链霉菌TOR3209对低温胁迫下番茄植株叶绿素合成和多胺含量的影响

doi:10.3864/j.issn.0578-1752.2025.15.010

摘要/Abstract

摘要：

【目的】研究链霉菌TOR3209缓解低温对番茄植株胁迫作用的生物学机制，主要从叶绿素合成代谢和多胺含量变化两方面入手揭示TOR3209对光系统中捕光色素蛋白复合体II（LHCII）的保护机制。【方法】培养番茄至4叶期于移栽时接种TOR3209菌剂，移栽30 d后进行低温胁迫（5 ℃）试验，设置常温接种TOR3209（TOR3209）、常温不接菌（NI）、低温接种TOR3209（TOR3209+C）、低温不接菌（NI+C）4个处理。采用蔗糖密度梯度离心法检测LHCII构象的变化，使用微型板酶检测技术测定叶绿素含量、叶绿素合成前体物质以及合成代谢关键酶活性，通过高效液相色谱法检测不同形态多胺含量，利用荧光定量PCR分析叶绿素和多胺合成代谢关键基因表达的差异。【结果】低温胁迫引起类囊体膜LHCII单体和三聚体丰度的降低，链霉菌TOR3209可有效缓解低温胁迫对LHCII单体及三聚体的降解。番茄叶片叶绿素含量受低温影响均显著降低，且叶绿素合成的前体物质胆色素原（PBG）大量积累并伴随着尿卟啉原III（Urogen III）、原卟啉IX（Proto IX）、镁-原卟啉IX（Mg-proto IX）和原叶绿素酸酯（Pchl）等下游前体物质的含量显著下降，叶绿素含量的降低主要是由PBG向Urogen III的转化受到阻碍导致的。链霉菌TOR3209能增强低温胁迫下胆色素原脱氨酶（PBGD）活性及其编码基因HemC的表达水平，促进PBG向Urogen III的转化，从而恢复受阻点后的叶绿素合成途径，同时降低叶绿素酶（Chlase）活性及其编码基因Clh的表达水平，促进叶绿素的积累。低温胁迫导致叶片游离态多胺和结合态多胺含量均显著降低，腐胺（Put）/精胺（Spm）比率下降；TOR3209在胁迫初期显著提升游离态和结合态多胺的积累水平；随着胁迫持续，通过上调Odc表达促进Put向亚精胺（Spd）转化，使游离态/结合态Put和结合态Spd持续积累，但Spm含量与低温对照无显著差异，因此Put/Spm比率显著提高，有助于增强低温胁迫下LHCII丰度并稳定LHCII的聚集状态。此外，TOR3209可提高因低温胁迫导致的转谷酰胺酶（TGase）活性下降，促进多胺从游离态向结合态转化。【结论】 TOR3209通过缓解因低温导致的PBG向Urogen III转化的受阻作用促进番茄叶片叶绿素的生物合成，并提高Put的含量和Put/Spm比率，来增强LHCII单体和三聚体的稳定性，保护光合器官免受低温胁迫的破坏。

关键词: 链霉菌, 番茄, 低温胁迫, 捕光色素蛋白复合体II, 叶绿素合成, 多胺

Abstract:

【Objective】 This study aims to elucidate the biological mechanisms underlying the mitigation of low temperature-induced damage in tomato plants by Streptomyces sp. TOR3209, with particular emphasis on evaluating the impact of TOR3209 inoculation on light-harvesting complex of PSII (LHCII). The investigation focuses primarily on the alterations in chlorophyll biosynthesis pathways and polyamine accumulation.【Method】 Tomato seedlings at the quadrifoliate stage were inoculated with Streptomyces sp. TOR3209 during transplantation into the pots. Low-temperature stress (5 ℃) was conducted 30 days post-transplantation. The experimental design comprised four treatment groups: TOR3209-inoculated plants (TOR3209), non-inoculated plants (NI), TOR3209-inoculated plants exposed to cold stress (TOR3209+C), and non-inoculated plants exposed to cold stress (NI+C). The conformational alterations in LHCII were analyzed through sucrose density gradient centrifugation. Chlorophyll content, precursor metabolite levels, and enzymatic activities in chlorophyll biosynthesis pathways were quantified using microplate-based enzymatic assays. Polyamine speciation profiles were determined by high performance liquid chromatography, while the expression differences of key genes involved in chlorophyll and polyamine biosynthesis metabolism were examined via quantitative real-time PCR.【Result】 Low temperature stress markedly decreased the abundance of LHCII monomers and trimers in thylakoid membranes, while TOR3209 inoculation effectively alleviated the degradation. The chlorophyll content in tomato leaves showed a pronounced decrease by low temperature, concurrent with substantial accumulation of the chlorophyll precursors porphobilinogen (PBG). This accumulation coincided with a marked decline in the level of downstream chlorophyll precursors, including uroporphyrinogen III (Urogen III), protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-proto IX), and protochlorophyll (Pchl). The reduction in chlorophyll content was mainly caused by the blocked site, which was from PBG to Urogen III in chlorophyll synthesis. Streptomyces sp. TOR3209 enhanced porphobilinogen deaminase (PBGD) enzymatic activity and upregulated expression of the encoded gene HemC. This regulatory effect mitigated the inhibitory impact of low temperature stress on the metabolic conversion from PBG to Urogen III, consequently facilitating the recovery of the previously impaired chlorophyll biosynthesis pathway. Concurrently, the inoculation suppressed both chlorophyllase (Chlase) activity and Clh gene expression levels, promoting chlorophyll accumulation. The contents of free and conjugated polyamines were all decreased significantly under low temperature stress, accompanied by a decreased putrescine (Put)/spermine (Spm) ratio. TOR3209 inoculation alleviated the decline of polyamines, and the levels of different forms of polyamines were significantly elevated at the early stage of stress. Extended stress duration activated the metabolic conversion from Put to spermidine (Spd) through Odc gene upregulation, resulting in progressive accumulation of free/conjugated Put and conjugated Spd. Notably, while Spm levels showed no significant variation relative to NI+C, the elevated ratio of Put/Spm contributed to enhancing the abundance of LHCII and stabilized the aggregation state of LHCII. In addition, TOR3209 effectively reversed the cold stress-induced reduction in transglutaminases (TGase) activity while stimulating polyamine conjugation processes.【Conclusion】 TOR3209 treatment enhanced chlorophyll biosynthesis in tomato leaves through relieving the hindrance on the transformation from PBG to Urogen III caused by low temperature stress, and elevating Put accumulation and the Put/Spm ratio. These biochemical modifications collectively stabilized monomeric and trimeric LHCII configurations, providing comprehensive protection for the photosynthetic apparatus against low temperature stress.

Key words: Streptomyces, tomato (Solanum lycopersicum), low temperature stress, light-harvesting complex of PSII (LHCII), chlorophyll synthesis, polyamine

马佳, 彭杰丽, 王旭, 贾楠, 李孟凯, 胡栋. 链霉菌TOR3209对低温胁迫下番茄植株叶绿素合成和多胺含量的影响[J]. 中国农业科学, 2025, 58(15): 3064-3080.

MA Jia, PENG JieLi, WANG Xu, JIA Nan, LI MengKai, HU Dong. Effects of Streptomyces sp. TOR3209 on Chlorophyll Synthesis and Polyamine Content in Tomato Plants Under Low Temperature Stress[J]. Scientia Agricultura Sinica, 2025, 58(15): 3064-3080.

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基因Gene	正向引物Forward primer (5′-3′)	反向引物Reverse primer (5′-3′)
HemC	TTCTCCACCGCTATCCCTCA	ATACCAATGGCACCCTGAGC
Clh	GCAAACTCCGCCATCAGTTC	TGCCTCGAATCCCTTTGGTC
Spds	GAGGAGGAGATGGTGGTGTC	ATGCAACTCCGTCACCAATG
Odc	ACTTCCACGACTTCCCTGAG	TTCGACATGCACGCTAATGG
Actin	TGTCCCTATTTACGAGGGTTATGC	CAGTTAAATCACGACCAGCAAGAT

叶绿素含量Chlorophyll content	处理Treatment	0 d	1 d	3 d	6 d	10 d
叶绿素a Chl a (mg·g^-1 FW)	TOR3209+C	11.67±0.49a	5.77±0.42b	6.07±0.26b	7.38±0.32b	6.23±0.57b
	NI+C	9.52±0.43b	4.61±0.42c	4.55±0.33c	4.68±0.65d	4.06±0.79c
	TOR3209	11.67±0.49a	8.11±0.36a	9.23±0.48a	9.03±0.66a	8.46±0.55a
	NI	9.52±0.43b	6.14±0.34b	6.54±0.54b	6.32±0.04c	5.78±0.06b
叶绿素b Chl b (mg·g^-1 FW)	TOR3209+C	4.65±0.40a	3.13±0.51c	2.74±0.39b	6.72±1.23b	2.77±0.11c
	NI+C	3.66±0.42b	2.66±0.37c	1.95±0.45b	3.28±0.97c	2.05±0.54c
	TOR3209	4.65±0.40a	9.01±0.53a	7.26±0.13a	9.02±0.20a	8.26±0.56a
	NI	3.66±0.42b	6.53±0.22b	7.29±0.14a	6.49±0.05b	5.54±0.58b
总叶绿素 Chl t (mg·g^-1 FW)	TOR3209+C	16.69±0.90a	9.11±0.54c	9.01±0.67c	14.45±1.58b	9.21±0.57c
	NI+C	13.47±0.61b	7.44±0.79d	6.65±0.23d	8.16±1.02c	6.25±1.34d
	TOR3209	16.69±0.90a	17.57±0.78a	16.89±0.62a	18.51±0.84a	17.14±1.14a
	NI	13.47±0.61b	13.00±0.51b	14.19±0.69b	13.15±0.09bc	11.60±0.66b

处理 Treatment	转谷酰胺酶活性TGase activity (mmol·h^-1·g^-1)
处理 Treatment	0 d	1 d	3 d	6 d	10 d
TOR3209+C	0.60±0.03a	0.51±0.00bc	0.49±0.02c	0.54±0.01c	0.53±0.02b
NI+C	0.55±0.02b	0.45±0.04c	0.45±0.01d	0.46±0.01d	0.36±0.02c
TOR3209	0.60±0.03a	0.73±0.03a	0.65±0.00a	0.63±0.02a	0.63±0.03a
NI	0.55±0.02b	0.53±0.04b	0.55±0.03b	0.58±0.02b	0.60±0.03a