CrMAPK3 regulates the expression of iron-deficiency-responsive genes in Chlamydomonas reinhardtii
© The Author(s). 2017
Received: 11 January 2017
Accepted: 28 April 2017
Published: 16 May 2017
Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas.
Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2–CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced.
CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.
Chlamydomonas reinhardtii (Volvocales, Chlorophyta) is a single-celled eukaryotic and flagellated green alga, whose three genetic systems located in the nucleus, chloroplast, and mitochondria can be used for transformation. This alga is regarded as a “photosynthetic yeast” because of its easy culturing process, rapid growth, short life cycle, and high photosynthetic efficiency. With its three genome sequences, this model organism is highly useful for cell and molecular biology research .
In phosphorylation cascades, mitogen-activated protein kinases (MAPKs) are eukaryotic signal proteins involved in extracellular signal amplification and intracellular signal transduction in yeasts, animals, and plants [2–4]. Combined with other signal molecules, MAPKs transfer external stimuli via successive phosphorylation reactions: MAPKKKs → MAPKKs → MAPKs. Progressively and continuously enlarged signals, such as environmental stress factors, including high salinity, high osmotic pressure, and low temperature, reach the nucleus and regulate downstream gene expression [5, 6]. In eukaryotic cells, phosphorylation cascades are composed of MAPKs, MAPKKs, and MAPKKKs. Homo sapiens possesses 15 MAPKs, 7 MAPKKs, and 16 MAPKKKs, while Arabidopsis contains 20 MAPKs, 10 MAPKKs, and 80 MAPKKKs. Few MAPK cascades have been described because of the complexity of genetic networks and pleiotropic and interaction effects. MAPK genes have been identified in plants, such as Arabidopsis, rice, corn, wheat, and barley [7–13]. MAPKs function through stress-response pathways [14, 15].
Iron is an essential trace element for most living organisms. A precise iron regulation system is necessary to maintain the dynamic equilibrium of iron  because iron overload and deficiency cause metabolic disorders. Following nitrogen and phosphate deficiencies, iron deficiency restricts plant growth and yield and consequently induces crop chlorosis and yields low productivity. In humans, insufficient iron concentrations trigger iron deficiency anemia or iron deficiency syndrome. Iron has also been considered a growth-limiting factor in some tumor cells. Therefore, iron chelators are clinically used for cancer suppression.
Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption that slightly differs from iron absorption in plants. Environmental ferric iron is reduced to ferrous iron via FRE1 (homology of Arabidopsis FRO2 ) on the plasma membrane and then putatively transferred to FOX1 by FEA1 . Afterward, FOX1 oxidizes ferrous iron to ferric iron, which is then transported to the cytoplasm by FTR1 on the plasma membrane [19–21]. The expression of the genes encoding these proteins is significantly increased under iron-deficient conditions, and this phenomenon indicates that iron deficiency signals in these genes are regulated. Nevertheless, the response, transmission, and regulation of downstream gene expression in algal cells have yet to be investigated. Considering that the MAPK pathway is essential for non-biological stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas. In this study, Arabidopsis MAPKs were used to search for the corresponding genes in the Chlamydomonas genome database (https://phytozome.jgi.doe.gov/pz/portal.html #), and 16 homologous genes, namely, CrMAPK2–CrMAPK17, were obtained. The mRNA expression level variation of these genes exposed to different stressors, such as –Fe, −N, and osmotic shock (150 mM NaCl), was also detected. Among these genes, CrMAPK3 is specifically functionally analyzed by RNA silencing.
Bioinformatics Analysis of MAPK Genes in Chlamydomonas
List of the 16 MAPK genes identified in C. reinhardtii and their sequence characteristics
Sub cellular location
Analysis of mRNA levels of MAPK gene under − Fe, −N, and 150 mM NaCl stress conditions
CrMAPK3 positively regulates the expression of CrFOX1 gene
CrMAPK3 positively regulates the expression levels of iron uptake-associated genes
MAPKs are widely distributed in eukaryotic organisms, such as yeast, humans, and plants, and are involved in phosphorylation signaling cascades in extracellular amplification and intracellular transduction . The MAPK pathway is responsive to biological and non-biological stress stimuli, hormones, or growth factors and to cell division and apoptosis. Moreover, the MAPK pathway comprises MAPKKK, MAPKK, and MAPK and amplifies signals via subsequent phosphorylation by using protein kinases and by migrating to the nucleus; thus, the extracellular stimuli of membrane receptors are connected to the molecular effectors of the cytoplasm and the nucleus [24, 25]. A few MAPKs, including 20 in Arabidopsis, 17 in rice, 19 in corn, 21 in aspen (Populus), 17 in tobacco, 16 in tomato, and 26 in apple, have been identified [26–28]. Proteins encoded by MAPKs in different species contain various domains. In Chlamydomonas, 3 of TEY, 7 of TDY, 4 of TSY, 1 of TPY, and 1 of TTY exist. In Arabidopsis, 8 of TDY and 12 of TEY are present. These diversities of types and kinase domains demonstrate that MAPKs participate in many metabolic activities. Through cluster analysis, we found that TDY and TEY of Chlamydomonas kinases are highly homologous to those of Arabidopsis kinases possibly because only TDY and TEY domains are found in Arabidopsis. Other domains are highly similar to human kinases.
Organisms need iron for respiration, DNA synthesis, and enzyme reactions. Transport systems have been developed for iron absorption because iron balance is vital. Iron regulation, especially iron absorption and transportation, has been extensively investigated, but iron signal response systems have been rarely explored. Iron deficiency in humans causes iron deficiency anemia and adolescent iron deficiency 1 syndrome. Iron is an important element required by the body; excessive or scarce amounts of iron likely cause metabolic disorders; therefore, organisms should have a sophisticated control system to regulate the dynamic balance of iron elements .
Iron deficiency is the third-most important limiting factor of plant growth and yield in agriculture. Photosynthetic plants reduce their chlorophyll synthesis and photosynthesis rate under iron-deficient conditions.
In humans, iron deficiency causes anemia. Conversely, excess iron increases the risk of liver disease, heart attack, and hypothyroidism. Iron is also a limiting factor in the growth of some tumor cells, and iron chelators are used clinically to inhibit tumor cell growth. Furthermore, studies on iron MAPK signal cascades have focused on human cancers. Iron deficiency inhibits the mitosis of lung carcinoma cells, melanoma cells, and dysembryoplastic neuroepithelial tumor cells and thus induces cell apoptosis [29–31]. Therefore, iron chelators, desferrioxamine (DFO), and Dp44mT are used to treat these cancers clinically [32, 33]. Iron deficiency signals are also transduced through the activation of JNA and P38 by ASK1 (MAPKKK) to regulate the suspension of the mitotic activity and apoptosis of cancer cells .
Plant MAPK gene responses to various stresses have also been detected. In our study, gene expression analysis revealed that 16 MAPK genes in Chlamydomonas were involved in response to stress. During iron deficiency, 8 MAPK genes, including CrMAPK3, were upregulated. Therefore, CrMAPK3 possibly responded to iron regulation. These findings were further verified by silencing CrMAPK3, and our results demonstrated that the mRNA levels of FOX1-ARS, the enzyme activities of ARS, and the endogenous mRNA level of CrFOX1 decreased. Therefore, CrMAPK3 positively regulated CrFOX1 expression. The mRNA levels of –Fe-inducing genes, including CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, and the expression of CrMAPK3 were reduced. These findings confirmed that CrMAPK3 positively regulated the expression of iron-absorption genes. However, the exact proteins upstream and downstream of CrMAPK3 should be identified to reveal the MAPK pathway of iron deficiency response in Chlamydomonas.
Algal strains and culture conditions
C. reinhardtii CC425 (mt) was purchased from the Chlamydomonas Genetics Center at Duke University. C. reinhardtii 2A38 is a transgenic strain with an integrated Fox1 promoter:ARS chimeric gene in C. reinhardtii CC425 genome. Under iron-deficient conditions, the CrFOX1 promoter in 2A38 strain stimulated the ARS gene expression and appeared blue when the XSO4 substrate was added. Liquid cultures were grown in the TAP medium at 26 °C with agitation at 220 rpm under 110 μmol⋅m−2s−1 of continuous light for 3 days and then to the TAP, TAP-Fe, TAP-N, or TAP + 150 mM NaCl media for various time periods (12, 24, and 36 h). Total RNA was extracted to prepare cDNA for gene cloning and real-time PCR assay. All Chlamydomonas strains were cultured in the TAP or deficiency medium of TAP with Hunter’s trace element mix.
Bioinformatics analysis of MAPK gene family of Chlamydomonas
Chlamydomonas MAPK homologous genes were retrieved from Chlamydomonas database (https://phytozome.jgi.doe.gov/pz/portal.html) by using the BLAST of Arabidopsis MAPK. Multiple sequence alignments were generated using ClustalX 2.1 and MEGA6. The following parameters were predicted: molecular weights and isoelectric points of proteins in Expasy (http://web.expasy.org/compute_pi/); protein structures in SMART; and conserved protein motifs in PROSITE (http://prosite.expasy.org/) and MEME (http://meme.nbcr.net/meme/). The structures of CrMAPK genes were generated online by using the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/), and the homologous chromosome segments were detected using a synteny plot in Plaza (http://bioinformatics.psb.ugent.be/plaza/versions/pico-plaza/synteny/index). The CrMAPK genes were subjected to BLAST analysis in Plaza, and their duplication patterns were detected using a synteny plot. The subcellular localization of Chlamydomonas MAPKs was performed using Euk-mPLoc2.0 (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/).
Data were presented as mean ± S.D. One-way ANOVA followed by Duncan’s post-test was performed to examine significant differences between means. In all cases, comparisons showing P < 0.05 were considered significant.
mRNA abundance detection
Primer sequences for amplifying the Chlamydomonas MAPK genes
Primer sequences for amplifying Chlamydomonas iron responsive genes
Construction of CrMAPK3 RNA interference vector and transformation of Chlamydomonas
Using Chlamydomonas cDNA as a template, we amplified the fragments through PCR with forward primer CrMAPK3-F: CGTCCGCAAAAGACAGTGTA and reverse primer CrMAPK3-R: CTTCGTCTACCAGGTGCTCC. We then inserted the amplified fragments into pMD18-T vector to generate CrMAPK3-18 T, which was further digested with HindIII and BamHI and ligated into the intermediate vector T282 to produce CrMAPK3-T282. CrMAPK3-T282-CrMAPK3 with inverted repeat sequence of CrMAPK3 (CrMAPK3IR) was developed by digesting CrMAPK3-18 T and CrMAPK3-T282. CrMAPK3IR was inserted into EcoRI-digested pMaa7/XIR to produce Maa7IR/CrMAPK3IR. Maa7IR/CrMAPK3IR was then transformed into C. reinhardtii 2A38 by applying the glass bead procedure .
ARS (arylsulfatase) activity detection
ARS activity was determined as described by Davies and Grossman . XSO4 (10 mM) was added to plates with –Fe TAP solid medium and scribed before clones were inoculated. After 1 day, the transformants that expressed ARS activity were identified using blue halos around their colonies. The cells were initially collected by centrifugation to quantify the ARS activity. The supernatant was mixed with 0.1 M glycine–NaOH at pH 9.0, 10 mM imidazole, and 4.5 mM p-nitrophenyl sulfate. The reaction mixture was incubated at 27 °C for 30 min. The reaction was terminated by adding 0.25 M NaOH, and its absorbance at 410 nm was determined. A standard curve of p-nitrophenol (Sigma Chemical Co.) was obtained using 0.2 M NaOH.
Silencing CrMAPK3 decreased the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced. Therefore, CrMAPK3 regulated the expression of iron-deficiency-responsive genes in C. reinhardtii.
This study was supported by the National Natural Science Foundation of China (31160050, 31360051), the Funds of Hainan Engineering and Technological Research (GCZX2011006, GCZX2012004, GCZX2013004), and the Key Projects of Hainan Province (ZDYF2016021).
Availability of data and materials
All of the data generated or analyzed in this study are included in this published article.
XW Fei and XD Deng designed experiments. XW Fei and JM Yu performed experiments. JM Yu and YJ Li analyzed data. XW Fei and XD Deng wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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