Promotion of testa rupture during garden cress germination involves seed compartment-specific expression and activity of pectin methylesterases

Pectin methylesterase (PME) controls the methylesterification status of pectins and thereby determines the biophysical properties of plant cell walls, which are important for tissue growth and weakening processes. We demonstrate here that tissue-specific and spatiotemporal alterations in cell wall pectin methylesterification occur during the germination of garden cress (Lepidium sativum). These cell wall changes are associated with characteristic expression patterns of PME genes and resultant enzyme activities in the key seed compartments CAP (micropylar endosperm) and RAD (radicle plus lower hypocotyl). Transcriptome and quantitative real-time reverse transcription-polymerase chain reaction analysis as well as PME enzyme activity measurements of separated seed compartments, including CAP and RAD, revealed distinct phases during germination. These were associated with hormonal and compartment-specific regulation of PME group 1, PME group 2, and PME inhibitor transcript expression and total PME activity. The regulatory patterns indicated a role for PME activity in testa rupture (TR). Consistent with a role for cell wall pectin methylesterification in TR, treatment of seeds with PME resulted in enhanced testa permeability and promoted TR. Mathematical modeling of transcript expression changes in germinating garden cress and Arabidopsis (Arabidopsis thaliana) seeds suggested that group 2 PMEs make a major contribution to the overall PME activity rather than acting as PME inhibitors. It is concluded that regulated changes in the degree of pectin methylesterification through CAP- and RAD-specific PME and PME inhibitor expression play a crucial role during Brassicaceae seed germination

Large-scale phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidative stress

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.Peer reviewe

Rôle du cyanure d'hydrogène dans la régulation de l'élimination de la dormance embryonnaire des semences de tournesol (Helianthus annuus L.)

Embryonic dormancy of freshly harvested sunflower (Helianthus annuus L.) seeds is expressed by their poor germination at low temperatures (10-15 °C). The research topic of the present work concerns the involvement of different endogenous factors such as hydrogen cyanide (HCN), reactive oxygen species (ROS), ethylene (C2H4) and abscisic acid (ABA) in the regulation of dormancy of sunflower embryos. The aims of the present work were: (i) to identify the effect of gaseous HCN on breaking of sunflower embryo dormancy, (ii) to determine whether HCN improving action is related with the ethylene synthesis or signaling pathways, and (iii) to investigate the mechanisms of action of HCN, particularly whether ROS are involved in this phenomenon. A short pre-treatment (3 hours) of dormant sunflower embryos by gaseous HCN released their dormancy and allowed their subsequent germination at 10-15 °C, temperatures which prevented germination of dormant embryos. This stimulatory effect did not result from an activation of the pentose phosphate or the cyanide-insensitive pathways, since other respiratory inhibitors such as sodium azide (NaN3) and salicylhydroxamic acid (SHAM), did not allow the germination of dormant embryos at 10-15 °C. In addition, b-cyanoalanine synthase (b-CAS) activity measurements excluded the role of HCN in stimulating amino acids synthesis (as alanine, serine, asparagine) during dormancy alleviation, by improving the activity of b-CAS, since no significant difference in the activity of this enzyme was observed in axes isolated from control non-treated and HCN or MV treated embryos. To identify the process by which dormancy of sunflower (non-cyanogenic) embryos is broken by HCN, the present work was focussed on its possible interaction with reactive oxygen species in this phenomenon. After HCN treatment, ROS (H2O2 - hydrogen peroxide and O2 .- - superoxide anions) progressively accumulated in cells of embryonic axis. However, application of HCN had only a slight effect on the in vitro activity of the main PDF stworzony przez wersje demonstracyjna pdfFactory Pro www.pdffactory.pl/ Summary in English 8 enzymes involved in the antioxidant defence system, such as catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), since these enzymes were potentially active. Imbibition of embryos in the presence of methylviologen (MV), as well as menadione (MD), ROS generating compounds, had the same stimulatory effect on germination as HCN, suggesting that HCN probably improved germination through accumulation of ROS. Increase in H2O2 and O2 .- concentration in the embryonic axis occurred concomitantly with an oxidation (carbonylation) of specific proteins. Obtained data allow us to propose a novel explanation for cyanide mode of action in alleviation of sunflower embryo dormancy, which involves ROS production and targets changes in protein oxidation patterns. In the group of carbonylated proteins specifically associated with breaking of dormancy by HCN and MV, some are identified as epoxide hydrolase and alcohol dehydrogenase (Oracz et al. 2007). In order to determine whether the stimulatory effect of HCN during germination of sunflower embryo might be related to ethylene biosynthesis and ethylene signaling pathway, transcription of genes involved in these proceses was studied simultaneously to HCN and MV treatments. The stimulatory effect of HCN and MV on germination of sunflower embryos, is not associated with a modification of transcription of genes involved in ethylene production such as ACS (ACC synthase) and ACO (ACC oxidase), and does not result from a stimulation of ethylene production. It was also demonstrated, that HCN and MV markedly increased the transcription of ERF1 (ethylene response factor 1) but not that of ETR2 (ethylene response 2) and CTR1 (constitutive triple response 1). This suggests that the transduction pathway of cyanide and MV, initiates the transcription factor ERF1, but through different mechanism of that involved by ethylene. The putative molecular mode of action of HCN on sunflower embryo germination is discussed, and its possible relationships with ROS and ethylene synthesis and signaling pathways are also evoked. The first time are presented the direct experimental data concerning HCN effect on the expression of genes related to (i) ROS production [NADPHox (NADPH oxidase), AO1 and AO2 (amine oxidase 1 and 2), POX (peroxidase)], (ii) ROS signaling [Ser/ThrPK (Ser/Thr protein kinase), MAPK6 (mitogen activated protein kinase 6), PTP (protein tyrosine phosphatase), CaM (calmodulin)], (iii) ethylene formation (ACS, ACO) and (iv) signal transduction induced by ethylene (ETR2, CTR1, ERF1). It is demonstrated, that HCN did not change the transcription of genes involved in ROS production (NADPHox, AO1, AO2, POX), and also did not seem to require the typical elements of ROS signaling pathway (MAPK6, SerThrPK, PTP, CaM), but can induce the physiological response, using some elements of other signal transduction pathways (e.g. ERF1 in ethylene signaling pathway). This work proposes HCN as a signal molecule, which via ROS as a second messenger, regulates many pathways leading in consequence to alleviation of embryo dormancy and stimulation of germination. These pathways probably do not operate independently, but rather are linked together in a network of interactions.Cette étude concerne la régulation de la dormance embryonnaire des semences de tournesol (Helianthus annuus L.) par le cyanure d'hydrogène (HCN), en relation avec les formes actives de l'oxygène (FAOs) et l'éthylène (C2H4). Les semences de tournesol fraîchement récoltées sont dormantes ce qui se caractérise par une germination difficile à des températures basses (10-15 °C). Un traitement de courte durée (3 h) des embryons dormants par du HCN gazeux élimine leur dormance et permet leur germination à 10°C. Cet effet stimulateur ne résulte pas d'une activation de la voie des pentoses phosphates ou de la voie insensible au cyanure, car d'autres inhibiteurs respiratoires ne permettent pas la germination des embryons dormants à 10°C. Après traitement par du HCN, les FAOs s'accumulent dans les cellules des axes embryonnaires ce qui s'accompagne d'une oxydation (carbonylation) de protéines spécifiques qui ont été en partie identifiées. Toutefois le cyanure ne modifie pas la transcription des gènes codant pour les enzymes impliqués dans la production et la signalisation des FAOs. L'effet stimulateur du cyanure sur la germination des embryons dormants n'est pas associé à une modification de la transcription des gènes impliqués dans la biosynthèse de l'éthylène, ACC synthétase et ACC oxydase, et ne résulte pas d'une augmentation de la production d'éthylène. En revanche, le HCN augmente nettement la transcription de ERF1 (ethylene response factor 1), mais n'a pas d'effet sur la transcription de ETR2 (ethylene response 2) et de CTR1 (constitutive triple response 1). Les résultats présentés permettent de proposer un nouveau mécanisme de régulation de la dormance des semences par le cyanure

phyB and HY5 are Involved in the Blue Light-Mediated Alleviation of Dormancy of Arabidopsis Seeds Possibly via the Modulation of Expression of Genes Related to Light, GA, and ABA

Light is one of the most important environmental factors regulating seed germination. It is known that light inhibits seed germination of some monocotyledonous species and that it is mostly related to the blue wavelength of the spectrum received by cryptochromes (cry). Research has also found that the red light (R) stimulates germination of dicotyledonous seeds and that this reaction involves mainly phytochromes (phy). Surprisingly, up to date, the role and the mechanism of action of blue light (BL) in seed biology of dicot plants is still very poorly understood and some questions are unexplained, e.g., whether BL plays a role in regulation of dicot seeds dormancy and/or germination? If, so what particular elements of light signaling pathway are involved in modulation of this(ese) process(es)? Also, is the BL action in regulation of dicot seeds dormancy and/or germination maybe due to changes of expression of genes related to metabolism and/or signaling of two phytohormones controlling seed-related events, such as gibberellins (GA) and abscisic acid (ABA)? To answer these intriguing questions, the combination of biological, transcriptomic, and genetic approaches was performed in this particular study. The germination tests show that freshly harvested wild type (WT) Arabidopsis thaliana Col-0 seeds are dormant and do not germinate in darkness (at 25 °C), while nondormant (after-ripened) seeds germinate well in these conditions. It is also proven that dormancy of seeds of this species is released in the presence of white and/or BL (λ = 447 nm) when placed at 25 °C. Presented here, novel results emphasize the role of BL in dormancy alleviation of dicot seeds, indicating that this wavelength of light spectrum received by phyB induces this process and that the sensitivity to this stimulus depends on the depth of seed dormancy. In addition, it is demonstrated that various elements of phy-mediated pathway can be used in response to the signal induced by BL in germinating dormant seeds of Arabidopsis. The quantitative real time PCR analysis supported by results of germination tests of WT, T-DNA insertion mutants (i.e., hy5, hfr1, and laf1) and overexpression transformants of Arabidopsis seeds (i.e., 35S:OE:HY5, 35S:OE:HYH, 35S:OE:HFR1, and 35S:OE:LAF1) revealed that the HY5 gene coding transcription factor is most probably responsible for the control of expression of genes involved in GA/ABA metabolism and/or signaling pathways during BL-dependent dormancy alleviation of Arabidopsis seeds, while biological functions of HYH and HFR1 are associated with regulation of germination. The model of BL action in regulation of dormancy alleviation and germination potential of Arabidopsis seeds is proposed

Allelopathy - new interpretations of plant - plant interactions

Allelopathy denotes biochemical interactions among all types of plants and microorganisms. Allelochemicals occur in the majority of secondary plant products and belong to terpenoids, phenolic compounds, phenylpropane derivatives, flavonoids, organic cyanides, long-chain fatty acids. In fields, allelopathy involves a complex of compounds, which complicates investigations of underlying mechanisms of its appearance. There is no single physiological function controlled by allelochemicals. The action of allelochemicals is diverse and affects a large number of physiological functions and biochemical reactions eg.: seed germination, cell division, cell elongation, membrane permeability and ion uptake. Significant effects of allelochemicals on photosynthesis and respiration have been also observed. The reduction of crop yields by weed competition is aggravated by the allelopathic effect of weeds on crop. Crop plants may inhibit their owngrowth and reduce the yield of subsequent crops due to the so called soil sickness. Crop rotation helps to cures soil sickness only when the subsequent crop is not affected by the accumulated allelochemicals of the previous crop or when they have been detoxified by soil microorganisms. Allelopatic weed control with crop plants has long been practised by farmers and horticulturists. The challenge is twofold: tominimize the negative impact of allelochemicals on crop growth and yield and to exploit allelopathic mechanisms for pest control and crop growth regulation strategies. Allelochemicals in new crop cultivars may provide naturally occurring pesticides that can limit or suppress weeds as well as prevent insect and nematode attack and damage. Biotechnology resources for the production of herbicide-resistant crops could then be channeled into the engineering of other desired crops qualities. Allelochemicals may furnish an entirely new generation of naturally produced weed-controlling compounds, replacing synthetic herbicides and other pesticides with nonacumulatting easy-degradable substances

The Mechanisms Involved in Seed Dormancy Alleviation by Hydrogen Cyanide Unravel the Role of Reactive Oxygen Species as Key Factors of Cellular Signaling during Germination[C][W]

The physiological dormancy of sunflower (Helianthus annuus) embryos can be overcome during dry storage (after-ripening) or by applying exogenous ethylene or hydrogen cyanide (HCN) during imbibition. The aim of this work was to provide a comprehensive model, based on oxidative signaling by reactive oxygen species (ROS), for explaining the cellular mode of action of HCN in dormancy alleviation. Beneficial HCN effect on germination of dormant embryos is associated with a marked increase in hydrogen peroxide and superoxide anion generation in the embryonic axes. It is mimicked by the ROS-generating compounds methylviologen and menadione but suppressed by ROS scavengers. This increase results from an inhibition of catalase and superoxide dismutase activities and also involves activation of NADPH oxidase. However, it is not related to lipid reserve degradation or gluconeogenesis and not associated with marked changes in the cellular redox status controlled by the glutathione/glutathione disulfide couple. The expression of genes related to ROS production (NADPHox, POX, AO1, and AO2) and signaling (MAPK6, Ser/ThrPK, CaM, and PTP) is differentially affected by dormancy alleviation either during after-ripening or by HCN treatment, and the effect of cyanide on gene expression is likely to be mediated by ROS. It is also demonstrated that HCN and ROS both activate similarly ERF1, a component of the ethylene signaling pathway. We propose that ROS play a key role in the control of sunflower seed germination and are second messengers of cyanide in seed dormancy release