Number of flower buds (or flowers) and biomass. The number of flowers in 2020–2021 was affected by N treatment (Fig. 2A and B). The flower bud (or flower) harvest in the S1-S3 continued to increase and then fell sharply in the S4. In addition, the performance of the N2-N4 treatments and the CK were essentially the same, and all were significantly higher than that of the N1 treatment (P < 0.05). The ranges of changes were 56.16–73.50 and 66.32–89.23 for the total number of flowers in the two growth stages, respectively. Notably, the total number of flowers in 2020 was greater than that in 2021.
The single-leaf biomass did not change significantly in N2-N4 compared with the CK and was significantly higher than that of the N1 treatment during all harvest stages of the two growing seasons (Fig. 2C, P < 0.05). The ranges of variation were 0.46–0.71 g·leaf− 1 and 0.44–0.68 g·leaf− 1 for the number of flowers in the two growth stages, respectively. Similarly, the total weight of functional leaves exhibited the same pattern as the biomass of a single leaf. The range of variation in functional leaves under all treatments was 17.71–69.58 g·plant− 1 in 2020, while it was 17.54–57.75 g·plant− 1 in 2021. In addition, the accumulation of functional leaves in 2020 was higher than that in 2021 (Fig. 2D).
Leaf N contents. There was a positive correlation between leaf N content and N concentration in four stages of the two growing seasons (Fig. 3A). In 2020, compared with that of the CK, the leaf N content of the four N treatments (N1, N2, N3, and N4) decreased by 24.40-62.71% during the four the harvest stages. In 2021, the leaf N content of the four N treatments (N1, N2, N3, and N4) decreased by 16.8-59.22% compared with that of the CK treatment.
Chloroplast pigment, chlorophyll fluorescence, and gas exchange parameters. N treatment affected the chloroplast pigment content of C. tinctoria leaves (P < 0.05). For the harvest period, the chlorophyll a, chlorophyll b and carotenoid contents peaked at S3 and then decreased in 2020–2021 (Fig. 3B, C and D).
The chlorophyll a content of N2, N3, N4, and the CK were not significantly different in S1, S2, and S4 during 2020–2021 (P > 0.05). However, the chlorophyll a content of the N3, N4 and CK treatments were significantly higher than those of the N2 and N1 treatments during S3 in both harvest seasons (P < 0.05). The chlorophyll b and carotenoids were unaffected by the N treatment in S1-S2, while the contents under the N2-N4 treatments and the CK in S3-S4 were higher than those under the N1 treatment in 2020–2021 (P < 0.05). The ranges of changes were 1.64–2.48 mg·g− 1 and 1.52–2.42 mg·g− 1 for chlorophyll a, 0.61–0.84 mg·g− 1 and 0.58–0.82 mg·g− 1 for chlorophyll b, and 0.32–0.43 mg·g− 1 and 0.33–0.47 mg·g− 1 for carotenoids in the two growth stages, respectively.
Changes in chlorophyll fluorescence parameters were monitored experimentally to determine the effect of low N conditions on these parameters (Fig. 4). The N starvation state (N1) significantly reduced the Fv/Fm, Fv'/Fm' and qP in leaves, while slight N deficiency conditions (N2-N4) did not affect these parameters under S1-S4 in 2020–2021. In contrast, significant increases in qN were achieved under N-starvation conditions (N1 treatment), with a trend of increasing under low N concentrations and decreasing under high N concentrations in four stages in 2020–2021. Furthermore, qN did not differ among N2-N4 and the CK at S1, while the N2 treatment caused significantly lower values than the N4 (or CK) treatment (P < 0.05) did at S2-S4 in the two harvest seasons.
The gas exchange parameters were similar to the chlorophyll fluorescence parameters (Fig. 5). Pn tended to increase during the S1-S3, followed by a substantial decrease at S4 in the two growth seasons. Compared with those under the CK treatment, the Pn levels decreased the under N1 treatment significantly decreased (P < 0.05), while those under the N2-N4 treatments remained consistent with those under the CK at S1 in 2020–2021. The performance patterns of Ci, Gs and Tr in the four harvest stages in the two growing seasons were similar to that of Pn. Overall, the performance of Ci at S1-S4 in 2020–2021 showed a trend of promotion by low N and inhibition by high N. Gs and Tr under the N2-N4 treatments and the CK were not different and were higher than the values measured under the N1 treatment at S1-S4 during 2020–2021.
Soluble proteins and free amino acids. The soluble protein content increased continuously as the N concentration increased (Fig. 6A) in the four harvest stages during 2020 and 2021. Compared with that under the CK, the content under N1 decreased 14.70% and 29.26% at S1, 39.58% and 43.10% at S2, 28.98% and 18.60% at S3, and 35.24% and 32.66% at S4 during 2020–2021, respectively. N reduction treatment significantly affected the content of total free amino acids in C. tinctoria leaves (Fig. 6B). For the two growing seasons, the content of total free amino acids under N2-N4 and the CK were not different but were significantly higher than those under N starvation (N1) except for S2 and S4 in 2020.
In addition, individual amino acid contents in the leaves in 2020–2021 were analyzed (Table 1). The contents and accumulation patterns of asparagine (Asn), glutamate (Glu), alanine (Ala), valine (Val), tyrosine (Tyr) and phenylalanine (Phe) were similar to those of the total amino acids. In 2020, compared with those of the CK treatment, the Asn, Glu, Ala, Val, Tyr and Phe contents of the N1 treatment decreased by 12.7–42.0% at S1, 11.6–27.8% at S2, 20.5–27.4% at S3, and 8.4–46.5% at S4, while the Asn, Glu, Ala, Val, Tyr, and Phe contents of the N1 treatment decreased by 11.4–21.1% at S1, 11.1–39.3% at S2, 4.7–19.2% at S3, and 13.4–22.7% at S4 in 2021. However, the accumulation patterns of threonine (Thr), serine (Ser), glycine (Gly), leucine (Leu), isoleucine (Ile), and lysine (Lys) decreased with increasing N concentration. In 2020, compared with those of the CK treatment, the Thr, Ser, Gly, Leu, Ile, and Lys contents of the N1 treatment increased by 20.4–48.8% at S1, 19.7–26.7% at S2, 9.0-28.6% at S3, and 8.9–27.7% at S4, the contents of Thr, Ser, Gly, Leu, Ile and Lys of the N1 treatment increased by 11.4–32.7% at S1, 10.7–38.5% at S2, 12.3–29.7% at S3, and 21.8–27.5% at S4 in 2021. Notably, the proline (Pro) content was the same as that in the CK at the level of slight N deficiency (N2-N4) and was higher than that under N-starvation conditions (N1) from S1 to S4 during 2020–2021. Arginine (Arg) levels were greatest in the slightly N-deficient treatments (N3 or N4 treatments), with significantly higher levels than those under N starvation (N1) or normal-N-supply levels (CK) (P < 0.05). In addition, little (or no) cysteine (Cys), methionine (Met), γ-aminobutyric acid (GABA), histidine (His), and ornithine (Orn) was detected, and the levels of these amino acids were not affected by N levels at S1-S4 in 2020–2021 (Table 2).
Table 1
Effect of N treatment on the free amino acid content (mg·g− 1) of C. tinctoria leaves (a).
Year | Harvest stage | Treatment | Asn | Thr | Ser | Glu | Gly | Ala | Val | Leu | Ile | Tyr | Phe | Lys | Arg | Pro |
2020 | | | | | | | | | | | | | | | | |
| S1 | N1 | 0.213b | 0.165a | 0.134b | 0.214b | 0.125a | 0.201b | 0.158b | 0.259a | 0.141a | 0.167c | 0.226d | 0.123a | 0.130c | 0.352a |
| N2 | 0.257a | 0.159a | 0.178a | 0.273a | 0.134a | 0.234ab | 0.167b | 0.231b | 0.145a | 0.221b | 0.274c | 0.118a | 0.234a | 0.331b |
| N3 | 0.261a | 0.143b | 0.173a | 0.303a | 0.114b | 0.244a | 0.198a | 0.232b | 0.121b | 0.225b | 0.317b | 0.119a | 0.242a | 0.336b |
| N4 | 0.272a | 0.138b | 0.169a | 0.261a | 0.117b | 0.256a | 0.189a | 0.226b | 0.122b | 0.262a | 0.314b | 0.096b | 0.148b | 0.331b |
| CK | 0.275a | 0.137b | 0.166a | 0.284a | 0.084c | 0.262a | 0.181a | 0.213c | 0.114b | 0.288a | 0.332a | 0.089b | 0.143b | 0.329b |
| S2 | N1 | 0.223b | 0.143a | 0.183c | 0.282c | 0.118a | 0.226b | 0.167b | 0.243a | 0.112a | 0.167d | 0.231d | 0.121a | 0.136b | 0.396a |
| N2 | 0.293a | 0.142a | 0.201b | 0.298b | 0.123a | 0.284a | 0.184a | 0.221b | 0.096b | 0.221c | 0.271c | 0.123a | 0.234a | 0.344b |
| N3 | 0.301a | 0.125b | 0.216a | 0.343a | 0.117a | 0.293a | 0.195a | 0.207b | 0.084c | 0.225c | 0.303b | 0.103a | 0.242a | 0.346b |
| N4 | 0.285a | 0.116b | 0.217a | 0.331a | 0.101b | 0.286a | 0.182a | 0.202b | 0.089c | 0.262b | 0.311b | 0.103b | 0.248a | 0.358b |
| CK | 0.293a | 0.113b | 0.212a | 0.319a | 0.093b | 0.293a | 0.183a | 0.203b | 0.086c | 0.288a | 0.320a | 0.097b | 0.223a | 0.302b |
| S3 | N1 | 0.257c | 0.194a | 0.243b | 0.326b | 0.114b | 0.279c | 0.127b | 0.271a | 0.114a | 0.194b | 0.365c | 0.147a | 0.179b | 0.675a |
| N2 | 0.302b | 0.173b | 0.251b | 0.321b | 0.137a | 0.316b | 0.156a | 0.251b | 0.112a | 0.273a | 0.374c | 0.113b | 0.223a | 0.495b |
| N3 | 0.296b | 0.169b | 0.294a | 0.381a | 0.128a | 0.342a | 0.163a | 0.248b | 0.102b | 0.281a | 0.412b | 0.110b | 0.222a | 0.449b |
| N4 | 0.325a | 0.174b | 0.288a | 0.348b | 0.113b | 0.354a | 0.153a | 0.233c | 0.982b | 0.267a | 0.417b | 0.102c | 0.225a | 0.475b |
| CK | 0.354a | 0.178b | 0.291a | 0.295c | 0.103b | 0.363a | 0.157a | 0.234c | 0.996b | 0.258a | 0.452a | 0.105c | 0182b | 0.485b |
| S4 | N1 | 0.201b | 0.124a | 0.193b | 0.223c | 0.122b | 0.309b | 0.196b | 0.249a | 0.135a | 0.183c | 0.376c | 0.141a | 0.179b | 0.682a |
| N2 | 0.245a | 0.109b | 0.213a | 0.243b | 0.148a | 0.324a | 0.191b | 0.219b | 0.121b | 0.284b | 0.389c | 0.135a | 0.243a | 0.566b |
| N3 | 0.254a | 0.105b | 0.217a | 0.268a | 0.134a | 0.342a | 0.224a | 0.208b | 0.119b | 0.296b | 0.415b | 0.127a | 0.232a | 0.531b |
| N4 | 0.234a | 0.104b | 0.223a | 0.283a | 0.106c | 0.332a | 0.222a | 0.201b | 0.115b | 0.325a | 0.412b | 0.107b | 0.234a | 0.496b |
| CK | 0.247a | 0.108b | 0.222a | 0.269a | 0.099c | 0.336a | 0.235a | 0.195b | 0.104c | 0.342a | 0.473a | 0.102b | 0.188b | 0.499b |
2021 | | | | | | | | | | | | | | | | |
| S1 | N1 | 0.256b | 0.183a | 0.124c | 0.235b | 0.136b | 0.194c | 0.179b | 0.261a | 0.112a | 0.237b | 0.285c | 0.134a | 0.146b | 0.427a |
| N2 | 0.261b | 0.157b | 0.136b | 0.274a | 0.146a | 0.213b | 0.184b | 0.234b | 0.110a | 0.258a | 0.279c | 0.131a | 0.256a | 0.411b |
| N3 | 0.281a | 0.156b | 0.135b | 0.285a | 0.128b | 0.216b | 0.203a | 0.242b | 0.105b | 0.251a | 0.324b | 0.119a | 0.225a | 0.413b |
| N4 | 0.282a | 0.146b | 0.149a | 0.282a | 0.127b | 0.236a | 0.201a | 0.237b | 0.102b | 0.262a | 0.345a | 0.106b | 0.215a | 0.406b |
| CK | 0.289a | 0.133c | 0.146a | 0.287a | 0.113c | 0.228a | 0.195a | 0.236b | 0.096b | 0.281a | 0.361a | 0.101b | 0.155b | 0.415b |
| S2 | N1 | 0.245b | 0.169a | 0.201b | 0.263b | 0.114b | 0.223c | 0.177b | 0.234a | 0.094a | 0.188c | 0.277c | 0.156a | 0.157c | 0.533a |
| N2 | 0.264a | 0.161a | 0.206b | 0.291a | 0.123a | 0.277b | 0.189a | 0.207b | 0.084b | 0.236b | 0.305b | 0.147a | 0.204a | 0.433b |
| N3 | 0.261a | 0.141b | 0.223a | 0.314a | 0.122a | 0.294a | 0.198a | 0.212b | 0.083b | 0.242b | 0.316b | 0.135a | 0.194a | 0.395c |
| N4 | 0.284a | 0.122c | 0.217a | 0.302a | 0.102b | 0.311a | 0.184a | 0.215b | 0.081b | 0.257a | 0.315b | 0.112b | 0.194a | 0.401c |
| CK | 0.295a | 0.122c | 0.227a | 0.296a | 0.103b | 0.303a | 0.193a | 0.219b | 0.074c | 0.263a | 0.342a | 0.106b | 0.178b | 0.411c |
| S3 | N1 | 0.294b | 0.221a | 0.264b | 0.334b | 0.122b | 0.265b | 0.127b | 0.274a | 0.112a | 0.194b | 0.365c | 0.153a | 0.173c | 0.633a |
| N2 | 0.323a | 0.193b | 0.265b | 0.372a | 0.149a | 0.295a | 0.136b | 0.253b | 0.109a | 0.257a | 0.374c | 0.142a | 0.194b | 0.538b |
| N3 | 0.314a | 0.187b | 0.291a | 0.378a | 0.143a | 0.332a | 0.163a | 0.254b | 0.102b | 0.267a | 0.412b | 0.133b | 0.213a | 0.521b |
| N4 | 0.311a | 0.191b | 0.283a | 0.356a | 0.120b | 0.341a | 0.153a | 0.257b | 0.101b | 0.273a | 0.417b | 0.121c | 0.191b | 0.485c |
| CK | 0.323a | 0.183b | 0.286a | 0.361a | 0.107c | 0.336a | 0.147a | 0.234c | 0.101b | 0.281a | 0.452a | 0.118c | 0.178c | 0.496c |
| S4 | N1 | 0.202b | 0.134a | 0.197b | 0.221b | 0.123b | 0.268b | 0.186b | 0.245a | 0.145a | 0.185b | 0.289c | 0.167a | 0.123b | 0.783a |
| N2 | 0.234a | 0.136a | 0.194b | 0.259a | 0.126b | 0.331a | 0.183b | 0.226b | 0.134b | 0.243a | 0.301c | 0.158a | 0.162a | 0.612b |
| N3 | 0.231a | 0.112b | 0.206a | 0.269a | 0.145a | 0.346a | 0.223a | 0.209c | 0.114c | 0.245a | 0.324b | 0.154a | 0.166a | 0.554c |
| N4 | 0.238a | 0.114b | 0.204a | 0.273a | 0.102c | 0.344a | 0.213a | 0.207c | 0.116c | 0.256a | 0.321b | 0.138b | 0.154a | 0.563c |
| CK | 0.232a | 0.109b | 0.217a | 0.274a | 0.101c | 0.342a | 0.216a | 0.201c | 0.108d | 0.259a | 0.374a | 0.131b | 0.129b | 0.541c |
a Means followed by a same letter within the column are not significantly different at (P < 0.05) probability level according to Tukey’s (HSD) test. |
Table 2
Effect of low nitrogen treatment on the free amino acid content (mg·g− 1) of C. tinctoria leaves (b).
Year | Harvest stage | Treatment | Cys | Met | GABA | His | Orn |
2020 | | | | | | | |
| S1 | N1 | / | 0.01a | / | 0.094a | 0.038a |
| N2 | / | 0.02a | / | 0.087a | 0.042a |
| N3 | / | 0.01a | / | 0.093a | 0.042a |
| N4 | / | 0.02a | / | 0.085a | 0.036a |
| CK | / | 0.01a | / | 0.102a | 0.041a |
| S2 | N1 | 0.01a | 0.04a | 0.02a | 0.103a | 0.044a |
| N2 | 0.01a | 0.04a | 0.02a | 0.112a | 0.046a |
| N3 | 0.01a | 0.03a | 0.02a | 0.109a | 0.048a |
| N4 | / | 0.04a | 0.01a | 0.113a | 0.042a |
| CK | / | 0.03a | 0.02a | 0.113a | 0.048a |
| S3 | N1 | / | / | / | 0.103a | 0.04a |
| N2 | / | / | / | 0.112a | 0.04a |
| N3 | / | / | / | 0.109a | 0.05a |
| N4 | / | / | / | 0.113a | 0.04a |
| CK | / | / | / | 0.113a | 0.04a |
| S4 | N1 | / | 0.04a | / | 0.116a | 0.045a |
| N2 | / | 0.04a | / | 0.121a | 0.051a |
| N3 | / | 0.03a | / | 0.125a | 0.046a |
| N4 | / | 0.04a | / | 0.117a | 0.046a |
| CK | / | 0.03a | / | 0.126a | 0.048a |
2021 | | | | | | | |
| S1 | N1 | / | 0.01a | / | 0.996a | 0.042a |
| N2 | / | 0.02a | / | 1.01a | 0.045a |
| N3 | / | 0.01a | / | 0.998a | 0.046a |
| N4 | / | 0.02a | / | 0.994a | 0.041a |
| CK | / | 0.01a | / | 0.984a | 0.042a |
| S2 | N1 | / | 0.02a | 0.01 | 0.102a | 0.043a |
| N2 | / | 0.03a | 0.02 | 0.105a | 0.046a |
| N3 | / | 0.03a | 0.01 | 0.106a | 0.041a |
| N4 | / | 0.02a | 0.01 | 0.114a | 0.046a |
| CK | / | 0.03a | 0.01 | 0.115a | 0.048a |
| S3 | N1 | / | / | / | 0.113a | 0.03a |
| N2 | / | / | / | 0.104a | 0.03a |
| N3 | / | / | / | 0.111a | 0.02a |
| N4 | / | / | / | 0.106a | 0.03a |
| CK | / | / | / | 0.109a | 0.02a |
| S4 | N1 | / | 0.02 | / | 0.128a | 0.045a |
| N2 | / | 0.03 | / | 0.129a | 0.047a |
| N3 | / | 0.02 | / | 0.133a | 0.047a |
| N4 | / | 0.03 | / | 0.134a | 0.041a |
| CK | / | 0.03 | / | 0.133a | 0.043a |
Nitrogen metabolism-related enzymatic activity. N levels significantly affected N metabolism in C. tinctoria leaves from S1-S3 in 2020 and 2021 (Fig. 7, P < 0.05). The activities of NR, NIR, GS, and GOGAT tended to increase with increasing N concentrations. The activities of NR, NIR, GS, and GOGAT from S1 to S4 were 137.66-370.56 µmol·min− 1·g− 1 and 124.12–365.70 µmol·min− 1·g− 1, 160.23-275.45 µmol·min− 1·g− 1 and 138.95-267.65 µmol·min− 1·g− 1, 27.87–51.21 U·g− 1 and 28.45–47.86 U·g− 1 and 751.16-1085.49 nmol·min− 1·g− 1 and 722.34-944.78 µmol·min− 1·g− 1 in 2020–2021, respectively. For S1-S2, the activities of NR and GS under the N2-N4 treatments and the CK were similar during 2020–2021, while the enzyme activity of NIR under the N1-N3 treatment remained consistent in the two growth seasons. GOGAT maintained a similar pattern of change under N2-N4 from S1-S2. Taken together, these results indicate that C. tinctoria may have a lower demand for N during the flower bud period and early flowering period. For the S3-S4 stage, the difference in NR, NIR, GS, and GOGAT activity was significantly increased between the N1 treatment and the other N treatments. At this point, N metabolism in C. tinctoria under N-starvation conditions was inhibited. However, GDH activity was significantly increased under low N conditions. Both the N1 and N2 treatments resulted in values that were significantly greater than those under the normal-N-supply levels (CK) during the four harvest periods in 2020–2021 (P < 0.05). The GDH activities of the two growth seasons were 1489.45-2276.33 nmol min− 1·g− 1 and 1422.38-2045.69 nmol min− 1·g− 1, respectively.
Correlation analysis. Correlation figures were plotted by integrating data from the four stages from 2020–2021 (Fig. 8). The leaf N content in 2020–2021 was significantly positively correlated with NR activity, NIR activity, GS activity, GOGAT activity, soluble protein content and total amino acid content but significantly negatively correlated with GDH activity (P < 0.05).