MeChlD encodes a magnesium chelatase subunit D
A magnesium chelatase subunit D, MeChlD, was successfully amplified and sequencing in cassava variety SC9 (Figure S1). The length of coding sequence is 2250 bp, encodes 749 amino acids. The information of MV, pI and positive/negative charge residues were identified and listed in Table S2. Bioinformatic analysis indicated that MeChlD belongs to stable protein and Hydrophilic protein (Figure S2).
To further characterize ChlD protein, we performed a study with ChlD of M. esculenta and homologues from HbChlD (Hevea brasiliensis), RcChlD (Ricinus communis), AtChlD (A. thaliana), NtChlD (N. tabacum), OsChlD (Oryza sativa), GsChlD (G. soja), CsChlD (Citrus sinensis) and GhChlD (Gossypium hirsutum). An amino acid sequence alignment of these proteins and a corresponding phylogenetic tree is shown (Fig. 1). Most of these ChlD proteins have conservative domains, ATPase domain without ATPase activity, and vWA structural domain with binding site for metal ion (Fig. 1a). MeChlD shows more than 81% identities with these proteins, especially has the closer relationship with HbChlD (96.74% identities) and RcChlD (90.60% identities) (Fig. 1b).
Mg 2+ affects the growth of the recombinant protein MeChlD Magnesium as an essential nutrient for plant growth and development, involved in the photosynthesis process. To investigate the roles of MeChlD in photosynthesis, ChlM and PrxQ were selected as positive controls [39–40]. These corresponding DNA were cloned into prokaryotic expression vector pET28a in order to expression them as recombinant proteins in E. coli BL21 (DE3). The five concentration gradients of Mg2+ (0 mM, 50 mM, 100 mM, 150 mM and 200 mM) were designed. The results indicated that the expression of ChlD, ChlM and PrxQ proteins were significantly increased under 200 mM MgCl2 treatment (Fig. 2). The growth curve and drip plate assay were also performed (Figure S3 and S4). These results suggest that Mg2+ highly affects the growth of the recombinant protein MeChlD. Therefore, we predicated MeChlD may be play an important role in photosynthesis.
MeChlD is involved in photosynthesis
What is the biological role of MeChlD during the photosynthesis? To answer this question, the expression level of MeChlD was firstly analyzed. In this study, we found that MeChlD was expressed in the leaves, mid-veins and stems in cassava gene expression atlas, especially highly expressed in the leaves (Fig. 3a). And then the correlation between the expression level of MeChlD and the net photosynthetic rate in different cassava accessions was also characterized. In this study, 13 cassava accessions were selected, and classified into two categories according to the value of net photosynthetic rate (Table 1). The expression levels of MeChlD were strongly increased in high net photosynthetic rate of cassava accessions (Fig. 3b), on the contrary, MeChlD with low expression profiles in low net photosynthetic rate of cassava accessions (Fig. 3c). These results indicated that the expression profile of MeChlD have a significantly positive correlation with the net photosynthetic rate.
Table 1
Average net photosynthetic rate of 13 cassava accessions of national cassava germplasm repository (Danzhou) was detected.
Cassava accessions | Accession numbers in NCGR database | Average net photosynthetic rate (µmol/(m2·s)) | Classification |
ZM99250 | MS000094 | 15.32977475 | Cassava accession with low average net photosynthetic rate |
TaiguoZhong | MS000626 | 15.5249856 |
GR024-2 | MS000245 | 15.66157154 |
Bra-home | MS000526 | 15.66793562 |
Yinnixiye | MS000021 | 15.87004708 |
HB60 | MS000623 | 16.01457674 |
SC8 | MS000129 | 16.9241 | Control Plant |
ZMG17 | MS000441 | 17.79893448 | Cassava accession with high average net photosynthetic rate |
SC13 | MS000421 | 17.7997385 |
KM21-2 | MS000133 | 17.90160996 |
R72 | MS000126 | 18.30468849 |
SC14 | MS000226 | 19.1704428 |
2–61 | GPMS1000L | 19.70053815 |
Mechld Was Chloroplast-localized Protein
To examined MeChlD localization, expressing MeChlD with C-terminal GFP tag was constructed. The fusion protein was transiently expressed in N. benthamiana leaves. Microscopy analysis indicated that MeChlD:GFP protein accumulated in the chloroplast after 3 dpi (Fig. 4).
Mechld Physically Interacts With Mechlm/prxq
ChlM and PrxQ play important functions in chlorophyll biosynthesis pathway and oxidative defense processes in chloroplasts, respectively. To analyze the interaction, the yeast two-hybrid system has been used. The vectors pGBKT7-MeChlD/pADT7, pGBKT7-MeChlM/pADT7 and pGBKT7-MePrxQ/pADT7 were constructed and transformed into yeast competent cell AH019. These combinations were able to grow in SD/-Trp-Leu dropout medium, but could not grow in SD/-Trp-Leu-Ade or SD/-Trp-Leu-Ade-His dropout medium. These results indicated that these vectors were expressed in yeast without auto-inducing (Figure S5).
The vectors pGBKT7-MeChlD/pADT7-MeChlM, pGBKT7-MeChlD/pADT7-MePrxQ and pGBKT7-MeChlM/pADT7-MePrxQ were constructed and transformed into yeast competent cell AH019. Specific interactions among MeChlD-MeChlM, MeChlD-MePrxQ, MeChlM-MePrxQ in yeast were confirmed (Fig. 5a), which were able to grow in SD/-Trp-Leu-Ade dropout medium. Remarkably, these interactions could not analyze in SD/-Trp-Leu-Ade-His dropout medium.
These vectors pGBKT7-MeChlM/pADT7-MeChlD, pGBKT7- MePrxQ/pADT7-MeChlD and pGBKT7-MePrxQ/pADT7-MeChlM were also constructed and transformed into yeast. The same results were observed (Figure S6). Therefore, we predicted that MeChlD-MeChlM, MeChlD-MePrxQ, MeChlM-MePrxQ showed weak interaction, respectively.
To further validate the interaction between MeChlD-MeChlM and MeChlD-MePrxQ observed in the Y2H system, the interaction of these proteins was tested using BiFC. The vectors pNC-BiFC-Enn-MeChlD, pNC-BiFC-Ecc-MeChlM and pNC-BiFC-Ecc-MePrxQ were constructed, and transformed into A. tumefaciens GV3101.
These fusion proteins were transiently expressed in tobacco leaves for 2–3 days. Microscopy analysis indicated co-expression of nYFP-MeChlD and MeChlM-cYFP, nYFP-MeChlD and MePrxQ-cYFP was observed yellow fluorescence signals at 3dpi (Fig. 5b), when only expressed nYFP-MeChlD, MeChlM-cYFP, MePrxQ-cYFP was not observed any fluorescence signals, respectively. These results were confirmed that the results of the Y2H system, MeChlD-MeChlM, MeChlD-MePrxQ were established the interaction relationship with each other.
Silence of MeChlD by VIGS affecting chlorophyll synthesis, photosynthesis and starch accumulation
To find out the role of MeChlD during the photosynthesis in cassava, virus-induced gene silencing (VIGS) technology was used. Cassava variety SC9 was inoculated with A. tumefaciens carrying pCsCMV-MeChlD. After three weeks post inoculation (wpi), the phenotype will be observed and taken a photo.
MeChlD expression was firstly analyzed by qRT-PCR in the leaves of VIGS-MeChlD plants at 3 wpi. These VIGS-MeChlD plants (MeChlD-1, MeChlD-2 and MeChlD-3) showed significantly reduced MeChlD transcript levels (from 30–60%) in comparison with plants inoculated with empty vector pCsCMV-NC in cassava (Fig. 6a). The upper leaves without inoculated from VIGS-MeChlD plants altered from green to yellow color. The VIGS-induce phenotype of MeChlD-1, MeChlD-2 and MeChlD-3 exhibited fully yellow leaves, yellow sectors from mosaic leaves and green sectors from mosaic leaves at 3 wpi (Fig. 6b). However, the appearance of the yellow phenotype increased with time and spread over the entire leaf.
To characterize these silenced plants, chlorophyll content was measured. The content of chlorophyll a, chlorophyll b and total chlorophyll of MeChlD-1, MeChlD-2 and MeChlD-3 were significantly reduced compared to plants inoculated with empty vector pCsCMV-NC (Fig. 6c).
To explore the role of MeChlD in retrograde signaling, the photosynthesis-associated nuclear genes (PhANGs) were also analyzed in these VIGS-silenced plants. The expression genes involved in photosynthesis (Rubisco small subunit, MeRbcS and phosphoenolpyruvate carboxylase, MePEPCase) and the tetrapyrrole biosynthesis pathway (ChlI and ChlM) were significantly down-regulated of MeChlD-2 plants (Fig. 6d), expect for MeChlH and MeRbcL. The transcript levels of PhANGs in the yellow leaves of MeChlD-1 and MeChlD-3 plants were also performed the similar results with MeChlD-2 plants (Figure S7). In conclusion, virus-induced gene silencing of MeChlD affects chlorophyll synthesis and photosynthesis in cassava.
To further investigate the biological role of MeChlD during the source-to-sink process, VIGS-MeChlD (MeChlD-2) plants were further used and transferred into the field, and then harvested after 4 months. The phenotype of yellow leaves or mosaic leaves was also observed and maintained in MeChlD-2 plants after 4 months in the field (Fig. 7a). The number and fresh weight of cassavas storage roots were significantly reduced in MeChlD-2 plants compared to the control plants (Fig. 7b). Furthermore, the content of total starch in cassava storage roots of these VIGS-MeChlD plants was also significantly reduced in comparison to VIGS-NC control plants (Fig. 7b). These findings indicate that MeChlD involved in source-to-sink by affecting starch accumulation.