AbhemC encoding porphobilinogen deaminase plays an important role in chlorophyll biosynthesis and function in albino Ananas comosus var. bracteatus leaves

Plant materials

Two-year-old chimeric A. comosus var. bracteatus plants were generated from the crown buds of the mother plant and grown at the experimental site of Sichuan Agricultural University.The mother plants were purchased from a supplier in Zhanjiang city, Guangdong province, China (21°12′N, 110°24′E). They were then grown at a temperature of 20 °C−30 °C during the day and 15 °C−18 °C at night, with 60–80% relative humidity. The normal green parts (Grs) and albino white parts (Whs) of the leaves were used in this study ( Fig. 1).

Measurement of photosynthetic pigments and synthetic precursors, and ALAD, PBGD, and uroporphyrinogen III synthase enzyme activity

The Whs and Grs of the chimeric leaves were used to determine Chl content and measure Chl biosynthetic precursors. Previously described methods were used to determine the contents of Chl a and Chl b (Holm, 1954), ALA (Dei, 1985), PBG (Bogorad, 1962), and Urogen III and Coprogen III (Czarnecki, Peter & Grimm, 2011). In addition, the contents of Proto IX, Mg-protoporphyrin IX (Mg-Proto IX), and protochlorophyllide (Pchlide) were assessed according to protocols reported by Rebeiz et al. (1975) and Lee et al. (1992). To measure enzyme activity, the following methods were applied: that of Mauzerall & Granick (1958) for ALAD, that of Lee et al. (1992) for PBGD, and that of Li et al. (2017) for uroporphyrinogen III synthase (UROS). Three independent biological replicates were used when measuring Chl a, Chl b, and the main Chl biosynthetic precursors, and the major enzyme activity.

Cloning and bioinformatics analysis of AbhemC

Specific primer pairs of AbhemC-F and AbhemC-R (Table S1) were designed according to the transcriptome sequence results (Ma et al., 2015) to amplify the full-length sequence of hemC from the leaves of A.comosus var. bracteatus. The ORF Finder (open reading frame finder) program was used to predict the ORF (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), for which the conserved domains were obtained using NCBI resources (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi and http://smart.embl-heidelberg.de/index2.cgi), and the secondary structure was predicted using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/). A three-dimension image of the protein and the multiple sequence alignment prediction were performed using SWISS-MODEL (http://swissmodel.expasy.org) and CLUSTX , respectively. The functional structure of the protein was predicted using InterProScan (http://www.ebi.ac.uk/interpro/search/sequence/) and ScanProsite (http://us.expasy.org/prosite) (Zdobnov & Apweiler, 2001). MEGA 5.0 was used with the neighbor-joining method to construct a phylogentic tree (Tamura et al., 2011).

RNA extraction and real-time quantitative PCR analysis

Total RNA from the Grs and Whs of chimeric leaves was carried out separately using TRIzol reagents (Invitrogen, USA ) following the recommendations of the manufacturer. The RNA was then reverse transcribed to cDNA using a PrimeScript RT Reagent kit. Using PCR system ABI prism 7900 Real-Time, the relative expression of AbhemC and hemC of tobacco plant were measured with SYBR Premix Ex TaqTM Kits (Takara), where 18S rRNA and Actin Tobacco housekeeping gene were used as an endogenous controls respectively (Li et al., 2014). The PCR amplification system was carried out as follows, heating for 30s at 95 °C, 40 cycles of denaturation at 95 °C for 15 s, annealing for 31 s at 58 °C, and extension at 72 °C for 35 s. Triplicate quantitative PCR experiments were performed for each sample, and the expression values obtained were normalized against the 18S rRNA gene. Data analysis of the relatively expressed gene was conducted using the Pfaffl method (Pfaffl, 2001). All the reactions were performed with three biological replicates. Primer pairs of AbhemC-1, Actin and hemC-1 were detailed in Table S1.

Prokaryotic expression of AbHEMC protein

After digestion with NdeI and XhoI, the full-length sequences of AbhemC were sub-cloned into pET15b vector to express the fusion protein. The purification of the protein involved ultrasonic fragmentation and nickel agarose affinity chromatography. The ultrasonic crushing of bacteria was performed in an ice bath twice at 20 min per session, with ultrasonic 2S suspended for 6 s as a cycle. Nickel agarose affinity chromatography was performed after ultrasound chromatography to purify the protein. The recombinant protein was expressed in E. coli Rosetta-gami (DE3) following the protocol of Ma et al. (2012), and its enzymatic activity was detected using the method of Lee et al. (1992).

Vector construction and tobacco transformation

AbhemC is highly homologous with hemC of tobacco; therefore, for the RNAi construct, the common conservative region of the two genes was amplified using primers for hemC-2 ( Table S1 ) and then subcloned into the binary vector pFGC5941. The pFGC5941 plasmid was then used to construct the RNAi vector (Deng et al., 2013). Conserved fragments were ligated into multiple cloning site 1 (MCS1) using NcoI and SwaI in the forward direction and into MCS2 using SmaI and XbaI in the reverse direction (Fig. 2). The recombinant plasmid was cloned into Agrobacterium strain EHA105 and then it was used to transform tobacco by Agrobacterium mediated method. The transformation of pFGC5941-AbhemC-RNAi vector to tobacco was conducted according to the protocol of Horch et al. (1985). Subsequently, the transgenic plants were screened and identified by the herbicide (PPT)-resistance gene Bar in the pFGC5941 vector. PCR identification of transgenic tobacco was performed using genomic DNA of resistant tobacco as template, wild-type tobacco genomic DNA template as negative control, and pFGC5941 plasmid as positive control. Physiological indicators were determined by analysis of three technical replicates, each of which was taken from three different transgenic plants.

Statistical analysis

SPSS was used as a statistical platform for the analysis of Chl contents ,precursors of Chl and enzyme activity. Two independent samples T test were used for statistical analysis.Figures were made with Excel 2016 in our experiments.

Assessment of Chl biosynthesis in the Whs and Grs of the chimeric leaves

Leaf color is usually associated with Chl contents (Kim & An, 2013). The concentrations of Chl a and Chl b in the Grs were significantly higher than those in the Whs of the chimeric leaves, respectively (Fig. 3A). To find out the key step of Chl biosynthesis in Whs which caused the albino of the leaves, the contents of the main Chl biosynthesis precursors were evaluated. Although there was no significant difference in the ALA and PBG content of the Whs and Grs of chimeric leaves, the content of Urogen III, Coprogen III, Proto IX, Mg-proto IX, and Pchlide was significantly lower in the Whs (Fig. 3A). These changes of the precursors suggested that the conversion of PBG to Urogen III was the first speed-limiting step in Chl biosynthesis in the Whs. The transition of ALA to PBG is catalyzed by ALAD, while that of PBG to Urogen III is catalyzed by PBGD and UROS. The enzymes activity of ALAD, PBGD and UROS are shown in Fig. 3B. The ALAD activity did not differ between the Whs and Grs, but the enzymes activity of PBGD and UROS were significantly reduced in the Whs. This suggests that decreased PBGD and UROS activity may have suppressed Urogen III formation in the Whs of leaves. The hemC gene encoding PBGD, which catalyses polymerization of PBG to produce 1-hydroxymethylbilane, may be the key functional gene in Chl biosynthesis and may play a role in the albino phenotype of chimeric A. comosus var. bracteatus.

Cloning and bioinformatics analysis of AbhemC

The full-length sequence of AbhemC was amplified, and the amplification products were purified, cloned and sequenced. The mRNA sequence obtained was determined to be that of AbhemC (GenBank accession number: KT377022); it comprised1135bp with an ORF of 1116bp. In addition, the AbHEMC protein was found to contain 371 amino acids; it belongs to the PBGD family (PLN02691) and had a high identity (78–82%), as well as a theoretical pI of 7.12 and molecular mass of 40.4 kD. The conserved domains of AbHEMC were identified using the NCBI Conserved Domain Search Service (Fig. 4B), and an uncharacterized subgroup of the PBGD family, namely the type 2 periplasmic binding protein fold (PBP2_PBGD_1), was found. Nine helices and nine strand structures were identified in the predicted secondary structure (Fig. 4C). Its 3D structure predicted a high sequence identity (78.37%) and similarity (0.53), and sequence coverage reached 0.86 with PBGD protein of Arabidopsis thaliana (Fig. 4D). Phylogenetic tree constructed with Molecular Evolutionary Genetics Analysis (MEGA) 5.0 showed that the phylogenetic tree was divided into three clades: temperate monocotyledons, tropical monocotyledons, and dicotyledons. It showed that AbHEMC was most closely related to AcHEMC in pineapple (Fig. 5). The predicted AbHEMC proteins of 21 other plants were derived from GenBank for phylogenetic analysis.

Expression of AbhemC in the Whs and Grs of the chimeric leaves

Expression of AbhemC in the Whs was 3.32-fold greater than in the Grs (Fig. 6). In previous transcriptomic (RNA-Seq) analysis, Xue et al. (2019) showed that hemC was significantly upregulated in the Whs of chimeric leaves from A. comosus var. bracteatus when compared with expression in the Grs, consistent with the results of qPCR. In the present study, AbhemC expression was upregulated whereas PDGD enzyme activity was reduced in the Whs. This indicates that the reduced function of the AbhemC-encoded PBGD protein disrupted Chl synthesis. Furthermore, it suggests regulation of the PBGD enzyme activity at the protein level or at the level of translation.

Prokaryotic expression and enzyme activity analysis of the AbHEMC protein

To verify the integrity of the mRNA sequence of the cloned hemC gene and the activity of the HEMC protein it encoded, prokaryotic expression and enzyme activity analysis were conducted. The ORF of AbhemC sequence was cloned into the expression vector pET-15b and expressed in E. coli Rosetta-gami (DE3) after induction with IPTG. SDS-PAGE analysis showed that the AbHEMC protein was expressed in E. coli and an obvious single band around 45 kD (lane 1–4 induced) was consistent with the expected molecular mass of AbHEMC (Fig. 7A). This confirmed that the mRNA sequence of AbhemC had been integrated and that it could translate a soluble fusion protein in E. coli cells. Further in vitro analysis showed that the prokaryotic-expressed AbHEMC protein had PBGD activity (Fig. 7B).

Functional analysis of AbhemC by transformation

To identify the function of AbHEMC, the RNA interference (RNAi) expression analysis was carried out. Agrobacterium EHA105 and a pFGC5941-AbhemC-RNAi fusion plasmid were transformed in tobacco leaves. The pFGC5941 vector in tobacco plants was used as a positive control while wild tobacco plants were used as a negative control. The resistant tobacco DNA obtained from PPT screening was extracted and identified by PCR with specific primers of Bar gene on pFGC5941 vector. The size of the target band amplified by PCR product electrophoresis was about 500bp, which was consistent with the expectation. Therefore, these resistant tobacco were identified as positive transgenic tobacco. According to RT-qPCR analysis of the tobacco plants, transformation of the pFGC5941 vector did not influence the expression of hemC significantly, whereas transformation of pFGC5941-AbhemC-RNAi significantly suppressed hemC expression (Fig. 8).

Furthermore, the leaf color of pFGC5941 vector-transformed tobacco plants closely resembled that of wild type tobacco plants, while the leaves of these two types were greener than the leaves of tobacco transformed with pFGC5941-AbhemC-RNAi vector (Fig. 9). In addition, the concentrations of Chl a and Chl b in tobacco plants transformed with pFGC5941-AbhemC-RNAi were significantly reduced relative to those in the wild type and pFGC5941 vector-transformed plants (Fig. 10A). These results indicate that Chl biosynthesis was significantly suppressed in tobacco plants transformed with the pFGC5941-AbhemC-RNAi vector. Thus, hemC is apparently a key gene in the Chl metabolism pathway and suppression of its expression inhibits Chl synthesis.

The enzyme activity of ALAD, PBGD, and UROS in transformed and wild tobacco plants is shown in Fig. 10B. ALAD and UROS activities did not differ among the three tobacco types, whereas PBGD activity was markedly lower in tobacco plants transformed with pFGC5941-AbhemC-RNAi than in the wild type and pFGC5941 vector-transformed plants. This suggests that inhibiting hemC expression results in decreased PBGD activity. In further analyses of the main Chl biosynthesis precursors, ALA and PBG concentrations did not differ among the three types of tobacco plant; however, Coprogen III, Proto IX, Mg-Proto IX, and Pchlide concentrations were markedly reduced in tobacco transformed with the pFGC5941-AbhemC-RNAi vector relative to the other two plant types (Fig. 10C). These results show that decreased PBGD activity in turn inhibits the transition of PBG to Urogen III and then the lack of Urogen III results in reduced biosynthesis of the four mentioned precursors of Chl biosynthesis.