Ectopic expression of miR396 suppresses GRF target gene expression and alters leaf growth in Arabidopsis

Abstract

MicroRNAs (miRNAs) are comprised of approximately 21 nucleotide (nt) RNAs that play important regulatory roles in growth and development by targeting mRNAs for cleavage or translational repression. In Arabidopsis, more than one hundred miRNAs have been identified but the biological functions of only a limited number of them have been determined by molecular genetic analysis. miR396 is a miRNA conserved among the dicot and monocot plants. In Arabidopsis, miR396 has two loci (MIR396a and MIR396b) and targets six Growth-Regulating Factor (GRF) genes encoding putative transcription factors with roles in plant leaf growth. Using a northern blot hybridizations approach, we have found that MIR396 is predominantly expressed in leaf and seedling. To further analyze the role of miR396 in the regulation of target genes and leaf growth, we have generated transgenic Arabidopsis plants that constitutively overexpress MIR396a or MIR396b. These transgenic plants have narrow-leaf phenotypes due to reduction in cell number. Ectopic overexpression of MIR396 represses expression of not only six GRF genes but also GIF1 encoding a GRF-interacting transcription coactivator with a role in cell proliferation in leaf. In addition, transgenic MIR396-overexpressing plants have lower densities of stomata and are more tolerant to drought than wild-type plants. These results strongly support the belief that miR396 plays an important role in plant leaf growth and development, most likely by repressing GRF gene expression.

Arabidopsis GROWTH-REGULATING FACTOR7 Functions as a Transcriptional Repressor of Abscisic Acid– and Osmotic Stress–Responsive Genes, Including DREB2A

“…In both drought and high salinity conditions, grf7-1 and amiG7 plants showed clearly enhanced tolerance compared with the wild type. A related result was reported by other researchers (Liu et al, 2009), who found that the overexpression of miR396, which targets seven members of the GRF family, including GRF7, led to enhanced dehydration tolerance in Arabidopsis plants. They interpreted this phenomenon as originating from a decrease in the transpiration rate of leaves of the transgenic plants due to decreased stomatal density (Liu et al, 2009).…”

“…A related result was reported by other researchers (Liu et al, 2009), who found that the overexpression of miR396, which targets seven members of the GRF family, including GRF7, led to enhanced dehydration tolerance in Arabidopsis plants. They interpreted this phenomenon as originating from a decrease in the transpiration rate of leaves of the transgenic plants due to decreased stomatal density (Liu et al, 2009). However, the GRF7-deficient plants showed enhanced tolerance of high salinity conditions, which is not likely affected by transpiration ( Figure 8D).…”

“…Therefore, the increased expression of osmotic stress-inducible genes seems to be the factor responsible for the osmotic stress tolerance, although it is possible that regulation of morphogenesis by GRF7 affects osmotic stress tolerance through other pathways. The regulation of plant size by GRF7 could be another such pathway, since most studies of GRF family members have revealed that GRF-deficient plants are smaller than the wild type (Kim et al, 2003;Horiguchi et al, 2005;Liu et al, 2009;Rodriguez et al, 2010;Wang et al, 2011). These reports suggest that the size reduction is due to direct regulation of plant morphogenesis by GRF family proteins.…”

Genome-Wide Characterization of Maize Small RNA Loci and Their Regulation in the required to maintain repression6-1 (rmr6-1) Mutant and Long-Term Abiotic Stresses

“…In Arabidopsis, miR156 was proposed to regulate leaf cell number and size (Usami et al, 2009), and its induction under stress conditions was shown to maintain the plant in the juvenile state for a relatively longer period of time, which helps it withstand unfavorable environmental conditions (Cui et al, 2014;Stief et al, 2014). Another two miRNAs involved in the control of normal leaf growth and development were altered by the abiotic stress treatments miR166 and miR396 (Nogueira et al, 2007;Liu et al, 2009). MiR399 controls inorganic phosphate (Pi) homeostasis and its response to Pi is highly specific (Bari et al, 2006).…”

Transcriptome and Small RNA Sequencing Analysis Revealed Roles of PaWB-Related miRNAs and Genes in Paulownia fortunei

“…In addition, it was also reported that SPL regulate cell proliferation in leaf primordial [57]. Leaf cell proliferation was positively controlled by functional transcriptional coactivators Ans and Grf-Interacting Factor 1 (GIF1) that affected the gene expression for leaf growth based on the interaction network [58,59]. In this study, pf-miR2646 was upregulated in the phytoplasma-infected seedlings, which might decrease the expression level of its target gene (SPL8).…”