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Brassinosteroids promote Arabidopsis pollen germination and growth

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Abstract

Pollen tubes are among the fastest tip-growing plant cells and represent an excellent experimental system for studying the dynamics and spatiotemporal control of polarized cell growth. However, investigating pollen tube tip growth in the model plant Arabidopsis remains difficult because in vitro pollen germination and pollen tube growth rates are highly variable and largely different from those observed in pistils, most likely due to growth-promoting properties of the female reproductive tract. We found that in vitro grown Arabidopsis pollen respond to brassinosteroid (BR) in a dose-dependent manner. Pollen germination and pollen tube growth increased nine- and fivefold, respectively, when media were supplemented with 10 µM epibrassinolide (epiBL), resulting in growth kinetics more similar to growth in vivo. Expression analyses show that the promoter of one of the key enzymes in BR biosynthesis, CYP90A1/CPD, is highly active in the cells of the reproductive tract that form the pathway for pollen tubes from the stigma to the ovules. Pollen tubes grew significantly shorter through the reproductive tract of a cyp90a1 mutant compared to the wild type, or to a BR perception mutant. Our results show that epiBL promotes pollen germination and tube growth in vitro and suggest that the cells of the reproductive tract provide BR compounds to stimulate pollen tube growth.

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Abbreviations

BRs:

Brassinosteroids

epiBL:

Epibrassinolide

PGM:

Pollen germination medium

CLSM:

Confocal laser scanning microscope

GFP:

Green fluorescent protein

NLS:

Nuclear localization signal

ECM:

Extracellular matrix

IQR:

Interquartile range

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Acknowledgments

We thank Mily Ron for providing the Gateway destination vector pGreenII-GW:NLS:3xEGFP and David Twell for sharing the Lat52p:GFP marker line. We are grateful to Frank Sprenger for assistance with the Spinning Disc Confocal Microscope and Thomas Dresselhaus for critical discussions. We thank Monika Kammerer for technical assistance. This work was funded by the German Research Council (DFG) with grants SFB 924 and SP 686/1-2 to SS.

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Correspondence to Stefanie Sprunck.

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Communicated by Tetsuya Higashiyama.

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497_2014_247_MOESM1_ESM.pdf

Figure S1 Scheme of the pollen application procedure. To balance out the time lags associated with pollen application and image acquisition, and to take varying pollen qualities into account, pollen was applied to the PGM in a standardized fashion. a By gently dabbing dehiscent anthers flowers on plates, spots of comparable size and pollen density were applied to the germination medium. Scale bar: 100 µm. b Seven pollen germination plates (A to G) containing PGM supplemented with varying epiBL concentrations (0 to 50 µM) were each subdivided in 8 sectors. Pollen was applied to the first sector of plate A (1.; yellow circle) and subsequently to the other 6 plates (2. – 7.). For the next sector, pollen application started with the second plate (B), and ended with plate A (pollination order 8. – 14.). For sector three, pollen application started with plate C (pollination order 15. – 21.), and so on. Images were acquired in the same order as the pollination was performed. (PDF 644 kb)

497_2014_247_MOESM2_ESM.pdf

Figure S2 BRI1-like (BRL) receptor kinases with BR binding activity have low expression values in reproductive tissues. Expression of BRI1-like 1 (BRL1) (light gray) and BRL3 (dark gray) in different reproductive and non-reproducti-ve tissues. Log2-fold expression values and gene expression levels displayed relative to the signal intensity on the 22 k ATH1 array according to the GENEVESTIGATOR microarray database (Zimmermann et al. 2004). Num-ber of samples used for calculating mean values ± 1SE: embryo, 38; endosperm, 11; pollen, 52; pistil, 29; car-pel, 2; stigma, 3; ovary, 4; ovule, 2; silique, 372; seed, 323; seedling, 2,330; shoot, 4,504; leaf, 2,849; roots, 1,063. (PDF 178 kb)

497_2014_247_MOESM3_ESM.pdf

Figure S3 CYP90A1/CPD promoter activity in developing pistils. af Pistils of plants expressing the NLS:3xEGFP reporter under control of the CYP90A1/CPD promoter (CYP90A1p:NLS:3xEGFP) were imaged at different developmental stages. Flower stages were defined according to Smyth et al. (1990). In pistils of flower stages 9 (a) and 10 (b), only very weak and ubiquitous promoter activity was detected. In flower stages 11 (c), 12 (d), and 13 (e), strong GFP signals appeared in the nuclei of reproductive tract cells, including the stigma, style and transmitting tract (tt). f After fertilization (flower stage 14), promoter activity in the cells of the reproductive tract started to diminish. Note that in flower stage 9 (a) the septum often ruptures while preparing the tiny pistils. Scale bars: ad 100 µm, e, f 150 µm. (PDF 11053 kb)

497_2014_247_MOESM4_ESM.pdf

Figure S4 Dwarf phenotypes of homozygous cyp90a1-1 and bri1-10. Compared to the wild type (Col-0), the BR biosynthesis mutant cyp90a1-1 and the BR perception mutant bri1-10 both exhibited severe dwarf phenotypes. (PDF 5709 kb)

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Vogler, F., Schmalzl, C., Englhart, M. et al. Brassinosteroids promote Arabidopsis pollen germination and growth. Plant Reprod 27, 153–167 (2014). https://doi.org/10.1007/s00497-014-0247-x

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