Mesophyll protoplasts: a key tool for deciphering plant nutrient starvation pathways.

The mesophyll protoplast transient expression system is an essential and robust methodology for examining gene expression regulation. Despite its potential, it has not been effectively employed to elucidate the genetic regulatory pathways and networks underlying plant responses to nutrient starvation, such as those involving phosphorus (Pi) and iron (Fe). In this study, we identified differentially expressed genes in response to Pi starvation in Arabidopsis using transcriptome analysis and RT-qPCR validation. Our findings revealed that Pi starvation significantly upregulated the expression of Pi-starvation induced (PSI) genes, including SPX1, SPX3, IPS1, and PS2, while simultaneously downregulating the expression of Fe starvation-responsive genes, such as FIT, IRT1, and FRO2. Additionally, through a dual luciferase transient expression assay in mesophyll protoplasts, we demonstrated that the transcription factor PHR1 serves as a crucial transcriptional regulator, modulating the expression of phosphate starvation response (PSR) genes. This regulation significantly enhances the transcriptional activity of the SPX1, SPX2, SPX3, IPS1, PS2, and PHT1;4 promoters. Upon the addition of the SPX1 protein, the activation of these gene promoters by PHR1 were alleviated. Concurrently, the transcriptional regulator FIT, which governs the expression of genes responsive to Fe starvation, markedly increased the transcriptional activity of the IRT1 and FRO2 promoters. Based on these findings, we propose the mesophyll protoplast transient expression system as a rapid and reliable method for investigating complex genetic networks. Overall, our study provides substantial evidence for understanding the role of the mesophyll protoplast transient expression system in elucidating the genetic regulatory pathways and networks involved in plant responses to nutrient starvation.