Plant Growth Regulation

AtGLK2, an Arabidopsis GOLDEN2-LIKE transcription factor, positively regulates

anthocyanin biosynthesis via HY5-mediated light signaling
--Manuscript Draft--

Manuscript Number: GROW-D-21-00313

Full Title: AtGLK2, an Arabidopsis GOLDEN2-LIKE transcription factor, positively regulates

anthocyanin biosynthesis via HY5-mediated light signaling

Article Type: Original Research

Keywords: Arabidopsis; Light signaling; AtGLK2; HY5; Anthocyanin biosynthesis

Abstract: Light serves as one of the major environmental signals that stimulates anthocyanin
biosynthesis in higher plants. In this study, we demonstrate that AtGLK2, an
Arabidopsis nuclear GARP transcription factor, plays a positive role in the regulation of
anthocyanin biosynthesis. The disruption of AtGLK2 decreased the anthocyanin
contents in Arabidopsis seedlings, whereas the overexpression of AtGLK2 resulted in
significant enhancements to the accumulation of anthocyanins. Gene expression
analysis demonstrated that the expression of late biosynthetic genes in the
anthocyanin biosynthetic pathway was dramatically reduced in glk2 but elevated in
AtGLK2 -overexpressing seedlings. It was found that the disruption of HY5
decreases the expression of AtGLK2 and that AtGLK2 exhibits light-responsive
expression patterns in an HY5-dependent manner. Further studies demonstrated that
HY5-mediated light signaling is required for AtGLK2-regulated anthocyanin
accumulation and photosynthetic development. Moreover, AtGLK2 and MYBL2
antagonistically regulate the expression of anthocyanin biosynthetic genes and the
subsequent anthocyanin accumulation process. Taken together, our data reveal that
AtGLK2 positively regulates anthocyanin biosynthesis through HY5-mediated light
signaling in Arabidopsis.

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1 AtGLK2, an Arabidopsis GOLDEN2-LIKE

2 transcription factor, positively regulates
3 anthocyanin biosynthesis via HY5-mediated light
4 signaling
5
6 Dong Liu1 · Dongming Zhao1 · Xuewei Li1 · Yongjun Zeng1*

8 1 Key Laboratory of Crop Physiology, Ecology and Genetic Breeding,

9 Ministry of Education, Jiangxi Agricultural University, Nanchang, 330045,
10 China

11 * Corresponding author
12
13
14
15 Author of correspondence:
16 Yongjun Zeng
17 Key Laboratory of Crop Physiology, Ecology and Genetic Breeding,
18 Ministry of Education
19 Jiangxi Agricultural University
20 Nanchang 330045
21 China
22 E-mail: zengyj2002@163.com

1
23 Abstract

24 Light serves as one of the major environmental signals that stimulates anthocyanin
25 biosynthesis in higher plants. In this study, we demonstrate that AtGLK2, an
26 Arabidopsis nuclear GARP transcription factor, plays a positive role in the
27 regulation of anthocyanin biosynthesis. The disruption of AtGLK2 decreased the
28 anthocyanin contents in Arabidopsis seedlings, whereas the overexpression of
29 AtGLK2 resulted in significant enhancements to the accumulation of anthocyanins.
30 Gene expression analysis demonstrated that the expression of late biosynthetic
31 genes in the anthocyanin biosynthetic pathway was dramatically reduced in glk2
32 but elevated in AtGLK2-overexpressing seedlings. It was found that the disruption
33 of HY5 decreases the expression of AtGLK2 and that AtGLK2 exhibits
34 light-responsive expression patterns in an HY5-dependent manner. Further studies
35 demonstrated that HY5-mediated light signaling is required for
36 AtGLK2-regulated anthocyanin accumulation and photosynthetic development.
37 Moreover, AtGLK2 and MYBL2 antagonistically regulate the expression of
38 anthocyanin biosynthetic genes and the subsequent anthocyanin accumulation
39 process. Taken together, our data reveal that AtGLK2 positively regulates
40 anthocyanin biosynthesis through HY5-mediated light signaling in Arabidopsis.
41
42 Keywords Arabidopsis · Light signaling · AtGLK2 · HY5 · Anthocyanin
43 biosynthesis

2
44 Introduction

45 Anthocyanins are broadly found in plant species and are responsible for different
46 colors in many plant organs, such as leaves, flowers, fruits, and nuts. The colors
47 of anthocyanins vary from red to blue, mainly depending on the environmental
48 pH and the linkages with heavy metals present in the plant cells and tissue
49 (Alappat and Alappat 2020). The presence of anthocyanin in flowers help attract
50 insects and animals to pollinate plants (Winkel-Shirley 2001). Anthocyanin, as
51 the predominant antioxidant component, also plays important roles in improving
52 plant stress tolerance (Nakabayashi et al. 2014; Moustaka et al. 2020).
53 Anthocyanins are biosynthesized on the cytosolic surface of the endoplasmic
54 reticulum and accumulate mainly in the vacuole. The biosynthesis of
55 anthocyanins is catalyzed by a series of enzymes in the specific branch of the
56 flavonoid pathway (Petroni and Tonelli 2011). These enzymes are encoded by
57 two groups of genes called early and late biosynthetic genes (Ferreyra et al.
58 2012). Early biosynthetic genes (EBGs) such as CHALCONE SYNTHASE (CHS),
59 CHALCONE ISOMERASE (CHI), and FLAVANONE 3-HYDROXYLASE (F3H)
60 are preferentially activated by MYB transcription factors such as MYB11,
61 MYB12, and MYB111 (Mehrtens et al. 2005; Stracke et al. 2007). In contrast,
62 late biosynthetic genes (LBGs), including FLAVONOID 3'-HYDROXYLASE
63 (F3'H), DIHYDROFLAVONOL 4-REDUCTASE (DFR),
64 LEUCOANTHOCYANIDIN OXYGENASE (LDOX), and
65 UDP-GLUCOSE:FLAVONOID 3-O-GLUCOSYLTRANSFERASE (UF3GT), are
66 primarily regulated by the MYB-bHLH-WD40 (MBW) transcription complex
67 regulatory network comprising several classes of proteins, including
68 R2R3-MYBs, bHLHs, and TRANSPARENT TESTA GLABROUS1 (TTG1;
69 Gonzalez et al. 2008; Feller et al. 2011; Dixon et al. 2013). The transcript levels
70 of these anthocyanin biosynthetic genes are closely related to changes in
71 anthocyanin content. Increasing or decreasing the expression of these genes
72 results in high or low anthocyanin accumulation, respectively.
3
 73 As one of the essential environmental signals to modulate diverse aspects of
74 plant growth and development throughout the plant life cycle, the light
75 environment plays an important role in the regulation of anthocyanin
76 biosynthesis (Cutuli et al. 2010). Light signals are perceived by specific
77 photoreceptors and are transduced to negatively regulate CONSTITUTIVE
78 PHOTOMORPHOGENIC1 (COP1), a key photomorphogenic negative regulator
79 (Osterlund et al. 1998, 1999). COP1 was shown previously to promote the
80 degradation of PAP1 and PAP2 to inhibit anthocyanin biosynthesis in the dark
81 (Maier et al. 2013). LONG HYPOCOTYL5 (HY5), a bZIP transcription factor,
82 functions as a crucial positive regulator in both light and UV-B signaling and is
83 activated by COP1 in response to UV-B (Oravecz et al. 2006). HY5 was the
84 earliest-discovered, and remains the most extensively studied, transcription factor
85 involved in photomorphogenesis (Oyama et al. 1997). A lack of HY5 weakens
86 photomorphogenic responses under all light conditions, indicating that HY5 acts
87 downstream of all photoreceptors and that HY5 is essential in light signaling
88 (Oyama et al. 1997; Lee et al. 2007). COP1 also interacts with HY5, which
89 positively regulates anthocyanin biosynthesis through directly binding to the
90 promoters of both EBGs and LBGs and activating their expression
91 (Chattopadhyay et al. 1998; Shin et al. 2007). Specifically, a series of previous
92 studies show that several downstream regulators of HY5 play distinctive roles in
93 anthocyanin biosynthesis. For example, the R2R3 MYB-type transcription factor
94 PAP1 is capable of mediating the activation of many genes involved in
95 anthocyanin biosynthesis (Solfanelli et al. 2006; Zvi et al. 2012; Mitsunami et al.
96 2014). Moreover, the expression of PAP1 is strongly activated upon light
97 exposure, and the light-dependent expression of anthocyanin biosynthetic genes
98 in Arabidopsis is regulated by both PAP1 and HY5 (Das et al. 2012; Shin et al.
99 2013). Further studies revealed that HY5 regulates light-induced anthocyanin
100 biosynthesis by inducing the transcriptional activation of PAP1 (Shin et al. 2013).
101 In addition to acting as a positive regulator, the transcription factor MYB-LIKE 2
102 (MYBL2) is reduced in response to high levels of light and acts downstream of
4
103 HY5 to negatively regulate anthocyanin biosynthesis (Dubos et al. 2008; Wang
104 et al. 2016).
105 The GOLDEN2-LIKE (GLK) genes are members of the GARP domain super
106 family of Myb transcription factors (Riechmann et al. 2000). The role of GLKs
107 in plant developmental processes has been suggested to regulate chloroplast
108 development (Yasumura et al. 2005). In the model plant Arabidopsis, AtGLK2
109 was found to be involved in the biosynthesis of chlorophyll in Arabidopsis roots
110 through combined action with HY5 (Kobayashi et al. 2012). An analysis of the
111 hy5 mutant overexpressing AtGLK2 revealed that AtGLK2 require HY5 to
112 maximize its regulation of chlorophyll biosynthesis in roots (Lee et al. 2007;
113 Kobayashi et al. 2012). Furthermore, predictive cis-element analysis in the
114 promoter regions of biosynthesis for key chlorophylls and their co-regulated
115 genes identified the coexistence of CCAATC and the G-box motif, which may be
116 targeted by AtGLK2 and HY5, respectively (Kobayashi et al. 2012).
117 Anthocyanin biosynthesis is induced by environmental light signals (Cutuli et al.
118 2010). It is known that HY5 contributes to the positive regulation of
119 light-induced anthocyanin accumulation in Arabidopsis (Chattopadhyay et al.
120 1998; Shin et al. 2007). However, how HY5 functions in this process is still not
121 fully understood. Given that AtGLK2 is involved in light-controlled singling,
122 whether AtGLK2 is involved in light-induced anthocyanin biosynthesis
123 represents a crucial question to be addressed. Here, the Arabidopsis
124 GOLDEN2-LIKE transcription factor AtGLK2 was identified as a possible
125 positive regulator of anthocyanin biosynthesis. Furthermore, the concerted
126 regulatory functions of the two transcription factors, AtGLK2 and HY5, acting
127 downstream of the light signaling pathway, are responsible for the regulation of
128 anthocyanin biosynthesis.
129

5
130 Materials and methods

131 Plant material and growth conditions

132 The Arabidopsis thaliana ecotype Col-0 was used for this study. The mutants hy5
133 (SALK_096651C) and glk2 (CS9806) were obtained from the Arabidopsis
134 Biological Resource Center (Ohio State University, Columbus, OH, USA). The
135 OEGLK2/hy5 plants were obtained by crossing 35S::GLK2 transgenic plants
136 with hy5 mutant. Double transgenic plants OEGLK2/OEMYBL2 were obtained
137 by crossing transgenic lines overexpressing AtGLK2 and MYBL2. The
138 surface-sterilized Arabidopsis seeds were refrigerated in the dark at 4 °C for 3
139 days and then germinated on 1/2 MS medium supplemented with 2% sucrose at
140 22 °C with a 16 hours light and 8 hours photoperiod.

141 Gene constructs and transgenic plants

142 In order to produce 35S::GLK2 transgenic plants, the coding sequence (CDS)
143 corresponding to the AtGLK2 gene (At5g44190) locus was amplified by PCR
144 and then cloned into the vector pCAMBIA 1301, in which AtGLK2 was expressed
145 under the control of the CaMV 35S promoter. In order to produce 35S::MYBL2
146 transgenic plants, the coding sequence (CDS) corresponding to the MYBL2 gene
147 (At1g71030) locus was amplified by PCR and then cloned into the vector
148 pCAMBIA 1301, in which MYBL2 was expressed under the control of the CaMV
149 35S promoter. The recombinant vectors were transformed into wild-type
150 Arabidopsis ecotype Col-0 plants by using Agrobacterium tumefaciens GV3101
151 and the floral-dip method (Clough and Bent 1998). Transformed seeds were
152 selected on 1/2 MS medium containing 50 μg ml-1 hygromycin. All primers used
153 are listed in supplementary Table S1.

154 Gene expression analysis

155 The total RNA of Arabidopsis samples were extracted using the Eastep® Super
156 Total RNA Isolation Kit (Promega, LS 1040) according to the manufacturer’s
157 instructions. The cDNA synthesis were performed according to the method
158 reported in the previous work (Yi et al. 2019). Quantitative PCR was conducted
159 using TB Green Premix Ex TaqTM II (TaKaRa) on the CFX96 real-time detection
160 system (Bio-Rad). The thermal treatment was 2 min at 95°C, then 40 cycles of 10
6
161 sec at 95°C, 30 sec at 60°C. Each sample was quantified at least in triplicate and
162 normalized using ACTIN2 as an internal control. The 2-ΔΔCt method was used for
163 relative quantification. Primers for genes of interest are listed in supplementary
164 Table S1.

165 Total anthocyanin contents measurement

166 Fresh Arabidopsis seedlings grown on the 1/2 MS medium (containing 2%

167 sucrose) or after light treatment were used for anthocyanin measurement. The
168 extraction and measurement of anthocyanin was performed as described by Wu
169 et al. (2019).

170 Transmission electron microscopy (TEM) analysis

171 The cotyledons of Arabidopsis seedlings were fixed in 2.5 % (w/v)

172 glutaraldehyde overnight at 4 ℃. Thereafter, the samples were post-fixed with
173 1 % (w/v) osmium tetroxide for 2 hours at 4 ℃. Then the fixed samples were
174 gradually dehydrated in a graded series of ethanol (v/v, 30 %, 50 %, 70 %, 90 %)
175 and acetone, embedded in Epon 812 and polymerized at 35 ℃ for 16 hours,
176 then 48 ℃ for 24 hours, and 65 ℃ for 48 hours. For observation, ultrathin sections
177 of the samples were done with a diamond knife, contrasted with uranyl acetate
178 and lead citrate, and examined with a JEM-2100 transmission electron
179 microscope.

180 Analysis of chlorophyll contents

181 Chlorophylls were determined as described previously with some slight

182 modifications (Lichtenthaler and Wellburn 1983). Briefly, fresh Arabidopsis
183 seedlings were homogenized and resuspended in 80 % acetone, and debris was
184 removed by centrifugation at 12000 rpm for 15 min. The absorbance of the
185 extract supernatant was measured at 663 and 645 nm with a spectrophotometer
186 (V-5000).

187 Statistical analysis

188 All data analyses were performed based on at least three biological replicates.
189 Value in each graph gives the mean value ± SD of replicates. Statistically
190 significant differences were analysed by the Student’s t test (*P < 0.05).

7
191 Results

192 AtGLK2 acts as an important transcription factor and positively

193 regulates anthocyanin biosynthesis in Arabidopsis seedlings

194 To explore whether the transcription factor AtGLK2 is involved in the regulation
195 of anthocyanin biosynthesis and accumulation in Arabidopsis seedlings, we
196 employed the glk2 mutant. The gene expression analysis results showed that this
197 mutant is a loss-of-function allele (Fig. 1a). Quantitative analysis demonstrated
198 that the anthocyanin levels were quite low when the wild-type and glk2 seedlings
199 grown on 1/2 MS medium without sucrose, and there were no obvious
200 differences observed between these two genotypes (Fig. 1b-c). As exogenous
201 sugars induce anthocyanin accumulation (Teng et al. 2005; Solfanelli et al. 2006),
202 we further examined the anthocyanin contents in these Arabidopsis seedlings
203 grown on 1/2 MS medium containing 2% sucrose. The results showed that the
204 anthocyanin contents of the glk2 mutant were dramatically lower than those of
205 the wild-type seedlings (Fig. 1b-c). As the loss-of-function mutant of AtGLK2
206 has an anthocyanin-less phenotype, we next determined the anthocyanin contents
207 in 35S::GLK2 (constitutive expression AtGLK2 with a wild-type background)
208 transgenic Arabidopsis seedlings (OEGLK2-1 and OEGLK2-2). As shown in Fig.
209 1b-c, the anthocyanin accumulation in the 35S::GLK2 transgenic lines was
210 indistinguishable from that in the untransformed wild-type seedlings in the
211 absence of sucrose. However, in the presence of 2% sucrose, these AtGLK2
212 overexpressors accumulated more anthocyanins. We also examined the transcript
213 levels of AtGLK2 under exogenous sucrose treatment. As expected, the
214 quantitative RT-PCR results displayed that the expression of AtGLK2 was
215 noticeably induced by treatment with sucrose (Fig. 1d).
216 The above results demonstrated that AtGLK2 is involved in the regulation of
217 anthocyanin biosynthesis. To better understand the molecular basis of
218 AtGLK2-regulated anthocyanin biosynthesis, the expression of EBGs such as
219 CHS and CHI was initially examined by quantitative RT-PCR. The results

8
220 displayed that the mRNA expression amounts of CHS were not significantly
221 altered in either the glk2 mutant or the AtGLK2 overexpressors. Although CHI
222 expression was slightly repressed in the glk2 mutant, there were no
223 dramatic differences in the expression of CHI between the wild-type and
224 AtGLK2-overexpressing seedlings (Fig. 1e). We also examined the transcript
225 levels of late biosynthetic genes (LBGs) such as F3′H, DFR, LDOX, UF3GT,
226 UGT75C1, and UGT78D2. The results revealed that the LBGs had similar
227 expression patterns, in which the mRNA expression amounts of these genes were
228 much lower in the loss-of-function mutant of AtGLK2 but higher in the AtGLK2
229 overexpressors when compared to the wild type (Fig. 1e). Next, we further
230 examined the transcript levels of several key regulatory genes of anthocyanin
231 biosynthesis, including PAP1, TT8, and MYB11. The results showed that the
232 transcript levels of PAP1 and TT8 were consistently and considerably lower in
233 the glk2 mutant but higher in the 35S::GLK2 transgenic lines when compared to
234 the levels in the wild-type controls, which was consistent with the expected
235 results. However, we found that the transcript levels of MYB11 were unchanged
236 between the 35S::GLK2 transgenic lines and wild type (Fig. 1e). Altogether,
237 these data indicate that AtGLK2 positively modulates anthocyanin biosynthesis
238 in Arabidopsis seedlings mainly by affecting the expression of late, but not early,
239 biosynthetic genes in the anthocyanin biosynthetic pathway.

240 AtGLK2 is involved in light-induced anthocyanin accumulation in

241 Arabidopsis seedlings
242 Anthocyanin biosynthesis is induced by environmental light signals. To
243 investigate the effects of light on the expression of AtGLK2, we used quantitative
244 RT-PCR to determine the AtGLK2 expression in etiolated wild-type Arabidopsis
245 seedlings exposed to light for 8 hours. As shown in Fig. 2a, the AtGLK2
246 transcript levels were much lower in etiolated seedlings, and exposure to light for
247 8 hours significantly increased the accumulation of AtGLK2 transcripts. Given
248 that HY5 is a regulatory switch for the light control of Arabidopsis development,

9
249 if the expression of AtGLK2 were regulated by HY5, then HY5 mutation should
250 affect AtGLK2 transcript levels. As expected, when the seedlings were grown in
251 the light for 4 days, AtGLK2 transcript levels in the hy5 mutant were statistically
252 significantly lower than those in the wild-type seedlings (Fig. 2b). We also
253 examined the expression of AtGLK2 in wild-type seedlings from dark to light
254 conditions. Compared to the levels in dark-grown seedlings, the AtGLK2
255 transcript levels increased 3.6-fold under light conditions. In contrast, mutation
256 in HY5 dramatically inhibited the light-dependent induction of AtGLK2
257 transcripts, and the AtGLK2 transcript levels were 43.2% lower in the hy5 mutant
258 seedlings than those in the light-grown wild-type seedlings (Fig. 2c). These
259 findings indicate that light mediates the regulation of AtGLK2 mRNA expression
260 in an HY5-dependent manner.
261 The activation of AtGLK2 transcription by light suggests that AtGLK2 is
262 likely to be involved in light-induced anthocyanin accumulation in Arabidopsis
263 seedlings. To verify this inference, we determined the anthocyanin contents of
264 wild type, glk2, and AtGLK2 overexpressors under dark and light conditions.
265 When grown in the dark for 3 days before transferring them to light, virtually no
266 anthocyanins were accumulated in these different genotypes of Arabidopsis
267 seedlings (data not shown). However, transferring these etiolated seedlings to
268 light significantly increased anthocyanin accumulation, with the glk2 mutant
269 seedlings accumulating fewer and the AtGLK2-overexpressing seedlings
270 accumulating more anthocyanins (Fig. 2d-e). Consistent with the data of
271 anthocyanin content, we also found that the mRNA expression amounts of the
272 anthocyanin biosynthetic and regulatory genes (F3′H, DFR, LDOX, UF3GT,
273 UGT75C1, UGT78D2, PAP1, and TT8) were consistently and significantly lower
274 in the loss-of-function mutant of AtGLK2 but higher in the AtGLK2
275 overexpressors when compared to the levels in the wild type (Fig. 2f). All the
276 above results indicated that AtGLK2 is involved in light-induced anthocyanin
277 biosynthesis in Arabidopsis seedlings.

10
278 AtGLK2 requires the presence of HY5 to positively regulate
279 anthocyanin biosynthesis
280 Based on the previous studies (Oyama et al. 1997; Lee et al. 2007; Shin et al.
281 2007) combined with our results presented here, we questioned whether
282 HY5-mediated light signaling is required for AtGLK2-regulated anthocyanin
283 accumulation. To address this question, the 35S::GLK2 construct was introduced
284 into the hy5 mutant background via genetic crossing (Fig. 3a), and the
285 anthocyanin contents of wild-type, 35S::GLK2 (OEGLK2, constitutive
286 expression AtGLK2 in the wild-type background), hy5, and 35S::GLK2/hy5
287 (OEGLK2/hy5, constitutive expression AtGLK2 in the hy5 background) seedlings
288 were examined. Compared to the wild type, the AtGLK2 overexpressor
289 accumulated more anthocyanins, while the hy5 mutant presented lower
290 anthocyanin contents. However, in the absence of HY5, AtGLK2 overexpression
291 did not result in largely enhanced anthocyanin accumulation compared to
292 AtGLK2 overexpression under a wild-type background (Fig. 3b-c).
293 To investigate whether the expression of anthocyanin biosynthetic and
294 regulatory genes is affected, the mRNA expression amounts of several
295 representative genes in the wild-type, 35S::GLK2, hy5 mutant, and
296 35S::GLK2/hy5 seedlings were determined. As shown in Fig. 3d, the transcript
297 levels of both EBGs (CHS and CHI) and LBGs (F3′H, DFR, LDOX, UF3GT,
298 UGT75C1, and UGT78D2) were significantly decreased in the hy5 mutant
299 seedlings. In contrast to HY5 regulation, AtGLK2 predominantly up-regulated
300 the transcript levels of the LBGs, as the expression of both the CHS and CHI was
301 not significantly influenced by stable AtGLK2 overexpression. Moreover, the
302 mRNA expression amounts of the anthocyanin regulatory genes PAP1 and TT8
303 were found to be significantly higher in the AtGLK2 overexpressor but lower in
304 the hy5 mutant when compared with the levels in the wild type (Fig. 3d).
305 Although the expression of the anthocyanin biosynthetic and regulatory genes
306 (F3′H, DFR, LDOX, UF3GT, UGT75C1, UGT78D2, PAP1, and TT8) was
307 strongly induced in the 35S::GLK2 transgenic seedlings, the induced expression
11
308 of these genes was much lower in the 35S::GLK2/hy5 seedlings (Fig. 3d). All of
309 these data indicate that AtGLK2 acts as a positive component in anthocyanin
310 biosynthesis but requires the presence of HY5.

311 AtGLK2-mediated positive regulation of photosynthetic development

312 requires functional HY5
313 Because the transcription factor HY5 plays a multifaceted role in light signal
314 transduction, we next explored whether the AtGLK2-mediated regulation of
315 photosynthetic development requires functional HY5. The glk2 mutant and
316 35S::GLK2 transgenic seedlings were investigated first for their responses to
317 light-induced photosynthetic development. After growing the seedlings in the
318 dark for 3 days before transferring them to light, no significant phenotypic
319 differences were observed between the different genotypes of Arabidopsis
320 seedlings (data not shown). However, transferring these 3-day-old etiolated
321 seedlings to light significantly increased chlorophyll accumulation, with the glk2
322 mutant seedlings accumulating less and the AtGLK2-overexpressing seedlings
323 accumulating more chlorophyll (Fig. 4a-b). We next examined the chloroplast
324 ultrastructure in the glk2 mutant and 35S::GLK2 transgenic seedlings after
325 transferring the seedlings to light. As shown in Fig. 4c, the chloroplast size of the
326 glk2 mutant seedling was slightly small, and the thylakoid components exhibited
327 a lower abundance and lower stacking degree than those of the wild-type controls.
328 In contrast, the 35S::GLK2 transgenic seedlings contained chloroplast with an
329 abundant defined thylakoid membrane structure and evidence of granal stacking.
330 The quantitative RT-PCR results also demonstrated that the expression of
331 photosynthesis-associated genes, including the genes that encode glutamyl-tRNA
332 reductase (HEMA1), chlorophyll a oxygenase (CAO), genomes uncoupled 4
333 (GUN4), magnesium chelatase (CHLH), a putative ZIP protein (CHL27), a
334 chlorophyll a/b-binding protein (LHCA4), light harvesting chlorophyll a/b
335 binding protein 1.2 (LHCB1.2), a component of light harvesting complex
336 photosystem II (LHCB4.1), oxygen evolving complex 33 (OE33), and a subunit

12
337 of photosystem I (PSAN), was dramatically reduced in glk2 but elevated in
338 AtGLK2-overexpressing seedlings (Fig. 4d). All these results demonstrated that
339 AtGLK2 plays a positive role in light-induced photosynthetic development of
340 Arabidopsis seedlings. To reveal the genetic interaction between HY5 and
341 AtGLK2, we next generated the hy5 mutant overexpressing AtGLK2 by crossing
342 hy5 with the 35S::GLK2 transgenic line. Although the chlorophyll levels of the
343 OEGLK2/hy5 seedlings were significantly higher than those in the parental hy5
344 mutant, these levels were still much lower than those in the 35S::GLK2 parental
345 lines (Fig. 4a-b). Consistent with the chlorophyll data, analysis of the
346 ultrastructure and gene expression also supported the notion that HY5 plays an
347 important role for AtGLK2 functions in the regulation of photosynthetic
348 development of etiolated Arabidopsis seedlings during greening (Fig. 4c-d).

349 AtGLK2 and MYBL2 antagonistically regulate anthocyanin

350 biosynthesis in Arabidopsis

351 It was previously reported that HY5 can bind to the promoter of MYBL2 and
352 repress its expression, thus indirectly up-regulating the expression of anthocyanin
353 biosynthetic genes and eventually resulting in increased anthocyanin biosynthesis
354 in Arabidopsis seedlings (Wang et al. 2016). Consistent with the previous
355 literature, we found that the transcript levels of MYBL2 were obviously higher in
356 the hy5 mutant than in wild-type seedlings. However, the transcript levels of
357 AtGLK2 were significantly lower in the hy5 mutant than in the wild-type
358 seedlings (Fig. 5a). The results presented here raised the possibility of whether
359 AtGLK2 and MYBL2, two factors modulated by HY5, could antagonistically
360 regulate anthocyanin biosynthesis. We, therefore, generated progenies of the
361 35S::GLK2 and 35S::MYBL2 transgenic plants via cross pollination and
362 examined the possible functional relationship of AtGLK2 and MYBL2 in the
363 regulation of anthocyanin biosynthesis (Fig. 5b). As shown in Fig. 5c-d, the
364 accumulation of anthocyanin was significantly increased in the 35S::GLK2
365 transgenic seedlings and drastically reduced in the 35S::MYBL2 transgenic
13
366 seedlings when compared to the wild-type seedlings. Quantification of the
367 anthocyanin contents in the 35S::GLK2/35S::MYBL2 double overexpressor
368 revealed that the level of anthocyanin was intermediate between the levels of the
369 35S::GLK2 overexpressor and the 35S::MYBL2 overexpressor, suggesting that
370 the additional overexpression of MYBL2 was able to suppress enhanced
371 anthocyanin accumulation in the AtGLK2-overexpressing seedlings. Likewise,
372 the expression levels of anthocyanin biosynthetic (F3′H, DFR, LDOX, UF3GT,
373 UGT75C1, and UGT78D2) and regulatory (PAP1 and TT8) genes in the
374 35S::GLK2/35S::MYBL2 double overexpressor were lower than those in the
375 35S::GLK2 overexpressor but higher than those in the 35S::MYBL2
376 overexpressor (Fig. 5e). These results indicate that AtGLK2 and MYBL2 act
377 antagonistically to regulate anthocyanin accumulation during Arabidopsis
378 seedling development.

14
379 Discussion

380 The regulation of anthocyanin biosynthesis by multiple external signals,

381 including temperature, light, and salinity, is mediated by a variety of
382 transcription factors directly acting at the transcriptional level (Eryilmaz 2006;
383 Nguyen et al. 2015; Kim et al. 2017; Fu et al. 2019). GLK transcription factors,
384 also known as GOLDEN2-Like and G2-Like, are widely present in the plant
385 kingdom and belong to the GARP-type transcription factor family (Riechmann et
386 al. 2000; Wang et al. 2013). In the present study, the role of the Arabidopsis
387 AtGLK2 in anthocyanin accumulation was explored. We discovered that
388 AtGLK2 plays a positive regulatory role in sucrose-induced anthocyanin
389 biosynthesis. The following three sets of experimental evidence support this
390 conclusion. First, exogenous sucrose treatment clearly enhanced the expression
391 of AtGLK2 in the wild-type Arabidopsis seedlings (Fig. 1d). Second, anthocyanin
392 accumulation in the wild type was indistinguishable from that in the glk2 mutant
393 and AtGLK2-overexpressing lines in the absence of sucrose. However, in the
394 presence of sucrose, the glk2 mutant accumulated fewer anthocyanins, while the
395 AtGLK2-overexpressing lines accumulated more anthocyanins (Fig. 1b-c). Third,
396 the quantitative RT-PCR analysis results demonstrated that the expression levels
397 of anthocyanin biosynthetic and regulatory genes were dramatically reduced in
398 glk2 but elevated in AtGLK2-overexpressing seedlings (Fig. 1e). The coding
399 genes of the enzymes involved in anthocyanin biosynthesis can be conveniently
400 divided into two categories: early biosynthetic genes (EBGs) and late
401 biosynthetic genes (LBGs; Ferreyra et al. 2012). It should be noted that the
402 expression of late (F3′H, DFR, LDOX, UF3GT, UGT75C1, and UGT78D2), but
403 not early (CHS and CHI), biosynthetic genes in the anthocyanin biosynthetic
404 pathway was positively correlated with AtGLK2 expression (Fig. 1e). In higher
405 plants, the expression of LBGs is primarily regulated via the MYB-bHLH-WD40
406 (MBW) transcription complex regulatory network comprising several classes of
407 proteins, including R2R3-MYBs, bHLHs, and TTG1 (Gonzalez et al. 2008;

15
408 Feller et al. 2011; Dixon et al. 2013). PAP1 belongs to the R2R3-MYB
409 transcription activator family and interacts with one of the three bHLH activators
410 (TT8, EGL3, or GL3) to form transcription complexes with the WD-repeat
411 transcription factor (Gonzalez et al. 2008; Matsui et al. 2008). Gene expression
412 analysis showed that the expression of some of the MBW transcription complex
413 genes, such as PAP1 and TT8, was significantly reduced in the glk2 mutant but
414 elevated in the AtGLK2-overexpressing seedlings (Fig. 1e), suggesting that
415 AtGLK2 positively modulated the expression of the late anthocyanin
416 biosynthetic genes, possibly through the MBW transcription complex. Given that
417 the expression of CHI was also altered (although to a lesser degree) in the glk2
418 seedlings (Fig. 1e), the possibility that AtGLK2 may also be involved in the
419 regulation of other regulator genes cannot be completely ruled out at present.
420 Light is one of the important environmental factors, not only provides the
421 energy used in photosynthesis, but also acts as an informational cue to control a
422 multitude of physiological responses throughout the plant life cycle. HY5 is a
423 basic leucine zipper (bZIP) transcription factor and has been widely studied for
424 its important regulatory roles in photomorphogenesis (Oyama et al. 1997).
425 Mutants impaired in HY5 display significantly elongated hypocotyls under all
426 light conditions, indicating that HY5 acts downstream of multiple photoreceptors
427 to promote photomorphogenesis (Oyama et al. 1997; Lee et al. 2007). It was
428 reported that light is an important elicitor that triggers anthocyanin biosynthesis
429 in higher plants (Cutuli et al. 2010). The light-signaling component HY5 also
430 mediates the regulation of anthocyanin biosynthesis by directly binding to the
431 promoters of anthocyanin biosynthetic and regulatory genes to induce their
432 expression in response to light (Shin et al. 2007, 2013). Because AtGLK2
433 functions as a positive regulator of anthocyanin biosynthesis (Fig. 1), we
434 determined the transcript levels of AtGLK2 in wild-type and hy5 mutant
435 seedlings from dark to light conditions. When the etiolated Arabidopsis seedlings
436 was exposed to light, AtGLK2 expression was highly up-regulated, while the
437 light-induced expression of AtGLK2 was partially abolished in the hy5 mutant
16
438 (Fig. 2c). These findings indicate that the light-mediated regulation of AtGLK2
439 acts in an HY5-dependent manner. Given that AtGLK2 is a light-induced gene
440 (Fig. 2a), it appears that the altered biosynthesis of anthocyanin in the AtGLK2
441 knockout or overexpression lines is dependent on the presence of light. Indeed,
442 transferring the etiolated Arabidopsis seedlings to light resulted in the glk2
443 mutant seedlings accumulating fewer anthocyanins and the
444 AtGLK2-overexpressing seedlings accumulating more anthocyanins (Fig. 2d-e).
445 The strong effect of AtGLK2 on anthocyanin biosynthesis suggests that AtGLK2
446 expression is subject to strict transcriptional control. Since the expression levels
447 of AtGLK2 were obviously changed under light irradiation (Fig. 2a), it is
448 reasonable to hypothesize that AtGLK2 expression requires HY5. As expected,
449 we found that the HY5 mutation had a minor but significant inhibitory effect on
450 AtGLK2 expression (Fig. 2b), suggesting that HY5 positively regulated AtGLK2
451 transcript levels. To further verify our hypothesis about the positive regulation of
452 AtGLK2 expression by HY5, we measured the anthocyanin contents in F3
453 seedlings of the 35S::GLK2/hy5 line. The results showed that the enhanced
454 accumulation of anthocyanin in the 35S::GLK2 transgenic seedlings was
455 dependent on the presence of HY5 because the 35S::GLK2/hy5 seedlings
456 presented lower anthocyanin contents than the 35S::GLK2 transgenic seedlings
457 (Fig. 3b-c). Consistent with these findings, the gene expression analysis also
458 demonstrated that the induced expression of the genes involved anthocyanin
459 biosynthesis and that regulation was much lower in the 35S::GLK2/hy5
460 transgenic seedlings than in the 35S::GLK2 seedlings (Fig. 3d). The genetic
461 evidence, combined with the other experimental data obtained in this study,
462 clearly indicates that HY5-mediated light signaling is required for
463 AtGLK2-regulated anthocyanin accumulation in Arabidopsis. Notably, mutation
464 in HY5 was not able to completely restore the anthocyanin contents of the
465 AtGLK2 overexpressor to hy5 levels (Fig. 3c). As HY5 and its homolog HYH
466 redundantly regulate light-mediated seedling development in Arabidopsis (Holm
467 et al. 2002), it is unsurprising that only limited contents of anthocyanin were
17
468 changed in the 35S::GLK2/hy5 seedlings. Indeed, the anthocyanin levels were
469 greatly reduced in the hy5 hyh double mutant compared to the single hy5 and hyh
470 mutants, for which only minor reductions in anthocyanin levels were observed
471 (Holm et al. 2002).
472 MYBL2, a transcription factor with a single MYB domain, plays a negative
473 regulatory role in anthocyanin biosynthesis in Arabidopsis (Dubos et al. 2008;
474 Matsui et al. 2008). It was reported that HY5 directly binds to the promoter
475 region of MYBL2 and positively regulates anthocyanin biosynthesis (Wang et al.
476 2016). Since AtGLK2 requires the presence of HY5 to positively regulate
477 anthocyanin biosynthesis (Fig. 3), the expression levels of MYBL2 were
478 examined in wild-type, glk2 mutant, and AtGLK2-overexpressing seedlings. We
479 found no significant changes in MYBL2 transcripts among these genotypes (data
480 not shown). Similarly, we also found no change in the AtGLK2 transcription
481 levels between the wild-type and MYBL2-overexpressing seedlings (data not
482 shown). These results suggest that the two transcription factors AtGLK2 and
483 MYBL2 do not regulate each other at the transcriptional level. Given that HY5
484 positively regulated the expression of AtGLK2 but negatively regulated the
485 expression of MYBL2 (Fig. 5a), we generated an OEGLK2/OEMYBL2 double
486 overexpressor via cross pollination and investigated the possible functional
487 relationship between AtGLK2 and MYBL2. The results demonstrated that
488 AtGLK2 and MYBL2 act antagonistically to regulate anthocyanin biosynthesis
489 by modulating the expression of anthocyanin biosynthetic and regulatory genes
490 (Fig. 5c-e), indicating that AtGLK2 and MYBL2 were transcriptionally regulated
491 by HY5 in the light to ensure the proper interplay of AtGLK2 and MYBL2
492 activities during the process of anthocyanin biosynthesis in Arabidopsis
493 seedlings.
494 Based on the experimental data obtained herein, we propose that the
495 transcriptional regulation of AtGLK2 by HY5 in response to light constitutes a
496 part of the mechanism by which light signals control anthocyanin biosynthesis.
497 The conclusions of previous studies (Wang et al. 2016) alongside the present
18
498 results indicate that AtGLK2 is positively regulated while MYBL2 is negatively
499 regulated by HY5 at the transcriptional level, thereby up-regulating the
500 expression of LBGs in the anthocyanin biosynthesis pathway and eventually
501 resulting in increased anthocyanin accumulation in Arabidopsis seedlings.
502 Moreover, AtGLK2 and MYBL2 antagonistically regulate the expression of
503 LBGs and the subsequent anthocyanin accumulation process. The experimental
504 results presented in our study reveal the fine-tuned regulation of anthocyanin
505 accumulation through the transcriptional regulation of AtGLK2 and MYBL2 by
506 HY5 and provide insights into the mechanism involving light-controlled
507 anthocyanin biosynthesis. However, we could not determine whether AtGLK2
508 regulates anthocyanin biosynthesis through directly binding to and activating the
509 promoters of LBGs. The possibility that AtGLK2 functions by affecting the
510 expression levels of other transcription factors and then, together with these
511 target transcription factors, co-regulates downstream anthocyanin biosynthetic
512 genes should also be considered.

19
513 Acknowledgments We would like to give our great thanks to the Arabidopsis
514 Biological Resource Center at The Ohio State University for providing the mutant
515 seeds relevant to our experiments. This research was funded by the National Natural
516 Science Foundation of China (grant nos. 31971847 and 31560077) and the Natural
517 Science Foundation of Jiangxi Province (grant nos. 20202BAB203023).
518
519 Author contributions DL, DZ and XL performed the experiments. YZ designed
520 and supervised the project. All authors discussed the results, revised the manuscript
521 and approved submission of this work.

522 Compliance with ethical standards

523 Conflict of interest The authors declare that they have no conflicts of interest.

20
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650 Figure legends
651
652 Fig. 1 AtGLK2 plays a positive regulatory role in anthocyanin biosynthesis. (a)
653 Quantitative RT-PCR analysis of the relative expression of AtGLK2 in the
654 4-day-old wild-type (Col), glk2, and two 35S::GLK2 transgenic seedlings
655 (OEGLK2-1 and OEGLK2-2). (b) Phenotypes of the wild-type, glk2, and
656 35S::GLK2 transgenic seedlings grown for 4 days on a 1/2 MS medium without
657 (-S) or with (+S) 2% sucrose. Arrows indicate the sites where anthocyanin
658 accumulated in 4-day-old seedlings. Scale bar=1 mm. (c) Anthocyanin
659 contents of the 4-day-old wild-type, glk2, and 35S::GLK2 transgenic seedlings
660 grown on a 1/2 MS medium without (-S) or with (+S) 2% sucrose. (d)
661 Accumulation of AtGLK2 mRNA under exogenous sucrose treatment.
662 Wild-type Arabidopsis seedlings were grown on a 1/2 MS medium without
663 sucrose for 4 days and then were transferred to a 1/2 MS medium without (-S)
664 or with (+S) 2% sucrose for an additional 24 hours of growth. Total RNA was
665 then extracted from each tissue sample and used for quantitative RT-PCR. (e)
666 Transcript levels of the anthocyanin biosynthetic and regulatory genes in the
667 4-day-old wild-type, glk2, and 35S::GLK2 transgenic seedlings. The asterisks
668 indicate statistically significant differences between wild-type and other
669 genotypes or between control and treatment of the samples.
670
671 Fig. 2 AtGLK2 is involved in light-induced anthocyanin biosynthesis. (a)
672 Quantitative RT-PCR analysis of the relative expression of AtGLK2 in the
673 4-day-old etiolated wild-type seedlings exposed to light for 8 hours. (b)
674 Quantitative RT-PCR analysis of the relative expression of AtGLK2 in the
675 4-day-old wild-type and hy5 mutant seedlings grown in the light. (c)
676 Quantitative RT-PCR analysis of the relative expression of AtGLK2 in the
677 4-day-old etiolated wild-type and hy5 mutant seedlings exposed to light for 8
678 hours. (d) Phenotypes and anthocyanin contents (e) of the wild-type, glk2, and
679 35S::GLK2 transgenic seedlings grown in the dark for 3 days followed by
680 continuous light for 24 hours. Scale bar=1 mm. (f) Transcript levels of the
681 biosynthetic and regulatory genes involved in anthocyanin biosynthesis in
682 wild-type, glk2, and 35S::AtGLK2 transgenic lines grown in the dark for 3 days
683 followed by continuous light for 24 hours. Asterisks indicate statistically
24
684 significant differences between the control and treatment samples or between
685 the wild type and other genotypes.
686
687 Fig. 3 HY5 is required for AtGLK2 to positively regulate anthocyanin
688 biosynthesis. (a) Quantitative RT-PCR analysis of the relative expression of
689 AtGLK2 and HY5 in the 4-day-old wild-type (Col), transgenic line
690 overexpressing AtGLK2 (OEGLK2), the hy5 mutant (hy5), and the transgenic
691 line overexpressing AtGLK2 in the hy5 background (OEGLK2/hy5). (b)
692 Phenotypes and anthocyanin contents (c) of the 4-day-old wild-type,
693 transgenic line overexpressing AtGLK2, the hy5 mutant, and the transgenic
694 line overexpressing AtGLK2 in the hy5 background. Scale bar=1 mm. (d)
695 Expression levels of biosynthetic and regulatory genes involved in anthocyanin
696 biosynthesis in the 4-day-old wild-type, transgenic line overexpressing AtGLK2,
697 the hy5 mutant, and the transgenic line overexpressing AtGLK2 in the hy5
698 background. Asterisks indicate statistically significant differences between the
699 wild type and other genotypes.
700
701 Fig. 4 HY5 plays a crucial role for AtGLK2’s functions in the regulation of the
702 photosynthetic development of etiolated Arabidopsis seedlings during
703 greening. (a) Phenotypes and chlorophyll contents (b) of the wild-type (Col),
704 glk2, 35S::GLK2 transgenic line (OEGLK2), hy5, and transgenic line
705 overexpressing AtGLK2 in the hy5 background (OEGLK2/hy5) grown in the
706 dark for 3 days followed by continuous exposure to light for 24 hours. Scale
707 bar=1 mm. (c) Chloroplast ultrastructure and the expression of
708 photosynthesis-associated genes (d) of the wild-type, glk2, 35S::GLK2
709 transgenic line, hy5, and transgenic line overexpressing AtGLK2 in the hy5
710 background grown in the dark for 3 days followed by continuous exposure to
711 light for 24 hours. Asterisks indicate statistically significant differences between
712 the wild type and other genotypes.
713
714 Fig. 5 AtGLK2 and MYBL2 function antagonistically to regulate anthocyanin
715 biosynthesis. (a) Quantitative RT-PCR analysis of MYBL2 and AtGLK2 in the
716 4-day-old wild-type (Col) and hy5 seedlings. (b) Quantitative RT-PCR analysis
717 of AtGLK2 and MYBL2 in the 4-day-old wild-type, 35S::GLK2 transgenic line
25
718 (OEGLK2), 35S::MYBL2 transgenic line (OEMYBL2), and
719 35S::GLK2/35S::MYBL2 double-overexpressing (OEGLK2/OEMYBL2)
720 seedlings. (c) Phenotypes and anthocyanin contents (d) of the 4-day-old
721 wild-type, 35S::GLK2 transgenic line, 35S::MYBL2 transgenic line, and
722 35S::GLK2/35S::MYBL2 double-overexpressing seedlings. Scale bar=1 mm.
723 (e) Transcript levels of biosynthetic and regulatory genes involved in
724 anthocyanin biosynthesis in the 4-day-old wild-type, 35S::GLK2 transgenic line,
725 35S::MYBL2 transgenic line, and 35S::GLK2/35S::MYBL2
726 double-overexpressing seedlings. Asterisks indicate statistically significant
727 differences between the wild type and other genotypes.

26
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