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 × + + + +
[1] ENGINEER C B, GHASSEMIAN M, ANDERSON J C, PECK S C, HU H, SCHROEDER J I. Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development. Nature, 2014, 513: 246-250.
doi: 10.1038/nature13452
[2] YANG J, LI C, KONG D, GUO F, WEI H. Light-mediated signaling and metabolic changes coordinate stomatal opening and closure. Front Plant Science, 2020, 4: 11: 601478.
[3] AGURLA S, GAHIR S, MUNEMASA S, MURATA Y, RAGHAVENDRA A S. Mechanism of stomatal closure in plants exposed to drought and cold stress. Advances in Experimental Medicine and Biology, 2018, 1081: 215-232.
[4] LUO D D, WANG C K, JIN Y. Stomatal regulation of plants in response to drought stress. Ying Yong Sheng Tai Xue Bao, 2019, 30(12): 4333-4343.
[5] FANOURAKIS D, ALINIAEIFARD S, SELLIN A, GIDAY H, KORNER O, REZAEI NEJAD A, DELIS C, BOURANIS D, KOUBOURIS G, KAMBOURAKIS E, NIKOLOUDAKIS N, TSANIKLIDIS G. Stomatal behavior following mid- or long-term exposure to high relative air humidity. Plant Physiology and Biochemistry, 2020, 153: 92-105.
doi: 10.1016/j.plaphy.2020.05.024
[6] HARA K, YOKOO T, KAJITA R, ONISHI T, YAHATA S, PETERSON K M, TORII K U, KAKIMOTO T. Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves. Plant Cell Physiology, 2009, 50: 1019-1031.
doi: 10.1093/pcp/pcp068
[7] OHKI S, TAKEUCHI M, MORI M. The NMR structure of stomagen reveals the basis of stomatal density regulation by plant peptide hormones. Nature Communication, 2011, 2: 512-512.
doi: 10.1038/ncomms1520
[8] SUGANO S S, SHIMADA T, IMAI Y, OKAWA K, TAMAI A, MORI M, HARA-NISHIMURA I. Stomagen positively regulates stomatal density in Arabidopsis. Nature, 2010, 463: 241-244.
doi: 10.1038/nature08682
[9] WANG Y L, XIE T, ZHANG C L, LI J J, WANG Z, LI H B, LIU X P, YIN L N, WANG S W, ZHANG S Q, DENG X P, KE Q B. Overexpression of the potato StEPF-2 gene confers enhanced drought tolerance in Arabidopsis. Plant Biotechnology Reports, 2020, 14(4): 479-490
doi: 10.1007/s11816-020-00627-4
[10] TPRII K U. Stomatal development in the context of epidermal tissues. Annals of Botany, 2021, 128(2): 137-148.
doi: 10.1093/aob/mcab052
[11] LIM S L, FKUTCH S, LIU J, DISTEFANO L, SANTELIA D, LIM B L. Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening. Nature Communication, 2022, 13(1): 652.
doi: 10.1038/s41467-022-28263-2
[12] HAAS K T, WIGHTMAN R, MEYEROWITZ E M, PWAUCELLE A. Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells. Science, 2020, 367(6481): 1003-1007.
doi: 10.1126/science.aaz5103
[13] CHEN Y, LI W, TURNER J A, ANDERSON C T. PECTATE LYASE LIKE12 patterns the guard cell wall to coordinate turgor pressure and wall mechanics for proper stomatal function in Arabidopsis. The Plant Cell, 2021, 33(9): 3134-3150.
doi: 10.1093/plcell/koab161
[14] GOU J, MILLER L M, HOU G, YU X, CHEN X, LIU C. Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen germination, and plant reproduction. The Plant Cell, 2012, 24: 50-65.
doi: 10.1105/tpc.111.092411
[15] WOLF S, MOUILLE G, PELLOUX J. Homogalacturonan methyl- esterification and plant development. Molecular Plant, 2009, 2(5): 851-860.
doi: 10.1093/mp/ssp066
[16] SENECHAL F, WATTIER C, RUSTERUCCI C, PELLOUX J. Homogalacturonan-modifying enzymes: structure, expression, and roles in plants. Journal of Experiment Botany, 2014, 65(18): 5125-5160.
doi: 10.1093/jxb/eru272
[17] KIM Y J, JEONG H Y, KANG S Y, SILVA J, KIM E J, PARK S K, JUNG K H, LEE C. Physiological importance of pectin modifying genes during rice pollen development. International Journal of Molecular Science, 2020, 21(14): 4840.
doi: 10.3390/ijms21144840
[18] PELLOUX J, RUSTERUCCI C, MELLEROWICZ E J. New insights into pectin methyl-esterase structure and function. Trends in Plant Science, 2007, 12: 267-277.
doi: 10.1016/j.tplants.2007.04.001
[19] CAMEJO D, MARTI M C, JIMENEZ A, CABRERA J C, OLMOS E, SEVILLA F. Effect of oligogalacturonides on root length, extracellular alkalinization and O-accumulation in alfalfa. Journal of Plant Physiology, 2011, 168: 566-575.
doi: 10.1016/j.jplph.2010.09.012
[20] DRAYE M, VAN CUTSEM P. Pectinmethylesterasesinduce an abrupt increase of acidic pectin during strawberry fruit ripening. Journal of Plant Physiology, 2008, 165: 1152-1160.
doi: 10.1016/j.jplph.2007.10.006
[21] LIU H, DAI T, CHEN J, LIU W, LIU C, DENG L, LIANG R. Extraction, characterization and spontaneous gelation mechanism of pectin from Nicandra physaloides (Linn.) Gaertn seeds. International Journal of Biology Macromolecules, 2022, 195: 523-529.
doi: 10.1016/j.ijbiomac.2021.12.032
[22] MERCED A, RENZAGLIA K. Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: Implications for function and evolution of stomata. Annals of Botany, 2014, 114(5): 1001-1010.
doi: 10.1093/aob/mcu165
[23] RUI Y, ANDERSON C T. Functional analysis of cellulose and xyloglucan in the walls of stomatal guard cells of Arabidopsis. Plant Physiology, 2016, 170(3): 1398-1419.
doi: 10.1104/pp.15.01066
[24] 撒世娟, 殷倩, 伍涵宇, 席云凤, 郑蕊, 姚新灵. 过量表达线粒体膜结合蛋白编码基因St536减少普通烟草的纤维素积累. 农业生物技术学报, 2021, 29(5): 915-923.
SA S J, YIN Q, WU H Y, XI Y F, ZHENG R, YAO X L. Effects of overexpression of St536 gene on cellulose accumulation in Nicotiana tabacum. Journal of Agricultural Biotechnology, 2021, 29(5): 915-923.
[25] SPANOS C, MOORE J B. Sample preparation approaches for iTRAQ labeling and quantitative proteomic analyses in systems biology. Methods of Molecular Biology, 2016, 1394: 15-24.
[26] VAUDEL M, BURKHART J M, ZAHEDI RP, MARTENS L, SICKMANN A. iTRAQ data interpretation. Methods of Molecular Biology, 2012, 893: 501-509.
[27] HAN S K, KWAK J M, QI X. Stomatal lineage control by developmental program and environmental cues. Front in Plant Science, 2021, 12: 751852.
doi: 10.3389/fpls.2021.751852
[28] SALA K, KARCZ J, RYPIEN A, KURCZYNSKA E U. Unmethyl- esterified homogalacturonan and extensins seal Arabidopsis graft union. BMC Plant Biology, 2019, 19(1): 151.
doi: 10.1186/s12870-019-1748-4
[29] RATHINAM M, RAO U, SREEVATHSA R. Novel biotechnological strategies to combat biotic stresses: Polygalacturonase inhibitor (PGIP) proteins as a promising comprehensive option. Applied Microbiology and Biotechnology, 2020, 104(6): 2333-2342.
doi: 10.1007/s00253-020-10396-3
[30] WARREN J G, KASUN G W, LEONARD T, KIRKPATRICK B C. A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase. Molecular Plant Pathology, 2016, 17(4): 480-486.
doi: 10.1111/mpp.12293
[31] BAKSHI G, ANANTHANARAYAN L. Isolation, purification, and characterization of pectin methylesterase inhibitor and polygalacturonase inhibitor protein from Indian lemon (Citrus limon L.). Phytochemistry, 2021, 189: 112802.
doi: 10.1016/j.phytochem.2021.112802
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