Transcriptional control of vitamin C defective 2 and tocopherol cyclase genes by light and plastid-derived signals: The partial involvement of GENOMES UNCOUPLED 1
Introduction
l-Ascorbic acid (vitamin C, AsA) and tocopherols (vitamin E; Toc) are vitamins, which are synthesized in plants and need to be taken up in the diet for human health [1]. AsA and Toc are important soluble and insoluble antioxidants, respectively, in higher plants. AsA has been identified as a substrate and cofactor for many enzymes, including ascorbate peroxidases (APXs), which are the main regulators of cellular H2O2 levels [2]. AsA also serves as an electron donor in the regeneration of oxidized Toc to its reduced form, which scavenges singlet oxygen and protect polyunsaturated fatty acids from lipid peroxidation [3], [4], [5]. Thus, these antioxidants play key roles in the regulation of cellular redox states, responses to stress and hormones, and growth and development in higher plants [2], [6]. Considering their biochemical interaction, the pool size of AsA and Toc are regulated to maintain cellular redox states.
Although several pathways have been proposed for the biosynthesis of AsA, the d-mannose/l-galactose (d-Man/l-Gal) pathway has been shown to play a predominant role in the biosynthesis of AsA in photosynthetic cells [2]. As shown in Fig. 1, AsA is sequentially synthesized by d-fructose-6P, d-Man-6P, d-Man-1P, GDP-d-Man, GDP-l-Gal, l-Gal-1P, l-Gal, and the final precursor, l-galactono-1,4-lactone (l-GalL), with catalytic reactions through phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-d-Man pyrophosphorylase (GMP), GDP-d-Man-3,5-epimerase (GME), GDP-l-Gal phosphorylase (GGP), l-Gal phosphatase (GPP), l-Gal dehydrogenase (GalDH), and l-GalL dehydrogenase (GalLDH) enzymes, respectively. l-GalL is synthesized in the cytosol, and the conversion of this compound to AsA is catalyzed in mitochondria [2], [7]. Although Arabidopsis has two PMI isoforms, PMI1 and PMI2, only PMI1 was found to be involved in this pathway [8]. Among the five vitamin C defective mutants identified, four mutants, vtc1, vtc2, vtc4, and vtc5 were shown to be defective in the d-Man/l-Gal pathway: VTC1, VTC2/5, and VTC4 encode GMP, GGP, and GPP, respectively [9], [10], [11], [12]. A double mutant lacking both VTC2 and VTC5 exhibited a seedling-lethal phenotype in the absence of a treatment with AsA or l-Gal, which suggested that AsA produced by the d-Man/l-Gal pathway was required for plant viability [12].
The biosynthetic pathway for Toc has also been completely characterized [13], [14], [15], [16]. In the biosynthesis of Toc, the shikimate and non-mevalonate (MEP) pathways synthesize the head group and hydrophobic tail, respectively (Fig. 1). Homogentisic acid (HGA), which is produced by the shikimate pathway, is prenylated with phytyldiphosphate (PDP) to yield the first committed intermediates in the synthesis of Toc, 2-methyl-6-phytylplastoquinone (MPBQ). MPBQ methyltransferase (MPBQMT) then adds a second methyl group to MPBQ to form 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ). Tocopherol cyclase (TC) converts MPBQ and DMPBQ to δ- and γ-Toc, respectively. Finally, γ-Toc methyltransferase (γ-TMT) converts δ- and γ-Toc to β- and α-Toc, respectively. All enzymes involved in the biosynthesis of Toc were shown to be localized in chloroplasts (Fig. 1).
Light is the most important environmental cue for the regulation of AsA biosynthesis. We previously reported that AsA levels were enhanced by illumination and suppressed by darkness in Arabidopsis [8], [17]. Consistent with these findings, the transcript levels of PMI1, GMP, VTC2, and GPP were up-and down-regulated by illumination and darkness, respectively [8], [17]. The biosynthetic capacity of AsA is also known to be regulated by light intensity. High light irradiation has been shown to increase the AsA pool size accompanied by upregulation of VTC2 [12], and similar findings were observed in the tomato and kiwifruit [18], [19]. The transient and constitutive overexpression of VTC2 has been shown to enhance AsA levels in Arabidopsis, tobacco, tomato, strawberry, and potato plants [20], [21], [22], [23]. α-Toc levels as well as the expression of TC were shown to be markedly increased under high light irradiation [24], [25]. The overexpression of TC has also been shown to increase Toc levels of Arabidopsis [24], tobacco [26], and lettuce plants [27]. These findings suggest that VTC2 and TC are rate-limiting enzymes in the biosynthesis of AsA and Toc, respectively, and the light-dependent transcriptional control of VTC2 and TC are crucial for regulating AsA and Toc levels in plants.
Previous studies demonstrated that the treatment of cells with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport (PET) chain, suppressed the light-dependent activation of AsA biosynthesis, which suggesting a role for PET in the regulation of AsA biosynthesis [8], [17]. The redox states of PET are known to regulate the expression of nuclear-encoded genes by acting as a retrograde signal from plastids to the nucleus. For example, the expression of photosynthesis-associated nuclear genes (PhANGs) and cytosolic APX2 under illumination was diminished when plants were treated with DCMU [28], [29]. Furthermore, the inhibition of plastid gene expression (PGE) and carotenoid biosynthesis by lincomycin (LIN) and norflurazon (NF), respectively, the latter of which affects the metabolism of tetrapyrroles, decreased the expression of PhANGs [30], [31]. Thus, PGE and the metabolism of tetrapyrroles as well as the redox states of PET were confirmed to be involved in retrograde signaling from plastids to the nucleus [30], [31]]. Previous studies reported that a nuclear-encoded plastidic protein, GENOMES UNCOUPLED 1 (GUN1), integrated PET-, PGE-, and tetrapyrrole metabolism-evoked signals within plastids, and transmitted these signals to the nucleus in order to regulate the expression of PhANGs, though it remains unclear how GUN1 is involved in the retrograde pathways [32], [33]. GUN1 has been found to affect cotyledon opening and expansion, anthocyanin biosynthesis, and hypocotyl elongation, and therefore, to be involved in stress response and photomorphogenesis in plants [34], [35]. Based on these findings, we hypothesized that the biosynthetic pathways of AsA and Toc may be regulated by plastid-derived signals through GUN1.
We herein investigated the effects of a light-dark environment and treatments with DCMU, LIN, and NF, all of which activate GUN1-dependent signaling, on the biosynthetic pathways of AsA and Toc in wild-type plants and GUN1-disrupted mutants. The results obtained suggest that the transcript levels of VTC2 and TC and the levels of AsA and Toc were regulated by light and plastid-derived signals in a similar manner, and also that GUN1 is at least partially involved in this regulation via the plastid-derived signals, but not light.
Section snippets
Plant material and growth conditions
Arabidopsis thaliana (Col-0) was used as the wild-type plant. The T-DNA insertion line of GUN1 (gun1-101, SAIL_33_D01) was donated from Dr Takehito Inaba (Miyazaki University) [36]. Surface-sterilized wild-type and mutant seeds were sown on solid Murashige and Skoog (MS) medium containing 3% sucrose. Plates were stratified in darkness for 2 days at 4 °C and then transferred to a growth chamber kept at 23 °C during 16 h of light (100 μmol photons m−2 s−1) and at 22 °C during 8 h of darkness (normal
The disruption of GUN1 did not influence the light-dark regulation of AsA and Toc biosynthesis
To clearify whether GUN1-mediated plastid signaling was involved in the light-dark regulation of AsA and Toc biosynthesis, we examined the effect of light-dark on the expression of AsA and Toc biosynthesis-associated genes and the levels of both antioxidants in a null mutant of GUN1, gun1-101. The gun1-101 mutation was demonstrated to inhibit plastid signaling [36]. Two-week-old wild type and gun1-101 grown under normal conditions (16 h of light at 100 μmol m−2 s−1 and 8 h of darkness), were
Discussion
The aim of this study was to clearify whether light and plastid signaling pathways regulated the biosynthetic pathways of AsA and Toc. We firstly examined the effects of light-dark on the expression of the biosynthesis-associated genes as well as the levels of both antioxidants. Consistent with our previous findings [17], the expression of VTC2 and GPP as well as levels AsA were enhanced and suppressed under continuous light and darkness, respectively (Fig. 2). We also showed that the
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research (A) (S.S.: 22248042) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and in part by the Strategic Project to Support the Formation of Research Bases at Private Universities: Matching Fund Subsidy from MEXT, 2011–2015 (S1101035). We are grateful to Dr. Takehito Inaba (Miyazaki University) for donating the gun1-101 seeds.
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