Proteomic analysis of oil body membrane proteins accompanying the onset of desiccation phase during sunflower seed development

Abstract

A noteworthy metabolic signature accompanying oil body (OB) biogenesis during oilseed development is associated with the modulation of the oil body membranes proteins. Present work focuses on 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based analysis of the temporal changes in the OB membrane proteins analyzed by LC-MS/MS accompanying the onset of desiccation (20–30 d after anthesis; DAA) in the developing seeds of sunflower (Helianthus annuus L.). Protein spots unique to 20–30 DAA stages were picked up from 2-D gels for identification and the identified proteins were categorized into 7 functional classes. These include proteins involved in energy metabolism, reactive oxygen scavenging, proteolysis and protein turnover, signaling, oleosin and oil body biogenesis-associated proteins, desiccation and cytoskeleton. At 30 DAA stage, exclusive expressions of enzymes belonging to energy metabolism, desiccation and cytoskeleton were evident which indicated an increase in the metabolic and enzymatic activity in the cells at this stage of seed development (seed filling). Increased expression of cruciferina-like protein and dehydrin at 30 DAA stage marks the onset of desiccation. The data has been analyzed and discussed to highlight desiccation stage-associated metabolic events during oilseed development.

Introduction

Proteomic analysis of seed development can help in providing insights into the temporal and spatial patterns of accumulation of seed proteins across different stages of seed development in plants.¹ Two dimensional electrophoretic (2-DE) proteomic analysis followed by protein identification either by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), Peptide mass fingerprinting (PMF) or Liquid chromatography–mass spectrometry (LC-MS/MS) in endosperm-dominant, starch-rich seeds of wheat,² barley,³ rice^4,5 and maize,^6,7 has earlier shown almost similar kinds of proteins identified and similar patterns of their distribution. Comparative proteomic analysis of the endosperms^2,4 and embryos of wheat and rice⁸ has highlighted noticeable differences among these 2 tissues. While the endosperm has relatively more abundant proteins involved in protein folding, proteins involved in stress response and hormonal signaling are in relatively high amount in the embryos, which indicates the likely metabolic control of the biochemical events in the endosperm by the embryo. Proteins of the major metabolic pathways are uniformly present both in endosperm and embryo of wheat and rice. Proteomic analysis of the endosperm development in the oilseed crop-Jatropha, has lead to the identification of total 522 proteins, of which most are seed storage proteins (SSPs). Apart from the SSPs in Jatropha, most of the proteins are from central metabolism, protein folding, stress response and cellular structure classes of proteins.⁹ In recent years, extensive proteomic research has been undertaken in Arabidopis thaliana,¹⁰ Brassica napus,¹¹ Lotus japonicus¹² and Glycine max.¹³ Proteomic analysis of Arabidopsis and Brassica seeds has revealed similar results, with proteins involved in central metabolism comprising the most abundant class of proteins.^10,11 The comparative proteomic analysis undertaken in Glycine max and Brassica has established a view on metabolic pathways involved in seed-filling processes in 2 distinct oilseeds, which is helpful in understanding the metabolic differences in the seed developmental processes of the protein-rich and oil-rich seeds.¹¹

Lipid reserves in oilseed plants are mainly concentrated in seeds as they provide them protection from lipid oxidation and hydrolysis until seed germination.¹⁴ ‘Orthodox seeds’ undergoing desiccation during the maturation phase have several oil bodies with smaller diameter as compared to ‘recalcitrant seeds’, which have larger and fewer oil bodies.¹⁵ In the exalbuminous seeds (like sunflower), lipid bodies are mainly located in seeds and embryo axis.¹⁶ There are many integral oil body membrane proteins, like oleosins, caleosins and steroleosins, along with some other enzymatic and non-enzymatic proteins located transiently on the oil body surface.¹⁷ These transiently expressed proteins find temporal and short or long association with the oil body membrane proteins during the course of oilseed development. They play important roles during oil body biogenesis. Oleosins are one of the most important structural protein constituents of oil body membranes in sunflower (and in other oilseeds) and constitute as much as 10% of the total oil body membrane proteins.¹⁵ They provide the steric hinderance and electronegative repulsion which in turn confer oil body stability.¹⁸ Analysis of proteomic and lipid composition of oil bodies from the 2 cultivars of Brassica napus illustrated the role of enzymes like dehydrogenase and a myrosinase-associated protein which are likely to be involved in oil body degradation.¹⁹ Studies performed on lipid and protein accumulation in Brassica revealed the differential accumulation of lipids and proteins during the seed development and the earlier and later stages of seed development had different lipid and protein compositions.²⁰

It would be significant to analyze the pattern of expression of intrinsic protein components of oil body membranes during different phases of seed development through proteome analysis. This has been achieved in the present work by subjecting the oil bodies to rigorous urea washings which reportedly removes all extrinsic protein constituents, majorly allowing the retention of intrinsic proteins in the preparation so obtained.²¹ The present proteomic analysis of oil body-associated membrane proteins, obtained from the 20 and 30 DAA stages of seed development, has been undertaken to identify the proteins associated with the oil body membranes accompanying the onset of desiccation phase during seed development in sunflower. Oil body membrane proteins obtained from the 40 DAA are difficult to resolve in the 2D SDS-PAGE because of the heavy accumulation of lipids at this stage which proves a hindrance in the electrophoretic resolution of proteins. Therefore, present analysis could not be taken up for seeds at 40 DAA stage. The present work is aimed at identifying various structural and functional proteins likely to play significant roles in lipid deposition, stabilization and dynamics of oil body biogenesis in the developing seeds of sunflower.

Results and Discussion

Seed development in sunflower can be divided into 3 phases: cell division, cell proliferation and cell enlargement, seed filling phase and desiccation phase leading to seed maturation.²² Seed development at 20, 30 and 40 d after anthesis (DAA) exhibited noteworthy changes in the degree of vacuolation, cell size and degree of abundance of oil bodies in the developing seeds (Fig. 1). Cotyledon cells at 20 DAA stage were observed to be longer, vacuolated and they showed a sparse distribution of large oil bodies. A gradual dehydration and oil body accumulation was evident in developing sunflower seeds during transition from 30–40 DAA stage of seed development.²² With seed maturation, cell size was relatively reduced and cells got filled with clusters of smaller oil bodies. Earlier investigations have revealed that oil body biogenesis in developing oilseeds starts within a few days after anthesis whereby oleosins and some other minor proteins are expressed on the oil body membrane.²³ Expression of 3 oleosin isoforms was also evident in the present analysis of OB membrane proteins from seeds at 20 and 30 DAA stages (Fig. 2). LC-MS/MS analysis of oil body membrane associated proteins from the developing seeds of sunflower (20 and 30 DAA), after 2-D PAGE analysis of urea-washed oil bodies (Fig. 3), revealed interesting difference in protein expression at the 2 stages of seed development. These unique expressions were tabulated (Table. 1) and discussed below under 7 subclasses.

Figure 1.

Model illustrating major events during sunflower (Helianthus annuus) seed development from early embryogenesis to maturity.

Figure 2.

Changes in the oil body membrane polypeptide pattern during seed devclopment stages. Oil bodies obtained after tissue homogenization in Tris-urea buffer (50 mM, pH 7.5 containing 9 M urea) were washed in the same buffer. 150 µg protein from each sample was loaded on a 10–20 % gradient gel containing 6 M urea. Lane 1 and 2: Oil body membrane proteins extracted from seeds at 20 and 30 DAA stages respectively. O represents oleosin.

Figure 3.

Two dimensional electrophoretic separation of urea-washed oil body membrane proteins from (A) 20 DAA and (B) 30 DAA stage of sunflower seed development, as visualized on 15% SDS-PAGE gel in the second dimension. The spots marked are qualitatively expressed in the stage and were picked up for LC-MS/MS analysis for protein identification.

Table 1.

Protein assignments from LC-MS/MS analysis of 2-D gel spots from oil body membrane proteins isolated from 20 and 30 DAA stages of seed development in sunflower

S. No.	Protein	EC No.	Species	Accession No.	Spot No.	Protein Score	MW (kDa)	pI
	PRIMARY METABOLISM
1	glyceraldehyde 3-phosphate dehydrogenase	EC 1.2.1.12	Scaevola procera	Q4VJ32	2	138.31	14.46	5.93
	glyceraldehyde 3-phosphate dehydrogenase	EC 1.2.1.13	Gynura bicolor	E0D6S1	3	193.41	36.95	6.52
	glyceraldehyde 3-phosphate dehydrogenase		Gynura bicolor	E0D6S1	5	166.16	36.95	6.52
	glyceraldehyde-3-phosphate dehydrogenase		Helianthus annuus	B5KUF5	8	122.26	21.4	8.68
	glyceraldehyde 3-phosphate dehydrogenase		Gynura bicolor	E0D6S1	9	185.72	36.95	6.52
	glyceraldehyde 3-phosphate dehydrogenase		Helianthus annuus	B5KUF5	10	390.32	21.4	7.51
	glyceraldehyde 3-phosphate dehydrogenase		Gynura bicolor	E0D6S1	11	166.16	36.95	6.52
	glyceraldehyde 3-phosphate dehydrogenase		Helianthus annuus	B5KUF5	12	390.32	21.4	7.51
	glyceraldehyde 3-phosphate dehydrogenase		Echinacea tennesseensis	U5TQS0	21	379.24	21.4	8.21
	glyceraldehyde 3-phosphate dehydrogenase		Scaevola procera	Q4VJ32	22	145.37	14.46	5.93
	glyceraldehyde 3-phosphate dehydrogenase		Scaevola procera	Q4VJ32	23	145.37	14.46	5.93
2	cytosolic phosphoglycerate kinase	EC 2.7.2.3	Helianthus annuus	I6LNU0	1	197.62	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	2	122.11	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	3	357.9	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	4	78.8	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	5	104.31	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	10	170.84	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	12	208.47	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	21	133.74	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	23	112.54	42.33	6.03
	cytosolic phosphoglycerate kinase		Helianthus annuus	I6LNU0	26	103.38	42.33	6.03
3	Nucleoside diphosphate kinase A	EC 2.7.46	Flaveria bidentis	P47920	3	770.21	16.12	6.26
	Nucleoside diphosphate kinase A		Flaveria bidentis	P47920	25	114.8	16.12	6.26
	Nucleoside diphosphate kinase A		Flaveria bidentis	P47920	26	982.2	16.12	6.26
	Nucleoside diphosphate kinase A		Flaveria bidentis	P47920	27	84.56	16.12	6.26
	ROS SCAVENGING
4	Mn-superoxide dismutase I	EC 1.15.1.1	Helianthus annuus	A6XIG5	1	234.95	25.32	7.86
	Mn-superoxide dismutase I		Helianthus annuus	A6XIG5	10	98.22	25.32	7.86
	Mn-superoxide dismutase I		Helianthus annuus	A6XIG5	12	160.69	25.32	7.86
	Mn-superoxide dismutase I		Helianthus annuus	A6XIG5	16	252.22	25.32	7.86
	Mn-superoxide dismutase I		Helianthus annuus	A6XIG5	18	189.06	25.32	7.86
	Mn-superoxide dismutase I		Helianthus annuus	A6XIG5	19	169.41	25.32	7.86
	Mn-superoxide dismutase I		Helianthus annuus	A6XIG5	21	125.39	25.32	7.86
	Mn-superoxide dismutase I		Helianthus annuus	A6XIG5	23	140.84	25.32	7.86
	Mn-superoxide dismutase I		Helianthus annuus	A6XIG5	26	158.79	25.32	7.86
5	cytosolic Cu/Zn-superoxide dismutase	EC 1.15.1.1	Helianthus annuus	Q6Y8C6	21	82.44	7.69	5.4
6	catalase	EC 1.11.1.6	Helianthus annuus	P45739	3	117.51	56.97	7.1
	PROTEOLYSIS AND PROTEIN TURNOVER
7	aspartic proteinase	EC 3.4.23	Helianthus annuus	Q9SSZ1	2	120.28	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	3	450.7	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	9	133.56	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	10	966.46	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	11	240.38	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	12	388.24	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	16	75.14	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	21	82.21	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	23	332	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	24	81.43	55.97	6.06
	aspartic proteinase		Helianthus annuus	Q9SSZ1	27	316.12	55.97	6.06
8	26S protease regulatory subunit 6B homolog	EC 3.4.25		P85200	12	106.62	46.79	5.25
9	Ubiquitin		Helianthus annuus	P69313	3	214.52	8.66	7.54
	Ubiquitin		Helianthus annuus	P69313	10	164.91	8.66	7.54
	Ubiquitin		Helianthus annuus	P69313	11	75.55	8.66	7.54
	Ubiquitin		Helianthus annuus	P69313	21	316.34	8.66	7.54
	Ubiquitin		Helianthus annuus	P69313	26	192.79	8.66	7.54
	SIGNALING
10	initiation factor eIF4A-15	EC 3.6.4.13	Helianthus annuus	Q6T8C6	3	293.51	46.8	5.1
	initiation factor eIF4A-16		Helianthus annuus	Q6T8C6	4	82.6	46.8	5.1
	initiation factor eIF4A-17		Helianthus annuus	Q6T8C6	7	81.34	46.8	5.1
	initiation factor eIF4A-18		Helianthus annuus	Q6T8C6	12	99.27	46.8	5.1
	initiation factor eIF4A-19		Helianthus annuus	Q6T8C6	21	211.93	46.8	5.1
	initiation factor eIF4A-20		Helianthus annuus	Q6T8C6	23	112.78	46.8	5.1
	initiation factor eIF4A-21		Helianthus annuus	Q6T8C6	26	341.32	46.8	5.1
11	Gly-rich RNA binding protein partial		Helianthus annuus	Q03250	3	78.44	126.28.02	6.95
12	14-3-3-like protein		Helianthus annuus	O65352	1	86.55	290.43	4.37
	14-3-3-like protein		Helianthus annuus	O65352	7	85.83	290.43	4.37
	14-3-3-like protein		Helianthus annuus	O65352	10	210.19	290.43	4.37
	14-3-3-like protein		Helianthus annuus	O65352	12	91.77	290.43	4.37
	14-3-3-like protein		Helianthus annuus	O65352	23	289.77	290.43	4.37
	Oil body Biogenesis and Lipid Metabolism
13	β-hydroxyacyl-ACP dehydratase	EC 4.2.1.59	Helianthus annuus	F2VYC9	2	84.64	24.19	8.23
	β-hydroxyacyl-ACP dehydratase		Helianthus annuus	F2VYC9	19	79.27	24.19	8.23
	β-hydroxyacyl-ACP dehydratase		Helianthus annuus	F2VYC9	27	85.23	24.19	8.23
14	ketoacyl-ACP reductase 1	EC 1.1.1.100	Helianthus annuus	F2VTN2	10	195.94	33.82	9.71
15	enoyl-ACP reductase 1	EC 1.3.1.9	Helianthus annuus	F2VTN4	11	79.84	41.77	9.35
	enoyl-ACP reductase 2		Helianthus annuus	F2VTN4	12	162.58	41.77	9.35
	enoyl-ACP reductase 3		Helianthus annuus	F2VTN4	25	82.8	41.77	9.35
16	Non-specific lipid-transfer protein AP10		Helianthus annuus	P82007	2	96.49	12.4	9.92
	Non-specific lipid-transfer protein AP11		Helianthus annuus	P82007	3	175.26	12.4	9.92
17	Oleosin		Helianthus annuus			142.89	19.7	9.63
	Dessication Tolerance
18	putative dehydrin		Helianthus annuus	Q8GUC8	9	466.73	25.79	7.15
19	cruciferina-like protein		Helianthus annuus	C1KLC1	10	322.21	7.13	7.37
	CYTOSKELETON
20	actin		Helianthus annuus	B8Y645	3	104.63	41.93	5.83
	actin		Helianthus annuus	B8Y645	4	173.11	41.93	5.83
	actin		Helianthus annuus	B8Y645	10	302.49	41.93	5.83
	actin		Helianthus annuus	B8Y645	12	292.87	41.93	5.83
	actin		Helianthus annuus	B8Y645	21	186.3	41.93	5.83
	actin		Helianthus annuus	B8Y645	22	125.63	41.93	5.83
	actin		Helianthus annuus	B8Y645	23	156.34	41.93	5.83
	actin		Helianthus annuus	B8Y645	25	204.07	41.93	5.83

Primary metabolism

At the primary metabolism level, seed development in sunflower at 30 DAA stage marks an enhancement of energy conserving phase of glycolysis whereby triosphosphate is converted to pyruvate involving the reduction of NAD⁺ TO NADH (through the action of glyceraldehyde-3-phosphate dehydrogenase) and ATP synthesis (through the action of phosphoglycerate kinase). Glyceraldehyde-3-phosphate dehydrogenase (GAPDHs) is involved in the energy conserving phase of the glycolytic cycle whereby it leads to the reduction of NAD⁺ into NADH. GAPDHs have also been detected in the cell wall fraction in Arabidopsis²⁴ and in the peribacteroid membrane of root nodules in Lotus japonica.²⁵ In Arabidopsis, 4 GAPDH isoforms encoded by a family of 4 genes encode putative phosphorylating glycolytic GAPDHs (GAPC1, GAPC2, GAPCp1, and GAPCp2). Two isoforms (GAPC1 and GAPC2) are cytosolic and the other 2 (GAPCp1, and GAPCp2) are plastidial in distribution. Cytosolic GAPDHs exhibit high gene expression as compared to the plastidial ones. In spite of low gene expression, down regulation of plastidial GAPDHs may lead to various abnormalities in plants, such as arrested root development,²⁶ male sterility in Arabidopsis²⁷ and altered ABA signal transduction in Arabidopsis by interfering with the sugar signaling and drastically altering sugar and amino acid balance in cells.²⁸ This indicates the importance of GAPDHs in establishing a coordination between complex metabolic and developmental interactions. Another important non-glycolytic function of plant GAPDHs involves suppression of H₂O₂-mediated cell death in protoplasts.²⁹ Modulation of this signaling function of plant GAPDHs, remains to be established. Cytosolic GAPDHs are also involved in the control of oxidative stress induced by H₂O₂ and suppression of ROS production in cells.²⁹ Increase in H₂O₂ in cells leads to modification of cysteine residues present at the catalytic sites of GAPDHs which results in its structural alterations, thereby inactivating the enzyme and rendering ineffective its participation in the signaling between H₂O₂ and phospholipases via protein-protein interactions. GAPDHs are also associated with the ROS signaling as they serve as inhibitory targets for H₂O₂. Inhibition of GAPDHs by H₂O₂ is reversible to a great extent and can be reversed by exogenous addition of glutathione.³⁰ Factors which are responsible for an increase in ROS generation in plants, like H₂O₂, ABA and dehydration, are known to contribute in an increase in the GAPDHs transcript levels in the cells.³¹

Phosphoglycerate kinase has been classified into 2 classes. One is cytosolic phosphoglycerate kinase (cPGK) and the other is plastidic phosphoglycerate kinase (pPGK). Cytosolic phosphoglycerate kinase catalyzes the reversible transfer of phosphate from the C1-position of 1,3-diphosphoglycerate (1,3-DPG) to ADP to form 3-phosphoglycerate (3-PG) and ATP, in the presence of Mg²⁺.³² It is also involved in DNA replication and repair in mammalian³³ and plant nuclei^34,35 by acting as an accessary protein to DNA polymerase-α. Chloroplastic phosphoglycerate kinase shows increased expression within 24 hr of exposure to salt in rice.³⁶

Neucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme which catalyzes the transfer of γ-PO₄²⁻ from ATP to NDP through autophosphorylation. Three types of NDPKs are found in plants. Out of these, type III is localized in the intermembranal spaces of mitochondria. It is an important enzyme for maintaining GTP levels through nucleotide homeostasis in various metabolic pathways and GTP-mediated signal transduction pathway. It also plays a role in cellular differentiation by forming complexes with β-tubulin.³⁷ During early stages of seedling growth and germination, there are marked changes in the levels of this enzyme.³⁸ It is also required for coleoptile elongation in rice.³⁹

ROS scavenging

Proteomic analysis has highlighted the stage-specific expression of ROS enzymes with catalase finding exclusive expression at the 20 DAA stage and the 2 isoforms of superoxide dismutase enzyme (Mn-SOD and Cu/Zn-SOD) expressing exclusively in 30 DAA stage of sunflower seed development. The two superoxide dismutase isoforms (Mn-SOD and Cu/Zn-SOD) possess antioxidative activity and are involved in combating the ill effects of oxidative stress induced by the formation and accumulation of ROS produced as a result of stress conditions or as a by-product of cellular metabolic events. Within a cell, they act as the first line of defense against oxidative stress by catalyzing suproxide ions and their breakdown into H₂O₂ and oxygen in different cellular loci.⁴⁰ Mn-SOD is mainly present in mitochondria⁴¹ and peroxisomes.^41,42 It is also present in the matrix of oilseed peroxisomes (glyoxysomes).^41,42 Seed longevity and germination under various environmental stress conditions can be improved by overexpression of the Cu/Zn-SOD.⁴³ Estimation of total SOD activity has revealed a peak at 30 DAA stage as compared to the 20 DAA stage.²² These observations indicate that the scavenging role of SOD at the mid-stage of seed development is more than any other ROS scavenging enzyme. Catalase (CAT) is a tetrameric enzyme responsible for the removal of the potentially toxic hydrogen peroxide produced under various stress conditions, by catalyzing the dissociation of H₂O₂ into water and oxygen.⁴⁴ CT is located mainly in glyoxysomes in mature seeds.⁴⁵ Eight catalase isoforms, (CAT1 to CAT8) have been identified in sunflower.⁴⁶ CAT plays an important role in seed development, specifically during the early stages of seed development, highlighting the modulation of its activity as a result of events associated with dehydration induced during the course of seed development as the seed starts attaining maturity. It has also been reported that CT activity is maximum in the early non-dehydrated stage of seed development which decreases with the seed maturity, when seeds are artificially dried.⁴⁴

Proteolysis and protein turnover

30 DAA stage is marked by the activation of proteolytic pathways and novel expression of associated proteins participating in these pathways for the proteolysis of unwanted proteins and protein turnover in the cell. Proteolysis and protein turnover take place via several proteases or through the ubiquitin-proteosome pathway which involves the complex of ubiquitin and 26S proteasome subunit. Among the proteases, aspartic proteinases represent one of the major enzymes involved in the proteolytic degradation of proteins. Since, aspartic proteinases are active at low pH, their expression at the 30 DAA stage marks the vacuolar targeting of proteins to be degraded. Plant aspartic proteinases are reported to be involved in the proteolysis of globular storage protein in the cocoa seeds⁴⁷ and castor beans.⁴⁸ They, thus, play an important role in protein turnover during seed development.⁴⁹

The 26S proteasome is a protease complex responsible for removing most short-lived intracellular proteins, thereby controlling many cellular processes.^50,51 It is also involved in exercising a pleotropic role in growth and development in Arabidopsis thaliana, as evident from the analysis of mutants defective in 26S proteasome and lacking rpn10-1 ubiquitin receptor. This mutant displays reduced seed germination, growth rate, stamen number, genetic transmission through the male gamete, and hormone-induced cell division, which can be explained partially by a constitutive downregulation of the key cell cycle gene CDKA;1.⁵² It has also been shown that rpn10-1 is more sensitive to abscisic acid (ABA), salt and sucrose stress and to DNA-damaging agents and has decreased sensitivity to cytokinin and auxin. Most of the phenotypes can be explained by a hypersensitivity to ABA. RPT2a, which is a component of 26S proteasome, is involved in the maintenance of root apical meristem, as has been established by mutating plants for a particular protein.⁵³

Ubiquitination of proteins is a versatile post-translational modification of protein which mediates growth and development of all eukaryotic species. Ubiquitin is a stable, highly conserved and universally expressed protein. The covalent attachment of ubiquitin to a lysine residue of select proteins can regulate stability, activity and trafficking of proteins in the cell. This post-translational modification of proteins allows efficient removal of abnormal proteins; regulate protein abundance and the activity of signaling proteins thereby rendering plant cells the ability to cope up with the various stressful conditions. It is known that plants utilize the ubiquitin proteasome pathway to combat various stressful conditions like salinity, heat, cold and drought.^54,55

Signaling

Proteomic analysis has revealed that at 20 DAA stage, developing seeds express glycine-rich RNA binding protein (GRP) which is involved in the signaling events whereas proteins like Initiation factor eIF4A and 14-3-3-like protein play a major role in the signaling events taking place in the cell in 30 DAA stage. GRP is a RNA-binding protein that harbors RRMs (RNA recognition motif) at N-terminus and a glycine rich region at C-terminus and is involved in the post-translational modification of proteins thereby regulating various biological and biochemical processes.^56,57 It functions in response to changing environmental conditions in plants by acting as RNA chaperon during cold adaption in plants. Its activity is also modulated under osmotic stress conditions. Eight GRP family members (AtGRP1–8) have been identified in Arabidopsis thaliana, with AtGRP2, −4 and −7 exercising an impact on seed germination and stress responses.^58,59 Several members of GRPs in Arabidopsis and rice have been shown to enhance seed germination, seedling growth and freeze tolerance by conferring freezing tolerance in the grp7 mutant in which the expression of AtGRP7 is suppressed and it is sensitive to cold and freezing stress.⁵⁹ Its stress responsive role has been characterized in Oryza sativa, Arabidopsis and Brassica napus.^60,61

14-3-3 protein is a phosphoserine binding protein and is involved in signal transduction and transcription, mainly facilitating protein-protein interaction. It is a monomeric protein having an acidic isoelectric point of 4–5. Important mechanisms of regulation by 14-3-3 include shuttling of proteins between different cellular locations or acting as a scaffold for larger signaling complexes thereby regulating many processes associated with plant development and their responses to various stresses.^62,63 It also acts as a heat shock related molecular chaperone and is also involved in drought stress in the regulation of 3 drought tolerant proteins in peanut (LEA protein-1, ascorbate peroxidase-1 and Ca⁺² binding protein).⁶⁴ 14-3-3 proteins have also been shown to be involved in generating the ABA response in plants thereby controlling ABA action and they are also under the control of ABA.⁶⁵ 14-3-3 proteins in plants are thought to be regulators of carbon and nitrogen metabolism.⁶⁶ They affect the carbon/nitrogen balance in plants by modulating the activities of 2 principle enzymes of the TCA cycle thereby, thereby modulating the levels of metabolites involved in TCA cycle.⁶⁷

Initiation factor eIF4A is a eukaryotic translation initiation factor involved in protein biosynthesis in the cells. It is an ATP-dependent RNA helicase and RNA-dependent ATPase in plants, animals and yeast.⁶⁸ They play a major role in initiating the translational process in plants in response to various stress conditions by forming a CAP-binding complex with other initiation factors and is also involved in removing secondary structure from the 5′ untranslated region of the mRNA, thus allowing 40S ribosome subunit binding and subsequent Kozak scanning to the first AUG codon.^69,70

Oil body biogenesis and lipid metabolism

Significant variations in the expression of proteins/enzymes associated with oil body biogenesis and modifications (elongation and desaturations) of fatty acid reserves are evident during seed development in sunflower. Oleosins are the principle oil body associated membrane proteins which are widely detected in both stages of seed development under investigation i.e. 20 and 30 DAA. Oleosins are alkaline and hydrophobic oil body membrane proteins responsible for maintaining the size and integrity of oil bodies.^71,72 They are known to exist in 2–4 isoforms of 15 to 26 kDa in Zea mays, Glycine max, and Brassica campestris.⁷³ They are involved in stabilizing the oil body structure and prevent oil body coalescence during seed desiccation phase of seed development, thereby, contributing to increasing oilseed viability during overwintering.⁷⁴ They are also involved in oil body formation and lipid accumulation.⁷⁵ One dimensional electrophoretic analysis of oleosins undertaken separately to observe the expression of oleosin isoforms accompanying seed development.

Non-specific lipid transfer proteins (LTP) are low molecular weight cysteine-rich, basic cytoplasmic proteins which facilitate transfer of phospholipids through lipid bilayers. LTP genes have earlier been found to be highly expressed early in development in embryo and cotyledons.⁷⁶ This explains their expression at 20 DAA stage in the present analysis as well. They play roles in diverse biological processes and are involved in long distance signaling, symbiosis between plants and fungi, as well as defense against fungal attacks. Their expression is induced by oxidative, cold and drought stresses^77,78 and they exhibit different developmental patterns in response to stress.⁷⁹

Fatty acid synthatase (FAS) is a complex system of fatty acid synthesis enzymes which are essential for the viability of organisms as it is required for membrane formation. Based on the enzymes involved, FAS system is subdivided into 2 categories (FAS I and FAS II). Plant fatty acid synthatase is a FAS II system where the acyl chain covalently attached to the acyl carrier protein (ACP) and is elongated with 4 enzymes catalyzing consecutively.^80,81 The principle enzymes, β-hydroxy acyl-ACP dehydratase, ketoacyl-ACP reductase and enoyl-ACP reductase, which are involved in fatty acid biosynthesis, have also been detected in the 30 DAA stage in present proteomic analysis of oil body-associated membrane proteins. Ketoacyl-ACP reductase is a member of short-chain dehydrogenase/reductase (SDR) superfamily and displays the amino acid signature of this family, Ser-X12-Tyr-X3-Lys.⁸² It catalyzes the pyridine-nucleotide-dependent reduction of a β-oxoacyl form of acyl carrier protein (ACP). β-hydroxy acyl-ACP dehydratase catalyzes the third step of FAS II system i.e., the conversion of 3-hydroxyacyl-ACP to trans-2-enoyl-AC⁸³ by 2 operating systems; FAB Z, catalyzing the dehydration of 3-hydroxyacyl-ACP into trans-2-acyl-ACP) and FAB A, catalyzing the isomerization of trans-2-acyl-ACP into trans-2-enoyl-AC).^84,85 Enoyl-ACP reductase catalyzes the last step of fatty acid elongation cycle⁸⁶ in the process of fatty acid biosynthesis. These observations indicate significant modulation of fatty acid elongation and desaturation enzymes during later phase of seed development.

Desiccation tolerance

Desiccation of cell contents during seed development is a major event at later stage of seed development. Consequently, it accompanies alterations in the expressions of dehydrin and cruciferina-like proteins. Proteomic analysis has shown stage specific expression of dehydrin and cruciferina-like proteins which are involved in desiccation tolerance at 30 DAA stage of sunflower seed development which marks the onset of dehydration so as to attain seed maturity (present work). Dehydrin belongs to group II LEA proteins and varies in amino acid composition and molecular mass. Dehydrin is widely distributed in plants. It is found in young plant organs and those exhibiting rapid cell division or cell elongation (root tips, elongating stems, and petioles). It is predominantly localized in the plant vascular system and apical meristems which is crucial for plant growth and survival. Dehydrin gets accumulated in maturing seeds or its expression is induced in vegetative tissues following salinity, dehydration, cold and freezing stress leading to cell dehydration.^87,88 Hence, it can be used as molecular marker for stress in plants tolerance.⁸⁹ Dehydrin interacts with membranes in the interior of cells and reduces dehydration induced damage⁹⁰ and prevents coagulation of macromolecules and maintains integrity of crucial cell structures by acting as chaperones to prevent the aggregation and/or inactivation of proteins under dehydration or high temperatures.^91,92,93 In addition to its role in vegetative stress tolerance, dehydrin is also important in seed survival as is evident from reduced levels of dehydrins in transgenic Arabidopsis seeds resulting in reduced seed longevity.⁹⁴

Cruciferina-like protein is a desiccation tolerant seed storage globulin and is a member of cupin-superfamily of proteins. It plays an important role in response to abiotic stress in plants. To sum up, significant and specific expression of dehydrin and cruciferina-like protein at 30 DAA stage of seed development marks the onset of dehydration phase.

Cytoskeleton

At 30 DAA stage, which is marked with seed filling, proteomic data and Confocal laser scanning microscopy (CLSM) imaging of seed sections from 2 stages of development (20 and 30 DAA) (Fig. 4) revealed enhanced expression of actin relative to 20 DAA stage (cell elongation and hydration). Actin is a protein encoded by multigene family. It is an important component of cytoskeleton which maintains the structural framework of the cell and helps in regulation of cytoplasmic streaming. The number of actin isotypes has been shown to increase with increasing complexity of the organisms. Thus, Chlamydomonas has only 2 isoforms of actin but many of the monocots (like, maize), dicots (Arabidopsis thaliana) and gymnosperms (Pinus) show presence of about 10 or more than 10 actin isoforms.⁹⁵ Actin is mainly categorized into 2 groups, vegetative and reproductive isovariants, which show variations in temporal and spatial expressions, thereby modulating different functions in various cell types. Vegetative isoforms of actin in plants mainly comprise 2 subclasses, subclass I comprised of ACT2, ACT8 and subclass II comprised of ACT7. ACT2 and ACT8 are constitutively expressed whereas; ACT7 is a highly regulated vegetative actin isoform.^96,97 ACT2 is the most abundant isoform constituting 50 % of total actin protein followed by ACT7 which constitutes 40–45 % of total actin protein and ACT8 is the weakly expressed isoform comprising of only 10–15 % of total actin protein.⁹⁷ ACT7 is the most strongly expressed actin in the aleurone layer of the seed, the seed coat, and tissues in young seedling (e.g. cotyledons, radical and hypocotyl) whereas the expression of ACT2 and ACT8 overlaps with ACT7 in most tissues, which indicates some functional redundancy among 3 actin family members. ACT7 is highly influenced by environmental and physiological cues and its levels are upregulated in response to various stress factors and hormones. It contains various conserved elements located upstream of the TATA box which are responsible for ACT7 expression in response to hormones.⁹⁸ ACT7 protein levels are up-regulated in single and double ACT2 and ACT8 mutants, thereby maintaining wild type protein levels in the mutants.

Figure 4.

(A) Localization of actin distribution by CLSM using anti-actin antibody in the 2 developmental stages (20 and 30 DAA) of seeds in sunflower. Magnification: 400X. Scale bar = 75 μm. (B) Quantification of actin signal from the marked areas of cotyledonary sections in (A).

Material and Methods

Plant material

Sunflower (Helianthus annuus L. cv KBSH-44) seeds were procured from the University of Agricultural Sciences, Bangalore (India) and plants were raised in the botanical garden of the Department of Botany, University of Delhi, during October to February. Developing seeds were harvested from the 2 peripheral whorls, 20, 30 and 40 d after anthesis (DAA). Collection of seeds was undertaken from a number of inflorescence maturing on specific dates for each stage of development. After the removal of hull, freshly harvested seeds were used for various analyses. For biochemical analyses, seeds were weighed and stored in liquid nitrogen until further use.

Extraction of urea-washed oil body membrane proteins

Urea-washed oil body membrane proteins were extracted according to David et al. (2010).⁹⁹ Seeds from 20 and 30 DAA stages (1 g each) were homogenized in Tris-urea buffer (50 mM Tris, pH 7.5, containing 9 M urea) and centrifuged at 10,000 g at 4°C for 20 min. The oil body pad obtained after centrifugation were resuspended in 2.5 ml of Tris-urea buffer and oil bodies were again recovered by centrifugation at 10,000 g at 20°C for 20 min by repeating the washing procedure thrice. Oil bodies obtained after third urea washing were resuspended in 2.5 ml of Tris-sucrose buffer (20 mM Tris, 0.2 M sucrose and pH 7.5), centrifuged at 10,000 g at 4 °C for 20 min and oil body pad was resuspended in 200 µl of the same buffer. One ml of diethyl ether was added to oil bodies suspended in Tris-sucrose buffer to wash out the lipidic constituents. Following centrifugation at 10,000 g for 15 min at 4°C, the top diethyl ether was removed and washing procedure was repeated 4 times. The residual diethyl ether was evaporated and the residue containing oil body membranes were suspended in Tris-sucrose buffer. 160 µl of each suspension was mixed with 40 µl of 10% SDS solution. Samples were then heated at 90°C in a water bath for 30 min in order to solubilize membrane proteins, and were centrifuged for 15 min at 7,000 g and at 4°C. Oil body membrane proteins in the supernatants were quantified by Markwell assay.¹⁰⁰ Each protein extract containing 150 µg of protein were mixed with reducing Laemmli sample buffer and heated in boiling water for 5 min prior to SDS-PAGE analysis. Proteins were resolved in 10–20% gradient SDS resolving gel containing 6 M urea for 1-D SDS gel electrophoresis and run at 75 V for 30 min followed by 450 V for the rest of the run time.

2D gel electrophoresis

Oil body membrane proteins were extracted as mentioned above and protein amount equivalent to 250 µg was mixed with loading buffer [8 M Urea, 2% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 200 mM Dithiothreitol (DTT), 0.002% Bromophenol blue (BPB) and 0.5% IPG Buffer] and loaded onto IPG strips (4–7 pH gradient, 11 cm, GE Healthcare, UK) which were then rehydrated with sample for 12–14 h at room temperature and subjected to isoelectric focusing (IEF) using a 6 step focusing protocol: 100 V for 4 h, 500 V for 4 h, 1000 V for 2 h, 3000 V and 6000 V for 2 h each in a gradient manner, and finally 6000 V for 2 h as a step. Following isoelectric focusing, the IPG strips were subjected to second dimensional SDS-PAGE in a 15% gel. Prior to SDS-PAGE, the strips were equilibrated for 15 min, first with equilibration buffer [50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2 % SDS and 0.002% bromophenol blue] containing 0.1% DTT and then with equilibration buffer containing 0.1% iodoacetamide. After polymerization of the resolving gel, the equilibrated IPG strip was slid on the vertical gel with the aid of electrode buffer and was sealed using 0.1% agarose solution prepared in electrode buffer. The second electrophoretic dimension was performed on a 14% SDS-polyacrylamide gel (at 75 V for 30 min followed by 450 V for the rest of the run time) using a vertical electrophoresis unit (LKB, Sweden). Proteins were visualized with colloidal Coomassie Brilliant blue G-250. The gels were analyzed by PD Quest software from Bio-Rad (USA).

Protein identification by LC-MS/MS analysis

Excised gel pieces of interest were digested with 7.5 μL of trypsin solution (Sequencing grade Modified Trypsin V511, Promega; 10 ng/μL in 5 mM ammonium bicarbonate/5% acetonitrile) for 5 h at 37°C. Digestion was stopped by the addition of 1 μL of 1% trifluoroacetic acid (TFA). The peptides were resolved using HPLC and eluted onto a resolving 10 cm analytical C18 column. The samples are loaded at 15 μL/min for 4 min, then the 44 min gradient is run at 400 nL/min starting from 2 to 35% B (98% acetonitrile, 0.1% formic acid), followed by 2 min linear gradient to 80%, and maintenance at 80% B for 4 min, and finally return to 2% in 1 min. LC-ESI-MS/MS analysis was based on Triple TOF 5600F. For database search, NCBInr database was used which is compiled by the NCBI (National Center for Biotechnology Information) as a protein database for Blast searches. It contains non-identical sequences from GenBank CDS translations, PDB, Swiss-Prot, PIR and PRF. MASCOT search engine (Matrix Science, http://www.matrixscience.com) was utilized for MS/MS ion search. The search parameters were 100–200 ppm tolerance for mass accuracy, one missed cleavage site, oxidation (Met), propionamide (Cys) and carbamidomethyl (Cys) allowed as variable modifications.

Immunohistochemical localization of actin in wax sections of seeds

Freshly harvested cotyledons of different developmental stages i.e. 20, 30 and 40 DAA, were fixed in a mixture of 0.05% glutaraldehyde and 4% paraformaldehyde (Kang et al., 2009). Fixed cotyledons were subjected to dehydration at 24°C for 1 h each in an increasing gradation of ethanol (70, 80, 90 and 100%) diluted in PBS. Cotyledons were dehydrated overnight in 100% ethanol followed by dehydration for 3 h each in 1:1 followed 1:3 proportion of ethanol: xylene, and finally in 100% xylene at 24°C for 2 hours. Tissues were cold infiltrated overnight with paraffin wax. Ten cold-infiltrated cotyledons of each stage were embedded in paraffin wax and serial sections (7 µm thick) were prepared using a rotary microtome. Slides containing dewaxed sections of different stages of development were incubated for 40 min in Blockaid blocking solution (Invitrogen, USA) and then incubated with a monoclonal Anti-Actin (plant) produced in mouse (Sigma-Aldrich Pvt Ltd, USA) diluted to 1:400 in PBS solution for 2 hours. Sections were subsequently washed thrice in PBS and then incubated for half hour in FITC (Santa-Cruz Biotechnology, USA). Sections were then mounted in glycerol and visualized using CLSM at an excitation 480 nm and emission was observed in 520 nm. The fluorescence was then quantified by ImageJ software 1.48 v (USA) for a fixed region.

All experiments were performed at least thrice.

Conclusions

Proteomic approach to analyze seed development stage specific qualitative expressions of various classes of proteins has put forward new information which can be explained further to investigate their respective physiological relevance. Recently published work from the author's laboratory (PSB, doi: 10.4161/psb.27900) has, in fact, provided significant supporting information on the modulation of ROS level and the activity of associated scavenging enzyme activities.

Support is further provided to the present proteomic analysis with regard to expression of actin (ACT8) isoforms accompanying seed development. Present work thus provides interesting information on future directions to facilitate a better biochemical understanding of seed development.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

Thanks are due to the Council of Scientific and Industrial Research, New Delhi, for the award of research fellowship to Anita Thakur.