Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study

Mao, Xuemei; Zhang, Yu; Wang, Xiaofei; Liu, Jin

doi:10.1186/s13068-020-01714-y

Research 研究
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Published: 已出版16 April 2020

Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study
盐度诱导油脂合成与类胡萝卜素生成机制的新见解：产虾青素富油微藻 Chromochloris zofingiensis 的多组学研究

Xuemei Mao¹^na1, 毛雪梅，
Yu Zhang¹^na1, 张宇
Xiaofei Wang¹^na1 &
王小飞
…
Jin Liu ORCID: orcid.org/0000-0002-4050-7207¹ 金刘

Biotechnology for Biofuels
生物燃料的生物技术 volume 13, Article number: 73 (2020) Cite this article
文章编号：73（2020）引用此文章

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A Correction to this article was published on 07 March 2023
本文章的更正于 2023 年 3 月 7 日发布

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Abstract 摘要

Background 背景

Chromochloris zofingiensis, a freshwater alga capable of synthesizing both triacylglycerol (TAG) and astaxanthin, has been receiving increasing attention as a leading candidate producer. While the mechanism of oleaginousness and/or carotenogenesis has been studied under such induction conditions as nitrogen deprivation, high light and glucose feeding, it remains to be elucidated in response to salt stress, a condition critical for reducing freshwater footprint during algal production processes.
**Chromochloris zofingiensis** 是一种淡水藻类，能够合成三酰甘油（TAG）和虾青素（astaxanthin），近年来因其作为潜在的高效生产者而备受关注。尽管人们已经研究了在氮限制、高光强和葡萄糖供给等诱导条件下，该藻类的产油特性和/或类胡萝卜素生成机制，但其在盐胁迫条件下的反应机制尚未得到充分阐明。而盐胁迫是减少淡水使用量、优化藻类生产过程的重要条件。

Results 结果

Firstly, the effect of salt concentrations on growth, lipids and carotenoids was examined for C. zofingiensis, and 0.2 M NaCl demonstrated to be the optimal salt concentration for maximizing both TAG and astaxanthin production. Then, the time-resolved lipid and carotenoid profiles and comparative transcriptomes and metabolomes were generated in response to the optimized salt concentration for congruent analysis. A global response was triggered in C. zofingiensis allowing acclimation to salt stress, including photosynthesis impairment, ROS build-up, protein turnover, starch degradation, and TAG and astaxanthin accumulation. The lipid metabolism involved a set of stimulated biological pathways that contributed to carbon precursors, energy and reductant molecules, pushing and pulling power, and storage sink for TAG accumulation. On the other hand, salt stress suppressed lutein biosynthesis, stimulated astaxanthin biosynthesis (mainly via ketolation), yet had little effect on total carotenoid flux, leading to astaxanthin accumulation at the expense of lutein. Astaxanthin was predominantly esterified and accumulated in a well-coordinated manner with TAG, pointing to the presence of common regulators and potential communication for the two compounds. Furthermore, the comparison between salt stress and nitrogen deprivation conditions revealed distinctions in TAG and astaxanthin biosynthesis as well as critical genes with engineering potential.
首先，研究了不同盐浓度对 **C. zofingiensis** 生长、脂质和类胡萝卜素的影响，结果表明，0.2 M NaCl 是最大化 TAG 和虾青素产量的最佳盐浓度。随后，在优化的盐浓度条件下，生成了随时间变化的脂质和类胡萝卜素含量数据，并通过比较转录组和代谢组进行了综合分析。研究发现，**C. zofingiensis** 在盐胁迫下触发了一种全局响应，使其能够适应盐胁迫环境，包括光合作用受损、活性氧（ROS）积累、蛋白质周转、淀粉降解，以及 TAG 和虾青素的积累。脂质代谢涉及一系列被激活的生物途径，这些途径为 TAG 的积累提供了碳前体、能量、还原分子、推动力和储存库。另一方面，盐胁迫抑制了叶黄素的生物合成，同时通过酮化反应显著促进了虾青素的生物合成，但对类胡萝卜素的总流量影响较小，导致虾青素以牺牲叶黄素为代价积累起来。虾青素主要以酯化形式积累，并与 TAG 的积累高度协调，表明两种化合物之间可能存在共同的调控因子和潜在的信号交流。此外，与氮限制条件相比，盐胁迫下 TAG 和虾青素的生物合成表现出显著差异，同时发现了一些具有工程改造潜力的关键基因。

Conclusions 结论

Our multi-omics data and integrated analysis shed light on the salt acclimation of C. zofingiensis and underlying mechanisms of TAG and astaxanthin biosynthesis, provide engineering implications into future trait improvements, and will benefit the development of this alga for production uses under saline environment, thus reducing the footprint of freshwater.
我们的多组学数据和综合分析揭示了 C. zofingiensis 的盐适应性及其三酰基甘油（TAG）和虾青素生物合成的潜在机制，为未来性状改良提供了工程启示，并将有助于在盐环境下开发该藻类的生产应用，从而减少淡水资源的占用。

Introduction 介绍

Alternative energy sources to petroleum-based fuels have long been pursued, of which biofuels from alga, the next-generation feedstock, have received increasing interest of both academia and industry [1,2,3]. Despite the progresses achieved during the past decades, challenges remain yet to be addressed for bringing down the production cost and realizing commercialization of algal biofuels [4,5,6]. Among the strategies proposed for addressing challenges, integrated production of lipids with value-added products from algae is believed to be promising to improve algal biofuel production economics [7]. These products include, but are not restricted to high-value proteins (e.g., phycobilins), ω-3 polyunsaturated fatty acids (e.g., eicosapentaenoic acid and docosahexaenoic acid), and carotenoids (e.g., β-carotene, fucoxanthin and astaxanthin), depending on the source of algal species/strains [7]. It is worth noting that, from a biorefinery point of view, concurrent synthesis of value-added products and lipids by algae is a prerequisite for implementation of the integrated production concept. Triacylglycerol (TAG), the ideal lipid for making biodiesel, generally accumulates in algae under stress conditions [1]. Among the above-mentioned value-added products, astaxanthin represents a high-value carotenoid with broad industrial applications and tends to accumulate in certain algae upon these TAG-induction stresses [8,9,10,11,12,13], pointing to the feasibility of using algae for integrated production of the two compounds.
长期以来，人们一直在寻找石油基燃料的替代能源，其中以藻类作为下一代原料来源的生物燃料受到学术界和工业界越来越多的关注[1, 2, 3]。尽管过去几十年间取得了一些进展，但在降低生产成本和实现藻类生物燃料商业化方面仍存在许多尚待解决的挑战[4, 5, 6]。在为应对这些挑战而提出的各种策略中，通过藻类综合生产脂类和高附加值产品被认为是改善藻类生物燃料生产经济性的有前景方法[7]。这些高附加值产品包括但不限于高价值蛋白质（如藻胆蛋白）、ω-3 多不饱和脂肪酸（如二十碳五烯酸和二十二碳六烯酸）以及类胡萝卜素（如β-胡萝卜素、岩藻黄素和虾青素），具体取决于藻类物种/菌株的来源[7]。值得注意的是，从生物炼制的角度来看，藻类同时合成高附加值产品和脂类是实施综合生产概念的前提条件。三酰基甘油（TAG）是生产生物柴油的理想脂质，通常在藻类受到胁迫条件下积累[1]。在上述高附加值产品中，虾青素是一种具有广泛工业应用的高价值类胡萝卜素，往往在某些藻类受到这些 TAG 诱导胁迫时积累[8, 9, 10, 11, 12, 13]，这表明利用藻类综合生产这两种化合物是可行的。

Chromochloris zofingiensis, also referred to as Chlorella zofingiensis, is a freshwater green alga capable of growing robustly under multiple trophic conditions, reaching up to 10 and 100 g L⁻¹ for photoautotrophic and heterotrophic modes, respectively [9, 11, 12, 14]. C. zofingiensis synthesizes a high level of intracellular TAG (up to 50% dry weight) and has been cited as a potential feedstock for biodiesel [11, 12, 14, 15]. The alga is also able to synthesize high-value ketocarotenoids and is thought to be a candidate astaxanthin producer alternative to Haematococcus pluvialis [16]. The robustness in concurrent accumulation of TAG and astaxanthin [11, 14, 17] and availability of chromosome-level genome sequence [18] enable C. zofingiensis as an emerging model alga for both fundamental studies and industrial applications. While many studies deal with the engineering of culture conditions for TAG and astaxanthin production by C. zofingiensis [9, 11, 12, 14, 17, 19,20,21,22], the molecular mechanisms underlying their biosynthesis are less touched and remain to be fully explored in a system-level manner.
Chromochloris zofingiensis，也被称为 Chlorella zofingiensis，是一种能够在多种营养条件下强劲生长的淡水绿藻，在光自养和异养模式下其生物量分别可达到 10 g/L 和 100 g/L [9, 11, 12, 14]。C. zofingiensis 能够合成高水平的细胞内三酰基甘油（TAG）（干重高达 50%），被认为是潜在的生物柴油原料 [11, 12, 14, 15]。此外，该藻类还能合成高价值的酮类类胡萝卜素，被认为是雨生红球藻（Haematococcus pluvialis）的替代虾青素生产候选者 [16]。其同时积累 TAG 和虾青素的能力 [11, 14, 17]，以及染色体水平基因组序列的可用性 [18]，使 C. zofingiensis 成为一个新兴的藻类模型，可用于基础研究和工业应用。尽管已有许多研究探讨了通过优化培养条件来促进 C. zofingiensis 生产 TAG 和虾青素 [9, 11, 12, 14, 17, 19, 20, 21, 22]，但其生物合成的分子机制研究较少，系统层面的探索仍有待深入。

Chromochloris zofingiensis has the capacity to synthesize both TAG and astaxanthin in response to such cues as the deprivation of nutrients (e.g., nitrogen and sulfur), high light, and glucose induction [11, 14, 17, 23, 24]. The shortage of freshwater resources has led to studying the response of freshwater algae to salt and potential of utilizing seawater for production applications [25,26,27,28,29]. It has been reported that C. zofingiensis can tolerate moderate salt concentrations and accumulate astaxanthin as a response [9]. By contrast, the effect of salt stress on TAG synthesis by the alga remains to be evaluated. Recently, several transcriptomic studies have been performed for C. zofingiensis under the conditions of nitrogen deprivation, high light and glucose feeding [15, 18, 24, 30], contributing to the understanding of biosynthesis of TAG and/or astaxanthin. However, the data under salt stress conditions are still lacking for C. zofingiensis and the underlying mechanisms are yet to be disclosed. To fill the gap, here we optimized the salt concentrations for maximizing both TAG and astaxanthin production by C. zofingiensis, generated comparative transcriptomes and metabolomes, and determined the time-course profiles of lipids, carotenoids and other compounds. The congruent analysis of these large data sets shed light on the mechanisms of salt stress-associated oleaginousness and carotenogenesis in the emerging model alga C. zofingiensis, identifies potential limiting factors for TAG and astaxanthin accumulation, and provides useful implications into future genetic engineering of this alga for trait improvements.
Chromochloris zofingiensis 能够在缺乏营养（如氮和硫）、高光照和葡萄糖诱导等刺激下合成甘油三酯（TAG）和虾青素 [11, 14, 17, 23, 24]。淡水资源的短缺促使人们开始研究淡水藻类对盐的响应以及利用海水进行生产的可能性 [25, 26, 27, 28, 29]。据报道，C. zofingiensis 能够耐受中等盐浓度，并以积累虾青素作为响应 [9]。相比之下，盐胁迫对该藻类 TAG 合成的影响仍需评估。最近，已有多项针对 C. zofingiensis 在氮缺乏、高光照和葡萄糖供给条件下的转录组学研究 [15, 18, 24, 30]，这些研究加深了对 TAG 和/或虾青素生物合成的理解。然而，目前对 C. zofingiensis 在盐胁迫条件下的数据仍然缺乏，其潜在机制尚未揭示。为填补这一空白，本研究优化了盐浓度以最大化 C. zofingiensis 的 TAG 和虾青素产量，生成了比较转录组和代谢组数据，并测定了脂类、类胡萝卜素及其他化合物的时间动态变化。对这些大规模数据集的综合分析揭示了盐胁迫相关的产油性和类胡萝卜素生成机制，识别了限制 TAG 和虾青素积累的潜在因素，并为未来通过遗传工程改良该藻类性状提供了有价值的启示。

Results 结果

Optimization of salinity levels for lipid and astaxanthin production by C. zofingiensis
优化盐度水平以提高 C. zofingiensis 的脂质和虾青素产量

To investigate the effect of salinity levels on growth and production of lipids and astaxanthin, sodium chloride (NaCl) was employed with five concentrations (0, 0.1, 0.2, 0.4, and 0.6 M). Apparently, the algal growth was impaired by salt treatment in a concentration-dependent manner: the higher the salt concentration, the lower the cell density and Fv/Fm value (Fig. 1a, b). Specifically, the growth was attenuated moderately by 0.1 and 0.2 M salt; by contrast, the alga showed nearly blocked proliferation in the presence of 0.4 and 0.6 M salt. Accordingly, the biomass concentration achieved after 4 days of cultivation was negatively associated with the salt concentration (Fig. 1c). Salt treatment also affected cell size as suggested by the elevated per cell weight (Fig. 1d). The salt-caused growth impairment to different degrees has been reported for many other freshwater algal strains [25,26,27,28,29, 31,32,33,34,35].
为了研究盐度水平对藻类生长及脂质和虾青素生产的影响，采用了五种浓度的氯化钠（NaCl）（0、0.1、0.2、0.4 和 0.6 M）。显然，盐处理以浓度依赖的方式抑制了藻类的生长：盐浓度越高，细胞密度和 Fv/Fm 值越低（图 1a, b）。具体而言，0.1 和 0.2 M 盐浓度对生长的抑制作用较为温和；相比之下，在 0.4 和 0.6 M 盐浓度下，藻类几乎完全停止增殖。因此，经过 4 天培养后获得的生物量浓度与盐浓度呈负相关（图 1c）。盐处理还影响了细胞大小，这从每个细胞重量的增加可以看出（图 1d）。不同程度的盐引起的生长抑制现象已在许多其他淡水藻类中有所报道 [25, 26, 27, 28, 29, 31, 32, 33, 34, 35]。

The content of total fatty acids (TFA) showed a considerable increase (over twofold) under the moderate salt concentration of 0.2 M, but did not increase further under high salt concentrations (Fig. 1e). Oleic acid (C18:1) that is believed to be beneficial to the quality of biodiesel [36], also increased in the presence of salt (Fig. 1f). Triacylglycerol (TAG), the most energy-dense lipid ideal for biodiesel production [1], exhibited a more drastic increase upon salt stress than TFA did and reached the highest content of 152 mg g⁻¹ (ca. threefold increase) under 0.2 M salt concentration (Fig. 1g). On the other hand, the secondary ketocarotenoid astaxanthin showed a considerable increase under higher salt concentrations (0.2–0.6 M) and its content reached the maximum under 0.4 M salt concentration (3.1 mg g⁻¹), which is 6.4-fold higher than that without salt treatment (Fig. 1h). From the production point of view, productivity is a more desirable parameter. Although the biomass productivity was attenuated by salt treatment (Fig. 1i), the productivities of TFA, TAG and astaxanthin were all promoted and reached the maximum under the moderate salt concentration of 0.2 M (Fig. 1j–l). These results together indicated that salt treatment is a simple and feasible strategy to boost both TAG and astaxanthin production, which is not only biotechnologically favorable, but also environmentally friendly, as it can reduce the usage of freshwater.
总脂肪酸（TFA）的含量在 0.2 M 的适度盐浓度下显著增加（超过两倍），但在更高的盐浓度下并未进一步增加（图 1e）。被认为对生物柴油质量有益的油酸（C18:1）在盐的作用下也有所增加（图 1f）。三酰基甘油（TAG）作为一种非常适合生物柴油生产的高能量密度脂质[1]，在盐胁迫下的增加幅度比 TFA 更显著，在 0.2 M 盐浓度下达到最高含量 152 mg/g（约增加三倍）（图 1g）。另一方面，次生酮类类胡萝卜素虾青素在较高盐浓度（0.2–0.6 M）下显著增加，其含量在 0.4 M 盐浓度下达到最大值（3.1 mg/g），是未处理盐条件下的 6.4 倍（图 1h）。从生产的角度来看，生产率是一个更理想的参数。尽管盐处理削弱了生物量生产率（图 1i），但 TFA、TAG 和虾青素的生产率均得到促进，并在适度盐浓度 0.2 M 下达到最大值（图 1j–l）。这些结果表明，盐处理是一种简单可行的策略，可以同时促进 TAG 和虾青素的生产，不仅具有生物技术优势，而且环保，可减少淡水的使用。

Time-resolved biochemical analysis of C. zofingiensis in response to salt treatment
盐处理下 C. zofingiensis 的时序生化分析

As demonstrated above, 0.2 M NaCl was the ideal salt concentration for both TAG and astaxanthin accumulation (Fig. 1). Under this concentration, the biochemical analysis was further investigated in a time-resolved manner to probe the dynamic changes of cellular compounds. Upon the salt treatment, protein showed little change (Fig. 2). This is different from nitrogen starvation (ND) treatment, where protein exhibited an immediate decrease [15]. Starch, the major carbohydrate reserve, maintained its content during the early treatment and decreased thereafter (Fig. 2). On the other hand, the total lipid content showed a gradual increase in response to salt treatment (Fig. 2). Specifically, the neutral lipid TAG had a basal level and was induced sharply by salt, accompanied by the decrease of polar lipids. A total of eight polar lipid classes were determined, namely, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG), diacylglycerol-N,N,N-trimethylhomoserine (DGTS), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). In response to salt treatment, MGDG, the main component of thylakoid membrane, was severely attenuated; DGDG, SQDG, and PG were also decreased, but occurring during the late stress period to a lesser extent; by contrast, the others showed no significant change during the whole period (Fig. 2). The content of individual fatty acids was altered by salt treatment and most of them increased, though to various extents. Notably, C18:1, the major fatty acid found in C. zofingiensis, exhibited the greatest increase. Furthermore, taking into account the relative abundance of fatty acids, while saturated fatty acids (SFA) showed little change, monounsaturated fatty acids (MUFA) particularly C18:1 increased considerably at the expense of polyunsaturated fatty acids (PUFA) (Additional file 1: Figure S1).
如上所示，0.2 M NaCl 是促进 TAG 和虾青素积累的理想盐浓度（图 1）。在此浓度下，对生化分析进行了时间分辨研究，以探究细胞化合物的动态变化。在盐处理下，蛋白质几乎没有变化（图 2）。这与氮饥饿（ND）处理不同，后者会导致蛋白质立即减少 [15]。淀粉作为主要的碳水化合物储备，在早期处理阶段保持不变，随后减少（图 2）。另一方面，总脂质含量在盐处理下逐渐增加（图 2）。具体而言，中性脂质 TAG 在基础水平上受到盐的显著诱导，同时伴随着极性脂质的减少。共检测到八种极性脂质类别，即单半乳糖二酰基甘油（MGDG）、双半乳糖二酰基甘油（DGDG）、磺基奎诺糖基二酰基甘油（SQDG）、二酰基甘油-N,N,N-三甲基高丝氨酸（DGTS）、磷脂酰甘油（PG）、磷脂酰胆碱（PC）、磷脂酰肌醇（PI）和磷脂酰乙醇胺（PE）。在盐处理下，作为类囊体膜主要成分的 MGDG 显著减少；DGDG、SQDG 和 PG 也减少，但主要发生在后期压力阶段且程度较轻；相比之下，其余脂质在整个过程中未发生显著变化（图 2）。盐处理改变了单个脂肪酸的含量，大多数脂肪酸均有所增加，但程度不同。值得注意的是，C18:1 作为 C. zofingiensis 中的主要脂肪酸，表现出最大幅度的增加。此外，考虑到脂肪酸的相对丰度，饱和脂肪酸（SFA）变化不大，而单不饱和脂肪酸（MUFA），特别是 C18:1，显著增加，伴随着多不饱和脂肪酸（PUFA）的减少（附加文件 1：图 S1）。

Upon salt treatment, total carotenoids showed little variation until the late culture period (96 h), where a slight increase was observed. Specifically, there was a considerable decrease for primary carotenoids, particular β-carotene and lutein; by contrast, secondary carotenoids exhibited a vast increase, with astaxanthin and canthaxanthin being the most elevated ones. In this context, it is likely that secondary carotenoids accumulated at the expense of primary carotenoids, as suggested by the strong negative correlation (Additional file 1: Figure S2). It is worth noting that astaxanthin was present predominantly in the form of ester (both mono-ester and di-ester), which accounted for more than 80% of total astaxanthin (Additional file 1: Figure S3). Apparently, astaxanthin correlated well with TAG in response to salt treatment (Additional file 1: Figure S2), which is consistent with the results under other induction conditions [11] and further implies the coordinated biosynthesis of TAG and astaxanthin in C. zofingiensis.
在盐处理下，总类胡萝卜素在早期培养阶段（96 小时之前）变化不大，但在后期培养阶段略有增加。具体来说，主要类胡萝卜素（尤其是β-胡萝卜素和叶黄素）显著减少；相反，次生类胡萝卜素大幅增加，其中虾青素和角黄素的升高最为显著。在这种情况下，次生类胡萝卜素可能以牺牲主要类胡萝卜素为代价进行积累，这从强负相关关系中得到了支持（附加文件 1：图 S2）。值得注意的是，虾青素主要以酯化形式存在（包括单酯和双酯形式），占总虾青素的 80%以上（附加文件 1：图 S3）。显然，在盐处理下，虾青素与 TAG（甘油三酯）相关性较高（附加文件 1：图 S2），这一点与其他诱导条件下的结果一致 [11]，进一步暗示了 C. zofingiensis 中 TAG 与虾青素的协同生物合成过程。

In addition, the algal samples from 0 and 12 h of salt treatment were subjected to untargeted metabolomics analysis, which identified a total of 141 metabolites (Additional file 2: Data S1). Upon salt treatment, 50 metabolites (34%) underwent a significant change (at least a 1.5-fold change and p < 0.05): 38 decreased and 12 increased. The former metabolites were enriched in amino acid metabolism and TCA cycle, while the latter ones showed no specific enrichment.
此外，经过 0 小时和 12 小时盐处理的藻类样本进行了非靶向代谢组学分析，共鉴定出 141 种代谢物（附加文件 2：数据 S1）。在盐处理过程中，有 50 种代谢物（34%）发生了显著变化（至少 1.5 倍变化，且 p < 0.05）：其中 38 种减少，12 种增加。前者主要富集于氨基酸代谢和三羧酸循环，而后者未显示特定的富集。

Transcriptomic analysis for global gene expression upon salt induction
盐诱导下全球基因表达的转录组分析

To understand the underlying mechanisms of lipogenesis and carotenogenesis induced by salt stress, the global gene expression was investigated by RNA-seq in parallel with the biochemical analysis in C. zofingiensis. The algal samples from 0 and 6 h were used for RNA-seq, with biological triplicates for each time point. Six high-quality transcriptomes were produced (Additional file 3: Table S1): the biological triplicates in each group had a high consistence and distinguished from the other group based on the Pearson correlation and principal component analysis (Fig. 3a, b). Furthermore, quantitative real-time PCR (qPCR) was employed to validate the transcriptomes by analyzing 24 genes (Additional file 3: Table S2), and the plotting results indicated a high coefficient of 0.88 (R²) (Additional file 1: Figure S4). More than 14,000 genes were mapped for each sample, most of which had a FPKM (fragments per kilobase of transcript per million mapped reads) value no less than 1 (Fig. 3c and Additional file 4: Data S2).
为了研究盐胁迫诱导的脂质生成和类胡萝卜素生成的潜在机制，通过 RNA 测序结合生化分析对 C. zofingiensis 的全基因表达进行了研究。RNA 测序使用了 0 小时和 6 小时的藻类样本，每个时间点设置了生物学三次重复。共生成了六个高质量的转录组（附加文件 3：表 S1）：每组的生物学三次重复具有较高的一致性，并通过 Pearson 相关性和主成分分析区分出了不同组（图 3a, b）。此外，通过对 24 个基因的分析（附加文件 3：表 S2），采用定量实时 PCR（qPCR）验证了转录组数据，绘图结果显示相关系数为 0.88（R ² ）（附加文件 1：图 S4）。每个样本中映射了超过 14,000 个基因，其中大多数的 FPKM 值不低于 1（图 3c 和附加文件 4：数据 S2）。

Based on the definition of differentially expressed gene (DEG) stated in “Methods”, 6473 genes were assigned as salt-responsive DEGs (Additional file 5: Data S3): 2878 genes were up-regulated and 3595 were down-regulated (Fig. 3d, e). KEGG pathway functional enrichment analysis indicated several up-regulated (amino acid biosynthesis, ribosome biogenesis, glycolysis, proteasome-related, etc.) and down-regulated (chlorophyll metabolism, DNA replication, photosynthesis-antenna proteins, etc.) pathways in response to salt stress (Fig. 3f; Additional file 6: Data S4). According to Liu et al. [15], the DEGs were also manually categorized into 12 groups (Fig. 3g; Additional file 7: Data S5). The genes in function unknown category have the greatest percentage for both up-regulated (46%) and down-regulated (58%) DEGs. This is not surprising as about half of the genes in C. zofingiensis were annotated with unknown function [18]. Metabolism represents the second largest category for both up-regulated and down-regulated DEGs, suggesting the complex regulation of metabolic pathways upon salt treatment. In the categories of photosynthesis, cell cycle, and cell structure and component, there are much more down-regulated DEGs than up-regulated DEGs, indicative of the repression of photosynthesis and cell division, which is consistent with the retarded cell proliferation caused by salt treatment (Fig. 1).
根据“方法”中对差异表达基因（DEG）的定义，6473 个基因被归类为盐响应性差异表达基因（附加文件 5：数据 S3）：其中 2878 个基因上调，3595 个基因下调（图 3d, e）。KEGG 通路功能富集分析表明，在盐胁迫下，上调的通路包括氨基酸生物合成、核糖体生物合成、糖酵解、蛋白酶体相关通路等，而下调的通路则包括叶绿素代谢、DNA 复制、光合作用天线蛋白等（图 3f；附加文件 6：数据 S4）。根据 Liu 等人的研究[15]，这些差异表达基因还被手动分为 12 类（图 3g；附加文件 7：数据 S5）。在功能未知类别中，无论是上调（46%）还是下调（58%）的差异表达基因比例都最高。这并不令人意外，因为约有一半的 C. zofingiensis 基因被注释为功能未知[18]。代谢是上调和下调差异表达基因的第二大类别，这表明盐处理会对代谢通路进行复杂的调控。在光合作用、细胞周期以及细胞结构与成分类别中，下调的差异表达基因显著多于上调的差异表达基因，这表明光合作用和细胞分裂受到了抑制，这与盐处理导致的细胞增殖减缓一致（图 1）。

Furthermore, considering the particular interest of this study in lipid metabolism and carotenoid synthesis, all putative genes involved in the pathways were manually identified and their expression profiles were listed in Additional file 8: Data S6 and Additional file 9: Data S7, respectively. Of the 192 lipid metabolism-related genes, 94 were DEGs: 61 (65%) were up-regulated and 33 (35%) were down-regulated. Of the 35 carotenoid synthesis-related genes, 16 belonged to DEGs. The regulation of lipid metabolism and carotenoid synthesis in response to salt stress are detailed in the subsequent sections by the congruent analysis of RNA-seq data and biochemical variations.
此外，鉴于本研究对脂质代谢和类胡萝卜素合成的特殊关注，所有涉及这些途径的推定基因均被手动鉴定，其表达谱分别列于附加文件 8：Data S6 和附加文件 9：Data S7。在 192 个与脂质代谢相关的基因中，有 94 个为差异表达基因（DEGs）：其中 61 个（65%）上调，33 个（35%）下调。在 35 个与类胡萝卜素合成相关的基因中，有 16 个属于差异表达基因（DEGs）。脂质代谢和类胡萝卜素合成在盐胁迫下的调控，通过 RNA-seq 数据与生化变化的联合分析，详述于以下章节。

Salt stress promotes fatty acid synthesis while attenuating its β-oxidation
盐胁迫促进脂肪酸合成，同时抑制其β-氧化

Resembling higher plants, the de novo fatty acid synthesis in algae occurs in the chloroplast and involves a set of enzymes [37]. Using acetyl-CoA as the substrate, acetyl-CoA carboxylase (ACCase) catalyzes the formation of malonyl-CoA, which is regarded as the first committed step for the de novo fatty acid synthesis [38]. Searching C. zofingiensis genome identified a prokaryotic form of ACCase consisting of four chloroplast-targeted subunits: carboxyltransferase subunits alpha (Cz02g12030) and beta (Cz02g17060), biotin carboxyl carrier protein (two isoforms, Cz03g28270 and Cz06g20040), and biotin carboxylase (Cz13g10110), and a eukaryotic multifunctional form (Cz19g10190) targeted to the cytosol (Fig. 4a; Additional file 8: Data S6). In response to salt stress, all subunits of the chloroplastic ACCase were considerably up-regulated (over eightfold), suggesting that the chloroplastic ACCase represents a committed enzyme controlling the biosynthesis of fatty acids in C. zofingiensis. The salt-induced expression of ACCase has also been reported in some other algae including Chlamydomonas [26, 39, 40], Chlorella [41], and Nitzschia [42]. Obviously, in C. zofingiensis the transcriptional expression of ACCase subunits correlated well with each other (Additional file 8: Data S6), supporting their coordinated regulation as stated by previous reports [43, 44]. The cytosolic ACCase, on the other hand, was down-regulated considerably by salt stress. The malonyl moiety of malonyl-CoA is transferred to an acyl carrier protein (ACP) by the action of a malonyl-CoA:acyl carrier protein transacylase (MCT) leading to the formation of malonyl-CoA for the subsequent condensation reactions to form C16 and/or C18 fatty acids. Both ACP and MCT genes were considerably up-regulated by salt stress (Fig. 4a; Additional file 8: Data S6). However, malonyl-CoA, the product of MCT, showed no change in response to the stress (Additional file 2: Data S1). This may be attributed to the considerable up-regulation of downstream fatty acid synthesis genes (Fig. 4a), which allows rapid consumption of malonyl-CoA and thus the maintaining of its homeostasis.
类似于高等植物，藻类中的从头脂肪酸合成发生在叶绿体中，并涉及一组酶[37]。以乙酰辅酶 A（acetyl-CoA）为底物，乙酰辅酶 A 羧化酶（ACCase）催化生成丙二酰辅酶 A（malonyl-CoA），这一过程被认为是从头脂肪酸合成的第一个限速步骤[38]。通过搜索**C. zofingiensis**基因组，发现了一种由四个叶绿体靶向亚基组成的原核形式的 ACCase：羧基转移酶α亚基（Cz02g12030）和β亚基（Cz02g17060）、生物素羧基载体蛋白（两种同工型，Cz03g28270 和 Cz06g20040）、以及生物素羧化酶（Cz13g10110）；还发现了一种靶向细胞质的真核多功能形式（Cz19g10190）（图 4a；附加文件 8：数据 S6）。在盐胁迫下，所有叶绿体 ACCase 亚基的表达显著上调（超过 8 倍），表明叶绿体 ACCase 是控制**C. zofingiensis**脂肪酸生物合成的限速酶。而在一些其他藻类中，包括衣藻（Chlamydomonas）[26, 39, 40]、小球藻（Chlorella）[41]以及硅藻（Nitzschia）[42]中，也曾报道过盐诱导的 ACCase 表达。在**C. zofingiensis**中，ACCase 各亚基的转录水平显著相关（附加文件 8：数据 S6），支持了之前研究所提到的其协同调控[43, 44]。另一方面，细胞质 ACCase 在盐胁迫下显著下调。丙二酰辅酶 A 的丙二酰基通过丙二酰辅酶 A:酰基载体蛋白转酰基酶（MCT）的作用转移到酰基载体蛋白（ACP）上，从而形成用于后续缩合反应以生成 C16 和/或 C18 脂肪酸的丙二酰-ACP。ACP 和 MCT 基因在盐胁迫下均显著上调（图 4a；附加文件 8：数据 S6）。然而，MCT 的产物丙二酰辅酶 A 在盐胁迫下未显示出变化（附加文件 2：数据 S1）。这可能归因于下游脂肪酸合成基因的显著上调（图 4a），这使得丙二酰辅酶 A 能够快速被消耗，从而维持其稳态。

Fatty acid synthesis from malonyl-ACP involves a set of fatty acid synthases consisting of 3-ketoacyl-ACP synthase (KAS), 3-ketoacyl-ACP reductase (KAR), 3-hydroxyacyl-ACP dehydratase (HAD), and enoyl-ACP reductase (ENR) [38]. In C. zofingiensis, four KAS, five KAR, one HAD, and one ENR genes were found (Fig. 4a; Additional file 8: Data S6). Three types of KAS are present in higher plants responsible for the condensation of acyl-ACP with acetyl-CoA: KAS III catalyzes the first condensation of malonyl-ACP and acetyl-CoA to form C4:0-ACP, KAS I catalyzes the subsequent condensation reactions up to the formation of C16:0-ACP, while KAS II catalyzes the formation of C18:0-ACP from C16:0-ACP, the last condensation step in the chloroplast. Both C. zofingiensis KAS III and KAS I have a single-copy gene encoding a chloroplastic form (Cz18g03070 and Cz02g14160); by contrast, KAS II have two genes, one (UNPLg00257) for a chloroplastic form and the other (Cz06g14030) for a mitochondrial form: all three chloroplastic genes were greatly up-regulated (over tenfold) upon salt stress. Of the five KAR genes, only one (Cz01g34370) encoded a chloroplastic form, which together with HAD (Cz01g09160) and ENR (Cz11g20040) had comparable transcript levels and were up-regulated to similar extent. Furthermore, BC, KAS III, and the chloroplastic KAR genes were chosen for time-resolved qPCR analysis to validate gene expression profiles. All three genes showed a dramatic increase and reached their maximum after 6 h of salt stress, highly consistent with the RNA-seq data (Fig. 4a). Taken together, salt stress strongly stimulated the entire pathway for de novo fatty acid synthesis in a well-coordinated manner, thus allowing the effective utilization of acetyl-CoA to accumulate C16:0, C18:0, and C18:1 in C. zofingiensis (Fig. 4a).
从丙二酰-ACP 合成脂肪酸涉及一组脂肪酸合酶，包括 3-酮酰基-ACP 合酶（KAS）、3-酮酰基-ACP 还原酶（KAR）、3-羟酰基-ACP 脱水酶（HAD）和烯酰基-ACP 还原酶（ENR）[38]。在**C. zofingiensis**中，发现了 4 个 KAS 基因、5 个 KAR 基因、1 个 HAD 基因和 1 个 ENR 基因（图 4a；附加文件 8：数据 S6）。高等植物中存在三种类型的 KAS，负责脂酰-ACP 与乙酰-CoA 的缩合：KAS III 催化丙二酰-ACP 与乙酰-CoA 的首次缩合，生成 C4:0-ACP；KAS I 催化随后的缩合反应，直至生成 C16:0-ACP；而 KAS II 催化从 C16:0-ACP 生成 C18:0-ACP，这是叶绿体内的最后一步缩合反应。**C. zofingiensis**的 KAS III 和 KAS I 各有一个编码叶绿体形式的单拷贝基因（Cz18g03070 和 Cz02g14160）；相比之下，KAS II 有两个基因，一个（UNPLg00257）编码叶绿体形式，另一个（Cz06g14030）编码线粒体形式：三个叶绿体基因在盐胁迫下均显著上调（超过 10 倍）。在五个 KAR 基因中，仅有一个（Cz01g34370）编码叶绿体形式，与 HAD（Cz01g09160）和 ENR（Cz11g20040）具有相似的转录水平，并在相同程度上被上调。此外，选择了 BC、KAS III 和叶绿体 KAR 基因进行时间分辨 qPCR 分析，以验证基因表达模式。这三个基因在盐胁迫 6 小时后显著上升并达到最高值，与 RNA-seq 数据高度一致（图 4a）。总之，盐胁迫显著刺激了整个新的脂肪酸合成途径，以良好的协调方式促进了乙酰-CoA 的有效利用，从而在**C. zofingiensis**中积累了 C16:0、C18:0 和 C18:1（图 4a）。

The de novo synthesized fatty acids (in the form of acyl-ACP) can be either directly incorporated into glycerolipids by chloroplast-localized acyltransferases or released as free fatty acids by an acyl-ACP thioesterase (FAT) [37]. A single-copy FAT gene (Cz04g05080) was identified in C. zofingiensis and its transcript was up-regulated considerably based on both RNA-seq and qPCR results (Fig. 4a). The released free fatty acids need to be exported across chloroplast envelopes, which is mediated by a fatty acid export 1 (FAX1) in Arabidopsis [45], and ligated to CoA via a long-chain acyl-CoA synthetase (LCAS), prior to utilization by the ER-localized acyltransferases for glycerolipid assembly. The FAX1 homologues in C. zofingiensis showed little variations upon salt stress (Fig. 4a). Among the four identified putative LCAS genes, all had a similar level of basal transcripts but only Cz11g20120 was up-regulated by salt stress (Fig. 4a; Additional file 8: Data S6). Similar to C. reinhardtii LCAS2 which contributes to TAG accumulation [46], Cz11g20120 may play a critical role in salt-induced TAG accumulation in C. zofingiensis.
新合成的脂肪酸（以酰基-ACP 形式）可以通过叶绿体定位的酰基转移酶直接掺入甘油脂中，或者通过酰基-ACP 硫酯酶（FAT）释放为游离脂肪酸 [37]。在**C. zofingiensis**中鉴定出一个单拷贝的 FAT 基因（Cz04g05080），并且根据 RNA-seq 和 qPCR 结果，其转录水平显著上调（图 4a）。释放的游离脂肪酸需要通过叶绿体膜输出，而这一过程在拟南芥中由脂肪酸输出蛋白 1（FAX1）介导 [45]，随后通过长链酰基辅酶 A 合成酶（LCAS）与 CoA 连接，才能被内质网定位的酰基转移酶利用以组装甘油脂。**C. zofingiensis**中的 FAX1 同源基因在盐胁迫下变化不大（图 4a）。在鉴定出的四个 LCAS 候选基因中，所有基因的基础转录水平相似，但只有 Cz11g20120 在盐胁迫下上调（图 4a；附加文件 8：数据 S6）。类似于**C. reinhardtii**中的 LCAS2 在 TAG 积累中的作用 [46]，Cz11g20120 可能在**C. zofingiensis**盐胁迫诱导的 TAG 积累中发挥关键作用。

The unsaturation degree of fatty acids is determined by a series of desaturases via an oxygen-dependent mechanism [37, 38]. Fatty acid desaturases are usually membrane-bound enzymes utilizing complex lipids as substrates [37]. An exception is the stearoyl-ACP desaturase (SAD), a chloroplast stroma-localized soluble enzyme catalyzing the insertion of a cis double bond to the ∆9 position of C18:0-ACP to form C18:1-ACP [47, 48]. Two SAD genes were found in C. zofingiensis: Cz04g09090 was considerably up-regulated (both RNA-seq and qPCR results), while Cz13g17200 had no change upon salt stress (Fig. 4a; Additional file 8: Data S6). Thus, it is likely the Cz04g09090 rather than Cz13g17200 that contributes to the tremendous increase in the cellular content of C18:1 (Fig. 2). Other identified desaturase genes included FAD2 (Cz03g33220), FAD4 (Cz12g10230), FAD5 (Cz07g00120, Cz06g00170, and Cz13g01140), FAD6 (Cz08g04110 and Cz11g21120), FAD7 (Cz06g28130 and Cz04g31180), and ∆4/∆6FAD (Cz06g12050 and UNPLg00012) (Fig. 4a; Additional file 8: Data S6). FAD2 is an ER-targeted desaturase responsible for introducing a double bond to the ∆12 position of C18:1 to form C18:2 bound to extrachloroplastic membrane lipids such as PE, PC, PI, and DGTS. By contrast, FAD6 is localized in the chloroplast and catalyzes the formation of C18:2 from C18:1 in the chloroplastic membrane lipids, e.g., MGDG, DGDG, SQDG, and PG. FAD7, on the other hand, resides likely in the chloroplast envelop and accesses both extrachloroplastic and chloroplastic lipids for the desaturation of C18:2 at the ∆15 position to form C18:3 [37, 49]. In C. zofingiensis, most FAD genes were up-regulated by salt stress (Fig. 4a; Additional file 8: Data S6), leading to enhanced cellular contents of unsaturated fatty acids (Fig. 2).
脂肪酸的不饱和程度是通过一系列去饱和酶通过氧依赖机制决定的[37, 38]。脂肪酸去饱和酶通常是利用复杂脂类作为底物的膜结合酶[37]。一个例外是硬脂酰-ACP 去饱和酶（SAD），它是位于叶绿体基质的可溶性酶，催化在 C18:0-ACP 的∆9 位置插入一个顺式双键，形成 C18:1-ACP[47, 48]。在**C. zofingiensis**中发现了两个 SAD 基因：**Cz04g09090**在盐胁迫下显著上调（RNA-seq 和 qPCR 结果均显示），而**Cz13g17200**则没有变化（图 4a；附加文件 8：数据 S6）。因此，可能是**Cz04g09090**而非**Cz13g17200**促成了 C18:1 细胞含量的显著增加（图 2）。其他已鉴定的去饱和酶基因包括**FAD2**（Cz03g33220）、**FAD4**（Cz12g10230）、**FAD5**（Cz07g00120、Cz06g00170 和 Cz13g01140）、**FAD6**（Cz08g04110 和 Cz11g21120）、**FAD7**（Cz06g28130 和 Cz04g31180），以及**∆4/∆6FAD**（Cz06g12050 和 UNPLg00012）（图 4a；附加文件 8：数据 S6）。**FAD2**是定位于内质网的去饱和酶，负责在 C18:1 的∆12 位置引入双键，形成 C18:2，绑定于如 PE、PC、PI 和 DGTS 等叶绿体外膜脂中。相比之下，**FAD6**定位于叶绿体中，催化叶绿体膜脂（如 MGDG、DGDG、SQDG 和 PG）中的 C18:1 转化为 C18:2。而**FAD7**可能位于叶绿体包膜，作用于叶绿体外膜和叶绿体膜脂，将 C18:2 在∆15 位置去饱和形成 C18:3[37, 49]。在**C. zofingiensis**中，大多数**FAD**基因在盐胁迫下被上调（图 4a；附加文件 8：数据 S6），从而导致细胞中不饱和脂肪酸含量的增加（图 2）。

Fatty acids can enter β-oxidation pathway for degradation, which involves a set of enzymes including LACS, acyl-CoA oxidase (AOX), enoyl-CoA hydratase (ECH), 3-hydroxyacyl-CoA dehydrogenase (HCD) and 3-ketoacyl-CoA thiolase (KATO) [37]. Overall, AOX and ECH genes were down-regulated (Fig. 4b; Additional file 8: Data S6), indicative of attenuated β-oxidation of fatty acids under salt stress conditions.
脂肪酸可以进入β-氧化途径进行降解，该过程涉及一组酶，包括 LACS、酰基-CoA 氧化酶（AOX）、烯酰基-CoA 水合酶（ECH）、3-羟基酰基-CoA 脱氢酶（HCD）和 3-酮酰基-CoA 硫解酶（KATO）[37]。总体而言，AOX 和 ECH 基因表现下调（图 4b；附加文件 8：数据 S6），表明在盐胁迫条件下脂肪酸的β-氧化受到抑制。

Salt stress likely stimulates the turnover of membrane lipids
盐胁迫可能会促进膜脂的代谢更新

It has been reported in algae that membrane lipids undergo turnover to provide fatty acyls for TAG assembly under abiotic stress conditions such as nitrogen deficiency (ND) [15, 50,51,52,53,54]. Similarly, salt stress also caused a decrease in polar lipids, but predominantly in the chloroplastic membrane lipids (MGDG, DGDG, SQDG and PG) (Figs. 2, 5a); this differs from the changes under ND conditions where all determined polar lipids were decreased [15]. However, the biosynthesis of polar lipids were not down-regulated at the transcriptional level; instead, some genes were even up-regulated by salt stress (Fig. 5b; Additional file 8: Data S6). It is likely that upon salt treatment C. zofingiensis maintained the synthesis of membrane glycerolipids, but up-regulated the expression of lipases responsible for membrane lipid degradation, thereby stimulating the turnover towards decreased polar lipids. There are a number of putative membrane lipase-encoding genes found in algae and many were considerably up-regulated under TAG induction conditions [15, 55,56,57]. Among these lipases, PGD1 from C. reinhardtii has been well investigated and showed to play a role in MGDG degradation [52]. Upon salt treatment, the PGD1 homologue in C. zofingiensis also showed a large up-regulation (Additional file 8: Data S6), suggesting its role in MGDG turnover. There are additional lipase genes that were up-regulated, such as Cz09g17170, Cz14g02110 and Cz19g09050 (Additional file 8: Data S6). They may be involved in the turnover of other membrane lipids in C. zofingiensis to provide fatty acids for TAG synthesis.
据报道，在藻类中，膜脂质会发生周转，在氮缺乏（ND）等非生物胁迫条件下为三酰甘油（TAG）的合成提供脂肪酰基[15, 50, 51, 52, 53, 54]。同样，盐胁迫也导致极性脂质的减少，但主要是在叶绿体膜脂质（MGDG、DGDG、SQDG 和 PG）中（图 2, 5a）；这与氮缺乏条件下的变化不同，后者表现为所有检测到的极性脂质均减少[15]。然而，极性脂质的生物合成在转录水平上并未下调；相反，一些基因在盐胁迫下甚至上调了（图 5b；附加文件 8：数据 S6）。这表明，在盐处理下，**C. zofingiensis** 维持了膜甘油脂的合成，但上调了负责膜脂质降解的脂肪酶的表达，从而通过周转导致极性脂质的减少。在藻类中发现了许多潜在的膜脂肪酶编码基因，其中许多在 TAG 诱导条件下显著上调[15, 55, 56, 57]。在这些脂肪酶中，来自**C. reinhardtii** 的 PGD1 已被深入研究，显示其在 MGDG 降解中发挥作用[52]。在盐处理下，**C. zofingiensis** 的 PGD1 同源基因也显著上调（附加文件 8：数据 S6），表明其在 MGDG 周转中的作用。此外，还有其他脂肪酶基因上调，例如 **Cz09g17170**、**Cz14g02110** 和 **Cz19g09050**（附加文件 8：数据 S6）。它们可能参与 **C. zofingiensis** 中其他膜脂质的周转，为 TAG 合成提供脂肪酸。

Salt stress induces the expression of TAG assembly and lipid droplet proteins for TAG accumulation
盐胁迫诱导 TAG 组装及脂质小滴相关蛋白的表达以促进 TAG 积累

TAG assembly in algae is thought to be mediated mainly by acyl-CoA-dependent Kennedy pathway and acyl-CoA-independent pathway [37, 58]. It is thought in C. reinhardtii that the former pathway contributes more than the latter one to abiotic stress-associated TAG synthesis [53, 59]. The acyl-CoA-dependent Kennedy pathway starts with glycerol-3-phosphate and involves a set of enzymes including glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), phosphatidate phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). C. zofingiensis possesses two GPATs, three LPAATs, three PAPs and ten DGATs (Fig. 5a; Additional file 8: Data S6). Upon salt stress, GPAT2 and LPAAT1 were up-regulated (Fig. 5a, b), consistent in principle with the results under ND conditions [15], supporting the role of the two acyltransferases in TAG synthesis under different abiotic stress conditions. Interestingly, under salt stress conditions, PAP3 rather than PAP1 was up-regulated (Fig. 5a, 5b), while under ND conditions, PAP1 instead of PAP3 was up-regulated [15]. This suggests that C. zofingiensis may adopt different PAPs to cope with salt and ND stresses for TAG assembly.
在藻类中，TAG 的组装主要被认为是通过依赖酰基辅酶 A（acyl-CoA）的 Kennedy 途径和非酰基辅酶 A 依赖的途径完成的 [37, 58]。在绿色单细胞藻**衣藻（C. reinhardtii）**中，前者（酰基辅酶 A 依赖途径）被认为比后者（非酰基辅酶 A 依赖途径）对非生物胁迫相关的 TAG 合成贡献更大 [53, 59]。酰基辅酶 A 依赖的 Kennedy 途径以甘油-3-磷酸（glycerol-3-phosphate）为起点，涉及一系列酶，包括甘油-3-磷酸酰基转移酶（GPAT）、溶血磷脂酸酰基转移酶（LPAAT）、磷脂酸磷酸酶（PAP）和二酰基甘油酰基转移酶（DGAT）。 **C. zofingiensis** 拥有两种 GPAT、三种 LPAAT、三种 PAP 和十种 DGAT（图 5a；附加文件 8：数据 S6）。在盐胁迫条件下，**GPAT2**和**LPAAT1**的表达上调（图 5a, 5b），这一点与缺氮（ND）条件下的结果基本一致 [15]，支持这两种酰基转移酶在不同非生物胁迫条件下参与 TAG 合成的作用。有趣的是，在盐胁迫条件下，**PAP3**的表达上调，而非**PAP1**（图 5a, 5b）；而在缺氮条件下，**PAP1**的表达上调，而非**PAP3** [15]。这表明**C. zofingiensis**可能通过选择不同的 PAP 酶来应对盐胁迫和缺氮胁迫，以完成 TAG 的组装。

DGAT catalyzes the last committed step and has been demonstrated to play a critical role in TAG accumulation in various algal species [53, 54, 60,61,62]. Recently, we showed by functional complementation in TAG-deficient yeast that seven out of the ten C. zofingiensis DGATs were able to restore TAG synthesis, with DGAT1A being most functional followed by DGTT5 and DGAT1B [63]. Upon salt treatment, only DGTT5 was up-regulated (Fig. 5a, 5b), indicative of its critical role in salt-induced TAG accumulation in C. zofingiensis. By contrast, seven DGAT genes including DGAT1A and DGTT5 were up-regulated under ND conditions [15, 63]. These may partially explain why salt stress induced less TAG than ND did in C. zofingiensis (Fig. 1g; [63]). Furthermore, DGAT1A preferred eukaryotic DAGs (sn-2 being C18:1) while DGTT5 preferred prokaryotic DAGs (sn-2 being C16:0) [63]. In consistence, salt-induced TAG had a lower percentage of eukaryotic TAG than ND-induced TAG (Additional file 1: Figure S5).
DGAT 催化了最后一个关键步骤，并被证明在多种藻类物种的 TAG 积累中起着重要作用[53, 54, 60, 61, 62]。最近，我们通过在 TAG 缺乏型酵母中的功能互补实验表明，C. zofingiensis 的 10 个 DGAT 中有 7 个能够恢复 TAG 合成，其中 DGAT1A 功能最强，其次是 DGTT5 和 DGAT1B[63]。在盐处理条件下，仅 DGTT5 被上调（图 5a, 5b），表明其在盐诱导的 C. zofingiensis TAG 积累中起关键作用。相比之下，在 ND 条件下，包括 DGAT1A 和 DGTT5 在内的 7 个 DGAT 基因均被上调[15, 63]。这可能部分解释了为什么盐胁迫诱导的 TAG 积累少于 ND 诱导的（图 1g；[63]）。此外，DGAT1A 更倾向于使用真核 DAG（sn-2 为 C18:1），而 DGTT5 更倾向于使用原核 DAG（sn-2 为 C16:0）[63]。与此一致，盐诱导的 TAG 中真核 TAG 的比例低于 ND 诱导的 TAG（补充文件 1：图 S5）。

The acyl-CoA-independent pathway for TAG synthesis is catalyzed by phospholipid:diacylglycerol acyltransferase (PDAT), which transfers an acyl moiety from phospholipids and/or other polar lipids to DAG for TAG synthesis [59]. C. zofingiensis PDAT was up-regulated upon salt stress, to the same extent as DGTT5 (Fig. 5a; Additional file 8: Data S6). Therefore, PDAT may also contribute essentially to TAG accumulation under salt conditions. It has been reported recently that PDAT interacts with DGAT for TAG assembly in higher plants [64]. This interaction may also occur for PDAT and DGTT5 in C. zofingiensis.
甘油三酯（TAG）合成的非酰基辅酶 A 依赖途径由磷脂：二酰基甘油酰基转移酶（PDAT）催化，PDAT 将酰基基团从磷脂和/或其他极性脂质转移到 DAG 以合成 TAG [59]。在盐胁迫下，*C. zofingiensis* 的 PDAT 与 DGTT5 的上调程度相同（图 5a；附加文件 8：数据 S6）。因此，PDAT 可能也对盐条件下的 TAG 积累起重要作用。最近有研究报道，PDAT 与 DGAT 在高等植物中协同作用以组装 TAG [64]。这种相互作用也可能存在于*C. zofingiensis* 的 PDAT 和 DGTT5 之间。

TAG, once synthesized, is packed into lipid droplets (LDs), the lipid-rich cellular organelles that regulate the storage and hydrolysis of neutral lipids [65]. LDs occur not only in cytosol, but also within chloroplast, which has been demonstrated in Chlamydomonas [66, 67]. Unlike Chlamydomonas, C. zofingiensis forms only cytoplasmic LDs, which are stimulated to grow in size under ND conditions [16]. The stabilization of LDs involves certain structural proteins, such as oleosin in Arabidopsis [68], MLDP in Chlamydomonas [69, 70] and Dunaliella [71], HOGP in Haematococcus [72], LDSP in Nannochloropsis [73], and StLDP in Phaeodactylum [74]. In C. zofingiensis, Cz04g29220, encoding a homologue to MLDP of green algal lineage, had a high transcript level (FPKM = 199) under non-stress conditions and exhibited a considerable up-regulation (~ 17-fold) upon salt stress (Additional file 8: Data S6). By contrast, Chlamydomonas had a much lower basal transcript level of MLDP [55], in correlation with the fact that under favorable conditions, Chlamydomonas contained trace amounts of TAG with no detectable LDs [53, 66], while C. zofingiensis accumulated TAG ~ 5% of dry weight with visible LDs peripherally scattered (Fig. 1; [16]). In addition to MLDP, C. zofingiensis harbored several caleosin genes (Cz16g16140, Cz09g31050, Cz09g11210 and Cz03g13150), which were up-regulated by salt stress (Fig. 6a; Additional file 8: Data S6). Unlike MLDP that lacks a specific hydrophobic core characteristic, caleosin has hydrophobic segments and a Ca²⁺-binding motif, possesses peroxygenase activities and is believed to be involved in oxylipin synthesis for combating stresses [75]. We recently demonstrated that C. zofingiensis MLDP and caleosins were localized in LDs with a comparable abundance [76]. The up-regulation of both MLDP and caleosins likely guarantee the stabilization of LDs as the sink for TAG storage and protect TAG against degradation under salt stress conditions.
TAG 一旦合成，就会被包装到脂滴（LDs）中，这是一种富含脂质的细胞器，负责调节中性脂质的储存和水解 [65]。脂滴不仅存在于细胞质中，还存在于叶绿体内，这一点已在衣藻中得到证实 [66, 67]。与衣藻不同，C. zofingiensis 只在细胞质中形成脂滴，并且在缺氮（ND）条件下脂滴的体积会增大 [16]。脂滴的稳定性涉及某些结构蛋白，例如拟南芥中的油胚素（oleosin）[68]，衣藻中的 MLDP [69, 70]，盐角草中的 MLDP [71]，血藻中的 HOGP [72]，小球藻中的 LDSP [73]，以及硅藻中的 StLDP [74]。在 C. zofingiensis 中，Cz04g29220 编码一种绿藻谱系中与 MLDP 同源的蛋白，其在非胁迫条件下具有较高的转录水平（FPKM = 199），并且在盐胁迫下显著上调（约 17 倍）（附加文件 8：数据 S6）。相比之下，衣藻的 MLDP 基础转录水平低得多 [55]，这与以下事实相关：在有利条件下，衣藻中仅含有微量的 TAG，且检测不到脂滴 [53, 66]，而 C. zofingiensis 的 TAG 积累量约为干重的 5%，且可见脂滴散布在细胞周围（图 1；[16]）。除了 MLDP，C. zofingiensis 还具有几个橙皮素基因（caleosin）（Cz16g16140、Cz09g31050、Cz09g11210 和 Cz03g13150），这些基因在盐胁迫下被上调（图 6a；附加文件 8：数据 S6）。与缺乏特定疏水核心特征的 MLDP 不同，橙皮素具有疏水片段和钙结合基序，具有过氧化物酶活性，并被认为参与氧脂合成以应对胁迫 [75]。我们最近证明，C. zofingiensis 的 MLDP 和橙皮素都定位于脂滴中，并具有相似的丰度 [76]。MLDP 和橙皮素的共同上调可能保证了脂滴的稳定性，使其成为 TAG 储存的场所，并在盐胁迫条件下保护 TAG 免于降解。

Salt stress enhances astaxanthin accumulation at the expense of primary carotenoids
盐胁迫通过牺牲初级类胡萝卜素来增强虾青素的积累

It has been suggested in green algae that the biosynthesis of carotenoids employs precursors derived from the chloroplastic methylerythritol phosphate (MEP) pathway rather than the cytosolic mevalonate (MVA) pathway [77,78,79]. In C. zofingiensis, all enzymes involved in the MEP pathway are present and appear to be encoded by single-copy genes, while some enzymes in the MVA pathway are missing (Additional file 9: Data S7), supporting that green algae may abandon the MVA pathway for supplying the building blocks for cellular isoprenoids [79]. The MEP pathway is initiated by 1-deoxy-d-xylulose 5-phosphate (DXP) synthase (DXS), which catalyzes the irreversible condensation of pyruvate and glyceraldehyde 3-phosphate (GAP) to form DXP. DXP is then converted to MEP mediated by DXP reductoisomerase (DXR), the first committed step of the MEP pathway towards isoprenoid synthesis. The last three steps involves 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS) and reductase (HDR), catalyzing the formation of 5-carbon isoprenoids isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In C. zofingiensis upon salt treatment, although the initial steps showed little changes, the last three steps were up-regulated (Fig. 7a; Additional file 9: Data S7), suggesting the stimulation of the MEP pathway.
研究表明，在绿色藻类中，类胡萝卜素的生物合成依赖于来自叶绿体甲基赤藓醇磷酸（MEP）途径的前体，而不是细胞质甲羟戊酸（MVA）途径的前体 [77, 78, 79]。在**C. zofingiensis**中，与 MEP 途径相关的所有酶均存在，并且似乎由单拷贝基因编码，而 MVA 途径中的某些酶缺失（附加文件 9：数据 S7）。这一发现支持绿色藻类可能放弃 MVA 途径为细胞异戊二烯类化合物提供构建模块的假说 [79]。MEP 途径由 1-脱氧-D-木酮糖-5-磷酸（DXP）合酶（DXS）启动，该酶催化丙酮酸和甘油醛-3-磷酸（GAP）的不可逆缩合反应，生成 DXP。随后，DXP 在 DXP 还原异构酶（DXR）的作用下转化为 MEP，这是 MEP 途径中通向异戊二烯类化合物合成的关键步骤。最后三个步骤涉及 2-C-甲基-D-赤藓糖-2,4-环二磷酸合酶（MCS）、4-羟基-3-甲基丁-2-烯-1-基二磷酸合酶（HDS）和还原酶（HDR），催化生成 5 碳异戊二烯类化合物——异戊烯基焦磷酸（IPP）和二甲基丙烯基焦磷酸（DMAPP）。在**C. zofingiensis**中，盐处理后，尽管初始步骤变化不大，但最后三个步骤被上调（图 7a；附加文件 9：数据 S7），表明 MEP 途径受到了刺激。

IPP and DMAPP, the building blocks of carotenoids, are converted to the 10-carbon geranyl diphosphate (GPP), followed by the condensation of two molecules of GPP to form geranylgeranyl pyrophosphate (GGPP). GGPP is further condensed to the first 40-carbon carotene phytoene, mediated by phytoene synthase (PSY). The colorless phytoene, after several desaturation and isomerization steps, is converted to lycopene. Interestingly, the genes encoding the enzymes involved in the formation of lycopene from 5-carbon isoprenoids were not up-regulated by salt stress; conversely, phytoene desaturase (PDS), which showed an up-regulation upon high light [80], was even down-regulated (Fig. 7a). This, together with up-regulation of the MEP pathway, may explain partially the build-up of IPP and DMAPP under salt stress conditions (Fig. 7a; Additional file 2: Data S1). They may be exported out of the chloroplast and serve as the precursors for sterol synthesis [79].
IPP 和 DMAPP 是类胡萝卜素的基本构件，它们被转化为 10 碳的牻牛儿二磷酸（GPP），随后两分子 GPP 结合形成牻牛儿基牻牛儿二磷酸（GGPP）。在类胡萝卜素合成酶（PSY）的作用下，GGPP 进一步缩合生成第一个 40 碳的类胡萝卜素——无色的番茄红素前体（phytoene）。无色的番茄红素前体经过多次脱饱和和异构化反应后被转化为番茄红素。有趣的是，编码从 5 碳异戊二烯类化合物合成番茄红素相关酶的基因并未因盐胁迫而上调；相反，番茄红素脱饱和酶（PDS）在强光下表现出上调[80]，但在盐胁迫下却表现为下调（图 7a）。这种现象，加上 MEP 途径的上调，可能部分解释了在盐胁迫条件下 IPP 和 DMAPP 的积累（图 7a；附加文件 2：数据 S1）。它们可能从叶绿体中输出，并作为甾醇合成的前体[79]。

Lycopene represents the branch point for α-carotene and β-carotene, which enter into the biosynthesis of lutein and astaxanthin, respectively. The severe down-regulation of lycopene ε-cyclase (LCYe, eightfold decrease) and up-regulation of lycopene β-cyclase (LCYb) likely divert the carotenoid flux away from lutein (Fig. 7a, b), thereby leading to the considerable decrease of lutein level under salt stress conditions (Fig. 2). Astaxanthin biosynthesis employs β-carotene as the direct precursor, involving multiple routes via a series of hydroxylation and ketolation steps mediated by β-carotene hydroxylase (CHYb) and ketolase (BKT). C. zofingiensis possesses one CHYb gene (Cz12g16080) and two BKT genes, BKT1 (Cz13g13100) and BKT2 (Cz04g11250). CHYb and BKT1 have been characterized by functional complementation in pathway-reconstructed E. coli cells [81, 82]. Upon salt stress, BKT1 rather than BKT2 or CHYb was up-regulated (Fig. 7), suggesting its contribution to salt-induced astaxanthin accumulation. The up-regulation of BKT1 has also been observed previously under various conditions [17, 18, 80, 82, 83], suggesting the critical role of BKT1 in astaxanthin biosynthesis regardless of induction conditions. This has been further confirmed recently through the characterization of bkt1 mutants, in which astaxanthin was almost abolished [18, 84]. Collectively, salt stress stimulated astaxanthin biosynthesis, particularly BKT1 while repressing lutein synthesis, therefore rerouting the carotenoid flux to accumulate secondary carotenoids including astaxanthin at the expense of primary carotenoids.
番茄红素是α-胡萝卜素和β-胡萝卜素的分支点，分别参与了叶黄素和虾青素的生物合成。番茄红素ε-环化酶（LCYe）被大幅下调（降低了 8 倍）以及番茄红素β-环化酶（LCYb）的上调，可能将类胡萝卜素的流动从叶黄素的合成中转移（图 7a, b），从而导致盐胁迫条件下叶黄素水平的显著下降（图 2）。虾青素的生物合成以β-胡萝卜素为直接前体，通过一系列由β-胡萝卜素羟化酶（CHYb）和酮化酶（BKT）介导的羟化和酮化步骤，涉及多条途径完成。**C. zofingiensis**（红发塔胞藻）中具有一个 CHYb 基因（Cz12g16080）和两个 BKT 基因，分别为 BKT1（Cz13g13100）和 BKT2（Cz04g11250）。通过在重建代谢途径的**E. coli**（大肠杆菌）细胞中的功能互补实验，已对 CHYb 和 BKT1 进行了功能验证[81, 82]。在盐胁迫下，BKT1 而非 BKT2 或 CHYb 被上调（图 7），表明其对盐胁迫诱导虾青素积累的贡献。在各种条件下，BKT1 的上调已被观察到[17, 18, 80, 82, 83]，表明 BKT1 在虾青素生物合成中的关键作用，无论诱导条件如何。通过对**bkt1**突变体的表征进一步证实了这一点，在这些突变体中，虾青素几乎完全消失[18, 84]。总体来看，盐胁迫促进了虾青素的生物合成，特别是 BKT1 的表达，同时抑制了叶黄素的合成，从而将类胡萝卜素的流动重新分配，以牺牲初级类胡萝卜素为代价积累次级类胡萝卜素，包括虾青素。

Discussion 讨论

Salt stress triggers a global response of C. zofingiensis
盐胁迫引发了 C. zofingiensis 的全局响应

Salt stress is a well-known abiotic stress that has multiplex effects on photosynthetic eukaryotes, such as photosynthesis impairment, ROS accumulation, protein turnover, glycerol build-up, oil accumulation, or carotenogenesis, depending on organisms [9, 28, 29, 40, 85,86,87]. The adverse impact of salt stress on photosynthesis has been observed for freshwater algae particularly the model alga Chlamydomonas reinhardtii, leading to attenuated biomass production in response to salt stress [28, 40, 88]. In C. zofingiensis, the vast majority of genes involved in chlorophyll biosynthesis, photosystem I and II, cytochrome b6/f complex and light harvest complexes showed a severe down-regulation upon salt stress (Additional file 10: Data S8). This, together with the decrease in photosynthetic pigments (chlorophylls, β-carotene, lutein, etc.) and chloroplast membrane lipids (MGDG, DGDG, PG, and SQDG) (Fig. 2; Additional file 1: Figure S6), firmly supports the impairment of the light reactions of photosynthesis. Similarly to higher plants, the Calvin–Benson cycle is believed to play a major role in algae for the photosynthetic fixation of CO₂ [89, 90]. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which catalyzes the carboxylation of ribulose 1,5-bisphosphate (RuBP), is a highly conserved, rate-limiting enzyme that initiates the cycle [91]. The activity of Rubisco, on the other hand, is regulated by Rubisco activase in an ATP-dependent manner [92]. The regeneration of RuBP also plays an important role in controlling the Calvin–Benson cycle for CO₂ fixation [93]. Upon salt stress, C. zofingiensis exhibited a considerable down-regulation for Rubisco (over 15-fold decrease), Rubisco activase and phosphoribulokinase (responsible for the last step of RuBP regeneration) at the transcriptional level (Additional file 11: Data S9); accordingly, a decrease was observed for 3-phosphoglycerate, the C3 product of carboxylation (Additional file 2: Data S1), indicative of the attenuation of Calvin–Benson cycle. In this case, upon salt treatment, the light reactions were severely repressed leading to less production of ATP and NADPH molecules for photosynthetic fixation of CO₂ and therefore the attenuated CO₂ fixation ability. As a consequence, C. zofingiensis had retarded cell growth and impaired biomass production (Fig. 1). This may also be a transient adaptive feature for survival as it allows algal cells to rely on more resources such as energy and building blocks to combat the salt stress.
盐胁迫是一种众所周知的非生物胁迫，对光合真核生物具有多重影响，例如光合作用受损、活性氧（ROS）积累、蛋白质周转、甘油积累、油脂堆积或类胡萝卜素生成，这些影响因生物体而异 [9, 28, 29, 40, 85, 86, 87]。盐胁迫对光合作用的负面影响已在淡水藻类中观察到，特别是在模式藻种莱茵衣藻（Chlamydomonas reinhardtii），表现为盐胁迫下生物质生产的减弱 [28, 40, 88]。在 C. zofingiensis 中，大多数参与叶绿素生物合成、光系统 I 和 II、细胞色素 b6/f 复合体和光捕获复合体的基因，在盐胁迫下表现出显著的下调（附加文件 10：数据 S8）。此外，光合作用色素（如叶绿素、β-胡萝卜素、叶黄素等）和叶绿体膜脂（MGDG、DGDG、PG 和 SQDG）的减少（图 2；附加文件 1：图 S6）进一步支持了光合作用光反应受到损害的结论。类似于高等植物，卡尔文–本森循环被认为在藻类中起主要作用，用于光合作用固定 CO₂ [89, 90]。核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）催化核酮糖-1,5-二磷酸（RuBP）的羧化反应，是启动这一循环的高度保守且限制速率的酶 [91]。另一方面，Rubisco 的活性由 Rubisco 活化酶在 ATP 依赖的过程中调控 [92]。RuBP 的再生对控制卡尔文–本森循环中的 CO₂固定也起着重要作用 [93]。在盐胁迫下，C. zofingiensis 在转录水平上表现出 Rubisco（降低超过 15 倍）、Rubisco 活化酶和磷酸核酮糖激酶（负责 RuBP 再生最后一步）的显著下调（附加文件 11：数据 S9）；同时，羧化产物 3-磷酸甘油酸的含量也有所减少（附加文件 2：数据 S1），表明卡尔文–本森循环被削弱。在这种情况下，盐处理导致光反应受到严重抑制，从而减少了光合作用固定 CO₂所需的 ATP 和 NADPH 的生成，因此 CO₂固定能力减弱。结果，C. zofingiensis 的细胞生长受阻，生物质生产受损（图 1）。这可能也是一种短暂的适应性特征，有助于藻类细胞将更多资源（如能量和结构成分）用于应对盐胁迫。

Salt stress has been demonstrated to exaggerate the generation of intracellular ROS in higher plants and algae [29, 40, 94]. Similarly, a considerable increase in ROS level was observed in salt-treated C. zofingiensis cells [83]. While serving as secondary messengers at basal levels, ROS in excess induces oxidative stress and is harmful to organisms [94]. To cope with the adverse effect of excess ROS, phototrophs have developed complex antioxidant mechanisms mediated by enzymatic components and non-enzymatic antioxidants; the former include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and ascorbate peroxidase (APX), while the latter are composed of ascorbic acid, reduced glutathione, proline, carotenoids, flavonoids, etc. [95]. Different algae may adopt various scavenging strategies for salt-induced ROS [28, 29, 40, 86]. In case of C. zofingiensis, upon salt stress, SOD genes were somewhat up-regulated, GPX genes were up-regulated, while CAT and APX genes were somewhat down-regulated (Additional file 12: Data S10). Therefore, SOD and GPX may contribute to enzymatic detoxification of ROS in C. zofingiensis under salt stress conditions. As for the non-enzymatic antioxidants, ascorbic acid, reduced glutathione, and proline that have been reported to accumulate upon salt treatment in other algae [28, 40], showed no increase in C. zofingiensis (Additional file 2: Data S1). By contrast, secondary carotenoids particularly astaxanthin were considerably promoted by salt stress (Fig. 2), pointing to the important role of astaxanthin in the non-enzymatic sequestration of ROS in C. zofingiensis, as is the case in H. pluvialis [96].
盐胁迫已被证明会加剧高等植物和藻类细胞内活性氧（ROS）的产生[29, 40, 94]。同样，在盐处理的**C. zofingiensis**细胞中，也观察到了 ROS 水平显著增加[83]。ROS 在基础水平上可以作为次级信使，但过量的 ROS 会引发氧化胁迫，对生物体有害[94]。为了应对过量 ROS 的不利影响，光养生物已经发展出复杂的抗氧化机制，这些机制由酶类成分和非酶类抗氧化剂介导；前者包括超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GPX）、谷胱甘肽还原酶（GR）和抗坏血酸过氧化物酶（APX），后者则由抗坏血酸、还原型谷胱甘肽、脯氨酸、类胡萝卜素、黄酮类等组成[95]。不同的藻类可能采用不同的清除盐诱导 ROS 的策略[28, 29, 40, 86]。对于**C. zofingiensis**来说，在盐胁迫下，SOD 基因有所上调，GPX 基因显著上调，而 CAT 和 APX 基因则有所下调（附加文件 12：数据 S10）。因此，SOD 和 GPX 可能在盐胁迫条件下通过酶促方式清除**C. zofingiensis**中的 ROS。至于非酶类抗氧化剂，尽管在其他藻类中已报道抗坏血酸、还原型谷胱甘肽和脯氨酸在盐处理下会积累[28, 40]，但在**C. zofingiensis**中未观察到这些物质的增加（附加文件 2：数据 S1）。相反，次级类胡萝卜素，特别是虾青素，在盐胁迫下显著增加（图 2），这表明虾青素在**C. zofingiensis**中非酶促 ROS 清除中起着重要作用，与**H. pluvialis**中的情况相似[96]。

Different algae have different salt tolerance capacities [9, 28, 39, 40]. Here were found that C. zofingiensis could tolerate NaCl up to 0.2 mM; further increase of salt concentration, however, nearly blocked the cell growth (Fig. 1). The adverse effect on algae imposed by salt stress may lie in the build-up of the excessive sodium ions in the cells and the hyperosmotic stress. Thus, organisms need to maintain both ionic and osmotic homeostasis [97]. Higher plants have evolved two main strategies to alleviate the build-up of Na⁺ in cytosol: efflux out of cells mediated by a plasma-membrane Na⁺/H⁺ antiporter encoded by the SOS1 gene and sequestration into vacuole by a vacuolar Na⁺/H⁺ exchanger encoded by NHX gene [98]. The corresponding genes in C. zofingiensis, however, showed no up-regulation in response to salt stress (Additional file 12: Data S10). In this case, the alga may adopt different mechanisms for Na⁺ detoxification. The synthesis and accumulation of glycerol is a strategy for cells to combat osmotic pressure, which has been observed in C. reinhardtii in response to salt stress [99]. In C. zofingiensis, glycerol-3-phosphatase (GPP) was up-regulated upon salt stress (Additional file 13: Data S11), but not upon ND [15], indicating that glycerol production might be stimulated under salt stress conditions.
不同种类的藻类具有不同的耐盐能力 [9, 28, 39, 40]。研究发现 **C. zofingiensis** 能够耐受高达 0.2 mM 的 NaCl；但进一步提高盐浓度几乎完全抑制了其细胞生长（图 1）。盐胁迫对藻类的不利影响可能在于细胞内过量钠离子的积累以及高渗透压应激。因此，生物体需要维持离子和渗透压的平衡 [97]。高等植物已经进化出两种主要策略以减轻细胞质中 Na⁺ 的积累：通过 SOS1 基因编码的质膜 Na⁺/H⁺ 逆向转运体将 Na⁺ 排出细胞外，以及通过 NHX 基因编码的液泡 Na⁺/H⁺ 交换体将 Na⁺ 隔离到液泡中 [98]。然而，在 **C. zofingiensis** 中，相应基因在盐胁迫下并未表现出上调表达（附加文件 12：数据 S10）。在这种情况下，该藻类可能采用不同的 Na⁺ 解毒机制。合成和积累甘油是细胞应对渗透压的一种策略，这在 **C. reinhardtii** 应对盐胁迫时已被观察到 [99]。在 **C. zofingiensis** 中，甘油-3-磷酸酶（GPP）在盐胁迫下表现出上调表达（附加文件 13：数据 S11），但在氮缺乏（ND）条件下并未上调 [15]，这表明甘油的生产可能是在盐胁迫条件下被刺激的。

Nitrogen metabolism was also affected severely by salt stress in C. zofingiensis. The transport and assimilation of various nitrogen sources including urea, nitrate, nitrite and ammonia were stimulated in response to salt stress (Additional file 14: Data S12). This may provide enough nitrogen sources for amino acid biosynthesis. In fact, the biosynthesis of many amino acids was up-regulated by salt stress (Additional file 14: Data S12). Nevertheless, the level of intracellular free amino acids did not increase but instead dropped to certain extents (Additional file 2: Data S1). Probably, the synthesized amino acids are quickly consumed by the algal cells for protein synthesis, as many ribosomal proteins, aminoacyl-tRNA synthetases, translation initiation factors and elongation factors were up-regulated (Additional file 15: Data S13). This is different from the results observed for C. zofingiensis under ND conditions [15]. The enhanced protein synthesis is likely a strategy of algal cells for replenishment, as proteins are vulnerable to denaturation caused by salt stress [40, 85, 86]. On the other hand, many chaperones particularly heat-shock proteins were up-regulated (Additional file 16: Data S14), probably preventing and/or reversing the protein denaturation. Notably, the protein catabolism was also stimulated, as indicated by the up-regulation of many proteases and proteasome proteins (Additional file 15: Data S13). This may facilitate the degradation of denatured or less needed proteins and contribute to nitrogen salvage for synthesizing desired proteins to cope with salt stress.
盐胁迫对 C. zofingiensis 的氮代谢产生了严重影响。尿素、硝酸盐、亚硝酸盐和氨等多种氮源的运输和同化在盐胁迫下受到刺激（附加文件 14：数据 S12）。这可能为氨基酸的生物合成提供了足够的氮源。事实上，许多氨基酸的生物合成在盐胁迫下被上调（附加文件 14：数据 S12）。然而，细胞内游离氨基酸的水平并未增加，反而在一定程度上下降（附加文件 2：数据 S1）。可能是因为合成的氨基酸被藻细胞迅速用于蛋白质合成，因为许多核糖体蛋白、氨酰-tRNA 合成酶、翻译起始因子和延伸因子均被上调（附加文件 15：数据 S13）。这与 C. zofingiensis 在氮缺乏条件（ND）下的观察结果不同[15]。增强的蛋白质合成可能是藻细胞的一种补偿策略，因为蛋白质在盐胁迫下容易发生变性[40, 85, 86]。另一方面，许多伴侣蛋白，特别是热休克蛋白被上调（附加文件 16：数据 S14），可能防止和/或逆转蛋白质变性。值得注意的是，蛋白质的分解代谢也被刺激，这表现在许多蛋白酶和蛋白酶体蛋白的上调（附加文件 15：数据 S13）。这可能有助于降解变性或不再需要的蛋白质，并通过氮回收为合成应对盐胁迫所需的蛋白质提供资源。

The salt stress-associated diversion of carbon from starch to storage lipids involves coordinated up-regulation of multiple biological pathways
盐胁迫相关的碳从淀粉到储存脂质的转移涉及多个生物途径的协调上调

Starch represents a major carbohydrate reserve in green algae and shares common carbon precursors with the storage lipid TAG. Seemingly, starch serves as a temporary carbon sink and is transformed to TAG for long storage, which has been observed in several algae under ND conditions [15, 89, 100]. It has also been reported that salt stress triggered TAG accumulation at the expense of starch in algae [26, 35, 39, 101], but the underlying mechanism remains rarely touched. We found in C. zofingiensis that coordinated up-regulation of multiple biological pathways contributed to the transition of starch to TAG upon salt stress, which are discussed as following.
淀粉是绿藻中的主要碳水化合物储备，与储存脂质 TAG 共享相同的碳前体。看起来，淀粉作为一种临时的碳汇，可以转化为 TAG 进行长期储存，这在几种藻类的缺氮（ND）条件下已有观察到[15, 89, 100]。还有研究报道，盐胁迫会以牺牲淀粉为代价触发藻类中 TAG 的积累[26, 35, 39, 101]，但其潜在机制鲜有涉及。我们在 C. zofingiensis 中发现，多条生物路径的协调上调促进了盐胁迫下淀粉向 TAG 的转化，具体内容如下所述。

Stimulation of starch catabolism
刺激淀粉分解

Starch biosynthesis involves a set of enzymes including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS, both soluble and granule bound) and starch branching enzyme (SBE), of which AGPase is considered as the first committed step [102]. Starch catabolism, on the other hand, has two main pathways leading to the formation of glucose and glucose 1-phosphate (G1P), respectively: the former pathway involves starch debranching enzyme (SDBE) and amylase while the latter is mediated by starch phosphorylase (SPL) (Fig. 8). In C. zofingiensis, upon salt stress, genes encoding the enzymes involved in starch biosynthesis had little variation (slight increase) at the transcriptional level; by contrast, the genes in both starch degradation pathways (particularly the SPL-mediated pathway) were up-regulated (Fig. 8; Additional file 11: Data S9). Thus, it is likely that salt stress stimulated starch catabolism while maintaining starch synthesis rate resulting in a decrease in starch content (Fig. 2). This is generally consistent with the phenomenon observed in ND-treated C. zofingiensis cells [15], providing carbon building blocks via glycolysis for storage lipids.
淀粉生物合成涉及一组酶，包括 ADP-葡萄糖焦磷酸化酶（AGPase）、淀粉合酶（SS，包括可溶性和颗粒结合型）以及淀粉支化酶（SBE），其中 AGPase 被认为是第一个关键步骤 [102]。另一方面，淀粉分解有两条主要途径，分别导致葡萄糖和葡萄糖-1-磷酸（G1P）的形成：前者途径涉及淀粉去支化酶（SDBE）和淀粉酶，而后者由淀粉磷酸化酶（SPL）介导（图 8）。在 C. zofingiensis 中，面对盐胁迫，与淀粉生物合成相关的酶编码基因在转录水平上的变化很小（略有增加）；相比之下，两种淀粉降解途径（特别是 SPL 介导的途径）的基因均被上调表达（图 8；附加文件 11：数据 S9）。因此，盐胁迫可能刺激了淀粉分解，同时维持了淀粉的合成速率，导致淀粉含量的下降（图 2）。这与在 ND 处理的 C. zofingiensis 细胞中观察到的现象基本一致 [15]，通过糖酵解为储存脂质提供碳骨架。

Stimulation of glycolysis and repression of gluconeogenesis
糖酵解的激活和糖异生的抑制

Similar to Chlamydomonas and Nannochloropsis [89, 90], C. zofingiensis has both chloroplastic and cytosolic glycolysis pathways [15]. Glycolysis is composed of three irreversible steps, namely, glucose phosphorylation catalyzed by hexokinase (HK) or glucokinase (GLK), fructose-6-phosphate (F6P) phosphorylation by 6-phosphofructokinase (PFK), and pyruvate formation by pyruvate kinase (PK). The PK-catalyzed reaction is also the last step of glycolysis and determines the rate of this pathway. In response to salt treatment, a considerable increase (~ 30-fold) in the transcript of PK genes was observed (Fig. 8; Additional file 11: Data S9), indicative of the stimulation of glycolysis. Accordingly, phosphoenolpyruvate, the substrate of PK, showed a drop upon salt treatment (Additional file 2: Data S1). Notably, overall, the chloroplastic PK genes showed little change while the cytosolic ones were strongly up-regulated (Additional file 11: Data S9), pointing to the dominant contribution of cytosolic glycolysis to pyruvate generation under salt stress conditions. It is worth noting that neither chloroplastic nor cytosolic glycolysis is complete (Fig. 8). Therefore, certain transporters for sugar intermediates are in need to facilitate the completion of glycolysis, such as triose phosphate/phosphate translocator (TPT) and phosphoenolpyruvate/phosphate translocator (PPT), which were up-regulated by salt (Additional file 12: Data S10). As an opposite pathway to glycolysis, gluconeogenesis has four irreversible steps, catalyzed by pyruvate carboxylase (PYC), phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (PBP), and G6P phosphatase. The genes encoding the first three enzymes were identified in C. zofingiensis and all showed a considerable decrease at the transcriptional level upon salt treatment (Fig. 8; Additional file 11: Data S9), suggesting the repression of gluconeogenesis. Thus, by stimulating glycolysis and repressing gluconeogenesis, salt stress drove carbon flux from starch degradation products to pyruvate for the downstream production of acetyl-CoA, the precursor of de novo fatty acid synthesis.
与**衣藻 (Chlamydomonas)** 和 **纳诺氯虫藻 (Nannochloropsis)** 相似 [89, 90]，**C. zofingiensis** 同样具有叶绿体和胞质两种糖酵解途径 [15]。糖酵解由三个不可逆的步骤组成，即由己糖激酶 (HK) 或葡萄糖激酶 (GLK) 催化的葡萄糖磷酸化、6-磷酸果糖 (F6P) 经 6-磷酸果糖激酶 (PFK) 催化的磷酸化，以及由丙酮酸激酶 (PK) 催化生成丙酮酸的反应。PK 催化的反应是糖酵解的最后一步，同时决定了该途径的速率。在盐处理下，PK 基因的转录水平显著增加了约 30 倍（图 8；附加文件 11：数据 S9），表明糖酵解受到激活。相应地，PK 的底物磷酸烯醇丙酮酸 (phosphoenolpyruvate, PEP) 在盐处理下出现了下降（附加文件 2：数据 S1）。值得注意的是，叶绿体中的 PK 基因整体变化不大，而胞质中的 PK 基因则被强烈上调（附加文件 11：数据 S9），表明在盐胁迫条件下，胞质糖酵解对丙酮酸生成的贡献占主导地位。需要指出的是，无论是叶绿体糖酵解还是胞质糖酵解都不完整（图 8）。因此，需要某些糖中间体的转运蛋白来促进糖酵解的完成，例如三羧酸磷酸盐/磷酸盐转运蛋白 (TPT) 和磷酸烯醇丙酮酸/磷酸盐转运蛋白 (PPT)，这些转运蛋白在盐条件下被上调（附加文件 12：数据 S10）。作为糖酵解的反向途径，糖异生包含四个不可逆的步骤，分别由丙酮酸羧化酶 (PYC)、磷酸烯醇丙酮酸羧激酶 (PEPCK)、果糖-1,6-二磷酸酶 (PBP) 和 G6P 磷酸酶催化。在 **C. zofingiensis** 中，编码前三种酶的基因已被鉴定，并且它们在盐处理下的转录水平显著下降（图 8；附加文件 11：数据 S9），表明糖异生受到抑制。因此，通过激活糖酵解和抑制糖异生，盐胁迫驱动碳流从淀粉降解产物流向丙酮酸，用于后续生成乙酰辅酶 A（acetyl-CoA），而乙酰辅酶 A 是新生脂肪酸合成的前体物质。

Enhanced biosynthesis of acetyl-CoA and G3P
增强乙酰辅酶 A（acetyl-CoA）和甘油三磷酸（G3P）的生物合成

Acetyl-CoA has multiple sources in different cell compartments (e.g., chloroplast, cytosol and mitochondria): (1) from pyruvate catalyzed by pyruvate dehydrogenase complex (PDHC); (2) from acetate by acetyl-CoA synthetase (ACS) or by acetate kinase (AK) and phosphate acetyltransferase (PAT); (3) from citrate via ATP-citrate lyase (ACL), etc. [103]. While mitochondrial acetyl-CoA feeds into the TCA cycle, chloroplastic acetyl-CoA is used directly for de novo fatty acid synthesis [103]. In C. zofingiensis, the production of chloroplastic acetyl-CoA relies on chloroplast-targeted PDHC and ACS [15]. Upon salt stress, the subunits of chloroplastic PDHC were up-regulated considerably (over 10-fold increase) in a well-coordinated manner; although ACS overall had little change, pyruvate decarboxylase (PDC) that provides acetate for ACS were up-regulated (Fig. 8 and Additional file 13: Data S11), pointing to the enhanced synthesis of chloroplastic acetyl-CoA. Probably, the synthesized acetyl-CoA is utilized quickly by the de novo fatty acid synthesis, which was stimulated by salt stress (Fig. 4), leading to no observed accumulation of acetyl-CoA (Additional file 2: Data S1).
乙酰辅酶 A（Acetyl-CoA）在不同的细胞区室（如叶绿体、细胞质和线粒体）有多种来源：(1) 由丙酮酸经丙酮酸脱氢酶复合体（PDHC）催化生成；(2) 由乙酸经乙酰辅酶 A 合成酶（ACS）或通过乙酸激酶（AK）和磷酸乙酰转移酶（PAT）生成；(3) 由柠檬酸经 ATP-柠檬酸裂解酶（ACL）生成等 [103]。线粒体中的乙酰辅酶 A 进入三羧酸循环（TCA 循环），而叶绿体中的乙酰辅酶 A 则直接用于新生脂肪酸的合成 [103]。在**C. zofingiensis**中，叶绿体乙酰辅酶 A 的生成依赖于靶向叶绿体的 PDHC 和 ACS [15]。在盐胁迫下，叶绿体 PDHC 的亚基被显著上调（增加超过 10 倍），并表现出高度协调的调控；尽管 ACS 整体变化不大，但为 ACS 提供乙酸的丙酮酸脱羧酶（PDC）被上调（图 8 和附加文件 13：数据 S11），表明叶绿体乙酰辅酶 A 的合成增强。可能合成的乙酰辅酶 A 被快速用于盐胁迫刺激下的新生脂肪酸合成（图 4），因此未观察到乙酰辅酶 A 的积累（附加文件 2：数据 S1）。

G3P, the backbone of glycerolipids, can be from dihydroxyacetone phosphate (DHAP) mediated by G3P dehydrogenase (GPDH). C. zofingiensis possesses four GPDH-encoded genes; the chloroplastic (Cz12g24180) and mitochondrial (Cz08g08240) ones were down-regulated, while the two cytosolic ones (Cz04g17090 and Cz10g29180) were up-regulated (fivefold increase for Cz04g17090) by salt stress (Fig. 8 and Additional file 13: Data S11). This is generally consistent with the results under ND conditions that Cz04g17090 was up-regulated [15], indicative of its critical role in G3P production for glycerolipid assembly. Interestingly, in C. reinhardtii, the chloroplastic GPDH genes rather than the cytosolic ones were up-regulated by ND or salt stress, and played a role in TAG accumulation [98, 104]. This may partially explain that under TAG induction conditions, C. zofingiensis activates transcriptional up-regulation of the extrachloroplastic GPAT (Fig. 6; [15]), while C. reinhardtii activates transcriptional up-regulation of the chloroplastic one [89]. G3P can also be derived from glycerol mediated by glycerol kinase (GK). Seemingly, the contribution of GK to G3P provision is marginal under salt stress, as C. zofingiensis showed little change in the overall transcript level of GK genes (Additional file 13: Data S11). Despite the stimulation of G3P synthesis, its level saw a drop upon salt stress (Additional file 2: Data S1), likely because it is consumed for TAG assembly and glycerol production (mediated by glycerol-3-phosphatase), which were both up-regulated (Fig. 6; Additional file 8: Data S6 and Additional file 13: S11).
G3P 是甘油脂的骨架，可以通过 G3P 脱氢酶（GPDH）介导从二羟丙酮磷酸（DHAP）生成。C. zofingiensis 拥有四个编码 GPDH 的基因；其中叶绿体（Cz12g24180）和线粒体（Cz08g08240）基因受到下调，而两个胞质基因（Cz04g17090 和 Cz10g29180）在盐胁迫下被上调（Cz04g17090 上调了五倍）（图 8 和附加文件 13：数据 S11）。这一结果与非氮（ND）条件下 Cz04g17090 被上调的情况基本一致[15]，表明其在 G3P 生成用于甘油脂组装过程中起着关键作用。有趣的是，在 C. reinhardtii 中，叶绿体 GPDH 基因而非胞质基因在 ND 或盐胁迫下被上调，并在 TAG 积累中发挥作用[98, 104]。这可能部分解释了在 TAG 诱导条件下，C. zofingiensis 激活了叶绿体外 GPAT 的转录上调（图 6；[15]），而 C. reinhardtii 则激活了叶绿体 GPAT 的转录上调[89]。G3P 还可以通过甘油激酶（GK）介导从甘油中生成。然而，在盐胁迫下，GK 对 G3P 提供的贡献似乎很小，因为 C. zofingiensis 的 GK 基因总体转录水平几乎没有变化（附加文件 13：数据 S11）。尽管 G3P 的合成受到刺激，但其水平在盐胁迫下有所下降（附加文件 2：数据 S1），可能是因为它被消耗用于 TAG 的组装和甘油的生成（由甘油-3-磷酸酶介导），而这两者均被上调（图 6；附加文件 8：数据 S6 和附加文件 13：S11）。

Stimulated generation of reductant and energy
刺激还原剂和能量的生成

De novo synthesis and desaturation of fatty acids require input of substantial amounts of reductant (e.g., NADPH) and energy (e.g., ATP). The considerable up-regulation of fatty acid synthetic pathways (Fig. 4) and increase of fatty acids (Fig. 2) suggested the need of stimulated provision of NADPH and ATP under salt stress conditions. Probably, multiple sources are stimulated to meet NADPH need in C. zofingiensis. Firstly, ferredoxin NADP reductase (FNR), catalyzing the NADPH-producing step in photosynthesis, was up-regulated (Additional file 13: Data S11). This is interesting and different from the results under ND conditions where FNR gene was down-regulated in algae [15, 56, 90]. Secondly, the enzymes responsible for the two NADPH-producing steps in the oxidative pentose phosphate (OPP) pathway, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGD), were all up-regulated at the transcriptional level (Additional file 13: Data S11), in agreement with the results under ND conditions [15]. Besides, some NADP⁺-dependent enzymes may contribute to the reductant generation, such as malic enzyme (ME; Cz06g32210), malate dehydrogenase (MDH; Cz01g16230) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cz06g05120), which were up-regulated mildly (Additional file 13: Data S11). Considering the abundance and up-regulation extent of these NADPH-producing enzymes at the transcriptional level (Additional file 13: Data S11), the OPP pathway may play a major role in NADPH supply in C. zofingiensis under salt stress conditions. As for ATP, it is likely contributed by substrate-level phosphorylation in glycolysis and the TCA cycle under salt stress conditions: (1) up-regulation of PKs that catalyze the last step of glycolysis for ATP generation; (2) up-regulation of isocitrate dehydrogenase (IDH) that produces ATP, and of 2-oxoglutarate dehydrogenase (OGDH) that produces NADH for ATP generation via oxidative phosphorylation (Fig. 8; Additional file 13: Data S11). Accordingly, the metabolites involved in the TCA cycle (e.g., isocitrate, 2-oxoglutarate and succinate) were decreased (Additional file 2: Data S1), which is generally consistent with the ND-induced results [15, 105]. Meanwhile, ATP transporters such as mitochondrial ATP/ADP carrier (AAC) that transports ATP from mitochondria to cytosol and chloroplastic ATP/ADP antiporter that transports ATP from cytosol to chloroplast [106], were up-regulated to meet the enhanced consumption of ATP for fatty acid synthesis in the chloroplast (Fig. 8; Additional file 12: Data S10).
脂肪酸的从头合成和去饱和化需要大量还原剂（如 NADPH）和能量（如 ATP）的输入。脂肪酸合成途径的显著上调（图 4）以及脂肪酸含量的增加（图 2）表明，在盐胁迫条件下，需要增强 NADPH 和 ATP 的供给。可能多种来源被激活以满足 C. zofingiensis 对 NADPH 的需求。首先，催化光合作用中 NADPH 生成步骤的铁氧还蛋白 NADP 还原酶（FNR）被上调（附加文件 13：数据 S11）。这一点很有趣，与非缺氮（ND）条件下的结果不同，在 ND 条件下，藻类的 FNR 基因被下调 [15, 56, 90]。其次，氧化磷酸戊糖途径（OPP）中两个 NADPH 生成步骤的关键酶，即 6-磷酸葡萄糖脱氢酶（G6PDH）和 6-磷酸葡糖酸脱氢酶（6PGD），在转录水平上均被上调（附加文件 13：数据 S11），这一结果与 ND 条件下的结果一致 [15]。此外，一些 NADP⁺ 依赖的酶也可能参与还原剂的生成，例如苹果酸酶（ME；Cz06g32210）、苹果酸脱氢酶（MDH；Cz01g16230）和甘油醛-3-磷酸脱氢酶（GAPDH；Cz06g05120），这些酶均有轻微上调（附加文件 13：数据 S11）。综合这些 NADPH 生成酶的转录水平丰度和上调程度（附加文件 13：数据 S11），在盐胁迫条件下，OPP 途径可能是 C. zofingiensis 中 NADPH 供应的主要来源。对于 ATP，在盐胁迫条件下可能通过糖酵解和 TCA 循环中的底物水平磷酸化贡献：（1）催化糖酵解最后一步生成 ATP 的丙酮酸激酶（PKs）上调；（2）生成 ATP 的异柠檬酸脱氢酶（IDH）和通过氧化磷酸化生成 ATP 的 2-氧戊二酸脱氢酶（OGDH）上调（图 8；附加文件 13：数据 S11）。相应地，TCA 循环相关的代谢物（如异柠檬酸、2-氧戊二酸和琥珀酸）减少（附加文件 2：数据 S1），这与 ND 条件下诱导的结果基本一致 [15, 105]。同时，一些 ATP 转运蛋白，如将 ATP 从线粒体转运到细胞质的线粒体 ATP/ADP 转运蛋白（AAC）以及将 ATP 从细胞质转运到叶绿体的叶绿体 ATP/ADP 逆向转运蛋白 [106]，被上调以满足叶绿体中脂肪酸合成对 ATP 消耗增加的需求（图 8；附加文件 12：数据 S10）。

C. zofingiensis synthesizes TAG and astaxanthin in a coordinated way
C. zofingiensis 以协调的方式合成甘油三酯和虾青素。

TAG and astaxanthin are secondary metabolites and generally accumulate under unfavorable growth conditions in algae. The simultaneous accumulation of TAG and astaxanthin has been observed in H. pluvialis [8, 13]. Similarly, C. zofingiensis synthesizes TAG and astaxanthin simultaneously under ND, high light, or the combination of the two stress conditions [11]. This was also observed in response to salt stress, with a high coefficient of 0.998 (Additional file 1: Figure S2), pointing to the coordinated synthesis of TAG and astaxanthin regardless of stress conditions in C. zofingiensis. Nevertheless, it is worth noting that TAG levels are much higher (ca. 100-fold) than astaxanthin levels (Additional file 1: Figure S2), suggesting the predominant carbon flux towards TAG compared to astaxanthin. The presence of crosstalk has been proposed between TAG and astaxanthin biosynthesis [8, 11]. First, algal TAG and astaxanthin may compete with each other for carbon precursors. It has been reported that impairing TAG accumulation via de novo fatty acid synthesis inhibitor led to enhanced astaxanthin level in C. zofingiensis [11]. Second, astaxanthin, predominantly esterified with fatty acids in algae, is stored in LDs that consist of a TAG-filled hydrophobic core [76]. Probably, a basal level of TAG is required to build LDs for astaxanthin storage [8, 13]. The astaxanthin-stored LDs, peripherally scattered within C. zofingiensis cells [16], likely function as a ‘sunscreen’ to alleviate photodamage. TAG and astaxanthin in LDs may also serve as the so-called compatible solutes to help algal cells cope with the osmotic stress caused by salt. The synthesis of TAG and astaxanthin in C. zofingiensis might be subjected to coordinated regulation by such regulators as transcription factors (TFs). In response to salt stress, of the 180 putative TFs, 19 were up-regulated and 48 were down-regulated (Additional file 17: Data S15). MYB (Cz10g24240) and bHLH (UNPLg00160), which might regulate both TAG and astaxanthin biosynthesis based on co-expression analysis [15], were up-regulated by salt stress (Additional file 17: Data S15), consistent with the expression pattern of key genes involved in TAG and astaxanthin synthesis, e.g., GPAT2, LPAAT1, PAP3, DGTT5, MLDP, LCYb, BKT1 (Additional file 8: Data S6 and Additional file 9: Data S7). In this context, these TFs are potential engineering targets for improving both TAG and astaxanthin in C. zofingiensis and worthy of further investigation.
TAG 和虾青素是次级代谢产物，通常在不利的生长条件下于藻类中积累。在雨生红球藻（H. pluvialis）中已观察到 TAG 和虾青素的同时积累 [8, 13]。类似地，C. zofingiensis 在氮缺乏（ND）、高光或两者结合的胁迫条件下同时合成 TAG 和虾青素 [11]。在盐胁迫条件下也观察到了这一现象，其相关系数高达 0.998（附加文件 1：图 S2），表明无论何种胁迫条件，C. zofingiensis 中 TAG 和虾青素的合成是协调进行的。然而需要注意的是，TAG 水平远高于虾青素水平（约高 100 倍）（附加文件 1：图 S2），这表明相较于虾青素，碳通量更倾向于 TAG 的合成。已有研究提出 TAG 和虾青素的生物合成之间可能存在互作关系 [8, 11]。首先，藻类 TAG 和虾青素可能会竞争碳前体。有报道指出，通过抑制新生脂肪酸合成来减少 TAG 积累，可提升 C. zofingiensis 中虾青素的水平 [11]。其次，虾青素在藻类中主要与脂肪酸酯化后储存在由 TAG 填充的疏水核心的脂滴（LDs）中 [76]。可能需要一定的基础水平的 TAG 以构建用于储存虾青素的脂滴 [8, 13]。C. zofingiensis 细胞内周边分布的虾青素储存脂滴 [16]，可能作为“防晒霜”以减轻光损伤。脂滴中的 TAG 和虾青素还可能作为所谓的兼容溶质，帮助藻类细胞应对盐胁迫导致的渗透胁迫。C. zofingiensis 中 TAG 和虾青素的合成可能受如转录因子（TFs）等调控因子的协调调节。响应盐胁迫时，180 个推测的转录因子中有 19 个上调，48 个下调（附加文件 17：数据 S15）。MYB（Cz10g24240）和 bHLH（UNPLg00160）可能通过共表达分析调控 TAG 和虾青素的生物合成 [15]，在盐胁迫下被上调（附加文件 17：数据 S15），与 TAG 和虾青素合成关键基因（如 GPAT2、LPAAT1、PAP3、DGTT5、MLDP、LCYb、BKT1）的表达模式一致（附加文件 8：数据 S6 和附加文件 9：数据 S7）。在此背景下，这些转录因子是改良 C. zofingiensis 中 TAG 和虾青素的潜在工程目标，值得进一步研究。

C. zofingiensis has distinctions in oleaginousness and carotenogenesis between salt stress and nitrogen deprivation conditions
C. zofingiensis 在盐胁迫和缺氮条件下在产油性和类胡萝卜素生成方面存在差异。

Both salt stress and ND conditions can activate certain biological pathways (Table 1) and trigger the accumulation of TAG and astaxanthin in C. zofingiensis (Fig. 2; [9, 11, 14]). Yet there are distinctions in oleaginousness for TAG synthesis between the two conditions (Table 1): (1) under salt stress conditions, the chloroplastic PDHC likely plays a major role in the production of acetyl-CoA in chloroplast for de novo fatty acid synthesis, while under ND conditions, ACS may also contribute; (2) as for the fatty acid desaturation, seemingly ND rather than salt stress activates up-regulation of all FADs; (3) of the enzymes involved in TAG assembly, only the chloroplastic LPAAT is up-regulated by salt stress while both chloroplastic and extrachloroplastic ones are up-regulated by ND, the extrachloroplastic PAP is up-regulated by salt stress while the chloroplastic one is up-regulated by ND, and only the type II DGAT (DGTT5) is up-regulated by salt stress while both type I and type II DGATs (DGAT1A, DGAT1B, and DGTT5 through DGTT8) are up-regulated by ND. In this context, salt stress seemingly stimulates less than ND in ‘pushing’ (pushes carbon flux to precursors for lipid metabolism) and ‘pulling’ (pulls fatty acids to glycerol backbone for TAG assembly), thus leading to a lower TAG level (Fig. 1; [11]). C. zofingiensis also shows variations in astaxanthin biosynthesis between salt stress and ND conditions. The conversion of β-carotene to astaxanthin involves two types of enzymes, BKT and CHYb [16]. Only BKT1 is activated upon salt stress while both BKTs (BKT1 and BKT2), CHYb and AAT are activated by ND (Table 1), which may explain why C. zofingiensis accumulates more astaxanthin under ND conditions (Fig. 1; [11]). Furthermore, the comparison reveals the critical genes that contribute to TAG and astaxanthin build-up regardless of stress conditions, such as GPAT2, LPAAT1, DGTT5, and BKT1, which are potential candidates for manipulation to improve the traits of C. zofingiensis once an advanced genetic toolbox is established.
盐胁迫和氮限制（ND）条件都可以激活某些生物路径（表 1），并触发 C. zofingiensis 中三酰甘油（TAG）和虾青素的积累（图 2；[9, 11, 14]）。然而，在这两种条件下，TAG 合成的含油性存在差异（表 1）：(1) 在盐胁迫条件下，叶绿体的丙酮酸脱氢酶复合体（PDHC）可能在叶绿体中乙酰辅酶 A（acetyl-CoA）的生成中起主要作用，以用于新生脂肪酸的合成，而在 ND 条件下，乙酰辅酶 A 合成酶（ACS）也可能参与其中；(2) 在脂肪酸的不饱和化方面，似乎是 ND 而非盐胁迫激活了所有脂肪酸脱饱和酶（FADs）的上调；(3) 在参与 TAG 组装的酶中，仅叶绿体的 LPAAT 在盐胁迫下被上调，而在 ND 条件下，叶绿体和叶绿体外的 LPAAT 都被上调；叶绿体外的 PAP 在盐胁迫下被上调，而叶绿体内的 PAP 在 ND 条件下被上调；只有 II 型 DGAT（DGTT5）在盐胁迫下被上调，而在 ND 条件下，I 型和 II 型 DGAT（DGAT1A、DGAT1B 以及 DGTT5 至 DGTT8）均被上调。在这一背景下，相较于盐胁迫，ND 条件似乎在“推动”（将碳流量推向脂质代谢前体）和“拉动”（将脂肪酸拉向甘油骨架以进行 TAG 组装）方面刺激更强，从而导致更高的 TAG 水平（图 1；[11]）。 C. zofingiensis 在盐胁迫和 ND 条件下的虾青素生物合成也表现出差异。β-胡萝卜素向虾青素的转化涉及两种酶：BKT 和 CHYb [16]。在盐胁迫下，仅 BKT1 被激活，而在 ND 条件下，BKT1 和 BKT2、CHYb 以及 AAT 均被激活（表 1），这可能解释了为什么 C. zofingiensis 在 ND 条件下积累更多的虾青素（图 1；[11]）。此外，这种比较揭示了一些在各种胁迫条件下均有助于 TAG 和虾青素积累的关键基因，例如 GPAT2、LPAAT1、DGTT5 和 BKT1，这些基因是潜在的改造目标，一旦建立了先进的遗传工具箱，就可以用来改善 C. zofingiensis 的性状。

Table 1 Comparison of the up-regulated genes involved in selected pathways of C. zofingiensis between salt stress and ND conditions
表 1 C. zofingiensis 在盐胁迫和非胁迫（ND）条件下参与特定通路的上调基因比较

Full size table 全尺寸表格

Conclusions 结论

Among the tested salt concentrations, 0.2 M was demonstrated to maximize both TAG and astaxanthin contents and their productivities in C. zofingiensis. Multi-omics analysis revealed a global response of C. zofingiensis to salt stress, including attenuated photosynthesis and CO₂ fixation, accelerated protein turnover, remodeled central carbon metabolism, oleaginousness and carotenogenesis, which provided a solid basis for better understanding TAG and astaxanthin synthesis in the alga (Fig. 9). The coordinated up-regulation of multiple pathways, such as carbon shunt from starch, acetyl-CoA production, fatty acid synthesis, membrane lipid turnover, G3P production, TAG assembly and LD formation, provided a strategic combination of ‘pushing’, ‘pulling’ and ‘protection’ to realize TAG accumulation. Astaxanthin, on the other hand, was induced to accumulate mainly by stimulating ‘pulling’ and ‘protection’ (up-regulation of LCYb, BKT1 and AAT) that diverted carotenoid flux from lutein to astaxanthin, which was esterified with fatty acids and packed into LDs for storage. TAG and astaxanthin accumulated in a coordinated manner, probably regulated by such TFs as MYB and bHLH. Comparative analysis between salt stress and ND conditions disclosed distinctions in addition to the common features with respect to oleaginousness and carotenogenesis of C. zofingiensis. Furthermore, critical enzymes and regulators for TAG and astaxanthin biosynthesis were identified. These results together (1) demonstrate the beneficial effect of salt on TAG and astaxanthin synthesis in C. zofingiensis and point to the potential of using diluted seawater for production uses; (2) shed light on the mechanisms of oleaginousness (for TAG boost) and carotenogenesis (for astaxanthin accumulation), and (3) provide candidate gene targets for future trait improvements via rational genetic engineering. It should be noted that the significant transcript change of a gene does not always guarantee its critical function and additional evidences (protein level, in vitro, in vivo, etc.) are needed.
在所测试的盐浓度中，0.2 M 被证明能够最大化 **C. zofingiensis**（小球藻）中 TAG 和虾青素的含量及其生产力。多组学分析揭示了 **C. zofingiensis** 对盐胁迫的全局响应，包括光合作用和 CO₂ 固定的减弱、蛋白质周转的加速、中心碳代谢的重塑、产油性和类胡萝卜素生物合成的调整，这为更好地理解该藻类中 TAG 和虾青素的合成提供了坚实的基础（图 9）。多条代谢途径的协调上调，例如来自淀粉的碳流分流、乙酰辅酶 A（acetyl-CoA）的生成、脂肪酸合成、膜脂质代谢、甘油-3-磷酸（G3P）的生成、TAG 组装和脂滴（LD）形成，提供了一种“推动”（pushing）、“拉动”（pulling）和“保护”（protection）相结合的策略来实现 TAG 的积累。而虾青素的积累主要通过刺激“拉动”和“保护”（LCYb、BKT1 和 AAT 的上调）来实现，这将类胡萝卜素的流向从叶黄素转向虾青素，虾青素随后与脂肪酸酯化并封装进脂滴中储存。TAG 和虾青素以协调的方式积累，可能由类似 MYB 和 bHLH 的转录因子（TFs）调控。盐胁迫与氮限制（ND）条件的比较分析表明，两种条件在 C. zofingiensis 的产油性和类胡萝卜素生物合成方面既有共同特点，也存在差异。此外，还鉴定出了对 TAG 和虾青素生物合成起关键作用的酶和调控因子。这些结果共同表明：（1）盐对 **C. zofingiensis** 中 TAG 和虾青素合成的积极作用，并指出使用稀释海水用于生产的潜力；（2）揭示了产油性（促进 TAG 增加）和类胡萝卜素生物合成（促进虾青素积累）的机制；（3）为未来通过合理的基因工程改良性状提供了候选基因靶点。需要注意的是，基因转录水平的显著变化并不总是能保证其关键功能，还需要其他证据（蛋白水平、体外实验、体内实验等）来进一步验证。

Methods 方法

Algal strain and growth conditions
藻株及生长条件

Chromochloris zofingiensis (ATCC 30,412) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). To recover algal activity, the cells from the maintaining alga was inoculated into flasks grown aerobically at 25 °C for 6 days with orbital shaking (150 rpm) and continuous illumination (30 µE m⁻² s⁻¹). The cells were then inoculated at 10% (v/v) into a 200-mL column (3-cm diameter), and grown to exponential phase under constant illumination of 70 µE m⁻² s⁻¹ and aeration of 1.5% CO₂ enriched air. The algal cells in exponential phase were harvested by centrifugation, resuspended in fresh medium supplemented with different sodium chloride concentrations (0, 0.1, 0.2, 0.4 and 0.6 M), and allowed to grow in columns under the same conditions mentioned above.
Chromochloris zofingiensis (ATCC 30,412) 从美国典型培养物保藏中心（ATCC, Rockville, MD, USA）购买。为恢复藻类活性，将保存的藻细胞接种到烧瓶中，在 25 °C 下进行有氧培养 6 天，设置 150 rpm 的摇床振荡和 30 µE m⁻² s⁻¹的连续光照。随后，将这些细胞以 10%（体积比）的比例接种到一个 200 mL 的柱状培养器（直径为 3 cm）中，并在 70 µE m⁻² s⁻¹的恒定光照和 1.5% CO₂富集空气的条件下培养至指数生长期。在指数生长期的藻细胞通过离心收集后，重新悬浮于不同氯化钠浓度（0、0.1、0.2、0.4 和 0.6 M）补充的新鲜培养基中，并在上述相同条件下继续在柱状培养器中生长。

Determination of physiological and biochemical changes
生理和生化变化的测定

Cell number was determined under a light microscope by using a hemocytometer. Dry weight was determined by weighing pre-dried Whatman GF/C filter papers (1.2 μm pore size). Fv/Fm, the potential quantum efficiency of PSII indicating the photosynthetic performance, was measured in a pulse amplitude-modulated (PAM) fluorometry (Walz, Germany) as previously stated [15].
细胞数量使用血球计数板在光学显微镜下测定。干重通过称量预先干燥的 Whatman GF/C 滤纸（孔径 1.2 μm）确定。Fv/Fm（PSII 的潜在量子效率，表明光合作用性能）使用脉冲幅度调制（PAM）荧光仪（德国 Walz）测量，方法如前所述 [15]。

Cell samples were harvested by centrifugation and lyophilized on a freeze-drier (Labconco, MO, USA) for subsequent biochemical analysis. Protein was extracted and determined as described by Liu et al. [15]. Starch was determined using the Starch Assay Kit (Sigma-Aldrich, MO, USA) according to the manual’s instructions.
细胞样本通过离心收集，并使用冷冻干燥机（Labconco，密苏里州，美国）进行冷冻干燥，以便进行后续的生化分析。蛋白质的提取和测定按照 Liu 等人[15]的方法进行。淀粉含量使用淀粉检测试剂盒（Sigma-Aldrich，密苏里州，美国）按照说明书的指示进行测定。

Lipids were extracted with chloroform–methanol (2:1) and the total lipids were determined gravimetrically [15]. Neutral lipids and polar lipids were separated on a Silica gel 60 TLC plate (EMD Chemicals, Merck, Germany) with different mobile phases: the former used a mixture of hexane/tert-butylmethyl ether (TBME)/acetic acid (80/20/2, by vol), while the latter used a mixture of chloroform/methanol/acetic acid/water (25/4/0.7/0.3, by vol) [53]. Total lipids or individual lipids recovered from TLC plates were transesterified with sulfuric acid in methanol [11]. Fatty acid methyl esters (FAMEs) were analyzed by using an Agilent 7890 capillary gas chromatograph equipped with a 5975 C mass spectrometry detector and a HP-88 capillary column (60 m × 0.25 mm) (Agilent Technologies, CA, USA) as detailed by Mao et al. [63]. Individual FAMEs were quantified with authentic standards in the presence of the internal standard heptadecanoic acid (Sigma-Aldrich). The content of TAG and polar lipids was expressed as the content of their corresponding fatty acids.
脂质通过氯仿-甲醇（2:1）的混合物提取，总脂质含量通过重量法测定[15]。中性脂质和极性脂质在硅胶 60 薄层色谱板（EMD Chemicals，默克公司，德国）上分离，分别使用不同的流动相：中性脂质使用己烷/叔丁基甲醚（TBME）/乙酸（80/20/2，按体积比），极性脂质使用氯仿/甲醇/乙酸/水（25/4/0.7/0.3，按体积比）[53]。从薄层色谱板上回收的总脂质或单个脂质通过在甲醇中加入硫酸进行酯化[11]。脂肪酸甲酯（FAMEs）通过 Agilent 7890 毛细管气相色谱仪进行分析，该仪器配备了 5975 C 质谱检测器和 HP-88 毛细管柱（60 m × 0.25 mm）（Agilent Technologies，加利福尼亚州，美国），具体方法参考 Mao 等人[63]。单个 FAMEs 的定量使用已知标准品，并加入内标庚烷酸（Sigma-Aldrich）。甘油三酯（TAG）和极性脂质的含量以其相应脂肪酸的含量表示。

Carotenoid extraction and determination followed the procedures described in our previous study [11]. Briefly, the lyophilized cell samples were homogenized vigorously in the presence of liquid nitrogen and extracted with acetone for three times under dim light. The carotenoid extracts were then separated on a high performance liquid chromatography system, which is composed of a Waters 2695 separation module, a Waters 2996 photodiode array detector and a Waters Spherisorb 5 µm ODS2 4.6 × 50 mm analytical column (Waters, MA, USA). For the ideal separation of lutein and zeaxanthin, a Waters YMC Carotenoid C30 column (5 μm, 4.6 × 250 mm) was used. Carotenoids were identified and quantified by comparing with authentic standards regarding the retention time, absorption spectra and peak area.
类胡萝卜素的提取和测定参照我们之前的研究方法[11]。简而言之，将冻干的细胞样品在液氮存在下充分匀浆，并在弱光条件下用丙酮提取三次。类胡萝卜素提取物随后使用高效液相色谱系统进行分离，该系统包括 Waters 2695 分离模块、Waters 2996 光二极管阵列检测器以及 Waters Spherisorb 5 µm ODS2 4.6 × 50 mm 分析柱（Waters, MA, USA）。为了实现叶黄素和玉米黄质的理想分离，使用了 Waters YMC Carotenoid C30 柱（5 μm，4.6 × 250 mm）。通过与标准品的保留时间、吸收光谱和峰面积进行比较来鉴定和定量类胡萝卜素。

Metabolites were extracted with cold 80% methanol by using a mini-beadbeater (Biospec Products, OK, USA) and analyzed by the Metabolomics Facility at Technology Center for Protein Sciences, Tsinghua University, using a TSQ Quantiva Triple Quadrupole liquid chromatography–mass spectrometry (Thermo Scientific) equipped with a 100 × 3 mm Synergi™ Hydro-RP 100A column (Phenomenex, CA, USA) according to our previously described procedures [15]. Relative quantification was employed between biological conditions (0 and 12 h of salt stress) wherein the data from 0 h served as the reference control. Significant difference was achieved when the relative value showed at least a 1.5-fold change with a p-value less than 0.05 (Student’s t-test).
代谢物使用冷的 80%甲醇通过迷你珠磨仪（Biospec Products, OK, USA）提取，并由清华大学蛋白质科学技术中心代谢组学平台使用 TSQ Quantiva 三重四极杆液相色谱-质谱仪（Thermo Scientific）配备 100 × 3 mm Synergi™ Hydro-RP 100A 柱（Phenomenex, CA, USA）按照我们之前描述的流程[15]进行分析。在生物条件（盐胁迫 0 小时和 12 小时）之间采用相对定量，其中 0 小时的数据作为参考对照。当相对值显示至少 1.5 倍变化且 p 值小于 0.05（Student's t 检验）时，认为差异具有显著性。

RNA-seq and analysis of differentially expressed genes
RNA 测序及差异表达基因分析

RNA was extracted from cell samples under the two conditions (0 and 6 h of salt stress) using the plant RNA extraction kit (TaKaRa, Japan) according to the manufacturer’s instructions. Contaminated DNA was removed by treating with RNase-free DNase I (TaKaRa). The RNA quality and concentration were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies) and a NanoDrop 2000C (Thermo Scientific, DE, USA). Around 10 mg total RNA was used for mRNA purification with Sera-mag Magnetic Oligo(dT) Beads (Thermo Scientific). The transcriptome libraries were prepared using the NEBNext mRNA Library Prep Reagent Set (New England Biolabs, MA, USA) according to the manual’s instructions and sequenced on a BGISEQ-500 platform (BGI, China). The RNA-seq raw data were deposited in the Gene Expression Omnibus under accession number GSE125419.
从两种条件（0 小时和 6 小时盐胁迫）下的细胞样本中，按照制造商说明使用植物 RNA 提取试剂盒（TaKaRa，日本）提取 RNA。通过使用无 RNase 的 DNase I（TaKaRa）处理去除污染的 DNA。使用 Agilent 2100 Bioanalyzer（Agilent Technologies）和 NanoDrop 2000C（Thermo Scientific，美国特拉华州）检测 RNA 的质量和浓度。约 10 mg 总 RNA 被用于使用 Sera-mag Magnetic Oligo(dT) Beads（Thermo Scientific）纯化 mRNA。转录组文库按照说明书使用 NEBNext mRNA Library Prep Reagent Set（New England Biolabs，美国马萨诸塞州）制备，并在 BGISEQ-500 平台（BGI，中国）上进行测序。RNA-seq 原始数据已上传至 Gene Expression Omnibus，编号为 GSE125419。

Clean reads (obtained by filtering the low-quality reads, reads with adaptors and reads with unknown bases) were aligned to C. zofingiensis genome [18] by using TopHat (version 2.0.4), allowed no more than two segment mismatches. Gene expression was measured as the numbers of aligned reads to annotated genes by Cufflinks (version 2.0.4) and normalized to the number of Fragments Per Kilobase Million (FPKM). Differentially expressed genes (DEGs) in response to salt stress were defined as follows: the FPKM value of at least one condition was no less than 1 and gene expression showed at least a twofold change with the false discovery rate (FDR) adjusted p-value less than 0.05. The DEGs were manually grouped into 12 categories to analyze the possible functional enrichment as previously described [90], in addition to KEGG pathway enrichment analysis.
通过过滤低质量读段、含接头序列的读段和含未知碱基的读段获得的高质量读段（Clean reads）使用 TopHat（版本 2.0.4）比对到 **C. zofingiensis** 基因组 [18]，允许不超过两个片段错配。基因表达量通过 Cufflinks（版本 2.0.4）计算为比对到注释基因的读段数，并标准化为每千碱基转录本的百万片段数（Fragments Per Kilobase Million, FPKM）。响应盐胁迫的差异表达基因（DEGs）的定义如下：至少一个条件的 FPKM 值不低于 1，且基因表达量变化至少两倍，同时假发现率（FDR）调整后的 p 值小于 0.05。通过手动将差异基因分为 12 类以分析可能的功能富集（参考以前的文献 [90]），并进行了 KEGG 通路富集分析。

Quantitative real-time PCR for the validation of RNA-seq data
用于验证 RNA-seq 数据的定量实时 PCR

Total RNA (1 μg) extracted from cell samples under different conditions (0, 6, 12, 24 and 48 h of salt stress) was reversely transcribed to cDNA by using the PrimeScript™ RT Master Mix (TaKaRa, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, MA, USA) with SYBR^® Premix Ex Taq™ II (TaKaRa, Japan). A total of 24 genes regarding lipid metabolism, astaxanthin biosynthesis and TFs were analyzed and their qPCR primers are listed in Additional file 3: Table S2. The housekeeping gene β-actin was used as the internal control, and the relative gene expression level was calculated based on the 2^−ΔΔCt method [107].
从不同条件（盐胁迫 0、6、12、24 和 48 小时）下的细胞样本中提取的总 RNA（1 μg），根据说明书使用 PrimeScript™ RT Master Mix（TaKaRa，日本）逆转录为 cDNA。定量实时 PCR（qPCR）使用 7500 Fast Real-Time PCR System（Applied Biosystems，美国马萨诸塞州）和 SYBR Premix Ex Taq™ II（TaKaRa，日本）进行。分析了与脂质代谢、虾青素生物合成和转录因子相关的 24 个基因，其 qPCR 引物列于附加文件 3：表 S2。以管家基因 β-actin 作为内参基因，基因的相对表达水平基于 2⁻ΔΔCt 方法计算 [107]。

Availability of data and materials
数据和材料的可用性

All data generated or analyzed during this study are included in this published article and its supplementary information files.
本研究中生成或分析的所有数据均包含在本文及其补充信息文件中。

Change history 变更历史

07 March 2023
A Correction to this paper has been published: https://doi.org/10.1186/s13068-023-02282-7

2023 年 3 月 7 日，本论文已发布更正：https://doi.org/10.1186/s13068-023-02282-7

Abbreviations 缩写

AACT:: Acetoacetyl-CoA thiolase
乙酰乙酰辅酶 A 硫解酶
AAH: AAH：: Allantoin amidohydrolase
尿囊素酰胺水解酶
AAT: AAT: Long-chain-alcohol O-fatty-acyltransferase
长链醇-O-脂肪酰基转移酶
AMI: AMI: 亚马逊机器映像: Formamidase 甲酰酰胺酶
AMT: 金额: Ammonium transporter 铵转运蛋白
AMX: AMX: Amine oxidase 胺氧化酶
ATDI:: Allantoate deiminase 尿囊酸脱亚胺酶
BKT:: Beta-carotenoid ketolase
β-胡萝卜素酮化酶
BPGA: BPGA: 位并行遗传算法: 1,3-Bisphosphoglycerate 1,3-二磷酸甘油酸
BUP: BUP: β-Ureidopropionase β-脲基丙酸酶
CDP-ME: CDP-ME: 4-Diphosphocytidyl-2-C-methylerythritol
4-二磷酸胞苷-2-C-甲基赤酰醇
CDP-MEP:: 4-Diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate
4-二磷酸胞苷-2-C-甲基-D-赤藓糖-2-磷酸
CHYb: CHYb: Beta-carotenoid hydroxylase
β-胡萝卜素羟化酶
CMK:: 4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase
4-二磷酸胞苷-2-C-甲基-D-赤藓糖醇激酶
CMS: 内容管理系统: 2-C-Methyl-d-erythritol 4-phosphate cytidylyltransferase
2-C-甲基-D-赤藓糖醇-4-磷酸胞苷酰转移酶
CRTISO: CRTISO: 类胡萝卜素异构酶: Carotenoid isomerase 类胡萝卜素异构酶
CYP97A: CYP97A: Cytochrome P450 beta hydroxylase
细胞色素 P450 β-羟化酶
CYP97C: CYP97C：: Cytochrome P450 epsilon hydroxylase
细胞色素 P450 ε-羟化酶
DEG: 度: Differentially expressed gene
差异表达基因
DHAP: 二羟基丙酮磷酸 (DHAP): Dihydroxyacetone phosphate
二羟基丙酮磷酸
DHDH:: Dihydrouracil dehydrogenase
二氢尿嘧啶脱氢酶
DHP:: Dihydropyrimidinase 二氢嘧啶酶
DMAPP:: Dimethylallyl pyrophosphate
二甲基烯丙基焦磷酸
DUR1:: Urea carboxylase 尿素羧化酶
DUR2:: Allophanate hydrolase 异氰酸酯水解酶
DUR3:: Urea active transporter 尿素主动转运蛋白
DXR: DXR: 实时光线追踪: 1-Deoxy-d-xylulose 5-phosphate reductoisomerase
1-脱氧-D-木酮糖-5-磷酸还原异构酶
DXP:: 1-Deoxy-d-xylulose 5-phosphate
1-脱氧-D-木酮糖-5-磷酸
DXS: DXS: 1-Deoxy-d-xylulose 5-phosphate synthase
1-脱氧-D-木酮糖-5-磷酸合酶
E4P: E4P：赤藓糖-4-磷酸: Erythrose 4-phosphate 赤藓糖-4-磷酸
FBA: 亚马逊物流: Fructose-bisphosphate aldolase
果糖二磷酸醛缩酶
FBPase: FBPase：果糖-1,6-二磷酸酶: Fructose-1,6-bisphosphatase
果糖-1,6-二磷酸酶
FBP: FBP：: Fructose 1,6-bisphosphate
果糖-1,6-二磷酸
FDR: FDR：: False discovery rate 假发现率
F6P: 果糖-6-磷酸: Fructose 6-phosphate 果糖-6-磷酸
Fd: 前馈：: Ferredoxin 铁氧还蛋白
FNR:: Ferredoxin-NADP (+) reductase
铁氧还蛋白-NADP(+)还原酶
FPP: 第一人称视角: Farnesyl diphosphate 法尼基二磷酸
FPPS: 异戊烯基焦磷酸合成酶: Farnesyl diphosphate synthase
法呢基二磷酸合酶
GAP: 间隙：: Glyceraldehyde 3-phosphate
甘油醛-3-磷酸
GAPDH: 甘油醛-3-磷酸脱氢酶: Glyceraldehyde 3-phosphate dehydrogenase
甘油醛-3-磷酸脱氢酶
GDA: GDA: 全日参考摄入量: Guanine deaminase 鸟嘌呤脱氨酶
GDH: GDH：: Glutamate dehydrogenase 谷氨酸脱氢酶
Glu: 谷氨酸: Glutamate 谷氨酸
Gln: 谷氨酰胺: Glutamine 谷氨酰胺
GOGAT: 谷氨酰胺合酶-谷氨酸合酶循环: Glutamate synthase 谷氨酸合成酶
GGPP: GGPP（牻牛儿基牻牛儿基焦磷酸）：: Geranylgeranyl diphosphate
甲基戊烯基焦磷酸
GGPPS: GGPPS: 烯丙基二磷酸合酶: Geranylgeranyl diphosphate synthase
香叶基香叶基二磷酸合酶
GPP: GPP：: Geranyl diphosphate 香叶基二磷酸
GPPS:: Geranyl diphosphate synthase
香叶基二磷酸合酶
GS:: Glutamine synthetase 谷氨酰胺合成酶
HCR: HCR: 健康风险评估: HMG-CoA reductase HMG-CoA 还原酶
HCS:: Hydroxymethylglutaryl-CoA synthase
羟甲基戊酰辅酶 A 合酶
HDR: 高动态范围: 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate reductase
4-羟基-3-甲基丁-2-烯-1-基二磷酸还原酶
HDS: HDS: 高密度存储: 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase
4-羟基-3-甲基丁-2-烯-1-基二磷酸合酶
HGM-CoA:: 3-Hydroxy-3-methylglutaryl-CoA
3-羟基-3-甲基戊二酰辅酶 A
HMB-PP:: (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate
(E)-4-羟基-3-甲基-丁-2-烯基焦磷酸
hν:: Photon energy 光子能量
IPP: IPP: 知识产权保护: Isopentenyl pyrophosphate
异戊烯基焦磷酸
IPPI:: Isopentenyl-diphosphate Delta-isomerase
异戊烯基二磷酸Δ-异构酶
LCYb: 镱掺杂的磷光体（LCYb）: Lycopene beta cyclase 番茄红素-β-环化酶
LCYe:: Lycopene epsilon cyclase
番茄红素ε环化酶
LHC: 大型强子对撞机: Light harvesting complex
光捕获复合体
MCS: MCS：: 2-C-Methyl-d-erythritol 2,4-cyclodiphosphate synthase
2-C-甲基-D-赤藓糖 2,4-环二磷酸合酶
MDH: MDH: Malate dehydrogenase 苹果酸脱氢酶
ME: 我：: Malic enzyme 苹果酸酶
MEcPP: MEcPP：: 2-C-Methyl-d-erythritol 2,4-cyclodiphosphate
2-C-甲基-D-赤藓糖 2,4-环二磷酸
MEP: 欧洲议会议员: 2-C-Methylerythritol 4-phosphate
2-C-甲基-D-赤藓糖醇-4-磷酸
MK:: Mevalonate-5-kinase 甲瓦龙酸-5-激酶
MPK:: Phosphomevalonate kinase
磷酸甲羟戊酸激酶
MPPD:: Mevalonate-5-pyrophosphate decarboxylase
甲瓦龙酸-5-焦磷酸脱羧酶
NAR1:: Nitrate transporter: NAR1 family
硝酸盐转运蛋白：NAR1 家族
NAR2:: Nitrate transporter: NAR2 family
硝酸盐转运蛋白：NAR2 家族
ND:: Nitrogen deprivation 氮剥夺
NIR: 近红外: Nitrite reductase 亚硝酸盐还原酶
NR: 编号:: Nitrate reductase 硝酸还原酶
NRT1: NRT1: Nitrate transporter: NRT1 family
硝酸盐转运蛋白：NRT1 家族
NRT2: NRT2: Nitrate transporter: NRT1 family
硝酸盐转运蛋白：NRT1 家族
NPQ: 非光化学淬灭: Non-chemical quenching 非化学淬灭
NXS: NXS：: Neoxanthin synthase 新黄素合酶
OAA: OAA: 草酰乙酸: Oxaloacetate 草酰乙酸
2OG: 2OG: α-酮戊二酸: 2-Oxoglutarate 2-Oxoglutarate 2-氧代戊二酸
PC: 个人电脑: Plastocyanin 质体蓝素
PDS:: Phytoene desaturase 植物烯脱氢酶
PEP:: Phosphoenolpyruvate 磷酸烯醇丙酮酸
PEPC: PEPC：: Phosphoenolpyruvate carboxylase
磷酸烯醇丙酮酸羧化酶
3PGA: 3-磷酸甘油酸: 3-Phosphoglycerate 3-磷酸甘油酸
PGK: 磷酸甘油酸激酶: Phosphoglycerate kinase 磷酸甘油酸激酶
PPDK: 丙酮酸磷酸二激酶: Pyruvate phosphate dikinase
丙酮酸磷酸二激酶
PQ: PQ：: Plastoquinone 质体醌
PRK: 朝鲜民主主义人民共和国: Phosphoribulokinase 磷酸核酮糖激酶
PS: 附注：: Photosystem 光系统
PSY: PSY：: Phytoene synthase 茄红素合成酶
RBCS:: Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit
核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基
ROS:: Reactive oxygen species 活性氧类（Reactive oxygen species）
R5P: R5P：: Ribose 5-phosphate 核糖-5-磷酸（Ribose 5-phosphate）
RPH:: Ammonium transporter: Rh family
铵转运蛋白：Rh 家族
RPI: RPI: 零售价格指数: Ribose 5-phosphate isomerase
核糖-5-磷酸异构酶
RPE: 视网膜色素上皮: Ribulose-phosphate 3-epimerase
核酮糖磷酸-3-差向异构酶
RuBP: 核酮糖-1,5-二磷酸: Ribulose 1,5-bisphosphate
核酮糖-1,5-二磷酸
Ru5P:: Ribulose 5-phosphate 核酮糖-5-磷酸
SBP:: Sedoheptulose 1,7-bisphosphate
庚糖醇-1,7-二磷酸
SBPase: SBPase：磷酸甘油酸脱氢酶: Sedoheptulose-1,7-bisphosphatase
七碳糖-1,7-二磷酸酶
S7P:: Sedoheptulose 7-phosphate
七碳糖磷酸 (庚酮糖-7-磷酸)
TAG: 标签: Triacylglycerol 三酰基甘油
TIM:: Triosephosphate isomerase
三磷酸异构酶
TRK:: Transketolase 转酮醇酶
UAH:: Ureidoglycolate amidohydrolase
尿素基乙醇酸酰胺水解酶
UIAH:: Ureidoglycine aminohydrolase
脲基甘氨酸氨基水解酶
UOX:: Urate oxidase 尿酸氧化酶
VDE:: Violaxanthin de-epoxidase
叶黄素脱环氧化酶
XDH:: Xanthine dehydrogenase 黄嘌呤脱氢酶
Xu5P: Xu5P（木酮糖-5-磷酸）：: Xylulose 5-phosphate 木酮糖-5-磷酸
ZDS:: Zeta-carotene desaturase
ζ-胡萝卜素脱氢酶
ZEP: 泽普: Zeaxanthin epoxidase 玉米黄质环氧化酶
ZISO:: Zeta-carotene isomerase ζ-胡萝卜素异构酶

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Acknowledgements

We thank Dr. Henri Gerken at Arizona State University for his kind comments on the manuscript.

Funding

This work is partially supported by grants from National Key R&D Program of China (2018YFA0902500), National Youth Thousand Talents Program of China and Peking University CCUS project supported by BHP Billiton.

Author information

Xuemei Mao, Yu Zhang and Xiaofei Wang contributed equally to this work

Authors and Affiliations

Laboratory for Algae Biotechnology & Innovation, College of Engineering, Peking University, Beijing, 100871, China
Xuemei Mao, Yu Zhang, Xiaofei Wang & Jin Liu

Authors

Xuemei Mao
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Yu Zhang
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Xiaofei Wang
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Jin Liu
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Contributions

JL conceived the study and designed the experiments. XM, YZ, and XW conducted the experiments and analyzed the data. JL wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Jin Liu.

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The authors declare that they have no competing interests.

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The original online version of this article was revised: The error in the grant number has been corrected in Funding section.

Supplementary information

Additional file 1: Figure S1.

Time course of percentage of SFA, UFA, and MUFA in response to 0.2 M NaCl. SFA, saturated fatty acids; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids. Figure S2. Correlation analysis between primary and secondary carotenoids, esterified astaxanthin and TAG, and esterified astaxanthin and oleic acid. The data were from the time-resolved experiment under 0.2 M salt concentration. Figure S3. Time course distribution of free astaxanthin, mono-ester, and di-ester in response to 0.2 M NaCl. Figure S4. Consistency between RNA-seq–based and qPCR–based transcript quantification. A total of 24 genes were chosen for qPCR validation. The genes and primer sequences for qPCR are listed in Additional file 3: Table S2. Figure S5. The relative abundance of C16:0 and C18:1 in sn-2 position of TAG from C. zofingiensis under ND and SS conditions. ND, nitrogen deprivation; SS, 0.2 M salt. Asterisk indicates the significant difference (t-test, p < 0.05) between ND and SS. Figure S6. Time course of chlorophyll content in C. zofingiensis in the presence of 0.2 M salt.

Additional file 2: Data S1.

Change of metabolites in C. zofingiensis in response to salt stress.

Additional file 3: Table S1.

Reads quality and mapping ratio of the transcriptomes. Table S2. The primer sequences of selected genes used in qPCR experiments.

Additional file 4: Data S2.

List of FPKM values of all genes.

Additional file 5: Data S3.

List of 6473 DEGs.

Additional file 6: Data S4.

KEGG pathway functional enrichment of DEGs.

Additional file 7: Data S5.

Manual curation of DEGs.

Additional file 8: Data S6.

RNA-seq data for genes involved in lipid metabolism.

Additional file 9: Data S7.

RNA-seq data for genes involved in carotenogenesis.

Additional file 10: Data S8.

RNA-seq data for genes involved photosynthesis.

Additional file 11: Data S9.

RNA-seq data for genes involved in CO₂ fixation and central carbon metabolism.

Additional file 12: Data S10.

RNA-seq data for genes involved in ROS scavenging and transport of certain metabolites.

Additional file 13: Data S11.

RNA-seq data for genes involved in producing acyl-CoAs, G3P, reductant and energy molecules.

Additional file 14: Data S12.

RNA-seq data for genes involved in amino acid metabolism.

Additional file 15: Data S13.

RNA-seq data for genes involved in protein metabolism.

Additional file 16: Data S14.

RNA-seq data for genes encoding putative chaperones.

Additional file 17: Data S15.

RNA-seq data for genes encoding putative transcription factors.

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Mao, X., Zhang, Y., Wang, X. et al. Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study. Biotechnol Biofuels 13, 73 (2020). https://doi.org/10.1186/s13068-020-01714-y

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Received: 17 October 2019
Accepted: 09 April 2020
Published: 16 April 2020
DOI: https://doi.org/10.1186/s13068-020-01714-y

Abstract 摘要

Background 背景

Results 结果

Conclusions 结论

Introduction 介绍

Results 结果

Optimization of salinity levels for lipid and astaxanthin production by C. zofingiensis优化盐度水平以提高 C. zofingiensis 的脂质和虾青素产量

Time-resolved biochemical analysis of C. zofingiensis in response to salt treatment盐处理下 C. zofingiensis 的时序生化分析

Transcriptomic analysis for global gene expression upon salt induction盐诱导下全球基因表达的转录组分析

Salt stress promotes fatty acid synthesis while attenuating its β-oxidation盐胁迫促进脂肪酸合成，同时抑制其β-氧化

Salt stress likely stimulates the turnover of membrane lipids盐胁迫可能会促进膜脂的代谢更新

Salt stress induces the expression of TAG assembly and lipid droplet proteins for TAG accumulation盐胁迫诱导 TAG 组装及脂质小滴相关蛋白的表达以促进 TAG 积累

Salt stress enhances astaxanthin accumulation at the expense of primary carotenoids盐胁迫通过牺牲初级类胡萝卜素来增强虾青素的积累

Discussion 讨论

Salt stress triggers a global response of C. zofingiensis盐胁迫引发了 C. zofingiensis 的全局响应

The salt stress-associated diversion of carbon from starch to storage lipids involves coordinated up-regulation of multiple biological pathways盐胁迫相关的碳从淀粉到储存脂质的转移涉及多个生物途径的协调上调

Stimulation of starch catabolism刺激淀粉分解

Stimulation of glycolysis and repression of gluconeogenesis**糖酵解的激活和糖异生的抑制**

Enhanced biosynthesis of acetyl-CoA and G3P增强乙酰辅酶 A（acetyl-CoA）和甘油三磷酸（G3P）的生物合成

Stimulated generation of reductant and energy刺激还原剂和能量的生成

C. zofingiensis synthesizes TAG and astaxanthin in a coordinated wayC. zofingiensis 以协调的方式合成甘油三酯和虾青素。

C. zofingiensis has distinctions in oleaginousness and carotenogenesis between salt stress and nitrogen deprivation conditionsC. zofingiensis 在盐胁迫和缺氮条件下在产油性和类胡萝卜素生成方面存在差异。

Conclusions 结论

Methods 方法

Algal strain and growth conditions藻株及生长条件

Determination of physiological and biochemical changes生理和生化变化的测定

RNA-seq and analysis of differentially expressed genesRNA 测序及差异表达基因分析

Quantitative real-time PCR for the validation of RNA-seq data用于验证 RNA-seq 数据的定量实时 PCR

Availability of data and materials数据和材料的可用性

Change history 变更历史

07 March 2023

Abbreviations **缩写**

References 参考文献

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Additional information

Publisher's Note

Supplementary information

Rights and permissions

About this article

Cite this article

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Keywords

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Optimization of salinity levels for lipid and astaxanthin production by C. zofingiensis
优化盐度水平以提高 C. zofingiensis 的脂质和虾青素产量

Time-resolved biochemical analysis of C. zofingiensis in response to salt treatment
盐处理下 C. zofingiensis 的时序生化分析

Transcriptomic analysis for global gene expression upon salt induction
盐诱导下全球基因表达的转录组分析

Salt stress promotes fatty acid synthesis while attenuating its β-oxidation
盐胁迫促进脂肪酸合成，同时抑制其β-氧化

Salt stress likely stimulates the turnover of membrane lipids
盐胁迫可能会促进膜脂的代谢更新

Salt stress induces the expression of TAG assembly and lipid droplet proteins for TAG accumulation
盐胁迫诱导 TAG 组装及脂质小滴相关蛋白的表达以促进 TAG 积累

Salt stress enhances astaxanthin accumulation at the expense of primary carotenoids
盐胁迫通过牺牲初级类胡萝卜素来增强虾青素的积累

Salt stress triggers a global response of C. zofingiensis
盐胁迫引发了 C. zofingiensis 的全局响应

The salt stress-associated diversion of carbon from starch to storage lipids involves coordinated up-regulation of multiple biological pathways
盐胁迫相关的碳从淀粉到储存脂质的转移涉及多个生物途径的协调上调

Stimulation of starch catabolism
刺激淀粉分解

Stimulation of glycolysis and repression of gluconeogenesis
糖酵解的激活和糖异生的抑制

Enhanced biosynthesis of acetyl-CoA and G3P
增强乙酰辅酶 A（acetyl-CoA）和甘油三磷酸（G3P）的生物合成

Stimulated generation of reductant and energy
刺激还原剂和能量的生成

C. zofingiensis synthesizes TAG and astaxanthin in a coordinated way
C. zofingiensis 以协调的方式合成甘油三酯和虾青素。

C. zofingiensis has distinctions in oleaginousness and carotenogenesis between salt stress and nitrogen deprivation conditions
C. zofingiensis 在盐胁迫和缺氮条件下在产油性和类胡萝卜素生成方面存在差异。

Algal strain and growth conditions
藻株及生长条件

Determination of physiological and biochemical changes
生理和生化变化的测定

RNA-seq and analysis of differentially expressed genes
RNA 测序及差异表达基因分析

Quantitative real-time PCR for the validation of RNA-seq data
用于验证 RNA-seq 数据的定量实时 PCR

Availability of data and materials
数据和材料的可用性

Abbreviations 缩写