Transcriptome analysis of Arabidopsis GCR1 mutant reveals its roles in stress, hormones, secondary metabolism and phosphate starvation

Abstract

The controversy over the existence or the need for G-protein coupled receptors (GPCRs) in plant G-protein signalling has overshadowed a more fundamental quest for the role of AtGCR1, the most studied and often considered the best candidate for GPCR in plants. Our whole transcriptome microarray analysis of the GCR1-knock-out mutant (gcr1-5) in Arabidopsis thaliana revealed 350 differentially expressed genes spanning all chromosomes. Many of them were hitherto unknown in the context of GCR1 or G-protein signalling, such as in phosphate starvation, storage compound and fatty acid biosynthesis, cell fate, etc. We also found some GCR1-responsive genes/processes that are reported to be regulated by heterotrimeric G-proteins, such as biotic and abiotic stress, hormone response and secondary metabolism. Thus, GCR1 could have G-protein-mediated as well as independent roles and regardless of whether it works as a GPCR, further analysis of the organism-wide role of GCR1 has a significance of its own.

Fig 1. (a) T-DNA insertion site/orientation in the mutated gene of GCR1.

The exons are represented as boxes and the introns are represented as lines. LB and RB represent the left and right border respectively. (b) qPCR validation of the mutant. The real time RT-PCR was performed in triplicate using independent samples of total RNA and the values are represented as relative quantity ± SE.

Fig 2. (a) Heat map of differentially expressed genes.

The background-subtracted microarray data was subjected to hierarchical clustering using Genespring software ver. 11.5 to generate the heatmap. Yellow represents the control data, while red and green represent up-regulation and down regulation respectively. (b) GO categorization of DEGs. The DEGs were categorized into GO classes using classification superviewer tool of Bioarray resource (www.bar.utoronto.ca)

Fig 3. Validation and comparison of microarray results using qPCR for a few genes selected from each of the important biological processes.

The experiment was carried out using biological triplicates and the values are presented as log2FC ± SE. (AT2G36690–2-OG; AT1G65390—ATPP2-A5; AT1G49570—peroxidase family protein; AT2G35710—PGSIP7; AT5G44420—PDF1.2; AT2G44840—ERF13; AT5G20550–2-oxoglutarate; AT4G01350—Cysteine/Hisidine-rich C1 domain family protein; AT5G20150—SPX1; AT3G26830—PAD3; AT5G40990—GLIP1; AT1G15520—PDR12; AT3G09922—IPS1; AT1G19250—FMO1; AT2G45130—SPX3; AT2G02160—CCCH type zinc finger family protein; AT5G50300—AZG2.

Fig 4. Mapman analysis of genes differentially regulated in gcr1 mutant.

Out of the total list of 350 DEGs, 119 mapped onto biotic stress response. The red dots represent the up-regulated genes, green dots represent the down-regulated genes and the grey dots represent the genes to which none of the DEGs were assigned. The level of differential regulation is according to the scale given.

Fig 5. Singular enrichment analysis (SEA) of the DEGs using AgriGO into important biological processes.

(a) Stress response (b) Hormone response (c) Response to Phosphate starvation (d) Protein modification.

Fig 6. Distribution of differentially regulated transcription factors into highly represented transcription factor families.