外源色氨酸对低氮胁迫下高粱苗期叶片碳氮平衡和衰老特性的影响

doi:10.3864/j.issn.0578-1752.2023.07.008

摘要/Abstract

摘要：

【目的】研究外源色氨酸对低氮胁迫下高粱叶片衰老的影响，探讨低氮条件下高粱叶片碳氮平衡与衰老的关系，旨在挖掘高粱耐低氮胁迫的有效调控手段。【方法】选用耐低氮高粱自交系398B和氮敏感自交系CS3541为试验材料，采用水培试验，设置正常氮（5 mmol·L^-1）和低氮（0.5 mmol·L^-1）2个氮水平，并进行50 mg·L^-1外源色氨酸喷施处理，喷施10 d后测定叶片形态、组织结构、光合特性、叶绿素荧光参数、叶片碳氮代谢相关物质含量和酶活性、C/N及衰老相关基因表达水平，并分析低氮胁迫下高粱幼苗叶片C/N与衰老相关基因的相关性。【结果】（1）与正常氮水平相比，低氮胁迫显著降低了398B和CS3541的叶面积，而外源色氨酸处理使398B和CS3541的叶面积显著增加了36.72%和52.06%；外源色氨酸显著增加了低氮胁迫下398B和CS3541的叶干重和叶鲜重。（2）与正常氮水平相比，低氮胁迫下，398B花环结构相对完整，而外源色氨酸处理使叶片细胞排列整齐，花环结构清晰；外源色氨酸显著增加了低氮胁迫下398B叶片叶绿素含量（36.85%），而未显著提高CS3541叶片各色素含量。（3）低氮胁迫下，与未喷施色氨酸相比，外源色氨酸处理使叶片光系统II（PSII）表现出更高的最大光化学效率（F_v/F_m）和热耗散（NPQ），增加了叶片光合速率，从而维持叶片更强的光合能力。（4）外源色氨酸提高了低氮胁迫下高粱叶片氮含量和碳氮代谢相关酶活性；降低了高粱叶片碳水平（可溶性糖、蔗糖、淀粉和总糖）和C/N，进而保证低氮胁迫下植株的碳氮生理平衡。（5）外源色氨酸正调控低氮胁迫下衰老相关基因SbLHCB和SbSGR-2的表达，负调控SbNAC6、SbPaO3、SbPPDK-2和SbSAG12-2的表达；另外，C/N与SbLHCB和SbSGR-2表达量呈正相关，与SbNAC6、SbPaO3、SbPPDK-2和SbSAG12-2表达量呈负相关。【结论】低氮胁迫下，外源色氨酸通过降低C/N和衰老基因的表达量，影响叶片形态和光合特性，同时通过调节叶片碳氮代谢，延缓了叶片衰老，增强高粱幼苗对低氮胁迫的耐性。因此，喷施外源色氨酸是缓解低氮胁迫的一种潜在策略。

关键词: 高粱, 衰老相关基因, C/N, 光合特性, 碳氮代谢

Abstract:

【Objective】 The purpose of this study was to investigate the effects of exogenous tryptophan on senescence of sorghum (Sorghum bicolor L.) seedling leaves under low nitrogen stress, and to explore the relationship between C/N balance and senescence of sorghum seedlings leaves, so as to provide effective regulation means for sorghum resistance to low nitrogen stress. 【Method】 In a hydroponic culture experiment, the low nitrogen tolerance sorghum line (398B) and the low nitrogen sensitive sorghum line (CS3541) were selected as the experimental materials. Two nitrogen levels were set at normal nitrogen (5 mmol·L^-1) and low nitrogen (0.5 mmol·L^-1), and 50 mg·L^-1 exogenous tryptophan was applicated by spraying. After 10 days application, leaf morphology, tissue structure, photosynthetic activity, chlorophyll fluorescence parameters, content of carbon and nitrogen metabolism-related substances and enzyme activities, C/N and senescence related gene expression levels were determined, and the correlation between C/N and senescence genes in sorghum seedlings under low nitrogen stress was analyzed.【Result】 (1) Compared with the normal nitrogen treatment, low nitrogen stress significantly reduced the leaf area of 398B and CS3541, while exogenous tryptophan significantly increased the leaf area of 398B and CS3541 by 36.72% and 52.06%. Meanwhile, leaf dry weight and leaf fresh weight of 398B and CS3541 were significantly increased by exogenous tryptophan under low nitrogen stress. (2) Compared with the normal nitrogen treatment, the rosette structure of 398B was relatively complete under low nitrogen stress, while exogenous tryptophan kept the leaf cells orderly and the rosette structure clear. In addition, exogenous tryptophan significantly increased the chlorophyll content of 398B leaves (36.85%), but did not significantly increase the pigment content of CS3541 leaves under low nitrogen stress. (3) Under low nitrogen stress, the exogenous tryptophan treatment resulted in higher PSII maximum photochemical efficiency (F_v/F_m) and non-photochemical quenching (NPQ) capacity, increased leaf photosynthetic rate, and maintained stronger photosynthetic capacity than that without tryptophan. (4) The treatment with exogenous tryptophan reduced the accumulation of sugar (soluble sugar, sucrose and starch) in leaves, but significantly increased the nitrogen content in leaves, correspondingly increased the carbon and nitrogen metabolism enzymes activities, and decreased the C/N in leaves. (5) Exogenous tryptophan positively regulated the expressions of senescence related genes SbLHCB and SBSGR-2, and negatively regulated the expressions of SbNAC6, SbPaO3, SbPPDK-2 and SbSAG12-2 under low nitrogen conditions. In addition, C/N was positively correlated with the expression of SbLHCB and SbSGR-2, and negatively correlated with the expression of SbNAC6, SbPaO3, SbPPDK-2 and SbSAG12-2.【Conclusion】 Under low nitrogen stress, exogenous tryptophan affected leaf morphology and photosynthetic characteristics by reducing C/N and senescence gene expression, and delayed leaf senescence by regulating leaf carbon and nitrogen metabolism, thus enhancing the tolerance of sorghum seedlings under low nitrogen stress. Tryptophan application would be a strategy to weaken low nitrogen stress in the future sustainable agricultural production.

Key words: Sorghum bicolor L., senescence related gene, C/N, photosynthetic activity, carbon and nitrogen metabolism

谷闻东, 刘春娟, 李邦, 刘畅, 周宇飞. 外源色氨酸对低氮胁迫下高粱苗期叶片碳氮平衡和衰老特性的影响[J]. 中国农业科学, 2023, 56(7): 1295-1310.

GU WenDong, LIU ChunJuan, LI Bang, LIU Chang, ZHOU YuFei. Effects of Exogenous Tryptophan on C/N Balance and Senescence Characteristics of Sorghum Seedlings Under Low Nitrogen Stress[J]. Scientia Agricultura Sinica, 2023, 56(7): 1295-1310.

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基因名称 Gene name	引物名称 Primer name	引物序列 Primer sequence (5′-3′)
SbGAPDH	GAPDH-F	GTGTTTGGACTTCTTGGGGGA
SbGAPDH	GAPDH-R	TCATCTCGGGGATCTCTGCC
SbPaO	PaO-F3	CTGCCACCAAGCTCTACCAA
SbPaO	PaO-R3	GAACAACCGACGTGAGCAAC
SbPPDK	PPDK-F2	CAGGCTTACCTGCATCACCA
SbPPDK	PPDK-R2	TTGAGCATCCTGCCACACAA
SbSAG12	SAG12-F2	CGCCTCTATGCTCCGCAATA
SbSAG12	SAG12-R2	CAACCAGTGCAGACAACAGC
SbSGR	SGR-F2	GGCCTCCGCTACTACATCTT
SbSGR	SGR-R2	GGGTCAGGTTGGAGTGGAAG
SbNAC6	NAC6-F	GGCCCTCTTGCTGATGAGTT
SbNAC6	NAC6-R	ACTGTCATCCACTTGGCGAG
SbLHCB	LHCB-F	CGCCAAGTTCGAGGAGTACA
SbLHCB	LHCB-R	TCTGGGGATGATGATGTCGC

处理 Treatment	自交系 Inbred line	叶绿素a Chl a (mg·g^-1)	叶绿素b Chl b (mg·g^-1)	类胡萝卜素 Car (mg·g^-1)	叶绿素含量 Chl (mg·g^-1)
NTrp	398B-NN	2.38±0.06c	0.51±0.01bc	0.49±0.01b	2.89±0.08b
	399B-LN	1.60±0.09e	0.33±0.10d	0.31±0.01d	1.93±0.18e
	CS3541-NN	2.12±0.05d	0.47±0.11bc	0.45±0.03c	2.59±0.14d
	CS3541-LN	1.27±0.05g	0.20±0.02e	0.30±0.01d	1.47±0.07f
Trp	398B-NN	2.50±0.04b	0.59±0.07bc	0.49±0.01b	3.09±0.10a
	399B-LN	2.19±0.06d	0.45±0.07c	0.48±0.01bc	2.64±0.13c
	CS3541-NN	3.41±0.06a	0.71±0.02a	0.70±0.01a	4.12±0.09a
	CS3541-LN	1.44±0.03f	0.19±0.01e	0.33±0.01d	1.63±0.04f