Plants have evolved several strategies to cope with the ever-changing environment. One example of this is given by seed germination, which must occur when environmental conditions are suitable for plant life. In the model system Arabidopsis thaliana seed germination is induced by light; however, in nature, seeds of several plant species can germinate regardless of this stimulus. While the molecular mechanisms underlying light-induced seed germination are well understood, those governing germination in the dark are still vague, mostly due to the lack of suitable model systems. Here, we employ Cardamine hirsuta, a close relative of Arabidopsis, as a powerful model system to uncover the molecular mechanisms underlying light-independent germination. By comparing Cardamine and Arabidopsis, we show that maintenance of the pro-germination hormone gibberellin (GA) levels prompt Cardamine seeds to germinate under both dark and light conditions. Using genetic and molecular biology experiments, we show that the Cardamine DOF transcriptional repressor DOF AFFECTING GERMINATION 1 (ChDAG1), homologous to the Arabidopsis transcription factor DAG1, is involved in this process functioning to mitigate GA levels by negatively regulating GA biosynthetic genes ChGA3OX1 and ChGA3OX2, independently of light conditions. We also demonstrate that this mechanism is likely conserved in other Brassicaceae species capable of germinating in dark conditions, such as Lepidium sativum and Camelina sativa. Our data support Cardamine as a new model system suitable for studying light-independent germination studies. Exploiting this system, we have also resolved a long-standing question about the mechanisms controlling light-independent germination in plants, opening new frontiers for future research.

• Keywords
• Cardamine hirsuta, DOF AFFECTING GERMINATION1, gibberellins, light, seed germination,
• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Cardamine * genetics   metabolism   physiology   radiation effects   growth & development   MeSH
• Gibberellins * metabolism   MeSH
• Germination * radiation effects   genetics   physiology   MeSH
• Arabidopsis Proteins metabolism   genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Seeds * growth & development   genetics   radiation effects   metabolism   MeSH
• Light MeSH
• Transcription Factors metabolism   genetics   MeSH
• Feedback, Physiological MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Gibberellins * MeSH
• Arabidopsis Proteins MeSH
• Transcription Factors MeSH

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.

• Keywords
• Desiccation, Legume, Maturation, Phytohormones, Pigmentation, Seed-coat,
• MeSH
• Arabidopsis * genetics   MeSH
• Domestication MeSH
• Gibberellins metabolism   MeSH
• Pisum sativum genetics   metabolism   MeSH
• Germination MeSH
• Abscisic Acid * metabolism   MeSH
• Seeds MeSH
• Plant Dormancy genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Gibberellins MeSH
• Abscisic Acid * MeSH

The view on the role of light during seed germination stems mainly from studies with Arabidopsis (Arabidopsis thaliana), where light is required to initiate this process. In contrast, white light is a strong inhibitor of germination in other plants, exemplified by accessions of Aethionema arabicum, another member of Brassicaceae. Their seeds respond to light with gene expression changes of key regulators converse to that of Arabidopsis, resulting in opposite hormone regulation and prevention of germination. However, the photoreceptors involved in this process in A. arabicum remain unknown. Here, we screened a mutant collection of A. arabicum and identified koy-1, a mutant that lost light inhibition of germination due to a deletion in the promoter of HEME OXYGENASE 1, the gene for a key enzyme in the biosynthesis of the phytochrome chromophore. koy-1 seeds were unresponsive to red- and far-red light and hyposensitive under white light. Comparison of hormone and gene expression between wild type and koy-1 revealed that very low light fluence stimulates germination, while high irradiance of red and far-red light is inhibitory, indicating a dual role of phytochromes in light-regulated seed germination. The mutation also affects the ratio between the 2 fruit morphs of A. arabicum, suggesting that light reception via phytochromes can fine-tune several parameters of propagation in adaptation to conditions in the habitat.

• MeSH
• Arabidopsis * metabolism   MeSH
• Brassicaceae * genetics   MeSH
• Phytochrome * genetics   metabolism   MeSH
• Hormones metabolism   MeSH
• Germination genetics   MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Seeds genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phytochrome * MeSH
• Hormones MeSH
• Arabidopsis Proteins * MeSH

Dormancy and heteromorphism are innate seed properties that control germination timing through adaptation to the prevailing environment. The degree of variation in dormancy depth within a seed population differs considerably depending on the genotype and maternal environment. Dormancy is therefore a key trait of annual weeds to time seedling emergence across seasons. Seed heteromorphism, the production of distinct seed morphs (in color, mass or other morphological characteristics) on the same individual plant, is considered to be a bet-hedging strategy in unpredictable environments. Heteromorphic species evolved independently in several plant families and the distinct seed morphs provide an additional degree of variation. Here we conducted a comparative morphological and molecular analysis of the dimorphic seeds (black and brown) of the Amaranthaceae weed Chenopodium album. Freshly harvested black and brown seeds differed in their dormancy and germination responses to ambient temperature. The black seed morph of seedlot #1 was dormant and 2/3rd of the seed population had non-deep physiological dormancy which was released by after-ripening (AR) or gibberellin (GA) treatment. The deeper dormancy of the remaining 1/3rd non-germinating seeds required in addition ethylene and nitrate for its release. The black seeds of seedlot #2 and the brown seed morphs of both seedlots were non-dormant with 2/3rd of the seeds germinating in the fresh mature state. The dimorphic seeds and seedlots differed in testa (outer seed coat) thickness in that thick testas of black seeds of seedlot #1 conferred coat-imposed dormancy. The dimorphic seeds and seedlots differed in their abscisic acid (ABA) and GA contents in the dry state and during imbibition in that GA biosynthesis was highest in brown seeds and ABA degradation was faster in seedlot #2. Chenopodium genes for GA and ABA metabolism were identified and their distinct transcript expression patterns were quantified in dry and imbibed C. album seeds. Phylogenetic analyses of the Amaranthaceae sequences revealed a high proportion of expanded gene families within the Chenopodium genus. The identified hormonal, molecular and morphological mechanisms and dormancy variation of the dimorphic seeds of C. album and other Amaranthaceae are compared and discussed as adaptations to variable and stressful environments.

• Keywords
• abscisic acid, coat-imposed dormancy, gibberellins, hormone metabolism, seed coat properties, seed heteromorphism, thermal time modelling, weed seed bank,
• 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

BACKGROUND: Karrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported. RESULTS: We present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the validation of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the successful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l). CONCLUSIONS: Our results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development.

• Keywords
• Karrikins, Smoke water, Stable isotope dilution method, Stable isotope labelled standard, Tandem mass spectrometry (MS/MS), Ultra-high performance liquid chromatography (UHPLC),
• Publication type
• Journal Article MeSH

Carotenoids are important isoprenoids produced in the plastids of photosynthetic organisms that play key roles in photoprotection and antioxidative processes. β-Carotene is generated from lycopene by lycopene β-cyclase (LCYB). Previously, we demonstrated that the introduction of the Daucus carota (carrot) DcLCYB1 gene into tobacco (cv. Xanthi) resulted in increased levels of abscisic acid (ABA) and especially gibberellins (GAs), resulting in increased plant yield. In order to understand this phenomenon prior to exporting this genetic strategy to crops, we generated tobacco (Nicotiana tabacum cv. Petit Havana) mutants that exhibited a wide range of LCYB expression. Transplastomic plants expressing DcLCYB1 at high levels showed a wild-type-like growth, even though their pigment content was increased and their leaf GA1 content was reduced. RNA interference (RNAi) NtLCYB lines showed different reductions in NtLCYB transcript abundance, correlating with reduced pigment content and plant variegation. Photosynthesis (leaf absorptance, Fv/Fm, and light-saturated capacity of linear electron transport) and plant growth were impaired. Remarkably, drastic changes in phytohormone content also occurred in the RNAi lines. However, external application of phytohormones was not sufficient to rescue these phenotypes, suggesting that altered photosynthetic efficiency might be another important factor explaining their reduced biomass. These results show that LCYB expression influences plant biomass by different mechanisms and suggests thresholds for LCYB expression levels that might be beneficial or detrimental for plant growth.

• Keywords
• Nicotiana tabacum cv. Petit Havana, RNAi, biomass, carotenoids, lycopene β-cyclase, photosynthesis, phytohormones, transplastomic, β-Carotene,
• MeSH
• Plants, Genetically Modified genetics   metabolism   MeSH
• Intramolecular Lyases * genetics   metabolism   MeSH
• Carotenoids MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Proteins genetics   metabolism   MeSH
• Nicotiana * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Intramolecular Lyases * MeSH
• Carotenoids MeSH
• lycopene cyclase-isomerase MeSH Browser
• Plant Proteins 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

Salinity and phosphorus (P) deficiency are among the most serious soil factors constraining crop productivity. A proposed strategy for alleviating these stresses is supporting plants by inoculation with growth-promoting rhizobacteria (PGPR). Here, a comparison of the ability of two maize composite and two F1 hybrid varieties to tolerate a P deficiency in either a saline or a non-saline environment showed that the uptake of nutrients by all four entries was significantly reduced by the imposition of both soil salinity and P deficiency, and that their growth was compromised to a similar extent. Subsequently, the ameliorative effect of inoculation with three strains of either Arthrobacter sp. or Bacillus sp. in an environment, which suffered simultaneously from salinity and P deficiency, was investigated. Inoculation with each of the strains was found to limit the plants' uptake of sodium cations, to increase their uptake of potassium cations, and to enhance their growth. The extent of the growth stimulation was more pronounced for the composite varieties than for the F1 hybrid ones, although the amount of biomass accumulated by the latter, whether the plants had been inoculated or not, was greater than that of the former varieties. When the bacterial strains were cultured in vitro, each of them was shown as able to produce the phytohormones auxin, abscisic acid, gibberellins, and cytokinins. The implication is that since the presence in the rhizospere of both Arthrobacter sp. and Bacillus sp. strains can support the growth of maize in salinity-affected and P deficient soils in a genotype-dependent fashion, it is important to not only optimize the PGPR strain used for inoculation, but also to select maize varieties which can benefit most strongly from an association with these bacteria.

• Keywords
• P deficiency, bacterial inoculation, maize, phytohormone production, plant growth, salinity stress,
• Publication type
• Journal Article MeSH