The sHSP22 heat-shock protein requires the ABI1 protein phosphatase to modulate polar auxin

81 Pages·2017·3.2 MB·English

Plant Physiology Preview. Published on December 29, 2017, as DOI:10.1104/pp.17.01206

1 Title Page
2 The sHSP22 heat-shock protein requires the ABI1 protein phosphatase to modulate
3 polar auxin transport and downstream responses
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6 Running title: sHSP22 modulates plant response to ABA and auxin
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8
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10 To whom correspondence should be addressed:
11 Qi Xie
12 State Key Laboratory of Plant Genomics
13 Institute of Genetics and Developmental Biology
14 Chinese Academy of Sciences
15 Beijing 100101,
16 China
17 TEL: [phone]
18 FAX: [phone]
19 E-mail: Qi Xie: [email protected]
20 Yaorong Wu: [email protected]
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21 The sHSP22 heat-shock protein requires the ABI1 protein phosphatase to modulate
22 polar auxin transport and downstream responses
23
24 Yanli Li1, 2, 4, Yaqiong Li1, 2, 4, Yongchang Liu3, Yaorong Wu1, *, and Qi Xie1, 2*
25 1State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental
26 Biology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District,
27 Beijing 100101, China
28 2University of Chinese Academy of Sciences, Beijing 100049, China
29 3College of Chemistry and Bioengineering, Hunan University of Science and
30 Engineering, Yongzhou, Hunan 425199, China
31 4These authors contributed equally to this work.
32 One sentence summary
33 Endoplasmic reticulum-localized small heat shock protein sHSP22 ingtegrates ABA
34 and auxin signaling in Arabidopsis
35
36 Author Contributions
37 Y.-L. L, Y.-R. W and Q.X. designed the research. Y.-L. L, Y.-Q. L and Y.-C. L
38 performed research. Y.-L. L, Y.-R. W and Q.X. analyzed data. Y.-L. L, Y.-R. W and
39 Q.X. wrote the article.
40 Funding information
41 This research was supported by the National Key R&D Program of China grants
42 2016YFA0500500 and 2015CB942900 and NSFC 31571441 and 91317308 from the
43 National Science Foundation of China.
44
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45 Abstract
46 The phytohormones abscisic acid (ABA) and indole-3-acetic acid (IAA) response
47 pathways interact synergistically or antagonistically to regulate plant development
48 and environmental adaptation. Here, we show that ABI1, a key negative regulator of
49 ABA signaling, is essential for auxin-modulated root development. We performed a
50 microarray analysis using the loss-of-function mutant abi1-3 and Col-0 seedlings
51 treated with IAA. For sHSP22, an endoplasmic reticulum (ER) small heat shock
52 protein-encoding gene, the induction by IAA was dependent on ABI1. shsp22
53 displayed enhanced sensitivity to ABA in primary root growth. In contrast,
54 overexpression of full-length, but not truncated sHSP22 lacking signal peptide and
55 ER-retention sequences, resulted in decreased ABA sensitivity. Overexpressed (OX)
56 sHSP22 partially rescued the ABA hypersensitivity of abi1-3. In addition, sHSP22 is
57 involved in auxin-regulated hypocotyl elongation at high temperature treatment.
58 sHSP22 also affected accumulation of auxin efflux carrier PIN proteins due to
59 potentiated intracellular trafficking. And sHSP22 OX lines initiated more lateral roots
60 after auxin application. Our results suggest that sHSP22 regulates auxin response
61 through modulating auxin polar transport, and ABI1-sHSP22 provides a novel module
62 orchestrating ABA and auxin signaling crosstalk in Arabidopsis.
63
64 Key words: sHSP22; ABI1; ABA; auxin polar transport; PINs
65
66 Introduction
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67 Plants exhibit tremendous plasticity and adaptability in growth and development to
68 coordinate with environmental conditions. Phytohormone levels and signaling are
69 fine-tuned to execute central roles in perceiving and initiating appropriate responses.
70 Mounting evidence supports the hypothesis that plant hormones act in the context of
71 large regulatory networks affecting each other’s synthesis and response pathways
72 (Kuppusamy et al., 2009). Among these hormones, indole-3-acetic acid (IAA) and
73 abscisic acid (ABA) interplay dynamically in diverse physiological processes
74 (Vanstraelen and Benkova, 2012).
75 IAA, the most abundant endogenous auxin, is the best-characterized hormone
76 affecting morphogenesis and tropisms in Arabidopsis. Spatiotemporal asymmetric
77 auxin distribution patterns within different tissues direct such developmental
78 processing (Vanneste and Friml, 2009; Sauer et al., 2013). Auxin metabolism,
79 including biosynthesis, conjugation, and cellular transport, determines its distribution
80 pattern (Ikeda et al., 2009; Zhao, 2010). The polar auxin transport facilitators, such as
81 membrane-localized ATAUX1/LIKE AUX1 (AUX1/LAX) influx transporters and
82 ATP-BINDING CASSETTE B (ABCB), PIN-FORMED (PIN) protein efflux carriers,
83 transport auxin in a cell-to-cell manner to establish the auxin gradients that is essential
84 for organogenesis (Bennett et al., 1996; Benkova et al., 2003; Swarup et al., 2008).
85 Auxin accumulation within an individual cell is interpreted by a short
86 nuclear-localized signaling pathway to induce transcriptional responses. Auxin binds
87 to the F-box protein TIR1/AFB, a subunit of ubiquitin E3 ligase SCFTIR1-AFBs
88 (Skp1-cullin-F box protein), in the presence of Aux/IAA repressor proteins, thus
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89 mediating Aux/IAA ubiquitylation and degradation by the 26S proteasome,
90 consequently releasing ARF (AUXIN RESPONSE FACTOR) transcriptional activity
91 (Gray et al., 2001; Dharmasiri et al., 2005; Kepinski and Leyser, 2005). The auxin
92 sensitive reporters DR5:β-glucuronidase (GUS) (Ulmasov et al., 1997) and
93 DR5:green fluorescent protein (GFP) (Friml et al., 2003) are extensively utilized to
94 monitor auxin responses.
95 ABA regulates many aspects of plant growth and development, such as seed
96 maturation and dormancy, root growth maintenance, vegetative development and
97 reproduction. In addition, ABA functions as a “stress hormone” that perceives and
98 reacts to environmental challenges such as drought, salinity, heat and cold (Zhu,
99 2002). The underlying mechanism relies on reprogramming the transcription of
100 stress-responsive protective genes and regulatory signaling components (Shinozaki
101 and Yamaguchi-Shinozaki, 2007). Elucidation of ABA signaling transduction pathway
102 highlights type 2C protein phosphatases (PP2Cs) such as ABI1, which act as
103 important upstream negative regulators. The second messengers phosphatidic acid
104 (PA) and hydrogen peroxide, both decrease the phosphatase activity of ABI1
105 (Meinhard and Grill, 2001; Zhang et al., 2004), emphasizing ABI1 as a hub that
106 integrates second messenger signaling and different effectors mediated stress
107 tolerance (Cutler et al., 2010).
108 Previous research shows that auxin and ABA have complex and extensive
109 cross-talk in seed germination, seedling establishment, and lateral root emergence.
110 The auxin reporter, DR5:GUS, exhibits asymmetric expression that is maximal at the
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111 meristems during Arabidopsis seed germination, which suggests that an auxin
112 gradient is essential for early seedling meristem establishment and maintenance
113 (Tanaka et al., 2006). ABA represses post-germination axis elongation through
114 combinatorial modulation of auxin transport facilitators AUX1, PIN2, and
115 down-regulation of AXR2/IAA7-mediated signal transduction (Belin et al., 2009).
116 Additionally, ARF10 repression by MIR160 has been proved to be important for seed
117 germination and post-embryonic development (Liu et al., 2007). Auxin Response
118 Factor2 (ARF2) modulates abscisic acid response in Arabidopsis by downregulating
119 the expression of homeodomain gene HB33 (Wang et al., 2011). DEXH box RNA
120 helicase is involved in the crosstalk between abscisic acid and auxin signaling by
121 mediating mitochondrial reactive oxygen species production in Arabidopsis (He et al.,
122 2012). All these findings demonstrate that ABA and auxin counteract each other at
123 these phases of development.
124 Both ABA and auxin are involved in lateral root development. The
125 ABA-insensitive 8 (abi8) mutant displays limited lateral roots due to a defect in root
126 meristem activity (Brocard-Gifford et al., 2004). Lateral root development 2 (LRD2)
127 plays an essential role in repressing lateral root development under osmotic stress, and
128 likely influences lateral root development by interfering with auxin signaling (Deak
129 and Malamy, 2005). Moreover, ABI3 modulated by farnesylation acts as an
130 interaction node that is involves auxin signaling and is required for lateral root
131 formation (Brady et al., 2003). The transcription factor WRKY46 acts upstream of
132 ABI4 to promote lateral root initiation under osmotic/salt conditions via regulation of
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133 auxin homeostasis (Ding et al., 2015). Additionally, ibr5, mutant of IBR5 encoding
134 dual-specificity phosphatase, shows the decreased sensitivity to both ABA and auxin.
135 Further research discovers that IBR5 interacts with and dephosphorylates MPK12 to
136 regulate auxin signaling, nevertheless, how ibr5 responds to ABA remains to be
137 determined (Monroe-Augustus et al., 2003; Strader et al., 2008; Lee et al., 2009).
138 Moreover, auxin signaling mutants axr1, axr2/iaa7, slr/iaa14, iaa16, and axr3/iaa17
139 all differentially respond to applied ABA compared with wild type plants, suggesting
140 that Aux/IAA-dependent auxin signaling also affects ABA activity (Fukaki et al.,
141 2002; Tiryaki and Staswick, 2002; Rinaldi et al., 2012). Despite description of
142 multiple genes mediating ABA and auxin crosstalk, the fundamental mechanisms
143 underlying their interconnection remain rudimentary.
144 Small heat shock proteins (sHSP) are ubiquitously distributed and comprise a
145 family of proteins that are characterized by a conserved -crystallin domain (ACD)
146 and range in size from 15-45 kDa (Jakob et al., 1993; Basha et al., 2006). The
147 representative ACD consists of about 90 amino acid sandwich, flanked by a variable
148 N-terminal and a short C-terminal extension (MacRae, 2000). sHSP expression is
149 induced under heat shock conditions as well as under other abiotic and biotic stresses.
150 A number of sHSPs are involved in plant development, for instance, seed maturation
151 and germination, embryogenesis as well as pollen development (Waters et al., 1996).
152 Crystal structures of an archaea HSP16.5 from M. jannaschii and a plant HSP16.9
153 from T. aestivum revealed large oligomers and the architectural basis for molecular
154 chaperone activity (Kim et al., 1998; van Montfort et al., 2001). sHSP complex bind
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155 and prevent irreversible denatured aggregation in an ATP-independent manner,
156 thereby conferring thermotolerance to heat shock (Haslbeck et al., 2005). While a
157 limited number of orthologs exist in other organisms, Arabidopsis has a relatively
158 large family of 19 sHSPs that are classified into cytosolic, chloroplastic, endoplasmic
159 reticulum, and mitochondrial members based on their subcellular localization (Scharf
160 et al., 2001). Although no clear explanation accounts for exact molecular function of
161 plant sHSPs in addition to heat acclimation, recent studies have shed light on sHSP’s
162 role in protein translocation, lipid interaction and maintenance of membrane integrity
163 (Torok et al., 2001; Chowdary et al., 2007; Balogi et al., 2008; Kim et al., 2011).
164 Distinct from other Arabidopsis sHSPs, sHSP22 is the only identified small heat
165 shock protein localized in ER with a specific signal peptide and the ER retention
166 tetrapeptide SKEL, that shares 64.5% amino acid similarity to class I cytosolic
167 sHSP17.6 (Helm et al., 1995). However, the molecular and physiological function of
168 sHSP22 remains to be elucidated.
169 In this study, we show that a loss-of-function allele in ABI1, a significant
170 negative regulator of ABA signal transduction, exhibits decreased sensitivity to
171 exogenous auxin in lateral root formation. In search of downstream targets of ABI1
172 essential for auxin-involved lateral root development, microarray analysis highlights
173 ABI-dependent sHSP22 expression. Further genetic and physiological examination of
174 sHSP22 loss-of-function and overexpression plants demonstrate that ER-localized
175 sHSP22 negatively regulates ABA signaling, whereas overexpression of sHSP22
176 enhances auxin-associated hypocotyl elongation at high temperature and increases
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177 sensitivity in root growth to auxin transport inhibitor naphthyl phthalamic acid (NPA).
178 sHSP22 affects intracellular vesicle trafficking of PIN proteins. As a consequence,
179 overexpression of sHSP22 promotes increased lateral root number when seedlings
180 were treated by auxin.
181
182 Results
183 ABI1 is involved in auxin-triggered lateral root development
184 Exogenous auxin application can promote lateral root development (Benkova et al.,
185 2003), which has been widely employed to identify mutants with altered auxin
186 responses. In order to identify more players in crosstalk between ABA and auxin
187 signal transduction pathways, we screened major ABA signaling mutants for changes
188 in lateral root formation of auxin application, including natural IAA and a synthetic
189 analog1-napthalene acetic acid (NAA). We show that abi1-3, a loss-of-function allele
190 of ABI1 which is hypersensitive to ABA in germination (Saez et al., 2006), decreased
191 sensitivity to exogenous auxin compared with Col-0 (Figure 1A). Although treatment
192 of Col-0 plants with 100 nM NAA or 100 nM IAA resulted in 10-fold and 4-fold more
193 lateral roots respectively, a reduced lateral roots number was observed in abi1-3
194 (Figures 1A and 1B). In support, another two abi1 mutants were also tested. As shown
195 in Figure S1, the knockdown mutant abi1-2 displayed similar phenotype with abi1-3,
196 whereas abi1-11, gain-of-function abi1-1 analogue in Col ecotype, has no dramatic
197 difference from WT. The less sensitivity of abi1 to auxin treatment in lateral root
198 development suggested that ABI1 is required for auxin-stimulated lateral root
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199 development. Given that ABI1 operates as a key repressor in the ABA signaling
200 pathway, we supposed that it plays a role in the interconnection between ABA and
201 auxin.
202
203 sHSP22 expression induced by IAA treatment is dependent on ABI1
204 To explore the function of ABI1 in response to IAA treatment, we performed a
205 microarray analysis using RNA prepared from abi1-3 and Col-0 seedlings treated with
206 or without IAA for a time course of 0, 2, and 6 hours. From the differential expressed
207 genes of the microarray data (Table S1), we searched for genes whose transcripts were
208 induced by IAA in Col-0, but were attenuated or absent in abi1-3. We identified
209 sHSP22 encoding a small heat shock protein. And no function of this gene was
210 reported. We further analyze in details of its function in plant response to IAA and
211 ABA treatments. As shown in Figure 2A, sHSP22 was strongly and transiently
212 induced at 2 hours of IAA treatment in Col-0, whereas this induction was completely
213 absent in abi1-3. Subsequent quantitative RT-PCR confirmed the microarray results
214 (Figure 2B), suggesting that IAA rapidly activated ABI1-dependent expression of
215 sHSP22. Because there is an adjacent gene mutation of MAP Kinase Kinase 1 (MKK1)
216 in abi1-3 (Wu et al., 2015), we detected the expression levels of sHSP22 in the
217 relevant materials to rule out the possibility that sHSP22 was influenced by MKK1.
218 The result showed that the expression levels of sHSP22 are not affected by the
219 additional mutation of MKK1 in abi1-3 (Figure S2). Moreover, we introduced sHSP22
220 fused with GUS reporter driven by its native promoter into Col-0 and abi1-3.
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