Decreased Pollen Viability and Thicken Pollen Intine in Antisense Silenced Brassica campestris Mutant of BcMF19

doi:10.1016/S2095-3119(13)60581-X

摘要/Abstract

摘要： Brassica campestris male fertility 19 (BcMF19; GenBank accession number GQ902048.1), a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was significantly affected in 35sbcmf19 compared with the control samples. 4´-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander’s staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These findings indicate that BcMF19 is essential to the pollen development and pollen tube extension of B. campestris ssp. chinensis.

关键词: Brassica rapa ssp. chinensis , PGIP , pollen , polygalacturonase-inhibiting protein , tapetum , pollen germination

Abstract: Brassica campestris male fertility 19 (BcMF19; GenBank accession number GQ902048.1), a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was significantly affected in 35sbcmf19 compared with the control samples. 4´-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander’s staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These findings indicate that BcMF19 is essential to the pollen development and pollen tube extension of B. campestris ssp. chinensis.

Key words: Brassica rapa ssp. chinensis , PGIP , pollen , polygalacturonase-inhibiting protein , tapetum , pollen germination

LIU Jin-long, GAO Ming-hui, LIU Ying , CAO Jia-shu. Decreased Pollen Viability and Thicken Pollen Intine in Antisense Silenced Brassica campestris Mutant of BcMF19[J]. Journal of Integrative Agriculture, 2014, 13(5): 954-962.

参考文献

Aguero C B, Uratsu S L, Greve C, Powell A L T, Labavitch J M, Meredith C P, Dandekar A M. 2005. Evaluation of tolerance to Pierce’s disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene. Molecular Plant Pathology, 6, 43-51

Albersheim P, Anderson A J. 1971. Protein from plant cell walls inhibit polygalacturonases secreted by plant pathogens. Proceedings of National Academy of Sciences of the United States of America, 68, 1815-1819

Baumberger N, Ringli C, Keller B. 2001. The chimeric leucine-rich repeat/extensin cell wall protein LRX1 is required for root hair morphogenesis in Arabidopsis thaliana. Gene & Development, 15, 1128-1139

Cao J S, Cao S C, Yi Q M. 1995. RAPD analysis on genomic dna of chinese cabbage and the other groups of brassica. Acta Horticulturae Sinica, 22, 47-52 (in Chinese)

Cao J S, Yu X L, Ye W Z, Lu G, Xiang X. 2006. CYP86MF gene in Chinese cabbage (Brassica campestris L. ssp chinensis makino). Plant Cell Reports, 24, 715-723

Coleman A W, Goff L J. 1985. Applications of fluorochromes to pollen biology. I. mithramycin and 4’,6-diamidino-2-pheenylindole (DAPI) as vital stains and for quantitation of nuclear DNA Stain Technology, 60, 145-154.

Czechowski T, Stitt M, Altmann T, Udvardi M K, Scheible W R. 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiology, 139, 5-17

D’Ovidio R, Mattei B, Roberti S, Bellincampi D. 2004. Polygalacturonases, polygalacturonase-inhibiting proteins and pectic oligomers in plant-pathogen interactions. Biochimica et Biophysica Acta (Proteins and Proteomics), 1696, 237-244

Devlin W S, Gustine D L. 1992. Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction. Plant Physiology, 100, 1189- 1195.

Federici L, di Matte A, Fernandez-Recio J, Tsernoglou D, Cervone F. 2006. Polygalacturonase inhibiting proteins: players in plant innate immunity? Trends in Plant Science, 11, 65-70

Ferrari S, Sella L, Janni M, de Lorenzo G, Favaron F, D’Ovidio R. 2012. Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum. Plant Biology, 14, 31-38

Ferrari S, Vairo D, Ausubel F M, Cervone F, de Lorenzo G. 2003. Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection. The Plant Cell, 15, 93-106

Forsthoefel N R, Dao T P, Vernon D M. 2010. PIRL1 and PIRL9, encoding members of a novel plant-specific family of leucine-rich repeat proteins, are essential for differentiation of microspores into pollen. Planta, 232, 1101-1114

Guyon V, Tang W H, Monti M M, Raiola A, de Lorenzo G, McCormick S, Taylor L P. 2004. Antisense phenotypes reveal a role for SHY, a pollen-specific leucine-rich repeat protein, in pollen tube growth. The Plant Journal, 39, 643-654

Guyon V N, Astwood J D, Garner E C, Dunker A K, Taylor L P. 2000. Isolation and characterization of cDNAs expressed in the early stages of flavonol-induced pollen germination in Petunia. Plant Physiology, 123, 699-710

Gotoh Y, Nalumpang S, Isshiki A, Utsumi T, Gomi K, Yamamoto H, Akimitsu K. 2002. A cDNA encoding polygalacturonase-inhibiting protein induced in citrus leaves by polygalacturonase of Alternaria citri. Journal of General Plant Pathology, 68, 57-61

Haas B J, Volfovsky H, Town C D, Troukhan M, Alexandrov N, Feldmann K A, Flavell R B, White O, Salzberg S L. 2002. Full-length messenger RNA sequences greatly improve genome annotation. Genome Biology, 3, 1-12

Hoekema A, Hirsch P R, Hooykaas P J J, Schilperoort R A. 1983. A binary plant vextor strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 303, 179-180

Huang L, Liu Y, Yu X L, Xiang X, Cao J S. 2011. A polygalacturonase inhibitory protein gene (BcMF19) expressed during pollen development in Chinese cabbage-pak-choi. Molecular Biology Reports, 38, 545- 552.

Ishiguro S, Kawai-Oda A, Ueda J, Nishida I, Okada K. 2001. The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. The Plant Cell, 13, 2191- 2209.

Janni M, Sella L, Favaron F, Blechl A E, de Lorenzo G, D’Ovidio R. 2008. The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana. Molecular Plant Microbe Interactions, 21, 171-177

Jiang J, Zhang Z, Cao J. 2013. Pollen wall development: The associated enzymes and metabolic pathways. Plant Biology, 15, 249-263

Kars I, Krooshof G H, Wagemakers L, Joosten R, Benen J A E, van Kan J A L. 2005. Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris. The Plant Journal, 43, 213-225

Kim H U, Cotter R, Johnson S, Senda M, Dodds P, Kulikauskas R, Tang W H, Ezcurra I, Herzmark P, McCormick S. 2002. New pollen-specific receptor kinases identified in tomato, maize and Arabidopsis: The tomato kinases show overlapping but distinct localization patterns on pollen tubes. Plant Molecular Biology, 50, 1-16

Lalanne E, Honys D, Johnson A, Borner G H H, Lilley K S, Dupree P, Grossniklaus U, Twell D. 2004. SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in Arabidopsis. The Plant Cell, 16, 229-240

Li R G, Rimmer R, Yu M, Sharpe A G, Seguin-Swartz G, Lydiate D, Hegedus D D. 2003. Two Brassica napus polygalacturonase inhibitory protein genes are expressed at different levels in response to biotic and abiotic stresses. Planta, 217, 299-308

Liu L C, Cao J S. 2007. Functional validation of BcMF4 gene related to male sterility of Chinese cabbage by antisense RNA. Journal of Hunan Agricultural University (Natural Sciences), 33, 412-418 (in Chinese)

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods, 25, 402-408

de Lorenzo G, D’Ovidio R, Cervone F. 2001. The role of polygalacturonase-inhibiting proteins (PGIPS) in defense against pathogenic fungi. Annual Review of Phytopathology, 39, 313-335

Manfredini C, Sicilia F, Ferrari S, Pontiggia D, Salvi G, Caprari C, Lorito M, de Lorenzo G. 2005. Polygalacturonase-inhibiting protein 2 of Phaseolus vulgaris inhibits BcPG1, a polygalacturonase of Botrytis cinerea important for pathogenicity, and protects transgenic plants from infection. Physiological and Molecular Plant Pathology, 67, 108-115

di Matteo A, Bonivento D, Tsernoglou D, Federici L, Cervone F. 2006. Polygalacturonase-inhibiting protein (PGIP) in plant defence: A structural view. Phytochemistry, 67, 528-533

Mizuno S, Osakabe Y, Maruyama K, Ito T, Osakabe K, Sato T, Shinozaki K, Yamaguchi-Shinozaki K. 2007. Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana. The Plant Journal, 50, 751-766

Mu J H, Lee H S, Kao T H. 1994. Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflata and the activity of its encoded kinase. The Plant Cell, 6, 709-721

Muschietti J, Eyal Y, McCormick S. 1998. Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2. The Plant Cell, 10, 319-330

Peterson R, Slovin J P, Chen C. 2010. A simplified method for differential staining of aborted and non-aborted pollen grains. International Journal of Plant Biology, 1, 66-69

Powell A L T, Kan J V, Have A T, Visser J, Greve L C, Bennett A B. 2000. Transgenic expression of pear PGIP in tomato limits fungal colonization. Molecular Plant Microbe Interactions, 13, 942-950

Ramanathan V, Simpson C G, Thow G, lannetta P P M, McNicol R J, Williamson B. 1997. cDNA cloning and expression of polygalacturonase-inhibiting proteins (PGIPs) from red raspberry (Rubus idaeus). Journal of Experimental Botany, 48, 1185-1193

Rubinstein A L, Broadwater A H, Lowrey K B, Bedinger P A. 1995. Pex1,a pollen-specific gene with an extensin- like domain. Proceedings of the Natitional Academy of Sciences of the United States of America, 92, 3086-3090

Tang W H, Kelley D, Ezcurra I, Cotter R, McCormick S. 2004. LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro. The Plant Journal, 39, 343- 353.

Veronico P, Melillo M T, Saponaro C, Leonetti P, Picardi E, Jones J T. 2011. A polygalacturonase-inhibiting protein with a role in pea defence against the cyst nematode Heterodera goettingiana. Molecular Plant Pathology, 12, 275-287

Wang J F, Zhu C L, Chen G X, Pan D M, Pan C B, Ye L Y. 2007. Cloning and sequencing of PGIP gene from Longyanmei, a cultivar of Prunus mume. Journal of Fruit Science, 24, 55-58. (in Chinese)

Willemse M T M, Keijzer C J. 1990. Tracing pollen nuclei in the ovary and ovule of Gasterria verrucosa (Mill.) H.Duval after pollination with DAPI-stained pollen. Sexual Plant Reproduction, 3, 219-224

Yu X L, Cao J S. 2004. Construction of plant expression plasmid vector containning the antisense gene CYP86MF and its transformation to Chinese cabbage-pak-choi. Journal of Zhejiang University (Agriculture and Life Sciences), 30, 179-184 (in Chinese)

Zhang Q, Huang L, Liu T T, Yu X L, Cao J S. 2008. BcMF6 in Chinese cabbage (Brassica campestris L. ssp chinensis Makino). Plant Cell Reports, 27, 1207-1215.

[1]	Elsheikh Y M Ahmed, ZHANG Yan-pei, YU Jian-ping, Rashid M A Rehman, ZHANG Hong-l. Mapping of three QTLs for seed setting and analysis on the candidate gene for qSS-1 in rice (Oryza sativa L.)[J]. Journal of Integrative Agriculture, 2016, 15(4): 735-743.
[2]	ZHANG Long-yu, ZHANG Gai-sheng, ZHAO Xin-liang , YANG Shu-ling. Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat[J]. Journal of Integrative Agriculture, 2013, 12(6): 941-950.
[3]	FU Guan-fu, SONG Jian, XIONG Jie, LI Yu-rong, CHEN Hui-zhe, LE Ming-kai and TAO Long-xing. Changes of Oxidative Stress and Soluble Sugar in Anthers Involve in Rice Pollen Abortion Under Drought Stress[J]. Journal of Integrative Agriculture, 2011, 10(7): 1016-1025.