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Agricultural and Environmental Chemistry

Protein cross-interactions for efficient photosynthesis in the cassava cultivar SC205 relative to its wild species Feifei An, Ting Chen, Qing X. Li, Jingjuan Qiao, Zhenwen Zhang, Luiz Joaquim Castelo Branco Carvalho, Kaimian Li, and Songbi Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00046 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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Journal of Agricultural and Food Chemistry

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Manuscript revised according to editor and reviewers’ comments for possible publication in

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Journal of Agriculture and Food Chemistry

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Protein cross-interactions for efficient photosynthesis in the cassava

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cultivar SC205 relative to its wild species

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Feifei An,a Ting Chen,b Qing X. Li,c Jingjuan Qiao,b Zhenwen Zhang,a Luiz JCB Carvalho,d

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Kaimian Li,a, Songbi Chen,a,

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aTropical

Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural

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Sciences/Key Laboratory of Ministry of Agriculture for Germplasm Resources Conservation

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and Utilization of Cassava, Danzhou 571737, China

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bCollege

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cDepartment

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Honolulu, Hawaii 96822, United States

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dGenetic

of Agronomy, Hainan University, Haikou, China of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa,

Resources and Biotechnology, Embrapa, Brazil

 The first two authors contributed equally to this work. Correspondence: Prof. Songbi Chen, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, China Tel: 86-898-23300289 Email: [email protected] Correspondence: Prof. Kiamian Li, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, China Tel: 86-898-23300295 Email: [email protected] 1

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Abstract

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The underlying mechanisms of higher photosynthetic efficiency of cultivated cassava relative

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to its wild species are poorly understood. In the present study, proteins in leaves and

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chloroplasts were analyzed to compare the differences among the cultivar SC205, its wild

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ancestor W14 and the related species Glaziovii. The functions of differential proteins are

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associated with 10 ontology groups including photosynthesis, carbohydrate and energy

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metabolism, as well as potential signal pathway. The protein-protein networks among 41

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differential proteins showed that PGK1 is a hub protein and protein cross-interactions

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affected the differentiation of photosynthetic rate. Anatomy patterns and PEPC detection

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suggested that SC205 has more C4 photosynthesis characteristics than Glaziovii and W14.

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Finally, a mechanism model of the efficient photosynthesis was proposed based on the

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remarkable variations in photosynthetic parameters and protein functions in the domestic

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cultivars.

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Key words: Cassava, Manihot esculenta, photosynthesis, protein network.

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Introduction

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Cassava (Manihot esculenta Crantz) is a major source of calories for more than 800

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million people in Africa, Asia and Latin America.(1–4) It plays a significant role in food

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security due to its beneficial traits, for example, its ability to grow in barren soil where many

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other crops perform poorly.(5,6) Cassava was initially considered as a “food for the poor”,

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while it is now a multipurpose crop in the world.(7,8) Cassava originates in South America

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and was domesticated less than 10000 years ago. Cultivated cassava (M. esculenta) may be

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derived from a wild progenitor M. esculenta ssp. Flabellifolia (W14).(9,10) Archaeological,

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ethnological, geographic and taxonomic records suggest that cassava might originate from 2


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several possible centers including southern Mexico (11), Guatemala (12), Colombia and

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Venezuela (13), and Amazon regions (14). The enormous diversity is a valuable source of

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genetic variability and useful in breeding to improve photosynthesis efficiency and enhance

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productivity.

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Cassava yields might increase largely via molecular breeding for photosynthetic

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efficiency increase.(15, 16) The relationships between leaf photosynthesis and productivity

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have been positively found in cassava (17). Elevated concentrations of carbon dioxide (CO2)

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increase net photosynthetic efficiency, and consequently largely increase the storage root

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yield (18). Cassava has very high leaf net photosynthetic rates in normal air, and under high

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photon flux density (>50 μmol CO2 m-2 s-1) and relatively low photorespiration (CO2

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compensation point around 25-35 ppm at 30-35 °C). The characteristics of leaf anatomy, high

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photosynthetic rates, low photorespiration and elevated phosphoenolpyruvate carboxylase

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(PEPC) activities in leaves of some wild Manihot species support the comment that cassava

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and its wild species stand for an intermediate photosynthesis between the typical C3 and C4

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species (17,19). Manihot may represent an evolutionary step towards C4 pathway. On the

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contrary, the others (9,20,21) have concluded that neither cassava nor wild relatives possess

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C4 photosynthesis, based on the dynamics of 14C labeling and CO2 compensation point.

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Calatayud et al. (9) reported that cassava wild species including W14 and the potential wild

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progenitor M. esculenta (22,23) exhibit typical traits of C3 photosynthesis, suggesting that

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cultivated cassava is not derived from C4 species.

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Although the evidence support the photosynthetic differences between the cultivated and

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wild cassava plants, the issue regarding their photosynthetic mechanism engenders

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controversy. The two dimensional electrophoresis (2-DE) – tandem mass spectrometry

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(MS/MS) analysis is a valid method to determine the changes of cassava under different

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stresses.(24,25) 2-DE-MS/MS analyses showed the correlation between high photosynthetic 3



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efficiency and low temperature stress.(25) It also showed the differences between cassava

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diploid and autotetraploid genotypes in global proteins and revealed the higher

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photosynthesis mechanism in autotetraploid leaves.(26) However, few studies were reported

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on the photosynthesis differences between cassava cultivars and wild species using

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proteomics. The objective of the present study was to determine the differentially expressed

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proteins in cassava leaves and chloroplasts, accounting for the photosynthetic difference

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between cultivated and wild species. Such results would reveal the protein patterns linked

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with the photosynthesis efficiency of cassava cultivars.

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Materials and methods Plant materials. The cassava cultivar SC205 (M. esculenta cv. SC205) (Supplementary

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Fig. S1-A1) and its wild progenitor (M. esculenta ssp. Flabellifolia) (Supplementary Fig.

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S1B1), and the related species India rubber (M. glaziovii) (Supplementary Fig. S1-C1)

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(herein after referred to as SC205, W14 and Glaziovii, respectively) were selected in the

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present study. SC205 was obtained from the Tropical Crops Genetic Resources Institute

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(TCGRI), Chinese Academy of Tropical Agricultural Sciences (CATAS). It was introduced

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from Philippine to China in 1820s. It adapted to grow in China after multi-generation

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selection and was named SC205 in 1970s. It is by far the most popular cassava cultivar

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planted in China. W14 and Glaziovii were introduced from Brazil and are currently being

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planted in the TCGRI experimental stations. W14 is the likely wild progenitor of cultivated

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cassava, and found throughout the Amazon basin. Glaziovii was domesticated in South

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America, and imported to East Africa in the early 20th century. The functional leaves

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(Supplementary Fig. S1-A2-C2) of three cassava species grown for 3 months were taken at

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11:00 am in a sunny day, immediately observed under microscopy, and frozen in liquid

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nitrogen for protein extraction. 4


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Microscopy of leaves using lyophilization. Small pieces in size of approximately

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0.7×0.5×0.3 cm3 were excised from the functional leaves with a razor blade, subjected to

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direct optimal cutting temperature (OCT) embedding solution, fixed on the machine (Leica

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CM1900, Germany). The slice thickness was 10 ~ 16 μm, and a slicer freezing temperature

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was -20 ~ -18°C. The slices were directly attached on a clean slide, with 5% glycerol

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mounting preparations, and then observed and photographed under the microscope (Leica

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DMLB, Germany).

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Photosynthetic activities measured with imaging pulse amplitude modulation. The

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selected functional leaves were detached from SC205, W14 and Glaziovii plants at 6 time

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points (7:00, 9:00, 11:00, 13:00, 15:00 and 17:00 o’clock) between 7:00 and 17:00 o’clock in

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a sunny day. All selected functional leaves were adjusted in the dark for 20 min prior to

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measurement. The Imaging PAM and Imaging WIN version 2.39 (both Heinz Walz GmbH,

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Effeltrich, Germany) were used to measure the photosynthetic activities.(26)

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Photosynthesis rates measured with LCPro-SD. The automatic portable photosynthesis

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analyzer (LCpro-SD, UK) was used to determine photosynthesis on the 3rd or 4th fully

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expanded leaf.(27) Each leaf was measured at 7:00, 11:00 and 14:00 o’clock in a sunny day,

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which the field temperature was 28, 37 and 43 °C, respectively. The leaf area was 6.25 mm2,

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and the net photosynthesis rate (Pn) (CO2 fixation), stomatal conductance (Cs), transpiration

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rate (Tr) and intercellular CO2 concentration (Ci) were measured for 6 individual plants.

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Isolation of chloroplasts. Chloroplasts were isolated from cassava functional leaves

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according to the protocol described by Fan et al.(28) Briefly, 50 g of cassava leaf were

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homogenized in the extraction buffer (0.3 M sorbitol, 1 mM MgCl2, 50 mM HEPES/KOH

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(pH7.8), 2 mM EDTA, 0.04% -mercaptoethanol, and 0.1% polyvinylpolypyrrolidone). The

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homogenate was filtered through 300 mesh muslin twice. The suspension containing

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chloroplasts was centrifuged at 500g for 3 min and 8000g for 10 min at 4 °C. Subsequently, 5



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the pellets were carefully resuspended in 20 mL of isolation buffer (0.3 M sorbitol, 1 mM

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MgCl2, 50 mM HEPES/KOH (pH 7.8), and 2 mM EDTA). The resuspended material was

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loaded on the top of a Percoll step gradient developed with 4 mL of 10%, 3 mL of 30%, and 2

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mL of 50% Percoll in isolation buffer, then centrifuged at 8000g for 15 min at 4 °C. Intact

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chloroplasts were collected from the interphase between 30 and 50% Percoll, washed twice

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with isolation buffer, and then directly applied to protein extraction. The purified chloroplasts

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were visualized under a Leica DMLN light microscope (Leica).

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Protein extraction, 2-DE separation and identification of leaves and chloroplasts. The

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proteins from functional leaves and chloroplasts of SC205, W14 and Glaziovii plants were

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extracted with phenol.(29) Protein 2-DE separation, image analysis, tryptic in gel digestion

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and protein identification of the spots were implemented.(26) The mass spectra were acquired

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on MALDI-TOF-TOF-MS (Ultraflex-TOF-TOF, Bruker, German) in the Analysis and

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Testing Center, Jiangsu University. The data were matched with the phytozome 11

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(http://phytozome.jpi.doe.gov/pz/) according to the plant genomic resources against M.

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esculenta V6.1.

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Western blot analyses. Functional leaves were collected at 7:00, 9:00, 11:00, 13:00,

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15:00 and 17:00 o’clock in a sunny day for protein extraction and western blot

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experiments.(30) Leaf proteins were detected by immuno-staining with

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anti-Rubisco-polyclonal antibody (AS07218), anti-OEC antibody (AS05092), anti-D1

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antibody (AS05084) and PEPC-polyclonal antibody (AS09458) from Agrisera (Vännäs,

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Sweden). Western blots were developed according to the method of NBT/BCIP kit

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(11681451001, Roche). The signals on the nitrocellulose were scanned immediately on

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Image Scanner III (GE healthcare, Wuxi, China) and the data were quantified with

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ChemImager 4400 software. All experiments were performed in triplicate.

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Establishment of protein-protein interaction network. All differential proteins 6


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identified from functional leaves and chloroplasts of three cassava species were used to

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generate the protein-protein interaction network via STRING, one of ELIXIR's Core Data

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Resources (www.string-db.org).(31)

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Protein-protein interaction detected with yeast two-hybrid (Y2H) assay. Yeast

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two-hybrid screening was performed according to the instructions of MatchmakerTM Gold

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Yeast Two-Hybrid System (Takara Bio, Inc., Beijing, China). PGADT7-FBA2 vectors,

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PGBKT7-RCA vectors were transfected into the yeast strains AH109 and Y2H Gold,

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respectively. Different constructs were cultured on a DDO (SD/-Leu/-Trp) plate for four

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days. The yeast cell transformed with AD-T and BD-Lam was served as a negative control,

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while the yeast cell transformed with AD-T and BD-53 was used as a positive control.

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Interaction screening was shown on QDO (SD/-Leu/-Trp/-His/-Ade) plates after five days.

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Bimolecular fluorescence complementation (BiFC) assays. The coding sequences of

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MeFBA2, MeRCA and MeCPN60B were cloned into serial pSAT6 vectors encoding either N-

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and C-terminal-enhanced with yellow fluorescence protein fragments. The resulting

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constructs were used for transient assays via polyethylene glycol transfection of tobacco

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leaves.(32) Transfected cells were imaged with a confocal spectral microscope (Fluo View

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FV1000, Olympus, Japan).

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Enzyme activity assay. The activities of phosphoglycerate kinase (PGK), ribulose

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bisphosphate carboxylase/oxygenase (Rubisco) and alcohol dehydrogenase (AOR) in leaves

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were assessed according to the manufacture’s protocol. All steps were carried out at 4 °C.

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The fully expanded leaves (0.1 g) were homogenized in 1 mL of reagent solution, centrifuged

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at different speeds. The supernatant was then used as the enzyme extract. All measurements

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were in biological triplicate.

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Results 7



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Morphological differences between cultivated and wild cassava species. SC205 was

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released from TCGRI to tropical and subtropical regions of China. W14 is regarded as the

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wild progenitor of cultivated cassava, while Glaziovii is regarded as the cassava related

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species. The Glaziovii and W14 plants were about 4.0-5.0 m tall, while SC205 was 2.0-2.5 m

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tall.(33) Shapes of central lobes of Glaziovii, W14 and SC205 were elliptic (Supplementary

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Fig. S1-A2), pandurate (Supplementary Fig. S1-B2) and linear (Supplementary Fig. S1-C2),

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respectively. Leaves of SC205, W14 and Glaziovii were 27.5 cm, 17.5 cm and 24.5 cm wide,

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respectively.

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Leaf anatomy between cultivar and wild species. Fig. 1 shows a leaf transverse section

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of SC205 (Fig. 1C) versus the wild species, W14 (Fig. 1B) and Glaziovii (Fig. 1A). The

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measurements of all leaf strata, including cuticle, epidermal, palisade parenchyma, and

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spongy mesophyll layers, showed significant differences among SC205, W14 and Glaziovii.

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SC205 and Glaziovii had a more distinctive bundle sheath with small and thin-walled cells.

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In W14, the vascular bundles were not distinctive and less uniform in shape than the other

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species. The palisade parenchyma layers in SC205 and Glaziovii were uniform and compact,

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but in W14 were loose and not uniform. In the three species, the vascular bundles occurred

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below the layers of elongated palisade cells. However, part of the bundle sheath was in

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contact with palisade cells, there was not a uniform layer of mesophyll cells around the

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bundle sheath as it is found in C4 plants. Additionally, the three cassava species examined

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had stomata on their abaxial surface.

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Photosynthetic activities in cassava cultivar and its wild relatives. Imaging-PAM

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studies were performed with the 3 month-old leaves of SC205, W14 and Glaziovii to

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understand the differences of cassava cultivar and wild species on the photosynthetic

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activities. The sensitive photosynthesis parameters Fv/Fm, PSII and NPQ/4 showed the

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changes of photosynthetic activities at different time points in photosystem II. Fig. 2 indicates 8


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significant differences among Glaziovii, W14 and SC205 on the efficiency of excitation

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energy capture by Fv/Fm, PSII and NPQ/4 (also Supplementary Table S1). The peak of

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PSII in three species was at 11:00 o’clock; and SC205 was 0.562, followed by Glaziovii

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and W14, 0.457, 0.332, respectively (Fig. 2A and B, Supplementary Table S1). The net

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photosynthetic rates were 19.93±0.82, 17.79±0.54, and 12.76±0.85 mmol CO2m-2s-1 in

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SC205, Glaziovii and W14, respectively (Table 1). The highest net photosynthetic rate was in

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SC205 (Table 1). These data imply that the photosynthesis activities of cassava cultivars were

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greater than those of wild species.

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Differential protein profiles between cassava cultivars and wild species. Proteome

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maps were compared with Delta 2D v4.0 software to identify variations in expression of

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individual proteins. Approximately 300 protein spots were analyzed (Supplementary Fig. S2,

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S3, and S4). In comparison with Glaziovii and W14, a total of 38 (greater abundance, 22

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spots; less abundance, 16 spots) and 70 spots (greater abundance, 39 spots; less abundance,

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31 spots) were detected in SC205 (Supplementary Fig. S5A and S5B, Supplementary Table

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S2), respectively. The spots with greater than  2.0-fold differences in intensity in all three

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replicate gels were found (Supplementary Table S4). Compared with Glaziovii, a total of 64

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differential protein spots (greater abundance, 30 spots; less abundance, 34 spots) were

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detected in W14 (Supplementary Fig. S5C, Supplementary Table S2).

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There were 3 replications for representative 2-D gel images of the whole chloroplast

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proteins extracted from Glaziovii, W14 and SC205 (Supplementary Fig. S6, S7, and S8,

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respectively). 2-D gel images of SC205, W14 and Glaziovii chloroplast proteins were

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wrapped (Supplementary Figure S9). In comparison with Glaziovii and W14, a total of 13

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(greater abundance, 8 spots; less abundance, 5 spots) and 23 spots (greater abundance, 17

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spots; less abundance, 6 spots) were detected in SC205 (Fig. S9A and S9B), of which 11

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spots and 20 spots were identified successfully (Supplementary Table S3), respectively. In 9



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comparison with Glaziovii, a total of 20 differential protein spots (greater abundance, 7 spot;

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less abundance, 13 spots) were detected in W14 (Supplementary Fig. S9C), of which 17 spots

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were successfully identified (Supplementary Table S3). The peptide sequences and spot

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volumes of differentially expressed proteins in triplicate were identified (Supplementary

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Table S5).

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Functional classification of identified proteins. Out of a total of 172 leaf protein spots

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extracted from 2-DE gels of three cassava species, 147 spots were successfully identified

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(Supplementary Table S4). Of these, a total of 34 different proteins (20 greater abundance

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and 14 less abundance proteins) were found in SC205 in comparison to Glaziovii, and

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annotated via the survey of phytozome 11. These proteins were involved in photosynthesis

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(14%), carbohydrate and energy metabolism (32%), detoxifying and antioxidant (9%),

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defense system (3%), protein biosynthesis (9%), chaperones (6%), HCN metabolism (12%),

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signal transduction mechanisms (3%) and DNA and RNA metabolism (3%); of which 4

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greater abundance proteins and 1 less abundance protein are related with the photosynthetic

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metabolism (Supplementary Fig. S10A). Compared to W14, a total of 60 differential proteins

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(37 greater abundance proteins and 23 less abundance proteins) were found in SC205. These

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proteins related with photosynthesis (28%), carbohydrate and energy metabolisms (35%)

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were among the largest group (63%), of these 13 greater abundance proteins and 4 less

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abundance proteins were involved with photosynthetic metabolism (Supplementary Fig.

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S10B). To further analyze the leaf characteristics of the two wild cassava species, we

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investigated their differentiation at the global protein-level. The result showed 53 differential

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proteins in W14 in comparison to Glaziovii (Supplementary Table S2), of which proteins

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were involved in 28% photosynthesis and 28% carbohydrate and energy metabolism. Four

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greater abundance proteins and 11 less abundance proteins were related with photosynthetic

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metabolism (Supplementary Fig. S10C). All fold changes of differentially expressed proteins 10


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in the leaves of three cassava species were noted (Supplementary Fig. S11A), indicating a

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closer genetic relationship of SC205 with Glaziovii than W14.

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Out of a total of 56 differential protein spots extracted from 2-DE gels of chloroplasts in

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three cassava species, 48 spots were successfully identified (Supplementary Table S5). Of

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these, a total of 11 different proteins (6 greater abundance and 5 less abundance proteins)

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were found in SC205 compared to Glaziovii, and annotated via the survey of phytozome 11.

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These proteins were associated with photosynthesis, carbohydrate and energy metabolism,

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protein biosynthesis and chaperones (6%); in which 3 greater abundance proteins and 1 less

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abundance protein were related with photosynthetic metabolism. Compared to W14, a total of

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20 differential proteins (15 greater abundance proteins and 5 less abundance proteins) were

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found in SC205 (Supplementary Table S3). Of these 7 greater abundance proteins were

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involved with photosynthetic metabolism (Supplementary Table S5). To further analyze the

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leaf characteristics of the two wild cassava species, the result showed 17 differential proteins

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in W14 compared to Glaziovii, of which 4 less abundance proteins were related with

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photosynthetic metabolism. All changes of differentially expressed proteins in the

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chloroplasts of three cassava species support that SC205 has a similar genetic relationship

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with Glaziovii (Supplementary Fig. S11B).

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Western blot validation of proteins. Images of Western blot for Glaziovii, W14 and

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SC205 were documented (Supplementary Fig. S12), of which Fig. 3 shows the results. SC205

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had the highest photosynthetic rate among the three species detected by IPAM. Key

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photosynthetic enzymes detected by Western blot included Rubisco large subunit, OEC, D1

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and PEPC. The results indicated that from 7:00 o’clock to 17:00 o’clock in the sunny day, the

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expression level of Rubisco large subunit (Fig. 3A), OEC (Fig. 3B) and D1 (Fig. 3C) were

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first increased and then decreased, and reaching the highest at 11 am. The expression was

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shown as SC205 > Glaziovii > W14. The data were consistent with photosynthetic activities 11



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described above. To further validate if the cultivated and wild cassava have characteristics of

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C4 plants, expression of PEPC at 11:00 o’clock was detected. The results showed that the

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PEPC expression was found in SC205, but was not found in Glaziovii and W14 (Fig. 3D).

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Construction and detection of protein-protein interaction network. All proteins

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identified in each genotype were used to construct a protein-protein interaction network via

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the STRING interface using the model plant Arabidopsis thaliana database. The network was

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constructed with 41 nodes and 210 edges (Fig. 4), while PGK1 (spot 76, phosphoglycerate

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kinase 1), RPE (spot 108, ribulose phosphate-3-epimerase), and RCA (spot 14, 15 and 75,

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ribulose bisphosphate carboxylase/oxygenase activase) were served as the hub proteins of

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cross-interaction propensity, with a degree of 29, 19, and 18 edges, respectively. In the

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network, the proteins associated with carbohydrate and energy metabolism, photosynthesis

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constituted the major nodes, followed by chaperones, protein synthesis, signal transduction,

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detoxifying and antioxidant etc. To confirm the reliability of the cross-interaction network

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between the differential proteins in the present study, the interaction relationships between

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proteins including RCA, CPN60B and FBA2 were detected with the yeast two-hybrid assay.

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As shown in Fig. 5A, RCA interacted with CPN60B as evidenced by the growth of

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transformed clones in selective SD (SD-Leu-Trp-His-Ade) medium. RCA interacted with

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FBA2, but the intensity was slightly weaker than that between RCA and CPN60B. However,

284

transformed clones with FBA2 and CPN60B were not grown in selective SD

285

(SD-Leu-Trp-His-Ade) medium, which demonstrated that CPN60B was not physically

286

interacted with FBA2. To further confirm the occurrence of these interactions in planta, a

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BiFC assay involving transient expression in tobacco leaves were performed (Fig. 5B).

288

Significant co-expression of the three combinations in chloroplasts of tobacco leaves

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suggested interactions among RCA, CPN60B and FBA2. The yeast hybridization data and

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BiFC agreed with the theoretical estimate from STRING online database. 12


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Discussion Proteomics methods were used to compare proteome patterns between cassava fibrous

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and storage roots (28), the proteome characteristics of cassava somatic embryos, plantlets and

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storage roots (28,30), and the protein changes during physiological deterioration of cassava

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storage root (35). However, the linkage of proteomic patterns and photosynthetic difference

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between cultivated cassava and wild species was poorly understood. The present study

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focused on the differentiation of leaf and chloroplast proteomic patterns from cassava

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cultivars and its wild species, and the linkage between proteome patterns and photosynthetic

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activities.

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Correlation between proteomic pattern changes among the three species and leaf

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photosynthetic differences. Li et al. (30) identified 110 proteins from plantlet leaves of SC8,

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of these the proteins involved in photosynthesis were among the largest group (21.8%) and

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Rubisco accounted for about 50% of the total protein content in leaves. Oxygen-evolving

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enhancer protein 1 (OEE1) may be synthesized in shoots and then transported to roots (30).

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In the present study, we observed the obvious changes in leaf proteomic patterns of SC205,

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Glaziovii and W14. The data implied that photosynthetic activity of SC205 is similar to that

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of Glaziovii, and obviously different from W14. The differential leaf proteomic data showed

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the photosynthetic rate in an order of SC205 > Glaziovii > W14. The data indicated that the

310

effects of leaf proteomic pattern changes on the photosynthetic activities significantly differ

311

between cassava cultivated and wild species. The results of photosynthetic activities using the

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Imaging-PAM method and LCPro-SD for the three species agreed with the changes of

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proteomic patterns (Fig. 2, Table 1 and Supplementary Table S1). The results of chlorophyll

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content measured at 11:00 o’clock in the sunny day for the three species also supported the

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conclusion (Supplementary Table S6). 13



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The maximum leaf net photosynthetic rates in cassava were between 40 and 50 μmol

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CO2 m-2s-1, grown under favorable field conditions, with high solar radiation (>1800 μmol

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m−2s−1 in the range of photosynthetically active radiation) and an optimum leaf temperature

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around 30-35 C. These high maximum photosynthetic rates may explain the observed high

320

productivity in cassava.(10) D1 protein is a key component of Photosystem II (36). The

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Western blot results showed that the expressions of Rubisco, OEC and D1 in SC205 were the

322

highest among the three species, followed by Glaziovii and W14 (Fig. 3A-C). These data

323

implied a close relationship between photosynthetic rates of three species and the expression

324

profiles of Rubisco, OEC and D1, confirming the photosynthetic activities measured by

325

Imaging-PAM (Fig. 2A).

326

Regulation of photosynthesis processing by RCA is directly linked with 14 proteins

327

detected (Fig. 4). The effects of the cross-interaction between the protein networks on the

328

regulation of photosynthetic pathways apparently result in the differentiation of

329

photosynthetic rates in different cassava species, and plausibly explain the mechanism of

330

high photosynthetic rates in SC205. Compared to Glaziovii and W14, 11 greater abundance

331

proteins and 3 less abundance proteins were detected in SC205, which are associated with the

332

photosynthetic acceleration in protein network. These proteomic profiles could be used in

333

cassava molecular breeding to enhance photosynthetic efficiency.

334

A model explaining photosynthetic networks in SC205 was proposed on the basis of the

335

abovementioned data and information in the literature (Fig. 6). It is well known that the

336

CO2-fixing enzyme Rubisco plays a crucial biological role as a primary determinant of plant

337

yield.(37,38) However, Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that

338

reactivates Rubisco by remodeling the conformation of inhibitor-bound sites.(39) RCA is

339

regulated by the ratio of ADP : ATP.(39) Within a certain range, the more ATP is produced,

340

the higher the activity of RCA becomes. In plants, ATP is produced through many pathways, 14


Page 14 of 32

Page 15 of 32


341

such as TCA cycle, glycolysis, and pentose phosphate pathway. PGK is crucial for glycolysis

342

and is a highly conserved enzyme in plants.(40) It links TCA cycle and pentose phosphate

343

pathway, and interacts with RCA. In Figure 6, PGK interacts with AOR and FBA2 which are

344

involved in TCA cycle as well as RPE associated with pentose phosphate pathway. AOR can

345

have a direct regulation to ATP, and the greater abundance of AOR in SC205 may result in

346

the higher accumulation of ATP. ATP could directly regulate Rubisco activity. These

347

examples of protein cross-interaction are proposed to promote photosynthesis in the cassava

348

cultivar relative to its wild species. The activities of PGK, Rubisco and AOR varied

349

significantly among SC205, W14 and Glaziovii (Table 2). Rubisco activity in SC205 leaves

350

was 9.1 times as W14 and 5.5 times as Glaziovii. PGK activity in SC205 leaves was 2.1

351

times as W14 and 1.7 times as Glaziovii. The highest activities of AOR, PGK and RCA in

352

SC205 leaves may give rise to high photosynthesis.

353

Association of proteome pattern changes with cassava domestication syndrome in wild

354

species. Wild species are still untapped resources for cassava genetic enhancement. Wild

355

cassava possess useful genes of which the incorporation into the cultigen would enrich its

356

gene pool with useful characters.(2) It would be of interest to know whether ancestors of

357

cultivated cassava possess C4 features, if so, it would suggest that this characteristic has been

358

selected out during the domestication, and that the potential for reintroduction of C4-like

359

feature exists.(9) Data on photosynthetic rates per unit of leaf area and the photosynthetic

360

response of cassava to CO2 are also consistent with C3 photosynthesis.(21) However, cassava

361

has several other characteristics typical of C4. For instance, its photosynthesis does not

362

saturate at a high irradiance condition. The photosynthetic rate is very high and

363

photosynthesis has a broad temperature optimum range between 20 and 45 C. Cassava

364

leaves also have distinct green bundle sheath cells, suggesting that cultivated cassava may be

365

derived from wild species possessing C4 photosynthesis.(20) On the contrary, Calatayud et al. 15



366

(9) reported that W14 exhibited the traits of typical C3 photosynthesis, suggesting that

367

cultivated cassava was not derived from wild C4 species. El-Sharkawy and Cock (41) showed

368

that cassava does not have the typical ‘C4-Kranz’ anatomy. It has a distinctive

369

chlorenchymatous vascular bundle sheath located below a single layer of palisade cells.

370

Unlike C3-C4 intermediates and C4 species, the bundle sheaths of cassava are not surrounded

371

by mesophyll cells. The bundle sheath cells that occur at a high frequency in cassava may

372

function in both photosynthesis and transportation of photosynthates in the leaf.(21) Leaf

373

anatomic structures of SC205, Glaziovii and W14 in the present study supported the opinion

374

provided (21, 41). However, it is possible that cassava and its wild relatives are evolving

375

biochemically towards C4 photosynthesis and may represent photosynthetic characteristics

376

intermediate between C3 and C4 species.(10) Hybridization between Manihot species occurs

377

naturally (42, 43) and could give rise to variation and diversity in different characters, either

378

of leaf photosynthesis. Moreover, knowledge of the anatomy of the cultivated plant in

379

comparison to the wild species may contribute to information on the domestication (44).

380

Cassava leaves are rich in proteins, vitamin A and flavonoids, it can probably be used as

381

functional food component like maize pollen (45).

382

The photosynthesis performance observed in the present study, together with the

383

expression of C3 photosynthetic enzymes including Rubisco, OEC and D1 (Fig. 3A-C),

384

indicates that SC205 and wild species are likely to be a C3 species. The anatomic structures

385

of the three species (Fig. 1) also confirm this opinion. A key enzyme of C4 plant, PEPC

386

detected by Western blot shows that the expression of PEPC is found in SC205, but not in the

387

wild species (Fig. 3D). However, Calatayud et al. (9) reported that PEPC activities in wild

388

cassava range from 1.5 to 5.5 μmol per mg chlorophyll per minute, compared to 6-12 μmol

389

per mg in sorghum (a C4 plant), and 0.2-0.4 μmol per mg in beans (a C3 plant), indicating

390

SC205 may have the characteristics typical of C4 more than those of Glaziovii and W14. It is 16


Page 16 of 32

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391

reported that W14 is the potential wild progenitor of M. esculenta.(23) In the present study,

392

34 and 60 differential leaf proteins in SC205 relative to Glaziovii and W14 (Supplementary

393

Table S2), respectively, imply a closer genetic relationship of SC205 with Glaziovii than

394

W14, suggesting cassava domestication syndrome in wild species. Such opinion is also

395

supported with the proteomic pattern changes in chloroplasts detected by the pairwise

396

comparison of SC205/Glaziovii and SC205/W14. Additionally, the comparative analysis of

397

anatomy structure and photosynthetic activities of the three species were consistent with the

398

above results.

399 400

Acknowledgements

401

The study was financially supported in part by the National Natural Science Foundation of

402

China (NSFC) Projects (No. 31601361 and No. 31371684), NSFC-CGIAR International

403

(Regional) Cooperation and Exchange Programs (31361140366), the Initial Fund of

404

High-level Creative Talents in Hainan Province, and TCGRI, CATAS Fundamental Research

405

projects (1630032015014). We acknowledge Shixin Zhang and Fei Qiao for their technical

406

assistance, and Ruili Xu for the collection and maintenance of the cassava clones. AF was a

407

China Scholarship Council scholarship recipient. The funding agencies had no role in the

408

study design, data collection and analysis, decision to publish, or preparation of the

409

manuscript.

17



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43. Nassar, N. (2001) Cassava, Manihot esculenta Crantz and wild relatives: Their relationships and evolution. Genet. Resour. Crop Ev. 00: 1–0 44. Bomfim, N. N., Graciano-Ribeiro, D., and Nassar, N. M. A. (2011) Genetic diversity of root anatomy in wild and cultivated Manihot species. Genet. Mol. Res. 10, 544–551

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45. Zhang, Z. Liang, Z. Yin, L. Li, Q. X. and Wu, Z. (2018) Distribution of four bioactive flavonoids in

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Chem. 66 (40), 1172-1178

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522

Figure Legends

523 524

Fig. 1. Photomicrographs of leaf transverse sections of Glaziovii (A), W14 (B) and

525

SC205 (C). Scale bar = 40 μm.

526 527

Fig. 2. Imaging pulse amplitude modulation (IPAM) of cassava leaves from Glaziovii,

528

W14 and SC205. A, The changes of PSII (effective PSII quantum yield) from 7:00 to

529

14:00 in Glaziovii, W14 and SC205. B, IPAM of Glaziovii, W14 and SC205 at 11:00am;

530

parameters shown were Fv/Fm [maximal photosystem II (PSII) quantum yield], ΦPSII (at

531

185 E m-2s-1), and NPQ/4 (non-photochemical quenching) (at 185 E m-2s-1). The color

532

gradient provided a scale from 0 to 100% for assessing the magnitude of the parameters.

533 534

Fig. 3. Expression of Rubisco (A), OEC (B), D1 (C) and PEPC (D) in leaves of cassava

535

Glaziovii, W14 and SC205 from 7:00 to 17:00 o’clock detected by Western blot. Their

536

expressed intensities were analyzed with ChemImager 4400 software.

537 538

Fig. 4. Protein-protein interaction network generated from online String software using

539

41 differential proteins. White arrows indicate less abundance proteins, and black arrows

540

indicated greater abundance proteins. The different color circle indicates different proteins.

541

The connection between two circles means protein-protein interaction. The heavy line

542

indicates the strong protein-protein interaction.

543 544

Fig. 5. Interactions between RCA, CPN60B and FBA2. A, Interaction of RCA, CPN60B

545

and FBA2 in the yeast cells. Yeast clones transformed with different constructs grown on

546

QDO for 5 days, yeast dilution fold was 10 and 100; B, BiFC analysis of the interaction of 22


Page 22 of 32

Page 23 of 32


547

RCA, CPN60B and FBA2. The bright field, YFP fluorescence (green), chlorophyll

548

autofluoresence (red) and combined images were visualized under a confocal microscope 16

549

h after transfection. Bars, 20m.

550 551

Fig. 6. A proposed model for photosynthesis networks in SC205 relative to its wild

552

species. The differential proteins identified in the present study were used to construct the

553

schematic representation. The interactions were based on the KEGG pathway database. Small

554

boxes with different colors displayed the fold changes in pairwise comparison of SC205/W14

555

(left) and SC205/Glaziovii (right).

23



Page 24 of 32

Table 1. Photosynthetic rate of cassava cultivated SC205, wild species Glaziovii and W14. Values were means  SE. Different letters in the same row indicated statistically significant differences according to Duncan test (P

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