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In Arabidopsis thaliana ( Arabidopsis ), the microRNA399 (miR399)/ PHOSPHATE2 ( PHO2 ) expression module is central to the response of Arabidopsis to phosphate (PO 4 ) stress. In addition, miR399 has been demonstrated to also alter in abundance in response to salt stress. We therefore used a molecular modification approach to alter miR399 abundance to investigate the requirement of altered miR399 abundance in Arabidopsis in response to salt stress. The generated transformant lines, MIM399 and MIR399 plants, with reduced and elevated miR399 abundance respectively, displayed differences in their phenotypic and physiological response to those of wild-type Arabidopsis (Col-0) plants following exposure to a 7-day period of salt stress. However, at the molecular level, elevated miR399 abundance, and therefore, altered PHO2 target gene expression in salt-stressed Col-0, MIM399 and MIR399 plants, resulted in significant changes to the expression level of the two PO 4 transporter genes, PHOSPHATE TRANSPORTER1;4 ( PHT1;4 ) and PHT1;9 . Elevated PHT1;4 and PHT1;9 PO 4 transporter levels in salt stressed Arabidopsis would enhance PO 4 translocation from the root to the shoot tissue which would supply additional levels of this precious cellular resource that could be utilized by the aerial tissues of salt stressed Arabidopsis to either maintain essential biological processes or to mount an adaptive response to salt stress.

Adequate phosphorous (P) is essential to plant cells to ensure normal plant growth and development. Therefore, plants employ elegant mechanisms to regulate P abundance across their developmentally distinct tissues. One such mechanism is PHOSPHATE2 (PHO2)-directed ubiquitin-mediated degradation of a cohort of phosphate (PO 4 ) transporters. PHO2 is itself under tight regulation by the PO 4 responsive microRNA (miRNA), miR399. The DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2 and DRB4, have each been assigned a specific functional role in the Arabidopsis thaliana ( Arabidopsis ) miRNA pathway. Here, we assessed the requirement of DRB1, DRB2 and DRB4 to regulate the miR399/ PHO2 expression module under PO 4 starvations conditions. Via the phenotypic and molecular assessment of the knockout mutant plant lines, drb1 , drb2 , we show here that; (1) DRB1 and DRB2 are required to maintain P homeostasis in drb4 , we show here that; (1) DRB1 and DRB2 are required to maintain P homeostasis in Arabidopsis shoot and root tissues; (2) DRB1 is the primary DRB required for miR399 production; (3) DRB2 and DRB4 play secondary roles in regulating miR399 production, and; (4) miR399 appears to direct expression regulation of the PHO2 expression module identifies the extreme importance of P homeostasis maintenance in PHO2 expression module identifies the extreme importance of P homeostasis maintenance in Arabidopsis to ensure that numerous vital cellular processes are maintained across Arabidopsis tissues under a changing cellular environment.

It is well established among interdisciplinary researchers that there is an urgent need to address the negative impacts that accompany climate change. One such negative impact is the increased prevalence of unfavorable environmental conditions that significantly contribute to reduced agricultural yield. Plant microRNAs (miRNAs) are key gene expression regulators that control development, defense against invading pathogens and adaptation to abiotic stress. Arabidopsis thaliana ( Arabidopsis ) can be readily molecularly manipulated, therefore offering an excellent experimental system to alter the profile of abiotic stress responsive miRNA/target gene expression modules to determine whether such modification enables Arabidopsis to express an altered abiotic stress response phenotype. Towards this goal, high throughput sequencing was used to profile the miRNA landscape of Arabidopsis whole seedlings exposed to heat, drought and salt stress, and identified 121, 123 and 118 miRNAs with a greater than 2-fold altered abundance, respectively. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was next employed to experimentally validate miRNA abundance fold changes, and to document reciprocal expression trends for the target genes of miRNAs determined abiotic stress responsive. RT-qPCR also demonstrated that each miRNA/target gene expression module determined to be abiotic stress responsive in Arabidopsis whole seedlings was reflective of altered miRNA/target gene abundance in Arabidopsis root and shoot tissues post salt stress exposure. Taken together, the data presented here offers an excellent starting platform to identify the miRNA/target gene expression modules for future molecular manipulation to generate plant lines that display an altered response phenotype to abiotic stress.