Nitrate-induced genes in tomato roots. Array analysis reveals novel genes that may play a role in nitrogen nutrition

Abstract

A subtractive tomato (Lycopersicon esculentum) root cDNA library enriched in genes up-regulated by changes in plant mineral status was screened with labeled mRNA from roots of both nitrate-induced and mineral nutrient-deficient (-nitrogen [N], -phosphorus, -potassium [K], -sulfur, -magnesium, -calcium, -iron, -zinc, and -copper) tomato plants. A subset of cDNAs was selected from this library based on mineral nutrient-related changes in expression. Additional cDNAs were selected from a second mineral-deficient tomato root library based on sequence homology to known genes. These selection processes yielded a set of 1,280 mineral nutrition-related cDNAs that were arrayed on nylon membranes for further analysis. These high-density arrays were hybridized with mRNA from tomato plants exposed to nitrate at different time points after N was withheld for 48 h, for plants that were grown on nitrate/ammonium for 5 weeks prior to the withholding of N. One hundred-fifteen genes were found to be up-regulated by nitrate resupply. Among these genes were several previously identified as nitrate responsive, including nitrate transporters, nitrate and nitrite reductase, and metabolic enzymes such as transaldolase, transketolase, malate dehydrogenase, asparagine synthetase, and histidine decarboxylase. We also identified 14 novel nitrate-inducible genes, including: (a) water channels, (b) root phosphate and K(+) transporters, (c) genes potentially involved in transcriptional regulation, (d) stress response genes, and (e) ribosomal protein genes. In addition, both families of nitrate transporters were also found to be inducible by phosphate, K, and iron deficiencies. The identification of these novel nitrate-inducible genes is providing avenues of research that will yield new insights into the molecular basis of plant N nutrition, as well as possible networking between the regulation of N, phosphorus, and K nutrition.

Figure 1

Comparative hybridization profiles for the 1280 cDNA array hybridized with root mRNA either from plants grown without N for 48 h or −N-grown plants resupplied with nitrate for 1 h. A, Portion of the array hybridized with labeled mRNA at 0 h (−N grown plants). B, Same part of the array hybridized with labeled mRNA from roots resupplied with 48 mm potassium nitrate for 1 h. DNA was denatured on the filter with 0.4 m NaOH and neutralized with 2× SSC. The root mRNA probes were labeled with ³²P-dATP via reverse transcription.

Figure 2

Expression profiles of genes encoding the nitrate transporters LeNRT12 and LeNRT21, NR, and NiR in response to nitrate resupply from 1 to 96 h. A, RNA gel blot. B, Quantitative expression data for the RNA gel blot depicted in A. In Figure 2B and the subsequent figures, the autoradiograph of the RNA gel blot was scanned and the image computer digitized. The gel blot intensities were quantified using the National Institutes of Health Image program. Changes in gene expression were quantified as a relative signal ratio, which was determined by dividing the quantified gel blot intensity at each nitrate exposure time point by the RNA blot intensity for the control plants (grown for 48 h without nitrate). LeNRT12, LeNRT21, and NR are tomato genes deposited in GenBank, whereas NiR is a homolog of a pepper NiR (GenBank accession no. AF065616) with a score of 291 and an E value of 6e-78. Approximately 1 μg of mRNA was loaded for each lane and α-tubulin was the loading control. Quantitated signals were weighted against the control in all the graphs.

Figure 3

Up-regulation of a transcription factor, and NT 16 and aquaporin gene homologs induced by nitrate resupply between 1 and 96 h. The tomato transcription factor EST is homologous to the tobacco (Nicotiana tabacum) TGA1a transcription factor with a score/E value of 238/4e-62. See Table I for additional details about all seven aquaporin genes induced by nitrate exposure. A, RNA gel blot. B, Quantitative expression data for the RNA gel blot depicted in A.

Figure 4

Changes in expression of ammonium, Pi, and K⁺ transporter genes induced by nitrate. HAK5 was not graphed. Time course used in the deficiency experiments was 1, 3, 6, 12, 24, and 48 h. A, RNA gel blot. B, Quantitative expression data for the RNA gel blot depicted in A.

Figure 5

Changes in expression of nitrate transporter, LeNRT12, induced by Pi, K, and Fe deficiencies. Time course used in the deficiency experiments was 1, 3, 6, 12, 24, and 48 h. A, RNA gel blot. B, Quantitative expression data for the RNA gel blot depicted in A.

Figure 6

Response of nitrate transporter, LeNRT21, to Pi, K, and Fe deficiencies. Time course used in the deficiency experiments was 1, 3, 6, 12, 24, and 48 h. A, RNA gel blot. B, Quantitative expression data for the RNA gel blot depicted in A.

Figure 7

Changes in expression of five different putative stress response genes to nitrate resupply. A, RNA gel blot. B, Quantitative expression data for the RNA gel blot depicted in A.

Figure 8

Changes in expression of homologs of the soybean Cim1 and tobacco pit1 genes in response to nitrate exposure for 1 to 96 h. pit1 was induced specifically by nitrate exposure and not by exposure to deficiencies of other mineral nutrients. A, RNA gel blot. B, Quantitative expression data for the RNA gel blot depicted in A.