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, 9 (4), e85991
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Comparison of Leaf Proteomes of Cassava (Manihot Esculenta Crantz) Cultivar NZ199 Diploid and Autotetraploid Genotypes

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Comparison of Leaf Proteomes of Cassava (Manihot Esculenta Crantz) Cultivar NZ199 Diploid and Autotetraploid Genotypes

Feifei An et al. PLoS One.

Abstract

Cassava polyploid breeding has drastically improved our knowledge on increasing root yield and its significant tolerance to stresses. In polyploid cassava plants, increases in DNA content highly affect cell volumes and anatomical structures. However, the mechanism of this effect is poorly understood. The purpose of the present study was to compare and validate the changes between cassava cultivar NZ199 diploid and autotetraploid at proteomic levels. The results showed that leaf proteome of cassava cultivar NZ199 diploid was clearly differentiated from its autotetraploid genotype using 2-DE combined MS technique. Sixty-five differential protein spots were seen in 2-DE image of autotetraploid genotype in comparison with that of diploid. Fifty-two proteins were identified by MALDI-TOF-MS/MS, of which 47 were up-regulated and 5 were down-regulated in autotetraploid genotype compared with diploid genotype. The classified functions of 32 up-regulated proteins were associated with photosynthesis, defense system, hydrocyanic acid (HCN) metabolism, protein biosynthesis, chaperones, amino acid metabolism and signal transduction. The remarkable variation in photosynthetic activity, HCN content and resistance to salt stress between diploid and autotetraploid genotypes is closely linked with expression levels of proteomic profiles. The analysis of protein interaction networks indicated there are direct interactions between the 15 up-regulation proteins involved in the pathways described above. This work provides an insight into understanding the protein regulation mechanism of cassava polyploid genotype, and gives a clue to improve cassava polyploidy breeding in increasing photosynthesis and resistance efficiencies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Standard chromosome counting and flow cytometric analysis of different ploidy levels in cassava cultivar NZ199 leaves.
A, in vitro plantlets of cassava diploid genotype; B, in vitro plantlets of cassava autotetraploid genotype; C, leaf of cassava diploid genotype; D, leaf of cassava autotetraploid genotype; E, chromosome number of diploid genotype (2n = 2x = 36); F, chromosome number of autotetraploid genotype (4n = 4x = 72); G, a fluorescence peak of diploid nuclei located at channel position 200; H, a fluorescence peak of autotetraploid nuclei located at channel position 400.
Figure 2
Figure 2. 2-D gel protein profiles of leaves from cassava NZ199 diploid (A) and autotetraploid genotypes (B) and wrapped 2-DE map from diploid and autotetraploid genotypes (C).
The white and black arrows in pane C indicated proteins that showed detectable changes (>2.0-fold of the normalized volume) in abundance compared with those observed in the control; white indicated a down-regulated match, and black indicated an up-regulated match. Small boxes indicated the gel regions to be amplified to highlight clearly detectable spots in Fig. 3.
Figure 3
Figure 3. Amplification of small boxes from Fig. 2C to highlight detectable spots that represent differentially abundant expression.
In I, II, and III: a, diploid genotype, b; autotetraploid genotype. White arrow indicated a down-regulated match, and black indicated an up-regulated match. The numbers correspond to the 2-DE gel in Fig. 2.
Figure 4
Figure 4. Functional categories of 52 differential proteins identified in cassava NZ199 autotetraploid leaves compared withdiploidgenotypes.
Number of spots altered in the expression in the leaves of cassava autotetraploid genotype. Unknown proteins included those whose functions had not been described.
Figure 5
Figure 5. Western blotting of Rubisco, APX and PrxQ.
The expression of Rubisco, APX and PrxQ in leaves of cassava NZ199 diploid (a) and autotetraploid (b) genotypes were detected by western blotting using antiRubisco-polyclonal antibody (AS07218), anti-APX antibody (AS08368) and anti-PrxQ antibody (AS05093) from Agrisera, respectively.
Figure 6
Figure 6. Imaging pulse amplitude modulation of cassava leaves from NZ199 diploid and autotetraploid genotypes.
A, diploid genotype; B, autotetraploid genotype; Parameters shown are Fv/Fm [maximal photosystem II (PSII) quantum yield], ΦPSII (effective PSII quantum yield) (at 185 µE m−2 s−1), and NPQ/4 (nonphotochemical quenching) (at 185 µE m−2 s−1). The color gradient provides a scale from 0 to 100% for assessing the magnitude of the parameters.
Figure 7
Figure 7. Effects of salt stress on the growth of cassava NZ199 diploid and autotetraploid genotypes.
Cassava NZ199 diploid and autotetraploid genotypes were grown at MS medium with 0.03/L NAA used as control, and salt-stressed medium contained MS medium with 0.03 mg/L and 50 mM sodium chloride. A1, C1 and E1 (roots), diploid control; A2, C2 and E2 (roots), salt-stressed diploid plantlets; B1, D1 and F1 (roots), autotetraploid control; B2, D2 and F2 (roots), salt-stressed autotetraploid plantlets.
Figure 8
Figure 8. Chromatograms of cyanogenic glucoside of cassava cultivar leaves from NZ199 diploid and autotetraploid genotypes.
I, HCN standard sample (0.5 ppm); II, NZ199 diploid genotypes; III, NZ199 autotetraploid genotype; IV, Extraction yield of cyanogenic glucoside from diploid and autotetraploid genotypes. Chromatographic conditions were: Kromasil 100-5C18 column (250×4.6 mm, 5 µm), gradient elution with aqueous acetonitrile, flow rate of 0.8 ml/min, UV detection at 215 nm, and column temperature at 30°C.
Figure 9
Figure 9. Biological networks generated for combination of twelve differential proteins.
Fifteen differentially up-regulated proteins including ATP synthase subunit beta, alcohol dehydrogenase, beta-glucosidase, phosphoglycerate kinase, triose phosphate isomerase, RCA, Rubisco, APX2, CDSP3, peroxiredoxin, thioredoxin translation elongation factor, glutamate-ammonia ligase, chaperone and 14-3-3 in cassava autotetraploid genotypes were used to generate a protein-protein interaction network through Pathway Studio analysis. Regulation is marked as an arrow with R, Chemical Reaction as an arrow with C and Binding as an arrow without any marks. The entity table, relation table and reference table data were presented in in Tables S1, S2, S3.

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Grant support

This work was supported by a Major Project of Chinese National Programs for Fundamental Research and Development Grants (2010CB126606), National Scientific and Technological Programs in Rural Fields (2012AA101204-2), the Earmarked Fund for Modern Agro-industry Technology Research System (nycytx-17) and the Initial Fund of High-level Creative Talents in Hainan Province. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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