Biochemical analysis of ‘kerosene tree’ Hymenaea courbaril L. under heat stress

Abstract

Hymenaea courbaril or jatoba is a tropical tree known for its medically important secondary metabolites production. Considering climate change, the goal of this study was to investigate differential expression of proteins and lipids produced by this tree under heat stress conditions. Total lipid was extracted from heat stressed plant leaves and various sesquiterpenes produced by the tree under heat stress were identified. Gas chromatographic and mass spectrometric analysis were used to study lipid and volatile compounds produced by the plant. Several volatiles, isoprene, 2-methyl butanenitrile, β ocimene and a numbers of sesquiterpenes differentially produced by the plant under heat stress were identified. We propose these compounds were produced by the tree to cope up with heat stress. A protein gel electrophoresis (2-D DIGE) was performed to study differential expression of proteins in heat stressed plants. Several proteins were found to be expressed many folds different in heat stressed plants compared to the control. These proteins included heat shock proteins, histone proteins, oxygen evolving complex, and photosynthetic proteins, which, we believe, played key roles in imparting thermotolerance in Hymenaea tree. To the best of our knowledge, this is the first report of extensive molecular physiological study of Hymenaea trees under heat stress. This work will open avenues of further research on effects of heat stress in Hymenaea and the findings can be applied to understand how global warming can affect physiology of other plants.

Keywords:

• biofuel
• heat stress
• Hymenaea
• protein
• sesquiterpene
• thermotolerance

Abbreviations

2D-DIGE

=

2D-difference in gel electrophoresis; GC-MS, gas chromatography – linked to mass spectrometer; GO, gene ontology; HS, heat stressed; HSPs, heat shock proteins; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MAPK, mitogen-activated protein kinase; OEC, oxygen evolving enhancer protein; PP2C, protein phosphatase 2C; PSI, photosystem I; PSII, photosystem II; RuBP, ribulose 1, 5-bisphosphate; ROS, reactive oxygen species; RT, retention time; SN, serial number; TMV, tobacco mosaic virus; VOC, volatile organic compounds

Introduction

Hymenaea courbaril L., popularly known as “jatoba” in Brazil, is a tropical leguminous tree species found commonly in semi-deciduous forests in South America. Out of 14 species of genus Hymenaea (Fabaceae), 9 are known to be found in different regions including lowland tropical ecosystem of Brazilian Amazon.^1,2 Almost all Hymenaea courbaril L. plant parts including roots, leaves, fruits and particularly stem barks are used traditionally for medicine and folk-medicine. Decoctions and infusions of this plant parts are used to treat wide range of diseases such as anemia, kidney problems, sore throat, bronchitis and asthma.³ This plant is also used as an important tree in timber industry.⁴

Climate change causing heat stressed conditions is a serious threat for agriculture and crop productivity. Oxygen, hydrocarbons, and chemicals emitted by the trees are collectively known as VOCs (Volatile Organic Compounds).⁵ The increasing environment temperature also affects the volatile compounds production by the trees. Plants can emit 400–800 Tg C/year (1 Tg = 10³² g) as hydrocarbons.⁶ Various terpene molecules produced by heat stressed Hymenaea plants were identified in this study. Isoprene, a hemiterpene, emitted by plants has environmental effects in creation of ozone molecules and growth of aerosols.⁷ It is well documented that heat stress in plants stimulate a cascade of biochemical reactions leading to ‘fright or flight’ mechanism, i.e., enhanced thermotolerance or succumb to heat stress (Reviewed by Wahid et al., 2007).⁸ In this study, we attempt a comprehensive study on biochemical responses of Hymenaea plant at biomolecular level. Here, we report a number of proteins that are differentially expressed during heat stress in Hymenaea plants. Moreover, we have also performed an analysis on total lipids and volatiles produced by this tree under the heat stress condition. The research data produced will help to understand molecular physiology of thermotolerance mechanisms in this economically important, but considerably understudied tree species. The understudied status of this tree is evidenced by PubMed search with the keyword ‘Hymenaea’, which resulted in only 76 hits.

Terpenes have potentials to be used as biofuel precursors^9,10 and in our study we have found out that Hymenaea produces many long chain sesquiterpene hydrocarbons. Nobel laureate Dr. Melvin Calvin while working on ‘diesel tree’ (Copaifera sp.)¹¹ reported that this tree produces sesquiterpenes, which could be used as diesel fuel in automobiles without any filtration. Meylemans et al. (2012)¹² produced high-density renewable fuel by dimerization of crude turpentines and other chemical compounds. Hymenaea is known as ‘kerosene tree’ for its ability to produce fuel-like compounds.¹³ We think that this tree may have potentials to be used as a future biodiesel-producing tree because of diversity of terpenes produced by this tree, and thus new knowledge generated via this study is of great interest. However, further research is needed to justify the biofuel potentiality of this tree.

Results

Volatiles study

Three different peaks were seen in case of heat stressed plant samples at retention time (RT) of 1.62, 2.32 and 5.29 min respectively (Fig. 1). The first, second and third peaks have shown a 95%, 90% and 92% similarity match with isoprene, 2-Methylbutanenitrile and β-ocimene respectively to NIST/EPA/NIH Mass Spectral Library (Data version: NIST 11, Software version 2.0). While in case of control plant only one peak at RT of 1.62 min has occurred (Fig. 1). This experiment was performed with triplicate of biological samples and all triplicates in both control and heat stressed have shown the first peak but peaks in heat stressed samples were significantly higher than in controls one. The second peak was observed only in 2 out of 3 heat stressed plant samples and the third peak occurred in all heat stressed plant samples but neither second, nor third peak was observed in the control plant samples.

Figure 1. Showing a comparison of volatiles production from control (black) and heat stressed (red). Peaks 1, 2, 3 are isoprene, 2-methyl butanenitrile and β-Ocimene respectively as compared with >90% similarity match to NIST/EPA/NIH Mass Spectral Library, Data version: NIST 11, Software version 2.0. Above peaks were confirmed with 3 biological samples.

Lipid analysis

Multiple peaks for total lipid analysis from both control and heat stressed plant were observed. Most of these peaks were found to be sesquiterpenes with few diterpenes in both cases except for a phenol peak at RT of 4.60 min. This peak was only observed in total lipid extract of control plant (Fig. 2). Although, the compounds were same, their production was found to be differential in both cases. Sesqueterpens, α-cubebene, copaene, germacrene D, gamma-elemene, cadina-1(10), 4-diene and germacrene-D-4-ol which appear at RT of 8.45, 8.76 9.77, 9.90, 10.02, 10.58 min respectively (Fig. 2) found to be produced in significantly higher amount in heat stressed than control plant. Other sesquiterpenes like β-elemene, caryophyllene, α-guaiene, humulene and α-Selinene which appear at RT of 8.85, 9.22, 9.28, 9.55 and 9.88 min respectively (Fig. 2) were found to be same in both conditions. An α-gurjunene peak at RT 9.10 minute was found to be reduced in heat stressed conditions. Two peaks at RT of 12.28 and 12.59 min (Fig. 2), which found to be phytol acetate and phytol were produced in higher amount in heat stressed than control plant. A peak for octacosane was also observed at RT of 17.32 min (Fig. 2), production of which found to be same in both case.

Figure 2. Showing comparison of peaks produced by total lipid extracted from control (black) and heat stressed plant (red) through GC-MS. Only the peaks having >90% similarity with the GC-MS NIST/EPA/NIH Mass Spectral Library, Data version: NIST 11, Software version 2.0 were shown here. In the above figure each part is with different magnification to clearly show the differences. The peaks 1 to 16 are phenol, α- Cubebene, Copaene, β-Elemene, α-Gurjunene, Caryophyllene, α-Guaiene, Humulene, Germacrene D, α-Selinene, gamma-Elemene, Cadina-1(10),4-diene, Germacrene-D-4-ol, Phytol acetate, Phytol and Octacosane. The differences in peaks were inferred with triplicate of technical replications.

Protein analysis

Differential protein expressions between control and heat stressed leaf tissue were examined based on 2D-DIGE analysis. Based on this in-gel analysis, a total of 151 spots were identified as differential expressed (Fig. 3). All the spots were put for student's t-test, and based on significance (p-value, see supplemental information) 57 spots were selected for protein identification via MALDI-TOF (Table 3). These 57 identified proteins were further evaluated by GO annotation using Uniprot and NCBI databases, and were categorized according to the various metabolic processes that they are predicted to be involved in (Fig. 4).

Table 1. Volatile compounds produced by heat stressed (HS) Hymenaea plants

SN	Compounds	Chemical formula	RT in min	Control	HS	% Similarity
1	Isoprene	C5H8	1.62	+	++	95
2	2-Methylbutanenitrile	C5H9N	2.32	−	+	90
3	β-ocimene	C10H16	5.29	−	+	92

Table 2. Lipid analysis of heat stressed (HS) Hymenaea plant as identified by GC MS

SN	Compounds	Chemical formula	RT in min	Control*	HS*	% Similarity
1	Phenol	C6H6O	4.60	+	−	92
2	α-Cubebene	C15H24	8.45	+	++	93
3	Copaene	C15H24	8.76	+	++	95
4	β-Elemene	C15H24	8.85	+	+	94
5	α-Gurjenene	C15H24	9.10	++	+	93
6	Caryophyllene	C15H24	9.22	+	+	96
7	α-Guaiene (Azulene)	C15H24	9.28	+	+	94
8	Humulene (α-caryophylene)	C15H24	9.55	+	+	96
9	Germacrene D	C15H24	9.77	+	++	94
10	α-Selinene	C15H24	9.88	+	+	94
11	gamma-Elemene	C15H24	9.90	+	++	92
12	Cadina-1(10),4-diene	C15H24	10.02	+	+	94
13	Germacrene-D-4-ol	C15H26O	10.58	+	+	94
14	Phytol actate	C22H42O2	12.28	+	++	91
15	3,7,11,15-tetramethyl-2-hexadece-1-ol (Phytol)	C20H40O	12.59	+	++	92
16	Octacosane	C28H58	17.32	+	+	96

* + and – signs denotes degree of up- or down- regulations.

Table 3. Differentially expressed proteins in Hymenaea plants after heat stress as identified by 2-D gel electrophoresis

SN	Spot#	Proteins	Fold change*
1	99	Oxygen-evolving enhancer protein 2, chloroplastic OS = Solanum tuberosum	28.5
2	97	Oxygen-evolving enhancer protein 2, chloroplastic OS = Solanum tuberosum	26.2
3	126	Histone H3-like 5 OS = Arabidopsis thaliana	19.1
4	47	glutamine synthase [Gossypium hirsutum]	13.6
5	129	Photosystem I reaction center subunit II, chloroplastic OS = Solanum lycopersicum	11.5
6	109	Germin-like protein [Pisum sativum]	9.8
7	49	glutamine synthetase isoform GSe1 [Triticum aestivum]	8.1
8	132	Ras-related protein RABB1b OS = Arabidopsis thaliana	8.0
9	70	Oxygen-evolving enhancer protein 1, chloroplastic OS = Spinacia oleracea	7.5
10	21	Ribulose bisphosphate carboxylase large chain (Fragment) OS = Aesculus pavia	7.4
11	127	Histone H2B.3 OS = Arabidopsis thaliana	7.2
12	98	20 kDa chaperonin, chloroplastic OS = Arabidopsis thaliana	6.2
13	6	PREDICTED: ATP-dependent zinc metalloprotease FTSH, chloroplastic [Vitis vinifera]	5.9
14	46	Fructose-bisphosphate aldolase 1, chloroplastic (Fragment) OS = Pisum sativum	5.8
15	87	caffeoyl-CoA-O-methyltransferase [Leucaena leucocephala]	5.6
16	50	Fructose-bisphosphate aldolase 1, chloroplastic (Fragment) OS = Pisum sativum	5.6
17	18	ATP synthase subunit α, chloroplastic OS = Manihot esculenta	5.5
18	55	Isoflavone reductase, putative [Ricinus communis]	5.3
19	110	Putative protein phosphatase 2C 22 OS = Oryza sativa subsp. japonica	5.1
20	52	ATP synthase gamma chain 1, chloroplastic OS = Arabidopsis thaliana	4.9
21	133	Ribulose bisphosphate carboxylase small chain 3, chloroplastic OS = Solanum tuberosum	4.7
22	25	Ribulose bisphosphate carboxylase/oxygenase activase A, chloroplastic OS = Hordeum vulgare	4.4
23	103	Carbonic anhydrase OS = Flaveria brownii	4.4
24	105	(3S,6E)-nerolidol synthase 1 OS = Fragaria ananassa	4.2
25	43	Sedoheptulose-1,7-bisphosphatase, chloroplastic OS = Spinacia oleracea	4.2
26	104	ATP synthase delta chain, chloroplastic OS = Spinacia oleracea	3.7
27	20	ATP synthase subunit β, chloroplastic OS = Morus indica GN = atpB	3.7
28	10	Stromal 70 kDa heat shock-related protein, chloroplastic OS = Pisum sativum	2.2
29	66	Ribulose bisphosphate carboxylase large chain (Fragment) OS = Sinapis alba	2.6
30	96	2-Cys peroxiredoxin BAS1-like, chloroplastic OS = Arabidopsis thaliana	−2.1
31	77	BTB/POZ domain-containing protein At5g48130 OS = Arabidopsis thaliana	−2.0
32	83	Oxygen-evolving enhancer protein 1–2, chloroplastic OS = Arabidopsis thaliana	−2.0
33	64	Ferredoxin–NADP reductase, leaf isozyme, chloroplastic OS = Pisum sativum	−2.1
34	81	Ribulose bisphosphate carboxylase large chain OS = Carica papaya	−2.1
35	23	Enolase 2 OS = Hevea brasiliensis	−2.1
36	63	lyase [Streptomyces sviceus ATCC 29083]	−2.2
37	13	Ribulose bisphosphate carboxylase large chain (Fragment) OS = Tasmannia insipida	−2.3
38	72	Coproporphyrinogen-III oxidase, chloroplastic OS = Oryza sativa subsp japonica	−2.3
39	128	Peptidyl-prolyl cis-trans isomerase OS = Catharanthus roseus	−2.4
40	90	Triosephosphate isomerase, chloroplastic OS = Spinacia oleracea	−2.6
41	65	Isoflavone reductase homolog OS = Solanum tuberosum	−2.8
42	1	Ribulose bisphosphate carboxylase/oxygenase activase, chloroplastic OS = Vigna radiata var. radiata G	−2.8
43	124	Ribulose bisphosphate carboxylase large chain (Fragment) OS = Humiria balsamifera	−3.0
44	89	Triosephosphate isomerase, cytosolic OS = Zea mays	−3.1
45	24	Glutamate–glyoxylate aminotransferase 1 OS = Arabidopsis thaliana	−3.6
46	3	Chaperone protein ClpC, chloroplastic OS = Pisum sativum	−3.9
47	74	Ribulose bisphosphate carboxylase large chain (Fragment) OS = Sinapis alba	−3.9
48	69	Ribulose bisphosphate carboxylase small chain, chloroplastic OS = Glycine tabacina	−4.1
49	14	Ribulose bisphosphate carboxylase large chain (Fragment) OS = Humiria balsamifera	−4.1
50	16	Ribulose bisphosphate carboxylase large chain (Fragment) OS = Pachira aquatica	−4.2
51	92	Carbonic anhydrase OS = Flaveria bidentis	−4.4
52	8	Ribulose bisphosphate carboxylase large chain OS = Viscum album	−4.5
53	34	Phosphoglycerate kinase, chloroplastic OS = Nicotiana tabacum	−5.0
54	135	putative cold-inducible protein-like protein [Helleborus orientalis]	−5.1
55	41	mucunain [Mucuna pruriens]	−7.8
56	119	Probable protein arginine N-methyltransferase 6.1 OS = Oryza sativa subsp indica	−9.4
57	82	Endochitinase A2 OS = Pisum sativum	−23.7

* Average ratio of 3 biological replications (Heat/control). “−”sign denotes down-regulation of particular protein expression.

Figure 3. 2D-DIGE analysis of Hymenaea leaf proteome. Protein samples of Hymenaea control plants and heat stressed plants were differentially labeled with Cy3 (green) and Cy5 (red) respectively. After mixing the 2 labeled proteins in equal ratios, they were first subjected to isoelectric focusing on a IPG strip, pH 3–10, and then on a 12.5% SDS-PAGE. The isoelectric point (pI) and molecular mass (kDa) are marked. Color coding: green spots indicates that the protein abundance is high in Cy3 (control), red spot indicates that the protein abundance is high in Cy5 (heat stressed) samples; yellow spots indicates that the protein abundance is similar in both the cases. Protein identifications of selected spots are shown in Table 3.

Figure 4 Graphical representation and functional cataloging of identified proteins in Hymenaea leaf samples. The protein cataloging is based on their predicted functions in biological processes. Functions of the proteins were identified using Uniprot and NCBI databases.

Discussion

Volatile compounds emission analysis from Hymenaea leaves

There are more than 1700 volatile compounds produced by more than 90 plant families.¹⁴ Our assay with GC-MS for volatiles emitted from the leaves yielded 3 compounds namely, isoprene, 2-methyl butanenitrile, and β ocimene (Table 1). Isoprene emission is directly correlated with thermotolerance in plants⁷ and this plays an important role in protecting photosynthetic units in plants during heat stress.⁷ 2-methyl butanenitrile was reported to be present in the volatile compounds produced by lima beans infested with spider mites.¹⁵ Also, Jardine et al. in 2010 reported production of 2-methyl butanenitrile from leaves of creosotebush (Larrea tridentata) under various environmental conditions.¹⁶ Another compound, which was produced differentially in heat stressed leaf sample, was β ocimene, a monoterpene. Dudareva et al., 2003 reported β ocimene as floral scent produced by snapdragon flowers.¹⁷ Beta ocimene is also produced by plants when attacked by insects¹⁸. We propose that production of 2-methyl butanenitrile and β ocimene was a mode of adaptation to abiotic stress. Production of β ocimene and 2-methyl butanenitrile can be justified as a method of thermotolerance.

Total lipid analysis: terpenes identified in heat stressed leaves

Total lipid extractions from heat stressed and control plant leaves yielded terpenes and sesquiterpenes (Fig. 2 and Table 2). It has been reported that terpenes may protect plants against herbivory and insect attack.¹⁹ Isoprenes, a hemiterepene also play important roles in protecting plants from heat stress^20,21 and water stress.²² Tingey et al. (1991)²³ reported high rate of monoterpene emission from plant leaves at elevated temperatures. Loreto et al. (1998) reported Quercus ilex L. emitted high amount of monoterpenes under heat stress.²⁴ They also showed Quercus ilex L. acquired increased thermotolerance, when the plants were fumigated with monoterpenes. Pateraki and Kanellis (2010)²⁵ studied several terpene biosynthetic genes in Cistus creticus subsp creticus and concluded that these genes are upregulated in abiotic stress situation like heat, drought, mechanical wounding. Our finding of abundance of sesquiterpene production in heat stress situation concurs with previous observations by other scientists and will help us to understand mechanism of thermotolerance in Hymenaea tree.

Proteins identified from 2-D gel electrophoresis

Heat shock proteins

Heat shock proteins (HSPs) are produced in plants including all other life forms in response to heat stress. They play important roles in improving thermotolerance in plants (review⁸). Transgenic plants overexpressing HSPs were successfully produced for improved thermotolerance (review²⁶). We have observed (Table 3) a stromal HSP differentially expressed in heat stressed Hymenaea leaves.

Histone proteins

Two histone-like-proteins (H3 and H2B) were found upregulated more than 19 times in heat stressed compared to control plant (Table 3). Histones have been reported to be involved in heat sensing in yeast cells and in Arabidopsis thaliana.^27,28 Kumar and Wigge (2010) showed that in Arabidopsis thaliana cells, alternative histone H2A.Z was essential in accurate sensing of ambient temperature²⁹. They have also shown H2A.Z played an important role in DNA unwrapping mechanism, which ultimately lead to heat sensing²⁹. In essence, warm temperature is directly associated with decrease in overall histone protein content. Weng et al. (2014) showed an interesting result, where, they showed histone and chaperone proteins can stimulate acetylation of histone H3K56 in Arabidopsis under heat stress.³⁰ Lang-Mladek et al., 2010 reported enhancement of heat-stress mediated histone acetylation events in plants.³¹ So, there is a possibility that heat stressed Hymenaea cells upregulated histone acetylation process to protect DNA from damage. Stress-induced histone modification is a complex phenomenon as reviewed by Chinnusami and Zhu (2009).³² Some histone modification including acetylation, phosphorylation and ubiquitination can enhance transcription,^33,34 whereas biotinylation and sumoylation may suppress histone gene expression.³⁴ So, there may be 3 explanations of enhanced production of histone proteins in Hymenaea cells: (1) heat stress induced histone chaperone proteins in cells, which in turn produced H3 and H2B at an increased rate, (2) histone acetylation is directly related to DNA repair in plants³⁵ and (3) there may be acetylation, phosphorylation or ubiquitination of histones in Hymenaea cells and hence there were surge of histone proteins production.

Oxygen evolving complex and other photosynthetic proteins

Oxygen evolving enhancer protein (OEC) is an important protein in photosysytem II (PSII) of plants involved in photo-oxidation of water. Heat stress may rapidly inactivate PSII complex³⁶ along with OEC.³⁷ Mathur et al. (2011) reported a higher temperature lead to irreversible damage of OEC in wheat (Triticum aestivum).³⁸ Heat stress also induces light harvesting complex (LSII) to aggregate to protect plants from damaging effects of heat stress.³⁹ However, Heckathorn et al., 1998 reported that OEC production may decrease within first 24 hours of heat stress, followed by a sharp increase in continued heat stress.⁴⁰ This increased expression of OEC may be needed to repair damaged PSII proteins.⁴¹ We observed OEC was upregulated 26 to 29 folds and downregulated 2 folds compared to control plant (Table 3). We speculate that OEC was dissociated following heat stress and then the cells upregulated OEC production to repair damaged PSII systems.

Heat stress directly impacts rate of photosynthesis in plants (reviewed by Wahid et al. 2007). Toth et al. (2005) showed heat stress can damage PSII units in barley leaves.⁴² However de novo synthesis of PSII can be accounted for gradual improvement of photosynthetic activities following heat stress.⁸ When spinach leaves were exposed to 40°C, electron flow through PSII was inhibited, but, activated cyclic electron transport in photosystem I (PSI).⁴³ Vasilikiotis and Melis (1994) showed PSII: PSI ratio increased from 1.4:1 to 15:1 when Dunaliella salina cells were transferred from low to high irradiance.⁴⁴ Accumulation of PSII following heat stress was proposed to play important roles in repairing and replacement of PSII.⁴⁴ It must be noted here that RuBP (ribulose 1,5-bisphosphate) and PSII reaction centers do not decreases with elevated levels of heat stress, although net photosynthesis is inhibitory at that temperature.⁴⁵ It has also been reported that RuBP concentration and electron transport may even increase following moderate heat stress in cotton (Gossypium hirsutum).⁴⁶ They concluded inhibition of photosynthesis in higher temperature may be caused by Rubisco inactivation under heat stress, and at the same time RuBP regeneration was observed to be increased in heat stressed plants. We observed activation of several photosynthesis related proteins (PSI reaction center subunit II, Rubisco) and at the same time we observed suppression of several similar proteins (Ferredoxin-NADP reducatse, Rubisco) (Table 3).

Glutamine synthase

El-Khatib et al (2004) showed that expression of a cytosolic glutamine synthetase (GS1) gene in poplar has improved tolerance to dehydaration.⁴⁷ Hoshida et al (2000) reported that overexpression of a chloroplast glutamine synthetase gene in rice has improved its tolerance to salinity.⁴⁸ Glutamine synthase protects plants from photo-oxidation under heat stress, resulting in increased photorespiration (as reported in tobacco⁴⁹). In this study, we observed that glutamine synthase was upregulated 14-fold in Hymenaea leaves during heat stress. We propose this enzyme plays an important role in protecting plants from various types of abiotic stresses including heat stress.

ATP synthase

ATP synthesis is critical for cells to survive in a stressful situation. However, increased ATP/ADP ratio was observed in spinach leaves, mainly due to decrease in demand for ATP in heat stressed leaves.⁵⁰ We observed ATP synthase was upregulated in heat stressed plants 5.54, 4.90, 3.70 and 3.65 folds compared to control plant leaves (Table 3). Crafts-Bradner and Law (2000) proposed that higher temperature inhibited Rubisco activation (not by suppressing RuBP regeneration) and we propose Rubisco inactivation suppressed Calvin cycle thereby creating less demand for ATP. Hence, we hypothesize that the fluctuations in ATP synthase is probably due to variations in demands of ATP as described above.

Others proteins

A putative protein phosphatase 2C (PP2C) was found to be overexpressed (5.09 folds) in heat stressed Hymenaea plant (Table 3). Rodriguez in 1998 had shown that PP2C, which function as a down regulator of mitogen-activated protein kinase (MAPK) pathway, play roles in various signal transductions pathway in alfalfa.⁵¹ We speculate PP2C may play an important role as a signal transduction protein during heat stress in Hymenaea. 

An isoflavone reductase like enzyme was also found upregulated by more than 5 folds in heat stressed plants. Isoflavone reductase belongs to isoflavonoid biosynthesis pathway. A study has shown that an overexpression of isoflavone reductase-like gene in rice provides a resistance against reactive oxygen species (ROS).⁵² This upregulation of isoflavone reductase-like gene is also in accordance with the fact that ROS activity generally elevated under different stresses including heat stress as reviewed before.^8,26 Our protein analysis also shows an enhanced expression (5.6 times) of Caffeoyl-CoA-O-methyltransferase. Caffeoyl-CoA-O-methyltransferases are enzymes involved in lignin biosynthesis and have shown to overexpress in response to tobacco mosaic virus (TMV) infection.^53-55 Our result of higher expression of caffeoyl-CoA-O-methyltransferase, again supports our hypothesis of cross interaction between genes against biotic stress (infection) and thermotolerance.

Materials and Methods

Hymenaea courbaril L. plants (Fig. S1) were purchased from Future Forests Nursery, Hawaii, USA. To study the heat stress responses, 18-months-old test plants were grown in a plant growth chamber with diurnal cycle of 12 hours light, and 12 hours dark. The control plants were kept at 25°C, but the heat stressed plants were kept at 40°C for 48 hours with similar photoperiod. The plants and the plant leaves for experiment were selected carefully to minimize any biasness due to size, shape, color and level of maturity.

Volatiles study

To study the volatiles produced by Hymenaea courbaril L. plant, 2 leaflets per plant were excised from 3 separate plants. The leaflets were immediately kept inside airtight glass vials (15 ml volume). The vials were kept at 30° angle and the adaxial surface of the leaf faced the light mimicking natural condition. The control vials were incubated at 25°C while test vials were kept at 40°C for 48 hours in a growth chamber with photoperiod similar to treatments explained above. After 48 hours of incubation, headspace air from each of the controls and heat stressed vials were sampled by an injection, and analyzed by gas chromatography linked to mass spectrometer (GC-MS). A GC-MS (GCMS-QP2010S, SHIMADZU, USA) equipped with a HP-5 column (crosslink 5% PhMe siloxane 30 m X 0.25 μM film thickness) was used in this study for volatiles and total lipids analysis. The following setup was used for GC-MS: an oven temperature from 65°C to 300°C for 18.67 min with a ramping of 15°C /min, with helium carrier gas at a flow rate of 1.2 ml/min and a split ratio of 1:20 (for volatiles) and 1:75 (for lipid). All the peaks in this study were analyzed by its comparison with NIST/EPA/NIH Mass Spectral Library (Data version: NIST 11, Software version 2.0) and only the peaks with more than 90% similarity matches were considered and mass spectra of peaks were compared (Fig. S2).

Lipid analysis

Total lipids were extracted from heat stressed and control plants to study lipids and terpene production. Hymenaea courbaril L plant was heat stressed as described in the above section. About 0.5 g of leaves (from heat stressed and control plants) were excised, mixed with 0.5 g of sands (EMD Chemicals, USA) and ground in liquid N₂. Sand was used for fine pulverization of leaf materials for improved lipid yield. The volume of leaf pastes were adjusted to 1 ml with sterile deionized water in glass tubes and total lipids were extracted following the Bligh and Dyer method.⁵⁶ Equal amount of lipid extracts were collected into Teflon capped glass vials, dried under N₂ gas and then dissolved into 500 μl of hexane (Sigma-Aldrich, USA). The extracted total lipids were analyzed by injecting 1 μl of samples into GC-MS.

Protein analysis

Three independent leaf samples were collected from heat stressed and control plants. The leaf tissue grinded in liquid nitrogen from above described experiment was homogenized in 300 μl of lysis buffer (30 mM Tris-HCl, pH 8.8, 7 M urea, 2 M thiourea and 4% CHAPS) under cold conditions. The homogenized sample was subjected to sonication on ice, followed by 30 minutes incubation on a rotary shaker at room temperature. The mixture was centrifuged at 25,000g (4°C) to collect the supernatant. Protein concentration in the supernatant was measured using protein assay kit (BioRad catalog# 500–0006). The lysate samples were diluted with cell lysis buffer described above at 5 μg/μl concentration for downstream experiments. Two-D DIGE and Mass Spectrometric protein identification were run by Applied Biomics (Hayward, CA) using the method essentially as described in Robbins et al. (2013).⁵⁷ Based on the in-gel analysis and statistical analysis (Table S3), protein spots of interest were picked up by Ettan Spot Picker (GE Healthcare). Individual excised gel spots were washed a couple of times in sterilized HPLC grade water, and in-gel protein digestion was performed with trypsin protease (Promega) using manufacturer's protocol. 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 onto the MALDI plate. MALDI-TOF (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). 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 UniProtKB_Viridiplantae accessed on March 2014 with 2,127,634 sequences present in the database. 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. The 3D-view of all the selected spots (Figure S3), and the protein ID summary (Table S1), and the peptide summaries for all the selected proteins spots (Table S2) is shown as supplemental information.

Conclusion

Hymenaea is one of the most important, but understudied tropical plants. Its importance as plant of medicinal value is well known. This plant is also found to be a rich source of different hydrocarbons and also referred as ‘kerosene tree.’ Some studies have shown that this plant exists at a very low density in the nature.⁵⁸ Global warming has a negative impact on normal life cycle of all life forms and can further be a factor in declining population of Hymenaea plant because of its low density growth habitat. How a Hymenaea plant responses to heat stress is still unknown. Study on heat stress response of this plant will be helpful in developing a heat tolerant Hymenaea in future. In this study, we have analyzed heat stress response of this plant at volatiles, total lipids and protein levels. We found differential response at these macromolecules under heat stressed condition. We identified several overexpressed proteins and terpenes (like isoprene) under heat stress condition, which are the same ones that have been previously reported to play important roles in plant heat stress sensing and tolerance. This study also showed that a number of different sesquiterpenes are produced by this plant. Interestingly, we have also found some of the terpenoid compounds and proteins involved in insect response in plants are overexpressed under heat stress in Hymenaea. Based upon our result, it is possible that both biotic and abiotic response system might have been working in parallel in Hymenaea plant with a possibility of cross-talk. This work will open future research avenues in the field of understanding molecular physiology or heat stress in plants, especially in tropical tree species.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

CB and DG acknowledge Dr. Michael Summers, CSUN Department of Biology for assistance with lipid extraction and partial student stipend provided to DG through Pamela M Klein, M.D. Scholarship in Biology at CSUN.

Funding

JSR acknowledges financial support from SDSU Agricultural Experiment Station, USDA/SunGrant, and Department of Biology & Microbiology.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.