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The role of photorespiration during astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae)
Accepted Manuscript The role of photorespiration during astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae) Chunhui Zhang, Litao Zhang, Jianguo Liu PII:
S0981-9428(16)30199-1
DOI:
10.1016/j.plaphy.2016.05.029
Reference:
PLAPHY 4560
To appear in:
Plant Physiology and Biochemistry
Received Date: 25 March 2016 Revised Date:
18 May 2016
Accepted Date: 18 May 2016
Please cite this article as: C. Zhang, L. Zhang, J. Liu, The role of photorespiration during astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae), Plant Physiology et Biochemistry (2016), doi: 10.1016/j.plaphy.2016.05.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The role of photorespiration during astaxanthin accumulation in Haematococcus
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pluvialis (Chlorophyceae)
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Chunhui Zhang1, 3, Litao Zhang1, 2, Jianguo Liu1, 2, *
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Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao
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266071, P.R. China
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National & Local Joint Engineering Laboratory of Ecological Mariculture, Key Laboratory of
National-Local Joint Engineering Research Center for Haematococcus pluvialis and
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Astaxanthin Products, Yunnan Alphy Biotech Co., Ltd., Chuxiong 675012, China
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University of Chinese Academy of Sciences, Beijing 100049, P.R. China
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7 Nanhai Road, Qingdao, Shandong, 266071, P.R. China
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Tel: +86-532-82898709, Fax: +86-532-82898612, E-mail: [email protected]
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Abstract Most previous studies on Haematococcus pluvialis have been focused on growth and
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astaxanthin accumulation. However, the relationships between photorespiration and astaxanthin
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accumulation have not been clarified. The purpose of this study was to examine the role of
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photorespiration during the process of astaxanthin accumulation in H. pluvialis. During
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astaxanthin
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photorespiration was inhibited by its specific inhibitor, carboxymethoxylamine. The inhibition of
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photorespiration did not change the dry weight, chlorophyll content and OJIP transients during the
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incubation; however, the inhibition of photorespiration significantly decreased the photochemistry
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of photosystem II and total photosynthetic O2 evolution capacity. Moreover, the restriction in
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photorespiration was synchronized with a decrease of astaxanthin accumulation. These results
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suggest that the photorespiratory pathway in H. pluvialis can accelerate astaxanthin accumulation.
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We speculate that photorespiration can enhance astaxanthin accumulation in the following ways: (i)
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photorespiration directly affects the glycerate-3-phosphate (PGA) level, which is intrinsically
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related to the accumulation of astaxanthin in H. pluvialis; (ii) the photorespiratory pathway
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indirectly affects the PGA level by effecting the dark reactions of photosynthesis, which then
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results in the enhancement of astaxanthin accumulation in H. pluvialis.
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astaxanthin
content
was
reduced
significantly
when
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accumulation,
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Keywords
Astaxanthin accumulation; Haematococcus pluvialis; Photorespiration; Photosynthesis
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1. Introduction Astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione), a ketocarotenoid, is commonly used in
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the poultry industry and in aquaculture (Johnson and Schroeder 1996; Benemann 1992). It also
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has potential clinical applications in medicine and in recent years has become popular as a
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functional food due to its powerful antioxidant properties (Goswami et al. 2010; Guerin et al. 2003;
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Liu et al. 2016). Haematococcus pluvialis, a unicellular green alga, is widely known as an
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important natural source, due to its suitability for mass production of astaxanthin (Chen et al.
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2015). It is far superior to other plants, bacteria or fungi that produce astaxanthin (Johnson and An
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1991; Yokoyama and Miki 1995; Tsubokura et al. 1999). Once growing conditions become
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unfavorable, cells of H. pluvialis start to increase their volume drastically, transform from green
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motile vegetative cells with flagella to red, non-motile, mature cysts (Boussiba 1991; Kobayashi
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2003), full of astaxanthin with no intermediates of the astaxanthin biosynthetic pathway remaining
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(Boussiba et al. 1999).
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Most of the work published to date on H. pluvialis has been focused on the growth and
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astaxanthin accumulation of this alga (Boussiba 2000; Liu et al. 2014). The astaxanthin
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biosynthetic pathway and regulatory mechanisms were studied during recent years by a number of
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groups (Han et al. 2013; Li et al. 2010; Lichtenthaler et al. 1997), while some reports
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demonstrated a correlation with fatty acid biosynthesis (Chen et al. 2015; Schoefs et al. 2001), and other reports indicated a correlation with the molecular oxygen evolved from photosynthesis (Li et
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al. 2008). However, far less attention has been paid to the interrelationship between other
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metabolic pathways and astaxanthin accumulation in H. pluvialis.
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Photorespiration is considered to be a carbon cycle system (Maurino and Peterhansel 2010) and 3
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is recognized as a key ancillary component of photosynthesis (Bauwe et al. 2012). It is caused by
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O2 substituting for CO2, which results in the production of the toxic product phosphoglycolate.
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Photorespiration involves conversion of the phosphoglycolate to glycine, followed by the
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conversion of glycine to serine, and finally serine is converted to glycerate which feeds into the
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Calvin cycle as glycerate-3-phosphate (PGA) (Thomas 2001; Wingler et al. 2000). In
plants,
PGA
is
converted
into
precursors
of
astaxanthin
biosynthesis,
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glyceraldehyde-3-phosphate (GAP) and pyruvate (Andrews et al. 1991). Photorespiration might
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affect the turnover of PGA (Bauwe et al. 2012) in H. pluvialis during astaxanthin accumulation.
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On the other hand, employment of the reactive oxygen species enhances astaxanthin formation in
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Chlorella zofingiensis (Ip and Chen 2005). Alteration in the rate of photorespiration could impact
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the probability of ROS accumulation in several organelles (Kangasjärvi et al. 2012). The role of
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photorespiration during the accumulation of astaxanthin needs to be clarified.
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This study examines the relationship between photorespiration and astaxanthin accumulation.
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Understanding the basic interactions between photorespiration and astaxanthin accumulation
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would provide some new insights into mechanisms impacting astaxanthin production in H.
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pluvialis. Determining the biological role of photorespiration during the accumulation of
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astaxanthin in H. pluvialis would help in optimizing CO2/air aeration during this stage of
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astaxanthin production.
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2. Materials and Methods
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2.1. Strains and culture conditions
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The alga Haematococcus pluvialis (strain H6) was obtained from the algal collection of Institute 4
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of Oceanology, Chinese Academy of Sciences. The algal cells were first pre-cultured in modified
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MCM medium (Sun et al. 2008) at 25 ± 1˚C indoors. Erlenmeyer flasks (5 L) with 3 L algal
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cultures were illuminated under a 14L/10D photoperiod. Erlenmeyer flasks (5 L) used in the
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pre-cultivation were 39.3 cm (height) × 20.8 cm (bottle bottom diameter) × 6 cm (inner diameter
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of bottle mouth), with 75° angle of the bottle bottom. Light at an intensity of 200 µmol photon m-2
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s-1 was provided from the top by red fluorescent lamps. And the aeration of CO2 was adjusted
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according to pH (7.5-8.5). During the pre-cultivation, the algal cultures were manually shaken 6-7
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times daily to avoid sticking.
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2.2. After 5 days of pre-cultivation, algal cells at stationary phase were divided into several 300
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mL algal cultures in Erlenmeyer flasks (500 mL), which were 18.3 cm (height) × 10.8 cm
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(bottle bottom diameter) × 3.1 cm (inner diameter of bottle mouth), with 75° angle of the
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bottle bottom. Carboxymethoxylamine (CM) has been widely used as an inhibitor of the
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glycine decarboxylase complex in the photorespiratory pathway (Corpas et al. 2004; Douce et
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al. 2001). To confirm the role of the photorespiratory pathway in H. pluvialis during
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astaxanthin accumulation, 0, 100 and 200 µM carboxymethoxylamine (CM) was added to the
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algal cultures at day 0, named as CK, CM100 and CM200, respectively. Each set of
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experiments was done in triplicate. Then the algal cultures transformed from indoor condition
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to outdoor condition at day 0. During the experiments, the flasks of algal cultures outdoors were manually shaken 6-7 times a day to avoid sticking. All experiments were done outdoors
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at the Yunnan Alphy Biotech Co., Ltd (Yunnan, China). The changes of temperature and solar
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radiation during the experimental period are shown in Table 1, as measured by an automatic
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weather station at Yunnan Alphy Biotech Co., Ltd.Analytical procedures 5
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For dry weight (DW) measurements, 10 ml samples were filtered through pre-washed, pre-dried
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and pre-weighed GF/C Whatman filter papers with 1.2 µm pore size (Whatman, Maidstone, UK).
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DWs were calculated in g/L after the filtrates were re-dried in an oven at 80˚C overnight. Total
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chlorophyll (chlorophyll a + b) and astaxanthin were extracted by methods described in Liu et al
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(Liu et al., 2002) and were assayed using a UV-visible spectrophotometer, according to
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Lichtenthaler (1987). The cellular morphology was observed under a microscope (37XB,
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Shanghai, China).
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Photosynthetic O2 evolution capacity and respiration rates (Zhang et al. 2016) were measured at
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room temperature (25˚C) with a Clark-type O2 electrode (Hansatech Instruments, Norfolk, UK)
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All measurements were performed at 0 d and 0.2 d (5 h).
The actual PSII photochemical efficiency (ΦPSII) of cultures during the process of incubation was measured using an FMS-2 pulse modulated fluorometer (Hansatech Instruments, Norfolk, UK)
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integrated with a modified adapter (Zhang et al. 2015). All measurements were performed at 0 d
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and 0.2 d (5 h).
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Chlorophyll a fluorescence (OJIP) transients of cells during the incubation were measured with
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a Handy PEA fluorometer (Hansatech Instruments, Norfolk, UK) according to Zhang and Liu
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(2016). All measurements were performed with cells that had been dark-adapted for 10 min at
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room temperature.
Data in the figures represent the mean±SD (n = 3) and were subjected to one-way ANOVA and
Tukey tests.
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3. Results 6
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The dry weight (DW) increased slightly (Fig. 1a) with increased time during the incubation, and
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the difference of DW between control and CM-treated cells was not statistically significant
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(P > 0.05), suggesting that inhibiting photorespiration did not change the biomass. The increase of
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dry weight during the incubation mainly depends on the net photosynthetic production (Torzillo et
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al. 1991). As shown in Fig. 3, the total photosynthetic O2 evolution capacity (c) was much higher
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than respiratory O2 consumption capacity (b) during the process of incubation, leading to
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accumulation of biomass and increase of dry weight. The increase of dry weight was also
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supported by the fact that the cell volume gradually increased, carbohydrates accumulated and cell
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lipid content increased during the accumulation of astaxanthin (Lee and Ding, 1994; Boussiba and
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Vonshak, 1991).
The chlorophyll content decreased gradually over time for the first 5 d (Fig. 1b), as astaxanthin
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accumulated slowly (Fig. 4b), then remained virtually stable at a low level (Fig. 1b), as
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astaxanthin accumulated rapidly (Fig. 4b). During the incubation, the chlorophyll content was not
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statistically different between control and CM-treated cells, and the two concentrations of CM also
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did not show any statistically significant difference (P > 0.05).
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The changes of chlorophyll a fluorescence (OJIP) transients during the incubation in H.
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pluvialis cells are depicted in Fig. 2. The H. pluvialis cells showed the typical OJIP chlorophyll
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fluorescence transient in the initial 3 d of incubation (Fig. 2) as astaxanthin accumulated slowly (Fig. 4b), and the fluorescence intensity of OJIP transients significantly decreased after 5 d (Fig 2)
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as astaxanthin accumulated rapidly (Fig. 4b). Any differences in the shape and intensity of OJIP
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transients between control and CM-treated cells during the incubation were not significant
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(P > 0.05), and the effects of different concentrations of CM on OJIP transients were not 7
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statistically significant (P > 0.05) either, indicating that the concentration of CM used in this study
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had no direct effect on the photosynthetic electron transport chain of H. pluvialis. The actual photochemical efficiency of PSII (ΦPSII) (Fig. 3a) provides an indication of the
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amount of energy used in photochemistry (Zhang et al. 2015). Treatment with CM decreased ΦPSII
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(Fig. 3a) significantly in H. pluvialis (P < 0.01) during the incubation. In addition, treatment with
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CM decreased ΦPSII quickly, by 0.2 d (5 h) of incubation, suggesting that inhibition of
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photorespiration decreased photochemical activity of PSII directly. The two concentrations of CM
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did not produce significantly different effects on ΦPSII (P > 0.05). The results obtained suggested
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that the photosynthetic carbon cycle might be restricted when the photorespiratory pathway was
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inhibited (Bykova et al. 2005) in H. pluvialis.
As shown in Fig. 3, the respiratory O2 consumption capacity (b) and total photosynthetic O2
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evolution capacity (c) declined gradually during the process of incubation. Treatment with CM
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inhibited the respiratory O2 consumption capacity and total photosynthetic O2 evolution capacity
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significantly after 2 d of incubation (P < 0.01). There was no obvious difference between
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CM100-treated and CM200-treated cells (P > 0.05). The decreased total photosynthetic O2
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evolution capacity when photorespiration was inhibited suggests that the photosynthetic carbon
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cycle might be restricted.
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As shown in the upper-left corner of every photo in Fig. 4a, a transition of cell morphology
from green motile cells to red non-motile cells in H. pluvialis was observed during the incubation.
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At 0 d of incubation, almost all cells in the Erlenmeyer flasks were green motile cells with two
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flagella. Green motile cells transformed to green non-motile cells during the first day. Astaxanthin
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started to accumulate by 2 d, and then accumulated in the mid-region of the cell around the cell 8
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nucleus after 3 d, forming an obvious red centre and a green periphery. The accumulation of
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astaxanthin in H. pluvialis was significant after 5 d, and gradually spread throughout the whole
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cell (7 d). Finally, at 10d, astaxanthin colored the whole cell. In contrast, the cells of the treatments
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with CM had a lower astaxanthin content as observed with a microscope. When photorespiration
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was inhibited, the cell morphology at 7 d was similar to the cell morphology of 3 d in control cells.
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In Erlenmeyer flasks, the color of the control algal culture was much redder than the CM-treated
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algal cultures after 3 d (Fig. 4a), and the color of CM-treated algal cultures at 7 d was similar to
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the control algal culture at 3 d. The accumulation of astaxanthin in H. pluvialis was significant
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after 5 d of incubation during the transition period (Fig. 4b). The astaxanthin content (% of dry
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weight) increased from 0.17% to 1.08% after 10 d of incubation in control cells, and 0.18% to
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0.60%, 0.18% to 0.58% in CM100-treated cells and CM200-treated cells, respectively. Treatment
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with CM decreased astaxanthin content significantly after 3 d of incubation (P < 0.05), with
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astaxanthin content in 7 d CM-treated cells nearly the same as control cells at 3 d, and with
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astaxanthin content in 10 d CM-treated cells only a little higher than control cells at 5 d. There
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was no significant difference between the two concentrations of CM during the accumulation of
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astaxanthin (P > 0.05), suggesting that astaxanthin accumulation was restricted when
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photorespiration was inhibited. The measured astaxanthin content (Fig. 4b) corresponded with the
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changing color of cells and whole algal cultures (Fig. 4a).
4. Discussion
4.1. The inhibition of photorespiration decreased photosynthesis during the accumulation of astaxanthin in H. pluvialis During the incubation, there was no obvious difference in dry weight and chlorophyll content 9
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between control and CM-treated cells (Fig. 1), suggesting that the inhibition of photorespiration
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by CM did not affect the biomass. Also, the shape and intensity of OJIP transients were similar
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between control and CM-treated cells (Fig. 2), indicating that the concentration of CM used in this
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study had no direct effect on the photosynthetic electron transport chain of H. pluvialis. However,
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treatment with CM significantly decreased the actual photochemical efficiency of PSII (ΦPSII), by
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0.2 d of incubation (Fig. 3a). These results suggest that the photosynthetic carbon cycle might be
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restricted when the photorespiratory pathway is inhibited (Bykova et al. 2005) in H. pluvialis,
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which is also demonstrated by the observation that the total photosynthetic O2 evolution capacity
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decreased in CM-treated cells compared with control cells (Fig. 3c).
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Treatment with CM suppressed the respiratory O2 consumption capacity significantly during the
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experiment (Fig. 3b), which indicates that consumption of organic compounds was reduced.
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Meanwhile, the total photosynthetic O2 evolution capacity of CM-treated cells decreased distinctly
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because of the photosynthetic carbon cycle being inhibited, which suggests that when the
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photorespiratory pathway was inhibited, the biosynthesis of organic compounds was reduced. The
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results suggest that the net product of the dark reactions of photosynthesis did not change, because
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the dry weight did not change, as shown in Fig. 1 a.
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A detailed analysis of the process showed that the inhibition of the photorespiratory pathway
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would impair photosynthetic carbon assimilation (Bykova et al. 2005), resulting in reduced actual efficiency of PSII photochemical activity and attenuation of total photosynthetic O2 evolution
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capacity in H. pluvialis. Nevertheless, the astaxanthin content increased (Fig. 4b) even while
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photosynthetic carbon assimilation was inhibited, indicating that other metabolic pathways likely
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contributed to astaxanthin accumulation. 10
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4.2. Photorespiration accelerated the accumulation of astaxanthin in H. pluvialis The inhibition of photorespiration by CM decreased astaxanthin content significantly during the
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incubation (Fig. 4b), which suggests that inhibition of photorespiration suppressed the
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biosynthesis of astaxanthin in H. pluvialis.
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Astaxanthin was proposed as the end-product of carotenoid accumulation in cytosolic lipid
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bodies under stress conditions (Johnson and An 1991; Johnson and Schroeder 1996). Isopentenyl
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pyrophosphate (IPP) is the precursor for carotenoid synthesis (Lichtenthaler 1999). In H. pluvialis,
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IPP is believed to be synthesized solely by the non-mevalonate 1-deoxy-D-xylulose- 5-phosphate
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pathway (DOXP pathway or MEP pathway) in the chloroplast (Lichtenthaler et al. 1997; Disch
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et al. 1998), and that the MEP pathway synthesized IPP using GAP (glyceraldehyde-3- phosphate)
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and pyruvate as precursors (Sprenger et al. 1997; Grolle et al. 2000). PGA (glycerate3-phosphate)
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could be converted to GAP and pyruvate (Andrews et al.1991). In other words, PGA is the
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primary substrate for astaxanthin biosynthesis. Therefore, the accumulation of astaxanthin in H.
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pluvialis is associated with PGA amounts.
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The photorespiratory pathway could affect the accumulation of astaxanthin in H. pluvialis in the following ways (Fig. 5):
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(1) Photorespiration results from the oxygenase reaction catalyzed by ribulose-1,5-bisphosphate
carboxylase/oxygenase. In this reaction phosphoglycolate is produced and subsequently
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metabolized in the photorespiratory pathway to form the Calvin cycle intermediate PGA (Wingler
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et al. 2000). The photorespiratory pathway would accelerate the regeneration of PGA. Therefore,
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photorespiration could directly increase the generation of PGA, which would increase the 11
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accumulation of astaxanthin in H. pluvialis. (2) PGA is the first product of photosynthetic carbon assimilation. The inhibition of the
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photorespiratory pathway would impair photosynthetic carbon assimilation. Therefore, the
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photorespiratory pathway could indirectly increase PGA amounts by accelerating the activity of
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photosynthetic carbon assimilation, which would then result in the accumulation of astaxanthin in
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H. pluvialis.
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In H. pluvialis, the accumulation of astaxanthin was reduced significantly when the
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photorespiratory pathway was inhibited by CM. Inhibition of the photorespiratory pathway did not
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change DW accumulation, chlorophyll content or OJIP transients during the incubation. In
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contrast, the inhibition of the photorespiratory pathway decreased the photochemical activity of
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PSII and total photosynthetic O2 evolution capacity significantly. The suppression of
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photorespiration correlated with the decrease of astaxanthin content. Taken together, these results
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suggest that the photorespiratory pathway plays an important role in astaxanthin accumulation in
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H. pluvialis.
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Contributions
Chunhui Zhang and Jianguo Liu designed the study and wrote the manuscript; Chunhui Zhang
and Litao Zhang performed the experiments and analyzed the data.
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Acknowledgements 12
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This work was supported by National Natural Science Foundation of China (No. 31572639) and
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Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine
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Science and Technology (No. Y62419101J). We thank Dr. John van der Meer for his English
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editing.
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isopentenyl diphosphate and isoprenoid biosynthesis in higher plants. Physiologia plantarum 15
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Liu JG, van der Meer JP, Zhang LT, Zhang Y (2016) Cultivation of Haematococcus pluvialis for
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astaxanthin production. In: Slocombe SP and Benemann JR, (eds.) Micro-algal production for
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biomass and high-value products. Taylor & Francis, New York, USA, pp 267-293.
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Liu JG, Yin MY, Zhang ZP et al (2002) Dynamic changes of inorganic nitrogen and astaxanthin
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accumulation in Haematococcus pluvialis. Chinese Journal of Oceanology and Limnology 20:
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358-364.
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Maurino VG and Peterhansel C (2010) Photorespiration: current status and approaches for metabolic engineering. Current Opinion in Plant Biology 13: 248-255.
Ogren WL (1984) Photorespiration: pathways, regulation and modification. Annual Review of
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Plant Physiology 35: 415–442.
Schoefs B, Rmiki NE, Rachadi J et al (2001) Astaxanthin accumulation in Haematococcus
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requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids. Febs Letters 500:
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Sprenger GA, Schorken U, Wiegert T et al (1997) Identification of a thiamin-dependent synthase
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Steinbrenner J and Linden H (2003) Light induction of carotenoid biosynthesis genes in the green
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alga Haematococcus pluvialis: regulation by photosynthetic redox control. Plant Mol. Biol.
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52:343-356. 16
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Sun YH, Liu JG, Zhang XL et al (2008) Strain H 2-419-4 of Haematococcus pluvialis induced by
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ethyl methanesulphonate and ultraviolet radiation. Chinese Journal of Oceanology and
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Limnology 26: 152-156.
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Sharkey TD (2001) Photorespiration. General Life Sciences. doi: 10.1038/npg.els.0001292
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Tolbert NE (1997) The C2 oxidative photosynthetic carbon cycle. Annual Review of Plant Physiology and Plant Molecular Biology 48: 1–25.
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Torzillo G, Sacchi A, Materassi R et al (1991) Effect of temperature on yield and night biomass
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loss in Spirulina platensis grown outdoors in tubular photobioreactors. Journal of Applied
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Phycology 3:103-109.
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Tsubokura A, Yoneda H, Mizuta H (1999) Paracoccus carotinifaciens sp. Nov., a new aerobic gram-negative astaxanthin-producing bacterium. Int J System Bacteriol 49: 277–282 Wingler A, Lea PJ, Quick WP et al (2000) Photorespiration: metabolic pathways and their role in
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stress protection. Philosophical Transactions of the Royal Society B: Biological Sciences
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355(1402): 1517-1529.
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Yokoyama A, Miki W (1995) Composition and presumed biosynthetic pathway of carotenoids in
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the astaxanthin-producing bacterium Agrobacterium aurantiacum. FEMS Microbiology Letters
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128: 139–144
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Zhang LT, Li L, He ML et al (2016) The role of photorespiration during H2 photoproduction in Chlorella protothecoides under nitrogen limitation. Plant cell reports 35: 1-4.
Zhang LT, He ML, Liu JG et al (2015) Role of the mitochondrial alternative oxidase pathway in hydrogen photoproduction in Chlorella protothecoides. Planta 241:1005-1014.
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Table 1 Characteristics of the temperature and solar radiation in the Yunnan region during astaxanthin accumulation in H. pluvialis
20 Incubation
Temperature
Solar radiation
Time (days)
°C
(W/m2) 18
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2 3
4
Mean
total solar
photosynthetically
radiation
active radiation
12.5
24.3
17.91
598.50
292.20
1
13.8
23.6
18.31
633.10
306.80
2
13.9
20.9
18.07
513.64
263.64
3
11.9
22.1
17.45
610.91
4
13.2
21.5
18.09
563.09
5
11.2
21.5
6
10.5
21.5
7
10.5
21.4
8
9.8
22.8
9
11.2
10
13.2
Mean
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0
294.73
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593.36
282.36
16.04
434.82
197.09
17.01
601.27
281.91
16.63
452.82
212.45
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16.98
16.81
425.27
208.00
20.1
16.56
424.22
210.78
21.93
17.26
531.91
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High
Average
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Low
Average
Figure caption
Fig. 1 The effects of CM (CM100: 100 µM; CM200: 200 µM) on the dry weight (a) and
5
chlorophyll content (b) in H. pluvialis with increased time during the incubation. Mean ± SE of
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three replicates are presented.
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Fig. 2 The effects of CM on the chlorophyll a fluorescence (OJIP) transients in H. pluvialis during
2
the incubation.
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3 Fig. 3 Changes of actual photochemical efficiency of PSII (ΦPSII) (a), the respiratory O2
5
consumption capacity (b), and total photosynthetic O2 evolution capacity (c), in H. pluvialis
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incubated in a gradient of CM. Mean ± SE of three replicates are presented.
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Fig. 4 The cell morphology (a) and astaxanthin content (b) in H. pluvialis exposed to CM. Mean ±
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SE of three replicates are presented.
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Fig. 5 Scheme of the possible interrelation between photorespiration and astaxanthin accumulation
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in H. pluvialis. PGA 3-phosphoglycerate, CEF-I cyclic electron flow around PSI, PSII/I
13
photosystem
14
carboxylase/oxygenase, GAP glyceraldehyde-3- phosphate, Pyr pyruvate, GGPP geranylgeranyl
15
diphosphate, Fdox/ Fdred ferredoxin, PQ plastoquinone, UQ ubiquinone, AOX alternative oxidase,
16
COX cytochrome oxidase, CM carboxymethoxylamine
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1,5-biphosphate,
Rubisco
ribulose
1,5-biphosphate
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ribulose
Highlights
Inhibition of the photorespiratory pathway did not change dry weight accumulation,
chlorophyll content or OJIP transients during the incubation.
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RuBP
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II/I,
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The inhibition of the photorespiratory pathway decreased the photochemical activity of photosystem II (PSII) and total photosynthetic O2 evolution capacity significantly. 20
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The suppression of photorespiration correlated with the decrease of astaxanthin content.
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The photorespiratory pathway plays an important role in astaxanthin accumulation in Haematococcus pluvialis.
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Contributions Chunhui Zhang and Jianguo Liu designed the study and wrote the manuscript; Chunhui Zhang
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and Litao Zhang performed the experiments and analyzed the data.
S0981-9428(16)30199-1
DOI:
10.1016/j.plaphy.2016.05.029
Reference:
PLAPHY 4560
To appear in:
Plant Physiology and Biochemistry
Received Date: 25 March 2016 Revised Date:
18 May 2016
Accepted Date: 18 May 2016
Please cite this article as: C. Zhang, L. Zhang, J. Liu, The role of photorespiration during astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae), Plant Physiology et Biochemistry (2016), doi: 10.1016/j.plaphy.2016.05.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The role of photorespiration during astaxanthin accumulation in Haematococcus
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pluvialis (Chlorophyceae)
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Chunhui Zhang1, 3, Litao Zhang1, 2, Jianguo Liu1, 2, *
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Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao
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266071, P.R. China
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National & Local Joint Engineering Laboratory of Ecological Mariculture, Key Laboratory of
National-Local Joint Engineering Research Center for Haematococcus pluvialis and
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Astaxanthin Products, Yunnan Alphy Biotech Co., Ltd., Chuxiong 675012, China
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University of Chinese Academy of Sciences, Beijing 100049, P.R. China
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7 Nanhai Road, Qingdao, Shandong, 266071, P.R. China
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Tel: +86-532-82898709, Fax: +86-532-82898612, E-mail: [email protected]
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Abstract Most previous studies on Haematococcus pluvialis have been focused on growth and
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astaxanthin accumulation. However, the relationships between photorespiration and astaxanthin
4
accumulation have not been clarified. The purpose of this study was to examine the role of
5
photorespiration during the process of astaxanthin accumulation in H. pluvialis. During
6
astaxanthin
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photorespiration was inhibited by its specific inhibitor, carboxymethoxylamine. The inhibition of
8
photorespiration did not change the dry weight, chlorophyll content and OJIP transients during the
9
incubation; however, the inhibition of photorespiration significantly decreased the photochemistry
10
of photosystem II and total photosynthetic O2 evolution capacity. Moreover, the restriction in
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photorespiration was synchronized with a decrease of astaxanthin accumulation. These results
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suggest that the photorespiratory pathway in H. pluvialis can accelerate astaxanthin accumulation.
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We speculate that photorespiration can enhance astaxanthin accumulation in the following ways: (i)
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photorespiration directly affects the glycerate-3-phosphate (PGA) level, which is intrinsically
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related to the accumulation of astaxanthin in H. pluvialis; (ii) the photorespiratory pathway
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indirectly affects the PGA level by effecting the dark reactions of photosynthesis, which then
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results in the enhancement of astaxanthin accumulation in H. pluvialis.
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astaxanthin
content
was
reduced
significantly
when
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accumulation,
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Keywords
Astaxanthin accumulation; Haematococcus pluvialis; Photorespiration; Photosynthesis
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1. Introduction Astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione), a ketocarotenoid, is commonly used in
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the poultry industry and in aquaculture (Johnson and Schroeder 1996; Benemann 1992). It also
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has potential clinical applications in medicine and in recent years has become popular as a
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functional food due to its powerful antioxidant properties (Goswami et al. 2010; Guerin et al. 2003;
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Liu et al. 2016). Haematococcus pluvialis, a unicellular green alga, is widely known as an
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important natural source, due to its suitability for mass production of astaxanthin (Chen et al.
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2015). It is far superior to other plants, bacteria or fungi that produce astaxanthin (Johnson and An
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1991; Yokoyama and Miki 1995; Tsubokura et al. 1999). Once growing conditions become
10
unfavorable, cells of H. pluvialis start to increase their volume drastically, transform from green
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motile vegetative cells with flagella to red, non-motile, mature cysts (Boussiba 1991; Kobayashi
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2003), full of astaxanthin with no intermediates of the astaxanthin biosynthetic pathway remaining
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(Boussiba et al. 1999).
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Most of the work published to date on H. pluvialis has been focused on the growth and
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astaxanthin accumulation of this alga (Boussiba 2000; Liu et al. 2014). The astaxanthin
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biosynthetic pathway and regulatory mechanisms were studied during recent years by a number of
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groups (Han et al. 2013; Li et al. 2010; Lichtenthaler et al. 1997), while some reports
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demonstrated a correlation with fatty acid biosynthesis (Chen et al. 2015; Schoefs et al. 2001), and other reports indicated a correlation with the molecular oxygen evolved from photosynthesis (Li et
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al. 2008). However, far less attention has been paid to the interrelationship between other
21
metabolic pathways and astaxanthin accumulation in H. pluvialis.
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Photorespiration is considered to be a carbon cycle system (Maurino and Peterhansel 2010) and 3
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is recognized as a key ancillary component of photosynthesis (Bauwe et al. 2012). It is caused by
2
O2 substituting for CO2, which results in the production of the toxic product phosphoglycolate.
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Photorespiration involves conversion of the phosphoglycolate to glycine, followed by the
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conversion of glycine to serine, and finally serine is converted to glycerate which feeds into the
5
Calvin cycle as glycerate-3-phosphate (PGA) (Thomas 2001; Wingler et al. 2000). In
plants,
PGA
is
converted
into
precursors
of
astaxanthin
biosynthesis,
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glyceraldehyde-3-phosphate (GAP) and pyruvate (Andrews et al. 1991). Photorespiration might
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affect the turnover of PGA (Bauwe et al. 2012) in H. pluvialis during astaxanthin accumulation.
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On the other hand, employment of the reactive oxygen species enhances astaxanthin formation in
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Chlorella zofingiensis (Ip and Chen 2005). Alteration in the rate of photorespiration could impact
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the probability of ROS accumulation in several organelles (Kangasjärvi et al. 2012). The role of
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photorespiration during the accumulation of astaxanthin needs to be clarified.
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This study examines the relationship between photorespiration and astaxanthin accumulation.
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Understanding the basic interactions between photorespiration and astaxanthin accumulation
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would provide some new insights into mechanisms impacting astaxanthin production in H.
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pluvialis. Determining the biological role of photorespiration during the accumulation of
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astaxanthin in H. pluvialis would help in optimizing CO2/air aeration during this stage of
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astaxanthin production.
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2. Materials and Methods
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2.1. Strains and culture conditions
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The alga Haematococcus pluvialis (strain H6) was obtained from the algal collection of Institute 4
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of Oceanology, Chinese Academy of Sciences. The algal cells were first pre-cultured in modified
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MCM medium (Sun et al. 2008) at 25 ± 1˚C indoors. Erlenmeyer flasks (5 L) with 3 L algal
3
cultures were illuminated under a 14L/10D photoperiod. Erlenmeyer flasks (5 L) used in the
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pre-cultivation were 39.3 cm (height) × 20.8 cm (bottle bottom diameter) × 6 cm (inner diameter
5
of bottle mouth), with 75° angle of the bottle bottom. Light at an intensity of 200 µmol photon m-2
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s-1 was provided from the top by red fluorescent lamps. And the aeration of CO2 was adjusted
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according to pH (7.5-8.5). During the pre-cultivation, the algal cultures were manually shaken 6-7
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times daily to avoid sticking.
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2.2. After 5 days of pre-cultivation, algal cells at stationary phase were divided into several 300
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mL algal cultures in Erlenmeyer flasks (500 mL), which were 18.3 cm (height) × 10.8 cm
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(bottle bottom diameter) × 3.1 cm (inner diameter of bottle mouth), with 75° angle of the
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bottle bottom. Carboxymethoxylamine (CM) has been widely used as an inhibitor of the
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glycine decarboxylase complex in the photorespiratory pathway (Corpas et al. 2004; Douce et
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al. 2001). To confirm the role of the photorespiratory pathway in H. pluvialis during
15
astaxanthin accumulation, 0, 100 and 200 µM carboxymethoxylamine (CM) was added to the
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algal cultures at day 0, named as CK, CM100 and CM200, respectively. Each set of
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experiments was done in triplicate. Then the algal cultures transformed from indoor condition
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to outdoor condition at day 0. During the experiments, the flasks of algal cultures outdoors were manually shaken 6-7 times a day to avoid sticking. All experiments were done outdoors
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at the Yunnan Alphy Biotech Co., Ltd (Yunnan, China). The changes of temperature and solar
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radiation during the experimental period are shown in Table 1, as measured by an automatic
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weather station at Yunnan Alphy Biotech Co., Ltd.Analytical procedures 5
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For dry weight (DW) measurements, 10 ml samples were filtered through pre-washed, pre-dried
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and pre-weighed GF/C Whatman filter papers with 1.2 µm pore size (Whatman, Maidstone, UK).
3
DWs were calculated in g/L after the filtrates were re-dried in an oven at 80˚C overnight. Total
4
chlorophyll (chlorophyll a + b) and astaxanthin were extracted by methods described in Liu et al
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(Liu et al., 2002) and were assayed using a UV-visible spectrophotometer, according to
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Lichtenthaler (1987). The cellular morphology was observed under a microscope (37XB,
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Shanghai, China).
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Photosynthetic O2 evolution capacity and respiration rates (Zhang et al. 2016) were measured at
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room temperature (25˚C) with a Clark-type O2 electrode (Hansatech Instruments, Norfolk, UK)
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All measurements were performed at 0 d and 0.2 d (5 h).
The actual PSII photochemical efficiency (ΦPSII) of cultures during the process of incubation was measured using an FMS-2 pulse modulated fluorometer (Hansatech Instruments, Norfolk, UK)
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integrated with a modified adapter (Zhang et al. 2015). All measurements were performed at 0 d
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and 0.2 d (5 h).
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Chlorophyll a fluorescence (OJIP) transients of cells during the incubation were measured with
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a Handy PEA fluorometer (Hansatech Instruments, Norfolk, UK) according to Zhang and Liu
17
(2016). All measurements were performed with cells that had been dark-adapted for 10 min at
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room temperature.
Data in the figures represent the mean±SD (n = 3) and were subjected to one-way ANOVA and
Tukey tests.
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3. Results 6
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The dry weight (DW) increased slightly (Fig. 1a) with increased time during the incubation, and
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the difference of DW between control and CM-treated cells was not statistically significant
3
(P > 0.05), suggesting that inhibiting photorespiration did not change the biomass. The increase of
4
dry weight during the incubation mainly depends on the net photosynthetic production (Torzillo et
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al. 1991). As shown in Fig. 3, the total photosynthetic O2 evolution capacity (c) was much higher
6
than respiratory O2 consumption capacity (b) during the process of incubation, leading to
7
accumulation of biomass and increase of dry weight. The increase of dry weight was also
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supported by the fact that the cell volume gradually increased, carbohydrates accumulated and cell
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lipid content increased during the accumulation of astaxanthin (Lee and Ding, 1994; Boussiba and
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Vonshak, 1991).
The chlorophyll content decreased gradually over time for the first 5 d (Fig. 1b), as astaxanthin
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accumulated slowly (Fig. 4b), then remained virtually stable at a low level (Fig. 1b), as
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astaxanthin accumulated rapidly (Fig. 4b). During the incubation, the chlorophyll content was not
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statistically different between control and CM-treated cells, and the two concentrations of CM also
15
did not show any statistically significant difference (P > 0.05).
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The changes of chlorophyll a fluorescence (OJIP) transients during the incubation in H.
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pluvialis cells are depicted in Fig. 2. The H. pluvialis cells showed the typical OJIP chlorophyll
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fluorescence transient in the initial 3 d of incubation (Fig. 2) as astaxanthin accumulated slowly (Fig. 4b), and the fluorescence intensity of OJIP transients significantly decreased after 5 d (Fig 2)
20
as astaxanthin accumulated rapidly (Fig. 4b). Any differences in the shape and intensity of OJIP
21
transients between control and CM-treated cells during the incubation were not significant
22
(P > 0.05), and the effects of different concentrations of CM on OJIP transients were not 7
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statistically significant (P > 0.05) either, indicating that the concentration of CM used in this study
2
had no direct effect on the photosynthetic electron transport chain of H. pluvialis. The actual photochemical efficiency of PSII (ΦPSII) (Fig. 3a) provides an indication of the
4
amount of energy used in photochemistry (Zhang et al. 2015). Treatment with CM decreased ΦPSII
5
(Fig. 3a) significantly in H. pluvialis (P < 0.01) during the incubation. In addition, treatment with
6
CM decreased ΦPSII quickly, by 0.2 d (5 h) of incubation, suggesting that inhibition of
7
photorespiration decreased photochemical activity of PSII directly. The two concentrations of CM
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did not produce significantly different effects on ΦPSII (P > 0.05). The results obtained suggested
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that the photosynthetic carbon cycle might be restricted when the photorespiratory pathway was
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inhibited (Bykova et al. 2005) in H. pluvialis.
As shown in Fig. 3, the respiratory O2 consumption capacity (b) and total photosynthetic O2
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evolution capacity (c) declined gradually during the process of incubation. Treatment with CM
13
inhibited the respiratory O2 consumption capacity and total photosynthetic O2 evolution capacity
14
significantly after 2 d of incubation (P < 0.01). There was no obvious difference between
15
CM100-treated and CM200-treated cells (P > 0.05). The decreased total photosynthetic O2
16
evolution capacity when photorespiration was inhibited suggests that the photosynthetic carbon
17
cycle might be restricted.
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As shown in the upper-left corner of every photo in Fig. 4a, a transition of cell morphology
from green motile cells to red non-motile cells in H. pluvialis was observed during the incubation.
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At 0 d of incubation, almost all cells in the Erlenmeyer flasks were green motile cells with two
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flagella. Green motile cells transformed to green non-motile cells during the first day. Astaxanthin
22
started to accumulate by 2 d, and then accumulated in the mid-region of the cell around the cell 8
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nucleus after 3 d, forming an obvious red centre and a green periphery. The accumulation of
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astaxanthin in H. pluvialis was significant after 5 d, and gradually spread throughout the whole
3
cell (7 d). Finally, at 10d, astaxanthin colored the whole cell. In contrast, the cells of the treatments
4
with CM had a lower astaxanthin content as observed with a microscope. When photorespiration
5
was inhibited, the cell morphology at 7 d was similar to the cell morphology of 3 d in control cells.
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In Erlenmeyer flasks, the color of the control algal culture was much redder than the CM-treated
7
algal cultures after 3 d (Fig. 4a), and the color of CM-treated algal cultures at 7 d was similar to
8
the control algal culture at 3 d. The accumulation of astaxanthin in H. pluvialis was significant
9
after 5 d of incubation during the transition period (Fig. 4b). The astaxanthin content (% of dry
10
weight) increased from 0.17% to 1.08% after 10 d of incubation in control cells, and 0.18% to
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0.60%, 0.18% to 0.58% in CM100-treated cells and CM200-treated cells, respectively. Treatment
12
with CM decreased astaxanthin content significantly after 3 d of incubation (P < 0.05), with
13
astaxanthin content in 7 d CM-treated cells nearly the same as control cells at 3 d, and with
14
astaxanthin content in 10 d CM-treated cells only a little higher than control cells at 5 d. There
15
was no significant difference between the two concentrations of CM during the accumulation of
16
astaxanthin (P > 0.05), suggesting that astaxanthin accumulation was restricted when
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photorespiration was inhibited. The measured astaxanthin content (Fig. 4b) corresponded with the
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changing color of cells and whole algal cultures (Fig. 4a).
4. Discussion
4.1. The inhibition of photorespiration decreased photosynthesis during the accumulation of astaxanthin in H. pluvialis During the incubation, there was no obvious difference in dry weight and chlorophyll content 9
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between control and CM-treated cells (Fig. 1), suggesting that the inhibition of photorespiration
2
by CM did not affect the biomass. Also, the shape and intensity of OJIP transients were similar
3
between control and CM-treated cells (Fig. 2), indicating that the concentration of CM used in this
4
study had no direct effect on the photosynthetic electron transport chain of H. pluvialis. However,
5
treatment with CM significantly decreased the actual photochemical efficiency of PSII (ΦPSII), by
6
0.2 d of incubation (Fig. 3a). These results suggest that the photosynthetic carbon cycle might be
7
restricted when the photorespiratory pathway is inhibited (Bykova et al. 2005) in H. pluvialis,
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which is also demonstrated by the observation that the total photosynthetic O2 evolution capacity
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decreased in CM-treated cells compared with control cells (Fig. 3c).
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Treatment with CM suppressed the respiratory O2 consumption capacity significantly during the
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experiment (Fig. 3b), which indicates that consumption of organic compounds was reduced.
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Meanwhile, the total photosynthetic O2 evolution capacity of CM-treated cells decreased distinctly
13
because of the photosynthetic carbon cycle being inhibited, which suggests that when the
14
photorespiratory pathway was inhibited, the biosynthesis of organic compounds was reduced. The
15
results suggest that the net product of the dark reactions of photosynthesis did not change, because
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the dry weight did not change, as shown in Fig. 1 a.
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A detailed analysis of the process showed that the inhibition of the photorespiratory pathway
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would impair photosynthetic carbon assimilation (Bykova et al. 2005), resulting in reduced actual efficiency of PSII photochemical activity and attenuation of total photosynthetic O2 evolution
20
capacity in H. pluvialis. Nevertheless, the astaxanthin content increased (Fig. 4b) even while
21
photosynthetic carbon assimilation was inhibited, indicating that other metabolic pathways likely
22
contributed to astaxanthin accumulation. 10
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4.2. Photorespiration accelerated the accumulation of astaxanthin in H. pluvialis The inhibition of photorespiration by CM decreased astaxanthin content significantly during the
4
incubation (Fig. 4b), which suggests that inhibition of photorespiration suppressed the
5
biosynthesis of astaxanthin in H. pluvialis.
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Astaxanthin was proposed as the end-product of carotenoid accumulation in cytosolic lipid
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bodies under stress conditions (Johnson and An 1991; Johnson and Schroeder 1996). Isopentenyl
8
pyrophosphate (IPP) is the precursor for carotenoid synthesis (Lichtenthaler 1999). In H. pluvialis,
9
IPP is believed to be synthesized solely by the non-mevalonate 1-deoxy-D-xylulose- 5-phosphate
10
pathway (DOXP pathway or MEP pathway) in the chloroplast (Lichtenthaler et al. 1997; Disch
11
et al. 1998), and that the MEP pathway synthesized IPP using GAP (glyceraldehyde-3- phosphate)
12
and pyruvate as precursors (Sprenger et al. 1997; Grolle et al. 2000). PGA (glycerate3-phosphate)
13
could be converted to GAP and pyruvate (Andrews et al.1991). In other words, PGA is the
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primary substrate for astaxanthin biosynthesis. Therefore, the accumulation of astaxanthin in H.
15
pluvialis is associated with PGA amounts.
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The photorespiratory pathway could affect the accumulation of astaxanthin in H. pluvialis in the following ways (Fig. 5):
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(1) Photorespiration results from the oxygenase reaction catalyzed by ribulose-1,5-bisphosphate
carboxylase/oxygenase. In this reaction phosphoglycolate is produced and subsequently
20
metabolized in the photorespiratory pathway to form the Calvin cycle intermediate PGA (Wingler
21
et al. 2000). The photorespiratory pathway would accelerate the regeneration of PGA. Therefore,
22
photorespiration could directly increase the generation of PGA, which would increase the 11
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accumulation of astaxanthin in H. pluvialis. (2) PGA is the first product of photosynthetic carbon assimilation. The inhibition of the
3
photorespiratory pathway would impair photosynthetic carbon assimilation. Therefore, the
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photorespiratory pathway could indirectly increase PGA amounts by accelerating the activity of
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photosynthetic carbon assimilation, which would then result in the accumulation of astaxanthin in
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H. pluvialis.
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7 5. Conclusion
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In H. pluvialis, the accumulation of astaxanthin was reduced significantly when the
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photorespiratory pathway was inhibited by CM. Inhibition of the photorespiratory pathway did not
11
change DW accumulation, chlorophyll content or OJIP transients during the incubation. In
12
contrast, the inhibition of the photorespiratory pathway decreased the photochemical activity of
13
PSII and total photosynthetic O2 evolution capacity significantly. The suppression of
14
photorespiration correlated with the decrease of astaxanthin content. Taken together, these results
15
suggest that the photorespiratory pathway plays an important role in astaxanthin accumulation in
16
H. pluvialis.
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Contributions
Chunhui Zhang and Jianguo Liu designed the study and wrote the manuscript; Chunhui Zhang
and Litao Zhang performed the experiments and analyzed the data.
21 22
Acknowledgements 12
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This work was supported by National Natural Science Foundation of China (No. 31572639) and
2
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine
3
Science and Technology (No. Y62419101J). We thank Dr. John van der Meer for his English
4
editing.
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Table 1 Characteristics of the temperature and solar radiation in the Yunnan region during astaxanthin accumulation in H. pluvialis
20 Incubation
Temperature
Solar radiation
Time (days)
°C
(W/m2) 18
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2 3
4
Mean
total solar
photosynthetically
radiation
active radiation
12.5
24.3
17.91
598.50
292.20
1
13.8
23.6
18.31
633.10
306.80
2
13.9
20.9
18.07
513.64
263.64
3
11.9
22.1
17.45
610.91
4
13.2
21.5
18.09
563.09
5
11.2
21.5
6
10.5
21.5
7
10.5
21.4
8
9.8
22.8
9
11.2
10
13.2
Mean
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294.73
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593.36
282.36
16.04
434.82
197.09
17.01
601.27
281.91
16.63
452.82
212.45
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16.98
16.81
425.27
208.00
20.1
16.56
424.22
210.78
21.93
17.26
531.91
256.19
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Average
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Average
Figure caption
Fig. 1 The effects of CM (CM100: 100 µM; CM200: 200 µM) on the dry weight (a) and
5
chlorophyll content (b) in H. pluvialis with increased time during the incubation. Mean ± SE of
6
three replicates are presented.
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Fig. 2 The effects of CM on the chlorophyll a fluorescence (OJIP) transients in H. pluvialis during
2
the incubation.
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3 Fig. 3 Changes of actual photochemical efficiency of PSII (ΦPSII) (a), the respiratory O2
5
consumption capacity (b), and total photosynthetic O2 evolution capacity (c), in H. pluvialis
6
incubated in a gradient of CM. Mean ± SE of three replicates are presented.
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Fig. 4 The cell morphology (a) and astaxanthin content (b) in H. pluvialis exposed to CM. Mean ±
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SE of three replicates are presented.
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Fig. 5 Scheme of the possible interrelation between photorespiration and astaxanthin accumulation
12
in H. pluvialis. PGA 3-phosphoglycerate, CEF-I cyclic electron flow around PSI, PSII/I
13
photosystem
14
carboxylase/oxygenase, GAP glyceraldehyde-3- phosphate, Pyr pyruvate, GGPP geranylgeranyl
15
diphosphate, Fdox/ Fdred ferredoxin, PQ plastoquinone, UQ ubiquinone, AOX alternative oxidase,
16
COX cytochrome oxidase, CM carboxymethoxylamine
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1,5-biphosphate,
Rubisco
ribulose
1,5-biphosphate
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ribulose
Highlights
Inhibition of the photorespiratory pathway did not change dry weight accumulation,
chlorophyll content or OJIP transients during the incubation.
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RuBP
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II/I,
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The inhibition of the photorespiratory pathway decreased the photochemical activity of photosystem II (PSII) and total photosynthetic O2 evolution capacity significantly. 20
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The suppression of photorespiration correlated with the decrease of astaxanthin content.
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The photorespiratory pathway plays an important role in astaxanthin accumulation in Haematococcus pluvialis.
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Contributions Chunhui Zhang and Jianguo Liu designed the study and wrote the manuscript; Chunhui Zhang
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and Litao Zhang performed the experiments and analyzed the data.