopsis thaliana ein2-1 and ctr1-1 mutants - A dissection of molecular mechanisms involved in acclimation.

To pinpoint ethylene-mediated molecular mechanisms involved in the adaptive response to salt stress we conducted a comparative study of Arabidopsis thaliana wild type (Col-0), ethylene insensitive (ein2-1), and constitutive signaling (ctr1-1) mutant plants. Reduced germination and survival rates were observed in ein2-1 plants at increasing NaCl concentrations. By contrast, ctr1-1 mutation conferred salt stress tolerance during early vegetative development, corroborating earlier studies. Аll genotypes experienced strong stress as evidenced by the accumulation of reactive oxygen species (ROS) and increased membrane lipid peroxidation. However, the isoenzyme profiles of ROS scavenging enzymes demonstrated a higher peroxidase (POX) activity in ctr1-1 individuals under control and salt stress conditions. A markedly elevated free L-Proline (L-Pro) content was detected in the ethylene constitutive mutant. This coincided with the increased levels of Delta-1-Pyrroline-5-Carboxylate Synthase (P5CS) which is the rate-limiting enzyme from the proline biosynthetic pathway. A stabilized upregulation of a stress-induced P5CS1 splice variant was observed in the ctr1-1 background, which was not documented in the ethylene insensitive mutant ein2-1. Transcript profiling of the major SALT OVERLY SENSITIVE (SOS) pathway players (SOS1, SOS2, and SOS3) revealed altered gene expression in the organs of the ethylene signaling mutants. Overall suppressed SOS expression was observed in the ein2-1 mutants while only the SOS transcript profiles in the ctr1-1 roots were similar to the wild type. Altogether, we provide experimental evidence for ethylene-mediated molecular mechanisms implicated in the acclimation response to salt stress in Arabidopsis, which operate mainly through the regulation of free proline accumulation and enhanced ROS scavenging.

The role of antioxidant defense in freezing tolerance of resurrection plant Haberlea rhodopensis.

Haberlea rhodopensis Friv. is unique with its ability to survive two extreme environmental stresses-desiccation to air-dry state and subzero temperatures. In contrast to desiccation tolerance, the mechanisms of freezing tolerance of resurrection plants are scarcely investigated. In the present study, the role of antioxidant defense in the acquisition of cold acclimation and freezing tolerance in this resurrection plant was investigated comparing the results of two sets of experiments-short term freezing stress after cold acclimation in controlled conditions and long term freezing stress as a part of seasonal temperature fluctuations in an outdoor ex situ experiment. Significant enhancement in flavonoids and anthocyanin content was observed only as a result of freezing-induced desiccation. The total amount of polyphenols increased upon cold acclimation and it was similar to the control in post freezing stress and freezing-induced desiccation. The main role of phenylethanoid glucoside, myconoside and hispidulin 8-C-(2-O-syringoyl-b-glucopyranoside) in cold acclimation and freezing tolerance was elucidated. The treatments under controlled conditions in a growth chamber showed enhancement in antioxidant enzymes activity upon cold acclimation but it declined after subsequent exposure to -10 °C. Although it varied under ex situ conditions, the activity of antioxidant enzymes was high, indicating their important role in overcoming oxidative stress under all treatments. In addition, the activity of specific isoenzymes was upregulated as compared to the control plants, which could be more useful for stress counteraction compared to changes in the total enzyme activity, due to the action of these isoforms in the specific cellular compartments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00998-0.

Proteomics, Holm Oak (Quercus ilex L.) and Other Recalcitrant and Orphan Forest Tree Species: How do They See Each Other?

Proteomics has had a big impact on plant biology, considered as a valuable tool for several forest species, such as Quercus, Pines, Poplars, and Eucalyptus. This review assesses the potential and limitations of the proteomics approaches and is focused on Quercus ilex as a model species and other forest tree species. Proteomics has been used with Q. ilex since 2003 with the main aim of examining natural variability, developmental processes, and responses to biotic and abiotic stresses as in other species of the genus Quercus or Pinus. As with the progress in techniques in proteomics in other plant species, the research in Q. ilex moved from 2-DE based strategy to the latest gel-free shotgun workflows. Experimental design, protein extraction, mass spectrometric analysis, confidence levels of qualitative and quantitative proteomics data, and their interpretation are a true challenge with relation to forest tree species due to their extreme orphan and recalcitrant (non-orthodox) nature. Implementing a systems biology approach, it is time to validate proteomics data using complementary techniques and integrate it with the -omics and classical approaches. The full potential of the protein field in plant research is quite far from being entirely exploited. However, despite the methodological limitations present in proteomics, there is no doubt that this discipline has contributed to deeper knowledge of plant biology and, currently, is increasingly employed for translational purposes.

Holm oak proteomic response to water limitation at seedling establishment stage reveals specific changes in different plant parts as well as interaction between roots and cotyledons.

Quercus ilex is a dominant tree species in the Mediterranean region with double economic and ecological importance and increasing use in reforestation. Seedling establishment is extremely vulnerable to environmental stresses, particularly drought. A time course study on physiological and proteomic response of holm oak to water limitation stress and recovery during early heterotrophic growth is reported. Applied stress led to diminution in plant water content and root growth, oxidative stress in roots and some alterations in the anti-oxidative protection. Plant parts differed substantially in soluble sugar and free phenolic content, and in their changes during stress and recovery. Proteomic response in holm oak roots and cotyledons was estimated using combined 1-DE/2-DE approach and protein identification by MALDI TOF-TOF PMF and MS/MS. A total of 127 differentially abundant protein species (DAPs) were identified. DAPs related to starch metabolism, lipid to sugar conversion, reserve proteins and their mobilization were typical for cotyledons. DAPs in roots were involved in sugar utilization, secondary metabolism and defense, including pathogenesis related proteins from PR-5 and PR-10 families. Results emphasize specific proteome signatures of separate plant parts as well as importance of sink-source interaction between root and cotyledon in the time course of stress and in recovery.

A year (2014-2015) of plants in Proteomics journal. Progress in wet and dry methodologies, moving from protein catalogs, and the view of classic plant biochemists.

The present review is an update of the previous one published in Proteomics 2015 Reviews special issue [Jorrin-Novo, J. V. et al., Proteomics 2015, 15, 1089-1112] covering the July 2014-2015 period. It has been written on the bases of the publications that appeared in Proteomics journal during that period and the most relevant ones that have been published in other high-impact journals. Methodological advances and the contribution of the field to the knowledge of plant biology processes and its translation to agroforestry and environmental sectors will be discussed. This review has been organized in four blocks, with a starting general introduction (literature survey) followed by sections focusing on the methodology (in vitro, in vivo, wet, and dry), proteomics integration with other approaches (systems biology and proteogenomics), biological information, and knowledge (cell communication, receptors, and signaling), ending with a brief mention of some other biological and translational topics to which proteomics has made some contribution.

2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage.

Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress.

Proteolytic activity and cysteine protease expression in wheat leaves under severe soil drought and recovery.

The involvement of acidic proteases in soil drought response of winter wheat (Triticum aestivum L.) at seedling stage in three cultivars differing in water stress tolerance was studied. Withholding irrigation for seven days resulted in severe drought stress corresponding to 60% leaf water deficit. Stressed plants were recovered by providing optimal water supply for 3 days. Reversible changes in leaf pigment and protein content were registered, being least expressed in the drought-resistant cultivar Katya. Protein loss was inversely related to the increase in total proteolytic activity at pH 5 and in aminopeptidase activity at pH 7. Quantitative differences among the cultivars were established only for azocaseinolytic activity (pH 5). The drought-resistant cultivar (Katya) showed relatively little increase in acid protease activity whereas the highest values of this activity were detected in cultivar Pobeda. In-gel staining for cysteine-activated proteases revealed four to five separate activity bands. The upper band, specifically inhibited by E-64, was raised at severe drought. Transcript abundance of two wheat cysteine proteases -Ta.61026 putative thiol protease, and WCP2 peptidase of papain type was analyzed by RT-PCR. Gene expression of the cysteine proteases under study was suppressed in the drought-tolerant cultivar, while in the less resistant ones it remained unchanged or augmented. The results suggest that lower proteolytic activity and decreased expression of certain cysteine protease genes under water deficit during early developmental stage could be regarded as an indicator for drought resistance of winter wheat cultivars.

Variety-specific response of wheat (Triticum aestivum L.) leaf mitochondria to drought stress.

The main objective of the present work was to examine leaf respiratory responses to dehydration and subsequent recovery in three varieties of winter wheat (Triticum aestivum L.) known to differ in their level of drought tolerance. Under dehydration, both total respiration and salicylhydroxamic acid (SHAM)-resistant cytochrome (Cyt) pathway respiration by leaf segments decreased significantly compared with well-watered plants. This decrease was more pronounced in the drought-sensitive Sadovo and Prelom genotypes. In contrast, the KCN-resistant SHAM-sensitive alternative (Alt) pathway became increasingly engaged, and accounted for about 80% of the total respiration. In the drought-tolerant Katya variety, increased contribution of the Alt pathway was accompanied by a slight decrease in Cyt pathway activity. Respiration of isolated leaf mitochondria also showed a variety-specific drought response. Mitochondria from drought-sensitive genotypes had low oxidative phosphorylation efficiency after dehydration and rewatering, whereas the drought-tolerant Katya mitochondria showed higher phosphorylation rates. Morphometric analysis of leaf ultrastructure revealed that mitochondria occupied approximately 7% of the cell area in control plants. Under dehydration, in the drought-sensitive varieties this area was reduced to about 2.0%, whereas in Katya it was around 6.0%. The results are discussed in terms of possible mechanisms underlying variety-specific mitochondrial responses to dehydration.