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Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis

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Abstract

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.

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Abbreviations

CalS:

Callose synthase

GSL:

Glucan synthase-like

MGU:

Male germ unit

RT-PCR:

Reverse transcription-polymerase chain reaction

DAPI:

4′6-Diamidino-2-phenylindole

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Acknowledgments

We thank the Arabidopsis Biological Resources Center (ABRC) for providing Arabidopsis seeds. This work was supported by grants from NSF (MCB 0548525 and IOB 0543923).

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Authors and Affiliations

  1. Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Gibb Hall 132, Moscow, ID, 83844-3052, USA

    Bo Xie, Xiaomin Wang & Zonglie Hong

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  1. Bo Xie
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  2. Xiaomin Wang
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  3. Zonglie Hong
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Corresponding author

Correspondence to Zonglie Hong.

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425_2009_1091_MOESM1_ESM.tif

Supplemental Fig. S1 Genomic PCR of cs9-5 and cs9-6 T-DNA insertion mutants. a Map of T-DNA insertion sites and primers used for genomic PCR reactions. There are two T-DNA inserts in cs9-5 (Salk_060111) and one in cs9-6 (Salk_124294). The directions of primers and T-DNA inserts are indicated by arrows. Black boxes represent exons and open boxes represent T-DNA. Primers used for genomic PCR reactions are LP 5′-ATA TAC CCT TGC ACC ACC ATG-3′, RP 5′-AAT ACT TAG CAG TTA GCA GGC G-3′ and LBb1 (Lb) 5′-GCG TGG ACC GCT TGC TGC AAC T-3′. b Genomic PCR products using two primers as indicated. In wild type, the amplified PCR products were a 1.05 kb genomic fragment from wild-type plants, two T-DNA specific fragments (0.8 and 0.5 kb) for cs9-5 and one T-DNA specific product (0.2 kb) for cs9-6. A nonspecific amplification product of 0.45 kb, indicated by star (*), was present in reactions with the LBb1 primer(TIFF 220 kb)

425_2009_1091_MOESM2_ESM.tif

Supplemental Fig. S2 Defects in reproduction in Arabidopsis plants expressing CalS5. a Phenotypes of newly opened anthers of wild type (WT), transgenic plants (S-CS5/WT, line 3) and cs5-1 mutant (cs5-1). Note that anthers of the transgenic plants and cs5-1 mutant were shrunken and contained fewer pollen grains. Bar, 0.5 mm b Silique phenotypes of wild type, S-CS5/WT transgenic plants (line 3) and cs5-1 mutant. Bar, 10 mm. c Embryos in the siliques of wild type, S-CS5/WT transgenic plants and cs5-1 mutant. Bar, 2.5 mm. d Differential interference contrast (DIC) microscope images of pollen grains of wild type (WT), S-CS5/WT plants (line 3) and cs5-1 mutant. Bar, 5 μm. e Scanning electron microscopy (SEM) images of pollen grains of wild type, and 3S-CS5 /WT plants (line 3). Bar, 5 μm (TIFF 3100 kb)

425_2009_1091_MOESM3_ESM.tif

Supplemental Fig. S3 Expression of CalS genes, callose contents and aborted embryos in transgenic plants expressing S-CS5. a-b RT-PCR analysis of the expression of CalS5 gene and the transgene S-CS5 in the leaf (a) and root (b) of 5 randomly selected S-CS5/WT transgenic plants. Primer CS5F2 and CS5R2 were used to detect the mRNA of CalS5. Primer CS5F1 corresponding to the CalS5 N-terminal region and the Strep specific primer StpR were used to detect the mRNA of S-CS5, Actin2 mRNA was used as an internal control. Three replications were conducted and one representative result is shown. c Callose contents of the tissues of wild type, cs5-1 mutant and S-CS5/WT (left-right: line 3, 5, 9, 11 and 14) plants. Quantitative measurements of callose content were performed as described previously (Shedletzky et al. 1997). Error bars were the means ± standard deviations from three replicate assays. In each plant tissues, Columns marked with different letters were significantly different according to Duncan multirange test, p≤ 0.05. d-e Percentages of short siliques (less than 5 mm), and aborted embryos in wild type, cs5-1 mutant and S-CS5/WT (left-right: line 3, 5, 9, 11 and 14) plants. Error bars were the means ± standard deviations from three replicate assays. Columns marked with different letters were significantly different according to Duncan multirange test, p≤ 0.05(TIFF 716 kb)

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Xie, B., Wang, X. & Hong, Z. Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis. Planta 231, 809–823 (2010). https://doi.org/10.1007/s00425-009-1091-3

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