Coordinated diurnal regulation of low- and high-affinity nitrate transporters in tomato

Two new members of the NRT2 gene family encoding high-affinity NO3- transporters were isolated from tomato by heterologous screening. Both cDNAs, LeNRT2;1 and LeNRT2;2, are highly similar in the coding region, however, significant nucleotide differences were found in the 3'-untranslated region. High-stringent Southern analysis with gene-specific probes indicated that the two genes are linked in the genome and are most probably derived from a recent gene duplication. Expression of both genes was restricted to roots. Although no difference in expression between both genes could be demonstrated by Northern analysis, LeNRT2;1 and LeNRT2;2 were isolated 56 and 3 times, respectively, from a cDNA library of root hairs, indicating a dominant expression of LeNRT2:1 in root hairs of tomato seedlings. Maximum transcript levels of LeNRT2 peaked 4h after NO3- induction and coincided with those of the putative low-affinity NO3- transporter gene, LeNRT1;2. In contrast, 4h after NO3- supply LeNRT1;1 transcription was maximally repressed, reflecting an opposite regulation. As transcript levels of LeNRT2 also increased independently from NO3- induction, diurnal gene expression was monitored. Ail investigated genes, LeNRT2, LeNRT1;1, LeNRT1;2 and LeNR, showed the highest peak in transcript levels 4 h after the onset of the light period and a second peak at the end of the light period. Although expression of these genes was highly coordinated at these times of the day, LeNRT1;1, LeNRT2;1 and LeNR showed additional peaks. This indicates that diurnal oscillation of these genes is most likely based on multiple factors, such as carbohydrate supply to the roots, feedback regulation from N assimilates and phytochrome-mediated regulation.