Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis

Abstract

In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.

Figure 1.

Levels of ASN1 mRNA were dramatically elevated in 35S-ASN1 lines. Nine-day-old seedlings grown on Murashige and Skoog agar plates under a regular day/night cycle (16 h of light/8 h of dark) were transferred to soil for a further growth of 14 d. Plants were subsequently treated under continuous light (L) or continuous dark (D) conditions for 48 h. Total leaf RNA was extracted as described in “Materials and Methods.” An aliquot of 20 μg of total RNA from each line was loaded onto each lane. Northern-blot analysis was performed as described in “Materials and Methods.”

Figure 2.

Elevation of amino acid levels in 35S-ASN1 lines grown under continuous light or continuous dark treatment. A, Leaves, plants were grown under the same treatment as in Figure 1. B, Siliques, 9-d-old seedlings were transferred to soil and grown under a regular day/night cycle (as described in Fig. 1) until most of the flower buds had turned into green siliques. One-half of the plants were subject to continuous light (L; white boxes) and the remaining one-half was subject to continuous dark treatment (D; black boxes), respectively, for 48 h. Leaves and siliques were harvested, and their amino acid levels were measured using amino acid analyzers (see “Materials and Methods”). Each bar represents an average of two samples. Error bars = ses. The data were analyzed by one-way ANOVA followed by lsd (Fisher's lsd) test. ^**, Corresponding lines were grouped distinctly from both control lines with P values less than 0.01. FW, Fresh weight.

Figure 3.

Elevation of soluble protein content in seeds of 35S-ASN1 lines. Seeds were prepared, harvested, and then assayed for soluble seed protein contents and seed weight as described in “Materials and Methods.” Each data point represents an average value determined from six to eight aliquots of 500 seeds randomly sampled from two seed pools of 12 plants each for each line. Soluble seed protein contents were expressed both on per milligram seeds (A) and per seed (B) basis. Weight data for the 500-seed aliquots were also shown (C). Error bars = ses. The data were analyzed by one-way ANOVA followed by lsd test. ^**, Corresponding lines were grouped distinctly from both control lines with P values less than 0.01. DW, Air-dried weight.

Figure 4.

Decrease of anthocyanin in young seedlings of 35S-ASN1 lines under high Suc and low nitrogen conditions. Anthocyanin contents (A₅₃₀ - 0.25A₆₅₇) of 10-d-old seedlings grown on nitrogen-free Murashige and Skoog agar plates containing 3% (w/v) Suc and 0.5 mm Gln were measured using a spectrometric method as described in “Materials and Methods.” Each data point represents an average value determined from four pools of 36 seedlings for each line. Seeds of different lines were sown onto the same agar plate to ensure homogeneity as described in “Materials and Methods.” Error bars = ses. The data were analyzed by one-way ANOVA followed by lsd test. ^**, Corresponding lines were grouped distinctly from both control lines with P values less than 0.01. FW, Fresh weight.

Figure 5.

Elevation of levels of ASN1 mRNA in sink versus source tissues of 35S-ASN1 lines grown under regular day/night cycle. Plants were grown under regular day/night cycle (16 h of light/8 h of dark) for about 5 to 6 weeks until flowers and development siliques emerged. Different tissues (rosette leaves, stems, cauline leaves, flowers, and siliques) were harvested on the same day after 8 h into the light period. The silique samples consisted of a mixture of siliques at the stage 5, 7, 9, and 11 d after flowering (DAF). Total RNA was extracted, and northern-blot analysis was performed using 20-μg total RNA aliquots as described in “Materials and Methods.”

Figure 6.

Changes in levels of free amino acid content in source versus sink tissues of 35S-ASN1 lines grown under regular day/night cycle. A to E, Free Asn, free Asp, free Gln, free Glu, and total free amino acids, respectively. The samples were harvested as described in Figure 5, except that siliques of different developmental stages were tagged, collected, and analyzed separately. Free amino acid analysis was performed as described in “Materials and Methods.” The contribution of Tyr was not included in the calculation of total free amino acids in E because the relative position of its peak recorded in the amino acid analyzer was very close to the internal standard nor-Leu and the absolute levels of Tyr were very low compared with other free amino acids. Each bar represents an average of two or three samples. Error bars = ses. The data were analyzed by one-way ANOVA followed by lsd test. ^* and ^**, Significant difference when compared with the wild-type Col-0, with a P value less than 0.05 or 0.01, respectively. FW, Fresh weight; Tot aa, total amino acids.

Figure 7.

Elevation of free Asn levels in phloem exudates of 35S-ASN1 lines. Plants were grown for 5 weeks under regular day/night cycle, and individuals that bore at least 10 to 15 developing siliques were used in the analysis. Phloem exudates were obtained using a scaled-down EDTA elution method, and free amino acid contents were determined as described in “Materials and Methods.” Each bar represents an average of three samples. Error bars = ses. The data were analyzed by one-way ANOVA followed by lsd test. ^**, Corresponding lines were grouped distinctly from both control lines with P values less than 0.01. DW, Dry weight after phloem exudate collection.