外源钙缓解小麦幼苗盐胁迫的作用机制

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

摘要/Abstract

摘要：

【目的】探究自噬参与调控外源钙维持盐胁迫下小麦幼苗稳态的机制，为进一步揭示植物盐胁迫应答机制提供分子证据。【方法】以小麦品种河农6425为材料，通过水培培养小麦幼苗，利用叶绿素荧光、组织原位染色、荧光定量PCR、激光共聚焦显微镜等生理及分子生物学技术开展研究。【结果】150 mmol/L NaCl胁迫显著抑制小麦幼苗生长，导致光系统II（photosystem II, PSII）受损，抗氧化能力减弱，细胞活力降低，自噬活性升高。随着NaCl处理时间的延长，小麦幼苗根和叶片中的自噬小体显著增多，细胞程序性死亡（programmed cell death, PCD）水平升高。正常条件下，外源施加Ca²⁺对小麦幼苗根和叶片中自噬小体数量没有明显影响。而在NaCl胁迫下，外源Ca²⁺处理能够在一定时间内维持NaCl胁迫诱导的自噬维持在促存活的水平，减少PCD。【结论】Ca²⁺通过调控小麦幼苗根和叶片的自噬水平限制PCD的规模，缓解NaCl胁迫对小麦幼苗的损伤。

关键词: 小麦, 盐胁迫, 外源Ca²⁺, 细胞自噬, 细胞程序性死亡

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

焦进兰, 王文文, 介欣芮, 王华忠, 岳洁瑜. 外源钙缓解小麦幼苗盐胁迫的作用机制[J]. 生物技术通报, 2024, 40(1): 207-221.

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.

图/表 19

表1 营养液配置表

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

表2 RT-qPCR引物

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

图1 不同浓度CaCl2对NaCl胁迫下小麦幼苗生长影响 A：不同浓度CaCl2对小麦幼苗生长的影响；B：不同浓度CaCl2对NaCl胁迫下小麦幼苗生长影响；C：NaCl胁迫下不同浓度CaCl2处理后小麦幼苗根长测量值； D：NaCl胁迫下不同浓度CaCl2处理后小麦幼苗叶宽测量值。不同字母表示各处理组数据差异显著（n=3，P<0.05）。CK表示对照；Ca表示外源添加CaCl2处理；Na表示外源添加NaCl处理。下同

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

图2 NaCl胁迫对小麦幼苗叶片生长机理影响 A：DAB染色检测小麦幼苗叶片H2O2积累情况；B：NBT染色检测小麦幼苗叶片O2·-积累量；C：伊文思蓝染色检测小麦幼苗叶片细胞活力。Bar=1 000 µm

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

图3 外源Ca2+对NaCl胁迫下小麦幼苗生长状况的影响

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

表3 外源Ca2+ 对NaCl胁迫下小麦幼苗根长、株高、叶长、叶宽的影响

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

图4 外源Ca2+对NaCl胁迫下小麦幼苗根和叶片中TaCDPK27表达情况的影响

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

图5 MDC染色检测NaCl胁迫下小麦幼苗根中自噬小体（标尺=100 µm）

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

图6 MDC染色检测外源Ca2+ 对NaCl胁迫下小麦幼苗根自噬活性影响（标尺=100 µm） A：MDC染色检测外源Ca2+ 对NaCl胁迫6 h小麦幼苗根自噬活性影响； B：MDC染色检测外源Ca2+ 对NaCl胁迫144 h小麦幼苗根自噬活性影响

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

图7 MDC染色检测外源Ca2+ 对NaCl胁迫144 h小麦幼苗叶片自噬活性的影响（标尺=100 µm）

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）

图8 RT-qPCR检测外源Ca2+对NaCl胁迫下小麦幼苗根和叶片中TaATGs表达的影响 A：外源Ca2+对NaCl胁迫下小麦幼苗根中TaATGs表达的影响；B：外源Ca2+对NaCl胁迫下小麦幼苗叶片TaATGs表达的影响

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

图9 伊文思蓝染色检测外源Ca2+对NaCl胁迫下小麦幼苗根和叶片细胞活力的影响（标尺=1 000 µm） A：外源Ca2+对NaCl胁迫下小麦幼苗叶片细胞活力的影响；B：外源Ca2+对NaCl胁迫下小麦幼苗根细胞活力的影响。图中箭头表示细胞死亡区域

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

图10 TUNEL染色检测外源Ca2+ 对NaCl胁迫下小麦幼苗根和叶片PCD的影响

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

图11 DAB与NBT染色检测外源Ca2+对NaCl胁迫下小麦幼苗H2O2与O2·-累积量的影响（标尺=1 000 µm） A和B分别为DAB染色检测小麦幼苗叶片和根中H2O2分布；C和D分别为NBT染色检测小麦幼苗叶片和根中O2·-分布

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

图12 外源Ca2+对NaCl胁迫下小麦幼苗根和叶片H2O2与O2·-含量的影响 A：小麦幼苗根与叶片中H2O2含量；B：小麦幼苗根与叶片中O2·-含量

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

图13 外源Ca2+ 对NaCl胁迫下小麦幼苗根和叶片POD活性影响

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

图14 外源Ca2+ 对NaCl胁迫下小麦幼苗SOD、CAT活性的影响 A：外源Ca2+对NaCl胁迫下小麦幼苗根与叶片中SOD含量的影响；B：外源Ca2+对NaCl胁迫下小麦幼苗根与叶片中CAT含量的影响

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

表4 外源Ca2+对NaCl胁迫5 d和10 d小麦幼苗叶绿素荧光参数的影响

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

表5 外源Ca2+对NaCl胁迫下小麦幼苗叶绿素含量的影响

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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