Molecular Manipulation of the miR396 and miR399 Expression Modules Alters the Response of Arabidopsis thaliana to Phosphate Stress.

Joseph L Pegler,Christopher P L Grof,Jackson M J Oultram,Duc Quan Nguyen,Andrew L Eamens

doi:10.3390/plants10122570

Abstract

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.

Highlights

The molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation, and the generation of considerable amounts of chemical energy in the form of ATP and GTP, all rely on an adequate supply of the essential macronutrient, phosphorous (P) [1,2]
Following experimental verification that miR396 and miR399 abundance was reduced in MIM396 and MIR399 plants, respectively, and that the abundance of these two miRNAs was elevated in the MIR396 and MIR399 transformant lines [53,58], 8-day-old MIM396, MIR396, MIM399 and MIR399 seedlings, together with unmodified
Col-0 seedlings of the same age, were exposed to a 7-day period of PO4 starvation. This approach was undertaken to attempt to establish a role for the miR396/GROWTH REGULATING FACTOR (GRF) expression module in the molecular response of Arabidopsis to PO4 starvation, as well as to compare any established requirement of the miR396/GRF expression module to this form of abiotic stress to that of the well documented and central role occupied by the miR399/PHO2 expression module

Summary

Introduction

Due to the limitation of available soil PO4 , combined with the absolute requirement of an adequate concentration of P in all plant cells for normal growth and development, plants employ elegant mechanisms to spatially regulate the cellular concentration of P across their developmentally distinct tissues [7,8]. To achieve such spatial variance in cellular P concentration, plants continually modulate P homeostasis via constant regulation of the rate of acquisition of external PO4 from the soil, in combination with the parallel adjustment of the degree of remobilization of their existing internal stores of

Methods

Results

Discussion

Conclusion