Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants

Abstract

Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as beta-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these "late" genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.

Figure 1.

The effect of P withdrawal on shoot fresh weight (A) and shoot P concentration expressed on a dry weight basis (B). Arabidopsis plants were grown hydroponically in a complete nutrient solution (•) or a solution lacking P (○). Plants were 28 d old at the beginning of the experiment. Data are expressed as mean ± SE (n = 3 experiments).

Figure 2.

Venn diagrams showing the numbers of genes differentially expressed in shoots of Arabidopsis plants in response to 4, 28, and 100 h of P starvation (A); 28 + 4 h of P starvation (late P-responsive genes), 28 h of K starvation, and 28 h N starvation (B); 4 h of P starvation (early P-responsive genes), 28 h of K starvation, and 28 h of N starvation (C). Differentially expressed genes were defined as those with a -fold difference in expression in two biological replicates of 2.5 between shoots of plants grown in solutions lacking specific elements and control plants grown in complete nutrient solutions and harvested at the same growth stage. The identities of genes that were differentially expressed in response to P, K, or N starvation are given in Tables I and II.

Figure 3.

The MIPS functional categories of genes whose expression had changed early (4 h; Table I) or late (28 or 100 h; Table II) after the withdrawal of P from 28-d-old hydroponically grown Arabidopsis. The AGI number for each gene was used to identify its functional category from the MIPS database (http://mips.gsf.de/proj/thal/db/tables/tables_func_frame.html). When no functional category was given by MIPS, the first predicted category identified in PENDANT was used.

Figure 4.

The activity of GUS in the leaves of two independent transgenic Arabidopsis lines bearing constructs containing the GUS marker gene under the control of the promoter sequence for the P-sensitive gene, SQD1. Plants were grown hydroponically and fully expanded leaves were harvested 20 h before and 4, 28, 100, and 220 h after withdrawing P. Excised leaves were vacuum infiltrated with staining solution containing 520 mg L^–1 5-bromo-4-chloro-3-indolyl β-d-glucopyranoside and incubated overnight at 37°C. To assist the visualization of the blue product, chlorophyll was removed from leaves by stepwise replacement of the staining solution with ethanol and mounted in a solution containing 50% (v/v) glycerol. The transgenic lines were GUS13/4 (top) and GUS22/1 (bottom).