Article Figures & Data
Figures
- Fig. 1.
Gene structure of the AtNRT2 family. Sequences were aligned using the Gap program of the GCG package. Sizes of the introns, indicated between brackets, are given in bp.
- Fig. 2.
Unrooted, bootstrapped tree of NRT2 proteins. ClustalW was used to create an alignment of the entire sequence of amino acids that was then bootstrapped (n = 1,000) to create the final tree using the Philip package. Values indicate the number of times (in percent) that each branch topology was found during bootstrap analysis. D, Dicotyledons; M, monocotyledons. GenBank protein ID numbers are: AtNRT2.1, AAC64170; AtNRT2.2, AAC35884; AtNRT2.3, BAB10099; AtNRT2.4, BAB10098; AtNRT2.5, AAF78499; AtNRT2.6, CAB89321; AtNRT2.7, CAB87624; Nicotiana plumbaginifolia NpNRT2.1, CAA69387; Lotus japonicusLjNRT2, CAC35729; soybean (Glycine max) GmNRT2,AAC09320; rice (Oryza sativa) OsNRT2, BAA33382; barley (Hordeum vulgare) HvNRT2.1, AAC49531; wheat (Triticum aestivum) TaNRT2.1, AAK19519;Chlamydomonas reinhardtii CrNRT2.1, CAA80925;Aspergillus nidulans CRNA, AAA62125; Hansenula polymorpha YNT1, CAA93631; and E. coli NARK,CAA34126.
- Fig. 3.
Comparison of NRT2 proteins. The amino acid sequences (identity nos. are indicated in Fig. 2) were analyzed with the ProDom 2001.2 program.
- Fig. 4.
Quantification of AtNRT2.7 expression in the linear range using the semiquantitative PCR method. Total RNA from roots (R), leaves (L), flowers (F), cauline leaves (CL), stem (S), young shoots (YS), and young roots (YR) of plants grown on 10 mm nitrate were isolated. A, mRNA was reverse transcribed and PCR amplified at different cycles using anAtNRT2.7- or AtAPT1-specific primer set. PCR products were blotted and specifically probed for quantification on a phosphor imager. B, Signal intensity was plotted versus cycle number of PCR reactions. The arrow indicates the cycle number within the linear phase that was chosen to standardize the treatments.
- Fig. 5.
Expression profiles of individualAtNRT2 genes (semiquantitative RT-PCR). For each gene, relative amounts of mRNA in different organs were added and then expressed as a percentage of the sum, in plants at the vegetative (A) or reproductive (B) stages of development. *, Below detection limit in the linear range of amplification.
- Fig. 6.
Effect of limiting nitrate conditions onAtNRT2 gene expression (semiquantitative method). Gene expression was analyzed on plants cultivated on non-limiting (10 mm KNO3, black bars) and limiting (0.5 mm KNO3, gray bars) conditions. Semiquantitative RT-PCR was performed on young roots (YR) and young shoots (YS) as described in Figure 4. *, Below detection limit in the linear range of amplification.
- Fig. 7.
Relative expression of the 7 AtNRT2genes in roots (R) and leaves (L; real-time PCR). Expression was studied by real-time RT-PCR in roots and leaves of flowering plants cultivated on 1 mm KNO3. mRNA levels of individual genes are given as percentage of the constitutive AtAPT1 gene expression level. *, Below 0.01% of the AtAPT1 expression level.
Tables
Nucleotide Identity Amino Acid Similarity AtNRT2.1 AtNRT2.2 AtNRT2.3 AtNRT2.4 AtNRT2.5 AtNRT2.6 AtNRT2.7 AtNRT2.1 – 90.4 76.4 87.7 67.1 76.4 55.6 AtNRT2.2 87.98 – 72.6 83.3 65.7 73.0 55.7 AtNRT2.3 67.12 65.94 – 78.9 64.6 91.5 55.2 AtNRT2.4 72.86 70.73 69.2 – 65.9 78.2 54.8 AtNRT2.5 60.85 57.83 55.75 52.26 – 65.5 60.0 AtNRT2.6 66.95 66.30 80.67 68.34 60.40 – 55.3 AtNRT2.7 50.97 51.50 56.18 48.68 57.89 54.09 – Gene Forward Primer Reverse Primer Semiquantitative PCR AtNRT2.1 5′-GAA ACG CCG GAG TTG CCT CTG TCT CTG GG 5′-CAC CAT AGC CAC AAC GGC AGT TAC AAG GG AtNRT2.2 5′-GAG CCG ACC AAC ACA AAG AAA AAG TAC 5′-TCT GCT GCT CCT CCT TAC TAT ATT CGC C AtNRT2.3 5′-AGG ACT AGT GAA CGG CTG TGC AGG CGG G 5′-AGA GAG CGG CGC GAG ACA AAA GGT GCA A AtNRT2.4 5′-ATG GTA ATA GAA GCA CAC TCG AGA AGA A 5′-TAA GGA GCG GCG TGA GAT GAA AGG TAT G AtNRT2.5 5′-CTC TCC ACC GCT CCC GCC GTC TAT TTC ACC 5′-CCT CCC GCA CCA GTC ATT CCC GAT ACC ACC AtNRT2.6 5′-AAC CGC CTT CTC ACT TAT GCT TAC AGC TCC 5′-GAG AAC GGC GGG AAA CAA AAG GAG CAA CAC AtNRT2.7 5′-CGG TAT CTC TCA GCT CCT TAT GCC TCT C 5′-GAT GAC GAA GGC CCA CAA CAC ATT CCA C AtApt1 5′-TCC CAG AAT CGC TAA GAT TGC C 5′-CCT TTC CCT TAA GCT CTG Real-time PCR AtNRT2.2 5′-GCT ATG CTT TCT CGG TAG ATG GTA G 5′-AAT GTC ATG TTT GGT GAG GTT AAG A AtNRT2.5 5′-CTC TGC TTT CGC CGT TCT CTT GTT C 5′-CGC TGC TAT AAT CCC TGC TGT CTG G AtNRT2.6 5′-TTG TTT CCC GCC GTT CTC TTG 5′-TCT GTC CAT TCC GCT CCA TAG AtNRT2.7 5′-CCT TCA TCC TCG TCC GTT TC 5′-AAT TCG GCT ATG GTG GAG TA
