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Downregulation of the auxin transporter gene SlPIN8 results in pollen abortion in tomato

  • Zengyu Gan
  • Yi Feng
  • Ting Wu
  • Yi Wang
  • Xuefeng Xu
  • Xinzhong Zhang
  • Zhenhai HanEmail author
Article

Abstract

Key message

SlPIN8 is expressed specifically within tomato pollen, and that it is involved in tomato pollen development and intracellular auxin homeostasis.

Abstract

The auxin (IAA) transport protein PIN-FORMED (PIN) plays key roles in various aspects of plant development. The biological role of the auxin transporter SlPIN8 in tomato development remains unclear. Here, we examined the expression pattern of the SlPIN8 gene in vegetative and reproductive organs of tomato. RNA interference (RNAi) transgenic lines specifically silenced for the SlPIN8 gene were generated to identify the role of SlPIN8 in pollen development. We found that SlPIN8 mRNA is expressed specifically within tomato pollen. In the anthers, the highest mRNA expression and β-glucuronidase (GUS) activity of promoter-SlPIN8-GUS was detected during late stages of anther development, when pollen maturation occurred. The downregulation of SlPIN8 did not drastically affect the vegetative growth of tomato. However, in SlPIN8-RNAi transgenic plants, approximately 80% of the pollen grains were identified to be abnormal and lack viability; they were shriveled and flattened. Furthermore, the downregulation of SlPIN8 affected the gene expression of some anther development-specific proteins. SlPIN8-RNAi transgenic plants induced seedless fruits because of defective pollen function rather than defective female gametophyte function. In addition, SlPIN8 was found to localize to the endoplasmic reticulum, consistent with the changes in the auxin levels of SlPIN8-RNAi lines, whereas the level of free IAA was increased in SlPIN8-overexpressing protoplasts, indicating that SlPIN8 is involved in intracellular auxin homeostasis.

Keywords

Auxin SlPIN8 Pollen RNAi Endoplasmic reticulum 

Notes

Acknowledgements

This work was supported by the Modern Agricultural Industry Technology System (CARS-27); Special Fund for Agro-Scientific Research in the Public Interest (201203075); Key Laboratory of Biology and Genetic Improvement of Horticultural Crop (Nutrition and Physiology) in Ministry of Agriculture, and Key Laboratory of Stress Physiology and Molecular Biology for Fruit Trees in Beijing Municipality.

Author Contributions

ZG designed and conducted the experiments, analyzed the data, accomplished pictures and wrote the manuscript. YF, YW, TW, XX contributed in design of the experiments. XZ designed the experiments and finalized the manuscript. ZH conceived and designed the experiments, and finalized the manuscript. All authors read and approved the final manuscript.

Supplementary material

11103_2019_836_MOESM1_ESM.docx (6.5 mb)
Figure S1. Expression analysis of tomato PIN genes in pollen at anthesis by qPCR. Error bars represent the means ± SD from three independent biological replicates. Figure S2. SlPIN8 expression patterns by qPCR. R root, S stem, L leaf, Fl flower, Se sepal, Pe petal, An anther, Po pollen. Figure S3. SlPIN8 gene promoter cis-element analysis. Figure S4. Inflorescence stem with GUS histochemical staining in Arabidopsis under the control of the SlPIN8 promoter. Bars = 0.5 mm. Figure S5. The construction and analysis of RNA interference (RNAi) SlPIN8 tomato lines. A, The RNAi tomato lines were generated by Agrobacterium tumefaciens-mediated transformation with the SlPIN8-RNAi T-DNA construct. B, Relative SlPIN8 mRNA levels in anthers collected from WT and SlPIN8-RNAi transgenic flowers at anthesis. Error bars represent the means ± SD from three independent biological replicates. Figure S6. The expression of SlPINs in SlPIN8-RNAi line and WT. Error bars represent the means ± SD from three independent biological replicates. Figure S7. Morphological observation of normal pollen germination in the WT and SlPIN8-RNAi transgenic plants. Bar = 25 μm. Figure S8. Paraffin section comparison of pollen development between the WT plants and SlPIN8-RNAi lines. Table S1. Primer for qRT-PCR. (DOCX 6627 KB)

References

  1. Abad M, Monteiro AA (1989) THE use of auxins for the production of greenhouse tomatoes in mild-winter conditions—a review. Sci Hortic 38:167–192CrossRefGoogle Scholar
  2. Aloni R, Aloni E, Langhans M, Ullrich CI (2006) Role of auxin in regulating Arabidopsis flower development. Planta 223:315–328CrossRefGoogle Scholar
  3. Bate N, Twell D (1998) Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements. Plant Mol Biol 37:859–869CrossRefGoogle Scholar
  4. Bender RL, Fekete ML, Klinkenberg PM, Hampton M, Bauer B, Malecha M, Lindgren K, Maki J, Perera MADN, Nikolau BJ, Carter CJ (2013) PIN6 is required for nectary auxin response and short stamen development. Plant J 74:893–904CrossRefGoogle Scholar
  5. Benkova E, Michniewicz M, Sauer M, Teichmann T, Seifertova D, Jurgens G, Friml J (2003) Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115:591–602CrossRefGoogle Scholar
  6. Bhadula SK, Sawhney VK (1991) Protein-analysis during the ontogeny of normal and male sterile stamenless-2 mutant stamens of tomato (Lycopersicon esculentum Mill.). Biochem Genet 29:29–41CrossRefGoogle Scholar
  7. Blakeslee JJ, Peer WA, Murphy AS (2005) Auxin transport. Curr Opin Plant Biol 8:494–500CrossRefGoogle Scholar
  8. Carlos Serrani J, Carrera E, Ruiz-Rivero O, Gallego-Giraldo L, Pereira Peres LE, Garcia-Martinez JL (2010) Inhibition of auxin transport from the ovary or from the apical shoot induces parthenocarpic fruit-set in tomato mediated by gibberellins. Plant Physiol 153:851–862CrossRefGoogle Scholar
  9. Cecchetti V, Altamura MM, Falasca G, Costantino P, Cardarelli M (2008) Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation. Plant Cell 20:1760–1774CrossRefGoogle Scholar
  10. Cecchetti V, Altamura MM, Brunetti P, Petrocelli V, Falasca G, Ljung K, Costantino P, Cardarelli M (2013) Auxin controls Arabidopsis anther dehiscence by regulating endothecium lignification and jasmonic acid biosynthesis. Plant J 74:411–422CrossRefGoogle Scholar
  11. Cecchetti V, Celebrin D, Napoli N, Ghelli R, Brunetti P, Costantino P, Cardarelli M (2017) An auxin maximum in the middle layer controls stamen development and pollen maturation in Arabidopsis. New Phytol 213:1194–1207CrossRefGoogle Scholar
  12. Chandler JW (2009) Local auxin production: a small contribution to a big field. Bioessays 31:60–70CrossRefGoogle Scholar
  13. Chen D, Zhao J (2008) Free IAA in stigmas and styles during pollen germination and pollen tube growth of Nicotiana tabacum. Physiol Plantarum 134:202–215CrossRefGoogle Scholar
  14. Cheng YF, Dai XH, Zhao YD (2006) Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Gene Dev 20:1790–1799CrossRefGoogle Scholar
  15. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefGoogle Scholar
  16. Dal Bosco C, Dovzhenko A, Liu X, Woerner N, Rensch T, Eismann M, Eimer S, Hegermann J, Paponov IA, Ruperti B, Heberle-Bors E, Touraev A, Cohen JD a, Palme K (2012) The endoplasmic reticulum localized PIN8 is a pollen-specific auxin carrier involved in intracellular auxin homeostasis. Plant J 71:860–870CrossRefGoogle Scholar
  17. David-Schwartz R, Weintraub L, Vidavski R, Zemach H, Murakhovsky L, Swartzberg D, Granot D (2013) The SlFRK4 promoter is active only during late stages of pollen and anther development. Plant Sci 199:61–70CrossRefGoogle Scholar
  18. de Jong M, Wolters-Arts M, Feron R, Mariani C, Vriezen WH (2009) The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development. Plant J 57:160–170CrossRefGoogle Scholar
  19. Ding Z, Wang B, Moreno I, Duplakova N, Simon S, Carraro N, Reemmer J, Pencik A, Chen X, Tejos R, Skupa P, Pollmann S, Mravec J, Petrasek J, Zazimalova E, Honys D, Rolcik J, Murphy A, Orellana A, Geisler M, Friml J (2012) ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis. Nat Commun 3:941CrossRefGoogle Scholar
  20. Dobrev PI, Kaminek M (2002) Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chromatogr A 950:21–29CrossRefGoogle Scholar
  21. Feng XL, Ni WM, Elge S, Mueller-Roeber B, Xu ZH, Xue HW (2006) Auxin flow in anther filaments is critical for pollen grain development through regulating pollen mitosis. Plant Mol Biol 61:215–226CrossRefGoogle Scholar
  22. Filichkin SA, Leonard JM, Monteros A, Liu PP, Nonogaki H (2004) A Novel Endo-β-mannanase gene in tomato LeMAN5 is associated with anther and pollen development. Plant Physiol 134:1080–1087CrossRefGoogle Scholar
  23. Ganguly A, Lee SH, Cho M, Lee OR, Yoo H, Cho HT (2010) Differential auxin-transporting activities of PIN-FORMED proteins in Arabidopsis root hair cells. Plant Physiol 153:1046–1061CrossRefGoogle Scholar
  24. Gillaspy G, Bendavid H, Gruissem W (1993) Fruits—a developmental perspective. Plant Cell 5:1439–1451CrossRefGoogle Scholar
  25. Goetz M, Hooper LC, Johnson SD, Rodrigues JCM, Vivian-Smith A, Koltunow AM (2007) Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato. Plant Physiol 145:351–366CrossRefGoogle Scholar
  26. Grunewald W, Friml J (2010) The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells. EMBO J 29:2700–2714CrossRefGoogle Scholar
  27. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300CrossRefGoogle Scholar
  28. Ingrosso I, Bonsegna S, De DS, Laddomada B, Blando F, Santino A, Giovinazzo G (2011) Over-expression of a grape stilbene synthase gene in tomato induces parthenocarpy and causes abnormal pollen development. Plant Physiol Biochem 49:1092–1099CrossRefGoogle Scholar
  29. Jefferson RA, Kavanagh TA, Bevan MW (1987) Gus fusions—beta-glucuronidase as a sensitive and versatile gene fusion marker in higher-plants. Embo J 6:3901–3907CrossRefGoogle Scholar
  30. Jeong HJ, Kang JH, Zhao M, Kwon JK, Choi HS, Bae JH, Lee HA, Joung YH, Choi D, Kang BC (2014) Tomato male sterile 10(35) is essential for pollen development and meiosis in anthers. J Exp Bot 65:6693–6709CrossRefGoogle Scholar
  31. Kawanabe T, Ariizumi T, Kawai-Yamada M, Uchimiya H, Toriyama K (2006) Abolition of the tapetum suicide program ruins microsporogenesis. Plant Cell Physiol 47:784–787CrossRefGoogle Scholar
  32. Krecek P, Skupa P, Libus J, Naramoto S, Tejos R, Friml J, Zazimalova E (2009) The PIN-FORMED (PIN) protein family of auxin transporters. Genome Biol 10:249CrossRefGoogle Scholar
  33. Lee TI, Young RA (2000) Transcription of eukaryotic protein-coding genes. Annu Rev Genet 34:77–137CrossRefGoogle Scholar
  34. Manning K (1994) Changes in gene-expression during strawberry fruit ripening and their regulation by auxin. Planta 194:62–68CrossRefGoogle Scholar
  35. McNeil KJ, Smith AG (2005) An anther-specific cysteine-rich protein of tomato localized to the tapetum and microspores. J Plant Physiol 162:457–464CrossRefGoogle Scholar
  36. Mounet F, Moing A, Kowalczyk M, Rohrmann J, Petit J, Garcia V, Maucourt M, Yano K, Deborde C, Aoki K, Berges H, Granell A, Fernie AR, Bellini C, Rothan C, Lemaire-Chamley M (2012) Down-regulation of a single auxin efflux transport protein in tomato induces precocious fruit development. J Exp Bot 63:4901–4917CrossRefGoogle Scholar
  37. Mravec J, Skupa P, Bailly A, Hoyerova K, Krecek P, Bielach A, Petrasek J, Zhang J, Gaykova V, Stierhof YD, Dobrev PI, Schwarzerova K, Rolcik J, Seifertova D, Luschnig C, Benkova E, Zazimalova E, Geisler M, Friml J (2009) Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature 459:1136CrossRefGoogle Scholar
  38. Nashilevitz S, Melamed-Bessudo C, Aharoni A, Kossmann J, Wolf S, Levy AA (2009) The legwd mutant uncovers the role of starch phosphorylation in pollen development and germination in tomato. Plant J 57:1–13CrossRefGoogle Scholar
  39. Nelson BK, Cai X, Nebenfuehr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136CrossRefGoogle Scholar
  40. Oka M, Miyamoto K, Okada K, Ueda J (1999) Auxin polar transport and flower formation in Arabidopsis thaliana transformed with indole acetamide hydrolase (iaaH) gene. Plant Cell Physiol 40:231–237CrossRefGoogle Scholar
  41. Okada K, Ueda J, Komaki MK, Bell CJ, Shimura Y (1991) Requirement of the auxin polar transport-system in early stages of Arabidopsis floral bud formation. Plant Cell 3:677–684CrossRefGoogle Scholar
  42. Paponov IA, Teale WD, Trebar M, Blilou K, Palme K (2005) The PIN auxin efflux facilitators: evolutionary and functional perspectives. Trends Plant Sci 10:170–177CrossRefGoogle Scholar
  43. Paponov IA, Paponov M, Teale W, Menges M, Chakrabortee S, Murray JAH, Palme K (2008) Comprehensive transcriptome analysis of auxin responses in Arabidopsis. Mol Plant 1:321–337CrossRefGoogle Scholar
  44. Pattison RJ, Catala C (2012) Evaluating auxin distribution in tomato (Solanum lycopersicum) through an analysis of the PIN and AUX/LAX gene families. Plant J 70:585–598CrossRefGoogle Scholar
  45. Petrasek J, Friml J (2009) Auxin transport routes in plant development. Development 136:2675–2688CrossRefGoogle Scholar
  46. Pina C, Pinto F, Feijó 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 138:744–756CrossRefGoogle Scholar
  47. Reinhardt D, Mandel T, Kuhlemeier C (2000) Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 12:507–518CrossRefGoogle Scholar
  48. Rogers HJ, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, Lonsdale DM, Twell D (2001) Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol 45:577–585CrossRefGoogle Scholar
  49. 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 11:297–322CrossRefGoogle Scholar
  50. Sato S, Peet MM, Gardner RG (2001) Formation of parthenocarpic fruit, undeveloped flowers and aborted flowers in tomato under moderately elevated temperatures. Sci Hortic 90:243–254CrossRefGoogle Scholar
  51. Shuai H, Meng Y, Luo X, Chen F, Qi Y, Yang W, Shu K (2016) The roles of auxin in seed dormancy and germination. Yichuan 38:314–322Google Scholar
  52. Simon S, Skupa P, Viaene T, Zwiewka M, Tejos R, Klima P, Carna M, Rolcik J, De Rycke R, Moreno I, Dobrev PI, Orellana A, Zazimalova E, Friml J (2016) PIN6 auxin transporter at endoplasmic reticulum and plasma membrane mediates auxin homeostasis and organogenesis in Arabidopsis. New Phytol 211:65–74CrossRefGoogle Scholar
  53. Singh S, Sawhney VK, Pearce DW (2010) Temperature effects on endogenous indole-3-acetic acid levels in leaves and stamens of the normal and male sterile ‘stamenless-2′ mutant of tomato (Lycopersicon esculentum Mill.). Plant Cell Environ 15:373–377CrossRefGoogle Scholar
  54. Sun HJ, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426–431CrossRefGoogle Scholar
  55. Sun Y, Ji K, Liang B, Du Y, Jiang L, Wang J, Kai W, Zhang Y, Zhai X, Chen P, Wang H, Leng P (2017) Suppressing ABA uridine diphosphate glucosyltransferase (SlUGT75C1) alters fruit ripening and the stress response in tomato. Plant J 91:574–589CrossRefGoogle Scholar
  56. Wan L, Xia X, Hong D, Li J, Yang G (2010) Abnormal vacuolization of the tapetum during the tetrad stage is associated with male sterility in the recessive genic male sterile Brassica napus L. J Plant Biol 53:121–133CrossRefGoogle Scholar
  57. Wang H, Jones B, Li ZG, Frasse P, Delalande C, Regad F, Chaabouni S, Latche A, Pech JC, Bouzayen M (2005) The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell 17:2676–2692CrossRefGoogle Scholar
  58. Wilson ZA, Morroll SM, Dawson J, Swarup R, Tighe PJ (2001) The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. Plant J 28:27–39CrossRefGoogle Scholar
  59. Wisniewska J, Xu J, Seifertova D, Brewer PB, Ruzicka K, Blilou I, Rouquie D, Benkova E, Scheres B, Friml J (2006) Polar PIN localization directs auxin flow in plants. Science 312:883–883CrossRefGoogle Scholar
  60. Wu JZ, Lin Y, Zhang XL, Pang DW, Zhao J (2008) IAA stimulates pollen tube growth and mediates the modification of its wall composition and structure in Torenia fournieri. J Exp Bot 59:2529–2543CrossRefGoogle Scholar
  61. Xing T, Malik K, Martin T, Miki BL (2001) Activation of tomato PR and wound-related genes by a mutagenized tomato MAP kinase kinase through divergent pathways. Plant Mol Biol 46:109–120CrossRefGoogle Scholar
  62. Xu SX, Liu GS, Chen RD (2006) Characterization of an anther- and tapetum-specific gene and its highly specific promoter isolated from tomato. Plant Cell Rep 25:231–240CrossRefGoogle Scholar
  63. Yang C, Vizcay-Barrena G, Conner K, Wilson ZA (2007) MALE STERILITY1 is required for tapetal development and pollen wall biosynthesis. Plant Cell 19:3530–3548CrossRefGoogle Scholar
  64. Zhang D, Yang L (2014) Specification of tapetum and microsporocyte cells within the anther. Curr Opin Plant Biol 17:49–55CrossRefGoogle Scholar
  65. Zhao Y (2008) The role of local biosynthesis of auxin and cytokinin in plant development. Curr Opin Plant Biol 11:16–22CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Zengyu Gan
    • 1
  • Yi Feng
    • 1
  • Ting Wu
    • 1
  • Yi Wang
    • 1
  • Xuefeng Xu
    • 1
  • Xinzhong Zhang
    • 1
  • Zhenhai Han
    • 1
    Email author
  1. 1.Institute of Horticultural Plants, College of HorticultureChina Agricultural UniversityBeijingPeople’s Republic of China

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