Abstract

Introduction

Drought is one of the major abiotic stresses affecting plant growth worldwide^1,2. Previous studies have found that drought stress decreased stomatal conductance (g_s), leaf transpiration rate (E), and net photosynthesis (Pn)^3,4. Drought also induced the generation of reactive oxygen species (ROS) and stimulated the activity of oxidative stress enzymes in leaf cells, e.g., increases in superoxidase^5,6. Photosynthesis is the most crucial process for plant growth^7,8. Many studies have used a decrease in the maximum quantum yield of photosynthesis (Fv/Fm) or a decrease in effective PSII quantum yield (Φ_PSII) as indicators of the extent to which environmental stress has damaged the photosynthetic apparatus^9–12. In addition, water use efficiency (WUE), the ability of the plant to produce dry matter/unit of water, is an important indicator of a plant’s resistance to drought stress. Drought stress generally increases WUE, which provides a fitness advantage in water-limited habitats^3,4,13–15.

Nitrogen (N) and phosphorus (P) are primary macronutrients that play critical roles in various processes such as photosynthesis^16–18 Nutrient addition plays a critical role in improving plant growth under drought stress because water deficit conditions constrain plants’ access to soil N¹⁹. Numerous studies have shown that fertilization may lessen the adverse effects of drought on plant growth by promoting the regulation of water use efficiency and enhancing the activities of antioxidant enzymes^20,21. The interactive effects of water and N availability on photosynthesis have been extensively studied^22–25. Previous studies found that nutrient combinations are more effective than the application of individual nutrients^26–31. However, the interactive effects of drought and nutrition have not been thoroughly investigated in bamboo.

Moso bamboo (Phyllostachys edulis) is widely distributed in southern China³². The Moso bamboo forest in China has an area of 3.87 million ha, comprising 70% of the total bamboo forest area in China^33,34. Moso bamboo is subjected to drought stress frequently, as irrigation water is not always available. Among the mineral nutrients used by plants nitrogen (N) demand is the highest³⁵ since N can increase the photosynthetic rate^36,37. There are many scholars concerned about the use of nitrogen fertilizer in Moso bamboo shoot production and its influence on Moso bamboo growth³⁸, photosynthetic physiology³⁹, soil and environmental effects⁴⁰ of Moso bamboo forest. Phosphorus (P) also plays an important role in Moso bamboo growth⁴¹, for the deficiency of soil phosphorus in Moso bamboo forest is a common problem in southern China⁴². Also, the change of N and P ratio has important physiological and ecological significance for Moso bamboo forest management⁴³. Inadequate fertilizer can potentially intensify the negative aspects of drought⁴⁴. Nevertheless, the effects of the combination of N and P nutrients on the morphological, physiological, and biochemical characteristics of Moso bamboo plants are not well documented. In this study, we compared the growth, leaf photosynthesis, photosynthetic pigments, chlorophyll fluorescence, water use efficiency, activities of antioxidant enzymes, and lipid membrane peroxidation under four fertilization treatments and two water regimes in order to explore the responses of bamboo population to water and nutrients from the physiological ecology and try to find the optimal way for the improvement of drought tolerance in Moso bamboo. The objectives were as follows: (1) to monitor the growth and physiological changes in Moso bamboo under drought, N and P application and their combination and (2) to determine whether the N and P application can reduce the detrimental effects of drought stress.

Results

Biomass accumulation and photosynthesis

The accumulation of biomass decreased under drought stress. Moreover, the total biomass accumulation (DMA) exhibited a significant increase under LNHP, HNHP and HNLP treatments. In contrast, under LNHP, HNHP and HNLP with drought treatments, the decrease in total biomass accumulation was less evident than in the LNLP treatment (Table 1). Pn, g_s and E significantly decreased under drought treatments. The treatments of LNHP, HNHP and HNLP also resulted in smaller decreases of Pn, g_s, and E under drought conditions (Table 1). Drought significantly increased the root to shoot ratio (RS) in LNLP plants, whereas the drought-induced increase in RS was less pronounced in HNHP, HNLP and LNHP plants (Table 1).

Table 1

Dry matter accumulation (DMA), root/shoot ratio (RS), net photosynthesis (Pn), stomatal conductance (g_s) and transpiration (E) in the seedlings of Moso bamboo, as affected by drought, N, and P and their combination.

Watering	N level	P level	DMA (g)	RS (root shoot ratio)	Pn (μmol m−2 s−1)	gs (mmol m−2 s−1)	E (mmol m−2 s−1)
30%	L	L	22.12±2.00f	2.42±0.21a	1.65±0.20g	24.75±1.93e	0.53±0.05e
30%	L	H	22.12±2.15f	2.27±0.12b	2.31±0.18f	33.27±3.11cd	0.70±0.06d
30%	H	H	26.51±2.08e	1.81±0.09c	2.99±0.12de	33.77±3.91cd	0.73±0.07d
30%	H	L	24.04±3.55fe	1.73±0.04c	2.57±0.08ef	30.62±1.85cd	0.67±0.05d
70%	L	L	30.53±2.05d	1.25±0.08e	3.16±0.23d	39.19±3.98c	1.27±0.11c
70%	L	H	34.83±2.54c	1.24±0.03e	3.91±0.41c	58.93±7.27b	1.51±0.03b
70%	H	H	45.54±1.40a	1.54±0.09d	6.43±0.36a	76.18±11.41a	2.19±0.02a
70%	H	L	39.84±2.00b	1.43±0.11d	4.56±0.50b	84.93±9.17a	1.46±0.13b
		P:Fw	***	***	***	***	***
		P:Fn	**	***	***	*	***
		P:Fp	***	NS	***	***	***
		P:Fw×n	*	***	*	NS	***
		P:Fw×p	**	*	NS	***	***
		P:Fn×p	NS	NS	NS	**	**
		P:Fw×n×p	NS	**	**	*	***

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Each value is the mean±SE (n=6–8). Values followed by the same letter in the same column are not significantly different at the P>0.05 level according to Tukey’s test. Fw, watering effect; Fn, nitrogen effect; Fp, phosphorus effect; Fw×n, watering and nitrogen interaction effect; Fw×p, watering and phosphorus interaction effect; Fn×p, nitrogen and phosphorus interaction effect; Fw×n×p, watering, nitrogen and phosphorus interaction effect, as determined by analyses of variance. NS, not significant; *P<0.05; **P<0.01; ***P<0.001.

Chloroplast pigments and photochemical parameters

Drought decreased total chlorophyll content, Φ_PSII, Fv/FM, and qP, but increased NPQ. Significant interaction effects of drought×nutrient were found on the total chlorophyll content, Φ_PSII, Fv/Fm, qP and NPQ. The LNHP, HNHP, and HNLP treatments resulted in significantly higher Φ_PSII, Fv/Fm, and qP than the LNLP treatments (Table 2). The increases in NPQ in drought-stressed plants were less evident in the LNHP, HNHP, and HNLP treatments than in the LNLP treatment (Table 2).

Table 2

Chlorophyll fluorescence characteristics of Moso bamboo under different treatments.

Watering	N level	P level	Chlorophyll content (mg g−1 FW)	ΦPSII	Fv/Fm	qP	NPQ
30%	L	L	2.43±0.17d	0.30±0.03d	0.65±0.04d	0.49±0.05c	0.80±0.03a
30%	L	H	2.31±0.22d	0.37±0.01c	0.62±0.01d	0.53±0.07c	1.13±0.04b
30%	H	H	2.72±0.06c	0.50±0.02b	0.72±0.03ab	0.67±0.06b	0.94±0.05c
30%	H	L	2.31±0.16d	0.39±0.04c	0.69±0.03c	0.56±0.05c	1.19±0.03a
70%	L	L	3.15±0.28b	0.56±0.04a	0.77±0.02a	0.76±0.02a	0.41±0.07e
70%	L	H	3.13±0.06b	0.56±0.02a	0.75±0.02ab	0.74±0.05a	0.74±0.08d
70%	H	H	3.73±0.13a	0.59±0.05a	0.74±0.02b	0.79±0.08a	0.42±0.05e
70%	H	L	3.20±0.08b	0.57±0.04a	0.76±0.02a	0.75±0.07a	0.62±0.08d
		P:Fw	***	***	***	***	***
		P:Fn	*	***	**	***	**
		P:Fp	**	NS	NS	NS	NS
		P:Fw×n	**	NS	***	NS	NS
		P:Fw×p	NS	NS	*	NS	***
		P:Fn×p	NS	**	***	NS	NS
		P:Fw×n×p	***	***	NS	**	***

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Each value is the mean±SE (n=6–8). Values followed by the same letter in the same column are not significantly different at the P>0.05 level according to Tukey’s test. Fw, watering effect; Fn, nitrogen effect; Fp, phosphorus effect; Fw×n, watering and nitrogen interaction effect; Fw×p, watering and phosphorus interaction effect; Fn×p, nitrogen and phosphorus interaction effect; Fw×n×p, watering, nitrogen and phosphorus interaction effect, as determined by analyses of variance. NS, not significant; *P<0.05; **P<0.01; ***P<0.001.

Relative water content (RWC), WUE_i, PNUE and PPUE

Drought stress significantly reduced the RWC, whereas the drought-induced decrease in RWC was less evident in HNHP, HNLP, and LNHP treatments than in the LNLP treatment (Table 2). LNHP, HNHP, HNLP alleviated the negative effect of drought effects on RWC (Table 2). A significant interaction between drought and nutrition application was detected for WUE_i and PNUE, indicating that the drought-induced reduction in these parameters was greater under the LNLP treatment than under the HNHP, HNLP, and LNHP treatments (Table 3). Drought stress significantly decreased the leaf N and P content. The leaf N content exhibited a smaller reduction under LNHP, HNHP and HNLP than under LNLP (Fig. 1).

Table 3

Instantaneous water use efficiency (WUE_i), photosynthetic N use efficiency (PNUE), photosynthetic P use efficiency (PPUE) and relative water content (RWC) of Moso bamboo under different treatments.

Watering	N level	P level	WUEi (μmolmmol−1)	PNUE (μmol g−1s−1)	PPUE (μmol g−1s−1)	RWC (%)
30%	L	L	3.14±0.42bc	6.39±0.87c	18.99±2.01e	66.31±7.83c
30%	L	H	3.70±0.29ab	7.74±0.59c	24.42±2.67de	75.89±4.70b
30%	H	H	4.10±0.36a	7.42±0.19c	30.56±2.22cd	80.86±1.50b
30%	H	L	3.85±0.27ab	6.77±0.14c	34.20±1.70cd	80.68±0.78b
70%	L	L	2.88±0.30bc	10.10±0.89b	38.19±4.02bc	90.82±3.11a
70%	L	H	2.89±0.34c	9.78±1.08b	30.87±4.18cd	93.97±2.44a
70%	H	H	2.93±0.14c	13.53±0.43a	44.60±4.10ab	92.09±1.43a
70%	H	L	3.13±0.15c	9.44±0.26b	48.32±7.81a	91.79±1.36a
		P:Fw	***	***	***	***
		P:Fn	NS	*	**	**
		P:Fp	NS	***	***	*
		P:Fw×n	NS	*	NS	**
		P:Fw×p	NS	NS	NS	NS
		P:Fn×p	NS	**	NS	*
		P:Fw×n×p	NS	**	NS	NS

Open in a separate window

Each value is the mean±SE (n=6–8). Values followed by the same letter in the same column are not significantly different at the P>0.05 level according to Tukey’s test. Fw, watering effect; Fn, nitrogen effect; Fp, phosphorus effect; Fw×n, watering and nitrogen interaction effect; Fw×p, watering and phosphorus interaction effect; Fn×p, nitrogen and phosphorus interaction effect; Fw×n×p, watering, nitrogen and phosphorus interaction effect, as determined by analyses of variance. NS, no significant; *P<0.05; **P<0.01; ***P<0.001.

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Figure 1

Leaf N content per leaf area (N) and leaf P content per leaf area of Moso bamboo under different treatments. Different letters above bars denote statistically significant differences between treatments at the P<0.05 level according to Tukey’s test. The significance of the factorial analysis (analysis of variance): W, watering effect; N, nitrogen effect; P, phosphorus effect; W×N, watering and nitrogen interaction effect; W×P, watering and phosphorus interaction effect; N×P, nitrogen and phosphorus interaction effect; W×N×P, watering, nitrogen and phosphorus interaction effect. Watering regime [30% (grey bars) or 70% (black bars) of the field capacity] is shown.

Enzyme activities and cellular damage

The oxidative stress resulting from drought was evident from the significant increases detected in MDA contents. Drought resulted in significantly higher POD and SOD activities and higher MDA. LNHP, HNHP, and HNLP plants had a significantly lower MDA content than LNLP plants. LNHP, HNHP and HNLP alleviated the damage resulting from drought stress on MDA. In addition, LNHP, HNHP and HNLP decreased the enhancement of SOD and POD under drought stress (Fig. 2).

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Figure 2

Superoxide dismutase (SOD), peroxidase activity (POD) and malondialdehyde (MDA) of Moso bamboo under different treatments. Different letters above bars denote statistically significant differences between treatments at the P<0.05 level according to Tukey’s test. The significance of the factorial analysis (analysis of variance): W, watering effect; N, nitrogen effect; P, phosphorus effect; W×N, watering and nitrogen interaction effect; W×P, watering and phosphorus interaction effect; N×P, nitrogen and phosphorus interaction effect; W×N×P, watering, nitrogen and phosphorus interaction effect. Watering regime [30% (grey bars) or 70% (black bars) of the field capacity] is shown.

Discussion

Drought stress caused a pronounced inhibition of growth in Moso bamboo. In contrast, N and P application significantly promoted growth under the two watering regimes. On the one hand, the drought stress decreased the biomass, which might be due to the reduced photosynthates². Drought significantly decreased gas exchange parameters, such as Pn, g_s and E, as reported in many previous studies^38,45,46. Furthermore, the decreases in maximum quantum yield of photosynthesis (Fv/Fm) and effective photosystem II quantum yield (Φ_PSII) indicated that structural injuries occurred in photosystem II primary photochemistry in Moso bamboo, as reported by Tezara et al. (2005) in two xerophytic shrubs. These studies have also shown that drought significantly decreased chlorophyll pigment contents, which coincides with the low photosynthesis rate under drought conditions. Therefore, it can be concluded that the reduction in photosynthesis rate under drought conditions may be due to stomatal limitation and PSII impairments. On the other hand, generally, drought can disrupt the electron transport chain, giving rise to oxidant stresses and inducing the production of ROS, which are harmful to organelles including the chloroplast, mitochondrion and peroxisome⁴⁷. Specific enzymes, such as SOD and POD, protect cells from oxidative stress. In the present study, SOD and POD activities were increased by drought stress^1,43,48. These findings suggest that drought alters enzymatic defence reactions to enhance the elimination system for ROS in Moso bamboo.

N and P may be involved in mitigating drought stress by improving dry matter production and its distribution between root and shoot. Previous studies on the interactive effects of N availability and stress conditions have produced various results, ranging from increased sensitivity to stress to decreased sensitivity to stress conditions⁴⁹. Greater growth with nitrogen supply was generally observed when water was less limiting due to a higher photosynthetic rate or water use efficiency (WUE). No growth response to N supply under water stress was observed, however, due to the physiological or structural changes⁵⁰. Such variation in results can be explained by the fact that the N effect on drought stresses depended mainly on the exposure duration, soil status, and nutrient requirements of species^49,51,52. In the present study, our results indicate that the effects of the interaction between water and fertilization on plant dry mass accumulation were significant. In addition, the results showed that fertilization treatments increase the growth of roots less than they promote the growth of shoots, as the values of the root to shoot ratio decreased in conditions with fertilization compared to conditions without fertilization despite the same watering regimes.

N and P addition may help regulate photoassimilates and membrane integrity to minimize the effects of drought stress. On the one hand, RWC and the photosynthesis rate were more reduced while MDA less reduced in LNLP plants than in HNHP, HNLP, or LNHP plants under drought conditions, suggesting a positive role of N and P addition in minimizing the effects of drought stress by monitoring photoassimilates through increasing membrane integrity and decreasing photo-oxidation, as proposed by Akram et al. 2009. Furthermore, the improved stomatal conductance, which was also observed in HNHP, HNLP, and LNHP plants under drought-stressed conditions in this study, indicates that N and P application can reduce stomatal resistance and enhance CO₂ assimilation². N addition and P addition resulted in significantly higher Φ_PSII, Fv/Fm, and qP, indicating that N and P application protects the photochemical efficiency from the damage caused by drought stress. In addition, the protection of photosynthesis and the tolerance of plants to drought stress can be stimulated by adopting strategies to increase the capacity for non-radiative dissipation of excitation energy as heat (NPQ)^53,54. Moreover, an increase in transpiration due to N and P supply might be caused by the accumulation of solutes, absorption and utilization of water use, thereby increasing metabolic activities⁵⁵. Our data showed that drought restrained the electron transport activity, with the restriction of PSII in HNHP, HNLP, and LNHP plants being less severe than in the LNLP plants, as reflected by the smaller decrease in qP. This result was consistent with the high CO₂ assimilation detected under the same treatment. On the other hand, the combined N and P application significantly resulted in lower oxidative damage, measured with MDA, which ultimately increased the biomass of Moso bamboo under water deficit conditions. HNHP, LNHP, and HNLP treatments caused a significant decrease in the activity of SOD and POD enzymes under water deficit conditions, which was in contrast with the other findings⁵⁶. Our results also highlight the lower pressure of oxidation and a reduced need for ROS removal (determined by low SOD and POD) in plants under HNHP, HNLP, LNHP treatments than in plants under LNLP treatments. The decreased activity of these enzymes suggested that Moso bamboo plants have the capacity to preserve the photosynthetic apparatus when grown under drought stress with N and P supplied. This fact is reinforced by the lower levels of lipid peroxidation and electrolyte leakage in the HNHP, HNLP, and LNHP treatments than in the LNLP treatment. Taken together, our findings reveal that the antioxidative system and cellular redox balance are more susceptible to disruption in LNLP than in HNHP plants.

Many studies have reported that water use efficiency increases with an increasing nutrient supply^57–60, while other experiments have shown that N supply had no or negative effects on water use efficiency^61–63. In our study, fertilization increased water use efficiency. This result is consistent with previous studies^58,59. Our result suggests that high N alleviates the negative effects of drought stress on WUE by preventing cell membrane damage and through the overall enhancement of plant photosynthesis. It has been reported that drought stress significantly decreased plant growth by reducing the uptake of water and nutrients⁶⁴. Similarly, our results show that drought stress significantly decreased leaf N and P concentrations, which might be attributed to the reduced N uptake by roots and its impaired transport from roots to shoots due to a restriction in transpiration rates⁶⁵. Furthermore, the inhibitory effects of drought on PPUE were greater in LNLP than in HNHP, HNLP, and LNHP plants, which implies that N and P addition may promote plants to absorb and transport water and mineral nutrition under drought stress conditions. Nevertheless, N and P addition increased the total N and P concentration of leaves under both well-watered and water-stressed treatments. Similar results have been reported in other studies^66–68.

In conclusion, drought stress reduced the total biomass, but N and P application, especially the combination of high N and high P, mitigated the adverse effects of drought. Under drought conditions, the reductions in total biomass were mainly caused by the reduction in photosynthesis rate, which may be due to stomatal limitation, which can alter enzymatic defence reactions to adapt to stress. Furthermore, N and P appear to be involved in mitigating drought stress by improving dry matter production driven by a higher photosynthetic rate, higher water use efficiency, and an increase in membrane integrity. Thus, the improved performance of Moso bamboo under nutrient treatments when exposed to drought may be mainly due to the maintenance of leaf water relations, oxidative damage alleviation and a greater capacity for the photoprotective process (indicated by increased RWC and decreased MDA). Therefore, in practice, the soil N and P status can be modified according to the background level of nutrients to improve drought tolerance. The combination of high N and high P may be an effective strategy to improve the drought tolerance of Moso bamboo seedlings. This study provides information useful for assessing seedling responses in nurseries, and certain ecophysiological responses of seedlings can be employed in modelling stand-scale performance. Such modelling is a key step needed to understand and improve the performance of Moso bamboo cultivation under different stresses in the field.

Materials and Methods

Acknowledgements

Author Contributions

Du. X.H., Wu. Z.Z. and Ying Y.Q. designed experiment and wrote the main manuscript text. Wu. Z.Z., Yu Fang Bi Y.F. and Wang A.K. exulted the experiment at outdoor and the test analysis in laboratory. Zhang Y.B. and proofed the manuscript.

Notes

Footnotes

References