Background

Cassava (Manihot esculenta Crantz) is an important food staple for raising nearly a billion people in the tropics, playing vital functions in food security [1]. Cassava not only used as industrial raw materials, but also act as edible food in China, attributed to its starchy roots [2, 3]. In recent decades, China became a major country of cassava production in the world during the long-term cultivation, the harvest area and yield were increased in China [4]. However, the production of cassava in China could not afford the domestic consumption, self-sufficiency rate was only 18%, most of cassava dry slices and starch dependent on import [5]. Therefore, it is an urgence to solve the disadvantaged situation by cultivating cassava varieties harboring the excellent characteristic with high-yield and high starch accumulation. Furthermore, cassava is a typical source-to-sink tuberous crop [6, 7]. Thus, to better understanding the molecular regulation mechanism of chlorophyll biosynthesis is the initial step for cassava breeding.

Chlorophyll is synthesized from Mg-protoporphyrin IX in the biosynthesis pathway. Magnesium chelatase is the first committed enzyme for Mg-porphyrin branch, which catalyzed Mg2+ in to protoporphyrin IX by consumption of ATP. Magnesium chelatase consists of at least three different subunits, ChlI, ChlH and ChlD subunits [8]. These subunits have been reported in plants, especially in Oryza sativa [9,10,11], Nicotiana benthamiana [12, 13], Arabidopsis thaliana [14, 15], Glycine max [16,17,18], Pisum sativum [19], Zea mays [20], which have specific functions in chlorophyll biosynthesis and photosynthesis. AtChlH protein involves in plastid-to-nucleus signal transduction [21, 22]. Mutations in ChlD also impairs retrograde signaling [23]. Additionally, ChlH and ChlI except ChlD also affect ABA signaling in stomatal guard cell in A. thaliana and N. benthamiana [24, 25]. These results indicated these subunits have multiple roles in plants. Thus, although the regulatory mechanism of chlorophyll biosynthesis is currently being elucidated in many species, there are still many details that required further investigation in cassava.

Virus-induced gene silencing (VIGS) system was widely and successfully used to characterized gene functions in many plants [26,27,28,29,30]. VIGS is mostly drive on RNA viruses, such as tobacco rattle virus (TRV), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), potato virus X (PVX) and cassava common mosaic virus (CsCMV). Especially, TRV and CsCMV are widely used in cassava for rapid gene function analysis [29,30,31,32], due to high silencing efficiency, long silencing duration, and obvious virus-induced disease symptoms. A magnesium chelatase subunit I (ChlI) was successfully silenced in tobacco and different cassava varieties by CsCMV-mediate VIGS system [30]. Therefore, CsCMV mediated-VIGS system can be used as a new tool for rapidly characterized gene functions in cassava.

In this study, MeChlD was successfully cloned and characterized, which encodes a magnesium chelatase subunit D. The tissue specific expression, subcellular localization, protein interactions and VIGS-inducing the phenotypes were characterized. These results suggested that MeChlD was chlorophyll-localized is required for chlorophyll biosynthesis, photosynthesis and starch metabolism in cassava.

Methods

Plant materials and growth conditions

Cassava variety South China 9 (SC9) was used in this study, which provided from National Cassava Germplasm Repository (Danzhou city, Hainan province). Tobacco (N. benthamiana) was provided by Prof. Yao from Institute of Tropical Bioscience and Biotechnology, Chinese academy of Tropical Agricultural Science, which was used in this study to analyzed subcellular localization and protein interactions. All plants were kept in an air-conditioned growth room (22 °C; 16-h photoperiod, 2000 lx light intensity).

Gene cloning and plasmid construction

Gene sequence of MeChlD was cloned using cDNA as a template. DNA sequences encoding proteins (MeChlD, MeChlM and MePrxQ) without signal peptides were cloned into prokaryotic expression vector pET28a (Novagen, Merck).

To analyze localization of GFP-tagged MeChlD, the coding sequence of MeChlD was PCR-amplified, and then inserted at the NcoI site of the binary vector pCAMBIA1302 (yielding a 35Spro-MeChlD:GFP construct).

To examine the interaction relationship between MeChlD and MeChlM/MePrxQ by using the yeast two-hybrid system, the vectors pGBKT7-MeChlD, pADT7-MeChlM, pADT7-MePrxQ, pGBKT7-MeChlM, pGBKT7-MePrxQ and pADT7-MeChlD were constructed and transformed into yeast competent cell AH109, respectively.

To further confirm the interaction by using BiFC, these vectors pNC-BiFC-Enn-MeChlD with N-terminal enhanced yellow fluorescence protein (nYFP), pNC-BiFC-Ecc-MeChlM and pNC-BiFC-Ecc-MePrxQ with C-terminal enhanced YFP (cYFP) were constructed by seamless cloning (ClonExpress II One Step Cloning Kit, Vazyme Biotech Co., Ltd), and transformed into Agrobacterium tumefaciens GV3101-pSoup-p19.

To investigate the role of MeChlD, 300 bp DNA fragment was PCR-amplified using cDNA of SC9 and finally cloned into the a VIGS vector pCsCMV-NC as described [30], yielding pCsCMV-MeChlD.

All constructs were verified by DNA sequencing. Primer sequences for plasmid construction are shown in Table S1.

Identification and bioinformatic analysis of MeChlD

The MW (molecular weight), pI (theoretical isoelectric point) and positive/negative charge residues of MeChlD were predicted by ExPASy [33]. The ProtScale program (http://web.expasy.org/ protscale/) was used to analyze the hydrophilicity of the ChlD protein. The gene expression of MeChlD was characterized in cassava expression atlas as described [34].

For the identification of MeChlD and related proteins, the amino acid sequence of MeChlD was used as a query sequence for blast at the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). MEGA X [35] was used to multiple alignment analysis of MeChlD and homologous proteins and a phylogenetic tree was constructed using the Neighbor-joining method.

Measurement of the net photosynthetic rate in cassava accessions

The net photosynthetic rate under nesting scenarios along with the temperature, CO2 concentration, and the photon flux density was measured by LI-6800 portable photosynthesis system (Li-COR company, Nebraska, USA). The required temperature was set 25 °C by temperature controlling module, the CO2 injection volume ratio was set 400 μL/L, the photo flux density was set to 1000 μmol/(m2·s) by LED light source module, the relative humidity was set 50–70% by water control module. Fully expanded functional leaves of cassava were randomly selected. The timing for measuring was selected at 09:00 and 12:00 am to avoid the effects of stomatal closure on this study.

The growth of the recombinant proteins under different concentrations of Mg2+ treatments

The vectors pET-28a-MeChlD, pET-28a-MeChlM, pET-28a-MePrxQ and pET-28a were transformed into E. coil (DE3). These recombinant proteins MeChlD, MeChlM and MePrxQ were treated by different concentrations of magnesium ion as described [36].

Subcellular localization of MeChlD in tobacco leaves

The vectors pCAMBIA1302-35Spro-MeChlD:GFP and pCAMBIA1302 were transformed into A. tumefaciens strain GV3101, and then infiltrated into N. benthamiana leaves as described. After 3 days post inoculation (dpi), GFP signals (395–509 nm) were detected using a laser confocal microscope.

Y2H assay

These vectors pGBKT7-MeChlD/pADT7, pGBKT7-MeChlM/pADT7 and pGBKT7-MePrxQ/pADT7, pGBKT7-MeChlD/pADT7-MeChlM, pGBKT7-MeChlD/pADT7-MePrxQ and pGBKT7-MeChlM/pADT7-MePrxQ were transformed into yeast competent cell AH109, and then the positive clones were screened on the selective SD dropout medium (SD-Leu-Trp and SD-Leu-Trp-His-Ade).

Bimolecular fluorescence complementation (BiFC) analysis

These vectors pNC-BiFC-Enn-MeChlD, pNC-BiFC-Ecc-MeChlM and pNC-BiFC-Ecc-MePrxQ were transformed into A. tumefaciens strain GV3101-psoup-p19, and then infiltrated into N. benthamiana leaves (growth of 30 days) as described [37]. The yellow fluorescence (514–527 nm) in the infiltrated leaf areas was examined using a laser confocal microscope.

VIGS assay in cassava

The best VIGS target region of MeChlD CDS sequence (Manes. 14G005700) was analyzed by online software (SGN VIGS Tool), and then we amplified this region (274–573 bp of CDS sequence) and constructed the vectors pCsCMV-MeChlD. The vectors pCsCMV-MeChlD and pCsCMV-NC were transformed into A. tumefaciens strain GV3101-pSoup-p19, and then infiltrated the leaves of cassava variety SC9 by a syringe injection as described [30].

The VIGS-mediated silent plants were grown in the air-condition room (22 °C; 16-h photoperiod). After 3 weeks post inoculation, the expression level of MeChlD in silent plants of cassava was measured by qRT-PCR to further calculate the silence efficient. Currently, chlorophyll content in different silent plants of cassava was performed as described [38]. The content of the total starch was measured as described [32].

RNA extraction and qRT-PCR verification

Total RNA was extracted from cassava leaves using a RNAprep Pure Plant plus Kit (Tiangen, China). One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, China) was used for first-strand cDNA synthesis. The reactions solutions were performed in 10 mL volume in Real-time Thermal Cycler fluorescence quantification apparatus (Thermo Fisher Scientific Inc., Göteborg, Sweden). MeActin was used as an internal reference gene. All samples were performed in triplicate per sample. The relative expression levels of genes were calculated by the formula 2−ΔΔCT method. The qRT-PCR primers were shown in Table S1.

Statistical analysis

Microsoft Excel 2010 was used for data processing; SPSS 23.0, DPS 7.0 and Kruskal–Wallis software was used for data analysis; GraphPad 6.0 and Origin2021 software were used to create experimental figures.

Results

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 82.27 kDa, 5.81, 100 and 92, respectively (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).

Fig. 1
figure 1

Alignment and phylogenetic analysis of MeChlD and MeChlD-related proteins in plants. a Alignment of amino acid sequences of MeChlD and MeChlD-related proteins. The alignment was performed with GENEIOUS software (https://www.geneious.com/). Identical amino acid residues are indicated with a black background, homologous residues with a grey background and dashes indicate gaps. The blue line and green line indicate ATPase domain and vWA structural domain, respectively. b A corresponding phylogenetic tree of MeChlD and related amino acid sequences. MeChlD characterized in this study are highlighted in blue. The tree was constructed by using the Neighbor-joining method. The horizontal bar represents a distance of 0.03 substitutions per site

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Mg2+ 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 MeChlD, MeChlM and MePrxQ proteins were significantly increased under 200 mM MgCl2 treatment (Fig. 2a). The growth curve showed that recombinant strain pET28a-MeChlD, pET28a-MeChlM and pET28a-MePrxQ were better than control strain pET28a under 200 mM MgCl2 treatment (Fig. 2b), and drip plate assay were also performed the growth of recombinant strain was better than control strain (Fig. 2c). These results suggest that Mg2+ highly affects the growth of the recombinant protein MeChlD in E. coli. Therefore, we predicated MeChlD may be play an important role in photosynthesis.

Fig. 2
figure 2

The growth of the recombinant proteins MeChlD, MeChlM and MePrxQ under different concentrations of MgCl2 treatment. a The empty vector pET28a was expressed in E. coli as a control. The five concentration gradients of Mg2+ (0 mM, 50 mM, 100 mM, 150 mM and 200 mM) were performed. Data indicate mean ± SD, asterisks indicate significantly different compared to the control (Kruskal–Wallis test, P < 0.05). b The growth curve of the recombinant proteins MeChlD, MeChlM and MePrxQ under magnesium ion treatment. The growth curve is obtained by determining the OD600 of the bacterial solution under conditions of 200 mM MgCl2. c Drop plate test of recombinant protein MeChlD, MeChlM and MePrxQ under magnesium ion stress conditions. The drop plate test is obtained by determining the OD600 of the bacterial solution under conditions of 200 mM MgCl2

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Biological function of MeChlD 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.

Fig. 3
figure 3

The profile of MeChlD expression in cassava. a Tissue-specific expression of MeChlD was analyzed in cassava gene expression atlas (http://shiny.danforthcenter.org/cassava_atlas/; Wilson et al., 2017). b MeChlD transcript levels in low photosynthetic rate of cassava accessions. c MeChlD transcript levels in high photosynthetic rate of cassava accessions. Leaves from cassava accessions were used for each RNA extraction (3 RNA extractions per variety; n = 3). Data indicate means ± SE. Statistically different transcript levels of different cassava accession compared with SC8 are marked by asterisks (the student’s test, P ≤ 0.05)

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Table 1 Average net photosynthetic rate of 13 cassava accessions of national cassava germplasm repository (Danzhou) was detected
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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). The result indicated that MeChlD was a chloroplast-localized protein.

Fig. 4
figure 4

Subcellular localization of MeChlD in transiently transformed into the tobacco leaves. Bright field and fluorescence images were taken and merged. Scale bars represent 20 μm

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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 S3).

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.

Fig. 5
figure 5

MeChlD interacts MeChlM and MePrxQ, respectively. a Analysis of the interaction between MeChlD-MeChlM and MeChlD-MePrxQ by the yeast two-hybrid assay. AD represents the pADT7-MeChlM and pADT7-MePrxQ; BD represents pGBKT7-MeChlD and pGBKT7-MeChlM. Interactions were assessed by growth on synthetic defined (SD) dropout medium lacking leucine and tryptophan (Leu/Trp), SD dropout medium lacking leucine, tryptophan and adenine (Leu/Trp/ Ade) and SD dropout medium lacking leucine, tryptophan, histidine and adenine (Leu/Trp/ Ade/His). b Bimolecular fluorescence complementation (BiFC) assays of the interaction between MeChlD-MeChlM and MeChlD-MePrxQ in tobacco leaves. Bright field and fluorescence images were taken and merged. Scale bars: 20 μm

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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 S4). 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 3 dpi (Fig. 5b), when only expressed nYFP-MeChlD, MeChlM-cYFP, MePrxQ-cYFP was not observed any fluorescence signals, respectively. This observation was consistent with Y2H results. In conclusion, these results indicated that MeChlD can interact with MeChlM and MePrxQ, respectively.

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 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 in the yellow phenotype increased with time and spread over the entire leaf.

Fig. 6
figure 6

Comparison of phenotype between VIGS-MeChlD plants and control plants. a qRT-PCR analysis of the expression of MeChlD in the yellow leaves of VIGS-MeChlD plants (MeChlD-1, MeChlD-2 and MeChlD-3). Leaves from VIGS-MeChlD plants were used for each RNA extraction (3 RNA extractions per silencing plants; n = 3). Data indicate means ± SE. Asterisks indicate significantly reduced compared to the control plants (the student’s test, P ≤ 0.05). b The VIGS-induce phenotype in leaves of VIGS-MeChlD plants in cassava at 3 wpi. Bars: 5 cm. c The content of total chlorophyll, chlorophyll a and chlorophyll b were measured in VIGS-MeChlD plants. Data indicate means ± SE. Asterisks indicate significantly reduced compared to the control plants (Kruskal–Wallis test, P  < 0.05). d qRT-PCR analysis of the transcript level of photosynthesis-associated nuclear genes in the yellow leaves of MeChlD-2 plants. Data indicate means ± SE. Asterisks indicate significantly different compared to the control plants (the student’s test, P ≤ 0.05)

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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 S5). 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.

Fig. 7
figure 7

VIGS-MeChlD-2 plants were transferred into the field and characterized after 4 months harvested. a The VIGS-induce phenotype in cassava leaves of MeChlD-2 plants in the field after 4 months harvested. Bars: 2 cm. b The storage root number, fresh weight and the content of total starch in cassava storage roots of MeChlD-2 plants in the field were measured after 4 months harvested. Data indicate means ± SE. Asterisks indicate significantly reduced compared to the control plants (Kruskal–Wallis test, P < 0.05)

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Discussion

Photosynthesis directly affects crop production and quality [41]. Chlorophyll content is essential for photosynthesis [42]. Mg-chelatase is the first enzyme in the chlorophyll biosynthesis pathway. It catalyzes the insertion of magnesium ions into protoporphyrin IX under ATP-dependent conditions [17]. A subunit D is indispensable for the enzymatic reaction of Mg-chelatase in the activation and insertion steps [8]. However, to date, no ChlD has been characterized in Euphorbiaceae. In this study, a subunit D called MeChlD was successfully cloned and characterized in cassava cultivar SC9 (Figure S1). Alignment analysis and phylogenetic tree indicated that MeChlD protein belongs to AAA+ protein superfamily, and shows higher homology with HbChlD and RcChlD (Fig. 1). These results suggested that ChlD proteins are highly conservative in Euphorbiaceae. However, the biological function of ChlD is still unclear, thus requiring further elucidation in Euphorbiaceae, especially in tuberous crop cassava.

ChlD encodes one of the key enzymes in chlorophyll biosynthesis. In this study, a higher concentration of Mg2+ affected the growth of the recombinant protein MeChlD (Fig. 2). MeChlD was found to be highly expressed in leaves (Fig. 3a and b). Furthermore, the MeChlD protein is localized in the chloroplast (Fig. 4). Similar results were observed in other Mg-chelatase subunits of different plant species [17, 19, 40], suggesting that these subunits are involved in chlorophyll biosynthesis. In addition, Y2H assay indicated that MeChlD interacts with MeChlM and MePrxQ (Fig. 5a). However, the relationship of these interactions was not very strong (Fig. 5a). Therefore, further study was performed to elucidate the interactions (Fig. 5b). The results showed that MeChlD interacts with MeChlM to mediate Mg2+ transport, which is involved in chlorophyll biosynthesis. Meanwhile, MeChlD interacts with MePrxQ, which is involved in the oxidative defense process in chloroplasts. These results suggested that MeChlD not only plays an essential role in chlorophyll biosynthesis, but also involves with oxidative defense process in cassava.

Gene function analysis depends on the perfect genome. Genome sequencing of cassava with good quality has been completed and recently released [43]. However, most studies on cassava paid increased attention to omics research, without validating the gene function in depth, which is limited by the genetic transformation system [44]. Therefore, developing a powerful method for rapid analysis of gene function in vivo is necessary. VIGS system based on cassava common mosaic virus is widely used to investigate gene function in cassava [30, 32]. In this study, VIGS-induced reduction of MeChlD expression resulted in mosaic leaves or fully yellow leaves of VIGS-MeChlD plants compared with VIGS-NC control plants (Fig. 6b). This finding could be attributed to the reduction in chlorophyll content (Fig. 6c). These changes were consistent with those in previous studies on VIGS in Mg-chelatase subunits [17, 19, 30]. Furthermore, the changes in chlorophyll biosynthesis were significantly correlated with the reduced expression of photosynthesis-related genes (Fig. 6d), but not of MeChlH silencing. In this study, the expression of MeChlI was significantly reduced in VIGS-MeChlD plants, consistent with the result of previous studies, which showed that ChlI stabilizes ChlD, and acts as a chaperone [45]. The transcript level of MeChlH was significantly up-regulated in VIGS-MeChlD plants, and similar results have been reported in Arabidopsis ChlM mutant [46]. However, the different results indicated that ChlH accumulation without changing the expression of VIGS-ChlD plants in pea [17]. These findings suggested that the regulation of ChlH is a complex process.

To our knowledge, cassava is a typical source-to-sink tuberous crop, and its sink capacity depends on the delivery of forms of sources [47]. In this study, the phenotype of yellow leaves or mosaic leaves was observed after 4 months of plantation in the field (Fig. 7a). The content of total starch, the number and fresh weight of cassava storage roots (4 months) in VIGS-MeChlD-2 plants significantly decreased compared with those in VIGS-NC control plants (Fig. 7b). This result indicated that the effect of VIGS could be maintained for at least 4 months in cassava storage roots, thus proving the hypothesis as described [30]. In conclusion, MeChlD not only possesses an important role in source biosynthesis, but also affects starch accumulation in cassava.