Two membrane-anchored Aspartic Proteases contribute to pollen and ovule development

56 Pages·2016·4.52 MB·English

Plant Physiology Preview. Published on November 21, 2016, as DOI:10.1104/pp.16.01719
1 Running title:
2 Aspartic proteases A36/39 in plant fertility
3 Corresponding Authors:
4 (1) Yi Guo
5 NO.20 Nan Er Huan East Road, College of Life Science, Hebei Normal University,
6 Shijia Zhuang, 050024, P.R.China
7 Phone number: 0086-0311-80787502
8 Email: [email protected]
9 (2) Rui Li
10 NO.20 Nan Er Huan East Road, College of Life Science, Hebei Normal University,
11 Shijia Zhuang, 050024, P.R.China
12 Phone number: 0086-0311-80787548
13 Email: [email protected]
14
15 Research Areas
16 Primary Research Area: Cell Biology
17 Secondary Research Area: Genes, Development and Evolution
18
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19 Two membrane-anchored Aspartic Proteases contribute to pollen and ovule
20 development
21 Hui Gao1,2,3,a, Yinghui Zhang1,2,3,a, Wanlei Wang1,2,3, Keke Zhao1,2,3,b, Chunmei Liu1,2,3, Lin
22 Bai1,2,3, Rui Li1,2,3,*& Yi Guo1,2,3,*
23 1Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Science, Hebei
24 Normal University, Shijia Zhuang, Hebei 050024, P.R. China
25 2Key Laboratory of Molecular and Cellular Biology of Ministry of Education, College of Life
26 Science, Hebei Normal University, Shijia Zhuang, Hebei 050024, P.R. China
27 3Hebei Collaboration Innovation Center for Cell Signaling, Shijia Zhuang, Hebei 050024,
28 P.R. China
29 a These authors contributed equally to this work.
30
31
32 AUTHOR CONTRIBUTIONS
33 Y.G., R.L. and H.G. conceived the project and designed the experiments. H.G. carried out
34 protein extraction, western blot, glycosylation analysis, CLSM imaging and GUS staining
35 assay; Y.H.Z. carried out the cross-section experiment, TUNEL assay, and transmission
36 electron microscopy (TEM) assay; W.L.W. carried out the plasmid constructions; K.K.Z.
37 carried out the phenotypic analysis, transmission efficiency analysis, SEM assay and
38 subcellular localization; C.M.L. carried out the reverse transcription PCR (RT-PCR)
39 analysis; L.B. carried out the transgenic experiments; Y.G., R.L. and H.G. wrote the
40 manuscript.
41
42
43
44 One-sentence Summary：Two putative GPI-anchored aspartic proteases, A36 and A39,
45 are involved in pollen and ovule development as well as directional growth of pollen tube
46 toward ovules in Arabidopsis.
47
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48 Footnotes
49 This work was supported by the National Science Foundation of China (Grant no.
50 31270357 and 31540005), Program for New Century Excellent Talents in University
51 (NECT-12-0687), Key Project of Chinese Ministry of Education (Grant no. 211019) and
52 Natural Science Foundation of Hebei Province (Grant no.C2013205160). Foundation for
53 High-level Talents in Higher Education of Hebei, and 100 Innovative Talents Program of
54 Higher Education of Hebei for Yi Guo. The funders had no role in study design, data
55 collection and analysis, decision to publish, or preparation of the manuscript.
56
57 b Present address: NO. 2 Middle School of Qiu County, Handan, Hebei 057450,
58 P.R.China
59
60 * Address correspondence to [email protected] or [email protected]
61
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62 Abstract
63 Aspartic proteases are a class of proteolytic enzymes with conserved aspartic acid
64 residues, which are implicated in protein processing, maturation and degradation.
65 Compared to yeast and animals, plants possess a larger aspartic protease family.
66 However, little is known about most of these enzymes. Here, we characterized two
67 Arabidopsis (Arabidopsis thaliana) putative GPI-anchored aspartic protease genes, A36
68 and A39, which are highly expressed in pollen and pollen tubes. a36 and a36 a39 mutants
69 display significantly reduced pollen activity. Transmission electron microscopy and
70 terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assays further
71 revealed that the unviable pollen in the a36 a39 may undergo unanticipated apoptosis-like
72 programmed cell death. The degeneration of female gametes also occurred in a36 a39.
73 Aniline blue staining, scanning electron microscopy and semi-in vitro guidance assays
74 indicated that the micropylar guidance of pollen tubes is significantly compromised in a36
75 a39. A36 and A39 that were fused with the green fluorescent protein (GFP) are localized
76 to the plasma membrane and display punctate cytosolic localization and co-localize with
77 the GPI-anchored protein COBRA-LIKE 10. Furthermore, in a36 a39, the abundance of
78 highly methlyestered homogalacturonans and xyloglucans was significantly increased in
79 the apical pollen tube wall. These results indicate that A36 and A39, two putative
80 GPI-anchored aspartic proteases, play important roles in plant reproduction in
81 Arabidopsis.
82 Keywords: Aspartic proteases, pollen, pollen tube guidance, programmed cell death,
83 Arabidopsis.
84
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85 INTRODUCTION
86 In higher flowering plants, the life cycle from the generation of haploid gametophytes
87 to the generation of diploid sporophytes is essential to reproduction(Dumas and
88 Mogensen, 1993). Through meiosis and mitosis, the male and female gametophytes, also
89 called the pollen grain and the embryo sac, respectively, are generated from anther tissue
90 and ovules. The pollen grain germinates on the stigma, forming a long pollen tube, which
91 rapidly grows through the maternal tissue and accurately reaches the female gametes.
92 Then, the pollen tube ruptures and releases the two sperm cells to complete double
93 fertilization with the egg and central cell(Palanivelu and Tsukamoto, 2012). Therefore,
94 successful reproduction relies on normal male and female gametogenesis, as well as on
95 precise communication between the pollen tube and the ovule.
96 Aspartic protease (EC3.4.23), one of the four major types of proteolytic enzymes,
97 widely exist in bacteria, fungi, animals and plants(Barrett et al., 1998). These enzymes
98 contain two aspartic acid residues within the conserved Asp-Thr/Ser-Gly (DT/SG) motifs
99 at the active site and are activated at acidic pH and specifically inhibited by pepstatin
100 A(Barrett et al., 2004; Takahashi et al., 2008). Aspartic proteases have been widely
101 studied in animals, including β-secretase, pepsin, cathepsin D, etc(Ingram, 1951; Vassar
102 et al., 1999; Bach et al., 2015). However, research on plant aspartic proteases remains
103 limited.
104 The first well-studied plant aspartic protease was phytepsin from barley, which
105 possess a plant-specific insert (PSI), is localized to the vacuole and may function in the
106 programmed cell death of tracheary elements and sieve cells(Runeberg-Roos and
107 Saarma, 1998; Kervinen et al., 1999). Rice oryzasin also contains a PSI domain, is highly
108 expressed two or three weeks after flowering, and may be involved in the processing of
109 storage proteins in seeds(Asakura et al., 1995; Asakura et al., 2000). In 2005, Faro and
110 Gal found fifty-one genes that may encode putative aspartic proteases in the Arabidopsis
111 genome and divided them into three types, including typical aspartic proteases,
112 nucellin-like aspartic proteases and atypical aspartic proteases, according to their putative
113 domains and active sites(Faro and Gal, 2005). In 2008, Kenji Takahashi enlarged the
114 aspartic protease family to approximately 69 members(Takahashi et al., 2008).
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115 In the last decade, emerging evidences have revealed important roles for aspartic
116 proteases in leaf senescence, pathogen resistance, stress responses, and reproduction.
117 Tobacco CND41 is a DNA-binding aspartic protease expressed in chloroplasts, which
118 plays an essential role in leaf aging by degrading the Rubisco protein(Kato et al., 2005). In
119 Arabidopsis and rice, the apoplastic aspartic protease CONSTITUTIVE DISEASE
120 RESISTANCE 1 (CDR1) is involved in salicylic acid-mediated disease resistance(Xia et
121 al., 2004; Prasad et al., 2009). Furthermore, the overexpression of ASPARTIC
122 PROTEASE INGUARD CELL1 (ASPG1) increased ABA sensitivity in guard cells,
123 conferring drought tolerance in Arabidopsis(Yao et al., 2012). Recently, aspartic protease
124 APCB1 is involved in processing of Bcl-2-associated athanogene 6(BAG6) to trigger
125 autophagy and defense resistance(Li et al., 2016).
126 Aspartic proteases also play vital roles in plant sexual reproduction, such as tapetum
127 degeneration and gamete development. The proper timing of programmed cell death
128 (PCD) in tapetum cells is crucial for pollen formation. AtMYB80 and ETERNAL TAPETUM
129 1 (EAT1) in rice are transcription factors that control PCD in the tapetum and target
130 downstream aspartic proteases genes AtUNDEAD, OsAP25 and OsAP37, respectively.
131 Silencing the expression of AtUNDEAD caused the tapetal programmed cell death to
132 occur earlier, leading to pollen abortion(Phan et al., 2011), and overexpressing OsAP25
133 or OsAP37 induced cell death in both plants and yeast(Niu et al., 2013). In addition, many
134 aspartic proteases are also involved in gametophyte development. In rice, S5 participates
135 in indica-japonica hybrid fertility and could stimulate endoplasmic reticulum stress, giving
136 rise to PCD in the embryo sac(Chen et al., 2008; Yang et al., 2012). OsAP65 was shown
137 to be essential for pollen germination and tube growth(Huang et al., 2013). In Arabidopsis,
138 PROMOTION OF CELL SURVIVAL1 (PCS1) encodes an ER-localized aspartic protease,
139 and loss function of PCS1 causes degeneration of gametophytes and embryos(Ge et al.,
140 2005). Therefore, plant aspartic proteases may be implicated in the regulation of
141 programmed cell death in plant reproduction, though the underlying mechanism is not
142 clear. Moreover, the functions of the majority of Arabidopsis aspartic proteases are still
143 unknown.
144 In the present study, we characterized A36 and A39, two putative GPI-anchored
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145 aspartic proteases that are preferentially expressed in the pollen and pollen tube in
146 Arabidopsis. Loss function of A36 and A39 results in the apoptosis-like programmed cell
147 death of microspores, the degeneration of female gametophytes and compromised pollen
148 tube guidance. Moreover, A36-GFP and A39-GFP are co-localized with COBRA-LIKE 10
149 (COBL10), which is a GPI-anchored protein involved in pollen tube guidance. Our results
150 indicated that A36 and A39 play important roles in plant fertility.
151
152
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153 RESULTS
154 A36 and A39 are highly expressed in pollen and pollen tube and displays aspartic
155 proteolytic enzyme activity
156 Our preliminary work on proteomic analyses of proteins in germinating Arabidopsis
157 thaliana pollen had identified 71 differentially expressed proteins(Ge et al., 2011). In the
158 present study, two aspartic proteases, A36 (At5G36260) and A39 (At1G65240), were
159 selected for further investigation. Evolutionary analysis indicates that A36 shares high
160 similarity with several proteins from different model organisms but also displays relatively
161 clear differentiation (Supplemental Fig. S1A). In addition, A39 shares 73% amino acid
162 identity with A36, both contain N-terminal signal peptide and hydrophobic C-terminus
163 (http://smart.embl-heidelberg.de/, http://www.cbs.dtu.dk/services/SignalP-2.0/) and are
164 predicted to be GPI-anchored proteins (GPI-APs) by big-PI Predictor
165 (http://mendel.imp.ac.at/gpi/gpi_server.html)(Eisenhaber et al., 1998) (Supplemental Fig.
166 S1B). Based on protein sequence alignments, A36 shows 23% and 22% identity with
167 yeast (Saccharomyces cerevisiae) ScYps1p and human BACE1, respectively. To explore
168 the detailed functions of A36 and A39 in plant growth, particularly in male gametophyte
169 development, reverse transcription-PCR (RT-PCR) was performed to examine the
170 expression pattern of A36 and A39 in different organs, including roots, stems, rosette
171 leaves, cauline leaves, inflorescence, open flowers, siliques and seedlings (Fig. 1A). As
172 shown in Fig. 1A, both A36 and A39 are highly expressed in inflorescence and open
173 flowers. A36 mRNA is also present at relatively high levels in the roots and has moderate
174 expression in the stems, siliques, and seedlings, while A39 transcripts were highly
175 detected in the siliques and showed little expression in the cauline leaves and seedlings.
176 To further explore the expression profiles of A36 and A39, pA36::GUS and pA39::GUS
177 transgenic plants were generated. In T3 transgenic lines, A36 is exclusively highly
178 expressed in the pollen (Fig. 1, B and D) and growing pollen tubes (Fig. 1C; Supplemental
179 Fig. S2E). During microspore development, the GUS signal of A36 could only be found in
180 tricellular pollen and not in uninuclear or bicellular pollen (Fig. 1, E-L and Supplemental
181 Fig. S2, H-K). The GUS signal of A36 was not detected in the seedlings, leaves, stems,
182 siliques or ovules (Supplemental Fig. S2, A-D and P). Compared with A36, A39 shows a
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183 similar but distinct expression profile. Similar to A36, A39 is also highly expressed in the
184 inflorescence and pollen tubes (Fig. 1, M and O), and its GUS signal is not detected in the
185 seedlings or leaves (Supplemental Fig. S2, F and G). However, different from A36, A39
186 was expressed in from tetrad to tricellular pollen (Fig. 1, P-W and Supplemental Fig. S2,
187 L-O), young siliques (Fig. 1N) and developing ovules (Supplemental Fig. S2Q). Therefore,
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188 both A36 and A39 are preferentially expressed in tricellular pollen and pollen tubes,
189 although A39 is also expressed in the developing microspores, ovules and young siliques.
190 To clarify whether A36 and A39 share aspartic protease activity, we first performed a
191 sequence alignment of their active site regions with the corresponding regions from PCS1
192 (PROMOTION OF CELL SURVIVAL 1, A35) and CDR1 (CONSTITUTIVE DISEASE
193 RESISTANCE, A3) (Supplemental Fig. S1C). Like other eukaryotic aspartic proteases,
194 both A36 and A39 contain two catalytic aspartic acid residues within two conserved active
195 sites with the sequence Asp-Thr/Ser-Gly-Ser/Thr. Next, we made four glutathione
196 S-transferase (GST)-fusion protein constructs, GST-A36-D (106-1347 bp of CDS),
197 GST-A36 (full CDS sequence), GST-A39-D (88-1275 bp of CDS) and GST-A39 (full CDS
198 sequence) (Supplemental Fig. S3). In addition, four mutations of A36 and A39 were
199 generated by site-directed mutagenesis: D96N and D310N in A36 and A36-D (two
200 aspartic acid residues at position 96 and 310 were replaced by asparagines) and D92N (a
201 single aspartic acid residue at position 92 is replaced by asparagines) in A39 and A39-D
202 (Supplemental Fig. S3). All of these GST-fusion proteins were expressed in Escherichia
203 coli and purified in vitro. Because additional bands existed in purified GST-fusion proteins,
204 we used different contents of BSA and the corresponding mean gray value to make a
205 standard curve for measuring the concentration of purified full-length GST-fusion proteins
206 (Supplemental Fig. S3). A semi-quantitative assay was performed using FITC-labeled
207 casein as the substrate (Fig. 1X). The same amounts of proteins were used to measure
208 the proteolytic activity. At pH 5.4, the proteolytic activity of GST-A36 was 45.6% of that of
209 pork pepsin, while GST-A36-D showed 41.5% relative activity (Fig. 1X). GST-A39 and
210 GST-A39-D displayed proteolytic activities that are 34% and 33.2% of those of pork
211 pepsin, respectively (Fig. 1X). However, GST-A36 D96N D310N, GST-A36-D D96N
212 D310N, GST-A39 D92N and GST-A39-D D92N have hardly any proteolytic activity (Fig.
213 1X). The proteolytic activity of all GST-fusion proteins is significantly inhibited by the
214 aspartic protease inhibitor pepstatin A (Fig. 1X). Thus, both A36 and A39 show aspartic
215 proteolytic activity in vitro.
216 Disruption of A36 results in decreased male transmission efficiency
217 To explore the biological functions of A36 and A39 in plant growth, particularly in male
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