D27-like carotenoid isomerases: at the crossroads of strigolactone and abscisic acid biosynthesis.

Strigolactones and abscisic acid (ABA) are apocarotenoid-derived plant hormones. Their biosynthesis starts with the conversion of trans-carotenes into cis forms, which serve as direct precursors. Iron-containing DWARF27 isomerases were shown to catalyse or contribute to the trans/cis conversions of these precursor molecules. D27 converts trans-ß-carotene into 9-cis-ß-carotene, which is the first committed step in strigolactone biosynthesis. Recent studies found that its paralogue, D27-LIKE1, also catalyses this conversion. A crucial step in ABA biosynthesis is the oxidative cleavage of 9-cis-violaxanthin and/or 9-cis-neoxanthin, which are formed from their trans isomers by unknown isomerases. Several lines of evidence point out that D27-like proteins directly or indirectly contribute to 9-cis-violaxanthin conversion, and eventually ABA biosynthesis. Apparently, the diversity of D27-like enzymatic activity is essential for the optimization of cis/trans ratios, and hence act to maintain apocarotenoid precursor pools. In this review, we discuss the functional divergence and redundancy of D27 paralogues and their potential direct contribution to ABA precursor biosynthesis. We provide updates on their gene expression regulation and alleged Fe-S cluster binding feature. Finally, we conclude that the functional divergence of these paralogues is not fully understood and we provide an outlook on potential directions in research.

Heat stress during male meiosis impairs cytoskeletal organization, spindle assembly and tapetum degeneration in wheat.

The significance of heat stress in agriculture is ever-increasing with the progress of global climate changes. Due to a negative effect on the yield of staple crops, including wheat, the impairment of plant reproductive development triggered by high ambient temperature became a restraint in food production. Although the heat sensitivity of male meiosis and the following gamete development in wheat has long been recognized, a detailed structural characterization combined with a comprehensive gene expression analysis has not been done about this phenomenon. We demonstrate here that heat stress severely alters the cytoskeletal configuration, triggers the failure of meiotic division in wheat. Moreover, it changes the expression of genes related to gamete development in male meiocytes and the tapetum layer in a genotype-dependent manner. 'Ellvis', a heat-tolerant winter wheat cultivar, showed high spikelet fertility rate and only scarce structural aberrations upon exposure to high temperature. In addition, heat shock genes and genes involved in scavenging reactive oxygen species were significantly upregulated in 'Ellvis', and the expression of meiosis-specific and major developmental genes showed high stability in this cultivar. In the heat-sensitive 'Mv 17-09', however, genes participating in cytoskeletal fiber nucleation, the spindle assembly checkpoint genes, and tapetum-specific developmental regulators were downregulated. These alterations may be related to the decreased cytoskeleton content, frequent micronuclei formation, and the erroneous persistence of the tapetum layer observed in the sensitive genotype. Our results suggest that understanding the heat-sensitive regulation of these gene functions would be an essential contribution to the development of new, heat-tolerant cultivars.

D27-LIKE1 isomerase has a preference towards trans/cis and cis/cis conversions of carotenoids in Arabidopsis.

Carotenoids contribute to a variety of physiological processes in plants, functioning also as biosynthesis precursors of ABA and strigolactones (SLs). SL biosynthesis starts with the enzymatic conversion of all-trans-ß-carotene to 9-cis-ß-carotene by the DWARF27 (D27) isomerase. In Arabidopsis, D27 has two closely related paralogs, D27-LIKE1 and D27-LIKE2, which were predicted to be ß-carotene-isomerases. In the present study, we characterised D27-LIKE1 and identified some key aspects of its physiological and enzymatic functions in Arabidopsis. d27-like1-1 mutant does not display any strigolactone-deficient traits and exhibits a substantially higher 9-cis-violaxanthin content, which is accompanied by a slightly higher ABA level. In vitro feeding assays with recombinant D27-LIKE1 revealed that the protein exhibits affinity to all ß-carotene isoforms but with an exclusive preference towards trans/cis conversions and the interconversion between 9-cis, 13-cis and 15-cis-ß-carotene forms, and accepts zeaxanthin and violaxanthin as substrates. Finally, we present evidence showing that D27-LIKE1 mRNA is phloem mobile and D27-LIKE1 is an ancient isomerase with a long evolutionary history. In summary, we demonstrate that D27-LIKE1 is a carotenoid isomerase with multi-substrate specificity and has a characteristic preference towards the catalysation of cis/cis interconversion of carotenoids. Therefore, D27-LIKE1 is a potential regulator of carotenoid cis pools and, eventually, SL and ABA biosynthesis pathways.

KARRIKIN INSENSITIVE2 regulates leaf development, root system architecture and arbuscular-mycorrhizal symbiosis in Brachypodium distachyon.

KARRIKIN INSENSITIVE2 (KAI2) is an α/ß-hydrolase required for plant responses to karrikins, which are abiotic butenolides that can influence seed germination and seedling growth. Although represented by four angiosperm species, loss-of-function kai2 mutants are phenotypically inconsistent and incompletely characterised, resulting in uncertainties about the core functions of KAI2 in plant development. Here we characterised the developmental functions of KAI2 in the grass Brachypodium distachyon using molecular, physiological and biochemical approaches. Bdkai2 mutants exhibit increased internode elongation and reduced leaf chlorophyll levels, but only a modest increase in water loss from detached leaves. Bdkai2 shows increased numbers of lateral roots and reduced root hair growth, and fails to support normal root colonisation by arbuscular-mycorrhizal (AM) fungi. The karrikins KAR1 and KAR2 , and the strigolactone (SL) analogue rac-GR24, each elicit overlapping but distinct changes to the shoot transcriptome via BdKAI2. Finally, we show that BdKAI2 exhibits a clear ligand preference for desmethyl butenolides and weak responses to methyl-substituted SL analogues such as GR24. Our findings suggest that KAI2 has multiple roles in shoot development, root system development and transcriptional regulation in grasses. Although KAI2-dependent AM symbiosis is likely conserved within monocots, the magnitude of the effect of KAI2 on water relations may vary across angiosperms.

Comprehensive Analysis of DWARF14-LIKE2 (DLK2) Reveals Its Functional Divergence from Strigolactone-Related Paralogs.

Strigolactones (SLs) and related butenolides, originally identified as active seed germination stimulants of parasitic weeds, play important roles in many aspects of plant development. Two members of the D14 α/ß hydrolase protein family, DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are essential for SL/butenolide signaling. The third member of the family in Arabidopsis, DWARF 14-LIKE2 (DLK2) is structurally very similar to D14 and KAI2, but its function is unknown. We demonstrated that DLK2 does not bind nor hydrolyze natural (+)5-deoxystrigol [(+)5DS], and weakly hydrolyzes non-natural strigolactone (-)5DS. A detailed genetic analysis revealed that DLK2 does not affect SL responses and can regulate seedling photomorphogenesis. DLK2 is upregulated in the dark dependent upon KAI2 and PHYTOCHROME INTERACTING FACTORS (PIFs), indicating that DLK2 might function in light signaling pathways. In addition, unlike its paralog proteins, DLK2 is not subject to rac-GR24-induced degradation, suggesting that DLK2 acts independently of MORE AXILLARY GROWTH2 (MAX2); however, regulation of DLK2 transcription is mostly accomplished through MAX2. In conclusion, these data suggest that DLK2 represents a divergent member of the DWARF14 family.

Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment.

Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid.

Molecular aspects of the antagonistic interaction of smoke-derived butenolides on the germination process of Grand Rapids lettuce (Lactuca sativa) achenes.

Smoke-derived compounds provide a strong chemical signal to seeds in the soil seed bank, allowing them to take advantage of the germination niche created by the occurrence of fire. The germination stimulatory activity of smoke can largely be attributed to karrikinolide (KAR(1) ), while a related compound, trimethylbutenolide (TMB), has been shown to have an inhibitory effect on germination. The aim of this study was to characterize the interaction of these potent fire-generated compounds. Dose-response analysis, leaching tests and a detailed transcriptome study were performed using highly KAR(1) -sensitive lettuce (Lactuca sativa cv 'Grand Rapids') achenes. Dose-response analysis demonstrated that the compounds are not competitors and TMB modulates germination in a concentration-dependent manner. The transcriptome analysis revealed a contrasting expression pattern induced by the compounds. KAR(1) suppressed, while TMB up-regulated ABA, seed maturation and dormancy-related transcripts. The effect of TMB was reversed by leaching the compound, while the KAR(1) effect was only reversible by leaching within the first 2 h of KAR(1) treatment. Our findings suggest that the compounds may act in concert for germination-related signaling. After the occurrence of fire, sufficient rainfall would contribute to post-germination seedling recruitment by reducing the concentration of the inhibitory compound.

Effect of light on the gene expression and hormonal status of winter and spring wheat plants during cold hardening.

The effect of light on gene expression and hormonal status during the development of freezing tolerance was studied in winter wheat (Triticum aestivum var. Mv Emese) and in the spring wheat variety Nadro. Ten-day-old plants (3-leaf stage) were cold hardened at 5°C for 12 days under either normal (250 µmol m(-2) s(-1) ) or low (20 µmol m(-2) s(-1) ) light conditions. Comprehensive analysis was carried out to explore the background of frost tolerance and the differences between these wheat varieties. Global genome analysis was performed, enquiring about the details of the cold signaling pathways. The expression level of a large number of genes is affected by light, and this effect may differ in different wheat genotypes. Photosynthesis-related processes probably play a key role in the enhancement of freezing tolerance; however, there are several other genes whose induction is light-dependent, so either there is cross-talk between signaling of chloroplast originating and other protective mechanisms or there are other light sensors that transduce signals to the components responsible for stress tolerance. Changes in the level of both plant hormones (indole-3-acetic acid, cytokinins, nitric oxide and ethylene precursor 1-aminocyclopropane-1-carboxylic acid) and other stress-related protective substances (proline, phenolics) were investigated during the phases of the hardening period. Hormonal levels were also affected by light and their dynamics indicate that wheat plants try to keep growing during the cold-hardening period. The data from this experiment may provide a new insight into the cross talk between cold and light signaling in wheat.

Salicylic acid treatment of pea seeds induces its de novo synthesis.

Salicylic acid (SA), which is known as a signal molecule in the induction of defense mechanisms in plants, could be a promising compound for the reduction of stress sensitivity. The aim of the present work was to investigate the distribution of SA in young pea (Pisum sativum L.) seedlings grown from seeds soaked in (3)H-labeled SA solution before sowing, and to study the physiological changes induced by this seed treatment. The most pronounced changes in SA levels occurred in the epicotyl and the seeds. Radioactivity was detected only in the bound form of SA, the majority of which was localized in the seeds, and only a very low level of radioactivity was detected in the epicotyl. SA pre-treatment increased the expression of the chorismate synthase and isochorismate synthase genes in the epicotyl. Pre-soaking the seeds in SA increased the activities of some antioxidant enzymes, namely ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) and the level of ortho-hydroxycinnamic acid, but decreased the level of polyamines. These results suggest that the increased level of free and bound SA detected in plants growing from seeds soaked in SA solution before sowing is the product of de novo synthesis, rather than having been taken up and mobilized by the plants.

Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR1.

BACKGROUND: Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR1), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR1 action. RESULTS: In this paper we demonstrate that although smoke-water and KAR1 treatment of maize kernels result in a similar physiological response, the gene expression and the protein ubiquitination patterns are quite different. Treatment with smoke-water enhanced the ubiquitination of proteins and activated protein-degradation-related genes. This effect was completely absent from KAR1-treated kernels, in which a specific aquaporin gene was distinctly upregulated. CONCLUSIONS: Our findings indicate that the array of bioactive compounds present in smoke-water form an environmental signal that may act together in germination stimulation. It is highly possible that the smoke/KAR1 'signal' is perceived by a receptor that is shared with the signal transduction system implied in perceiving environmental cues (especially stresses and light), or some kind of specialized receptor exists in fire-prone plant species which diverged from a more general one present in a common ancestor, and also found in non fire-prone plants allowing for a somewhat weaker but still significant response. Besides their obvious use in agricultural practices, smoke and KAR1 can be used in studies to gain further insight into the transcriptional changes during germination.

Stress-related genes define essential steps in the response of maize seedlings to smoke-water.

Smoke from burning vegetation is widely recognised as a germination cue for seed germination and recent reports suggest that smoke treatments can improve seedling vigour also. We investigated the effect of smoke-water on seedling vigour and changes of the global transcriptome in the early post-germination phase in maize. Application of smoke-water improved the germination characteristics and seedling vigour. The transcriptional response of embryos and emerging radicles 24 and 48 h after the onset of smoke treatment was investigated. The microarray study revealed a number of smoke-responsive genes amongst which stress- and abscisic acid (ABA)-related genes were over-represented. The global promoter analysis of the smoke-responsive genes revealed a tight correlation with the results obtained from Gene Ontology annotations. This concerted over-expression shows that smoke treatment induces stress and ABA-related responses in the early post-germination phase which leads to better adaptation to environmental stress factors occurring during germination, eventually resulting in greater seedling vigour.

Ferritin2 gene in paraquat-susceptible and resistant biotypes of horseweed Conyza canadensis (L.) Cronq.

Ferritins, the multimeric iron storage proteins, are the main regulators of the cellular level of uncomplexed iron. Ferritins are encoded by small gene families and expressed differentially under various developmental conditions depending on iron availability, effect of hormones or oxygen radical generating agents. In the present work the primary structure of the ferritin2 gene from resistant and susceptible biotypes of horseweed Conyza canadensis was determined. This gene was found to exhibit great similarity and possess all the structural characteristics of known plant ferritin2 genes. The C. canadensis ferritin2 genes had identical primary structure in the two biotypes and were upregulated by paraquat (Pq) in both susceptible and resistant plants. The enhanced expression level was probably connected with defence reactions in the plants after Pq treatment.