Plant Physiology Preview. Published on September 8, 2016, as DOI:10.1104/pp.16.00698
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Optimization of Light Harvesting Pigment Improves
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Photosynthetic Efficiency
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Honglei Jin, Mengshu Li, Sujuan Duan, Mei Fu, Xiaoxiao Dong, Bing Liu,
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Dongru Feng, Jinfa Wang1&Hong-Bin Wang1
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State Key Laboratory of Biocontrol and Collaborative Innovation Center of
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Genetics and Development, Guangdong Provincial Key Laboratory of Plant
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Resources, School of Life Sciences, Sun Yat-sen University, 510275
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Guangzhou, People’s Republic of China
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ORCID ID: 0000-0003-4957-0509 (H.-B.W.)
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[email protected] (Hong-Bin Wang)
Address correspondence to [email protected] (Jinfa Wang) or
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Tel: 8620-84039179
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Running Title: Light Harvesting Pigment Optimization
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One Sentence Summary
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Characterization of Arabidopsis hpe1 mutants revealed a novel strategy to
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optimize light harvesting pigments which improved photosynthetic efficiency
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and biomass production.
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Copyright 2016 by the American Society of Plant Biologists
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AUTHOR CONTRIBUTIONS
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H.-B.W., J.W. and H.J. designed the study. H.J., M.L. and S.D. performed
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research. H.J., J.W., H.-B.W., analyzed data. H.-B.W. and H.J. wrote the
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article. B.L., D.F., M. F., and X.D. revised the article. All authors read and
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approved the final manuscript.
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FUNDING INFORMATION
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This work was supported by the grants from the National Natural Science
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Foundation of China (No. 31425003 and No. 31500195), the Natural Science
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Foundation of Guangdong Province, PR China (No. 2014A030310491), the
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China postdoctoral Science Foundation (No. 2015M572399 and No.
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2016T90808), the National Science and Technology Major Project Foundation
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of China (No. 2016ZX08009003-005-005), and the Fundamental Research
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Funds for the Central Universities.
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ABSTRACT
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Maximizing light capture by light harvesting pigment optimization represents
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an attractive but challenging strategy to improve photosynthetic efficiency.
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Here, we report that loss of a previously uncharacterized gene HIGH
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PHOTOSYNTHETIC EFFICIENCY 1 (HPE1) optimizes light harvesting
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pigments leading to improved photosynthetic efficiency and biomass
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production. Arabidopsis hpe1 mutants show faster electron transport and
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increased contents of carbohydrates. HPE1 encodes a chloroplast protein
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containing a RNA recognition motif which directly associates with and
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regulates the splicing of target RNAs of plastid genes. HPE1 also interacts with
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other plastid RNA splicing factors, including CAF1 and OTP51 which share
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common targets with HPE1. Deficiency of HPE1 alters the expression of
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nuclear-encoded chlorophyll-related genes probably through plastid to nucleus
53
signaling, causing decreased total content of chlorophyll (a+b) in limited range
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but increased ratio of chlorophyll a/b. Interestingly, this adjustment of light
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harvesting pigment reduces antenna size, improves light capture, decreases
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energy loss, mitigates photodamage, and enhances photosynthetic quantum
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yield during photosynthesis. Our findings suggest a novel strategy to optimize
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light harvesting pigments which improves photosynthetic efficiency and
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biomass production in higher plants.
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INTRODUCTION
The
tremendous
increase
in
world
population
and
environmental
64
deterioration pose serious challenges to agricultural production and food
65
security (Ray et al., 2013). To meet this challenge, crops with high yield
66
potential need to be developed (Long et al., 2015). However, the yield traits
67
which have played key roles during the green revolution have had their
68
potential nearly exhausted, thus new strategies are needed. Photosynthesis,
69
the unique biological process responsible for the conversion of light energy to
70
chemical forms, is the ultimate basis of crop yield (Zhu et al., 2010).
71
Theoretically, enhancing photosynthetic efficiency should be an excellent
72
strategy to increase crop yield. However, improvement of photosynthetic
73
efficiency has played only a minor role in the remarkable crop productivity
74
improvement achieved in the last half-century (Zhu et al., 2010; Ort et al.,
75
2015).
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In the light reactions of photosynthesis, light energy is used by chlorophyll
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and associated pigments, water is split, and electron transport on the
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chloroplast membrane reduces NADP, resulting in a proton gradient that
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powers the phosphorylation of ADP. NADPH and ATP power the Calvin cycle,
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which assimilates and reduces carbon dioxide to carbohydrate (Ort et al.,
81
2015). Strategies to improve photosynthesis mainly include optimization of
82
light capture, light energy conversion in the light reaction, carbon capture and
83
conversion in the dark reaction (Ort et al., 2015). Previous research mainly
84
focused on the optimization of dark reactions through improvement of carbon
85
capture and conversion to directly increase biomass (Miyagawa et al., 2001;
86
Kebeish et al., 2007; Lin et al., 2014; Ort et al., 2015). However, less effort has
87
been spent to optimize light capture and light energy conversion in the light
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reactions to improve the whole photosynthetic efficiency (Ort et al., 2015).
89
Maximizing light capture by adjustment of antenna size can optimize light
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capture and light energy conversion, but is difficult to achieve (Blankenship
91
and Chen, 2013). Antenna in photosynthetic systems typically consist of
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92
pigments specifically bound to membrane-associated proteins. These antenna
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pigment-protein complexes closely associate with the reaction center
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complexes, and deliver absorbed energy to the reaction centers where some
95
of the energy originally in the photon is captured by electron transfer
96
processes (Blankenship, 2002; Green and Parson, 2003). However, light
97
saturation could take place at intensities much lower than would be expected if
98
every chlorophyll was able to carry out photosynthesis by itself (Blankenship,
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2002). The light saturation problem has also been addressed from the antenna
100
perspective, and many efforts are underway to truncate the antenna system in
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photosynthetic microorganisms. A smaller antenna associated with each
102
reaction center will in principle also shift the light response curve, so that light
103
saturation sets in at higher intensities, thereby reducing excess light and
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increasing productive light. While the concept of increased efficiency due to
105
reduced antenna size is simple, reaching this goal has not yet been achieved
106
(Blankenship and Chen, 2013). In green algae, the reduction of light harvesting
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pigments by decreasing the expression of Chl a oxygenase (CAO) gene, which
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is responsible for the synthesis of Chl b via the oxidation of Chl a (Czarnecki
109
and Grimm, 2012), led to efficient photosynthesis due to the balance between
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captured light and photochemical reactions (Perrine et al., 2012). However,
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there is still no success in higher plants.
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In this study, we performed a large scale genetic screen using the model
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organism Arabidopsis and identified two independent alleles of an
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uncharacterized
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EFFICIENCY1 (HPE1) whose mutation confers improved photosynthetic
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efficiency by optimizing of light harvesting pigment. Deficiency of HPE1 shows
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higher light reaction activity of photosynthesis, more efficient carbon fixation
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and significantly increased biomass production. Interestingly, HPE1 encodes a
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chloroplast protein containing RNA recognition motif and regulates splicing of
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RNAs of plastid genes by directly associating with target RNAs. HPE1
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mutation results in the splicing deficiency of plastid genes which may alter the
gene
which
we
named
HIGH
PHOTOSYNTHETIC
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expression of chlorophyll-related genes probably through plastid to nucleus
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signaling. Altered expression of chlorophyll-related genes changes the content
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of light harvesting pigments and optimizes the light harvesting system. Our
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characterization of HPE1 mutants suggests a novel strategy to optimize light
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harvesting and improve photosynthetic efficiency in higher plants.
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RESULTS
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Loss
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Photosynthesis
of
HPE1
Confers
Improved
Light
Reaction
Activity
of
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To better understand the regulatory mechanism of photosynthesis and
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identify mutants with increased photosynthetic efficiency, we screened many
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Arabidopsis mutant pools by using a chlorophyll fluorescence video imaging
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system (Jin et al., 2014). We identified high photosynthetic efficiency (hpe)
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mutants which exhibit high maximum photochemical efficiency of PSII (Fv/Fm)
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under growth light. One hpe1 mutant (SALK_012657), At1g70200, showed
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higher photosynthetic efficiency than wild type plants (Figure 1A-1C). This
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allele contains a T-DNA element inserted into the 5’-UTR of HPE1 gene
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(Figure 1A). Interestingly, we also obtained another independent hpe1 allele
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(SALK_092951) which harbors a T-DNA element inserted into the first exon of
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HPE1 gene and exhibits a phenotype identical to that of hpe1 mutants (Figure
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1A-1C). The results were confirmed with twice-backcrossed mutants and imply
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that deficiency of HPE1 results in higher light reaction activity during
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photosynthesis. We subsequently named SALK_012657 and SALK_092951
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as hpe1-1 and hpe1-2, respectively. Next, to further precisely determine the
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light reaction activity of photosynthesis in hpe1 mutants, we analyzed the light
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intensity
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light-response curves of PSII quantum yield (ΦPSII), photochemical quenching
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(qP), and electron transport rate (ETR). ΦPSII, qP, and ETR were much higher
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in the hpe1 mutants than in the wild type (Figure 1D), together with higher
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Fv/Fm in the hpe1 mutants (Figure 1C), which confirms that the light reaction
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activity of photosynthesis is higher in hpe1 mutants than in the wild type.
dependence
of
three
chlorophyll
fluorescence
parameters,
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The Content of Carbohydrates and Biomass Production Are Increased in
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hpe1 Mutants
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Because NADPH and ATP from the light reaction are used to power the
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Calvin cycle, we speculated that improved light reaction performance can
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promote carbon fixation during the dark reaction of photosynthesis in the hpe1
160
mutants. To test this, we first determined the contents of representative
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carbohydrates in leaves by using gas chromatography/mass spectrometry
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(GC-MS). The hpe1 mutant plants accumulated higher contents of
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carbohydrates per unit leaf area, including glucose and fructose. The contents
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of glucose and fructose were approximately 51.1% and 49.9% higher in the
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hpe1 mutants than in the wild type plants, respectively (Figure 2A, 2B). To
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determine whether the more accumulation of carbohydrates in hpe1 mutants is
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due to decrease in carbohydrate turnover, we measured the degradation rate
168
of wild type and the mutant plants after inhibiting the photosynthetic light
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reactions using DCMU treatment. Interestingly, after 5-h DCMU treatment, the
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content of glucose was decreased by approximately 39.9% in wild type, 40.3%
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in hpe1-1, and 42.5% in hpe1-2 plants. The content of fructose was decreased
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by approximately 38.4% in wild type, 39.1% in hpe1-1, and 41.2% in hpe1-2
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plants (Supplemental Figure S1). These results indicate that the turnover rate
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of carbohydrates in hpe1 mutants is comparable to that in wild type plants,
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implying that the more accumulation of carbohydrates is due to more efficient
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carbon fixation in hpe1 mutants. More interestingly, the content of proline, the
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only proteinogenic secondary amino acid which responds to metabolic stress
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and participates in metabolic signaling (Phang et al., 2010), was significantly
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higher in the hpe1 mutants than in the wild type plants (Figure 2C,
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Supplemental Figure S1), implying that the hpe1 mutants may be resistant to
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adverse stress.
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To evaluate whether more efficient photosynthesis could result in increased
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biomass, we compared the growth phenotype of the hpe1 mutants and wild
184
type. Interestingly, 5-week-old mature hpe1 mutants showed a substantial
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increase in biomass (Figure 2D), including both greater fresh weight (Figure
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2E) and dry weight (Figure 2F), although the hpe1 mutants and wild type
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controls showed no obvious differences at the young plant stage. Rosette size
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and leaf number of the hpe1 mutants and wild type showed no obvious
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differences (Supplemental Figure S2), which indicates that the increased
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biomass may be due to more fleshy leaves of the hpe1 mutants than the wild
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type controls. Together, these results suggest that carbon fixation is more
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efficient and biomass production is increased in hpe1 mutants.
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HPE1 Is Specifically Localized to Chloroplast and Affects RNA Splicing
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of Plastid Genes
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To elucidate the mechanism underlying the improved photosynthetic
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efficiency and biomass of the hpe1 mutants, we analyzed the molecular
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functions of HPE1. HPE1 encodes a protein of 538 amino acids of unknown
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function
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(www.Arabidopsis.org). HPE1 protein contains a predicted N-terminal
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chloroplast transit peptide (1-49) (Figure 3A), based on TargetP prediction
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(Emanuelsson et al., 2000). Analysis of the subcellular localization of
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HPE1-GFP fusion proteins by confocal laser scanning microscopy revealed
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that HPE1 is specifically localized to the chloroplast (Figure 3B). Notably,
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HPE1 contains an RNA recognition motif (RRM, 188-267) (Figure 3A), also
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known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP),
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which is one of the most abundant protein domains involved in RNA binding
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and is found in all life kingdoms (Maris et al., 2005), suggesting that HPE1 may
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be involved in regulation of plastid RNA metabolism. To test this, we first
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examined the transcription of plastid genes by using real-time PCR.
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Transcription of plastid genes regulated by nuclear-encoded RNA polymerase,
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plastid-encoded RNA polymerase, or both was comparable in the hpe1
213
mutants and wild type (Supplemental Figure S3). Furthermore, we analyzed
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plastid RNA processing in the hpe1 mutants, and found that no obvious
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differences in RNA editing or stability were detected between the hpe1
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mutants and wild type controls. Finally, we determined RNA splicing efficiency
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of the intron-containing plastid genes. Interestingly, we found increased
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accumulation of unspliced precursors of Lysine tRNA (trnK), ATPase F subunit
according
to
The
Arabidopsis
Information
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Resource
219
(atpF),
and
RNA
polymerase
beta'
subunit-1(rpoC1),
but
reduced
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accumulation of spliced RNAs of trnK, atpF and rpoC1 in the hpe1 mutants in
221
varying degrees (Figure 3C and Supplemental Figure S4B). Notably, the group
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II intron in the trnK gene of land plants encodes a conserved protein called
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MatK, which shares sequence similarity with canonical group II maturases in
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domains that may also assist the splicing of its own and other chloroplast
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group II introns (Stern et al., 2010). However, there are no significant effects of
226
HPE1 deficiency on splicing of other plastid genes which contain introns
227
(Supplemental Figure S4A). These results suggest that HPE1 only regulates
228
the splicing of some chloroplast RNAs.
229
We further assessed whether HPE1 is associated with target RNAs by using
230
RNA immunoprecipitation (RIP), an analysis that detects the presence of the
231
corresponding RNA in the protein immunoprecipitates by reverse transcription
232
(RT)-PCR. The immunoprecipitated HPE1 complexes were found to
233
specifically co-precipitate the RNAs of trnK, atpF, and rpoC1 as revealed by
234
comparative RT-PCR analysis of the corresponding samples derived from the
235
hpe1 mutants and wild type plants (Figure 3D). These results thus indicate that
236
HPE1 associates in vivo with target RNA, including trnK, atpF, and rpoC1,
237
which suggests that HPE1 is a novel chloroplast RNA splicing factor. Introns
238
and multiple splicing factors assemble into ribonucleoprotein (RNP) complexes
239
in land plants and in Chlamydomonas to regulate RNA splicing (Perron et al.,
240
2004; Stern et al., 2010). Previous studies reported that RNA splicing of trnK,
241
atpF and rpoC1 is also regulated by CAF1 (Asakura and Barkan, 2006),
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OTP51 (de Longevialle et al., 2008), OTP70 (Chateigner-Boutin et al., 2011) or
243
WTF1 (Kroeger et al., 2009). To determine whether HPE1 assembles into
244
RNP complexes with other RNA splicing factors in chloroplasts, we analyzed
245
the interaction between HPE1 and CAF1, OTP51, OTP70 or WTF1. Interaction
246
of HPE1 with CAF1 or OTP51 were detected by bimolecular fluorescence
247
complementation (BiFC) analysis (Figure 4A), which was further confirmed by
248
co-immunoprecipitation assays (Co-IP) (Figure 4B). However, interaction of
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249
HPE1 with OTP70 and WTF1 were not detected (Supplemental Figure S6).
250
These observations indicate that HPE1 forms a complex specifically with
251
CAF1 and OTP51 to co-regulate chloroplast RNA splicing.
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Light Harvesting Pigment Are Optimized through Downregulation of the
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Expression of Nuclear-encoded Chlorophyll-related Genes in hpe1
255
Mutants
256
Biochemical analysis indicated that HPE1 is involved in post-transcription
257
regulation of gene expression in plastids, but it is still unclear how loss of
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HPE1
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Interestingly, we found that the leaves of the hpe1 mutants had a
260
chlorophyll-deficient phenotype (Figure 2D). Notably, the content of chlorophyll
261
was also decreased in mutants of other chloroplast RNA splicing factors,
262
including CAF1 and OTP51 (Asakura and Barkan, 2006; de Longevialle et al.,
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2008). Both chlorophyll a and chlorophyll b contents were decreased in the
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hpe1 mutants (Figure 5B, Supplemental Figure S9), consistent with other
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chloroplast RNA splicing mutants (Asakura and Barkan, 2006; de Longevialle
266
et al., 2008; Kroeger et al., 2009; Chateigner-Boutin et al., 2011). Interestingly,
267
different from mutants of other chloroplast RNA splicing regulators, the extent
268
of chlorophyll reduction in hpe1 mutants was smaller, which may be related to
269
less contribution of HPE1 to plastid RNA splicing. The reduced chlorophyll is
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probably still able to meet the need of light capture but meanwhile reduces the
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light-harvesting antenna size, thus ameliorating the loss of light energy. In
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addition, the reduction in chlorophyll b content was greater than the reduction
273
in chlorophyll a content (Figure 5B, Supplemental Figure S9), resulting in a
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higher chlorophyll a/b ratio in the hpe1 mutants than in the wild type (Figure 5B,
275
Supplemental Figure S9). Taken together, these results suggest that
276
downregulation of chlorophyll content may be a common theme in response to
277
impaired plastid RNA splicing.
improved
photosynthetic
efficiency
and
biomass
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production.
278
The plastid-encoded gene trnE-UUC encodes tRNAGlu which functions in
279
plastid protein synthesis. Apart from this function, tRNA-Glu is a precursor for
280
the biosynthesis of tetrapyrroles including chlorophyll in plants, archaea and
281
most bacteria (Levican et al., 2005). However, trnE-UUC gene does not
282
contain an intron and thus its expression should not be affected in hpe1
283
mutants. Consistently, quantitative RT-PCR assay indicated that the
284
expression of trnE-UUC was indeed not affected in hpe1 mutants
285
(Supplemental Figure S5). Thus the decreased chlorophyll content in hpe1
286
mutants should not be due to alteration in the expression of trnE-UUC which is
287
required for the biosynthesis of chlorophyll.
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Chlorophyll synthesis is carried out by enzymes encoded by nuclear genes
289
(Czarnecki and Grimm, 2012). Real-time PCR showed that multiple key genes
290
in the chlorophyll synthesis pathway, including 5-aminolevulinic acid (ALA)
291
formation-related genes, protoporphyrin IX (Proto IX) formation-related genes,
292
and chlorophyll formation-related genes, were all down-regulated 3 to 4 folds
293
in the hpe1 mutants compared to the wild type (Figure 5A), suggesting that
294
defects in chlorophyll accumulation in the hpe1 mutants may be due to the
295
down-regulation of chlorophyll synthesis-related genes. Given the role of
296
HPE1 in plastid RNA splicing, we wondered whether mutants of other
297
regulators of plastid RNA splicing also show decreased expression of
298
nuclear-encoded chlorophyll-related genes. Therefore, we analyzed the
299
mutants of CAF1 and OTP51 (Supplemental Figure S7). The results showed
300
that the expression of nuclear-encoded chlorophyll synthesis-related genes
301
mutants were also repressed in caf1 and otp51. Notably, the downregulation of
302
these nuclear-encoded genes was more significant in caf1 and otp51 mutants
303
than in hpe1 mutants, probably due to more severe plastid RNA splicing
304
deficiency in caf1 and otp51 mutants (Supplemental Figure S7). These results
305
indicate that decreased plastid RNA splicing can repress the expression of
306
nuclear-encoded chlorophyll-related genes, but how this regulation is achieved
307
remains unclear.
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One possibility is that a decrease in plastid RNA splicing elicits a retrograde
309
plastid-to-nucleus signaling to repress the expression of nuclear-encoded
310
chlorophyll-related genes. There are multiple distinct putative plastid
311
retrograde signaling pathways based on the sources of the signals, and plastid
312
gene expression is a key signal in plastid to nuclear signaling (Chi et al., 2013),
313
which is mediated by the PPR motif-containing protein GUN1 (Cottage et al.,
314
2007). ABI4, an Apetala 2(AP2)–type transcription factor, is common to all
315
retrograde
316
retrograde-regulated gene through a conserved motif found in close proximity
317
to a light-regulatory element to regulate gene expression such as light
318
harvesting complex subunit b (Lhcb) (Koussevitzky et al., 2007). However,
319
Lhcb4 which is regulated by the GUN1 pathway was unaffected before and
320
after Lincomycin treatment (Supplemental Figure S8), implying that the
321
down-regulation of chlorophyll-related genes in hpe1 mutants is independent
322
of the GUN1-ABI4 pathway. Thus further investigation is warranted to
323
understand how impaired splicing of plastid RNA leads to down-regulation of
324
nuclear-encoded chlorophyll-related genes.
signaling
pathways.
ABI4
binds
the
promoter
of
a
325
326
The Coordination of Light Capture with Conversion Is Improved in hpe1
327
Mutants
328
We further compared the utility and loss of light energy during the light
329
reactions in the hpe1 mutants. Heat, including regulatable non-photochemical
330
quenching yield [Y(NPQ)] (Figure 6A) and non-regulatable non-photochemical
331
quenching yield [Y(NO)]) (Figure 6B), and chlorophyll fluorescence (Figure 6C)
332
were significantly reduced in the hpe1 mutants when compared with the wild
333
type, together with faster photochemical quenching and electron transport rate
334
(Figure 1D). These results suggest that the optimized chlorophyll decreases
335
the loss of light energy in hpe1 mutants. The preponderance of light harvesting
336
pigment optimization should be more significant under excess light
337
(Blankenship and Chen, 2013). We found hpe1 mutants still show higher
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338
photosynthetic efficiency under high light stress (Figure 6D) consistent with the
339
greater accumulation of proline in the hpe1 mutants (Figure 2C), suggesting
340
that loss of light energy and photodamage was reduced in hpe1 mutants under
341
high light.
342
To examine the effect of the optimized light-harvesting system on the
343
reaction center in hpe1 mutants, we determined the levels of photosystem
344
complexes located in the specialized thylakoid membrane through blue native
345
(BN)-PAGE analysis. Interestingly, on an equal chlorophyll basis, the
346
abundance of the photosystem complexes that specifically associate with
347
light-harvesting pigments, namely PSI and PSII, was approximately 44.7% and
348
74.4% greater in the hpe1 mutants than in the wild type plants (Figure 7). In
349
particular, the abundance of the PSII core subunits D1, D2, CP43, and CP47,
350
was approximately 120.9%, 71.1%, 67.2%, and 50.9% higher in the hpe1
351
mutants than in the wild type plants, respectively. PSI subunits PsaA, PsaC,
352
and PsaD were approximately 168.9%, 120.1% and 120.4% higher in hpe1
353
mutants than in the wild type plants, respectively (Figure 8). However, the
354
amounts of cytb6/f and ATPase complex, which are not associated with
355
light-harvesting pigments, showed no obvious difference between the hpe1
356
mutants and wild type plants (Figure 7, Figure 8).
357
In addition, considering the lower chlorophyll content in the hpe1 mutants,
358
we calculated the relative abundance of PSII complexes and proteins on an
359
equal leaf area or fresh weight basis. On an equal fresh weight basis, the
360
abundance of PSII and PSI complexes was still approximately 15.8% and 39.5%
361
greater in hpe1 mutants than in the wild type plants, respectively
362
(Supplemental Figure S10A). The abundance of PSII subunits D1, D2, CP43
363
and CP47 was approximately 100.8%, 59.2%, 56.0% and 42.4% greater in
364
hpe1 mutants than in the wild type plants, respectively. The abundance of PSI
365
subunits PsaA, PsaC, and PsaD was approximately 140.8%, 100.1% and
366
100.3% higher in hpe1 mutants than in the wild type plants, respectively
367
(Supplemental Figure S10B). However, the abundance of Cytb6/f, LHC and
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368
ATPase complexes was approximately 20.9% and 17.6% lower in hpe1
369
mutants than in the wild type plants, respectively (Supplemental Figure S10A).
370
The abundance of Cytf, Lhca1, Lhcb1 and ATPB was approximately 12.8%,
371
24.1%, 20.0% and 19.8% lower in hpe1 mutants than in the wild type plants,
372
respectively
373
chlorophyll-associated photosystem complexes including both PSII and PSI
374
may be due to photodamage mitigation in hpe1, which suggests that the
375
balance between captured light and photochemical reactions may be improved
376
in these mutants.
(Supplemental
Figure
S10B).
Thus,
the
increase
377
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
in
378
DISCUSSION
379
Maximizing light capture by optimizing light harvesting pigments to improve
380
photosynthetic efficiency, although very appealing, is very challenging. While
381
the principle of increasing efficiency by reducing antenna size is simple, this
382
goal has not yet been achieved (Blankenship and Chen, 2013). In this study,
383
we revealed that chloroplast RNA splicing factor HPE1 provides a means to
384
improve photosynthetic efficiency through optimization of light harvesting
385
pigments in higher plants.
386
387
Photosynthetic Efficiency Can be Improved through Optimization of
388
Light Harvesting Pigment in Higher Plants
389
There are several lines of evidence supporting that loss of HPE1 confers
390
improved photosynthetic efficiency. First, two independent mutants of the hpe1
391
gene show greater maximum and actual photochemical efficiency of PSII
392
(Fv/Fm) (Figure 1C), more PSII quantum yield and faster electron transport
393
rates (ETR) (Figure 1D), indicating that the activity of the light reaction was
394
higher in hpe1 mutants than in wild type plants. Second, the significant
395
reduction in chlorophyll fluorescence (Figure 6C) and heat, including
396
regulatable non-photochemical quenching yield [Y(NPQ)] (Figure 6A) and
397
non-regulatable non-photochemical quenching yield [Y(NO)]) (Figure 6B) in
398
the hpe1 mutants, indicates that the energy loss is mitigated in hpe1 mutants.
399
Third, the content of carbohydrates, including glucose and fructose was higher
400
in hpe1 mutants (Figure 2A, 2B, Supplemental Figure S1), implying that
401
improved light reaction performance can promote carbon fixation. Finally,
402
increased fresh weight and dry weight indicated that efficient photosynthesis
403
leads to improved biomass production (Figure 2D-2F).These results suggest
404
that light capture and light energy conversion in light reaction are optimized in
405
hpe1 mutants.
406
Antenna of photosynthetic systems consist of pigments specifically bound to
407
membrane-associated proteins, and are responsible for photon absorption,
16
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
408
while excitation transfer delivers energy to the reaction centers where some of
409
the energy in the photon is captured by electron transfer processes
410
(Blankenship, 2002; Green and Parson, 2003). Maximizing light capture by
411
reducing the antenna size represents another strategy to optimize light capture
412
and light energy conversion (Blankenship and Chen, 2013). In algal,
413
decreasing light harvesting pigments is an optional approach to reduce
414
antenna size (Perrine et al., 2012). We found that levels of both chlorophyll a
415
and chlorophyll b were reduced in hpe1 mutants (Figure 5B). Moreover, the
416
decrease of chlorophyll b content was more significant than that in chlorophyll
417
a content, resulting in a higher chlorophyll a/b ratio in the hpe1 mutants than in
418
the wild type (Figure 5B), which is reversely related to antenna size (Kirst et al.,
419
2012; Perrine et al., 2012; Blankenship and Chen, 2013). In addition, the level
420
of other light harvesting pigment, carotenoids, was also slightly decreased
421
(Supplemental Figure S9), altogether with less LHCII complex as revealed by
422
in BN-gel in hpe1 mutants (Figure 7A), implying that the antenna size is also
423
reduced in hpe1 mutants. Moreover, adjustment of light harvesting pigments
424
optimizes light capture (Figure 1C), decreases energy loss (Figure 6A-6C),
425
ameliorates photodamage of photosystem (Figure 6D, Figure 7 and Figure 8),
426
and improves carbon fixation during photosynthesis. These results suggest
427
that photosynthetic efficiency can be improved through optimization of light
428
harvesting pigments in higher plants.
429
430
The Regulation of Light Harvesting Pigment
431
In plants, light harvesting pigments are regulated both transcriptionally and
432
post-translationally. Most of the genes encoding enzymes involved in
433
chlorophyll biosynthesis have been identified in plants, which catalyze the
434
formation of ALA, Pro IX and mature chlorophyll. The transcriptional regulation
435
of these genes remains unclear although post-translational regulation
436
including enzyme activity control are well understood (Czarnecki and Grimm,
437
2012). To explore the possible cause underlying the optimization of light
17
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
438
harvesting pigment in hpe1 mutants, we first determined that HPE1 is a
439
chloroplast protein (Figure 3B), consistent with the predicted chloroplast
440
targeting signal in this protein (Figure 3A). In addition, HPE1 also contains a
441
RNA recognition motif (RRM) (Figure 3A). The RRM motif is one of the most
442
abundant protein domains in RNA binding proteins and is found in all life
443
kingdoms (Maris et al., 2005), suggesting that HPE1 is involved in the
444
regulation of chloroplast RNA processing. RT-PCR and sequencing analysis
445
showed that the unspliced pre-mRNAs, including trnK, ropC1 and atpF, are all
446
increased in hpe1 mutants (Figure 3C, Supplemental Figure S4), suggesting
447
that HPE1 regulates plastid RNA splicing. RIP analysis indicates that HPE1
448
associates with target RNAs directly (Figure 3D). Altogether these results
449
suggest that HPE1 is a novel chloroplast RNA splicing regulator. In addition,
450
BiFC and CoIP analysis showed that HPE1 interacts with other plastid RNA
451
splicing factors, including CAF1 and OTP51 (Figure 4A, 4B), which share
452
common target RNAs with HPE1 (Asakura and Barkan, 2006; de Longevialle
453
et al., 2008), suggesting HPE1 may form a complex specifically with CAF1 and
454
OTP51 to co-regulate chloroplast group II intron splicing.
455
Interestingly, the chlorophyll contents in mutants of chloroplast RNA splicing
456
regulators, including CAF1 and OTP51 (Asakura and Barkan, 2006; de
457
Longevialle et al., 2008), are decreased. The contents of chlorophyll are also
458
decreased in hpe1 mutants, very similar to CAF1 and OTP51 mutants.
459
However, the degree of chlorophyll decrease is different in mutants of different
460
chloroplast RNA splicing regulators. Deficiency in some chloroplast RNA
461
splicing events results in albinism, but some also result in yellow or light-green
462
leaves (Kroeger et al., 2009).The leaves of hpe1 mutants are light-green, and
463
show less of a decrease in chlorophyll compared to mutants of other
464
chloroplast RNA splicing factors (Figure 2D, Figure 5B, Supplemental Figure
465
S9). The slight chlorophyll decrease in hpe1 mutants may optimize the light
466
harvesting antenna. The plastid-encoded gene trnE-UUC encodes tRNAGlu
467
whose amino acyl form tRNA-Glu is a precursor for the biosynthesis of
18
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
468
tetrapyrroles (e.g., heme and chlorophyll) in plants, archaea and most bacteria
469
(Levican et al., 2005). Quantitative RT-PCR assay indicates that the transcript
470
of trnE-UUC is not affected in hpe1 mutants (Supplemental Figure S5),
471
suggesting the down-regulation of chlorophyll content in hpe1 mutants may not
472
be due to alteration of trnE-UUC expression.
473
Interestingly, we found that multiple nuclear-encoded key genes of the
474
chlorophyll synthesis pathway were markedly down-regulated in hpe1 mutants
475
(Figure 5A), suggesting that defects in chlorophyll accumulation may be
476
caused by the down-regulation of chlorophyll synthesis-related genes.
477
Downregulation of chlorophyll-related genes reduces the size of the
478
light-harvesting complex, ensuring optimized light capture and energy
479
conversion during light reaction. Finally, optimization of light reactions
480
improves the efficiency of carbon fixation, which leads to greater
481
photosynthetic efficiency and biomass production (Supplemental Figure S11).
482
It is seemingly perplexing that loss of a plastid RNA splicing regulator affects
483
the expression of nuclear genes. One probability is that loss of hpe1 elicits
484
plastid-to-nucleus signaling leading to down-regulation of these nuclear genes.
485
A significant consequence of hpe1 mutation is the accumulation of unspliced
486
RNAs, however the spliced RNAs and the encoded proteins are only slightly
487
reduced (Supplemental Figure S4B and S10). Thus we speculate that such
488
plastid-to-nucleus signaling is more likely triggered by an accumulation of
489
unspliced mRNAs (Figure 5A, Supplemental Figure S7), which is reminiscent
490
of the well-known Unfolded Protein Response (UPR) which is triggered by an
491
accumulation of unfolded proteins in endoplasmic reticulum (Bernales et al.,
492
2006; Howell, 2013; Popp and Maquat, 2013). However, we cannot introduce
493
exogenous unspliced pre-mRNAs to plastids to directly test this hypothesis
494
due to technological limits, and we are not able to exclude the possibility that
495
such plastid-to-nucleus signaling is triggered by the down-regulation of plastid
496
proteins.
497
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
498
METHODS
499
Plant Materials and Growth Conditions
500
T-DNA Arabidopsis thaliana mutants used in this study were in the Col-0
501
background. The hpe1-1 and hpe1-2 mutants were obtained from the
502
Arabidopsis Biological Resource Center (stock nos. SALK_012657C and
503
SALK_092951C). Arabidopsis thaliana was grown in the soil in a growth
504
chamber (100 μmol photons m-2 s-1, 12-h/12-h photoperiod, 21°C, and 60%
505
relative humidity). Plants for the chlorophyll fluorescence assays and protein
506
analysis were three and five weeks of age, respectively. To study the effects
507
of high light, plants were placed in a high light growth chamber (1200–1500
508
μmol photons m-2 s-1).
509
510
Determination of Carbohydrate and Amino Acids Levels
511
Plant leaf tissues of the same area were placed into liquid N2 immediately and
512
then ground using a pestle. Quantification of monosaccharides (glucose and
513
fructose) and amino acids were performed using GC-MS-based methods as
514
described (Lisec et al., 2006). The degradation of carbohydrate was analyzed
515
after 5-h DCMU treatment according the methods described (Kowallik and
516
Schatzle, 1980).
517
518
Pigment Analysis
519
Chlorophyll from three-week-old plants was extracted with 80% acetone in
520
2.5 mM HEPES-KOH (pH 7.5), and the amount of chlorophyll was
521
determined as previously described (Wellburn, 1994). Carotenoids were
522
extracted and analyzed as previously described by using spectrofluorometry
523
(Yang et al., 2012) and HPLC (Pogson et al., 1996; Li et al., 2009). Pigments
524
were identified by comparing retention times to reference standards.
525
526
Chlorophyll Fluorescence
527
Chlorophyll fluorescence parameters were measured with the MAXI version
20
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
528
of
the
IMAGING-PAM
M-Series
chlorophyll
fluorescence
system
529
(Heinz-Walz Instruments). Plants were dark-adapted for 30 min before
530
measurements were made, and light-response curves were determined as
531
described (Lu et al., 2011).
532
533
Isolation of Thylakoid Membranes
534
Thylakoid membranes were prepared as previously described (Howard H.
535
Robinson, 1980). Isolated thylakoid membranes were quantified on the basis
536
of total chlorophyll as described (Porra et al., 1989).
537
538
Production of Anti-HPE1 Polyclonal Antibodies
539
Affinity-purified anti-HPE1 polyclonal antibodies were prepared by GenScript.
540
A 15-amino-acid peptide (corresponding to amino acids 467–481 of HPE1)
541
with an additional N-terminal Cys residue, CFDKPEAKPARVEGK, was
542
synthesized, conjugated with keyhole limpet hemocyanin, and used to
543
induce antibodies against HPE1.
544
545
RT-PCR and Quantitative Real-time RT-PCR
546
Total RNA was extracted from Arabidopsis rosette leaves by using the
547
RNeasy Plant Mini Kit (Qiagen). The RNA samples were reverse-transcribed
548
into first-strand cDNA by using the PrimeScript RT Reagent Kit (TaKaRa).
549
Both
550
reverse-transcribe the first-strand cDNA. For RT-PCR, UBQ10 was used as
551
the control gene. Quantitative real-time RT-PCR was performed using
552
gene-specific primers and SYBR Premix ExTaq reagent (TaKaRa) on a
553
real-time RT-PCR System (RoChe-LC480), according to the manufacturer’s
554
instructions. Reactions were performed in triplicate for each sample, and
555
expression levels were normalized against ACTIN and UBQ4.
random
and
oligo
dT-contained
mix primers
were
556
557
BN-PAGE and Immunoblot Analyzes
21
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
used
to
558
BN-PAGE was performed as described (Schagger et al., 1994) with
559
modifications (Peng et al., 2006). For quantification of thylakoid proteins,
560
gels were loaded on an equivalent chlorophyll basis in amounts ensuring
561
that immunodetection was in the linear range. Primary antibodies and
562
antisera were induced in rabbits. Antisera against photosynthetic proteins
563
were purchased from Agrisera.
564
565
Subcellular Localization of GFP Fusions and BiFC
566
Subcellular localization of GFP fusion proteins and BiFC was performed as
567
previously described (Zhang et al., 2011).
568
569
Immunoprecipitation
570
Immunoprecipitation was performed as described (Zhang et al., 2015) with
571
minor modifications. 4 mL Arabidopsis protoplasts was transfected with 400
572
μg plasmid of 35S:CAF1-HA or 35S:OTP51-HA. Total proteins were extracted
573
with 500 μL protein extraction buffer and 20 μL protein extract was used as
574
input. The total cell extracts were further incubated with 30 μL anti-HA affinity
575
gel (Roche) for 6 h at 4°C with rotation. After washing five times with ice cold
576
PBS buffer (pH 7.4), the bound proteins were eluted by boiling the gel using
577
30 μL SDS-PAGE sample buffer without β-mercaptoethanol and loaded onto
578
SDS-PAGE for immunoblotting.
579
580
Analysis of RNA Splicing
581
Analysis of RNA splicing by RT-PCR was performed as described (Valkov et
582
al., 2009). The RT-PCR products of unspliced and spliced RNA were
583
identified by sequencing. Quantitative RT-PCR analysis of RNA splicing was
584
performed as described (de Longevialle et al., 2008). The primers used are
585
listed in Supplemental Table S1.
586
587
RNA Immunoprecipitation
22
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
588
RNA immunoprecipitation assays were performed (Garcia-Andrade et al.,
589
2013) as described with minor revisions. Two grams of leaf tissue from
590
Arabidopsis plants (4 weeks of age) was ground to a fine powder with a mortar
591
and pestle in liquid N2 and homogenized in 12.5 mL/g lysis buffer (50 mM
592
Tris-HCl, pH 7.4, 2.5 mM MgCl2, 100 mM KCl, 0.1% Nonidet P-40, 1 μg/mL
593
leupeptin, 1 μg/mL aprotonin, 0.5 mM phenylmethylsulfonyl fluoride, one tablet
594
of Complete proteinase inhibitor tablet (Roche), and 50 units/mL RNase OUT;
595
Invitrogen). Cell debris were pelleted by centrifugation for 5 min at 12,000 rcf at
596
4°C. Clarified lysates were incubated with 4 μg/mL of the anti-HPE1 antibody
597
for 15 min at 4°C and then with 100 μL of Protein A agarose (Roche) per
598
milliliter for 30 min at 4°C. Beads were washed six times for 10 min with lysis
599
buffer at 4°C and then divided for protein and RNA analyses. RNAs were
600
recovered by incubating the beads in 0.5 volumes of proteinase K buffer (0.1 M
601
Tris-HCl, pH 7.4, 10 mM EDTA, 300 mM NaCl, 2% SDS, and 1 μg/μL
602
proteinase K; Roche) for 15 min at 65°C; extracted with saturated phenol,
603
phenol:chloroform:isoamyl alcohol, and chloroform; and precipitated with
604
ethanol. For RT-PCR assays, 1 μg of total RNA was used for the input fraction,
605
and 20% of the RNA immunoprecipitate was used for immunoprecipitation.
606
607
608
Supplemental Data
609
Supplemental FigureS1 Degradation of metabolite production in hpe1 mutant
610
and wild type plants.
611
Supplemental FigureS2 Growth parameters of the wild type and hpe1 mutant
612
plants.
613
Supplemental FigureS3 Transcript levels of representative genes that
614
encode chloroplast proteins were analyzed using quantitative real-time
615
RT-PCR.
616
Supplemental FigureS4 RNA splicing analysis in hpe1 mutants and wild type
617
plants.
23
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
618
Supplemental FigureS5 The level of TrnE transcript was analyzed using
619
quantitative real-time RT-PCR.
620
Supplemental FigureS6 Interaction analysis of HPE1 with other splicing
621
factors involved in the splicing of trnK, rpoC1, and atpF introns by using
622
bimolecular fluorescence complementation (BiFC).
623
Supplemental FigureS7 Analysis of the expression of chlorophyll-related
624
genes in other plastid splicing mutants.
625
Supplemental FigureS8 Analysis of the expression of plastid to nucleus
626
signaling-related genes.
627
Supplemental Figure S9 HPLC analysis of pigments in the wild type (Col-0),
628
hpe1-1, and hpe1-2 plants.
629
Supplemental Figure S10 Proteins amount of photosystem were calculated
630
based on fresh weight.
631
Supplemental Figure S11 Presumed working model of HPE1 in the regulation
632
of photosynthesis.
633
Supplemental Table S1. A list of primers used in this study.
634
635
Acknowledgments
636
We are grateful to Yonggang Zheng (The Johns Hopkins University) for
637
critical reading of the manuscript. We thank ABRC for providing plant
638
materials. This research was supported by the grants from the National
639
Natural Science Foundation of China (No. 31425003 and No. 31500195),
640
the Natural Science Foundation of Guangdong Province, PR China (No.
641
2014A030310491), the China postdoctoral Science Foundation (No.
642
2015M572399 and No. 2016T90808), the National Science and Technology
643
Major Project Foundation of China (No. 2016ZX08009003-005-005), and the
644
Fundamental Research Funds for the Central Universities.
24
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
645
Figure Legends
646
Figure1 Isolation of hpe1 Arabidopsis mutant plants.
647
(A) Schematic diagram of the HPE1 gene inferred using DNA sequence
648
analysis. Exons (black boxes) and introns (lines) are indicated. Positions of the
649
T-DNA insertions corresponding to hpe1-1 and hpe1-2 are shown. ATG start
650
codon and TGA stop codon are shown. (B) RT-PCR analysis of HPE1
651
transcription in the wild type (Col-0) and hpe1 mutants. (C) False-color images
652
representing Fv/Fm under growth light conditions in 3-week-old wild type and
653
hpe1 mutant plants. Red pixels indicate that Fv/Fm is above the cutoff value
654
(0.800). (D) Light-response curves of PSII quantum yield (ΦPSII),
655
photochemical quenching (qP), and electron transport rate (ETR) in the wild
656
type and hpe1 mutants. Measurements were performed at the following light
657
intensities: 0, 81, 145, 186, 281, 335, 461, 701, and 926 μmol photons m-2 s-1.
658
Each data point represents at least twenty independent plants.
659
660
Figure 2 Metabolite and biomass production of hpe1 Arabidopsis mutant
661
plants.
662
(A)-(C) Contents analysis of glucose (A), fructose (B), and proline (C) in the
663
wild type and hpe1 mutant plants by GC-MS. (D) Representative photographs
664
of selected 5-week-old wild type and hpe1 mutant plants. (E) Fresh weight of
665
5-week-old wild type and hpe1 mutant plants. (F) Dry weight of 5-week-old wild
666
type and hpe1 mutant plants. FW, fresh weight; DW, dry weight. Each data
667
point represents at least twenty independent plants. Significant differences
668
were identified at 5% (*) and 1% (**) probability levels by using Student’s t-test.
669
670
Figure 3 Confirmation of subcellular location and molecular function of
671
HPE1.
672
(A) Schematic diagram of the HPE1 protein including chloroplast transit
673
peptide (CTP) and RNA recognition motif (RRM). (B) Subcellular localization
674
of HPE1 within the chloroplast by using the GFP assay. GFP, control with
25
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
675
empty vector; Nuc-GFP, nuclear control; Chl-GFP, chloroplast control;
676
HPE1-GFP, HPE1-GFP fusion. Bars = 10 μm. (C) Analysis of splicing defects
677
in the hpe1 mutants by using RT-PCR. Products of RT-PCR were isolated and
678
sequenced. “＊,” unspliced mRNA precursors; “★,” spliced mature mRNA. (D)
679
Confirmation of association of HPE1 with target RNA by using RNA
680
immunoprecipitation (RIP). The upper panel shows western blot of proteins
681
present in crude leaf extracts derived from Col-0 and hpe1 mutant plants and
682
proteins immunoprecipitated (IP) with the anti-HPE1 antibody. In the lower
683
panel, RT-PCR was used to detect the association of trnK, rpoC1, and atpF
684
introns with HPE1. Five additional independent biological replicates were
685
performed, and similar results were obtained.
686
687
Figure 4 Interaction analysis of HPE1 with other RNA splicing factors of
688
plastid genes.
689
(A) Interaction of HPE1 with other splicing factors involved in the splicing of
690
trnK,
691
complementation (BiFC) analysis. HPE1 fused with the N terminus of YFP (YN)
692
and CAF1 and OTP51 fused with the C terminus of YFP (YC) were
693
co-transfected into protoplasts and visualized using confocal microscopy. As a
694
positive control, both HHL1 fused with YN and LQY1 fused with YC were
695
co-transfected into protoplasts. As a negative control, HPE1 was fused with
696
YN and empty vector YC. Bars = 10 μm. (B) Confirmation of the interaction
697
between HPE1 and chloroplast splicing factors CAF1 and OTP51 by using the
698
Co-IP assay. Fusion proteins of CAF1-HA and OTP51-HA were expressed in
699
Arabidopsis protoplasts and precipitated with anti-HA–coupled agarose. The
700
immunoprecipitates were probed with anti-HPE1 antibodies, and protoplasts
701
expressing the empty HA vector were used as controls. All experiments were
702
repeated three times with similar results.
rpoC1,
and
atpF
introns
by
using
bimolecular
fluorescence
703
704
26
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
705
Figure 5 The analysis of chlorophyll-related genes expression and
706
chlorophyll contents.
707
(A) Effect of HPE1 deficiency on the expression of chlorophyll-related genes in
708
three different steps. Left panel indicates 5-aminolevulinic acid (ALA)
709
formation-related genes; middle panel indicates protoporphyrin IX (Proto IX)
710
formation-related
711
formation-related genes. (B) Analysis of chlorophyll a (Chl a) content,
712
chlorophyll b (Chl b) content, total chlorophyll a and chlorophyll b (Chl a+b)
713
content, and the ratio of chlorophyll a to chlorophyll b (Chl a/b). FW, fresh
714
weight. All experiments were repeated three times with similar results.
715
Significant differences were identified at 5% (*) and 1% (**) probability levels
716
by using Student’s t-test.
genes;
and
right
panel
indicates
chlorophyll
(Chl)
717
718
Figure 6 Loss of light energy of photosynthesis in the hpe1 mutants.
719
(A)-(C) Loss of light energy in the hpe1 mutants. Light-response curves of
720
regulatable non-photochemical quenching yield [Y(NPQ)] (A), non-regulatable
721
non-photochemical quenching yield [Y(NO)] (B), and chlorophyll fluorescence
722
yield [Y(Chl fluor)] (C). Each data point represents at least twenty independent
723
plants. (D) Analysis of tolerance to high light stress of the hpe1 mutants. Plants
724
were exposed to high light at 1200 μmol photons m–2 s–1 at 0 h, 3 h, and 6 h.
725
The fraction of active PSII (Fv/Fm) was measured after dark incubation for 30
726
min. Data represent means ± SE values (n = 20). Significant differences were
727
identified at 5% (*) and 1% (**) probability levels by using Student’s t-test.
728
Differences between the hpe1 mutants and wild type plants were more
729
significant after high light treatment.
730
731
Figure 7 Analysis of photosystem complexes from the wild type and the
732
hpe1 mutant plants.
733
(A) BN-PAGE analysis of chlorophyll-protein complexes. Equal thylakoid
734
membrane (10 μg chlorophyll) from the leaves of the wild type and hpe1-1
27
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
735
mutants were solubilized by treatment with 1% (w/v) DM and separated by
736
BN-PAGE. The assignments of macromolecular protein complexes of thylakoid
737
membranes indicated at left were identified according to Jin et al. (2014). (B)
738
2D BN/SDS-PAGE fractionation of thylakoid membrane protein complexes.
739
After separation in the first dimension in a nondenaturing gel, the protein lanes
740
were subjected to a denaturing 2D gel (2D BN/SDS-PAGE) followed by
741
Coomassie blue staining. The identity of relevant proteins is indicated by
742
arrows. The PSII and PSI protein, which appeared more in hpe1 mutants, are
743
circled in red. (C) BN-PAGE for immunoblot analysis (3 μg chlorophyll). (D) A
744
representative immunoblot with anti-CP43 antiserum used to probe the
745
photosystem II (PSII) complex. (E) A representative immunoblot with anti-PsaA
746
antiserum used to probe the photosystem I (PSI) complex. (F) A representative
747
immunoblot with anti-Cytf antiserum used to probe the Cytb6/f complex. (G) A
748
representative immunoblot with anti-ATPB antiserum used to probe the ATP
749
synthase (ATPase) complex. Three independent biological replicates for all
750
experiments were performed and a representative one is shown. (H) Proteins
751
immunodetected from (D)-(G) were analyzed with Phoretix 1D Software
752
(Phoretix International, UK). The values (mean ± SE, n = 3 independent
753
biological replicates) are given as the ratio to protein amount of the wild type
754
(Col-0) and hpe1-1 mutants. (Student’s t-test; *, p < 0.05; **, p < 0.01).
755
756
Figure 8 Analysis of thylakoid membrane protein accumulation in the
757
wild-type and hpe1 mutants.
758
(A) Thylakoid membrane proteins from wild type (Col-0) and hpe1 mutants
759
were separated by 15% SDS-urea-PAGE, transferred onto PVDF membranes,
760
and probed with antibody against known thylakoid membrane proteins
761
obtained from Agrisera. Samples were loaded on an equal chlorophyll basis.
762
PSII, photosystem II complex; PSI, photosystem I complex; Cytb6/f,
763
cytochrome b6/f complex; LHC, light harvesting complex; ATPase, ATP
764
synthase complex. Three independent biological replicates for all experiments
28
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
765
were performed and a representative one is shown. (B) Proteins
766
immunodetected from (A) were analyzed with Phoretix 1D Software (Phoretix
767
International, UK). The values (mean ± SE, n = 3 independent biological
768
replicates) are given as the ratio to protein amount of the wild type (Col-0) and
769
hpe1 mutants. (Student’s t-test; *, p < 0.05; **, p < 0.01).
770
771
29
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
A
D
1
hpe1-1 (SALK_012657)
ATG
ФPSII
TGA
5’UTR
hpe1-2 (SALK_092951)
B
hpe1-1
hpe1-2
0.4
0.2
0
1.2
Col-0 hpe1-1 hpe1-2
HPE1
0.9
qP
UBQ10
C
Col-0
0.8
0.6
0.6
0.3
Col-0
hpe1-1
hpe1-2
0
125
ETR
Image
100
75
50
25
Fv/Fm
0
0
(Cutoff=0.80)
200
400
600
800
1000
PPDF(umol photons m-2s-1)
Figure1 Isolation of hpe1 Arabidopsis mutant plants.
(A) Schematic diagram of the HPE1 gene inferred using DNA sequence analysis. Exons (black
boxes) and introns (lines) are indicated. Positions of the T-DNA insertions corresponding to hpe1-1
and hpe1-2 are shown. ATG start codon and TGA stop codon are shown. (B) RT-PCR analysis of
HPE1 transcription in the wild type (Col-0) and hpe1 mutants. (C) False-color images representing
Fv/Fm under growth light conditions in 3-week-old wild type and hpe1 mutant plants. Red pixels
indicate that Fv/Fm is above the cutoff value (0.800). (D) Light-response curves of PSII quantum
yield (ΦPSII), photochemical quenching (qP), and electron transport rate (ETR) in the wild type
and hpe1 mutants. Measurements were performed at the following light intensities: 0, 81, 145,
186, 281, 335, 461, 701, and 926 μmol photons m-2 s-1. Each data point represents at least twenty
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
independent plants.
2.0
**
1.5
1
0.5
**
**
1.5
1
0.5
0
0
E
hpe1-1
hpe1-2
*
0.8
FW/Line (g line-1)
D
*
0.6
0.4
0.2
0
Col-0 hpe1-1 hpe1-2
**
**
3
2
1
0
Col-0 hpe1-1 hpe1-2
Col-0 hpe1-1 hpe1-2
Col-0
4
F
Col-0 hpe1-1 hpe1-2
**
0.2
DW/Line (g line-1)
**
C
Relative content
of fructose
Relative content
of glucose
2.0
B
Relative content
of proline
A
0.15
**
0.1
0.05
0
Col-0 hpe1-1 hpe1-2
Figure2 Metabolite and biomass production of hpe1 Arabidopsis mutant plants.
(A)-(C) Contents analysis of glucose (A), fructose (B), and proline (C) in the wild type and hpe1
mutant plants by GC-MS. (D) Representative photographs of selected 5-week-old wild type and
hpe1 mutant plants. (E) Fresh weight of 5-week-old wild type and hpe1 mutant plants. (F) Dry
weight of 5-week-old wild type and hpe1 mutant plants. FW, fresh weight; DW, dry weight. Each
data point represents at least twenty independent plants. Significant differences were identified at
5% (*) and 1% (**) probability levels by using Student’s t-test.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
A
C
1
49
188
CTP
267
trnK
538 (AA)
Col-0 hpe1-1
atpF
rpoC1
Col-0 hpe1-1
Col-0 hpe1-1
RRM
*
GFP
B
Chlorophyll
Merged
★
*
Bright
*
★
★
GFP
D
Input
Col-0
Nuc-GFP
IP
hpe1-1
Col-0
hpe1-1
HPE1
Input
Col-0
Chl-GFP
RIP
hpe1-1
Col-0
hpe1-1
trnK
atpF
HPE1-GFP
rpoC1
Figure 3 Confirmation of subcellular location and molecular function of HPE1.
(A) Schematic diagram of the HPE1 protein including chloroplast transit peptide (CTP) and RNA recognition motif (RRM). (B) Subcellular
localization of HPE1 within the chloroplast by using the GFP assay. GFP, control with empty vector; Nuc-GFP, nuclear control; Chl-GFP,
chloroplast control; HPE1-GFP, HPE1-GFP fusion. Bars = 10 μm. (C) Analysis of splicing defects in the hpe1 mutants by using RT-PCR.
Products of RT-PCR were isolated and sequenced. “＊,” unspliced mRNA precursors; “★,” spliced mature mRNA. (D) Confirmation of
association of HPE1 with target RNA by using RNA immunoprecipitation (RIP). The upper panel shows western blot of proteins present in crude
leaf extracts derived from Col-0 and hpe1 mutant plants and proteins immunoprecipitated (IP) with the anti-HPE1 antibody. In the lower panel,
RT-PCR was used to detect the association of trnK, rpoC1, and atpF introns with HPE1. Five additional independent biological replicates were
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
2016 American Society of Plant Biologists. All rights reserved.
performed,Copyright
and ©similar
results were obtained.
A
B
YFP
Chlorophyll
Merged
Bright
HHL1-YN
+
LQY1-YC
HPE1
CAF1-HA
+
+
+
IP
Anti-HPE1
Input
HPE1-YN
+
CAF1-YC
Anti-HA
HPE1
OTP51-HA
HPE1-YN
+
OTP51-YC
Input
+
+
+
IP
Anti-HPE1
Input
HPE1-YN
+
YC
Anti-HA
Input
Figure 4 Interaction analysis of HPE1 with other RNA splicing factors of plastid
genes.
(A) Interaction of HPE1 with other splicing factors involved in the splicing of trnK, rpoC1,
and atpF introns by using bimolecular fluorescence complementation (BiFC) analysis.
HPE1 fused with the N terminus of YFP (YN) and CAF1 and OTP51 fused with the C
terminus of YFP (YC) were co-transfected into protoplasts and visualized using confocal
microscopy. As a positive control, both HHL1 fused with YN and LQY1 fused with YC
were co-transfected into protoplasts. As a negative control, HPE1 was fused with YN and
empty vector YC. Bars = 10 μm. (B) Confirmation of the interaction between HPE1 and
chloroplast splicing factors CAF1 and OTP51 by using the Co-IP assay. Fusion proteins of
CAF1-HA and OTP51-HA were expressed in Arabidopsis protoplasts and precipitated with
anti-HA–coupled agarose. The immunoprecipitates were probed with anti-HPE1
antibodies, and protoplasts expressing the empty HA vector were used as controls. All
experimentsDownloaded
werefromrepeated
three times with similar results.
on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
A
ALA formation
**
**
**
**
Col-0
hpe1-1
hpe1-2
1.0
1.2
0.8
0.6
0.4
0.2
1.0
0.8
0.6
0.4
GSA2
HEME2
CHLI2
0.6
0.4
0.2
*
**
*
*
*
2.5
Chl a+b (mg/g FW)
Chl b (mg/g FW)
CHLI1
0.8
HEMF1
**
0.6
0.5
0.4
0.3
0.2
0.1
0
Col-0 hpe1-1 hpe1-2
**
**
0
HEMC
*
**
**
1.0
Col-0 hpe1-1 hpe1-2
2.0
1.5
Chl a/b
GSA1
*
Chl a (mg/g FW)
1.2
0
GATB
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
**
**
0.2
0
B
**
**
**
**
Chl formation
Relative expression
**
**
Relative expression
Relative expression
1.2
Proto IX formation
1.0
0.5
0
Col-0 hpe1-1 hpe1-2
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Col-0 hpe1-1 hpe1-2
Figure 5 The analysis of chlorophyll-related genes expression and chlorophyll contents.
(A) Effect of HPE1 deficiency on the expression of chlorophyll-related genes in three different steps. Left panel indicates 5-aminolevulinic acid
(ALA) formation-related genes; middle panel indicates protoporphyrin IX (Proto IX) formation-related genes; and right panel indicates
chlorophyll (Chl) formation-related genes. (B) Analysis of chlorophyll a (Chl a) content, chlorophyll b (Chl b) content, total chlorophyll a and
chlorophyll b (Chl a+b) content, and the ratio of chlorophyll a to chlorophyll b (Chl a/b). FW, fresh weight. All experiments were repeated three
times with similar results. Significant differences were identified at 5% (*) and 1% (**) probability levels by using Student’s t-test.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
C
A
0.6
Col-0
hpe1-1
hpe1-2
0.4
0.3
Y (Chl fluor)
Y(NPQ)
0.5
0.4
0.3
0.2
0.2
0.1
0.1
0
0
0
200
400
600
800
-2
-1
PPDF(μmol photons m s )
0
1000
B
200
400
600
800
-2
-1
PPDF(μmol photons m s )
D
*
1.0
*
0.5
Fv/Fm
0.3
**
**
0.8
0.4
Y(NO)
1000
**
**
0.6
Col-0
hpe1-1
hpe1-2
0.4
0.2
0.1
0.2
0
0
200
400
600
800
-2
-1
PPDF(μmol photons m s )
1000
0
0h
3h
6h
Time after high light stress
Figure 6 Loss of light energy of photosynthesis in the hpe1 mutants.
(A)-(C) Loss of light energy in the hpe1 mutants. Light-response curves of regulatable non-photochemical quenching yield [Y(NPQ)]
(A), non-regulatable non-photochemical quenching yield [Y(NO)] (B), and chlorophyll fluorescence yield [Y(Chl fluor)] (C). Each
data point represents at least twenty independent plants. (D) Analysis of tolerance to high light stress of the hpe1 mutants. Plants
were exposed to high light at 1200 μmol photons m–2 s–1 at 0 h, 3 h, and 6 h. The fraction of active PSII (Fv/Fm) was measured
after dark incubation for 30 min. Data represent means ± SE values (n = 20). Significant differences were identified at 5% (*) and
1% (**) probability levels by using Student’s t-test. Differences between the hpe1 mutants and wild type plants were more
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
significant after highCopyright
light© 2016
treatment.
American Society of Plant Biologists. All rights reserved.
A
Col-0 hpe1-1
B
BN-PAGE
PSII-LHCII (Ⅰ)
ⅠⅡⅢⅣⅤ Ⅵ
Ⅶ
ⅠⅡⅢⅣⅤ Ⅵ
Ⅶ
PSII dimer/PSI monomer (Ⅱ)
SDS-PAGE
PSI monomer/CF1 (Ⅲ)
PSII monomer (Ⅳ)
PSII-CP43 monomer (Ⅴ)
LHCII trimer (Ⅵ)
hpe1-1
Col-0
Unassembled protein (Ⅶ)
E
D
C
F
G
Col-0 hpe1-1
Col-0 hpe1-1
Col-0 hpe1-1
Col-0hpe1-1
Col-0 hpe1-1
BN-PAGE
Anti-PSII
Anti-PSI
Anti-Cytb6/f
Anti-ATPase
PSII-LHCII (Ⅰ)
PSII dimer/PSI monomer (Ⅱ)
PSI monomer/CF1 (Ⅲ)
PSII monomer (Ⅳ)
PSII-CP43 monomer (Ⅴ)
LHCII trimer (Ⅵ)
Unassembled protein (Ⅶ)
H
**
Relative amount of
photosystem complex
2
**
Col-0
hpe1-1
1.5
1
0.5
0
PSII
PSI
Cytb6/f
ATPase
Figure 7 Analysis of photosystem complexes from the wild type and the hpe1 mutant plants.
(A) BN-PAGE analysis of chlorophyll-protein complexes. Equal thylakoid membrane (10 μg chlorophyll) from the
leaves of the wild type and hpe1-1 mutants were solubilized by treatment with 1% (w/v) DM and separated by BNPAGE. The assignments of macromolecular protein complexes of thylakoid membranes indicated at left were
identified according to Jin et al. (2014). (B) 2D BN/SDS-PAGE fractionation of thylakoid membrane protein
complexes. After separation in the first dimension in a nondenaturing gel, the protein lanes were subjected to a
denaturing 2D gel (2D BN/SDS-PAGE) followed by Coomassie blue staining. The identity of relevant proteins is
indicated by arrows. The PSII and PSI protein, which appeared more in hpe1 mutants, are circled in red. (C) BNPAGE for immunoblot analysis (3 μg chlorophyll). (D) A representative immunoblot with anti-CP43 antiserum used
to probe the photosystem II (PSII) complex. (E) A representative immunoblot with anti-PsaA antiserum used to
probe the photosystem I (PSI) complex. (F) A representative immunoblot with anti-Cytf antiserum used to probe
the Cytb6/f complex. (G) A representative immunoblot with anti-ATPB antiserum used to probe the ATP synthase
(ATPase) complex. Three independent biological replicates for all experiments were performed and a
representative one is shown. (H) Proteins immunodetected from (D)-(G) were analyzed with Phoretix 1D Software
(Phoretix International, UK). The values (mean ± SE, n = 3 independent biological replicates) are given as the
ratio to protein amount of the wild type (Col-0) and hpe1-1 mutants. (Student’s t-test; *, p < 0.05; **, p < 0.01).
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
A
Col-0 hpe1-1 hpe1-2
Col-0 hpe1-1 hpe1-2
Col-0 hpe1-1 hpe1-2
D1
PsaA
D2
PSI PsaC
Cytf
Cytf
Lhca1
LHC
PSII
PsaD
CP43
Lhcb1
CP47
B
3.5
ATPase
**
**
**
**
Relative amount of
photosystem subunits
3
**
**
2.5
**
**
**
**
ATPB
**
**
Col-0
hpe1-1
**
**
hpe1-2
2
1.5
1
0.5
0
D1
D2
CP43
CP47
PsaA
PsaC
PsaD
Cytf
Lhca1
Lhcb1
ATPB
Figure 8 Analysis of thylakoid membrane protein accumulation in the wild-type and hpe1 mutants.
(A) Thylakoid membrane proteins from wild type (Col-0) and hpe1 mutants were separated by 15% SDS-ureaPAGE, transferred onto PVDF membranes, and probed with antibody against known thylakoid membrane proteins
obtained from Agrisera. Samples were loaded on an equal chlorophyll basis. PSII, photosystem II complex; PSI,
photosystem I complex; Cytb6/f, cytochrome b6/f complex; LHC, light harvesting complex; ATPase, ATP synthase
complex. Three independent biological replicates for all experiments were performed and a representative one is
shown. (B) Proteins immunodetected from (A) were analyzed with Phoretix 1D Software (Phoretix International,
UK). The values (mean ± SE, n = 3 independent biological replicates) are given as the ratio to protein amount of
the wild type (Col-0) and hpe1 mutants. (Student’s t-test; *, p < 0.05; **, p < 0.01).
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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32
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905
906
907
908
909
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911
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913
914
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916
917
918
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Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D,
Wang J, Wang H (2011) A highly efficient rice green tissue protoplast
system
for
transient
gene
expression
and
studying
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Zhu XG, Long SP, Ort DR (2010) Improving Photosynthetic Efficiency for
Greater Yield. Annual Review of Plant Biology, Vol 61 61: 235-261
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