Differential Proteome Analysis of Mature and Germinated Embryos of Araucaria Angustifolia ⇑ Tiago S

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Phytochemistry 72 (2011) 302–311

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Phytochemistry

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Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia ⇑ Tiago S. Balbuena a,d, , Leonardo Jo a, Fernanda P. Pieruzzi a, Leonardo L.C. Dias a, Vanildo Silveira b, Claudete Santa-Catarina b, Magno Junqueira c, Jay J. Thelen d, Andrej Shevchenko c, Eny I.S. Floh a

a Department of Botany, Institute of Biosciences, University of Sao Paulo, 05422-970 Sao Paulo, SP, Brazil b Biotechnology and Biosciences Center, North Fluminense State University, 28013-602 Campos dos Goytacazes, RJ, Brazil c Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, SN, Germany d Department of Biochemistry and Interdisciplinary Plant Group, University of Missouri, 65211 Columbia, MO, USA

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Article history: Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to Received 10 April 2010 identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, Received in revised form 30 July 2010 using one and two dimensional gel electrophoresis approaches followed by protein identiﬁcation by tan- Accepted 6 December 2010 dem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional Available online 27 January 2011 gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, Keywords: resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identiﬁed pro- Araucaria angustifolia teins were mainly related to energetic metabolism pathways, translational processes, oxidative stress Araucariaceae Conifer regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive pro- Mass spectrometry tein expression overview of changes in germinated embryos in relation to matures, providing insights Plant proteomics into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring Recalcitrant seed and control of in vitro somatic embryos were discussed. Seed germination Published by Elsevier Ltd. Open access under the Elsevier OA license.

1. Introduction million hectares; however, due to predatory exploration between 1930 and 1990, the natural resources were reduced to 1–2% of In most flowering plants, zygotic embryos suffer a desiccation the original area (Guerra et al., 2000). Many efforts have been car- process at the maturation phase, followed by a reduced metabolic ried out in order to propagate and conserve this species. However, state prior to germination (Bewley and Black, 1994). Also, some conventional strategies are hampered mostly due to the recalci- seeds present dormancy to avoid early plantlet development if trant nature of the seeds (Panza et al., 2002; Salmen-Espindola not stimulated by environmental signals, allowing plants to restrict et al., 1994). In vitro somatic embryo propagation has only resulted timing of their establishment to certain seasons, rainfall or to the in mature somatic embryos unable to germinate (Guerra et al., removal of vegetation cover (Penfield and King, 2009). However, 2002; dos Santos et al., 2002). Knowledge of the underlying bio- in nature, there are plants in which embryos perish under desicca- chemistry and metabolic status before and after the germination tion and, thus, do not stand long storage periods as they are shed at process could be important for the development and optimization high moisture content (Berjak and Pammenter, 2008). Although of strategies for large scale propagation and germplasm conserva- different authors suggest the use of an intermediary nomenclature, tion in this species, such as in vitro somatic embryogenesis and historically, seeds containing embryos that tolerate desiccation are cryopreservation, respectively. called orthodox seeds and those which do not tolerate desiccation Proteomics is a global analysis strategy that provides informa- are recalcitrants (Roberts, 1973). tion of a multitude of processes in complex events, such as germi- Araucaria angustifolia is a Brazilian native conifer tree, classified nation (Gallardo et al., 2001). Despite the increasing interest in as a recalcitrant seed species. Originally, it covered an area of 20 plant proteomics, woody plant seed germination studies are still very limited. To date, the only attempts to understand the prote- ⇑ ome changes during the germination of woody plants were carried Corresponding author at: Life Sciences Center, Department of Biochemistry, out in Fagus sylvatica (Pawlowski, 2007), Prunus campanulata (Lee University of Missouri, 65211 Columbia, MO, USA. E-mail addresses: [email protected], [email protected] (T.S. et al., 2006), Jatropha curcas (Yang et al., 2009) and Phoenix Balbuena). dactylifera (Sghaier-Hammami et al., 2009a,b). Changes in the pro-

0031-9422 Published by Elsevier Ltd. Open access under the Elsevier OA license. doi:10.1016/j.phytochem.2010.12.007 T.S. Balbuena et al. / Phytochemistry 72 (2011) 302–311 303 teome profile during embryogenesis and maturation of somatic during germination and early seedling development. Vicilin-like embryos of woody species have also been studied (Marsoni et al., proteins are abundant storage proteins in A. angustifolia seeds, in 2008; Pan et al., 2009; Sghaier-Hammami et al., 2010). However, which accumulation occurs parallel to cotyledon differentiation to our knowledge, there are no comparative analyses of protein (Balbuena et al., 2009; Silveira et al., 2008). These proteins may changes on zygotic or somatic embryos of a recalcitrant seed spe- also have a protective role against insect predation (Sales et al., cies before and after germination. 2001). Cleavage of the N terminus of this protein also led to pro- We are interested in determining the mechanisms that are in- duction of antimicrobial peptide fragments, as reported for volved in seed development and germination of A. angustifolia to Macadamia integrifolia and Theobroma cacao (Marcus et al., 2008). better understand the molecular and physiological basis of embryo However, the protective role of the vicilin-like storage proteins, differentiation and development in this species. Identification of conferring protection to A. angustifolia embryo during germination, metabolic changes during these processes may also be utilized still needs to be explored. for the establishment of a correct quality control, through monitor- Vicilin-like proteins were detected in 10 different specific spots ing of stage specific markers, and maximal plant conversion rates in mature embryo gels, indicating the existence of protein isoforms from somatic embryos. To this end, we previously evaluated the and an active storage protein catabolism. Exclusive protein spots composition of amino acids, proteins and plant hormones in zygo- with this same accession were also detected in germinated embryo tic and somatic embryos (Astarita et al., 2003a,b; dos Santos et al., gels. During germination, storage proteins are degraded to provide 2006, 2008a,b; Silveira et al., 2006, 2008; Steiner et al., 2007). In nutrients and amino acids for de novo synthesis of proteins, a fun- the present study, we aimed the identification of differentially ex- damental process in this event (Rajjou et al., 2004). The fact that pressed proteins between mature and germinated embryos of A. exclusive vicilin-like protein spots were detected in mature and angustifolia, using one and two dimensional gel electrophoresis ap- germinated embryo gels indicates that they probably correspond proaches followed by protein identification by tandem mass to protein fragments or possibly modifications leading to degrada- spectrometry. tion products, with different two dimensional gel migration patterns. 2. Results and discussion Carbamoyl phosphate synthase (spot 03) was specifically detected in mature embryo gels. This enzyme is responsible for bio- Seed germination is a complex process comprising events from synthesis of carbamoyl phosphate, which is utilized in the first seed imbibition to growth. Morphologically, initiation of growth cor- step of two biosynthetic pathways, one leading to pyrimidines responds to radicle emergence; subsequent growth is generally de- and the other to arginine (Kolloffel and Verkerk, 1982). The two fined as seedling growth (Rajjou et al., 2004). A. angustifolia most abundant amino acids in the vicilin-like proteins are the glu- germination, under the conditions used here, ended after 8 days of tamic acid and arginine, comprising 10.7% and 10.5% of its primary sowing when in more than 50% of the seeds the radicle had broken sequence, respectively. A. angustifolia mature embryo axis and down the seed coat marking the end of germination and start of cotyledons accumulate 1795 and 1576 nmol/g, respectively, of seedling growth phase (Fig. 1). Moisture content presented no signif- arginine. This being the most abundant pool of free amino acids icant changes during the germination process (see Supplementary after aspartic and glutamic acids (Astarita et al., 2003a). The fact Fig. 1S), corroborating the recalcitrant nature of the species. To en- that the carbamoyl phosphate synthase spot was only identified sure a good overview of the metabolic changes that may be present in mature embryo gels, suggests that the arginine amino acid flux during this very short time frame of germination and identify poten- to protein biosynthesis should continue up to the end of the zygo- tial candidates for in vitro germination screening, two different pro- tic embryogenesis, when embryos reach the mature stage, but not teomic approaches were conducted. persist until the end of germination. Besides proteins, A. angustifolia seeds accumulate starch as a 2.1. Two dimensional gel electrophoresis and specifically enriched main storage compound (Panza et al., 2002). Polyglucans, like spots identification starch, are main repositories of carbon and energy reserves for many organisms, being the starch phosphorylase, an analog of In this approach, specifically expressed protein spots in mature the a-glucan phosphorylase, responsible for reversible conversion and germinated embryo gels were identified using stringent and of a-1,4-glucan and inorganic phosphate into glucose-1-phos- sequence similarity database searches. Eighteen and 14 proteins phate. Spot 04 was specifically detected in mature embryo gels were confidently identified in mature and germinated embryo gels, and corresponds to the plastidic form of the enzyme starch phos- respectively (Fig. 2). From the 32 identified spots, 14 corresponded phorylase. Although this enzyme synthesizes and degrades starch, to a vicilin-like protein. Although seeds of A. angustifolia store the important role of the plastidic starch phosphorylase has been starch as the main carbon source for plantlet growth (Panza described to be in starch degradation, primarily due to the low et al., 2002), proteins play an important role as storage metabolites physiological glucose-1-phosphate concentration and high

Fig. 1. Morphology of mature (A) and germinated (B) A. angustifolia embryos at 0 and 8 days after sowing. Seed coat was removed and megagametophytes (m) can be observed surrounding intact embryos (e), containing cotyledons and embryo axis. 304 T.S. Balbuena et al. / Phytochemistry 72 (2011) 302–311

pH pH 3 10 3 10 Mr (kDa) 6 4 206 3 A 202 B 97

201 199 66 151 135136 171 145 136 45 126 122

93 30 72 11 9 61 78 60 70 56 57 55

47 48 20

25 14

Fig. 2. Master 2-DE gels for mature (A) and germinated (B) embryos of Araucaria angustifolia (pH 3–10 linear gradient). In silico master gels only contain protein spots presented in all three biological repetitions. Proteins that were identiﬁed are numbered as speciﬁed in Table 1.

inorganic phosphate/glucose-1-phosphate ratio found in vivo (Rat- translocation of substrates by pulling the substrate out of the tran- hore et al., 2009). Identification of this enzyme in mature embryo slocon in an ATP-dependent manner (Kovacheva et al., 2007; gels corroborates the hypothesis of early storage mobilization. On Rutschow et al., 2008). The C1pC peptidase is part of the complex the other hand, as no direct in vivo evidence is available yet to sup- C1p plant plastid proteolytic system. In Escherichia coli, C1p prote- port phosphorolytic degradation of starch, the role of this enzyme ase machinery can be divided into two main biochemical compo- in the biosynthesis of A. angustifolia starch cannot be discarded, nents, a proteolytic core complex formed by two serine-type especially due to its phosphorylation capacity, and consequently peptidases and a hexameric ring formed by the C1pA or C1pX that possible changes in two dimensional spot migration patterns be- interact with the core complex and ‘‘feed’’ it with substrates (Adam tween phosphorylated and non-phosphorylated isoforms. et al., 2006). Although the plastid proteolytic system is much more The most abundant features in germinated embryo gels were complex, including 15 different proteins, C1pC acts as the C1pA in those related with protein degradation processes (Table 1). Spots E. coli (Adam et al., 2006) ‘‘feeding’’ the system with substrates, 122 and 151 were identified as proteins involved in the 26S protea- such as storage proteins, for degradation. some system, the dominant proteolytic pathway in plants (Smalle and Vierstra, 2004). Components of this pathway comprise approx- 2.2. One dimensional gel electrophoresis and detection of mature and imately 5% of Arabidopsis thaliana proteome (Vierstra, 2003) and germinated enriched proteins are mostly responsible for the protein turnover and degradation of stored proteins. Cysteine proteinases are also one of the favored The second approach used in the present work allowed for iden- candidates for initiating and mediating storage proteins degrada- tification of proteins having a higher expression level in mature tion in storage tissues of cereals and dicotyledonous plants (mature enriched proteins) or germinated (germinated enriched (Schlereth et al., 2000). In A. angustifolia embryos, this role seems proteins) embryos (Table 2). From the 387 unique identifications to be performed by serine proteases, as the spots 25, 199 and (see Supplementary Table 1S), 10 and 14 proteins were confidently 202 from the germinated embryo gels, corresponded to the ATP- assigned to be germinated and mature enriched, respectively dependent C1pC protease. In Vicia sativa, stored proteinases start (Fig. 3). The most preeminent mature enriched protein was the thi- globulin mobilization (Muntz et al., 2001). However, the absence azole biosynthetic enzyme (TA15741_3330). This protein is in- of the C1pC proteases in A. angustifolia mature embryos suggests volved in the biosynthesis of thiamine, an essential compound this enzyme is synthesized during germination, which is in accor- that is accumulated through thiamine-binding proteins in the aleu- dance with identification of an eukaryotic initiation factor (spot rone layer of grain germs during maturation (Watanabe et al., 201) in germinated embryo gels and the physiological requirement 2004). Down regulation of the thiazole biosynthetic enzyme during of de novo protein synthesis during germination in orthodox seeds germination may indicate the importance of high levels of thia- (Rajjou et al., 2004). Co-immunoprecipitation and cross-linking mine in mature embryos in comparison to germinated embryos. experiments established that the caseinolytic protease C (C1pC), Besides the nutritional role (Golda et al., 2004), thiamine may also also known as HSP93, acts in close proximity of the TIC (translocon confer systemic acquired resistance (SAM) through priming, or at the inner envelope membrane of chloroplasts) complex assisting elicitation competency, mobilizing plants in a state of enhanced Table 1 List of identified proteins from mature (-M-) and germinated (-G-) A. angustifolia embryos 2-DE gels via combined MASCOT stringent search and MS BLAST sequence-similarity searches.

SPOTa MASCOT MS BLAST Alignment g ID Species Acc No. b Scorec Peptides d ID Species Acc No. b Cove Queries f 003 -M- Carbamoyl phosphate synthase B A. thaliana 18397283 668 17 Carbamoyl phosphate synthase B A. thaliana 18397283 16 16 100 004 -M- Unknown P. sitchensis 116787220 370 8 Starch phosphorylase S. tuberosum 130173 29 25 91 006 -M- Os02g0519900 O. sativa 115446385 312 9 Elongation factor A. thaliana 6056373 5 5 95 018 –M- Unknown P. sitchensis 116781417 223 5 CBS domain-containing protein S. scutellarioides 118162023 42 8 86 047 -M- Vicilin-like protein A. angustifolia 21913852 599 16 Vicilin-like protein A. angustifolia 21912852 15 6 100 048 -M- Vicilin-like protein A. angustifolia 21913852 505 17 Vicilin-like protein A. angustifolia 21912852 21 10 100 053 -M- Vicilin-like protein A. angustifolia 21913852 747 23 Vicilin-like protein A. angustifolia 21912852 28 10 100 055 -M- Vicilin-like protein A. angustifolia 21913852 477 15 Vicilin-like protein A. angustifolia 21912852 26 10 100

056 -M- Vicilin-like protein A. angustifolia 21913852 240 18 Vicilin-like protein A. angustifolia 21912852 30 12 100 302–311 (2011) 72 Phytochemistry / al. et Balbuena T.S. 057 -M- Vicilin-like protein A. angustifolia 21913852 458 18 Vicilin-like protein A. angustifolia 21912852 31 12 100 070 -M- Vicilin-like protein A. angustifolia 21913852 803 26 Vicilin-like protein A. angustifolia 21912852 20 9 100 072 -M- Vicilin-like protein A. angustifolia 53618 886 26 Vicilin-like protein A. angustifolia 21912852 29 11 100 078 -M- Vicilin-like protein A. angustifolia 21913852 458 14 Vicilin-like protein A. angustifolia 21912852 5 2 100 092 -M- Glyceraldehyde-3P dehydrogenase P. sitchensis 462140 512 10 Glyceraldehyde-3P dehydrogenase A. thaliana 15219206 28 10 84 126 -M- Hypothetical protein V. vinifera 147800876 231 5 19S proteosome A. thaliana 3450889 14 6 91 136 -M- Vicilin-like protein A. angustifolia 21913852 840 24 Vicilin-like protein A. angustifolia 21913852 20 9 100 145 -M- Unknown P. sitchensis 116787113 380 10 UDP-glucose P P. tremula 32527831 9 4 89 171 -M- Unknown P. trichocarpa 118486341 842 16 RBP-60 kDa chaperone P. sativum 2506277 32 16 92 009 -G- Vicilin-like protein A. angustifolia 21913852 376 10 Vicilin-like protein A. angustifolia 21913852 9 4 100 011 -G- Aldolase P. patens 22759856 90 2 NI - ---- S. oleracea 4105131 492 11 ATP-dependent C1p protease A. thaliana 18423214 10 9 92 025 -G- C1pC protease, endopeptidase 060 -G- Vicilin-like protein A. angustifolia 21913852 343 14 NI – –––– 061 -G- Vicilin-like protein A. angustifolia 21913852 736 25 Vicilin-like protein A. angustifolia 21913852 22 11 100 093 –G- Unknown P. trichocarpa 118486146 255 5 Glutamine synthase A. glutinosa 2811030 12 4 94 135 -G- ATPase alpha subunit G. biloba 6561613 732 17 ATPase subunit alpha G. max 231585 31 15 95 136 –G- Vicilin-like protein A. angustifolia 21913892 442 14 Vicilin-like protein A. angustifolia 21913852 22 11 100 151 –G- AAA ATPase;26S proteasome M. truncatula 87241031 614 15 AAA ATPase;26S proteasome M. truncatula 87241031 27 11 100 199 –G- Hypothetical protein V. vinifera 147866973 1113 30 ATP-dependent C1p protease A. thaliana 18423214 23 18 92 122 –G- Predicted protein P. patens 168018023 734 20 26S protease regulatory S. oleracea 2492519 27 10 97 201 –G- Eukariotic initiation factor A. thaliana 21555870 963 23 Eukaryotic initiation factor N. tabacum 2500520 36 12 98 202 –G- Hypothetical protein V. vinifera 147866973 744 20 ATP-dependent C1p protease A. thaliana 18423214 17 15 92 206 –G- Cell division protein 48 G. max 1705678 1411 31 Cell division cycle protein G. max 1705678 38 26 100

NI: non-identified protein. a Spot numbers correspond to the numbers indicated in Fig. 1. Spots identified in mature and germinated embryo gels are indicated as -M- and -G-, respectively. b Accession number in NCBI protein database. c Probability based MOWSE score of MASCOT software for the hit. d Number of unique peptide sequences identified by MASCOT. e Percentage of predicted protein sequence covered by matched query sequence via MSBLAST according to [ Rpositive queries (aa) 100]/predicted protein (aa). f Number of unique sequences identified via MS BLAST that had a significant sequence-alignment score superior or equal to 55. g Sequence coverage between MASCOT and MS BLAST accessions. 305 306 T.S. Balbuena et al. / Phytochemistry 72 (2011) 302–311

Table 2 List of mature and germinated enriched proteins identiﬁed in Araucaria angustifolia embryos.

a b c d e Identified protein Homologous identification Identity Accession RSC Mature enriched proteins Thiazole biosynthetic enzyme (Pseudotsuga menziesii) – – TA15741_3330 2.065 Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone:MFH8 Abscisic acid receptor PYL8 (Arabidospsi thaliana) 100% TA17905_3330 1.759 (Arabidopsis thaliana) Hypothetical protein At1g04080 (Arabidopsis thaliana) Pre-mRNA-processing protein prp39, putative 63% TA15876_3330 1.759 (Ricinus communis) OSJNBa0041A02.8 protein (Oryza sativa) DAL1 protein 75% TA15264_3330 1.683 60S ribosomal protein L23A (Fritillaria agrestis) – – TA14655_3330 1.683 NPR1 (Carica papaya) – – TA22391_3330 1.372 CO254760 – – CO254760 1.372 CO242339 – – CO242339 1.372 Proteasome subunit beta type 1 (Petunia hybrida) – – TA15208_3330 1.216 Histone H2B (Arabidopsis thaliana) – – TA13796_3330 1.152 Germinated enriched proteins Expressed protein (Arabidopsis thaliana) RD2 protein (Arabidopsis thaliana) 96% TA22405_3330 1.231 26S protease regulatory subunit 7 (Arabidopsis thaliana) – – TA17399_3330 1.231 26S proteasome subunit RPN1b (Arabidopsis thaliana) – – TA16735_3330 1.231 Hypothetical protein (Solanum tuberosum) Fructose-bisphosphate aldolase (Solanum tubersum) 100% TA14395_3330 1.231 Csf-1 protein (Cucumis sativus) 60S ribosomal protein L14 (Ricinus communis) 86% TA14393_3330 1.231 Hypothetical protein (Oryza sativa) Stem-specific protein TSJT1 (Oryza sativa) 99% TA14667_3330 1.354 TA24361_3330 Transmembrane CLPTM1 family protein (Arabidopsis 75% TA24361_3330 1.387 thaliana) Translation initiation factor (Pisum sativum) – – TA28693_3330 1.387 Thioredoxin M-type (Spinacia oleracea) – – TA19161_3330 1.387 Mitogen-activated protein kinase D5 (Pisum sativum) – – TA19016_3330 1.387 Histone H2B (Arabidopsis thaliana) – – TA16260_3330 1.387 TA18889_3330 Eukaryotic initiation factor3 (Zea mays) 56% TA18889_3330 1.775 Putative phosphoglycerate mutase (Oryza sativa) – – TA19531_3330 2.080 Succinyl-CoA ligase alpha 1 subunit (Lycopersicon esculentum) – – TA17031_3330 2.547

a UniProt-UniRef protein annotation with the best alignment to the TIGR transcript assembly. b Homologous protein identified by BLAST sequence alignment. c Identity score for the alignment between identified protein and proposed homologous. d TIGR transcript assembly identifier or GenBank accession number for the identified protein. e Relative spectral counts for a protein, calculated as RSC = log2[(n2 + f)/(n1 + f)] + log2[(t1 n1 + f)/(t2 n2 + f)], where n1 and n2 correspond to the sum of spectral counts, across the biological replicates, for a given protein in mature and germinated embryos gels, respectively; t1 and t2 correspond to the total number of spectra over all proteins in the mature and germinated samples, respectively; and f is a correction factor set to 1.25.

ability to suppress future attacks (Ahn et al., 2007). However, thi- angustifolia embryos from mature to germinated stage, allowing amine per se does not induce cellular and molecular responses, the growing plantlet to switch from heterotrophy to autotrophy requiring the presence of hydrogen peroxide and the non-expres- status. sor pathogen related protein 1 (NPR1) for successful systemic ac- Protein synthesis from pools of stored mRNAs is essential for quired resistance (Ahn et al., 2007). Balbuena et al. (2009) radicle protrusion and, thus, germination (Rajjou et al., 2004). In detected high expression of ascorbic peroxidase isoforms in pro- the present work, two translation initiation factors were germi- embyo, globular and torpedo stages in comparison to cotyledonary nated enriched indicating higher regulation in germinated em- A. angustifolia embryo and a higher number of proteins involved in bryos than in mature ones (TA28693_3330 and TA18889_330). the stress responses only at early seed development, indicating These identifications may mark the restart of growing events after that a high oxidative metabolism and, consequently, high hydro- seed filling and maturation. However, high rates of de novo synthe- gen peroxide production may be present in mature embryos. In sis of proteins for germination require not only activation of an the present work, it was observed that the NPR1 protein efficient system for translation, but also degradation of amino acids (TA22391_3330) was mature enriched in comparison to the accu- stored pools. In the previous approach, the most abundant identi- mulation level observed in germinated embryos, corroborating the fied protein spots were those related to protein degradation pro- hypothesis of a defense role of thiamine by enhancing the capacity cess (Section 2.1). One dimensional gel electrophoresis strategy to express pertinent defense mechanisms in A. angustifolia mature confirmed previous identification of the 26S proteasome subunits embryos. (TA17399_3330 and TA16735_3330), highlighting the importance The thiazole biosynthetic enzyme may also play a role in the of this system in providing free amino acids probably through deg- photosynthetic process of A. angustifolia germinating embryos. radation of vicilin-like stored proteins. Many molecules inhibit photosynthetic electron transport (PET) From the 14 germinated embryos enriched proteins, three were by binding to the D-1 protein of photosystem II (PSII) reaction cen- involved in primary energetic metabolism. Homologous fructose- ter complex (Dayan et al., 2000). The practical approach of this bisphosphate aldolase (TA14395_3330) and phosphoglycerate mu- property is the use of these molecules as herbicides, because they tase (TA19531_3330) are glycolytic enzymes, while succinyl-CoA li- apparently compete with the natural electron acceptor plastoqui- gase catalyses formation of succinate from succinyl-CoA at the none for the QB binding site of PSII center complex (Dayan et al., tricarboxylic acid cycle (TCA). Higher detection of these enzymes 2000). Although no thiazole has been developed as a PET-inhibiting after germination indicates high rates of energy releasing processes herbicide, thiazole and thiazole-like derivatives have been known for growth and development of A. angustifolia plantlet. Activation of to inhibit PSII activity (Kluth et al., 1991; Tietjen et al., 1991). Down glycolytic pathway is dependent on availability of its primary pre- regulation of the thiazole biosynthetic enzyme throughout germi- cursor, D-glucose. Although no enzymes involved in starch mobiliza- nation may indicate an increase in the photosynthetic activity of A. tion were germination-enriched, vicilin-like stored protein T.S. Balbuena et al. / Phytochemistry 72 (2011) 302–311 307

MATURE GERMINATED ifolia, defense related proteins were enriched in germinated embryos. Although the precise role of these proteins during germination is unclear, in A. thaliana, the universally expressed protein RD2 was induced by desiccation (Yamaguchi-Shinozaki et al., 379 381 1992). Also, expression of the homologous transmembrane CLPTM1 protein was increased in Malus domestica after application of the non-pathogenic bacterium Pseudomonas fluorescens (Kurkcuoglu et al., 2007). Mitogen-activated protein kinases (MAPKs) have been RSC determination and distribution described as the major components of cellular signal transduction pathways mediating various biotic and abiotic stress responses including hormone signaling, cell division and developmental processes (Brock et al., 2010). MAPKs have also been implicated in ABA signaling (Hirayama and Shinozaki, 2007). In A. thaliana, MAPK3, MAPK4 and MAPK6 are the best characterized members, being transiently activated after ABA application (Ichimura et al., mature germinated 2000; Lu et al., 2002). Recently, Brock et al. (2010) identified a regu- enriched enriched latory mechanism of ABA-induced MAPKs via mitogen-activated protein kinase phosphatases (MKPs), as depletion of the phosphatases showed enhanced activation of the MAPKs, while ectopic expression of the MKPs resulted in inactivation of MAPKs and - insensibility to ABA stimulus. In A. angustifolia embryos, MAPK (TA19016_3330) and the abscisic acid receptor PYL8 (TA17905_ 3330) showed an unexpected opposite expression pattern. How- ever, in the present work, stringent database searches were per- -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 formed without considering phosphorylation modification which Rsc may occur in serine/threonine/tyrosine amino acid residues of the differentially enriched proteins identified MAPK resulting in m/z shift in the acquired spectra. Iden- at 95% confidence level tification of this protein in germinated but not in mature embryos may indicate existence of a phospho-regulation of this protein (phosphorylated form in mature embryo and unphosphorylated in germinated), which is in accordance with the MKP phosphorylation 10 363 14 regulatory mechanism (Brock et al., 2010) and with the high ABA concentration in mature embryos of A. angustifolia (Pieruzzi et al., unpublished data).

2.3. Implications on somatic embryogenesis Fig. 3. Diagram of total and differentially enriched proteins identified from mature and germinated A. angustifolia embryos. Upper panel: total number of proteins Storage proteins are known to accumulate in plant embryogenic indentified in each embryo state; middle panel: frequency distribution of the tissues during the maturation of zygotic embryos. Although in relative spectral counts (RSC) for all identified proteins; lower panel: number of common and differentially enriched proteins at 95% confidence level. some plant systems these type of proteins are not observed during somatic embryogenesis, mature somatic embryos of Picea abies mobilization and regulation of enzymes involved in energetic contain all the major storage protein classes found in zygotic em- metabolism corroborates the idea of an early storage mobilization. bryos (Lipert et al., 2005), including the most abundant protein Another germinated enriched protein involved in energetic spots observed for A. angustifolia mature zygotic embryos, the vic- metabolism was thioredoxin (TA19161_3330). This enzyme has ilin-like storage protein. Interestingly, this class of proteins has an important role in activation of TCA cycle enzymes and in regu- been used as a marker in the investigation of culture conditions lation of thylakoid electron transport chain through redox control for oil palm somatic embryo maturation (Morcillo et al., 2001). (Johnson, 2003). This dual role ensures both activation of TCA en- During germination of A. angustifolia, two dimensional gel electro- zymes under reduction conditions and avoidance of over-reduction phoresis analyses permitted the identification of early degradation of ferredoxin, the terminal acceptor of the electron transport chain, of vicilin-like stored proteins, that accumulated during maturation when exposed to light stimulus (Johnson, 2003). Although A. of zygotic embryos. Although still not reported in A. angustifolia, angustifolia seeds do not show desiccation (see Supplementary the protective role of the vicilin seed proteins can not be ruled Fig. 1S) and stored proteins are not expected to be in an oxidative out and formation of vicilin fragments may have antimicrobial/ form, thioredoxins appear to play a key role in reducing oxidized insecticide function in embryos and be required for successful ger- stored proteins in cereals and Medicago truncatula (Alkhalfioui mination. Enzymes involved in protein mobilization were also de- et al., 2007). This enzyme acts as a signal in early germination to tected in both strategies used here in. Among the 14 germinated facilitate mobilization of reserves by reducing storage proteins, enriched protein spots, five were involved in protein degradation. enhancing their solubility and susceptibility to proteolysis, reduc- It is known that proteasome activity is closely aligned with cell ing and inactivating disulfide proteins inhibit specific amylases and proliferation processes (Amsterdam et al., 1993) and regulation proteases, thereby facilitating breakdown of stored starch and pro- of proteasome subunits in mature tissues was also observed in P. teins, and reductively activating individual enzymes functional in abies somatic embryos (Lipert et al., 2005). Degradation of storage germination (Alkhalfioui et al., 2007). proteins and expression of enzymes involved in germination may Seeds of rainforest trees tend to germinate soon after dispersal, as be of a great value for monitoring the conversion and development seed fungal attack can confer a strong selective pressure in an envi- of embryos to plantlets in this species. ronment characterized by high humidity and temperature (Tweddle Enrichment of the thiazole biosynthetic enzyme and the DAL1 et al., 2003; Vazquez-Yanes and Orozco-Segovia, 1993). In A. angust- protein in mature embryos of A. angustifolia may interfere in 308 T.S. Balbuena et al. / Phytochemistry 72 (2011) 302–311

plastidial mRNA processing and chloroplast differentiation. The (embryo axis and cotyledons) grounded to a fine powder under N2. expression pattern of these proteins may be related to a mecha- Samples (300 mg) of each ground and mixed powders were trans- nism to avoid early germination, which is particularly useful in ferred into 2 ml microtubes containing 1.5 ml of extraction buffer plants that do not have dormancy or decreases in metabolic rates (7 M urea, 2 M thiourea, 1% dithiothreitol (DTT), 2% triton X-100, after maturation. Down regulation of these proteins after somatic 1 mM phenylmethylsulphonyl fluoride, 5 lM pepstatin). All ex- embryos maturation may be useful to monitor development and tracts were briefly vortexed and kept in extraction buffer, standing formation of in vitro A. angustifolia plantlets. Also, germinated em- on ice, for 30 min followed by centrifugation at 12,000g for 5 min bryos enriched proteins, although presenting an unclear role in at 4 °C. Supernatants were transferred to clear tubes and proteins conifer zygotic embryogenesis, may also be used as potential can- were precipitated in ice, for 1 h, in Cl3CO2H (10%) and washed three didates, such as the homologous transmembrane CLPTM1 protein times with cold acetone. Finally, proteins were resuspended and and the stem-specific protein TSJT1. concentrated in 0.5 ml of the same extraction buffer with the addi- Redox homeostasis proteins have roles in regulation and main- tion of 2% immobilized pH gradient (IPG) buffer (pH 3–10) (GE tenance of cell differentiation status (Pan et al., 2009). A number of Healthcare, Freiburg, Germany) for two dimensional electrophore- data indicates that stress itself could play an important role as an sis samples. Protein concentration was estimated by 2-D Quant Kit embryogenic switch (Feher et al., 2003; Ikeda-Iwai et al., 2003). (GE Healthcare) and samples were stored at 20 °C until In Citrus sinensis, three out of five protein spots related to oxidative electrophoresis. stress were down regulated during somatic embryo maturation, indicating that oxidative stress may stimulate cell differentiation 4.3. Protein fractionation and sample processing and promote somatic embryo formation, whereas other antioxida- tive proteins may serve a different function(s) such as protecting 4.3.1. One dimensional electrophoresis (1-DE) and in gel protein the cultures cells from toxicity caused by long term in vitro cultur- digestion ing (Pan et al., 2009). Balbuena et al. (2009) suggested a higher oxi- Sample aliquots (100 lg of proteins) were mixed with an equal dative control in early zygotic embryogenesis of A. angustifolia in volume of loading buffer containing 0.5 M Tris (pH 6.8), 20% glyc- comparison to late events, which is in accordance with results erol, 2% SDS, 5% 2-mercaptoethanol and traces of bromophenol identified in the present work. Oxidative stress caused by in- blue. In this experiment, two biological replicates were evaluated creased levels of radical oxygen species due to addition of different and reproducibility of data acquired was quantitatively evaluated agents introduced in in vitro cultures has been reported to enhance through spectral counting of each protein identified between rep- somatic embryogenesis in many plant species (Caliskan et al., licates (see Supplementary Fig. 2S). Protein separations were per- 2004; Ganesan and Jayabalan, 2004; Luo et al., 2001; Pasternak formed in 11 cm 1.5 mm gels at 20 mA per gel. With protein et al., 2002). Further analysis focusing on changes of the redox sta- bands visualized using Colloidal Coomassie Blue Stain (Neuhoff tus of somatic embryos may be useful to successfully achieve an et al., 1988). After gel electrophoresis, mature and germinated em- efficient protocol for in vitro embryo conversion and development. bryo gel lanes were sliced into 9 different segments across the molecular weight ranges, diced into approximately 1 mm cubes with a clean scalpel and transferred into new sterile microtubes 3. Concluding remarks (1.5 mL) for in gel trypsin digestion. The latter was carried out according to Shevchenko et al. (2007). Firstly, gel pieces were Examination of differentially expressed proteins between ma- washed three times with a solution containing CH CN:100 mM ture and germinated A. angustifolia embryos suggests an active deg- 3 (NH )HCO for 30 min and dehydrated in CH CN (500 L) for radation metabolism, activation of energetic metabolism pathways, 4 3 3 l 10 min. Reduction was performed in 400 L of a DTT solution initiation of translational processes and oxidative stress regulation l (10 mM DTT in 100 mM (NH )HCO ) for 30 min at 56 C, followed during the germination process. Signaling regulation analysis still 4 3 ° by a dehydration step in CH CN for 10 min. For alkylation 55 mM need to be explored during the zygotic embryo germination, espe- 3 iodoacetamide (400 L) in 100 mM (NH )HCO was added and cially in terms of monitoring phosphorylation sites for the MAPK l 4 3 gel pieces were incubated at room temperature for 30 min in the identified in germinated embryos. Also, further functional analysis dark, followed by a dehydration step in CH CN for 10 min. Protein of the differentially expressed proteins identified in this study and 3 digestion was performed by addition of sequencing grade porcine comparisons with the differential protein expression pattern pre- trypsin (200 L) (Promega, Madison, USA) at 15 ng/ L. After sented in somatic embryos will clarify their exact roles in embryo l l 120 min of cold incubation at 4 C, samples were placed into an germination and may be explored for improving in vitro growth con- ° air circulation thermostat and proteins were digested overnight ditions for large scale propagation of this species. at 37 °C. Then, gel pieces were saturated with extraction buffer

(700 lL) 5% HCO2H(FA):CH3CN (1:2, v/v) and incubated for 4. Experimental 30 min at 37 °C. Supernatants were collected, dried down in a vac- uum centrifuge and kept at 80 °C until LC-MS/MS analyses. 4.1. Plant material and germination 4.3.2. Two dimensional electrophoresis (2-DE) and spot matching Mature seeds of A. angustifolia were harvested in the Santa- Sample aliquots (150 lg of proteins) were used for 2-DE. Prior 0 0 Catarina State, Brazil (27°47 S, 49°29 W), in May 2007 and horizon- to loading in 11 cm IPG strips, small volumes of rehydration buffer tally placed in sterilized vermiculite:soil (1:1) mixture. Seeds were (7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer (pH 3–10), 1% then cultivated in a plant growth chamber at 27 °C and 16 h pho- DTT and 0.002% bromophenol blue) were added to the sample in toperiod. Embryo axis and cotyledons of mature and germinated order to achieve the final volume (250 lL). After 12 h in gel rehy- embryos were isolated from the megagametophytes at 0 and dration, isoeletric focusing was performed on an IPGPhor II appara- 8 days after sowing and stored at 80 °C until analysis. tus (GE Healthcare) for a total of 15 kVh at 20 °C. IPG strips were then subjected to reduction, alkylation by 2 15 incubations with 4.2. Protein extraction buffer (50 mM Tris–HCl, 6 M urea, 30% glycerol, 2% SDS and 0.002% bromophenol blue) containing 125 mM DTT for the first incubation Protein extracts were prepared in biological triplicates. Each and 125 mM iodoacetamide for the second. Then, strips were ap- biological replicate was prepared from a bulk of 10 intact embryos plied to the top of a 12% polyacrylamide gel. Second dimension T.S. Balbuena et al. / Phytochemistry 72 (2011) 302–311 309 electrophoresis was carried out at 25 mA per gel and Colloidal the close evolutionary relationship shared between studied species Coomassie Blue stained. In this experiment, three biological repli- and P. glauca, as both belong to the Pinales order. Also, protein se- cates were evaluated to detect specifically expressed proteins in quences derived from a single species were used to avoid protein mature and germinated embryos. For this, experimental gels from redundancy and double counting of spectra associated with orthol- three biological replicates were aligned using the Image Master ogous proteins that could lead to false interpretations of protein Platinum v. 6 software (GE Healthcare). Spot detection parameters expression changes. P. glauca nucleotide sequences were translated were optimized by checking different protein spots in certain re- and the open reading frames (ORFs) scanned using the Virtual gions of the gel and then automatically detected, followed by vi- Ribosome software (Wernersson, 2006). For each nucleotide se- sual inspection for removal or addition of undetected spots. In quence, one amino acid sequence was obtained, corresponding to order to attribute a common spot identity for the same spot de- the longest ORF reported. In order to estimate the false discovery rived from different repetitions, experimental gels were automati- rate of the peptides that could randomly match the database, ran- cally matched, based on obvious three common spots used as domized (decoy) sequences were combined to the forward data- anchors, and again visually inspected for improper spot matches. base, resulting in a concatenated database of 98,824 entries. In silico master gels containing spots present in all three biological Protein identification was performed using the SEQUEST search repetitions were then created and used to detect specifically ex- algorithm under the BioWorks 3.3 software package (Thermo Fish- pressed proteins in mature and germinated embryos through gel er Scientific). The search parameters for the searches were set as matching as described above. Spots of interest were excised using follows: oxidation of methionine was allowed as a variable modi- a clean scalpel and in gel digested as described in the Section 4.3.1. fication and carbamidomethylation of cysteine as a static modification; enzyme: trypsin; number of allowed missed cleavages: 1; 4.4. Liquid chromatography and mass spectrometry analysis mass range: 300–1600; threshold: 500; minimum ion count: 10; precursor mass tolerance 10 ppm; fragment mass tolerance 4.4.1. 1-DE fractionated proteins 0.6 Da. After searches, SEQUEST output files were uploaded and Tryptic digests from segments were injected into an Ultimate analyzed by Scaffold 3.0 software (Proteome Software, Portland, 3000 nanoLC system (Dionex, Sunnyvale, USA) interfaced on-line USA). Each 9 SEQUEST outputs from searches of the 9 gel lane seg- to a LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, ments of each biological replicate were combined and the number Bremen, Germany). Peptides were firstly loaded onto a 5 mm of assigned peptides and spectra in each biological replicates used 300 lm i.d. trapping micro column packed with C18 PepMap100 for confident protein identification and quantification. To obtain 5 lm particles in 0.1% FA at the flow rate of 20 ll/min. After high-confidence and unambiguous protein assignments, shared 4 min of washing, they were back-flushed and separated on a peptides were removed from the analysis, and ProteinProphet 15 cm 75 lm i.d. nanocolumn packed with C18 PepMAP100 and PeptideProphet Probabilities (Keller et al., 2002) equal to 99% 3 lm particles at the flow rate of 200 nl/min using the following and 95%, respectively, and at least 2 unique matched peptides mobile phase gradient of solvent A, 95:5 H2O/CH3CN (v/v) with per protein were required for confident protein identification. False 0.1% HCO2H, and solvent B, 20:80 H2O/CH3CN (v/v) with 0.1% discovery rate was 1.28%. HCO2H: from 5% to 20% of solvent B in 20 min, 20–50% B in Spectral counts were used to estimate the protein amount in 16 min, 50–100% B in 5 min, 100% B during 10 min and back to each biological sample. To assess differences in the relative protein 5% B in 5 min. After LC separation, peptides were eluted into the abundance between mature and germinated embryos, we used a mass spectrometer via robotic nanoflow ion source TriVersa (Adv- metric named relative spectral counting (Old et al., 2005), defined ion BioSystems, Ithaca, USA) with 1.7 kV ionization voltage and as capillary transfer temperature at 180 °C. Each data dependent R ¼ log ½ðn þ f Þ=ðn þ f Þ þ log ½ðt n þ f Þ=ðt n þ f Þ ð1Þ acquisition cycle consisted of a survey scan covering the range of SC 2 2 1 2 1 1 2 2 m/z 300–1600 performed at the Orbitrap analyzer, followed by where n1 and n2 correspond to the sum of spectral counts, across MS/MS fragmentation of the four most abundant precursor ions the biological replicates, for a given protein in mature and germi- under normalized collision energy of 35% in the ion trap. The m/z nated embryos gels, respectively; t1 and t2 correspond to the total of fragmented precursor ions were excluded for a further 90 s. number of spectra over all proteins in the mature and germinated samples, respectively; and f is a correction factor set to 1.25, as pro-

4.4.2. 2-DE fractionated proteins posed by Old et al. (2005). Positive RSC values suggest enrichment of Tryptic digests from 2-DE gel spots were injected into an Ulti- the proteins of interest in germinated embryos, whereas negative mate nanoLC system (Dionex) interfaced on-line to a linear ion trap values suggest enrichment in mature embryos. To determine statis-

LTQ (Thermo Fisher Scientific). Prior to mass spectrometry analy- tically significant cutoff values for the RSC, we tested the normaliza- sis, peptides were firstly separated in using C18 column as de- tion of the 387 identified proteins (see Supplementary Fig. 3S) and scribed in Section 4.4.1. Peptides were eluted into the mass we chose the 95th percentile of the observed distribution to deter- spectrometer via a dynamic nanospray probe (Thermo Fisher mine the RSC cutoffs and the confidence intervals (95% confidence Scientific) using a silica tip uncoated needle with a spray voltage level). Protein was considered germination-enriched if the calcu- of 4.5 kV and the transfer capillary temperature was set to lated RSC was equal or higher than 1.2314 and considered mature 200 °C. Data dependent acquisition cycle consisted of a survey scan enriched if RSC was equal or lower than 1.012. covering the range of m/z 350–1500 followed by MS/MS fragmentation of the four most abundant precursor ions under normalized 4.5.2. 2-DE fractionated proteins collision energy of 35% in the ion trap. The m/z of fragmented pre- Comprehensive database search was carried out against the cursor ions were excluded for a further 60 s. NCBI database by MASCOT v.2.2 (Matrix Science, Boston, USA). Mass tolerance for precursor and fragmented ions were 4.5. Database search and data analysis 2000 ppm and 0.6 Da for precursor and fragmented peptides, respectively. Carbamidomethylation of cysteine was set as fixed 4.5.1. 1-DE fractionated proteins modification, and oxidation of methionine as variable modifica- In this experiment, the TIGR transcript assembly database tion. Identifications of proteins were considered confident if hits (http://plantta.jcvi.org) for Picea glauca (release 2) was used to were produced by matching of at least three MS/MS spectra with query all MS/MS acquired data. This database was chosen due to peptide ions scores above 20. For hits matched by one or two 310 T.S. Balbuena et al. / Phytochemistry 72 (2011) 302–311 spectra, it was required that at least one spectrum should be dos Santos, A.L.W., Silveira, V., Steiner, N., Vidor, M., Guerra, M.P., 2002. Somatic matched with score of 50 or better. Also, complementary protein embryogenesis in Parana Pine (Araucaria angustifolia (Bert.) O. Kuntze). Braz. Arch. Biol. Technol. 45, 97–106. identification of protein spots was performed through de novo dos Santos, A.L.W., Steiner, N., Guerra, M.P., Zoglauer, K., Moerschbacher, B.M., mass spectra interpretation, via PepNovo software (Frank and 2008b. Somatic embryogenesis in Araucaria angustifolia. Biol. Plantarum 52, Pevzner, 2005). For each interpretation seven partially redundant 195–199. dos Santos, A.L.W., Wietholter, N., Gueddari, N.E., Moerschbacher, B.M., 2006. candidate sequences were produced, each of them containing a Protein expression during seed development in Araucaria angustifolia: transient quality score, which stands for the expected number of confidently accumulation of class IV chitinases and arabinogalactan proteins. Physiol. Plant. determined amino acid residues in the most accurate sequence 127, 138–148. Feher, A., Pasternak, T.P., Dudits, D., 2003. Transition of somatic plant cells to an proposal. Candidate sequences with score higher than six were embryogenic state. Plant Cell Tissue Organ Cult. 74, 201–228. merged into a single search string in arbitrary order and MS BLAST Frank, A., Pevzner, P., 2005. PepNovo: de novo peptide sequencing via probabilistic searches were performed against NCBInr database at http://genet- network modeling. Anal. Chem. 77, 964–973. 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