3.1. Protein Expression on SDS-PAGE
SDS-PAGE was performed in order to profile proteins from the pollen and pistil. Pollen and pistil were collected from wild type W22 (ga1) and near-isogenic lines W22 (Ga1), respectively. Pollen lanes were well separated whereas pistil lanes were not clearly visual (
Figure 1,
Supplementary Figure 1). However, high performance LTQ-FTICR MS was used to excise the gel into 10 pieces.
Figure 1.
SDS-PAGE pattern in the pollen and pistil of W22 (ga1; Ga1) in maize. Samples were analyzed triplicate as described in the method section and gels were stained using Coomassie Brilliant Blue (CBB) staining. Standard molecular weight (kDa) is on the left.
Figure 1.
SDS-PAGE pattern in the pollen and pistil of W22 (ga1; Ga1) in maize. Samples were analyzed triplicate as described in the method section and gels were stained using Coomassie Brilliant Blue (CBB) staining. Standard molecular weight (kDa) is on the left.
3.5. The Implication of Differentially Expressed Proteins from Pollen and Pistil of Maize
A total of 44 proteins were identified from the pollen and pistil of W22 (ga1) and W22 (Ga1) of which 24 proteins were confirmed from the pollen of W22 (ga1) and W22 (Ga1) and 20 proteins from the pistil of W22 (ga1) and W22 (Ga1). However, two proteins were identified from the pollen of W22 (ga1) whereas albumin b-32 (32.4 kDa, p
I 5.38) regarded as the protein of maize endosperm that is a monomeric albumin with an apparent molecular weight of about 32 kDa with a p
I of 5.38. Di Fonzo
et al., 1988 [
21] found that the two variants expose similar amino acid composition but minor differences are appeared by their tryptic peptide maps. They also noticed that the protein is localized in the soluble part of the cytoplasm and does not bind to any particular structure.
In addition, 12 proteins were identified only from the pollen of W22 (Ga1). However, ATP synthase subunit alpha (55.1 kDa, p
I 5.85) and ATP synthase subunit beta (59.0 kDa, p
I 6.01) were confirmed in our investigation. ATP synthesis is membrane-bound enzyme complexes/ion transporters that accelerate ATP synthesis and/or hydrolysis with the transport of protons across a membrane. It can harness the energy from a proton gradient, using the flux of ions across the membrane via the ATPase proton channel to drive the synthesis of ATP. The alpha/A and beta/B subunits can each be divided into three regions, or domains, centered on the ATP-binding protein, and based on structure and function. The central domain contains the nucleotide-binding residues that make direct contact with the ADP/ATP molecule [
22]. 1-Cys peroxiredoxin PER1 (24.8 kDa, p
I 6.31) was considered as the antioxidant protein which seems to contribute to the inhibition of germination during stress. It was prevailed that overexpression of rice 1-cys-peroxiredoxin in transgenic tobacco accelerated oxidative stress tolerance, but dormancy was not affected [
23]. Glucose-6-phosphate isomerase (62.1 kDa, p
I 6.96) was identified in the pollen of W22 (Ga1) that catalyzes the conversion of glucose-6-phosphate into fructose 6-phosphate in the second step of glycolysis. This protein has various functions inside and outside the cell. This protein is also involved in the glycolysis and gluconeogenesis within the cytoplasm, while outside the cell it acts as a neurotrophic factor for spinal and sensory neurons. In
Ananas comosus, it was prevailed that the mitochondria may produce this protein to allow cytoplasmic conversion of glucose-6 phosphate into fructose-6 phosphate in the second step of glycolysis [
24]. ADP, ATP carrier protein 2 (42.3 kDa, p
I 9.85) was found in the pollen of W22 (Ga1) that catalyzes the exchange of ADP and ATP over the mitochondrial inner membrane. An ADP/ATP carrier protein was found in
K. pinnata mitochondria. The protein may be employed in the mitochondrial energy synthesis in which ATP synthase provides ATP via oxidative phosphorylation, and may work in reverse as a proton-pumping ATPase. It was revealed at
K. pinnata that ADP and ATP could sustain via ADP/ATP carrier proteins between mitochondrial membranes and other organelles [
24]. Cysteine synthase (CS) was identified in the pollen of W22 (Ga1) with molecular weight 34.1 kDa and p
I 5.91. CS catalyzes the biosynthesis of cysteine in plants [
25]; cysteine acts as a precursor for the synthesis of various sulfur containing metabolites [
26], whereas glutathione represents the most important one which employed as a universal antioxidant and detoxifier for coping with various stresses [
27]. Ferredoxin-dependent glutamate synthase (175 kDa, p
I 6.21) was identified in the pollen of W22 (Ga1) that is involved in metabolic function especially in nitrogen assimilation. In Arabidopsis, the increase of ferredoxin dependent glutamate synthase is probably a consequence of limited electron transport and may affect feedback regulation to compete for electrons required for nitrogen assimilation [
28].
In pistil, 10 proteins were identified in the W22 (ga1). However, catalase isozyme 1 (24.8 kDa, p
I 6.31) was detected in the W22 (ga1) that occurs in almost all aerobically respiring organisms and serves to protect cells from the toxic effects of hydrogen peroxide. In the early stage of drought stress, catalase activities were found to increase or be stable, and then decrease with further increase in magnitude of water stress [
29]. Furthermore, catalase isozyme 1 was only increased at 10 DPA under drought stress in Kauz which indicated that catalase might be activated to diminish toxic compounds during the early stage while the plant acclimatize the drought stress [
30]. However, the catalase isozyme 3 (56.7 kDa, p
I 6.47) was identified in the pistil of W22 (ga1) and it occurs also in almost all aerobically respiring organisms and serves to protect cells from the toxic effects of hydrogen peroxide. Its levels are highest in the light period and are lowest in the dark period. Therefore, it may be important for scavenging hydrogen peroxide at night, rather than during the day. Acetolactate synthase 2 (68.9 kDa, p
I 6.48) was confirmed in the pistil of W22 (ga1). The acetolactate synthase (ALS) enzyme is a protein observed in plants and micro-organisms. ALS catalyzes the first step in the synthesis of the branched-chain amino acids (valine, leucine, and isoleucine) [
31]. This protein is well known enzyme that is involved in catalytic activity, especially a part of the biosynthesis of various amino acids. However, in plants, it is located in the chloroplasts to assist the metabolic processes. It has been found in several experiments that mutated strands of
Escherichia coli K-12 without the enzyme were not able to grow in the presence of only acetate as the only carbon sources [
32]. Asparagine synthetase (66.5 kDa, p
I 5.83) is an enzyme that generates asparagine from aspartate and arises only in the pistil of W22 (ga1). This reaction is similar to that accelerated by glutamine synthetase. It is also possible that asparagine synthetase poses its effects by fulfilling an as yet unknown function in the cell that is independent of its catalytic activity [
33]. 14-3-3 like protein GF14-6 (29.6 kDa, p
I 4.76) was confirmed in the pistil of W22 (ga1) that is associated with a DNA binding complex to bind to the G box, a well-characterized
cis-acting DNA regulatory element found in plant genes. The functional properties of 14-3-3s are to bind and activate tyrosine and tryptophane hydroxylase in bovine brain in the presence of Ca
2+/calmodulin-dependent protein kinase type II [
34].
However, seven proteins were detected only in the pistil of W22 (Ga1). Glyceraldehyde 3-phosphate dehydrogenase (36.4 kDa, p
I 7.01) were identified from the pistil of W22 (ga1) that catalyzes the conversion of glyceraldehyde 3-phosphate to D-glycerate 1, 3-bisphosphate. In soybean, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as down-regulated at both the mRNA and protein levels in response to NaCl treatment, suggesting that it plays a role in salt stress and can be used as a target gene in soybean seedlings [
35]. The main role of this gene is the tolerance and its relationship to improving salt tolerance in plants [
36]. It is revealed that the ATP production will be reduced by the down-regulation of glyceraldehydes-3-phosphate dehydrogenase and eventually there will be a decrease in plant growth under salt stress. Late embryogenesis abundant protein EMB564 (9.6 kDa, p
I 6.6) constitutes a set of proteins that participate in plant stress responses. During exposure to abiotic challenges, late embryogenesis abundant (LEA) proteins accumulate naturally in desiccation-tolerant structures, such as seed or pollen grains, and in plant vegetative tissues. However, Emb564 acts for displaying a complex combination of different PTMs, including phosphorylation, acetylation, methylation and deamination in the native protein, which may be relevant for its seed-specific role [
37].
Furthermore, 10 proteins were detected from the pollen which shared both W22 (ga1) and W22 (Ga1) and three proteins were shared from both W22 (ga1) and W22 (Ga1) of pistil. Profilin-3 (14.2 kDa, p
I 4.91) was confirmed in the pollen of both W22 (ga1) and W22 (Ga1). Profilins generate a large and diverse protein family. Multiple isoforms of profilins are available in many species, being encoded by separate genes, or in some cases translated from mRNA splice variants. In the case of animals and higher plants, isoforms may be exposed in a tissue-specific manner. Moreover, profilins are identified at different subcellular locations [
38]; in particular, enrichment of the dynamic plasma membranes was ascertained for various cells types. Also, profilins were investigated in association with internal membranes that implicated in vesicular transport [
39]. It revealed that the overall functional properties of different profilins are similar and eventually, one isoform can be interchanged with another one from quite a distant source [
40]. Endochitinase A (29.3 kDa, p
I 8.3) was identified from both of the pollen of W22 (ga1) and W22 (Ga1) that defends against chitin containing fungal pathogens. In
Arabidopsis thaliana, a basic endochitinase (At3g12500) was confirmed that was involved in the ethylene/jasmonic acid-mediated signaling pathway during systemic acquired resistance [
41].