The Differential Responses of Chlorophyll Fluorescence Parameters and Lipid Metabolism to Low Temperature between Cycas Bida and C. panzhihuaensis

Background: Our previous work showed that freezing tolerance of Cycas panzhihuaensis was higher than that of C. bida. However, the mechanisms underlying the differential freezing tolerance of the two species is not clear. Photosynthesis is one of the most temperature-sensitive processes. Lipids play important roles in membrane structure, signal transduction and energy storage which are closely related to stress response of plants. Hence, the chlorophyll uorescence parameters and lipid proles of the two species were characterized to explore the dynamic changes of photosynthetic activity and lipid metabolism following low temperature and subsequent recovery. Results: The photosynthetic activity decreased signicantly with the decrease of temperatures in C. bida, reaching to zero after recovery, which however, was little affected in C. panzhihuaensis. Lipid composition of C. bida was more affected by cold and freezing treatments than C. panzhihuaensis. Compared to the control, the proportions of all the lipid categories recovered to the original level for C. panzhihuaensis but those of most lipid categories changed signicantly for C. bida after 3 d of recovery. Particularly, the glycerophospholipids and prenol lipids of C. bida degraded severely during recovery period for C. bida. The changes of acyl chain length and double bond index (DBI) occurred in more lipid classes immediately after low temperatures in C. panzhihuaensis than those in C. bida. DBI of the total main membrane lipids of C. panzhihuaensis was signicantly higher than that of C. bida following all the treatments. Conclusions: The results of chlorophyll uorescence parameters conrmed that the freezing tolerance of C. panzhihuaensis was higher than that of C. bida. The lipid metabolism of the two species had differential responses to low temperatures. The homeostasis and plastic adjustment of lipid metabolism and the higher level of DBI of the main membrane lipids might contribute to the higher tolerance of C. panzhihuaensis to low temperature.


Background
Low temperature is a major threat to plants whose geographical distribution and development are limited. It has been shown that membrane systems are particularly sensitive to low temperature [1]. Freezing induced extracellular ice formation could lead to the membrane rupture due to the mechanical stress and dehydration of the living cells [2]. Thylakoids possess the most abundant membranes of plant leaves wherein the lightdependent reactions of photosynthesis occur. Under low temperature, the balances between light harvesting and light utilization for assimilation are liable to be broken [3], and the excessive absorbed energy can lead to oxidative stress by overproducing reactive oxygen species (ROS) [4]. The chloroplast is proven to be the main site of ROS production and ROS attack under stress [5]. Therefore, photosynthesis is extremely sensitive to cold/freezing stress in plants [6]. However, many plant species have evolved various adaptive mechanisms to minimize the negative effects of freezing temperatures [2]. Observations have demonstrated that some of the molecular, metabolic and physiological characteristics are modulated to enhance plant freezing tolerance [2,7].
Lipids have important functions in membrane structure, signal transduction and energy storage. Lipid composition varys among species, tissues and membranes which is affected by developmental stages and environmental conditions [8,9]. Therefore, lipid metabolism is closely related to the development and stress response of plants. Membrane is the primary site of low-temperature-induced injury in plants [10,11]. Therefore, the membrane properties such as integrity and temperature-compatible uidity are crucial to maintain plant function and survival under varying temperatures [12]. Glycerophospholipids and saccharolipids are the main membrane lipids, the unsaturation level and acyl chain length of which affect the membrane uidity [13]. To cope with the adverse effects of low temperature, lipid compositions are modulated to increase the membrane uidity and the amount of bilayer-stabilizing lipids such as phosphatidylcholine (PC) and digalactosyldiacylglycerol (DGDG) is also increased [13,14]. Besides membrane lipids, glycerolipids are also involved in the tolerance of plants to low temperature due to their key functions in intracellular homeostasis and energy balance [15,16].
Studies show that the conversion of diacylglycerol (DAG) to triacylglycerol (TAG) contributes to freezing tolerance of some plant species [1].
Cycads are long considered the living fossils. Considering the primitiveness and persistence of cycads, their study is of great interest in terms of evolution and ecological adaptation of plants and global environmental changes. However, 62% of the known species of cycads are now in danger of extinction [17]. Cycas is the oldest genus of cycads which are restricted to the tropical and subtropical areas of Asia, Eastern Africa and Madagascar islands and Australia Paci c islands [18,19]. More than 20 species of Cycas are distributed in China and all of them has been proposed as rst-ranked plants for national protection in China [20]. The Cycas species are generally considered to be sensitive to low temperature. However, few studies have focused on the adaptation of these species to cold and freezing temperatures.
Cycas bi da is one of the most endangered cycads in China and the distribution of the species is restricted to some areas of Yunnan and Guangxi provinces. C. panzhihuaensis is endemic to the dry-hot valleys of the Jinsha River in southwest China and its natural distribution is in the northernmost limit areas and at the highest altitude among the Cycas species. The natural distribution of C. bi da and C. panzhihuaensis in China is restricted to subtropical zone, our previous work showed that the freezing tolerance of C. panzhihuaensis is higher than that of C. bi da [21]. However, their responses to low temperature and subsequent recovery conditions are not clear. The aim of this study was to dissect the effects of cold, freezing and subsequent recovery on photosynthetic activities and lipid metabolism of C. bi da and C. multipinnata. The results can provide theoretical bases for the freezing sensitivity and introduction of the two species.

Changes of chlorophyll uorescence parameters
Chlorophyll uorescence is a non-invasive and highly sensitive probe in monitoring the effects of environmental stresses on photosynthesis. Fv/Fm, Y(II) and ETR of C. bi da decreased signi cantly with the decrease of temperature, reaching the lowest level (near zero) after 3 d of recovery (Table 1). However, Y(NO) of C. bi da presented the opposite change trend, increasing by 323.21% for the recovered seedlings compared to the control. Y(NPQ) of C. bi da decreased signi cantly for seedlings subjected to freezing and recovery treatments, by 75.87% and 83.49%, respectively. qP and qN of C. bi da decreased signi cantly for seedlings subjected to freezing treatment, then qP increased to the control level and qN increased signi cantly following recovery. All these suggested that cold diminished the photosynthetic activity but freezing severely damaged the photosynthetic apparatus of C. bi da. Fv/Fm of C. panzhihuaensis decreased signi cantly for seedlings subjected to cold, freezing and recovery treatments, by 4.96%, 5.54% and 5.07%, respectively. Y(NO) increased signi cantly but qN decreased signi cantly for seedlings subjected to freezing treatment. Except for Fv/Fm, Y(NO) and qN, all other parameters maintained unchanged following low temperature treatments. Both Y(II) and ETR of C. panzhihuaensis decreased signi cantly but Y(NPQ) increased signi cantly for recovered seedlings. Fv/Fm, Y(II) and ETR of C. panzhihuaensis were signi cantly higher than those of C. bi da after various treatments. These results demonstrated that the phothosynthesis of C. panzhihuaensis was slightly affected by cold and the photosynthetic apparatus was damaged under freezing to a much lesser extent than that C. bi da. Lipids pro ling in the leaves of C. bi da and C. panzhihuaensis 26 lipid classes including 613 lipid species were identi ed from the leaves of C. bi da and C. panzhihuaensis (Additional le 1). The lipids contained two neutral glycerolipid classes, eight classes of glycerophospholipids (excluding lysophospholipids here), four of lysophospholipids, four of saccharolipids, four of sphingolipids, two of sterol lipids, one of prenol lipids (coenzyme Q) and one of fatty acyls (wax esters). The total lipid content of C. bi da was not signi cantly different from that of C. panzhihuaensis. Neutral glycerolipids, glycerophospholipids and saccharolipids were the main lipid categories for both the species, accounting for more than 90% of the total lipids ( Table 2). The absolute content of neutral glycerolipids, glycerophospholipids and saccharolipids did not differ but the proportions of glycerophospholipids and saccharolipids differed signi cantly between the two species (Fig 1; Table 2). The ratio of saccharolipids to phospholipids was signi cantly higher in C. panzhihuaensis (Additional le 2). Phosphatidic acid (PA), PC, phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidylinositol (PI) were the main glycerophospholipids.
Monogalactosyldiacylglycerol (MGDG), DGDG and sulphoquinovosyldiacylglycerol (SQDG) were the main saccharolipids (Table 3). Changes of the composition of the lipid categories For C. bi da, the content of total lipid, glycerophospholipids, lysophospholipids, saccharolipids, prenol lipids and sterol lipids increased after cold and freezing treatments. However, glycerophospholipids and prenol lipids decreased signi cantly and sterol lipids increased signi cantly after 3 d of recovery compared to those of the control (Fig. 1). The content of fatty acyls decreased signi cantly after cold treatment but recovered to the original level after freezing and recovery treatments. The ratio of saccharolipids to glycerophospholipids of C. bi da increased signi cantly after recovery treatments which was mainly due to the severe degradation of glycerophospholipids (Additional le 2). It also suggested that glycerophospholipids of C. bi da were more easily affected by freezing temperature than saccharolipids. Compared to those of the control in C. bi da, the relative contents of all the lipid categories except saccharolipids, lysophospholipids and fatty acyls changed signi cantly after recovery ( Table 2). This showed that the lipid metabolic balances of C. bi da were disturbed by low temperatures. For C. panzhihuaensis, the content of total lipids and saccharolipids increased signi cantly after cold treatment and fatty acyls increased signi cantly after freezing treatment (Fig. 1). The ratio of saccharolipids to glycerophospholipids of C. panzhihuaensis increased signi cantly after cold treatment which was mainly due to the accumulation of saccharolipids (Additional le 2). Although the relative contents of neutral glycerolipids, glycerophospholipids, saccharolipids, prenol lipids and fatty acyls changed signi cantly following cold or freezing treatment, they all returned to the control level after recovery ( Table 2). This showed that the lipid metabolism of C. panzhihuaensis could positively respond to low temperature stress and recover to the normal state after recovery. It can also be seen that freezing and subsequent thawing did not disturb the lipid metabolic system.
Compared between the two species, the contents of glycerophospholipids and lysophospholipids were signi cantly lower and those of saccharolipids and fatty acyls were signi cantly higher in C. panzhihuaensis after cold treatment. The contents of total lipids, glycerophospholipids, lysophospholipids, sphingolipids and sterol lipids were signi cantly lower and those of neutral glycerolipids and fatty acyls were signi cantly higher in C. panzhihuaensis following freezing treatment. After recovery treatment, the contents of total lipids, glycerophospholipids, saccharolipids and prenol lipids were signi cantly higher and those of lysophospholipids and sphingolipids were signi cantly lower in C. panzhihuaensis.

Changes of the composition of main lipid classes and lipid species of glycerophospholipids and saccharolipids
Phosphatidylinositol (PIP) and cardiolipin (CL) were also detected but their contents were extremely low (Additional le 3). Therefore, the two lipid classes were not analyzed separately but included in the analysis of total glycerophospholipids. PA, PC, PE, PG, PI and PS contents of C. bi da increased signi cantly after cold and/or freezing treatments (Table 3). However, PA, PC, PG and PI contents recovered to the original level and PE and PS contents decreased signi cantly after 3 d of recovery. Compared to those of the control, PC and PE contents of C. panzhihuaensis increased signi cantly after cold treatment (Table 3). PG content decreased signi cantly but PI content increased signi cantly after freezing treatment. After recovery, PG content increased signi cantly compared to that of the freezing-treated seedlings but did not reach the original level. PA, PG and PI contents of C. panzhihuaensis were signi cantly lower than those of C. bi da after cold. PA, PC, PE and PG contents of C. panzhihuaensis were signi cantly lower than those of C. bi da after freezing treatment. After recovery, PG, PI and PS contents of C. panzhihuaensis were signi cantly higher than those of C. bi da.

Changes of the ACL and DBI of glycerophospholipids and saccharolipids
The ACL of total glycerophospholipids, PE and PI of C. bi da were not affected by various treatments (Table 4).
Although the ACL of PA and PC was not affected by low temperature but increased signi cantly after recovery.
The ACL of PG decreased signi cantly after low temperature treatments but then increased to the control level after recovery. The ACL of PS increased signi cantly after low temperature and recovery treatments. C. panzhihuaensis showed signi cantly different responses to various treatments with C. bi da in the ACL of PC, PE, PI and PS ( Table 4). The ACL of PA and PE decreased and that of PS increased signi cantly after cold treatments. The ACL increased signi cantly for PC, PE, PI and total glycerophospholipids and decreased for PA and PS after freezing treatments. Compared to those in C. bi da, the ACL of most glycerophospholipid classes in C. panzhihauensis were higher following cold or/and freezing treatments but that of PS was lower following freezing. The ACL of total glycerophospholipids of C. panzhihuaensis was signi cantly higher than that of C. bi da after low temperature treatments (Table 4). Except for decreasing signi cantly in DGDG after recovery, the ACL of other classes of saccharolipids in C. bi da did not vary with the treatments ( Table 4). The ACL of total saccharolipids of C. bi da showed no signi cant change after various treatments. For C. panzhihauensis, the ACL of MGMG, MGDG and DGDG increased signi cantly after cold or/and freezing treatment and that of SQDG decreased signi cantly after freezing treatment (Table 4). Except for SQDG, the ACL of all the classes of saccharolipids increased signi cantly after 3 d of recovery, compared to those of the control. The ACL of total saccharolipids in C. panzhihauensis showed signi cant decrease after cold treatment, which was signi cantly lower than that of C. bi da. The ACL of the total main membrane lipids (glycerophospholipids and saccharolipids) decreased signi cantly after low temperature treatments in C.bi da and increased signi cantly after freezing treatment in C. panzhihuaensis, being signi cantly higher than that of C. bi da.
Except for increasing signi cantly in PS after low temperature treatments, DBI of every glycerophospholipid class and total glycerophospholipids in C. bi da did not change (Table 5). For C. panzhihauensis, DBI of all the glycerophospholipid classes increased signi cantly after cold treatment and those of PG and PI also increased signi cantly after freezing and recovery treatments but the unsaturation level of PS decreased signi cantly after freezing treatment. The DBI of total glycerophospholipids in C. panzhihauensis increased signi cantly after cold treatment. Compared to those of C. bi da, the DBI of all the glycerophospholipid classes except PG of C. panzhihuaensis were signi cantly higher in cold-treated seedlings, which of PA, PE, PG and PI were signi cantly higher in freezing-treated and recovered seedlings ( Table 5). The DBI and DBI/ACL of total glycerophospholipids of C. panzhihuaensis was signi cantly higher than that of C. bi da after various treatments (Table 5; Additional le 3). Except that DBI of DGDG increased signi cantly after recovery, the DBI of every classes of saccharolipids and total saccharolipids in C. bi da were not affected by low temperatures. For C. panzhihuaensis, the DBI of MGDG in low-temperature-treated seedlings and those of MGMG and DGDG in cold-treated seedlings increased signi cantly compared to the control. Compared to those of C. bi da, the DBI of MGDG and DGDG in lowtemperature-treated seedlings of C. panzhihuaensis and that of total saccharolipids in freezing-treated seedlings were signi cantly higher. The DBI and DBI/ACL of total main membrane lipids was signi cantly higher in C. panzhihuaensis than that in C. bi da following low temperature and recovery treatments (

Discussion
Low temperature is one of the key factors limiting the introduction of tropical and subtropical plants including cycads to the areas at higher latitude and altitude. Our previous study showed that the freezing tolerance of C. panzhihuaensis was higher than that of C. bi da [21]. However, there is still a lack of systematic and in-depth study on the adaptation of the two species to low temperature. Lipids play key roles in diverse cellular processes and lipid metabolism is closely related to freezing tolerance of some plants [1,22]. However, how they adjust under low temperature to regulate the tolerance of C. panzhihuaensis and C. bi da to low temperature stress are poorly understood. Photosynthesis is one of the most temperature-sensitive processes [6]. Therefore, it is often used to re ect the adaptability of plants to temperature change. In the present study, the chlorophyll uorescence parameters and lipid pro les of the two species subjected to cold, freezing and subsequent recovery were characterized.

The reduction and loss of photosynthetic activities
Chlorophyll uorescence could sensitively re ect the physiological status of plants. The signi cant decrease of Fv/Fm, Y(II) and ETR and signi cant increase of Y(NO) with the decrease of temperatures in C. bi da demonstrated that photosynthetic activities of C. bi da were affected, particularly severely by freezing treatment. That Fv/Fm, Y(II) and ETR reached to zero after 3 d of recovery suggested the photosynthetic apparatuses of C. bi da were severely damaged and cannot recover. For C. panzhihuaensis, only Fv/Fm of the cold-treated seedlings decreased by 3.85% and Fv/Fm, Y(II) and ETR of the recovered seedlings decreased only by 3.97%, 17.63% and 19.25% respectively, compared to those of the control. These showed that C. panzhihuaensis were relatively little affected by cold and freezing temperatures in comparison with C. bi da. The damaging effects of freezing on plant morphology might not appear immediately after treatments, which however, can be more obvious after a period of recovery. According to our observations, leaves of C. bi da gradually became yellow and dry but those of C. panzhihuaensis maintained green after 10 d of recovery. These results con rmed that C. bi da was more sensitive to low temperatures than C. panzhihuaensis.
The changes of composition of the lipid categories The metabolism of neutral glycerolipids are affected by low temperature which are related to the tolerance of plants to low temperature [22]. For example, DAG and TAG accumulated and DAG/TAG ratio decreased under freezing in Arabidopsis [14,22]. It has been reported that the accumulation of TAG due to the conversion of DAG contributes to the freezing tolerance of plants [1,22]. However, the neutral glycerolipid content maintained unchanged and the DAG-TAG ratios of the treated seedlings were not signi cantly different from the control for both the species ( Fig. 1; Additional le 3). These showed that the freezing sensitivity of the two species had little relation with the neutral glycerolipid metabolism under freezing. Glycerophospholipids and saccharolipids are the main extraplastidic and plastidic membrane lipids respectively in plants. Some ndings suggested that the two categories of lipids degraded under low temperatures [13,23]. However, the glycerophospholipids and saccharolipids contents increased signi cantly after cold and freezing treatments in C. bi da and saccharolipids increased after cold treatment in C. panzhihuaensis. The sources of these accumulated lipids under low temperatures were not clear. As the metabolic pathways of carbohydrates and lipids undergo cross talk to regulate energy homeostasis [24], whether the increased lipids are ascribed to the conversion of the stored carbohydrates needs to be veri ed. For all the treatments, the saccharolipids/glycerophospholipids ratio of C. panzhihuaensis was always signi cantly higher than that of C. bi da (Additional le 2). Whether this was related to the higher freezing tolerance of C. panzhihuaensis was not clear.
Except that saccharolipid and fatty acyl content increased signi cantly following cold and freezing treatment respectively, the absolute contents of all the lipid categories of C. panzhihuaensis did not change after various treatments (Fig. 1). For C. bi da, the absolute contents of all the lipid categories except neutral glycerolipids and sphingolipids varied with the treatments to different extent (Fig. 1). The results suggested that lipid metabolism of C. bi da was more affected by cold and freezing treatments than C. panzhihuaensis. The proportion of some lipid categories changed after low temperature treatments for both C. bi da and C. panzhihuaensis (Table 2). However, the proportions of all the lipid categories recovered to the original level after 3 d of recovery for C. panzhihuaensis, which of most lipid categories changed signi cantly for C. bi da in comparison with the control. This showed the plastic adjustment of lipid metabolism in C. panzhihauensis which might be related to the more tolerance of the species to low temperature. Phospholipids are major structural components of cell membranes and play roles in signal transduction and energy storage [25]. Prenol lipids (coenzyme Q here) are essential for energy metabolism in the electron transport system and also function as antioxidants within membrane systems [26]. The disorders of lipid metabolism after recovery such as the degradation of phospholipids and prenol lipids might contribute to the ultimate death of aboveground parts of C. bi da seedlings.
Lysophospholipids, sphingolipids and sterols are not only the structural components of membranes but also important signaling moleculars involved in plant development and environmetal responses [27][28][29]. It has been reported that the accumulation of lysophospholipids and sphingolipids under stresses might be detrimental to the cells [30,31]. Besides sterol contents, the contents of lysophospholipids and sphingolipids of C. bi da were signi cantly higher than those of C. panzhihuaensis. Whether the differential tolerance of the two species to freezing is related to their different contents and change patterns of these lipids needs to be explored. Cuticular waxes are the primary structures of the cuticle and play crucial roles in plant defense against biotic and abiotic stress including drought and frost [32]. The signi cantly higher content of wax esters of C. panzhihuaensis following cold and freezing treatments might contribute to its higher freezing tolerance than C. bi da.
The changes of composition of main lipid classes and lipid species of glycerophospholipids and saccharolipids Increasing evidence suggests that PA can form nonbilayer lipid structure with MGDG or DAG during low temperature, disrupting the integrity of cell membrane [33,34]. Some studies showed that PA content increased dramatically under stresses including freezing [13,33]. PA content maintained unchanged in C. panzhihuaensis after various treatments but increased by 144.59% and 71.62% in C. bi da after cold and freezing treatments, respectively ( Table 3). The maintenance of PA content in C. panzhihuaensis was conducive to keep the membrane stability but the increase of PA in C. bi da after low temperature treatments might pose potential threat to membrane integrity. Meanwhile, the signi cantly lower level of PA after cold and freezing treatments might confer higher freezing tolerance to C. panzhihuaensis.
Some studies showed that glycerophospholipid composition of various plants presented different responses to stresses [9,13,35]. For C. bi da, the content of each glycerophospholipid class increased signi cantly after cold or/and freezing treatment but the PE, PS and total glycerophospholipid contents decreased signi cantly after 3 d of recovery ( Fig. 1; Table 3). These results suggested that glycerophospholipid metabolism of C. bi da was dramatically affected by low temperature and the membrane was severely damaged following freezing treatment. For C. panzhihuaensis, different classes of glycerophospholipids showed different change trends following low temperatute treatments. However, all the classes except for PG recovered to the original level. It demonstrated that PG was more sensitive to freezing temperature than other phospholipids in C. panzhihuaensis. Studies have suggested that the level of high-melting-point PG moleculars such as 32:0 and 32:1 are related to the sensitivity of plants to low temperatures [36,37]. The high-melting-point PG moleculars were much lower in C. panzhihuaensis and showed decrease and increase after low temperature for C. panzhihuaensis and C. bi da, respectively (Fig. 2). This might be an important factor underlying the difference of freezing tolerance between the two species.
Saccharolipids are the main lipids of chloroplast envelope and thylakoid membrane which play key roles in the photosynthetic process [38]. Studies have suggested that these lipids are likely to tend to be degraded under some stresses [9,39]. However, all the classes of saccharolipids in C. bi da increased signi cantly after low temperature treatments and all with the exception of DGDG and SQDG recovered to the control level after recovery ( Table 3). The increase of saccharolipids after low temperatures might imply that seedlings of C. bi da positively resist the adverse effects of cold and freezing stresses on photosynthetic apparatus by stabilizing plastidic membranes. High DGDG/MGDG is proven to be more conducive to maintain bilayer membrane structure [14,38]. The substantial degradation of DGDG during post-freezing recovery suggested that the membrane integrity of C. bi da was damaged following freezing temperature. This was consistent with the results of chlorophyll uorescence parameters which showed the severe loss of photosynthetic activity of C. bi da. By contrast, the saccharolipids of C. panzhihuaensis including MGMG, MGDG and DGDG only responded to cold treatment. This might be the adaptive mechanisms of C. panzhihuaensis to cold temperature but the relevance of these ndings with the freezing tolerance of the species remained to be further explored. MGDG and DGDG are the main components of saccharolipids in most plants [8,40]. However, SQDG contents of C. bi da and C. panzhihuaensis accounted for 58.21% and 44.69% of the total saccharolipids respectively, surpassing the MGDG and DGDG contents ( Fig. 1; Table 3). Similar phenomenon was found in some algaes and lichens [41,42]. As SQDG were also found in cyanobacteria and non-photosynthetic bacteria [41]. The so high content of SQDG in the two Cycas species might be related to the highly diverse endophytic microbiome such as cyanobacteria and actinomycetes [43,44]. It might also be the long-term adaptation of these species to nutrient-poor environments as P-starvation could promote SQDG accumulation [45].
The changes of ACL and DBI of glycerophospholipids and saccharolipids ACL and DBI are two important determinants of membrane uidity which are related to the development and environmental adaptability of plants [13]. The decrease of ACL and increase of DBI under low temperature enable the membranes to be more uid which contribute to the acquirement of plant tolerance to low temperatures [13].
The ACL and DBI of different classes of glycerophospholipids and saccharolipids showed different change trends following various treatments for either species. Among glycerophospholipids, PG is the major component in chloroplast membranes. ACL of PG in C. bi da decreased signi cantly and DBI of PG in C. panzhihuaensis increased signi cantly after low temperature treatments. It suggested that the two species adopted different strategies to increase the membrane uidity. Our previous work suggested that ACL of PS was related to the plant lifespan which can be accelerated by promoted senescence but ceased to increase in plants being on the verge of death [46]. In the present study, ACL of PS increased signi cantly after low temperatures and did not return to the control level after 3 d of recovery in C. bi da. ACL of PS varied with the decrease of temperature but recovered to the control level subsequently in C. panzhihuaensis. This implied that freezing-treated seedlings of C. bi da might gradually lose viability, which of C. panzhihuaensis, however, were not severely damaged.
In general, the ACL and DBI changes occurred in more lipid classes immediately after low temperatures in C. panzhihuaensis than those in C. bi da (Table 4 and 5). The ACL and DBI of glycerophospholipids and saccharolipids maintained unchanged in C. bi da. However, ACL and DBI of glycerophospholipids and ACL of saccharolipids responded to cold or freezing treatment and they all recovered to the original level subsequently in C. panzhihuaensis (Table 4 and 5). The results showed that C. panzhihuaensis could better adjust the membrane uidity to respond to the decreasing temperature. Based on the higher level of DBI/ACL (Additional le 3), the higher level of DBI of total glycerophospholipids could maintain higher uidity of extraplastidic membranes under low temperature in C. panzhihuaensis, although the ACL being signi cantly higher. For total saccharolipids, the ACL of C. panzhihuaensis was shorter after cold temperature and DBI was higher after freezing temperature compared to those of C. bi da. These could enable the seedlings of C. panzhihauensis to obtain more uidity of plastidic membranes which are apt to be damaged under low temperature. The results were consistent with the chlorophyll uorescence parameters that photosynthetic activity following treatments severely lost in C. bi da but little changed in C. panzhihuaensis. The DBI of the total main membrane lipids of C. panzhihuaensis was signi cantly higher than that of C. bi da following all the treatments. The higher level of DBI after low temperature treatments might contribute to the higher freezing tolerance of C. panzhihuaensis.

Conclusions
The photosynthetic activity of C. bi da was more severely affected by low temperature than that of C. panzhihuaensis. The differential effects of freezing temperature were more obvious after 3 d of recovery that seedlings of C. bi da almost lost photosynthetic capacity but C. panzhihuaensis were little affected. The results con rmed our previous work that the freezing tolerance of C. panzhihuaensis was higher than that of C. bi da.
Lipid composition of C. bi da was more affected by cold and freezing treatments than C. panzhihuaensis. The proportions of all the lipid categories recovered to the original level for C. panzhihuaensis but those of most lipid categories changed signi cantly for C. bi da after 3 d of recovery. The homeostasis and plastic adjustment of lipid metabolism in C. panzhihauensis might be related to the more tolerance of the species to low temperature than C. bi da. However, the severe degradation of glycerophospholipids and prenol lipids might be an important determinant of seedling death during recovery period for C. bi da. The changes of ACL and DBI occurred in more lipid classes immediately after low temperatures in C. panzhihuaensis than those in C. bi da. The higher level of DBI of the main membrane lipids following low temperature treatments might contribute to the higher freezing tolerance of C. panzhihuaensis. It can be seen that the lipid metabolism underwent different changes between seedlings of C. panzhihuaensis and C. bi da which might lead to the differential tolerance of the two species to low temperature.

Plant materials and treatments
Seeds of C. panzhihuaensis were collected in Panzhihua which was approved by Administration Bureau of Panzhihua Cycas National Nature Reserve, Sichuan province. Seeds of C. bi da were collected from horticultural sources in Gejiu, Yunnan province which was permitted by the private land owners. The germinated four-year-old seedlings of C. bi da and C. panzhihuaensis grown in a greenhouse in Southwest Forestry University were used to conduct the experiments. The seedlings of the two species were identi ed based on the morphological characteristics by Shuangzhi Li of Southwest Forestry University, taxonomist expert. The voucher specimens of C. panzhihuaensis (No. ZYL-001) and C. bi da (No. ZYL-002) were prepared and deposited in the Southwest Forestry University. The average temperature of the greenhouse was about 30±1 °C and the daytime photosynthetic photon ux density was about 250-300 μmol m -2 s -1 . All the seedlings were planted in plastic pots containing humus and laterite soil (1:1 v/v). For cold treatment, seedlings were transferred to arti cial chambers at constant temperature of 4 °C for 3 d. The procedure of freezing and thaw treatments were referred to Zhang et al (2016) [47] and Arisz et al (2018) [1] with some modi cations. For freezing treatment, the cold-treated seedlings were put in a programmable temperature incubator set at 0 °C. Freezing was initiated by spraying ice cold water, and the temperature of the chamber was lowered at a rate of 2 °C h -1 until -5 °C was reached. They were then maintained for 1.5 h at -5 °C, after which the temperature was increased up to 4 °C at a rate of 2 °C h -1 .
The seedlings were thawed at 4 °C for 12 h and then recovered for 3 d at 30 °C. The seedlings which are not subjected to cold and freezing treatments are as the control. There were 16 seedlings for each treatment.
The chlorophyll uorescence parameters Four seedlings were selected from each treatment, and one leaf was sampled from each seedling. Fluorescence parameters were tested at indoor temperature using a chlorophyll uorometer (PAM-2500, Walz, Germany). Seedlings were dark-adapted for 30 min before measurements were conducted. The maximum quantum yield of photosystem II (PSII) (Fv/Fm), effective quantum yield of PS II (Y(II)), photochemical quenching coe cient (qP), non-photochemical quenching coe cient (NPQ), non-regulated (Y(NO)) and regulated (Y(NPQ)) nonphotochemical energy loss in PS II as well as electron transport rate (ETR) were measured.

Sample preparation and lipid extraction
Lipids were extracted according to MTBE method. Brie y, samples were rst spiked with appropriate amount of internal lipid standards and then homogenized with 200 µL water and 240 µL methanol. After that, 800 µL of MTBE was added and the mixture was ultrasound 20 min at 4 °C followed by sitting still for 30 min at room temperature. The solution was centrifuged at 14000 g for 15 min at 10 °C and the upper organic solvent layer was obtained and dried under nitrogen. Five seedlings were selected from each treatment, and one leaf was sampled from each seedling for lipid extraction.

LC-MS/MS method for lipid analysis
Reverse phase chromatography was selected for LC separation using CSH C18 column (1.7 µm, 2.1 mm × 100 mm, Waters). The lipid extracts were re-dissolved in 200 µL 90% isopropanol/ acetonitrile, centrifuged at 14000 g for 15 min, nally 3 µL of sample was injected. Solvent A was acetonitrile-water (6:4, v/v) with 0.1% formic acid and 0.1 Mm ammonium formate and solvent B was acetonitrile-isopropanol (1:9, v/v) with 0.1% formic acid and 0.1 Mm ammonium formate. The initial mobile phase was 30% solvent B at a ow rate of 300 μL/min. It was held for 2 min, and then linearly increased to 100% solvent B in 23 min, followed by equilibrating at 5% solvent B for 10 min. Calculation of lipid double bond index (DBI) and acyl chain length (ACL)