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. 2018 Aug 3;178(1):118–129. doi: 10.1104/pp.18.00539

Starch Deficiency Enhances Lipid Biosynthesis and Turnover in Leaves1,[OPEN]

Linhui Yu 1, Jilian Fan 1, Chengshi Yan 1,2, Changcheng Xu 1,3,4
PMCID: PMC6130009  PMID: 30076222

Starch deficiency enhances the biosynthesis and turnover of membrane lipids and triacylglycerol, which contributes to energy homeostasis and plant growth in Arabidopsis.

Abstract

Starch and lipids represent two major forms of carbon and energy storage in plants and play central roles in diverse cellular processes. However, whether and how starch and lipid metabolic pathways interact to regulate metabolism and growth are poorly understood. Here, we show that lipids can partially compensate for the lack of function of transient starch during normal growth and development in Arabidopsis (Arabidopsis thaliana). Disruption of starch synthesis resulted in a significant increase in fatty acid synthesis via posttranslational regulation of the plastidic acetyl-coenzyme A carboxylase and a concurrent increase in the synthesis and turnover of membrane lipids and triacylglycerol. Genetic analysis showed that blocking fatty acid peroxisomal β-oxidation, the sole pathway for metabolic breakdown of fatty acids in plants, significantly compromised or stunted the growth and development of mutants defective in starch synthesis under long days or short days, respectively. We also found that the combined disruption of starch synthesis and fatty acid turnover resulted in increased accumulation of membrane lipids, triacylglycerol, and soluble sugars and altered fatty acid flux between the two lipid biosynthetic pathways compartmentalized in either the chloroplast or the endoplasmic reticulum. Collectively, our findings provide insight into the role of fatty acid β-oxidation and the regulatory network controlling fatty acid synthesis, and they reveal the mechanistic basis by which starch and lipid metabolic pathways interact and undergo cross talk to modulate carbon allocation, energy homeostasis, and plant growth.

Plant metabolism, maintenance, growth, and development are fueled directly or indirectly by photosynthesis, a process that plants use to convert carbon dioxide into carbohydrates with sunlight as the sole energy source. In photosynthetic source tissues such as mature leaves, a large portion of the carbon assimilated during daytime photosynthesis is stored transiently in the form of starch inside the chloroplast or exported into the cytosol as precursors for Suc synthesis (Smith and Stitt, 2007; Graf and Smith, 2011; Stitt and Zeeman, 2012). The Suc synthesized during photosynthesis can be transported to sink organs via phloem, stored in the vacuole, or broken down to release energy to fuel growth and cellular metabolic processes. At night, when photosynthesis does not occur, starch accumulated during the day is remobilized to provide a continuous supply of sugars for energy production or for Suc synthesis. Both the rate of starch synthesis during the day and the rate of starch degradation during the subsequent night are tightly regulated under a wide range of conditions such that almost all of the carbon stored in starch is directed toward metabolism and growth by the dawn (Smith and Stitt, 2007; Graf and Smith, 2011; Stitt and Zeeman, 2012). Exhaustion of starch at the end of night leads to an inhibition of growth and even an induction of catabolism of proteins, lipids, and other molecules (Gibon et al., 2004; Bläsing et al., 2005; Usadel et al., 2008). On the other hand, incomplete remobilization of starch reduces carbon investment into growth and biomass production (Smith and Stitt, 2007; Stitt and Zeeman, 2012). The importance of starch metabolism in plant growth is clearly illustrated in mutants defective in starch synthesis, such as ADP-glucose pyrophosphorylase1 (adg1; Lin et al., 1988) and phosphoglucomutase1 (pgm1; Caspar et al., 1985). In both cases, mutant plants grow similarly to the wild type under continuous light, but growth is gradually inhibited as night lengths increase until it becomes arrested under short days.

In addition to carbohydrates such as starch and sugars, a substantial fraction of the carbon assimilated during the day is used in the chloroplast to synthesize fatty acids (FAs), the building blocks of membrane lipids and storage triacylglycerol (TAG). For example, in growing soybean (Glycine max) leaves, as much as 25% of 14C-labeled photoassimilates accumulated in lipids (Dickson and Larson, 1975), of which more than half were glycerolipids (Li-Beisson et al., 2013). In many plants, including Arabidopsis (Arabidopsis thaliana), FAs synthesized in the chloroplast are assembled into glycerolipids by two parallel pathways that are located in the chloroplast or in the endoplasmic reticulum (ER; Ohlrogge and Browse, 1995). While the chloroplast pathway produces photosynthetic membrane lipids, the ER pathway is capable of synthesizing both membrane lipids and TAG. Under normal growth conditions, the vast majority of FAs exported from the chloroplast are used for membrane lipid synthesis (Fan et al., 2013b), and less than 1% of total leaf FAs accumulate in TAG (Yang and Ohlrogge, 2009).

In vegetative tissues such as leaves, as much as 4% of total FAs degrade per day (Bao et al., 2000; Bonaventure et al., 2004), and the rate of FA loss increases under carbon starvation conditions such as extended darkness (Fan et al., 2017). In plants, the metabolic breakdown of FAs occurs in peroxisomes via the β-oxidation pathway (Graham, 2008), and the uptake of FAs across the peroxisomal membrane requires PEROXISOME ABC TRANSPORTER1 (PXA1; Footitt et al., 2002). In mature leaves of Arabidopsis, acetyl-CoA, the end product of FA β-oxidation, is used for energy production via mitochondrial respiration (Buchanan-Wollaston et al., 2005; Kunz et al., 2009). Disruption of SUGAR-DEPENDENT1 (SDP1), a key cytosolic lipase responsible for the initiation of TAG hydrolysis into FAs (Eastmond, 2006; Kelly et al., 2013), results in a severely reduced rate of FA degradation, causing an increased accumulation of membrane lipids and TAG in vegetative tissues, indicating an intermediate role of TAG metabolism in FA respiration (Fan et al., 2014, 2017).

Carbohydrate accumulation competes with FA synthesis not only for the same carbon precursors derived from carbon assimilation but also for ATP produced in the photosynthetic light reactions. Although the role of starch metabolism in diurnal sugar homeostasis and plant growth is well established (Smith and Stitt, 2007; Stitt and Zeeman, 2012), little is known about the interaction between starch and lipid metabolism. We recently showed that increasing sugar accumulation by combined disruption of sugar loading into phloem and starch synthesis enhances FA and TAG accumulation in Arabidopsis leaves, particularly in the background of a mutant defective in an importer of lipids into the chloroplast (Zhai et al., 2017). The results reported in this study reveal a functional cross talk between starch and lipid metabolic pathways and a role of lipid turnover in regulating FA synthesis, carbon balance, energy homeostasis, and plant growth and development.

RESULTS

Nighttime Carbon Starvation Triggers Increases in FA Synthesis via Posttranslational Regulation of the Plastidic Acetyl-CoA Carboxylase

Deficiency in starch synthesis and turnover causes temporal carbon starvation at night (Gibon et al., 2004; Thimm et al., 2004; Usadel et al., 2008), which has a profound impact on many aspects of metabolism and physiology (Smith and Stitt, 2007; Stitt and Zeeman, 2012). To test whether the disruption of starch metabolism also affects FA synthesis, we carried out [14C]acetate labeling experiments with rapidly expanding detached leaves of starchless mutants grown in soil. We found that the rate of FA synthesis, estimated as the rate of [14C]acetate incorporation into total lipids, was significantly higher in adg1 and pgm1 compared with the wild type (Fig. 1A). On the other hand, there were no significant differences in the rates of [14C]acetate incorporation into total lipids between the wild type and starchless mutants grown in sugar-supplemented Murashige and Skoog (MS) medium (Supplemental Fig. S1A) or in soil under continuous light (Supplemental Fig. S1B).

Figure 1.

Figure 1.

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Disruption of starch metabolism enhances FA synthesis via posttranslational activation of plastidic ACCase. A, FA synthesis measured as the rate of [14C]acetate incorporation into total leaf lipids in leaves of 4-week-old wild-type (WT) and starchless mutant plants. B, Rate of 3H from 3H2O incorporation into total lipids in leaves of 4-week-old plants. Radioactivity in total lipids was measured on a fresh weight basis and expressed as a percentage of radioactivity in total lipids in the wild type. C, ACCase activity in isolated chloroplasts. ACCase activity was calculated on an equal chlorophyll basis and expressed as a percentage of ACCase activity in the wild type. D, Immunoblot analysis of individual subunits of the plastidic ACCase. Proteins were separated on an equal protein basis. Ponceau S staining of Rubisco was used as a loading control. Asterisks indicate statistically significant differences from the wild type based on Student’s t test (P < 0.05). Data are means of three independent replicates ± sd.

An alternative method for quantifying FA synthesis is to measure the incorporation of 3H from 3H2O into total lipids (Bonaventure et al., 2004). The results obtained using this approach showed that rates of FA synthesis were 50.3% and 64.1% higher in adg1 and pgm1, respectively, compared with the wild type (Fig. 1B). These rates are comparable to the rates obtained with [14C]acetate labeling (Fig. 1A). Together, these results demonstrate that nighttime carbon starvation enhances FA synthesis in leaves.

Previous studies have indicated that the plastidic acetyl-CoA carboxylase (ACCase) is the key regulatory enzyme in FA synthesis in plants (Ohlrogge and Jaworski, 1997; Bates et al., 2013). To understand the mechanism underlying the increased FA synthesis in mutants defective in starch synthesis, we first compared ACCase activity in chloroplasts isolated from leaves of the wild type and starchless mutants. We found that the ACCase activity was 2.7- and 2.4-fold higher in adg1 and pgm1, respectively (Fig. 1C). Plastidic ACCase is a heteromeric bacteria-type enzyme consisting of biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), α-carboxylase (α-CT), and β-carboxylase (β-CT). To determine whether the increased ACCase activity is associated with changes in protein abundance, immunoblot analyses with antisera against individual subunits of plastidic ACCase were performed. There were no obvious changes in protein levels of all four subunits in starchless mutants compared with the wild type (Fig. 1D). Analysis of the relative signal intensity of the individual subunits using ImageJ confirmed that there were no significant differences in protein abundance of all four subunits between the starchless mutants and the wild type (Supplemental Fig. S2). These results suggest that plastidic ACCase activity in leaves of starchless mutants is most likely regulated at the posttranslational level, similar to the situation found in embryo cells of oilseeds (Andre et al., 2012; Bates et al., 2014).

Starch Deficiency Enhances FA Flux through Both the Chloroplast and ER Pathways of Glycerolipid Synthesis

The glycerolipids monogalactosyldiacylglycerol (MGDG) and phosphatidylcholine (PC) represent two major entry points for nascent FAs into the chloroplast and ER pathways of glycerolipid biosynthesis, respectively (Li-Beisson et al., 2013). To assess how the increased FA synthesis affects glycerolipid assembly, we incubated detached leaves with [14C]acetate and determined the rate of [14C]acetate incorporation into MGDG, PC, and TAG. As shown in Figure 2, rapidly expanding leaves incorporated [14C]acetate-derived FAs linearly into MGDG, PC, and TAG over a 1-h period. The initial rate of 14C-labeled FA incorporation was slightly greater for MGDG than for PC in both the wild type and adg1 (Fig. 2A). TAG labeling was low at all time points, accounting for less than 6% of the label found in MGDG (Fig. 2B). No significant differences in MGDG, PC, or TAG labeling were noted between the wild type and adg1 at 15 and 30 min of the incubation period. However, all the three lipids examined contained significantly higher radioactivity in adg1 than in the wild type at the end of the labeling experiment. These results suggest that starch deficiency results in an increase in FA flux through both the chloroplast and ER pathways of glycerolipid biosynthesis.

Figure 2.

Figure 2.

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Starch deficiency enhances FA flux through both the chloroplast and ER pathways of glycerolipid synthesis. Shown is the incorporation of 14C-labeled FAs into MGDG and PC (A) and TAG (B) during [14C]acetate labeling of rapidly expanding detached leaves. FW, Fresh weight. Asterisks indicate statistically significant differences from the wild type (WT) based on Student’s t test (P < 0.05). Data are means of three independent replicates ± sd.

Next, we carried out pulse-chase experiments to determine the potential impact of the increased FA synthesis on FA flux through the two parallel pathways of glycerolipid synthesis. Immediately following incubation for 1 h with [14C]acetate, most of the radiolabel was associated with PC, MGDG, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) in both the wild type and adg1 (Supplemental Fig. S3). Compared with the wild type, the relative label in MGDG was increased significantly, while the relative label in PC was decreased in adg1 (Fig. 3A). During the chase, the label in MGDG increased in the wild type but decreased in adg1, such that the percentage of label in MGDG was comparable between the wild type and adg1 after 3 d of chase (Fig. 3A). In both adg1 and the wild type, a marked decrease in label associated with PC was accompanied by a 10% increase in labeled digalactosyldiacylglycerol (DGDG) (Fig. 3A), reflecting a precursor-product relationship between these two major membrane lipids (Ohlrogge and Browse, 1995). The label in PG decreased in both the wild type and adg1, while the label associated with phosphatidylinositol/sulfoquinovosyldiacylglycerol (PI/SL) and PE increased (Fig. 3B). The label in TAG accounted for less than 4% of total label after the pulse in both the wild type and the mutant (Fig. 3C). During the chase, TAG label decreased slightly over time in the wild type but increased in adg1 following 3 d of chase (Fig. 3C), coinciding with the decline in MGDG label in adg1 (Fig. 3A).

Figure 3.

Figure 3.

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Changes in radioactivity associated with leaf lipids during 3 d of chase. A, Relative radioactivity in PC, MGDG, and DGDG. B and C, Relative radioactivity in PE, PG, phosphatidylinositol/sulfoquinovosyldiacylglycerol (PI/SL; B), and TAG (C). Detached expanding leaves of the wild type (WT) and starchless mutants were pulsed with [14C]acetate for 1 h. Following three washes with water, the leaves were incubated in unlabeled solution for 3 d (chase). Data are means of two independent experiments ± sd.

In pulse-chase experiments using radiolabeled acetate, MGDG is first labeled by the chloroplast pathway and PC is labeled following FA export from the chloroplast via the ER pathway. Therefore, the increase in initial label in MGDG (Fig. 3A) points to an increase in the chloroplast pathway of galactolipid synthesis in starchless mutants. Lipids assembled via the chloroplast pathway are enriched for 16-carbon (C16) FAs at the sn-2 position of the glycerol backbone, whereas lipids assembled via the ER pathway are enriched for 18-carbon FAs at the same position (Li-Beisson et al., 2013). Analysis of acyl group distribution following the position-specific lipase digestion of galactolipids indeed showed that there was a significant increase in 16:3 at the expense of 18:3 at the sn-2 position of MGDG in starchless mutants (Fig. 4A), such that the total C16 FAs at the sn-2 position of MGDG increased from 75.8% ± 1.3% in the wild type to 80% ± 0.2% and 83% ± 2.7% in adg1 and pgm1, respectively. On the other hand, the FA positional distribution of DGDG, which is produced predominantly via the eukaryotic pathway (Li-Beisson et al., 2013), was not altered substantially in starchless mutants (32.2% ± 2.4% for adg1 and 38.2% ± 2.5% for pgm1) compared with the wild type (34.6% ± 3.9%). These results suggest that starch deficiency results in an increase in the chloroplast pathway of MGDG synthesis.

Figure 4.

Figure 4.

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Disruption of starch synthesis alters the FA composition of MGDG and PC. A, FA composition at the sn-2 position of MGDG. B, FA composition of PC. Asterisks indicate statistically significant differences from the wild type (WT) based on Student’s t test (P < 0.05). Data are means of three independent replicates ± sd.

In addition to changes in the relative proportion of MGDG assembled via the two parallel pathways of galactolipid synthesis, disruption of starch synthesis also altered the FA composition of other major membrane lipids. Notably, there were significant increases in 18:1 and 18:2 with a concomitant decrease in 18:3 in PC in starchless mutants compared with the wild type (Fig. 4B). PC is not only the main entry point for FAs synthesized in the chloroplast into the ER pathway (Bates et al., 2007) but also the dominant site of FA desaturation in the ER (Browse et al., 1993; Okuley et al., 1994). Therefore, it is possible that the increased FA synthesis and, hence, an overall increase in FA flux through PC may overwhelm the FA desaturation machinery, thus resulting in an increase in 18:1 and 18:2 at the expense of more desaturated 18:3 in the ER.

The Rate of FA Turnover Is Increased in Starchless Mutants

As shown in Figure 1, mutants deficient in starch synthesis showed about a 50% increase in FA synthesis compared with the wild type. However, the total leaf FA content measured at the end of the night period was significantly lower in starchless mutants compared with the wild type (Fig. 5A). These results suggest an increase in FA turnover in starchless mutants. To confirm this, we analyzed the decay rate of total labeled FAs in pulse-chase experiments using [14C]acetate. To this end, we first incubated detached leaves with [14C]acetate for 1 h, and then the label was chased for 3 d. In the wild type, the total radiolabel loss during the 3-d chase was 18% (Fig. 5B), which corresponded to an average decay rate of 6% per day. By contrast, the starchless mutants lost as much as 38% of the total label during the same period, which corresponded to an average label decay rate of 12.6% per day, an approximately 2-fold increase in the rate of FA loss in starchless mutants compared with the wild type.

Figure 5.

Figure 5.

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FA turnover is important for the growth and development of starchless mutants. A, Total FA content in leaves (based on dry weight [DW]) of wild-type (WT) and starchless mutant plants measured at the onset of the light period. B, Radiolabel loss during 3 d of chase following 1 h of pulse of detached leaves with [14C]acetate. C, Five-week-old wild-type and single and double mutant plants grown in soil. D, Shoot biomass of 4-week-old plants grown in soil. Asterisks indicate statistically significant differences from the wild type (A and B) or between the double mutants and adg1 (D) based on Student’s t test (P < 0.05). Data are means of five independent replicates ± sd.

FA Turnover Is Important for the Growth and Development of Starchless Mutants

To test the physiological significance of the increased FA turnover in the starchless mutants, we constructed double mutants between adg1 and pxa1-2 or sdp1-4 (Fan et al., 2017). We confirmed that, under our growth conditions, adg1, pgm1, adg1 sdp1-4, and adg1 pxa1-2 contained very limited amounts of starch at the end of day (Supplemental Fig. S4). Compared with adg1, both adg1 sdp1-4 and adg1 pxa1-2 double mutants showed significantly reduced growth and delayed bolting and flowering under long days (Fig. 5, C and D). Thus, for example, the shoot fresh weights of adg1 sdp1-4 and adg1 pxa1-2 were 66% and 54% of that of adg1 plants, respectively, after 40 d of growth (Supplemental Fig. S5C). However, at week 4 after sowing, adg1 sdp1-4 and adg1 pxa1-2 double mutants had the same number of leaves but a reduced rosette size compared with the single mutants and the wild type (Supplemental Fig. S5, A and B). The growth and development of double mutants, in particular adg1 pxa1-2, was stunted under short days (Supplemental Fig. S6). While the adg1 sdp1-4 double mutant was able to generate a few siliques with viable seeds, the adg1 pxa1-2 double mutant failed to bolt and produce flowers after 4 months of growth under an 8-h-light/16-h-dark photoperiod. No major differences in growth phenotypes were found among the wild type, sdp1-4, and pxa1-2 under either long or short days, while adg1 showed reduced growth and slower development under both photoperiods compared with the wild type (Fig. 5C; Supplemental Fig. S6), as observed in previous studies (Caspar et al., 1985; Lin et al., 1988).

Disruption of SDP1 or PXA1 Alters Lipid and FA Composition in adg1

To investigate how defects in FA turnover affect the plant growth and development of starchless mutants, we first compared the leaf lipid profiles of the single and double mutants. Figure 6A shows total leaf FA content measured in mature leaves 4 h into the light period. There was no significant difference in total FA levels among sdp1-4, pxa1-2, and the wild type. Disruption of SDP1 or PXA1 in adg1 resulted in a 22% or 18% increase in total leaf FAs, respectively, due primarily to increases in MGDG (Fig. 6A) and TAG (Fig. 6B) levels in the double mutants compared with adg1.

Figure 6.

Figure 6.

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Disruption of SDP1 or PXA1 alters lipid and FA composition in adg1. A, Total leaf FA and membrane lipid levels (based on dry weight [DW]) in leaves of wild-type (WT) and single and double mutant plants measured at the onset of the light period. B, TAG content in leaves of 6-week-old plants grown in soil. C, FA composition of PC in leaves of wild-type and single and double mutant plants grown in soil. D, C16 FA composition at the sn-2 position of MGDG from leaves of wild-type and starchless mutant plants. Asterisks indicate statistically significant differences between the double mutants and adg1 based on Student’s t test (*, P < 0.05 and **, P < 0.01). Data are means of three independent experiments ± sd.

The FA composition of membrane lipids in the double mutants was altered compared with that in adg1. Notably, the relative proportion of 18:1 in PC was decreased markedly, whereas 18:3 was increased significantly in adg1 sdp1-4 and adg1 pxa1-2 double mutants (Fig. 6C). In addition, there was a significant decrease in the relative proportion of C16 FAs at the sn-2 position of MGDG (Fig. 6D), suggesting a decrease in the chloroplast pathway or an increase in the ER pathway of galactolipid biosynthesis in the double mutants compared with adg1.

Disruption of SDP1 or PXA1 Significantly Reduces FA Turnover in adg1

Compared with the wild type, there was no significant increase in total FA content in adg1 sdp1-4 and adg1 pxa1-2 double mutants (Fig. 6A), despite a marked increase in FA synthesis in adg1 (Fig. 1). A likely explanation for this discrepancy is that, in the absence of FA turnover, FA synthesis is reduced in adg1. However, radiotracer labeling experiments using 3H2O showed that rates of FA synthesis remained largely unchanged in adg1 sdp1-4 and adg1 pxa1-2 double mutants compared with adg1 (Fig. 7A). To test whether FA turnover is affected in the double mutants, a [14C]acetate pulse-chase labeling with detached leaves was again carried out. Surprisingly, disruption of PXA1 or SDP1 in adg1 had only a slight, albeit significant, effect on the rate of FA turnover, measured as the loss of 14C-labeled FAs during the 3-d chase period, compared with adg1 (Fig. 7B).

Figure 7.

Figure 7.

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FA synthesis and turnover in adg1 sdp1-4 and adg1 pxa1-2. A, FA synthesis measured as the rate of 3H from 3H2O incorporation into total lipids in expanding leaves of starchless mutant plants. Radioactivity in total lipids was measured on an equal fresh weight basis and expressed as a percentage of radioactivity in total lipids in adg1. B, FA turnover measured as a percentage of radiolabeled FA loss during 3 d of chase following incubation with [14C]acetate for 1 h. Asterisks indicate statistically significant differences between the double mutants and adg1 based on Student’s t test (P < 0.05). Data are means of three independent experiments ± sd.

Double Mutants Defective in Starch Synthesis and FA Turnover Accumulate Sugars

To test whether carbon fixation is affected in adg1 sdp1-2 and adg1 pxa1-2 double mutants, a number of parameters associated with photosynthesis were compared. The relative rate of 14CO2 fixation was similar among the wild type, sdp1-4, pxa1-2, adg1, adg1 sdp1-4, and adg1 pxa1-2 (Supplemental Fig. S7A). In addition, there were no significant differences in the maximum quantum efficiency of PSII, linear photosynthetic electron transport rate (ETR), and quantum yield of photochemical energy conversion (ΦPSII) among the wild type and single and double mutants (Supplemental Fig. S7, B–D).

On the other hand, total leaf sugar levels measured at the end of the light period were 1.8- and 2-fold higher in adg1 sdp1-4 and adg1 pxa1-2, respectively, compared with adg1 (Fig. 8A). In the wild type, sdp1-4, and pxa1-2, Glc and Suc each accounted for 45% of the total sugars measured, with a much lower percentage contributed by Fru (10%; Fig. 8B). In adg1, adg1 sdp1-4, and adg1 pxa1-2 mutants, Glc was the most abundant sugar (45%), followed by Suc (40%), and again Fru content was much lower (15%) compared with Glc and Suc. Additionally, as shown in Figure 8, there also were substantial significant increases in sugar contents in sdp1-4 and pxa1-2 single mutants compared with the wild type. Sugar content was significantly higher in adg1 relative to the wild type, consistent with the results of previous analyses (Lin et al., 1988).

Figure 8.

Figure 8.

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Disruption of SDP1 or PXA1 in adg1 enhances sugar accumulation in leaves. A, Total sugar levels (based on dry weight [DW]) in leaves of wild-type (WT) and mutant plants. B, Glc (Glu), Fru, and Suc content in the same samples as in A. Data are means of at least three independent replicates ± sd.

DISCUSSION

The ability to adjust metabolism in response to environmental cues or metabolic perturbations is vital for the survival of organisms. In mammalian cells, a key aspect of metabolic adaptability is the capacity to switch between using carbohydrates and fats as a source of energy. In plants, carbohydrates are the dominant respiratory substrates (Plaxton and Podesta, 2006). Lipids serve as a major carbon and energy source during seed germination and early seedling establishment of oilseeds (Graham, 2008) but are rarely used for respiration during the rest of the life cycle (Plaxton and Podesta, 2006; Araújo et al., 2011), except under stress conditions such as extended darkness (Kunz et al., 2009; Fan et al., 2017). Here, we show that, like mammalian cells, plants also possess the ability to switch between carbohydrates and FAs as respiratory substrates during normal growth. We found that, in mutants defective in starch accumulation, rates of both FA synthesis and turnover are increased significantly. Blocking FA turnover caused an up to 40% reduction in the growth of starchless mutants and a significant delay of bolting and flowering, suggesting that FA breakdown plays a previously unrecognized role in the growth and development of mutants defective in starch metabolism.

Lipids contain more than twice as much energy per gram as carbohydrates. Yet, the role of lipids in supporting growth at night is limited, as evident from the retarded growth phenotype of starchless mutants under normal growth conditions. This limitation is likely due to the inability of vegetative tissues of plants, such as Arabidopsis, to convert lipids to sugars due to the lack of several key enzymes associated with the glyoxylate cycle (Buchanan-Wollaston et al., 2005). In addition, while the rates of both starch synthesis and degradation are exquisitely regulated to ensure a constant supply of carbon at night (Stitt and Zeeman, 2012), lipids are turned over rapidly during the day, causing a futile cycle of FA synthesis and breakdown and, thus, wasting carbon and energy derived from photosynthesis.

Sugar levels increase significantly in starchless mutants and more so in starchless mutants deficient in FA respiration (Fig. 8). Sugar respiration is known to be determined mainly by substrate availability (Azcón-Bieto et al., 1983; Peraudeau et al., 2015; O’Leary et al., 2017). Indeed, it was reported that increased sugar accumulation stimulated respiration in starchless mutants (Caspar et al., 1985; Lin et al., 1988), such that sugars accumulated in the day were depleted quickly upon entering the night period, leaving the mutant plants starved for carbon for most of the subsequent night (Gibon et al., 2004; Thimm et al., 2004; Usadel et al., 2008). In addition, increased respiration during the day may enhance respiratory carbon loss, leading to a reduced carbon economy and, thus, reduced growth. This may explain at least in part the growth retardation phenotype of double mutants defective in starch synthesis and FA turnover (Fig. 5C; Supplemental Fig. S6). Sugar accumulation is known to cause a feedback inhibition of photosynthesis (Sun et al., 1999). However, under our growth conditions, the efficiency of photosynthesis appeared to be unaffected in the starchless mutants or in double mutants lacking starch synthesis and FA turnover (Supplemental Fig. S7).

Peroxisomal β-oxidation is the sole pathway for the metabolic breakdown of FAs in plants (Graham, 2008). However, the disruption of PXA1 had only a slight influence on the rate of radiolabeled FA loss in adg1 leaves (Fig. 7B). This result suggests that, in the absence of FA transport mediated by PXA1, FA β-oxidation still occurs in starchless mutants. In yeast (Saccharomyces cerevisiae), PXA proteins are only responsible for transporting long-chain FAs such as 18:1, but not for medium-chain FAs such as laureate (12:0) and myristate (14:0), into peroxisomes (Hettema et al., 1996). Therefore, it is possible that, in the absence of PXA1, medium-chain rather than long-chain FAs are imported into peroxisomes to support β-oxidation and, hence, carbon flux through FA synthesis in starchless mutants. Alternatively, additional pathways are activated and responsible for FA breakdown in starchless mutants lacking PXA1.

We recently demonstrated that TAG is a key intermediate in the peroxisomal breakdown of FAs of membrane lipids (Fan et al., 2014, 2017). In line with this view, there were no major differences between adg1 sdp1-4 and adg1 pxa1-2 with regard to shoot growth, sugar accumulation, leaf FA content, and membrane lipid FA composition. However, under short days, disruption of PXA1 caused more pronounced growth retardation than inactivation of SDP1 in the adg1 mutant background (Supplemental Fig. S6). Recent studies in yeast and mammals have established that, in addition to cytosolic TAG lipases, a vacuolar/lysosomal acid lipase is involved in the selective breakdown by autophagy of lipid droplet-stored TAG, termed lipophagy under conditions of nutrient starvation (Jaishy and Abel, 2016; Wang, 2016). A homolog of mammalian acid lipase has been shown to harbor TAG lipase activity (El-Kouhen et al., 2005). In addition, it was demonstrated recently that autophagy is activated under short days and contributes to nighttime energy supply for growth in starchless mutants (Izumi et al., 2013). Therefore, a simple explanation for the growth difference between adg1 sdp1-4 and adg1 pxa1-2 and for the occurrence of FA β-oxidation in adg1 sdp1-4 is that, in the absence of SDP1 and starch accumulation, lipophagy is activated to release FAs from TAG stores for energy production via peroxisomal β-oxidation and mitochondrial respiration.

In Arabidopsis leaves, about 40% of the FAs synthesized in the chloroplast are used for membrane lipid assembly at the chloroplast envelope and the remainder are exported into cytosol to enter the ER pathway (Browse et al., 1986). TAG accumulated in adg1 sdp1-4 or adg1 pxa1-2 contained mostly C18 FAs (Supplemental Fig. S8), suggesting that TAG accumulation and FA β-oxidation directly draw FAs from the ER pathway. Therefore, increasing TAG storage in cytosol and FA β-oxidation in peroxisomes causes a decrease in FA flux into the ER pathway of membrane synthesis and, hence, a relative increase in the MGDG and DGDG species made via the chloroplast pathway in starchless mutants. On the other hand, blocking FA turnover in the starchless mutants results in an increase in the trafficking of ER-derived lipids back into the chloroplast, causing an increase in the proportion of galactolipids made via the ER pathway in the double mutants. Taken together, these results not only demonstrate a remarkable flexibility of two parallel glycerolipid biosynthetic pathways, as observed previously in other Arabidopsis mutants (Kunst et al., 1988; Xu et al., 2003) as well as in plants’ adaptation to temperature stress (Li et al., 2015), but also suggest an important role of FA β-oxidation and TAG accumulation in glycerolipid pathway adjustments in response to metabolic perturbations and environmental challenges.

From a biotechnological perspective, lipids in the form of TAG are not only the major source of dietary calories but also important raw materials for numerous industrial applications. Therefore, enhancing TAG accumulation in plant biomass has been a major goal in many research endeavors (Xu and Shanklin, 2016). Our results suggest that blocking starch synthesis and/or FA turnover, common strategies used in engineering TAG accumulation in vegetative plant tissues (Xu and Shanklin, 2016), has a major negative impact on FA accumulation. Future studies to uncover the molecular mechanism controlling ACCase activity and, hence, FA synthesis in mutants disrupted in starch metabolism may provide new strategies to enhance FA accumulation in plants for food, feed, industrial chemicals, and fuels.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) plants used in this study were of the Columbia ecotype. The adg1 mutant was described previously by Lin et al. (1988), pgm1 by Caspar et al. (1985), and sdp1-4 and pxa1-2 by Fan et al. (2017). The adg1 and pgm1 mutants were ordered from the Arabidopsis Biological Research Center at Ohio State University (Alonso et al., 2003). The primers used for genotyping pxa1-2 and sdp1-4 in double mutant constructions were as described previously (Fan et al., 2014).

For growth on plates, surface-sterilized seeds of Arabidopsis were germinated on 0.6% (w/v) agar-solidified one-half-strength MS medium (Murashige and Skoog, 1962) supplemented with 1% (w/v) Suc in an incubator with a photon flux density of 50 to 80 μmol m–2 s–1, a light period of 16 h (22°C), and a dark period of 8 h (18°C). For growth in soil, plants were first grown on MS medium for 10 d, transferred to soil (Arabidopsis Growth Medium; Lehle Seeds), and then grown under a photosynthetic photon flux density of 80 to 150 μmol m−2 s−1 at 22°C/18°C (day/night) with a 16-h-light/8-h-dark period, unless sepcified. Nutrition solution (Miracle-Gro; Scotts) was applied once at the time of transplanting within the first 4 weeks and then once every 2 weeks starting from week 5 after sowing.

Plant Growth Analysis

For plant growth analysis, seeds of the wild type and single and double mutants were sown on one-half-strength MS medium as described above. Ten-day-old seedlings were transplanted to pots (12 seedlings per pot) containing the soil mixture described above. Pots were then distributed randomly in a walk-in growth chamber under conditions as described above. After additional growth for 18 or 30 d, shoot fresh weight was measured from 20 to 30 plants per genotype. In each experiment, three random pots were used as three biological replicates, and the growth analysis experiment was conducted at least five times using independent trials with similar designs and similar results.

Lipid and FA Analyses

Samples for lipid analysis as well as for other assays were taken from plants approximately 4 h into the light period, unless stated otherwise. Lipids were extracted and analyzed as described previously (Fan et al., 2013a). The FA composition at the sn-2 position of the glycerol backbone was determined by Rhizopus arrhizus lipase (Sigma-Aldrich) digestion (Fan et al., 2013b).

Immunoblotting

The total soluble proteins from homogenized leaves were extracted with protein extraction buffer (50 mm HEPES-KOH, pH 7.6, 150 mm NaCl, 1 mm EDTA, 10% (v/v) glycerol, 1 mm 2-mercaptoethanol, 0.25% (v/v) Triton X-100, and 1× complete protease inhibitor cocktail). Protein content was quantified using a Bio-Rad DC protein assay kit according to the manufacturer’s instructions. The same amount of proteins was separated by a 10% (v/v) SDS-PAGE gel and blotted to a PVDF membrane. The BC, BCCP, and α-CT subunits of plastidic ACCase were immunologically detected with antisera that had been generated and characterized (Shorrosh et al., 1996). Anti-β-CT antibody was obtained from Agrisera Antibodies (AS15 2880).

Assays for FA Synthesis and Degradation

In vivo labeling experiments with [14C]acetate were done according to Koo et al. (2005). Briefly, rapidly growing leaves of 4-week-old plants were incubated in light at 80 µmol m−2 s−1 at 22°C in 10 mL of medium containing 20 mm MES, pH 5.5, one-tenth-strength MS salts, and 0.01% (v/v) Tween 20. The assay was started by the addition of 0.1 mCi of [14C]acetate (106 mCi mmol−1; American Radiolabeled Chemicals) or 0.2 mCi of 3H2O (American Radiolabeled Chemicals). After incubation for 1 h, leaves were washed two times with water and used immediately for lipid extraction. For pulse-chase labeling experiments, leaves were labeled for 1 h with [14C]acetate. After washing three times with water, the leaves were incubated further with unlabeled solution under a 16-h-light/8-h-dark cycle for 3 d. Total lipids were extracted and separated as described (Fan et al., 2013a), and radioactivity associated with total lipids or different lipid classes was determined by liquid scintillation counting or phosphor imaging. Radioactivity was calculated by correcting for the dilution of specific activity caused by tissue growth during the chase period.

ACCase Activity Assay

Intact chloroplasts from 4-week-old Arabidopsis plants were isolated by a discontinuous Percoll gradient according to Fan et al. (2015) and suspended in incubation buffer (330 mm sorbitol and 50 mm HEPES-KOH, pH 8). Chlorophyll was extracted with 80% (v/v) acetone and quantified according to Lichtenthaler (1987). ACCase activity was determined as described previously (Shorrosh et al., 1996). Briefly, the same amount of chloroplast suspension, corresponding to 10 μg of chlorophyll, was assayed in a 0.5-mL Eppendorf tube with 50 μL of reaction buffer containing 100 mm Tricine (pH 8.2), 50 mm KCl, 5 mm MgCl2, 5 mm DTT, 3 mm ATP, 0.5 mm acetyl-CoA, 0.2% (v/v) Triton X-100, and 3.4 mm NaH14CO3 (54 mCi mmol−1; American Radiolabeled Chemicals) for 20 min at 30°C. Assays omitting acetyl-CoA served as controls. Reactions were stopped by adding 15 μL of 10 m HCl. The reaction mixture was dried at 80°C, and the radioactivity was determined by liquid scintillation counting.

Assay for 14CO2 Fixation

To examine CO2 fixation, plants were grown on plates containing agar-solidified one-half-strength MS medium without Suc for 4 weeks. The plates were then transferred to a transparent plastic bag (approximately 1.5-L gas space) preloaded with a shallow vial containing 0.1 mCi of NaH14CO3 (54 mCi mmol−1; American Radiolabeled Chemicals). Plants were illuminated with a photon flux density of 80 μmol m–2 s–1 at 22°C. To generate 14CO2, 100 µL of 10% (v/v) H2SO4 was injected into the vial of the sealed bag. After further incubation for 1 h, the bag was removed and shoots were harvested and extracted with boiling 80% (v/v) ethanol. The radioactivity of ethanol-soluble materials was determined by liquid scintillation counting.

Measurements of Chlorophyll Fluorescence Parameters

Chlorophyll fluorescence measurements were performed with a portable pulse amplitude fluorometer (PAM-2000; Walz) on 4-week-old plants grown in soil. The maximum quantum efficiency of PSII was recorded after 15 min of dark adaptation. The fluorescence kinetic parameters for ETR and ΦPSII were recorded during steady-state photosynthesis, which was achieved after 15 min of illumination with an actinic light at a photon flux density similar to that experienced by the plants during growth. ΦPSII was calculated according to the following equation: ΦPSII = (Fm′ − F′)/Fm′ (Baker, 2008), where Fm′, and F′ are maximum and steady-state fluorescence yields from light-adapted leaves, respectively. Photosynthetic ETR was calculated as the product of ΦPSII × actinic light intensity × 0.84 × 0.5, according to Genty et al. (1989).

Quantification of Starch and Soluble Sugars

The homozygosity of the starchless lines used in this study was verified by the absence of starch at the end of day by iodine staining according to Lin et al. (1988) and coupled enzymatic assays according to Stitt et al. (1989). Soluble sugars were extracted twice with 80% (v/v) ethanol at 80°C, and extracts were dried and dissolved in distilled water. Levels of soluble sugars (Glc, Fru, and Suc) in ethanol extracts were quantified by the sequential addition of Glc-6-P dehydrogenase, hexokinase, Glc-6-P isomerase, and invertase and measuring the increase in A340 as described by Stitt et al. (1989).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: ADG1, At5g48300; PDAT1, At5g13640; PGM1, At5g51820; PXA1, At4g39850; and SDP1, At5g04040.

Supplemental Data

The following supplemental materials are available.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

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.

Footnotes

1

This work was supported by the U.S. 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.

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