CrMAPK3 regulates the expression of iron-deficiency-responsive genes in Chlamydomonas reinhardtii

Background 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. Results 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. Conclusion CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

Iron is an essential trace element for most living organisms. A precise iron regulation system is necessary to maintain the dynamic equilibrium of iron [16] 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 [17]) on the plasma membrane and then putatively transferred to FOX1 by FEA1 [18]. Afterward, FOX1 oxidizes ferrous iron to ferric iron, which is then transported to the cytoplasm by FTR1 on the plasma membrane [19][20][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 nonbiological 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, CrMA PK2-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
Sixteen homologous genes (Table 1), which are localized in chromosomes 1, 2, 3, 8, 12, 13, 16, and 17, were identified by searching the Chlamydomonas genome database with Blast. The predicted open reading frames of these genes were 1062-5301 bp in length, and their protein products contained 353-1766 amino acids with molecular weights of 39.8-178.76 kD and isoelectric points of 5.68-9.5. Fourteen of the MAPKs located in the cytosome were predicted by Euk-mPLoc2.0 except CrMAPK6 and CrMAPK14, which exist in the nucleus. Using PROSITE predictions, we verified that the 16 CrMAPKs were mitogen-activated protein kinases. Multi-sequence alignment of the MAPK-specific F and R represent the forward and reverse directions on the chromosome, respectively. In total, 16 CrMAPK proteins were obtained by BLASTP search using the C. reinhardtii V5.5 proteome database and MAPK proteins from Arabidopsis thaliana as queries. The 16 CrMAPK genes were named based on their name annotated in JGI database. The molecular weights and pIs of the 16 CrMAPK proteins were predicted using ExPASy. The CrMAPK sub-cellular locations were predicted using the Euk-mPLoc2.0 program TXY motifs in the CrMAPK proteins revealed that the T(D/E/T/S/P)Y activation loop motifs were conserved in the serine-threonine kinase (S-Tkc) domain in the 16 CrMAPKs (Fig. 1). CrMAPK3, CrMAPK6, and CrMAPK8 contain TEY; CrMAPK2, CrMAPK4, CrMAPK5, CrMAPK7, CrMAPK9, CrMAPK10, and CrMAPK13 comprise TDY; CrMAPK11, CrMAPK12, CrMAPK15, and CrMAPK16 possess TSY; CrMAPK17 is composed of TPY; and CrMAPK14 consists of TTY. Chlamydomonas MAPKs were divided into two groups by using MEGA6. Group I contained CrMAPK2 to CrMAPK10, whereas Group II comprised CrMAPK11 to CrMAPK17 (Fig. 2a). All of the MAPK genes contained 6 to 10 exons. The gene length ranged from 2.8 kb to 10.5 kb. Among these genes, CrMAPK3 is the shortest and CrMAPK11 is the longest (Fig. 2b). In addition to the S-Tkc-conserved region, 8 other protein domains/ motifs, such as Syn N(Syntaxin N-terminal domain), RIO (RIO-like kinase), CUE (domain that may be involved in binding ubiquitin-conjugating enzymes), and Tyr-kc (tyrosine kinase catalytic domain) motifs ( Fig. 2c), are present in CrMAPKs. The annotated CrMAPK1(Cre13.g582650) in JGI database is a small protein with 149 aa. After the alignment was compared with the other CrMAPKs(CrMAPK2-CrMAPK17), the results revealed that the conserved domain (T(D/E/T/S/P)YXTRWYRAPEL(V)) in the MAPK family could not be found in CrMAPK1. As such, CrMAPK1 is not included in Table 1. were affected by iron deficiency, nitrogen deficiency, and high salt concentration. Compared with the expression of the gene in the TAP medium, the mRNA expression levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were increased by iron deficiency to various degrees. CrMAPK3, CrMAPK5, and CrMAPK11 respectively increased by 43-, 5-, and 40-fold after cultivation for 48 h. However, iron deficiency decreased the mRNA levels of CrMAPK7, CrMAPK12, CrMAPK13, CrMAPK15, CrMAPK16, and CrMAPK17 after cultivation for 48 h. In nitrogen deficiency, the mRNA expression levels of CrMAPK6 and CrMAPK14 were significantly increased, whereas the expression levels of most MAPKs, such as those of CrMAPK2, CrMAPK3, CrMAPK5, CrMAPK5, CrMAPK8, CrMAPK9, CrMAPK10, CrMAPK11, CrMAPK12, CrMAPK13, CrMAPK15, CrMAPK16, and CrMAPK17, of C. reinhardtii were inhibited, and the mRNA expression levels of these genes were significantly decreased. The mRNA expression levels of all MAPK genes were also inhibited under high salt (150 mM NaCl) condition, and the mRNA expression of CrMAPK17 was reduced by 10E10.

CrMAPK3 positively regulates the expression of CrFOX1 gene
The C. reinhardtii 2A38 strain was prepared by using the integrated FOX1 promoter:ARS box into the chromosome of the CC425 strain. Under iron-deficient conditions, CrFOX1 promoted the ARS reporter expression and appeared deep blue when this gene was mixed with XSO4 substrate or yellow when this gene was mixed with p-nitrophenylsulfate. A total of 133 colonies were obtained after Maa7IR/CrMAPK3IR was transformed into C. reinhardtii 2A38 and then transferred onto - Fig. 1 Multiple alignments of T(D/E/T/S/P)Y domains from MAPK proteins. The U-box domains in CrPUB proteins were predicted using MEME programs. Their sequences were aligned using ClustalX 2.1, and the alignments were edited using GeneDoc 2.7 sequence editor. Black, gray, and light gray shades indicate the identities and similarities among these sequences as 100, 80, and 60%, respectively Fe plates with XSO4 to determine the ARS activities.
Only 44 colonies and the control sample of 2A38 appeared blue, whereas the 99 other colonies were colorless or light blue. Furthermore, 74.4% of chromogenic reactions indicated that CrMAPK3 silencing affected the FOX1 promoter function in -Fe. The transformants of RNAi11, RNAi37, and RNAi62 appeared colorless in both + Fe (16 uM) and -Fe except the non-transgenic C. reinhardtii 2A38 control, which appeared deep blue under -Fe conditions (Fig. 4a). These results were further confirmed by the ARS activity in transgenic strains. The ARS activities of RNAi11, RNAi37, and RNAi62 respectively decreased by 82, 85, and 83% compared with those of the C. reinhardtii 2A38 control (Fig. 4b). The mRNA of the ARS2 of the transgenic stains decreased by more than 97% in -Fe (Fig. 4c). The mRNA levels of the target CrMAPK3 of the transgenic strains RNAi11, RNAi37, and RNAi62 decreased remarkably by 97, 97, and 98%, respectively (Fig. 4d). These data implied that CrMAPK3 silencing positively regulated FOX1:ARS expression. CrMAPK3 silencing also decreased the gene expression of endogenous FOX1 in Chlamydomonas. The mRNA levels of CrFOX1 of the RNAi11 strain decreased by 63, 54, and 71% when this strain was cultured for 12, 24, and 48 h under the -Fe condition, respectively. CrMAPK3 silencing also repressed the iron-induced upregulation of CrFOX1 gene expression. Therefore, CrMAPK3 positively regulated the endogenous expression of CrFOX1 (Fig. 4e).
CrMAPK3 positively regulates the expression levels of iron uptake-associated genes Chlamydomonas exhibits an iron uptake pattern similar to Type I plants. During iron deficiency, Chlamydomonas cells undergo the affinity iron absorption mechanism by inducing the expression of CrFOX1 [19], CrNRAMP2 [18], CrATX1 [22], CrFTR1 [20], and CrFEA1 to enhance iron absorption [18]. CrFEA1 is located in the cell walls and responsible for the transport of reduced Fe 2+ via FRE1 (homolog of Arabidopsis FRO2) to CrFOX1, which is found in the plasma membrane (homolog of yeast FET3 [23]), and reoxidizes Fe 2+ to Fe 3+ . CrFOX1 is then transported inside the cells through the plasma membrane protein CrFTR1, and ATX1 of yeast transports Cu 2+ to the cytoplasm. Thus far, direct evidence supporting iron transmission has yet to be obtained, but studies have shown that ATX1 is an iron-deficiency-inducible protein.
The NRAMP gene family is located on the vacuole membrane, and it shuttles Fe 2+ between vacuole membranes to maintain the iron concentration in the cytoplasm. The mRNA levels of iron absorption-related genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, in the CrMAPK3 RNAi transgenic strain RNAi11 are shown in In the CrMAPK3-silenced strain RNAi11, the mRNA levels of the genes, including CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also decreased when the mRNA level of CrMAPK3 was decreased. Thus, CrMAPK3 might positively regulate the expression of iron-uptake-associated genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1.

Discussion
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 [23]. 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] Fig. 3 Results of qPCR analysis of the CrMAPK genes under − Fe, −N, or 150 mM NaCl conditions. C. reinhardtii CC425 was pre-cultured in TAP to the mid-logarithmic phase, followed by centrifugation and resuspension in TAP, TAP-Fe, TAP-N, and TAP with 150 mM NaCl. All the samples continued culturing for 12, 36, and 48 h. The cells were collected, and RNA samples were isolated. The gene transcript levels were determined using Real Time quantitative PCR. All expression values were normalized to the value of the 18S rRNA gene. The relative amounts were calibrated based on the number of transcripts of the corresponding genes in cells maintained in TAP for 12 h 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 [16].
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][30][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 [34].
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.  Fe (a, b). Moreover, the mRNA levels of ARS were significantly decreased (c). The mRNA level of target gene CrMAPK3 was decreased by 97-98% compared with the control (d), indicating that CrMAPK3 in transgenic strains of RNAi11, RNAi37, and RNAi62 has been effectively silenced. The mRNA levels of endogenous CrFOX1 were reduced by 63, 54, and 71%, respectively, at 12, 24, and 48 h post-incubation in -Fe, indicating that CrMAPK3 positively regulates the expression of CrFOX1 gene (e). The data are shown as the means (±SD, n = 3). Significance is indicated as *P < 0.05, **P < 0.01 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.

Methods
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 −2 s −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.

Statistical analyses
Data were presented as mean ± S.D. One-way ANOVA followed by Duncan's post-test was performed to examine In the CrMAPK3-silenced strain RNAi11, the mRNA levels of genes, including CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were decreased when the mRNA level of CrMAPK3 was decreased and after they were incubated with -Fe after 48 h. This finding indicated that CrMAPK3 may positively regulate the expression of iron uptake-associated genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1. The data are shown as means ± SD (n = 3). Significance is indicated as *P < 0.05, **P < 0.01 significant differences between means. In all cases, comparisons showing P < 0.05 were considered significant.

mRNA abundance detection
Three independent cell populations exposed to different stress conditions in various periods were collected. Total RNA was extracted and real-time PCR was performed as described previously [35]. The primers used in this study are listed in Tables 2 and 3. All the products were 300 +/-50 nt The amplification rate of each transcript (Ct) was calculated using the PCR Base Line Subtracted method performed in the iCycler software at a constant fluorescence level. Cts were determined with three repeats. Relative fold differences were calculated on the basis of the relative quantification analytical method (2 -ΔΔCT ) using 18 s rRNA amplification as internal standard [36].

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: CTTCGTCTACCAGGTGC TCC. 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. CrMAP K3IR 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 [37].

ARS (arylsulfatase) activity detection
ARS activity was determined as described by Davies and Grossman [38]. 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

Conclusions
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.