Abstract
Plant intracellular Ras-group-related leucine-rich repeat proteins (PIRLs) are a plant-specific class of leucine-rich repeat (LRR) proteins related to animal and fungal LRRs that take part in developmental signaling and gene regulation. As part of a systematic functional study of the Arabidopsis thaliana PIRL gene family, T-DNA knockout mutants defective in the closely related PIRL1 and PIRL9 genes were identified and characterized. Pirl1 and pirl9 single mutants displayed normal transmission and did not exhibit an obvious developmental phenotype. To investigate the possibility of functional redundancy, crosses to generate double mutants were carried out; however, pirl1;pirl9 plants were not recovered. Reciprocal crosses between wild type and pirl1/PIRL1;pirl9 plants, which produce 50% pirl1;pirl9 gametophytes, indicated male-specific transmission failure of the double-mutant allele combination. Scanning electron microscopy and viability staining showed that approximately half of the pollen produced by pirl1/PIRL1;pirl9 plants was inviable and severely malformed. Tetrad analyses with qrt1 indicated that pollen defects segregated with the double-mutant allele combination, thus demonstrating that PIRL1 and PIRL9 function after meiosis. Pollen development was characterized with bright field, fluorescence, and transmission electron microscopy. Pirl1;pirl9 mutants stopped growing as microspores, failed to initiate vacuolar fission, aborted, and underwent cytoplasmic degeneration. Development consistently arrested at the late microspore stage, just prior to pollen mitosis I. Thus, PIRL1 and PIRL9 have redundant roles essential at a key transition point early in pollen development. Together, these results define a functional context for these two members of this distinct class of plant LRR genes.
Similar content being viewed by others
Abbreviations
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- LRR:
-
Leucine-rich repeat
- PIRL:
-
Plant intracellular Ras-group-related LRR protein
- RT-PCR:
-
Reverse transcription PCR
- SEM:
-
Scanning electron microscopy
- TEM:
-
Transmission electron microscopy
References
Alexander MP (1969) Differential staining of aborted and non-aborted pollen. Stain Technol 41:117–122
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657
Baez JM, Riveros M, Lehnback C (2002) Viability and longevity of pollen of Notofagus species in south Chile. N Z J Bot 40:671–678
Belkhadir Y, Subramaniam R, Dangl JL (2004) Plant disease resistance protein signaling: NBS-LRR proteins and their partners. Curr Opin Plant Biol 7:391–399
Boavida LC, Shuai B, Yu HJ, Pagnussat GC, Sundaresan V, McCormick S (2009) A collection of Ds insertional mutants associated with defects in male gametophyte development and function in Arabidopsis thaliana. Genetics 181:1369–1385
Borg M, Brownfield L, Twell D (2009) Male gametophyte development: a molecular perspective. J Exp Bot 60:1465–1478
Brownfield L, Hafidh S, Durbarry A, Khatab H, Sidorova A, Doerner P, Twell D (2009) Arabidopsis DUO POLLEN3 is a key regulator of male germline development and embryogenesis. Plant Cell 21:1940–1956
Buchanan SG, Gay NJ (1996) Structural and functional diversity in the leucine-rich repeat family of proteins. Prog Biophys Mol Biol 65:1–44
Chaiwongsar S, Otegui MS, Jester PJ, Monson SS, Krysan PJ (2006) The protein kinase genes MAP3K epsilon 1 and MAP3K epsilon 2 are required for pollen viability in Arabidopsis thaliana. Plant J 48:193–205
Chen YC, McCormick S (1996) Sidecar pollen, an Arabidopsis thaliana male gametophytic mutant with aberrant cell divisions during pollen development. Development 122:3243–3253
Claudianos C, Campbell HD (1995) The novel flightless-I gene brings together two gene families, actin-binding proteins related to gelsolin and leucine-rich-repeat proteins involved in Ras signal transduction. Mol Biol Evol 12:405–414
Cushing DA, Forsthoefel NR, Gestaut DR, Vernon DM (2005) Arabidopsis emb175 and other ppr knockout mutants reveal essential roles for pentatricopeptide repeat (PPR) proteins in plant embryogenesis. Planta 221:424–436
Cutler ML, Bassin RH, Zanoni L, Talbot N (1992) Isolation of rsp-1, a novel cDNA capable of suppressing v-Ras transformation. Mol Cell Biol 12:3750–3756
da Costa-Nunes JA, Grossniklaus U (2003) Unveiling the gene-expression profile of pollen. Genome Biol 5:205
Dafini A, Devan P, Husband B (2005) Practical pollination biology. Enviroquest Ltd, Canada, p 590
Dai P, Xiong WC, Mei L (2006) Erbin inhibits RAF activation by disrupting the sur-8-Ras-Raf complex. J Biol Chem 281:927–933
De Smet I, Voss U, Jurgens G, Beeckman T (2009) Receptor-like kinases shape the plant. Nat Cell Biol 11:1166–1173
Dievart A, Clark SE (2004) LRR-containing receptors regulating plant development and defense. Development 131:251–261
Dong X, Hong Z, Sivaramakrishnan M, Mahfouz M, Verma DP (2005) Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis. Plant J 42:315–328
Dougherty GW, Jose C, Gimona M, Cutler ML (2008) The Rsu-1-PINCH1-ILK complex is regulated by Ras activation in tumor cells. Eur J Cell Biol 87:721–734
Durbarry A, Vizir I, Twell D (2005) Male germ line development in Arabidopsis. duo pollen mutants reveal gametophytic regulators of generative cell cycle progression. Plant Physiol 137:297–307
Eitas TK, Dangl JL (2010) NB-LRR proteins: pairs, pieces, perception, partners, and pathways. Curr Opin Plant Biol 13(4):472–477
Feldmann KA, Coury DA, Christianson ML (1997) Exceptional segregation of a selectable marker (KanR) in Arabidopsis identifies genes important for gametophytic growth and development. Genetics 147:1411–1422
Fluhr R (2001) Sentinels of disease. Plant resistance genes. Plant Physiol 127:1367–1374
Forsthoefel NR, Cutler K, Port MD, Yamamoto T, Vernon DM (2005) PIRLs: a novel class of plant intracellular leucine-rich repeat proteins. Plant Cell Physiol 46:913–922
Guan YF, Huang XY, Zhu J, Gao JF, Zhang HX, Yang ZN (2008) RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis. Plant Physiol 147:852–863
Guyon V, Tang WH, Monti MM, Raiola A, Lorenzo GD, McCormick S, Taylor LP (2004) Antisense phenotypes reveal a role for SHY, a pollen-specific leucine-rich repeat protein, in pollen tube growth. Plant J 39:643–654
Hellmann H, Estelle M (2002) Plant development: regulation by protein degradation. Science 297:793–797
Henry IM, Dilkes BP, Young K, Watson B, Wu H, Comai L (2005) Aneuploidy and genetic variation in the Arabidopsis thaliana triploid response. Genetics 170:1979–1988
Honys D, Twell D (2003) Comparative analysis of the Arabidopsis pollen transcriptome. Plant Physiol 132:640–652
Honys D, Twell D (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol 5:R85
Howden R, Park SK, Moore JM, Orme J, Grossniklaus U, Twell D (1998) Selection of T-DNA-tagged male and female gametophytic mutants by segregation distortion in Arabidopsis. Genetics 149:621–631
Jeong KW, Lee YH, Stallcup MR (2009) Recruitment of the SWI/SNF chromatin remodeling complex to steroid hormone-regulated promoters by nuclear receptor coactivator flightless-I. J Biol Chem 284:29298–29309
Johnson SA, McCormick S (2001) Pollen germinates precociously in the anthers of raring-to-go, an Arabidopsis gametophytic mutant. Plant Physiol 126:685–695
Johnson MA, von Besser K, Zhou Q, Smith E, Aux G, Patton D, Levin JZ, Preuss D (2004) Arabidopsis hapless mutations define essential gametophytic functions. Genetics 168:971–982
Johnson-Brousseau SA, McCormick S (2004) A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed genes. Plant J 39:761–775
Kajava AV (1998) Structural diversity of leucine-rich repeat proteins. J Mol Biol 277:519–527
Kim HU, Cotter R, Johnson S, Senda M, Dodds P, Kulikauska R, Tang W, Ezcura 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 Mol Biol 50:1–16
Kobe B, Deisenhofer J (1994) The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 19:415–421
Kobe B, Kajava AV (2001) The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11:725–732
Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11:2283–2290
Lalanne E, Twell D (2002) Genetic control of male germ unit organization in Arabidopsis. Plant Physiol 129:865–875
Lee YH, Campbell HD, Stallcup MR (2004) Developmentally essential protein flightless I is a nuclear receptor coactivator with actin binding activity. Mol Cell Biol 24:2103–2117
Lee Y, Kim ES, Choi Y, Hwang I, Staiger CJ, Chung YY (2008) The Arabidopsis phosphatidylinositol 3-kinase is important for pollen development. Plant Physiol 147:1886–1897
Leon G, Holuigue L, Jordana X (2007) Mitochondrial complex II is essential for gametophyte development in Arabidopsis. Plant Physiol 143:1534–1546
Letunic I, Doerks T, Bork P (2009) SMART 6: recent updates and new developments. Nucleic Acids Res 37:D229–D232
Li W, Han M, Guan KL (2000) The leucine-rich repeat protein SUR-8 enhances MAP kinase activation and forms a complex with Ras and Raf. Genes Dev 14:895–900
McCormick S (2004) Control of male gametophyte development. Plant Cell 16(Suppl):S142–S153
McHale L, Tan X, Koehl P, Michelmore RW (2006) Plant NBS-LRR proteins: adaptable guards. Genome Biol 7:212
Meinke DW (1995) Molecular genetics of plant embryogenesis. Annu Rev Plant Physiol Plant Mol Biol 46:369–394
Morillo SA, Tax FE (2006) Functional analysis of receptor-like kinases in monocots and dicots. Curr Opin Plant Biol 9:460–469
Morris ER, Walker JC (2003) Receptor-like protein kinases: the keys to response. Curr Opin Plant Biol 6:339–342
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. Plant Cell 10:319–330
Park SK, Twell D (2001) Novel patterns of ectopic cell plate growth and lipid body distribution in the Arabidopsis gemini pollen1 mutant. Plant Physiol 126:899–909
Park SK, Howden R, Twell D (1998) The Arabidopsis thaliana gametophytic mutation gemini pollen1 disrupts microspore polarity, division asymmetry and pollen cell fate. Development 125:3789–3799
Paxson-Sowders DM, Dodrill CH, Owen HA, Makaroff CA (2001) DEX1, a novel plant protein, is required for exine pattern formation during pollen development in Arabidopsis. Plant Physiol 127:1739–1749
Pina C, Pinto F, Feijo JA, Becker JD (2005) Gene family analysis of the Arabidopsis pollen transcriptome reveals biological implications for cell growth, division control, and gene expression regulation. Plant Physiol 138:744–756
Preuss D, Rhee SY, Davis RW (1994) Tetrad analysis possible in Arabidopsis with mutation of the QUARTET (QRT) genes. Science 264:1458–1460
Procissi A, de Laissardiere S, Ferault M, Vezon D, Pelletier G, Bonhomme S (2001) Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana. Genetics 158:1773–1783
Regan SM, Moffatt BA (1990) Cytochemical analysis of pollen development in wild-type Arabidopsis and a male-sterile mutant. Plant Cell 2:877–889
Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu YC, Lee PY, Truong MT, Beals TP, Goldberg RB (1999) Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod 11:297–322
Sieburth DS, Sun Q, Han M (1998) SUR-8, a conserved Ras-binding protein with leucine-rich repeats, positively regulates Ras-mediated signaling in C. elegans. Cell 94:119–130
Somers DE, Fujiwara S (2009) Thinking outside the F-box: novel ligands for novel receptors. Trends Plant Sci 14:206–213
Sternberg PW, Alberola-Ila J (1998) Conspiracy theory: RAS and RAF do not act alone. Cell 95:447–450
Stratford S, Barne W, Hohorst DL, Sagert JG, Cotter R, Golubiewski A, Showalter AM, McCormick S, Bedinger P (2001) A leucine-rich repeat region is conserved in pollen extensin-like (Pex) proteins in monocots and dicots. Plant Mol Biol 46:43–56
Sussman MR, Amasino RM, Young JC, Krysan PJ, Austin-Phillips S (2000) The Arabidopsis knockout facility at the University of Wisconsin-Madison. Plant Physiol 124:1465–1467
Tang W, 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. Plant J 39:343–353
Tax FE, Vernon DM (2001) T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics. Plant Physiol 126:1527–1538
Taylor PE, Glover JA, Lavithis M, Craig S, Singh MB, Knox RB, Dennis ES, Chaudhury AM (1998) Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants. Planta 205:492–505
Trump BF, Smuckler EA, Benditt EP (1961) A method for staining epoxy sections for light microscopy. J Ultrastruct Res 5:343–348
Vernon DM, Forsthoefel NR (2002) Leucine-rich repeat proteins in plants: diverse roles in signaling and development. In: Pandali SG (ed) Research signpost: recent research developments in plant biology, pp 202–214
Vernon DM, Meinke DW (1994) Embryogenic transformation of the suspensor in twin, a polyembryonic mutant of Arabidopsis. Dev Biol 165:566–573
Vernon DM, Hannon MJ, Le M, Forsthoefel NR (2001) An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae). Am J Bot 88:570–582
Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV et al (2007) An electronic fluorescent pictograph browser for exploring and analyzing large-scale biological data sets. PLoS One 2:e718
Yamamoto Y, Nishimura M, Hara-Nishimura I, Noguchi T (2003) Behavior of vacuoles during microspore and pollen development in Arabidopsis thaliana. Plant Cell Physiol 44:1192–1201
Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–2632
Acknowledgments
This work was supported by the National Science Foundation [award # 06016166 to D.M.V.], and by NSF DBI-0922978. We thank Barbara Simeles for her contributions to pollen developmental studies, and Michelle Shafer and Heidi Geiser for assistance with imaging and SEM.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Forsthoefel, N.R., Dao, T.P. & Vernon, D.M. 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 (2010). https://doi.org/10.1007/s00425-010-1242-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-010-1242-6