Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (17): 3278-3288.doi: 10.3864/j.issn.0578-1752.2022.17.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Leaf Stomatal Close and Opening Orchestrate Rhythmically with Cell Wall Pectin Biosynthesis and Degradation

ZHANG XiaoPing(),SA ShiJuan,WU HanYu,QIAO LiYuan,ZHENG Rui,YAO XinLing()   

  1. Life Science College, Ningxia University/Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia/ Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in West China, Yinchuan 750021
  • Received:2022-03-09 Accepted:2022-06-15 Online:2022-09-01 Published:2022-09-07
  • Contact: XinLing YAO E-mail:1101608186@qq.com;chinanoahl@163.com

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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

Table 1

DEGs involving in pectin metabolism under light and darkness"

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*

Fig. 1

StEPF-2 transformation, positive transgenic line selection and their stomata observation A: RT-PCR for StEPF-2 cDNA, 1-2: 541F/R primer products, M: DL2000 DNA Marker; B: Transformed explants and callus regeneration; C: StEPF-2 overexpression lines; D-F: GUS staining for inhibition, control and overexpression lines of StEPF-2 expression; G: RT-qPCR of StEPF-2 in transgenic lines leaf; H: Count on stomatal density (No./mm) of transgenic lines; I-K: Stomata in leaves of transgenic lines. **: P<0.01"

Table 2

DEPs of pectin metabolism in response to rising and lowing stomatal density"

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*

Fig. 2

Construction of fusing gene 6×His-StEPF-2, purification of the gene expression product and pulldown with 6×His-StEPF-2 A: Diagram for 6×His-StEPF-2 construction in vector pTrcHis; B: SDS-PAGE for expression product of 6×His-StEPF-2; 1 and 2: Cell disruption and outflow; 3: 6×His-StEPF-2 protein after elution; C: Western Blot outcome; 1: 6×His-StEPF-2 protein; D: SDS-PAGE for 6×His-StEPF-2 pulldown with the leaf total protein"

Table 3

IEPs identified by pulldown-LC-MS/MS with labeled His-StEPF-2"

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

Table 4

Comparison outcome on pectin metabolism DEPs in response to stomatal close and various densities"

序号
No.		 蛋白家族
Protein family		 功能
Function		 气孔密度 Stomatal density Pulldown
LC-MS/MS
0*
0		 对照
Control		 低
Low		 高
High
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 × + + + +
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