I V Pokotylo

S V Kretynin

V A Khripach

E Ruelland

Ya B Blume

V S Kravets
email: kravets@bpci.kiev.ua

Á S V Kretynin

Influence of 24-epibrassinolide on lipid signalling and metabolism in Brassica napus

Keywords: 24-Epibrassinolide, Oil productivity, Salt stress, Fatty acids, Phospholipase D

Due to the increasing demand for biofuel production, it is an important goal to optimize the seed productivity and quality of oilseed plants even in adverse conditions. Acting on signalling mechanisms might provide means to attain such goals. In this study, we were interested in the effect of a brassinosteroid hormone 24-epibrassinolide (24-EBR) on Brassica napus cultivated in salt stress condition. We show that salt stress leads to a 60 % decrease in seed production in B. napus. This is accompanied by a 50 % decrease in seed oil content. Treatment with 24-EBR had no effect on seed and oil productivity in control plants. However, it could rescue half of the seed production and all the oil production in salt-treated plants. The fatty acid composition of seed oil in B. napus was selectively affected by salt stress, 24-EBR or combined treatment. Besides these long-term actions of 24-EBR, we have also investigated its short-term actions in cell signalling. We did so by in vivo labelling of plantlets with fluorescently labelled phosphatidylcholine. A treatment of 2 h with 24-EBR was sufficient to induce a substantial increase in the content of diacylglycerol and phosphatidic acid, two lipid mediators. Nonspecific phospholipases C and phospholipases D are involved in these increases. Therefore, brassinosteroid treatments appear as promising way to gain oil productivity when plants have to grow in unfavourable conditions such as salt stress. The link between long-term actions and shortterm signalling of 24-EBR is discussed.

Introduction

Plant oil is now considered as one of the main sources of biorenewable fuels. In the majority of plants, oil is stored in seeds as triacylglycerols (TAG) that will serve as an energy reserve required for the initial growth of seedlings in the absence of established photosynthesis. In plants, TAG biosynthesis pathway is known to be complex (Fig. 1). All fatty acids (FA) initially originate from a pool of acetylcoenzyme A (CoA) that is located in the plastids. Then, plastid-localized and endoplasmic reticulum-localized enzymes participate in the biosynthesis of all main cell glycerolipids, including storage TAGs [START_REF] Voelker | Variations in the biosynthesis of seed-storage lipids[END_REF][START_REF] Chapman | Compartmentation of triacylglycerol accumulation in plants[END_REF]. Glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic-acid acyltransferase (LPAAT) and diacylglycerol acyltransferase (DGAT), that catalyse the consecutive acylation steps of the and cellular responses [START_REF] Clouse | Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulating plant development[END_REF]. Receptor-independent effects of lipophilic BRs on plant metabolism and plant membranes have also been suggested [START_REF] Albrecht | Brassinosteroids inhibit pathogen-associated molecular patterntriggered immune signaling independent of the receptor kinase BAK1[END_REF][START_REF] Zhang | Brassinosteroids regulate plasma membrane anion channels in addition to proton pumps during expansion of Arabidopsis thaliana cells[END_REF]. BRs stimulate plant growth and photosynthesis [START_REF] Xia | Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in Cucumis sativus[END_REF]) and affect activity of antioxidant systems [START_REF] Zhang | ZmMPK5 is required for the NADPH oxidase-mediated selfpropagation of apoplastic H 2 O 2 in brassinosteroid-induced antioxidant defence in leaves of maize[END_REF]. BRs can also increase plant stress tolerance [START_REF] Divi | Brassinosteroid-mediated stress tolerance in arabidopsis shows interactions with abscisic acid, ethylene and salicylic acid pathways[END_REF]) and improve B. napus germination and growth in drought and saline conditions [START_REF] Kagale | Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses[END_REF]). The role of BRs in stomata development has also been demonstrated [START_REF] Kim | Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway[END_REF] and may be crucial for water-use efficiency in salt-stressed plants. However, the exact mechanisms of BR-induced plant regulation are unclear.

The intracellular action of BRs may be functionally associated with phospholipases that control many plant growth reactions [START_REF] Wang | Plant phospholipases: an overview. Lipases and phospholipases[END_REF]. Phospholipases hydrolyse membrane lipids to produce second messengers essential for regulatory signalling. Phospholipases also contribute to generic lipid metabolism and lipid turnover [START_REF] Li | Quantitative profiling of Arabidopsis polar glycerolipids in response to phosphorus starvation. Roles of phospholipases Df1 and Df2 in phosphatidylcholine hydrolysis and digalactosyldiacylglycerol accumulation in phosphorus-starved plants[END_REF]) including TAG biosynthesis [START_REF] Lee | Phospholipid and triacylglycerol profiles modified by PLD suppression in soybean seed[END_REF]). In addition, phospholipases are well known to participate in stress and hormone signalling [START_REF] Janda | Phosphoglycerolipids are master players in plant hormone signal transduction[END_REF]. As an example, phosphatidic acid (PA) production via phospholipase D activity was rapidly induced in saltstressed tobacco [START_REF] Pokotylo | Role of phospholipase D in metabolic reactions of transgenic tobacco cax1 cells under the influence of salt stress[END_REF]) while overexpression of phospholipase Da resulted in improved drought and salt tolerance of plants [START_REF] Peng | Overexpression of a PLDa1 gene from Setaria italica enhances the sensitivity of Arabidopsis to abscisic acid and improves its drought tolerance[END_REF][START_REF] Zhang | Overexpression of phospholipase Da gene enhances drought and salt tolerance of Populus tomentosa[END_REF]. Interestingly, plant non-specific phospholipases C (NPC), which are able to directly cleave structural membrane phospholipids to produce diacylglycerol (DAG) [START_REF] Pokotylo | The plant non-specific phospholipase C gene family. Novel competitors in lipid signalling[END_REF], were activated after BR treatment in tobacco BY-2 cells [START_REF] Wimalasekera | Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana[END_REF].

Therefore, the aim of this study was to investigate the influence of 24-EBR on oil productivity and fatty acid composition of seed oil from B. napus plants subjected to salt stress. The role of phospholipases in mediating BR effects is also analysed.

Materials and methods

Plant material and treatments

Brassica napus cv. Magnat plants and Arabidopsis thaliana

Col-1 plants were used in this study. Seeds were imbibed and cold treated at 4 °C for 4 days before germination and growth. B. napus plants used in cultivation experiments were grown open-air in isolated vessels in alfisol-type soil subjected to temperate climate conditions with controlled watering. Arabidopsis plants were grown in soil under long day conditions (16 h light 125 lE m -2 s -1 ; 8 h dark) at 23 °C. Salt stress was induced by watering with 0.5 M NaCl solution to achieve final concentration of 50 mM Metabolic steps affected by salt stress (S), brassinosteroids (BR) or controlled by phospholipase activity are highlighted [START_REF] Kocourkova | The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress[END_REF][START_REF] Wimalasekera | Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana[END_REF][START_REF] Pokotylo | Role of phospholipase D in metabolic reactions of transgenic tobacco cax1 cells under the influence of salt stress[END_REF]). DAG, diacylglycerol; DGAT, DAG acyltransferase; DGK, diacylglycerol kinase; G3P, glycerol 3-phosphate; FFA, free fatty acids; G3P, glycerol 3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPAAT, lysophosphatidic acid acyltransferase; PA, phosphatidic acid; PAP, phosphatidic acid phosphohydrolase; PC, phosphatidylcholine; PLD, phospholipase D; NPC, non-specific phospholipase C; PLA 2 , phospholipase A2; TAG, triacylglycerol; CPT, choline phosphotransferase; PDAT, phospholipid:diacylglycerol acyltransferase glycerol-3-phosphate backbone, are all critical for TAG biosynthesis. Alternatively, the sn-3 acylation of DAG can be achieved by the combined actions of plant-specific choline phosphotransferases (CPT) and phospholipid:diacylglycerol acyltransferases (PDAT). Most of these pathways have been characterized both at biochemical and molecular levels [START_REF] Beisson | Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a webbased database[END_REF]). Yet, how plants achieve an efficient metabolic biosynthesis in adverse conditions is not entirely understood.

Nevertheless, there have been numbers of attempts to develop crop plants with improved oil yield or providing oils with specific FA composition. Several biotechnological approaches were implemented including genetic engineering aimed at manipulating the lipid/FA biosynthesis pathways [START_REF] Tan | Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds[END_REF]Ruiz-López et al. 2012;[START_REF] Bates | The significance of different diacylglycerol synthesis pathways on plant oil composition and bioengineering[END_REF]. However, it is becoming clearer and clearer that oil productivity is a complex trait controlled by several enzymatic complexes [START_REF] Tang | Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation[END_REF]. That is why affecting individual enzymes in lipid biosynthesis pathways may not be as effective as originally expected [START_REF] Ohlrogge | Regulation of fatty acid synthesis[END_REF]. In contrast, treatments of plant with hormones, that orchestrate plant development and influence major metabolic fluxes, might be a promising way of modulating oil quantity and quality in seeds. Indeed, synthetic cytokinins were shown to affect oil accumulation in Lavandula dentata plantlets [START_REF] Sudria | Effect of benzyladenine and indolebutyric acid on ultrastructure, glands formation, and essential oil accumulation in Lavandula dentata plantlets[END_REF]) and rhizobacteria-derived auxins increased the oil quantity in Brassica juncea [START_REF] Asghar | Relationship between in vitro production of auxins by rhizobacteria and their growth-promoting activities in Brassica juncea L[END_REF]. Brassinosteroids (BRs) constitute a unique group of plant steroid growth-promoting hormones that, at low concentrations, act as important metabolism regulators [START_REF] Kim | Brassinosteroid signal transduction from receptor kinases to transcription factors[END_REF]. BRs, that include 24-epibrassinolide (24-EBR), are sensed by BRASSINOSTEROID-INSENSITIVE 1 receptor kinase and activate distinct signal transduction cascade NaCl in the soil. Chemically synthesized 24-epibrassinolide was obtained from the Institute of Bioorganic Chemistry of the National academy of Sciences of Belarus [START_REF] Khripach | Synthesis of brassinosteroids[END_REF]). Leaves of B. napus plants were sprayed with 0.1 lM solution of 24-EBR twice during the course of cultivation.

Brassica napus plants used in short-term signalling experiments were grown hydroponically in half-strength MS solution. N-ethylmaleimide and n-butanol were used at final concentrations of 5 mM and 0.5 % (v/v), respectively.

Analysis of seed oil content and FA composition

Total seed oil was extracted by the addition of 10 ml of hexane to 500 mg of freshly ground seeds followed by vacuum evaporation. FA composition of lipid samples was assessed by HPLC analysis using the method described by [START_REF] Chaytor | Analysis of fatty acids in lipids by high performance liquid chromatography (HPLC)[END_REF] with modifications. In brief, lipids were saponificated with butylated hydroxytoluene in 1 M solution of KOH in methanol over a period of 1 h at 80-90 °C. The solution thus obtained was diluted with tetrahydrofuran or acetonitrile and neutralized with 1 M orthophosphoric acid. Then, 0.2 M 18-crown-6 and 0.2 M n-bromophenacyl bromide were added to form FA conjugates. FA separation was performed on Agilent 1100 series HPLC with UV-Vis diode array detector. Linear gradient elution with 0.05 M orthophosphoric acid and methanol on Agilent ZORBAX Eclipse XDB-C 18 5 lm and Thermo Scientific Hypersil BDS, C 18 3 lm reverse phase columns was used. Absorbance peaks corresponding to individual FAs were identified using standards.

Analysis of fluorescently-labelled products of phosphatidylcholine cleavage

Roots of B. napus plantlets were preincubated with 0.66 lg ml Invitrogen) for 10 min at 24 °C on an orbital shaker prior to treatment with reagents. At the end of the experiment lipids were extracted by the addition of methanol:chloroform 2:1 (v/v). Changes in the production of phospholipidderived second messengers were monitored using a thin layer chromatography (TLC). Unmodified silica gel 60 TLC plates (Merck) were developed with upper organic phase of the ethyl acetate/isooctane/formic acid/water (12:2:3:10, v/v) mixture. Lipid spots were visualized and counted by Pharos FX (Biorad) molecular imager. Products were identified according to [START_REF] Pejchar | Aluminium ions inhibit the formation of diacylglycerol generated by phosphatidylcholine-hydrolysing phospholipase C in tobacco cells[END_REF] and using standards.

-1 boron-dipyrromethene (BODIPY) labelled phosphatidylcholine (BODIPY-2-decanoyl-1-(O-(11-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- propionyl)amino)undecyl)-sn-glycero-3-phosphocholine,

Results and discussion

24-Epibrassinolide application alleviates productivity loss in salt-stressed B. napus plants Brassica napus are nowadays accounted to be the third most grown oil crop. We have analysed seed productivity and seed oil content of B. napus cv. Magnat plants under normal growth conditions and under salt stress. Plants were germinated and grown for 7 days. Then, a designated group of plants were watered with NaCl solution to achieve a final soil salt concentration of 50 mM NaCl. Seeds were collected in dry siliques and weighted. The seed oil was extracted and assayed for quantity and quality. Our results indicate that seed productivity of B. napus was reduced by two thirds when cultivated in salt stress conditions. Oil content of seeds obtained from plants grown in salt conditions was also significantly decreased (Fig. 2), suggesting severe inhibition of lipid metabolic pathways.

It is well known that salt stress causes an inhibition of photosynthesis, of generic metabolism and of viability [START_REF] Evers | Towards a synthetic view of potato cold and salt stress response by transcriptomic and proteomic analyses[END_REF]. Though it was shown that salt stress can trigger oil accumulation in growth-inhibited Chlamydomonas reinhardtii algae [START_REF] Siaut | Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves[END_REF], in case of oilseed plants its action is typically manifested by a dramatic loss of oil productivity [START_REF] Francois | Growth, seed yield, and oil content of canola grown under saline conditions[END_REF].

In contrast, BRs are known to enhance seed oil accumulation. For instance, end-point increase in oil content induced by tissue-specific photosynthesis in silique walls can be mimicked by application of BRs [START_REF] Hua | Maternal control of seed oil content in Brassica napus: the role of silique wall photosynthesis[END_REF]. In our studies, application of 24-EBR during germination and vegetative growth did not sizeably affected seed productivity or oil content of seeds obtained from control B. napus plants (Fig. 2). However, 24-EBR application restored the seed yield of salt-stressed plants to 60 % of that of control plants. Besides, seed oil content in such conditions was matching that of normally-grown plants (Fig. 2).

This indicates a role for 24-EBR in modulating plant metabolic reactions, thus leading to alleviation of stress influence and increased plant productivity. Two explanations of the observed effect of 24-EBR should be considered. One possibility is that 24-EBR optimises plant growth and increase photosynthesis efficiency during stress. Another possibility is that exogenous BR treatment might subdue some of the plant basal activities or developmental programmes for sake of increased productivity. However, germination rate of seeds obtained from BR-treated plants was unaffected (data not shown). Being lipophilic compounds, BRs may also be targeted to membranes where they contribute to stress-induced adaptation.

Influence of salt stress and 24-EBR treatments on fatty acid composition of seed oil in B. napus and A. thaliana model plant

The FA composition of seed oil is a decisive factor of oil quality and usability both for biodiesel production and food purposes. We have analysed FA composition of seed oil from Arabidopsis model plants and B. napus crop plants belonging to the same Brassicaceae family (Fig. 3). FA compositions of seed oil obtained from both plant species were not significantly affected by 24-EBR treatment when grown in auspicious conditions. In turn, in the salt-stressed Arabidopsis, an increase in 16:0 and 18:1 FAs content was accompanied by the decrease in other FAs abundance (Fig. 3). In B. napus changes of FA composition induced by salt stress were less pronounced with the slight decrease of 18:1 and 16:1 FAs content notable (Fig. 3). Several plant hormones are known to affect lipid FA composition. Auxins increased FA polyusnaturation ratio in phosphatidylcholine, triglycerides and free fatty acids, but not in glycolipids in cotyledons of soybean embryo [START_REF] Liu | Auxin-regulated changes of fatty acid content and composition in soybean zygotic embryo cotyledons[END_REF]. Gibberellins had influence on the level of saturated FA in phospholipids of heat-shocked aleurone layers of barley [START_REF] Grindstaff | Effect of gibberellin and heat shock on the lipid composition of endoplasmic reticulum in barley aleurone layers[END_REF]. In contrast, 24-EBR was previously reported to not influence FA composition in plant seeds [START_REF] Janeczko | 24-Epibrassinolide modifies seed composition in soybean, oilseed rape and wheat[END_REF]. However, in another study, BRs affected FA profile of polar lipids in mango fruits resulting in higher unsaturation degree beneficial in cold stress conditions [START_REF] Li | Brassinolide enhances cold stress tolerance of fruit by regulating plasma membrane proteins and lipids[END_REF]. We observed that 24-EBR application to plants subjected to salt stress have affected content of several FAs (Fig. 4). Hence, in the saltstressed B. napus 24-EBR treatment induced over-accumulation of 18:2 FA. In the salt-stressed Arabidopsis application of 24-EBR resulted in partial reversion of 18:1 and 20:1 FAs content to that of a control plants. This may possibly indicate weakening of stress impact in 24-EBRtreated plants, as also suggested by increased productivity (Fig. 1).

It is known that the relationship between fatty acid biosynthesis and stress responses is reciprocal in plants. As an example, FA desaturase was reported to be involved in salt tolerance in Arabidopsis [START_REF] Zhang | Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth[END_REF]) and changes in FA composition were demonstrated to be critical for maintaining membrane properties and fluidity in Lipid signalling is activated by 24-EBR and upon salt stress in B. napus Both abiotic and biotic stresses are known to evoke complex responses and defence reactions in plants [START_REF] Atkinson | The interaction of plant biotic and abiotic stresses: from genes to the field[END_REF]). Here we investigated whether 24-EBR and stress conditions stimulate lipid signalling pathways in B. napus. Roots of 7-day-old plantlets were labelled with fluorescently labelled BODIPY-phosphatidylcholine for 10 min [START_REF] Pejchar | Aluminium ions inhibit the formation of diacylglycerol generated by phosphatidylcholine-hydrolysing phospholipase C in tobacco cells[END_REF]. Then 100 mM NaCl or 1 lM 24-EBR were added in cultivation media for 2 h before extracting and separating lipids by thin layer chromatography (Fig. 4). Both treatments led to a substantial increase in fluorescent DAG and PA accumulation. This may suggest the activation of plant non-specific phospholipase C and/or phospholipase D enzymes that catalyse the hydrolysis of phosphatidylcholine (PC) into DAG and PA, respectively [START_REF] Hong | Phospholipase Da3 is involved in the hyperosmotic response in Arabidopsis[END_REF][START_REF] Kocourkova | The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress[END_REF][START_REF] Krinke | Phospholipase D activation is an early component of the salicylic acid signalling pathway in Arabidopsis cell suspensions[END_REF]. In our experiments we have also studied the effects of two substances that affect lipid signalling. N-ethylmaleimide (NEM) is a thiol-reactive inhibitor of phosphatidate phosphatases that perform PA dephosphorylation to DAG [START_REF] Furukawa-Stoffer | Identification of N-ethylmaleimide-sensitive andinsensitive phosphatidate phosphatase activity in microsporederived cultures of oilseed rape[END_REF]. In turn, n-butanol (But) is used by PLDs as a substrate in the so-called transphosphatidylation reaction [START_REF] Rainteau | Acyl chains of phospholipase D transphosphatidylation products in Arabidopsis cells: a study using multiple reactions monitoring mass spectrometry[END_REF]) thus leading to the production of phosphatidylbutanol (PBut) detrimentally to PA production. PBut production is thus considered as a marker of PLD activity [START_REF] Kravets | Rapid activation of specific phospholipase(s) D by cytokinin in Amaranthus assay system[END_REF]). In our investigations, we have observed limited or no effects of NEM on DAG and PA accumulation (Fig. 4). This indicates that DAG is likely to arise via a direct action of NPC on PC rather than via the dephosphorylation of PA. Treatment with n-butanol invoked slight decrease in PA production in salt stressed plants but not in plants treated with 24-EBR (Fig. 4). More importantly, these changes were markedly accompanied by the significant accumulation of PBut. To our knowledge, this is the first report demonstrating PLD activation in response to brassinosteroid treatment. Whether a post-translational [START_REF] Clouse | Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulating plant development[END_REF]. It was recently reported that expression of DREB2A is negatively regulated by the basal activities of phospholipase D and phospholipase C coupled to DGK [START_REF] Djafi | The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositidedependent phospholipase C coupled to diacylglycerol kinase in Arabidopsis thaliana[END_REF]. This is an indication of the links between DREB2A genetic pathway and lipid signalling. This opens the possibility that BRs affect DREB2A expression through altering lipid signalling. In support, expression of several phospholipase genes (namely PLDa1 and NPC4) is also responsive to BRs and salt stress stimuli, which suggests their implication in the control of plant stress responses. Biochemical cues are also indicating that NPC activity is sensitive to BRs application in plants [START_REF] Wimalasekera | Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana[END_REF]. It was also suggested that BRs act in concert with phospholipase A 2 -derived lysophosphatidylethanolamine phospholipid to regulate growth of primary roots and the formation of lateral roots in Arabidopsis [START_REF] Yoon Jeong | Effect of lysophosphatidylethanolamine and brassinosteroids on development of Arabidopsis roots[END_REF].

The integration of phospholipases to lipid metabolism is now well ascertained. PA derived presumably from PC, as a major constituent of plant membranes, is important for lipid turnover and tobacco pollen tube growth [START_REF] Pleskot | Turnover of phosphatidic acid through distinct signalling pathways affects multiple aspects of tobacco pollen tube tip growth[END_REF]. In turn, DAG molecules produced by NPC are common precursors to both membrane PC and storage TAGs [START_REF] Pokotylo | The plant non-specific phospholipase C gene family. Novel competitors in lipid signalling[END_REF]. Interestingly, phospholipases are abundant in seeds. PLD was shown to be active thaliana genes implicated to lipid metabolism and signalling that show responsiveness to salinity conditions or treatment with brassinosteroids (as shown in circle intersections). Genes that potentially facilitate BR-mediated regulation of lipid metabolism in salt stress conditions are marked in red [START_REF] Hruz | Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes[END_REF]. Common gene names are shown (for details see Electronic supplementary material Table 1). Experimental data used (GEO numbers): salt treatment AT-00120, brassinosteroid treatment AT-00110. Representative lipid metabolism genes were selected from [START_REF] Beisson | Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a webbased database[END_REF] activation of PLD or a regulation of PLD gene expression both contribute to the increased PLD activity in response to BRs is a question of further studies. It is also remain to be investigated whether BR treatment affects activity of diacylglycerol kinases that were identified as major contributors to PA production in plants [START_REF] Arisz | Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase[END_REF].

Lipid signalling is often regarded to be critical for plant stress-tolerance as was demonstrated for salt-sensitive PLD-deficient Arabidopsis mutants [START_REF] Hong | Phospholipase Da3 is involved in the hyperosmotic response in Arabidopsis[END_REF]. Our results indicate that both BR and salt stress activate unidirectional lipid signalling. The way plant cell distinguish related signals evoked by dissimilar stimuli is not well understood. It is thought that spatial and temporal parameters of the second-messenger production by different phospholipases isozymes are important. Different composition of fatty acid residues in such signalling molecules may also stand for dissimilarity in evoked cellular responses [START_REF] Maatta | Levels of Arabidopsis thaliana leaf phosphatidic acids, phosphatidylserines, and most trienoate-containing polar lipid molecular species increase during the dark period of the diurnal cycle[END_REF][START_REF] Rainteau | Acyl chains of phospholipase D transphosphatidylation products in Arabidopsis cells: a study using multiple reactions monitoring mass spectrometry[END_REF].

Plant lipid metabolism and oil accumulation is linked with BR/phospholipid signalling and plant stress responses

Here we provide an in silico analysis pointing at integration of plant lipid metabolism with BR/phospholipid signalling and stress responses at transcriptional level using Genevestigator interface [START_REF] Hruz | Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes[END_REF]. Rapeseed genome has been reported to be sequenced by Bayer Crop science. However, the size and complexity of allotetraploid B. Napus genome and the lack of available array data make it challenging to conduct efficient in silico studies for this species. Instead, we have analysed gene expression data of the taxonomically related model plant A. thaliana (Fig. 5). Numerous genes that are involved in the lipid metabolism have their expression modified in response to BR and salt stress. As an example, KCS1 and FAR1, genes encoding proteins implicated in FA biosynthesis, were identified to be BR-responsive [START_REF] Goda | Microarray analysis of brassinosteroid-regulated genes in Arabidopsis[END_REF]. Interestingly, the expression of 3-KETOACYL-COA SYN-THASE 9 (KCS9), GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED LIPID PROTEIN TRANSFER GENE 2 (LTPG2) and GDSL-LIKE LIPASE genes were repressed in salt stressed plants but activated after BR treatment. FATTY ACID REDUCTASE 1 (FAR1) gene demonstrated reverse regulatory pattern. In addition two key genes of fatty acid metabolism -FATA ACYL-ACP THIOESTERASE (FATA1) and FATTY ACID ELONGATION1 (FAE1) were affected by BR and salt stress unidirectional. These available data provide hints at putative targets of BR for controlling the lipid productivity in stress conditions.

BRs also affect the expression of several genes that are directly implicated in plant stress responses. Among them are DREB2A, RD29B and RAB18. This may suggest that BRs can affect plant lipid productivity indirectly by boosting throughout most parts of seed development while germination and storage TAGs composition was significantly affected in PLDa-deficient mutants of soybean [START_REF] Lee | Enhanced seed viability and lipid compositional changes during natural ageing by suppressing phospholipase Da in soybean seed[END_REF]. It is becoming apparent that not only phospholipases participate in immediate stress signalling but actively contribute to lipid turnover in plant leaves and, more importantly, seeds.

Conclusions

In summary, our results suggest that treatment of B. napus plants with BRs leads to the increased seed oil productivity in stress conditions. We have also observed that BRs and salt stress activate phospholipid signalling. This data suggest that phospholipases are among the cell components that mediate BRs signal. Gene expression analysis in publicly available database also provided a hint at an association of lipid metabolic processes with BRs and phospholipases. The question how BR signal is mediated downstream of lipid signalling implicating PLD and NPC phospholipases in plant cells is currently unanswered. Though some clues can be provided by the identification of DAG-sensitive or PA-sensitive intracellular targets [START_REF] Mcloughlin | Identification of novel candidate phosphatidic acid-binding proteins involved in the salt-stress response of Arabidopsis thaliana roots[END_REF].

Not only our results provide evidences for BRs role in the control of lipid signalling and lipid metabolism, but they grant basis for further research required for steady development of biofuel production.

Fig. 1

 1 Fig.1Simplified scheme of plant TAG biosynthesis pathways. Metabolic steps affected by salt stress (S), brassinosteroids (BR) or controlled by phospholipase activity are highlighted[START_REF] Kocourkova | The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress[END_REF][START_REF] Wimalasekera | Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana[END_REF][START_REF] Pokotylo | Role of phospholipase D in metabolic reactions of transgenic tobacco cax1 cells under the influence of salt stress[END_REF]). DAG, diacylglycerol; DGAT, DAG acyltransferase; DGK, diacylglycerol kinase; G3P, glycerol 3-phosphate; FFA, free fatty acids; G3P, glycerol 3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPAAT, lysophosphatidic acid acyltransferase; PA, phosphatidic acid; PAP, phosphatidic acid phosphohydrolase; PC, phosphatidylcholine; PLD, phospholipase D; NPC, non-specific phospholipase C; PLA 2 , phospholipase A2; TAG, triacylglycerol; CPT, choline phosphotransferase; PDAT, phospholipid:diacylglycerol acyltransferase

Fig. 2

 2 Fig. 2 Seed yield and seed oil content of Brassica napus cv. Magnat plants subjected to salt stress and 24-EBR. Plants were grown in soil substrate in individual containers. Salt stress was conditioned by adjusting soil NaCl concentration to 50 mM by watering. Selected plants were treated with 100 nM solution of 24-EBR by spraying during the course of germination and after 2 weeks of growth. At the end of plant vegetation, seeds were harvested, dried for 2 weeks and weighted. Seed oil was extracted from aliquot seed samples by solvents, evaporated and measured. Each seed aliquot was obtained from six plants grown in specified conditions. A statistical comparison (Student's t test) was performed for data obtained from salt stressed-plants and salt stressed-plants treated by 24-EBR. Data represent the mean ± SE; * , P \ 0.05, Student's t test, n = 10 discrete samples from two biological experiments

Fig. 3

 3 Fig.3Changes in fatty acid composition of seed oil from A. thaliana and B. napus subjected to different cultivation conditions. Oil samples were extracted from seeds gathered at the end of vegetative period and analysed by HPLC. Plant treatment conditions were identical to those described previously. A statistical comparison (Student's t test)

Fig. 4

 4 Fig. 4 Activation of phospholipid signalling in B. napus seedlings subjected to salt stress and 24-EBR. B. napus seedlings were hydroponically grown for 7 day. Seedlings were labelled with BODIPY-PC for 10 min and then 1 lM 24-EBR (BR) or 100 mM NaCl (Salt) were added as indicated. Control plants were treated with buffer. N-ethylmaleimide (NEM) and n-butanol (But) were added to the incubation buffer prior to stress treatment. After 2 h lipids were extracted and separated by TLC. a Chromatography plate.

Fig. 5

 5 Fig.5Genetic association of lipid metabolism with BR-signalling and salt stress responses. Given here is a diagram representing A. thaliana genes implicated to lipid metabolism and signalling that show responsiveness to salinity conditions or treatment with brassinosteroids (as shown in circle intersections). Genes that potentially facilitate BR-mediated regulation of lipid metabolism in salt stress conditions are marked in red[START_REF] Hruz | Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes[END_REF]. Common gene names are shown (for details see Electronic supplementary material Table1). Experimental data used (GEO numbers): salt treatment AT-00120, brassinosteroid treatment AT-00110. Representative lipid metabolism genes were selected from[START_REF] Beisson | Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a webbased database[END_REF]

Acknowledgments We thank Institute of Agriculture NAAS for providing seeds of B. napus cv. Magnat plants. We are also grateful to V.P. Grahov for the help with HPLC analysis. This work was supported by NAS of Ukraine .