Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis

<div><p>The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L^-1) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L^-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus.</p></div

Unique proteins that were identified in embryogenic (E) or non-embryogenic (NE) sugarcane callus under maturation treatment.

<p>Confidence scores were calculated by ProteinLynx Global Server (PLGS).</p><p>Unique proteins that were identified in embryogenic (E) or non-embryogenic (NE) sugarcane callus under maturation treatment.</p

Pie charts showing the functional classification of the co-expressed proteins.

<p>Functional classification of the co-expressed proteins from embryogenic (E) and non-embryogenic (NE) callus before (0) and after 21 days of maturation treatment (E-0, E-21, NE-0 and NE-21).</p

Venn diagram and pie charts displaying the numbers and functions of unique and co-expressed proteins.

<p>The number of unique and co-expressed proteins from embryogenic(E) and non-embryogenic (NE) callus during maturation treatment and the functional classification of unique proteins from embryogenic (E) and non-embryogenic (NE) callus at 0 and 21 days of maturation treatment (E-0, E-21, NE-0 and NE-21).</p

Influence of heme from meal on the α-glucosidase expression (A) and activity (B) in the <i>R. prolixus</i> midgut.

<p>P – control insects fed on plasma; P + H – insects fed on hemin-enriched plasma (500 μM). Insects were fed on rabbit plasma with and without hemin. Four days after feeding, midguts (n = 20) were dissected in cold 100 mM NaCl. The α-glucosidase activity was determined by measuring the release of ρ-nitrophenolate from ρ-nitrophenyl α-D-glucopyranoside. Results shown are representative of three independent experiments run in triplicate. Plasma plus hemin is significantly different from plasma *(<i>P</i><0.05).</p

Analysis of α-glucosidase genes using Blastx.

<p>Analysis of α-glucosidase genes using Blastx.</p

Hz formation activity in the presence or absence of maltose <i>in vitro</i>.

<p>Ctl - hemin; Mal - Maltose; CF - chromatographic fraction; CF + Mal → H - chromatographic fraction + maltose and hemin added 4 h after starting assay; CF + H → Mal - chromatographic fraction + hemin and maltose added 4 h after starting assay. The assays of Hz formation were carried out for 24 h at 28°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006966#s4" target="_blank">materials and methods</a>. Hz formation activity was expressed as nmol of aggregated heme, during 24 h, for 8 µg protein. The results are the mean and standard deviation of one experiment run in triplicate. The experiment where maltose was added before hemin was significantly different from that with protein alone or that with hemin being added first *(<i>P</i><0.05).</p

Physiological effects of dsRNA-mediated silencing of α-glucosidase.

<p>The insects were injected with 2 µL of 100 mM PBS pH 7.4 or dsLacZ (controls) and dsα-glu (2 or 10 µg/female); mortality and oviposition were monitored 4 days after feeding. In all panels, results are means ±SEM (n = 70). The insects injected with 10 µg dsα-glu, analyzed 4 days feeding, were significantly different from control insects injected with both PBS and dsLacZ and also analyzed 4 days after feeding ^*(<i>P</i><0.05).</p>a<p>Access number to GenBank database.</p>b<p>Function expected according the Blastx sequence results similarity in GenBank database.</p>c<p>Protein Data Bank.</p

Analysis of the α-glucosidase sequence.

<p>A. Alignment of amino acid sequences of α-glucosidase from <i>R. prolixus</i> (AgluRp), <i>Culex quinquefasciatus</i> (AGluCulex, AGluCulex2, AGluCulex3), α-amylase from <i>Aedes aegypti</i> (AmAeds), maltase-like Agm2 from <i>Anopheles gambiae</i> (MaltaseAgm2), maltase 2 from <i>Drosophila virilis</i> (Maltase2Dv), α-Glucosidase from Gsj (AgluGsj), Oligo-1,6-Glucosidase from <i>Bacillus cereus</i> (GluOligo) and α-glucosidase from <i>Saccharomyces cerevisiae</i> (AgluSc). Identical residues are indicated by “*”; conserved and semiconserved residues are indicated by “:” and “.”, respectively. Residues of aspartic acid and histidine present in AGluRp and also present in AGluSc are marked in gray. The secondary structure prediction using the JPred server is represented in red (α-helices), green (β-sheets) and blue (loops). B. Partial nucleotide sequence of the <i>R. prolixus</i> α-glucosidase cDNA and its deduced amino acid sequence. The amino acid sequences used for the design of specific Real Time-PCR primers are underlined.</p

Hz formation and α-glucosidase activities in the presence or absence of inhibitors <i>in vitro</i>.

<p>A. C. and D. Hz formation. B. α-glucosidase activity. Ctl - hemin; PE - protein extract of midgut epithelium; PE + Eri - protein extract + erythritol; PE + DEPC - protein extract + diethypyrocarbonate; PE + Cs - protein extract + castanospermine; PE + AB - protein extract + anti <i>D. peruvianus</i> α-glucosidase antibody; Ctl + AB - hemin + antibody; Ctl + Cs - hemin + castanospermine; Ctl + DEPC - hemin + diethypyrocarbonate; PE → AB - protein extract + antibody 10 hours after starting assay; PE → DEPC - protein extract + diethypyrocarbonate 10 hours after starting assay; PE 100°C - protein extract boiled for 10 min before starting assay; PE → 100°C - protein extract boiled 10 hours after starting assay. The assays of Hz formation were carried out for 24 h at 28°C as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006966#s4" target="_blank">materials and methods</a>. Hz formation activity was expressed as nmol heme aggregated in 24 h for 15 µg protein extract. The assays of α-glucosidase activity were determined using a colorimetric method. Unless otherwise indicated, activity was expressed as nmol <i>ρ</i>-nitrofenolate released in 1 min. Results shown are means ±SEM (n = 4) of two experiments run in triplicate. The experiments with inhibitors were significantly different from protein extract alone *(<i>P</i><0.05).</p