Contents of endogenous brassinosteroids and the response to drought and/or exogenously applied 24-epibrassinolide in two different maize leaves.

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.

Constitutive water-conserving mechanisms are correlated with the terminal drought tolerance of pearl millet [Pennisetum glaucum (L.) R. Br.].

Pearl millet, a key staple crop of the semi-arid tropics, is mostly grown in water-limited conditions, and improving its performance depends on how genotypes manage limited water resources. This study investigates whether the control of water loss under non-limiting water conditions is involved in the terminal drought tolerance of pearl millet. Two pairs of tolerant x sensitive pearl millet genotypes, PRLT 2/89-33-H77/833-2 and 863B-P2-ICMB 841-P3, and near-isogenic lines (NILs), introgressed with a terminal drought tolerance quantitative trait locus (QTL) from the donor parent PRLT 2/89-33 into H77/833-2 (NILs-QTL), were tested. Upon exposure to water deficit, transpiration began to decline at lower fractions of transpirable soil water (FTSW) in tolerant than in sensitive genotypes, and NILs-QTL followed the pattern of the tolerant parents. The transpiration rate (Tr, in g water loss cm(-2) d(-1)) under well-watered conditions was lower in tolerant than in sensitive parental genotypes, and the Tr of NILs-QTL followed the pattern of the tolerant parents. In addition, Tr measured in detached leaves (g water loss cm(-2) h(-1)) from field-grown plants of the parental lines showed lower Tr values in tolerant parents. Defoliation led to an increase in Tr that was higher in sensitive than in tolerant genotypes. The differences in Tr between genotypes was not related to the stomatal density. These results demonstrate that constitutive traits controlling leaf water loss under well-watered conditions correlate with the terminal drought tolerance of pearl millet. Such traits may lead to more water being available for grain filling under terminal drought.

Terminal drought-tolerant pearl millet [Pennisetum glaucum (L.) R. Br.] have high leaf ABA and limit transpiration at high vapour pressure deficit.

It was previously shown that pearl millet genotypes carrying a terminal drought tolerance quantitative trait locus (QTL) had a lower transpiration rate (Tr; g cm(-2) d(-1)) under well-watered conditions than sensitive lines. Here experiments were carried out to test whether this relates to leaf abscisic acid (ABA) and Tr concentration at high vapour pressure deficit (VPD), and whether that leads to transpiration efficiency (TE) differences. These traits were measured in tolerant/sensitive pearl millet genotypes, including near-isogenic lines introgressed with a terminal drought tolerance QTL (NIL-QTLs). Most genotypic differences were found under well-watered conditions. ABA levels under well-watered conditions were higher in tolerant genotypes, including NIL-QTLs, than in sensitive genotypes, and ABA did not increase under water stress. Well-watered Tr was lower in tolerant than in sensitive genotypes at all VPD levels. Except for one line, Tr slowed down in tolerant lines above a breakpoint at 1.40-1.90 kPa, with the slope decreasing >50%, whereas sensitive lines showed no change in that Tr response across the whole VPD range. It is concluded that two water-saving (avoidance) mechanisms may operate under well-watered conditions in tolerant pearl millet: (i) a low Tr even at low VPD conditions, which may relate to leaf ABA; and (ii) a sensitivity to higher VPD that further restricts Tr, which suggests the involvement of hydraulic signals. Both traits, which did not lead to TE differences, could contribute to absolute water saving seen in part due to dry weight increase differences. This water saved would become critical for grain filling and deserves consideration in the breeding of terminal drought-tolerant lines.

Drought-tolerant and drought-sensitive genotypes of maize (Zea mays L.) differ in contents of endogenous brassinosteroids and their drought-induced changes.

The contents of endogenous brassinosteroids (BRs) together with various aspects of plant morphology, water management, photosynthesis and protection against cell damage were assessed in two maize genotypes that differed in their drought sensitivity. The presence of 28-norbrassinolide in rather high quantities (1-2 pg mg-1 fresh mass) in the leaves of monocot plants is reported for the first time. The intraspecific variability in the presence/content of the individual BRs in drought-stressed plants is also described for the first time. The drought-resistant genotype was characterised by a significantly higher content of total endogenous BRs (particularly typhasterol and 28-norbrassinolide) compared with the drought-sensitive genotype. On the other hand, the drought-sensitive genotype showed higher levels of 28-norcastasterone. Both genotypes also differed in the drought-induced reduction/elevation of the levels of 28-norbrassinolide, 28-norcastasterone, 28-homocastasterone and 28-homodolichosterone. The differences observed between both genotypes in the endogenous BR content are probably correlated with their different degrees of drought sensitivity, which was demonstrated at various levels of plant morphology, physiology and biochemistry.

Recovery of maize (Zea mays L.) inbreds and hybrids from chilling stress of various duration: photosynthesis and antioxidant enzymes.

The differences between two maize (Zea mays L.) inbred lines and their F1 hybrids in their response to chilling periods of various duration (1, 2, 3 or 4 weeks) and subsequent return to optimum temperatures were analysed by the measurement of the photosystem (PS) 1 and 2 activity, the photosynthetic pigments' content and the activity of antioxidant enzymes. The PS2 activity and the chlorophyll content decreased in plants subjected to 3 or 4 weeks of chilling, but not in those subjected to 1 or 2 weeks of chilling. This decrease was more pronounced in inbreds compared to their hybrids. The activity of superoxide dismutase did not much change with the increasing length of chilling period in the inbreds but decreased in the hybrids, the glutathione reductase activity increased in both types of genotypes but more in the inbred lines, while for ascorbate peroxidase and catalase the changes in parents-hybrids relationship did not show any specific trend. The PS1 activity and the carotenoids' content was not much affected.

The disadvantages of being a hybrid during drought: A combined analysis of plant morphology, physiology and leaf proteome in maize.

A comparative analysis of various parameters that characterize plant morphology, growth, water status, photosynthesis, cell damage, and antioxidative and osmoprotective systems together with an iTRAQ analysis of the leaf proteome was performed in two inbred lines of maize (Zea mays L.) differing in drought susceptibility and their reciprocal F1 hybrids. The aim of this study was to dissect the parent-hybrid relationships to better understand the mechanisms of the heterotic effect and its potential association with the stress response. The results clearly showed that the four examined genotypes have completely different strategies for coping with limited water availability and that the inherent properties of the F1 hybrids, i.e. positive heterosis in morphological parameters (or, more generally, a larger plant body) becomes a distinct disadvantage when the water supply is limited. However, although a greater loss of photosynthetic efficiency was an inherent disadvantage, the precise causes and consequences of the original predisposition towards faster growth and biomass accumulation differed even between reciprocal hybrids. Both maternal and paternal parents could be imitated by their progeny in some aspects of the drought response (e.g., the absence of general protein down-regulation, changes in the levels of some carbon fixation or other photosynthetic proteins). Nevertheless, other features (e.g., dehydrin or light-harvesting protein contents, reduced chloroplast proteosynthesis) were quite unique to a particular hybrid. Our study also confirmed that the strategy for leaving stomata open even when the water supply is limited (coupled to a smaller body size and some other physiological properties), observed in one of our inbred lines, is associated with drought-resistance not only during mild drought (as we showed previously) but also during more severe drought conditions.

Genetic variability and heritability of chlorophyll a fluorescence parameters in Scots pine (Pinus sylvestris L.).

Current knowledge of the genetic mechanisms underlying the inheritance of photosynthetic activity in forest trees is generally limited, yet it is essential both for various practical forestry purposes and for better understanding of broader evolutionary mechanisms. In this study, we investigated genetic variation underlying selected chlorophyll a fluorescence (ChlF) parameters in structured populations of Scots pine (Pinus sylvestris L.) grown on two sites under non-stress conditions. These parameters were derived from the OJIP part of the ChlF kinetics curve and characterize individual parts of primary photosynthetic processes associated, for example, with the exciton trapping by light-harvesting antennae, energy utilization in photosystem II (PSII) reaction centers (RCs) and its transfer further down the photosynthetic electron-transport chain. An additive relationship matrix was estimated based on pedigree reconstruction, utilizing a set of highly polymorphic single sequence repeat markers. Variance decomposition was conducted using the animal genetic evaluation mixed-linear model. The majority of ChlF parameters in the analyzed pine populations showed significant additive genetic variation. Statistically significant heritability estimates were obtained for most ChlF indices, with the exception of DI0/RC, φD0 and φP0 (Fv/Fm) parameters. Estimated heritabilities varied around the value of 0.15 with the maximal value of 0.23 in the ET0/RC parameter, which indicates electron-transport flux from QA to QB per PSII RC. No significant correlation was found between these indices and selected growth traits. Moreover, no genotype × environment interaction (G × E) was detected, i.e., no differences in genotypes' performance between sites. The absence of significant G × E in our study is interesting, given the relatively low heritability found for the majority of parameters analyzed. Therefore, we infer that polygenic variability of these indices is selectively neutral.

The effect of exogenous 24-epibrassinolide on the ecdysteroid content in the leaves of Spinacia oleracea L.

The aim of this study was to show whether/how the application of exogenous 24-epibrassinolide can affect the content of ecdysteroids in spinach leaves. Brassinosteroids and ecdysteroids, structurally related phytosterols, show effect on a range of processes in plants. Brassinosteroids increase biomass yield in some species, photosynthesis and resistance to stress, and ecdysteroids show effect on proteins responsible for binding of CO2 or water cleavage. The mutual interaction of these sterols in plants is unclear. The UPLC-(+)ESI-MS/MS analyses of extracts of treated and untreated spinach (Spinacia oleracea L.) leaves show that the application of exogenous 24-epibrassinolide does influence the ecdysteroid content in plant tissues. The response differs for the major ecdysteroids and also differs from that for the minor ones and is dependent on the developmental stage of the leaves within the same plant or the 24-epibrassinolide concentration applied.

24-epibrassinolide and 20-hydroxyecdysone affect photosynthesis differently in maize and spinach.

The aim of the work was to examine the effect of brassinosteroid (24-epibrassinolide; 24E) and ecdysteroid (20-hydroxyecdysone; 20E) on various parts of primary photosynthetic processes in maize and spinach. Additionally, the effect of steroids on gaseous exchange, pigment content and biomass accumulation was studied. The efficiency of the photosynthetic whole electron-transport chain responded negatively to the 24E or 20E treatment in both species, but there were interspecific differences regarding Photosystem (PS) II response. A positive effect on its oxygen-evolving complex and a slightly better energetical connectivity between PSII units were observed in maize whereas the opposite was true for spinach. The size of the pool of the PSI end electron acceptors was usually diminished due to 24E or 20E treatment. The treatment of plants with 24E or 20E applied individually positively influenced the content of photosynthetic pigments in maize (not in spinach). On the other hand, it did not affect gaseous exchange in maize but resulted in its reduction in spinach. Plants treated with combination of both steroids mostly did not significantly differ from the control plants. We have demonstrated for the first time that 20E applied in low (10nM) concentration can affect various parts of photosynthetic processes similarly to 24E and that brassinosteroids regulate not only PSII but also other parts of the photosynthetic electron transport chain - but not necessarily in the same way.

The physiology and proteomics of drought tolerance in maize: early stomatal closure as a cause of lower tolerance to short-term dehydration?

Understanding the response of a crop to drought is the first step in the breeding of tolerant genotypes. In our study, two maize (Zea mays L.) genotypes with contrasting sensitivity to dehydration were subjected to moderate drought conditions. The subsequent analysis of their physiological parameters revealed a decreased stomatal conductance accompanied by a slighter decrease in the relative water content in the sensitive genotype. In contrast, the tolerant genotype maintained open stomata and active photosynthesis, even under dehydration conditions. Drought-induced changes in the leaf proteome were analyzed by two independent approaches, 2D gel electrophoresis and iTRAQ analysis, which provided compatible but only partially overlapping results. Drought caused the up-regulation of protective and stress-related proteins (mainly chaperones and dehydrins) in both genotypes. The differences in the levels of various detoxification proteins corresponded well with the observed changes in the activities of antioxidant enzymes. The number and levels of up-regulated protective proteins were generally lower in the sensitive genotype, implying a reduced level of proteosynthesis, which was also indicated by specific changes in the components of the translation machinery. Based on these results, we propose that the hypersensitive early stomatal closure in the sensitive genotype leads to the inhibition of photosynthesis and, subsequently, to a less efficient synthesis of the protective/detoxification proteins that are associated with drought tolerance.