Volume 6, Issue 4, Pages 1163-1175 (July 2013) LlSR28 Is Involved in Pollen Germination by Affecting Filamentous Actin Dynamics  Li-Juan Cao, Meng-Meng Zhao, Chang Liu, Huai-Jian Dong, Wang-Cheng Li, Hai-Yun Ren  Molecular Plant  Volume 6, Issue 4, Pages 1163-1175 (July 2013) DOI: 10.1093/mp/sst097 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Predicted Domain Organization of LlSR28 and Phenotypic Analysis of the LlSR28 Complementing atsr45-1 Mutant. (A) Schematic representation of the predicted domain organization of LlSR28. LlSR28 consisted of two arginine and serine repeats (RS) sequences in addition to the RNA recognition motif (RRM) domain. The gray rounded rectangles represent the RS repeats (from positions 13 to 50, and from 140 to 229). The black rectangle represents the RRM (from position 54 to 127). (B) pAtSR45–LlSR28GFP complemented the root length of the atsr45-1 mutant phenotype. WT represents wild-type in this study, COMP represents complemented plant, L represents different lines of transformant. (C) Showing the recovery of the primary root. Data are the mean ± SE (** P < 0.01, by t-test, n > 40). DAP, d after planting. Three repeats. (D) The overall shape of complementary plants was rescued to the wild-type (30 d after planting). (E) pAtSR45–LlSR28GFP complemented the atsr45-1 mutant phenotype under 3% glucose treatments. Except for atsr45-1 mutant, the cotyledon of other plants turned green at 7 d after planting. Molecular Plant 2013 6, 1163-1175DOI: (10.1093/mp/sst097) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 LlSR28 Expression Pattern and Subcellular Localization. (A) The expression level of LlSR28 was analyzed by semi-quantitative RT–PCR in different tissues. S, stem; L, leaf; F, flower filament of stamen; P, petal; R, root; St, style; P0, ungerminated pollen; P3, pollen tube after germinated for 3h; P6, pollen tube after germinated for 6h. LlEF1α was used as the control. (B) Real-time quantitative RT–PCR analysis of the relative amount of LlSR28 mRNA at different germination stages of lily pollen. P0, P3, and P6 are identical to Figure 2A. (C) Vegetative nucleus localization of LlSR28 in lily and Arabidopsis pollens. Lily pollen was transformed with the Zm13–LlSR28GFP plasmid (1.0 μg) by microprojectile bombardment (a–d). The bombarded pollen was cultured for 3h in liquid medium and then fixed with 4% paraformaldehyde for 1h and stained with 0.4 μg ml–1 4’,6-diamidino-2-phenylindole (DAPI; b). The Lat52–LlSR28GFP plasmid was stably transformed into Arabidopsis and pollen grain was fixed and stained same to lily pollen grain (e–h). (a–d, bar = 50 μm; e–h, bar = 10 μm). Individual pictures, merged pictures, and pictures taken under white light (differential interference contrast (DIC)) are presented. Molecular Plant 2013 6, 1163-1175DOI: (10.1093/mp/sst097) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Overexpression of Lat52–LlSR28GFP Completely Inhibited Pollen Germination in Arabidopsis and the atsr45-1 Mutant Enhanced the Pollen Germination Rate in Early Stage and Pollen Tube Length. (A) Arabidopsis pollen expressing Lat52–LlSR28GFP could not germinate (16 transformed lines were screened and showed the same phenotype). White dots represent the fluorescence of LlSR28GFP (black arrow denotes). Bar = 20 μm. (B) Trypan blue was used to test pollen viability. The pollen viability of ungerminated pollen grains was not different from that of the wild-type. OX represents Lat52–LlSR28GFP overexpressing pollen. Standard errors for three independent experiments are shown (n > 500, P > 0.05 by t-test). (C) Histogram showing the germination rate of pollen germinated for 3, 5, and 9h from wild-type, atsr45-1 mutant, and complemented plants L4 (n > 500 pollen grains for each genotype). Standard errors for three independent experiments are shown (* P < 0.05 by t-test). COMP represents complemented pollen. (D) Histogram showing the pollen tube length of pollen germinated for 3 and 5h from wild-type, atsr45-1 mutant, and complemented plants (error bars mean ± SE, ** P < 0.01, * P < 0.05, n ≥ 100), three repeats. (E, F) Measurement of growth rates was performed by tracking individual pollen tubes. Photos of pollen tube growth were from wild-type, atsr45-1 mutant, and complementary plants. Bar = 50 μm. (F) Statistic analysis of pollen tube growth rate shows that there were no differences among wild-type, atsr45-1 mutant, and complemented pollen (error bars mean ± SE, P > 0.05, by t-test, n = 40). Molecular Plant 2013 6, 1163-1175DOI: (10.1093/mp/sst097) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Actin Dynamics during Pollen Germination. (A) F-actin dynamics of the wild-type and atsr45-1 mutant during pollen germination. a and d represent unhydrated pollen, b and e represent pollen hydrated for 10min, c and f represent just-germinated pollen (hydrated for 120min). The upper row shows wild-type pollen and the lower row shows atsr45-1 mutant pollen. The fresh pollen grains of different genotypes were harvested and cultured for corresponding times (0, 10, and 120min) on solid cultured medium. Afterwards, pollen was fixed by 300 μM MBS and stained by 200 nM Alexa-488 phalloidin (see the ‘Methods’ section). Bar = 10 μm. (B) Quantification of relative F-actin of Figure 4A (error bars mean ± SE, ** P < 0.01, by t-test, n > 40). The corresponding lines were used to compare fluoresce value between two different hydration times. (C) Lifeact–mEGFP reveals F-actin dynamics of living pollen cell during pollen germination. The fresh pollen grains were placed on solid germination medium and scanned within 40min. The premise was that pollen grains would germinate within 40min. Bar = 10 μm. (D) Quantification of relative F-actin amount during pollen germination. The relative amount of F-actin of ungerminated pollen grains was normalized to 100%. Error bars mean ± SD (n = 17). (E) F-actin dynamics during pollen germination in the wild-type and LlSR28GFP-overexpressing transformant. Hydration time of a–f is identical to (A). The upper row shows the F-actin of wild-type pollen, the middle row shows the F-actin of LlSR28GFP-overexpressing pollen, and the lower row shows LlSR28GFP fluorescence. Pollen grains from the wild-type and LlSR28GFP-overexpressing transformants were stained with Alexa-568 phalloidin. Bar = 10 μm. (F) Quantification of relative F-actins of Figure 4C (Error bars represent mean ± SE, ** P < 0.01, by t-test, n > 40). OX represents LlSR28GFP-overexpressing. (G) Total actin amount in pollen extracts was measured with an immunoassay. The amount of actin after pollen germination significantly increased compared with unhydrated pollen. a–c shows the total proteins of different hydration stages (0, 10, and 60min) by Coomassie brilliant blue staining. d–f shows the protein immunoblotting that antiactin antibody was used to assess the actin amount during pollen hydration (0, 10, and 60min). The pollen hydration was processed on the liquid pollen germination medium. M represents protein marker (kDa). Molecular Plant 2013 6, 1163-1175DOI: (10.1093/mp/sst097) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 LlSR28 and AtSR45 Affected Alternative Splicing of AtVLN1. (A) Nucleic acid and amino acid sequences of AtVLN1 and AtVLN1.b.VHP represents Villin headpiece; GEL represents Gelsolin domain. Arrows represent the position of primers used to amplify AtVLN1 two transcripts (Figure 5B). (B) RT–PCR analysis of AtVLN1 and AtVLN1.b in wild-type, atsr45-1, LlSR28-overexpressing, and complementary pollen. (C) Real-time quantitative PCR showing the expression level of AtVLN1 and AtVLN1.b. In atsr45-1 mutant, AtVLN1.b amount significantly decreased and AtVLN1 increased compared with other genotypes. (D) RT–PCR analysis of the expression level of ABPs known to be present in pollen grains. AtEIF4A was used as the loading control. Amplification bands were semi-quantified. Results are representative of at least three independent experiments. Each COMP represents complemented pollen. Molecular Plant 2013 6, 1163-1175DOI: (10.1093/mp/sst097) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions