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. 2020 Nov 18;185(1):94–107. doi: 10.1093/plphys/kiaa013

Chloroplast lipid biosynthesis is fine-tuned to thylakoid membrane remodeling during light acclimation

Linhui Yu 1, Jilian Fan 1, Chao Zhou 1,1, Changcheng Xu 1,✉,b
PMCID: PMC8133659  PMID: 33631801

Abstract

Reprogramming metabolism, in addition to modifying the structure and function of the photosynthetic machinery, is crucial for plant acclimation to changing light conditions. One of the key acclimatory responses involves reorganization of the photosynthetic membrane system including changes in thylakoid stacking. Glycerolipids are the main structural component of thylakoids and their synthesis involves two main pathways localized in the plastid and the endoplasmic reticulum (ER); however, the role of lipid metabolism in light acclimation remains poorly understood. We found that fatty acid synthesis, membrane lipid content, the plastid lipid biosynthetic pathway activity, and the degree of thylakoid stacking were significantly higher in plants grown under low light compared with plants grown under normal light. Plants grown under high light, on the other hand, showed a lower rate of fatty acid synthesis, a higher fatty acid flux through the ER pathway, higher triacylglycerol content, and thylakoid membrane unstacking. We additionally demonstrated that changes in rates of fatty acid synthesis under different growth light conditions are due to post-translational regulation of the plastidic acetyl-CoA carboxylase activity. Furthermore, Arabidopsis mutants defective in one of the two glycerolipid biosynthetic pathways displayed altered growth patterns and a severely reduced ability to remodel thylakoid architecture, particularly under high light. Overall, this study reveals how plants fine-tune fatty acid and glycerolipid biosynthesis to cellular metabolic needs in response to long-term changes in light conditions, highlighting the importance of lipid metabolism in light acclimation.

Lipid metabolism is fine-tuned to cellular metabolic demands during thylakoid membrane remodeling in response to long-term changes in light intensity.

Introduction

As sessile organisms, plants must respond and acclimate to environmental changes to maximize their chances of survival and reproduction. Light is the most dynamic environmental factor, varying in intensity and spectral quality across a wide range of time scales from seconds to seasons, but it is also a key resource, as light is the driving force for photosynthesis and hence for plant growth, development, and biomass production. Changes in light intensity result in an imbalance between light energy absorbed by light-harvesting complex II (LHCII) versus the use of the absorbed energy to drive photosynthetic electron transport and carbon assimilation (Rochaix, 2014; Allahverdiyeva et al., 2015; Derks et al., 2015; Pinnola and Bassi, 2018). Such an energy imbalance can lead to a decrease in photosynthetic efficiency under light-limiting conditions, and to irreversible oxidative damage to components of the photosynthetic machinery, particularly photosystem II (PSII) under high-light conditions due to excessive production of reactive oxygen species (ROS; Takahashi and Badger, 2011; Li et al., 2018). During acclimation to changing light conditions, plants adjust the composition of the photosynthetic electron transport chain, regulate light energy harvesting and utilization, reprogram cellular metabolism, and modify chloroplast structure and leaf architecture. These acclimation responses serve to optimize photosynthesis under light-limiting conditions while alleviating and repairing oxidative damage to PSII under high-light environments (Kirchhoff, 2014; Rochaix, 2014; Allahverdiyeva et al., 2015; Pinnola and Bassi, 2018; Kirchhoff, 2019; Liu et al., 2019).

At the ultrastructural level, one of the most conspicuous photoacclimation responses involves changes in the structural organization of the thylakoid membrane network inside chloroplasts (Anderson and Andersson, 1988; Lee, 2000; Anderson et al., 2012; Herbstova et al., 2012; Kirchhoff, 2014, 2019; Lambrev and Akhtar, 2019). Land plant thylakoids are characterized by their stacking to form tightly appressed layers, called grana, which are interconnected to each other by a single nonappressed but continuous membrane network of stromal lamellae (Anderson et al., 2012; Herbstova et al., 2012; Kirchhoff, 2019). These two thylakoid domains house different protein components of the photosynthetic electron transport chain, with PSII and LHCII concentrated in the grana, whereas photosystem I and ATP synthase are found mainly in the unstacked regions (Anderson et al., 2012; Kirchhoff, 2019; Koochak et al., 2019). Plants respond to low light (LL) by increasing grana diameter, the number of thylakoids per granum, and the relative amount of LHCII to maximize light capture in order to optimize limiting light levels for photosynthesis. Conversely, high-light intensities cause decreases in grana size and partial or complete unstacking of grana membranes (Walters, 2005; Li et al., 2009; Herbstova et al., 2012; Kirchhoff, 2014). Such high light (HL)-responsive structural changes have been linked to photoprotective thermal dissipation of absorbed light energy that exceeds the capacity of optimal photosynthetic metabolism (Demmig-Adams et al., 2015; Schumann et al., 2017) and are thought to be crucial for the rapid removal and/or repair of photodamaged PSII proteins under excess light (Khatoon et al., 2009; Herbstova et al., 2012; Yoshioka-Nishimura and Yamamoto, 2014). To date, however, the mechanisms underlying thylakoid stacking are still not fully understood and the physiological role of thylakoid stacking has not been rigorously tested. Major factors implicated in grana formation include LHCII (Day et al., 1984), protein phosphorylation (Fristedt et al., 2009), curvature thylakoid1 protein complexes (Luque and Ochoa de Alda, 2014), and major thylakoid membrane lipids including two galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG; Lee, 2000; Deme et al., 2014; Seiwert et al., 2018; Mazur et al., 2019).

In plants, fatty acids, the building blocks of thylakoid lipids, are synthesized in the plastid (Ohlrogge and Jaworski, 1997). The first committed step of fatty acid synthesis is catalyzed by the heteromeric, bacterial-type enzyme acetyl-CoA acetyltransferase (ACCase), which consists of four different subunits: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), alpha-carboxylase (α-CT), and beta-carboxylase (β-CT). Plastidic ACCase is subject to transcriptional and biochemical regulation by various mechanisms and is widely considered as the key regulatory enzyme controlling fatty acid synthesis (Ohlrogge and Jaworski, 1997; Li-Beisson et al., 2013; Salie and Thelen, 2016). Light controls plastidic ACCase activity to coordinate photosynthesis and fatty acid synthesis via redox regulation and changes in stromal pH and Mg2+ concentrations (Ohlrogge and Jaworski, 1997), but whether long-term changes in light levels affect ACCase activity and fatty acid synthesis in leaves and the mechanism of regulation have not been examined.

In many plants including Arabidopsis, two parallel pathways compartmentalized in the plastid or the endoplasmic reticulum (ER) contribute to membrane lipid assembly (Browse and Somerville, 1991; Ohlrogge and Browse, 1995). The plastid pathway begins with the stepwise acylation of glycerol-3-phosphate (G-3-P) with nascent fatty acids, leading to the generation of phosphatidic acid (PA). PA and its dephosphorylated product diacylglycerol (DAG) are used to assemble membrane lipids including major thylakoid lipids MGDG and DGDG inside the plastid. Alternatively, fatty acids are exported from the plastid and first incorporated into phosphatidylcholine (PC) via acyl editing to enter the ER pathway of glycerolipid assembly (Bates et al., 2007). Acyl groups derived from PC acyl editing are used to synthesize PA and DAG, which serve as precursors for the synthesis of major phospholipids such as PC and phosphatidylethanolamine (PE). Part of the phospholipids assembled via the ER pathway return to plastids to serve as substrates for thylakoid lipid synthesis (Li-Beisson et al., 2013). A transenvelope complex consisting of five trigalactosyldiacylglycerol (TGD) proteins is involved in transferring the ER-derived phospholipids to the chloroplast (Xu et al., 2003; Awai et al., 2006; Lu et al., 2007; Xu et al., 2008; Fan et al., 2015). The Arabidopsis plastidic glycerol-3-phosphate acyltransferase1 (act1) is defective in the first step of G-3-P acylation in the plastid (Kunst et al., 1988; Xu et al., 2006). Consequently, this mutant lacks thylakoid glycolipids assembled via the plastid pathway and instead synthesizes its thylakoid lipids almost exclusively via the ER pathway. On the other hand, the trigalactosyldiacylglycerol1 (tgd1) mutant is deficient in the ER pathway of thylakoid lipid synthesis, and most of the photosynthetic membrane lipids in this mutant are assembled via the plastid pathway (Xu et al., 2003, 2005).

Because the substrate specificity of acyltransferases in the two compartments differs, glycerolipids made via the plastid or ER pathway are characterized by the presence of a 16- or an 18-carbon (C16 or C18) fatty acid at the sn-2 position of the glycerol backbone, respectively (Li-Beisson et al., 2013). Plants adjust the balance of the two parallel pathways in response to temperature stress (Li et al., 2015) or genetic perturbations (Kunst et al., 1988; Xu et al., 2003). Short-term changes in light levels affect membrane lipid synthesis and transcript abundance of some genes involved in lipid synthesis (Burgos et al., 2011; Szymanski et al., 2014), but whether growth light levels also affect the balance between two pathways of thylakoid lipid synthesis remains unknown.

This study was undertaken to examine the role of lipids in long-term acclimation to different growth light intensities in Arabidopsis. Our results showed that changes in growth irradiance affect fatty acid synthesis and fatty acid flux between two parallel pathways of membrane lipid synthesis. Taking advantage of tgd1 and act1 mutants defective in the ER or the plastid pathway of thylakoid lipid biosynthesis, respectively, we demonstrated that glycerolipid pathway adjustments contribute to thylakoid ultrastructural reorganization and hence plant acclimation to changes in growth irradiance.

Results

Extensive physiological and structural modifications in response to changes in light levels

To examine the effect of light intensity on lipid synthesis, 10-d-old wild-type Arabidopsis seedlings grown on agar plates were transferred to soil and grown under normal light (NL; 200 μmol m−2 s−1) for 1 week. Seedlings were then divided into three groups and each group was maintained under different light levels but at the same temperature with a 16-h photoperiod for an additional 10 d. One group was illuminated with LL (LL; 25 μmol m−2 s−1); a second, control group was maintained under NL; and a third group under HL (HL; 1000 μmol m−2 s−1). As shown in Figure 1, plants grown under LL showed drastic reductions in plant growth (Figure 1A) and leaf dry weight/fresh weight (DW/FW) ratio (Figure 1D) while showing higher chlorophyll content (Figure 1E) compared with equivalent plants grown under NL. HL-grown plants, on the other hand, showed increased growth and accelerated development as evidenced by early bolting (Figure 1C), along with a higher DW/FW ratio (Figure 1D) and lower chlorophyll content (Figure 1E) compared with NL-grown plants. The leaf thickness, as revealed by microscopic analysis of leaf cross-sections, was significantly lower in LL-grown plants but much higher in HL-grown plants compared with plants grown under NL (Figure 1F). Ultrastructural examination by transmission electron microscopy (TEM) revealed alterations in thylakoid structure (Figure 1, G–I). In particular, growth under HL caused a swelling of thylakoid membranes and a lower degree of thylakoid stacking (Figure 1I). Strikingly, whereas plastoglobules (also known as plastid lipid droplets) in chloroplasts of LL- and NL-grown plants were small and highly osmiophilic (Figure 1, G and H), those in chloroplasts of HL-grown plants were much larger in size and became much less osmiophilic (Figure 1I). 

Figure 1.

Figure 1

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Plant growth and chloroplast ultrastructure under different light conditions. (A–C) Four-week-old wild-type plants grown under LL (A), NL (B), or HL (C) conditions. (D) Leaf DW to FW ratio. (E) Total leaf chlorophyll content. (F) Leaf thickness. Data are means of three independent biological replicates with SD. Asterisks indicate statistically significant differences from NL-grown plants based on Student’s t test (**P < 0.01). (G–I) Electron micrographs of representative chloroplasts in leaves of plants grown under LL (G), NL (H), and HL (I) conditions. Arrows indicate plastoglobuli. S, starch granules. Bars = 2 cm in A–C; 1 µm in G–I.

Fatty acid synthesis is regulated via modulating plastidic ACCase activity during light acclimation

To assess the rate of fatty acid synthesis, leaf discs derived from rapidly growing leaves of 4-week-old plants were incubated with 14C-acetate, which enabled the labeling of nascent fatty acids with 14C during the initial steps of fatty acid synthesis (Browse et al., 1981). When calculated on a DW basis, the rate of 14C incorporation into total fatty acids was significantly higher in leaves grown under LL compared with plants grown under NL, whereas those grown under HL showed a significantly lower rate of fatty acid synthesis (Figure 2A).

Figure 2.

Figure 2

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Fatty acid synthesis and its regulation under different light conditions. (A) Fatty acid synthesis measured as rate of 14C-acetate incorporation into total leaf lipids in leaves of 4-week-old wild-type plants grown under LL, NL, or HL conditions. (B) Relative ACCase activity expressed as a percentage of ACCase activity in isolated chloroplasts of NL-grown plants. Data are means of three independent biological replicates with SD. Asterisks indicate statistically significant differences from NL-grown plants based on Student’s t test (*P < 0.05, **P < 0.01). (C) Immunoblot analysis of BC, BCCP, α-CT, and β-CT subunits of the plastidic ACCase. Proteins were separated on an equal protein basis. Actin is used as loading control.

To test the role of plastidic ACCase in the regulation of fatty acid synthesis in response to changes in light intensity, intact chloroplasts were isolated from leaves of 4-week-old plants and the ACCase activity in isolated chloroplasts was assessed by radiotracer labeling using 14C-NaHCO3 (Hunter and Ohlrogge, 1998). Plastidic ACCase activity was more than six-fold higher in LL-grown plants compared with NL-grown plants (Figure 2B). Plants grown under HL, in contrast, showed significantly lower ACCase activity relative to plants grown under NL.

To dissect the mechanistic basis underlying changes in the activity of plastidic ACCase, the transcript and protein levels of plastidic ACCase subunits were determined. There were no significant differences in transcript levels of BC, α-CT, and BCCPs between plants grown under LL or HL compared with those grown under NL conditions (Supplemental Figure S1A). Likewise, there were no consistent changes in protein abundance of all four ACCase subunits α-CT, β-CT, BC, and BCCPs when compared on either an equal protein (Figure 2C) or an equal chlorophyll (Supplemental Figure S1B) basis. Together, these results suggest that posttranslational mechanisms are involved in the regulation of plastidic ACCase activity in response to long-term changes in light intensity.

Changes in radiolabeling distribution among leaf lipids under different light conditions

To test whether changes in growth light levels alter labeled fatty acid distribution among different lipid species, total lipids were extracted from 14C-acetate labeled leaves and separated by thin layer chromatography (TLC). Radioactivity associated with major membrane lipids were quantified by scintillation counting. The overall radiolabeling distribution among major membrane lipids was similar between LL and NL plants, except that there was slightly higher radioactivity associated with MGDG in LL plants compared with plants grown under NL (Figure 3). In contrast, plants grown under HL showed higher radioactivity associated with PC and PE/phosphatidylinositol (PI)/sulfoquinovosyldiacylglycerol (SL) at the expense of radioactivity associated with MGDG, whereas radiolabeling in DGDG and phosphatidylglycerol (PG) remained largely unchanged (Figure 3). Radiolabeling associated with triacylglycerol (TAG) was low in LL and NL plants, but was four-fold higher in plants grown HL compared with plants grown under NL (Figure 3).

Figure 3.

Figure 3

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Changes in radiolabeling distribution among leaf lipids under different light conditions. Detached leaves of 4-week-old wild-type plants grown under LL, NL, or HL conditions were incubated with 14C-acetate for 1 h. Total lipids were separated by TLC. Radioactivity associated with individual lipids was quantified by liquid scintillation counting. Data are means of three independent biological replicates with SD. Asterisks indicate statistically significant differences from NL-grown plants based on Student’s t test (*P < 0.05, **P < 0.01).

Leaf lipid content and fatty acid compositions are altered under different light conditions

Consistent with altered fatty acid synthesis, plants grown under LL or HL also showed a higher or lower level of total leaf fatty acids, respectively, compared with NL-grown plants (Supplemental Figure S2A). Quantification of major individual lipid species revealed higher relative amounts of MGDG and PG in LL-grown plants compared with NL-grown plants, whereas other major membrane lipid levels remained largely unchanged (Figure 4A). There were higher DGDG and PC levels but lower PG content in plants grown under HL compared with plants grown under NL. Consistent with radiotracer labeling experiments shown in Figure 3, TAG levels were very low in leaves of plants grown under LL and NL growth conditions but significantly higher in plants grown under HL compared with plants grown under NL (Supplemental Figure S2B).

Figure 4.

Figure 4

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Changes in membrane lipid content and fatty acid composition under different light conditions. (A) Major membrane lipid content in leaves of wild-type plants grown under LL, NL, or HL conditions. (B–D) Fatty acid composition of MGDG (B), DGDG (C), and PC (D). Data are means of three independent biological replicates with SD. Asterisks indicate statistically significant differences from NL-grown plants based on Student’s t test (*P < 0.05, **P < 0.01).

In addition to changes in lipid content, alterations in growth light levels caused significant changes in fatty acid composition of major membranes including MGDG, DGDG, and PC. Specifically, in MGDG, there were higher levels of C16 unsaturated fatty acids and a lower level of 18:3 in LL-grown plants, whereas in HL-grown plants, there was a lower level of 16:3 and higher relative amounts of 16:0 and 18:2 compared with plants grown under NL (Figure 4B). The fatty acid composition of DGDG remained largely unchanged in LL-grown plants, but there was a higher level of 16:0 and lower levels of 16:3 and 18:3 in HL-grown plants compared with NL-grown plants (Figure 4C). In PC, the relative 18:1 level was higher whereas 18:2 and 18:3 levels were lower in LL-grown plants compared with plants grown under NL. HL-grown plants also showed higher 16:0 content but lower 18:0, 18:1, and 18:2 levels in PC compared with NL-grown plants (Figure 4D).

The balance between two glycerolipid biosynthetic pathways is altered under different light conditions

C16 fatty acids in galactolipids are distributed predominantly at the sn-2 position of the glycerol backbone (Li-Beisson et al., 2013). Therefore, changes in relative levels of C16 fatty acids in MGDG suggest adjustments in thylakoid lipid synthesis pathways in response to alterations in light intensity. To confirm this, we analyzed the positional distribution of acyl chains in MGDG and DGDG following position-specific lipase digestion. The results revealed significantly higher levels of C16 fatty acids and a lower amount of 18:3 at the sn-2 position of MGDG (Figure 5A) and DGDG (Figure 5B) in plants grown under LL compared with those grown under NL. As a result, the sum of C16 fatty acids at the sn-2 position was higher, whereas the total C18 fatty acids at the same position were lower in MGDG and DGDG (Figure 5C), suggesting an increase in the relative contribution of the plastid pathway to galactolipid assembly in response to LL. In contrast, plants grown under HL showed a significantly lower level of 16:3 or 16:0 and a higher level of 18:3 at the sn-2 position of MGDG or DGDG, respectively (Figure 5, A and B), indicative of an increase in the relative contribution of the ER pathway to thylakoid lipid biosynthesis in response to HL.

Figure 5.

Figure 5

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Position-specific analysis of galactolipid acyl groups. (A and B), Fatty acid composition at the sn-2 position of the glycerol backbone of MGDG (A) and DGDG (B) extracted from leaves of wild-type plants grown under LL, NL, or HL conditions. (C) Sums of 16:0, 16:1, 16:2, and 16:3 (C16) and 18:0, 18:1, 18:2, and 18:3 (C18). Data are means of three independent biological replicates with SD. Asterisks indicate statistically significant differences from NL-grown plants based on Student’s t test (*P < 0.05, **P < 0.01).

Lipid pathway adjustments are important for growth under different light conditions

To test the physiological significance of glycerolipid pathway adjustments in plant response to changing growth light, we again grew the wild-type, tgd1, and act1 plants first under NL for 17 d. They were then exposed to LL, NL, or HL for 10 d. Like the wild type, both act1 and tgd1 mutants showed reduced growth under LL (Figure 6A) and early bolting (Figure 6C) under HL growth conditions. Interestingly, when normalized to shoot FW under NL, the relative shoot biomass in act1 was significantly lower under LL but significantly higher under HL compared with the wild type (Figure 6D). The tgd1 mutant, on the other hand, grew better under LL but its relative growth was severely compromised under HL compared with the wild type. In addition, a large portion of tgd1 leaves was bleached in contrast to the wild type and act1, which did not show the chlorotic phenotype (Figure 6C). Consistent with these visual phenotypes, the relative chlorophyll content was significantly lower in leaves of tgd1 compared with the wild type under HL growth conditions (Figure 6E). These results suggest that defects in lipid biosynthetic pathways compromise the ability of plants to acclimate to altered growth light intensities.

Figure 6.

Figure 6

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Plant growth and chlorophyll content under different growth light conditions. (A) Four-week-old plants grown under LL (A), NL (B), or HL (C) conditions. (D) Shoot FW. (E) Total leaf chlorophyll (Chl) content. Data are means of three to five independent biological replicates with SD. Asterisks indicate statistically significant differences from NL-grown plants based on Student’s t test (*P < 0.05, **P < 0.01).

To test how defects in the prokaryotic or eukaryotic pathway affect light acclimation, we first quantified galactolipid content in the wild-type, act1, and tgd1 mutant plants. The relative level of MGDG was similar among the wild type, act1, and tgd1 under NL (Figure 7A). However, under LL and HL, both act1 and tgd1 mutants showed lower levels of MGDG compared with the wild type. The relative level of DGDG was significantly lower in tgd1 grown under NL and HL compared with the wild type (Figure 7B). In act1, DGDG content was significantly higher under LL, whereas no differences in DGDG levels were found under NL or HL compared with the wild type. As a result of changes in galactolipid levels, the MGDG/DGDG ratio was significantly higher in tgd1 under both NL and HL but slightly lower under LL compared with the wild type (Figure 7C). The MGDG/DGDG ratio in act1 was significantly lower under both LL and HL but remained largely unchanged under NL compared with the corresponding wild type.

Figure 7.

Figure 7

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Changes galactolipid content under different light conditions. (A and B) MGDG (A) and DGDG (B) content in leaves of wild-type, act1 and tgd1 plants grown under LL, NL, or HL conditions. (C) The MGDG to DGDG ratio. Data are means of three independent replicates with SD. Asterisks indicate statistically significant differences from WT based on Student’s t test (*P < 0.05, **P < 0.01).

We next used TEM to examine thylakoid membrane organization with respect to grana number per chloroplast, grana width, and thylakoid number per granum in the wild type, act1, and tgd1. As previously reported (Kunst et al., 1989), under NL growth conditions, chloroplasts of act1 plants showed a strong decrease in the overall degree of grana stacking relative to the wild type (Figure 8,  B and E). The overall thylakoid structure of tgd1 (Figure 8H) was similar to that of the wild type (Figure 8B). On average, about 4.9 thylakoids per granum were present in NL-grown wild-type plants and 5.7 in tgd1 plants (Figure 8J), this number was reduced to 3.9 in NL-grown act1. The decrease in the number of thylakoids per granum in act1 was accompanied by an increase in the number of grana per chloroplast (Supplemental Figure S3A) and a decrease in grana width (Supplemental Figure S3B) compared with the NL-grown wild type. In NL-grown tgd1 plants, there were no significant changes in the number of grana per chloroplast (Supplemental Figure S3A) and grana width (Supplemental Figure S3B) compared with the NL-grown wild-type plants.

Figure 8.

Figure 8

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TEM analysis of chloroplasts ultrastructure. (A–I) TEM images of the thylakoid architecture of 4-week-old plants grown under LL, NL, or HL conditions. Arrows indicate plastoglobules. Bars = 0.5 µm. (J) Number of thylakoid layers per granum in chloroplasts of 4-week-old wild-type plants grown under different light levels. (K) Distribution of thylakoid layers in grana stacks. Data are means of quantitation of 10 images obtained from different chloroplasts of 2 different leaves.

The number of thylakoids per granum was higher in LL-grown wild-type and lower in LL-grown tgd1, but remained largely unchanged in LL-grown act1 compared with the respective genotype grown under NL conditions (Figure 8J), whereas the grana number per chloroplast (Supplemental Figure S3A) and grana width (Supplemental Figure S3B) remained unchanged in all three genotypes grown under LL compared with those under NL. Under HL growth conditions, there were marked decreases in grana stacking as evident from decreases in thylakoid number per granum (Figure 8, J  and K) and in grana width (Supplemental Figure S3B) in the wild-type and act1. In contrast, there was no major change in thylakoid stacking in HL-grown tgd1 compared with NL-grown tgd1. In addition, HL growth caused a swelling of thylakoids in all three genotypes, particularly in act1 (Figure 8, C, F, and I). Like HL-grown wild-type plants (Figures 1, G and 8, C), plastoglobules in chloroplasts of HL-grown act1 (Figure 8F) and tgd1 (Figure 8I) plants were much larger in size and became less osmiophilic compared with those in LL-grown (Figure 8D) and NL-grown (Figure 8, B and H) plants.

Discussion

Adaptation to changes in light conditions is critical for plant growth, development, and survival. One of the key acclimatory responses in this respect involves remodeling of the photosynthetic apparatus to maintain the balance between light capture and light energy utilization. This study investigated the role of lipid metabolism in plant acclimation to changing growth irradiance. We found that long-term changes in light levels modulate the glycerolipid composition via regulating fatty acid synthesis and fatty acid flux through two parallel pathways of glycerolipid assembly. We demonstrated that ACCase, the key regulatory enzyme in the pathway of plastidic fatty acid synthesis (Ohlrogge and Jaworski, 1997), is regulated at the posttranslational level, consistent with previous findings suggesting a crucial role of posttranscriptional mechanisms in plant acclimation to changing growth irradiance (Flachmann and Kuhlbrandt, 1995; Bruick and Mayfield, 1999; Walters, 2005; Miller et al., 2017). In addition, analysis of Arabidopsis mutants defective in one of the two glycerolipid biosynthetic pathways suggests that light intensity-induced lipid pathway adjustments are important for the structural reorganizations of thylakoid membrane systems and hence for plant growth and biomass production under changing growth irradiance.

A working model for the role of lipids in plant acclimation to different growth light levels is shown in Figure 9. Under LL conditions, plants increase the number of thylakoids per granum and total thylakoid membranes. In contrast, HL growth causes unstacking of grana thylakoids as is evident from decreases in grana size, the number of thylakoids per granum, and total membrane lipid content. These acclimation responses alter cellular demands for fatty acids, whose synthesis is feedback-regulated in the short term by 18:1-acyl carrier protein (ACP; Andre et al., 2012) or the longer term by biotin attachment domain-containing proteins (Keereetaweep et al., 2018). Therefore, it is possible that the increased cellular need for fatty acids for thylakoid membrane biosynthesis under LL conditions cause relief of feedback inhibition of ACCase, leading to increases in fatty acid synthesis and hence total lipid content. It should be noted, however, that the increased ACCase activity and fatty acid synthesis under LL are not proportional: a six-fold increase in ACCase activity (Figure 2B) only resulted in a small increase in the rate of fatty acid synthesis (Figure 2A). A possible explanation for this discrepancy is that under LL conditions other factors such as carbon precursors, reductants, and ATP limit fatty acid synthesis. It is also possible that differences in stromal pH, Mg2+ concentrations, ATP/ADP ratios, and reductant levels among plants grown under different light levels cause the discrepancy between the ACCase activity and the rate of fatty acid synthesis.

Figure 9.

Figure 9

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A working model for the role of lipid metabolism in plant acclimation to different growth light levels. Growth under LL or HL promotes thylakoid stacking or unstacking, respectively. These changes in thylakoid architecture alter cellular demands for fatty acids, which trigger post-translational mechanisms that regulate ACCase activity and hence fatty acid synthesis. In addition, long-term changes in growth light levels also modulate fatty acid flow through two parallel pathways of glycerolipid assembly, with LL promoting the plastid but suppressing the eukaryotic pathway, whereas HL causes opposite effects on fatty acid flux through the two pathways. Growth under HL conditions enhances TAG synthesis in the chloroplast and the ER, manifested as increased accumulation of lipid droplets in both compartments. The increased TAG accumulation in HL may increase fatty acid flux toward the peroxisomal β-oxidation pathway. Blue and red arrows indicate increased or decreased enzymatic and pathway activities, respectively, under LL or HL compared with those under NL growth conditions. Dash arrows indicate limited pathway activities. Question marks denote the steps that remain to be experimentally verified. Chl, chloroplast; FA, fatty acid; FAS, fatty acid synthase; GL, glycolipid; PL, phospholipid.

Under HL conditions, there is a decrease in fatty acid demands for thylakoid membrane biosynthesis, which causes an increase in 18:1-ACP, leading to decreased ACCase activity and hence reduced fatty acid synthesis. In addition, there was a higher accumulation of TAG, lipid droplets, and plastoglobules. Since both lipid droplets (Xu and Shanklin, 2016) and plastoglobules (van Wijk and Kessler, 2017) are buffers for toxic lipids during membrane maintenance and dismantling, and TAG is one of the major structural components of plastoglobules (van Wijk and Kessler, 2017;Xu et al., 2020), an increase in accumulation lipid droplets and plastoglobulels suggests an increase in the conversion of membrane lipids into TAG under HL growth conditions. TAG is a key intermediate in the mobilization of fatty acids from membrane lipids for β-oxidation in peroxisomes (Fan et al., 2014, 2017). Therefore, increases in TAG and lipid droplet accumulation under HL growth conditions may suggest an increase in fatty acid flux through the peroxisomal β-oxidation pathway (Figure 9), though this remains to be experimentally verified.

In addition to changes in fatty acid synthesis, growth under LL promotes the plastid pathway but suppresses the ER pathway of thylakoid lipid synthesis, whereas HL growth causes opposite effects on fatty acid flux through the two pathways. Although the physiological relevance of these metabolic changes still needs to be further explored, the finding that mutants defective in the plastid or ER pathway showed opposite growth patterns to changes in light intensity suggests that the observed lipid pathway adjustments are an integral part of plant light acclimation responses.

According to our current understanding of lipid metabolism and transport, thylakoid membrane lipids assembled via the ER pathway have a higher energy cost than those assembled via the plastid pathway because the ER pathway requires additional ATP for both trafficking of fatty acids and complex lipids between the ER and the plastid and the ATP-dependent activation of free fatty acids at the outer chloroplast envelope (Li-Beisson et al., 2013). Therefore, increasing fatty acid flux through the plastid pathway reduces energy demand for lipid synthesis, which may confer a performance advantage under LL conditions, when energy is limiting for growth and a massive expansion of the thylakoid membrane system is occurring. On the other hand, redirection of acyl chains though the ER pathway may be of adaptive benefit under HL conditions because it increases energy consumption through glycerolipid synthesis, thus reducing harmful ROS production due to the light energy transfer to oxygen (Allahverdiyeva et al., 2015; Liu et al., 2019). In addition, because the sole metabolic pathway for breakdown of fatty acids occurs in peroxisomes in plants (Graham, 2008), increasing lipid flux through the ER pathway may represent an important adaptive mechanism to permit the direction of excess membrane lipids towards the synthesis of cytosolic TAG, a key intermediate in peroxisomal β-oxidation (Fan et al., 2014, 2017), during thylakoid remodeling in response to changes in growth light conditions.

Previous studies have shown that HL-induced thylakoid structural changes such as unstacking of grana thylakoids and thylakoid swelling are critical for efficient PSII repair and thus represent key aspects of plant adaptation to high-light stress (Khatoon et al., 2009; Anderson et al., 2012; Kirchhoff, 2014; Yoshioka-Nishimura and Yamamoto, 2014; Allahverdiyeva et al., 2015; Derks et al., 2015; Kirchhoff, 2019; Liu et al., 2019). Both MGDG content and the MGDG/DGDG ratio have been suggested as key contributing factors to thylakoid stacking (Lee, 2000; Deme et al., 2014; Seiwert et al., 2018). In Arabidopsis, MGDG is predominantly assembled via the plastid pathway, whereas DGDG is mainly synthesized via the ER pathway (Li-Beisson et al., 2013). Therefore, disruption of one of the two parallel pathways may be expected to affect the ability of plants to reorganize their thylakoid architecture via adjusting galactolipid content and composition and hence their ability to acclimate to changing growth irradiance. Indeed, whereas the increased growth in act1 (Figure 6D) was accompanied by reduced thylakoid stacking (Figure 8J) under HL growth conditions, tgd1 mutant plants showed significantly increased thylakoid stacking (Figure 8J) and reduced growth (Figure 6D) under HL conditions compared with wild-type plants. However, under NL conditions, an increase in the MGDG/DGDG ratio in tgd1 (Figure 7C) was not accompanied by alterations in thylakoid structural organization (Figure 8, J and K). In addition, thylakoid stacking was increased to a similar extent in wild-type plants as well as mutants disrupted in the plastid or the ER pathway of thylakoid lipid synthesis under LL conditions (Figure 8J). Together, these results suggest a specific role of galactolipids in thylakoid architectural reorganizations in response to HL stress. Under LL and NL growth conditions, however, factors such as LHCII (Day et al., 1984; Anderson and Andersson, 1988; Anderson et al., 2012) and curvature thylakoid1 (Luque and Ochoa de Alda, 2014) rather than galactolipid content may play more important roles in thylakoid stacking.

Materials and methods

Plant materials

The Arabidopsis thaliana Columbia ecotype was used in this study. The mutant act1 was previously described by Kunst et al. (1988) and tgd1 mutant by Xu et al. (2003). Arabidopsis seeds were surface-sterilized for 12 min in 10% bleach (v/v), washed five times with sterile water, and plated on one-half-strength Murashige and Skoog (MS) solid medium containing 1% (w/v) sucrose and 0.6% (w/v) agar. After stratification at 4°C for 2 d, the plates were transferred to an incubator with a photon flux density of 50–80 μmol m−2 s−1, a light period of 16 h (22°C), and a dark period of 8 h (18°C). Ten-day-old seedlings were transferred to soil and grown in a growth chamber for light treatments.

Cultivation conditions and experimental design

Ten-day-old seedlings were transferred from MS plates to soil and grown under NL with a photosynthetic photon flux density of 200 μmol m−2 s−1 at 22/18°C (day/night) with a 16-h photoperiod for 1 week. Seedlings were then divided into three groups and each group was maintained under different light levels but at the same temperature of 22/18°C (day/night) and a 16-h photoperiod for an additional 10 d. One group was illuminated with LL of 25 μmol m−2 s−1; a second control group was maintained under NL; and a third group under HL of 1,000 μmol m−2 s−1. After an additional 10-d growth under different light conditions, plants were used for all physiological, biochemical, and ultrastructural analyses. All the experiments were performed with at least three biological repeats. The growth analysis experiment was conducted at least three times using independent trials with similar designs and similar results.

Growth measurement and pigment quantification

For DW measurement, fresh mature leaves were first dried at 100°C for 2 h, then dried at 80°C for 2 d until the weight of the leaves was constant. Chlorophyll content was measured as previously described (Yu et al., 2016).

ACCase activity assay

Intact chloroplasts form plants grown under different light conditions were isolated by discontinuous Percoll gradient according to Fan et al. (2015) and suspended in incubation buffer (50 mM Hepes/potassium hydroxide (KOH), pH 8.0; 330 mM Sorbitol). Chlorophyll was extracted with 80% (v/v) acetone and quantified according to Arnon (1949). Same amounts of chloroplast suspension, corresponding to 10 μg of chlorophyll, were used for ACCase activity assay as previously described (Yu et al., 2018).

Lipid and FA analyses

Lipids of rapidly growing leaves from 5-week-old grown plants under different light conditions were extracted and analyzed as described previously (Fan et al., 2013). Polar lipids were seperated on silica plates (Silica Gel 60, EMD Millipore Corporation) by TLC using acetone–toluent–water (91:30:7, by volume) as a developing solvent. Leaf TAG was extracted and separated according to Fan et al. (2019). Individual lipids were quantified by gas chromatograph–mass spectrometry as described by Fan et al. (2013a). The fatty acid composition at the sn-2 position of the glycerol backbone was determined by Rhizopus arrhizus lipase (Sigma-Aldrich) digestion and quantified as previous described (Fan et al., 2013a).

Assays for FA synthesis

FA synthesis was investigated by 14C-acetate labeling experiments according to Fan et al. (2013). Briefly, leaf discs of mature leaves were incubated in 10 mL labeling medium (20 mM MES, pH5.5, one-tenth strength of MS salts, and 0.01% Tween-20 (v/v)) with shaking. The assay was started by the addition of 0.1 mCi of 14C-acetate (106 mCi/mmol; American Radiolabeled Chemicals, St Louis) and incubated at 22°C under 80 µmol m−2 s−1. After 1-h incubation, leaf discs were washed three times with water, blotted dry, and weighed. Total lipids were extracted using chloroform:methanol:formic acid (1:2:0.1, by volume). The radioactivity associated with total lipids was determined by liquid scintillation counting. Different lipid classes were separated by two-phase TLC, with the first phase using a solvent system of acetone–toluent–water (91:30:7, by volume) and the second phase using a solvent system of hexane:diethyl ether:acetic acid (70:30:1, by volume). The radioactivity associated with individual lipids was quantified by liquid scintillation counting.

Immunoblot analysis

The total soluble proteins from mature leaves of 5-week-old plants under different light conditions were extracted and BC, BCCP, α-CT, and β-CT subunits of plastidic ACCase were immunologically detected as described by Yu et al. (2018).

RNA extraction and reverse-transcription quantitative PCR

Total RNAs were extracted from leaves of wild-type Arabidopsis grown under different light levels using the TRIzol reagent (Thermo Fisher Scientific). Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (New England Biolabs) was used for first-strand cDNA synthesis. Quantitative polymerase chain reaction (qPCR) amplifications were performed on each cDNA dilution with SYBR green master mix (Bio-Rad). qPCR analysis was performed using the iCycler Real-Time PCR System (Bio-Rad). PCR cycles were as follows: one cycle of 95°C for 30 s, followed by 45 cycles of 95°C for 5 s, 58°C for 10 s, and 72°C for 15 s. The primers used in the reverse-transcription quantitative PCR analysis are listed in Supplemental Table S1 and the results were normalized to expression levels of UBQ10. All measurements were carried out in three independent replicates.

Transmission electron microscopy

Mature leaves from 5-week-old plants grown under different light conditions were used for preparation of transmission electron microscopy. Leaf tissues were fixed at room temperature for 2 h in 2.5% glutaraldehyde (v/v) and 0.1 M sodium phosphate buffer (pH 7.2) followed by a secondary fixation with 1% (w/v) osmium tetroxide in the same buffer for 2 h. Samples were then dehydrated in a graded series of ethanol, embedded in EPON812 resin (Electron Microscopy Sciences), and sectioned. After stained with 2% uranyl acetate (w/v) and 2% lead citrate (w/v), the thin sections were viewed under a JEM-1400 LaB6 120 KeV transmission electron microscope (JEOL Inc., http://www.jeolusa.com). Quantitative measurements of membrane profiles on electron micrographs were performed using ImageJ software (Abràmoff et al., 2004). Micrographs used for thylakoid membrane stacking analysis are shown in Supplemental Figures S4–S6.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: ACT1, At1g32200; BCCP1 (AT5G16390); BCCP2 (AT5G15530); BC (AT5G35360); α-CT (AT2G38040); β-CT (ATCG00500) TGD1, At1g19800.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Transcript levels and protein abundance of plastidic ACCase.

Supplemental Figure S2. Changes in levels of total leaf lipids and TAGs under different light conditions.

Supplemental Figure S3. Quantitative analysis of chloroplast ultrastructural changes during light acclimation.

Supplemental Figure S4. Micrographs of leaf cross-section demonstrating changes in thylakoid structure during light acclimation in wild-type plants.

Supplemental Figure S5. Micrographs of leaf cross-section demonstrating changes in thylakoid structure during light acclimation in act1 plants.

Supplemental Figure S6. Micrographs of leaf cross-section demonstrating changes in thylakoid structure during light acclimation in tgd1 plants.

Supplemental Table S1. Primers used in this study.

Supplementary Material

kiaa013_Supplementary_Data
Click here for additional data file. (55.9MB, zip)

Aknowledgments

We thank John Shanklin for critical reading of the article. We also thank John Ohlrogge for antisera against BC, BCCP, and α-CT subunits of the plastidic ACCase.

Funding

This work was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract number DE-SC0012704—specifically through the Physical Biosciences program of the Chemical Sciences, Geosciences, and Biosciences Division.

Conflict of interest statement. None declared.

 

C.X. conceived this project and designed the experiments. L.Y., J.F., C.Z., and C.X. performed the research and participated in data analysis. C.X. wrote the article with contributions from all authors.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys) is: Changcheng Xu (cxu@bnl.gov).

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Supplementary Materials

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