Improving Plant Nitrogen Use Efficiency through Alteration of Amino Acid Transport Processes

Abstract

Improving the efficiency of nitrogen (N) uptake and utilization in plants could potentially increase crop yields while reducing N fertilization and, subsequently, environmental pollution. Within most plants, N is transported primarily as amino acids. In this study, pea (Pisum sativum) plants overexpressing AMINO ACID PERMEASE1 (AAP1) were used to determine if and how genetic manipulation of amino acid transport from source to sink affects plant N use efficiency. The modified plants were grown under low, moderate, or high N fertilization regimes. The results showed that, independent of the N nutrition, the engineered plants allocate more N via the vasculature to the shoot and seeds and produce more biomass and higher seed yields than wild-type plants. Dependent on the amount of N supplied, the AAP1-overexpressing plants displayed improved N uptake or utilization efficiency, or a combination of the two. They also showed significantly increased N use efficiency in N-deficient as well as in N-rich soils and, impressively, required half the amount of N to produce as many fruits and seeds as control plants. Together, these data support that engineering N allocation from source to sink presents an effective strategy to produce crop plants with improved productivity as well as N use efficiency in a range of N environments.

Figure 1.

Shoot biomass and root-to-shoot amino acid transport in AAP1-OE and wild-type (WT) pea plants grown in low (2 g of N per week), moderate (4 g of N per week), or high N (8 g of N per week) environments. A, Image of two AAP1-OE lines (OE1 and OE2) and wild-type plants after 8 weeks of growth. B, Shoot dry weight of plants 8 weeks after germination (WAG; n ≥ 6). C, Shoot dry weight of desiccated plants at harvest (16-week-old plants; n ≥ 6). D, Total free amino acids in xylem (n ≥ 7). Data are means ± sd. Significant differences are indicated by letters (ANOVA; P ≤ 0.05).

Figure 2.

Analysis of source-to-sink amino acid transport in two AAP1-OE lines (AAP1-OE1 and AAP1-OE2) and wild-type (WT) pea plants. Eight-week-old plants grown under low (2 g of N per week), moderate (4 g of N per week), or high N (8 g of N per week) supply were examined. A, Expression analysis of the amino acid transporters AAP1 (Zhang et al., 2010) and CAT6 (Tan et al., 2010) in source leaves using reverse transcription-quantitative PCR. Results are shown as fold change compared with the wild type (n = 5 biological replicates). A mean fold change of greater than 2 between the wild type and mutants was used to identify differentially expressed genes. B, Total amino acids in leaf exudates (n ≥ 7). C, Individual amino acids (AA) in leaf exudates (n ≥ 7). Data are means ± sd. Significant differences for each AAP1-OE line are indicated by letters (A and B) or asterisks (C; ANOVA; P ≤ 0.05).

Figure 3.

Analyses of sink number and seed N levels of AAP1-OE1 and wild-type (WT) plants grown with low (2 g of N per week), moderate (4 g of N per week), or high N (8 g of N per week) supply. Plants were grown for 16 weeks and analyzed after desiccation. A, Total number of pods per plant (n ≥ 6). B, Total seed number per plant (n ≥ 6). C, Seed elemental N content (n ≥ 5 plants). D, Seed protein content (n ≥ 5). E, Total seed N yield per plant (n ≥ 5). F, Total seed protein yield per plant (n ≥ 5). Data are means ± sd. Significant differences are indicated by letters (ANOVA; P ≤ 0.05). DW, Dry weight.

Figure 4.

Analyses of stubble biomass and shoot N content of AAP1-OE1 and wild-type (WT) plants grown with low (2 g of N per week), moderate (4 g of N per week), or high N (8 g of N per week) supply. Stubble biomass includes stem, leaves, and pod walls and excludes seeds. A, Stubble dry weight (n ≥ 5 plants). B, Percentage of N in stubble (n ≥ 5). C, Total stubble N (n ≥ 5). D, Total shoot N as a sum of the total amount of N in stubble and seeds per plant (n ≥ 5). E, N harvest index, which presents the percentage of shoot N in seeds (n ≥ 5). Data are means ± sd. Significant differences are indicated by letters (ANOVA; P ≤ 0.05).

Figure 5.

Analysis of NUE in AAP1-OE1 and wild-type (WT) plants grown with low (2 g of N per week), moderate (4 g of N per week), or high N (8 g of N per week) supply. NUE is determined by plant seed yield relative to the amount of N applied and is generally composed of both NUpE and NUtE (see “Materials and Methods”). A, Plant NUpE (n ≥ 5). B, Plant NUtE (n ≥ 5). C, Seed yield per plant (n ≥ 5). D, Plant NUE (n ≥ 5). Data are means ± sd. Significant differences are indicated by letters (ANOVA; P ≤ 0.05). Numbers above the columns indicate percentage changes between AAP1-OE and wild-type plants grown under the same (solid brackets) or different (dashed brackets) N conditions.

Figure 6.

Overview model of AAP1 overexpression in pea and the effects on N uptake, allocation, and use under high N and N-deficient growth conditions. Left, In previous work, it was found that increased AAP1 transporter expression in the phloem and seeds of pea plants grown under very high N supply results in (1) increased N partitioning from source leaves to sinks, affecting seed development, and (2) improved N import into seed cotyledons, influencing seed N/protein level (Zhang et al., 2015). Enhanced leaf-to-seed N transport positively affected root-to-shoot N allocation in the xylem, probably due to feedback regulation through root-shoot signaling via the phloem (large white arrow). Arrows with red circles point to increased expression of AAP1 transporters in the phloem and seeds. Right, AAP1-OE and wild-type plants were grown with low (2 g of N per week), moderate (4 g of N per week), or high N (8 g of N per week) supply. Arrows indicate relative change in AAP1-OE plants compared with the wild type. Under high N nutrition, shoot N supply, seed yield, and seed N content were increased in AAP1-OE pea plants compared with the wild type. The transgenic plants displayed improved NUE due to increased NUpE, but NUtE was unchanged. When N fertilization was reduced by half (moderate N), AAP1-OE plants continued to outperform wild-type plants with respect to N allocation to sinks and seed yield, but seed N/protein levels were unchanged. Under these strongly reduced N conditions, NUE of AAP1-OE plants was increased significantly due to increases in both NUpE as well as NUtE. Under extreme N deficiency (low N), AAP1-OE plants still produced higher seed yields, although with lower N content. NUE was increased significantly in the N-starved plant due to more efficient N utilization for seed production, but NUpE was not changed. Overall, this model supports that manipulation of source-to-sink N transport is an effective strategy to improve plant NUE and plant productivity independent of the N treatment.