Arabidopsis thaliana (Arabidopsis) has been used extensively as a heterologous system for molecular manipulation to genetically characterize both dicotyledonous and monocotyledonous plant species. Here, we report on Arabidopsis transformant lines molecularly manipulated to over-accumulate the small regulatory RNA microRNA397 (miR397) from the emerging C₄ monocotyledonous grass model species Setaria viridis (S. viridis). The generated transformant lines, termed SvMIR397 plants, displayed a range of developmental phenotypes that ranged from a mild, wild-type-like phenotype, to a severe, full dwarfism phenotype. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based profiling of the SvMIR397 transformant population revealed a strong correlation between the degree of miR397 over-accumulation, repressed LACCASE (LAC) target gene expression, reduced lignin content, and the severity of the developmental phenotype displayed by SvMIR397 transformants. Further, exposure of SvMIR397 transformants to a 7-day regime of salt stress revealed the SvMIR397 transformant lines to be more sensitive to the imposed stress than were wild-type Arabidopsis plants. Taken together, the findings reported here via the use of Arabidopsis as a heterologous system show that the S. viridis miR397 small regulatory RNA is able to repress the expression of three Arabidopsis LAC genes which led to reduced lignin content and increased salt stress sensitivity.

Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens-mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis (S. viridis). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE/THERONINE-PROTEIN PHOSPHATASE 2A (PP2A), 5′-ADENYLYLSULFATE REDUCTASE 6 (ASPR6) and DUAL SPECIFICITY PHOSPHATASE (DUSP) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A, ASPR6 and DUSP, were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes across the same tissues. This approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C4 crop species.

In recent decades, the presence of cadmium (Cd) in the environment has increased significantly due to anthropogenic activities. Cd is taken up from the soil by plant roots for its subsequent translocation to shoots. However, Cd is a non-essential heavy metal and is therefore toxic to plants when it over-accumulates. MicroRNA (miRNA)-directed gene expression regulation is central to the response of a plant to Cd stress. Here, we document the miRNA-directed response of wild-type Arabidopsis thaliana (Arabidopsis) plants and the drb1, drb2 and drb4 mutant lines to Cd stress. Phenotypic and physiological analyses revealed the drb1 mutant to display the highest degree of tolerance to the imposed stress while the drb2 mutant was the most sensitive. RT-qPCR-based molecular profiling of miRNA abundance and miRNA target gene expression revealed DRB1 to be the primary double-stranded RNA binding (DRB) protein required for the production of six of the seven Cd-responsive miRNAs analyzed. However, DRB2, and not DRB1, was determined to be required for miR396 production. RT-qPCR further inferred that transcript cleavage was the RNA silencing mechanism directed by each assessed miRNA to control miRNA target gene expression. Taken together, the results presented here reveal the complexity of the miRNA-directed molecular response of Arabidopsis to Cd stress.

Setaria viridis has recently emerged as an ideal model species to genetically characterize the C₄ monocotyledonous grasses via a molecular modification approach. Soil salinization has become a compelling agricultural problem globally with salinity adversely impacting the yield potential of many of the major cereals. Small regulatory molecules of RNA, termed microRNAs (miRNAs), were originally demonstrated crucial for developmental gene expression regulation in plants, however, miRNAs have since been shown to additionally command a central regulatory role in abiotic stress adaptation. Therefore, a small RNA sequencing approach was employed to profile the salt stress responsive miRNA landscapes of the shoot and root tissues of two Setaria viridis accessions (A10 and ME034V) amenable to molecular modification. Small RNA sequencing-identified abundance alterations for miRNAs, miR169, miR395, miR396, miR397, miR398 and miR408, were experimentally validated via RT-qPCR. RT-qPCR was further applied to profile the molecular response of the miR160 and miR167 regulatory modules to salt stress. This analysis revealed accession- and tissue-specific responses for the miR160 and miR167 regulatory modules in A10 and ME034V shoot and root tissues exposed to salt stress. The findings reported here form the first crucial step in the identification of the miRNA regulatory modules to target for molecular manipulation to determine if such modification provides S. viridis with an improved tolerance to salt stress.

In plant cells, the molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation and the generation of the vast amount of chemical energy required to drive these processes relies on an adequate supply of the essential macronutrient, phosphorous (P). The requirement of an appropriate level of P in plant cells is evidenced by the intricately linked molecular mechanisms of P sensing, signaling and transport. One such mechanism is the posttranscriptional regulation of the P response pathway by the highly conserved plant microRNA (miRNA), miR399. In addition to miR399, numerous other plant miRNAs are also required to respond to environmental stress, including miR396. Here, we exposed Arabidopsis thaliana (Arabidopsis) transformant lines which harbor molecular modifications to the miR396 and miR399 expression modules to phosphate (PO₄) starvation. We show that molecular alteration of either miR396 or miR399 abundance afforded the Arabidopsis transformant lines different degrees of tolerance to PO₄ starvation. Furthermore, RT-qPCR assessment of PO₄-starved miR396 and miR399 transformants revealed that the tolerance displayed by these plant lines to this form of abiotic stress most likely stemmed from the altered expression of the target genes of these two miRNAs. Therefore, this study forms an early step towards the future development of molecularly modified plant lines which possess a degree of tolerance to growth in a PO₄ deficient environment.

Rice is the second-most important primary crop in the world and one of the most susceptible crops to salt stress. Soil salinization hinders seedling growth and decreases crop yield by inducing ionic and osmotic imbalances, photosynthesis disturbances, cell wall alterations, and gene expression inhibition. Plants have developed a range of defense mechanisms to adapt to salt stress. One of the most effective means is to make use of plant microRNAs (miRNAs) as post-transcriptional regulators to regulate the expression of developmental genes in order to mitigate the detrimental effects of salt stress. In this study, the miRNA sequencing data between two contrasting rice cultivars, salt-tolerant Doc Phung (DP) and salt-sensitive IR28 seedlings, were compared under control and salt stress (150 mM NaCl) conditions to determine the salt stress-responsive miRNAs. Comparative analysis of miRNA sequencing data detected a total of 69 differentially expressed miRNAs in response to salt stress treatment. Among them, 18 miRNAs from 13 gene families, MIR156, MIR164, MIR167, MIR168, MIR171, MIR396, MIR398, MIR1432, MIR1846, MIR1857, MIR1861, MIR3979, and MIR5508, were identified to be specifically and significantly expressed in the shoot and root tissues of DP seedlings. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses further revealed that these detected miRNAs regulate a range of essential biological and stress response processes, including gene transcription, osmotic homeostasis, root formation, ROS scavenger synthesis, and auxin and abscisic acid signaling pathways. Our findings provide more insight into the miRNA-mediated responsive mechanisms of rice under salt stress and should benefit the improvement of salt stress tolerance in rice.