Domestication has altered the ABA and gibberellin profiles in developing pea seeds.

MAIN CONCLUSION: We showed that wild pea seeds contained a more diverse combination of bioactive GAs and had higher ABA content than domesticated peas. Although the role of abscisic acid (ABA) and gibberellins (GAs) interplay has been extensively studied in Arabidopsis and cereals models, comparatively little is known about the effect of domestication on the level of phytohormones in legume seeds. In legumes, as in other crops, seed dormancy has been largely or entirely removed during domestication. In this study, we have measured the endogenous levels of ABA and GAs comparatively between wild and domesticated pea seeds during their development. We have shown that wild seeds contained more ABA than domesticated ones, which could be important for preparing the seeds for the period of dormancy. ABA was catabolised particularly by an 8´-hydroxylation pathway, and dihydrophaseic acid was the main catabolite in seed coats as well as embryos. Besides, the seed coats of wild and pigmented cultivated genotypes were characterised by a broader spectrum of bioactive GAs compared to non-pigmented domesticated seeds. GAs in both seed coat and embryo were synthesized mainly by a 13-hydroxylation pathway, with GA29 being the most abundant in the seed coat and GA20 in the embryos. Measuring seed water content and water loss indicated domesticated pea seeds´ desiccation was slower than that of wild pea seeds. Altogether, we showed that pea domestication led to a change in bioactive GA composition and a lower ABA content during seed development.

The Anti-Senescence Activity of Cytokinin Arabinosides in Wheat and Arabidopsis Is Negatively Correlated with Ethylene Production.

Leaf senescence, accompanied by chlorophyll breakdown, chloroplast degradation and inhibition of photosynthesis, can be suppressed by an exogenous application of cytokinins. Two aromatic cytokinin arabinosides (6-benzylamino-9-ß-d-arabinofuranosylpurines; BAPAs), 3-hydroxy- (3OHBAPA) and 3-methoxy- (3MeOBAPA) derivatives, have recently been found to possess high anti-senescence activity. Interestingly, their effect on the maintenance of chlorophyll content and maximal quantum yield of photosystem II (PSII) in detached dark-adapted leaves differed quantitatively in wheat (Triticum aestivum L. cv. Aranka) and Arabidopsis (Arabidopsisthaliana L. (Col-0)). In this work, we have found that the anti-senescence effects of 3OHBAPA and 3MeOBAPA in wheat and Arabidopsis also differ in other parameters, including the maintenance of carotenoid content and chloroplasts, rate of reduction of primary electron acceptor of PSII (QA) as well as electron transport behind QA, and partitioning of absorbed light energy in light-adapted leaves. In wheat, 3OHBAPA had a higher protective effect than 3MeOBAPA, whereas in Arabidopsis, 3MeOBAPA was the more efficient derivative. We have found that the different anti-senescent activity of 3OHBAPA and 3MeOBAPA was coupled to different ethylene production in the treated leaves: the lower the ethylene production, the higher the anti-senescence activity. 3OHBAPA and 3MeOBAPA also efficiently protected the senescing leaves of wheat and Arabidopsis against oxidative damage induced by both H2O2 and high-light treatment, which could also be connected with the low level of ethylene production.

Estimating heat tolerance of plants by ion leakage: a new method based on gradual heating.

Heat tolerance of plants related to cell membrane thermostability is commonly estimated via the measurement of ion leakage from plant segments after defined heat treatment. To compare heat tolerance of various plants, it is crucial to select suitable heating conditions. This selection is time-consuming and optimizing the conditions for all investigated plants may even be impossible. Another problem of the method is its tendency to overestimate basal heat tolerance. Here we present an improved ion leakage method, which does not suffer from these drawbacks. It is based on gradual heating of plant segments in a water bath or algal suspensions from room temperature up to 70-75°C. The electrical conductivity of the bath/suspension, which is measured continuously during heating, abruptly increases at a certain temperature TCOND (within 55-70°C). The TCOND value can be taken as a measure of cell membrane thermostability, representing the heat tolerance of plants/organisms. Higher TCOND corresponds to higher heat tolerance (basal or acquired) connected to higher thermostability of the cell membrane, as evidenced by the common ion leakage method. The new method also enables determination of the thermostability of photochemical reactions in photosynthetic samples via the simultaneous measurement of Chl fluorescence.

The interplay between cytokinins and light during senescence in detached Arabidopsis leaves.

Light and cytokinins are known to be the key players in the regulation of plant senescence. In detached leaves, the retarding effect of light on senescence is well described; however, it is not clear to what extent is this effect connected with changes in endogenous cytokinin levels. We have performed a detailed analysis of changes in endogenous content of 29 cytokinin forms in detached leaves of Arabidopsis thaliana (wild-type and 3 cytokinin receptor double mutants). Leaves were kept under different light conditions, and changes in cytokinin content were correlated with changes in chlorophyll content, efficiency of photosystem II photochemistry, and lipid peroxidation. In leaves kept in darkness, we have observed decreased content of the most abundant cytokinin free bases and ribosides, but the content of cis-zeatin increased, which indicates the role of this cytokinin in the maintenance of basal leaf viability. Our findings underscore the importance of light conditions on the content of specific cytokinins, especially N6 -(Δ2 -isopentenyl)adenine. On the basis of our results, we present a scheme summarizing the contribution of the main active forms of cytokinins, cytokinin receptors, and light to senescence regulation. We conclude that light can compensate the disrupted cytokinin signalling in detached leaves.

Chloroplast avoidance movement as a sensitive indicator of relative water content during leaf desiccation in the dark.

In the context of global climate change, drought is one of the major stress factors with negative effect on photosynthesis and plant productivity. Currently, chlorophyll fluorescence parameters are widely used as indicators of plant stress, mainly owing to the rapid, non-destructive and simple measurements this technique allows. However, these parameters have been shown to have limited sensitivity for the monitoring of water deficit as leaf desiccation has relatively small effect on photosystem II photochemistry. In this study, we found that blue light-induced increase in leaf transmittance reflecting chloroplast avoidance movement was much more sensitive to a decrease in relative water content (RWC) than chlorophyll fluorescence parameters in dark-desiccating leaves of tobacco (Nicotiana tabacum L.) and barley (Hordeum vulgare L.). Whereas the inhibition of chloroplast avoidance movement was detectable in leaves even with a small RWC decrease, the chlorophyll fluorescence parameters (F V/F M, V J, Ф PSII, NPQ) changed markedly only when RWC dropped below 70 %. For this reason, we propose light-induced chloroplast avoidance movement as a sensitive indicator of the decrease in leaf RWC. As our measurement of chloroplast movement using collimated transmittance is simple and non-destructive, it may be more suitable in some cases for the detection of plant stresses including water deficit than the conventionally used chlorophyll fluorescence methods.

Anthocyanin contribution to chlorophyll meter readings and its correction.

Leaf chlorophyll content is an important physiological parameter which can serve as an indicator of nutritional status, plant stress or senescence. Signals proportional to the chlorophyll content can be measured non-destructively with instruments detecting leaf transmittance (e.g., SPAD-502) or reflectance (e.g., showing normalized differential vegetation index, NDVI) in red and near infrared spectral regions. The measurements are based on the assumption that only chlorophylls absorb in the examined red regions. However, there is a question whether accumulation of other pigments (e.g., anthocyanins) could in some cases affect the chlorophyll meter readings. To answer this question, we cultivated tomato plants (Solanum lycopersicum L.) for a long time under low light conditions and then exposed them for several weeks (4 h a day) to high sunlight containing the UV-A spectral region. The senescent leaves of these plants evolved a high relative content of anthocyanins and visually revealed a distinct blue color. The SPAD and NDVI data were collected and the spectra of diffusive transmittance and reflectance of the leaves were measured using an integration sphere. The content of anthocyanins and chlorophylls was measured analytically. Our results show that SPAD and NDVI measurement can be significantly affected by the accumulated anthocyanins in the leaves with relatively high anthocyanin content. To describe theoretically this effect of anthocyanins, concepts of a specific absorbance and a leaf spectral polarity were developed. Corrective procedures of the chlorophyll meter readings for the anthocyanin contribution are suggested both for the transmittance and reflectance mode.

High cytokinin levels induce a hypersensitive-like response in tobacco.

BACKGROUND AND AIMS: Cytokinins are positive regulators of shoot development. However, it has previously been demonstrated that efficient activation of the cytokinin biosynthesis gene ipt can cause necrotic lesions and wilting in tobacco leaves. Some plant pathogens reportedly use their ability to produce cytokinins in disease development. In response to pathogen attacks, plants can trigger a hypersensitive response that rapidly kills cells near the infection site, depriving the pathogen of nutrients and preventing its spread. In this study, a diverse set of processes that link ipt activation to necrotic lesion formation were investigated in order to evaluate the potential of cytokinins as signals and/or mediators in plant defence against pathogens. METHODS: The binary pOp-ipt/LhGR system for dexamethasone-inducible ipt expression was used to increase endogenous cytokinin levels in transgenic tobacco. Changes in the levels of cytokinins and the stress hormones salicylic, jasmonic and abscisic acid following ipt activation were determined by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). Trends in hydrogen peroxide content and lipid peroxidation were monitored using the potassium iodide and malondialdehyde assays. The subcellular distribution of hydrogen peroxide was investigated using 3,3'-diaminobenzidine staining. The dynamics of transcripts related to photosynthesis and pathogen response were analysed by reverse transcription followed by quantitative PCR. The effects of cytokinins on photosynthesis were deciphered by analysing changes in chlorophyll fluorescence and leaf gas exchange. KEY RESULTS: Plants can produce sufficiently high levels of cytokinins to trigger fast cell death without any intervening chlorosis - a hallmark of the hypersensitive response. The results suggest that chloroplastic hydrogen peroxide orchestrates the molecular responses underpinning the hypersensitive-like response, including the inhibition of photosynthesis, elevated levels of stress hormones, oxidative membrane damage and stomatal closure. CONCLUSIONS: Necrotic lesion formation triggered by ipt activation closely resembles the hypersensitive response. Cytokinins may thus act as signals and/or mediators in plant defence against pathogen attack.

Urea derivative MTU improves stress tolerance and yield in wheat by promoting cyclic electron flow around PSI.

Increasing crop productivity under optimal conditions and mitigating yield losses under stressful conditions is a major challenge in contemporary agriculture. We have recently identified an effective anti-senescence compound (MTU, [1-(2-methoxyethyl)-3-(1,2,3-thiadiazol-5yl)urea]) in in vitro studies. Here, we show that MTU delayed both age- and stress-induced senescence of wheat plants (Triticum aestivum L.) by enhancing the abundance of PSI supercomplex with LHCa antennae (PSI-LHCa) and promoting the cyclic electron flow (CEF) around PSI. We suppose that this rarely-observed phenomenon blocks the disintegration of photosynthetic apparatus and maintains its activity as was reflected by the faster growth rate of wheat in optimal conditions and under drought and heat stress. Our multiyear field trial analysis further shows that the treatment with 0.4 g ha-1 of MTU enhanced average grain yields of field-grown wheat and barley (Hordeum vulgare L.) by 5-8%. Interestingly, the analysis of gene expression and hormone profiling confirms that MTU acts without the involvement of cytokinins or other phytohormones. Moreover, MTU appears to be the only chemical reported to date to affect PSI stability and activity. Our results indicate a central role of PSI and CEF in the onset of senescence with implications in yield management at least for cereal species.

Diethyl ether anesthesia induces transient cytosolic [Ca2+] increase, heat shock proteins, and heat stress tolerance of photosystem II in Arabidopsis.

General volatile anesthetic diethyl ether blocks sensation and responsive behavior not only in animals but also in plants. Here, using a combination of RNA-seq and proteomic LC-MS/MS analyses, we investigated the effect of anesthetic diethyl ether on gene expression and downstream consequences in plant Arabidopsis thaliana. Differential expression analyses revealed reprogramming of gene expression under anesthesia: 6,168 genes were upregulated, 6,310 genes were downregulated, while 9,914 genes were not affected in comparison with control plants. On the protein level, out of 5,150 proteins identified, 393 were significantly upregulated and 227 were significantly downregulated. Among the highest significantly downregulated processes in etherized plants were chlorophyll/tetrapyrrole biosynthesis and photosynthesis. However, measurements of chlorophyll a fluorescence did not show inhibition of electron transport through photosystem II. The most significantly upregulated process was the response to heat stress (mainly heat shock proteins, HSPs). Using transgenic A. thaliana expressing APOAEQUORIN, we showed transient increase of cytoplasmic calcium level [Ca2+]cyt in response to diethyl ether application. In addition, cell membrane permeability for ions also increased under anesthesia. The plants pre-treated with diethyl ether, and thus with induced HSPs, had increased tolerance of photosystem II to subsequent heat stress through the process known as cross-tolerance or priming. All these data indicate that diethyl ether anesthesia may partially mimic heat stress in plants through the effect on plasma membrane.

SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement.

Non-destructive assessment of chlorophyll content has recently been widely done by chlorophyll meters based on measurement of leaf transmittance (e.g. the SPAD-502 chlorophyll meter measures the leaf transmittance at 650 and 940 nm). However, the leaf transmittance depends not only on the content of chlorophylls but also on their distribution in leaves. The chlorophyll distribution within leaves is co-determined by chloroplast arrangement in cells that depends on light conditions. When tobacco leaves were exposed to a strong blue light (about 340 µmol of photons m⁻² s⁻¹), a very pronounced increase in the leaf transmittance was observed as chloroplasts migrated from face position (along cell walls perpendicular to the incident light) to side position (along cell walls parallel to the incoming light) and the SPAD reading decreased markedly. This effect was more pronounced in the leaves of young tobacco plants compared with old ones; the difference between SPAD values in face and side position reached even about 35%. It is shown how the chloroplast movement changes a relationship between the SPAD readings and real chlorophyll content. For an elimination of the chloroplast movement effect, it can be recommended to measure the SPAD values in leaves with a defined chloroplasts arrangement.

Aromatic Cytokinin Arabinosides Promote PAMP-like Responses and Positively Regulate Leaf Longevity.

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth to the regulation of biotic and abiotic stress-related responses and senescence. The components of the plant immune system can learn from past elicitations by microbial pathogens and herbivores and adapt to new threats. It is known that plants can enter the primed state of enhanced defense induced by either natural or synthetic compounds. While the involvement of cytokinins in defense priming has been documented, no comprehensive model of their action has been provided to date. Here, we report the functional characterization of two aromatic cytokinin derivatives, 6-benzylaminopurine-9-arabinosides (BAPAs), 3-methoxy-BAPA and 3-hydroxy-BAPA, that proved to be effective in delaying senescence in detached leaves while having low interactions with the cytokinin pathway. An RNA-seq profiling study on Arabidopsis leaves treated with 3-methoxy-BAPA revealed that short and extended treatments with this compound shifted the transcriptional response markedly toward defense. Both treatments revealed upregulation of genes involved in processes associated with plant innate immunity such as cell wall remodeling and upregulation of specific MAP kinases, most importantly MPK11, which is a MAPK module involved in stress-related signaling during the pathogen-associated molecular patterns (PAMPs) response. In addition, elevated levels of JA and its metabolites, jasmonate/ethylene-driven upregulation of PLANT DEFENSIN 1.2 (PDF1.2) and other defensins, and also temporarily elevated levels of reactive oxygen species marked the plant response to 3-methoxy-BAPA treatment. Synergistic interactions were observed when plants were cotreated with 3-hydroxy-BAPA and the flagellin-derived bacterial PAMP peptide (flg22), leading to the enhanced expression of the PAMP-triggered immunity (PTI) marker gene FRK1. Our data collectively show that some BAPAs can sensitively prime the PTI responses in a low micromolar range of concentrations while having no observable negative effects on the overall fitness of the plant.

Exogenous application of cytokinin during dark senescence eliminates the acceleration of photosystem II impairment caused by chlorophyll b deficiency in barley.

Recent studies have shown that chlorophyll (Chl) b has an important role in the regulation of leaf senescence. However, there is only limited information about senescence of plants lacking Chl b and senescence-induced decrease in photosystem II (PSII) and photosystem I (PSI) function has not even been investigated in such plants. We have studied senescence-induced changes in photosynthetic pigment content and PSII and PSI activities in detached leaves of Chl b-deficient barley mutant, chlorina f2f2 (clo). After 4 days in the dark, the senescence-induced decrease in PSI activity was smaller in clo compared to WT leaves. On the contrary, the senescence-induced impairment in PSII function (estimated from Chl fluorescence parameters) was much more pronounced in clo leaves, even though the relative decrease in Chl content was similar to wild type (WT) leaves (Hordeum vulgare L., cv. Bonus). The stronger impairment of PSII function seems to be related to more pronounced damage of reaction centers of PSII. Interestingly, exogenously applied plant hormone cytokinin 6-benzylaminopurine (BA) was able to maintain PSII function in the dark senescing clo leaves to a similar extent as in WT. Thus, considering the fact that without BA the senescence-induced decrease in PSII photochemistry in clo was more pronounced than in WT, the relative protective effect of BA was higher in Chl b-deficient mutant than in WT.

Ultra-structural and functional changes in the chloroplasts of detached barley leaves senescing under dark and light conditions.

Changes in the chloroplast ultra-structure and photochemical function were studied in detached barley (Hordeum vulgare L. cv. Akcent) leaf segments senescing in darkness or in continuous white light of moderate intensity (90 mumol m-2 s-1) for 5 days. A rate of senescence-induced chlorophyll degradation was similar in the dark- and light-senescing segments. The Chl a/b ratio was almost unchanged in the dark-senescing segments, whereas in the light-senescing segments an increase in this ratio was observed indicating a preferential degradation of light-harvesting complexes of photosystem II. A higher level of thylakoid disorganisation (especially of granal membranes) and a very high lipid peroxidation were observed in the light-senescing segments. In spite of these findings, both the maximal and actual photochemical quantum yields of the photosystem II were highly maintained in comparison with the dark-senescing segments.

Photosynthetic alterations of pea leaves infected systemically by pea enation mosaic virus: A coordinated decrease in efficiencies of CO(2) assimilation and photosystem II photochemistry.

We have investigated photosynthetic changes of fully expanded pea leaves infected systemically by pea enation mosaic virus (PEMV) that often attacks legumes particularly in northern temperate regions. A typical compatible virus-host interaction was monitored during 40 post-inoculation days (dpi). An initial PEMV-induced decrease in photosynthetic CO(2) assimilation was detected at 15 dpi, when the virus appeared in the measured leaves. This decrease was not induced by stomata closure and corresponded with a decrease in the efficiency of photosystem II photochemistry (Φ(PSII)). Despite of a slight impairment of oxygen evolution at this stage, PSII function was not primarily responsible for the decrease in Φ(PSII). Chlorophyll fluorescence imaging revealed that Φ(PSII) started to decrease from the leaf tip to the base. More pronounced symptoms of PEMV disease appeared at later stages, when a typical mosaic and enations appeared in the infected leaves and oxidative damage of cell membranes was detected. From 30 dpi, a degradation of photosynthetic pigments accelerated, stomata were closing and corresponding pronounced decline in CO(2) assimilation was observed. A concomitant photoprotective responses, i.e. an increase in non-photochemical quenching and accumulation of de-epoxidized xanthophylls, were also detected. Interestingly, alternative electron sinks in chloroplasts were not stimulated by PEMV infection, which is in contradiction to earlier reports dealing with virus-induced plant stresses. The presented results show that the PEMV-induced alterations in mature pea leaves accelerated leaf senescence during which a decrease in Φ(PSII) took place in coordinated manner with an inhibition of CO(2) assimilation.

Low-light-induced violaxanthin de-epoxidation in shortly preheated leaves: uncoupling from Delta pH-dependent nonphotochemical quenching.

Plants protect themselves against excessive light by the induction of Delta pH-dependent nonphotochemical quenching (qE) that is associated with de-epoxidation of violaxanthin (V) to zeaxanthin (Z) in thylakoid membranes. In this work, we report that low light (12 micromol photons m(-2) s(-1)) is sufficient for a marked stimulation of the V to Z conversion in shortly preheated wheat leaves (5 min, 40 degrees C), but without a substantial increase in qE. Re-irradiation of these leaves with high light led to a rapid induction of nonphotochemical quenching, implying a potential photoprotective role of low-light-induced Z in preheated leaves. On the contrary to low light conditions, preheated leaves exposed to high light behaved similar to nonheated leaves with respect to the V to Z conversion and qE induction. The obtained results indicate that low-light-induced lumen acidification in preheated leaves is high enough to activate V de-epoxidation, but not sufficiently high to induce the formation of quenching centers.

Photosynthetic responses of lettuce to downy mildew infection and cytokinin treatment.

Changes in primary metabolism of lettuce, Lactuca sativa L. (cv. Cobham Green), induced by compatible interaction with the biotrophic oomycete pathogen Bremia lactucae Regel (race BL 16), under two intensities of illumination in the presence and absence of exogenous cytokinins were studied by chlorophyll fluorescence imaging. Thirteen days post-inoculation leaf discs infected by B. lactucae exhibited impairments of photosynthesis associated with biotrophic infections, including: reductions in photosynthetic pigment contents and the maximum quantum yield of photosystem II photochemistry (F(V)/F(M)), inhibition of electron transport (Phi(PSII)) and increased non-photochemical chlorophyll fluorescence quenching (NPQ). Detected changes in photosynthetic parameters correlated with the leaf area colonized by the pathogen's intercellular hyphae. Applications of two cytokinins, benzylaminopurine and meta-topolin, previously shown to suppress B. lactucae sporulation if applied 24 h prior to inoculation at a concentration of 200 microM, retarded the pathogen's asexual reproduction with no apparent negative effects on the host's photosynthetic apparatus. However, long-lasting treatment of healthy tissues with this high concentration of exogenous cytokinin led to effects parallel to pathogenesis: reductions in photosynthetic pigment contents accompanied by inhibition of photosystem II photochemistry and electron transport. These effects of both prolonged exposure to cytokinins and the pathogenesis were weaker in discs exposed to the lower photosynthetic photon flux density. The role of cytokinins in plant-biotrophic pathogen interactions and their potential as disease control agents are discussed.

Electrical and chemical signals involved in short-term systemic photosynthetic responses of tobacco plants to local burning.

Short-term (up to 1 h) systemic responses of tobacco (Nicotiana tabacum cv. Samsun) plants to local burning of an upper leaf were studied by measuring the following variables in a distant leaf: extracellular electrical potentials (EEPs); gas exchange parameters; fast chlorophyll fluorescence induction; and endogenous concentrations of three putative chemical signaling compounds-abscisic (ABA), jasmonic (JA), and salicylic (SA) acids. The first detected response to local burning in the distant leaves was in EEP, which started to decline within 10-20 s of the beginning of the treatment, fell sharply for ca. 1-3 min, and then tended to recover within the following hour. The measured gasometric parameters (stomatal conductance and the rates of transpiration and CO(2) assimilation) started to decrease 5-7 min after local burning, suggesting that the electrical signals may induce stomatal closure. These changes were accompanied by systemic increases in the endogenous ABA concentration followed by huge systemic rises in endogenous JA levels started after ca. 15 min, providing the first evidence of short-term systemic accumulation of these plant hormones in responses to local burning. Furthermore, JA appears to have an inhibitory effect on CO(2) assimilation. The correlations between the kinetics of the systemic EEP, stomatal, photosynthetic, ABA, and JA responses suggest that (1) electrical signals (probably induced by a propagating hydraulic signal) may trigger chemical defense-related signaling pathways in tobacco plants; (2) both electrical and chemical signals are interactively involved in the induction of short-term systemic stomatal closure and subsequent reductions in the rate of transpiration and CO(2) assimilation after local burning events.