Plant Cell Advance Publication. Published on May 18, 2017, doi:10.1105/tpc.16.00484
RESEARCH ARTICLE
Carbonic Anhydrases Function in Anther Cell Differentiation Downstream of the Receptor-like Kinase EMS1 Jian Huang,a Zhiyong Li,a Gabriel Biener,b Erhui Xiong,a,c Shikha Malik,a Nathan Eaton,a Catherine Z. Zhao,d Valerica Raicu,a,b Hongzhi Kong,e and Dazhong Zhaoa,f,1 a
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA Department of Physics, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA c College of Life Science, Henan Agricultural University, Zhenzhou 450002, China d Whitefish Bay High School, Whitefish Bay, WI 53217, USA e State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China f College of Life Science, Shandong Normal University, Jinan 250014, China b
1
Corresponding Author:
[email protected].
Short title: Signaling Role of Carbonic Anhydrases One-sentence summary: β-carbonic anhydrases are post-translationally modified by EMS1 and act as direct downstream effectors of this receptor-like kinase, highlighting their crucial role in cell differentiation. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Dazhong Zhao (
[email protected]).
ABSTRACT Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS MICROSPOROCYTES1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive microsporocytes during early anther development in Arabidopsis thaliana. Here, we report the identification of β-carbonic anhydrases (βCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with βCA proteins. Loss-of-function of βCA genes caused defective tapetal cell differentiation, while overexpression of βCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates βCA1 at four sites, resulting in increased βCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of βCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. βCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of βCA in controlling cell differentiation and provide insights into the post-translational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation.
1 ©2017 American Society of Plant Biologists. All Rights Reserved
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INTRODUCTION
2 3
In flowering plants, successful sexual reproduction depends on the normal development of
4
anthers, which produce the male gametophyte, pollen. Within each of the four lobes
5
(microsporangia) of a mature anther, the central reproductive microsporocytes (or pollen mother
6
cells) are surrounded by four concentrically organized somatic cell layers: the epidermis,
7
endothecium, middle layer, and tapetum (from the outside to inside) (Goldberg et al., 1993; Scott
8
et al., 2004; Zhao, 2009; Walbot and Egger, 2016). Microsporocytes give rise to pollen via
9
meiosis, while the somatic cell layers, particularly the tapetum, are required for pollen
10
development and release. Due to the central importance of anthers for plant yield and breeding, it
11
is imperative to obtain an in-depth understanding of anther cell differentiation.
12
In Arabidopsis thaliana, the tapetum consists of a single layer of endopolyploid cells,
13
which enclose microsporocytes, tetrads, microspores, and developing pollen grains as anther
14
development progresses (Goldberg et al., 1993; Scott et al., 2004; Walbot and Egger, 2016).
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Tapetum development comprises three stages: differentiation, maturation, and programmed cell
16
death (PCD). Early differentiated tapetal cells secrete enzymes required for the release of haploid
17
microspores from meiotic tetrads (Pacini et al., 1985; Clement and Pacini, 2001; Ishiguro et al.,
18
2001; Hsieh and Huang, 2007; Parish, 2010). During endoreduplication, mature tapetal cells,
19
characterized by multiple nuclei, non-photosynthetic plastids (elaioplasts), and tapetosomes, are
20
highly metabolically active, and thereby serve as a nutritional source to provide energy and
21
materials (polysaccharides, lipids, and proteins) for the development of microspores and pollen
22
grains. In the later stage, tapetal cells are degenerated via PCD and the resulting materials are
23
deposited onto pollen to form the pollen coat (tryphine). Mutants lacking tapetum or plants with
24
genetically ablated tapetum fail to produce pollen grains (Mariani et al., 1990; Zhao et al., 2002;
25
Yang et al., 2003; Huang et al., 2016b). In addition, precocious or delayed tapetum degeneration
26
causes abnormal pollen development (Zhang et al., 2006; Zhang and Yang, 2014). Furthermore,
27
stress-induced male sterility is mainly ascribed to alterations in tapetum development (Parish et
28
al., 2012; De Storme and Geelen, 2014; Smith and Zhao, 2016). Although the tapetum is
29
essential for pollen development, the molecular mechanisms underlying tapetal cell
30
differentiation and functional maintenance are not clear.
31
We previously demonstrated that the EMS1 (EXCESS MICROSPOROCYTES1) 2
32
leucine-rich repeat receptor-like kinase (LRR-RLK)-linked signaling pathway plays a
33
fundamental role in the differentiation of somatic tapetal cells and reproductive microsporocytes
34
during early anther development. In Arabidopsis, anthers in ems1 [also known as exs (extra
35
sporogenous cells)] and tpd1 (tapetum determinant1) mutants have no tapetal cells; instead, they
36
produce excess microsporocytes (Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003). We
37
identified TPD1 as a putative small protein ligand of EMS1 (Jia et al., 2008). Our results further
38
showed that TPD1 is secreted from precursors of microsporocytes and then activates EMS1,
39
which is localized at the plasma membrane of tapetal precursor cells/tapetal cells. The
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TPD1-EMS1 signaling pathway initially promotes periclinal division of parietal cells to form
41
tapetal precursor cells, and later determines and maintains the fate of functional tapetal cells.
42
Additionally, tapetal cells suppress microsporocyte proliferation (Huang et al., 2016c). We
43
recently also found that the SERK1/2 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE
44
KINASE) LRR-RLK acts as a potential co-receptor of EMS1 (Li et al., 2017). However, the
45
downstream signaling molecules of TPD1-EMS1/SERK1/2 are currently unclear.
46
In this study, we identified β-carbonic anhydrases (βCAs) as EMS1-interacting proteins.
47
CAs catalyze the rapid, reversible reaction CO2+H2O↔HCO3‾+H+ (Tripp et al., 2001; Alterio et
48
al., 2009; Becker et al., 2011). In animals, CAs are important for various processes in normal and
49
especially pathological states, such as gluconeogenesis, lipogenesis, ureagenesis, and tumor
50
formation (Chegwidden et al., 2000; Supuran, 2008; Davis et al., 2010). In plants, CAs
51
principally belong to three independently evolved families: α, β, and γ, with the β family being
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predominant (Badger, 2003; Rowlett, 2014). βCAs function in photosynthesis mainly by
53
concentrating CO2 and facilitating the movement of CO2 and HCO3–. Arabidopsis has six βCAs
54
(βCA1 to βCA6), and each βCA has various splice forms (Wang et al., 2014). βCA1
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loss-of-function mutants show reduced seedling establishment (Ferreira et al., 2008). βCA1 and
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βCA4 coordinate with EPIDERMAL PATTERNING FACTORS2 (EPF2) and CO2 RESPONSE
57
SECRETED PROTEASE (CRSP) to control stomatal development and movement (Hu et al.,
58
2010; Engineer et al., 2014). Furthermore, βCA4 and its interactor protein PIP2;1 aquaporin are
59
required for CO2-induced stomatal movement (Wang et al., 2016a). A recent study demonstrated
60
that βCA2 and βCA4 are important for optimal plant growth via affecting amino acid
61
biosynthesis but not photosynthesis (DiMario et al., 2016). Thus, βCAs may possess many
62
unidentified non-photosynthetic roles in plant growth and development. 3
63
In this study, we showed that βCA1, βCA2 and βCA4 biochemically interact with EMS1.
64
Loss-of-function of βCA genes led to abnormal tapetal cell differentiation, whereas
65
overexpression of βCA1 caused the formation of extra tapetal cells. EMS1 phosphorylates βCA1,
66
βCA2 and βCA4. The phosphorylation of βCA1 significantly increases its activity. Moreover,
67
phosphorylation-blocking mutations caused the failure of βCA1 to recover tapetal cell
68
differentiation in the βca1 βca2 βca4 mutant; however, a phosphorylation mimic mutation
69
promoted the formation of tapetal cells. βCAs likely regulate tapetal cell pH. Our results suggest
70
that βCAs serve as the direct downstream targets of EMS1, which highlights a role for βCAs in
71
controlling cell differentiation and provides a paradigm for LRR-RLK-linked signal transduction
72
pathways. Furthermore, our research sheds light on the post-translational modification of CA via
73
receptor-like kinase-mediated phosphorylation.
74 75 76
RESULTS
77 78
EMS1 Interacts With βCAs
79 80
To identify the downstream targets of EMS1, we performed yeast two-hybrid (Y2H) screening
81
for potential EMS1-interacting proteins using the EMS1 kinase domain (852-1192 amino acids)
82
as the bait (Figure 1). We generated a cDNA library using young buds with stage-5 and -6
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anthers, in which EMS1 expression is at its peak. Out of 129 selected positive clones, 79 cDNAs
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encoded β-carbonic anhydrase 1 (βCA1, At3g01500). Three splice variants for βCA1 were found
85
in The Arabidopsis Information Resource (TAIR). Our Y2H screening only identified βCA1.3
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(16/79) and a new splice variant (63/79) which lacks the first and last exon, named βCA1.4
87
(Supplemental Figure 1). Y2H tests showed that βCA1.3 and βCA1.4 interacted with EMS1 at a
88
higher level than βCA1.1 and βCA1.2 (Supplemental Figure 2). Phylogenetic analysis
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demonstrated that βCA1 and βCA2 share the highest similarity and are closely related to βCA3
90
and βCA4, whereas βCA5 and βCA6 form a different clade (Supplemental Figure 3,
91
Supplemental File 1). All βCA genes except βCA3 have different numbers of splice variants
92
(TAIR). Further Y2H assays showed that βCA2.2, βCA3, βCA4.1, βCA5.2, and βCA6.1, which
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have the closest structures to βCA1.4 compared with other splice forms, also interacted with 4
94 95
EMS1, possibly at different levels (Figure 1A). We then examined interactions between EMS1 and βCAs by performing a bimolecular
96
fluorescence complementation (BiFC) assay with Arabidopsis mesophyll protoplasts. EMS1 and
97
βCAs were fused to the amino- and carboxy-terminal halves of Enhanced Yellow Fluorescent
98
Protein (EYFP), respectively. EYFP signals were observed at the plasma membrane when
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EMS1-cEYFP was co-transfected with βCA1.4-nEYFP (Figure 1B), βCA2.2-nEYFP (Figure
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1C), and βCA4.1-nEYFP (Figure 1E); however, no EYFP signal was observed at the plasma
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membrane when EMS1-cEYFP was co-transfected with βCA3-nEYFP (Figure 1D),
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βCA5.2-nEYFP (Figure 1F), or βCA6.1-nEYFP (Figure 1G). Protein gel blot analysis showed
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EMS1-cEYFP and βCAs-nEYFP were expressed at similar levels in the BiFC assay
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(Supplemental Figure 4). As controls, no EYFP signal was observed from EMS1-cEYFP and
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αCA1.1-nEYFP or BRI1-cEYFP and βCA1.4-nEYFP combinations (Figure 1H and 1I). Our
106
results support the notion that EMS1 specifically interacts with βCA1.4, βCA2.2 and βCA4.1 at
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the plasma membrane in living cells.
108
We also performed a Förster Resonance Energy Transfer (FRET) experiment to quantify
109
the EMS1-βCA1.4 interaction using a two-photon micro-spectroscope (Raicu et al., 2009; Biener
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et al., 2014). FRET efficiencies were calculated using images generated by unmixing measured
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spectra of the donor (EMS1-CFP) at 860 nm (Figure 1J) and acceptor (βCA1.4-EYFP) following
112
excitation at 860 nm (Figure 1K), as well as 960 nm (Figure 1L). A FRET efficiency of 13±4%
113
(Supplemental Table 1) was obtained from 29 regions of interest at the plasma membrane,
114
indicating that the EMS1-βCA1.4 interaction occurs at the plasma membrane.
115
Finally, we carried out co-immunoprecipitation (co-IP) experiments to examine whether
116
EMS1 interacts with βCAs by representatively testing βCA1 in planta. Membrane proteins
117
extracted from young buds of ProEMS1:EMS1-4xcMyc ProβCA1:βCA1-Flag double transgenic
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plants, where both EMS1 and βCA1 were expressed in early anthers, were immunoprecipitated
119
with an anti-cMyc antibody. Protein gel blot analysis using anti-cMyc and anti-Flag antibodies
120
against the immunoprecipitated proteins showed that βCA1-Flag was co-immunoprecipitated by
121
EMS1-4xcMyc (Figure 1M), indicating that EMS1 interacts with βCA1 in planta. To test
122
whether the EMS1 ligand TPD1 affects the EMS1 and βCA1 interaction, we performed co-IP
123
using a protoplast transient expression system. βCA1.4-Flag was expressed in protoplasts
124
prepared from the leaves of Pro35S:EMS1-cMyc and Pro35S:EMS1-cMyc 5
125
Pro35S:TPD1p-GFP-∆TPD1 transgenic plants (Huang et al., 2016c). Protein gel blot analysis
126
using an anti-Flag antibody against membrane proteins immunoprecipitated by anti-cMyc
127
detected a similar level of βCA1.4-Flag in the presence and absence of TPD1 (Supplemental
128
Figure 5), suggesting that TPD1 is not essential for the interaction between βCA1.4 and EMS1.
129
Collectively, our results show that EMS1 biochemically interacts with βCA1, βCA2 and
130
βCA4, suggesting that βCA1, βCA2 and βCA4 may act as downstream signaling effectors of
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EMS1.
132 133 134
βCAs Are Expressed in Tapetal Cells
135 136
To investigate whether βCAs serve as EMS1 downstream signaling molecules, we examined the
137
expression of βCAs in anthers and tapetal cells (Figure 2). The βCA1 gene has 10 exons, which
138
leads to four alternative splice variants (Supplemental Figure 1). Our RT-PCR results showed
139
that the expression of βCA1.3 was dominant in leaf, seedling, silique and stem tissues, while
140
βCA1.4 was only weakly expressed in seedlings (Supplemental Figure 6). All βCA1 variants
141
except βCA1.1 were expressed in young buds (Figure 2A); however, only βCA1.3 and βCA1.4
142
were detected in wild-type stage-5/6 anthers (Figure 2A). In ems1 stage-5/6 anthers, the
143
expression of βCA1.3 was not altered compared to wild type. Conversely, the expression of
144
βCA1.4 was not detected (Figure 2A), suggesting that βCA1.4 might be a major downstream
145
molecule of EMS1. We also detected the expression of βCA2 and βCA4 in wild-type stage-5/6
146
anthers, and their expression was reduced in ems1 stage-5/6 anthers (Figure 2B). We did not
147
observe the expression of βCA3 in wild-type or ems1 stage-5/6 anthers (Figure 2B). Our results
148
suggest that βCA1 (mainly βCA1.4), together with βCA2 and βCA4, are potential downstream
149
molecules of EMS1.
150
Using the βCA1 1.4 kb promoter region and transcribed region without the last intron and
151
exon, we generated ProβCA1:βCA1-GFP transgenic plants expressing βCA1.3-GFP and
152
βCA1.4-GFP proteins. We observed a weak GFP signal in epidermal cells in stage-4 anthers
153
(Figure 2C). At stage 5 and stage 6, two key stages for tapetal cell differentiation, GFP signals
154
strongly accumulated in tapetal cells (Figure 2D and 2E); however, GFP signals became
155
gradually reduced in tapetal cells of stage-7 (Figure 2F) and stage-8 (Figure 2G) anthers. GFP 6
156
signals were not detectable in tapetal cells at stage 10, when tapetal cell degeneration initiates
157
(Figure 2H). Furthermore, we found that βCA1 was localized at the plasma membrane and in the
158
cytoplasm of tapetal cells in stage-5 anthers (Figure 2I to 2K). We also generated
159
ProβCA2:βCA2-GFP, ProβCA3:βCA3-GFP and ProβCA4:βCA4-GFP transgenic plants. Our
160
results showed that instead of βCA3 (Figure 2M), βCA2 (Figure 2L) and βCA4 (Figure 2N) were
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detected in both tapetal cells and microsporocytes of stage-5 anthers.
162
Taken together, similar to EMS1 (Huang et al., 2016c), βCA1, βCA2 and βCA4 are
163
localized in tapetal cells, supporting the notion that βCA1, βCA2, and βCA4 are downstream
164
signaling partners of EMS1.
165 166 167
βCAs Are Required for Tapetal Cell Differentiation
168 169
To investigate the function of βCAs in anther cell differentiation, we analyzed the phenotypes of
170
βCAs loss-of-function mutants (Figure 3). We did not detect mutant phenotypes in βca1
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(SALK_106570), βca2 (CS303346: identified in this study, Supplemental Figure 7), βca3
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(Salk_144106) or βca4 (CS859392) single mutant anthers, nor in βca1 βca2, βca1 βca3, βca1
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βca4, βca2 βca4 double or βca1 βca2 βca3 triple mutant anthers. Compared to wild-type plants
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(Figure 3A and 3H), βca1 βca2 βca4 triple mutant plants were smaller and did not produce
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pollen grains in anthers (Figure 3B and 3I). Employing the artificial miRNA approach (Schwab
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et al., 2006), we used the 35S promoter to knock down βCA1 to βCA4 genes. Among the 80
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Pro35S:amirβCA1-4 transgenic plants examined, 52.5% (42/80) of plants were small and only
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formed a few dead pollen grains (Figure 3C and 3J). To rule out the possibility that the pollen
179
production defect was caused by abnormal vegetative growth, we specifically knocked down
180
βCA1 to βCA4 in tapetal cells using the tapetum-specific promoter A9 (Paul et al., 1992; Feng
181
and Dickinson, 2010). Among the 90 examined ProA9:amirβCA1-4 transgenic plants, all of
182
which showed normal vegetative growth (Figure 3D), 68.9% (62/90) of plants produced
183
completely empty anthers (Figure 3K), indicating that βCAs are required for pollen formation.
184
To confirm that βCAs are responsible for pollen development, we conducted
185
complementation experiments. Our results showed that 64.0% (16/25) of ProβCA1:βCA1/βca1
186
βca2 βca4 plants (Figure 3E), 60.6% (20/33) of ProβCA2:βCA2/βca1 βca2 βca4 plants 7
187
(Supplemental Figure 8A) and 55.0% (22/40) of ProβCA4:βCA4/βca1 βca2 βca4 (Supplemental
188
Figure 8B) plants had normal growth and development. Although some plants were still smaller
189
than the wild type, 80.0% (20/25) of ProβCA1:βCA1/βca1 βca2 βca4 plants (Figure 3L), 75.8%
190
(25/33) of ProβCA2:βCA2/βca1 βca2 βca4 plants (Supplemental Figure 8C) and 82.5% (33/40)
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of ProβCA4:βCA4/βca1 βca2 βca4 plants (Supplemental Figure 8D) produced normal pollen
192
grains. Moreover, although 70.0% (14/20) of ProA9:βCA1.4/βca1 βca2 βca4 plants were similar
193
to βca1 βca2 βca4 plants in terms of vegetative growth, their seed production was restored
194
(Figure 3F). Further analysis revealed that these plants produced normal pollen grains (Figure
195
3M). By contrast, 100% (22/22) of ProA9:βCA1.3/βca1 βca2 βca4 plants exhibited short siliques
196
(Figure 3G) and had no pollen grains (Figure 3N), suggesting that βCA1.4 but not βCA1.3 is
197
mainly responsible for early anther development. In conclusion, our results support the notion
198
that the disruption of βCA1, βCA2 and βCA4 caused the failure of pollen formation.
199
To further investigate the function of βCAs in anther development, we analyzed anther
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cell differentiation in semi-thin sections (Figure 4). Our results showed that at stage 6, wild-type
201
anther lobes contained four somatic cell layers (epidermis, endothecium, the middle layer and
202
tapetum) and microsporocytes in the center (Figure 4A); however, tapetal-like cells were
203
vacuolated in βca1 βca2 βca4 mutant anthers (Figure 4B). Similar defects were observed in
204
anthers of Pro35S:amirβCA1-4 (Figure 4C) and ProA9:amirβCA1-4 (Figure 4D) plants in which
205
βCA1 to βCA4 were knocked down, with defects observed throughout the plant and specifically
206
in the tapetum, respectively. At stage-7 wild-type anthers, tetrads had formed (Figure 4F). By
207
contrast, in βca1 βca2 βca4 (Figure 4G), Pro35S:amirβCA1-4 (Figure 4H) and
208
ProA9:amirβCA1-4 (Figure 4I) anthers, tetrads had not formed. Instead, tapetal-like cells
209
continuously expanded and microsporocytes were degenerating. In stage-9 wild-type anthers,
210
tapetal cells were still present and the microspore wall was becoming thickened, indicating
211
normal pollen development (Figure 4K). Conversely, in βca1 βca2 βca4 (Figure 4L),
212
Pro35S:amirβCA1-4 (Figure 4M) and ProA9:amirβCA1-4 (Figure 4N) anthers, both tapetal-like
213
cells and microsporocytes were degenerated, resulting in empty anther lobes. In
214
ProA9:βCA1.4/βca1 βca2 βca4 plants, anther cell differentiation was the same as that of
215
wild-type plants (Figure 4E, 4J and 4O).
216 217
We then examined the expression of A9, a tapetum-specific marker gene, via in situ hybridization and qRT-PCR. At stage 6, the A9 gene was strongly expressed in tapetal cells in 8
218
wild-type anthers (Figure 4P and 4Y), but the expression levels of A9 were significantly reduced
219
in tapetal-like cells in βca1 βca2 βca4 (Figure 4Q and 4Y), Pro35S:amirβCA1-4 (Figure 4R and
220
4Y) and ProA9:amirβCA1-4 (Figure 4S and 4Y) anthers. As expected, A9 expression was
221
normal in ProA9:βCA1.4/βca1 βca2 βca4 tapetal cells (Figure 4T).
222
Elaioplasts (non-photosynthetic plastids) and tapetosomes, which are tapetum-specific
223
lipid-accumulating organelles, serve as nutrient sources to provide energy and materials
224
(polysaccharides, lipids, and proteins) for the development of microspores and pollen grains
225
(Dickinson, 1973; Owen and Makaroff, 1995; Clement and Pacini, 2001; Hsieh and Huang,
226
2007). Thus, we introduced the elaioplast marker FIB1a-GFP and the tapetosome marker
227
GRP17-GFP (Suzuki et al., 2013) into ProA9:amirβCA1-4 plants. Confocal microscopy analysis
228
revealed strong FIB1a-GFP signals in tapetal cells of stage-8 FIB1a-GFP (wild-type) anthers
229
(Figure 4U), but in the anthers from the 12 ProA9:amirβCA1-4 FIB1a-GFP plants examined, the
230
number and size of FIB1a-GFP signals were strongly reduced (Figure 4V). Similarly, compared
231
to the wild type (Figure 4W), GRP17-GFP signals were strongly reduced in stage-10 anthers
232
from the 17 analyzed ProA9:amirβCA1-4 GRP17-GFP plants (Figure 4X). Our results indicate
233
that βCAs are involved in the formation of elaioplasts and tapetosomes during tapetal cell
234
differentiation.
235
In summary, tapetal cell differentiation is impaired in βCA loss-of-function mutants,
236
resulting in the early degeneration of tapetal cells and consequently failed pollen formation. Thus,
237
our results support the notion that βCAs are required for tapetal cell differentiation.
238 239 240
Overexpression of βCA1 Leads to Extra Tapetal Cells
241 242
To further investigate whether βCAs are essential for tapetal cell differentiation, we
243
overexpressed the βCA1 gene by generating Pro4x35S-βCA1:βCA1 transgenic plants using four
244
cauliflower mosaic virus (CaMV) 35S enhancers (Weigel et al., 2000) and the βCA1 genomic
245
fragment (Figure 5). Among the 178 transgenic plants analyzed, 25.8% (46/178) of plants
246
showed short siliques (Supplemental Figure 9A and 9B), and a majority of pollen grains were
247
dead in the anthers (Supplemental Figure 9D and 9E). Moreover, 10.1% (18/178) of plants were
248
completely sterile and did not produce any pollen grains (Supplemental Figure 9C and 9F). 9
249
Further analysis of semi-thin sections showed that the 4x35S-ProβCA1:βCA1 anthers formed
250
extra tapetal-like cells at stage 6 (Figure 5A, 5B and 5O). At stage 7, extra vacuolated
251
tapetal-like cells were observed in Pro4x35S-βCA1:βCA1 anthers (Figure 5C, 5D and 5P).
252
However, when we introduced Pro4x35S-βCA1:βCA1 into the ems1 mutant,
253
Pro4x35S-βCA1:βCA1/ems1 anthers had the same phenotype as ems1 mutant anthers at stage 6
254
(Figure 5E and 5F), suggesting that the βCA1 gain-of-function effect is dependent on the normal
255
functioning of EMS1.
256
To confirm the cellular identity of the observed extra tapetal-like cells, we introduced
257
ProA9:mGFP5er, a tapetal cell marker, into Pro4x35S-βCA1:βCA1 plants. Confocal microscopy
258
analysis showed that the GFP signal was observed in a single layer of tapetal cells in wild-type
259
anthers at stage 6 (Figure 5G) and 7 (Figure 5I); however, in Pro4x35S-βCA1:βCA1 anthers,
260
additional cells expressed ProA9:mGFP5er at stage 6 (Figure 5H) and 7 (Figure 5J). Moreover,
261
the in situ hybridization results, which agreed with the results of confocal microscopy, showed
262
stronger and expanded expression of A9 in Pro4x35S-βCA1:βCA1 anthers (Figure 5L and 5N)
263
compared with wild type (Figure 5K and 5M). Furthermore, qRT-PCR showed that the
264
expression of A9 (Figure 5Q) and another tapetum-specific gene, ATA7 (Figure 5R), was
265
significantly higher in Pro4x35S-βCA1:βCA1 anthers compared to wild type. In conclusion, our
266
results suggest that overexpression of βCA1 promotes tapetal cell formation.
267 268 269
βCA1 is Phosphorylated by EMS1
270 271
Based on the well-established paradigm for the action of receptor-like kinases, EMS1 should
272
phosphorylate its interacting proteins. Therefore, we tested whether βCA1 can be phosphorylated
273
by EMS1 (Figure 6). Our in vitro phosphorylation assay showed that βCA1.4 was strongly
274
phosphorylated by EMS1-KD-GST (kinase domain) (Figure 6A), as was βCA2.2 and βCA4.1
275
(Supplemental Figure 10). We then performed protein gel blot analysis to investigate the in vivo
276
phosphorylation of βCA1. Membrane proteins extracted from young ProβCA1:βCA1-GFP and
277
ProβCA1:βCA1-GFP/ems1 buds were immunoprecipitated with an anti-GFP antibody. We
278
detected a weak signal from young ProβCA1:βCA1-GFP/ems1 buds but a strong signal from
279
young ProβCA1:βCA1-GFP buds using an anti-phospho-(Ser/Thr) antibody (Figure 6B). To 10
280
further confirm whether EMS1 can phosphorylate βCA1, we transiently expressed βCA1.4-EYFP
281
in protoplasts prepared from wild-type and Pro35S:EMS1 Pro35S:TPD1 leaves (Huang et al.,
282
2016c). The phosphorylation of βCA1.4 was detected in the presence of EMS1 and TPD1
283
(Figure 6C). Collectively, our data suggest that βCA1 can be phosphorylated by EMS1 both in
284
vitro and in vivo.
285 286 287
Phosphorylation of βCA1 by EMS1 Enhances Its Activity
288 289
We examined how phosphorylation mediated by EMS1 affects βCA enzyme activity (Figure 7).
290
Four phosphorylation sites (Thr35, Thr54, Thr69 and Ser189) were identified by mass
291
spectrometry (Figure 7A; Supplemental Figure 11; Supplemental Table 2). Thr35, Thr54 and
292
Thr69 are upstream of the β_CA_cladeB domain (NCBI Accession: cd00884; the CA domain for
293
short), while only Ser189 is located in the CA domain (Figure 7A). Although βCA2.2 and
294
βCA4.1 can also be phosphorylated by EMS1, only Thr35 is conserved (Supplemental Figure
295
11).
296
We found that EMS1 significantly enhanced βCA1.4 activity (Figure 7B to 7E). To
297
further analyze the significance of identified phosphorylation sites for the βCA1 function, we
298
produced mutated βCA1.4 in which Thr35, Thr54, Thr69 and Ser189 were substituted by alanine
299
(A) to block Thr (T)/Ser (S) phosphorylation and by aspartate (D) to functionally mimic
300
phosphorylated Thr (T)/Ser (S). βCA1.4T35A/D lost CA enzyme activity, and EMS1 treatment did
301
not increase its activity (Figure 7B). By contrast, the activities of βCA1.4T54A/D and βCA1.4T69A/D
302
were similar to that of βCA1.4, which were significantly enhanced by EMS1 treatment, although
303
they were lower than that of EMS1-treated wild-type βCA1.4 (Figure 7C and 7D). The activity
304
of βCA1.4S189A was similar to that of βCA1.4, but EMS1 treatment did not increase its activity
305
(Figure 7E). Interestingly, βCA1.4S189D activity was significantly higher than that of wild type,
306
and EMS1 treatment increased its activity more strongly than that of EMS1-treated βCA1.4
307
(Figure 7E).
308
To test the in vivo effect of EMS1 signaling on CA activity, we measured CA activity in
309
Pro35S:EMS1 Pro35S:TPD1 plants. Our results showed that CA activity was significantly
310
higher in both leaves and young buds of Pro35S:EMS1 Pro35S:TPD1 plants compared to wild 11
311
type (Figure 7F).
312
In summary, our results suggest that the phosphorylation of βCA1 by the kinase EMS1
313
increases its enzyme activity. Phosphorylation of Thr54, Thr69 and Ser189 likely contribute to
314
the enhancement of βCA1 activity, although Ser189 is more critical.
315 316 317
Phosphorylation of βCA1 Is Not Required for Its Subcellular Localization, Dimerization or
318
Interaction with EMS1
319 320
We investigated whether the phosphorylation of βCA1 affects its subcellular localization,
321
dimerization or interaction with EMS1 (Figure 8). The subcellular localization of a protein is
322
strongly associated with its function. βCA1 is localized to chloroplasts and the vicinity of the
323
plasma membrane (Fabre et al., 2007; Hu et al., 2010; Hu et al., 2015). We transiently expressed
324
βCA1.3-EYFP in Arabidopsis leaf protoplasts and detected EYFP signals in chloroplasts and at
325
the plasma membrane (Figure 8A and 8B), whereas βCA1.4-EYFP was localized at the plasma
326
membrane and in the cytoplasm (Figure 8C). We did not detect changes in the localization of
327
βCA1.4T35A (Figure 8D) or βCA1.4S189A (Figure 8E).
328
Studies on the structure of βCAs have revealed that the βCA dimer serves as the
329
functional unit of βCAs (Kimber and Pai, 2000; Strop et al., 2001). Our BiFC assay results
330
support the formation of homodimers of βCA1.4 at the plasma membrane (Figure 8F). Similarly,
331
we found dimerization between βCA1.4 and βCA1.4T35A (Figure 8G), βCA1.4T35A and
332
βCA1.4T35A (Figure 8H), βCA1.4 and βCA1.4 S189A (Figure 8I), as well as βCA1.4S189A and
333
βCA1.4 S189A (Figure 8J). In addition, βCA1.4T35A and βCA1.4S189A were still able to interact with
334
EMS1 (Figure 8K and 8L). Together, our results indicate that mutation of Thr35A or Ser189A
335
does not affect βCA1.4 localization, dimerization, or interaction with EMS1. Therefore, the
336
phosphorylation of βCA1 by EMS1 appears to mainly affect the enzyme activity of βCA1, which
337
might be required for its function in tapetal cell differentiation.
338 339 340
Phosphorylation of βCA1 by EMS1 Is Important for Tapetal Cell Differentiation
341
12
342
To investigate the functional significance of βCA1 phosphorylation in anther development, we
343
used ProA9:βCA1.4T35A, ProA9:βCA1.4T54A, ProA9:βCA1.4T69A and ProA9:βCA1.4S189A to
344
complement the βca1 βca2 βca4 phenotype (Figure 9). Seventy percent (14/20) of
345
ProA9:βCA1.4/βca1 βca2 βca4 plants (Figure 3F), 60.0% (12/20) of ProA9:βCA1.4T54A/βca1
346
βca2 βca4 plants (Figure 9A and 9D) and 63.3% (19/30) of ProA9:βCA1.4T69A/βca1 βca2 βca4
347
plants (Figure 9A and 9E) produced seeds. Similar to the wild type (Figure 9G and 9M), all of
348
these complemented plants showed viable pollen grains (Figure 3M; Figure 9J and 9K) and
349
normal tapetal cell differentiation (Figure 4E; Figure 9P and 9Q). However, all examined
350
ProA9:βCA1.4T35A/βca1 βca2 βca4 (13 total) and ProA9:βCA1.4S189A/βca1 βca2 βca4 (15 total)
351
plants showed the same defects in seed production (Figure 9B, 9C and 9F), pollen viability
352
(Figure 9H, 9I and 9L) and tapetal cell differentiation (Figure 9N, 9O and 9R) as those of βca1
353
βca2 βca4 plants. The expression levels of these mutated βCA1.4 transgenes were similar to that
354
of the βCA1.4 transgene in the βca1 βca2 βca4 background (Supplemental Figure 12). Therefore,
355
our results suggest that Thr35 and Ser189 are critical for the function of βCA1.4 in anther cell
356
differentiation.
357
Our in vitro studies showed that the enzyme activity of βCA1.4S189D was significantly
358
increased compared to wild type in both the absence and presence of EMS1 (Figure 7E). Thus,
359
we further studied the effect of phosphorylation of βCA1.4 on tapetal cell differentiation by
360
generating ProA9:βCA1.4S189D plants (Figure 10). Forty percent (32/80) of ProA9:βCA1.4S189D
361
plants showed reduced fertility (indicated by shorter siliques) compared with wild type (Figure
362
10A and 10B), as well as a reduced number of viable pollen grains (Figure 10D and 10E).
363
Moreover, 15.0% (12/80) of plants were completely sterile (Figure 10C) and did not produce
364
pollen grains (Figure 10F). Like the Pro4x35S-βCA1:βCA1 lines, anther sections from
365
completely sterile plants revealed an increased number of tapetal cells at stage 7 (Figure 10G to
366
10I). The expression levels of βCA1.4S189D and βCA1.4 in the transgenic plants were similar
367
(Supplemental Figure 13). Thus, our results suggest that phosphorylation of βCA1 by EMS1 is
368
essential for tapetal cell differentiation.
369 370 371
βCAs Regulate the pH of Tapetal Cells
372
13
373
To examine the possible mechanism by which βCAs control anther cell differentiation, we
374
investigated the pH of tapetal cells using the Carboxy SNARF®-1 pH indicator (Zhang et al.,
375
2001; Leshem et al., 2006; Sano et al., 2009). Tapetal cells had a lower pH than epidermal cells
376
in wild-type anthers (Figure 11). In βca1 βca2 βca4 anthers, the pH of epidermal cells was not
377
significantly altered; however, the pH of tapetal cells was significantly reduced compared to wild
378
type (Figure 11). Our findings suggest that βCAs might control tapetal cell differentiation by
379
regulating the pH of tapetal cells.
380
Taken together, our results provide several lines of evidence supporting the notion that
381
β-carbonic anhydrases serve as the direct downstream signaling molecules of the receptor-like
382
kinase EMS1 to control anther cell differentiation in Arabidopsis.
383 384 385
DISCUSSION
386 387
In this study, we identified βCAs as direct downstream players of the LRR-RLK EMS1 to
388
control anther cell differentiation in Arabidopsis. LRR-RLKs, with 223 members in Arabidopsis
389
(McCarty and Chory, 2000; Shiu et al., 2004; Torii, 2004; Zhao, 2009; Ma et al., 2016; Li et al.,
390
2017), are involved in various growth and developmental processes as well as defense responses,
391
including the regulation of shoot and root meristem sizes (Clark et al., 1997; Bommert et al.,
392
2013; Shinohara et al., 2016), cell fate determination and patterning (Canales et al., 2002; Zhao
393
et al., 2002; Kwak et al., 2005; Shpak et al., 2005; Jia et al., 2008), steroid hormone signaling (Li
394
and Chory, 1997; Li et al., 2002; Nam and Li, 2002; Hothorn et al., 2011; She et al., 2011;
395
Santiago et al., 2013), vascular patterning (Clay and Nelson, 2002; Fisher and Turner, 2007),
396
organ size and shape regulation (Torii et al., 1996; Xu et al., 2008), organ abscission (Jinn et al.,
397
2000; Leslie et al., 2010; Kumpf et al., 2013), pollen tube reception (Takeuchi and Higashiyama,
398
2016; Wang et al., 2016b), defense responses (Song et al., 1995; Gomez-Gomez and Boller,
399
2000; Lee et al., 2011; Sun et al., 2013; Halter et al., 2014; Zorzatto et al., 2015), plant
400
transpiration (Masle et al., 2005), nodulation (Endre et al., 2002; Searle et al., 2003), and
401
nitrogen acquisition (Tabata et al., 2014). To date, only a few direct downstream signaling
402
molecules have been identified for an increasing number of functionally investigated
403
LRR-RLKs. 14
Brassinosteroid (BR) signaling is one of the best-understood LRR-RLK-linked signal
404 405
transduction pathways in plants. Unlike animals, which use nuclear receptors to perceive steroids,
406
plants employ plasma membrane LRR-RLKs, the BRI1 receptor and its four redundant
407
co-receptors, SERK1-4, for BR signaling (Li et al., 2002; Nam and Li, 2002; Wang et al., 2008;
408
Hothorn et al., 2011; She et al., 2011; Gou et al., 2012; Santiago et al., 2013; Sun et al., 2013). In
409
the absence of BRs, BRI1 exists as an inhibitory homodimer via associating with the anchor
410
protein BKI1 in the cytoplasm (Wang and Chory, 2006). When BRs are present, the binding of
411
BRs to the extracellular LRR domain of BRI1 causes the release of BKI1 and consequent
412
heterodimerization between BRI1 and one of the SERK co-receptors (Santiago et al., 2013). The
413
activated BRI1 then activates BSK (BR signaling kinase), the BSU1 phosphatase, and the BIN2
414
GSK3-like kinase cascade (Mora-Garcia et al., 2004; Vert and Chory, 2006; Tang et al., 2008;
415
Kim et al., 2011; Sreeramulu et al., 2013). Finally, the BIN2-regulated BES1/BZR1/HAT1
416
transcription factors transmit BR signaling to the nucleus via activating the expression of BR
417
target genes (Yin et al., 2002; He et al., 2005; Vert and Chory, 2006; Oh et al., 2012; Zhang et al.,
418
2013).
419
Perception of the oligo-peptide ligand flg22 by FLAGELLIN SENSITIVE2 (FLS2) triggers
420
the association of FLS2 with its co-receptor BRI1-ASSOCIATED RECEPTOR KINASE1
421
(BAK1), followed by the phosphorylation of BOTRYTIS-INDUCED KINASE1 (BIK1)
422
(Gomez-Gomez and Boller, 2000; Chinchilla et al., 2007; Lu et al., 2010; Sun et al., 2013). The
423
phosphorylated BIK1 phosphorylates and activates the NADPH oxidase RBOHD, leading to the
424
production of reactive oxygen species (ROS) (Kadota et al., 2014). BAK1 also phosphorylates
425
two closely related E3 ubiquitin ligases, PUB12 and PUB13, which polyubiquitinate FLS2 and
426
promote flagellin-induced degradation of FLS2 (Lu et al., 2011). The ERECTA (ER/ERL)
427
family and TOO MANY MOUTHS (TMM) perceive the secreted cysteine-rich peptides
428
EPIDERMAL PATTERNING FACTOR (EPF)/EPF-LIKE to control the proper density and
429
spacing of stomata (Shpak et al., 2005; Sugano et al., 2010; Lee et al., 2015). ER/ERL signaling
430
activates the downstream YODA (YDA), MKK4/5 and MPK3/6 MAPK cascade, resulting in the
431
inhibition of the SPEECHLESS (SPCH)/SCREAM (SCRM) transcriptional module (Lampard et
432
al., 2008; Lee et al., 2012; Torii, 2015). Moreover, ER/ERL and MPK3/MPK6 function in anther
433
lobe formation and anther cell differentiation (Hord et al., 2008). In the presence of the
434
CLAVATA3 (CLV3) ligand, CLV1 acts together with CORYNE (CRN)/CLV2, possibly via a 15
435
MAPK cascade, to inhibit the expression of WUSCHEL (WUS), WUSCHEL-RELATED
436
HOMEOBOX (WOX), and HAIRY MERISTEM (HAM) (Clark et al., 1997; Fletcher et al., 1999;
437
Schoof et al., 2000; Muller et al., 2008; Ogawa et al., 2008; Zhou et al., 2015). In the current
438
study, we established βCAs as downstream effectors of LRR-RLK, shedding light on
439
LRR-RLK-linked signal transduction pathways. EMS1 directly targets an enzyme rather than the
440
MAPK cascade, which might be required for rapid responses to environmental and
441
developmental cues. Moreover, our results indicate that phosphorylation mediated by a
442
receptor-like kinase is essential for the functional regulation of βCA.
443
Although decades of research have implicated CAs in various metabolic, cellular and
444
physiological processes in both plants and animals, the role of CAs in plant cell signaling
445
remains a mystery. CAs regulate the levels of CO2, protons and bicarbonate (HCO3‾), which are
446
important for carboxylation/decarboxylation, pH regulation, inorganic carbon transport, ion
447
transport, as well as water and electrolyte balance. In animals, CAs, which are found in the
448
plasma membrane, cytosol and mitochondria, are essential for gluconeogenesis, lipogenesis,
449
ureagenesis, and tumor formation. For example, the membrane-localized CA IX, an established
450
marker for tumor hypoxia and aggressive cancers, plays a critical role in tumor cell proliferation
451
by regulating pH (Alterio et al., 2009; Swietach et al., 2009). Besides their photosynthetic
452
functions (Hatch and Burnell, 1990; Badger and Price, 1994; Smith and Griffiths, 2000; Studer et
453
al., 2014; Clement et al., 2016; Ho et al., 2016; Ludwig, 2016), recent studies found that βCAs
454
are critical for CO2-induced stomatal development and movement (Hu et al., 2010; Engineer et
455
al., 2014; Hu et al., 2015; Matrosova et al., 2015; Wang et al., 2016a) as well as for amino acid
456
biosynthesis (DiMario et al., 2016).
457
Arabidopsis has six βCAs, each with a variable number of splice variants (Ferreira et al.,
458
2008; Wang et al., 2014). Our studies identified the βCA1 splice variant βCA1.4. Protein
459
localization studies show that βCA1 and βCA5 are found in plastids, including chloroplasts,
460
while βCA2 and βCA3 are localized to the cytosol, βCA4 is found at the plasma membrane, and
461
βCA6 is localized in mitochondria (Fabre et al., 2007). Furthermore, βCA1 is found in the
462
vicinity of the plasma membrane and chloroplasts (Hu et al., 2010) or near the plasma membrane
463
and cytoplasm when the first 65 amino acids (chloroplast signal peptide) are removed (Hu et al.,
464
2015). We found that βCA1.3 was localized in chloroplasts and at the plasma membrane,
465
whereas βCA1.4 was localized at the plasma membrane and in the cytoplasm. The multiplicity of 16
466
βCA isoforms and their diverse localizations suggest that βCAs might possess additional
467
functions.
468
CA activity in animals is regulated by phosphorylation. Phosphorylation stimulated by
469
3’-5’-cyclic adenosine monophosphate (cAMP) increases the activity of CAs from rat gastric
470
tissue (Bersimbaev et al., 1975) and rat astroglial cell cultures (Church et al., 1980). When
471
phosphorylated by protein kinase A (PKA) (Narumi and Miyamoto, 1974) and protein kinase G
472
(PKG) (Carrie and Gilmour, 2016), the activity of CA is enhanced in bovine erythrocytes and
473
rainbow trout gill, respectively. Human CA IX is a tumor-associated transmembrane carbonic
474
anhydrase. Phosphorylation on Thr443 is required for the function of CA IX in hypoxic tumor
475
cells (Ditte et al., 2011). In the single-cell algae, Chlamydomonas reinhardtii, Cah3, an
476
intracellular α-CA, is localized in the thylakoid lumen and its activity is also regulated by
477
phosphorylation (Blanco-Rivero et al., 2012). In this study, we found that phosphorylation by the
478
receptor-like kinase increases the activity of CAs in flowering plants. We also identified four
479
phosphorylation sites (Thr35, Thr54, Thr69, and Ser189) in βCA1.
480
Both the phosphorylation-blocking mutation T35A and the phosphomimic mutation
481
T35D in βCA1.4 caused the loss of enzyme activity, even after EMS1 treatment. T54A, T69A or
482
S189A mutation did not significantly alter βCA1 activity, but the enhancement of activity by
483
phosphorylation was significantly affected by these mutations. In particular, the activity of
484
βCA1.4T189A remained unchanged without or with EMS1 treatment; however, the S189D
485
mutation resulted in a significant increase in βCA1.4 activity. In addition, EMS1 treatment
486
further enhanced the activity of βCA1.4S189D, suggesting that phosphorylation of S189 is critical
487
for the regulation of βCA1 activity. It would be worthwhile to investigate how phosphorylation
488
of these residues affects βCA activity in the future.
489
Our loss- and gain-of-function studies of βCAs showed that βCAs are required for
490
tapetal differentiation. Tapetal cells produce elaioplasts (tapetum-specific plastids) and
491
tapetosomes (an ER-derived organelle rich in triacylglycerols and oleosins) (Dickinson, 1973;
492
Wu et al., 1997; Hsieh and Huang, 2007). Plastids in tapetal cells are essential for pollen wall
493
and pollen coat formation (Owen and Makaroff, 1995; Pacini, 1997; Clement and Pacini, 2001).
494
Lipids are the main precursors for components of pollen exine, such as sporopollenin. In tapetal
495
cells, pro-plastids undergo division during early tapetum development and subsequently develop
496
into non-green plastids (elaioplast) that are involved in the biosynthesis of tapetal lipids as well 17
497
as starch accumulation and/or mobilization (Dickinson, 1973; Pacini et al., 1992; Weber, 1992;
498
Clement et al., 1998; Wu et al., 1999; Clement and Pacini, 2001). In Brassicaceae species
499
including Arabidopsis, fully differentiated tapetal cells accumulate elaioplasts and tapetosomes.
500
In the male sterile1 mutant, tapetal cells produce greatly reduced numbers of elaioplasts and
501
tapetosomes (Ito et al., 2007; Yang et al., 2007). Mutations in the Arabidopsis MS2 and rice
502
DEFECTIVE POLLEN WALL (DPW) genes, which encode plastid-localized fatty acid
503
reductases, result in abnormal tapetum and pollen development (Aarts et al., 1997; Shi et al.,
504
2011). Disruption of phosphoenolpyruvate/phosphate translocator1 (PPT1) and the
505
plastid-localized enolase1 (ENO1) affect sporopollenin formation (Prabhakar et al., 2010). We
506
found that elaioplast and tapetosomes production was reduced when the function of βCAs was
507
disrupted. In animals, the importance of CAs increases in pathological states. Hypoxia-induced
508
CA IX facilitates cancer cell survival and proliferation by combating the high rate of glycolytic
509
metabolism to keep up with the increased energy demand for ATP and biosynthetic precursors
510
(Parks et al., 2013). Like tumor cells, tapetal cells might require high βCA activity to maintain
511
their highly active metabolic state. HCO3‾ is important for lipid formation. Based on our results,
512
the phosphorylation of βCA1 by EMS1 significantly enhances its activity. The highly active
513
βCAs might be required for tapetum development via affecting the formation of elaioplasts and
514
tapetosomes.
515
It is also possible that βCAs regulate tapetal cell pH, which might be important for tapetal
516
cell differentiation and the maintenance of tapetal function. The regulation of extracellular (pHe)
517
and intracellular pH (pHi) is critical for cell division, differentiation, and survival. In animals,
518
CAs play a key role in buffering cellular pH via regulating HCO3‾ and H+ concentrations (Alterio
519
et al., 2009; Chiche et al., 2009; Swietach et al., 2009; Swietach et al., 2010; Parks et al., 2011;
520
Benej et al., 2014). In plants, in addition to H+ pumps, such as P-type H+-adenosine
521
triphosphatase, vacuolar H+-ATPase, and H+-pyrophosphatase (Li et al., 2005), EMS1-regulated
522
βCAs might be especially important for moderating pH in tapetal cells, because they are highly
523
active in metabolism. In fact, our data revealed that the pH of epidermal cells and tapetal cells
524
differed in wild-type anthers. Moreover, loss-of-function of βCAs caused a significant decrease
525
in tapetal cell pH. Auxin signaling is highly active in the tapetum (Aloni et al., 2006; Cecchetti et
526
al., 2017), suggesting that auxin might be important for tapetal cell differentiation. Auxin
527
represses chloroplast and amyloplast development (Miyazawa et al., 1999; Kobayashi et al., 18
528
2012). Therefore, auxin might regulate tapetal cell differentiation and function via affecting the
529
formation of elaioplasts (Sakata et al.; Miyazawa et al., 1999; Cecchetti et al., 2008; Kobayashi
530
et al., 2012). Furthermore, auxin is essential for pollen development and filament elongation
531
(Sakata et al.; Cecchetti et al., 2008). The H+ gradient maintained by βCAs might be important
532
for auxin transport during anther development. Normal tapetum development is required for
533
sexual reproduction and high yield in plants under both normal and stress conditions (Smith and
534
Zhao, 2016). Future research should be focused on investigating the molecular mechanisms by
535
which βCAs control tapetal cell differentiation and pollen development.
536 537 538
METHODS
539 540
Plant Materials and Growth Condition
541 542
The Arabidopsis thaliana Landsberg erecta (Ler) and Columbia (Col-0) ecotypes were used in
543
this study. Plants were grown in Metro-Mix 360 (Sun-Gro Horticulture) in growth chambers
544
(Philips PLUS T8 High Output 8-foot cool white fluorescent lamps and 100μmol m−2 s−1
545
photon density) under a 16-hr light/8-hr dark photoperiod at 22°C and 50% humidity.
546 547
Phylogenetic Analysis
548 549
Alignment of βCA1 to βCA6 protein sequences was carried out with MUSCLE, followed by
550
manual adjustment. Phylogenetic analysis was performed by PhyML using the Maximum
551
Likelihood method with default parameters (Dereeper et al., 2008). TreeDyn was used to display
552
the phylogenetic tree.
553 554 555
Yeast Two-Hybrid (Y2H) Screening
556 557
The ProQuest Two-Hybrid system with Gateway technology (Invitrogen) was employed to
558
identify EMS1 interacting proteins. Briefly, the EMS1 kinase domain (852-1192), which was 19
559
cloned into the pDEST 32 vector, was used as the bait. To enrich the potential EMS1-interacting
560
proteins, mRNA was prepared from young buds containing stage-5/6 anthers in the Landsberg
561
erecta (Ler) background. A cDNA library was then constructed using the SuperScript™ Plasmid
562
System with the Gateway® Technology. According to the manufacturer’s manual,
563
protein-protein interactions were assayed on the synthetic drop-out medium minus Leu, Trp and
564
His, as well as containing 25 mM 3-Amino-1,2,4-Triazole (3AT), using the yeast strain MaV203.
565 566 567
Generation of Constructs and Transgenic Plants
568 569
All DNAs and cDNAs were amplified using Phusion High-Fidelity DNA Polymerase (New
570
England Biolabs). To test interactions between EMS1 and βCAs by Y2H, βCA1.4, βCA2.2,
571
βCA3 and βCA4.3 were cloned into the pENTR/D-TOPO vector (Invitrogen, Cat#: K240020),
572
respectively, followed by introduction into the pDEST22 vector using Gateway LR recombinase
573
II enzyme mix (Invitrogen, Cat#: 11791100). pSAT vectors (Tzfira et al., 2005) were used for
574
the subcellular localization study and the BiFC assay in the protoplast system. To generate the
575
BiFC constructs, cEYFP (C-terminus of EYFP; pSAT1-cEYFP-C1-B) was fused to the
576
full-length EMS1 and BRI1, and the nEYFP (N-terminal of EYFP; pSAT4-nEYFP-N1) was
577
fused to βCA1.4, βCA2.2, βCA3, βCA4.1, βCA5.2, βCA6.1 and αCA1.1. To generate constructs
578
for the Förster Resonance Energy Transfer (FRET) assay, the βCA1.4 was fused to
579
pSAT6-EYFP-N1 to produce βCA1-EYFP. The EYFP in pSAT6-EYFP-N1 was replaced by
580
CFP to generate pSAT6-CFP-N1. Full-length EMS1 was then inserted into pSAT6-CFP-N1 to
581
generate EMS1-CFP. To investigate subcellular localization, βCA1, βCA1.3 and βCA1.4 were
582
cloned into pSAT6--EYFP-N1. βCA1.4T35A, βCA1.4T54A, βCA1.4T69A, βCA1.4S189A, βCA1.4T35D,
583
βCA1.4T54D, βCA1.4T69D and βCA1.4S189D were generated by overlapping PCR and cloned into
584
the pENTR/D-TOPO vector. βCA1.4T35A and βCA1.4S189A were cloned into pSAT6--EYFP-N1
585
for subcellular localization analysis. To test the dimerization of βCA1.4 and its mutated versions,
586
βCA1.4, βCA1.4T35A and βCA1.4S189A were fused to nEYFP and cEYFP, respectively.
587
For the co-immunoprecipitation assay using protoplasts, βCA1.4-Flag was
588
PCR-amplified and inserted into pSAT6-EYFP-N1 after removing the EYFP tag to generate
589
pSAT6- βCA1.4-Flag-N1. 20
590
For βCA protein localization studies in planta, genomic DNA fragments including
591
promoters, introns and exons were cloned into the pENTR/D-TOPO vector. The βCA1 genomic
592
fragment without the last intron and exon was used to construct ProβCA1:βCA1-GFP. Genomic
593
DNA fragments with all exons were used to construct ProβCA2:βCA2-GFP,
594
ProβCA3:βCA3-GFP and ProβCA4:βCA4-GFP, respectively.
595
For the complementation experiments, 4884-bp, 5865-bp and 5207-bp genomic DNA
596
fragments of βCA1, βCA2 and βCA4 were cloned into the pENTR/D-TOPO vector, respectively.
597
The A9 (At5g07230) gene promoter (Feng and Dickinson, 2010) was amplified and cloned into
598
the pENTR/D-TOPO vector. The βCA1.4 cDNA was inserted after the A9 promoter to produce
599
ProA9:βCA1.4. The same method was used to produce the ProA9:βCA1.4T35A,
600
ProA9:βCA1.4T54A, ProA9:βCA1.4T69A, ProA9:βCA1.4S189A, ProA9:βCA1.4T35D and
601
ProA9:βCA1.4S189D constructs. AimrβCA1-4 for generating artificial microRNAs targeting βCA1
602
to βCA4 transcripts (βCA1-4) was designed as described previously (Schwab et al., 2006). The
603
aimrβCA1-4 fragment was cloned into the pENTR/D-TOPO vector to generate
604
Pro35S:aimrβCA1-4 and ProA9:aimrβCA1- 4, respectively.
605
For produce the Pro4x35S-βCA1:βCA1 construct, the CaMV 35S enhancer was amplified
606
from the pSK1015 vector (Weigel et al., 2000) and cloned into the pENTR/D-TOPO vector. A
607
4884-bp fragment of genomic DNA, including a 2-kb βCA1 promoter region, was inserted after
608
the CaMV 35S enhancer.
609
For the phosphorylation assays, a 950-bp cDNA fragment of EMS1 was amplified and
610
cloned into the pGEX-4T-2 vector (GE Healthcare, Cat#: 27-1542-01) to generate EMS1-KD
611
(kinase domain)-GST. βCA1.4 was amplified and cloned into the pET28a vector (EMD Millipore,
612
Cat#: 69864-3) to produce the βCA1.4-His protein. βCA1.4, βCA2.2, βCA4.1, βCA1.4T35A,
613
βCA1.4T54A, βCA1.4T69A, βCA1.4S189A, βCA1.4T35D, βCA1.4T54D, βCA1.4T69D and βCA1.4S189D were
614
PCR-amplified and cloned into the pGEX-4T-2 vector to produce GST fusion proteins,
615
respectively.
616
All constructs generated with pENTR/D-TOPO vectors were introduced into Gateway
617
Binary vectors (pGBWs or pEARLEYs) using Gateway LR recombinase II enzyme mix
618
(Invitrogen). Detailed information for all constructs and primers is shown in Supplemental Data
619
Set 1 and 2. The resulting constructs were transformed into Agrobacterium strain GV3101. Plant
620
transformation was performed using the floral dip method (Clough and Bent, 1998). 21
621
Transformants were screened on 50 µg/mL of kanamycin and 25 µg/mL of hygromycin or 1%
622
Basta.
623 624 625
Protoplast Transfection and BiFC assay
626 627
For high-quality plasmid preparation, the PureYieldTM Plasmid Midiprep System (Promega, Cat#:
628
A2495) was used to isolate plasmids. A pair of constructs was used to co-transfect Arabidopsis
629
protoplasts prepared from 4-week-old leaves (Yoo et al., 2007). The Pro35S:EYFP construct was
630
used to monitor transfection efficiency. At least three replicates were performed for each assay.
631
Samples were observed after 16 hours under a Leica TCS SP2 laser scanning confocal
632
microscope using a 63×/1.4 water immersion objective lens (Huang et al., 2016c).
633 634 635
Co-immunoprecipitation
636 637
The Co-immunoprecipitation assay was performed as previously described (Jia et al., 2008; Li et
638
al., 2017). Young buds were harvested from wild-type, ProEMS1:EMS1-4xcMyc,
639
ProβCA1:βCA1-Flag and ProEMS1:EMS1-4xcMyc ProβCA1:βCA1-Flag plants. βCA1.4-Flag
640
was also transiently expressed in protoplasts from Pro35S:EMS1-cMyc and Pro35S:EMS1-cMyc
641
Pro35S:TPD1sp-GFP-∆TPD1 leaves. Non-transfected wild-type and Pro35S:EMS1-cMyc
642
protoplasts were used as controls. Membrane proteins were extracted and incubated with 30 µl of
643
Myc-Trap® beads (Chromotek, Cat# yta-10) overnight at 4°C. After three washes, the eluted
644
immunoprecipitates were used for protein gel blot analysis to detect EMS1-4xcMyc (anti-cMyc,
645
Roche; Cat. #: 11667149001; 1:500 dilution), TPD1sp-GFP-∆TPD1 (anti-GFP, Torrey Pines
646
Biolabs) and βCA1-Flag (anti-Flag, GeneScript; Cat#: A01428-100; 1:1000 dilution),
647
respectively.
648 649 650
FRET Assay
651
22
652
For the FRET assay, EMS1-CFP (donor) and βCA1.4-EYFP (acceptor) were co-transfected into
653
Arabidopsis protoplasts. Control reactions included single-plasmid transfections and mock
654
transfections (without plasmid). Acquisition of spectrally resolved fluorescence images, spectral
655
unmixing of donor, acceptor, and autofluorescence signals, as well as determination of FRET
656
efficiency were performed as previously described (Raicu and Singh, 2013; King et al., 2016;
657
Stoneman et al., 2016). Briefly, an optical micro-spectroscope (OptiMiS) was used to capture
658
images with linear unmixing of CFP and EYFP from cells co-expressing EMS1-CFP and
659
βCA1-EYFP. OptiMiS is featured with the line-scan excitation powered by a femtosecond laser
660
emitting near-IR light pulses with tunable emission wavelength between 780 nm and 1040 nm
661
(Raicu et al., 2009; Biener et al., 2014). The spectral images were used to generate
662
two-dimensional fluorescence maps of donor and acceptor (Raicu and Singh, 2013). The CFP,
663
EYFP and auto-fluorescence spectra were acquired from cells expressing EMS1-CFP only,
664
βCA1-EYFP only, and that were devoid of any fluorescent protein, respectively. The unmixing
665
protocol has been optimized for cells presenting substantial autofluorescence emission with the
666
final goal to determine donor and acceptor emission images free from any autofluorescence
667
(Mannan et al., 2013). Since the laser wavelength of 860 nm (selected as optimal for donor
668
excitation) also caused slight excitation of the acceptor, we did not attempt to compute the FRET
669
efficiency for every image pixel from the donor and acceptor fluorescence maps (Raicu and
670
Singh, 2013), or else the FRET efficiency map would have been contaminated by such spurious
671
excitation. Instead, we used a method introduced recently, which employed two excitation
672
wavelengths (860 nm and 960 nm) to account for the direct excitation of acceptor and to
673
compute the average FRET efficiency for regions of interest (at the plasma membrane) (King et
674
al., 2016; Stoneman et al., 2016). In order to reduce noise and avoid singularities at pixels with
675
zero intensity values, a signal-to-noise threshold of 1.5 SD was applied to FRET efficiency
676
calculations.
677 678 679
Pollen Staining and Semi-thin Sectioning of Anthers
680 681
To identify viable pollen grains, mature anthers prior to dehiscence were collected for Alexander
682
staining of pollen (Zhao et al., 2002; Xin et al., 2017). To study anther cell differentiation, 23
683
semi-thin sectioning was carried out essentially as previously described (Zhao et al., 2002;
684
Huang et al., 2016b). For semi-thin sectioning, dissected young buds were fixed in 2.5% of
685
(vol/vol) glutaraldehyde in 0.1 M HEPES (N-2-Hydroxyethyl pipera-zine-N-2-ethanesulfonic
686
acid) buffer (pH 7.2) and 0.02% of TritonX-100 overnight at 4°C. Samples were washed twice in
687
0.1 M HEPES buffer (pH 7.2) and post-fixed in 1% of OsO4 overnight. The samples were
688
dehydrated in a graded acetone series (10% of increments), infiltrated, and embedded in Spurr’s
689
resin. Semi-thin (1 µm) sections were made using an Ultracut E ultramicrotome (Reichert-Jung)
690
and stained with 0.25% of Toluidine Blue O. The number of tapetal cells was counted under a
691
microscope from central sections of upper two lobes. Sections from 20 individual buds were
692
examined.
693 694 695
Phosphorylation Assay and Identification of βCA1 Phosphorylation Sites
696 697
EMS1-KD-GST, βCA2.2-GST and βCA4.1-GST were expressed in BL21DE3 according to the
698
manufacturer’s instructions (GE Healthcare) and protein extraction was performed using the
699
Pierce™ Glutathione Agarose (Thermo Scientific™, Cat#: 16100). . The expression and
700
purification of βCA1.4-His protein were performed as previously described (Idrees et al., 2016).
701
βCA1.4-His protein was extracted by the HIS-Select® Nickel Affinity Gel (Sigma, Cat#: P6611)
702
and further purified by Hi Trap DEAE FF column (GE healthcare, Cat#: 17-5055-01). For the
703
phosphorylation reaction, 1 µg of EMS1-KD-GST and 5 µg of βCA1.4-His were incubated with
704
[γ-32P] ATP in the kinase buffer (HEPES at pH7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT)
705
for 30 min at 25 oC (Zhao et al., 2002; Li et al., 2017). Proteins were separated by 10% of
706
SDS-PAGE and the gel was then analyzed by autoradiography. Additionally, 1 µg of
707
EMS1-KD-GST and 5 µg of βCA2.2-GST and βCA4.1-GST were incubated in kinase buffer for
708
30 min at 25 oC. Proteins were separated by 10% SDS-PAGE and protein phosphorylation was
709
detected with anti-phospho-(Ser/Thr) antibody (Abcam, Cat#: ab17464, 1:400 dilution).
710
For mass spectrometer analysis, the purified recombinant βCA1.4-His, βCA2.2-GST and
711
βCA4.1-GST proteins were trans-phosphorylated by EMS1-KD-GST in vitro in the presence of
712
ATP. After SDS-PAGE, protein bands were excised from the gels, followed by in-gel digestion
713
using trypsin. Samples were analyzed with an Agilent 1100 series LC/MSD Trap SL coupled to 24
714
the LTQ Orbitrap XL (Thermo Scientific) mass spectrometer (University of Wisconsin-Madison
715
Biotechnology Center).
716 717 718
Carbonic Anhydrase Activity Assay
719 720
Phosphorylation of βCA1.4, βCA1.4T35A, βCA1.4T54A, βCA1.4T69A, βCA1.4S189A, βCA1.4T35D,
721
βCA1.4T54D, βCA1.4T69D and βCA1.4S189D was performed as described above. Four-week-old
722
leaves and young buds from wild-type and Pro35S:TPD1 Pro35S:EMS1 transgenic plants
723
(Huang et al., 2016d) were harvested. The CA activity assay was performed as previously
724
described (Wilbur and Anderson, 1948; Hu et al., 2010). CA activity was defined as WA unit/mg
725
protein. Protein concentration was determined by the Bradford method.
726 727 728
RT-PCR, qRT-PCR, and RNA in situ Hybridization
729 730
Total RNA was isolated from various plant tissues/organs using an RNeasy Plant Mini Kit
731
(Qiagen, Cat#: 74904). RNA quantification, reverse transcription, PCR, qRT-PCR (DNA Engine
732
Opticon 2 system), and data analysis were performed as described previously (Liu et al., 2009;
733
Huang et al., 2016a). Expression of βCA1.1, βCA1.2, βCA1.3 and βCA1.4 was determined by
734
amplifying each full-length CDS. RNA in situ hybridization was performed using anthers from
735
wild-type, βca1 βca2 βca4, Pro35S:amirβCA1-4, ProA9:amirβCA1-4, ProA9:βCA1.4/βca1 βca2
736
βca4 and Pro4x35S-βCA1:βCA1 plants (Zhao et al., 2002; Liu et al., 2010). An SP6/T7 DIG
737
RNA Labeling Kit (Roche, Cat#: 11175025910) was used to generate A9 sense and antisense
738
probes. Primers for PCR, qRT-PCR and in situ hybridization are listed in Supplemental Data Set
739
2.
740 741 742
Microscopy
743 744
Images of pollen staining and semi-thin sections were photographed under an Olympus BX51 25
745
microscope equipped with an Olympus DP 70 digital camera (Jia et al., 2008; Huang et al.,
746
2016d). For confocal microscopy analysis, samples were observed under a Leica TCS SP2 laser
747
scanning confocal microscope. Samples were mounted in water and observed with a 20× lens or
748
a 63×/1.4 water immersion objective lens. Protoplasts were observed using a 63×/1.4 water
749
immersion objective lens. A 488-nm laser was used to excite GFP, EYFP, and chlorophyll. The
750
emission was captured using PMTs set at 505–530 nm, 500–550 nm, and 644–719 nm,
751
respectively.
752 753 754
pH Measurement
755 756
Anthers were dissected from flower buds and stained with 20 µm of SNARF-1-AM (Invitrogen,
757
Cat#: c1271) in MES/KCl buffer (5mM KCl, 50 µM CaCl2, 10mM MES buffered to pH 6.15
758
with KOH) for 30 minutes (Zhang et al., 2001). The anthers were washed three times with
759
SNARF-1-AM-free buffer. Confocal imaging was performed on a Leica SP2 confocal laser
760
scanning microscope (Leica Microsystems). The excitation was set at 488 nm. The emission was
761
set at 540-590 nm for channel 1 and 610-700 nm for channel 2 (Sano et al., 2009). Images were
762
analyzed by ImageJ. The intensity ratio of channel 1/channel 2 was converted to pH according to
763
the calibration graph (Zhang et al., 2001; Leshem et al., 2006).
764 765
Accession Numbers
766 767
Sequence data from this article can be found in The Arabidopsis Information Resource
768
(http://www.arabidopsis.org/) under the following accession numbers: EMS1 (At5G07280),
769
TPD1 (AT4G24972), BRI1 (AT4G39400), βCA1 (At3g01500), βCA2 (AT5G14740), βCA3
770
(AT1G23730), βCA4 (AT1G70410), βCA5 (AT4G33580), βCA6 (AT1G58180), αCA1
771
(AT3G52720), A9 (At5G07230), and ATA7 (At4g28395).
772 773 774
Supplemental Data
775
26
776
Supplemental Figure 1. Alternative splice variants of βCA1.
777
Supplemental Figure 2. EMS1 interacts with four βCA1 variants, possibly at different levels.
778
Supplemental Figure 3. Phylogenetic analysis of βCAs in Arabidopsis.
779
Supplemental Figure 4. Expression of EMS1-cEYFP and βCAs-nEYFP in a BiFC experiment.
780
Supplemental Figure 5. TPD1 is not required for EMS1 and βCA1 interaction.
781
Supplemental Figure 6. Expression of four βCA1 splice variants in different tissues.
782
Supplemental Figure 7. Identification of the βca2 mutant.
783
Supplemental Figure 8. βCA2 and βCA4 complement the βca1 βca2 βca4 mutant phenotype.
784
Supplemental Figure 9. Overexpression of βCA1 affects male fertility.
785
Supplemental Figure 10. βCA2.2 and βCA4.1 are phosphorylated by EMS1 in vitro.
786
Supplemental Figure 11. Phosphorylation sites identified in βCA1.4.
787
Supplemental Figure 12. Expression of wild-type and mutated βCA1.4 transgenes in
788
complementation experiments.
789
Supplemental Figure 13. Expression of βCA1.4 and βCA1.4S189D in transgenic plants.
790
Supplemental Table 1. Calculation of FRET efficiency using a two-excitation wavelength
791
scheme.
792
Supplemental Table 2. Phosphorylation sites in βCA1.4 identified by LC/MS/MS.
793
Supplemental Data Set 1. Constructs generated in this study.
794
Supplemental Data Set 2. Primers used in this study.
795
Supplemental File 1. Alignment used to produce the phylogenetic tree shown in Supplemental
796
Figure 3.
797 798
ACKNOWLEDGMENTS
799 800
We thank T. Schuck, J. Gonnering and P. Engevold for plant care, P. He, and J. Sheen for the
801
protoplast transfection, H. Owen for confocal microscopy, the ABRC for EMS1, TPD1, A9,
802
βCA1, βCA2, βCA3, βCA4, βCA5, βCA6, BRI1, αCA1 BAC clones and cDNAs, T. Nakagawa for
803
the pGBW vectors, C. Pikaard for the pEARLEY vectors, J. Haseloff for the pBIN Gal4–
804
mGFP5er vector, D. Weigel for the pRS300 and pSK1015 vectors, S. Gelvin for the pSAT
805
vectors, and S. Ishiguro for the FIB1a-GFP and GRP17-GFP vectors. This work was supported
806
by the National Science Foundation grants IOS-0721192 and IOS-1322796 to D.Z., the Research 27
807
Growth Initiative (RGI) at the University of Wisconsin-Milwaukee to D.Z. and V.R., and the
808
UW-Madison/UW-Milwaukee Intercampus Research Incentive Grants Program to D.Z. D.Z.
809
also gratefully acknowledges the support of the Shaw Scientist Award from the Greater
810
Milwaukee Foundation, the Bradley Catalyst Award from the UWM Research Foundation, and
811
the CAS/SAFEA International Partnership Program for Creative Research Teams. The optical
812
microspectroscopy imaging facility used for this study was developed with financial support
813
from the National Science Foundation, Major Research Instrumentation Program (Grant:
814
PHY-1126386 awarded to V.R.). The FRET assays were partially funded by the National
815
Science Foundation, Physics of Living Systems Program (Grant: PHY-1058470 awarded to
816
V.R.).
817 818 819
Competing Finacial Interests
820 821
Dr. Raicu is a co-founder of Aurora Spectral Technologies LLC (AST), which commercializes
822
the OptiMiS technology used in this work. All the other authors declare no competing financial
823
interests.
824 825 826
AUTHOR CONTRIBUTIONS
827 828
J.H. and D.Z. designed the research. J.H., Z.L., G.B., E.X., S.M., N.E., C.Z., H.K. and V.R.
829
performed research. All authors analyzed data. J.H. and D.Z. wrote the article with contributions
830
from the other authors.
831 832 833 834
FIGURE LEGENDS
835 836 837
Figure 1. EMS1 interacts with βCAs. 28
838 839
(A) Yeast two-hybrid (Y2H) assay using the EMS1 kinase domain (EMS1-KD) fused to BD
840
(DNA binding domain, BD-EMS1-KD) and six AD (DNA activation domain)-βCA fusions.
841
Yeast cells were grown on synthetic drop-out medium lacking Leu, Trp, and His with 25 mM of
842
3-Amino-1,2,4-Triazole (3AT).
843
(B) to (I) Bimolecular fluorescence complementation (BiFC) assay using Arabidopsis mesophyll
844
protoplasts. Confocal images showing that EMS1 interacts with βCA1.4 (B), βCA2.2 (C), and
845
βCA4.1 (E) at the plasma membrane; EMS1 does not interact with βCA3 (D), βCA5.2 (F),
846
βCA6.1 (G), or αCA1.1 (H, control); and no interaction between BRI1 and βCA1.4 (I, control).
847
(J) to (L) Förster Resonance Energy Transfer (FRET) assay showing fluorescence images of the
848
donor (EMS1-CFP) obtained only following excitation at 860 nm (J) and the acceptor
849
(βCA1.4-EYFP) only following excitation at 860 nm (K) and 960 nm (L). Signals were obtained
850
by spectral unmixing of images in cells co-expressing EMS1-CFP and βCA1.4-EYFP.
851
(M) Membrane proteins were extracted from young buds of the wild type (Lane 1),
852
ProEMS1:EMS1-4xcMyc (Lane 2), ProβCA1:βCA1-Flag (Lane 3) and ProEMS1:EMS1-4xcMyc
853
ProβCA1:βCA1-Flag transgenic plants (Lane 4). After immunoprecipitation by anti-cMyc
854
antibody, βCA1-Flag was detected from proteins of ProEMS1:EMS1-4xcMyc
855
ProβCA1:βCA1-Flag plants.
856 857
Figure 2. Expression analyses of βCAs in anthers.
858 859
(A) RT-PCR showing the expression of four splice variants of βCA1 in wild-type (WT) young
860
buds as well as in WT and ems1 anthers.
861
(B) RT-PCR showing the expression of βCA2, βCA3, and βCA4 in WT and ems1 anthers. PCR
862
products represent total transcripts of βCA2, βCA3, and βCA4. The ACTIN2 gene was used as an
863
internal control.
864
(C) to (H) Confocal images showing the localization of βCA1 protein in ProβCA1:βCA1-GFP
865
anthers. Green: GFP signal, red: autofluorescence from chloroplasts, S: anther stage. βCA1 was
866
detected at low levels in the epidermis at stage 4 (C) and at high levels in tapetal cells at stage 5
867
(D) and stage 6 (E), which are two key stages for tapetal cell differentiation. βCA1 levels in
868
tapetal cells gradually decreased at stage 7 (F) and stage 8 (G). No βCA1 was observed in 29
869
stage-10 anthers (H). Bars = 50 µm.
870
(I) to (K) βCA1 was detected at the plasma membrane and in the cytoplasm of tapetal cells in
871
stage-5 anthers (I). (J) FM4-64 stained image of (I). (K) Merged image of (I) and (J). Insets
872
showing tapetal cell at high magnification (Arrowheads: plasma membrane). Bars = 10 µm.
873
(L) to (N) Confocal images showing relatively weak GFP signals in both tapetal cells and
874
microsporocytes in ProβCA2:βCA2-GFP (L) and ProβCA4:βCA4-GFP (N) anthers at stage 5,
875
but no GFP signals in ProβCA3:βCA3-GFP anthers (M). E: epidermis, M: microsporocyte, and
876
T: tapetal cell. Bars = 50 µm.
877 878 879
Figure 3. Downregulation of βCAs impairs male fertility.
880 881
(A) to (G) Wild type (A), small and sterile βca1 βca2 βca4 (B), Pro35S:amirβCA1-4 (C) and
882
ProA9:βCA1.3/βca1 βca2 βca4 (G) plants; normal growth but sterile ProA9:amirβCA1-4 plants
883
(D), normal growth and fertile ProβCA1:βCA1/βca1 βca2 βca4 plants (E), and fertile but small
884
ProA9:βCA1.4/βca1 βca2 βca4 plants (F) (All six weeks old). Arrows: fertile siliques,
885
Arrowheads: sterile siliques; Bars = 2 cm.
886
(H) to (N) Alexander staining of pollen in mature anthers showing viable pollen grains in
887
wild-type (H), ProβCA1:βCA1/βca1 βca2 βca4 (L) and ProA9:βCA1.4/βca1 βca2 βca4 (M)
888
anthers, but no viable pollen grains in βca1 βca2 βca4 (I), Pro35S:amirβCA1-4 (J),
889
ProA9:amirβCA1-4 (K) and ProA9:βCA1.3/βca1 βca2 βca4 (N) anthers. Bars = 50 µm.
890 891 892
Figure 4. Downregulation of βCAs causes abnormal tapetal cell differentiation.
893 894
(A) to (E) Semi-thin sections of stage-6 anther lobes showing four somatic cell layers, including
895
epidermis (E), endothecium (En), the middle layer (ML) and tapetum (T), as well as reproductive
896
microsporocytes (M) in the centers of wild-type (A) and ProA9:βCA1.4/βca1 βca2 βca4 (E)
897
anthers, but vacuolated tapetal-like (TL) cells in βca1 βca2 βca4 (B), Pro35S:amirβCA1-4 (C),
898
and ProA9:amirβCA1-4 (D) anthers.
899
(F) to (J) Semi-thin sections of stage-7 anther lobes showing normal tapetum and tetrads (Tds) in 30
900
wild-type (F) and ProA9:βCA1.4/βca1 βca2 βca4 (J) anthers, but highly vacuolated tapetal-like
901
cells and degenerating microsporocytes (DM, no tetrads formed) in βca1 βca2 βca4 (G),
902
Pro35S:amirβCA1-4 (H), and ProA9:amirβCA1-4 (I) anthers.
903
(K) to (O) Semi-thin sections of stage-9 anther lobes showing a thin tapetum and normal
904
microspores (Ms) in wild-type (K) and ProA9:βCA1.4/βca1 βca2 βca4 (O) anthers, but
905
degenerated tapetal-like cells and microsporocytes (anther lobes are empty) in βca1 βca2 βca4
906
(L), Pro35S:amirβCA1-4 (M), and ProA9:amirβCA1-4 (N) anthers.
907
(P) to (T) At stage 6, in situ hybridization results showing that the tapetal cell marker gene A9
908
was expressed strongly in a monolayer of tapetal cells in wild-type (P) and ProA9:βCA1.4/βca1
909
βca2 βca4 (T) anthers, but weakly expressed in vacuolated tapetal-like cells of βca1 βca2 βca4
910
(Q), Pro35S:amirβCA1-4 (R) and ProA9:amirβCA1-4 (S) anthers.
911
(U) and (V) Confocal images showing that the expression of the elaioplast marker gene
912
FIB1a-GFP in tapetal cells of wild-type anthers (U) was higher than that in tapetal-like cells of
913
ProA9:amirβCA1-4 anthers (V) at stage 8. Arrows indicate GFP signals in tapetal cells. Bars =
914
10 µm.
915
(W) and (X) Confocal images showing that expression of the tapetosome marker gene
916
GRP17-GFP was higher in tapetal cells of wild-type anthers (W) than in tapetal-like cells of
917
ProA9:amirβCA1-4 anthers (X) at stage 10. Arrows indicate GFP signals in tapetal cells. Bars =
918
10 µm.
919
(Y) qRT-PCR results showing the expression of A9 in wild-type, βca1 βca2 βca4,
920
Pro35S:amirβCA1-4, and ProA9:amirβCA1-4 anthers. Numbers indicate independent lines of
921
Pro35S:amirβCA1-4 and ProA9:amirβCA1-4. Three independent transgenic lines were used for
922
each transgenic plant and three samples were collected from each transgenic line. Bars indicate
923
SD. Asterisks indicate significant difference (P
