Physiologia Plantarum 154: 28–38. 2015
© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Thylakoid membrane oxidoreductase LTO1/AtVKOR is involved in ABA-mediated response to osmotic stress in Arabidopsis Ying Lua,† , Jun-Jie Penga,† , Zhi-Bo Yua , Jia-Jia Dua , Jia-Ning Xua and Xiao-Yun Wanga,b,* a College b
of Life Science, Shandong Agricultural University, Taian 271018, China State Key Laboratory of Crop Biology, Shandong Agricultural University, Taian 271018, China
Correspondence *Corresponding author, e-mail: [email protected] Received 7 June 2014; revised 4 July 2014 doi:10.1111/ppl.12268
†
Arabidopsis lumen thiol oxidoreductase 1 (LTO1) – the At4g35760 gene product – was previously found to be related to reactive oxygen species (ROS) accumulation. Here, we show that ROS accumulated in a mutant Arabidopsis line (lto1-2, mutant of LTO1/AtVKOR) under osmotic stress at a higher level than that observed in wild-type and transgenic complemented plants of the lto1-2 mutant (lto1-2C, transgenic complemented plants of lto1-2). Because ROS accumulation in osmotic stress is triggered by abscisic acid (ABA), an ABA-responsive gene, Annexin 1 (AnnAt1), was selected to study the response. Osmotic stress or exogenous ABA can significantly upregulate the transcription of AnnAt1 in wild-type and lto1-2C plants. Only a slight change in the transcriptional abundance of AnnAt1 was observed under osmotic stress in the lto1-2 mutant, but exogenous ABA application could increase the expression of AnnAt1, which suggested that exogenous ABA had a partial complementation role. Because the transcription of AnnAt1 is regulated by ABRE (ABA-responsive elements) binding proteins (AREBs)/ABRE binding factors (ABFs), the expression of AREBs/ABFs was also analyzed. The transcription of AREBs/ABFs in the lto1-2 mutant was not induced by osmotic stress but was significantly upregulated by exogenous ABA, which significantly differs from the wild-type and lto1-2C plant responses. Similarly, the expression of another ABA-responsive gene, RD29B (responsive to desiccation stress gene 29B), in the lto1-2 mutant was also upregulated by exogenous ABA. The partial complementation of mutants by ABA indicated that the ABA signal transduction pathway was not significantly affected in the lto1-2 mutant. Taken together, these results suggest that LTO1 is involved in ABA-mediated response to osmotic stress, possibly by affecting the biosynthesis of endogenous ABA.
These authors contributed equally to the work.
Abbreviations – ABA, abscisic acid; ABFs, ABRE binding factors; ABREs, ABA-responsive elements; AREBs, ABRE binding proteins; DABHCl, diaminobenzidine tetrahydrochloride; DPBF5, Dc3 promoter binding factor 5; DRE, dehydration-responsive element; DsbA, disulfide bond protein A; LTO1, lumen thiol oxidoreductase1; NBT, nitroblue tetrazolium; NCED, 9-cis-epoxycarotenoid dioxygenase; qRT-PCRs, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; Trx, thioredoxin; VKOR, vitamin K epoxide reductase; VKORC1L1, VKOR complex subunit 1-like 1 protein.
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Introduction Plants have evolved a variety of sophisticated mechanisms to respond to abiotic and biotic environmental changes (Zhu 2002, Gill and Tuteja 2010). Abscisic acid (ABA) is an essential hormone for plant growth and development and also plays an important role in the physiological regulation of water stress. The role of ABA in osmotic stress mainly involves the maintenance of the water balance via the regulation of guard cells and the induction of ABA-responsive genes that encode dehydration-tolerance proteins (Zhu 2002). The latter role is achieved by the binding of various activated transcription factors to many cis-regulatory elements of the genes in order to regulate the expression of a large number of genes involved in stress tolerance (Busk and Pages 1998). ABA-responsive elements (ABREs) are typical cis-regulatory elements with the conserved sequence PyCGTGGC that are found in many genes involved in stress tolerance (Marcotte et al. 1989, Mundy et al. 1990). ABREs can be recognized and bound by the ABRE binding proteins (AREBs), which are also called ABRE binding factors (ABFs) (Choi et al. 2000, Uno et al. 2000). AREBs/ABFs belong to the basic domain/leucine zipper (bZIP) class of transcription factors, and nine homologues have been identified in Arabidopsis thaliana (Fujita et al. 2005). Among them, the expressions of AREB1/ABF2, AREB2/ABF4 and ABF3/Dc3 promoter binding factor 5 (DPBF5) are induced by ABA, drought and high salinity in plant cells (Choi et al. 2000, Fujita et al. 2005). AREB1/ABF2, AREB2/ABF4 and ABF3/DPBF5 are referred to hereafter as ABF2, ABF4 and ABF3, respectively. Phosphorylated AREBs/ABFs bind to the ABRE region to activate the expression of ABA-responsive genes (Furihata et al. 2006), such as annexin 1 (AnnAt1), in A. thaliana (Konopka-Postupolska et al. 2009). AnnAt1, a member of the multigene family of Annexins, contains one ABRE and one dehydration-responsive element (DRE) in its promoter region. The expression of AnnAt1 is enhanced by treatment with ABA, hydrogen peroxide, salicylic acid, cold, drought and salinity. The over-expression of AnnAt1 in Arabidopsis confers enhanced tolerance to drought stress (Konopka-Postupolska et al. 2009). The in vivo regulation of the reactive oxygen species (ROS) levels by AnnAt1 has been demonstrated by the fact that a lower level of H2 O2 is observed in AnnAt1-overexpressing plants and a higher level of H2 O2 is observed in AnnAt1-knockout plants (Konopka-Postupolska et al. 2009). The two active cysteines of the three sulfur-containing cluster (Cys-Met-Cys), which is conserved as a redox reactive center in cotton Physiol. Plant. 154, 2015
annexin, are also found in AnnAt1 and are oxidized to S-glutathionylation by glutathione after ABA treatment (Hofmann et al. 2003, Konopka-Postupolska et al. 2009). These results suggest that redox regulation may be involved in the ABA-mediated response to abiotic stresses. A thylakoid membrane oxidoreductase was initially found to possess the ability to catalyze the formation of a disulfide bond (Lu et al. 2013). The full-length protein is a fusion protein that contains an integral membrane domain homologous to the mammalian vitamin K epoxide reductase (VKOR) and a soluble disulfide bond protein A (DsbA)-like/thioredoxin (Trx)-like domain in Arabidopsis (Furt et al. 2010, Feng et al. 2011). This protein is named AtVKOR or lumen thiol oxidoreductase 1 (LTO1) (Karamoko et al. 2011). The VKOR domain can oxidize the DsbA-like/Trx-like domain as the acceptor of electrons (Furt et al. 2010). The electrons may be further transferred to phylloquinone or other components that bind to the thylakoid membrane (Lu et al. 2013). Similar to human VKOR, which acts as a vitamin K-dependent antioxidant (Westhofen et al. 2011), LTO1/AtVKOR was found to be involved in the ROS metabolism via the use of a homozygous knockout mutant of lto1-2 (mutant of LTO1/AtVKOR) (Lu et al. 2013). In addition, deficiency in LTO1/AtVKOR can affect the activity of zeaxanthin, which is a component of the xanthophyll cycle that participates in the thermal dissipation of excess absorbed light energy (Yu et al. 2014). Zeaxanthin is also the original source for the biosynthesis of ABA under osmotic stress conditions (Marin et al. 1996, Zhu 2002, Yu et al. 2014). The relationship of the role of LTO1/AtVKOR in ROS metabolism with the ABA-mediated response to osmotic stresses needs to be elucidated. In this study, an lto1-2 mutant, transgenic lto1-2C (transgenic complemented plants of lto1-2), and wild-type plants were used to investigate their ABA-mediated responses to osmotic stress.
Materials and methods Plant materials and growth conditions The lto1 T-DNA insertion mutant line of A. thaliana, CS858849 (lto1-2), was obtained from the Arabidopsis Biological Resource Center. Homozygous lto1-2 lines were screened, and complemented plants were obtained according to a previous study (Lu et al. 2013). Arabidopsis was grown in vermiculite under short-day conditions (8-h illumination at 120 μmol·m−2 ·s−1 and 16-h dark cycle) at a constant temperature of 22∘ C. For growth on agar plates, the seeds were surface-sterilized with 70% ethanol for 5 min and 2.6% bleach for 10 min and then washed five times with sterilized distilled 29
water. The washed seeds were plated on Murashige and Skoog (MS) medium containing 3% sucrose according to a previous study. To ensure synchronized germination, the seeds were sown in the dark for 48 h at 4∘ C before being transferred to a growth chamber. Stress treatments The stress treatments were carried out according to a previous study (Choi et al. 2000, Konopka-Postupolska et al. 2009). Wild-type, transgenic and lto1-2 mutant plants were grown to 6 weeks of age. Fully expanded leaves were detached, laid on a surface with 150 mM NaCl and 100 μM ABA, and incubated under these conditions for 12, 24, 36 and 48 h. For the drought stress treatment, fully expanded leaves were detached, rapidly laid in petri dishes and incubated at the conditions noted above for 1.5, 2, 3 and 5 h. At the indicated time points, the treated leaves were harvested, frozen in liquid nitrogen and stored at −80∘ C until use. RNA extraction and qRT-PCR The harvested leaves were used for total RNA isolation according to a previous study (Jing et al. 2011). The cDNA was synthesized according to the standard procedures of the RevertAid Fist Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA). Quantitative real-time polymerase chain reaction (qRT-PCRs) was performed for each cDNA dilution using SYBR Premix Ex Taq™ according to the manufacturer’s protocol (TaKaRa, Dalian, Japan). The expression levels of specific genes were standardized to the level of the housekeeping gene Tubulin. All of the primers are listed in Table S1, Supporting information. The qRT-PCR cycles were defined as follows: 1 cycle of 30 s at 95∘ C and 45 cycles of 5 s at 95∘ C, 10 s at 58∘ C and 15 s at 72∘ C. Each reaction was conducted using three biological replicates. Cloning, expression and purification of AnnAt1 and antiserum production The sequence encoding AnnAt1 was cloned by PCR amplification from an Arabidopsis cDNA library. The PCR products were sequenced by Sunny Biotechnology Company (Shanghai, China) and confirmed via alignment with the At1g35720 gene. The primers are listed in Table S1. The amplified DNA fragments were cloned into the polyclone sites of pET30a (+) (Merck, Darmstadt, Germany), and the DNA sequences were confirmed. The recombinant AnnAt1 was expressed in Escherichia coli BL21 (DE3) cells. The cells were grown in Luria-Bertani (LB) medium to an OD600 of 0.4–0.6 at 37∘ C, and 30
the expression of the recombinant protein was induced for 8 h with 0.4 mM isopropyl-D-thiogalactopyranoside at 37∘ C. The cells were harvested by centrifugation at 6000 g and 4∘ C for 10 min and were resuspended in ice-cold lysis buffer containing 50 mM Tris–HCl pH 8.0, 0.1 M NaCl and 1 mM ethylenediaminetetraacetic acid (EDTA). After sonication, the crude lysate was centrifuged at 12 000 g and 4∘ C for 15 min, and the supernatant fraction was applied to a HisTrap™FF column (GE Healthcare, Pittsburgh, PA). The fused AnnAt1 protein was eluted with a 20–200 mM linear gradient of imidazole in 20 mM Tris–HCl pH 7.4 and 0.5 M NaCl. The peak fractions containing AnnAt1 were collected and stored at −20∘ C. The polyclonal antibody was raised in mice with purified antigen. Preparation of thylakoid membranes and immunoblotting analysis The thylakoid membrane proteins were prepared as previously described (Peng et al. 2006). The chlorophyll content was determined according to a previous study (Arnon 1949). For the western blot assay, protein samples with equal amounts of chlorophyll were separated on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The proteins were then transferred onto Immobilon-P membranes (Millipore, Billerica, MA) and blotted with antibodies specific to AnnAt1. The signals were detected with SuperSignal West Pico (ThermoFisher). In situ detection of ROS Leaves of wild-type, transgenic and lto1-2 mutant plants treated with 150 mM NaCl for 24 h, drought for 2 h and 100 μM ABA for 12/24 h were used to detect the accumulation of the superoxide anion radical (O2 ·− ) and H2 O2 . The level of O2 ·− was detected in situ by treating plants with nitroblue tetrazolium (NBT) as previously described (Lu et al. 2013). The aerial portions of the plants were detached from the roots and incubated in 0.1% NBT (in 10 mM potassium phosphate buffer, pH 7.6) for 2 h in the dark. The stained leaves were boiled in lactic acid: glycerol: ethanol [1:1:3 (v/v/v)] for 5 min and photographed. The level of H2 O2 was detected in situ by treating plants with diaminobenzidine tetrahydrochloride (DABHCl) as previously described by (Lu et al. 2013). The aerial portions of the plants were detached from the roots and incubated in 50 mM Tris-acetic acid, pH 3.8 with 0.5 mg ml−1 DABHCl for 24 h in the dark. The stained leaves were boiled in lactic acid: glycerol: ethanol solution and photographed (see above). Leaves of wild-type, transgenic and lto1-2 mutant plants treated with 150 mM NaCl for 24 h, drought for
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Fig. 1. Analysis of ROS in wild-type, transgenic lto1-2C (transgenic complemented plants of lto1-2) and lto1-2 (mutant of LTO1/AtVKOR) mutant plants after treatment with osmotic stress and ABA. (A) DABHCl staining of H2 O2 in wild-type, lto1-2C and lto1-2 plants under normal growth conditions. (B) NBT staining of O2 ·− in wild-type, lto1-2C and lto1-2 plants under normal growth conditions. (C) DABHCl staining of H2 O2 in wild-type, lto1-2C and lto1-2 plants after treatment with 150 mM NaCl for 24 h. (D) NBT staining of O2 ·− in wild-type, lto1-2C and lto1-2 plants after treatment with 150 mM NaCl for 24 h. (E) DABHCl staining of H2 O2 in wild-type, lto1-2C and lto1-2 plants after treatment with drought for 2 h. (F) NBT staining of O2 ·− in wild-type, lto1-2C and lto1-2 plants after treatment with drought for 2 h. (G) DABHCl staining of H2 O2 in wild-type, lto1-2C and lto1-2 plants after treatment with 100 μM ABA for 12 and 24 h. (H) NBT staining of O2 ·− in wild-type, lto1-2C and lto1-2 plants after treatment with 100 μM ABA for 12 and 24 h. Quantitative analysis of (I) O2 ·− and (J) H2 O2 in the investigated plants, each column represents an average of three replicates and error bars indicate standard deviation.
2 h and 100 μM ABA for 24 h were used to quantify the amount of the superoxide anion radical (O2 ·− ) and H2 O2 . The quantitative assay of O2 ·− and H2 O2 was performed as previously described (Huang et al. 2013). Leaves from osmotic stress and ABA treatments were ground to a fine power in liquid nitrogen and extracted using 50 mM phosphate buffer saline (PBS). The detection wavelengths were 436 and 530 nm for O2 ·− and H2 O2 , respectively.
Results More ROS were accumulated in LTO1/AtVKOR-deficient mutant plants under osmotic stress or exogenous ABA treatment Abiotic stresses, such as salt, drought and high light, often lead to the overproduction of ROS, particularly O2 ·− and H2 O2 , via the deregulation of electron transport in the chloroplast and mitochondria (Mittler et al. 2004). Our previous study demonstrated that mutant A. thaliana plants with LTO1/AtVKOR deficiency accumulate more ROS than wild-type and transgenic complemented lto1-2C plants under strong light stress (Lu et al. 2013). Osmotic stress or ABA treatment also
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induces significant ROS accumulation in mutant lines, as shown in Fig. 1. The levels of O2 ·− and H2 O2 were not observed to significantly increase in leaves of the wild-type and lto1-2C plants after 150 mM NaCl treatment for 24 h or draught treatment for 2 h. However, the lto1-2 mutant accumulated much more O2 ·− and H2 O2 than the wild-type and lto1-2C plants after these treatments (Fig. 1C–F). The accumulation of O2 ·− and H2 O2 is related to the enhancement of endogenous ABA (Jiang and Zhang 2002). Salt and drought stress can induce endogenous ABA accumulation in plants, which can increase the expression of genes involved in scavenging ROS and upregulates the activities of several antioxidant enzymes (Jiang and Zhang 2002, Konopka-Postupolska et al. 2009). Treatment with 100 μM exogenous ABA did not induce a significant accumulation of ROS in leaves of the lto1-2 mutant plants; however, more ROS, especially O2 ·− , accumulated in the lto1-2 mutant with treatment for 24 h, while only a comparatively slight increase in the O2 ·− and H2 O2 levels was observed in the wild-type and lto1-2C plants (Fig. 1G, H). The levels of O2 ·− and H2 O2 were further quantitatively assessed in wild-type, lto1-2C and lto1-2 mutant plants after osmotic stress and ABA treatment. The quantities of O2 ·− and H2 O2 were consistent with the results of NBT staining and DAB 31
staining, respectively (Fig. 1I, J). These results indicated that a deficiency in LTO1/AtVKOR in Arabidopsis limited the plant’s ability to scavenge ROS mediated by ABA. LTO1/AtVKOR was related to the expression of the ABA-responsive gene AnnAt1 at the transcriptional level under osmotic stress AnnAt1 is a typical ABA-responsive gene and can be induced by various abiotic stresses. More H2 O2 is accumulated in the annAt1 mutant plants than the wild-type and over-expressing transgenic plants after treatment with ABA (Konopka-Postupolska et al. 2009). To detect whether the higher ROS accumulation in the lto1-2 mutant under osmotic stress is related to the expression of ABA-responsive genes, the transcriptional levels of AnnAt1 were determined in the wild-type, lto1-2C and lto1-2 mutant leaves. The levels of AnnAt1 mRNA in the wild-type and lto1-2C plants were upregulated after treatment with 150 mM NaCl or drought, and the transcription levels were maximized after 24 and 2 h of stimulation, respectively (Fig. 2A, B). However, the amount of AnnAt1 mRNA only slightly changed in the lto1-2 mutant in response to salt or draught stress. These results suggested that AnnAt1 was insensitive to osmotic stress in the lto1-2 mutant. The ABA-induced changes in the AnnAt1 mRNA level were very similar to those in response to osmotic stress in the wild-type and lto1-2C plants (Fig. 2C). ABA treatment led to the upregulation of the transcriptional level of AnnAt1 in lto1-2 mutant plants (Fig. 2C), and the transcription level was maximized after 12 h of ABA treatment in these plants. These results suggested that exogenous ABA could complement, at least in part, the role of osmotic stress in the lto1-2 mutant. The transcript level of AnnAt1 affected the accumulation of AnnAt1 protein under osmotic stress and ABA Plant Annexin proteins are present in different organelles and have amphipathic properties due to their insertion into the membrane (Clark et al. 2012). Arabidopsis AnnAt1 is primarily localized in the cytosol but has also been found in chloroplasts (Mortimer et al. 2008). Western blot analysis was performed to further investigate whether the transcription of AnnAt1 affected the accumulation of its coding product AnnAt1. The amount of AnnAt1 was elevated to the maximum level in the wild-type and lto1-2C plants after treatment with 150 mM NaCl for 24 h. However, the amount of AnnAt1 in the lto1-2 mutant did not significantly change under salt stress (Fig. 3A). A similar trend was observed 32
Fig. 2. qRT-PCR analysis of mRNA of AnnAt1 in wild-type, transgenic 1to1-2C (transgenic complemented plants of lto1-2) and lto1-2 (mutant of LTO1/AtVKOR) mutant plants after treatment with osmotic stress and ABA. (A) Plants were incubated on a surface with 150 mM NaCl for 12, 24, 36 and 48 h. (B) Detached, fully expanded leaves were treated with drought for 1.5, 2, 3 and 5 h. (C) Plants were incubated on a surface with 100 μM ABA for 12, 24, 36 and 48 h. Each column represents the mean values of three replicates, and the error bars indicate the standard deviation.
when plants were treated with drought stress (Fig. 3B). Exogenous ABA application for 12 h induced a significant accumulation of AnnAt1 in lto1-2 mutant plants, which is consistent with the change observed in the transcription of AnnAt1 (Fig. 3C). A significant enhancement of AnnAt1 was also observed in the wild-type and lto1-2C plants. These results demonstrate that the protein level of AnnAt1 was consistent with its transcription level in response to osmotic stress and ABA. A deficiency in LTO1/AtVKOR affected the expression of ABA-responsive genes in response to osmotic stress. Deficiency of LTO1/AtVKOR led to an insensitive response of AREBs/ABFs to osmotic stress but a sensitive response to exogenous ABA The promoter of AnnAt1 contains a ‘classical’ ABRE element and a putative DRE from the DRE family, which are typical elements in the promoters of stress-inducible
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Fig. 3. Accumulation of AnnAt1 after treatment with osmotic stresses and ABA. The accumulation of AnnAt1 was detected in leaves treated with 150 mM NaCl (A), drought (B), and 100 μM ABA (C) when the transcription of AnnAt1 was maximized. (D) The proteins were stained with Coomassie brilliant blue as the loading control.
genes (Konopka-Postupolska et al. 2009). The insensitive response of AnnAT1 in the lto1-2 mutant to osmotic stress may be related to the inactivation of transcription, which is mainly regulated by three master transcription factors, namely ABF2, ABF3 and ABF4. These three transcription factors cooperatively regulate the expression of ABRE-dependent genes for ABA signaling under water stress conditions (Yoshida et al. 2010). The transcript levels of ABF2, ABF3 and ABF4 in response to salt, drought stress and exogenous ABA were assayed. The mRNA levels of the three transcription factors in the wild-type and lto1-2C plants were persistently upregulated after treatment with 150 mM NaCl, and the transcriptional level was maximized after 24 h of stimulation. However, the transcriptional levels of the three transcription factors gradually decreased during the assay process in the lto1-2 mutant, although the initial mRNA levels were higher than those observed in the wild-type and lto1-2C plants (Fig. 4A). A similar result was obtained after drought treatment. A significant increase in the transcription of the three transcription factors was observed in the wild-type and lto1-2C plants, whereas only a slight change was observed in the lto1-2 mutant (Fig. 4B). These results showed that the deficiency in LTO1/AtVKOR led to an insensitive response of AREBs/ABFs to osmotic stress. Because the three AREBs/ABFs are also induced by ABA, we examined the effect of exogenous ABA on the
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transcriptional levels of AREBs/ABFs. Different from the changes observed under osmotic stress, the transcriptional levels of the three transcription factors were all upregulated in the wild-type, lto1-2C and lto1-2 mutant plants after 100 μM ABA treatment (Fig. 4C), but the times at which the maximum transcriptional levels were attained slightly differed among these plants. In the lto1-2 mutant plants, exogenous ABA treatment stimulated the expression of the three transcription factors at an earlier time than in the wild-type and lto1-2C plants. In other words, exogenous ABA partially complemented the inducing role of osmotic stress in lto1-2 mutant. A wild-type (WT)-like pattern of gene expression restoration by the exogenous ABA treatment suggested that the ABA signal transduction pathway was minimally affected in the mutant. We hypothesize that a deficiency in LTO1/AtVKOR may affect the biosynthesis of ABA to limit the expression of ABA-responsive genes in response to stress conditions in the mutant plants.
Deficiency of LTO1/AtVKOR led to an insensitive response of RD29B to osmotic stresses but a sensitive response to exogenous ABA The RD29B (responsive to desiccation stress gene 29B) gene is sensitive to various abiotic stresses and is typically induced by AREBs/ABFs and exogenous ABA (Msanne et al. 2011). The expression of AREBs/ABFs 33
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Fig. 4. Analysis of the transcriptional levels of ABF2, ABF3 and ABF4 in wild-type, transgenic lto1-2C (transgenic complemented plants of lto1-2) and lto1-2 (mutant of LTO1/AtVKOR) mutant plants after treatment with osmotic stress and ABA by qRT-PCR. The total RNA that was isolated to detect the mRNA level of AnnAt1 as well as the transcriptional levels of ABF2, ABF3 and ABF4. (A) The transcriptional levels of ABF2, ABF3 and ABF4 in wild-type, lto1-2C and lto1-2 plants after treatment with 150 mM NaCl. (B) The transcriptional levels of ABF2, ABF3 and ABF4 in wild-type, lto1-2C and lto1-2 plants after treatment with drought. (C) The transcriptional levels of ABF2, ABF3 and ABF4 in wild-type, lto1-2C and lto1-2 plants after treatment with 100 μM ABA. Each column represents the mean values of three replicates, and the error bars indicate the standard deviation.
Discussion
Fig. 5. Analysis of RD29B transcription in wild-type, transgenic lto1-2C (transgenic complemented plants of lto1-2) and lto1-2 (mutant of LTO1/AtVKOR) mutant plants after treatment with osmotic stress and ABA by qRT-PCR. The total RNA that was isolated to detect the mRNA level of AnnAt1 as well as the transcriptional levels of RD29B. (A) The transcriptional levels of RD29B in wild-type, lto1-2C and lto1-2 plants after treatment with 150 mM NaCl. (B) The transcriptional levels of RD29B in wild-type, lto1-2C and lto1-2 plants after treatment with drought. (C) The transcriptional levels of RD29B in wild-type, lto1-2C and lto1-2 plants after treatment with 100 μM ABA. Each column represents the mean values of three replicates, and the error bars indicate the standard deviation.
changed little in the lto1-2 mutant after the osmotic stress treatments. We implied that the expression of the RD29B gene was also affected due to the deficiency of LTO1 in the lto1-2 mutant. After the osmotic stress treatments, the transcriptional level of RD29B slightly changed in mutant plants, which markedly differed from the large increase in the wild-type and lto1-2C plants (Fig. 5A, B). However, ABA treatment upregulated the transcriptional level of RD29B in the wild-type, lto1-2C and lto1-2 mutant plants (Fig. 5C). The trend in the RD29B mRNA level changes was consistent with that of AREBs/ABFs after osmotic stress and ABA treatments in the wild-type, lto1-2C and lto1-2 mutant plants. The result confirmed that AtVKOR is involved in ABA-mediated osmotic stress.
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ROS were originally known as toxic by-products of aerobic metabolism induced in cells in response to various stress conditions (Mittler 2002). Many defense machineries protect an organism against the oxidative damage induced by ROS (Gill and Tuteja 2010). Human VKOR complex subunit 1-like 1 protein (VKORC1L1) was found to mediate the vitamin K-dependent intracellular antioxidation function (Westhofen et al. 2011). In HEK239T cells, the expression of VKORC1L1 was increased approximately 5.5-fold after treatment with 75 μM H2 O2 , which can induce intracellular oxidative stress. The over-expression of VKORC1L1 increases the VKOR enzymatic activity, which increases the intracellular levels of reduced vitamin K cofactors and thereby limits the intracellular ROS levels and ROS-induced protein damage (Westhofen et al. 2011). The thylakoid membrane oxidoreductase LTO1/AtVKOR is a homolog of mammalian VKOR and can regulate the oxidoreduction change in proteins that are involved in photosynthesis and ROS metabolism (Karamoko et al. 2011, Lu et al. 2013). In this study, we found that LTO1/AtVKOR is also involved in the ABA-mediated response to osmotic stress. A higher level of ROS was accumulated in lto1-2 mutant leaves than in the wild-type and lto1-2C plants after osmotic stress treatments. The increased generation of ROS due to osmotic stress triggered by the accumulation of ABA and biosynthesis of ABA can upregulate the activities of antioxidant enzymes (Jiang and Zhang 2002). The high level of ROS accumulation in the lto1-2 mutant may be related to ABA-mediated osmotic stress. Thus, the expression of ABA-responsive genes was detected. AnnAt1 is a typical gene that can be induced by ABA, hydrogen peroxide, drought and salt (Konopka-Postupolska et al. 2009, Clark et al. 2012). In this study, we detected the upregulation of AnnAt1 mRNA after treatment with drought and high salt in the wild-type and lto1-2C plants. However, the transcriptional level of AnnAt1 in the lto1-2 mutant plants only slightly changed. At the translational level, the change in the amount of AnnAt1 in the chloroplasts was consistent with the change observed at the transcriptional level. Unlike the changes induced by stresses, ABA significantly induced the expression of AnnAt1 in the lto1-2 mutant, and the application of exogenous ABA led to an increase in the expression of AREBs/ABFs. RD29B is another marker gene of plant stress and ABA response (Fujita et al. 2005, Msanne et al. 2011). The changes in the transcriptional level of RD29B mRNA were very similar to those of AnnAt1 and AREBs/ABFs. These results show that exogenous ABA can partly complement the role of the osmotic stress response in the 35
lto1-2 mutant and that a deficiency in LTO1/AtVKOR impairs the expression of ABA-responsive genes. Osmotic stress induces the biosynthesis of ABA and then initiates ABA-dependent signaling pathways to respond to the stress by regulating the expression of stress-related genes (Lee and Luan 2012). Two models exist for the ABA signaling pathway in plant cells that contain at least three classes of ABA receptors that localize in the plasma membrane, chloroplasts and soluble in cytoplasm and nucleus (Finkelstein 2013). One ABA signaling pathway contains the ABA receptors PYR/PYL/RCAR, PP2C/SnRK2 and AREBs/ABFs (Fujii et al. 2009, Ma et al. 2009, Park et al. 2009). AREBs/ABFs are phosphorylated to activate the expression of ABRE-dependent genes (Furihata et al. 2006). This model can well explain the increased expression of AnnAt1 and RD29B after exogenous ABA treatment in the lto1-2 mutant observed in this study. Our data suggested that the downstream transduction pathways might not be affected in the mutant plants. Thus, we propose that LTO1/AtVKOR may affect the initiatory stage of the ABA signaling pathways to limit the expression of ABA-responsive genes under stress conditions in the mutant plants. The other ABA signaling pathway involves the transcription factor WRKYs and an ABA receptor, the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR) (Shen et al. 2006, Shang et al. 2010). In response to a low level of ABA, WRKYs combine the W-box sequences harbored in the promoters of genes due to weak interaction with ABAR and inhibit the expression of the ABF2, ABF4, ABI4 (ABA Insensitive 4) and ABI5 genes (Ren et al. 2010, Shang et al. 2010, Antoni et al. 2011). We suppose that LTO1/AtVKOR deficiency impairs the biosynthesis of ABA after osmotic stress treatment, which subsequently leads to the failure of ABA acceptor perception and ultimately influences the expression of ABA-responsive genes by repressing the expression of AREBs/ABFs. Oxidation stimulates the biosynthesis of ABA. Zeaxanthin (Z) is the original resource for the biosynthesis of ABA (Marin et al. 1996, Zhu 2002). Zeaxanthin is converted by zeaxanthin epoxidase to violaxanthin (V) via antheraxanthin (A), which also participates in the xanthophyll cycle (Jahns et al. 2009). The products are then catalyzed by a series of enzymes, including 9-cis-epoxycarotenoid dioxygenase (NCED), ABA2, ABA3 and ABA aldehyde oxidase, in order to synthesize ABA (Tan et al. 1997, Seo et al. 2000, Xiong et al. 2001). LTO1/AtVKOR, which is the only oxidoreductase found in thylakoids to date, can catalyze the disulfide bond formation of luminal proteins (Kieselbach 2013), and recent studies have discovered that LTO1/AtVKOR is involved in the xanthophyll cycle (Kieselbach 2013, Yu 36
et al. 2014). The de-epoxidation state of the xanthophyll cycle pigments is lower in the lto1-2 mutant line than in the wild-type and complemented plants, suggesting that the de-epoxidation of violaxanthin to zeaxanthin via antheraxanthin is suppressed in the lto1-2 mutant (Yu et al. 2014). Although the zeaxanthin pool may primarily participate in photoprotection via its action as an antioxidant (Peguero-Pina et al. 2013), deficiency in LTO1/AtVKOR causes severe photoinhibition (Yu et al. 2014), and higher levels of ROS were accumulated in the lto1-2 mutant after stress treatment. Zeaxanthin is apparently continually consumed without complement in the lto1-2 mutant. In addition, NCED is also a key enzyme that catalyzes a rate-limiting step in the biosynthesis of ABA, and NCED contains a disulfide bond (Messing et al. 2010). According to our data, a deficiency in LTO1/AtVKOR may affect the initiatory stage of the ABA signaling pathways and thus affect the expression of ABA-responsive genes in response to stress conditions in the mutant plants. LTO1/AtVKOR may act as a novel regulator between ABA and ROS in the chloroplast.
Author contributions X.-Y. W., Y. L. and J.-J. P. designed the experiments. Y. L., J.-J. P., Z.-B. Y. and J.-J. D. performed the experiments. Y. L., J.-J. P., Z.-B. Y., J.-J. D., J.-N. X. and X.-Y. W. analyzed the data. Y. L. and X.-Y. W. wrote the article. Acknowledgements – This study was supported by the Special Research Fund of Public Welfare of China Agricultural Ministry (201303093).
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Primers used in this work.
Edited by C. Foyer
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© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Thylakoid membrane oxidoreductase LTO1/AtVKOR is involved in ABA-mediated response to osmotic stress in Arabidopsis Ying Lua,† , Jun-Jie Penga,† , Zhi-Bo Yua , Jia-Jia Dua , Jia-Ning Xua and Xiao-Yun Wanga,b,* a College b
of Life Science, Shandong Agricultural University, Taian 271018, China State Key Laboratory of Crop Biology, Shandong Agricultural University, Taian 271018, China
Correspondence *Corresponding author, e-mail: [email protected] Received 7 June 2014; revised 4 July 2014 doi:10.1111/ppl.12268
†
Arabidopsis lumen thiol oxidoreductase 1 (LTO1) – the At4g35760 gene product – was previously found to be related to reactive oxygen species (ROS) accumulation. Here, we show that ROS accumulated in a mutant Arabidopsis line (lto1-2, mutant of LTO1/AtVKOR) under osmotic stress at a higher level than that observed in wild-type and transgenic complemented plants of the lto1-2 mutant (lto1-2C, transgenic complemented plants of lto1-2). Because ROS accumulation in osmotic stress is triggered by abscisic acid (ABA), an ABA-responsive gene, Annexin 1 (AnnAt1), was selected to study the response. Osmotic stress or exogenous ABA can significantly upregulate the transcription of AnnAt1 in wild-type and lto1-2C plants. Only a slight change in the transcriptional abundance of AnnAt1 was observed under osmotic stress in the lto1-2 mutant, but exogenous ABA application could increase the expression of AnnAt1, which suggested that exogenous ABA had a partial complementation role. Because the transcription of AnnAt1 is regulated by ABRE (ABA-responsive elements) binding proteins (AREBs)/ABRE binding factors (ABFs), the expression of AREBs/ABFs was also analyzed. The transcription of AREBs/ABFs in the lto1-2 mutant was not induced by osmotic stress but was significantly upregulated by exogenous ABA, which significantly differs from the wild-type and lto1-2C plant responses. Similarly, the expression of another ABA-responsive gene, RD29B (responsive to desiccation stress gene 29B), in the lto1-2 mutant was also upregulated by exogenous ABA. The partial complementation of mutants by ABA indicated that the ABA signal transduction pathway was not significantly affected in the lto1-2 mutant. Taken together, these results suggest that LTO1 is involved in ABA-mediated response to osmotic stress, possibly by affecting the biosynthesis of endogenous ABA.
These authors contributed equally to the work.
Abbreviations – ABA, abscisic acid; ABFs, ABRE binding factors; ABREs, ABA-responsive elements; AREBs, ABRE binding proteins; DABHCl, diaminobenzidine tetrahydrochloride; DPBF5, Dc3 promoter binding factor 5; DRE, dehydration-responsive element; DsbA, disulfide bond protein A; LTO1, lumen thiol oxidoreductase1; NBT, nitroblue tetrazolium; NCED, 9-cis-epoxycarotenoid dioxygenase; qRT-PCRs, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; Trx, thioredoxin; VKOR, vitamin K epoxide reductase; VKORC1L1, VKOR complex subunit 1-like 1 protein.
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Introduction Plants have evolved a variety of sophisticated mechanisms to respond to abiotic and biotic environmental changes (Zhu 2002, Gill and Tuteja 2010). Abscisic acid (ABA) is an essential hormone for plant growth and development and also plays an important role in the physiological regulation of water stress. The role of ABA in osmotic stress mainly involves the maintenance of the water balance via the regulation of guard cells and the induction of ABA-responsive genes that encode dehydration-tolerance proteins (Zhu 2002). The latter role is achieved by the binding of various activated transcription factors to many cis-regulatory elements of the genes in order to regulate the expression of a large number of genes involved in stress tolerance (Busk and Pages 1998). ABA-responsive elements (ABREs) are typical cis-regulatory elements with the conserved sequence PyCGTGGC that are found in many genes involved in stress tolerance (Marcotte et al. 1989, Mundy et al. 1990). ABREs can be recognized and bound by the ABRE binding proteins (AREBs), which are also called ABRE binding factors (ABFs) (Choi et al. 2000, Uno et al. 2000). AREBs/ABFs belong to the basic domain/leucine zipper (bZIP) class of transcription factors, and nine homologues have been identified in Arabidopsis thaliana (Fujita et al. 2005). Among them, the expressions of AREB1/ABF2, AREB2/ABF4 and ABF3/Dc3 promoter binding factor 5 (DPBF5) are induced by ABA, drought and high salinity in plant cells (Choi et al. 2000, Fujita et al. 2005). AREB1/ABF2, AREB2/ABF4 and ABF3/DPBF5 are referred to hereafter as ABF2, ABF4 and ABF3, respectively. Phosphorylated AREBs/ABFs bind to the ABRE region to activate the expression of ABA-responsive genes (Furihata et al. 2006), such as annexin 1 (AnnAt1), in A. thaliana (Konopka-Postupolska et al. 2009). AnnAt1, a member of the multigene family of Annexins, contains one ABRE and one dehydration-responsive element (DRE) in its promoter region. The expression of AnnAt1 is enhanced by treatment with ABA, hydrogen peroxide, salicylic acid, cold, drought and salinity. The over-expression of AnnAt1 in Arabidopsis confers enhanced tolerance to drought stress (Konopka-Postupolska et al. 2009). The in vivo regulation of the reactive oxygen species (ROS) levels by AnnAt1 has been demonstrated by the fact that a lower level of H2 O2 is observed in AnnAt1-overexpressing plants and a higher level of H2 O2 is observed in AnnAt1-knockout plants (Konopka-Postupolska et al. 2009). The two active cysteines of the three sulfur-containing cluster (Cys-Met-Cys), which is conserved as a redox reactive center in cotton Physiol. Plant. 154, 2015
annexin, are also found in AnnAt1 and are oxidized to S-glutathionylation by glutathione after ABA treatment (Hofmann et al. 2003, Konopka-Postupolska et al. 2009). These results suggest that redox regulation may be involved in the ABA-mediated response to abiotic stresses. A thylakoid membrane oxidoreductase was initially found to possess the ability to catalyze the formation of a disulfide bond (Lu et al. 2013). The full-length protein is a fusion protein that contains an integral membrane domain homologous to the mammalian vitamin K epoxide reductase (VKOR) and a soluble disulfide bond protein A (DsbA)-like/thioredoxin (Trx)-like domain in Arabidopsis (Furt et al. 2010, Feng et al. 2011). This protein is named AtVKOR or lumen thiol oxidoreductase 1 (LTO1) (Karamoko et al. 2011). The VKOR domain can oxidize the DsbA-like/Trx-like domain as the acceptor of electrons (Furt et al. 2010). The electrons may be further transferred to phylloquinone or other components that bind to the thylakoid membrane (Lu et al. 2013). Similar to human VKOR, which acts as a vitamin K-dependent antioxidant (Westhofen et al. 2011), LTO1/AtVKOR was found to be involved in the ROS metabolism via the use of a homozygous knockout mutant of lto1-2 (mutant of LTO1/AtVKOR) (Lu et al. 2013). In addition, deficiency in LTO1/AtVKOR can affect the activity of zeaxanthin, which is a component of the xanthophyll cycle that participates in the thermal dissipation of excess absorbed light energy (Yu et al. 2014). Zeaxanthin is also the original source for the biosynthesis of ABA under osmotic stress conditions (Marin et al. 1996, Zhu 2002, Yu et al. 2014). The relationship of the role of LTO1/AtVKOR in ROS metabolism with the ABA-mediated response to osmotic stresses needs to be elucidated. In this study, an lto1-2 mutant, transgenic lto1-2C (transgenic complemented plants of lto1-2), and wild-type plants were used to investigate their ABA-mediated responses to osmotic stress.
Materials and methods Plant materials and growth conditions The lto1 T-DNA insertion mutant line of A. thaliana, CS858849 (lto1-2), was obtained from the Arabidopsis Biological Resource Center. Homozygous lto1-2 lines were screened, and complemented plants were obtained according to a previous study (Lu et al. 2013). Arabidopsis was grown in vermiculite under short-day conditions (8-h illumination at 120 μmol·m−2 ·s−1 and 16-h dark cycle) at a constant temperature of 22∘ C. For growth on agar plates, the seeds were surface-sterilized with 70% ethanol for 5 min and 2.6% bleach for 10 min and then washed five times with sterilized distilled 29
water. The washed seeds were plated on Murashige and Skoog (MS) medium containing 3% sucrose according to a previous study. To ensure synchronized germination, the seeds were sown in the dark for 48 h at 4∘ C before being transferred to a growth chamber. Stress treatments The stress treatments were carried out according to a previous study (Choi et al. 2000, Konopka-Postupolska et al. 2009). Wild-type, transgenic and lto1-2 mutant plants were grown to 6 weeks of age. Fully expanded leaves were detached, laid on a surface with 150 mM NaCl and 100 μM ABA, and incubated under these conditions for 12, 24, 36 and 48 h. For the drought stress treatment, fully expanded leaves were detached, rapidly laid in petri dishes and incubated at the conditions noted above for 1.5, 2, 3 and 5 h. At the indicated time points, the treated leaves were harvested, frozen in liquid nitrogen and stored at −80∘ C until use. RNA extraction and qRT-PCR The harvested leaves were used for total RNA isolation according to a previous study (Jing et al. 2011). The cDNA was synthesized according to the standard procedures of the RevertAid Fist Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA). Quantitative real-time polymerase chain reaction (qRT-PCRs) was performed for each cDNA dilution using SYBR Premix Ex Taq™ according to the manufacturer’s protocol (TaKaRa, Dalian, Japan). The expression levels of specific genes were standardized to the level of the housekeeping gene Tubulin. All of the primers are listed in Table S1, Supporting information. The qRT-PCR cycles were defined as follows: 1 cycle of 30 s at 95∘ C and 45 cycles of 5 s at 95∘ C, 10 s at 58∘ C and 15 s at 72∘ C. Each reaction was conducted using three biological replicates. Cloning, expression and purification of AnnAt1 and antiserum production The sequence encoding AnnAt1 was cloned by PCR amplification from an Arabidopsis cDNA library. The PCR products were sequenced by Sunny Biotechnology Company (Shanghai, China) and confirmed via alignment with the At1g35720 gene. The primers are listed in Table S1. The amplified DNA fragments were cloned into the polyclone sites of pET30a (+) (Merck, Darmstadt, Germany), and the DNA sequences were confirmed. The recombinant AnnAt1 was expressed in Escherichia coli BL21 (DE3) cells. The cells were grown in Luria-Bertani (LB) medium to an OD600 of 0.4–0.6 at 37∘ C, and 30
the expression of the recombinant protein was induced for 8 h with 0.4 mM isopropyl-D-thiogalactopyranoside at 37∘ C. The cells were harvested by centrifugation at 6000 g and 4∘ C for 10 min and were resuspended in ice-cold lysis buffer containing 50 mM Tris–HCl pH 8.0, 0.1 M NaCl and 1 mM ethylenediaminetetraacetic acid (EDTA). After sonication, the crude lysate was centrifuged at 12 000 g and 4∘ C for 15 min, and the supernatant fraction was applied to a HisTrap™FF column (GE Healthcare, Pittsburgh, PA). The fused AnnAt1 protein was eluted with a 20–200 mM linear gradient of imidazole in 20 mM Tris–HCl pH 7.4 and 0.5 M NaCl. The peak fractions containing AnnAt1 were collected and stored at −20∘ C. The polyclonal antibody was raised in mice with purified antigen. Preparation of thylakoid membranes and immunoblotting analysis The thylakoid membrane proteins were prepared as previously described (Peng et al. 2006). The chlorophyll content was determined according to a previous study (Arnon 1949). For the western blot assay, protein samples with equal amounts of chlorophyll were separated on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The proteins were then transferred onto Immobilon-P membranes (Millipore, Billerica, MA) and blotted with antibodies specific to AnnAt1. The signals were detected with SuperSignal West Pico (ThermoFisher). In situ detection of ROS Leaves of wild-type, transgenic and lto1-2 mutant plants treated with 150 mM NaCl for 24 h, drought for 2 h and 100 μM ABA for 12/24 h were used to detect the accumulation of the superoxide anion radical (O2 ·− ) and H2 O2 . The level of O2 ·− was detected in situ by treating plants with nitroblue tetrazolium (NBT) as previously described (Lu et al. 2013). The aerial portions of the plants were detached from the roots and incubated in 0.1% NBT (in 10 mM potassium phosphate buffer, pH 7.6) for 2 h in the dark. The stained leaves were boiled in lactic acid: glycerol: ethanol [1:1:3 (v/v/v)] for 5 min and photographed. The level of H2 O2 was detected in situ by treating plants with diaminobenzidine tetrahydrochloride (DABHCl) as previously described by (Lu et al. 2013). The aerial portions of the plants were detached from the roots and incubated in 50 mM Tris-acetic acid, pH 3.8 with 0.5 mg ml−1 DABHCl for 24 h in the dark. The stained leaves were boiled in lactic acid: glycerol: ethanol solution and photographed (see above). Leaves of wild-type, transgenic and lto1-2 mutant plants treated with 150 mM NaCl for 24 h, drought for
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Fig. 1. Analysis of ROS in wild-type, transgenic lto1-2C (transgenic complemented plants of lto1-2) and lto1-2 (mutant of LTO1/AtVKOR) mutant plants after treatment with osmotic stress and ABA. (A) DABHCl staining of H2 O2 in wild-type, lto1-2C and lto1-2 plants under normal growth conditions. (B) NBT staining of O2 ·− in wild-type, lto1-2C and lto1-2 plants under normal growth conditions. (C) DABHCl staining of H2 O2 in wild-type, lto1-2C and lto1-2 plants after treatment with 150 mM NaCl for 24 h. (D) NBT staining of O2 ·− in wild-type, lto1-2C and lto1-2 plants after treatment with 150 mM NaCl for 24 h. (E) DABHCl staining of H2 O2 in wild-type, lto1-2C and lto1-2 plants after treatment with drought for 2 h. (F) NBT staining of O2 ·− in wild-type, lto1-2C and lto1-2 plants after treatment with drought for 2 h. (G) DABHCl staining of H2 O2 in wild-type, lto1-2C and lto1-2 plants after treatment with 100 μM ABA for 12 and 24 h. (H) NBT staining of O2 ·− in wild-type, lto1-2C and lto1-2 plants after treatment with 100 μM ABA for 12 and 24 h. Quantitative analysis of (I) O2 ·− and (J) H2 O2 in the investigated plants, each column represents an average of three replicates and error bars indicate standard deviation.
2 h and 100 μM ABA for 24 h were used to quantify the amount of the superoxide anion radical (O2 ·− ) and H2 O2 . The quantitative assay of O2 ·− and H2 O2 was performed as previously described (Huang et al. 2013). Leaves from osmotic stress and ABA treatments were ground to a fine power in liquid nitrogen and extracted using 50 mM phosphate buffer saline (PBS). The detection wavelengths were 436 and 530 nm for O2 ·− and H2 O2 , respectively.
Results More ROS were accumulated in LTO1/AtVKOR-deficient mutant plants under osmotic stress or exogenous ABA treatment Abiotic stresses, such as salt, drought and high light, often lead to the overproduction of ROS, particularly O2 ·− and H2 O2 , via the deregulation of electron transport in the chloroplast and mitochondria (Mittler et al. 2004). Our previous study demonstrated that mutant A. thaliana plants with LTO1/AtVKOR deficiency accumulate more ROS than wild-type and transgenic complemented lto1-2C plants under strong light stress (Lu et al. 2013). Osmotic stress or ABA treatment also
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induces significant ROS accumulation in mutant lines, as shown in Fig. 1. The levels of O2 ·− and H2 O2 were not observed to significantly increase in leaves of the wild-type and lto1-2C plants after 150 mM NaCl treatment for 24 h or draught treatment for 2 h. However, the lto1-2 mutant accumulated much more O2 ·− and H2 O2 than the wild-type and lto1-2C plants after these treatments (Fig. 1C–F). The accumulation of O2 ·− and H2 O2 is related to the enhancement of endogenous ABA (Jiang and Zhang 2002). Salt and drought stress can induce endogenous ABA accumulation in plants, which can increase the expression of genes involved in scavenging ROS and upregulates the activities of several antioxidant enzymes (Jiang and Zhang 2002, Konopka-Postupolska et al. 2009). Treatment with 100 μM exogenous ABA did not induce a significant accumulation of ROS in leaves of the lto1-2 mutant plants; however, more ROS, especially O2 ·− , accumulated in the lto1-2 mutant with treatment for 24 h, while only a comparatively slight increase in the O2 ·− and H2 O2 levels was observed in the wild-type and lto1-2C plants (Fig. 1G, H). The levels of O2 ·− and H2 O2 were further quantitatively assessed in wild-type, lto1-2C and lto1-2 mutant plants after osmotic stress and ABA treatment. The quantities of O2 ·− and H2 O2 were consistent with the results of NBT staining and DAB 31
staining, respectively (Fig. 1I, J). These results indicated that a deficiency in LTO1/AtVKOR in Arabidopsis limited the plant’s ability to scavenge ROS mediated by ABA. LTO1/AtVKOR was related to the expression of the ABA-responsive gene AnnAt1 at the transcriptional level under osmotic stress AnnAt1 is a typical ABA-responsive gene and can be induced by various abiotic stresses. More H2 O2 is accumulated in the annAt1 mutant plants than the wild-type and over-expressing transgenic plants after treatment with ABA (Konopka-Postupolska et al. 2009). To detect whether the higher ROS accumulation in the lto1-2 mutant under osmotic stress is related to the expression of ABA-responsive genes, the transcriptional levels of AnnAt1 were determined in the wild-type, lto1-2C and lto1-2 mutant leaves. The levels of AnnAt1 mRNA in the wild-type and lto1-2C plants were upregulated after treatment with 150 mM NaCl or drought, and the transcription levels were maximized after 24 and 2 h of stimulation, respectively (Fig. 2A, B). However, the amount of AnnAt1 mRNA only slightly changed in the lto1-2 mutant in response to salt or draught stress. These results suggested that AnnAt1 was insensitive to osmotic stress in the lto1-2 mutant. The ABA-induced changes in the AnnAt1 mRNA level were very similar to those in response to osmotic stress in the wild-type and lto1-2C plants (Fig. 2C). ABA treatment led to the upregulation of the transcriptional level of AnnAt1 in lto1-2 mutant plants (Fig. 2C), and the transcription level was maximized after 12 h of ABA treatment in these plants. These results suggested that exogenous ABA could complement, at least in part, the role of osmotic stress in the lto1-2 mutant. The transcript level of AnnAt1 affected the accumulation of AnnAt1 protein under osmotic stress and ABA Plant Annexin proteins are present in different organelles and have amphipathic properties due to their insertion into the membrane (Clark et al. 2012). Arabidopsis AnnAt1 is primarily localized in the cytosol but has also been found in chloroplasts (Mortimer et al. 2008). Western blot analysis was performed to further investigate whether the transcription of AnnAt1 affected the accumulation of its coding product AnnAt1. The amount of AnnAt1 was elevated to the maximum level in the wild-type and lto1-2C plants after treatment with 150 mM NaCl for 24 h. However, the amount of AnnAt1 in the lto1-2 mutant did not significantly change under salt stress (Fig. 3A). A similar trend was observed 32
Fig. 2. qRT-PCR analysis of mRNA of AnnAt1 in wild-type, transgenic 1to1-2C (transgenic complemented plants of lto1-2) and lto1-2 (mutant of LTO1/AtVKOR) mutant plants after treatment with osmotic stress and ABA. (A) Plants were incubated on a surface with 150 mM NaCl for 12, 24, 36 and 48 h. (B) Detached, fully expanded leaves were treated with drought for 1.5, 2, 3 and 5 h. (C) Plants were incubated on a surface with 100 μM ABA for 12, 24, 36 and 48 h. Each column represents the mean values of three replicates, and the error bars indicate the standard deviation.
when plants were treated with drought stress (Fig. 3B). Exogenous ABA application for 12 h induced a significant accumulation of AnnAt1 in lto1-2 mutant plants, which is consistent with the change observed in the transcription of AnnAt1 (Fig. 3C). A significant enhancement of AnnAt1 was also observed in the wild-type and lto1-2C plants. These results demonstrate that the protein level of AnnAt1 was consistent with its transcription level in response to osmotic stress and ABA. A deficiency in LTO1/AtVKOR affected the expression of ABA-responsive genes in response to osmotic stress. Deficiency of LTO1/AtVKOR led to an insensitive response of AREBs/ABFs to osmotic stress but a sensitive response to exogenous ABA The promoter of AnnAt1 contains a ‘classical’ ABRE element and a putative DRE from the DRE family, which are typical elements in the promoters of stress-inducible
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Fig. 3. Accumulation of AnnAt1 after treatment with osmotic stresses and ABA. The accumulation of AnnAt1 was detected in leaves treated with 150 mM NaCl (A), drought (B), and 100 μM ABA (C) when the transcription of AnnAt1 was maximized. (D) The proteins were stained with Coomassie brilliant blue as the loading control.
genes (Konopka-Postupolska et al. 2009). The insensitive response of AnnAT1 in the lto1-2 mutant to osmotic stress may be related to the inactivation of transcription, which is mainly regulated by three master transcription factors, namely ABF2, ABF3 and ABF4. These three transcription factors cooperatively regulate the expression of ABRE-dependent genes for ABA signaling under water stress conditions (Yoshida et al. 2010). The transcript levels of ABF2, ABF3 and ABF4 in response to salt, drought stress and exogenous ABA were assayed. The mRNA levels of the three transcription factors in the wild-type and lto1-2C plants were persistently upregulated after treatment with 150 mM NaCl, and the transcriptional level was maximized after 24 h of stimulation. However, the transcriptional levels of the three transcription factors gradually decreased during the assay process in the lto1-2 mutant, although the initial mRNA levels were higher than those observed in the wild-type and lto1-2C plants (Fig. 4A). A similar result was obtained after drought treatment. A significant increase in the transcription of the three transcription factors was observed in the wild-type and lto1-2C plants, whereas only a slight change was observed in the lto1-2 mutant (Fig. 4B). These results showed that the deficiency in LTO1/AtVKOR led to an insensitive response of AREBs/ABFs to osmotic stress. Because the three AREBs/ABFs are also induced by ABA, we examined the effect of exogenous ABA on the
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transcriptional levels of AREBs/ABFs. Different from the changes observed under osmotic stress, the transcriptional levels of the three transcription factors were all upregulated in the wild-type, lto1-2C and lto1-2 mutant plants after 100 μM ABA treatment (Fig. 4C), but the times at which the maximum transcriptional levels were attained slightly differed among these plants. In the lto1-2 mutant plants, exogenous ABA treatment stimulated the expression of the three transcription factors at an earlier time than in the wild-type and lto1-2C plants. In other words, exogenous ABA partially complemented the inducing role of osmotic stress in lto1-2 mutant. A wild-type (WT)-like pattern of gene expression restoration by the exogenous ABA treatment suggested that the ABA signal transduction pathway was minimally affected in the mutant. We hypothesize that a deficiency in LTO1/AtVKOR may affect the biosynthesis of ABA to limit the expression of ABA-responsive genes in response to stress conditions in the mutant plants.
Deficiency of LTO1/AtVKOR led to an insensitive response of RD29B to osmotic stresses but a sensitive response to exogenous ABA The RD29B (responsive to desiccation stress gene 29B) gene is sensitive to various abiotic stresses and is typically induced by AREBs/ABFs and exogenous ABA (Msanne et al. 2011). The expression of AREBs/ABFs 33
34
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Fig. 4. Analysis of the transcriptional levels of ABF2, ABF3 and ABF4 in wild-type, transgenic lto1-2C (transgenic complemented plants of lto1-2) and lto1-2 (mutant of LTO1/AtVKOR) mutant plants after treatment with osmotic stress and ABA by qRT-PCR. The total RNA that was isolated to detect the mRNA level of AnnAt1 as well as the transcriptional levels of ABF2, ABF3 and ABF4. (A) The transcriptional levels of ABF2, ABF3 and ABF4 in wild-type, lto1-2C and lto1-2 plants after treatment with 150 mM NaCl. (B) The transcriptional levels of ABF2, ABF3 and ABF4 in wild-type, lto1-2C and lto1-2 plants after treatment with drought. (C) The transcriptional levels of ABF2, ABF3 and ABF4 in wild-type, lto1-2C and lto1-2 plants after treatment with 100 μM ABA. Each column represents the mean values of three replicates, and the error bars indicate the standard deviation.
Discussion
Fig. 5. Analysis of RD29B transcription in wild-type, transgenic lto1-2C (transgenic complemented plants of lto1-2) and lto1-2 (mutant of LTO1/AtVKOR) mutant plants after treatment with osmotic stress and ABA by qRT-PCR. The total RNA that was isolated to detect the mRNA level of AnnAt1 as well as the transcriptional levels of RD29B. (A) The transcriptional levels of RD29B in wild-type, lto1-2C and lto1-2 plants after treatment with 150 mM NaCl. (B) The transcriptional levels of RD29B in wild-type, lto1-2C and lto1-2 plants after treatment with drought. (C) The transcriptional levels of RD29B in wild-type, lto1-2C and lto1-2 plants after treatment with 100 μM ABA. Each column represents the mean values of three replicates, and the error bars indicate the standard deviation.
changed little in the lto1-2 mutant after the osmotic stress treatments. We implied that the expression of the RD29B gene was also affected due to the deficiency of LTO1 in the lto1-2 mutant. After the osmotic stress treatments, the transcriptional level of RD29B slightly changed in mutant plants, which markedly differed from the large increase in the wild-type and lto1-2C plants (Fig. 5A, B). However, ABA treatment upregulated the transcriptional level of RD29B in the wild-type, lto1-2C and lto1-2 mutant plants (Fig. 5C). The trend in the RD29B mRNA level changes was consistent with that of AREBs/ABFs after osmotic stress and ABA treatments in the wild-type, lto1-2C and lto1-2 mutant plants. The result confirmed that AtVKOR is involved in ABA-mediated osmotic stress.
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ROS were originally known as toxic by-products of aerobic metabolism induced in cells in response to various stress conditions (Mittler 2002). Many defense machineries protect an organism against the oxidative damage induced by ROS (Gill and Tuteja 2010). Human VKOR complex subunit 1-like 1 protein (VKORC1L1) was found to mediate the vitamin K-dependent intracellular antioxidation function (Westhofen et al. 2011). In HEK239T cells, the expression of VKORC1L1 was increased approximately 5.5-fold after treatment with 75 μM H2 O2 , which can induce intracellular oxidative stress. The over-expression of VKORC1L1 increases the VKOR enzymatic activity, which increases the intracellular levels of reduced vitamin K cofactors and thereby limits the intracellular ROS levels and ROS-induced protein damage (Westhofen et al. 2011). The thylakoid membrane oxidoreductase LTO1/AtVKOR is a homolog of mammalian VKOR and can regulate the oxidoreduction change in proteins that are involved in photosynthesis and ROS metabolism (Karamoko et al. 2011, Lu et al. 2013). In this study, we found that LTO1/AtVKOR is also involved in the ABA-mediated response to osmotic stress. A higher level of ROS was accumulated in lto1-2 mutant leaves than in the wild-type and lto1-2C plants after osmotic stress treatments. The increased generation of ROS due to osmotic stress triggered by the accumulation of ABA and biosynthesis of ABA can upregulate the activities of antioxidant enzymes (Jiang and Zhang 2002). The high level of ROS accumulation in the lto1-2 mutant may be related to ABA-mediated osmotic stress. Thus, the expression of ABA-responsive genes was detected. AnnAt1 is a typical gene that can be induced by ABA, hydrogen peroxide, drought and salt (Konopka-Postupolska et al. 2009, Clark et al. 2012). In this study, we detected the upregulation of AnnAt1 mRNA after treatment with drought and high salt in the wild-type and lto1-2C plants. However, the transcriptional level of AnnAt1 in the lto1-2 mutant plants only slightly changed. At the translational level, the change in the amount of AnnAt1 in the chloroplasts was consistent with the change observed at the transcriptional level. Unlike the changes induced by stresses, ABA significantly induced the expression of AnnAt1 in the lto1-2 mutant, and the application of exogenous ABA led to an increase in the expression of AREBs/ABFs. RD29B is another marker gene of plant stress and ABA response (Fujita et al. 2005, Msanne et al. 2011). The changes in the transcriptional level of RD29B mRNA were very similar to those of AnnAt1 and AREBs/ABFs. These results show that exogenous ABA can partly complement the role of the osmotic stress response in the 35
lto1-2 mutant and that a deficiency in LTO1/AtVKOR impairs the expression of ABA-responsive genes. Osmotic stress induces the biosynthesis of ABA and then initiates ABA-dependent signaling pathways to respond to the stress by regulating the expression of stress-related genes (Lee and Luan 2012). Two models exist for the ABA signaling pathway in plant cells that contain at least three classes of ABA receptors that localize in the plasma membrane, chloroplasts and soluble in cytoplasm and nucleus (Finkelstein 2013). One ABA signaling pathway contains the ABA receptors PYR/PYL/RCAR, PP2C/SnRK2 and AREBs/ABFs (Fujii et al. 2009, Ma et al. 2009, Park et al. 2009). AREBs/ABFs are phosphorylated to activate the expression of ABRE-dependent genes (Furihata et al. 2006). This model can well explain the increased expression of AnnAt1 and RD29B after exogenous ABA treatment in the lto1-2 mutant observed in this study. Our data suggested that the downstream transduction pathways might not be affected in the mutant plants. Thus, we propose that LTO1/AtVKOR may affect the initiatory stage of the ABA signaling pathways to limit the expression of ABA-responsive genes under stress conditions in the mutant plants. The other ABA signaling pathway involves the transcription factor WRKYs and an ABA receptor, the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR) (Shen et al. 2006, Shang et al. 2010). In response to a low level of ABA, WRKYs combine the W-box sequences harbored in the promoters of genes due to weak interaction with ABAR and inhibit the expression of the ABF2, ABF4, ABI4 (ABA Insensitive 4) and ABI5 genes (Ren et al. 2010, Shang et al. 2010, Antoni et al. 2011). We suppose that LTO1/AtVKOR deficiency impairs the biosynthesis of ABA after osmotic stress treatment, which subsequently leads to the failure of ABA acceptor perception and ultimately influences the expression of ABA-responsive genes by repressing the expression of AREBs/ABFs. Oxidation stimulates the biosynthesis of ABA. Zeaxanthin (Z) is the original resource for the biosynthesis of ABA (Marin et al. 1996, Zhu 2002). Zeaxanthin is converted by zeaxanthin epoxidase to violaxanthin (V) via antheraxanthin (A), which also participates in the xanthophyll cycle (Jahns et al. 2009). The products are then catalyzed by a series of enzymes, including 9-cis-epoxycarotenoid dioxygenase (NCED), ABA2, ABA3 and ABA aldehyde oxidase, in order to synthesize ABA (Tan et al. 1997, Seo et al. 2000, Xiong et al. 2001). LTO1/AtVKOR, which is the only oxidoreductase found in thylakoids to date, can catalyze the disulfide bond formation of luminal proteins (Kieselbach 2013), and recent studies have discovered that LTO1/AtVKOR is involved in the xanthophyll cycle (Kieselbach 2013, Yu 36
et al. 2014). The de-epoxidation state of the xanthophyll cycle pigments is lower in the lto1-2 mutant line than in the wild-type and complemented plants, suggesting that the de-epoxidation of violaxanthin to zeaxanthin via antheraxanthin is suppressed in the lto1-2 mutant (Yu et al. 2014). Although the zeaxanthin pool may primarily participate in photoprotection via its action as an antioxidant (Peguero-Pina et al. 2013), deficiency in LTO1/AtVKOR causes severe photoinhibition (Yu et al. 2014), and higher levels of ROS were accumulated in the lto1-2 mutant after stress treatment. Zeaxanthin is apparently continually consumed without complement in the lto1-2 mutant. In addition, NCED is also a key enzyme that catalyzes a rate-limiting step in the biosynthesis of ABA, and NCED contains a disulfide bond (Messing et al. 2010). According to our data, a deficiency in LTO1/AtVKOR may affect the initiatory stage of the ABA signaling pathways and thus affect the expression of ABA-responsive genes in response to stress conditions in the mutant plants. LTO1/AtVKOR may act as a novel regulator between ABA and ROS in the chloroplast.
Author contributions X.-Y. W., Y. L. and J.-J. P. designed the experiments. Y. L., J.-J. P., Z.-B. Y. and J.-J. D. performed the experiments. Y. L., J.-J. P., Z.-B. Y., J.-J. D., J.-N. X. and X.-Y. W. analyzed the data. Y. L. and X.-Y. W. wrote the article. Acknowledgements – This study was supported by the Special Research Fund of Public Welfare of China Agricultural Ministry (201303093).
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Primers used in this work.
Edited by C. Foyer
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Physiol. Plant. 154, 2015
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