Exogenously applied brassinosteroids (BRs) improve plant response to drought. However, many important aspects of this process, such as the potential differences caused by different developmental stages of analyzed organs at the beginning of drought, or by BR application before or during drought, remain still unexplored. The same applies for the response of different endogenous BRs belonging to the C27, C28-and C29- structural groups to drought and/or exogenous BRs. This study examines the physiological response of two different leaves (younger and older) of maize plants exposed to drought and treated with 24-epibrassinolide (epiBL), together with the contents of several C27, C28-and C29-BRs. Two timepoints of epiBL application (prior to and during drought) were utilized to ascertain how this could affect plant drought response and the contents of endogenous BRs. Marked differences in the contents of individual BRs between younger and older maize leaves were found: the younger leaves diverted their BR biosynthesis from C28-BRs to C29-BRs, probably at the very early biosynthetic steps, as the levels of C28-BR precursors were very low in these leaves, whereas C29-BR levels vere extremely high. Drought also apparently negatively affected contents of C28-BRs (particularly in the older leaves) and C29-BRs (particularly in the younger leaves) but not C27-BRs. The response of these two types of leaves to the combination of drought exposure and the application of exogenous epiBL differed in some aspects. The older leaves showed accelerated senescence under such conditions reflected in their reduced chlorophyll content and diminished efficiency of the primary photosynthetic processes. In contrast, the younger leaves of well-watered plants showed at first a reduction of proline levels in response to epiBL treatment, whereas in drought-stressed, epiBL pre-treated plants they were subsequently characterized by elevated amounts of proline. The contents of C29- and C27-BRs in plants treated with exogenous epiBL depended on the length of time between this treatment and the BR analysis regardless of plant water supply; they were more pronounced in plants subjected to the later epiBL treatment. The application of epiBL before or during drought did not result in any differences of plant response to this stressor.

• Keywords
• OJIP analysis, brassinosteroids, drought, endogenous content, exogenous application, leaf age, proline,
• Publication type
• Journal Article MeSH

BACKGROUND: Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. RESULTS: A high-throughput sample preparation method for liquid chromatography-tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. CONCLUSION: The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

• Keywords
• 3D printing, Evolutionarily distant plant species, High-throughput, In-tip microSPE, Liquid chromatography, Mass spectrometry, Miniaturisation, Plant hormones,
• Publication type
• Journal Article MeSH

Acclimation to salt stress in plants is regulated by complex signaling pathways involving endogenous phytohormones. The signaling role of salicylic acid (SA) in regulating crosstalk between endogenous plant growth regulators' levels was investigated in barley (Hordeum vulgare L. 'Ince'; 2n = 14) leaves and roots under salt stress. Salinity (150 and 300 mM NaCl) markedly reduced leaf relative water content (RWC), growth parameters, and leaf water potential (LWP), but increased proline levels in both vegetative organs. Exogenous SA treatment did not significantly affect salt-induced negative effects on RWC, LWP, and growth parameters but increased the leaf proline content of plants under 150 mM salt stress by 23.1%, suggesting that SA enhances the accumulation of proline, which acts as a compatible solute that helps preserve the leaf's water status under salt stress. Changes in endogenous phytohormone levels were also investigated to identify agents that may be involved in responses to increased salinity and exogenous SA. Salt stress strongly affected endogenous cytokinin (CK) levels in both vegetative organs, increasing the concentrations of CK free bases, ribosides, and nucleotides. Indole-3-acetic acid (IAA, auxin) levels were largely unaffected by salinity alone, especially in barley leaves, but SA strongly increased IAA levels in leaves at high salt concentration and suppressed salinity-induced reductions in IAA levels in roots. Salt stress also significantly increased abscisic acid (ABA) and ethylene levels; the magnitude of this increase was reduced by treatment with exogenous SA. Both salinity and SA treatment reduced jasmonic acid (JA) levels at 300 mM NaCl but had little effect at 150 mM NaCl, especially in leaves. These results indicate that under high salinity, SA has antagonistic effects on levels of ABA, JA, ethylene, and most CKs, as well as basic morphological and physiological parameters, but has a synergistic effect on IAA, which was well exhibited by principal component analysis (PCA).

• Keywords
• Hordeum vulgare, barley, phytohormones, salicylic acid, salt stress,
• Publication type
• Journal Article MeSH

Cross-talk between exogenous salicylic acid (SA) and endogenous phytohormone pathways affects the antioxidant defense system and its response to salt stress. The study presented here investigated the effects of SA treatment before and during salt stress on the levels of endogenous plant growth regulators in three barley cultivars with different salinity tolerances: Hordeum vulgare L. cvs. Akhisar (sensitive), Erginel (moderate), and Kalaycı (tolerant). The cultivars' relative leaf water contents, growth parameters, proline contents, chlorophyll a/b ratios, and lipid peroxidation levels were measured, along with the activities of enzymes involved in detoxifying reactive oxygen species (ROS) including superoxide-dismutase, peroxidase, catalase, ascorbate-peroxidase, and glutathione-reductase. In addition, levels of several endogenous phytohormones (indole-3-acetic-acid, cytokinins, abscisic acid, jasmonic acid, and ethylene) were measured. Barley is known to be more salt tolerant than related plant species. Accordingly, none of the studied cultivars exhibited changes in membrane lipid peroxidation under salt stress. However, they responded differently to salt-stress with respect to their accumulation of phytohormones and antioxidant enzyme activity. The strongest and weakest increases in ABA and proline accumulation were observed in Kalaycı and Akhisar, respectively, suggesting that salt-stress was more effectively managed in Kalaycı. The effects of exogenous SA treatment depended on both the timing of the treatment and the cultivar to which it was applied. In general, however, where SA helped mitigate salt stress, it appeared to do so by increasing ROS scavenging capacity and antioxidant enzyme activity. SA treatment also induced changes in phytohormone levels, presumably as a consequence of SA-phytohormone salt-stress cross-talk.

• MeSH
• Antioxidants metabolism   MeSH
• Biomass MeSH
• Time Factors MeSH
• Chlorophyll A metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Hordeum drug effects   growth & development   physiology   MeSH
• Salicylic Acid pharmacology   MeSH
• Thiobarbituric Acid Reactive Substances metabolism   MeSH
• Plant Leaves drug effects   physiology   MeSH
• Proline metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Plant Growth Regulators pharmacology   MeSH
• Salt Stress drug effects   MeSH
• Water MeSH
• Plant Shoots drug effects   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antioxidants MeSH
• Chlorophyll A MeSH
• Chlorophyll MeSH
• chlorophyll b MeSH Browser
• Salicylic Acid MeSH
• Thiobarbituric Acid Reactive Substances MeSH
• Proline MeSH
• Reactive Oxygen Species MeSH
• Plant Growth Regulators MeSH
• Water MeSH

Soil salinity is severely affecting crop productivity in many countries, particularly in the Mediterranean area. To evaluate early plant responses to increased salinity and characterize tolerance markers, three important Brassica crops - Chinese cabbage (Brassica rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata) and kale (B. oleracea var. acephala) were subjected to short-term (24 h) salt stress by exposing them to NaCl at concentrations of 50, 100, or 200 mM. Physiological (root growth, photosynthetic performance parameters, and Na+/K+ ratio) and biochemical parameters (proline content and lipid peroxidation as indicated by malondialdehyde, MDA, levels) in the plants' roots and leaves were then measured. Photosynthetic parameters such as the total performance index PItotal (describing the overall efficiency of PSI, PSII and the intersystem electron transport chain) appeared to be the most salinity-sensitive parameter and informative stress marker. This parameter was decreased more strongly in Chinese cabbage than in white cabbage and kale. It indicated that salinity reduced the capacity of the photosynthetic system for efficient energy conversion, particularly in Chinese cabbage. In parallel with the photosynthetic impairments, the Na+/K+ ratio was highest in Chinese cabbage leaves and lowest in kale leaves while kale root is able to keep high Na+/K+ ratio without a significant increase in MDA. Thus Na+/K+ ratio, high in root and low in leaves accompanying with low MDA level is an informative marker of salinity tolerance. The crops' tolerance was positively correlated with levels of the stress hormone abscisic acid (ABA) and negatively correlated with levels of jasmonic acid (JA), and jasmonoyl-L-isoleucine (JA-Ile). Furthermore, salinity induced contrasting changes in levels of the growth-promoting hormones brassinosteroids (BRs). The crop's tolerance was positively correlated with levels of BR precursor typhasterol while negatively with the active BR brassinolide. Principal Component Analysis revealed correlations in observed changes in phytohormones, biochemical, and physiological parameters. Overall, the results show that kale is the most tolerant of the three species and Chinese cabbage the most sensitive to salt stress, and provide holistic indications of the spectrum of tolerance mechanisms involved.

• Keywords
• Chinese cabbage, brassinosteroids, kale, photosynthetic performance, salinity stress, stress hormones, tolerance, white cabbage,
• Publication type
• Journal Article MeSH