马铃薯叶片气孔的开张与关闭同步伴随果胶的降解与合成

doi:10.3864/j.issn.0578-1752.2022.17.002

摘要/Abstract

摘要：

【目的】比较气孔关闭与不同气孔密度开张之间叶片分化表达蛋白质及其积累变化,揭示果胶代谢如何调控气孔开张和关闭,为深入理解气孔的环境适应功能提供依据。【方法】构建体内过量和抑制StEPF-2（EPIDERMAL PATTERNING FACTOR 2 from Solanum tuberosum）表达载体,以茎段为外植体,转化马铃薯克新1号植株,获得不同气孔密度的转化株系,以黑暗条件下气孔关闭为对照,采用RNA-seq和iTRAQ分别检测不同处理和对照叶片的基因和蛋白质表达谱,比较不同气孔密度分化表达的蛋白质,并通过StEPF-2标记的pulldown和LC-MS/MS结果确认,明确不同气孔密度特异诱导表达的胞壁果胶代谢参与的蛋白质。【结果】叶片成熟过程中,随着气孔的关闭和开张,至少有14个蛋白质家族参与了保卫细胞壁果胶的代谢;黑暗条件下,气孔关闭时有5个蛋白质家族特异表达,分别是阻止HGs水解的多聚半乳糖醛酸酶抑制子（polygalacturonase inhibitor,PGI）、RG侧链合成的UDP-鼠李糖合成酶（rhamnose synthase,RHM）、半乳聚糖β-1,4半乳糖基转移酶（galactan β-1,4-galactosyltransferase,GALS）、UDP-D-葡萄糖醛-4-异构酶（UDP-D-glucuronate 4-epimerase,GAE）和聚半乳糖4-α-半乳糖转移酶（polygalacturonate 4-α-galacturonosyltransferase,GAUT）;光照条件下,在不同气孔密度的叶片中检出4个蛋白质家族特异表达,它们分别是降解果胶C6甲酯的果胶甲基酯酶（pectinmethylesterase,PME）、内切和末端水解果胶的多聚半乳糖醛酸酶（polygalacturonase,PG）、α-半乳糖苷酶（α-galactosidase,AGAL）和类果胶裂解酶（pectate lyase-like,PLL）。另有5个蛋白质家族同时参与了气孔的开张和关闭,它们分别是阿拉伯半乳聚糖-蛋白质（aabinogalactan-protein,AGP）、果胶乙酰酯酶（pectinacetylesterase,PAE）、β-半乳糖苷酶（β-galactosidase,BGAL）、类枯草杆菌蛋白酶（subtilase-like,SBT）和果胶甲基酯酶抑制子（pectinesterase inhibitor,PMEI）。【结论】光照条件下,PME催化果胶去甲酯,其后PG和PLL及AGAL分别内切和末端水解果胶,丧失结构的果胶在膨压下开裂,打开气孔;黑暗条件下,PGIP抑制果胶水解,RHM增强果胶侧链合成,结构完整的果胶自行膨胀,保持气孔关闭。

关键词: 马铃薯, 气孔密度, 果胶, RNA-Seq, iTRAQ, 蛋白质

Abstract:

【Objective】 Comparing on differential expression proteins between stomatal closing and opening at different leaf stomata-densities, it is to be revealed how pectin metabolism regulates stomata closing and opening. The result will play an essential role in understanding how stomata functions to environment adaptation.【Method】 Vectors, either over- or inhibiting-expression of StEPF-2 (Solanum tuberosum EPIDERMAL PATTERNING FACTOR 2) in vivo were constructed. The fusing genes were transformed into Solanum tuberosum cultivar Kexin 1. Transgenic potato lines, either rise or lower at leaf stomatal density were generated. Gene and protein expression profiles of leaves at various stomatal densities were assayed via RNA-seq and iTRAQ. Comparing differentiation expression proteins, pectin metabolic enzymes driving stomatal movement under light and darkness were identified and confirmed by the Pulldown and LC-MS/MS. A pectin metabolism pathway regulating stomatal movement was to be proposed.【Result】 At least 14 protein families, driving stomata closing and opening involved in pectin metabolism of the guard cell wall during stomatal mature. Five protein families were detected and confirmed only in the stomatal-closed leaves under darkness, including polygalacturonase inhibitor proteins (PGIP) and rhamnose synthase (RHM) for RG side-chain biosynthesis. Four protein families, polygalacturonase (PG), pectate lyase-like (PLL), pectinmethylesterase (PME) and α-galactosidase (AGAL) were identified only in leaves at various stomatal densities under light. Additionally, five protein families were concurrently identified in both leaves of stomata closing and opening, including pectinacetylesterase (PAE) and subtilase (SBT). 【Conclusion】 Under light, PMEs catalyze pectin demethylesterification, afterwards, pectin was exo- and endo-hydrolyzed by PG, PLL and AGAL. Pectin losing structure was split under turgor, results in stomatal opening. Reversely, under darkness, PGI inhibited pectin hydrolysis. Pectin side-chain biosynthesis was promoted by RHM. Therefore, stomata kept closing due to structurally-complete pectin with voluntary expending function.

Key words: potato, stomatal density, pectin, RNA-Seq, iTRAQ, protein

张晓萍,撒世娟,伍涵宇,乔丽媛,郑蕊,姚新灵. 马铃薯叶片气孔的开张与关闭同步伴随果胶的降解与合成[J]. 中国农业科学, 2022, 55(17): 3278-3288.

ZHANG XiaoPing,SA ShiJuan,WU HanYu,QIAO LiYuan,ZHENG Rui,YAO XinLing. Leaf Stomatal Close and Opening Orchestrate Rhythmically with Cell Wall Pectin Biosynthesis and Degradation[J]. Scientia Agricultura Sinica, 2022, 55(17): 3278-3288.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.17.002

https://www.chinaagrisci.com/CN/Y2022/V55/I17/3278

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基因 Gene	注释（已校对） Annotation (reviewed)	蛋白质ID Protein ID
AGAL	α-半乳糖苷酶 α-galactosidase (EC:3.2.1.22) ^#	DMP400043893
GAUT	聚半乳糖4-α-半乳糖转移酶 Polygalacturonate 4-α-galacturonosyltransferase (EC:2.4.1.43)	DMP400018139*
PGI	多聚半乳糖醛酸酶抑制子 Polygalacturonase inhibitor	DMP400038422*
GALS	半乳聚糖β-1,4-半乳糖基转移酶 Galactan β-1,4-galactosyltransferase	DMP400024112*
RHM	鼠李糖合成酶 Rhamnose synthase (EC:4.2.1.76)	DMP400036826*
PAE	果胶乙酰酯酶 Pectinacetylesterase	DMP400031769, DMP400034673
AGP	阿拉伯半乳聚糖-蛋白质 Arabinogalactan-protein	DMP400043555, DMP400000448, DMP400031527, DMP400016609, DMP400010345, DMP400031060, DMP400031034, DMP400031033
BGAL	β-半乳糖苷酶 β-galactosidase (EC:3.2.1.23)	DMP400008590, DMP400006312, DMP400056145, DMP400038759, DMP400016280, DMP400004620, DMP400000658
PLL	类果胶裂解酶 Pectin lyase-like	DMP400055840, DMP400040644, DMP400026712, DMP400019050, DMP400006877
PME	果胶甲基酯酶 Pectinesterase (EC 3.1.1.11)	DMP400033460, DMP400033459, DMP400031280, DMP400008185, DMP400000585, DMP400016181, DMP400016180, DMP400033458, DMP400006476, DMP400008439, DMP400008438
PMEI	果胶甲基酯酶抑制子 PME inhibitor	DMP400021262, DMP400033225, DMP400019111, DMP400021265
PG	多聚半乳糖醛酸酶 Polygalacturonase	DMP400054735, DMP400054734, DMP400037553, DMP400037552, DMP400037551, DMP400023906, DMP400012937, DMP400001218, DMP400023905, DMP400042576
SBT	类枯草杆菌蛋白酶 Subtilase	DMP400037294, DMP400058901, DMP400041860, DMP400001181, DMP400027005, DMP400013996, DMP400016092, DMP400060436, DMP400043338*
GAE	UDP-葡萄糖-4-异构酶 UDP-D-glucuronate 4-epimerase	DMP400019858, DMP400039878, DMP400008248, DMP400005486, DMP400013781*

基因 Gene	注释（已校对） Annotation (reviewed)	蛋白质ID Protein ID
AGAL	α-半乳糖苷酶 α-galactosidase (EC:3.2.1.22) ^#	DMP400006168, DMP400006167*
BGAL	β-半乳糖苷酶 β-galactosidase (EC:3.2.1.23)	DMP400053911*, DMP400053910, DMP400056145 DMP400045895
PME	果胶甲基酯酶 Pectinesterase (EC 3.1.1.11)	DMP400031280, DMP400016183, DMP400017593
PLL	类果胶裂解酶 Pectate lyase-like (EC:4.2.2.2)	DMP400041370, DMP400041370*
PG	多聚半乳糖醛酸酶 Polygalacturonase	DMP400034980, DMP400034981, DMP400055029 DMP400055030, DMP400055031, DMP400027737
SBT	类枯草杆菌蛋白酶 Subtilase-like	DMP400059687, DMP400053771, DMP400017566*

基因 Gene	注释（已校对） Annotation (reviewed)	蛋白质ID Protein ID
AGAL	α-半乳糖苷酶 α-galactosidase 1 (EC:3.2.1.22) ^#	DMP400043893
BXL	β-D-木糖酶 β-D-xylosidase (EC:3.2.1.55)	DMP400039773
PAE	果胶乙酰酯酶 Pectinacetylesterase	DMP400041742
PME	果胶甲基酯酶 Pectinesterase (EC 3.1.1.11)	DMP400033460, DMP400016183
PMEI	果胶甲基酯酶抑制子 Pectinesterase inhibitor	DMP400016182, DMP400033458
SBT	类枯草杆菌蛋白酶 Subtilase-like	DMP400037294, DMP400008705

序号 No.	蛋白家族 Protein family	功能 Function	气孔密度 Stomatal density				Pulldown LC-MS/MS
序号 No.	蛋白家族 Protein family	功能 Function	0* 0	对照 Control	低 Low	高 High	Pulldown LC-MS/MS
1	PGI	多聚半乳糖醛酸酶抑制子 Polygalacturonase inhibitor	+	×	×	×	×
2	GAE	UDP-葡萄糖-4-异构酶 UDP-D-glucuronate 4-epimerase	++	×	×	×	×
3	RHM	鼠李糖合成酶Rhamnose synthase	+	×	×	×	×
4	GAUT	聚半乳糖4-α-半乳糖转移酶 Polygalacturonate 4-α-galacturonosyltransferase	+	×	×	×	×
5	GALS	半乳聚糖β-1,4-半乳糖基转移酶 Galactan β-1,4-galactosyltransferase	+	×	×	×	×
6	AGP	阿拉伯半乳聚糖-蛋白质 Aabinogalactan-protein	++	++	×	×	×
7	PAE	果胶乙酰酯酶 Pectinacetylesterase	++	×	×	×	+
8	BGAL	β-半乳糖苷酶 β-galactosidase	++	++	+	++	+
9	SBT	类枯草杆菌蛋白酶 Subtilase	++	++	+	++	++
10	PMEI	果胶甲基酯酶抑制子 Pectinesterase inhibitor	++	++	++	~	++
11	PME	果胶甲基酯酶 Pectinmethylesterase	×	++	~	+	++
12	PG	多聚半乳糖醛酸酶 Polygalacturonase	×	++	~	++	~
13	PLL	类果胶裂解酶 Pectate lyase-like	×	++	+	+	~
14	AGAL	α-半乳糖苷酶 α-galactosidase	×	+	+	+	+