Asparagus IRX9, IRX10 and IRX14A are essential for xylan biosynthesis in the Golgi apparatus of ...

57 Pages·2016·7.57 MB·English

Plant Physiology Preview. Published on March 7, 2016, as DOI:10.1104/pp.15.01919

1 Short title: Asparagus xylan synthase complex
2
3 To whom correspondence should be addressed:
4 Prof. Antony Bacic
5 ARC Centre of Excellence in Plant Cell Walls,
6 School of BioSciences,
7 The University of Melbourne,
8 Melbourne, Victoria 3010, Australia
9
10 Email: [email protected]
11 Ph: [phone]
12 Fax: [phone]
13
14 Research area: Biochemistry and Metabolism
1
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Copyright 2016 by the American Society of Plant Biologists
15 Asparagus IRX9, IRX10 and IRX14A are essential for xylan biosynthesis in the Golgi
16 apparatus of Asparagus
17
18 Wei Zeng†a, Edwin R. Lampugnani†a, Kelsey L. Picard†a, Lili Songb, Ai-Min Wuc, Isabela M.
19 Fariona, Jia Zhaoa, Kris Forda, Monika S. Doblina, Antony Bacic*a
20
21 † These authors contributed equally to this work.
22
23 a ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, The University of
24 Melbourne, Parkville, Victoria 3010, Australia
25 b Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F
26 University, Lin’an, Hangzhou, 311300, China
27 c State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,
28 South China Agricultural University, Guangzhou, 510642, China
29
30 Summary: Asparagus IRX9, IRX10 and IRX14A are involved in xylan biosynthesis in the
31 Golgi apparatus.
32
33 Footnotes:
34 Financial Source: This work was supported by Australian Research Council (ARC) Centre of
35 Excellence in Plant Cell Walls grant (WZ, ERL, KLP, JZ, IMF, KF, MSD, AB
36 (CE110001007)), China National Natural Science Foundation (AW (31170165)) and
37 Zhejiang Provincial Natural Science Foundation of China (LS (LR15C160001)).
38
39 Author contributions:
40 WZ, ERL, KLP, MSD, AB conceived and designed the experiments:
41 WZ, ERL, KLP, LS, AW, IMF, JZ, KF performed the experiments.
42 WZ, ERL, KLP, MSD, AB wrote the paper and provided the conceptual framework for the
43 work.
44
45 * To whom correspondence should be addressed:
46 Antony Bacic
47 [email protected]
48
2
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
49
3
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
50 Abstract
51
52 Heteroxylans are abundant components of plant cell walls and provide important raw
53 materials for the food, pharmaceutical and biofuel industries. A number of studies in
54 Arabidopsis have suggested that the IRREGULAR XYLEM (IRX)9, IRX10 and IRX14
55 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localised
56 complex termed the xylan synthase complex (XSC). However, both the biochemical and cell
57 biological research lags the genetic and molecular evidence. In this study, we characterised
58 Asparagus officinalis (garden asparagus) stem xylan biosynthesis genes (AoIRX9, AoIRX9L,
59 AoIRX10, AoIRX14A and AoIRX14B) by heterologous expression in Nicotiana benthamiana.
60 We reconstituted and partially purified an active XSC and showed that three proteins,
61 AoIRX9, AoIRX10 and AoIRX14A, are necessary for xylan xylosyltranferase (XylT) activity
62 in planta. To better understand the XSC structure and its composition, we carried out co-
63 immunoprecipitation (Co-IP) and Bimolecular Fluorescence Complementation (BiFC)
64 analysis to show the molecular interactions between these three IRX proteins. Using a site-
65 directed mutagenesis (SDM) approach, we showed that the DxD motifs of AoIRX10 and
66 AoIRX14A are crucial for the catalytic activity. These data provide the first lines of
67 biochemical and cell biological evidence that AoIRX9, AoIRX10 and AoIRX14A are core
68 components of a Golgi-localised XSC, each with distinct roles for effective heteroxylan
69 biosynthesis.
70
71
4
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
72 Introduction
73 Heteroxylans are abundant non-cellulosic matrix polysaccharides of plant cell walls. They are
74 the main component of the primary wall of commelinid monocots (including grasses) and
75 also the secondary wall of most woody dicot species (Faik 2010; Rennie and Scheller 2014).
76 Heteroxylans have a linear backbone of β-1,4-linked D-xylopyranosyl residues (Xylp) with
77 backbone decorations that vary between cell types and tissues, developmental stages and
78 species. The dominant decorations are α-1,2 and/or α-1,3-linked L-arabinofuranosyl residues
79 (Araf) in grass primary walls and α-1,2-linked (methyl)glucuronopyranosyl residues
80 ((Me)GlcAp) in woody tissues (Bastawde 1992; Ebringerova et al. 2005). In commelinid
81 monocots, the Araf residues can be further decorated with a ferulic acid residue which,
82 through dimerization, can crosslink heteroxylans into the wall (Ishii 1997). The α-1,2-GlcA
83 residues can be further modified with α-1,2- linked galactopyranosyl residues (Galp) found in
84 heteroxylans of Eucalyptus globulus (Shatalov et al. 1999) and Arabidopsis (Zhong et al.
85 2014). Recently, an ‘atypical’ xylan was found with a 1,2- pentosyl residue (not determined if
86 Ara or Xyl) linked to the α-1,2-GlcA sidechain of the xylan backbone in the primary wall
87 from Arabidopsis (Chong et al. 2015; Mortimer et al. 2015). Additionally, the reducing end
88 (RE) sequence of heteroxylans from dicots and gymnosperms differs from that of grasses. In
89 the dicots/gymnosperms it is a characteristic tetrasaccharide glycosyl sequence; -β-D-Xylp-
90 (1→3)-α-L-Rhap-(1→2)-α-D-GalpA-(1→4)-D-Xylp that is absent in the grasses (for a
91 review see Hao and Mohnen (2014). Recently Ratnayake et al (2014) reported that the RE
92 glycosyl sequence of wheat endosperm arabinoxylan comprises a linear (1→4)-β-D-Xylp
93 backbone which may be mono-substituted with either an α-L-Araf residue at the RE β-D-
94 Xylp residue and/or penultimate RE β-D-Xyl residue or with an α-D-GlcpA residue at the RE
95 β-D-Xylp residue. These studies, and many others, highlight the diversity of heteroxylan
96 structures and therefore the complex nature of heteroxylan biosynthesis.
5
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
97 Most non-cellulosic plant cell wall polysaccharides are predicted to be synthesised in the
98 Golgi apparatus (GA) by glycosyltransferase (GT) enzyme complexes (Oikawa et al. 2013)
99 with the exception of cellulose, callose and (1,3;1,4)-β-glucans that are assembled at the
100 plasma membrane (Doblin et al. 2010; Wilson et al. 2015). Some examples of Golgi-located
101 polysaccharide biosynthesis complexes include GAUT1/GAUT7 involved in pectin
102 biosynthesis (Atmodjo et al. 2011), CSLC4/XylT in xyloglucan biosynthesis (Chou et al.
103 2012; Cocuron et al. 2007) and ARAD1/ARAD2 in arabinan biosynthesis (Harholt et al.
104 2012). Genetic studies in Arabidopsis have identified several GTs that are predicted to be
105 involved in a xylan backbone biosynthesis complex, namely AtIRX9, AtIRX10, AtIRX14,
106 AtPARVUS, AtIRX8, as well as their homologs (Brown et al. 2007; Brown et al. 2009;
107 Keppler and Showalter 2010; Lee et al. 2007a; Lee et al. 2007b; Pena et al. 2007; Wu et al.
108 2010; Wu et al. 2009; Zhong et al. 2005). Among these, AtIRX9 and AtIRX14 (both GT43
109 family members) and AtIRX10 (GT47), as well as their functionally redundant homologs, are
110 believed to be directly involved in xylan backbone biosynthesis and form a complex in the
111 GA (Rennie and Scheller 2014). Orthologs of the AtIRX proteins have been identified in
112 many other species including Triticum aestivum (wheat) (Zeng et al. 2010), Populus
113 trichocarpa (poplar) (Lee et al. 2012b), Physcomitrella patens (moss) (Hornblad et al. 2013),
114 Plantago ovata (psyllium) (Jensen et al. 2013), Oryza sativa (rice) (Chen et al. 2013;
115 Chiniquy et al. 2013), Gossypium hirsutum (cotton) (Li et al. 2014), Neolamarckia cadamba
116 (kadam) (Zhao et al. 2014) and Asparagus officinalis (garden asparagus) (Song et al. 2015).
117 Enzymes involved in xylan backbone biosynthesis as well as sidechain decorations, such as
118 the arabinosyltransferases (AraTs), glucuronosyltransferases (GlcATs) and acetylation
119 enzymes (TBL29) were recently reviewed by Rennie and Scheller (2014) and Hao and
120 Mohnen (2014).
6
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
121 Several biochemical studies have contributed towards unravelling the core partners
122 involved in xylan biosynthesis, but the issue is complex and the full suite of proteins has not
123 yet been elucidated (Rennie and Scheller 2014). Nevertheless, some progress has been made.
124 Microsomes extracted from tobacco BY2 cells co-expressing AtIRX9 and AtIRX14 have
125 been shown to have xylan synthase activity in vitro (Lee et al. 2012a). Furthermore, two
126 independent studies have demonstrated using a heterologous expression approach that
127 AtIRX10L (Urbanowicz et al. 2014) and IRX10 from plantago and P. patens (Jensen et al.
128 2014) also have distributive xylan XylT activity. Interestingly Ren et al.(2014), using site-
129 directed mutagenesis (SDM) and genetic approaches, showed that AtIRX9 is not directly
130 involved in catalytic activity, because mutant variants of potential catalytic domains of
131 AtIRX9 were still able to complement Arabidopsis irx9 mutants. Taken together, these
132 observations are consistent with the hypothesis that AtIRX9, AtIRX10 and AtIRX14 form a
133 xylan synthase complex (XSC) (Rennie and Scheller 2014). However, unequivocal
134 biochemical (or cell biological) proof of the nature of their interaction(s)/stoichiometry in
135 planta is still lacking, predominantly because of the low abundance of these Golgi-localised
136 proteins which makes the purification and characterisation of the enzyme complexes
137 challenging.
138 In order to define the biochemical activity of these Golgi-localised GTs, different
139 heterologous expression systems are routinely used for functional characterisation and
140 therefore overcome the inherent difficulties of purifying these low abundance membrane-
141 bound proteins. Nicotiana benthamiana is widely used as an efficient and high-level
142 expression system for functional characterisation of candidate genes (Voinnet et al. 2003). It
143 has been utilised to examine the biochemical activities of several plant cell wall
144 polysaccharide biosynthetic GTs, including xylogalacturonan (pectin) xylosyltransferase
145 (Jensen et al. 2008), xylan glucuronosyltransferase (Rennie et al. 2012), β-(1,4)-galactan
7
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
146 synthase (Liwanag et al. 2012), arabinogalactan-protein (AGP) galactosyltransferase (Geshi
147 et al. 2013) and (1,3;1,4)-β-glucan CSLF glucan synthase (Wilson et al. 2015). Other
148 heterologous expression systems (e.g. either yeast (Pichia pastoris) or mammalian cell lines)
149 have also been used to characterise the functions of GTs such as xyloglucan
150 xylosyltransferase (Faik et al. 2002) and pectin homogalacturonan galacturonosyltransferase
151 (Sterling et al. 2006).
152 We previously demonstrated a high level of xylan XylT activity (around 10 fold higher than
153 any other native in vitro system, including Arabidopsis) in vegetative spears of Asparagus
154 officinalis, a non-commelinid monocot species, and also identified five putative xylan
155 backbone biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A and AoIRX14B)
156 (Song et al 2015). Furthermore, we also showed that the xylan content, the xylan XylT
157 activity and the expression of the backbone biosynthesis genes changed along the
158 developmental gradient of the spear as it transitioned from an elongation zone to a maturation
159 zone (Song et al 2015). To characterise the precise biochemical function(s) of these genes, we
160 now show that three of these proteins (AoIRX9, AoIRX10 and AoIRX14A) must be
161 heterologously co-expressed for xylan XylT activity equivalent to that observed using
162 asparagus microsomal membranes (MMs). We also show that all three proteins are required
163 for GA retention. By using SDM, we demonstrated that the DxD motifs of AoIRX10 and
164 AoIRX14A are essential for catalytic activity. BiFC analysis indicated that AoIRX14A
165 directly interacts with AoIRX9 while AoIRX10 likely indirectly interacts with AoIRX9 and/or
166 AoIRX14A. These data provide the first lines of direct evidence that AoIRX9, AoIRX10 and
167 AoIRX14A are part of a core XSC together with potentially unknown partner(s) in the GA.
168
169
8
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
170 Results
171 Expression of Asparagus xylan biosynthesis genes in N. benthamiana
172 We have previously demonstrated that garden asparagus spears have strikingly high XylT
173 enzyme activity and identified five predicted xylan biosynthesis genes (AoIRX9, AoIRX9L,
174 AoIRX10, AoIRX14A and AoIRX14B) (Song et al. 2015). In the current study we sought to
175 test which of these genes are essential for the observed native activity by expressing
176 candidates heterologously in N. benthamiana, since an efficient transformation procedure for
177 asparagus is not available. The full-length open reading frames of AoIRX9, AoIRX9L,
178 AoIRX14A, AoIRX14B and AoIRX10 were cloned into a binary vector under the control of the
179 strong, constitutively active CaMV 35S promoter and expressed either singly or in
180 combinations in N. benthamiana leaves.
181 To verify the expression of these asparagus IRX proteins, we developed antibodies against
182 AoIRX9, AoIRX10 and AoIRX14A for protein detection and pull-down assays. Using
183 Western blot analysis we demonstrated that the antibodies detect the protein that they were
184 raised against (Figure 1). The bands detected by the AoIRX10- and AoIRX14A-directed
185 antibodies matched the predicted molecular mass (47.4 kDa and 57.4 kDa, respectively). A
186 weak band above 100 kDa was also detected with anti-AoIRX14A, possibly indicating the
187 existence of the homodimer (Figure 1). However, other bands were also visible suggesting
188 that this antibody is less specific than that of AoIRX10. Interestingly, a band at approximately
189 55 kDa was detected with anti-AoIRX9, which is higher than the predicted molecular mass of
190 40.8 kDa for this protein (Figure 1), suggesting that there may be post-translational
191 modifications occurring. The AoIRX9 antibody detected a single band in MMs from N.
192 benthamiana expressing either AoIRX9 or the triple combination of AoIRX9/10/14A, but no
193 signal was detected in MMs expressing either AoIRX9L, AoIRX10 and/or AoIRX14A (Figure
194 1). Similarly, AoIRX10 and separately AoIRX14A antibodies were only able to detect signal
9
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
195 in MMs that expressed their cognate protein (Figure 1). The expression levels of each AoIRX
196 protein are not significantly affected when co-expressed with other proteins since the Western
197 blot signal intensities of singly expressed and co-expressed AoIRX proteins are similar
198 (Figure 1).
199 Xylan synthase activity in MMs prepared from N. benthamiana leaves co-expressing
200 predicted xylan biosynthesis genes from Asparagus
201 As heteroxylans are synthesised in the GA, xylan XylT activity was assayed using MMs
202 prepared from N. benthamiana leaves expressing either single or combinations of asparagus
203 IRX proteins. Asparagus spear MMs were used as a positive control for the XylT activity. As
204 previously demonstrated by Song et al. (2015), asparagus MMs are capable of incorporating
205 up to 6 Xyl residues onto the Xyl -AA fluorescent acceptor within 1 hr under standard assay
5
206 conditions, producing β-1,4-xylo-oligosaccharides up to a degree of polymerisation (DP) of
207 11 (Figure 2). MMs prepared from N. benthamiana leaves infiltrated with a VENUS control
208 contained only the Xyl -AA acceptor, indicating that there is no detectable endogenous XylT
5
209 activity in N. benthamiana leaf MMs under these assay conditions. Individual expression of
210 each of the five AoIRXs showed no significant incorporation of Xyl onto the Xyl -AA
5
211 acceptor except for AoIRX10 and AoIRX14A which produced a small peak of Xyl -AA
6
212 (Figure 2). To quantify the XylT activity, we used UDP-[14C]Xyl as a donor to monitor Xyl
213 incorporation (Figure 2). Consistent with the RP-HPLC results, single expression of either
214 AoIRX9 or AoIRX9L leads to negligible Xyl incorporation. The expression of either
215 AoIRX10, AoIRX14A or AoIRX14B individually results in a very low level of incorporation,
216 equivalent to less than 7% of the XylT activity of native asparagus MMs.
217 Previous studies have proposed that AtIRX9, AtIRX10 and AtIRX14 are part of an active
218 xylan XylT complex in the GA for xylan backbone biosynthesis during Arabidopsis
10
Downloaded from on November 22, 2018 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.