Mechanism of Exogenous Calcium Alleviating Salt Stress Toxicity in Wheat Seedlings

doi:10.13560/j.cnki.biotech.bull.1985.2023-0583

Abstract

Abstract:

【Objective】This paper aims to explore the mechanism that exogenous calcium maintains the homeostasis of wheat seedlings under salt stress when autophagy participates in regulating. These results will provide molecular evidence for revealing the response mechanism of plants to salt stress. 【Method】The wheat variety ‘Henong 6425' was used as experimental material. The wheat seedlings were cultured hydroponically. And then some physiological, biochemical, and molecular biology methods were adopted to carry out the research, including chlorophyll fluorescence parameter analysis, in-situ staining of tissues, quantitative real-time PCR（RT-qPCR）, and confocal laser scanning microscope technique. 【Result】The results indicated that 150 mmol/L NaCl stress significantly inhibited the growth of wheat seedlings, led to damage to photosystem II（PSII）, weakened antioxidant capacity, decreased cell viability, and increased autophagy activity. With the extension of NaCl treatment time, the number of autophagosomes in the roots and leaves of wheat seedlings significantly increased, and the level of programmed cell death（PCD）in the roots and leaves also increased. Exogenous application of Ca²⁺alleviated the damage of NaCl stress to wheat seedlings and regulated the autophagy level in the wheat roots and leaves. Under normal growth conditions, exogenous application of Ca²⁺treatment had no significant effect on the number of autophagosomes in the roots and leaves of wheat seedlings. Under NaCl stress, exogenous Ca²⁺treatment maintained autophagy in the roots and leaves of wheat seedlings at a pro-survival level induced by NaCl stress, and reduced the PCD level in the roots and leaves of wheat seedlings, and slowed down the toxic effect of NaCl stress on wheat seedlings. 【Conclusion】These results indicate that Ca²⁺controls the scale of PCD by regulating autophagy levels in the roots and leaves of wheat seedlings, alleviating the damage of NaCl stress to wheat seedlings.

Key words: wheat, NaCl stress, exogenous Ca²⁺, autophagy, programmed cell death

JIAO Jin-lan, WANG Wen-wen, JIE Xin-rui, WANG Hua-zhong, YUE Jie-yu. Mechanism of Exogenous Calcium Alleviating Salt Stress Toxicity in Wheat Seedlings[J]. Biotechnology Bulletin, 2024, 40(1): 207-221.

Figures/Tables 19

Table 1 Nutrient solution formula

成分Content	浓度Concentration/（mg·L^-1）
Ca（NO₃）₂·4H₂O	165
KNO₃	51
NH₄NO₃	8
KH₂PO₄	27
MgSO₄	49
微量元素	1
铁盐溶液	1

Table 2 Primers used for RT-qPCR

基因Gene	引物序列Primer's sequence（5'-3'）
TaCDPK27	F: GCCGCCTTCCAATACTTT R: TTATCCTGATCTACTTCGCCTA
TaATG2	F:TGTATCCAGATGGGGGTGTT R:GGAACTTAAGCTGCCCTTGA
TaATG5	F: CCAGAAAGGCCATGGAATCTAAC R: GCCTCTTTCAGGGAATTGTTGTA
TaATG7	F: TGACGTTATCGCTCCTGTTG R: ACAGCTGCTCGAGGAATAGC
TaATG10	F: TATTACTCGAGAGGAGCATCCCCAC R: GATTTTCAATCCTACTGCCTGACCG
TaATG8	F: GGAAAGGAGGCAAGCTGAA R: GCATCTCGTTAGGGACAAGGTA
Tubulin	F: GTGGAACTGGCTCTGGC R: CGCTCAATGTCAAGGGA

Fig. 1 Effects of different concentrations of CaCl2 on wheat seedling growth under NaCl stress A: Effects of different concentrations of CaCl2 on wheat seedling growth. B: Effects of different concentrations of CaCl2 on wheat seedling growth under NaCl stress. C: Root length of wheat seedlings treated with different concentrations of CaCl2 under NaCl stress. D: Leaf width of wheat seedlings treated with different concentrations of CaCl2 under NaCl stress. Different letters indicate significant difference in data of each treatment group（n=3, P<0.05）. CK in the figure indicates the control check. Ca indicates the treatment with exogenous addition of CaCl2. Na indicates the treatment with exogenous addition of NaCl. The same below

Fig. 2 Effects of NaCl stress on the growth mechanism of wheat seedling leaves A: DAB staining was used to detect H2O2 accumulation in wheat seedling leaves. B: NBT staining was used to detect O2·- accumulation in wheat seedling leaves. C: Evans Blue staining was used to detect cell viability in wheat seedling leaves. Bar=1 000 µm

Fig. 3 Effects of exogenous Ca2+ on the growth of wheat seedlings under NaCl stress cm

Table 3 Effects of exogenous Ca2+ on the root length, plant height, leaf length and leaf width of wheat seedlings under NaCl stress

不同处理组 Different treatment group	根长Root length	株高Plant height	叶长Leaf Length	叶宽Leaf width
CK	31.17±1.144a	27.07±0.249a	14.68±0.130a	0.40±0.012a
Ca	31.83±0.450a	27.33±0.170a	14.80±0.294a	0.41±0.005a
La	29.83±0.464b	25.60±0.616b	14.27±0.125b	0.38±0.008b
Na	21.70±0.497d	21.73±0.403d	13.47±0.047d	0.33±0.014d
Ca+Na	23.43±0.205c	23.60±0.216c	13.90±0.082c	0.35±0.005c
La+Na	18.20±0.589e	19.60±1.393e	13.01±0.084e	0.31±0.005e

Fig. 4 Effects of exogenous Ca2+ on TaCDPK27 expression in the roots and leaves of wheat seedlings under NaCl stress

Fig. 5 Autophagy activity of wheat seedling roots under NaCl stress detected by MDC staining（Bar=100 µm）

Fig. 6 Effects of exogenous Ca2+ on the autophagy activities of wheat seedling roots under NaCl stress by MDC staining（Bar=100 µm） A: MDC staining was used to detect the effects of exogenous Ca2+ on autophagy activity in the roots of wheat seedlings under NaCl stress for 6 h. B: MDC staining was used to detect the effects of exogenous Ca2+ on the autophagy activity in the roots of 144 h wheat seedlings under NaCl stress

Fig. 7 Effects of exogenous Ca2+ on the autophagy activities of wheat seedling leaves under NaCl stress at 144 h by MDC staining（Bar=100 µm）

Fig. 8 Effects of exogenous Ca2+ on TaATGs expression in the roots and leaves of wheat seedlings under NaCl stress by RT-qPCR A: Effects of exogenous Ca2+ on TaATGs expression in wheat seedling roots under NaCl stress. B: Effects of exogenous Ca2+ on TaATGs expression in wheat seedling leaves under NaCl stress

Fig. 9 Effects of exogenous Ca2+ on cell viability in the roots and leaves of wheat seedlings under NaCl stress by Evans Blue staining（Bar=1 000 µm） A: Effects of exogenous Ca2+ on the cell viability of wheat seedling leaves under NaCl stress. B: Effects of exogenous Ca2+ on the cell viability of wheat seedling roots under NaCl stress. The arrows in the figures indicatethe area of cell death

Fig. 10 Effects of exogenous Ca2+ on PCD in the roots and leaves of wheat seedlings under NaCl stress by TUNEL staining

Fig. 11 Effects of exogenous Ca2+ on H2O2 and O2·- accumulation in wheat seedlings under NaCl stress by DAB and NBT staining（Bar=1 000 µm） A and B showed the distribution of H2O2 in the leaves and roots of wheat seedlings detected by DAB staining. C and D showed the distribution of O2·- in the leaves and roots of wheat seedlings detected by NBT staining

Fig. 12 Effects of exogenous Ca2+ on H2O2 and O2·- contents in the roots and leaves of wheat seedlings under NaCl stress A: The contents of H2O2 in the roots and leaves of wheat seedlings. B: The contents of O2·- in the roots and leaves of wheat seedlings

Fig. 13 Effects of exogenous Ca2+ on the POD activities of wheat seedlings under NaCl stress

Fig. 14 Effects of exogenous Ca2+ on the SOD and CAT activities of wheat seedlings under NaCl stress A: The effects of exogenous Ca2+ on SOD content in the roots and leaves of wheat seedlings under NaCl stress. B: The effects of exogenous Ca2+ on CAT content in the roots and leaves of wheat seedlings under NaCl stress

Table 4 Effects of exogenous Ca2+ on the chlorophyll fluorescence parameters of wheat seedlings after 5 d and 10 d of NaCl stress

处理天数 Treatment days/d	处理组 Treatment group	Fv/Fm	Y（Ⅱ）	qP	F₀	NPQ	ETR
5	CK	0.796±0.001a	0.393±0.011a	0.621±0.044a	0.046±0.001a	1.227±0.039e	59.2±3.767a
	Ca	0.797±0.004a	0.392±0.004a	0.624±0.007a	0.046±0.002a	1.229±0.107e	58.6±2.546a
	La	0.792±0.004b	0.386±0.048b	0.608±0.054b	0.044±0.003b	1.363±0.151d	55.9±1.543b
	Na	0.787±0.001d	0.281±0.007d	0.474±0.008d	0.038±0.003d	1.731±0.099b	40.2±0.834d
	Ca+Na	0.789±0.007c	0.309±0.037c	0.497±0.045c	0.041±0.001c	1.566±0.036c	43.2±3.982c
	La+Na	0.784±0.003e	0.254±0.036e	0.383±0.019e	0.035±0.002e	1.782±0.037a	35.2±4.626e
10	CK	0.794±0.001a	0.379±0.007a	0.628±0.002a	0.055±0.003a	1.341±0.129e	53.3±1.296a
	Ca	0.792±0.001a	0.383±0.025a	0.633±0.016a	0.053±0.007a	1.339±0.024e	54.6±1.964a
	La	0.779±0.001b	0.342±0.025b	0.55±0.011b	0.047±0.003b	1.404±0.099d	46.9±1.438b
	Na	0.778±0.002d	0.265±0.024d	0.459±0.033d	0.035±0.002d	1.595±0.117b	36.8±3.432d
	Ca+Na	0.782±0.002c	0.321±0.033c	0.546±0.045c	0.039±0.002c	1.541±0.043c	44.3±4.613c
	La+Na	0.765±0.003e	0.238±0.017e	0.454±0.035e	0.033±0.003e	2.056±0.125a	32.9±2.286e

Table 5 Effects of exogenous Ca2+ on the chlorophyll contents of wheat seedlings under NaCl stress

处理组Treatment group	5 d	10 d
CK	0.785±0.021a	0.966±0.027a
Ca	0.793±0.019a	0.985±0.061a
La	0.686±0.009b	0.872±0.048b
Na	0.531±0.037d	0.640±0.012d
Ca+Na	0.620±0.008c	0.746±0.032c
La+Na	0.336±0.048e	0.538±0.014e

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