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Sexual Plant Reproduction

, Volume 22, Issue 4, pp 263–275 | Cite as

Ectopic expression of S-RNase of Petunia inflata in pollen results in its sequestration and non-cytotoxic function

  • Xiaoying Meng
  • Zhihua Hua
  • Ning Wang
  • Allison M. Fields
  • Peter E. Dowd
  • Teh-hui KaoEmail author
Original Article

Abstract

The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature “S-RNase”, the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S2-RNase, S3-RNase and non-glycosylated S3-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin–myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.

Keywords

Compartmentalization Glycosylation Petunia inflata Pollen Self-incompatibility S-RNase 

Notes

Acknowledgments

We thank Simon Gilroy and Richard Cyr for advice and suggestions on confocal experiments. This work was supported by National Science Foundation grants IOB 05-43201 and IOS 08-43195 to T.-h.K.

Supplementary material

497_2009_114_MOESM1_ESM.doc (22 kb)
(DOC 24 kb)
497_2009_114_MOESM2_ESM.tif (262 kb)
Supplementary Fig. 1 Schematic drawing of the constructs, pBI LAT52-S 2 -RNase:GFP, pBI LAT52-S 3 -RNase:GFP, and pBI LAT52-S 3 -RNase(N29D):GFP, used in transformation. The region between the right border (RB) and the left border (LB) is integrated into transgenic plants. Nos, the gene encoding nopaline synthase; pro, promoter; ter, transcription terminator; P1 and P2 denote the locations of the forward and reverse primers, respectively, used in PCR for each transgene (see text for sequences); NPTII, the gene encoding neomycin phosphotransferase II (conferring kanamycin resistance). (TIFF 261 kb)
497_2009_114_MOESM3_ESM.zip (5 mb)
Supplementary Movies 1 Trafficking of S3-RNase:GFP-containing compartments in living in vitro-germinated pollen tubes and the effect of BDM and oryzalin on trafficking. The movies show in sequence: 1. GFP in in vitro-germinated pollen tubes of a transgenic S 2 S 3 plant carrying the GFP transgene expressed by the LAT52 promoter; 2. PiSLF2:GFP in in vitro-germinated pollen tubes of a transgenic S 2 S 3 plant carrying the PiSLF 2 :GFP transgene expressed by the LAT52 promoter; 3. Trafficking of S3-RNase:GFP-containing compartments in living in vitro-germinated pollen tubes of a transgenic S 2 S 3 plant carrying the S 3 -RNase:GFP transgene expressed by the LAT52 promoter; 4. Effect of 30 mM BDM on the trafficking of S3-RNase:GFP-containing compartments in living in vitro-germinated pollen tubes; 5. Effect of 50 mM BDM on the trafficking of S3-RNase:GFP-containing compartments in living in vitro-germinated pollen tubes; 6. Effect of 50 μM oryzalin on the trafficking of S3-RNase:GFP-containing compartments in living in vitro-germinated pollen tubes. In each movie, the left panel is the GFP channel and the right panel is the bright-field channel. (TAR 5,924 kb)
497_2009_114_MOESM4_ESM.zip (4.7 mb)
Supplementary Movies 2 Trafficking of S3-RNase(N29D):GFP-containing compartments in living in vitro-germinated pollen tubes and the effect of BDM and oryzalin on the trafficking. The movies show in sequence: 1. GFP in in vitro-germinated pollen tubes of a transgenic S 2 S 3 plant carrying the GFP transgene expressed by the LAT52 promoter; 2. PiSLF2:GFP in in vitro-germinated pollen tubes of a transgenic S 2 S 3 plant carrying the PiSLF 2 :GFP transgene expressed by the LAT52 promoter; 3. Trafficking of S3-RNase(N29D):GFP-containing compartments in living in vitro-germinated pollen tubes of a transgenic S 2 S 3 plant carrying the S 3 -RNase(N29D):GFP transgene expressed by the LAT52 promoter; 4. Effect of 30 mM BDM on the trafficking of S3-RNase(N29D):GFP-containing compartments in living in vitro-germinated pollen tubes; 5. Effect of 50 mM BDM on the trafficking of S3-RNase(N29D):GFP-containing compartments in living in vitro-germinated pollen tubes; 6. Effect of 50 μM oryzalin on the trafficking of S3-RNase(N29D):GFP-containing compartments in living in vitro-germinated pollen tubes. In each movie, the left panel is the GFP channel and the right panel is the bright-field channel. (TAR 5,720 kb)

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Xiaoying Meng
    • 1
  • Zhihua Hua
    • 1
    • 2
  • Ning Wang
    • 1
  • Allison M. Fields
    • 3
  • Peter E. Dowd
    • 3
    • 4
  • Teh-hui Kao
    • 1
    • 3
    Email author
  1. 1.Intercollege Graduate Degree Program in Plant BiologyThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Department of GeneticsUniversity of WisconsinMadisonUSA
  3. 3.Department of Biochemistry and Molecular BiologyThe Pennsylvania State UniversityUniversity ParkUSA
  4. 4.Department of BotanyUniversity of WisconsinMadisonUSA

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