COPII components Sar1b and Sar1c play distinct yet interchangeable roles in pollen development

COPII components Sar1b and Sar1c play distinct yet interchangeable roles in pollen 1 development 2 Xin Liang, Shan-Wei Li, Li-Min Gong, Sha Li, Yan Zhang* 3 State Key laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural 4 University, Tai’an, 271018, China 5 1 Contributed equally 6 * To whom correspondence should be addressed (email: yzhang@sdau.edu.cn) 7 8 Research area: Cell biology 9 Short title: Sar1 isoforms mediate pollen development 10 11 For correspondence: 12 Yan Zhang, 13 State Key laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural 14 University, Tai’an, 271018, China 15 Tel: (86)538-8246365 16 Email: yzhang@sdau.edu.cn 17 18 Author contributions 19 X. L. performed most of the experiments with the assistance of S.-W.L., L.-M.G.; Y.Z. 20 initiated and supervised the project together with S.L.; Y.Z. and S.L. secured the 21 funding; X.L. and Y.Z. analyzed the data; Y.Z. wrote the article with contributions from 22 all authors. 23 24 Footnotes: 25 This work was supported by Natural Science Foundation of China (31625003, 26 31471304, and 31871422 to Y.Z.; 3190020286 to S.-W. L.; 31970332 and 31771558 to 27 S.L.). Y.Z.’s laboratory is partially supported by Tai-Shan Scholar Program by Shandong 28 Provincial Government. 29 Plant Physiology Preview. Published on April 23, 2020, as DOI:10.1104/pp.20.00159


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Functional loss of Sar1b but not Sar1a and Sar1c results in male sterility 126 To examine the function of the three Sar1 isoforms in plant development, we took a 127 reverse genetic approach. We isolated T-DNA insertion mutants of Sar1a and Sar1c, 128 i.e. sar1a and sar1c (Supplemental Figure 1). Both mutants are null mutants based on 129 transcript analysis. Single mutants or the sar1a;sar1c double mutant grew 130 comparably to wild type in both vegetative and reproductive stages (Supplemental 131 Figure 1). Because there were no valid T-DNA insertion mutants of Sar1b, we 132 generated sar1b mutants by CRISPR/Cas9 ( Figure 1A). Two mutant alleles were 133 identified from transformants, sar1b-1 and sar1b-2, both of which expressed 134 mutated Sar1b transcripts with pre-stop codons resulted either from an insertion or 135 from a deletion in the Sar1b coding region ( Figure 1A). Vegetative growth of both 136 sar1b mutants was comparable to that of wild type (Supplemental Figure 2).

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However, siliques of the homozygous sar1b mutants did not elongate ( Figure 1C To determine what caused the male sterility of sar1b mutants, we performed 148 sectioning and light microscopy of developing anthers ( Figure 2). The specification of 149 anthers was comparable between wild type and sar1b such that both contain 150 epidermis, endothecium, middle layer, tapetum, and microspore mother cells ( Figure   151 2A). At anther developmental stage 6-7, i.e. when tetrads are released in pollen sac 152 (Sanders et al., 1999), sar1b started to differ from wild type (Figure 2A  However, starting from stage 7 when tetrads were formed normally in wild type 167 ( Figure 3C), tetrads in sar1b anthers seemed detached from the callose wall that 168 encased them ( Figure 3D). Later on, unicellular microspores with reticular pollen coat 169 structures, presumably sporopollenins, were developed in wild type ( Figure 3E). By 170 contrast, a large amount of electron-dense materials was deposited to the 171 intercellular space between the tapetum and the middle layer in sar1b ( Figure 3F).

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There were also aggregates of electron-dense materials deposited in pollen sacs 173 without attaching to the surface of microspores in sar1b ( Figure 3F). Microspores

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To determine the exact defects occurred in sar1b;sar1c male gametophytes, we 268 performed sectioning and light microscopy of developing sar1b/+;sar1c anthers.

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We report here that Sar1s, and by inference, COPII-mediated ER to Golgi trafficking, 286 are essential for both sporophytic and gametophytic control of pollen development. 287 We show that Sar1b is essential for sporophytic male fertility. It has been widely 288 accepted that sporopollenin components of the pollen exine, electron-dense 289 materials in TEM images, are transported via vesicles before being deposited to 290 developing microspores. AGCG26, whose mutation compromised sporophytic pollen

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The CRISPR construct used to generate the sar1b mutants was as previously  Supplemental Table S1.  Table S1. Oligos used in this study.