水杨酸调控盐胁迫下羽衣甘蓝种子萌发的机理

doi:10.11983/CBB19047

摘要/Abstract

摘要： 盐胁迫是植物种子萌发与植株生长的重要限制因子。以羽衣甘蓝(Brassica oleracea var. acephala)名古屋为材料, 研究不同盐分对其种子萌发的影响, 探索水杨酸(SA)及其合成抑制剂氨基茚磷酸(AIP)处理对羽衣甘蓝种子萌发的调控效应。实验结果表明, 150与200 mmol·L ^-1 NaCl处理后的羽衣甘蓝种子活力显著降低。盐胁迫显著降低种子的吸水速率、种子活力与幼苗质量, 降低苯丙氨酸裂解酶活性与内源SA含量, 提高过氧化氢(H₂O₂)与超氧阴离子(O₂ ^-.)含量。SA可以缓解盐胁迫对羽衣甘蓝种子活力的抑制作用, 通过促进内源SA合成, 从而提高种子吸水率与种子活力, 促进种子对K ⁺、Mg ²⁺的吸收, 降低Na ⁺含量。此外, 外源施加SA能够显著增强超氧化物歧化酶和过氧化物酶活性, 降低H₂O₂与O₂ ^-.的积累。相反, 氨基茚磷酸(AIP)处理能够增强盐胁迫对种子萌发的抑制作用, 推测这与AIP处理能够显著降低种子内源SA含量密切相关。研究表明外源SA主要通过提高保护酶活性、降低活性氧积累和维持体内离子平衡来增强羽衣甘蓝的耐盐性。

关键词: 羽衣甘蓝, 种子萌发, 盐胁迫, 水杨酸, 生理特性

Abstract: Salinity is a major abiotic constraint affecting seed germination and plant growth. The effects of salt stress and salicylic acid (SA) and 2-aminoindan-2-phosphonic acid (SA synthesis inhibitor, AIP) on seed germination were investigated using kale (Brassica oleracea var. acephala) variety ‘Nagoya’. The results showed that the seed vigor of kale was significant decreased under 150 or 200 mmol·L ^-1 NaCl treatment by significantly reducing seed water uptake rate, seed vigor, and seedling quality, inhibited phenylalanine ammonia and lyase activity, decreasing endogenous SA content, and increasing hydrogen peroxide (H₂O₂) and superoxide anion (O₂ ^-.) contents. SA treatment effectively alleviated the inhibition of salt stress on seed germination, promoted endogenous SA anabolism, increased seed water absorption rate and seed vigor, promoted K ⁺ and Mg ²⁺ absorption, and reduced the Na ⁺ content. Furthermore, SA treatment significantly increased superoxide dismutase and peroxidase activities and inhibited H₂O₂ and O₂ ^-.accumulation. By contrast, AIP treatment significantly inhibited the germination process under salt stress, likely due to the reduced level of endogenous SA. This study demonstrates that exogenous SA enhances salt tolerance in kale seeds primarily through increasing the protective enzyme activities, reducing reactive oxygen species accumulation, and maintaining ion homeostasis during seed germination.

Key words: kale, seed germination, salt stress, salicylic acid, physiological characteristics

曹栋栋,陈珊宇,秦叶波,吴华平,阮关海,黄玉韬. 水杨酸调控盐胁迫下羽衣甘蓝种子萌发的机理. 植物学报, 2020, 55(1): 49-61.

Dongdong Cao,Shanyu Chen,Yebo Qin,Huaping Wu,Guanhai Ruan,Yutao Huang. Regulatory Mechanism of Salicylic Acid on Seed Germination Under Salt Stress in Kale. Chinese Bulletin of Botany, 2020, 55(1): 49-61.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19047

https://www.chinbullbotany.com/CN/Y2020/V55/I1/49

图/表 11

表1 qRT-PCR反应所用引物序列

Table 1 Primers used for qRT-PCR

Gene name	No. of accession	Primer sequence (5'-3')
18srRNA	NC_016118.1	F: GGAAGGACTTGTACGGTAACATTG R: TGGACCTGCCTCATCATACTCA
BoPAL1	XM_013781025.1	F: TCGATCTTCCACAAGATTGGT R: TCCACTTCGTCAGGAAGCA
BoPAL2	XM_013749252.1	F: AATCAGCTGAGCAACATAACCA R: GACGTTTTGCGAGACGAGA
BoCAT1	XM_013742034.1	F: ATCCTCGTGGTTTTGCTGTC R: TGCCAACAAGATCAAAGTTCC
BoCAT2	XM_013774413.1	F: ATGGAAGGCTCAGGTGTCAA R: CGTAGTGAGCTTTTCCGGATT
BoSOD1	XM_013783393.1	F: ACATCATTGTTGGAGATGATGG R: GAGGGATCTGGCAGTCAGTG
BoSOD3	XM_013750980.1	F: TGGTGATCCTGATGACCTTG R: CCTGCGTTTCCTGTTGATTT
BoPOD2	XM_013729485.1	F: GGTGGTTTCTTGCGCTGA R: CCATGGTCCGTTGATCACTA
BoPOD3	XM_013728218.1	F: CGTCAGGAAACACGGAGAA R: CCTCAATGAAGCCAAATCCT

图1 盐胁迫对羽衣甘蓝种子萌发的影响 (A) 盐胁迫下种子萌发进度曲线; (B) 盐胁迫96小时种子萌发情况

Figure 1 Effects of salt stress on seed germination of kale (A) Time course of seed germination under salt stress; (B) Seed germination after 96 hours of NaCl treatments with different concentration

表2 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对羽衣甘蓝种子萌发过程中吸水率的影响

Table 2 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on water uptake rate during seed germination of kale under salt stress

Treatments	Water uptake rate (%)
Treatments	0-4 h	4-8 h	8-16 h	16-24 h	24-36 h	36-48 h
Control-1	47.18±3.52 a	42.56±2.19 a	19.14±0.81 a	18.33±2.04 a	18.21±1.33 a	27.26±2.67 a
Control-2	37.11±4.31 b	31.67±3.15 c	14.62±1.09 b	12.62±0.09 c	10.21±1.02 b	17.64±0.08 b
SA	42.27±2.89 a	37.75±3.09 b	14.31±1.59 b	14.24±0.15 b	16.77±1.44 a	25.81±1.77 a
AIP	32.12±2.71 c	30.41±4.93 c	13.41±1.07 c	12.78±0.21 c	8.21±0.06 c	7.55±1.23 c

表3 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对羽衣甘蓝种子发芽的影响

Table 3 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on seed germination of kale under salt stress

Treatments	Germination energy (%)	Germination percentage (%)	Germination index	Vigor index	Seedling length (cm)	Seedling dry weight (g)
Control-1	85.333±3.270 a	85.333±3.272 a	15.742±0.742 a	1.610±0.892 a	8.241±0.711 a	0.083±0.010 a
Control-2	53.667±3.208 c	56.333±4.044 c	8.231±0.810 c	0.908±0.071 c	7.822±0.635 b	0.076±0.008 b
SA	62.333±4.615 b	68.667±4.730 b	10.558±0.556 b	1.167±0.064 b	7.837±0.706 b	0.079±0.009 b
AIP	41.000±3.331 d	46.667±2.516 d	7.267±0.597 d	0.762±0.079 d	7.156±0.523 c	0.064±0.004 c

图2 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对羽衣甘蓝发芽5天幼苗生长的影响 Control-1、Control-2、SA和AIP同表2。

Figure 2 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on kale seedling growth at 5 days post-germination under salt stress Control-1, Control-2, SA and AIP are the same as Table 2.

图3 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对萌发的羽衣甘蓝种子中水杨酸含量(A)、苯丙氨酸裂解酶活性(B)及其合成基因表达量(C, D)的影响 PAL: 苯丙氨酸裂解酶。Control-1、Control-2、SA和AIP同表2。不同小写字母表示不同处理间差异显著(P<0.05)。

Figure 3 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on SA content (A), PAL activity (B) and BoPAL1-2 expression (C, D) in kale seeds during germination under salt stress PAL: Phenylalanine ammonia-lyase. Control-1, Control-2, SA and AIP are the same as Table 2. Different lowercase letters above the bars indicate significant differences (P<0.05) among treatments at the same sown time.

表4 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对羽衣甘蓝种子发芽过程中离子含量的影响

Table 4 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on ions contents during seed germination of kale under salt stress

Germination time (h)	Treatments	Ion content (mg·g^-1 FW)
Germination time (h)	Treatments	Na⁺	K⁺	Ca²⁺	Mg²⁺
12	Control-1	2.553±0.219 c	1.471±0.134 a	0.221±0.018 a	0.431±0.026 a
	Control-2	3.711±0.431 a	1.227±0.203 b	0.192±0.012 b	0.313±0.033 c
	SA	3.292±0.331 b	1.435±0.053 a	0.183±0.009 b	0.372±0.042 b
	AIP	3.809±0.504 a	1.170±0.077 c	0.211±0.018 a	0.211±0.018 d
24	Control-1	3.951±0.412 c	1.682±0.117 a	0.148±0.017 a	0.477±0.033 a
	Control-2	5.587±0.581 a	1.427±0.109 b	0.123±0.017 b	0.385±0.049 b
	SA	5.108±0.432 b	1.623±0.152 a	0.112±0.006 b	0.462±0.053 a
	AIP	5.717±0.367 a	1.301±0.083 c	0.118±0.008 b	0.321±0.019 c
36	Control-1	3.762±0.319 c	1.677±0.092 a	0.176±0.074 a	0.597±0.055 a
	Control-2	6.193±0.419 a	1.486±0.106 c	0.093±0.012 b	0.511±0.022 b
	SA	5.654±0.384 b	1.583±0.201 b	0.101±0.004 b	0.590±0.044 a
	AIP	6.202±0.609 a	1.472±0.013 c	0.095±0.011 b	0.367±0.048 c
48	Control-1	4.188±0.381 b	1.769±0.208 a	0.132±0.008 a	0.646±0.054 a
	Control-2	5.772±0.720 a	1.311±0.011 c	0.073±0.003 c	0.491±0.060 c
	SA	5.790±0.364 a	1.422±0.015 b	0.072±0.009 c	0.582±0.015 b
	AIP	5.831±0.519 a	1.226±0.064 d	0.094±0.014 b	0.401±0.008 d

表5 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对羽衣甘蓝种子发芽过程中渗透调节物质含量的影响

Table 5 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on osmotic adjustment substances during seed germination of kale under salt stress

Germination time (h)	Treatments	Osmotic adjustment substance
Germination time (h)	Treatments	Soluble sugar (mg·g^-1 FW)	Soluble protein (mg·g^-1 FW)	Free proline (μg·g^-1 FW)
12	Control-1	13.56±0.81 d	3.57±0.40 d	108.19±1.23 c
	Control-2	23.19±1.73 b	8.11±0.71 b	143.77±1.35 b
	SA	29.28±2.00 a	12.17±1.03 a	156.53±1.17 a
	AIP	19.13±1.23 c	6.59±0.33 c	157.11±0.90 a
24	Control-1	17.11±0.92 c	6.17±0.55 c	115.64±0.87 c
	Control-2	26.35±1.74 b	9.27±0.49 b	167.59±2.11 a
	SA	33.27±4.02 a	13.56±0.84 a	142.13±1.33 b
	AIP	25.77±3.19 b	6.59±0.30 c	122.64±2.16 c
36	Control-1	23.85±1.39 d	6.12±0.41 c	117.26±1.55 c
	Control-2	37.50±2.47 b	11.73±1.23 a	141.33±1.46 b
	SA	43.59±4.33 a	12.19±1.19 a	172.57±1.15 a
	AIP	30.66±2.20 c	9.13±0.52 b	109.33±0.82 c
48	Control-1	22.74±3.10 c	5.80±0.37 b	133.52±0.81 c
	Control-2	34.19±2.79 a	8.62±0.79 a	167.29±1.31 b
	SA	36.82±2.12 a	8.93±0.64 a	128.19±1.55 c
	AIP	26.70±2.34 b	6.18±0.56 b	186.53±2.08 a

图4 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对萌发的羽衣甘蓝种子中过氧化氢(A)与超氧阴离子(B)含量的影响 Control-1、Control-2、SA和AIP同表2。不同小写字母表示不同处理间差异显著(P<0.05)。

Figure 4 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on H2O2 (A) and O2-. (B) contents of kale seed during germination under salt stress Control-1, Control-2, SA and AIP are the same as Table 2. Different lowercase letters above the bars indicate significant differences (P<0.05) among treatments at the same sown time.

图5 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对萌发的羽衣甘蓝种子中过氧化氢酶(CAT) (A)、超氧化物歧化酶(SOD) (B)与过氧化物酶(POD) (C)活性的影响 Control-1、Control-2、SA和AIP同表2。

Figure 5 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on the activities of catalase (CAT) (A), superoxide dismutase (SOD) (B), and peroxidase (POD) (C) in kale seeds during germination under salt stress Control-1, Control-2, SA and AIP are the same as Table 2.

图6 盐胁迫下水杨酸(SA)及其抑制剂氨基茚磷酸(AIP)对羽衣甘蓝种子发芽过程中BoCAT1 (A)、BoCAT2 (B)、BoSOD1 (C)、BoSOD3 (D)、BoPOD2 (E)和BoPOD3 (F)表达量的影响 Control-1、Control-2、SA和AIP同表2。不同小写字母表示不同处理间差异显著(P<0.05)。

Figure 6 Effects of salicylic acid (SA) and SA synthesis inhibitor (2-aminoindano-2-phosphonic acid, AIP) on gene expression of BoCAT1 (A), BoCAT2 (B), BoSOD1 (C), BoSOD3 (D), BoPOD2 (E), and BoPOD3 (F) during seed germination of kale under salt stress Control-1, Control-2, SA and AIP are the same as Table 2. Different lowercase letters above the bars indicate significant differences (P<0.05) among treatments at the same sown time.

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