The Photosynthetic Physiological Response And Its Transcriptomic Analysis Of Oleaginous Greenalga Scenedesmus Acuminatus To Different Initial Nitrogen Supplies

There are currently several serious issues associated with fossil fuels,including ongoing depletion and uneven distribution of reserves and increased consumption,all of which are leading to a worldwide energy crisis.Thus,there is significant interest in the development of biobased fuels.Many microalgae have the potential to serve as alternatives to oil crops,owing to advantages that include more rapid growth rates,higher photosynthetic efficiencies and greater intracellular lipid contents.These algae are therefore considered an ideal feedstock for biodiesel production.Scenedesmus acuminatus is a single-celled green alga that can contain up to 50%lipids when grown under limited nitrogen conditions,and so is a promising biodiesel source.However,the photosynthetic,physiological and metabolic processes that occur during lipid accumulation in this alga,especially the distribution of excitation energy and the carbon partitioning mechanism under low nitrogen conditions,are poorly understood.To obtain a better understanding of lipid metabolism in S.acuminatus,and more generally the lipid synthesis mechanism in microalgae,it is necessary to elucidate certain processes in these organisms.These processes include the excitation energy distribution between photosystemⅡ（PSⅡ）and photosystemⅠ（PSⅠ）photosystems,the distribution of electron flow,the carbon partitioning mechanism and the transcriptomic profiles.The main findings of the present work can be summarized as follows.The effects of the initial nitrogen supplies(susing 0.1,0.2,0.3,0.4,0.5,0.6,0.75,0.8,1.0 or 1.5 g L^–1 sodium nitrate)on the growth of S.acuminatus cultured under photoautotrophic conditions were assessed.The 0.1 g L^–1 group exhibited the highest carbon and lipid contents,together with the highest heating value and lowest lipid productivity,with values of 61.86%,44.24%,3.10 MJ kg^-1 and 0.09 g L^–1 d^–1,respectively.The maximum lipid productivities(0.13–0.14 g L^–1 d^–1)of S.acuminatus were obtained in the 0.4–0.75 g L^–1 groups.In the case of the 0.1–0.3 g L^–1 groups,the excitation energy distributions between PSⅡand PSⅠshowed a state transition（state 1 to state 2）on days 0–2,with a gradual return to state 1 after 3 days of culturing.In the 0.4-1.5 g L^–1 groups,the excitation energy distributions of S.acuminatus were found to undergo a pronounced three stage alteration.This consisted of（i）the regulation of state transitions after 3 days of culturing,（ii）the dissociation of LHCⅡfrom PSⅡwithout the binding of PSⅠbetween days 3 and 5,and（iii）a return to state1 after 5 days of culturing.The photosynthetic activity of PSⅡof S.acuminatus was significantly affected by the level of Q_A reduction.As the culture progressed,the light energy absorbed by the photochemical reaction in PSⅡwas reduced,while that for the non–photochemical quenching was increased.In the 0.1–0.6 g L^–1 groups,the light energy absorbed by PSⅡwas dissipated by the photoprotection mechanism（yield of non–photochemical quenching,Y（NPQ）>0.4）to a greater extent,while light energy in the 0.75–1.5 g L^–1groups was primarily released by dissipation as heat（yield of constitutive thermal dissipation,Y（NO）>0.4）.The low nitrogen(the 0.1–0.6 g L^–1groups)also showed increased tolerance to high light stress.After 3 days of culturing,the 0.3 and 1.5 g L^–1 groups produced a greater degree of cyclic electron flow,while the 0.75 g L^–1 and 0.8 g L^–1 groups did not show any post–illumination increase in Chl fluorescence.As the culturing progressed,each group demonstrated an increase in the proportion of cyclic electron flow.During the early stage of these trials（1–24 h）,the O₂^–production rate and H₂O₂content decreased,while superoxide dismutase,ascorbate peroxidase and glutathione peroxidase were activated and the extent to which the water–water cycle proceeded decreased.After 24 h of culturing,the O₂^–production rate and H₂O₂ level in the nitrogen starvation group were found to have increased and the activities of superoxide dismutase,ascorbate peroxidase and glutathione peroxidase were also increased.These results indicate that nitrogen deficiency enhanced the water–water cycle.During culturing,the 1.5 g L^–1 group accumulated more H₂O₂ than the 0.75 g L^–1 group.The catalase activity in the 0.75 and 1.5 g L^–1 groups gradually decreased over time,and there was evidently a positive correlation between the catalase and ascorbate peroxidase activities.The nitrogen starvation group showed inhibited growth and lower pigment content but increases in the individual cell weights.Lutein and zeaxanthin are the main pigments generated in response to nitrogen stress in S.acuminatus.The total soluble protein contents in the S.acuminatus decreased with increases in the culturing time and in the initial nitrogen supply.In addition,the total carbohydrate content initially increased and then decreased over time,while the total lipid content increased with time.A reduced nitrogen supply was associated with higher lipid contents.The inhibition of fatty acid synthesis in such organisms reduces their carbohydrate contents,and the inhibition of synthesis and degradation of carbohydrate can also reduce the total lipid contents.The former effect has a profound impact on lipid accumulation.S.acuminatus uses carbohydrates as its the main carbon and energy storage unit,and so the conversion of carbohydrates to lipids is essential to increase lipid contents.In the case of the nitrogen starvation group,the differentially expressed genes involved in glycolysis,the pentose phosphate and fatty acid synthesis pathways,nitrogen uptake and assimilation,and amino acid synthesis（except for sulfur-containing amino acids）were significantly up-regulated.With increases in the nitrogen supply(that is,in the 0.75 and 1.5 g L^–1 groups),the number of differential genes up-regulated in the above pathways gradually decreased,although the absolute value of the differential multiple of the partial genes in the 0.75 g L^–1group was higher than that in the 1.5 g L^–1 group.The differentially expressed genes associated with the PSⅡcore and oxygen-releasing complex proteins,and with the Cytb₆f complex involved in photosystem structure repair,were up-regulated in the nitrogen starvation group.In this same sample,the encodes gene of phospholipid:diacylglycerol acyltransferase（PDAT）was significantly up-regulated,indicating that S.acuminatus cultured in the absence of nitrogen primarily accumulates neutral lipids through an acetyl-Co A-independent TAG synthesis pathway.At low nitrogen levels(that is,in the case of the 0.75 g L^–1 group),the differentially expressed genes involved in the pentose phosphate and fatty acid synthesis pathways were significantly up-regulated,and the genes encoding DGAT and PDAT were up-regulated,indicating that the TAG synthesis required both DGAT and PDAT.Low nitrogen levels evidently have a significant regulatory effect on pathways associated with nitrogen metabolism.In addition,the differentially expressed genes involved in nitrogen transport and assimilation,and in the amino acid,carbamyl phosphate synthesis and urea cycle pathways,are significantly up-regulated,thus improving nitrogen utilization efficiency.