Due to their sessile life style, plants are unavoidably exposed to environmental stresses throughout their life cycle. To deal with such constraints, plants have evolved mechanisms that allow for efficient adaption to an ever-changing environment. Fatty acids (FAs) are pivotal constituents of cellular membranes, suberin, and cutin waxes that provide structural barriers to the environment (Beisson et al., 2007). Cellular membranes are a major target of environmental signals, and the maintenance of their integrity, permeability, fluidity and functionality of transport proteins by recalibrating lipid composition is critical for the resistance to different stresses (Rodriguez-Vargas et al., 2007; Huynh et al., 2012; Maejima et al., 2014).

Acyl carrier proteins (ACPs) play a central role in de novo FA synthesis. Fatty acid synthases (FASs) can be separated into two distinct classes. TypeI FASs consist of a single, large, multifunctional polypeptide and are common to mammals, fungi, and some bacteria. Type II FASs are found in archaea, bacteria and plants and are characterized by the involvement of discrete, mono-functional enzymes for FA synthesis. ACP exists as a separated domain (Jenke-Kodama et al., 2005). Plant ACPs are small (9 kD) separated polypeptides with 70–80 mostly acidic residues, modified by the covalent attachment of 4′-phosphopantetheine to a centrally localized serine (Wakil et al., 1983; Marrakchi et al., 2002). De novo biosynthesis of FAs proceeds via a conserved set of reactions, which are carried out during the elongation cycle (Smith and Sherman, 2008). ACPs are central components of FASs, which covalently bind all fatty acyl intermediates. In the first step, ACP synthase (ACPS) attaches a phosphopantetheine group from CoA on a serine residue of ACP in a conserved Asp-Ser-Leu motif to form holo-ACP (Mofid et al., 2002). The initial substrate of FASs, malonyl-CoA, is transferred to ACP and the acetyl-CoA unit (C2) is expanded to a butyryl group (C4). The synthetic cycle is repeated multiple times until saturated C16 or C18 acyl-ACPs are produced for utilization in membrane biosynthesis (Chan and Vogel, 2010).

Besides the production of saturated FAs (SFAs), ACPs are also involved in the biosynthesis of unsaturated FAs (UFAs), which proceeds via a slightly different reaction scheme. In Escherichia coli, most of the C₁₀ acyl chain substrates are processed by β-hydroxyacyl dehydratase (FabZ) to form trans-2-decenoyl-ACP. Subsequently, enoyl reductase (FabI), β-Oxoacyl synthase I (FabB) or β-Oxoacyl synthase II (FabF) catalyzes the formation of SFAs. Alternatively, this fatty acyl intermediate can be converted through dehydration and isomerization to cis-3-decenoyl-ACP catalyzed by β-hydroxyacyl dehydratase (FabA) (Heath and Rock, 1996; Leesong et al., 1996), which can be further extended to UFAs by FabB (Feng and Cronan, 2009). Therefore, in E. coli FabBs and FabIs compete for cis-3- and trans-2-decenoyl-ACP, respectively, and the relative abundance of these two enzymes determines the ratio of saturated to UFAs in the membrane. The vast majority of UFAs in E. coli are cis-9-hexadecenoic acid (palmitoleic acid) and cis-11-octadecenoic acid (vaccenic acid) (Cronan, 1967; Morein et al., 1996).

The levels of UFAs can be changed to adjust membrane lipid fluidity to environmental conditions, mainly by regulating the activity of FA desaturases. For example, enhancement of polyunsaturated FA levels in chloroplast lipids was shown to be an effective means to resist chilling stress, while an opposite trend was observed during heat stress (Iba, 2002). Free linolenic acid itself is a stress signal and the precursor for phyto-oxylipin biosynthesis (Blee, 2002). Increasing evidence suggests that chloroplast oleic acid (18:1) levels are critical for normal pathogen defense responses in Arabidopsis, including programmed cell death and systemic acquired resistance (SAR) (Kachroo et al., 2001). Also, the Arabidopsis FAD2, an endoplasmic reticulum (ER)-localized ω-6 desaturase, which converts oleic acid (18:1) to linoleic acid (18:2) by inserting a double bond at the ω-6 position, is required for chilling and salt tolerance (Miquel et al., 1993; Zhang et al., 2012). All higher plants studied so far contain multiple isoforms of ACP, some of which are expressed ubiquitously while others appear to be expressed in a tissue-specific manner (Battey and Ohlrogge, 1990; Hlousekradojcic et al., 1992). The genome of the model plant Arabidopsis thaliana harbors three genes encoding mitochondrial ACPs, namely mtACP1 (At2g44620), mtACP2 (At1g65290), and mtACP3 (At5g47630), and five plastidial ACPs, AtACP1 (At3g05020), AtACP2 (At1g54580), AtACP3 (At1g54630), AtACP4 (At4g25050), and AtACP5 (At5g27200) (Meyer et al., 2007). Based on protein analysis, AtACP1, AtACP2, and AtACP3 are expressed in all tissues examined (Hlousekradojcic et al., 1992), whereas AtACP4 is expressed predominantly in leaves and the mRNA levels are increased by light (Shintani, 1996; Bonaventure and Ohlrogge, 2002). AtACP1 and AtACP3 are regulated by the transcription factor WRINKLED1 (WRI1) and are required for the accumulation of triacylglycerols in Arabidopsis seeds (Ruuska et al., 2002; Maeo et al., 2009). The expression of both genes followed a bell-shaped pattern that increased through the early seed development stages and peaked between 8 and 11 days after flowering (Ruuska et al., 2002). AtACP2 was associated with the cell cycle (Menges et al., 2002), and its expression was up-regulated in developing siliques (Moire et al., 2004) and in cytokinin receptor mutants (Rashotte et al., 2003). AtACP4 was required to perceive the mobile SAR signal in distal tissues of Arabidopsis (Xia et al., 2009). Except for AtACP5, all plastid AtACPs showed increased protein abundance upon phosphate deficiency (Lan et al., 2012).

Generally, the expression levels of ACPs relate to the FA composition. Overexpression (OE) of AtACP1 resulted in decreased 16:3 and increased 18:3 FAs (Branen et al., 2001). Compromised expression of AtACP4 by both antisense AtACP4 and T-DNA insertion led to reduced proportion of 16:3 FAs (Branen et al., 2003; Ajjawi et al., 2010) and caused a chlorotic phenotype and compromised photosynthetic competence (Ajjawi et al., 2010). Although all Arabidopsis ACP isoforms have been characterized except AtACP5, little is known regarding their putative involvement in the acclimation to abiotic stresses. Understanding the role of plastidial ACPs in environment stresses in general, and the molecular function of AtACP5, are the two main aims of the present study. We here described the transcriptional responses of five plastidial ACPs to various abiotic stresses such as salt, drought and deficiencies in nitrogen, phosphorus, potassium, and iron in Arabidopsis. It was found that the expression of AtACP1-3 was induced by drought, AtACP4 was down-regulated in nitrogen- and iron-deficient plants, and AtACP5 was dramatically inhibited by salt stress. Loss of AtACP5 function resulted in hypersensitivity to salt stress, while AtACP5 OE lines showed increased salt tolerance. The results showed that alterations in FA composition were causative for the different phenotypes observed under salt stress. Specifically, decreased oleic acid and increased palmitic acid levels in AtACP5 OE plants were shown to contribute to increased salt tolerance, possibly by maintaining the cellular homeostasis of Na⁺ and K⁺.

Acyl carrier proteins are a family of universal, relatively abundant and highly conserved carriers of acyl, representing the core of the FAS system. Most species encode at least one ACP, but multiple isoforms and even dozens of homologous carrier proteins involved in FAS and secondary metabolism have been reported, suggesting functional diversity. For instance, more than 85 ACPs are encoded in the genome of Streptomyces avermitilis (Ikeda et al., 2003), the soybean (G. max, complex allotetraploid) genome harbors 25 ACPs (Wang et al., 2014). Arabidopsis, with its relatively simple genome, encodes five plastidial and three mitochondrial ACPs (Meyer et al., 2007). Phylogenetic analysis of 517 putative ACP proteins from 43 plant species identified a legume-specific subclade (Wang et al., 2014), supporting the assumption of functional diversification.

In the present investigation, phylogenetic analysis of Arabidopsis ACPs, regardless using neighbor-joining, maximum likelihood or minimum-evolution methods, revealed a subclade comprising all Arabidopsis plastidial ACP proteins (except AtACP4) and putative ACPs from other Brassicaceae (Figure 1), indicating divergence between different families during evolution. The formation of another subclade of AtACP4 suggests that AtACP4 and ACPs from other species unrelated to Brassicaceae might be derived from a different ancient gene. Apart from gene duplication and de novo gene generation, horizontal gene transfer (HGT) is an important way of acquiring new genes. Most HGT events have been occurred in prokaryotes (Gogarten et al., 2002) and unicellular eukaryotes (Keeling and Palmer, 2008), while the HGT is also involved in the evolution of high plant (Bock, 2010). The first example of nuclear HGT in seed plant is a Mu-like element (MULE) transposon between in Setaria species (millets) and in the rice (O. sativa) (Diao et al., 2006). Taken together, the exact mechanism of AtACP4 evolution awaits more evidence. Nevertheless, all Arabidopsis ACPs display the basic features of ACP proteins. Sequence alignment with homologous ACP proteins identified the characteristic and highly conserved LGADSLDTVEIVM motif comprising the DSL motif (Supplementary Figure 1), which represents the recognition site for ACPs (Mofid et al., 2002).

In plants, ACPs are expressed both ubiquitously and in a tissue-specific manner (Battey and Ohlrogge, 1990; Hlousekradojcic et al., 1992). The presence of multiple ACP isoforms and their differential responsiveness to external stimuli indicate that individual ACPs may play different roles in the adaptation to environmental conditions. For example, it was shown that the expression of AtACP4 was induced by light, whereas the abundance of AtACP2 or AtACP3 mRNA was not responsive to the light regime (Bonaventure and Ohlrogge, 2002). In the present study, we found that the expression of AtACP4, but not of the other ACPs under study was down-regulated by nitrogen and iron deficiency (Figure 2D). AtACP4 antisense plants (Branen et al., 2003) and T-DNA insertion mutants (acp4-1 and acp4-2) showed a different degree of leaf bleaching appearance, reduced photosynthetic efficiency, and a decreased concentrations of 16:3 FAs. The lower concentrations of 16:3 FAs are attributed to the lower concentration of 16:3 FAs on the monogalactosyldiacylglycerol (MGDG) moiety, and most of the 16:3 FAs in Arabidopsis leaf tissue are found on the sn-2 position of MGDG (Ajjawi et al., 2010). AtACP4 thus is a major player controlling the biosynthesis of FAs for chloroplast membrane lipids. Similarly, plant experiencing nitrogen or iron deficiency developed similar chlorotic phenotypes. Iron deficiency has been well documented to impair chlorophyll biosynthesis and chloroplast development (Mori, 1999), while N-starvation changes the chloroplast structure associated with alterations in thylakoid membrane structure and degradation of plastidial membrane lipids, leading to a damaged photosynthetic systems (Siaut et al., 2011; Wang et al., 2011). These results suggest that the expression level of AtACP4 positively correlated with the photosynthetic systems status, and probably functions as a hub among light, nitrogen and iron deficiency. The expression of AtACP5 was significantly repressed under salt stress (Figure 2B); while no significant differences in transcript levels were observed under other stresses, especially under drought stress (Figures 2C–G). These results thus indicated that the down-regulated expression of AtACP5 under salt stress might be due to ion toxicity rather than osmotic stress. Consistent with these results, a knock-out T-DNA insertion mutant exhibited increased salt sensitivity whereas AtACP5 OE lines showed increased tolerance to salt treatment (Figures 3, 4), indicating a possible role of AtACP5 in salt stress tolerance.

AtACP5 OE lines accumulated more palmitic acid 16:0 and less oleic acid 18:1 FAs compared with wild-type plants (Figures 5A, B). The major fate of 16:0 and 18:l acyl chains produced in the plastid is to form the hydrophobic portion of glycerolipid molecules, which are components of all cellular membranes (Ohlrogge and Browse, 1995). Surprisingly, atacp5 loss-of-function did not contain less FAs, which could be expected from the observed increase in the OE lines. One possible reason is genetic redundancy; the Arabidopsis genome harbors five plastidial ACPs carrying a highly conserved consensus motif. Overexpression of AtACP1 resulted in decreased concentration of 16:3 and increased levels of 18:3 FAs (Branen et al., 2001); inhibiting the expression of AtACP4 led to reduced 16:3 FA concentration (Branen et al., 2003; Ajjawi et al., 2010). These genes may function in concert with AtACP5 to regulate FA homeostasis. An alternative explanation is that the modification of FA composition is local rather than systemic. The detected FAs in the present study were from 10-day-old intact seedlings rather than from roots, which will dilute the contributions of the roots to the total FAs. Furthermore, AtACP5 is primary expressed in root and could be functional mainly in the roots. In summary, although no significant differences in FA profiles were found between atacp5 and the wild-type, the results from the AtACP5 OE lines suggest that a complex regulatory network of FA metabolism exists in plants and AtACP5 is a key player in FA homeostasis. Future study focusing on the root FA profiles among the three genotypes would expand our understandings toward the functions of AtACP5 in plants.

Fatty acids are major components of membranes and as such part of the mechanisms by which cellular processes are adapted to environmental constraints. Changes in plasma membrane lipids are an adaptation mechanism to salinity in broccoli roots (Lopez-Perez et al., 2009). Studies in cucumber, soybean, and borage (Borago officinalis L.) have reported that salinity caused a significant increase of SFAs and UFAs in thylakoid membranes, plasma membrane, and leaf, respectively (Surjus and Durand, 1996; Jaffel-Hamza et al., 2013; Shu et al., 2015). Similarly, increased salinity was shown to reduce the plasma membrane fluidity in the halophyte Spartina patens calli, which was associated with increased FA saturation (Wu et al., 2005). The amount of 18:3 FAs was found to be reduced under salt stress in salt-tolerant, but not salt-sensitive citrus cells (Gueta-Dahan et al., 1997), wheat (Triticum aestivum) (Mohamed et al., 1994), and soybean (G. max) (Surjus and Durand, 1996). Due to their role in membrane rigidity, SFAs in the membrane bilayer reduces its permeability and fluidity (Van Blitterswijk et al., 1981). Moller et al. (2007) reported that reduction of polyUFAs in membranes contribute to salt tolerance via amelioration of the oxidative effects of salt. On the contrary, some studies reported that increasing UFAs resulted in salt tolerance. In Synechococcus, increasing the expression of the desA gene, which encodes the Δ12 acyl-lipid desaturase involving in the synthesis of diunsaturated FAs, enhanced NaCl tolerance due to an increased ability to repair the photosynthetic and Na⁺/H⁺ antiport systems (Allakhverdiev et al., 2001). Application of exogenous linoleic acid exhibited protective effects on tonoplast function in barley seedlings under salt stress (Zhao and Qin, 2005). An increase of UFAs in membrane lipids contributed to an alleviated salt-induced photoinhibition of PSII in Thellungiella halophile (Sui and Han, 2014). However, Chalbi et al. (2013), also revealed that the ability to increase unsaturation in FAs was not a determinant factor for salt resistance in barley species. Nevertheless, in Arabidopsis, the FA desaturases FAD2 and FAD6, which enhance the degree of unsaturation of FAs, were found to be required for salt tolerance (Zhang et al., 2009, 2012). ER-localized FAD2 and plastid-localized FAD6 encode two ω-6 desaturases that convert oleic acid (18:1) to linoleic acid (18:2) by inserting a double bond at the ω-6 position, FAD3 (ER) and FAD7 and FAD8 (plastids) encode three ω-3 desaturases, which convert linoleic acid (18:2) to linolenic acid (18:3) by inserting a double bond at the ω-3 position. Both fad2 and fad6 mutants showed increased cytoplasmic Na⁺ accumulation and enhanced sensitivity to salt stress due to increased 18:1 and decreased 18:2 FA levels. In agreement with the decreased drought tolerance of transgenic FAD7 antisense tobacco plants (Im et al., 2002), OE of either FAD3 or FAD8 increased tolerance to drought in transgenic tobacco plants (Zhang et al., 2005). Since increased 18:1 FAs resulted in increased sensitivity to salt, it is reasonable to assume that decreased amounts of 18:1 FA levels contribute to salt tolerance. In agreement with previous studies in Arabidopsis mentioned above, in the present study, although the ratio between SFAs and UFAs was increased under salt stress in all genotypes (Figure 5C), OE of AtACP5 further led to an altered composition of FAs, mainly a decrease of oleic acid (C18:1) and an increase of palmitic acid (C16:0), which could explain why OE of AtACP5 can increase salt tolerance. ACPs have been documented to be involved in the biosynthesis of both SFAs and UFAs (Heath and Rock, 1996; Leesong et al., 1996; Feng and Cronan, 2009; Chan and Vogel, 2010). Thus, manipulating the expression of ACPs could be a strategy to increase the tolerance of plants to environmental stresses including salt.

Under saline conditions, rapid influx of Na⁺ from the soluble phase of the soil into the cytoplasm of cortical cells in plant roots occurs through non-selective cation channels (NSCCs) rather than through the high-affinity K⁺ transporter HKT1 (Essah et al., 2003). The relatively high permeability for Na⁺ of many depolarization-activated NSCCs (DA-NSCCs) suggests they are also important contributors to the uptake and translocation of Na⁺, and thus their function impacts on salt tolerance. Increased 3′,5′-cyclic guanosine monophosphate (cGMP) affected monovalent cation homeostasis during salt stress and reduced Na⁺ uptake through NSCCs such as CNGC3 (Gobert et al., 2006). Some NSCCs respond to physical stimuli, relaying external signals into electrical and/or Ca²⁺ signals through the action of mechanosensitive channels (MCs) whose gating depends on changes in tension forces on the membrane. Maintenance of appropriate tension forces depends on membrane integrity and fluidity, which are largely affected by the composition of lipids in general and FAs in particular (Mikami and Murata, 2003). To maintain ion homeostasis, plants could secrete Na⁺ out of the cell via plasma membrane-bound Na⁺/H⁺ antiporters (e.g., SOS1) and sequester Na⁺ into the vacuole via Na⁺/H⁺ antiporters (e.g., NHX1) on the tonoplast (Wu et al., 1996; Apse et al., 1999). In the present study, no significant changes in the transcripts of AtSOS1 and AtNHX1 were detected (Supplementary Figure 6), suggesting that both secreting and sequestering Na⁺ might not be predominated among genotypes. However, Na⁺ concentration was slightly reduced while K⁺ concentration was unchanged in OE lines, leading to a lower Na⁺/K⁺ ratio, which might confer OE lines more tolerant to salt stress. The exact mechanisms by which overexpressing AtACP5 increases salt tolerance awaits further study, although it could be possible that ectopic expression of AtACP5 alters the membrane integrity and fluidity through the alterations of FA composition (Figure 5).

We found that Arabidopsis plastidial ACPs are a highly conserved family of FAS system proteins, which respond disparately to different stresses. The atacp5 mutant plants showed increased salt sensitivity associated with a higher mortality rate, weaker growth and lower FW when compared with wild-type plants. By contrast, AtACP5 OE lines performed better than the wild-type in each aspect. The primary difference between OE lines and wild-type plants were cytosolic Na⁺ concentration and FA composition, which indicates an involvement of AtACP5 in the homeostasis of FAs. These results suggest that the modification of FAs contribute to the maintenance of membrane integrity, which might lead to improved salt tolerance. The findings presented here will facilitate the understanding of the relationship between cellular microenvironment and ever-changing environmental conditions.

PL designed and supervised this study; RS supervised this study; JH, CX, HW, and LW performed the experiments; WS polished the writing.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.