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BcPME37c is involved in pollen intine formation in Brassica campestris
Biochemical and Biophysical Research Communications 517 (2019) 63e68
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BcPME37c is involved in pollen intine formation in Brassica campestris Xingpeng Xiong a, Dong Zhou a, Liai Xu a, Tingting Liu a, Xiaoyan Yue a, Weimiao Liu a, Jiashu Cao a, b, c, * a
Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou, 310058, China Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou, 310058, China c Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Hangzhou, 310058, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 June 2019 Accepted 2 July 2019 Available online 16 July 2019
Pollen wall development is one of the key processes of pollen development. Several pectin methylesterase (PME) genes participate in pollen germination and pollen tube growth. However, the relationship between PME genes and pollen intine formation remains unclear. In this study, we investigated the expression and subcellular localization of the PME gene BcPME37c in Brassica campestris. Furthermore, morphology and cytology methods were used to examine the phenotype of the CRISPR/Cas9 system-induced BcPME37c mutant. We found that BcPME37c is predominately expressed in mature stamen and located at the cell wall. BcPME37c mutation causes the abnormal thickening of the pollen intine of B. campestris. Our study indicated that BcPME37c is required for pollen intine formation in B. campestris. © 2019 Published by Elsevier Inc.
Keywords: Brassica campestris PME37c Pollen development Intine
1. Introduction Pollen development is an important event in the sexual reproduction process of seed plants. Normal pollen wall formation is essential for the maintenance of the pollen structure and function. Despite the importance of pollen wall development, the molecular mechanisms implicated in the formation of the pollen wall are not fully known [1,2]. The pollen wall of angiosperms consists of three layers from outside to inside: pollen coat, exine, and intine [3,4]. The main components of intine are pectin, cellulose, hemicellulose, and structural proteins, and pectin is the most abundant component [1]. The deviant synthesis or modification of pectin can cause abnormalities in the structure of intine, thereby affecting its function. A series of genes is required for intine development; these genes include FLA3 [5], AtUSP [6], and pectin methylesterase 48 (PME48) [7] in Arabidopsis; OsGT1 [8] and CAP1 [9] in rice; and BcPLL9 [10], BcPLL10 [11], BcMF8 [12], BcMF9 [13], BcMF18 [14], and BcMF23a (a PME gene) [15] in Brassica campestris (syn. Brassica rapa). All the
* Corresponding author. Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou, 310058, China. E-mail addresses: [email protected] (X. Xiong), 15574994586@163. com (D. Zhou), [email protected] (L. Xu), [email protected] (T. Liu), [email protected] (X. Yue), [email protected] (W. Liu), jshcao@zju. edu.cn (J. Cao). https://doi.org/10.1016/j.bbrc.2019.07.009 0006-291X/© 2019 Published by Elsevier Inc.
mutants or gene silencing plants of these genes display disrupted intine [5e15]. Pectin is a complex polysaccharide whose main component is 1, 4-a-D-galacturonic acid. The main components of pectin are divided into four categories based on their structures, namely, homogalacturonan (HG), xylose galacturonan, and rhamnogalacturonan I and II [16,17]. PME (EC3.1.1.11) is a key enzyme in pectin metabolism that mainly catalyzes HG demethylation. After demethylation, pectin is subsequently degraded by polygalacturonase and pectin lyase; the cell wall is loosened or forms cross links through calcium ions; and the cell wall is finally strengthened [18,19]. PME is involved in important physiological processes related to plant vegetative growth and reproductive development, including cell wall elongation and sclerosis, cell adhesion and isolation, fruit ripening, wood development, stem elongation, leaf growth, microsporogenesis, pollen tube growth, and seed germination [19e21]. PMEs are a large family containing dozens of members [22]. A total of 67 and 110 PME genes are present in Arabidopsis thaliana and B. campestris, respectively [23]. At least 14 [18] and 25 (unpublished data) PME genes show specific or prominent expression in pollen or inflorescence in A. thaliana and B. campestris, respectively. Some of these genes are involved in pollen development and pollen tube growth. For example, the tetrad in the QRT1 mutant is not normally separated [24]. In VGD1 mutant, PME activity is
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reduced, and pollen tube growth is blocked, resulting in decreased fertility [25]. AtPPME1 mutation affects the shape and elongation of pollen tubes [26]. The pollen of pme48 show delayed or impaired germination in vitro and in vivo [7]. The PME gene BcMF23a, which was identified by our laboratory, is also involved in intine formation. After BcMF23a silencing, the intine of B. campestris is abnormally thickened at the germination aperture, which affects pollen germination and pollen tube growth [15]. In other plants, some PME genes are also important for pollen development and pollen tube growth. NtPPME1 silencing in tobacco decreases the growth rate of pollen tube in the stigma [27]. A recent study reported that the maize PME gene ZmGa1P (Zm00001d048936) is associated with unilateral cross-incompatibility [28]. Aside from the abovementioned genes, the function of most PME genes related to pollen development, especially pollen wall development, remains unknown. Bra003491 (BcPME37c) encodes a PME gene and is predominantly expressed in the mature pollen stage of the flower [29]. Bra003491 is one of the three syntenic genes of At3g62170 (PME37), and the other two are Bra007665 and Bra014410. These three PME genes belong to the three subgenomes of B. campestris; thus, we named Bra007665, Bra014410, and Bra003491 as BcPME37a, BcPME37b, and BcPME37c, respectively. The BcPME37c mutant has been created through the CRISPR/Cas9 system [29], but its function remains unclear. In this study, we investigated the expression pattern of BcPME37c in different tissues and organs and the subcellular localization of BcPME37c in onion epidermal cells. Furthermore, the phenotype of bcpme37c was examined. Results showed that BcPME37c was expressed specifically in the stamen, and it was involved in pollen intine formation in B. campestris. Our work provides good evidence for an in-depth understanding of the role of the PME gene in pollen intine development. 2. Materials and methods 2.1. Plant materials and growth condition The BcPME37c mutant was obtained via the CRISPR/Cas9 system [29]. B. campestris ssp. chinensis var. parachinensis cv. Youqing 49 (bought from Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China) was used as the control plant. The seeds of the control plant and bcpme37c were sown on wet filter paper in 90 mm Petri dishes and then placed into an incubator at 28 C for 2e3 days to accelerate germination. When seedling roots reached 2e3 cm in length, the seedlings were carefully planted into pots with mixed medium (3:2:1 mixed peat, vermiculite, and perlite) and then cultivated in a biotron at 22 C with 16 h of light and at 18 C with 8 h of darkness. 2.2. qRT-PCR and subcellular localization analysis Total RNA of the root, stem, leaf, inflorescence, and silique at flowering stage; buds at flower stage I to V; and sepal, petal, stamen, and pistil of buds at flower stage V of the control plants was prepared using Trizol® reagent (Invitrogen, USA) following the manufacturer's instructions. Flower stage I to V correspond to five stages of pollen development, namely, pollen mother cell, tetrad, uninucleate pollen, binucleate pollen, and trinucleate pollen stages (mature pollen stage) [30]. The first-strand cDNA was then synthesized using PrimeScript™ RT reagent Kit (TAKARA, Japan). Quantitative real-time PCR (qRT-PCR) was performed on a Bio-Rad CFX96 Real-time PCR Detection System (Bio-Rad, USA) using QTaq for Real-Time qPCR (TAKARA, Japan). BcUBC10 was used as the reference gene. The following primers were used for qRT-PCR:
BcPME37c-F (50 -ATAGGTGACAAAGGTCCCAAG-30 ), BcPME37c-R (50 -GGTTCAATCGAAGCACTAATC-30 ), BcUBC10-F (50 -GGGTCCTACAGACAGTCCTTAC-30 ), and BcUBC10-R (50 -ATGGAACACCTTCGTC CTAAA-30 ). The relative expression levels of BcPME37c were analyzed using the 2DDCt method [31]. Three biological repeats were performed. The BcPME37c:eGFP construct and subcellular localization analysis were as described previously [12].
2.3. Phenotypic characterization Alexander staining was used to analyze pollen viability [32]. The stained pollen was observed, and the pollen diameter was analyzed under Nikon ECLIPSE 90i (Nikon, Japan). Aniline blue staining was performed as described by Lin et al. [12]. 4’,6-Diamidino-2phenylindole (DAPI) was used to stain pollen nuclei [33]. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and semi-thin section examination of pollen were performed as previously described [30]. In vitro pollen germination was performed as previously described with minor adjustments [12]. In brief, pollen grains were collected and cultured in medium containing 15% sucrose, 0.4 mM HBO3, 0.4 mM Ca(NO3)2, and 0.1% agarose. The pH of the medium was adjusted to 5.8. The pollen grains were grown at 22 C with 100% relative humidity for 4 h in the dark.
3. Results 3.1. Expression pattern and subcellular localization of BcPME37c As described in the introduction, BcPME37c is expressed predominantly at the mature pollen stage of the flower. To further determine the expression pattern, we used qRT-PCR to investigate the expression level of BcPME37c in different organs. The results showed that the expression level of BcPME37c was relatively higher in inflorescences and relatively lower in roots, stems, leaves, and siliques (Fig. 1A). BcPME37c was expressed predominantly in the buds at the mature pollen stage of the flower (Fig. 1B). Further analysis indicated that BcPME37c was expressed predominantly in stamens and showed low expression level in sepals, petals, and gynoecium (Fig. 1C). At the pollen tube growth stage, BcPME37c showed low expression level (Fig. 1D). The subcellular localization of BcPME37c was investigated using the transient expression of BcPME37c:eGFP fusion protein. The results showed that BcPME37c was localized to the cell wall (Fig. 2).
3.2. Mutation of BcPME37c did not affect plant growth or flower formation The BcPME37c mutant was obtained through the CRISPR/Cas9 system, as reported in a previous study. The vegetative growth of bcpme37c showed no remarkable differences compared with that of the control plants (Supplementary Figs. 1G and 1H). Given that BcPME37c was mainly expressed in the mature pollen stage, we focused on whether the mutant exhibits differences in the reproductive growth process from the control plants. The morphology and size of the open flowers of bcpme37c were not significantly different from those of the control plants (Supplementary Figs. 1A and 1B). Further observation of the shape and size of the sepals, petals, stamens, and pistils showed no evident abnormalities (Supplementary Figs. 1Ce1F).
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Fig. 1. qRT-PCR analysis of BcPME37c expression patterns. R, Ste, L, Inf, and Si represent root, stem, leaf, inflorence, and silique, respectively. B1eB5 indicates the buds at flower stage I to stage V, respectively. Se, Pe, Sta, and Gy correspond to the sepal, petal, stamen, and gynoecium, respectively; pollinated, pollinated pistil; unpollinated, unpollinated pistil; 1, 3, 10, and 24 h represent the number of hours after pollination.
Fig. 2. Subcellular localization analysis of BcPME37c-encoded protein. A, B, C, and D are the pFGC-eGFP empty vector; and E, F, G, and H are BcPME37c:eGFP. A, B, E, and F are GFP fields of view; and C, D, G, and H are bright fields. The tablets of A, C, E, and G are made from distilled water; and B, D, F, and H are cells under plasmolysis. The bars are 100 mm.
3.3. Diameter of a portion of bcpme37c pollen evidently increased after imbibition To determine whether BcPME37c mutation shows any phenotype in pollen formation, we performed Alexander staining and SEM observation. The results of Alexander staining showed that the pollen viability of bcpme37c was not notably different from that of the control plants, but the pollen diameter of a small part of bcpme37c pollen remarkably increased (Fig. 3A, B, E, and F). After
imbibition, the pollen diameter of 99.24% of the control plants was not more than 30 mm, whereas only 94.64% of the pollen diameter of bcpme37c pollen was in this interval. The percentage of pollen of the control plants with a diameter larger than 30 mm was only 0.76%, whereas that of bcpme37c was 5.36% (Fig. 3A, B, E, and F and Supplementary Fig. 2A). SEM examination results demonstrated that approximately 5.73% of bcpme37c pollen showed malformation compared with 0.65% of the control pollen (Fig. 3C, D, G, and H and Supplementary Fig. 2B).
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Fig. 3. Cytological staining and electron microscope scanning analysis of bcpme37c and the control plants. A, B, E, and F are Alexander staining results; C, D, G, and H are scanning electron micrographs; I, J, M, and N are aniline blue staining diagrams; and K, L, O, and P are DAPI staining results. AeD and IeL are the pollen samples from the control plants; EeH and M P are bcpme37c pollen. J, L, N, and P are the bright-field images of I, K, M, and O, respectively. The bars are 50 (A, E, IeP), 25 (B and F), 100 (C and G), and 30 mm (D and H). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.4. Intine of bcpme37c mature pollen showed abnormal thickening To further clarify the reason for the increased diameter and malformed pollen formation after pollen imbibition in bcpme37c, we observed the pollen of the mutant and control plants by DAPI staining, aniline blue staining, semi-thin section, and TEM. DAPI and aniline blue staining results showed that the pollen nuclei formation and callose degradation of bcpme37c were normal compared with those of control plants (Fig. 3IeP). The results of semi-thin section showed that the pollen of bcpme37c was normal at all five pollen development stages compared with that of the control plants (Supplementary Fig. 3). TEM examination results showed no significant difference between bcpme37c and the control plants in the pollen mother cell, tetrad, and uninucleate pollen stages (figures of pollen mother cell and tetrad stages are not listed; Fig. 4). Sections in the binucleate and trinucleate pollen stages showed the abnormal thickening of the intine of bcpme37c pollen, but the tectum and bacula of the pollen exine and pollen coat were normal (Fig. 4H, I, K, and L). The above results showed that the abnormal thickening of the intine of bcpme37c pollen occurred after the uninucleate pollen stage.
3.5. In vitro pollen germination and the number of seeds per silique of bcpme37c were normal To further explore the roles of BcPME37c in pollen germination, we conducted in vitro pollen germination. The results showed that the percentages of germinated and ungerminated pollen and no viability pollen from bcpme37c and the control plants were almost the same (Supplementary Fig. 4). These data suggested that the abnormal thickening of intine did not affect the germination of bcpme37c pollen. To determine whether BcPME37c mutation affects female gamete fertility, we employed self-cross in the control plants and bcpme37c. The number of seeds per silique of the control plants and bcpme37c was almost the same (data not shown). Thus, BcPME37c mutation did not impair female gamete fertility.
4. Discussion Predecessors labeled mature pollen with John Innes Monoclonal 5 (JIM5) antibody, which specifically binds to low methylated HG; JIM5 exhibits a strong signal in the inner wall, indicating that the degree of HG methylation in the intine of pollen is low [34]. These findings also showed that PME is active in the construction of
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Fig. 4. Cytological staining and electron microscope scanning analysis of bcpme37c and the control plants. AeC and GeI are pollen sections of the control plants; DeF and JeL are the pollen sections of bcpme37c. Pollen in the first column (A, D, G, and J) are at the mononucleate pollen stage; pollen in the second column (B, E, H, and K) are at the binucleate pollen stage; and pollen in the third column (C, F, I, and L) are at the trinucleate pollen stage. AeF show the cut surface of whole pollen, and GeL are the enlarged view of the pollen wall. Arrows in H, I, K, and L point to the intine. The bars are 5 (AeF) and 0.5 mm (GeL).
pollen intine. Proteome analysis indicated that at least PME37, PPME1, and PME48 in Arabidopsis mature pollen had high PME contents [35]. PME genes are involved in pollen development and pollen germination [13e19]. However, the relationship between the PME gene and pollen intine development is rarely studied. For example, PME48 mutation and BcMF23a silencing impair pollen germination and pollen tube growth [7,15]. Our study further proved that the PME gene was involved in pollen intine formation. Pectin deesterification catalyzed by PMEs can free carboxyl groups on pectin chains, thereby loosening or strengthening the cell wall [1]. PME activity can modulate the degree of methylesterification of HG at pollen intine and affect the balance of plasticity and mechanical strength of intine. In pme48 mature pollen, the degree of methyl esterification of HG was nearly 20% higher than that of the wild type, indicating that PME activity in intine
decreased after PME48 mutation; thus, the degree of methyl esterification of HG increased and eventually caused abnormal pollen germination [7]. In bcpme37c pollen, approximately 5.36% showed larger size than the control plant pollen after imbibition. The results of SEM analysis also showed that approximately 5.73% of the bcpme37c pollen were deformed. TEM analysis results indicated that the intine of bcpme37c pollen were thick at the binucleate and trinucleate pollen stages. The abnormal thickening of the intine of the mature pollen of bcpme37c was possibly caused by the abnormal degree of methyl esterification of HG. However, to verify this speculation, we must study the phenotype of bcpme37c pollen in detail. AtPME37 is highly homologous to VGD1 and exhibits the same expression profile as VGD1 but fails to complement the vgd1 mutant [25]. These characteristics indicate that AtPME37 and VGD1 exhibit
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different biological functions [25]. BcPME37c is the syntenic gene of AtPME37, and their expression patterns are similar. These two genes may function similarly, but numerous studies must be conducted before reaching this conclusion. Aside from BcPME37c, BcPME37a and BcPME37b are also the syntenic genes of AtPME37 (http:// brassicadb.org/brad/index.php). More than 20 PME genes showed specific expression at mature pollen stage of the flower in B. campestris (unpublished data). The phenotypes of bcpme37c were not obvious due to gene function redundancy, but the relationship between these pollen development-related PME genes must be further studied. In summary, BcPME37c is a stamen-specific gene, which encodes a secreted protein. BcPME37c mutation causes abnormal pollen intine formation in B. campestris. Our work suggested that BcPME37c is involved in pollen intine development in B. campestris. Conflicts of interest All authors declare that they have no conflict of interest. Ethical approval Research did not involve human participants and/or animals. Acknowledgments This research was supported by grants from the National Natural Science Foundation of China (No. 31471877 to J. C.).The authors are grateful to all the members of the Cao lab for technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.009. References [1] T.D. Quilichini, E. Grienenberger, C.J. Douglas, The biosynthesis, composition and assembly of the outer pollen wall: a tough case to crack, Phytochemistry 113 (2015) 170e182. https://doi.org/10.1016/j.phytochem.2014.05.002. [2] J. Shi, M. Cui, L. Yang, Y.J. Kim, D. Zhang, Genetic and biochemical mechanisms of pollen wall development, Trends Plant Sci. 20 (2015) 741e753. https://doi. org/10.1016/j.tplants.2015.07.010. [3] Z.A. Wilson, D.B. Zhang, From Arabidopsis to rice: pathways in pollen development, J. Exp. Bot. 60 (2009) 1479e1492. https://doi.org/10.1093/jxb/ erp095. [4] U.M. Hesse, D. Halbritter, U.M. Weber, D.B.R. Buchner, A. Frosch-Radivo, M.S. Ulrich, Pollen Terminology, Springer, Vienna, 2009. https://doi.org/10. 1007/978-3-211-79894-2. [5] J. Li, M. Yu, L.L. Geng, J. Zhao, The fasciclin-like arabinogalactan protein gene, FLA3, is involved in microspore development of Arabidopsis, Plant J. 64 (2010) 482e497. https://doi.org/10.1111/j.1365-313X.2010.04344.x. [6] J.A. Schnurr, K.K. Storey, H.J. Jung, D.A. Somers, J.W. Gronwald, UDP-sugar pyrophosphorylase is essential for pollen development in Arabidopsis, Planta 224 (2006) 520e532. https://doi.org/10.1007/s00425-006-0240-1. [7] C. Leroux, S. Bouton, M.C. Kiefer-Meyer, T.N. Fabrice, A. Mareck, S. Guenin, F. Fournet, C. Ringli, J. Pelloux, A. Driouich, P. Lerouge, A. Lehner, J.C. Mollet, PECTIN METHYLESTERASE48 is involved in Arabidopsis pollen grain germination, Plant Physiol. 167 (2015) 367e380. https://doi.org/10.1104/pp.114. 250928. [8] S. Moon, S. Kim, G. Zhao, J. Yi, Y. Yoo, P. Jin, S. Lee, K. Jung, D. Zhang, G. An, Rice GLYCOSYLTRANSFERASE1 encodes a glycosyltransferase essential for pollen wall formation, Plant Physiol. 161 (2013) 663e675. https://doi.org/10.1104/ pp.112.210948. [9] K. Ueda, F. Yoshimura, A. Miyao, H. Hirochika, K. Nonomura, H. Wabiko, COLLAPSED ABNORMAL POLLEN1 gene encoding the arabinokinase-like protein is involved in pollen development in rice, Plant Physiol. 162 (2013) 858e871. https://doi.org/10.1104/pp.113.216523. [10] J. Jiang, L. Yao, Y. Yu, Y. Liang, J. Jiang, N. Ye, Y. Miao, J. Cao, PECTATE LYASE-LIKE 9 from Brassica campestris is associated with intine formation, Plant Sci. 229 (2014) 66e75. https://doi.org/10.1016/j.plantsci.2014.08.008. [11] J. Jiang, L. Yao, Y. Yu, M. Lv, Y. Miao, J. Cao, PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris, J. Integr. Plant Biol. 56
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BcPME37c is involved in pollen intine formation in Brassica campestris Xingpeng Xiong a, Dong Zhou a, Liai Xu a, Tingting Liu a, Xiaoyan Yue a, Weimiao Liu a, Jiashu Cao a, b, c, * a
Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou, 310058, China Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou, 310058, China c Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Hangzhou, 310058, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 June 2019 Accepted 2 July 2019 Available online 16 July 2019
Pollen wall development is one of the key processes of pollen development. Several pectin methylesterase (PME) genes participate in pollen germination and pollen tube growth. However, the relationship between PME genes and pollen intine formation remains unclear. In this study, we investigated the expression and subcellular localization of the PME gene BcPME37c in Brassica campestris. Furthermore, morphology and cytology methods were used to examine the phenotype of the CRISPR/Cas9 system-induced BcPME37c mutant. We found that BcPME37c is predominately expressed in mature stamen and located at the cell wall. BcPME37c mutation causes the abnormal thickening of the pollen intine of B. campestris. Our study indicated that BcPME37c is required for pollen intine formation in B. campestris. © 2019 Published by Elsevier Inc.
Keywords: Brassica campestris PME37c Pollen development Intine
1. Introduction Pollen development is an important event in the sexual reproduction process of seed plants. Normal pollen wall formation is essential for the maintenance of the pollen structure and function. Despite the importance of pollen wall development, the molecular mechanisms implicated in the formation of the pollen wall are not fully known [1,2]. The pollen wall of angiosperms consists of three layers from outside to inside: pollen coat, exine, and intine [3,4]. The main components of intine are pectin, cellulose, hemicellulose, and structural proteins, and pectin is the most abundant component [1]. The deviant synthesis or modification of pectin can cause abnormalities in the structure of intine, thereby affecting its function. A series of genes is required for intine development; these genes include FLA3 [5], AtUSP [6], and pectin methylesterase 48 (PME48) [7] in Arabidopsis; OsGT1 [8] and CAP1 [9] in rice; and BcPLL9 [10], BcPLL10 [11], BcMF8 [12], BcMF9 [13], BcMF18 [14], and BcMF23a (a PME gene) [15] in Brassica campestris (syn. Brassica rapa). All the
* Corresponding author. Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou, 310058, China. E-mail addresses: [email protected] (X. Xiong), 15574994586@163. com (D. Zhou), [email protected] (L. Xu), [email protected] (T. Liu), [email protected] (X. Yue), [email protected] (W. Liu), jshcao@zju. edu.cn (J. Cao). https://doi.org/10.1016/j.bbrc.2019.07.009 0006-291X/© 2019 Published by Elsevier Inc.
mutants or gene silencing plants of these genes display disrupted intine [5e15]. Pectin is a complex polysaccharide whose main component is 1, 4-a-D-galacturonic acid. The main components of pectin are divided into four categories based on their structures, namely, homogalacturonan (HG), xylose galacturonan, and rhamnogalacturonan I and II [16,17]. PME (EC3.1.1.11) is a key enzyme in pectin metabolism that mainly catalyzes HG demethylation. After demethylation, pectin is subsequently degraded by polygalacturonase and pectin lyase; the cell wall is loosened or forms cross links through calcium ions; and the cell wall is finally strengthened [18,19]. PME is involved in important physiological processes related to plant vegetative growth and reproductive development, including cell wall elongation and sclerosis, cell adhesion and isolation, fruit ripening, wood development, stem elongation, leaf growth, microsporogenesis, pollen tube growth, and seed germination [19e21]. PMEs are a large family containing dozens of members [22]. A total of 67 and 110 PME genes are present in Arabidopsis thaliana and B. campestris, respectively [23]. At least 14 [18] and 25 (unpublished data) PME genes show specific or prominent expression in pollen or inflorescence in A. thaliana and B. campestris, respectively. Some of these genes are involved in pollen development and pollen tube growth. For example, the tetrad in the QRT1 mutant is not normally separated [24]. In VGD1 mutant, PME activity is
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reduced, and pollen tube growth is blocked, resulting in decreased fertility [25]. AtPPME1 mutation affects the shape and elongation of pollen tubes [26]. The pollen of pme48 show delayed or impaired germination in vitro and in vivo [7]. The PME gene BcMF23a, which was identified by our laboratory, is also involved in intine formation. After BcMF23a silencing, the intine of B. campestris is abnormally thickened at the germination aperture, which affects pollen germination and pollen tube growth [15]. In other plants, some PME genes are also important for pollen development and pollen tube growth. NtPPME1 silencing in tobacco decreases the growth rate of pollen tube in the stigma [27]. A recent study reported that the maize PME gene ZmGa1P (Zm00001d048936) is associated with unilateral cross-incompatibility [28]. Aside from the abovementioned genes, the function of most PME genes related to pollen development, especially pollen wall development, remains unknown. Bra003491 (BcPME37c) encodes a PME gene and is predominantly expressed in the mature pollen stage of the flower [29]. Bra003491 is one of the three syntenic genes of At3g62170 (PME37), and the other two are Bra007665 and Bra014410. These three PME genes belong to the three subgenomes of B. campestris; thus, we named Bra007665, Bra014410, and Bra003491 as BcPME37a, BcPME37b, and BcPME37c, respectively. The BcPME37c mutant has been created through the CRISPR/Cas9 system [29], but its function remains unclear. In this study, we investigated the expression pattern of BcPME37c in different tissues and organs and the subcellular localization of BcPME37c in onion epidermal cells. Furthermore, the phenotype of bcpme37c was examined. Results showed that BcPME37c was expressed specifically in the stamen, and it was involved in pollen intine formation in B. campestris. Our work provides good evidence for an in-depth understanding of the role of the PME gene in pollen intine development. 2. Materials and methods 2.1. Plant materials and growth condition The BcPME37c mutant was obtained via the CRISPR/Cas9 system [29]. B. campestris ssp. chinensis var. parachinensis cv. Youqing 49 (bought from Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China) was used as the control plant. The seeds of the control plant and bcpme37c were sown on wet filter paper in 90 mm Petri dishes and then placed into an incubator at 28 C for 2e3 days to accelerate germination. When seedling roots reached 2e3 cm in length, the seedlings were carefully planted into pots with mixed medium (3:2:1 mixed peat, vermiculite, and perlite) and then cultivated in a biotron at 22 C with 16 h of light and at 18 C with 8 h of darkness. 2.2. qRT-PCR and subcellular localization analysis Total RNA of the root, stem, leaf, inflorescence, and silique at flowering stage; buds at flower stage I to V; and sepal, petal, stamen, and pistil of buds at flower stage V of the control plants was prepared using Trizol® reagent (Invitrogen, USA) following the manufacturer's instructions. Flower stage I to V correspond to five stages of pollen development, namely, pollen mother cell, tetrad, uninucleate pollen, binucleate pollen, and trinucleate pollen stages (mature pollen stage) [30]. The first-strand cDNA was then synthesized using PrimeScript™ RT reagent Kit (TAKARA, Japan). Quantitative real-time PCR (qRT-PCR) was performed on a Bio-Rad CFX96 Real-time PCR Detection System (Bio-Rad, USA) using QTaq for Real-Time qPCR (TAKARA, Japan). BcUBC10 was used as the reference gene. The following primers were used for qRT-PCR:
BcPME37c-F (50 -ATAGGTGACAAAGGTCCCAAG-30 ), BcPME37c-R (50 -GGTTCAATCGAAGCACTAATC-30 ), BcUBC10-F (50 -GGGTCCTACAGACAGTCCTTAC-30 ), and BcUBC10-R (50 -ATGGAACACCTTCGTC CTAAA-30 ). The relative expression levels of BcPME37c were analyzed using the 2DDCt method [31]. Three biological repeats were performed. The BcPME37c:eGFP construct and subcellular localization analysis were as described previously [12].
2.3. Phenotypic characterization Alexander staining was used to analyze pollen viability [32]. The stained pollen was observed, and the pollen diameter was analyzed under Nikon ECLIPSE 90i (Nikon, Japan). Aniline blue staining was performed as described by Lin et al. [12]. 4’,6-Diamidino-2phenylindole (DAPI) was used to stain pollen nuclei [33]. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and semi-thin section examination of pollen were performed as previously described [30]. In vitro pollen germination was performed as previously described with minor adjustments [12]. In brief, pollen grains were collected and cultured in medium containing 15% sucrose, 0.4 mM HBO3, 0.4 mM Ca(NO3)2, and 0.1% agarose. The pH of the medium was adjusted to 5.8. The pollen grains were grown at 22 C with 100% relative humidity for 4 h in the dark.
3. Results 3.1. Expression pattern and subcellular localization of BcPME37c As described in the introduction, BcPME37c is expressed predominantly at the mature pollen stage of the flower. To further determine the expression pattern, we used qRT-PCR to investigate the expression level of BcPME37c in different organs. The results showed that the expression level of BcPME37c was relatively higher in inflorescences and relatively lower in roots, stems, leaves, and siliques (Fig. 1A). BcPME37c was expressed predominantly in the buds at the mature pollen stage of the flower (Fig. 1B). Further analysis indicated that BcPME37c was expressed predominantly in stamens and showed low expression level in sepals, petals, and gynoecium (Fig. 1C). At the pollen tube growth stage, BcPME37c showed low expression level (Fig. 1D). The subcellular localization of BcPME37c was investigated using the transient expression of BcPME37c:eGFP fusion protein. The results showed that BcPME37c was localized to the cell wall (Fig. 2).
3.2. Mutation of BcPME37c did not affect plant growth or flower formation The BcPME37c mutant was obtained through the CRISPR/Cas9 system, as reported in a previous study. The vegetative growth of bcpme37c showed no remarkable differences compared with that of the control plants (Supplementary Figs. 1G and 1H). Given that BcPME37c was mainly expressed in the mature pollen stage, we focused on whether the mutant exhibits differences in the reproductive growth process from the control plants. The morphology and size of the open flowers of bcpme37c were not significantly different from those of the control plants (Supplementary Figs. 1A and 1B). Further observation of the shape and size of the sepals, petals, stamens, and pistils showed no evident abnormalities (Supplementary Figs. 1Ce1F).
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Fig. 1. qRT-PCR analysis of BcPME37c expression patterns. R, Ste, L, Inf, and Si represent root, stem, leaf, inflorence, and silique, respectively. B1eB5 indicates the buds at flower stage I to stage V, respectively. Se, Pe, Sta, and Gy correspond to the sepal, petal, stamen, and gynoecium, respectively; pollinated, pollinated pistil; unpollinated, unpollinated pistil; 1, 3, 10, and 24 h represent the number of hours after pollination.
Fig. 2. Subcellular localization analysis of BcPME37c-encoded protein. A, B, C, and D are the pFGC-eGFP empty vector; and E, F, G, and H are BcPME37c:eGFP. A, B, E, and F are GFP fields of view; and C, D, G, and H are bright fields. The tablets of A, C, E, and G are made from distilled water; and B, D, F, and H are cells under plasmolysis. The bars are 100 mm.
3.3. Diameter of a portion of bcpme37c pollen evidently increased after imbibition To determine whether BcPME37c mutation shows any phenotype in pollen formation, we performed Alexander staining and SEM observation. The results of Alexander staining showed that the pollen viability of bcpme37c was not notably different from that of the control plants, but the pollen diameter of a small part of bcpme37c pollen remarkably increased (Fig. 3A, B, E, and F). After
imbibition, the pollen diameter of 99.24% of the control plants was not more than 30 mm, whereas only 94.64% of the pollen diameter of bcpme37c pollen was in this interval. The percentage of pollen of the control plants with a diameter larger than 30 mm was only 0.76%, whereas that of bcpme37c was 5.36% (Fig. 3A, B, E, and F and Supplementary Fig. 2A). SEM examination results demonstrated that approximately 5.73% of bcpme37c pollen showed malformation compared with 0.65% of the control pollen (Fig. 3C, D, G, and H and Supplementary Fig. 2B).
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Fig. 3. Cytological staining and electron microscope scanning analysis of bcpme37c and the control plants. A, B, E, and F are Alexander staining results; C, D, G, and H are scanning electron micrographs; I, J, M, and N are aniline blue staining diagrams; and K, L, O, and P are DAPI staining results. AeD and IeL are the pollen samples from the control plants; EeH and M P are bcpme37c pollen. J, L, N, and P are the bright-field images of I, K, M, and O, respectively. The bars are 50 (A, E, IeP), 25 (B and F), 100 (C and G), and 30 mm (D and H). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.4. Intine of bcpme37c mature pollen showed abnormal thickening To further clarify the reason for the increased diameter and malformed pollen formation after pollen imbibition in bcpme37c, we observed the pollen of the mutant and control plants by DAPI staining, aniline blue staining, semi-thin section, and TEM. DAPI and aniline blue staining results showed that the pollen nuclei formation and callose degradation of bcpme37c were normal compared with those of control plants (Fig. 3IeP). The results of semi-thin section showed that the pollen of bcpme37c was normal at all five pollen development stages compared with that of the control plants (Supplementary Fig. 3). TEM examination results showed no significant difference between bcpme37c and the control plants in the pollen mother cell, tetrad, and uninucleate pollen stages (figures of pollen mother cell and tetrad stages are not listed; Fig. 4). Sections in the binucleate and trinucleate pollen stages showed the abnormal thickening of the intine of bcpme37c pollen, but the tectum and bacula of the pollen exine and pollen coat were normal (Fig. 4H, I, K, and L). The above results showed that the abnormal thickening of the intine of bcpme37c pollen occurred after the uninucleate pollen stage.
3.5. In vitro pollen germination and the number of seeds per silique of bcpme37c were normal To further explore the roles of BcPME37c in pollen germination, we conducted in vitro pollen germination. The results showed that the percentages of germinated and ungerminated pollen and no viability pollen from bcpme37c and the control plants were almost the same (Supplementary Fig. 4). These data suggested that the abnormal thickening of intine did not affect the germination of bcpme37c pollen. To determine whether BcPME37c mutation affects female gamete fertility, we employed self-cross in the control plants and bcpme37c. The number of seeds per silique of the control plants and bcpme37c was almost the same (data not shown). Thus, BcPME37c mutation did not impair female gamete fertility.
4. Discussion Predecessors labeled mature pollen with John Innes Monoclonal 5 (JIM5) antibody, which specifically binds to low methylated HG; JIM5 exhibits a strong signal in the inner wall, indicating that the degree of HG methylation in the intine of pollen is low [34]. These findings also showed that PME is active in the construction of
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Fig. 4. Cytological staining and electron microscope scanning analysis of bcpme37c and the control plants. AeC and GeI are pollen sections of the control plants; DeF and JeL are the pollen sections of bcpme37c. Pollen in the first column (A, D, G, and J) are at the mononucleate pollen stage; pollen in the second column (B, E, H, and K) are at the binucleate pollen stage; and pollen in the third column (C, F, I, and L) are at the trinucleate pollen stage. AeF show the cut surface of whole pollen, and GeL are the enlarged view of the pollen wall. Arrows in H, I, K, and L point to the intine. The bars are 5 (AeF) and 0.5 mm (GeL).
pollen intine. Proteome analysis indicated that at least PME37, PPME1, and PME48 in Arabidopsis mature pollen had high PME contents [35]. PME genes are involved in pollen development and pollen germination [13e19]. However, the relationship between the PME gene and pollen intine development is rarely studied. For example, PME48 mutation and BcMF23a silencing impair pollen germination and pollen tube growth [7,15]. Our study further proved that the PME gene was involved in pollen intine formation. Pectin deesterification catalyzed by PMEs can free carboxyl groups on pectin chains, thereby loosening or strengthening the cell wall [1]. PME activity can modulate the degree of methylesterification of HG at pollen intine and affect the balance of plasticity and mechanical strength of intine. In pme48 mature pollen, the degree of methyl esterification of HG was nearly 20% higher than that of the wild type, indicating that PME activity in intine
decreased after PME48 mutation; thus, the degree of methyl esterification of HG increased and eventually caused abnormal pollen germination [7]. In bcpme37c pollen, approximately 5.36% showed larger size than the control plant pollen after imbibition. The results of SEM analysis also showed that approximately 5.73% of the bcpme37c pollen were deformed. TEM analysis results indicated that the intine of bcpme37c pollen were thick at the binucleate and trinucleate pollen stages. The abnormal thickening of the intine of the mature pollen of bcpme37c was possibly caused by the abnormal degree of methyl esterification of HG. However, to verify this speculation, we must study the phenotype of bcpme37c pollen in detail. AtPME37 is highly homologous to VGD1 and exhibits the same expression profile as VGD1 but fails to complement the vgd1 mutant [25]. These characteristics indicate that AtPME37 and VGD1 exhibit
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different biological functions [25]. BcPME37c is the syntenic gene of AtPME37, and their expression patterns are similar. These two genes may function similarly, but numerous studies must be conducted before reaching this conclusion. Aside from BcPME37c, BcPME37a and BcPME37b are also the syntenic genes of AtPME37 (http:// brassicadb.org/brad/index.php). More than 20 PME genes showed specific expression at mature pollen stage of the flower in B. campestris (unpublished data). The phenotypes of bcpme37c were not obvious due to gene function redundancy, but the relationship between these pollen development-related PME genes must be further studied. In summary, BcPME37c is a stamen-specific gene, which encodes a secreted protein. BcPME37c mutation causes abnormal pollen intine formation in B. campestris. Our work suggested that BcPME37c is involved in pollen intine development in B. campestris. Conflicts of interest All authors declare that they have no conflict of interest. Ethical approval Research did not involve human participants and/or animals. Acknowledgments This research was supported by grants from the National Natural Science Foundation of China (No. 31471877 to J. C.).The authors are grateful to all the members of the Cao lab for technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.009. References [1] T.D. Quilichini, E. Grienenberger, C.J. Douglas, The biosynthesis, composition and assembly of the outer pollen wall: a tough case to crack, Phytochemistry 113 (2015) 170e182. https://doi.org/10.1016/j.phytochem.2014.05.002. [2] J. Shi, M. Cui, L. Yang, Y.J. Kim, D. Zhang, Genetic and biochemical mechanisms of pollen wall development, Trends Plant Sci. 20 (2015) 741e753. https://doi. org/10.1016/j.tplants.2015.07.010. [3] Z.A. Wilson, D.B. Zhang, From Arabidopsis to rice: pathways in pollen development, J. Exp. Bot. 60 (2009) 1479e1492. https://doi.org/10.1093/jxb/ erp095. [4] U.M. Hesse, D. Halbritter, U.M. Weber, D.B.R. Buchner, A. Frosch-Radivo, M.S. Ulrich, Pollen Terminology, Springer, Vienna, 2009. https://doi.org/10. 1007/978-3-211-79894-2. [5] J. Li, M. Yu, L.L. Geng, J. Zhao, The fasciclin-like arabinogalactan protein gene, FLA3, is involved in microspore development of Arabidopsis, Plant J. 64 (2010) 482e497. https://doi.org/10.1111/j.1365-313X.2010.04344.x. [6] J.A. Schnurr, K.K. Storey, H.J. Jung, D.A. Somers, J.W. Gronwald, UDP-sugar pyrophosphorylase is essential for pollen development in Arabidopsis, Planta 224 (2006) 520e532. https://doi.org/10.1007/s00425-006-0240-1. [7] C. Leroux, S. Bouton, M.C. Kiefer-Meyer, T.N. Fabrice, A. Mareck, S. Guenin, F. Fournet, C. Ringli, J. Pelloux, A. Driouich, P. Lerouge, A. Lehner, J.C. Mollet, PECTIN METHYLESTERASE48 is involved in Arabidopsis pollen grain germination, Plant Physiol. 167 (2015) 367e380. https://doi.org/10.1104/pp.114. 250928. [8] S. Moon, S. Kim, G. Zhao, J. Yi, Y. Yoo, P. Jin, S. Lee, K. Jung, D. Zhang, G. An, Rice GLYCOSYLTRANSFERASE1 encodes a glycosyltransferase essential for pollen wall formation, Plant Physiol. 161 (2013) 663e675. https://doi.org/10.1104/ pp.112.210948. [9] K. Ueda, F. Yoshimura, A. Miyao, H. Hirochika, K. Nonomura, H. Wabiko, COLLAPSED ABNORMAL POLLEN1 gene encoding the arabinokinase-like protein is involved in pollen development in rice, Plant Physiol. 162 (2013) 858e871. https://doi.org/10.1104/pp.113.216523. [10] J. Jiang, L. Yao, Y. Yu, Y. Liang, J. Jiang, N. Ye, Y. Miao, J. Cao, PECTATE LYASE-LIKE 9 from Brassica campestris is associated with intine formation, Plant Sci. 229 (2014) 66e75. https://doi.org/10.1016/j.plantsci.2014.08.008. [11] J. Jiang, L. Yao, Y. Yu, M. Lv, Y. Miao, J. Cao, PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris, J. Integr. Plant Biol. 56
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