Cytological and Transcriptomic Analyses Reveal Important Roles of CLE19 in Pollen Exine Formation.

Plant Physiology Preview. Published on September 15, 2017, as DOI:10.1104/pp.17.00439

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Running title: CLE19 regulates pollen wall formation

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*Corresponding authors: Fang Chang

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Address：School of Life Sciences, Fudan University,

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Shanghai 200438, China

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Tel：086-021-51630534

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Fax：086-021-51630504

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E-mail：[email protected]

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*Corresponding authors: Hong Ma

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Address：School of Life Sciences, Fudan University,

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Shanghai 200438, China

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Tel：086-021-51630532

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Fax：086-021-51630504

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Downloaded from on September 16, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Copyright 2017 by the American Society of Plant Biologists

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Cytological and Transcriptomic Analyses Reveal Important Roles of CLE19 in

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Pollen Exine Formation

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Shuangshuang Wang, Jianan Lu, Xiu-Fen Song, Shi-Chao Ren, Chenjiang You, Jie Xu, Chun-Ming

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Liu, Hong Ma*, Fang Chang*

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State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering and Institute of Biodiversity Sciences, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200438, China (S.W., J.L., C.Y., H.M., F.C.); Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China (X.S., S.R., C.L.); Institute of Crop Science, Chinese Academy of Agricultural Science, Beijing, 100081, China (C.L.); Collaborative Innovation Center for Genetics and Development, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China (J.X.)

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*Address correspondence to [email protected].

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Summary: The proper amount of CLE19 is required for normal formation of pollen exine through

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regulating the expression of AMS and its downstream networks.

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Footnotes:

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This work was supported by grants from the National Natural Science Foundation of China

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(31130006, 31470282, 31670316, and 31370029) and the State Key Laboratory of Genetic

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Engineering at Fudan University.

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S.W., H.M. and F.C. designed the research. S.W. performed the RNA in situ hybridization analysis,

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the phenotypic observation, the real time PCR experiments, the SEM and TEM observations, and

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tissue collection for transcriptome; S-C. R. X-F. S. and C-M. L. generated the DN-CLEs transgenic

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mutants; J.L. and C.Y. together carried out the transcriptome analysis; S.W., H.M. and F.C. analyzed

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the data and wrote the article.

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ABSTRACT：

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The CLAVATA3/ESR-RELATED (CLE) peptide signals are required for cell-cell communication in

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several plant growth and developmental processes. However, little is known regarding possible

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functions of the CLEs in the anther. Here, we show that a T-DNA insertional mutant, and

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dominant-negative (DN) and overexpression (OX) transgenic plants of the CLE19 gene exhibited

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significantly reduced anther size and pollen grain number, and abnormal pollen wall formation.

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Interestingly, the DN-CLE19 pollen grains showed a more extensively covered surface but

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CLE19-OX pollen exine exhibited clearly missing connections in the network and lacked separation

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between areas that normally form the lacunae. With a combination of cell biological, genetic, and

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transcriptomic analyses on cle19, DN-CLE19, and CLE19-OX plants, we demonstrated that

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CLE19-OX plants produced highly vacuolated and swollen ams-like tapetal cells, lacked lipidic

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tapetosomes and elaioplasts, and had abnormal pollen primexine without obvious accumulation of

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sporopollenin precursors. Moreover, CLE19 is important for the normal expression of > 1,000 genes,

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including transcription factor gene AMS, 280 AMS-downstream genes, and other genes involved in

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pollen coat and pollen exine formation, lipid metabolism, pollen germination, and hormone

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metabolism. In addition, the DN-CLE19(+/+) ams(-/-) plants exhibited the ams anther phenotype and

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ams(+/-) partially suppressed the DN-CLE19 transgene-induced pollen exine defects. These findings

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demonstrate that the proper amount of CLE19 signal is essential for the normal expression of AMS

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and its downstream gene networks in the regulation of anther development and pollen exine

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formation.

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Key words: anther development, CLE19, pollen exine formation, transcriptome, Arabidopsis

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thaliana

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INTRODUCTION

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Pollen grains are generated in the male reproductive organ anther of flowering plants and are

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essential for plant fertility. In the model plant Arabidopsis thaliana, pollen grains are ellipsoidal, and

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the pollen surface is covered by a reticulate exine. In contrast, Oryza sativa pollen grains are globular

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and have a smooth surface without the reticulate structure. A well-organized pollen wall structure is

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essential for the physical and chemical stability of the mature pollen by providing protection for

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pollen grains from environmental stresses, such as desiccation and microbial attacks (Scott et al.,

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2004). The pollen wall is mainly composed of three layers: the intine layer, the exine layer, and the

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pollen coat (Zinkl et al., 1999; Edlund et al., 2004; Blackmore et al., 2007). The intine is the

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innermost layer of the pollen wall immediately adjacent to the plasma membrane of the pollen

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vegetative cell and is mainly composed of pectin, cellulose, hemicellulose, hydrolytic enzymes and

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hydrophobic proteins (Scott et al., 2004). Both the exine layer and the pollen coat layer are basically

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of a lipidic nature. The exine is largely formed from acyl lipid and phenylpropanoid precursors,

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which together form the mixed stable biopolymer known as sporopollenin (Guilford et al., 1988;

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Bedinger et al., 1994; Thom et al., 1998). The pollen exine has many important functions in the

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prevention of dehydration of male gametophytes, the facilitation of pollen dispersal and

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pollen-stigma recognition and adhesion (Zinkl et al., 1999). The pollen coat fills the cavities of the

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pollen exine during the late stages of pollen development, and previous studies have demonstrated

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that developmental defects of pollen wall structure or lack of pollen coat lipids in Arabidopsis lead to

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a failure or delay of pollen hydration and subsequent male sterility (Preuss et al., 1993; Mayfield and

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Preuss, 2000; Wilson and Zhang, 2009; Li and Zhang, 2010).

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Anther development is precisely regulated temporally by transcriptional networks, receptor-like

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protein kinase-mediated signal pathways, and other pathways (Ma, 2005; Ge et al., 2010; Chang et

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al., 2011). The basic helix-loop-helix (bHLH) transcriptional factor DYSFUNCTIONAL

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TAPETUM1 (DYT1) is among the earliest-acting male-specific regulators and is required for the

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normal development of tapetum by regulating the normal expression of more than 1,000 anther genes,

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and dyt1 mutants form abnormal prematurely vacuolated tapetal cells and lack mature pollen (Zhang

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et al., 2006; Feng et al., 2012; Gu et al., 2014; Zhu et al., 2015). In addition, the bHLH010,

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bHLH089, and bHLH091 together are required for the normal tapetum development and 4


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transcriptome and pollen fertility, forming both feed-forward and feed-back loops with DYT1 (Zhu

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et al., 2015; Cui et al., 2016). Another bHLH transcription factor ABORTED MICROSPORES

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(AMS) is a key regulator of anther transcriptome, sporopollenin biosynthesis and secretion and

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pollen wall formation (Sorensen et al., 2003; Xu et al., 2010; Feng et al., 2012; Ma et al., 2012; Xu et

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al., 2014). DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION (TDF1)/MYB35 encodes a

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putative R2R3 MYB transcription factor, and loss of TDF1 function due to mutation or transgene

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causes tapetal hypertrophy extending into the locule and results in sporophytic male sterility (Zhu et

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al., 2008; Feng et al., 2012). MALE STERILITY1 (MS1) is a PHD-finger motif-containing nuclear

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protein and is essential for tapetum development at the post-meiotic phase and regulates genes

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important for exine formation (Wilson et al., 2001; Ito et al., 2007). In addition, another R2R3 MYB

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gene, MS188/MYB103/MYB80, is required for sporopollenin biosynthesis and sexine formation

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(Phan et al., 2011; Xiong et al., 2016). Further molecular genetic and bioinformatics studies revealed

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that these transcription factors likely form a genetic pathway as follows: DYT1, MYB35/TDF1,

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AMS, MS188/MYB103/MYB80, MS1 and MYB99 from upstream to downstream during

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anther/pollen development (Ito et al., 2007; Feng et al., 2012).

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Extracellular ligands and their receptor-like protein kinase (RLK)-mediated signaling pathways play

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important roles in the regulation of anther development. In particular, the RLKs EMS1/EXS and

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SERK1/2 and secreted peptide TPD1 are required for tapetum formation and function (Canales et al.,

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2002; Zhao et al., 2002; Yang et al., 2003; Colcombet et al., 2005; Albrecht et al., 2006). Furthermore,

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the TPD1 protein from microsporocyte precursors/microsporocyte interacts with the LRR domain of

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the RLK EMS1/EXS expressed in the tapetal precursor/tapetal cells to promote the phosphorylation

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of EMS1, thereby regulating anther tapetum development and function (Canales et al., 2002; Zhao et

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al., 2002; Yang et al., 2003; Yang et al., 2005; Jia et al., 2008; Huang et al., 2016; Li et al., 2016). In

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addition, brassinosteroids (BRs) and BR cell surface receptor BRI1 (BRASSINOSTEROID

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INSENSITIVE 1) are required for pollen exine formation and pollen release through the regulation

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of key genes in tapetum and pollen development, e.g., AMS, MYB103, MS1, and MS2 (Ye et al.,

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2010). In addition, several other RLKs are required for normal anther development, but their ligands

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remain unknown. For instance, ERECTA (ER), ER-LIKE 1 (ERL1) and ERL2 are important for

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anther lobe formation and normal cell patterning (Torii et al., 1996; Shpak et al., 2003; Hord et al., 5


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2008). BARELY ANY MERISTEM1 (BAM1) and BAM2 regulate the formation of anther somatic

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cell layers, and the bam1 bam2 double mutant anther forms many pollen mother-like cells but lacks

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the three subepidermal somatic cell layers (Deyoung et al., 2006; Hord et al., 2006).

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RECEPTOR-LIKE PROTEIN KINASE2 (RPK2) is essential for the formation of both the tapetum

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and middle layer (Mizuno et al., 2007). On the other hand, besides TPD1, no other peptide signals

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have been implicated in anther or pollen development.

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CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-RELATED (CLE) genes encode a

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family of putative peptide ligands, with at least 32 CLE members in Arabidopsis (Cock and

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McCormick, 2001). Mature CLE peptide signals are 12 to 14 amino acids long, and many of them

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play important roles in various plant developmental processes. For instance, CLAVATA3 is necessary

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to restrict stem cell numbers in the shoot apical meristem (SAM) (Fletcher et al., 1999) by promoting

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the formation of the CLV1/CLV2 protein complex to inhibit the expression of WUSCHEL (WUS) in

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the organizing center (OC) of the SAM (Laux et al., 1996; Fiers et al., 2004). CLE8 regulates the size

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of embryos and seeds and is expressed in the endosperm and the early embryo to promote WOX8

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expression (Fiume and Fletcher, 2012). CLE40 regulates the activity of root apical meristem (RAM)

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and is expressed in differentiating cells in roots and likely acts through the receptor-like kinase

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CRINKLY4 (ACR4) to restrict the expression and position of WOX5 (Stahl and Simon, 2009). The

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CLE19

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(Casamitjana-Martinez et al., 2003; Fiers et al., 2005; Al-Refu et al., 2009). However, whether the

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CLE genes are required for normal anther development still remains unknown.

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One difficulty in studying the endogenous functions of these small CLE genes is the shortage of

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genetic materials, namely the mutant plants. In addition, previous studies revealed that T-DNA

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insertion mutants of CLE1, CLE7, CLE10, CLE16, CLE18, or CLE19 in Arabidopsis exhibited no

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visible abnormal phenotype (Fiers et al., 2004; Jun et al., 2010), but overexpression of CLE genes 2,

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3, 4, 5, 6, 7, 10, 11, and 13 all resulted in pleiotropic phenotypes similar to that of CLV3 or CLE40

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overexpression plants and in vitro application or overexpression of one of CLE genes 19, 21, 25, 42,

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and 44 caused similar dwarf and short-root phenotypes (Fiers et al., 2004; Fiers et al., 2005; Strabala

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et al., 2006), suggesting a high level of functional redundancy or overlap among CLE members.

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Fortunately, an antagonistic peptide technology (Song et al., 2013) was recently developed as an

peptide

triggers

root

meristem

consumption

in

a

CLV2-dependent

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manner

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effective tool to investigate the endogenous functions of these functionally redundant secreted

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peptides. Using CLV3 as a test case, Song and coworkers examined the antagonistic peptide

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technology by transformations of wild-type Arabidopsis with constructs carrying the full-length

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CLV3 with every residue in the peptide coding region replaced one at a time by alanine, to probe the

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effectiveness of each mutation. They found that the Gly-to-Ala substitution in the core CLE motif

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caused a dominant-negative (DN) clv3-2-like phenotype. Further substitutions of the glycine residue

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individually with the other 18 possible proteinaceous amino acids determined that the

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glycine-to-threonine substitution resulted in the strongest antagonistic effect in the wild type (Song et

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al., 2012; Song et al., 2013). With this strategy, the peptide signal CLE19 was revealed to be

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important for cotyledon establishment in embryos and endosperm development (Xu et al., 2015).

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Here, we tested CLE genes for expression in the anther and show that CLE9, CLE16, CLE17, CLE19,

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CLE41, CLE42 and CLE45 were preferentially expressed in tapetal and reproductive cells in the

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anther. Using the antagonistic peptide technology, we generated dominant negative (DN) mutants for

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these seven genes and our analyses of these mutant transgenic plants indicated that they are

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important for normal pollen development, especially for the development of pollen exine. Using

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CLE19 as a representative, we further characterized its function in the regulation of pollen exine

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formation using a combination of morphological molecular and transcriptomic analyses of cle19,

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DN-CLE19 and CLE19-OX mutants. We finally revealed that CLE19 acts as a negative regulatory

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element in tapetum development, sporopollenin biosynthesis, and exine formation likely through the

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regulation of the transcription factor AMS and downstream genes. Thus, we propose a novel

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signaling pathway starting from an extracellular peptide signal and eventually affecting the

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intracellular transcriptional network that is crucial for the regulation of pollen wall development.

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RESULTS

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Seven CLE Genes Are Preferentially Expressed in Arabidopsis Tapetal and Reproductive Cells

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The expression of CLE genes in Arabidopsis was first examined by searching the public microarray

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database (http://bar.utoronto.ca/~jwaese/EFP/ and http://www.arabidopsis.org/portals/expression/

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microarray/ATGenExpress.jsp) and using quantitative real-time PCR and RNA in situ hybridization 7


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analyses to detect the expression of these CLE genes in the developing anther. Among the fourteen

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CLE genes that were included in the microarray expression databases, CLE9, CLE10, CLE21, CLE26,

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CLE40, CLE44, and CLV3 exhibited relatively high expression levels in the anther, whereas CLE6

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and CLE12 are relatively highly expressed in the mature pollen (Supplemental Fig. S1A).

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Quantitative real-time PCR analyses using Col-0 inflorescence and stage 4 to 12 anthers revealed

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that CLE9, CLE16, CLE17，CLE25, CLE27, CLE41, CLE42, and CLE45 were relatively highly

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expressed in both inflorescence and stages 4 to 12 anthers (Supplemental Fig. S1B), suggesting that

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they might be involved in male reproductive development.

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Further RNA in situ hybridization analyses were performed to investigate the expression patterns of

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these anther-expressed CLE genes. Strong CLE9, CLE16, CLE17, CLE19, CLE41, CLE42, and

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CLE45 signals were detected in the anther at various developmental stages (Fig. 1). For instance,

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CLE9 and CLE16 were both expressed in the tapetum and microspores at anther stages 7 to 10 but

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not expressed in either cell layer at stage 5 (Fig. 1, A-H). CLE19 was preferentially expressed in both

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tapetal and male reproductive cells at anther stages 5 to 7 and CLE17, CLE41, CLE42, and CLE45

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were preferentially expressed in both tapetal and male reproductive cells at anther stages 5 to 10 (Fig.

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1, I-AB), suggesting their involvement in regulating tapetum function and pollen development. In

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addition, their highly similar spatial-temporal expression pattern also suggested that these seven CLE

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genes possibly participate in similar anther developmental processes.

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Dominant-Negative (DN) Mutant Transgenic Plants of CLE9, CLE16, CLE17, CLE19, CLE41,

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CLE42, and CLE45 Exhibited Abnormal Anther Development

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To further investigate the functions of the CLE genes in anther development, the T-DNA insertional

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mutants of CLE9, CLE17, CLE19, or CLE42 and the RNAi mutant of CLE16 were obtained

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(Supplemental Fig. S2), and the transcript level of these genes in mutant plants were analyzed using

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real-time quantitative PCR. The cle9-1, cle16-1, cle17-1, cle19-1 (in qrt1-2 background), cle19-2,

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and cle42-1 mutants exhibited drastically reduced expression of the relevant genes (Supplemental

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Fig. S2) and were further examined phenotypically. Both cle19-1 qrt1-2 and cle19-2 produced

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abnormally short silique, and had slightly decreased anther size and some aborted pollen grains (Fig.

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2, A-E, J-N, S-W, AB-AF). Considering the cle19-1 was in a qrt1-2 background, we observed the 8


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phenotype of silique, flower, anther, and pollen in the qrt1-2 plants and did not find any defects

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except for the pollen phenotype (Fig. 2, C, L, U, and AD). The above results suggested that CLE19 is

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possibly involved in anther development. In contrast, the cle9-1, cle16-1, cle17-1, and cle42-1 single

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mutants all exhibited normal plant growth, and flower and anther development (Supplemental Fig.

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S3, and Supplemental Table 1).

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Based on the high amino acid similarity between the functional domain of these Arabidopsis CLE

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members (Ni and Clark, 2006) and no/weak defects in anther development observed in the available

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cle single mutants, we proposed possible functional redundancy among these anther-expressed CLE

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genes in the regulation of anther development. Therefore, we generated the double, triple mutants of

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these CLE genes by crossing the cle16-1, cle17-1, cle19-1, and cle42-1. The qrt1-2 mutation in

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cle19-1 plants segregated away during this process. Compared to the cle19-1 mutant, our phenotypic

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and statistic analyses showed that the cle16 cle19, cle17 cle19, and cle19 cle42 double mutants had

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fewer pollen grains in the anther (Supplemental Fig. S4, A-AA; Supplemental Table 1). Moreover,

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the cle17 cle19 cle42 triple mutant showed more barren siliques, less pollen amount, and a larger

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fraction of defective pollen in the anther (Supplemental Fig. S4, A-AA; Supplemental Table 1). In

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addition, we generated DN mutant plants for CLE9, CLE16, CLE17, CLE19, CLE41, CLE42, and

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CLE45 by transforming wild-type (WT) plants separately with fusions the CLE promoter and the

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coding region for the CLEG6T point mutation (DN-CLE). The DN-CLE transgenic plants exhibited

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obviously reduced silique length (Fig. 2, F and G; Supplemental Fig. S5, B-G). Alexander staining

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and scanning electron microscope (SEM) experiments revealed abnormal anther development with

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aborted pollen in the anthers of these DN-CLEs plants (Fig. 2, X and Y, AG and AH; Supplemental

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Fig. S5, H-U). These results suggested that the normal functions of these genes are important for

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anther development and that the anther-expressed CLE9, CLE16, CLE17, CLE19, CLE41, and

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CLE42 might function in a redundant manner. Although the DN transgenic plants of all 7 CLE genes

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exhibited abnormal anther development, only the cle19 KO mutants showed weak anther

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developmental defects; therefore, we focused the subsequent detailed functional analyses on CLE19,

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to gain insights into its role in anther/pollen development.

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9


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CLE19 Is Required for Normal Pollen Development, Pollen Germination, and Pollen Tube

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Elongation

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In addition to the above mentioned cle19 and DN-CLE19 mutants, we also wanted to investigate

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whether an increase of CLE19 expression affects the male reproductive development, using the 35S

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promoter-driven CLE19 overexpression transgenic lines (CLE19-OX) (Fiers et al., 2004). Two

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CLE19-OX transgenic lines with different CLE19 expression level, CLE19-OX-S and CLE19-OX-M

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(Figure 3D), were selected for analysis of anther developmental phenotypes. Both lines exhibited

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obviously reduced fertility, including aborted siliques, small anthers, and abnormal pollens (Fig. 2, H

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and I, Q and R, Z and AA, and AI and AJ).

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As detected using real-time PCR, the expression level of CLE19 was found to be elevated up to over

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100 fold in the CLE19-OX-S line (Fig. 3D), and to a lesser extent in DN-CLE19 anthers (Fig. 3C).

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The anther expression pattern of CLE19 in the transgenic plants was further examined using RNA in

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situ hybridization analysis (Fig. 3, E-X). Consistently, the expression of CLE19 was obviously

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increased in CLE19-OX-S and decreased in cle19-2 in both tapetal and male reproductive cells (Fig.

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3, J-S). However, the expression in DN-CLE19 anthers showed no obviously difference from that in

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the wild type (Fig. 3, T-X), suggesting that the 35S promoter is not highly active in the tapetum and

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in situ hybridization might not be quantitative enough to detect relatively small difference in

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expression level.

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We further performed statistical analysis for various male fertility processes of the cle19-2,

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DN-CLE19-23, and CLE19-OX-S plants, including the pollen number in each anther, pollen

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germination and pollen tube elongation, silique length and silique number in each main stem, and

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seed number in each silique. Compared with the WT, DN-CLE19-23 produced obviously fewer

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pollen grains (48.4% of that in each WT anther), a reduced pollen germination rate (15.7% of the rate

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for WT), defective pollen tube elongation, a decreased number of siliques from one reproductive

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shoot, reduced silique length, and a lower seed number in each silique (Table 1). The cle19-1 and

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cle19-2 plants exhibited similar but weaker phenotypes compared with those of DN-CLE19,

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suggesting a functional redundancy among the anther-expressed CLE family members, as other

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members might also be inhibited by the mutated CLE peptide. CLE19-OX-S anther produced only 9%

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of the amount of pollen in one WT anther (Table 1). All these results demonstrated the important 10


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function of CLE19 in male reproduction processes and that the correct level of CLE19 expression is

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important for normal function.

280

To further investigate the function of CLE19 in anther development, particularly to identify the

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earliest stages when morphological differences can be observed, anther transverse semi-thin sections

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of cle19-2, DN-CLE19-23, and CLE19-OX mutants were examined. At anther stage 5, the WT anther

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formed well-organized 5-cell layers in each anther lobe, the epidermis, endothecium, middle layer,

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tapetum, and the microsporocytes (Ms) from outer to inner. Afterward, the microsporocytes

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underwent meiosis and formed the microspores that further developed into the pollen in the WT at

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stages 6 to 13.

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Compared with the WT (Fig. 4, A-E), the cle19-2 mutant anther showed no obvious cytological

288

defects during anther stage 5-10 except for fewer pollen grains in the anther at stage 12 (Fig. 4, F-J).

289

However, the DN-CLE19-23 anthers tended to produce more than normal tapetum cells surrounding

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the Ms at stages 5, and aborted pollens were observed at stage 10 and onward (Fig. 4, K-O). In

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comparison, change of the tapetal cell number was not obvious in either CLE19-OX-S or

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CLE19-OX-M anthers; nevertheless, most of the tapetal cells were enlarged and excessively

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vacuolated at stages 5 to 7, and the CLE19-OX-S tapetum soon degenerated at stage 8 (Fig. 4, P-R).

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Therefore, the development of most pollen grains was defective (Fig. 4, S and T). The CLE19-OX-M

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tapetum exhibited a weaker vacuolated morphology compared with CLE19-OX-S but also

296

disappeared at stage 10. A small portion of defective pollens was observed at stage 10 and onward

297

(Fig. 4, U-Y). These results further strengthened the idea that a proper amount of CLE19 is required

298

for normal anther development.

299 300

Pollen Exine Formation Was Affected in CLE19 Mutants

301

We then further investigated whether CLE19 is involved in pollen exine formation. The

302

morphological pollen exine structure of the WT, DN-CLE19-23, and CLE19-OX-S plants were first

303

observed using scanning electron microscopy (SEM). Pollen grains with either moderate or severe

304

defects were analyzed, respectively, in both DN-CLE19-23 and CLE19-OX-S plants. Results showed

305

that the external surface of WT pollen exine had a network-like structure formed with well-organized 11


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lacunae and three narrow apertures (Fig. 5, A and F). In comparison, both the moderately defective

307

(DN-CLE19-moderate) and the severely defective pollen (DN-CLE19-severe) of DN-CLE19-23

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exhibited a more extensively covered surface with smaller lacunae filled with additional materials

309

(Fig. 5, B and C, G and H). In contrast, the CLE19-OX pollen exine exhibited clearly missing

310

connections in the network and lack of separation between what spaces that would form the lacunae

311

(Fig. 5, D and E, I and J), suggesting that the proper amount of CLE19 is required for the formation

312

of normal pollen exine, possibly promoting the proper organization of the network and lacunae.

313

Given that the pollen wall is composed of the exine and the intine layers, we further examined the

314

characteristics of these two pollen wall layers through a diethyloxadicarbocyanine iodide (DiOC2)

315

and Tinopal-based staining method. As shown in Fig. 5K, the WT pollen exine was stained as red,

316

and the intine was stained as purple. In comparison, stronger red exine signals but minimal purple

317

intine signals were observed in the DN-CLE19-23 pollen grains (Fig. 5, L and M), especially in the

318

DN-CLE19-severe pollen grain, where little or no intine signal was observed (Fig. 5M), suggesting

319

that CLE19 is required for the normal formation of both pollen exine and intine. Consistently, the

320

CLE19-OX pollen exhibited discrete red exine signals and irregular inward-folded purple intine

321

fluorescence (Fig. 5, N and O). These data suggested that CLE19 possibly negatively and positively

322

regulates pollen exine and intine formation, respectively.

323

To further analyze the function of CLE19 in pollen exine formation, transmission electron

324

microscope (TEM) analyses were performed. WT pollen grains exhibited a uniformly structured cell

325

wall with clearly visible exine (T-shaped bacula and tectum) (Fig. 5P, U). However, although the

326

exine T-shape structure of the DN-CLE19-23 pollens appeared normal, the lacunae were filled with

327

extra sporopollen-like materials (Fig. 5, Q and R, V and W). In contrast, the CLE19-OX exine-like

328

layer lacked the complete bacula/tectum structure (Fig. 5, S and T, X and Y), demonstrating that

329

CLE19 is a negative regulator of pollen exine biogenesis, especially for the construct of the T-shape

330

structure.

331 332

DN-CLE19 Transgenic Plant Is Deficient in Pollen Coat Formation

333

As the DN-CLE19 mutant pollen grains exhibited germination defects (Table 1) and the pollen coat 12


334

was filled with extra unknown substances, we assessed whether these substances result from

335

additional synthesis or defective degradation/exploitation. Thus the pollen coat structures of WT and

336

DN-CLE19 at anther stages 10 to 13 were examined. Both wild-type and DN-CLE19 pollen in stage

337

10 to 11 anthers were covered with pollen coat materials, which filled the reticulate networks that

338

surrounded the wild-type pollen; such materials were reduced and were almost invisible in stage 12

339

to 13 WT pollen; therefore, the well-organized reticulate exine networks were clearly visible on the

340

surface of the wild-type pollen. However, these pollen coat materials were still present on

341

DN-CLE19 mutant pollen at anther stages 12 to 13 (Supplemental Fig. 6C), suggesting the defect

342

was in the degradation or removal of these materials.

343 344

Expression of AMS and Downstream TF Genes Was Affected in cle19 Mutants

345

As the transcription factor genes DYT1, MYB35/TDF, AMS, MS188/MYB103/MYB80, and MS1 are

346

required for normal anther development in a DYT1-MYB35/TDF1-AMS-MS188/MYB103/MYB80-

347

MS1 regulatory pathway (Ge et al., 2010; Chang et al., 2011; Zhu et al., 2011) and AMS was

348

reported to act as a master regulator of coordinating pollen wall development (Xu et al., 2014), we

349

further examined the expression levels of genes encoding these five transcription factors in

350

DN-CLE19 and CLE19-OX anthers using quantitative real-time PCR analyses. The expression levels

351

of AMS, MS188/MYB103/MYB80, and MS1 showed over 2-fold increase in DN-CLE19 but over

352

2-fold decrease in CLE19-OX anthers (Fig. 6A-B), suggesting these genes are negatively regulated

353

by CLE19. However, the expression of either DYT1 or MYB35 exhibited no obvious difference

354

between the DN-CLE19 and WT anthers, but both showed 40% reduction in CLE19-OX anthers (Fig.

355

6, A and B), suggesting expression of these two genes possibly was not or only slightly affected by

356

CLE19. These results suggested that CLE19 probably functions as a negative regulator of the

357

AMS-dependent transcriptional networks for normal pollen wall formation.

358

The expression of AMS in DN-CLE19 and CLE19-OX anthers was further tested using RNA in situ

359

hybridization analysis. The results showed that the AMS signal was hardly seen at anther stage 5 (Fig.

360

6C-D); and was detected in both the tapetum and microsporocytes at stage 7-10, albeit weakly

361

(Supplemental Fig. S7, B-C); the signal then disappeared at anther stage 12 (Supplemental Fig. S7,

362

B-C). In comparison, AMS expression of was obviously increased in the DN-CLE19 transgenic 13


363

anthers at anther stage 7-10 (Fig. 6E-F), suggesting inhibition of the CLE19 function caused an

364

increase in AMS expression. However, no AMS signal was detected in CLE19-OX transgenic anthers

365

at all examined stages, similar to the results in the ams mutant (Supplemental Fig. S7, G-P),

366

revealing that the increased CLE19 function suppresses AMS expression. These data demonstrated

367

that CLE19 acts as a negative upstream regulator of AMS.

368 369

CLE19-OX Anthers Had ams-Like Swollen Tapetal Cells

370

It is known that tapetal cells secrete sporopollenin precursors onto the primexine of the developing

371

pollen for the formation of the pollen exine and AMS plays an important role in this process with

372

ams showing severely swollen tapetal cells with large vacuoles and few lipidic tapetosomes and

373

elaioplasts (Xu et al., 2014). We postulated that, if CLE19 acts as a negative upstream regulator of

374

AMS, tapetal cells of the CLE19-OX anther would exhibit similar abnormal phenotypes to that of the

375

ams. Therefore, we performed transmission electron microscope (TEM) analysis of CLE19-OX

376

tapetal cells. As expected, in contrast to the condensed and degenerated cytoplasm of wild-type

377

tapetal cells with evident disintegration of cell membrane and accumulation of lipidic tapetosomes

378

and elaioplasts (Fig. 6G), CLE19-OX tapetal cells were highly vacuolated and swollen, with large

379

vacuoles and lack of lipidic tapetosomes and elaioplasts (Fig. 6H). In addition, sporopollenin

380

precursors on the primexine were clearly observed in the outer surface of WT pollen grains; however,

381

no obvious accumulation of sporopollenin precursors was seen on the primexine of the CLE19-OX

382

(Fig. 6I to J) and ams pollen grains (Fig. 6K to L).

383 384 385

Altered Expression of 1,711 and 2,188 Genes in DN-CLE19 and CLE19-OX Anthers, Respectively

386

To further investigate the CLE19 effects on anther transcriptomes, those of stage 4 to 10 anthers were

387

assessed (Supplemental Fig. S8, A and B). Compared with the WT, 578 (34% of a total of 1,711

388

differentially expressed genes) were down-regulated and 1,133 (66%) were up-regulated in the

389

DN-CLE19 anthers (Fig. 7, A and B; Supplemental dataset 1). Moreover, the expression of 1,809

390

genes was down-regulated and 379 up-regulated (83% and 17% of the 2,188 total altered genes,

391

respectively) in the CLE19-OX anthers (Fig. 7, A and B; Supplemental dataset 2). The fact that most 14


392

of the differentially expressed genes were up-regulated in the loss-of-function DN-CLE19 mutant

393

and down-regulated in the gain-of-function CLE19-OX mutant suggests that CLE19 negatively

394

regulates anther gene expression. Interestingly, the expression of 742 genes was both up-regulated in

395

DN-CLE19 and down-regulated in CLE19-OX (Fig. 7C; Supplemental dataset 3), while only 52

396

genes were both down-regulated in DN-CLE19 and up-regulated in CLE19-OX transcriptomic data

397

(Fig. 7D; Supplemental dataset 4), further supporting CLE19 functioning as a negative regulator of

398

many anther genes.

399 400

Expression of 280 AMS-Downstream Genes Was Affected in Both DN-CLE 19 And CLE19-OX

401

Mutants

402

Considering the similar developmental defect of tapetum of CLE19-OX and ams mutant, we further

403

compared the 923 genes that were commonly regulated in DN-CLE19 and CLE19-OX in our

404

transcription datasets to the microarray-based transcriptomic data of the ams stage 4-7 anther and

405

RNA-seq-based transcriptomic data of ams buds (Xu et al., 2010; Feng et al., 2012; Ma et al., 2012).

406

The results revealed that the expression of 135 out of the 923 CLE19 downstream genes were also

407

altered in the ams stage 4-7 anther and 201 were also altered in various stages of ams buds (Fig. 6E;

408

Supplemental dataset 5). The combined set included a total of 280 genes commonly affected by both

409

CLE19 and AMS; the putative functions of these genes are enriched in various biological processes,

410

including pollen exine formation, sexual reproduction, lipid transport, lipid storage, and lipid

411

metabolism (Fig. 7F), suggesting that CLE19 likely functions through AMS-MYB103/MYB80-MS1

412

pathways, but probably not through the more upstream regulators DYT1 and/or MYB35 to regulate

413

the transcriptional networks important for pollen exine formation (Fig. 7G).

414 415

CLE19 Controls the Synthesis of Lipidic and Phenolic Components and Flavonoids Required

416

for Pollen Wall Formation

417

Sporopollenins are the major components of pollen exine. Increasing evidence indicates that

418

sporopollenin mainly consists of complex aliphatic monomers, including very-long chain fatty acids

419

(VLCFAs) and their polyhydroxylated derivatives as well as phenolic compounds (Kim and Douglas, 15


420

2013). Lipid metabolism and transport genes are highly expressed in the pollen compared with

421

vegetative tissues, suggesting important roles of lipid metabolism in pollen development (Honys and

422

Twell, 2004). Among the differentially expressed genes, many are involved in the pollen coat and

423

exine formation, as determined by previous studies (Wilson and Zhang, 2009; Ariizumi and

424

Toriyama, 2011), contributing to the synthesis of pollen coat precursors and sporopollenin. It is

425

generally understood that sterol esters and saturated acyl groups are the major components of pollen

426

coat, similar to those of lipid or oil droplet but different from lipids of biological membranes.

427

As developmental defects were observed in both tapetal cells and pollen wall in CLE19 mutants, we

428

analyzed the expression of genes implicated in lipid acyl metabolism, including the metabolism of

429

flavonoids, phenolics, free fatty acids, wax ester and cutin (Fig. 8A), and found that 36 lipid acyl

430

metabolism-related genes showed more that 2-fold difference in expression between DN-CLE19

431

and/or CLE19-OX mutant anther. Among these, 20 genes were also altered in expression in ams

432

anthers and/or floral buds (Ma et al., 2012; Xu et al., 2014). Importantly, 19 out of the 20

433

co-regulated genes showed changes in expression with opposite direction: up-regulated in

434

CLE19-DN mutant but down-regulated in CLE19-OX and ams mutant (Fig. 8B-F). These results

435

suggest that CLE19 mediates tapetum and pollen wall developmental signaling pathway by

436

negatively regulating the expression of these genes, with at least some dependence on AMS function.

437 438

ams Mutation Partially Suppressed DN-CLE19 Induced Abnormal Anther Development and

439

Pollen Exine Formation

440

To test the hypothesis that CLE19 functions with at least some dependence on AMS function, we

441

generated the DN-CLE19(+/+) ams(+/-) and DN-CLE19(+/+) ams(-/-) plants by crossing the

442

DN-CLE19-23 homozygous plant (DN-CLE19-23+/+) with the ams mutant and observed the pollen

443

exine structure from each genotype. Compared with the WT anther containing pollen with

444

well-organized pollen exine structure (Fig. 9A and F), ams produced smaller anther without any

445

pollen (Fig. 9B), and DN-CLE19-23 plant produced slightly smaller anther, fewer pollen grains, with

446

some pollen grains being aborted (Fig. 9C, G-I). Interestingly, the DN-CLE19(+/+) ams(+/-) anthers

447

were even smaller that the DN-CLE19 anthers, with few pollen grains (Fig. 9D); the DN-CLE19(+/+)

448

ams(-/-) anthers were similar to the ams anthers (Fig. 9E). 16


449

To further investigate the genetic interaction between CLE19 and AMS, we then compared the pollen

450

exine structure between pollen grains from the DN-CLE19 and DN-CLE19(+/+) ams(+/-) plants. As

451

both the DN-CLE19 and DN-CLE19(+/+) ams(+/-) plants produced pollen grains with pollen exine

452

defects with different degrees of severity, we classified the defects into three levels: the slight (Fig.

453

9G and J), moderate (Fig. 9H and K), and severe (Fig. 9I and L) defects. Statistic analysis showed

454

that, after the ams(+/-) genotype was introduced into the DN-CLE19 plants, the percentage of

455

severely defective pollen was obviously reduced (from 32.9% to 22.4%; Fig. 9M) and the percentage

456

of pollen with moderate defects and slight defects were increased (from 26.5% to 35.5% and 40.6%

457

to 42.1%, respectively; Fig. 9M), demonstrating that CLE19 works in a way dependent on the AMS

458

function.

459 460

DISCUSSION

461

A Proper Amount of CLE19 Is Important for Pollen Wall Formation

462

Cell-cell communication is often mediated by the extracellular ligands and their receptors and

463

essential for the development of multicellular organisms, including anther development in

464

angiosperms (Zhao et al., 2002; Yang et al., 2003; Jia et al., 2008; Huang et al., 2016; Yang et al.,

465

2016). During pollen development, the adjacent tapetal layer provides metabolites and sporopollenin

466

precursors for pollen coat and exine formation as well as signals essential for pollen development

467

(Mariani et al., 1990; Goldberg et al., 1993; Sanders et al., 1999). The pollen mother cells surrounded

468

by the tapetum undergo meiosis and form a tetrad enveloped by a thick protective callose

469

(1,3-β-glucan) wall that is subsequently digested by glucanases secreted from the tapetum, resulting

470

in the release of microspores. Then, the microspores develop into mature pollen grains with the

471

deposition of lipidic precursors/components secreted by the tapetal cells onto the pollen surface to

472

form the pollen exine and pollen coats (Mascarenhas, 1975; Stieglitz, 1977; Pacini and Juniper,

473

1979). The signaling communication between tapetal cells and the reproductive cells (including

474

pollen mother cell and developing pollen grains) is important for normal anther development and

475

pollen formation. For instance, Arabidopsis reproductive cell-preferential TPD1 and the

476

tapetum-expressed EMS1 and SERK1/2 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE

477

KINASE1/2) complex interact to regulate the normal tapetum differentiation and pollen development, 17


478

and plants with mutations in TPD1, EMS1, or SERK1/2 produce no microspores (Zhao et al., 2002;

479

Yang et al., 2003; Albrecht et al., 2005; Huang et al., 2016; Li et al., 2016). However, the functions

480

of extracellular ligand and RLK-mediated signaling pathways in the regulation of pollen

481

development are rarely reported.

482

In this study, using combined strategies of cell biology, genetics, and transcriptomics, we found that

483

CLE19 is required for the normal development of pollen, especially for the normal biogenesis of

484

pollen exine. All CLE19 mutant lines exhibited abnormal pollen development with a portion of

485

pollen grains aborted; however, pollen exine structures exhibited differences among mutants with

486

different CLE19 expression levels. Unlike the wild-type pollen grains that are covered by a

487

well-organized network-like lacunae structure, the cle19-2 and DN-CLE19 pollen grains had a more

488

extensively covered surface with broader muri and smaller lacunae units, and most of lacunae units

489

were filled with extra sporopollenin-like substances (Fig. 5). In contrast, the CLE19-OX pollen grains

490

exhibited a shortage of the characteristic reticulate structures composed of bacula and tectum (Fig. 5),

491

suggesting that the lacunae of pollen exine were incomplete. Further transcriptomic analyses

492

revealed that the expression of numerous genes involved in pollen wall assembly, lipid metabolism,

493

transport and localization were up-regulated in DN-CLE19 but down-regulated in CLE19-OX anthers

494

(Fig. 7 and 8), demonstrating that CLE19 functions as a negative regulator of these genes. These data

495

suggested that a proper amount of the CLE19 signal is essential for the normal organization of the

496

pollen wall, and either more or less than normal level of CLE19 would destroy the balance of both

497

expression of pollen developmental genes and construction of normal pollen exine.

498 499

CLE19 Functions as a Negative Regulator of AMS-regulated Pollen Wall Developmental

500

Pathways

501

Previous studies have demonstrated that the transcription factors DYT1, MYB35/TDF1, AMS,

502

MYB103/MYB80, and MS1 are required for anther development and they form a genetically defined

503

pathway/regulatory cascade: DYT1-MYB35/TDF1-AMS-MYB103/MYB80-MS1 (Zhu et al., 2011).

504

AMS is a key regulator of sporopollenin biosynthesis, secretion and pollen wall formation and can

505

form a complex with MS188/MYB103/MYB80 that likely activates the expression of CYP703A2

506

and MS1 encoding a transcription factor (Xu et al., 2010; Zhu et al., 2011; Feng et al., 2012; Gu et al., 18


507

2014; Xu et al., 2014; Xiong et al., 2016; Ferguson et al., 2017). DYT1 and MYB35/TDF1 are

508

required for the development of tapetum and reproductive cells at early stages, as supported by the

509

findings that DYT1 and MYB35/TDF1 mutants exhibit abnormal tapetal cells at anther stage 5 and

510

onward and their anthers lack any pollen (Zhang et al., 2006; Zhu et al., 2008; Gu et al., 2014). These

511

two genes are important for normal anther and pollen development, at least in part by activating the

512

normal expression of AMS. In our study, we showed that CLE19 negatively regulates the expression

513

of AMS and downstream genes MYB103/MYB80 and MS1 to prevent their deleterious overexpression,

514

but does not obviously affect the normal expression of DYT1 and MYB35/TDF1 (Fig. 6),

515

demonstrating that CLE19 probably functions to modulate the proper level of the activity of the

516

AMS-regulated network and the appropriate amount of sporopollenin biosynthesis and pollen wall

517

formation. The cytological results that both CLE19-OX transgenic anthers and the ams mutant

518

anthers showed similar abnormal tapetum cells and pollen primexine developmental defects, and the

519

genetic results that ams (+/-) partially suppressed the DN-CLE19 transgene-induced pollen exine

520

defects (Fig. 9) together supporting the idea that AMS works downstream of CLE19 signaling

521

pathway and the full effect of CLE19 depends on the normal level of AMS.

522

Therefore,

523

membrane-localized receptor(s), negatively regulates the AMS-MYB103-MS1 transcriptional

524

cascades to maintain their functional homeostasis and ensure the normal development of pollen (Fig.

525

10A). However, in the DN-CLE19 mutants, reduced function of CLE19 releases the transcriptional

526

inhibition and caused the deleterious overexpression of AMS and downstream networks; such

527

overexpression then subsequently affected the normal organization of pollen exine (Fig. 10B). In

528

contrast, in the CLE19-OX anthers, CLE19 synthesized at abnormally high levels excessively inhibits

529

the expression and function of AMS and downstream networks, thereby inducing abnormal pollen

530

development (Fig. 10C).

531

Functional Redundancy Between Anther-expressed CLE Genes

532

As an extracellular peptide hormone, CLE19 was expressed at relatively low levels in the

533

inflorescence and stage 4-12 anthers in our quantitative real-time PCR analysis using EF1α as the

534

internal control (Supplemental Fig. S1B). Nevertheless, both CLE19-OX and DN-CLE19 transgenic

we

propose

that

the

CLE19

peptide,

probably

through

19


unknown

plasma

535

plants showed obviously male fertility defects, even though the cle19 single mutants showed weak

536

anther developmental defects (Figure 2). We would like to offer the following thoughts: Firstly, RNA

537

in situ hybridization analysis results showed that CLE19 was only expressed in the tapetal and male

538

reproductive cells at anther stages 5 to 7 (Figure 1M-P), suggesting strong cell-specificity and a short

539

duration of CLE19 expression in the anther. We believe that both the high cell specificity and short

540

duration of CLE19 expression contribute to its signal being diluted by non-expressing cells in the

541

inflorescence or stage 4 to 12 anthers. Secondly, as supported by the results here, CLE19 acts as a

542

negative regulator of AMS and additional downstream genes, which are required for normal tapetum

543

function and pollen development. If CLE19 is allowed to be expressed at very high levels, normal

544

pollen development would be inhibited, as seen in the CLE19-OX plants.

545

It is challenging to obtain mutants of CLE genes because they are all small genes; in addition, the

546

anther-expressed CLE genes might have overlapping (redundant) functions. In this study, knock-out

547

(KO) mutants of 5 anther-expressed CLE genes，CLE9, CLE16, CLE17, CLE19, and CLE42, were

548

briefly characterized, and only CLE19 KO mutants exhibited very weak anther developmental

549

defects whereas mutants of the other four genes showed no obviously development or fertility

550

defects. Nevertheless, our statistic analysis results showed that, comparing with the WT plants, their

551

double and triple mutants showed several detectable defects: increased barren silique number,

552

reduced total pollen number but increased aborted pollen number in each anther, and increased

553

percentage of pollen with abnormal pollen exine structure (Supplemental Fig. S4 and Supplemental

554

Table 1). In addition, the DN transgenic mutants of these 7 anther-expressed CLE members,

555

DN-CLE9, DNCLE16, DN-CL17, DNCLE19, DN-CLE41, DNCLE42, and DN-CLE45, all showed

556

severe anther developmental and pollen exine formation defects (Supplemental Fig. S5). All these

557

results suggested that these anther-expressed CLE members possibly function in a redundant way in

558

the regulation of male fertility, especially in the regulation of pollen exine formation. We believe that

559

further studies with various multiple mutants may uncover the detail redundant and distinctive

560

functions on different developmental aspects between these CLE members.

561

In addation, further studies are also needed to uncover additional signaling components in the

562

complex network that regulates pollen wall formation. For example, what is the RLK that perceives 20


563

the CLE peptide signal and transduces the signal into the cytoplasm? What are the downstream

564

effectors, such as kinase and other factors, that respond to the CLE-RLK signaling module and how

565

do they activate the AMS transcription factor? What is the proper amount of CLE peptide? Further

566

investigations using a combination of omics, biochemistry, cell biology, and genetic strategies may

567

answer these questions and further advance the study on male fertility.

568 569

CONCLUSION

570

In summary, our observations demonstrated that CLE19 is essential for normal anther development

571

and pollen wall formation, likely and in large part by negatively regulating the expression of AMS

572

and downstream genes MYB103/MYB80 and MS1 to prevent their deleterious overexpression, and to

573

modulate the proper level of the activity of AMS-regulated network and the appropriate anther

574

development and pollen wall formation.

575

576

MATERIALS AND METHODS

577

Plant Growth and Genotyping

578

Arabidopsis thaliana plants were grown on soil in greenhouse conditions (21℃) under long-day

579

conditions (16-h of light/8-h of dark). T-DNA insertion mutant alleles of cle19 (CS879453 and

580

CS321816), cle9 (SALK_018122C), cle 17 (SALK_103714C), cle 42 (SALK_057407C) mutants

581

and RNAi plants of CLE16 (CS201209) in Col-0 background were all obtained from the ABRC

582

stocks. The T-DNA insertion of CLE19 alleles were verified using the T-DNA border primer

583

SALKLB1.3 in combination with, gene-specific primers listed in Supplemental Table 2.

584

Molecular Cloning and Generation of Transgenic Plants and Genotyping

585

Genomic sequences of CLE9, CLE16, CLE17, CLE19, CLE41, CLE42, and CLE45 were cloned into

586

the pDONR221 vector (Life Technologies, Shanghai) with primers list in the Supplemental Table 3.

587

Using a PCR-based site-directed mutagenesis kit (Transgene, Beijing) and specific primers

588

(Supplemental Table 3), the codon of glycine at the sixth position of the CLE motif of these CLE 21


589

genes

was

replaced

by

the

codon

of

threonine,

producing

pDONR221-CLE9G6T,

590

pDONR221-CLE16G6T, pDONR221-CLE17G6T, pDONR221-CLE19G6T, pDONR221-CLE41G6T,

591

pDONR221-CLE42G6T and pDONR221-CLE45G6T entry clones. These entry clones were then

592

sub-cloned into the pBGWFS7 binary vector (Karimi et al., 2002) to produce the CLE19G6T,

593

CLE16G6T, CLE17G6T, CLE41G6T, CLE42G6T and CLE45G6T. Transformation was performed via an

594

Agrobacterium tumefaciens-mediated floral dip method (Clough and Bent, 1998). Transgenic plants

595

were obtained under the selection of 25 mg Basta (Sigma-Aldrich).

596

Quantitative Real-time PCR Analysis

597

To analyze the expression level of the CLEs and other downstream genes, stage 1-11 flower buds of

598

Col-0, each related T-DNA or RNAi mutant, DN-CLEs transgenic plants were collected and total

599

RNA was extracted according to a Trizol-based (Sigma-Aldrich) method. After DNase I digestion

600

and first-strand cDNA synthesis, the quantitative real-time PCR was performed with SYBR premix

601

Ex Taq II (Takara) on the ABI StepOnePlus real-time system (Life Technologies) using primers

602

listed in Supplemental Table 4 and with EF1a (AT5G60390) as the internal control.

603

RNA In situ Hybridization

604

RNA in situ hybridization analysis was performed using the Digoxigenin RNA Labeling Kit (Roche,

605

USA) and the PCR DIG Probe Synthesis (Roche, USA). cDNA fragments of target genes about

606

400bp were amplified using specific primers listed in Supplemental Table 5. PCR products were

607

cloned into the pGEM-T vector and confirmed by sequencing. Completely digested plasmid DNAs

608

were used as the template for transcription with T7 or SP6 RNA polymerase.

609

Phenotypic Analysis of Flowers and Anthers

610

Plants were photographed with digital camera (Canon, Japan). Flower images were taken with an

611

SPOT FLEX digital camera (DIAGNOSTIC instruments, Sterling Heights, MI, USA) under a

612

SteREO Discovery V8 dissecting microscope (Carl Zeiss Microimaging Inc., Germany).

613

Stage 12 anthers were collected and stained overnight at room temperature using the Alexander

614

solution prepared following the published protocols (Alexander, 1969) and further anthers were

615

pressed to release the stained pollen grains and photographed by an AXIO ScopeA1 microscope 22


616

(Carl Zeiss Microimaging Inc., Germany) with a Axio Cam HRc camera (Carl Zeiss Microimaging

617

Inc., Germany).

618

Light and Electron Microscopy Observation

619

Inflorescences of WT and mutant plants were collected and fixed in FAA solution and embedded in

620

Technovit 7100 resin (Heraeus Kulzer GmbH, Germany) as described (Jin et al., 2013). Semi-thin

621

sections were prepared by cutting inflorescence materials into 1.0µm thick sections using a

622

motorized RM2265 rotary microtome (Leica Microsystems IR GmbH, Germany), and then were

623

stained with 0.05% Toluidine Blue O for 15 to 30 minutes and photographed by bright-field

624

microscopy.

625

For pollen wall observation, sections were stained with Toluidine blue for 5 min (10 mg/ml), Tinapol

626

for 15 min (10 ug/ul) (Sigma, USA), and DiOC2 for 5 min (5 ul/ml) (Sigma, USA), samples were

627

taken photographs under AXIO Scope with390-440 nm excitation filter and 478nm blocking filter.

628

For scanning electron microscopic examination, fresh pollen grains released from stage 13 anthers

629

were coated with gold and observed under SU8010 microscopy (HITACHI, Japan).

630

For TEM observation, different stage buds were fixed in glutaric dialdehyde buffer and embedded

631

into the fresh mixed resin. Ultrathin sections (70-100 nm thick) were observed with a Tecnai G2

632

Spirit TWIN TEM (FEI, Czech Republic).

633

RNA Sequencing and Data Analysis

634

More than 1000 of stage 4-10 anthers from the WT and mutant plants were collected and

635

immediately frozen in the liquid nitrogen, respectively. Total RNA from each sample was extracted

636

and purified using the ZR plant RNA Miniprep™ kit (Zymo Research, USA). 2 μg total RNA of each

637

sample was used for deep sequencing by an Illumina™ Hi-seq 2000 system (Illumina Ins., USA). All

638

sequencing data were mapped and analyzed following the previous reported method (Zhu et al.,

639

2015).

640

Accession Numbers

641

The original RNA-seq data from this article has been submitted to NCBI GEO database under

642

accession number GSE94607. Sequence data from this article can be found in the Arabidopsis 23


643

Genome Initiative database under the following accession numbers: CLE9 (AT1G26600), CLE16

644

(AT2G01505), CLE17 (AT1G70895), CLE19 (At3g24225), CLE42 (AT2g34925), CLE45

645

(AT1g69588), DYT1 (At4g21330), AMS (At2g16910), MYB35/TDF1 (At3g28470), MS1

646

(At5g22260), MYB103 (At1g63910), EF1α (AT5G60390), and ACT2 (AT3G18780). Germplasm

647

used included cle9-1 (SALK_018122C), cle16-1 (CS201209), cle17-1 (SALK_103714C), cle19-1

648

(CS879453), qrt1-2 (SALK_CS8846), cle19-2 (CS321816), and cle42-1 (SALK_057407C).

649 650

SUPPLEMENTAL DATA

651

The following supplemental materials are available.

652

Supplemental Figure S1. Expression patterns of CLE genes in various Arabidopsis organs.

653

Supplemental Figure S2. The T-DNA locations or RNAi targeting sites and relative expression of

654

detected genes in the cle9-1, cle16-1, cle17-1, qrt1-2, cle19-1 qrt1-2, cle19-2, cle42-1 mutants.

655

Supplemental Figure S3. Phenotypic analyses of plant growth, anthers and pollens of the WT and

656

the cle9-1, cle16-1, cle17-1, qrt1-2, cle19-1 qrt1-2, cle19-2, cle42-1 plants.

657

Supplemental Figure S4. Phenotypic analyses of siliques, flowers, anthers and pollen grains in WT

658

and the cle single, double, and triple mutants.

659

Supplemental Figure S5. Phenotypic analyses of plant growth, anthers and pollen grains of the WT

660

and DN-CLE transgenic plants.

661

Supplemental Figure S6. Comparison of exine pattern among cle19-2, DN-CLE19, DNCLE41,

662

DNCLE42, and the wild type.

663

Supplemental Figure S7. RNA in situ hybridization analysis to detect the spatial and temporal

664

expression of AMS gene in the WT, ams, DN-CLE19 and CLE19-OX transgenic anthers.

665

Supplemental Figure S8. Scatter plots for replicates of transcriptome data.

666

Supplemental Table 1. Statistically phenotypic analysis result of the cle single, double, and triple

667

mutants.

668

Supplemental Table 2. Primers used for genotyping.

669

Supplemental Table 3. Primers used for DN-CLE transgenic constructs in this work. 24


670

Supplemental Table 4. Primers used for quantitative real time PCR analysis.

671

Supplemental Table 5. Primers used for in situ constructs in this work.

672

Supplemental Data Set 1. List of 578 genes down-regulated and 1133 genes up-regulated in

673

DN-CLE19-23 transcriptomic analysis in this study.

674

Supplemental Data Set 2. List of 1809 genes down-regulated and 379 genes up-regulated in

675

CLE19-OX-S transcriptomic analysis in this study.

676

Supplemental Data Set 3. List of 742 genes both up-regulated in DN-CLE19-23 and

677

down-regulated in CLE19-OX-S transcriptomic data.

678

Supplemental Data Set 4. List of 52 genes both down-regulated in DN-CLE19-23 and up-regulated

679

in CLE19-OX-S transcriptomic data.

680

Supplemental Data Set 5. List of overlap genes differentially expressed in both DN-CLE19-23 and

681

CLE19-OX-S transcription data and the microarray-based transcriptomic data of the ams stage 4-7

682

anther and 404 RNA-seq-based transcriptomic data of ams buds.

683 684

Acknowledgements

685

The authors would like to thank the Salk Institute Genomic Analysis Laboratory and the Ohio State

686

University Arabidopsis Biological Resources Center for providing the sequence-indexed Arabidopsis

687

thaliana T-DNA insertion mutants. This work was supported by grants from the National Natural

688

Science Foundation of China (31130006, 31470282, 31670316, and 31370029) and the State Key

689

Laboratory of Genetic Engineering at Fudan University.

690 691 692 693 694

25


695

Table 1. Phenotypic analysis of cle19-2, DN-CLE19-23, and CLE19-OX-S. Table 1. Phenotypic analysis of cle19-2, DN-CLE19-23, and CLE19-OX-S.

silique number Col-0 68.9±3.9 cle19-2 55.9±3.9 DNCLE19-23 30.6±3.0 C24 67.2±3.7 CLE19-OX-S 12.9±3.1

silique length (mm) 14.3±0.7 13.0±1.9 10.7±2.5 14.6±1.0 9.6±2.4

seed number (per silique) 54.8±3.6 41.58±10.1 30.6±11.5 48.8±4.5 22.0±9.4

pollen number (per anther) 640.3±22.0 413.6±40.1 310.2±99.6 617.3±34.9 54.5±26.7

pollen germination rate(%) 99.97±5.25 41.67±5.16 15.67±4.72 99.96±7.20 98.53±15.18

Pollen tube length(μm) 208.0±11.8 137.0±25.0 82.8±6.5 205.1±9.9 35.5±8.7

Data are the means ±SD. For silique number, three replicates using 30 branches were analyzed. For silique length, three replicates and 30 siliques in each replicates were analyzed. For seed number, three replicates and 30 siliques in each replicates were analyzed. For pollen number, three replicates and more than 30 anthers in each replicates were analyzed. For pollen germination rate and pollen tube length analyses, three replicates and more than 100 pollen for each replicate were observed.

696 697

FIGURE LEGENDS

698

Figure 1. Spatial and temporal expression analysis of CLE genes.

699

A to AB, Expression patterns of CLE9 (A to D), CLE16 (E to H), CLE17 (I to L), CLE19 (M to P),

700

CLE41 (Q to T), CLE42 (U to X) and CLE45 (Y to AB) in WT anthers by RNA in situ hybridization.

701

Sections are from anthers of stage 5 (A, E, I, M, Q, U and Y), stage 7 (B, F, J, N, R, V and Z), stage

702

10 (C, G, K, O, S, W and AA) and controls (D, H, L, P, T, X and AB) using sense probe on WT stage

703

7 anthers. T, tapetum; Ms, microsporocytes; Tds, tetrads; Msp, microspores. Scale bars = 10 μm.

704 705

Figure 2. Phenotypic analyses of the WT, cle19 T-DNA insertion mutants, DN-CLE19

706

transgenic and CLE19-OX transgenic plants.

707

A to I, Phenotypic analysis of plant growth of the WT, cle19 T-DNA insertion mutants, DN-CLE19

708

transgenic and CLE19-OX transgenic plants. J to R, Phenotypic analysis of flowers of the WT,

709

CLE19 T-DNA insertion mutants，DN-CLE19 transgenic and CLE19-OX transgenic plants. S to AA, 26


710

Phenotypic analysis of anthers of the WT, CLE19 T-DNA insertion mutants，DN-CLE19 transgenic

711

and CLE19-OX transgenic plants. AB to AJ, Phenotypic analysis of pollens of the WT, CLE19

712

T-DNA insertion mutants，DN-CLE19 transgenic and CLE19-OX transgenic plants. Red arrows

713

indicated aborted pollen grains. Scale bar for plants: 2cm; for flowers: 500μm; for anthers: 200μm;

714

and for pollens: 25 μm.

715 716

Figure 3. Spatial and temporal expression of CLE19 in the WT, CLE19-OX, cle19-2, and

717

DN-CLE19 anthers.

718

A, A diagram showing the T-DNA insertion sites of cle19-1 and cle19-2. B, a diagram showing the

719

amino acid mutation site in the DN-CLE19. C to D, Quantitative real-time PCR analysis to detect

720

CLE19 expression in stage 4-12 anthers of WT, DN-CLE19 (C) and CLE19-OX (D), using EF1α as

721

an internal control. DN-23 and DN-30 indicates two individual CLE19::DN-CLE19 transgenic lines.

722

19OX-M and 19OX-S indicate two independent 35S::CLE19 transgenic lines. Data from three

723

biological replicates were collected. E to X, RNA in situ hybridization results showing the expression

724

of CLE19 in WT, CLE19-OX-S, cle19-2, and DN-CLE19-23 mutant anthers. Scale bar for anther

725

lobes: 10μm.

726 727

Figure 4. Cell biological analyses of WT, cle19-2, DN-CLE19, and CLE19-OX anthers.

728

A to E, Wild-type anther morphology using semi-thin transverse sections. F to J, Morphology of

729

cle19-2 anthers using semi-thin transverse sections. K to O, Morphology of DN-CLE19 anthers using

730

semi-thin transverse sections. P to T, Morphology of CLE19-OX-S anthers using semi-thin transverse

731

sections. U to Y, Morphology of CLE19-OX-M anthers using semi-thin transverse sections. Z to AA,

732

Statistic analyses of the tapetal or reproductive cell numbers in DN-CLE19, CLE19-OX and

733

wild-type anthers, respectively. AB, indicates how the tapetal and reproductive cells were calculated.

734

Numbers of tapetal or reproductive cell in each lobe in transverse sections was calculated. E,

735

epidermis; En, endothecium; T, tapetum; Ms, meiocytes; PG, pollen grains; DPG, degenerated pollen

736

grains. Scale bars = 20μm.

737 27


738

Figure 5. Microscopic analyses of WT, DN-CLE19, and CLE19-OX pollen exine.

739

A to J, Pollen grains from the WT, DN-CLE19, and CLE19-OX plants visualized under scanning

740

electron microscope (SEM). K to O, Cytochemical staining of semi-thin sections of the WT,

741

DN-CLE19, and CLE19-OX pollen. P to Y, The WT, DN-CLE19, and CLE19-OX pollen visualized

742

under the transmission electron microscopy (TEM). (A), (F), (K), (P), and (U) Wild type; (B), (G),

743

(L), (Q), and (V) DN-CLE19-M pollen; (C), (H), (M), (R), and (W) DN-CLE19-S pollen; (D), (I), (N),

744

(S), and (X) CLE19-OX-M pollen; (E), (J), (O), (T), and (Y) CLE19-OX-S pollen. Ex, exine; In,

745

intine. Scale bars = 10 μm for A-E; scale bars = 2 μm for F-J; scale bars = 5 μm for K-O; scale bars =

746

2 μm for P-T; scale bars = 0.5 μm for U-Y.

747 748

Figure 6. qRT-PCR and RNA in situ hybridization analyses to test the expression of AMS and

749

downstream transcription factor genes in DN-CLE19 and CLE19-OX transgenic anthers, and

750

TEM observation of the CLE19-OX and ams tapetal cell and microspore.

751

A and B, The relative expression levels of transcription factor gene DYT1, MYB35, AMS, MYB103,

752

and MS1 in DN-CLE19 and CLE19-OX mutants compared to related wild type plants, with ACTIN

753

expression as internal control. C and D, Expression of AMS in the WT anthers. E and F, Expression

754

of AMS in the DN-CLE19 anthers. C and E, anther stage 7; D and F, anther stage 10. G-L, Tapetal

755

cells of WT and CLE19-OX-S anther visualized under the transmission electron microscopy (TEM).

756

G, Wild type; H, CLE19-OX-S. I to L, Microspore cell of CLE19-OX-S and ams anther visualized

757

under the transmission electron microscopy (TEM). I and J, CLE19-OX-S; K and L, ams. T, tapetum;

758

Ela, elaioplasts; Ta, tapetosomes; dMsp, degenerated microspores; Pm, primexine. Scale bars = 1 μm

759

for C to H.

760 761

Figure 7. Transcriptomic comparison analyses between CLE19 and AMS.

762

A, Percentage of up- and down-regulated genes in the DN-CLE19 and CLE19-OX transcriptome,

763

respectively. B, Comparison of differentially expressed genes between DN-CLE19 and CLE19-OX

764

transcriptome. C, Comparison of differentially expressed genes between DN-CLE19 up-regulated

765

and CLE19-OX down-regulated gene groups. D, Comparison of differentially expressed genes

766

between DN-CLE19 down-regulated and CLE19-OX up-regulated gene groups. E, Venn diagram 28


767

showing the overlap between genes affected in CLE19 anthers, ams floral buds, and ams anthers. F,

768

Enriched biological process categories for transcriptome analysis of genes affected in both CLE19

769

and ams mutants. G, The CLE19 genetic pathways controlling anther development.

770 771

Figure 8. Transcriptomic analyses genes involved in the metabolic of flavonoids, phenolics, wax,

772

cutin, and free fatty acids in WT and CLE19 mutant.

773

A, Metabolic processes of the main compounds of pollen wall. B to F, Fold change in expression

774

levels of genes involved in flavonoids metabolic, cutin metabolic, wax easter metabolic, free fatty

775

acids metabolic, and phenolics metabolic processes.

776 777

Figure 9. Phenotypic analysis of anthers from the WT, ams mutant，DN-CLE19 transgenic，

778

DN-CLE19+/+ ams+/- and DN-CLE19+/+ ams-/- plants.

779

A to E, Alexander stained anthers from the WT (A), ams mutant (B), DN-CLE19 transgenic (C)，

780

DN-CLE19+/+ ams+/- (D) and DN-CLE19+/+ ams-/- (E) plants. F to L, pollen grains from the WT

781

(F), DN-CLE19 transgenic (G to I), and DN-CLE19+/+ ams+/- (J to L) plants. M, Statistic analysis

782

data to show the percentage of pollens with abnormal exine in the WT, DN-CLE19 transgenic and

783

DN-CLE19+/+ ams+/- plants. Scale bar for anthers: 200μm; and for pollens: 10 μm.

784 785

Figure 10. Proposed working model for CLE19 in the regulation of anther development.

786

A, In WT anthers, CLE19 peptides interact with their unknown receptor to negatively regulate the

787

AMS-MYB103-MS1 transcriptional cascades to prevent their deleterious overexpression, which

788

triggers the normal expression of downstream exine formation related genes, subsequently regulate

789

the formation of normally well-organized pollen exine. B, In DN-CLE19 anthers, the CLE19

790

peptides without function competitively interact with the unknown CLE19 receptor, which block or

791

obviously reduce the signal transduction from the functional CLE19 to its downstream molecules,

792

therefore leading to the deleterious overexpression of AMS-MYB103-MS1 transcriptional cascades

793

and downstream exine formation related genes, and subsequently affected the normal pollen exine

794

formation. C, In CLE19-OX anthers, over-expression of CLE19 peptide strengthen the suppression of

795

the transcription factors gene expression, resulting in abnomal pollen exine formation. 29


796 797

Supplemental Figure S1. Expression patterns of CLE genes in various Arabidopsis organs.

798

A, Heat map of expression level of 14 Arabidopsis thaliana CLE genes in seedlings, roots, leaves,

799

shoots, seeds, flowers, carpels, sepals, stamens and pollens. petals 1, petals of stage15 flowers; sepals

800

1, sepals of stage12 flowers; petals 2, petals of stage12 flowers; stamens 1, stamens of stage12

801

flowers; sepals 2, sepals of stage 15 flowers; stamens 2, stamens of stage15 flowers; carpels 1,

802

carpels of stage12 flowers; carpels 2, carpels of stage15 flowers. B, Quantitative real-time PCR

803

analysis for CLE genes in Col-0 wild type inflorescence and stage 4-12 anthers using EF1α as the

804

internal control. Values are means (±SE) from three biological replicates.

805 806

Supplemental Figure S2. The T-DNA locations and relative expression of detected genes in the

807

cle9-1, cle16-1, cle17-1, qrt1-2, cle19-1 qrt1-2, cle19-2, cle42-1 mutants. EF1α was used as the

808

internal control. Data from three biological replicates were collected. Values are means (±SE) from

809

three biological replicates.

810 811


812

the cle9-1, cle16-1, cle17-1, qrt1-2, cle19-1 qrt1-2, cle19-2, cle42-1 plants. Scale bar for plants: 2cm;

813

for flowers: 500μm; and for anthers: 25μm.

814 815

Supplemental Figure S4. Phenotypic analyses of siliques, flowers, anthers and pollen grains in WT

816

and the cle single, double, and triple mutants. Scale bar for plants: 2cm; for anthers: 200μm; for

817

pollens: 10μm.

818 819


820

DN-CLE transgenic plants.

821

A to U, Transgenic plants of CLE9::DN-CLE19, CLE16::DN-CLE16, CLE17::DN-CLE17,

822

CLE41::DN-CLE41, CLE42::DN-CLE42 and CLE45::DN-CLE45 were analyzed. Red arrows

823

indicated aborted pollen grains. Scale bar for plants: 2cm; for anthers: 200μm; and for pollens: 25

824

μm.

825 30


826

Supplemental Figure S6. Comparison of exine pattern among cle19-2, DN-CLE19, DN-CLE41,

827

DN-CLE42, and the wild type.

828

A, Percentage of Lacunes filled with materials in WT and CLE19 mutants. B, Number of lattices per

829

50 μm of WT and CLE19-OX pollen exine. C, Morphology of pollen exine in the WT and

830

DN-CLE19 mutant anthers at stage10-13 visualized by a scanning electron microscope. Scale bar:

831

25μm.

2

832 833

Supplemental Figure S7. RNA in situ hybridization analysis to detect the spatial and temporal

834

expression of AMS gene in the WT, ams, DN-CLE19 and CLE19-OX transgenic anthers.

835

A to C, Expression of AMS in WT anthers; D to F, Expression of AMS in DN-CLE19 transgenic

836

anthers; G to K, Expression of AMS in CLE19-OX transgenic anthers; L to P, Expression of AMS in

837

and ams mutant anthers. T, tapetum; Ms, microsporocytes; Tds, tetrads; Msp, microspores. Scale bar

838

for anther lobes:10 μm; anthers: 200μm; and for pollens: 25 μm.

839 840

Supplemental Figure S8. Scatter plots for replicates of transcriptome data.

841

The coordinate of each dot refers to the RNA expression level in log2 (RPKM) of two replicates in

842

(A) DN-CLE19 and (B) CLE19-OX backgrounds. The R square value for the replicate pair is shown

843

under the corresponding figure.

844

31


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chimaeric ribonuclease gene. Nature 347: 737-741 Mascarenhas JP (1975) The biochemistry of angiosperm pollen development. Bot. Rev, 41: 259-314 Mayfield J, Preuss D (2000) Rapid initiation of Arabidopsis pollination requires the oleosin-domain protein GRP17. Nat. Cell Biol. 2: 128-130 Mizuno S, Osakabe Y, Maruyama K, Ito T, Osakabe K, Sato T, Shinozaki K, Yamaguchi-Shinozaki K (2007) Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana. Plant J. 50: 751–766 Ni J, Clark SE (2006) Evidence for functional conservation, sufficiency, and proteolytic processing of the CLAVATA3 CLE domain. Plant Physiol 140: 726-733 Pacini E, Juniper BE (1979) The ultrastructure of pollen-grain development in the OLIVE(OLEA EUROPAEA). 2. Secretion by the tapetal cells. New Phytol. 83: 165-174 Phan HA, Iacuone S, Li SF, Parish RW (2011) The MYB80 transcription factor is required for pollen development and the regulation of tapetal programmed cell death in Arabidopsis thaliana. Plant Cell 23: 2209-2224 Preuss D, Lemieux B, Yen G, Davis RW (1993) A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization. Gene Dev. 7: 974-985 Sanders PM, Bui AQ, Weterings K, Mcintire KN, Hsu YC, Pei YL, Mai TT, Beals TP, Goldberg RB (1999) Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Plant Reprod. 11: 297-322 Scott RJ, Spielman M, Dickinson HG (2004) Stamen structure and function. Plant Cell 16 Suppl: S46-60 Shpak ED, Lakeman MB, Torii KU (2003) Dominant-negative receptor uncovers redundancy in the Arabidopsis ERECTA Leucine-rich repeat receptor-like kinase signaling pathway that regulates organ shape. Plant Cell 15: 1095-1110 Song X, Guo P, Ren S, Xu T, Liu C (2013) Antagonistic peptide technology for functional dissection of CLV3/ESR genes in Arabidopsis. Plant Physiol. 161: 1076-1085 Song X, Yu D, Xu T, Ren S, Guo P, Liu C (2012) Contributions of individual amino acid residues to the endogenous CLV3 function in shoot apical meristem maintenance in Arabidopsis. Mol. Plant 5: 515-523 Sorensen AM, Kröber S, Unte US, Huijser P, Dekker K, Saedler H (2003) The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor. Plant J. 33: 413-423 Stahl Y, Simon R (2009) Is the Arabidopsis root niche protected by sequestration of the CLE40 signal by its putative receptor ACR4? Plant Signal. Behav. 4: 634-635 Stieglitz H (1977) Role of β-1,3-glucanase in postmeiotic microspore release. Dev. Biol. 57: 87-97 Strabala TJ, O'Donnell PJ, Smit AM, Ampomah-Dwamena C, Martin EJ, Netzler N, Nieuwenhuizen NJ, Quinn BD, Foote HC, Hudson KR (2006) Gain-of-function phenotypes of many CLAVATA3/ESR genes, including four new family members, correlate with tandem variations in the conserved CLAVATA3/ESR domain. Plant Physiol. 140: 1331-1344 Thom I, Grote M, Abraham-Peskir J, Wiermann R (1998) Electron and X-ray microscopic analyses of reaggregated materials obtained after fractionation of dissolved sporopollenin. Protoplasma 204: 13-21 Torii KU, Mitsukawa N, Oosumi T, Matsuura Y, Yokoyama R, Whittier RF, Komeda Y (1996) The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular Leucine-rich repeats. Plant Cell 8: 735-746 Wilson ZA, Morroll SM, Dawson J, Swarup R, Tighe PJ (2001) The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. Plant J. 28: 27-39 Wilson ZA, Zhang DB (2009) From Arabidopsis to rice: pathways in pollen development. J. Exp. Bot. 60: 1479-1492 Xiong SX, Lu JY, Lou Y, Teng XD, Gu JN, Zhang C, Shi QS, Yang ZN, Zhu J (2016) The transcription factors MS188 and AMS form a complex to activate the expression of CYP703A2 for sporopollenin biosynthesis in Arabidopsis thaliana. Plant J. 88: 936-946 34


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1010

35


Wang_Figure 1 stage 5

A

stage 7

stage 10

B

sense

C

D

T

T

T

T

Ms

Tds

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CLE42 Ms

Y CLE45

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Z T Ms

T Tds

Tds

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AA

AB T

T

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Figure 1. Spatial and temporal expression analysis of CLE genes. A to AB, Expression patterns of CLE9 (A to D), CLE16 (E to H), CLE17 (I to L), CLE19 (M to P), CLE41 (Q to T), CLE42 (U to X) and CLE45 (Y to AB) in WT anthers by RNA in situ hybridization. Sections are from anthers of stage 5 (A, E, I, M, Q, U and Y), stage7 (B, F, J, N, R, V and Z), stage10 (C, G, K, O, S, W and AA) and controls (D, H, L, P, T, X and AB) using sense probe on WT stage 7 anthers. T, tapetum; Ms, microsporocytes; Tds, tetrads; Msp, microspores. Scale bars = 10 µm.


Wang_Figure 2

A

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E

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HF

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AJ

Figure 2. Phenotypic analyses of plant growth, flowers, anthers and pollens of the WT, CLE19 T-DNA insertion mutants, DN-CLE19 transgenic and CLE19-OX transgenic plants. A to I, Phenotypic analysis of plant growth of the WT, CLE19 T-DNA insertion mutants, DN-CLE19 transgenic and CLE19-OX transgenic plants. J to R, Phenotypic analysis of flowers of the WT, CLE19 T-DNA insertion mutants，DN-CLE19 transgenic and CLE19-OX transgenic plants. S to AA, Phenotypic analysis of anthers of the WT, CLE19 T-DNA insertion mutants，DN-CLE19 transgenic and CLE19-OX transgenic plants. AB to AJ, Phenotypic analysis of pollens of the WT, CLE19 T-DNA insertion mutants，DN-CLE19 transgenic and CLE19-OX transgenic plants. Red arrows indicated aborted pollen grains. Scale bar for plants: 2cm; for flowers: 500µm; for anthers: 200µm; and for pollens: 25 µm.


Wang_Figure 3

Figure'3 stage5 S5

C CLE19 promoter

5`UTR CLE Motif

cle19-2

cle19-1

B

CLE19 expression level

A

DN-CLE19 RVIPTGPNPLHNR T

CLE19 expression level

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20 15 10 5 0

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Figure 3. Spatial and temporal expression of CLE19 in the WT, CLE19-OX, cle19-2, and DN-CLE19 anthers. A, A diagram showing the T-DNA insertion sites of cle19-1 and CLE25 cle19-2. B, a diagram showing the amino acid mutation site in the DN-CLE19. C to D, Quantitative real-time PCR analysis to detect CLE19 expression in stage 4-12 anthers of CLE27 WT, DN-CLE19 (C) and CLE19-OX (D), using EF1α as an internal control. DN-23 and DN-30 indicates two individual CLE19::DN-CLE19 transgenic lines. 19OX-M and 19OXS indicate two independent 35S::CLE19 transgenic lines. Data from three biological CLE41 replicates were collected. E to X, RNA in situ hybridization results showing the expression of CLE19 in WT, CLE19-OX-S, cle19-2, and DN-CLE19-23 mutant anthers. Scale bar for anther lobes: 10µm.


Wang_Figure 4 Stage5

B

A WT

F

Ms

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Figure 4. Cell biological analyses of WT, cle19-2, DN-CLE19, and CLE19-OX anthers. A to E, Wild-type anther morphology using semi-thin transverse sections. F to J, Morphology of cle19-2 anthers using semi-thin transverse sections. K to O, Morphology of DN-CLE19 anthers using semi-thin transverse sections. P to T, Morphology of CLE19-OX-S anthers using semi-thin transverse sections. U to Y, Morphology of CLE19-OX-M anthers using semi-thin transverse sections. Z to AA, Statistic analyses of the tapetal or reproductive cell number in DN-CLE19, CLE19OX and wild type anthers, respectively. AB, indicates how the tapetal and reproductive cells were calculated. Numbers of tapetal or reproductive cell in each lobe in transverse sections was calculated. E, epidermis; En, endothecium; T, Downloaded on September 2017 -degenerated Published by www.plantphysiol.org tapetum; Ms, meiocytes; PG, from pollen grains;16, DPG, pollen grains. Scale Copyright © 2017 American Society of Plant Biologists. All rights reserved. bars = 20 µm.

Wang_Figure 5 A

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Figure 5. Microscopic analyses of WT, DN-CLE19, and CLE19-OX pollen exine. A to J, Pollen grains from the WT, DN-CLE19, and CLE19-OX plants visualized under scanning electron microscope (SEM). K to O, Cytochemical staining of semi-thin sections of the WT , DN-CLE19, and CLE19-OX pollen. P to Y, The WT, DN-CLE19, and CLE19-OX pollen visualized under the transmission electron microscopy (TEM). (A), (F), (K), (P), and (U) Wild type; (B), (G), (L), (Q), and (V) DN-CLE19-M pollen; (C), (H), (M), (R), and (W) DN-CLE19-S pollen; (D), (I), (N), (S), and (X) CLE19-OXM pollen; (E), (J), (O), (T), and (Y) CLE19-OX-S pollen. Ex, exine; In, intine. Scale bars = 10 µm for A-E; scale bars = 2 µm for F-J; scale bars = 5 µm for K-O; scale bars = 2 µm for P-T; scale bars = 0.5 µm for U-Y. Downloaded from on September 16, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Wang_Figure 6 Relative Expression Level

Col-0 DN

B

C24 OX

1.2 1.2 1 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 0

Relative Expression Level

8 8 6 6 4 4 2 2 0 0

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Ela Ta J

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Figure 6. qRT-PCR and RNA in situ hybridization analyses to test the expression of AMS and downstream transcription factor genes in DN-CLE19 and CLE19-OX transgenic anthers, and TEM observation of the CLE19-OX and ams tapetal cell and microspore. A and B, The relative expression levels of transcription factor genea DYT1, MYB35, AMS, MYB103, and MS1 in DN-CLE19 and CLE19-OX mutants compared to related wild type plants, with ACTIN expression as internal control. C and D, Expression of AMS in the WT anthers. E and F, Expression of AMS in the DN-CLE19 anthers. C and E, anther stage 7; D and F, anther stage 10. G-L, Tapetal cells of WT and CLE19-OX-S anther visualized under the transmission electron microscopy (TEM). G, Wild type; H, CLE19-OX-S. I to L, Microspore cell of CLE19-OX-S and ams anther visualized under the transmission electron microscopy (TEM). I and J, CLE19-OX-S; K and L, ams. T, tapetum; Ela, elaioplasts; Ta, tapetosomes; dMsp, degenerated microspores; Pm, primexine. Scale bars = 1 µm for C to H.



ACP

ACP

Malonyl-ACP

Acetyl-ACP

Fatty acyl-ACP

7-OH-C12:0-CoA

Fatty acyl-CoA (C20-C36) Free fatty acids

4

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-2 -4

CER6 AT1G02190 AT5G55320

Free fatty acids

KCS21 KCS7 KCS15

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Cutin

CYP703A2 CYP98A8 CYP86C3 CYP704B1

Wax ester

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UGT79B6 CHS LAP6 CHIL AT4G00040 LAP5 BEN1 DRL1

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Cutin

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Acetyl-CoA Malonyl-CoA

Phenolics

A

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Wang_Figure 8

UGT72E2 SHT TSM1 CAD2 PAL4 CAD3 PAL1 4CL3 SDT AT1G76470 CCRL6 PAL2 AT4G30470 ACOS5 AT2G23910 4CL5 OPCL1 CRL1

Figure 8. Transcriptomic analyses genes involved in the metabolic of flavonoids, phenolics, wax, cutin, and free fatty acids in WT and CLE19 mutant. A, Metabolic processes of the main compounds of pollen wall. B to F, Fold change in expression levels of genes involved in flavonoids metabolic, cutin metabolic, wax easter metabolic, free fatty acids metabolic, and phenolics metabolic processes.


Wang_Figure 9

Figure 9. Phenotypic analysis of anthers from the WT, ams mutant，DN-CLE19 transgenic， DN-CLE19+/+ ams+/- and DN-CLE19+/+ ams-/- plants. A to E, Alexander stained anthers from the WT (A), ams mutant (B)，DN-CLE19 transgenic (C)，DN-CLE19+/+ ams+/(D) and DN-CLE19+/+ ams-/- (E) plants. F to L, Pollen grains from the WT (F), DNCLE19 transgenic (G to I)，and DN-CLE19+/+ ams+/- (J to L) plants. M, Statistic analysis data to show the percentage of pollens with abnormal exine in the WT, DN-CLE19 transgenic and DN-CLE19+/+ ams+/- plants. Scale bar for anthers: 200µm; and for pollens: 10 µm.


Wang_Figure 10 C

B

A

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DN-CLE19

WT

AMS

MYB103

MS1

AMS

genes involved in pollen wall formation

MS1


Normal CLE19

MYB103

Exine material increased

AMS

MYB103

MS1


Exine material decreased

CLE19DN

Figure 10. Proposed working model for CLE19 in the regulation of anther development. A, In WT anthers, CLE19 peptides interact with their unknown receptor to negatively regulate the AMS-MYB103-MS1 transcriptional cascades to prevent their deleterious overexpression, which triggers the normal expression of downstream exine formation related genes, subsequently regulate the formation of normally well-organized pollen exine. B, In DNCLE19 anthers, the CLE19 peptides without function competitively interact with the unknown CLE19 receptor, which block or obviously reduce the signal transduction from the functional CLE19 to its downstream molecules, therefore leading to the deleterious overexpression of AMS-MYB103-MS1 transcriptional cascades and downstream exine formation related genes, and subsequently affected the normal pollen exine formation. C, In CLE19-OX anthers, over-expression of CLE19 peptide strengthen the suppression of the transcription factors gene expression, resulting in abnomal pollen exine formation.


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