Ascophyllum nodosum extract mitigates salinity stress in Arabidopsis thaliana by modulating the expression of miRNA involved in stress tolerance and nutrient acquisition

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.