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\begin{document}

\title{Tapetal 3-Ketoacyl-Coenzyme A synthases are involved in pollen coat
lipid accumulation for pollen-stigma interaction in Arabidopsis}



\author[1]{Zaibao Zhang}%
\author[2]{Huadong Zhan}%
\author[2]{Jieyang Lu}%
\author[2]{Shuangxi Xiong}%
\author[2]{Naiying Yang}%
\author[1]{Hongyu Yuan}%
\author[2]{Zhong-Nan Yang}%
\affil[1]{Xinyang Normal University}%
\affil[2]{Shanghai Normal University}%


\vspace{-1em}



  
  \date{May 05, 2024}


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\selectlanguage{english}
\begin{abstract}
Pollen coat lipids form an outer barrier to protect pollen itself and
play essential roles in pollen-stigma interaction. However, the precise
molecular mechanisms underlying pollen coat lipids production,
deposition, regulation and function during anther development remain
largely elusive. 3-ketoacyl-coenzyme A synthases (KCS) are involved in
fatty acid elongation or very long chain fatty acid (VLCFA) synthesis.
Here, we identified six members of Arabidopsis KCS family expressed in
anther. Of them, KCS7, KCS15 and KCS21 express in tapetal cells at
stages 8-10. Further analysis demonstrated that they act downstream of
Male sterility1 (MS1), a regulator for late tapetum development. The
kcs7/15/21 triple-mutant is fertile. Both cellular observation and lipid
staining showed pollen coat lipid was decreased in triple mutant. After
landing on stigma, the wild-type pollen was hydrated in about 5 min
while the triple mutant pollen took about 10 min. Pollen tube growth of
the triple mutant was also delayed. These results demonstrate the
pathway in tapetum to produce pollen coat lipid, and reveal the roles of
tapetal-derived pollen coat lipid for pollen-stigma interaction.%
\end{abstract}%



\sloppy


\textbf{Summary Statement}

Pollen coat lipids play essential roles in pollen-stigma interaction.
Mutation of tapetal-derived KCS proteins (KCS7, KCS15 and KCS21) affects
pollen hydration and tube growth. A critical point of tapetal-derived
lipids in pollen coat is required for pollen hydration.

\textbf{Key words:} pollen coat, lipid, 3-ketoacyl-coenzyme A synthase,
pollen hydration

\textbf{INTRODUCTION}

Pollen-pistil interaction is critical for the successful fertilization
of flowering plants. This interaction is a crucial step in preventing
inbreeding and maintaining species identity, thus contributing to
angiosperm diversity. Pollen-pistil interaction consists of multiple
selective steps, including pollen adhesion, hydration, germination and
polarized tube growth (Bedinger, Broz, Tovar-Mendez, \& McClure, 2017;
Edlund, Swanson, \& Preuss, 2004; Zheng, Lin, Liang, Wang, \& Chen,
2018). Pollen adhesion and hydration are two earliest events in
pollination among species with dry stigmas. These highly regulated
processes require proteins and lipids deriving from pollen wall (Elleman
\& Dickinson, 1990; Safavian \& Goring, 2013).

The mature pollen wall of flowering plants includes three main layers,
intine, exine and pollen coat. Intine is produced by microspores and is
mainly composed of pectin, cellulose and hemicellulose. Exine is
constituted of sexine and nexine (Lou et al., 2014). The main
composition of sexine is sporopollenin. Its precursors are synthesized
by multiple enzymes regulated by a MYB transcription factor MS188 in
tapetum (K. Wang et al., 2018; Z. B. Zhang et al., 2007). Pollen coat
contains many sticky and heterogenous material composed of lipids,
proteins, carotenoids and polysaccharides (Hern\selectlanguage{ngerman}ández-Pinzón, Ross,
Barnes, Damant, \& Murphy, 1999; Piffanelli, Ross, \& Murphy, 1998),
which is crucial to pollen protection from abiotic stresses, successful
pollen contact, hydration and subsequent pollen germination on dry
stigma (Dickinson, 1995; Elleman \& Dickinson, 1990). The pollen coat
contains various components including pollen coat proteins (PCP) and
pollen coat lipids. PCPs are derived from tapetum after its programmed
cell death (PCD) alongside gametophytically derived proteins. In
Arabidopsis, several lipases and lipid-binding oleosin proteins were
identified in PCPs (Mayfield, Fiebig, Johnstone, \& Preuss, 2001). Male
sterility1 (MS1) is a PHD transcription factor for late tapetum
development (Wilson, Morroll, Dawson, Swarup, \& Tighe, 2001). These
sporophyte-derived PCPs are regulated by MS1 in tapetum (Lu et al.,
2020). They may play roles in the early events of pollen-stigma
recognition including pollen adhesion and hydration. S-locus
cysteine-rich protein (SCR), SLR1-BP1/2, PCP-A1(PCP7) and PCP-Bs have
been identified as gametophyte-derived PCPs (Doughty et al., 1998;
Nasrallah \& Nasrallah, 2014; Takayama et al., 2000; L. Wang et al.,
2017). They are likely to play important roles in pollen germination and
pollen tube growth.

Lipids are one of the major subcellular components, and comprise
different combinations and positional distributions of fatty acids. They
play important roles in plants by forming membrane structures, acting as
storage lipids, signaling molecules, and surface coverings (Li-Beisson,
Nakamura, \& Harwood, 2016). The pollen coat lipids display a semi-solid
state and may guide the water transfer from the stigma to pollen during
pollen-stigma interaction (Edlund et al., 2004). In lipid metabolism,
3-ketoacyl CoA synthase (KCS) are involved in fatty acid elongation or
very long chain fatty acid (VLCFA) synthesis (Haslam \& Kunst, 2013;
Joubès et al., 2008). Eceriferum 2 (Cer2) and cer2-like 2 (Cer2l2)
encode BAHD acyltransferase and enhance the elongation of VLCFA from 28
to 30 carbon atoms catalyzed by KCS6 (Fiebig et al., 2000; Haslam et
al., 2015). The \emph{kcs6} single mutant and \emph{cer2cer2l2} double
mutant can produce mature pollen. However, they fail to hydrate (Fiebig
et al., 2000; Haslam et al., 2015; Hulskamp, Schneitz, \& Pruitt, 1995;
Preuss, Lemieux, Yen, \& Davis, 1993). KCS6, CER2 and CER2L2 express in
endothecium suggesting that endothecium plays a role to synthesize
pollen coat lipids for pollen hydration (Zhan, Xiong, Wang, \& Yang,
2018). It was generally considered that pollen coat lipids are derived
from tapetum (Hernández-Pinzón et al., 1999). It is not clear how
tapetum contributes to pollen coat lipids synthesis.

The KCS family in Arabidopsis contains 21 \emph{KCS} members
(Costaglioli et al., 2005; Joubès et al., 2008). In this study, we
reported KCS7, KCS15 and KCS21 were expressed in tapetum. They act
downstream of MS1, a regulator for late tapetum development. The reduced
pollen coat lipid in the triple mutant (\emph{kcs7/15/21} ) and
defective pollen hydration demonstrate that they play redundant roles
for pollen coat lipid synthesis for pollen hydration.

\textbf{RESULTS}

\textbf{Six \emph{KCS} genes expressed in anther of \emph{Arabidopsis}}

\emph{Arabidopsis} genome contains 21 \emph{KCS} genes. Of
them,\emph{KCS1/5/6/7/9/10/11/13/15/20/21} were reported to express in
flowers (Joubès et al., 2008). We performed reverse
transcription-polymerase chain reaction (RT-PCR) analysis, and confirmed
their expression in flowers (Fig. S1). Among them, \emph{KCS5}
,\emph{KCS7} and \emph{KCS15} were specifically expressed in flowers,
while \emph{KCS6, KCS9, KCS10, KCS13, KCS20} and \emph{KCS21} were
relatively highly expressed in flowers (Fig. S1). To further understand
the expression patterns of these KCS proteins in the developing anthers,
the \emph{KCS} genomic sequences were fused to green fluorescent protein
gene (GFP) to make the reporter constructs (\emph{pKCS::KCS-GFP} ) to
transform WT Arabidopsis plants. Transgenic lines
expressing\emph{pKCS::KCS-GFP} in anthers were identified (Fig.1a). The
GFP signals of KCS7-GFP, KCS15-GFP and KCS21-GFP proteins displayed
similar expression patterns (Fig.1a). Their GFP fluorescence was
detected in tapetum at anther stage 8-10 (Fig.1a). No GFP signals were
detected at stage 11 when tapetum programmed cell death (PCD) occur. The
expression of these \emph{KCS} s is quite similar with that of tapetal
sporopollenin synthesis genes (K. Wang et al., 2018). KCS20 was
expressed in tapetum at anther stage 9, and distributed within the
anther locule and pollen at stage 9-11. KCS5-GFP signal was localized in
epidermis at anther stages 10 to 12, while KCS10-GFP signal was
preferentially detected in both epidermis and endothecium at anther
stages 10 and 11 (Fig.1b).

\emph{KCS7, KCS15} \textbf{and \emph{KCS21} act downstream of\emph{MS1}}

Dysfunctional tapetum1 (DYT1), Defective in tapetal development and
function 1 (TDF1), Aborted microspore (AMS), MS188, and MS1 are
essential tapetum regulators which form a genetic pathway (Sorensen et
al., 2003; Wilson et al., 2001; W. Zhang et al., 2006; Z. B. Zhang et
al., 2007; Zhu et al., 2008; Zhu, Lou, Xu, \& Yang, 2011). Based on the
microarray data, \emph{KCS7} , \emph{KCS15} and \emph{KS21} act
downstream of these regulators (Fig. 2a) (Zhu et al., 2011). We
collected the inflorescences of \emph{dyt1} , \emph{tdf1} , \emph{ams}
,\emph{ms188} and \emph{ms1} for quantitative real-time PCR analysis.
These results showed that the expressions of \emph{KCS7} ,
\emph{KCS15}and \emph{KCS21} were downregulated while \emph{KCS20} was
not affected in these mutants (Fig. 2b).

MS1 is the key regulator for late tapetum development in the genetic
pathway (Zhu et al., 2011). The downregulation of \emph{KCS7}
,\emph{KCS15} and \emph{KCS21} in \emph{ms1} mutant suggested that these
three \emph{KCS} genes were regulated by MS1. To further analyze the
molecular regulation between \emph{MS1} and \emph{KCS7} ,
\emph{KCS15}and \emph{KCS21} , we introduced the \emph{pKCS::KCS-GFP}
construct into the \emph{ms1} plants and analyzed the distribution of
KCS-GFP signals (Fig. 1a). The GFP signals could not be detected in
\emph{ms1} mutant while they were clearly observed in the tapetal cells
of wild type (Fig. 1a). These results were consistent with the
microarray and qPCR results (Fig. 2a,b), which further showed that
\emph{KCS7} , \emph{KCS15} and\emph{KCS21} act downstream of \emph{MS1}
. \emph{MYB99} is a direct target of MS1 and its mutant displays a
slight defect in pollen development (Alves-Ferreira et al., 2007). The
expressions of\emph{KCS7,} \emph{KCS15} and \emph{KCS21} were not
changed in\emph{myb99} (Fig. 2c). MS188 is an upstream regulator of MS1
(Zhu et al., 2011). Our recent work showed that many \emph{PCPs} were
downregulated in both \emph{ms188} and \emph{ms1} , and expression
of\emph{MS1} driven by the \emph{MS188} promoter in \emph{ms188} mutant
(\emph{ms188/pMS188::MS1} ) restored the expression of \emph{PCPs} (Lu
et al., 2020). We performed RT-qPCR in the same transgenic line
(\emph{ms188/pMS188::MS1} ), and the expressions of \emph{KCS7}
and\emph{KCS15} were fully restored in \emph{pMS188::MS1} transgenic
plants (Fig. 2d). However, the expression of \emph{KCS21} did not show
any recovery in \emph{pMS188::MS1} transgenic plants compared with that
in\emph{ms1} (Fig. 2d).

\textbf{The pollen coat is reduced in \emph{kcs7/15/21} triple mutant}

To investigate the functions of the \emph{KCS7} , \emph{KCS15}
and\emph{KCS21} in anther development, T-DNA insertion lines
of\emph{KCS7} , \emph{KCS15} and \emph{KCS21} were identified (Fig. S2).
These mutants exhibited drastically reduced expression of the relevant
genes (Fig. S2). No obvious vegetative or reproductive morphological
abnormalities were observed in any of the single (\emph{kcs7, kcs15,
kcs21} ), double (\emph{kcs7/15, kcs7/21, kcs15/kcs21} ) and triple
mutants of \emph{KCS7} , \emph{KCS15} and
\emph{KCS21}(\emph{kcs7/15/21,} Fig. S3). Aborted pollen grains were
occasionally observed in \emph{kcs15} single mutant (70 of 1030), and
the percentage of aborted pollen grains was increased to 13\%
in\emph{kcs7/kcs15/kcs21} triple mutant (110 of 853, Fig. S3).

Lipids were deposited in pollen coat during pollen development, and KCS
proteins were responsible for lipid synthesis. To analysis whether
mutation in \emph{KCS7} , \emph{KCS15} and \emph{KCS21} affect the
pollen coat lipids, the scanning electron microscopy (SEM) and
transmission electron microscope (TEM) observations were used. WT pollen
grains displayed a well-organized exine and a smooth pollen coat.
However, the well-smoothed pollen coat was disturbed, with many
particles in the cavity of triple mutant pollen exine (Fig. 3). In
addition, the severely defective pollen of \emph{kcs7/kcs15/kcs21} was
small and displayed dis-organized exine covered with additional
materials (Fig. 3). The diethyloxadicarbocyanine iodide
(DiOC\textsubscript{2}) was used to stain the fatty acid content of
pollen wall and fatty acid exhibit a red fluorescence of
DiOC\textsubscript{2} staining (Gu et al., 2014). A strong red
fluorescence of DiOC\textsubscript{2} staining was observed in exine and
pollen coat of the WT mature pollen (Fig. 3b,c).
Whereas,\emph{kcs7/kcs15/kcs21} mature pollen showed weaker red signals
with little or no signals observed in the pollen coat (Fig. 3b,c).
Further TEM analysis showed \emph{kcs7/kcs15/kcs21} pollen coat is
disordered and thinner than the wild type (Fig. 3d,e,f,g). Therefore,
these results indicate that lipid content may be reduced in the pollen
coat of

\emph{kcs7/kcs15/kcs21.}

\textbf{Pollen hydration was delayed in \emph{kcs7/15/21} triple mutant}

Pollen coat is generally considered to involve in pollen-stigma
interaction. A pollen hydration assay was carried out to analyze if the
reduced pollen coat in \emph{kcs7/kcs15/kcs21} affect the pollen-stigma
interaction. \emph{Ms188} is a male sterile line without any pollen
inside anther while its stigmas are not affected (Z. B. Zhang et al.,
2007). The pollen hydration assay was carried out by pollinating stigmas
of \emph{ms188} with pollen grains derived from WT and
the\emph{kcs7/15/21} triple mutant (Fig. 4). After landing on stigma,
the wild-type pollen began to absorb water and became spherical (pollen
hydration) in about 5 min (Fig. 4a). However, the hydration of
the\emph{kcs7/15/21} triple mutant pollen occurred in 10 min after
pollination. Accordingly, pollen germination of the triple mutant is
also delayed (Fig. 4a). To determine if the hydration delay is derived
from a defect in water absorption, an \emph{in vitro} hydration of WT
and \emph{kcs7/15/21} triple mutant pollen in PEG 3350 series was
carried out (Fig. S4). The \emph{in vitro} hydration was not
significantly different between WT and \emph{kcs7/15/21} triple mutant
pollen in PEG 3350 series, indicating that the absence of KCS7, KCS15,
and KCS21 proteins did not impair the ability of pollen to absorb water.
To test whether the subsequent steps of pollination were also affected
in \emph{kcs7/15/21} triple mutant, we monitored pollen tube initiation
and growth as previously described (Mayfield \& Preuss, 2000) (Fig.
S5,4b). 20\% of WT pollen grains showed a pollen-tube emergence within
20 min of pollination, while, \emph{kcs7/15/21} triple mutant
pollen-tube emergence was postponed to 30 min (Fig. 4c,S5). After 2 h,
WT pollen produced significantly longer tubes (470 um) and penetrated
into the style, while pollen tubes of \emph{kcs7/15/21} triple mutant
were much shorter (266 um) (Fig. S5). Therefore, the elongation of
pollen tube was postponed in \emph{kcs7/15/21} triple mutant (Fig.
4b,c,S5). Despite the observed defects in pollen hydration and pollen
tube elongation in \emph{kcs7/15/21} triple mutant, there was no
significant difference in seed set in \emph{kcs7/15/21} triple mutant
compared with WT plants.

\textbf{The expression of KCS6 in endothecium persist much later than
KCS7/15/21 in tapetum during anther development}

Our recent work showed that KCS6 interacts with CER2/CER2L2 for pollen
coat very-long-chain fatty acids (VLCFAs) synthesis required for pollen
hydration (Zhan et al., 2018). Therefore, both KCS6 in endothecium and
KCS7/15/21 in tapetum contribute to the synthesis of pollen coat lipids.
Currently no efficient techniques are available to distinguish the
difference of lipids synthesized by the tapetum and endothecium. We
compared the expression of KCS and CER proteins during anther
development to understand the accumulation of the lipids in pollen coat.
In previous work, we obtained transgenic lines of\emph{pKCS6::KCS6-GFP}
, \emph{pCER2::CER2-GFP} and\emph{pCER2L2::CER2L2-GFP} (Zhan et al.,
2018). All the GFP transgenic plants were grown in the same pot and
cultured in the same condition. The expression of KCS6, CER2 and CER2L2
displayed a long period in endothecium from anther stage 8 to 12 (Fig.
5a). KCS7, KCS15 and KCS21 were expressed in tapetum (stage 8 to 10)
(Fig. 1,5). They displayed a shorter time period than KCS6, CER2 and
CER2L2 (Fig. 5a). These results not only suggested the different
functions between KCS7/15/21 and KCS6 in anther development, but also
indicating the different roles of tapetum- and endothecium-derived
lipids. Tapetum-derived lipids may synthesize and deposited in pollen
coat much earlier than endothecium-derived lipids. MS1 protein
accumulates in tapetal cells at anther stage 7 and stage 8 (Yang,
Vizcay-Barrena, Conner, \& Wilson, 2007), overlapping with that of KCS7,
KCS15 and KCS21 (Fig. 5a), further indicating that the expression of
\emph{KCS7} , \emph{KCS15} and\emph{KCS21} were regulated by MS1 (Fig.
2).

\textbf{DISCUSSION}

\textbf{Pollen coat lipids derived from both tapetum and endothecium}

Pollen coat lipids are major part of pollen coat which constitutes the
outer layer of pollen. Previous investigation suggests that some pollen
coat lipids are derived from endothecium (Zhan et al., 2018). It was
generally considered that pollen coat lipids are mainly derived from
tapetum (Hernández-Pinzón et al., 1999). In this work, the reduced
pollen coat lipids in the triple mutant of tapetum expressed genes
(\emph{KCS7} , \emph{KCS15} and \emph{KCS21} ) suggest that
these\emph{KCS} s play a role in tapetum to provide pollen coat lipids
(Fig. 3, 5). KCS7, KCS15 and KCS21 were expressed in tapetum from stage
8 to 10 while tapetum programmed cell death (PCD) occurred after stage
10 (Fig. 1). It is likely that lipids begin to deposit to the outside of
developing microspores before tapetum PCD. With tapetum PCD, all tapetum
compounds including lipids deposit to the pollen coat. The expression of
KCS6, CER2, and CER2L2 in endothecium is much later than the expression
of KCS7, KCS15 and KCS21 in tapetum (Fig. 5a). This further supports the
previous prediction that lipids from endothecium deposit outside of the
lipids from tapetum (Zhan et al., 2018). KCSs catalyze the synthesis of
C20 to C28 lipids, while CER2 and CER2L2 are required for the production
of C28 to C34 lipids (Fiebig et al., 2000; Haslam et al., 2015). This
indicates that the inner pollen coat lipid from tapetum is likely
medium- and long-chain fatty acids (\textless{} 28 carbon atoms), while
outer pollen coat lipid from endothecium is very-long-chain fatty acids
(\textgreater{} 30 carbon atoms).

\textbf{Pollen coat lipids function in pollen-stigma interaction}

The pollen-stigma interaction includes pollen adhesion, hydration and
pollen tube growth. The pollen coat lipids may display a semi-solid
state and waterproof the pollen grain since from its dispersal until its
capture on a compatible stigma (Wheeler, Franklin-Tong, \& Franklin,
2001). Following the pollen-stigma interactions, a `foot-like' structure
is established to enhance pollen-stigma adhesion (Elleman \& Dickinson,
1990). During `foot' formation, the lipids may create a capillary system
to facilitate water transfer from the stigma cell to desiccated pollen,
and pollen hydration occurs. However, it is difficult to clearly
separate the processes of pollen adhesion and hydration in most reports.
The very-long-chain fatty acids from endothecium is predicted to locate
outer pollen coat (Zhan et al., 2018). In the mutant of \emph{kcs6}
and\emph{cer2cer2l2} , the pollen could not hydrate on stigma and the
plants are male sterility (Fiebig et al., 2000; Haslam et al., 2015;
Zhan et al., 2018). This hydration defects might result from adhesion
defect. The delayed hydration in the triple mutant of \emph{kcs7/15/21}
suggest that the tapetum-derived lipids might assist in establishing a
water gradient between the pollen and stigma after pollen adhesion (Fig.
4), and its mutation only postpone the pollen hydration (Fig. 5b). We
propose that the endothecium-derived lipid might function early in
pollen adhesion, while tapetum-derived lipid might function later in
pollen hydration. This hypothesis need to be further studied. Hydration
defects have also been reported in mutants of pollen coat proteins, such
as EXL4, GRP17, PCP-B (Updegraff, Zhao, \& Preuss, 2009; L. Wang et al.,
2017; Wolters-Arts, Lush, \& Mariani, 1998), indicating that lipids and
proteins of pollen coat may work cooperatively to facilitate pollen
hydration.

\textbf{The tapetum regulatory cascade for pollen coat lipid formation}

Tapetum provides nutrition for microspore development, secrets
hydrolases for tetrad wall dissolution, and supply materials for pollen
wall and pollen coat formation during anther development. In
Arabidopsis, the genetic regulatory pathway DYT1-TDF1-AMS-MS188-MS1 is
important for tapetum development and function (Gu et al., 2014; Lou et
al., 2014; Lou et al., 2018; Zhu et al., 2011). In this pathway, AMS
directly regulates ABCG26 for sporopollenin transportation (J. Xu et
al., 2010) and MGT5 to provide the Mg2\textsuperscript{+} for
microspores development (X. F. Xu et al., 2015). MS188 directly
regulates CYP703A2 and other sporopollenin biosynthesis genes for
sporopollenin synthesis and sexine formation (K. Wang et al., 2018;
Xiong et al., 2016). Sexine is the outer pollen wall where pollen coat
(including pollen coat lipids) are deposited. MS1 is a transcription
factor for late tapetum development directly regulated by MS188. It
regulates the expression of multiple pollen coat proteins (Lu et al.,
2020). MS1 also regulates the expression of \emph{KCS7} ,\emph{KCS15}
and \emph{KCS21} for pollen coat lipid synthesis (Fig. 2, 5). This
pollen coat lipid may provide a matrix for assembly of pollen coat PCPs
(Fig. 5b). After tapetum PCD, the endothecium-derived lipids deposited
outside of the pollen coat (Fig. 5b). Together, this work reveals that
following outer pollen wall formation regulated by MS188, its directly
regulating transcription factor MS1 regulates the downstream gene
expression for pollen coat formation. This regulatory cascade is helpful
to make sure that pollen wall and pollen coat are orderly synthesized
during anther development.

\textbf{MATERIALS AND METHODS}

\textbf{Plant materials and growth conditions}

\emph{Arabidopsis thaliana} plants were grown in greenhouse at 21°C and
16h- light/8h-dark photoperiod. T-DNA insertion mutant of
\emph{kcs7}(At1g71160, SALK\_023400), \emph{kcs15} (At3g52160,
SALK\_209946C) and\emph{kcs21} (At5g49070, SALK\_089611) in Col-0
background were all obtained from the ABRC stocks. T-DNA insertion
status of \emph{KCS7} ,\emph{KCS15} and \emph{KCS21} were confirmed
using the T-DNA border primer SALK LBb1.3 in combination with
gene-specific primers listed in Table S1.

\textbf{RNA extraction and RT-qPCR}

Total RNA was isolated using TRIzol reagent (Invitrogen) according to
the manufacturer's instructions. RNA quantity and purity were assessed
with NanoQuant spectrophotometer. First strand cDNAs were synthesized
from DNase I-treated total RNA using cDNA Synthesis Kit (Takara). Gene
expression was normalized using \emph{tubulin} (At5g23860) as the
reference gene, and relative gene expression was calculated as the mean
of three biological replicates and three technical replicates. Gene
specific primers used for RT-qPCR are listed in Table S1.

\textbf{Microscopy and phenotypic characterization}

Pollen viability was assessed using Alexander's staining. For scanning
electron microscopic (SEM) analysis, mature pollen grains from freshly
dehisced anthers were mounted and coated with gold on stubs. The samples
were then immediately observed using FESEM (SU8010). TEM (transmission
electron microscopy) analysis was performed as described previously (Z.
B. Zhang et al., 2007). The procedure of semi-thin section and lipid
staining were performed as described previously (Lou et al., 2014; Z. B.
Zhang et al., 2007).

\textbf{Protein localization analysis}

The transgenic plants of \emph{KCS5-GFP, KCS7-GFP, KCS10-GFP, KCS15-GFP}
, \emph{KCS20-GFP} and \emph{KCS21-GFP} were constructed by fusing
\emph{GFP} with genomic sequences of \emph{KCS5, KCS7, KCS10, KCS15} ,
\emph{KCS20} and \emph{KCS21} driven by their native promoters. The
flower buds of the transgenic plants were harvested and the anthers were
isolated. The isolated anthers were scanned without fixation with Zeiss
LSM510 confocal scanning microscope.

\textbf{Semi\emph{-in vivo} pollen hydration and pollen tube growth
assay}

For pollen hydration, freshly opened \emph{ms188} pistil was cut and
attached upright on agar (1.0\%). Then pollen grains (n =
50\textasciitilde{}80) from a freshly dehiscing anther were transferred
onto the stigmatic papilla. The behavior of the pollen was captured
under a microscope immediately after pollinations were initiated.
For\emph{in vivo} pollen tube growth analysis, pollinations were
initiated on \emph{ms188} stigmas and allowed to proceed for 2 h as
described previously (L. Wang et al., 2017). Then stigmas were fixated
overnight (60\% v/v ethanol, 30\% v/v chloroform, 10\% v/v acetic acid),
incubated in 8 M NaOH for 20 min, and washed in dH\textsubscript{2}O
three times (5 min/each). Samples were dyed in 0.1\% decolourized
aniline blue (0.1\% w/v aniline blue in 0.1 M
K\textsubscript{3}PO\textsubscript{4}, pH11) for 1 h and images were
captured with an Olympus BX51 microscope. Pollen tube growth was
calculated as the mean of three biological replicates. All statistical
analyses were carried out using Microsoft Excel.

\textbf{ACKNOWLEDGMENTS}

This work was supported by the grants from National Natural Science
Foundation of China (31870296, 31700277), Natural Science Foundation of
Henan (No. 182300410063), Nanhu Scholars Program for Young Scholars of
Xinyang Normal University, the Shanghai Science and Technology Committee
(18DZ2260500, 17DZ2252700), and the Collaborative Innovation Center for
Modern Crop Production to H.Z.

\textbf{Author contributions}

Z.Y., Z.Z. and H.Z. planned and designed the research and wrote the
manuscript; Z.Z. and H.Z. were involved with all aspects of the research
with S.X., J.L. and N.Y. contributing to expression analysis, SEM and
TEM work; H.Y. assisted with critical assessment of the manuscript.

\textbf{Figure legend}

\textbf{Figure 1. Expression analysis of KCS proteins during anther
development.} a, The expression of KCS7-GFP, KCS15-GFP and KCS21-GFP
fusion proteins were initially detected in tapetum at stage 8 and
subsequently accumulated in tapetum and anther locule at stages 9 and
10. After stage 10, the GFP signal was disappeared. The expression of
KCS20-GFP fusion protein was initially detected in tapetum at stage 9
and subsequently accumulated in anther locule and pollen at stages 10
and 11. GFP signal in tapetum was shown in enlarged view. No GFP signals
of KCS7-GFP, KCS15-GFP and KCS21-GFP were identified in
\emph{ms1}background. T, tapetum. Bars= 20 \selectlanguage{greek}μ\selectlanguage{english}m. b, The expression of
KCS10-GFP fusion protein was detected in epidermis and endothecium at
stage 10 and stage 11, while the GFP signal of KCS5 was only detected in
epidermis from anther stage 10 to 12. Bars= 10 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 2. MS1 regulates the expression of \emph{KCS7, KCS15 and
KCS21}.} a, Heatmap showing the expression of \emph{KCS7, KCS15,}
and\emph{KCS21} genes were downregulated in \emph{dyt1, tdf1, ams,
ms188,}and \emph{ms1} mutants, while the expression of \emph{KCS20} was
not affected in these mutants. b, qPCR analysis of \emph{KCS} genes
in\emph{dyt1, tdf1, ams, ms188,} and \emph{ms1} mutants. c, RT-PCR
analysis of \emph{KCS7, KCS15,} and \emph{KCS21} genes in \emph{ms1,}and
\emph{myb99} mutants. d, Expression of \emph{KCS7} , \emph{KCS15}
and\emph{KCS21} genes was analyzed in \emph{pMS188::MS1} transgenic
plants in the \emph{ms188} homozygous background (rescue).

\textbf{Figure 3.} \textbf{Pollen coat was affected
in\emph{kcs7}/\emph{15}/\emph{21} triple mutant.} a, Scanning electron
microscopic (SEM) analysis of exine and pollen coat morphology in WT
and\emph{kcs7} /\emph{15} /\emph{21} triple mutant plants. Red arrows
and blue arrows indicated irregular pollen coat and particles,
respectively. b and c, Lipid staining of semi-thin sections of WT
and\emph{kcs7} /\emph{15} /\emph{21} triple mutant plants at anther
stage 12. The red fluorescence indicates the lipid staining on mature
pollen grains (white arrow). Bars= 10 \selectlanguage{greek}μ\selectlanguage{english}m. d, e, f and g, Analysis of
pollen wall structure by transmission electron microscopy (TEM). The
ultrastructure of the pollen coat of \emph{kcs7} /\emph{15}
/\emph{21}triple mutant showed decreased and disordered compared with
the wild type. Bars= 5 \selectlanguage{greek}μ\selectlanguage{english}m in d, e, and bars= 1 \selectlanguage{greek}μ\selectlanguage{english}m in f, g.

\textbf{Figure 4. \emph{In vivo} pollen hydration and pollen tube growth
analysis in \emph{kcs7}/\emph{15}/\emph{21} triple mutant.} a, Diagram
of pollen hydration in WT and \emph{kcs7} /\emph{15} /\emph{21} triple
mutant. b, Diagram of pollen germination on stigma. c, \emph{In
vivo}pollen tube emergence after manual pollination at 10 min, 20 min,
30 min, 45 min, 1 h and 2 h post-pollination. Pollen tubes were emerged
in WT pollen after manual pollination at 20 min, while pollen tubes were
identified in \emph{kcs7} /\emph{15} /\emph{21} triple mutant at 30 min.
Pollen grains were applied to stigmas of \emph{ms188} . Bars= 10 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 5. A proposal model for anther derived lipids in pollen
wall development.} a, Expression pattern of KCS6, KCS15, CER2, and
CER2L2 proteins during anther development. The expression of KCS15-GFP
was detected in tapetum, while the expression of KCS6-GFP, CER2-GFP and
CER2L2-GFP fusion proteins were detected in endothecium. Bars= 20 \selectlanguage{greek}μ\selectlanguage{english}m. b,
A proposed model for tapetum and endothecium in biosynthesis of lipids
for pollen wall development. In tapetum, MS1 regulates the expression
of\emph{KCS7} , \emph{KCS15} and \emph{KSC21} , and the KCS-mediated
lipids were deposited in pollen coat. In endothecium, CER2 and CER2L2
interact with KCS6 to modulate the biosynthesis of pollen coat VLCFAs
for pollen hydration.

\textbf{Supplement Data}

\textbf{Figure S1.} Reverse transcription polymerase chain reaction
(RT-PCR) expression analysis of eleven \emph{KCS} genes in root, stem,
leaf and flowers.

\textbf{Figure S2.} RT-PCR analysis of \emph{kcs7} , \emph{kcs15}
,\emph{kcs21} single mutants and \emph{kcs7/15/21} triple mutant.

\textbf{Figure S3.} Phenotypes of single, double and triple mutants
of\emph{kcs7} , \emph{kcs15} and \emph{kcs21} . a and b, Plants, anthers
and pollen grains of \emph{kcs7} , \emph{kcs15} , \emph{kcs21} single
mutants and \emph{kcs7} /\emph{15} /\emph{21} triple mutant. Black
arrows indicated aborted pollen grains in \emph{kcs15} and\emph{kcs7}
/\emph{15} /\emph{21} triple mutants. Bars= 20 \selectlanguage{greek}μ\selectlanguage{english}m. c, Cell biological
analyses of WT and \emph{kcs7} /\emph{15} /\emph{21} triple mutant
anthers using semi-thin transverse sections. Bars= 20 \selectlanguage{greek}μ\selectlanguage{english}m. d, Pollen
grains from the single, double and triple mutants of the\emph{KCS7} ,
\emph{KCS15} and \emph{KCS21} genes visualized under scanning electron
microscope. Scale bar for huge pollen 50 \selectlanguage{greek}μ\selectlanguage{english}m; for single pollen 10 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure S4.} Width of \emph{kcs7} /\emph{15} /\emph{21} triple
mutant and wild-type pollen when placed in a PEG 3350 series. Each point
represents the average width of 100 pollen grains.

\textbf{Figure S5.} \emph{kcs7} /\emph{15} /\emph{21} triple mutant and
wild-type \emph{in vivo} pollen tube growth. a, Emergence of pollen tube
with aniline blue staining. Scale bar represents 10 um in figures of 10,
20, 30 and 45 min, 80 um in figure of 1 hour, 100 um in figure of 2
hour. b, The length of pollen tubes at different hours.

\textbf{Supplemental Table 1.} Primers used in this study

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