Suppression of leaf growth and photosynthetic capacity as an acclimation strategy to nitrogen deficiency in a nitrogen-sensitive and shade-tolerant plant Panax notoginseng

ABSTRACT

Photosynthesis is susceptible in response to nitrogen (N) deficiency. However, the acclimation of shade-tolerant and high-N sensitive species to N deficiency is unclear. Leaf morpho-physiological traits, photosynthetic performance related parameters were examined in a shade-tolerant and high-N sensitive species P. notoginseng grown under different N levels. Lower N content and Chl content were recorded in the N₀-grown P. notoginseng. The maximum values of leaf morpho-physiological traits, photosynthetic rate, and photosynthetic N use efficiency (PNUE) were obtained in the N₁₅-grown P. notoginseng. Coefficients for leaf N allocation into the carboxylation and light-harvesting system components in the N₀-grown plants were significantly higher than others. N₀ and N_7.5 plants showed higher K phase. N addition decreased the absorption and capture of the light energy per unit area (ABS/RC and TR_O/RC) and non-photochemical quenching (NPQ). Photochemical quenching (qP), electron transport rate (ETR), and effective quantum yield of photosystem II (ϕ_PSII) were reduced in the N₀-grown plants. The reduction of light-harvesting and utilization capacity not only leads to a decrease in PNUE, but also induces the damage of PSII reaction center. Overall, the inhibition of leaf growth and photosynthetic capacity is an essential strategy for high-N sensitive and shade-tolerant plants in response to N deficiency.

KEYWORDS:

• Nitrogen deficiency
• photosynthetic nitrogen allocation
• photosynthesis
• Panax notoginseng

1. Introduction

Nitrogen (N) is an essential constituent of nucleic acid, proteins, and chlorophyll (Chl) (Jian et al. 2017). Chl content, plant growth, and crop yield are positively correlated with N application within a certain range of N supply (Maheswari et al. 2017; Alves Negrini et al. 2020). N deficiency inhibits plant growth and development, reduces leaf area, Chl content and photosynthesis, and ultimately decreases crop yield, and this suppression has been confirmed in N-deficient crop Zea mays and Glycine max (Mu et al. 2018; Yazaki et al. 2021). The imbalance between the vegetative and reproductive growth results in a decrease in biomass and yield when Cucumis sativus and Lycopersicon esculentum are exposed to N-excess conditions (Zhang et al. 2008; Yuan et al. 2012). However, N utilized by plants accounts for only about 30% of the applied N (Coskun et al. 2017). It is urgent for us to reduce crop dependence on N fertilizer and to improve N use efficiency (Wei et al. 2022). Therefore, more efforts should be made to understand the tolerance or acclimation of plants to N deficiency, and thus to improve crop productivity.

N use efficiency can increase plant fitness (Rütting et al. 2018). However, the majority of leaf N in some plants is allocated to the photosynthetic system (Onoda et al. 2017; Evans and Clarke 2019). Thus, photosynthetic N use efficiency (PNUE) is a major indicator for evaluating low-N tolerance in plants (Kumar et al. 2002). PNUE is positively related to photosynthetic efficiency (Lei et al. 2021). However, significant decline in specific leaf N (SLN), Chl content and net photosynthetic assimilation rate (P_n) with the increasing degree of N deficiency results in a decreased PNUE in sun-demanding species Cannabis sativa, Panicum virgatum, and Saccharum spp. (Zhu et al. 2014; Saloner and Bernstein 2020; Tofanello et al. 2021). Meanwhile, PNUE is described as photosynthetic capacity per unit leaf N (Rotundo and Cipriotti 2017). Thus, the allocation of leaf N into photosynthetic apparatus significantly affects the PNUE (Onoda et al. 2004). The decrease of proportion for leaf N allocation into the carboxylation (P_C) and bioenergetics (P_B) components results in an inhibition to photosynthetic capacity and PNUE in shade-tolerant species Torreya jackii grown under N-deficient condition (Lu et al. 2021). The sun-demanding species Z. mays tends to allocate more N in the bioenergetics components under low N condition to reduce light-harvesting capacity, thus maintaining electron transport and increasing PNUE by 54% (Mu et al. 2016). Nevertheless, it is still unclear whether low-N-driven allocation of leaf N is correlated with the change of PNUE.

Chl fluorescence is closely associated with photosynthesis and is strongly influenced by low N stress (Tantray et al. 2020). N deficiency significantly suppress the maximum and the effective quantum yield of PSII (F_v/F_m and ϕ_PSII), consequently inducing PSII photoinhibition and decreasing photosynthetic capacity (Carpenter et al. 2014; Bascuñán-Godoy et al. 2018). Light-harvesting and photosynthetic efficiency are considerably reduced in N-deficient Spinacia oleracea, while high xanthophyll cycle and non-photochemical quenching (NPQ) enhances the dissipation of excess light energy (Verhoeven et al. 1997). Meanwhile, N deficiency inhibits the photochemical quenching (qP) and enhances NPQ in plants (Wang et al. 2022). The previous studies confirm that N deficiency is closely related to PSII activity and functions (Kalaji et al. 2014). A fast Chl a fluorescence (JIP-test) could assess the structure and function of PSII (Stirbet and Govindjee 2012; Murchie and Lawson 2013). The ability of the JIP method to uncover changes in PSII photochemistry is influenced by environmental factors, e.g. Cd stress, heat stress, and salt stress (Jedmowski and Brüggemann 2015; Chen et al. 2021b; Wang et al. 2022). There are some examples of the application of JIP method in high-N-stressed plant (Dudeja and Chaudhary 2005; Cun et al. 2021). However, relatively few studies have been conducted to assess low-N tolerance of PSII in terms of OJIP.

Panax notoginseng (Burkill) F. H. Chen (Sanqi in Chinese) is a perennial herb (the Panax genus, Araliaceae), which is a shade-tolerant species (Wen and Zimmer 1996; Jiang et al. 2020). The root of P. notoginseng has been used as a traditional Chinese medicinal material for thousands of years (Pharmacopoeia of the People’s Republic of China 2020). Photosynthetic capacity and accumulation of root biomass peaked in response to 9.6–11.5% light transmittance (Chen et al. 2016). Additionally, N surplus lead maximum root yield, but excessive N increases the incidence of root rot in P. notoginseng (Chen et al. 2018; Zhang et al. 2020b). PNUE, photosynthetic efficiency, photoprotection capacity, root biomass, and yield are significantly suppressed in P. notoginseng grown under high N condition (450 kg·ha⁻¹) (Zhang et al. 2020a, 2020b; Cun et al. 2021). Thus, P. notoginseng has also been a high-N sensitive species or a low-N tolerant species. Nevertheless, previous studies have mainly elucidated the effect of high N on photosynthetic performance in the N-sensitive and shade-tolerant species P. notoginseng. Low-N-grown P. notoginseng possess a higher survival rate and higher PNUE than high-N-grown individuals (Ou et al. 2020; Zhang et al. 2020a). SLN, Chl content, photosynthetic efficiency are reduced in P. notoginseng grown under low N condition, but the economic yield is significantly elevated (Ou et al. 2020; Zhang et al. 2020a, 2020b; Cun et al. 2021). However, it is not very well known about the acclimation of shade-tolerant and high-N sensitive species to N deficiency. In the present study, leaf morpho-physiological traits, leaf N allocation and photosynthetic efficiency, PNUE, and OJIP kinetics curves were examined in P. notoginseng grown under different nitrogen deficiency levels. We anticipated that: (i) leaf growth is limited in P. notoginseng grown under N-deficient condition; (ii) N deficiency inhibits leaf N allocation into the carboxylation and bioenergetics components; (iii) N deficiency induces photodamage to PSII of P. notoginseng.

2. Materials and methods

2.1. Experiment design

The pot experiment was carried out from January 2021 to November 2021 in Kunming (102°45'E, 25°08'N), Yunnan Province, China. An environmentally controlled house with about 10% full sunlight irradiance was used for the pot experiments (Zhang et al. 2021). The raw soil has the physical and chemical properties as showed in Table 1.

Table 1. Analysis of soil physical and chemical properties.

Variables	Values (mean ± SD)
pH	6.02 ± 0.11
Organic matter (g·kg^−1)	11.07 ± 0.33
Hydrolysable nitrogen (mg·kg^−1)	54.58 ± 3.79
Available phosphorus (mg·kg^−1)	0.46 ± 0.07
Available potassium (mg·kg^−1)	36.67 ± 3.67
Total nitrogen (%)	0.10 ± 0.003
Total phosphorus (%)	0.10 ± 0.003
Total potassium (g·kg^−1)	0.79 ± 0.10

Note: Different letters in the same row indicate significant difference (P < 0.05), values are means ± SD (n = 3).

Three replicates were used in a complete randomized block design, including three dosages of N fertilizer: (i) without N fertilizer addition (severe N deficiency), N₀; (ii) N fertilizer applied at 7.5 kg·N·667 m⁻² (mild N deficiency), N_7.5; and (iii) N fertilizer applied at 15 kg·N·667 m⁻² (normal N), N₁₅. Each N level was replicated by three plot (n = 3), there were 40 pots in each plot, 120 pots were used for each N levels, and a total of 360 pots were arranged. All treatments except N fertilizer received the same phosphate (15 kg·P₂O₅·667 m⁻²) and potassium (30 kg·K₂O·667 m⁻²) fertilizers. Compound fertilizer (32% N, 4% P₂O₅), calcium superphosphate (52% P₂O₅, 34% K₂O), and potassium sulfate (52% K₂O) were used. Fertilizer was applied in four splits: mid-May, -June, -July, and -August 2021, respectively. The use of chemicals was used to control weeds, pests, and diseases. In November 2021, after the measurement of photosynthetic parameters, the leaf area, dry matter, Chl, and N content were determined.

2.2. Measurement of gas exchange parameters

Between 08:30 am and 11:30 am, selected leaves were measured for P_n – PPFD (photosynthetic photon flux density) and P_n - C_i (internal leaf CO₂ concentrations) response curves using a photosynthesis system (LI-6400XT, Li-Cor, USA). The leaf chamber temperature and the air flow rate were set as 25°C and 400 μmol·s⁻¹, respectively. For the measurement of P_n – PPFD response curves, the CO₂ concentrations of leaf chamber was set at 400 μmol·CO₂·mol⁻¹ (with 10% blue light). It was adjusted based on the following light intensity regimes: 800, 500, 300, 250, 200, 150, 100, 80, 60, 40, 20, and 0 μmol·photons·m⁻²·s⁻¹; for the measurement of the P_n – C_i response curves, the PPFD of leaf chamber was set 500 μmol·photons·m⁻²·s⁻¹ (with 10% blue light). It was adjusted based on the following CO₂ concentrations regimes: 400, 300, 250, 200, 150, 100, 50, 400, 500, 600, 800, 1000, 1200, and 1500 μmol·CO₂·mol⁻¹. The maximum net photosynthetic rate (P_max), the light compensation point (LCP), the light saturating point (LSP), the dark respiration rate (R_d), the maximum carboxylation rate (V_cmax), and the maximum electron transport rate (J_max), were calculated using the P_n − PPFD/P_n − C_i response curve according to the method suggested by Long and Bernacchi (2003).

2.3. Determination of chlorophyll fluorescence characteristics

Dual-PAM 100 chlorophyll (Chl) fluorometer (Heinz Walz Gmbh, Effeltrich, Germany) was used to determine Chl fluorescence parameters and fast Chl fluorescence kinetics curves (OJIP kinetics curves). After dark adaptation of 3 h, the leaf of P. notoginseng was exposed to saturating (8000 μmol·photons·m⁻²·s⁻¹) red (652 nm) actinic light for 1 s, and fluorescence intensity at the O (20 μs), J (2 ms), I (30 ms), and P (300 ms) step were quantified along with the initial slope of the fluorescence transient (M_O). Immediately after that, in accordance with the method suggested by Strasser et al. (2000, 2004), several additional parameters would be calculated.

2.4. Pigment measurements

Chl was extracted as described by Pérez-Patricio et al. (2018). A Li-3000 leaf-area meter (Li-Cor, USA) was used to determine leaf area. 0.5 g of fresh leaves were immersed in a 15 mL extraction mixture [99% acetone was mixed with ethanol (2:1 v/v)]. 3 h of standing in the dark were followed by a 10 min centrifugation at 3000 rpm. Absorbance readings were performed at wavelengths of 665 and 649 nm. Chl a and b content were calculated based on the method of Gu et al. (2016). Total Chl content was the sum of Chl a and b.

2.5. Calculation of N allocation in photosynthetic apparatus

After completing photosynthetic measurements, the leaf was oven-dried separately, at 60°C for 96 h. SLA (specific leaf area, cm²·g⁻¹), LAR (leaf area ratio, cm²·g⁻¹), and LMA (leaf mass per unit area, g·cm⁻²) were calculated based on dry leaf matter and leaf area. The leaf N content was determined using Kjeldahl's method (Lynch and Barbano 1999). Photosynthetic N use efficiency (PNUE, μmol·g⁻¹·N·s⁻¹) was calculated as the ratio of P_max and Specific leaf N (SLN, g·m⁻²) (Gao et al. 2020). Based on the method suggested by Niinemets and Tenhunen (1997), N allocation were calculated for each component of the photosynthetic apparatus. $P_{\mathrm{C}}=V_{\mathrm{c}\mathrm{m}\mathrm{a}\mathrm{x}}/(6.25\times V_{\mathrm{c}\mathrm{r}}\times \mathrm{S}\mathrm{L}\mathrm{N})\times 100\text{\%}$ $P_{\mathrm{B}}=J_{\mathrm{m}\mathrm{a}\mathrm{x}}/(8.06\times J_{\mathrm{m}\mathrm{c}}\times \mathrm{S}\mathrm{L}\mathrm{N})\times 100\text{\%}$ $P_{\mathrm{L}}=C_{\mathrm{C}}/(C_{\mathrm{B}}\times \mathrm{S}\mathrm{L}\mathrm{N})\times 100\text{\%}$ $P_{\mathrm{p}\mathrm{s}\mathrm{n}}=(P_{\mathrm{C}}+P_{\mathrm{B}}+P_{\mathrm{L}})\times 100\text{\%}$ $P_{\mathrm{n}\mathrm{o}\mathrm{n}\text{-}\mathrm{p}\mathrm{s}\mathrm{n}}=\left(1-P_{\mathrm{p}\mathrm{s}\mathrm{n}}\right)\times 100\text{\%}$ $N_{\mathrm{C}}=P_{\mathrm{C}}\times \mathrm{S}\mathrm{L}\mathrm{N}$ $N_{\mathrm{B}}=P_{\mathrm{B}}\times \mathrm{S}\mathrm{L}\mathrm{N}$ $N_{\mathrm{L}}=P_{\mathrm{L}}\times \mathrm{S}\mathrm{L}\mathrm{N}$ $N_{\mathrm{p}\mathrm{s}\mathrm{n}}=N_{\mathrm{C}}+N_{\mathrm{B}}+N_{\mathrm{L}}$ $N_{\mathrm{n}\mathrm{o}\mathrm{n}\text{-}\mathrm{p}\mathrm{s}\mathrm{n}}=1-N_{\mathrm{p}\mathrm{s}\mathrm{n}}$

where P_C is the coefficients for leaf N allocation into the carboxylation component. P_B is the coefficient for leaf N allocation into the bioenergetics component. P_L is the coefficient for leaf N allocation into the light-harvesting system component. P_psn is the coefficient for leaf N allocation in photosynthetic components. P_non-psn is the coefficient for leaf N allocation in non-photosynthetic components. N_C is the N content in the carboxylation component. N_B is the N content in bioenergetics component. N_L is the N content in the light-harvesting system component. N_psn is the N content in photosynthetic components. N_non-psn is the N content in non-photosynthetic components. V_cr is the specific activity of Rubisco (20.78 μmol·CO₂·g⁻¹·Rubisco·s⁻¹) at 25°C. J_mc is the potential rate of photosynthetic electron transport (155.65 μmol·electrons·mmol⁻¹·Cyt f·s⁻¹) at 25°C. C_C is the Chl content (mmol·m⁻²). C_B is the ratio of Chl to organic N in light-harvesting components (2.15 mmol·g⁻¹) at 25°C (Nolan and Smillie 1977; Jordan and Ogren 1984).

2.6. Statistical analysis

One-way ANOVA was applied to assess the differences in each parameter among the treatments with the SPSS 20.0 statistical software packages. Significant differences (P < 0.05) among treatments are indicated by different letters using the least significant difference test (n = 3). GraphPad 8.0 software was used to make a plot.

3. Results

3.1. Effects of N supply on leaf Chl and N content

The minimum values of leaf N content and SLN were recorded in N₀-grown P. notoginseng (Figure 1, P < 0.05), but there was no significant difference in the leaf N content between N_7.5 and N₁₅ conditions (Figure 1A, P > 0.05). Chl content was increased with the N application, and Chl Index (Chlldx) and Chl content in N₀-grown plants were significantly lower than that in N_7.5- and N₁₅-grown individuals (Figure 2, P < 0.05).

Figure 1. The contents of leaf nitrogen in Panax notoginseng grown under different nitrogen levels. (A) Nitrogen content in leaf (%); (B) SLN is the specific leaf nitrogen (g·m⁻²). Green represents N₀, bule represents N_7.5, red represents N₁₅. Values for each point were means ± SD (n = 3). Significant differences are indicated by letters (ANOVA; P < 0.05).

Figure 2. The contents of chlorophyll (Chl) in P. notoginseng grown under different nitrogen levels. Chl Index (Chlldx) imaging were obtained using a multifunctional plant photosynthetic phenotyping system (Plant Explorer Pro⁺) (A–C); (D) Chl contents (mmol·m⁻²). Values for each point were means ± SD (n = 3). Significant differences are indicated by letters (ANOVA; P < 0.05).

3.2. Leaf structural traits in response to N levels

Leaf phenotypic and structural traits differed significantly among N levels (Figure 3A). The maximum values of SLA and LAR were obtained in P. notoginseng grown under N₁₅ conditions (Figures 3B,D; P < 0.05). Lower LAM was recorded in N₁₅-grown plants than the N₀ and N_7.5 plants (Figure 3C, P < 0.05). There was no significant difference in SLA, LAR, and LAM of P. notoginseng between N₀ and N_7.5 conditions (Figures 3B–D, P > 0.05).

Figure 3. Leaf phenotypic traits in P. notoginseng grown under different nitrogen deficiency levels (A). (B) SLA is the specific leaf area (cm²·g⁻¹); (C) LAM is the leaf mass per unit area (g·cm⁻²); (D) LAR is the leaf area ratio (cm²·g⁻¹). Green represents N₀, bule represents N_7.5, red represents N₁₅. Values for each point were means ± SD (n = 3). Significant differences are indicated by letters (ANOVA; P <  0.05).

3.3. Responses of photosynthetic gas exchange parameters to N regimes

In the P_n – PPFD response curves, P_n, P_max, LCP, and LSP were higher in the N₁₅-grown plants compared with N₀- and N_7.5-grown P. notoginseng (Figure 4A and Table 2; P < 0.05). The minimum values of P_n, P_max, LCP, LSP, and R_d were shown in N₀-grown plants (Figure 4A and Table 2; P < 0.05). In the P_n – C_i response curves, P_n, CE (carboxylation efficiency), R_L (light respiration rate), V_cmax, J_max, and J_max / V_cmax were not significantly different among N levels (Figure 4B and Table 2; P > 0.05).

Figure 4. The response of net photosynthetic rate (P_n) to nitrogen regimes. (A) Response of P_n to photosynthetic photon flux density (PPFD) in P. notoginseng grown under different N levels; (B) The change of P_n with intercellular CO₂ concentration (C_i) in P. notoginseng grown under different nitrogen levels. Green represents N₀, bule represents N_7.5, red represents N₁₅. Values for each point were means ± SD (n = 3).

Table 2. The effect of nitrogen deficiency on leaf photosynthetic-related parameters of P. notoginseng.

Variables	Nitrogen deficiency levels
	N0	N7.5	N15
Pmax	2.60 ± 1.03b	4.50 ± 1.14ab	5.49 ± 0.88a
LSP	44.06 ± 12.76b	231.12 ± 8.94a	226.93 ± 32.08a
LCP	2.92 ± 2.29b	3.95 ± 1.93a	4.54 ± 1.65a
Rd	−0.28 ± 1.11b	0.16 ± .0.07a	−0.27 ± 0.10b
CE	0.0084 ± 0.0041a	0.0050 ± 0.0025a	0.0041 ± 0.0026a
RL	2.47 ± 0.28a	1.88 ± 1.00 a	1.46 ± 1.34a
Vcmax	8.53 ± 5.20a	19.00 ± 11.55a	8.70 ± 6.49a
Jmax	30.45 ± 15.32a	106.65 ± 77.72a	44.63 ± 43.01a
Jmax / Vcmax	22.05 ± 1.36a	4.75 ± 0.79a	0.03 ± 4.43a

Note: Different letters in the same row indicate significant difference (P < 0.05), values are means ± SD (n = 3).

P_max, maximum net photosynthetic rate; LCP, light compensation point; LSP, light saturation point; R_d, dark breathing rate; CE, carboxylation efficiency; J_max, maximum electron transfer rate; V_cmax, maximum carboxylation efficiency; R_L, Light respiration rate.

3.4. N-driven changes in photosynthetic N allocation

Different proportions of leaf N were allocated among carboxylation, bioenergetics, and light-harvesting components (Table 3). P_C, P_L, and P_psn in the N₀-grown plants were significantly higher than others (Table 3 and Figure 5; P < 0.05). P_B was positively correlated with N levels, and the minimum values of P_B and P_non-psn were recorded in N₀-grown P. notoginseng (Table 3 and Figure 5).

Figure 5. Nitrogen allocation in leaves of P. notoginseng at different nitrogen levels. (A) Leaf nitrogen allocation under N₀; (B) Leaf nitrogen allocation under N_7.5; (C) Leaf nitrogen allocation under N₁₅. The data outside of the brackets are the nitrogen content of components, the units is g·m⁻²; the data in the brackets are the allocation of the nitrogen content, the unit is percentage (%); the data of inner-circle indicate the leaf total nitrogen content, the units is g·m⁻². Values for each point were means (n = 3). See an explanation of trait abbreviations in Abbreviations.

Table 3. Effects of different N deficiency levels on N allocation within the photosynthetic apparatus of P. notoginseng.

N levels	N allocation within the photosynthetic				Pnon-psn (g·g^−1)
	PC (g·g^−1)	PB (g·g^−1)	PL (g·g^−1)	Ppsn (g·g^−1)
N0	0.15 ± 0.003 a	0.015 ± 0.0003 c	0.19 ± 0.004 a	0.36 ± 0.007 a	0.64 ± 0.007 c
N7.5	0.06 ± 0.001c	0.018 ± 0.0006 b	0.14 ± 0.003 c	0.22 ± 0.004 c	0.78 ± 0.004 a
N15	0.09 ± 0.0007 b	0.04 ± 0.0002 a	0.18 ± 0.001 b	0.30 ± 0.020 b	0.69 ± 0.001 b

Note: Values followed by different letters are significantly different at P < 0.05 (n = 3).

P_C, coefficients for the leaf N allocation into carboxylation system; P_B, coefficients for the leaf N allocation into bioenergetics; P_L, coefficients for the leaf N allocation into light harvesting system; P_psn, coefficients for the leaf N allocation into photosynthetic apparatus; P_non-psn, coefficients for the leaf N allocation into non-photosynthetic apparatus.

The obvious differences in N_psn were not observed within N₀ and N_7.5 treatments (Figure 5). N_L was 17.6% and 20.0% higher in the N_7.5 and N₁₅ individuals than in the N₀ individuals, respectively (Figure 5). N_C was increased by 46.67% and 22.22% in N₀-grown plants compared with N_7.5- and N₁₅-grown P. notoginseng, respectively (Figure 5). N_B was increased with N levels, and N_B was increased by 71.43% in N₁₅-grown plants compared with N₀-grown plants (Figure 5). PNUE in N₁₅-grown plants was significantly higher than that in the N₀- and N_7.5-grown P. notoginseng (Figure 6, P < 0.05), and the obvious differences in PNUE were not observed within N_7.5 and N₁₅ conditions (Figure 6, P > 0.05). Moreover, PNUE firstly increases and then decreases with the increase of P_C & P_B (R² = 0.9485, Figure 7A), P_psn (R² = 0.9057, Figure 7B), and P_non-psn (R² = 0.9057, Figure 7C).

Figure 6. The response of photosynthetic nitrogen use efficiency (PNUE) to nitrogen regimes. Green represents N₀, bule represents N_7.5, red represents N₁₅. Values for each point were means ± SD (n = 3). Significant differences are indicated by letters (ANOVA; P < 0.05).

Figure 7. Relationship between photosynthetic nitrogen use efficiency and N allocation of P. notoginseng under N regimes. (A) Correlation between the PNUE and P_C & P_B in P. notoginseng. (B) Correlation between the PNUE and P_psn in P. notoginseng. (C) Correlation between the PNUE and P_non-psn in P. notoginseng. Green represents N₀, bule represents N_7.5, red represents N₁₅. Values for each point were means (n = 3).

3.5. Chl fluorescence characteristics in response to N levels

Chl fluorescence induction kinetics showed an ‘S’ shape (OJIP type) in all treatments (Figure 8A). Compared with the N₁₅-grown plants, Chl fluorescence was higher in N₀ and N_7.5-grown individuals when the time was 300 μs, and the N₀- and N_7.5-grown plants showed higher K phase (ΔK > 0, Figure 8B). Interestingly, the levels of ΔI was increased and decreased in the N_7.5 and N₀ plants, respectively, compared with the N₁₅ plants (Figure 8B).

Figure 8. Effects of nitrogen levels on chlorophyll fluorescence transients of P. notoginseng. (A) O, J, I, and P phase represent the fluorescence at T = 20 μs, 2, 30 and 300 ms, respectively. (B) Effects of nitrogen levels on relative variable fluorescence (ΔV_t). ΔV_t= V_(treatment)-V_(control), ΔK, ΔJ and ΔI represent the relative variable fluorescence at T = 300 μs, 2, 30 ms. (C) The radar diagram of JIP-test index under different N levels. Green represents N₀, bule represents N_7.5, red represents N₁₅. Values for each point were means (n = 3).

As showed in Figures 8C and 9, there were no significant differences in V_J and DI_O/RC among N levels (P > 0.05). N addition decreased the absorption and capture of light energy per unit area (ABS/RC and TR_O/RC) and electron transport of quantum yield per unit area (ET_O/RC), thus showing significant differences (Figures 8C,9). However, the maximum value of PI_ABS (performance index on an absorption basis) was recorded in N₁₅-grown P. notoginseng (Figure 8C). F_v/F_m and qN exhibited no significant changes under N regimes (Figures 10A,D; P > 0.05). ϕ_PSII, qP, and ETR in N₀-grown plants were significantly lower than ones in N_7.5- and N₁₅-grown P. notoginseng (Figures 10B,C,F; P < 0.05). Moreover, ϕ_PSII, qP, and ETR were not significantly different between N_7.5 and N₁₅ conditions (Figures 10B,C,F; P > 0.05). NPQ was lower in the N₁₅-grown plants compared with the N_7.5 and N₀ plants (Figure 10E; P < 0.05).

Figure 9. Chlorophyll fluorescence transients in P. notoginseng grown under different N levels. Green represents N₀, bule represents N_7.5, red represents N₁₅. Values for each point were means ± SD (n = 3). Significant differences are indicated by letters (ANOVA; P < 0.05).

3.6. The sensitivity of different parameters in response to N deficiency

Multivariate comparisons were carried out with the mean values of photosynthetic-related parameters in P. notoginseng grown under different N levels. The first two axes of the principal component analysis (PCA) explained 66.2% (PC1, 37.9%; PC2, 28.3%) of the total variation of the 48 photosynthetic-related parameters (Figure 11). Leaf N content, N_L, LSP, and N_B parameters showed larger weighting coefficients, and demonstrated a positive correlation with PC1 and contributed more to PC1 (Figure 11 and Table S1). Loading coefficients showed that W_K (F_O and the redox degree of the PSII donor side), TR_O/RC, and ABS/RC have a negative correlation with PC1 and contributed more to PC1 (Figure 11 and Table S1).

Figure 11. A principal component analysis for 48 photosynthetic-related parameters in P. notoginseng grown under different nitrogen deficiency levels.

4. Discussion

4.1. Plants adapt to N deficiency by reducing the light-harvesting area and inhibiting leaf growth

Changes in leaf structure are commonly quantified by SLA, LMR, and LAR (Evans 1989; Díaz et al. 2016). The maximum values of the SLA, LAR, and Chl content were obtained in N₁₅-grown P. notoginseng (Figure 3B,D; P < 0.05). This has been confirmed by the fact that Chl content, SLA, and LAR of Rauvolfia vomitoria is reduced with a decrease in N application (Li et al. 2010). Low SLA and LAR indicate that light-harvesting leaf area per unit biomass is reduced (Weraduwage et al. 2015; Wu et al. 2018). Thus, photosynthetic capacity (as reflected by P_n and P_max) was inhibited in P. notoginseng grown under N₀ and N_7.5, partly due to a decrease in light-harvesting area (Table 2 and Figure 4). This interpretation is consistent with the previous study that photosynthesis is considerably decreased in Oryza sativa grown under N deficiency (Shao et al. 2020). Meanwhile, P_n, SLN, and Chl declined in N-deficient P. notoginseng (Figures 1,2,4), and this might result in the reduced light energy absorption and utilization as suggested by Niinemets (2010). Additionally, biomass allocation is an essential strategy for plants to respond to N stress (Mccarthy and Enquist 2007; Chen et al. 2021a). The decrease of SLA and LAR indicates that N deficiency force more biomass to be allocated to roots, thus promotes N and water uptake as observed in Malus domestica (Figure 3; Wang et al. 2019).

4.2. Lower N content of photosynthetic components reduces PNUE in N-deficient plants

N allocation affects the photosynthesis-N relationship and the acclimation to N deficiency (Zhong et al. 2019). Lower leaf N is allocated to the carboxylation and bioenergetics components in Betula alnoides and T. jackii with the low N input, eventually resulting in lower PNUE (Tang et al. 2019; Lu et al. 2021). In the present study, P_C, P_L, and P_psn in N₀-grown plants were significantly higher than ones in the N_7.5- and N₁₅-grown plants (Table 3 and Figure 5; P < 0.05), but N_B, N_C, and PNUE were reduced with the increase of N levels (Figures 5,6). These results imply that a larger proportion of leaf N would be allocated to photosynthetic components under severe N deficiency. However, the N content in carboxylation and bioenergetics components were reduced, as a corollary to this, also decreases PNUE and photosynthetic efficiency. This is consistent with the results reported by Mao et al. (2012) that photosynthetic N allocation between carboxylation and bioenergetics components is the vital factor limiting PNUE, as reflected by the decrease in P_net, P_max, PNUE, LSP, and LSP under the N₀ and N_7.5 conditions (Table 2). However, the optimized allocation of leaf N in carboxylation and bioenergetics components would lead to an increased PNUE of P. notoginseng (N₁₅ conditions) and Catalpa bungee (Figure 7A; Xiao et al. 2019).

Higher SLN and Chl induces the enhancement of light-harvesting capacity (Niinemets 2010). Evidence is accumulating that Chl content, SLN, and light-harvesting capacity are decreased in N-deficient Camellia sinensis and Mitragyna speciosa (Zhang et al. 2020c; Lin et al. 2021). Chl content, SLN, and PNUE were declined in N₀-grown plants (Figures 1,2,6), but higher N_L and P_L were recorded in P. notoginseng grown under N₁₅ conditions (Table 3; Figures 5,11). This has also been confirmed by the previous study that PNUE is positively correlated with N_L in O. sativa and C. bungee (Xiao et al. 2019; Zhong et al. 2019; Figure 11). It might be speculated that leaf N deficiency might cause the imbalance between the absorption and utilization of light energy, thus leading to a depressed PNUE and photosynthetic capacity. Furthermore, the increase of N allocation to leaves is an effective strategy for improving carbon fixation and PNUE (Perchlik and Tegeder 2018). The previous observation is consistent with our results that N_psn, PNUE and photosynthetic capacity were increased in N₁₅-grown P. notoginseng (Table 2; Figures 2,5,7B). More leaf N is allocated to structural and defense components, and thus promoting the growth of Ulmus americana (Reich et al. 1989). Hence, higher PNUE might be the underlying reason for the superior photosynthesis and the growth of N₁₅-grown plants (Figures 6,7). In short, the reduction of N allocation in carboxylation and bioenergetics components leads to a decrease in PNUE and photosynthetic capacity of P. notoginseng under N deficiency.

4.3. N deficiency induces photodamage of PSII reaction centers

N deficiency leads to a decline in photosynthetic protein synthesis, resulting in photoinhibition or photodamage of the PSII (Chen et al. 2003; Cisse et al. 2020). PI_ABS could reflect the activity of PSII compared with F_v/F_m (Crafts-Brandner and Salvucci 2002). In the present study, F_v/F_m did not change, and PI_ABS was obviously decreased in N₀- and N_7.5-grown P. notoginseng (Figure 8C and 10A). PSII activity is suppressed in N-deficient P. notoginseng during the process of light reaction, and this may lead to PSII photoinhibition or photodamage. A similar effect has been observed in N-deficient Tritium aestivum and Coffea arabica (Pompelli et al. 2010; Gao et al. 2018; Li et al. 2021). The inability to utilize and dissipate excess light energy rapidly is a main cause of photoinhibition or photodamage in N-deficient plants (Raven 2011; Zhang et al. 2015). N deficiency induced a significant reduction in ϕ_PSII (Figure 10B), indicating a decline in the utilization efficiency of light energy as reflected by TR_O/RC, ETR, and qP (Wang et al. 2016). However, NPQ was significantly improved under N deficiency (Figure 10). The proportion of light energy allocated to thermal dissipation, PSII photochemistry and fluorescence would be imbalanced in plants grown under N-deficient condition (Demmig-Adams et al. 1996; Shuang et al. 2022). A similar effect has also been observed in Petiveria alliacea grown under N deficiency condition (Zuluaga et al. 2004). Meanwhile, K phases were raised in N₀- and N_7.5-grown plants (Figures 8B,11), and N deficiency reduced the OEC activity and prevented the release of oxygen and electrons, thus resulting in a decrease in electron transport (Figure 10; Oukarroum et al. 2007). The photochemical efficiency of PSII and the activity of PSII reaction centers would be decreased, and photodamage of PSII would occur in Porphyridium cruentum grown under N starvation (Zhao et al. 2017). N deficiency might induce the accumulation of ROS, and this can directly damage the thylakoid membranes of the chloroplasts, consequently resulting in the destruction of the PSII reaction center and rendering these membranes unable to carry out electron transport (Figure 10; Xiang et al. 2021). These results imply that light-harvesting capacity and electron flux are decreased in high-N sensitive P. notoginseng grown under N deficiency condition, and however thermal dissipation is enhanced. In a word, an imbalance of light energy allocation between thermal dissipation and electron transports might be the main reason for the damage of PSII reaction centers in high-N sensitive P. notoginseng grown under N deficiency condition.

Figure 10. Chlorophyll fluorescence transients in P. notoginseng grown under different N levels. (A) F_v/F_m is the maximum photochemical efficiency of PSII under dark adaptation; (B) ϕ_PSII is the effective quantum yield of PSII photochemistry; (C) qP is the photochemical quenching; (D) qN is the non-photochemical quenching coefficient; (E) NPQ is the non-photochemical quenching; (F) ETR is the electron transport rate. Green represents N₀, bule represents N_7.5, red represents N₁₅. Values for each point were means ± SD (n = 3). Significant differences are indicated by letters (ANOVA; P < 0.05).

5. Conclusion

Leaf growth, N allocation to the photosynthetic system were obviously inhibited in high-N sensitive and shade-tolerant plants grown under N deficiency, leading to a decrease in PNUE and photosynthetic capacity. To further analyze the mechanism of suppressed photosynthesis under N deficiency condition, the damaged model diagrams were proposed for the PSII reaction center in the high-N sensitive and shade-tolerant species represented by P. notoginseng under N-deficient conditions (Figure 12). N deficiency reduces Chl and N content in leaf. Light-harvesting capacity and electron flux are reduced in high-N sensitive and shade-tolerant species grown under N deficiency, and thermal dissipation is enhanced. The imbalanced allocation of light energy leads to a damage of PSII reaction centers in N-deficient plants. Overall, the inhibition of leaf growth as well as the suppression of photosynthetic capacity by reducing leaf N allocation to photosynthetic components and by damaging PSII reaction centers are the essential strategy for the high-N sensitive and shade-tolerant plants to respond to N deficiency

Figure 12. A damage model diagrams were proposed for the PSII reaction center in the N-sensitive and shade-tolerant species P. notoginseng under N-deficient conditions. The light capture capacity was significantly reduced under N-deficient stress (there were many black sites in TR/ABS), electron flux decreased (ET/ABS decreased), and thermal dissipation increased (DI/ABS increased), indicating the imbalance of the light energy allocation strategy. The shade of leaf color represents the level of leaf nitrogen content. ABS/ABS is the absorbed light energy; TR/ABS is the quantum yield of PSII photochemistry; ET/ABS is the quantum yield of electron transport; DI/ABS is the quantum yield of thermal dissipation. Each part of the graphic element represents a value of the activity of the photoreaction center. Changes in shape and size all represent that the activity is affected, and the black dot represents damage. (A) N₀; (B) N_7.5; (C) N₁₅.

Author contributions

Jun-Wen Chen: conceiving and designing the project. Zhu Cun: analyzing the data and writing the paper. Hong-Min Wu, Jin-Yan Zhang, Sheng-Pu Shuang, Jie Hong: performing data analysis and correcting the manuscript. Hong-Chao Zhao, Jing Yang, Li-Lin Gao: participating in the construction of shade-house and pot experiments. All authors agree to the publication of this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was supported by the National Natural Science Foundation of China (32160248 and 81860676), the Major Special Science and Technology Project of Yunnan Province (202102AA310048), the National Key Research and Development Plan of China (2021YFD1601003), and the Innovative Research Team of Science and Technology in Yunnan Province (202105AE160016).

Notes on contributors

Zhu Cun

Zhu Cun PhD at Yunnan Agricultural University, Kunming, China. Research interests: plant ecophysiology and plant nutrition, mainly including photosynthesis of plants, interaction between plants and light/nitrogen, and the effects of nitrogen on the accumulation of secondary metabolites in medicinal plants. Published in international journals as first author: Photosynthesis research, Frontiers in plant science.

Sheng-Pu Shuang

Sheng-Pu Shuang a master candidate at Yunnan Agricultural University, Kunming, China. Research interests: photosynthesis of plants, mainly including interaction between plants and light intensities. Published in international journals as first author: Published in international journals as first author: Frontiers in plant science.

Jin-Yan Zhang

Jin-Yan Zhang PhD at Yunnan Agricultural University, Kunming, China. Research interests: plant ecophysiology and plant nutrition, mainly including photosynthesis of plants, interaction between plants and light/nitrogen, and the effects of nitrogen on the accumulation of secondary metabolites in medicinal plants. Published in international journals as first author: Industrial crops and products, BMC plant biology, Plant physiology and biochemistry, Frontiers in plant science, Acta physiologiae plantarum, et al.

Jie Hong

Jie Hong a master candidate at Yunnan Agricultural University, Kunming, China. Research interests: plant ecophysiology, mainly including interaction between soil microorganism and light/nitrogen.

Hong-Min Wu

Hong-Min Wu a master candidate at Yunnan Agricultural University, Kunming, China. Research interests: plant ecophysiology and plant nutrition, mainly including the effects of nitrogen on the accumulation of secondary metabolites in medicinal plants.

Jing Yang

Jing Yang a master candidate at Yunnan Agricultural University, Kunming, China. Research interests: photosynthesis of plants, interaction between plants and light/nitrogen.

Hong-Chao Zhao

Hong-Chao Zhao PhD at Yunnan Agricultural University, Kunming, China. Research interests: photosynthesis of plants, interaction between plants and light/nitrogen.

Li-Lin Gao

Li-Lin Gao a master candidate at Yunnan Agricultural University, Kunming, China. Research interests: photosynthesis of plants, mainly including interaction between plants and light intensities.

Jun-Wen Chen

Jun-Wen Chen The professor of Yunnan Agricultural University, Kunming, China. He has studied in the plant ecophysiology field for over 15 years. He has published more than 60 scientific papers.