2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

Abstract

The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves.

Introduction

Radiation exposure, both of internal and external origin, has been shown to be a highly damaging stress factor to most organisms, particularly in high doses.¹ Although some extremophile organisms (such as Deinococcus radiodurans) are known to survive in conditions of high ambient radiation, most higher-order organisms, such as plants and animals, are much more vulnerable to elevated background radiation levels. Acute radiation doses are often fatal or extremely damaging, but elevated radiation exposure over long periods can also lead to detrimental effects in the growth and development of cells, tissues/organs, and organisms as a whole. Although much effort has been applied to the study of radiation effects in mammalian models,² the molecular effects of radiation exposure on plants have not been so thoroughly studied, and most of it has been focused on plant absorption of radioactive substances and the consequences of their consumption by humans.

The multidisciplinary group of Agrawal and Rakwal and co-workers, working across disciplines, has been studying the effects of ultra-low doses of gamma radiation on plants, and our first few studies.^3,4 were focused on the morphological and molecular-genetic effects in the cereal crop/grass genome model, rice – Oryza sativa L.^5-9 Rice has also been a focus of our studies due to its tremendous importance as a food crop and nutrient source, especially in Asia. Those initial studies examined the effects of short-time (72 h) external radiation exposure to cut leaf segments from 2-week-old rice plants, using a rice seedling model system that was established to demonstrate the stress responses at molecular level.¹⁰ Our first study was a genome-wide analysis on the transcription profile of rice leaf segments exposed to ultra-low level gamma radiation emitted from a contaminated soil sample from the area surrounding the Chernobyl nuclear accident site, which revealed 516 differentially expressed genes.³ The second study replicated the experiment using an in-lab controlled source of radiation to confirm the previous study results of ultra-low level gamma radiation affecting rice self-defense mechanisms.⁴ Combined these studies provided the first evidence for ultra-low level gamma radiation triggering changes at the molecular level in the multi-layered defense/stress-related biological processes in rice leaves, laying the foundation for future studies. Meanwhile, our group has continued to look into the rice plant responses, at the physiological and molecular levels, against high dose ionizing radiation such as carbon ion beams,¹¹ gamma rays and X-rays [unpublished data by Rakwal et al.[. These published.¹¹ and unpublished data have revealed a wide ranging response (defense/stress-related) at the level of the genome in rice leaves following exposure to high-dose radiation, and which is to be expected.

The events following the 2011, 3.11 Great Tohoku Earthquake and the subsequent nuclear accident at Fukushima Daiichi Nuclear Power Plant (FDNPP) not only made research into the effects of radiation in plants significantly more relevant, but at the same time provided an opportunity to do collaborative research with fellow scientists (physicists/radiation experts) at the highly contaminated field in Iitate village of Fukushima prefecture, Japan.¹² The contaminated Iitate Farm (hereafter abbreviated as ITF) field, which was used for the re-examination of low-level gamma radiation experiments using rice as a model system, is located 31 km from the damaged nuclear power plant and presented at the time of this study, an ambient radiation level of ∼5 µSv/h, around 100 times higher than natural background radiation for Japan (∼0.05 µSv/h).¹² The experiment was performed in the summer of 2012, where the rice plants were exposed to low-level gamma radiation, being emitted from the contaminated ground without causing internal contamination, followed by an investigation into the morphological and molecular genetic changes in leaves under a dose-dependent manner.¹³ In that study, gamma radiation-exposed samples were examined by RT-PCR to reveal the differential gene expression in leaves in a time-dependent manner over 3 days. Further, the genome-wide transcriptome profiling of changed genes in the leaf was carried out in a time-dependent manner using a custom-rice whole genome 4×44K DNA microarray chip. That omics-level approach thus provided an inventory of a large number of gamma radiation-responsive genes in rice.¹³

Following on that major genome-wide study, we defined the objective of the current study as to analyze the differential expression of proteins in irradiated rice leaves at the level of the proteome. For this purpose, we employed the highly sensitive 2-D differential gel electrophoresis (2-DIGE) technique, which is a very useful and reliable method for detecting changes in expression between 2 samples of the same genome.^14,15 The general experimental strategy for proteomics.¹⁶ followed in this current study is depicted in Figure. 1.

Figure 1.

Experimental design and strategy for determining the effect of low-level dose of gamma radiation on protein expression in rice plants.

Results presented here provide not only a support to but also confirm our previous researches conducted genome-wide, in the laboratory using cut rice leaf segments (in-vitro experiments), which revealed ultra-/low-level gamma radiation induced self-defense responses in rice.^3,4 Secondly, the current research complements the first genome-wide data¹³ on rice plants response to low-level gamma radiation in a radioactively contaminated field environment by presenting new proteome-wide analysis data.

Results and Discussion

2D-DIGE analysis revealed 91 differentially expressed spots in response to low-level gamma radiation in rice seedling leaves

A side by side comparison of the 2D-DIGE images representing the control sample (RF1) and the irradiated sample (RF3), as well as a composite image of both (RF1/RF3), is shown in Figure 2A. 2D-DIGE revealed 91 spots with absolute value for RF3/RF1 higher than 1.0 (60 positive, 31 negative), and these are marked in Figure 2B. Figure 3 shows 2 examples of image comparison and quantification of the differential expression for both an up- and down-accumulated spot. The proteins, identified by mass spectrometry (MS), are listed in Table 1. Most proteins accounted for more than one spot in the gel. In all a total of 59 non-identical protein matches were found as having at least one significant differentially expressed spot. Among them, 50 of those had an overall (mean result of all spots) up-accumulation, and 9 showed down-accumulation. Further, we divided the non-identical proteins into 10 categories, according to their general protein function.^17,18 These are metabolism, energy, cell growth/division, transcription, protein synthesis/destination, transporters, cell structure, signal transduction, stress response, and unclassified, with some proteins belonging to more than one category (Fig. 4). The sum of the RF3/RF1 values for all the proteins in each category was also compiled to show how levels of expression were changed according to the metabolic function (Fig. 5). Additionally, the sum of the absolute values for RF3/RF1 was also compiled, to give an indication of how much each category was affected in its expression, independently of the positive or negative influence or expression (Fig. 6). In the case of proteins present in more than one spot, the mean value for all the individual spots was used, and proteins that belonged to more than one category contributed their value to each of them.

Figure 2.

The 2D-DIGE fluorescent labeled proteins. A) Side by side comparison of gel images showing the differently-labeled proteins from the control sample (RF1) and the irradiated sample (RF3), as well as a composite image of 2 (RF1/RF3). B) The labeled composite RF1/RF3 2D gel. Differentially expressed spots (91 in total) that were selected for extraction and MS analysis are marked.

Figure 3.

Two examples of image comparison and quantization of differential expression of a gel spot. A) Refers to Spot 11 (up-accumulated), and B) to spot 62 (down-accumulated) spot.

Table 1.

List of differentially expressed proteins identified from the protein lysate of leaves of rice plants subjected to low-level gamma radiation at Iitate, Fukushima, Japan

Protein	Accession1	Category2	RF3/RF1 (mean)3	Spots (RF3/RF1)4
Putative thiamin biosynthesis protein	gi|13435255	I	8.51	21(13.48), 22(3.54)
DEAD-box ATP-dependent RNA helicase 3, chloroplastic	RH3_ORYSJ	IV	6.22	11
Chalcone synthase 1	CHS1_ORYSJ	I/IX	4.92	41
Os02g0285800	gi|115445587	V	4.19	9
Putative protein ABIL2	ABIL2_ORYSJ	VII	4.13	43
Chloroplast 29 kDa ribonucleoprotein	gi|149392545	IV	3.44	58
Tubulin α−2 chain	TBA2_ORYSJ	VII	3.27	84(3.11), 85(3.13), 86(3.56)
RuBisCO large subunit-binding protein subunit β, chloroplastic	gi|2506277	I	3.08	28
Phenylalanine ammonia-lyase	PAL1_ORYSJ	I	2.95	23
Proteasome subunit α type-1	PSA1_ORYSJ	I	2.94	52
Cysteine protease 1	CYSP1_ORYSJ	I	2.82	73
putative chaperonin 21 precursor	gi|51090752	IX	2.66	61
Os06g0308000	gi|115467746	VI/IX	2.56	37
Lactoylglutathione lyase	LGUL_ORYSJ	IX	2.52	88
Putative NAD-malate dehydrogenase	gi|42407501	II	2.49	47
RuBisCO large subunit-binding protein subunit α, chloroplastic	gi|134102	I	2.46	26
Endoribonuclease Dicer homolog 1	DCL1_ORYSJ	IV	2.36	8(2.21), 79(2.50)
S-adenosylmethionine synthase 2	METK2_ORYSJ	I/III/IV	2.32	39
Phosphoglucomutase, cytoplasmic 2, putative, expressed	gi|108710732	II	2.27	19
Chloroplast inner envelope protein, putative, expressed	gi|110289317	X	2.17	6
Protein STAR1	STAR1_ORYSJ	VI	2.16	1(1.78), 18(2.51), 25(2.18)
Abscisic stress ripening protein 2	gi|149391461	IX	2.14	78
Tubulin α−1 chain	TBA1_ORYSJ	VII	2.10	87
RecName: Full=Heat shock cognate 70 kDa protein	gi|123650	IX	2.08	15
Ketol-acid reductoisomerase, chloroplastic	ILV5_ORYSJ	I	2.07	31(2.39), 32(1.75)
5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, putative, expressed	gi|108862992	I	2.02	12
Os02g0519900	gi|115446385	V	2.02	10
Cyanate hydratase	CYNS_ORYSJ	IX	1.99	75
Heat shock protein 70	gi|21664287	IX	1.98	14
Heat shock protein 81-1	HSP81_ORYSJ	IX	1.96	3
Nucleic acid-binding protein-like	gi|42407940	X	1.94	49
ATP-dependent zinc metalloprotease FTSH 1, chloroplastic	FTSH1_ORYSJ	IX	1.92	16
Triosephosphate isomerase, cytosolic	TPIS_ORYSJ	VII	1.87	60
Endoribonuclease Dicer homolog 3a	DCL3A_ORYSJ	IV	1.86	27
Fructose-1,6-bisphosphatase, chloroplastic	F16P1_ORYSJ	II	1.85	36
Translationally-controlled tumor protein homolog	TCTP_ORYSJ	VII/IX	1.78	70
Thioredoxin reductase NTRC	NTRC_ORYSJ	VIII/IX	1.77	33
OSJNBa0091D06.15	gi|38567873	V	1.74	4
Os01g0372700	gi|115436616	I	1.70	20
Cell division cycle protein 48, putative, expressed	gi|110289141	III	1.64	5
Os04g0118400	gi|115456914	V	1.63	13
Putative heat shock 70 KD protein, mitochondrial precursor	gi|27476086	IX	1.62	17
GSH-dependent dehydroascorbate reductase 1	gi|6939839	I/IX	1.54	64
Calreticulin	CALR_ORYSJ	V	1.53	35
ATP synthase subunit β, chloroplastic	ATPB_ORYSI	I	1.52	30
Elongation factor 1-delta 1	EF1D1_ORYSJ	V	1.51	50
Ribulose bisphosphate carboxylase small chain, chloroplastic	RBS1_ORYSJ	I	0.95	2(1.85), 24(5.36), 80(−1.59), 82(−1.84)
ATP synthase subunit α, chloroplastic	ATPA_ORYSI	I	0.61	29(1.95), 34(−2.01), 91(−1.78)
B3 domain-containing protein Os03g0120900	Y1237_ORYSJ	IV	0.52	74(−3.93), 83(4.97)
S-adenosylmethionine synthase 1	METK1_ORYSJ	I//III/IV	0.18	40(2.95), 46(−2.60)
Ribulose bisphosphate carboxylase/oxygenase activase, chloroplastic	RCA_ORYSJ	I	−0.98	45(−2.30), 48(1.51), 51(−2.14)
Pyruvate, phosphate dikinase 2	PPDK2_ORYSJ	II	−1.76	7
Ribulose bisphosphate carboxylase large chain	RBL_ORYSI	I	−1.79	42(2.18), 53(−2.14), 54(−2.44), 55(−2.36), 56(−2.18), 57(−2.20), 63(−1.80), 65(−2.04), 66(−1.85), 67(−1.96), 68(−2.04), 69(−1.59), 71(−2.57), 77(−1.76), 90(−2.07)
Germin-like protein 8-14	GL814_ORYSJ	IX	−1.86	76
Eukaryotic initiation factor 4A-1	IF4A1_ORYSJ	V	−2.16	38
Chlorophyll a-b binding protein 2, chloroplastic	CB22_ORYSJ	II	−2.38	59
Chloroplast 23 kDa polypeptide of photosystem II	gi|164375543	II	−2.55	62
2-Cys peroxiredoxin BAS1, chloroplastic	BAS1_ORYSJ	IX	−2.85	72(−2.25), 89(−3.44)
Os02g0240300	gi|115445243	IX	−6.14	44

¹ Protein accession number (GI or UniProt). ² I: metabolism, II: energy, III: cell growth/division, IV: transcription, V: protein synthesis/destination, VI: transporters, VII: cell structure, VIII: signal transduction, IX: disease/stress defense, and X: unclassified. ³ RF3/RF1 ratio as a mean of the individual values from all spots of the same protein (if more than one). ⁴ Spots in which the protein was identified, as well as RF3/RF1 ratio for each spot (if more than one).

Figure 4.

Distribution into 10 functional categories of the non-identical differentially expressed protein matches found in the gamma irradiated rice samples. Categories are I: metabolism, II: energy, III: cell growth/division, IV: transcription, V: protein synthesis/destination, VI: transporters, VII: cell structure, VIII: signal transduction, IX: stress response, and X: unclassified. Some proteins are classified in more than one category.

Figure 5.

Cumulative sum of the RF3/RF1 values for all the differentially expressed proteins each in each functional category. For proteins present in more than one spot, the mean value for all the individual spots was used. Proteins that belong to more than one category contributed their value to each of them.

Figure 6.

Cumulative sum of the absolute values of RF3/RF1 ratios for all the differentially expressed proteins each in each functional category. This visualization indicates how much each category was affected, whether positively or negatively, compared to control sample expression levels. For proteins present in more than one spot, the mean value for all the individual spots was used. Proteins that belong to more than one category contributed their value to each of them.

Functional categorization of the differential up- and down-accumulated changes in protein expressions observed in low-level gamma ray exposed rice leaves

Metabolism

The proteins involved in general cell metabolism seemed to be the ones most affected by the gamma radiation exposure, including having the most instances of up-accumulated proteins among all protein functional categories (Figs. 5 and 6). Among the up-accumulated proteins, the most significant increase in expression for an individual protein, in both the cell metabolism related protein and other category was for the putative thiamin biosynthesis protein (gi: 13435255). This protein has a role in producing thiamin (vitamin B₁), which is critical to plant metabolism. RuBisCO proteins also showed a general increase in expression. For example, the chloroplastic large subunit-binding protein, subunit α (gi:134102) and β (gi:2506277) were found to be the most up-accumulated, while the chloroplastic RuBisCO small chain (RBS1_ORYSJ) was either up- or down-accumulated depending upon the different spots from which they were identified, with lower molecular mass spots (equivalent to a small chain monomer) being down-accumulated, and higher-mass ones (in polymer form or possibly attached to large chain fragments) being substantially up-accumulated. It is possible that this is a consequence of overall increased RuBisCO accumulation and activity.

Chalcone synthase 1 (CHS1_ORYSJ) is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway, and has been shown to have a strong expression in plants under a variety of stress conditions.¹⁹ Our study also identified it as having a significantly enhanced expression in the leaves. Another protein, the GHS-dependent dehydroascorbate reductase 1 (gi:6939839) also showed an increase in expression. This enzyme, which is a part of the ascorbate and aldarate metabolism, glutamate metabolism, and glutathione metabolism, is also responsible for regenerating ascorbic acid from an oxidized state, regulating its role in responding to oxidative stress and maintaining photosynthetic function.²⁰ S-adenosylmethionine synthase 1 (METK1_ORYSJ) and 2 (METK2_ORYSJ), which is also involved in cell growth and transcription, was found to have an increased expression level.

Other metabolism-related proteins that showed increased expression were the chloroplastic ATP synthase, subunits α (ATPA_ORYSI) and β (ATPB_ORYSI); cysteine protease 1 (CYSP1_ORYSJ); homocysteine methylase (gi:108862992); chloroplastic ketol-acid reductoisomerase (ILV5_ORYSJ); Os01g0372700 (gi:115436616), a nucleotide synthetase; phenylalanine ammonia-lyase (PAL1_ORYSJ), and  proteasome subunit α type-1 (PSA1_ORYSJ).

Only two proteins in the metabolism category showed a slight down-accumulation, namely the ribulose bisphosphate carboxylase large chain (RBL_ORYSI) and the chloroplastic ribulose bisphosphate carboxylase/oxygenase activase (RCA_ORYSJ).

Energy

Three proteins involved in energy metabolism were found to be up-accumulated, though not a very high level of expression. Those were a putative NAD-malate dehydrogenase (gi: 42407501), a putative cytoplasmic phosphoglucomutase (gi:108710732), and chloroplastic fructose-1,6-bisphosphatase, (F16P1_ORYSJ). On the contrary, the category of proteins related to energy processes was the only one among other protein functions to see an overall decrease in expression. Three proteins related to energy metabolism and related to photosynthetic processes had reduced expression. The chloroplast 23 kDa polypeptide of photosystem II (gi:164375543), chloroplastic chlorophyll a-b binding protein 2 (CB22_ORYSJ), and pyruvate, phosphate dikinase 2 (PPDK2_ORYSJ) showed significant down-accumulation. We could speculate that the considerable reduction in expression of photosynthesis proteins could be linked with the withering observed at the tips of the irradiated leaves.¹³

Cell growth

Of proteins related to cellular growth, a putative cell division cycle protein 48 (gi:110289141), responsible for mediating a variety of degradative and regulatory processes.²¹ was also found to be slightly up-accumulated. To note, the aforementioned S-adenosylmethionine synthases 1 and 2, which also take part in general metabolism, also showed increased expression levels.

Transcription

Of proteins related to the transcription processes, the DEAD-box ATP-dependent RNA helicase 3, chloroplastic, showed the highest up-accumulation. DEAD-box helicases have been shown to be induced in plants under stress conditions.²² Two endoribonuclease dicer homologs, 1 (DCL1_ORYSJ) and 3a (DCL3A_ORYSJ), also showed increased expression. Interestingly, these proteins have a function to terminate the transcription at sites damaged by stress conditions.²³ A chloroplast 29 kDa ribonucleoprotein (gi:164375543) was also found to be up-accumulated, and the B3 domain-containing protein Os03g0120900 (Y1237_ORYSJ) had a small overall increase in its expression level.

Protein synthesis and destination

Of the proteins involved in protein synthesis and destination, 2 hypothetical proteins Os02g0285800 (gi:1154455870), which is homologous to tyrosine phosphorylated protein A (TypA)/BipA family (belongs to ribosome-binding GTPases) and Os02g0519900 (gi:115446385), which is similar to elongation factor EF-2 Os04g0118400 (gi:115456914), had the largest increase in expression. Another not well characterized rice protein, OSJNBa0091D06.15 (gi:38567873) (Q7XQQ7_ORYSJ; having GTPase activity) was also found to be slightly up-accumulated. Calreticulin (CALR_ORYSJ), a protein responsible for tagging misfolded proteins as well as regulating transcription, was also slightly up-accumulated. Interestingly, elongation factor 1-delta 1, of which the human variant has been shown to be up-accumulated in the presence of electromagnetic radiation,²⁴ also showed increased expression level.

The only protein related to protein synthesis processes that showed down-accumulation was the eukaryotic initiation factor 4A-1 (IF4A1_ORYSJ). The gene expressing this protein has been shown to be stably expressed during drought and salt stress, but not necessarily during oxidative or other types of stress.²⁵

Transport

Of the proteins related to transport activity, the STAR1 protein (STAR1_ORYSJ), a transmembrane protein usually associated with response to aluminum toxicity,²⁶ and the Os06g0308000 protein (gi: 115467746; also involved in the stress response), were found to be up-accumulated.

Cell structure

Our results revealed that several structural proteins were up-accumulated. The highest up-accumulation was found for the putative protein ABIL2 (ABIL2_ORYSJ) and the tubulin α−1 (TBA1_ORYSJ) and −2 (TBA2_ORYSJ) proteins. Cytosolic triosephosphate isomerase (TPIS_ORYSJ) and a translationally-controlled tumor protein homolog (TCTP_ORYSJ), which has been shown to be up-regulated in response to DNA damage,²⁷ also showed increased expression levels.

Signaling

Thioredoxin reductase (NTRC_ORYSJ), a protein involved in cell signaling and oxidative stress defense,²⁸ was found to be up-accumulated.

Stress response

After the general metabolism related proteins, proteins involved in stress responses were the next most affected functional category both in the overall positive increase (Fig. 5) and in total change of expression levels (Fig. 6). Several heat shock proteins, the most common stress/defense-related proteins in plants, including 3 70 kDa proteins (gi:123650, gi:21664287, and gi:27476086), and a 81 kDa protein (HSP81_ORYSJ) were up-accumulated. Abscisic stress ripening protein 2 (gi:149391461), a protein responsible for ripening and biotic and abiotic stress response,²⁹ and the chloroplastic ATP-dependent zinc metalloprotease FTSH 1, which has been suggested to promote the cleavage of photosystem II proteins under heat stress,³⁰ were also up-accumulated. Another induced protein was the cyanate hydratase (CYNS_ORYSJ). The finding of this protein is interesting because the plant cyanases may play diverse roles under different physiological conditions; it has been suggested to be involved in physiological processes of ethylene, arginine and pyrimidine and thus having potential roles in developmental processes and stress responses.³¹ The individual proteins with the highest comparative increase in expression, however, were a putative chaperonin 21 (gi: 51090752) precursor and lactoylglutathione lyase (LGUL_ORYSJ).

Of the down-accumulated stress response proteins, the most strongly affected – and in fact the most down-accumulated protein identified in this study – was the Os02g0240300 (gi:115445243), and which appears to be a type of peroxidase. Additionally, the 2-cys peroxiredoxin BAS1 (BAS1_ORYSJ), an enzyme tasked with protecting the chloroplast against oxidative damage,^32,33 was also significantly down-accumulated. The down-accumulation of proteins involved in protecting the cell, especially the chloroplasts against oxidative damage, a very serious consequence of ionizing radiation, is curious, but consistent with the reduced levels of photosynthesis proteins previously mentioned, as well as the withering of leaf tips after 3 days of gamma radiation exposure, it is also consistent with the findings of our previous study, where a significant down-regulation of genes responsible for peroxide stress response was found.¹⁰ The germin-like protein 8–14 (GL814_ORYSJ), which might play a role in plant defense, was also found to be slightly down-accumulated.

Unclassified

Other unclassified, and up-accumulated proteins were a putative chloroplast inner envelope protein (gi:110289317), and a nucleic acid-binding protein (gi:42407940).

Concluding remarks

By using the high-throughput proteomic 2D-DIGE approach, a novelty of this study, we were able to identify 91 differentially expressed protein spots in the leaves of rice seedling subjected to ambient low-level gamma radiation from a site (field) heavily contaminated by radionuclides from the nuclear (FDNPP) accident site (Fukushima). Utilizing MS analysis, the proteins in these spots were identified, resulting in a total of 59 non-identical proteins among the differentially expressed proteins. The identified proteins were divided into 10 functional categories, as a means to better visualize which aspects of the rice plant (leaf) metabolism was most affected by the exposure to gamma radiation. The results of this analysis complement the ones previously obtained in the context of genome and transcriptome.¹³ by adding a proteome dimension as well.

From the identification and analysis of the differentially expressed proteins, it was shown that overall the majority of the differentially expressed proteins were up-accumulated, and that general cell metabolism (nucleotide metabolism, etc.) proteins were the most significantly up-accumulated, while energy metabolism (carbohydrate metabolism, photosynthesis, etc.) ones were more negatively affected, particularly those in the photosynthesis process. Moreover, stress and defensive proteins were generally up-accumulated, but curiously, some key proteins for defense against oxidative stress had their expression greatly reduced, even though this is one of the most likely types of stress damage likely to occur in the presence of ionizing radiation. This, however, would be consistent with the resulting reduction in levels of photosynthesis and the withering observed at the tip of the gamma irradiated leaves, possibly indicating that rice seeding is not very apt at responding efficiently to long term low-level gamma radiation conditions. However, we are cautious to this interpretation as long-term exposure experiments over the life cycle of the rice plant would need to be performed. This is the second paper in a series of research reports that will examine and present data on how rice plants behave to low-level gamma radiation directly in the field (in Iitate village, Fukushima). The first paper was focused on gene expression,¹⁰ and many of the results found in that work at the transcriptome level were confirmed at proteome level here, such as an increase in expression of stress-/defense-related proteins, with the particular exception of the peroxide response. An increase in general and secondary metabolism, which could be seen as supporting the stress-/defense-related activity, is also observed in both the data, as well as an apparent increase in the presence of RuBisCO. One differing result was the down-accumulation found for several photosynthesis proteins, which might being negatively affected at translational or metabolic level, although not all chloroplast proteins (such as RuBisCO) seem to be as much affected. Moreover, it should be also pointed out that although increased in relation to natural levels, the low levels of gamma radiation present here (at ITF) are not expected to cause dramatic metabolic changes that may result in major visible changes, and that other than some small amount of withering at the tip of the 3^rd leaves, the sample rice plants remained in an overall healthy state.

Finally, we would like to reiterate the reason for conducting such an experimental under field conditions at a highly contaminated village, Iitate in Fukushima prefecture, in that rice plant can serve as a human model for examining the effects of short and/or long-term effects of low-level gamma radiation consequent to a nuclear accident (at FDNPP), such as the present case of 3.11 Great Tohoku Earthquake. Moreover, current and previous study,¹³ serves as a baseline for future research on clarifying the effects of radiation exposure to plants and maybe through the obtained molecular information, clues to how human tissues may react or respond at the level of the gene, protein and metabolite as the biological pathways and networks in organism such as rice in general may be similar, but not identical, to that in other living organisms, like humans. Nevertheless, it should be emphasized that the relevance of our results will not be easy to correlate with humans as that will require controlled experiments in humans, and which cannot be done. Therefore, we continue to look deeply into the biological effects via omics approaches to get new information on how living organisms, like our grass model rice, respond to ionizing radiation at the molecular level. The next step in our research currently underway is the investigation of the effects in the next generation, the rice seed.

Materials and Methods

Rice seedling growth, transport, and low-level gamma ray exposure

Preparation and irradiation of the rice samples was carried out as detailed by Hayashi and coworkers.¹³ Briefly, seeds of Japonica-type rice (Oryza sativa L.) cv Nipponbare were grown in the greenhouse facility National Institute for Environmental Studies (NIES, Tsukuba, Japan) during July, August, and September 2012. Fourteen days after start of the germination protocol, healthy rice seedlings were transported to the designated experimental site at ITF (in Iitate village, Fukushima, Japan) for initiating the experiment. Leaves were sampled upon reaching the experiment site (referred to as the start of the experiment when the rice plants were first directly exposed to the gamma radiation (0 h ITF sample) at the test field. The low-level gamma field was defined as a level-ground area of cesium-contaminated soil overlaid with a blue tarpaulin sheet, where ground radiation levels were measured at a constant dose of ∼5 µSv/h.

Briefly, 3 cylindrical boxes containing the rice seedlings were placed at a distance of 2 m apart, 2 of the boxes were shielded to control the amount of radiation reaching inside to the rice seedling. The gamma ray dose was recorded by 2 MYDOSE mini electronic pocket dosimeters placed near the 3^rd fully formed leaves. The 3^rd leaf has been an established and stable sampling tissue in all our studies involving the 2-week-old rice seedling model system.¹⁰ The second box, a single shield resulting in exposing rice to 190 µSv/3 days (∼2.6 µSv/h), was the sampled material (3^rd leaf) used in this study. Further details are in Hayashi et al.¹³

Sample collection and preparation

Several gamma ray exposure times for the rice leaf samples were set as described in Hayashi et al.,¹³ but for this study only the 72 h samples were used to specifically investigate the proteome-wide changes. Six to 10 seedlings were sampled, by cutting the 3^rd fully formed leaf at the base of attachment to the sheath. Cut leaves were placed in aluminum foil and stored packed in dry ice and placed in a deep freezer set at −30°C. Control rice leaves were sampled from from healthy rice seedlings at the time of arrival at ITF. Further details are in Hayashi et al.¹³ The samples were prepared as described in Agrawal et al.¹⁶ Individual leaves taken from each 72 h exposed seedling and the 0 h control were separately ground into a fine powder with a pre-chilled mortar and pestle in liquid nitrogen, and stored at −80°C until further analysis was performed.

Protein lysate preparation

Standard LB-TT extraction.¹⁶ was used, and lyophilized powders were prepared (Fig. 1). The lyophilized powders were then dissolved in 300 µL 2-D cell lysis buffer (30 mM Tris-HCl, pH 8.8, containing 7 M urea, 2 M thiourea and 4% CHAPS) was added, and then subjected to sonication while on ice, followed by keeping the tubes on the shaker for 30 min at room temperature. The tubes were then spun for 30 min at 4°C at 25000 g and the supernatant was collected. Protein concentration was determined using the Bio-Rad protein assay method. The lysate samples were diluted with the 2-D cell lysis buffer to the same protein concentration at 5 mg/mL.

Minimal Cy dye labeling

The 2D-DIGE work was performed by Applied Biomics (Hayward, CA). Briefly, to 30 µg of protein lysate, 1.0 µL of diluted CyDye (1:5, diluted with DMF from 1 nmol/µL stock) was added, followed by vortex mixing and then keeping the tube in the dark on ice for 30 min. Next, 1.0 µL of 10 mM lysine was added to each of the samples, vortexed and left to incubate under darkness on ice for an additional 15 min. Cy2 and Cy3 labels were added to the samples, followed by 2× concentrated 2-D sample buffer (8 M urea, 4% CHAPS, 20 mg/mL DTT, 2% pharmalytes and trace amount of bromophenol blue, BPB), and then by 100 µL destreak solution and rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/mL DTT, 1% pharmalytes and trace amount of BPB) to 350 µL for application to the 18 cm IPG strip. Labeled samples were mixed by vortexing and spun down before loading into the strip holder.

IEF and SDS-PAGE

After loading the labeled samples into the strip holder, the 18 cm strip was laid facing down, and 1.5 mL mineral oil was added on top of strip. The provided protocol (Amersham BioSciences) was then followed, letting the IEF run under dark at 20°C. Upon completing the IEF, the IPG strips were incubated in freshly made equilibration buffer 1 (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of BPB and 10 mg/mL DTT) for 15 min with slow shaking. Then the strips were rinsed in freshly made equilibration buffer 2 (50 mM Tris-HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of BPB and 45 mg/mL iodacetamide) for 10 min with slow shaking. The IPG strips were then rinsed once in the SDS-gel running buffer before transferred into the SDS-gel (12% SDS-gel prepared using low fluorescence glass plates) and sealed with 0.5% (w/v) agarose solution (in SDS-gel running buffer). The SDS-gels were run at 15°C until the dye front starts running out of the gels.

Image scan and data analysis

Image scans were carried out immediately following the SDS-PAGE using Typhoon TRIO (GE Healthcare) following the protocols provided. The scanned images were then analyzed by Image QuantTL software (GE-Healthcare), and then subjected to in-gel analysis and cross-gel analysis using the DeCyder software version 6.5 (GE-Healthcare). The intensity change of the protein differential expression was obtained from in-gel DeCyder software analysis. Proteins with changes in expression levels of at least 1.5-fold were selected. Comparing the 2 samples, an RF3/RF1 value consisting of the absolute intensity of a spot in RF3 minus the intensity of its counterpart in RF1, was obtained for each spot. Therefore, a positive value indicates an increase in expression, and a negative value indicates a decrease in expression. The average ratio value indicates the standardized volume ratio between the 2 groups or populations. Values are displayed in the range of −∞ to –1 for decreases in accumulation and +1 to +∞ for increases in accumulation. Values between −1 and 1 are not represented, hence a 2-fold increase and decrease is represented by 2 and –2, respectively (not 2 and 0.5).

Spot picking and trypsin digestion

The spots of interest were picked up by Ettan Spot Picker (GE Healthcare) based on the in-gel analysis and spot picking design by DeCyder software. The gel spots were washed a few times, and digested in-gel with modified porcine trypsin protease (Trypsin Gold, Promega). The digested tryptic peptides were desalted by Zip-tip C18 (Millipore). Peptides were eluted from the Zip-tip with 0.5 µL of matrix solution (-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25 mM ammonium bicarbonate) and spotted on the MALDI plate.

Mass spectrometry

MALDI-TOF (MS) and TOF/TOF (tandem MS/MS) were performed on a 5800 mass spectrometer (AB Sciex). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 2000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 2000 laser shots per fragmentation spectrum on each of the 10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions).

Database search

Both the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer version 3.5 equipped with MASCOT search engine (Matrix science) to search the database of National Center for Biotechnology Information non-redundant (NCBInr). Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage allowed in the search parameters. Candidates with either protein score C.I.% or Ion C.I.% greater than 95 were considered significant.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors appreciate the help of Mr. Katsumi Matsumoto (NIES, Tsukuba) for managing the growth of the rice seedlings used in these experiments. The authors thank the people of Iitate village (Fukushima) and all other people involved in this study at various parts of the experiment for their support and encouragement, without which this work could not have seen light. The authos also appreciate the support of Iitate-mura Society for Radioecology (IISORA) (http://iitate-sora.net/).