A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation

Abstract

Phosphorus, one of the essential elements for plants, is often a limiting nutrient because of its low availability and mobility in soils. Significant changes in plant morphology and biochemical processes are associated with phosphate (Pi) deficiency. However, the molecular bases of these responses to Pi deficiency are not thoroughly elucidated. Therefore, a comprehensive survey of global gene expression in response to Pi deprivation was done by using Arabidopsis thaliana whole genome Affymetrix gene chip (ATH1) to quantify the spatio-temporal variations in transcript abundance of 22,810 genes. The analysis revealed a coordinated induction and suppression of 612 and 254 Pi-responsive genes, respectively. The functional classification of some of these genes indicated their involvement in various metabolic pathways, ion transport, signal transduction, transcriptional regulation, and other processes related to growth and development. This study is a detailed analysis of Pi starvation-induced changes in gene expression of the entire genome of Arabidopsis correlated with biochemical processes. The results not only enhance our knowledge about molecular processes associated with Pi deficiency, but also facilitate the identification of key molecular determinants for improving Pi use by crop species.

Fig. 1.

Effects of Pi deficiency on some of the traits of Arabidopsis. (A) Soluble Pi in whole plant after transfer to low (open bars) or high (filled bars) Pi. (B) Anthocyanin accumulation in the leaves in response to Pi deficiency. (C) secondary/primary root length ratio in low-Pi (open bars) or high-Pi (filled bars). Macroelements (D) and microelements (E) in the high Pi (black bars) and low Pi (white bars) leaves and high Pi (dark gray bars) and low Pi (light gray bars) roots. (F) ¹³C-NMR analysis. fum, fumarate; Glu, glutamate; Gln, glutamine; Arg, arginine; ref, reference (500 μmol maleate). Upper traces correspond to the enlargement of the corresponding spectrum in the rectangle in the lower trace.

Fig. 2.

Number of genes induced (A) or repressed (B) in low Pi. Comparison of short-term (diamonds), medium-term (squares), and long-term (circles) experiments. The whole plant was analyzed to monitor the regulation of Pi-responsive genes during short- and medium-term experiments. Results from leaves and roots analyzed separately were mixed as long term experiment.

Fig. 3.

Validation of ATH1 results. (A) Comparison of chip results and q-PCR. P(-)/P(+) ratio in leaves and roots for some selected genes are shown. Two different scales were used for the genes that were up- or down-regulated. Q-PCR and ATH1 results are means and SD of three assays performed on triplicates. (B) Northern blot analysis and chip results [ratio P(-)/P(+)] of Pi-responsive genes in leaves (L) and roots (R). Plants were harvested after 5, 10, and 15 days of transfer in low [P(-)] or high Pi [P(+)], and 10 μg of total RNA, isolated from the whole plant, was hybridized with ³²P-labeled cDNA fragments of the genes. Equivalence of RNA loading in all of the lanes is shown by ³²P-labeled tubulin hybridization and ethidium bromide-stained rRNA (Lower). (C) Expression of GFP fused to AtS6K2 (At3g08720) promoter in the stele and emerging lateral roots of transgenic plants grown under high [P(+)] and low Pi [P(-)]. (Scale bar, 50 μm.)

Fig. 4.

Transcriptional regulation in pathway of glycosylglyceride biosynthesis in short-term (squares), medium-term (diamonds), and long-term [leaves (circles) and roots (triangles) analyzed separately] experiments. Fold change: red, >10; orange, 4–10; yellow, 2–4; white, 0.5–2; green, 0.25–0.5; pale blue, 0.1–0.25; dark blue, <0.1. *, Significant, one-way ANOVA, P = 0.05. (Insets) Metabolite quantification [% of total lipids in P(+) and P(-) and ratio P(-)/P(+)] in short-, medium- and long- (leaf, root) term treatments. DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SQDG, sulfoquinovosyldiacyl glycerol; MGDG, galactosyl-1,2-diacylglycerol; DGDG, digalactosyl-diacylglycerol.