1. Introduction
Plants break down sugars through respiration to fuel metabolism, growth, storage and maintenance throughout the day/night cycle. During the day, leaves carry out photosynthesis, which converts carbon dioxide and water into organic compounds using sunlight as an energy source. The prime end-product of photosynthetic carbon assimilation, triose phosphate, is partitioned into several central metabolic pathways, including those leading to the generation of carbohydrates, proteins and lipids [
1]. In many plants, including Arabidopsis, a large fraction of triose phosphate formed during photosynthesis is used to synthesize starch, a glucose polymer, in the form of semi-crystalline insoluble granules in the chloroplast. At night, when photosynthesis ceases, starch is hydrolyzed to provide a constant supply of sugars for respiration [
2,
3,
4,
5]. The physiological importance of diurnal starch metabolism is clearly illustrated in mutants that are defective in starch synthesis [
6,
7] or degradation [
8,
9]. For example, an Arabidopsis starchless mutant defective in
ADP-glucose pyrophosphorylase1 (
adg1) shows growth retardation under long days, stunted growth under short days, but similar growth rates to wild type under continuous night [
6]. The growth inhibition of starchless mutants in day/night conditions has been attributed to nighttime carbon starvation [
4,
5], increased root respiration [
10] and sugar-induced feedback inhibition of photosynthesis [
11].
Lipids serve as the building blocks of cellular membranes, and the storage lipid triacylglycerol (TAG) is amongst the most energy-rich compounds that occur in nature. In Arabidopsis and many other plants, two parallel pathways compartmentalized either in the plastid or the endoplasmic reticulum (ER) are responsible for synthesizing the vast majority of cellular lipids, principally glycerolipids [
12]. Fatty acids, the predominant components of glycerolipids are synthesized in the plastid. They can be incorporated into thylakoid membranes, including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in the plastid via the endogenous plastid pathway. Alternatively, fatty acids can be exported from the plastid and metabolized in the ER to generate both membrane phospholipids and TAG storage lipids via the ER pathway. A fraction of phospholipids assembled in the ER re-enter the plastid to support the plastid pathway of thylakoid lipid synthesis. Because the substrate specificity of acyltransferases in the ER and plastids differs, lipids assembled via either the ER or the plastid pathway can be distinguished by the presence of acyl groups with chain lengths of 16 carbons (C16) or 18 carbons (C18) at the
sn-2 position, respectively [
13].
In vegetative tissues such as leaves, most of the fatty acid flux through the ER pathway is directed toward membrane lipid synthesis [
14]. Consequently, TAG does not accumulate to significant levels in leaves [
15], despite the presence of high TAG synthetic [
16] and hydrolytic [
17] activities. In plants, the last step of TAG synthesis is mediated by two distinct types of acyltransferases: acyl-CoA:diacylglycerol acyltransferase and phospholipid:diacylglycerol acyltransferase (PDAT) [
18]. Overexpressing PDAT1 caused an increase in fatty acid synthesis, an increased diversion of fatty acids from membrane lipids to TAG and hence a significant increase in TAG levels in leaves [
14].
The metabolic breakdown of TAG is catalyzed by lipases including SUGAR-DEPENDENT 1 (SDP1) [
19], and the resultant free fatty acids are imported into the peroxisome by PEROXISOMAL ABC TRANSPORTER 1 (PXA1) where they are metabolized via β-oxidation to acetyl-CoAs, which represent key metabolites for energy production in mitochondria [
20]. Disruption of SDP1 or PXA1 significantly compromises the growth and development of
adg1 [
21], suggesting that in the absence of starch, lipids are used as an alternative respiratory substrate for energy production. Here, we show that overexpression of
PDAT1 enhances carbon partitioning into lipids and improves the growth of the
adg1 starchless mutant. Together, our studies reveal a previously unrecognized role of lipid metabolism in diurnal energy homeostasis and plant growth under normal growth conditions.
3. Discussion
We recently showed that deficiency in starch synthesis in
adg1 results in increased rates of fatty acid synthesis and turnover without impacting overall membrane content [
21]. Results from the present study show that overexpression of PDAT1, a critical enzyme catalyzing the last step in TAG synthesis in leaves, enhances fatty acid and TAG synthesis at the expense of soluble sugars and improves the growth of the starchless mutant
adg1. Lipids contain more than twice as much energy per gram as carbohydrates and proteins. In addition, unlike sugars, which can be metabolized easily and quickly as a source of immediate energy, TAG breakdown is complex and slow, requiring the coordinated actions of several different subcellular organelles including lipid droplets, peroxisomes and mitochondria. The difference in the rate at which sugars and TAG are metabolized is clearly illustrated in
adg1 lines overexpressing PDAT1. Although, leaves of the PDAT1-overexpressing lines accumulated up to two-fold more sugars than TAG on a dry weight (DW) basis at the end of the day, sugars were completely depleted within the first 4 h into the night period (
Figure 6), whereas as much as 30% TAG remained at the end of the dark period (
Figure 1). Sugars were almost completely consumed within the initial 4 h of the night period in
adg1 (
Figure 6), leaving
adg1 plants starved for carbon and energy for the rest of the night period, which has been shown to cause severe reductions in plant growth during the following light period [
24]. Overexpression of PDAT1 diverts about half of carbon destined for sugar synthesis into TAG. The slow breakdown of TAG accumulated during the day allows a constant supply of fatty acids for energy production throughout the night period, which alleviates nighttime energy deficiency in the absence of transient starch, likely explaining why overexpression of PDAT1 improves the growth of
adg1.
Leaf photosynthetic capacity is known to be regulated by the rate of utilization of photoassimilates in the rest of the plants [
25]. Indeed, increasing sink strength has been demonstrated to increase the rate of photosynthetic carbon assimilation in wide variety of species [
26,
27]. Fatty acid synthesis is a highly energy-demanding process, consuming chemical energy stored in seven molecules of ATP and reducing power in 14 molecules of NADPH for every molecule of 16:0 produced [
28], in addition to carbon precursors in the form of triose phosphate, the primary end product of photosynthetic carbon fixation. Therefore, it has be hypothesized that increasing TAG accumulation in leaves may lead to an increase in photosynthesis [
29]. Our study shows that the overexpression of PDAT1 had no significant effects on photosynthesis as assessed by chlorophyll fluorescence kinetic parameters, and that TAG accumulation in PDAT1 overexpressing lines was at the expense of sugars. These results imply that under our growth conditions, photosynthesis is not limited by sink activity, but possibly by available light. Future studies will test whether increasing TAG accumulation affects photosynthesis and plant fitness and performance under high light conditions or in natural environments. Since leaves analyzed in this study may contain chloroplasts at different levels of development, further studies are also needed to assess the difference in the pathway of carbon flow as well as in response to changing light intensities between developing and mature chloroplasts.