实验海洋生物学重点实验室知识库

雨生红球藻（Haematococcus pluvialis，简称红球藻）作为天然虾青素生产的最佳生物来源备受关注。红球藻的规模化培养中广泛采用的是二步法，即首先维持红球藻在绿色游动阶段增加生物量，然后改变培养条件，胁迫诱导虾青素大量积累，最终成为红色不动细胞。以往红球藻中的研究集中于游动细胞转变为不动细胞并积累虾青素的过程，缺乏对其生长繁殖机制的探讨。相较于其他经济微藻如小球藻，红球藻绿色游动细胞分裂速度依然较为缓慢，导致整个培养周期长。而红色不动细胞在适宜条件下可在短时间内萌动分裂快速释放大量游动细胞，细胞扩繁效率远高于绿色游动细胞的分裂。作为一种有潜力的提高培养效率的手段，对于不动细胞快速萌动分裂转为游动细胞的上述过程，目前的认知仍然十分浅显。影响萌动分裂的必要外部条件以及内部物质能量代谢的调控机制尚不清楚。

本研究主要从氮营养调控以及能量角度入手，借助叶绿素荧光分析，转录组和代谢组等手段，通过检测孢子囊形成、游动孢子释放、光合和呼吸初生代谢过程以及线粒体呼吸电子传递链中的交替氧化酶（AOX）途径，重点对该藻不动细胞萌动分裂过程中的物质与能量流的调控机制展开研究。获得的主要结果如下：

（1）研究明确了氮营养是红球藻不动细胞萌动分裂的必要营养条件。当氮营养的浓度低于10 mg/L时，不动细胞萌动分裂和游动孢子的释放受到限制。红色不动细胞在仅含有氮营养的培养基中即可正常萌动分裂，释放大量游动孢子。而在缺氮条件下，尽管在72 h小部分不动细胞可以释放少量游动孢子，但随后孢子囊形成和游动孢子释放均被限制，萌动分裂进程被阻断。结合叶绿素荧光参数及初生代谢组分析发现，缺氮主要限制光合碳同化速率，但对光合电子传递速率影响较小。同时，缺氮条件下，糖酵解途径（EMP），磷酸戊糖途径（PPP），三羧酸循环（TCA）等呼吸碳代谢途径被显著干扰。其中TCA循环中柠檬酸、异柠檬酸、α-酮戊二酸和延胡索酸等中间碳骨架的含量上升，但是甘氨酸、丙氨酸、鸟氨酸、瓜氨酸和精氨酸等氨基酸含量显著下降。综上所述，缺氮条件下，碳氮代谢出现失衡，无法为DNA复制中核苷酸的合成提供足够的底物。此外，由于谷氨酸的下调，光呼吸途径中有毒物质2-磷酸乙醇酸无法被及时代谢，上调了2.6倍，从而限制萌动分裂过程中呼吸与光合碳代谢进程。

（2）研究了萌动分裂过程中各呼吸途径活性的变化，包括AOX途径，细胞色素（COX）途径和剩余呼吸途径。在不动细胞萌动分裂之初的能量主要由COX途径和AOX途径两条呼吸电子传递链提供，其对总呼吸速率的贡献比例相近，分别为48.6%和44.4%。随着萌动分裂过程的进行，AOX途径活性逐渐升高，培养48 h后AOX途径活性比0 h增加82.8%。COX途径作为一种更高效的ATP生成途径，其在48 h内的稳定活性保证了萌动分裂基本能量ATP的供给。随后在72 h，游动孢子被大量释放，所有呼吸途径的活性都呈下降趋势，表明萌动分裂完成后，释放后的游动孢子对从呼吸代谢中获取碳骨架和ATP的需求减少。

（3）AOX途径的上调反映其在萌动分裂过程中发挥着积极作用。在以上研究基础上，使用AOX特异性抑制剂水杨基羟肟酸研究其在萌动分裂过程中内部能量平衡与代谢调控的关键作用。抑制AOX后，不动细胞萌动分裂随即停滞，且检测到的AOX途径活性与总呼吸速率均显著降低。在AOX途径受抑制的条件下，一方面细胞内还原当量（NADH，NADPH，FADH₂）的含量显著上调，与NADPH相关的氧化还原系统过度激活。另一方面红色不动细胞中与NADH、NADPH、和FADH₂产生相关，用于碳水化合物分解的EMP，TCA，PPP途径以及用于三酰甘油分解的脂肪酸β-氧化和乙醛酸途径的转录水平整体下调。此外，脱落酸途径也参与了AOX调控的代谢激活促进萌动分裂的过程。本研究证实AOX途径可以保证呼吸电子传递链中还原当量的快速消耗，从而加速淀粉及三酰甘油等储存物质通过呼吸代谢途径进行有效分解，为萌动分裂过程中的孢子形成提供能量和碳骨架。

综上，本文研究表明氮营养对萌动分裂过程中光合碳同化以及呼吸碳代谢的正常运行具有重要作用，其次揭示了AOX在萌动分裂过程中通过调节能量代谢来驱动物质快速流动的关键调控机制。本研究有利于加深对红球藻细胞周期调控的理解，同时有利于发展以红色不动细胞为规模种源快速扩繁，有效提高生产效率，革新生产周期的培养模式，为红球藻产业可持续发展提供理论和技术支持。

Haematococcus pluvialis is highly regarded as the best biological source for natural astaxanthin production. In large-scale cultivation of H. pluvialis, a two-step method is widely employed. Initially, green motile cells are cultivated to accumulate biomass, followed by the transformation into red non-motile cells under stress conditions accompanied by the induction of astaxanthin accumulation. Previous studies have largely focused on the astaxanthin accumulation process during the transition from motile to non-motile cells, with little in-depth exploration of their growth and reproduction mechanisms. Compared to other economic microalgae like Chlorella, green motile cells of H. pluvialis divide at a relatively slow pace, leading to a lengthy cultivation cycle. However, under suitable conditions, red non-motile cells can germinate and rapidly release a large number of motile cells, significantly outpacing the reproduction efficiency of green motile cell division. As a potential means to improve cultivation efficiency, the knowledge of the quick germination and division process from non-motile to motile cells is superficial, and the necessary external conditions and internal material and energy metabolism regulation mechanisms affecting germination are still unclear.

This study delved into the dynamics of material and energy flow during H. pluvialis germination, with a particular emphasis on nitrogen nutrition and energy flow. Employing a suite of analytical tools including chlorophyll fluorescence analysis, transcriptomics, and metabolomics, the research explored various aspects such as the formation of sporangia, the release of zoospores, and the primary processes of photosynthesis and respiration. Furthermore, it examined the role of the alternative oxidase (AOX) pathway within the mitochondrial respiratory electron transport chain, aiming to unravel the regulatory mechanisms governing these vital biological processes.

The main contents and results are listed as follows:

(1) The study clarified that nitrogen nutrition is an essential nutrition condition for the germination of red non-motile cells. When the concentration of nitrogen nutrition was lower than 10 mg/L, it significantly affected the release of zoospores. Red non-motile cells could normally germinate and release a large number of motile spores in the medium only containing nitrogen nutrition. Under nitrogen deficiency, an initial small portion of red non-motile cells could release a few zoospores, however, subsequent sporangium formation and motile spore release were limited, blocking the germination process. The analysis of chlorophyll fluorescence parameters and primary metabolites revealed that nitrogen deficiency mainly suppressed the rate of photosynthetic carbon assimilation but had little effect on the rate of photosynthetic electron transport. Also, under nitrogen deficiency, pathways of respiratory carbon metabolism, such as the Embden-Meyerhof-Parnas pathway (EMP), the pentose phosphate pathway (PPP), and the tricarboxylic acid cycle (TCA), were significantly disturbed. Among these, the content of intermediate carbon skeletons like citrate, isocitrate, α-ketoglutarate, and succinate in the TCA cycle increased, while the content of amino acids like glycine, alanine, ornithine, citrulline, and arginine significantly decreased. Thus, carbon and nitrogen metabolism became imbalanced due to nitrogen deficiency, failing to provide sufficient substrates for nucleotide synthesis in DNA replication. Moreover, the downregulation of glutamate resulted in the accumulation of the toxic compound, 2-phosphoglycolate, within the photorespiratory pathway, as it was not metabolized efficiently. This accumulation, which displayed a 2.6-fold increase, subsequently hampered the respiratory and photosynthetic carbon metabolism processes during germination red non-motile cells of H. pluvialis.

(2) The study investigated the changes in the activity of various respiratory pathways during germination, including the AOX pathway, the cytochrome oxidase (COX) pathway, and residual respiration pathways. Initially, the energy for red non-motile cell germination was mainly provided by both the COX pathway and the AOX pathway in the respiratory electron transport chain, contributing similarly to the total respiration rate, at 48.6% and 44.4%, respectively. As the germination process progressed, the activity of AOX pathway gradually increased, with an 82.8% increase at 48 h compared to 0 h mark. The COX pathway, as a more efficient ATP-producing pathway, maintained stable activity within 48 h to ensure the basic energy supply of ATP for germination. After 72 h, when a large number of zoospores were released, the activity of all respiratory pathways showed a declining trend, indicating that after the completion of germination, the released zoospores had a reduced demand for carbon skeletons and ATP from respiratory metabolism.

(3) The upregulation of the AOX pathway reflected its positive role during germination. Further research, employing salicylhydroxamic acid, a specific AOX inhibitor—delved into its crucial function in regulating the internal energy balance and metabolism during germination. Upon inhibiting AOX, germination of non-motile cells was immediately arrested. Both detected AOX pathway activity and total respiration rate were significantly lower. When the AOX pathway was suppressed, on the one hand, there was a notable increase in the levels of reducing equivalents (NADH, NADPH, FADH₂), triggering an overactivation of the NADPH-dependent redox system. On the other hand, in red non-motile cells, there was an overall downregulation of pathways associated with the production of NADH, NADPH, and FADH₂. This downregulation encompassed the transcription levels of pathways involved in carbohydrate breakdown (EMP, TCA, PPP) and the decomposition of triacylglycerols via fatty acid β-oxidation and the glyoxylate cycle. Additionally, the abscisic acid pathway also participated in the AOX-regulated metabolic activation during germination. This indicated that the AOX pathway ensures the rapid consumption of reducing equivalents in the respiratory chain, thus guaranteeing the effective decomposition of stored materials, providing energy and carbon skeletons for the germination of red non-motile cells.

In summary, this study highlighted the important role of nitrogen nutrition in the normal operation of photosynthetic carbon assimilation and respiratory carbon metabolism during germination. Furthermore, it also revealed the key regulatory mechanism of the AOX pathway in modulating energy metabolism to facilitate swift material turnover during germination. This research contributes to a deeper understanding of cell cycle regulation in H. pluvialis and aids in developing new methods and potential approaches for rapidly propagating using red non-motile cells as a scalable seed source, effectively improving production efficiency and innovating the cultivation cycle model. These insights offer theoretical and technical support for the sustainable growth of the H. pluvialis industry.