ELONGATED HYPOCOTYL 5 mediates blue light-induced starch degradation in tomato
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Journal of Experimental Botany, Vol. 72, No. 7 pp. 2627–2641, 2021
doi:10.1093/jxb/eraa604 Advance Access Publication 29 December 2020
RESEARCH PAPER
ELONGATED HYPOCOTYL 5 mediates blue light-induced
starch degradation in tomato
Han Dong1,2, Chaoyi Hu2, Chaochao Liu2, Jiachun Wang1,2, Yanhong Zhou2,3, and Jingquan Yu1,2,3,*,
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1
College of Horticulture, Northwest Agriculture & Forestry University, Yangling, Shaanxi 712100, China
2
Department of Horticulture, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China
3
Key Laboratory of Horticultural Plants Growth, Development and Quality Improvement, Agricultural Ministry of China, Zijingang Road
866, Hangzhou 310058, China
* Correspondence: jqyu@zju.edu.cn
Received 14 October 2020; Editorial decision 30 November 2020; Accepted 24 December 2020
Editor: Diane Beckles, University of California, Davis, USA
Abstract
Starch is the major storage carbohydrate in plants, and its metabolism in chloroplasts depends mainly on light.
However, the mechanism through which photoreceptors regulate starch metabolism in chloroplasts is unclear. In this
study, we found that the cryptochrome 1a (CRY1a)-mediated blue light signal is critical for regulating starch accumula-
tion by inducing starch degradation through the transcription factor HY5 in chloroplasts in tomato. cry1a mutants and
HY5-RNAi plants accumulated more starch and presented lower transcript levels of starch degradation-related genes
in their leaves than wild-type plants. Blue light significantly induced the transcription of starch degradation-related
genes in wild-type and CRY1a- or HY5-overexpressing plants but had little effect in cry1a and HY5-RNAi plants. Dual-
luciferase assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation–qPCR revealed that HY5
could activate the starch degradation-related genes PWD, BAM1, BAM3, BAM8, MEX1, and DPE1 by directly binding
to their promoters. Silencing of HY5 and these starch degradation-related genes in CRY1a-overexpressing plants led
to increased accumulation of starch and decreased accumulation of soluble sugars. The findings presented here not
only deepen our understanding of how light controls starch degradation and sugar accumulation but also allow us to
explore potential targets for improving crop quality.
Keywords: Blue light, cryptochrome, HY5, Solanum lycopersicum, starch degradation, sugar accumulation, tomato.
Introduction for sucrose synthesis, maintenance of leaf respiration, plant me-
tabolism, growth, and development (Smith and Stitt, 2007; Stitt
As the principal storage carbohydrate, starch plays an indis- and Zeeman, 2012; Zeeman et al., 2007). In guard cells, how-
pensable role in the growth, development, and stress response ever, starch degrades in the light (Santelia and Lunn, 2017).
of plants. Photosynthetic products of higher plants are stored Starch not only is important in regulating carbohydrate allo-
in the chloroplasts of mesophyll cells in the form of transitory cation, energy homeostasis, and plant growth as a carbohy-
starch during the day, and are hydrolyzed to maltose and glu- drate source, but also acts as a carbohydrate reserve in other
cose at night for subsequent transport out of the chloroplasts organs, such as fruits, seeds, or tubers. In addition, starch is a
© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: journals.permissions@oup.com
2628 | Dong et al.
determinant of plant fitness under abiotic stress, and starch re- accumulation are usually explained by dynamic changes in
serves can be remobilized to release energy, sugars, and derived photosynthesis because light provides plants with energy
metabolites, thereby improving plant adaptability and resist- for photosynthesis (Sulpice et al., 2014). Several studies have
ance (Thalmann and Santelia, 2017; Yano et al., 2005; Zanella characterized the crucial enzymes in the processes of starch
et al., 2016; Zhuang et al., 2019). Increasing starch biosynthesis synthesis and degradation during the natural daily light/dark
could be one way to increase starch accumulation in a range of cycle (D’Hulst et al., 2015; Graf et al., 2010; Graf and Smith,
storage organs, including tubers and roots, as well as in cereal 2011; Lu et al., 2005; Smith et al., 2004). However, the role
seed products, while manipulation of starch breakdown is a of photoreceptor-dependent light signaling in starch metab-
potential strategy for increasing sugar accumulation in fleshy olism is poorly understood.
fruit products. Plants have evolved a series of photosensory receptors as
Starch is synthesized and degraded via an intricate net- signal factors to initiate a variety of physiological and bio-
work of reactions involving the synergistic action of mul- chemical reactions (Kami et al., 2010). The photoreceptors
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tiple enzymes. In the chloroplast, triose phosphate, which is in plants mainly include red/far-red photoreceptor phyto-
a product of photosynthetic carbon assimilation formed by chrome, blue/ultraviolet A photoreceptor cryptochrome
the Calvin–Benson cycle, is catalyzed by aldolase to produce (CRY), phototropin, and ultraviolet B (UV-B) receptor UV
fructose-6-phosphate. Then, fructose-6-phosphate is con- RESISTANCE LOCUS8 (UVR8) (Chory, 2010; Galvao and
verted to glucose-6-phosphate by plastid phosphoglucose Fankhauser, 2015). Previous studies demonstrated that HY5,
isomerase (PGI or GPI), and glucose-6-phosphate is con- which is a bZIP transcription factor, plays a pivotal role in light
verted to glucose-1-phosphate by phosphoglucomutase signaling and mediates photoreceptor responses to promote
(PGM). Glucose-1-phosphate and ATP are catalyzed by photomorphogenesis (Gangappa and Botto, 2016). HY5 can
ADP glucose pyrophosphorylase to produce ADP-glucose activate the transcription of a large number of light-responsive
(ADPG) and inorganic pyrophosphate. Subsequently, starch genes by directly binding to the G-box (CACGTG) elements
is formed as a result of the activities of enzymes including or ACGT-containing elements (ACEs) of their promoters in
granule-bound starch synthase (GBSS), soluble starch syn- the model plant Arabidopsis (Lee et al., 2007). Recently, HY5
thase (SSS), and starch branching enzyme (Orzechowski, was found to be a regulator of several physiological processes,
2008; Stitt and Zeeman, 2012). For starch catabolism, the such as nutrient uptake and pigmentation, in Arabidopsis and
initial steps involve the phosphorylation of the starch granule tomato (Solanum lycopersicum) (Binkert et al., 2014; Chen et al.,
surface by glucan water dikinase (GWD) and phosphoglucan 2016; Liu et al., 2018b; Shin et al., 2007). However, whether
water dikinase (PWD) enzymes. Then, as a result of co- HY5-dependent light signaling participates in starch metab-
operation between β-amylase (BAM), α-amylases (AMY), olism in plants remains unclear.
transglucosidase 1 [also known as disproportionating en- In tomato, four CRY genes have been identified: CRY1a,
zyme 1 (DPE1)], and other enzymes, glucose and maltose are CRY1b, CRY2, and CRY-DASH (Chaves et al., 2011; Facella
produced. Maltose and glucose are transferred from chloro- et al., 2012; Lopez et al., 2012). In our previous study, transgenic
plasts into the cytoplasm by maltose excess protein (MEX) tomato plants overexpressing CRY1a showed increased soluble
and the plastid glucose transporter (Edner et al., 2007; Fettke solid contents in the fruits, while mutants of CRY1a had re-
et al., 2009; Orzechowski, 2008; Stettler et al., 2009; Streb duced soluble solid contents in the fruits (Liu et al., 2018a), sug-
and Zeeman, 2012). gesting a possible role of CRY1a in carbohydrate metabolism.
In plants, starch metabolism is regulated by the intrinsic To gain insights into the mechanism of blue light-regulated
carbon status, circadian rhythm, redox homeostasis, hor- starch metabolism, we used cry1a and CRY1a-overexpressing
mones, and environmental factors such as light, tempera- (CRY1a-OE) tomato plants and compared the accumulation
ture, water, and the nutrient supply (Bhatia and Singh, 2002; of starch and sugars, the transcription and activity of starch
Geigenberger, 2011; Graf et al., 2010; Hendriks et al., 2003; synthesis- and degradation-related genes and enzymes, and the
Lu et al., 2005; Monroe et al., 2014; Petra et al., 1998; Seiler accumulation of HY5 with those of wild-type (WT) plants.
et al., 2011; Smith et al., 2004; Weise et al., 2006). Plant hor- We also examined whether HY5 mediates blue light-induced
mones such as abscisic acid (ABA) stimulate starch breakdown starch accumulation by transcriptional activation of genes in-
under osmotic stress (Thalmann et al., 2016). Low temperat- volved in starch metabolism. The results of the present study
ures induce the degradation of starch and increase the ac- demonstrated that blue light plays a critical role in starch
cumulation of soluble sugars in leaves (Zhuang et al., 2019). breakdown by inducing HY5. By binding to the promoters of
In response to nitrogen starvation, plants initiate autophagy several starch degradation-related genes, HY5 prevents plants
to induce starch degradation in the chloroplasts during leaf from accumulating starch in the leaves.These findings not only
senescence (Masclaux-Daubresse, 2014; Wang and Liu, 2013; deepen our understanding of the light regulation of carbohy-
Wang et al., 2013). Notably, light is the most influential envir- drate metabolism in plants but also aid in exploring potential
onmental factor in starch metabolism. Daily changes in starch targets for improving crop quality.
HY5 regulates starch metabolism in tomato | 2629
Materials and methods vectors were also co-infiltrated as controls (pTRV). VIGS plants were
grown at 23 °C/21 °C (day/night) in a growth chamber with a 12 h
Plant materials and growth conditions day length. qRT–PCR was performed to determine the gene silencing
CRY1a-OE, HY5-OE, and HY5-RNAi plants were generated as de- efficiency when pTRV-PDS plants (with silencing of the gene encoding
scribed previously (Liu et al., 2004, 2018a; Wang et al., 2019a). Seeds of phytoene desaturase) showed strong bleaching (Supplementary Table S3).
cv. Moneymaker, the cry1a mutant in the cv. Moneymaker background, Leaflets of the fourth fully expanded leaves that exhibited ~20–40% of
and cv. Ailsa Craig were obtained from the Tomato Genetics Resource the transcript levels of the control plants were used in the experiments
Center at the University of California, Davis, USA (https://tgrc.ucdavis. (Wang et al., 2016, 2019b).
edu). Seedlings were grown in pots containing a mixture of peat and
vermiculite (2:1, v/v) and received Hoagland’s nutrient solution. The Iodine staining and carbohydrate measurements
growth conditions were maintained as follows: white light (photosyn-
thetic photon flux density 300 μmol m−2 s−1) with a 12 h light/12 h The fourth leaves from the bottom of plants at the seven-leaf stage were
dark photoperiod, and temperature of 25 °C/20 °C (day/night). For the harvested at the end of the light phase (19.00 h) or at the end of the dark
blue light treatment, plants at the seven-leaf stage were pre-acclimated in phase (07.00 h). Iodine staining of the leaves was performed as described
previously (Wang et al., 2013). Specifically, the chlorophyll of the plant
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the dark for 12 h and were then transferred to blue light for 12 h. Blue
light (460 nm) was applied at a photosynthetic photon flux density of material was decolorized in 80% (v/v) ethanol at 95 °C for 30 min twice
300 μmol m−2 s−1. and rinsed twice with water to remove excess ethanol. Plant materials
were then stained in Lugol solution (Solarbio, Beijing, China) for 15 min,
destained with ultrapure water five times, and then photographed. The
Characterization of plant growth and development phenotypes starch content was determined with a Starch Content Detection Kit
and determination of photosynthetic indices (BC0705, Solarbio, Beijing, China) as previously described (Chen et al.,
2019; Zhuang et al., 2019). The soluble sugar content in the leaves was
The plant height and stem diameter were measured at the seven-leaf stage. assayed by using methods described by Kong et al. (2011). Sucrose, fruc-
The photosynthetic parameters were determined in the fifth fully ex- tose, glucose, and maltose were extracted using the method described by
panded leaves with a LI-6400 Portable Photosynthesis System (LI-COR, Niu et al. (2015) and determined using an ACQUITY UPLC® I-Class
Lincoln, NE, USA).The air temperature (25 °C), relative humidity (60%), system coupled to a Waters XevoTM TQ-XS triple quadruple mass spec-
CO2 concentration (400 μmol mol−1), and photosynthetic photon flux trometer. ADPG was extracted according to Lunn et al. (2006) and ana-
density (1000 μmol m−2 s−1) were controlled by the automatic control lyzed by an Agilent 1290 UHPLC coupled to a 6460 triple quadruple
device of the instrument (Liu et al., 2018a). The days to flowering were mass spectrometer.The activities of α-amylase and β-amylase in the leaves
counted from sowing to the opening of the first flower in the plants. were analyzed using assay kits (K-CERA and K-BETA, Megazyme, Bray,
Ireland) and determined by the method described by Scheidig et al.
Total RNA extraction and quantitative real-time PCR analyses (2002). GBSS and SSS activity assays were performed with a Granule-
Bound Starch Synthase Assay Kit and a Soluble Starch Synthase Assay Kit
The total RNA was extracted from tomato leaves by using an RNA ex- (Cat# BC3295 and Cat# BC1855, Solarbio, Beijing, China) as previously
traction kit (Tiangen, Shanghai, China) according to the manufacturer’s described (Nakamura et al., 1989; Jiang et al., 2003).
instructions. The total RNA (1 µg) was reverse transcribed using the
ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative real-
time PCR (qRT–PCR) analyses were performed using a LightCycler 480 Transmission electron microscopy
II Real-Time PCR Detection System (Roche, Basel, Switzerland). The To determine the development of leaf starch granules, small pieces
PCR program included pre-denaturation at 95 °C for 3 min, followed by (~4 mm×1 mm) of tomato leaves were excised, immediately fixed with
45 cycles of 95 °C for 30 s, 57 °C for 20 s, and 72 °C for 30 s. The gene- 2.5% glutaraldehyde, and then post-fixed with 1% OsO4 in phosphate
specific primers used in this study are presented in Supplementary Table buffer. Tissues were dehydrated in an ethanol series and embedded in
S1. The relative expression levels were normalized to the expression level Epon 812. Ultrathin sections were cut with an ultramicrotome (Leica,
of the tomato ACTIN2 and UBI3 genes (Livak and Schmittgen, 2001). Wetzlar, Germany) and stained with uranyl acetate and lead citrate.
Transmission electron microscopy (TEM) was performed on the stained
sections with an H7650 transmission electron microscope (Hitachi,Tokyo,
Virus-induced gene silencing constructs and Agrobacterium-
Japan) (Chi et al., 2020). The image analysis software ImageJ (National
mediated viral infiltration Institutes of Health, USA) was used to analyze the area of chloroplasts,
Tobacco rattle virus (TRV) virus-induced gene silencing (VIGS) con- and the number of starch granules in each leaf sample was counted in at
structs were used for silencing the HY5, PWD, BAM1/BAM3/BAM8, least 10 different visual fields.
MEX1, and DPE1 genes in tomato plants. Target gene fragments of
~300 bp were amplified from tomato cDNA by PCR using gene-specific
primers containing EcoRI and BamHI restriction sites or BamHI and Protein extraction and western blot
SmaI restriction sites (Supplementary Table S2) and then ligated into the For extraction of the HY5 protein, frozen leaf tissue (0.3 g) was
corresponding sites of the pTRV2 vector using the ClonExpress II One ground in liquid nitrogen in 1 ml of extraction buffer (100 mM Tris–
Step Cloning Kit (C112, Vazyme, Nanjing, China). The empty pTRV2 HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM
vector was used as a control. The resulting plasmids were electroporated phenylmethylsulfonyl fluoride, and 0.2% β-mercaptoethanol). The ex-
into Agrobacterium tumefaciens strain GV3101.VIGS was performed twice tracts were centrifuged at 13 000 g for 20 min at 4 °C, after which the ex-
by infiltration of germinated seeds, followed by infiltration of the fully tracted proteins were denatured at 95 °C for 10 min. For western blotting,
expanded cotyledons of 15-day-old tomato seedlings with A. tumefaciens the denatured protein extracts were separated using 12% SDS-PAGE and
harboring a mixture of pTRV1 and pTRV2-target genes in a 1:1 ratio. then transferred to nitrocellulose membranes (Millipore, Saint-Quentin,
For co-silencing of BAM1, BAM3, and BAM8, a mixture of pTRV1, France). The membranes were blocked for 1 h in TBST buffer (20 mM
pTRV2-BAM1, pTRV2-BAM3, and pTRV2-BAM8 in a ratio of 3:1:1:1 Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% (w/v)
was used. Cultures of A. tumefaciens carrying empty pTRV1 and pTRV2 BSA (Sigma) for 1 h at room temperature and then incubated overnight2630 | Dong et al. in TBST buffer with 1% BSA containing a rabbit antibody against HY5 test or Student’s t-test was performed to compare the means. Significance (Shanghai Jiayuan Bio Co., Shanghai, China) to detect the HY5 protein. was accepted at P
HY5 regulates starch metabolism in tomato | 2631
A C
120000
(μnumber mm-2 chloroplasts)
cry1a
Starch granules number
WT
100000 a
CRY1a-OE
07.00h
80000
b
60000 b
c c
40000
19.00h 20000 d
0
07.00h 19.00h
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cry1a WT CRY1a-OE
B D
90 cry1a
07.00h a
mol glucose g FW)
75 WT
CRY1a-OE b
Starch content
-1
60
45 c
19.00h 30 d d
15 e
(
0
07.00h 19.00h
cry1a WT CRY1a-OE
Fig. 1. Starch accumulation in the leaves is influenced by CRY1a expression. (A) Qualitative analysis of leaf starch accumulation using iodine staining. (B)
TEM observation of starch granules. (C) Number of starch granules per mm2 area of chloroplasts. The image-analysis software ImageJ (National Institutes
of Health, USA) was used to analyze the area of chloroplasts, and the number of starch granules in different tissues in each leaf sample was counted in
at least 10 different visual fields. (D) Leaf starch contents. The fourth leaf at the seven-leaf stage was sampled at 07.00 h (the end of the dark phase) and
19.00 h (the end of the light phase). FW, fresh weight. Values are the mean ±SD (n=4). Different letters indicate significant differences (P2632 | Dong et al.
A Relative expression B
0.0 1.0 4.0 600 cry1a
min-1 g-1 FW)
WT
Amylase activity
PWD
GWD
a
450 CRY1a-OE
BAM3 a
BAM8
BAM1
300 b b
DPE1 c
150 c
DPE2
(nmol
MEX1
AMY2 0 α-Amylase β-Amylase
cry1a WT CRY1a-OE
C
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Relative expression
0.0 1.0 6.0
PWD
GWD
BAM3
BAM8
BAM1
DPE1
DPE2
MEX1
AMY2
0 3 6 12 0 3 6 12 0 3 6 12 h
cry1a WT CRY1a-OE
D E
800 cry1a 600 cry1a
(nmol min-1g-1 FW)
α- Amylase activity
WT
(nmol min-1g-1 FW)
β- Amylase activity
WT
640 CRY1a-OE 480 CRY1a-OE
a a
480 c b b 360 b
c
d
320 de d 240 e
def def efg ef ef
160 efg 120 fgh
g g fg h gh h
0 0 0h
0h 3h 6h 12 h 3h 6h 12 h
Fig. 2. Transcript levels of starch degradation-related genes and activity of starch degradation enzymes as influenced by CRY1a expression and blue
light illumination. (A) Transcript analysis of starch degradation-related genes in plants with altered CRY1a transcripts. (B) Activities of α-amylase and β-
amylase in plants with altered CRY1a transcripts. (C) Transcript response to blue light illumination in plants with altered CRY1a transcripts. (D, E) Activity
of α-amylase (D) and β-amylase (E) in plants exposed to blue light illumination. For all experiments, the fourth leaf of tomato plants at the seven-leaf stage
was used for the analysis. For (A) and (B), plants were grown under white light with a 12 h light regime, and samples were collected at 07.00 h. For the
qRT–PCR analysis (A), ACTIN2 and UBI3 were used as reference genes, and the gene expression in the WT leaves at 07.00 h was assigned a value of
1. For (C–E), the plants were pre-acclimated in the dark for 12 h and then transferred to blue light for 12 h. Samples were collected at 0, 3, 6, and 12 h
after exposure to blue light. The gene expression level was determined relative to that in the WT at 0 h (set at a value of 1) by qRT–PCR using ACTIN2
and UBI3 as the reference genes in (C). Blue light was applied at 300 μmol m−2 s−1 at 460 nm. Values are the mean ±SD (n=4). Different letters indicate
significant differences (PHY5 regulates starch metabolism in tomato | 2633
phenotype of HY5-OE plants was similar to that of CRY1a-OE with the greatest accumulation observed in the CRY1a-OE
plants (Supplementary Fig. S1B, Supplementary Table S8). plants and the lowest in the cry1a mutants (Fig. 3B).
HY5-OE plants showed delayed flowering relative to the WT The induction of HY5 transcription and HY5 protein ac-
plants. Moreover, no significant differences in the net photo- cumulation by blue light prompted us to investigate whether
synthetic rate were observed among the HY5-RNAi, WT, and HY5 participates in the regulation of starch accumulation.
HY5-OE plants (Supplementary Table S8). Iodine staining revealed that HY5-RNAi leaves were black,
To investigate whether HY5 plays a role in CRY1a-induced while WT and HY5-OE leaves were respectively blue and
starch accumulation, we examined the transcription of HY5 brown at 07.00 h and 19.00 h, suggesting that the transcrip-
and the accumulation of HY5 in tomato leaves in response to tion of HY5 is negatively related to the accumulation of starch
blue light. We found that the transcript level of HY5 showed (Fig. 4A). Consistently, TEM observations demonstrated that
a strong response to blue light treatment, reaching the highest the HY5-RNAi leaves accumulated more and larger starch
level at 3 h after illumination in all plants; moreover, the tran- granules in the chloroplasts compared with the WT leaves.
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script level of HY5 was higher in the CRY1a-OE plants, and In contrast, fewer and smaller starch granules were observed
lower in the cry1a mutants, compared with that in the WT plants in the chloroplasts of the HY5-OE leaves compared with the
after blue light illumination (Fig. 3A). Consistent with this WT leaves (Fig. 4B). Furthermore, the number of starch gran-
finding, blue light induced the accumulation of HY5 protein, ules in the chloroplasts of mesophyll cells of the HY5-RNAi
A 40 cry1a
HY5
Relative expression
WT
30 CRY1a-OE
a
20
b b b
10 c
c
fg
d
0 fg
g
ef e
B
0h 3h 6h 12 h
HY5
Actin
cry1a WT CRY1a-OE cry1a WT CRY1a-OE
0h 6h
Fig. 3. Time course of the induction of HY5 transcript and HY5 protein accumulation in response to blue light. (A) Transcript accumulation of HY5
in tomato leaves in response to blue light. Plants at the seven-leaf stage were pre-acclimated in the dark for 12 h and were then transferred to blue
light (300 μmol m−2 s−1 at 460 nm) for 12 h. Samples were collected at 0, 3, 6, and 12 h after exposure to blue light. The gene expression level was
determined relative to that in the WT at 0 h (set at a value of 1) by qRT–PCR using ACTIN2 and UBI3 as the reference genes. Values are the mean± SD
(n=4). Different letters indicate significant differences (P2634 | Dong et al.
C
A
chloroplasts)
60000 HY5-RNAi
a
Starch granules number
WT
07.00h 50000
HY5-OE b
40000 c
30000
d
-2
(number mm
20000 e
19.00h f
10000
0 07.00h 19.00h
HY5-RNAi WT HY5-OE
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B D
75
HY5-RNAi
a
mol glucose g FW)
07.00h
60 WT
Starch content
-1
HY5-OE
b
45
c
30 d
19.00h e e
( 15
0
07.00h 19.00h
HY5-RNAi WT HY5-OE
Fig. 4. Starch accumulation in the leaves as influenced by HY5 expression. (A) Qualitative analysis of leaf starch contents using iodine staining. (B) TEM
observation of starch granules. (C) Number of starch granules per mm2 area of chloroplasts. The image analysis software ImageJ (National Institutes
of Health, USA) was used to analyze the area of chloroplasts, and the numbers of starch granules in each leaf sample were counted with at least 10
different visual fields. (D) Leaf starch contents. The fourth leaf at the seven-leaf stage was sampled at 07.00 h (the end of the dark phase) and 19.00 h
(the end of the light phase). FW, fresh weight. Values are the mean± SD (n=4). Different letters indicate significant differences (PHY5 regulates starch metabolism in tomato | 2635
A Relative expression B
0.0 1.0 3.0 500 HY5-RNAi
PWD
min-1g-1 FW)
Amylase activity
GWD 400 WT
BAM3 HY5-OE
a
a
BAM8 300 b b
BAM1
DPE1 200 c c
(nmol
DPE2
100
MEX1
AMY2 0
HY5-OE
-Amylase -Amylase
HY5-RNAi WT
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C Relative expression
0.0 1.0 6.0
PWD
GWD
BAM3
BAM8
BAM1
DPE1
DPE2
MEX1
AMY2
0 3 6 12 0 3 6 12 0 3 6 12 h
HY5-RNAi WT HY5-OE
D E
750 HY5-RNAi 800 HY5-RNAi
(nmol min-1g-1 FW)
-Amylase activity
WT
FW)
-Amylase activity
WT
600 HY5-OE
600 HY5-OE
a a
min-1g-1
450 a b a
b b
cd c 400 c
300 d cd d de
e e
150 200
(nmol
ef ef f e f f
0 f f
0h 3h 6h 12 h 00h 6h
3h 12 h
Fig. 5. Transcript levels of starch degradation-related genes and activity of starch degradation enzymes as influenced by HY5 expression and blue light
illumination. (A) Transcript analysis of starch degradation-related genes in plants with altered HY5 transcripts. (B) Activities of α-amylase and β-amylase
in plants with altered HY5 transcripts. (C) Transcript response to blue light illumination in plants with altered HY5 transcripts. (D, E) Activity of α-amylase
(D) and β-amylase (E) in plants under blue light illumination. For all experiments, the fourth leaf of tomato plants at the seven-leaf stage was used for the
analysis. For (A) and (B), the plants were grown under white light with a 12 h light regime, and samples were collected at 07.00 h. The gene expression
level was determined relative to that in the WT at 07.00 h (set at a value of 1) by qRT–PCR using ACTIN2 and UBI3 as the reference genes in (A). For
(C–E), plants were pre-acclimated in the dark for 12 h and then transferred to blue light for 12 h. Samples were collected at 0, 3, 6, and 12 h after
exposure to blue light. The gene expression level was determined relative to that in the WT at 0 h (set at a value of 1) by qRT–PCR using ACTIN2 and
UBI3 as the reference genes in (C). Blue light was applied at 300 μmol m−2 s−1 at 460 nm. Values are the mean± SD (n=4). Different letters indicate
significant differences (P2636 | Dong et al.
transcription. A sequence analysis showed that G-box and/or A B C
ACE motifs occur in the promoters of the starch degradation-
related genes PWD, BAM3, BAM1, BAM8, DPE1, and MEX1,
with the total numbers of these motifs ranging from two to
Protein-HY5
five for each promoter. Two E-box [CA(T/C)GTG] mo- Probe
PWD-A BAM3-A BAM1-B
tifs were found in the PWD and BAM3 promoters, and five Mut Mut Mut
G-box motifs were found in the BAM1 promoter. In addition, D E F
two G-box motifs and two E-box motifs occur in the BAM8
promoter, one A-box motif (TACGTA), one G-box motif,
and one E-box motif occur in the DPE1 promoter, and two Protein-HY5
Probe
E-box motifs and one G-box motif occur in the MEX1 pro- BAM8-A DPE1-C MEX1-A
moter (Supplementary Fig. S7A). The EMSA results showed Mut Mut Mut
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that the HY5 protein could bind to the promoters of PWD- 8 PWD BAM3 BAM1 BAM8 DPE1 MEX1
G
IgG
% Chip signal/input
A, BAM3-A, BAM3-B, DPE1-C, MEX1-A, and MEX1-C
6 Ab
a
containing the E-box motif, as well as BAM8-A, BAM1-B,
BAM1-C, BAM1-D, DPE1-B, and MEX1-B containing the a
4 a
G-box motif. In contrast, when these motifs were mutated a a a
b b b b
from CACGTG/CAGTG to TTTTTT, the HY5 protein lost 2 bb b bbb b
bb b b b b b
its ability to bind to them (Fig. 6A–F, Supplementary Fig. S8).
To examine the in vivo binding ability of HY5 to the pro- 0
E
Y T
Y T
E
E
Y T
Y T
E
Y T
E
E
Y T
H W
H W
moters of the starch degradation-related genes, we subjected
O
O
O
H W
H W
H W
O
H W
O
O
5-
5-
5-
5-
5-
5-
transgenic HY5-3HA-OE plants to continuous blue light H
10
for 12 h and performed ChIP–qPCR experiments using an
Relative LUC activity
8 a
anti-HA antibody or an anti-IgG antibody as a negative con- a
(LUC/REN)
trol. Using the immunoprecipitated HY5-3HA products and 6 a
a
the anti-HA antibody, the PWD, BAM3, BAM8, BAM1, 4
a
DPE1, and MEX1 promoter fragments in the 35S:HY5-HA a
(HY5-OE) samples were found to be enriched by 1.9-, 2.1-, 2 b b b b b b
2.1-, 2.1-, 4.5-, and 3.4-fold, respectively, compared with those 0
of the WT plants (Fig. 6G). In contrast, the IgG control anti-
body failed to pull down any of these promoter DNA seg-
ments. Dual-luciferase assays showed that HY5 can significantly
induce the promoter activity of PWD, BAM3, BAM8, BAM1,
Fig. 6. HY5 binds to the G-box and ACGT-containing elements (ACEs)
DPE1, and MEX1 by 5.2-, 1.6-, 3.2-, 6.1-, 3.4-, and 1.1-fold, in the promoters of starch degradation-related genes. (A–F) EMSAs
respectively (Fig. 6H, Supplementary Fig. S7B). Collectively, showing that HY5 binds to the G-box and/or ACE motifs present in the
these findings suggested that HY5 can bind to the promoters promoters of PWD (A), BAM3 (B), BAM1 (C), BAM8 (D), DPE1 (E), and
of the starch degradation-related genes PWD, BAM3, BAM8, MEX1 (F) in vitro. The assays were repeated three times, and similar results
were obtained. (G) ChIP–qPCR assay to test the ability of HY5 to bind to
BAM1, DPE1, and MEX1 at sites containing the G-box or
the promoters of PWD, BAM3, BAM1, BAM8, DPE1, and MEX1 in vivo.
E-box motif and then activate the transcription of these genes, ChIP was performed using leaves of transgenic tomato plants steadily
ultimately promoting starch degradation in leaves. overexpressing the HY5-HA fusion protein. Anti-HA antibody (Ab) was
used to immunoprecipitate HY5-HA and associated DNA fragments, and
anti-IgG antibody (IgG) was used as the negative control. The resultant
HY5 mediates CRY1a-regulated starch degradation DNA fragments were assayed by qPCR using primers specific to the
fragments containing PWD-A, BAM3-A, BAM1-B, BAM8-A, MEX1-A, and
Taking the above results into account, it is reasonable to specu- DPE1-C. Bars indicate the SD of three parallel samples. Different letters
late that CRY1a-induced starch degradation is at least par- indicate significant differences (PHY5 regulates starch metabolism in tomato | 2637
A study allow us to propose a working model for CRY1a-
induced starch degradation in tomato (Fig. 8).
Starch accumulation in plants changes with the light condi-
tions (Hendriks et al., 2003; Ma et al., 2017). In this study, we
provide several lines of evidence for the critical role of blue
light signaling in starch degradation. First, the cry1a mutant
WT CRY1a-OE leaves accumulated more starch and showed lower transcript
B levels of starch degradation-related genes than the WT leaves.
( mol glucose g-1 FW)
40 a In contrast, the CRY1a-OE leaves had a lower accumulation
of starch and a higher transcript level of starch degradation-
Starch content
b
30 related genes compared with the WT leaves (Figs 1, 2). Second,
b b
exposure to blue light increased the transcript and activity
20 c
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c c levels of starch degradation-related genes and enzymes in the
10
WT and CRY1a-OE plants, but had little effect in the cry1a
mutant (Fig. 2C–E). Third, the ADPG content in the cry1a
0 leaves was lower than that in the WT (Supplementary Fig.
8 1
RV V 5 /B D
PE EX1 S4C). Moreover, no significant changes were found in the
pT pTR -HY /B3 W
-P V-D
R V 1 V -M transcript and activity levels of genes and enzymes involved in
pT V -B
pTR pTR TRV
TR p starch synthesis among the cry1a mutant, WT, and CRY1a-OE
p
plants (Supplementary Fig. S4A, B). Fourth, silencing of starch
C
WT CRY1a-OE
15 degradation-related genes, such as PWD, BAM1, BAM3, and
Soluble sugar content
a BAM8, in the CRY1a-OE plants significantly increased starch
12
accumulation and decreased soluble sugar accumulation in the
(mg g FW)
b
9 cd c leaves (Fig. 7). Collectively, these results demonstrated that blue
d
-1
e e light and its receptor CRY1a are essential for light-regulated
6
starch accumulation in response to changes in the growth en-
3 vironment. The results also revealed that blue light signaling
0 alters starch accumulation by influencing the starch degrad-
RV RV Y5 B/ D E1 X1
8 ation that takes place in chloroplasts in mesophyll cells, rather
pT pT V-H 1/B3 V-PW -DP -ME
R V than by influencing starch biosynthesis. The mild induction of
pT RV- pTR pTR TRV
B
pT p starch degradation-related genes in the cry1a plants relative to
WT CRY1a-OE the HY5-RNAi plants by blue light suggests that other CRYs,
such as CRY1b and CRY-DASH, are potentially involved in
Fig. 7. Silencing of HY5 and starch degradation-related genes suppresses
starch degradation in CRY1a-overexpressing plants. (A) Qualitative the regulation of starch catabolism. It was reported that to-
analysis of leaf starch content using iodine staining. (B, C) Starch (B) and mato CRY2 affects the genes and proteins involved in starch
soluble sugar (C) contents in the leaves. FW, fresh weight. Values are the accumulation, sucrose biosynthesis, and secondary metabolism
mean ±SD (n=4). Different letters indicate significant differences (P2638 | Dong et al.
Maltose Glucose
BL
MEX
Chloroplast
Glucose
CRY1a
Triose -P Calvin
DPE1 MEX1
cycle
DPE2 HY5
Fru6P DPE1
GPI Maltose
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BAMs
Glc6P
PGM AMY
PWD Nucleus
ATP Glc1P ADPGase BAMs
ISA
PPi ADPG
GBSS
SSS GWD
DBE PWD
SBE
Starch AMP
Pi
ATP
Fig. 8. Working model showing that HY5 mediates CRY1a-induced expression of PWD, BAM1/BAM3/BAM8, DPE1, and MEX1, and thereby regulates
starch degradation in leaves. ADPG, ADP-glucose; ADGPase, ADP glucose pyrophosphorylase; AMY, α-amylase; BAMs, β-amylases (here, this means
BAM1, BAM3, and BAM8); DBE, debranching enzyme; DPE1, disproportionating enzyme 1; DPE2, disproportionating enzyme 2; Fru6P, fructose-6-
phosphate; GBSS, granule-bound starch synthase; Glc1P, glucose-1-phosphate; Glc6P, glucose-6-phosphate; GPI, plastid phosphoglucose isomerase;
GWD, glucan water dikinase; ISA, isoamylase; MEX1, maltose excess protein 1; PGM, phosphoglucomutase; PGT, plastid glucose transporter; PPi,
inorganic pyrophosphate; PWD, phosphoglucan water dikinase; SBE, starch branching enzyme; SSS, soluble starch synthase.
transcript levels and activity of these enzymes in the HY5- Previous studies demonstrated that HY5 acts as the center
RNAi plants (Fig. 5C–E). Third, the content of ADPG did of the transcriptional network hub in different plant signaling
not differ between the HY5-RNAi, WT, and HY5-OE plants processes, such as light, hormone, nutrient, anthocyanin bio-
(Supplementary Fig. S6C). Fourth, silencing of HY5 abolished synthesis, sucrose metabolism, abiotic stress, and reactive
CRY1a-mediated degradation of starch (Fig. 7).We also found oxygen species signaling (Gangappa and Botto, 2016; Wang
that the accumulation of the HY5 transcript and HY5 pro- et al., 2019a). HY5 could regulate the transcription of a large
tein is light-responsive, as observed in earlier studies (Liu et al., number of genes by directly binding to the cis-regulatory elem-
2018b; Wang et al., 2019a). The protein abundance of HY5 is ents, for example, the G-box element or ACEs (Binkert et al.,
under the control of COP1, which is a central switch in light 2014; Chattopadhyay et al., 1998; Hajdu et al., 2018; Lee et al.,
signal transduction, via interactions with upstream light recep- 2007).The sequence analysis revealed that six of the nine starch
tors and downstream target proteins. In darkness, COP1 is lo- degradation-related genes examined in tomato plants, namely
cated in the nucleus and continuously degrades HY5 via the PWD, BAM1, BAM3, BAM8, MEX1, and DPE1, contained
26S proteasome. When exposed to light, photoreceptors are G-box or ACE motifs in their promoters (Supplementary Fig.
activated, which can relocate COP1 to the cytoplasm, thereby S7A). In vitro and in vivo experiments using EMSA, ChIP–
releasing HY5 (Hoecker, 2017). In agreement with this finding, qPCR, and dual-luciferase assays revealed that HY5 could
we found that the HY5 transcript and HY5 protein were in- directly recognize and bind to the G-box and ACE motifs in
duced in response to blue light (Fig. 3). We found that the the promoters of these starch degradation-related genes and
HY5-RNAi plants showed more significant changes relative to activate their transcription (Fig. 6, Supplementary Fig. S8).
the WT plants in terms of the accumulation of starch between Consistent with this finding, the transcripts of PWD, BAM1,
night and day; by contrast, limited changes in the accumulation BAM3, BAM8, MEX1, and DPE1 were highly regulated by
of starch were observed in the HY5-OE plants between night both CRY1a and HY5, and silencing of these genes abolished
and day (Fig. 4). Taken together, these results provided convin- CRY1a-induced degradation of starch, which led to increased
cing evidence for HY5 as a regulator of starch metabolism in starch accumulation and decreased soluble sugar accumulation
response to environmental light fluctuations. (Fig. 7).These results collectively indicate that CRY1a-inducedHY5 regulates starch metabolism in tomato | 2639
starch breakdown is mediated by HY5 through activation of Fig. S5. Sugar content in the WT, HY5-RNAi, and
the transcription of these degradation-related genes. HY5-OE leaves.
Our finding that blue light regulates starch–sugar intercon- Fig. S6. Gene expression and activity of starch synthesis-
version is interesting and has several implications. Sugars are related enzymes and ADPG content in the WT, HY5-RNAi,
not only important for horticultural products but could also and HY5-OE leaves.
function as signals for plant growth, development, and stress re- Fig. S7. Schematic representation of the G-box and ACE
sponses. For example, cold induces the accumulation of HY5, motifs in the PWD, BAM1, BAM3, BAM8, DPE1, and MEX1
which may contribute to the increased accumulation of sol- promoters and the vector construction principle used in the
uble sugars to adapt to cold episodes (Wang et al., 2019a). Blue dual-luciferase assays.
light was also found to induce the degradation of starch in Fig. S8. EMSA results of the capacity of HY5 to bind to the
guard cells within 30 min of light exposure to promote sto- BAM3, BAM1, DPE1, and MEX1 promoters in several other
matal opening (Horrer et al., 2016), and it will be of great binding motifs.
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interest to study whether sugars are involved in this process. Fig. S9.VIGS efficiency analysis.
In addition to increasing sugar accumulation in the fruits of Table S1. List of primer sequences used for the qRT–PCR
CRY1a-OE tomato plants (Liu et al., 2018a), treatment with analysis.
blue light activated the expression of cryptochrome genes and Table S2. PCR primer sequences used for vector construc-
enhanced sugar production in broad bean leaves (Talbott and tion in VIGS.
Zeiger, 1993) and Chinese bayberry fruit (Shi et al., 2016), sug- Table S3. qRT–PCR primer sequences used for verification
gesting that light manipulation could be a potential way to in- of the gene silencing efficiency in VIGS.
crease sugar accumulation in agricultural products. Finally, this Table S4. Probes used in the EMSAs.
HY5-regulated starch–sugar interconversion is likely involved Table S5. Primers used for the ChIP–qPCR assays.
in carbohydrate resource allocation and the maintenance of a Table S6. PCR primer sequences used for vector construc-
certain C/N ratio, because HY5 has been found to participate tion in the dual-luciferase assays.
in the transcriptional activation of several nitrogen uptake- and Table S7. Changes in the plant phenotype and photosyn-
metabolism-related genes (Chen et al., 2016). thetic capacity as influenced by CRY1a.
The data presented here provide new insights into the regu- Table S8. Changes in the plant phenotype and photosyn-
lation of carbohydrate metabolism in plants. Evidence is pre- thetic capacity as influenced by HY5.
sented to show that starch accumulation is largely controlled by
blue light, partially via a CRY1a-associated pathway that pro-
motes HY5 accumulation.This process is achieved by CRY1a- Acknowledgements
regulated starch degradation rather than by starch synthesis. As We are grateful to Prof. Jim Giovannoni from Cornell University and
a result, HY5 regulates starch accumulation by activating the the Tomato Genetics Resource Center at the University of California
transcription of starch degradation-related structural genes in for providing the tomato HY5-RNAi seeds. This work was supported by
response to blue light. These mechanisms highlight gaps in our grants from the National Key Research and Development Program of
knowledge and point to research areas that show promise for China (2019YFD1000300) and the Modern Agro-industry Technology
the bioengineering and manipulation of starch metabolism by Research System of China (CARS-25-02A), the National Natural
light to achieve more desirable phenotypes, such as high starch Science Foundation of China (31825023), and the Key Research and
accumulation in cereal seeds, tubers, or roots, or high sugar ac- Development Program of Zhejiang (2018C0210). We are thankful to Dr
cumulation in fleshy fruits or leafy vegetables. X.D. Wu for help in the sugar analysis and Dr Q.Z. Yu for the mainten-
ance of the growth chambers. We thank Mr C.X. Liu for the placement
of the blue light tubes.
Supplementary data
The following supplementary data are available at JXB online.
Author contributions
Fig. S1. Effect of genetic manipulation of the cryptochrome1a
(CRY1a) and elongated hypocotyl 5 (HY5) genes on plant growth JY conceived, designed, and supervised the experiments; HD, CH, and
development in tomato plants. JW conducted the experiments; CL constructed the materials and pre-
Fig. S2. Sugar content in the WT, cry1a mutant, and sented the ideas, HD analyzed the data and prepared the first draft; JY and
YZ contributed to the final editing of the manuscript.
CRY1a-OE leaves.
Fig. S3. Phylogenetic analysis of tomato BAM1, BAM3, and
BAM8 genes and AtBAMs.
Fig. S4. Gene expression and activity of starch synthesis- Data availability
related enzymes and ADPG content in the WT, cry1a mutant, All data supporting the findings of this study are available within the
and CRY1a-OE leaves. paper and within its supplementary data published online.2640 | Dong et al.
Conflict of interest Hajdu A, Dobos O, Domijan M, Bálint B, Nagy I, Nagy F, Kozma-
Bognár L. 2018. ELONGATED HYPOCOTYL 5 mediates blue light signalling
The authors declare that they have no conflict of interest. to the Arabidopsis circadian clock. The Plant Journal 96, 1242–1254.
Hendriks JH, Kolbe A, Gibon Y, Stitt M, Geigenberger P. 2003.
ADP-glucose pyrophosphorylase is activated by posttranslational redox-
modification in response to light and to sugars in leaves of Arabidopsis and
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