The paralogous R3 MYB proteins CAPRICE, TRIPTYCHON and ENHANCER OF TRY AND CPC1 play pleiotropic and partly non-redundant roles in the phosphate starvation response of Arabidopsis roots

Abstract

Phosphate (Pi) deficiency alters root hair length and frequency as a means of increasing the absorptive surface area of roots. Three partly redundant single R3 MYB proteins, CAPRICE (CPC), ENHANCER OF TRY AND CPC1 (ETC1) and TRIPTYCHON (TRY), positively regulate the root hair cell fate by participating in a lateral inhibition mechanism. To identify putative targets and processes that are controlled by these three transcription factors (TFs), we conducted transcriptional profiling of roots from Arabidopsis thaliana wild-type plants, and cpc, etc1 and try mutants grown under Pi-replete and Pi-deficient conditions using RNA-seq. The data show that in an intricate interplay between the three MYBs regulate several developmental, physiological and metabolic processes that are putatively located in different tissues. When grown on media with a low Pi concentration, all three TFs acquire additional functions that are related to the Pi starvation response, including transition metal transport, membrane lipid remodelling, and the acquisition, uptake and storage of Pi. Control of gene activity is partly mediated through the regulation of potential antisense transcripts. The current dataset extends the known functions of R3 MYB proteins, provides a suite of novel candidates with critical function in root hair development under both control and Pi-deficient conditions, and challenges the definition of genetic redundancy by demonstrating that environmental perturbations may confer specific functions to orthologous proteins that could have similar roles under control conditions.

Keywords: Gene regulation; RNA-seq; genetic redundancy; phosphate starvation; root hairs; transcriptional profiling..

Fig 1.

Root hair phenotypes of the wild type and mutants. (A) Root hair number. (B) Root hair length. (C) Compiled confocal micrographs of the various genotypes. The number of asterisks denotes statistically significant differences to Pi-replete wild-type plants based on Student’s t-test (* P<0.05, ** P<0.01, *** P<0.001). Bar, 100 μM.

Fig. 2.

Genes differentially expressed between cpc, etc1 and try mutants and the wild-type plants. Pie charts show genes that are preferentially expressed in root hairs (RH; Lan et al., 2013) and in the phosphate starvation response (PSR) genes. Numbers represent gene counts. In the pie charts, red and blue colour represents the percentage of up- and down-regulated genes, respectively. (This figure is available in colour at JXB online.)

Fig. 3

Effect of Pi deficiency on the expression of R3 MYB protein. (A) Abundance changes in the transcripts of CPC, ETC1, ETC3 and TRY determined by RNA-seq analysis. Values are given in RPKM. (B, C) CPC promoter activity. Cross-sections are from the meristematic region of pCPC-GUS plants grown under (B) control and (C) Pi-deficient conditions. (This figure is available in colour at JXB online.)

Fig. 4.

Over-represented GO categories (biological function) of genes that were differentially expressed in roots of cpc, etc1 or try under (A) control and (B) Pi-deficient conditions. (This figure is available in colour at JXB online.)

Fig. 5.

Co-expression network of PSR genes that were differentially expressed in roots of cpc, etc1 and try. Genes were clustered based on their co-expression relationships with a Pearson’s coefficient of ≥0.60 using the MACCU software package. Pink nodes denote genes that were up-regulated in the mutants, green nodes indicate genes with decreased transcript abundance and purple nodes represent genes that are regulated in opposite directions in different mutants.

Fig. 6.

Scheme depicting the regulation and putative roles of genes involved in membrane lipid remodelling by CPC, ETC1 and TRY in Pi-deficient Arabidopsis roots. PC, phosphatidylcholine; LPC, lysophosphatidylcholine; G3P, glycerol-3-phosphate; PA, phosphatidic acid; P-Cho, phosphocholine; Cho, cholin; DAG, diacylglycerol. (This figure is available in colour at JXB online.)

Fig. 7.

Regulation of transition metal transporters by CPC, ETC1 and TRY. The transport proteins are likely localized in different cell types and tissues.