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

Full size image

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 [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.