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BackgroundWe have previously shown that lipophilic components (LPC) of the brown seaweed Ascophyllum nodosum (ANE) improved freezing tolerance in Arabidopsis thaliana. However, the mechanism(s) of this induced freezing stress tolerance is largely unknown. Here, we investigated LPC induced changes in the transcriptome and metabolome of A. thaliana undergoing freezing stress.ResultsGene expression studies revealed that the accumulation of proline was mediated by an increase in the expression of the proline synthesis genes P5CS1 and P5CS2 and a marginal reduction in the expression of the proline dehydrogenase (ProDH) gene. Moreover, LPC application significantly increased the concentration of total soluble sugars in the cytosol in response to freezing stress. Arabidopsis sfr4 mutant plants, defective in the accumulation of free sugars, treated with LPC, exhibited freezing sensitivity similar to that of untreated controls. The 1H NMR metabolite profile of LPC-treated Arabidopsis plants exposed to freezing stress revealed a spectrum dominated by chemical shifts (δ) representing soluble sugars, sugar alcohols, organic acids and lipophilic components like fatty acids, as compared to control plants. Additionally, 2D NMR spectra suggested an increase in the degree of unsaturation of fatty acids in LPC treated plants under freezing stress. These results were supported by global transcriptome analysis. Transcriptome analysis revealed that LPC treatment altered the expression of 1113 genes (5%) in comparison with untreated plants. A total of 463 genes (2%) were up regulated while 650 genes (3%) were down regulated.ConclusionTaken together, the results of the experiments presented in this paper provide evidence to support LPC mediated freezing tolerance enhancement through a combination of the priming of plants for the increased accumulation of osmoprotectants and alteration of cellular fatty acid composition.

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Ascophyllum nodosum extract (ANE) contains bioactive compounds that improve the growth of Arabidopsis in experimentally-induced saline conditions; however, the molecular mechanisms through which ANE elicits tolerance to salinity remain largely unexplored. Micro RNAs (miRNAs) are key regulators of gene expression, playing crucial roles in plant growth, development, and stress tolerance. Next generation sequencing of miRNAs from leaves of control Arabidopsis and from plants subjected to three treatments (ANE, NaCl and ANE+NaCl) was used to identify ANE-responsive miRNA in the absence and presence of saline conditions. Differential gene expression analysis revealed that ANE had a strong effect on miRNAs expression in both conditions. In the presence of salinity, ANE tended to reduce the up-regulation or the down-regulation trend induced caused by NaCl in miRNAs such as ath-miR396a-5p, ath-miR399, ath-miR2111b and ath-miR827. To further uncover the effects of ANE, the expression of several target genes of a number of ANE-responsive miRNAs was analyzed by qPCR. NaCl, but not ANE, down-regulated miR396a-5p, which negatively regulated the expression of AtGRF7 leading to a higher expression of AtDREB2a and AtRD29 in the presence of ANE+NaCl, as compared to ANE alone. ANE+NaCl initially reduced and then enhanced the expression of ath-miR169g-5p, while the expression of the target genes AtNFYA1 and ATNFYA2, known to be involved in the salinity tolerance mechanism, was increased as compared to ANE or to NaCl treatments. ANE and ANE+NaCl modified the expression of ath-miR399, ath-miR827, ath-miR2111b, and their target genes AtUBC24, AtWAK2, AtSYG1 and At3g27150, suggesting a role of ANE in phosphate homeostasis. In vivo and in vitro experiments confirmed the improved growth of Arabidopsis in presence of ANE, in saline conditions and in phosphate-deprived medium, further substantiating the influence of ANE on a variety of essential physiological processes in Arabidopsis including salinity tolerance and phosphate uptake.

We studied the mechanism of Ascophyllum nodosum (a brown macroalga) induced resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000. Root treatment of A. thaliana Col-0 plants with extracts of A. nodosum [aqueous (ANE), chloroform (C-ANE) and ethylacetate fractions, (E-ANE)] reduced the development of disease symptoms on the leaves. These extracts also induced resistance in salicylic acid deficient NahG and ics1 plants. However, the extracts did not elicit an effect on jar1 (jasmonic acid resistance 1) mutant. A. nodosum extract induced resistance to Pst DC3000 correlated with increased expression of jasmonic acid related gene transcripts PDF1.2 while PR1 and ICS1 expression were less affected. Additionally, pretreatment of Arabidopsis plants with ANE, protected the plants from a necrotroph, Sclerotinia sclerotiorum. The results suggest that the A. nodosum extracts can induce resistance in Arabidopsis to different pathogens which is largely jasmonic acid dependent.

Ascophyllum nodosum, a brown macroalga, is the most widely used seaweed in agriculture. We report a rapid method for the detection of cytokinin-like activity in plants treated with a commercial A. nodosum liquid concentrate (Stimplex®) using a transgenic line of Arabidopsis carrying the ARR5 promoter fused to ß-glucuronidase (GUS) reporter gene. Based on GUS activity assay, an increase in cytokinin-like activity was detected in plants grown in vitro treated with 3 mL L −1 Stimplex®, whereas foliar spray treatments showed similar cytokinin-like activity at a concentration of 5 mL L −1 . Histochemical staining showed Stimplex®-induced GUS activity in leaf as well as in the root tissues. Taken together, our results suggest that Stimplex® contains compounds that may elicit endogenous cytokinin-like activity. Furthermore, it is shown that this bioassay can be used for rapid screening of extracts that can stimulate cytokinin-like activities using Arabidopsis AAR5::GUS reporter transgenic plants.

Successful development of plants resistant to salinity stress is problematic as a result of the complex polygenic natures of salt tolerance. Previously, alkaline extracts of the brown seaweed Ascophyllum nodosum have shown promise in enhancing plant tolerance toward abiotic stresses. To understand the underlying molecular mechanisms, the whole genome transcriptome of Arabidopsis undergoing salt stress was analyzed by microarray analysis after treatment with the chemical components of A. nodosum extracts (ANE). Treatment with ANE induced many positive regulators of salt tolerance in addition to downregulating numerous other genes. Using T-DNA insertion mutants within these downregulated genes, we examined the potential for a novel source of enhanced NaCl tolerance through removal of negative regulators of NaCl stress responses within Arabidopsis. Several potential target mutations were identified with enhanced salt-tolerant phenotypes. A T-DNA insertion within the promoter of a putative Pectin Methyl Esterase Inhibitor (PMEI) gene (At1g62760) was found to be resistant to salinity stress and was further characterized. This T-DNA insertion mutant was designated as pmei1-1. The phenotype of pmei1-1 seedlings included increased primary root growth in vitro and improved biomass accumulation under NaCl stress. Additionally, modified transcript levels of dehydration-responsive genes, including RD29A, were observed in pmei1-1 plants. Taken together, these results suggest a role for PMEI as a negative regulator of NaCl resistance and that chemical stress-induced transcriptome analysis may lead to identification of additional novel regulators of abiotic stress tolerance in plants, the use of which would have significant implications for agriculture globally.

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