Haematococcus pluvialis (Chlorophyceae, Order Volvocales), a freshwater green microalgal species, has commercial value owing to its ability to accumulate high concentrations of astaxanthin (up to 5% of dry weight). Astaxanthin (3,3′-dihydroxy-β, β-carotene-4,4′-dione) is a red ketocarotenoid which has many important biologi-cal functions, including antioxidant activity, regulation of immune responses, and disease resistance, and has the potential for application in the aquacultural, nutraceutical, pharmaceutical, and cosmetic industries. H. pluvialis has a distinctive lifecycle as it exhibits a green motile stage and a red non-motile resting stage called an aplano-spore. In general, astaxanthin accumulation in H. pluvialis is restricted to the aplanospore stage. Astaxanthin is accumulated in extra-plastidic lipid vesicles as a secondary carotenoid, and it is believed to be synthesized in re-sponse to oxidative stress in the red aplanospore stage under unfavorable environmental conditions such as high light, temperature, and salinity, or low nutrient availability. Several enzymes such as lycopene β-cyclase (Lcy),β-carotenoid hydroxylase (CrtR-B) and β-carotene ketolase (Bkt) are involved in the astaxanthin biosynthesis pathway in H. pluvialis. Lcy catalyzes the formation of β-carotene from lycopene, and CrtR-B and Bkt catalyze further steps leading to astaxanthin synthesis. Changes in expression of the genes encoding these three enzymes can criti-cally affect the biosynthesis and accumulation of astaxanthin in H. pluvialis. In addition, various antioxidant en-zymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) also have important protective effects for combating oxidative stress under unfavourable environmental conditions. We aimed to explore the effect of salt stress on astaxanthin accumulation in H. pluvialis, by examining the mechanism of astaxanthin synthesis and the relationship between the different antioxidant mechanisms in H. pluv-ialis at high salinity. We examined its growth rate; astaxanthin content; Lcy, CrtR-B, and Bkt gene expression lev-els; SOD, CAT, and GSH-Px activities; and malondialdehyde (MDA) content at four salinity levels (0.04 mol/L, 0.08 mol/L, 0.12 mol/L, and 0.16 mol/L) over three timescales (3 days, 6 days, and 9 days). Our results showed that the density of H. pluvialis decreased under increasing salinity over different periods of time, while its mortality rate and aplanospore proportion increased with increasing salt stress concentration by the ninth day of stress. Asta-xanthin content, Lcy, CrtR-B, and Bkt gene expressions increased over time with increasing salinity. SOD, CAT, and GSH-Px activities and MDA content also increased in comparison to H. pluvialis grown at the control level of 0 mol/L NaCl, and significantly increased at the 0.12 mol/L NaCl level (P<0.05). Astaxanthin content and Lcy, CrtR-B, and Bkt gene expressions were lower during the early and mid-stages of salt stress (e.g., on the third and sixth days of observation), and increased by the ninth day. Meanwhile, SOD, CAT, and GSH-Px activities and MDA content were higher during early and mid-stage stress, but were lower by the ninth day. These results suggest that salt stress can improve astaxanthin accumulation over time in H. pluvialis at the appropriate level of salt stress, despite its negative effects on growth. Astaxanthin synthesis in H. pluvialis is promoted mainly through an increase in the transcription level of astaxanthin synthesis-related enzyme genes under salt stress, and the antioxidant activities of astaxanthin and antioxidant enzymes complement one other to protect from oxidative damage under salt stress. This study provides a new insight into the astaxanthin synthesis and antioxidant mechanisms in H. pluvialis.%为探讨盐胁迫对雨生红球藻(Haematococcus pluvialis)虾青素合成的影响与机理,以及雨生红球藻各抗氧化机制之间的关系,本研究采用生化和分子生物学方法研究了不同浓度(0.04 mol/L、0.08 mol/L、0.12 mol/L和0.16 mol/L)和不同时间(3 d、6 d和9 d)的盐(NaCl)胁迫对雨生红球藻生长、虾青素积累、番茄红素 β-环化酶(Lcy)、β-胡萝卜素羟化酶(CrtR-B)和 β-胡萝卜素酮化酶(Bkt)基因表达、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)的活性以及丙二醛(MDA)含量的影响.结果表明,各胁迫时间的雨生红球藻的密度均随着盐胁迫浓度的增加而不断下降,在盐胁迫的第9天,雨生红球藻的死亡率和孢子比例均随着盐胁迫浓度的增加而不断升高;雨生红球藻虾青素含量、Lcy、CrtR-B和Bkt基因表达量均随着盐胁迫浓度和时间的增加而不断提高;雨生红球藻SOD、CAT和GSH-Px活性以及MDA含量在不同浓度和不同时间的盐胁迫下与对照组(0.00 mol/L NaCl)相比均升高,且在不同时间的0.12 mol/L NaCl胁迫下与对照组相比均显著升高(P<0.05);雨生红球藻虾青素含量、Lcy、CrtR-B和Bkt基因表达量在盐胁迫的早期(第3天)和中期(第6天)阶段较低,在盐胁迫的后期(第9天)阶段较高,而SOD、CAT和GSH-Px活性以及MDA含量在盐胁迫的早期和中期阶段较高,在盐胁迫的后期阶段较低.实验结果说明了适当浓度和时间的盐胁迫能促进雨生红球藻累积虾青素,雨生红球藻在盐胁迫下主要是通过提高虾青素合成相关酶基因的转录水平来促进虾青素的合成,其虾青素和抗氧化酶的抗氧化活性可能互为补充,共同保护雨生红球藻免受盐胁迫的氧化损伤.
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