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2013 Comparative proteomic analysis for assessment of the ecological significance of maize


J O U RN A L OF P ROT EO M IC S 7 8 ( 2 01 3 ) 4 4 7 –4 60

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Comparative proteomic analysis for assessment of the ecological significance of maize and peanut intercropping
Hongchun Xiong1 , Hongyun Shen1 , Lixia Zhang, Yanxiang Zhang, Xiaotong Guo, Pengfei Wang, Penggen Duan, Chunqiao Ji, Lina Zhong, Fusuo Zhang, Yuanmei Zuo?
Key Laboratory of Plant-Soil Interactions, MOE, Centre for Resource, Environment and Food Security, China Agricultural University, Beijing 100193, China

AR TIC LE I N FO
Article history: Received 10 July 2012 Accepted 14 October 2012 Available online 24 October 2012 Keywords: Peanut/maize intercropping Proteomic analysis Ecological significance Iron nutrition Rhizosphere interaction

ABS TR ACT
Intercropping is an important and sustainable cropping practice in agroecosystems. Peanut/ maize intercropping is known to improve the iron (Fe) content of peanuts in calcareous soils. In this study, a proteomic approach was used to uncover the ecological significance of peanut/ maize intercropping at the molecular level. We demonstrate that photosynthesis-related proteins accumulated in intercropped peanut leaves, suggesting that the intercropped peanuts had a stronger photosynthetic efficiency. Moreover, stress-response proteins displayed elevated expression levels in both peanut and maize in a monocropping system. This indicated that intercropping contributes to resistance to stress conditions. Allene oxide synthase and 12-oxo-phytodienoic acid reductase, two key enzymes in jasmonate (JA) biosynthesis, increased in abundance in the maize roots of the intercropping system, consistent with the upregulation of JA-induced proteins shown by microarray analysis. These results imply that JA may act as a signaling molecule, playing an important role in intercropping through rhizosphere interaction. This study suggests that peanut/maize intercropping results in high Fe availability in the rhizosphere, leading to variation in the proteins related to carbon and nitrogen metabolism. The advantages of intercropping systems may improve the ecological adaptation of plants to environmental stress. ? 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Intercropping, the mixed growth of two or more crops, is practiced in >28 million hectares of areas sown annually in China [1] and is common in other parts of the world, such as India, Southeast Asia, Latin America and Africa [2]. In general, multiple-cropping systems in China, including intercropping and related practices, have contributed to increased crop productivity due to a more effective utilization of resources compared to monoculture crops [3]. Facilitative root interactions in mixed cropping systems are of importance, since they enable nutritional improvement of crops grown in nutrient-poor soils
? Corresponding author. Tel.: + 86 10 62733407; fax: +86 10 62731016. E-mail address: zuoym@cau.edu.cn (Y. Zuo). 1 These authors contribute equally to this work. 1874-3919/$ – see front matter ? 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2012.10.013

and low-input agroecosystems [4]. Adapting such cropping systems through more efficient use of soil nutrients and a lower reliance on chemical fertilizers, is a promising strategy for the development of sustainable crop production while maintaining future food security. Peanut (Arachis hypogaea), is an important food legume capable of symbiotic N2 fixation. Peanut seeds contain a rich source of edible protein and represent the major oilseed crop in China, accounting for 30% of the total production. However, iron (Fe) deficiency frequently limits both crop yields and the quality of monocropped peanuts grown in the calcareous soils of Northern China. Peanut/maize intercropping is a successful

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crop management strategy that results in more effective and sustainable practice for farmers due to improvement in the Fe content of the peanut plants. Peanut/maize intercropping is known to improve Fe nutrition in all peanut tissues [5,6]. Fe is an essential micronutrient for plants as it participates in fundamental biological redox processes such as photosynthesis and respiration. However, Fe bioavailability to plants is low, due to its sparing solubility under aerobic conditions. Fe deficiency represents a yield-limiting factor with major implications for field crop production in many agricultural areas [7]. To combat this, plants have evolved both strategy I and strategy II mechanisms in response to Fe deficiency [8]. In strategy I, an acidification/reduction mechanism, based on the secretion of protons into the rhizosphere, and the reduction of Fe(III) to Fe(II) by root ferric chelate reductase [9,10], is used to enhance Fe solubility prior to uptake. The response of strategy II plants to Fe shortage includes the biosynthesis and secretion of phytosiderophores (PS) which specifically bind Fe(III) with high affinity, resulting in the efficient uptake of Fe(III)-PS complexes from the rhizosphere by specific cellular transport systems [11]. In the peanut/maize intercropping system, peanuts employ strategy I whilst maize employs strategy II. The secretion of PS from maize in the peanut/maize intercropping system may contribute to the improved Fe nutrition of the peanut [12]. Importantly, through inter-specific root interactions, peanut/maize intercropping not only enhances the Fe nutrition of peanuts, but also contributes to other nutrient elements, including improvements in shoot zinc (Zn), phosphorus (P), and potassium (K) concentrations [13]. The nitrogen (N) efficiency in both peanut and maize plants also improves [14]. Moreover, intercropping greatly enhances Fe and Zn concentrations in the seeds of peanut [12]. Over the past decade, progress has been made in our understanding of the physiological basis of the maize/ peanut intercropping system. However, to our knowledge, the ecological significance of this intercropping practice has not been reported at a molecular level. Proteomic analyses provide a powerful tool to address the biochemical and physiological aspects of plant responses to abiotic and biotic stresses [15]. Recently, changes in the root protein profile induced by direct Fe deficiency from tomato (Lycopersicon esculentum) [16,17], cucumber (Cucumis sativus) [18], sugar beet (Beta vulgaris) [19] and Medicago truncatula [20] have been investigated by 2-DE proteomic profiling. However, there is little information on the proteomic response of the leaves of Fe-deficient plants. The proteomic profiles of thylakoid membranes of sugar beet [21] and spinach plants [22] in response to Fe deficiency suggest that Fe deficiency causes changes in the relative abundance of several Fe-containing protein species, especially those in photosystem I (PS I) that ultimately affect photosynthetic efficiency. However, all reports were analyzed based on solution culture conditions. The effects of the intercropping system and its ecological significance are complex in calcareous soils and are therefore difficult to study. Proteomic profiling represents a promising tool for ecological studies and will be helpful to illustrate the ecological significance of the maize-peanut intercropping system [23]. In the present study, we used proteomics to investigate the physiology and molecular phenotype of peanut and maize under monocropping and intercropping, based on 2-DE and

MALDI MS/MS approaches. The combination of 2-DE-based protein separation with tandem mass spectrometry for peptide sequencing is an attractive method for the characterization of organisms for which the genome sequence is not yet available. We utilized a variety of physiological, transcriptome and proteome techniques to evaluate the responses of peanuts and maize to intercropping and monocropping conditions in an environmentally controlled greenhouse. The overall objective of this research was to develop a protein reference map of the ecological significance of peanut/maize intercropping.

2.
2.1.

Materials and methods
Plant materials and growth conditions

Peanut (A. hypogaea L. cv. Luhua14) and maize (Zea mays L. cv. Nongda108) were used in this study. Pot experiments were conducted in a calcareous sandy soil in a greenhouse. The soil sample was enhanced with basal fertilizers [composition (mg·kg? 1 soil): N 100 (Ca (NO3)2·4H2O), P 150 (KH2PO4), K 100 (KCl), Mg 50 (MgSO4·7H2O), Cu 5(CuSO4·5H2O), and Zn 5(ZnSO4·7H2O)]. The experiment consisted of three cropping treatments with six replicates of each treatment. Six peanut plants or three maize plants were grown in a single pot as monocrops. Three peanut plants and three maize plants were grown in a single pot for the intercropping treatments. Maize plants were sown 10 days after peanuts were germinated. After 63 days of growth for the peanut plants, plant samples of maize and peanut were harvested due to obvious Fe deficiency chlorosis in the young leaves of the peanuts. For active Fe content analysis, six replicates from the young leaves of peanuts were taken. For protein isolation, root samples of maize and young leaf samples of maize and peanut from three different pots for each treatment were harvested. The remaining three pots provided samples for RNA extraction. For protein and RNA analyses, six peanut plants or three maize plants per sample from the monocropping system and, three peanut plants and three maize plants from the intercropping system were sampled, frozen in liquid N, and kept at ?80 °C for further analyses.

2.2.

Plant analysis

Soil and Plant Analyzer Development (SPAD) values were measured in the middle area of young leaves in peanuts to determine the relative chlorophyll content according to previous reports [24,25]. A Minolta SPAD-502 chlorophyll meter (Minolta Camera Co. Ltd., Osaka, Japan) was used to take the SPAD readings. An average of 30 leaves per pot was recorded. The plant samples were washed several times in distilled water. Young leaves from peanuts in each treatment (six replicates thereof) were taken for the assessment of HCl-extractable Fe (so-called ‘active Fe’), according to the procedure developed by Takkar and Kaur [26] and determined by Inductively-Coupled Plasma Atomic Emission Spectrometry (ICP, Perkin-Elmer Optima 3300DV).

2.3.

Protein extraction and analysis

The protein from three replicates of each treatment was extracted using trichloroacetic acid (TCA) precipitation

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according to Toorchi et al. [27], with slight modifications. Briefly, fresh tissue samples were ground with liquid N and added to an acetone buffer containing 10% TCA, 0.07% ?-mercaptoethanol, and maintained at ? 20 °C for 1 h. The homogenate was then centrifuged twice at 15,000 ×g for 45 min at 4 °C. Supernatants were then dried and acetone purified. The lysis buffer consisted of 7 M urea, 2 M Thiourea, 2% CHAPS, 0.5% IPG buffer, 40 m MDDT. The total protein concentration of the lysates was measured using the 2D-Quant kit (GE Healthcare Life Sciences, USA). First-dimension isoelectric focusing (IEF) separation was performed on ReadyStrip Linear IPG strips (pH 4–7, 24 cm; GE) and the Strips were loaded into an Ettan? IPGphor? 3 IEF System (GE). For second-dimension polyacrylamide gel electrophoresis (SDS-PAGE), IPG strips were placed onto 12.5% SDS-PAGE gels to separate proteins of between 10 and 100 kDa. Proteins were stained with Coomassie Blue G-250 (Sigma, Barcelona, Spain). Three independent gels from each treatment were produced. Image Master 2D Plantinum v5, v6 software (GE) was used for spot detection, gel matching, and interclass analysis. The average spot intensity was normalized to the total spot volume with a multiplication factor of 100. Each spot showing a ratio above 1.3 was manually checked to ensure a high level of reproducibility between normalized spot volumes of gels produced in triplicate. The spots detected in at least two of the triplicates from one treatment were selected as consistent spots with a matching degree of more than 85%. After estimation of the missing spot volumes by a sequential K-nearest neighbor algorithm, a second normalization was conducted to compensate for gel replicate variation. Statistical significance was assessed using the Student's t-test (P ≤0.05). In-gel digestion, sample preparation, MALDI TOF and MALDI TOF-TOF peptide mass fingerprint and database searching was also performed [17]. The principles for the identification of candidate proteins were: protein mass and pI matched between the measured and theoretical values; mascot scores above 95% confidence interval; scores above or equal to 70; coverage was above or equal to 13%. A two-fold change in spot signal intensity between treatments was taken as a threshold for differentially accumulated proteins. Proteins expressed in only one treatment were taken as new or lost proteins. Positive matches were BLAST searched against the UniProt database (http://www. uniprot.org) and/or NCBI protein database (http://www.ncbi. nlm.nih.gov) for updated annotation and the identification of homologous proteins.

2 min at 50 °C and 1 min at 95 °C followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 45 s. The quantitative variation between samples was evaluated by the delta-delta threshold cycle method. All reactions were performed in duplicate and a no-template control was included in each reaction. For the microarray analysis of maize roots in intercropping versus monocropping, total RNA was extracted using Trizol (Invitrogen) and purified using an RNeasy MinElute Cleanup Kit (Qiagen). The GeneChip Maize Genome Array (Affymetrix), containing 17,555 probes, was used in this study. Hybridization, washing and scanning were performed according to the Affymetrix GeneChip System Protocols (http://www.cbse.ucsc. edu/sites/default/files/affymetrix_protocol050404.pdf). Data was analyzed using GeneChip? Operating SoftwareVersion1.4 (Affymetrix) and SAM Software (http://en.bio-soft.net/chip/ SAM.html) for background adjustment, normalization, and summarization. Three biological replicates were analyzed and significance levels were adjusted using the false discovery rate (FDR) method [28]. Genes showing an FDR less or equal to 5% and with changes in signal intensity between intercropping and monocropping of twofold or higher were considered differentially expressed genes.

3.

Results and discussion

3.1. Intercropping of peanut/maize improves iron nutrition in calcareous soil
To gain a better understanding of the ecological significance of peanut/maize intercropping, pot experiments were performed in calcareous soil in a greenhouse environment. As shown in Fig. 1a, peanuts intercropped with maize did not display the chlorosis symptoms of Fe deficiency compared with the monocropped ones. In accordance with the symptoms observed, both the SPAD value and active Fe concentration in the young leaves of the intercropped peanut were significantly higher than those from the monocropping system (Fig. 1b and c). Intercropping with maize did not affect the fresh weight of peanuts compared with monocropping (Fig.1d), suggesting that the Fe-deficient chlorosis observed during monocropping was not due to a dilution of Fe concentration. Taken together, these results strongly suggest that peanut intercropping with maize effectively improved the Fe nutrition in calcareous soil, as shown in previous studies [5,6,13,29,30].

2.4.

Gene expression analysis

For real-time PCR, gene specific primers were designed using Primer Premier v5.0 (Biosoft). The 18S ribosomal RNA was used as an internal control. The following primers were used: 18S-F, 5′-CCGTCTCAAACAAGAACAAAACC-3′; 18S-R, 5′-TCACACCAA GTATCGCATTTCG-3′; IRT-F, 5′-TTCTCTGCCTTATTCACGCT-3′; IRT-R, 5′-ACGAGTTGTGTGTCTCCATCTT-3′; FRO-F, 5′-TTTGAG TCAGGGCTGTCTTG-3′; FRO-R, 5′-TTCTTGAGTCGTGCTTGGT3′. Total RNA was extracted from peanut tissues using Trizol (Invitrogen) and treated with RNase-free DNaseI (Takara). cDNA was prepared with M-MLV reverse transcriptase (Promega). Real-time PCR was performed using 2 × SYBR Green PCR Master Mix reagent (Toyobo) using an ABI7500 Fast Real-Time PCR System (Applied Biosystems) under the following conditions:

3.2. High-resolution 2-DE protein patterns from peanut and maize in monocropping and intercropping systems
To investigate the mechanism and underlying ecological significance of the peanut and maize intercropping system, a proteomics approach involving 2-DE followed by the identification of isolated proteins by tandem MS was applied. Changes in protein abundance were identified in young peanut leaves, maize roots and young leaves from both inter- and monocropping systems. High quality 2-DE synthesis gel maps from young peanut leaves, maize roots and the young leaves of monocropped (left) or intercropped (right) plants are shown in Fig. 2. The isoelectric points (pIs) of the spots ranged from pH 4

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(a)

(b)
40

*
30

SPAD

20 10 0

Mono-P

Inter

Mono-M

Mono

Inter

(c)
Active Fe content (mg kg-1)
3.5 3 2.5 2 1.5 1 0.5 0 Mono Inter

(d)
90

*

Biomass (g plant-1 FW)

75 60 45 30 15 0 Mono Inter

Fig. 1 – Peanut/maize intercropping improves the Fe nutrition of peanuts in calcareous soils. Experiments consisted of three treatments with six replicates. (a) Intercropping and monocropping. Mono-P, peanut monocropping; Inter, peanut/maize intercropping; Mono-M, maize monocropping. (b) SPAD value (chlorophyll content) of the young leaves of intercropped and monocropped peanuts. Inter, intercropping; Mono, monocropping. (c) Active Fe content of young peanut leaves. (d) Fresh weight of peanut. Asterisks indicate statistical significance: *P < 0.05.

to 7. Molecular masses ranged from 14 to 94 kDa, with the majority of spots (57%) ≤40 kDa. Each experiment was performed in triplicate and showed highly reproducible results. These high quality 2-DE gel maps were essential for detailed quantitative analyses and protein identification.

3.3. Differences in the protein profiles of maize and peanut plants between monocropping and intercropping
The average numbers of detected protein spots (means± SD) were 242 ± 20 and 310 ± 27 (intercropped and monocropped young peanut leaves), 983 ± 53 and 982 ± 64 (intercropped and monocropped maize roots), 534 ± 26 and 584 ± 4 (intercropped and monocropped young maize leaves). More than 85% of the detected spots were consistently found in triplicates. Among these, the number of protein species showing changes in relative abundance in intercropping when compared to monocropping was 29, 23 and 20 in young peanut leaves, maize roots and young maize leaves, respectively (Table 1). It is noteworthy that, of the 29 proteins that displayed altered abundance in young peanut leaves, 21 were in higher abundance in monocropping, including 12 proteins detected only in monoculture young peanut leaves. In contrast, 8 proteins were in higher abundance through intercropping, including 3 detected in intercropping alone. For maize, a total of 43 proteins displayed significantly altered abundance levels between intercropping and monocropping in roots and young leaves.

Among them, 21 proteins had a higher abundance following intercropping, and 22 proteins displayed increased abundance following monocropping (Table 1). The identified proteins were further categorized on the basis of their putative functions (Fig. 3). Proteins showing a higher abundance in the young leaves of the monocropped peanuts were primarily involved in stress tolerance (23.8%), photosynthesis (19.0%) and metabolism (19.0%) (Fig. 3a). However, the more prolific proteins seen in the intercropped peanut leaf were primarily involved in photosynthesis (75.0%) (Fig. 3b). From the maize root protein profiles, the proteins with a higher abundance in monocropping were mainly involved in carbon (C) metabolism (38.5%) and stress tolerance (30.8%) (Fig. 3c), and only 10.0% were among the proteins that exhibited increased levels in intercropped maize (Fig. 3d). In line with the protein profile of maize roots, the percentages of accumulated proteins related to stress (25.0%) were also higher in the monocropped young maize leaves compared to those of the intercropped plants (11.1%) (Fig. 3e and f). These variable protein expression profiles indicate that a number of metabolic processes are altered in both plants during intercropping.

3.4.

Photosynthesis-related proteins

Fe is one of the essential elements for photosynthesis in plants. In line with the improvements in Fe nutrition of peanuts intercropped with maize on calcareous soil, most of the

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Fig. 2 – High-resolution 2-DE pattern of the proteins with three independent replicates in young leaves of peanut (a) and (b), maize root (c) and (d), young maize leaves (e), and (f) in monocropping (left) and intercropping (right). Labels indicate spot numbers which are listed in Tables 2, 3, and 4. Red arrows represent a higher accumulation under intercropping, and blue arrows represent a higher accumulation under monocropping.

photosynthesis-related proteins were in higher abundance in the young leaves of intercropped peanuts compared to those in monocropped plants. Our results indicate that Rubisco small unit (spot 1), Rubisco large unit (spot 2 and 3), and Rubisco activase (spot 7) accumulated more in young peanut leaves during intercropping (Table 2). Rubisco is the primary photosynthetic CO2 assimilation enzyme in plants and is composed of Rubisco small subunits and large subunits [31]. The activity of Rubisco is functionally regulated by Rubisco activase [32]. Transketolase 1 (spot 9), an enzyme involved in the Calvin cycle during photosynthesis, was also expressed in higher levels

during intercropping compared to monocropping (Table 2). Moreover, light-harvesting chlorophyll a/b-binding (LHC) protein (spot 5), a protein associated with higher plant lightharvesting antenna, was expressed most abundantly during intercropping (Table 2). Winder and Nishio [33] reported that a shortage of Fe caused a marked reduction in leaf chlorophyll, which decreased carbon dioxide fixation linearly. In this regard, Fe deficiency drastically disrupted photosynthesis [34], changed the chloroplast ultrastructure [35] and decreased the expression of the small and large subunits of Rubisco, chlorophyll a/b-binding proteins, and chlorophyll,

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Table 1 – Summary of differentially accumulated proteins in young peanut leaves, maize roots, and leaves in monocropping or intercropping systems. Mono-PL, young leaves of monocropped peanut; Inter-PL, young leaves of intercropped peanut; Mono-MR, roots of monocropped maize; Inter-MR, roots of intercropped maize; Mono-ML, young leaves of monocropped maize; Inter-ML, young leaves of intercropped maize. The numbers of total spots are expressed as means ± SD from three independent experiments. Treatments Total spots
310 ± 27 242 ± 20 982 ± 64 983 ± 53 584 ± 4 534 ± 26

Increased proteins abundance
21 8 13 10 9 11

Only expressed proteins
12 3 6 1 5 11

Proteins > 2 fold
9 5 7 9 4 0

Mono-PL Inter-PL Mono-MR Inter-MR Mono-ML Inter-ML

among other proteins [22,33,36]. Hence, those proteins show lower abundance in young leaves of monocropped peanuts which suffer from Fe deficiency. This suggests that intercropping

of maize and peanut could induce stronger photosynthetic efficiency in intercropped peanut leaves due to the higher Fe concentration and SPAD value (Fig. 1b and c). The photosystem II (PS II) stability/assembly factor HCF136 (spots 6 and 10) and thioredoxin (spot 4) were detected only during monocropping (Table 2). HCF136 is specifically required for the assembly of the PS II reaction center [37]. Thioredoxins in chloroplasts regulate the activity of a number of enzymes involved in photosynthetic C metabolism [38,39]. Meanwhile, the Rubisco subunit binding protein beta subunit (Chaperonin60b, Cpn60b, spot 8) accumulated significantly in young monocropped peanut leaves (Table 2). Cpn60b is involved in the folding and assembly of chloroplast proteins such as Rubisco [40,41] and plays a role in the acclimation of photosynthesis to heat stress, possibly by protecting thermally labile proteins such as Rubisco activase from thermal denaturation [42]. It has been reported that Fe deficiency leads not only to a pronounced degradation of PS I, but also induces remodeling of the photosynthetic apparatus [43,44]. The proteins involved in maintaining the stability of the photosynthetic apparatus, which accumulated in the young leaves of monocropped peanuts, may also indicate that Fe deficiency in monocropped peanuts is more intense, and thus can induce the accumulation of these proteins. These results highlight the adaptive nature of

Fig. 3 – Functional category distribution of the identified proteins. Proteins more highly expressed in monocropped (a) or intercropped (b) young peanut leaves, monocropped (c) or intercropped (d) maize roots, and monocropped (e) or intercropped (f) young maize leaves.

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Table 2 – Young peanut leaf proteins differentially expressed in mono- and inter-cropping systems as identified by MALDI-TOF/TOF. SP a Accession no. b Homologous protein name Species MW/pI exp c MW/pI MOWSE CV. f Fold theo d score e change g IP/MP
20.7/8.3 19.2/5.5 43.3/5.6 17.5/5.6 28.1/5.3 44.1/6.8 34.9/5.6 64.5/5.6 80.4/6.2 45.5/9.0 72 79 74 316 96 172 145 151 112 91 34% 47% 26% 83% 29% 26% 47% 45% 18% 34% 3.2 New 2.6 Lost 2.8 Lost 2.5 0.4 2.2 Lost

Photosynthesis 1 gi|417593 2 gi|156143081 3 gi|15212192 4 gi|115187464 5 gi|2804572 6 gi|15237225 7 gi|94549022 8 gi|223537354 9 gi|3559814 10 gi|75252730 Stress and defense 11 gi|187940332 12 gi|82567813 13 gi|145904610 14 gi|37693104 15 gi|223546177 Metabolism 16 gi|7417008 17 gi|218194708 18 gi|218963652 19 gi|1708924

Rubisco small subunit 3 Rubisco large subunit Rubisco large subunit Thioredoxin fold Chlorophyll a/b-binding protein PS II stability/assembly factor HCF136 Rubisco activase Rubisco subunit binding-protein beta subunit Transketolase 1 PS II stability/assembly factor HCF136

Solanum tuberosum Asclepias tuberosa Kyllingiella microcephala Arachis hypogaea Fagus crenata Ricinus communis Pachysandra terminalis Ricinus communis Capsicum annuum Oryza sativa

16.9/6.8 17.1/4.8 21.9/6.2 17.7/5.9 34.1/5.0 37.9/5.7 46.3/5.2 58.9/5.5 97.2/6.8 66.7/6.3

Pathogenesis-related class 10 protein Pathogenesis-related class 10 protein Ara h 8 allergen isoform Putative lectin precursor Heat shock protein, putative

Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Ricinus communis

15.7/5.1 16.3/5.2 16.9/5.4 32.5/4.6 95.7/4.9

16.9/5.0 16.9/5.0 16.4/5.1 30.7/5.9 73.0/4.8

76 98 90 107 137

46% 28% 51% 28% 30%

0.24 Lost Lost Lost 0.21

Cell death associated protein Hypothetical protein OsI_15572 T-anol/isoeugenol synthase NADP-dependent malic enzyme

Nicotiana tabacum 33.9/5.0 Oryza sativa Indica Group 39.5/6.3 Pimpinella anisum 36.7/5.6 Vitis vinifera 30.73/5.1

37.8/5.3 31.8/9.5 36.4/6.0 65.7/6.3

75 72 76 101

42% 32% 28% 24%

0.4 0.5 Lost 0.5

Secondary metabolism 20 gi|223530620 Cytochrome P450 Transcription related 21 gi|62733385 Transposase family tnp2 Protein synthesis 22 gi|223533763 Eukaryotic translation initiation factor 3f Cell division 23 gi|223541365 Transitional endoplasmic reticulum ATPase 24 gi|223541363 Transitional endoplasmic reticulum ATPase Protein destination 25 gi|12229923 Unknown 26 gi|116786904 27 gi|226520957 28 gi|194466181 29 gi|115473129
a

Ricinus communis

25.5/5.8

54.0/9.1

74

38%

New

Oryza sativa

96.7/6.5 58.5/9.6

80

36%

New

Ricinus communis

32.7/4.8

31.6/5.0

117

25%

Lost

Ricinus communis Ricinus communis

100.5/5.4 90.2/5.2 101.1/5.4 90.4/5.1

126 123

29% 31%

0.2 0.3

Proteasome subunit alpha type-5

Glycine max

29.6/4.7 26.1/4.7

74

35%

Lost

Unknown Predicted protein Unknown Os07g0592600

Picea sitchensis Micromonas sp. Arachis hypogaea Oryza sativa

20.2/5.1 30.0/4.9 33.9/5.9 55.7/6.4

38.1/7.6 51.2/9.9 22.4/8.4 67.6/5.6

83 82 139 75

34% 24% 56% 27%

Lost Lost Lost 0.2

Spot number in 2-DE gel as shown in Fig. 2. Accession number of Uniport and/or NCBI database. c MW/pI exp: Experimental molecular weight and pI. d MW/pI theo: Theoretical molecular weight and pI. e MOWSE score: statistical probability of true positive identification of predicted proteins calculated by MASCOT (http://www.matrixscience.com) with 150 ppm masses tolerance and one allowed missed cleavage (MOWSE score ≥70). f Cv.: sequence coverage. g Fold change IP/MP denotes the ratio of accumulation of a particular protein from young leaves of peanut plants under intercropping versus monocropping condition. IP: intercropped peanut, MP: monocropped peanut. New means the protein only detected in intercropping. Lost indicates the protein only detected in monocropping.
b

the response to Fe deficiency that is directed towards optimizing the photosynthetic architecture to conditions in which Fe is a limiting cofactor.

Meanwhile, chlorophyll a–b binding protein 8 (spot 30) and ferredoxin NADP + reductase (spot 31) were only detected in young maize leaves of intercropped plants (Table 3),

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Table 3 – Young maize leaf proteins differentially expressed in mono- and inter-cropping systems as identified by MALDI-TOF/TOF. SP a Accession no. b Homologous protein name Species MW/pI exp c
21.7/5.4 33.2/5.5 32.9/6.9

MW/pI theo d
29/8.9 37.9/8.4 39.6/8.5

MOWSE score e
99 107 100

CV. f

Fold change g IM/M
New New Lost

Photosynthesis 30 gi|195613254 31 gi|195627630 32 gi|162459168 Carbon metabolism 33 gi|195634659 34 gi|226498182 35 gi|162461856 36 gi|162457930 Stress and defense 37 gi|226533502 38 gi|226494943 39 gi|195612768 40 gi|226500882

Chlorophyll a–b binding protein 8 Ferredoxin NADP + reductase Ferredoxin

Zea mays Zea mays Zea mays

47% 52% 47%

Fructose-bisphosphate aldolase Phosphoribulokinase GADPH 1 Phosphoenolpyruvate carboxykinase

Zea mays Zea mays Zea mays Zea mays

35.3/6.2 40.3/4.7 40.7/6.9 110/6.5

41.9/7.6 45.1/5.8 43.2/7.0 73.8/6.6

109 128 134 122

44% 53% 44% 33%

New Lost New Lost

Ascorbate peroxidase Glutathione S-transferase parA IN2-1 protein Lactoylglutathione lyase

Zea mays Zea mays Zea mays Zea mays

24.3/5.9 27.9/6.8 28.2/5.2 31.1/5.2

27.5/5.6 25.4/6.8 27.2/5.2 37.6/5.87

91 119 158 88

50% 30% 67% 41%

New Lost 0.3 0.24

Amino acid metabolism 41 gi|226508814 Aspartate aminotransferase 42 gi|195627092 Glutamine synthetase Phosphate metabolism 43 gi|195610706 Inorganic pyrophosphatase ATP synthesis 44 gi|50812525 45 gi|50812525 Transcription related 46 gi|226503569 Protein synthesis 47 gi|226508704 Unknown 48 gi|225441104 49 gi|51535232
a

Zea mays Zea mays

39.8/6.5 41.4/5.7

50.5/8.2 47.2/7.6

118 108

40% 35%

New New

Zea mays

28.2/4.6

31.7/5.8

90

29%

New

ATP synthase CF1 alpha subunit ATP synthase CF1 alpha subunit

Saccharum officinarum Saccharum officinarum

30.3/5.9 53.8/6.4

55.7/5.9 55.7/5.9

76 254

25% 55%

Lost 0.4

Transcription factor BTF3

Zea mays

20.6/6.7

17.7/6.6

80

41%

0.3

Elongation factor Tu

Zea mays

41.8/5.4

50.8/6.1

109

30%

New

Hypothetical protein Hypothetical protein

Vitis vinifera Oryza sativa

17.5/4.6 25.0/6.8

26.8/5.6 11.9/11.6

89 71

13% 62%

New New

Spot number in 2-DE gel as shown in Fig. 2. Accession number of Uniport and/or NCBI database. c MW/pI exp: Experimental molecular weight and pI. d MW/pI theo: Theoretical molecular weight and pI. e MOWSE score: statistical probability of true positive identification of predicted proteins calculated by MASCOT (http://www.matrixscience.com) with 150 ppm masses tolerance and one allowed missed cleavage (MOWSE score≥70). f CV.: sequence coverage. g Fold change IM/M denotes the ratio of accumulation of a particular protein from young leaves of maize plants under intercropping versus monocropping condition. IM: intercropped maize, M: monocropped maize. New means the protein only detected in intercropping. Lost indicates the protein only detected in monocropping.
b

indicating the positive intercropping effect in maize with regard to Fe nutrition. Taken together, these results suggest that photosynthesisrelated proteins are associated in a co-ordinated fashion with the rate of photosynthesis, through regulating the expression and assembly of Rubisco subunits. The altered levels of photosynthesis-related proteins with the improved Fe nutrition in peanuts induced by intercropping was clearly observed in our greenhouse model, which may imply that intercropping of maize and peanuts would show an ecologically significant

increase in photosynthesis efficiency in the field on calcareous soils.

3.5. Reduced abundance of stress-related proteins in intercropped plants
Our proteomic study between intercropping and monocropping on calcareous soils indicated that most stress and defenserelated proteins increased in young peanut leaves and young maize leaves and roots after monocropping. In young peanut

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leaves, the pathogenesis-related class 10 proteins (spot 11 and 12), Ara h 8 allergen isoform (spot 13) and putative lectin precursor (spot 14) all accumulated during monocropping (Table 2). In addition, the pathogenesis-related class 10 proteins (spot 11 and 12) and heat shock protein (spot 15) accumulated to levels at least fourfold higher in monocropping compared to intercropping (Table 2). The pathogenesis-related class 10 (PR-10) proteins are found in various species in the plant kingdom and play a role in general plant defense mechanisms, as their induction occurs after pathogen or elicitor treatment wounding [45,46], and other environmental stresses, including drought and salt stress [47]. Lectins bind glycans of glycoproteins, glycolipids, or polysaccharides with high affinity [48], and serve in plant defense, carbohydrate metabolism, and the packaging of storage proteins [49]. The lectin concentration (activity) increases in plant tissues after infection by pathogens, in response to abiotic stress, as well as during tissue growth and development. In young maize leaves, the glutathione S-transferase parA (spot 38) and IN2-1(spot 39) proteins, two homologs belonging to the Glutathione S-transferase (GST) superfamily, were found in higher abundance in monocropping (Table 3). GST, which catalyzes the conjugation of glutathione to electrophilic substrates, plays a crucial role in cell detoxification and stress tolerance in plants [50–55]. Moreover, lactoylglutathione lyase (spot 40), known as glyoxalase I, which catalyzes isomerization of hemithioacetal from methylglyoxal and reduced glutathione, is important for glutathione-based detoxification. Previous studies suggested that various stress conditions, such as salt and water stress, up-regulated the expression of glyoxalase I [56,57], which showed increased accumulation in monocropped young maize leaves (Table 3). In addition, ascorbate peroxidase (spot 37) had a higher abundance in the young leaves of intercropped maize (Table 3). Ascorbate plays an important role in the protection of plants from various stresses by regulating cellular H2O2 levels, [58] and ascorbate peroxidase is an Fe-containing protein. The increase in ascorbate peroxidase accumulation in intercropped maize leaves may be due to the higher Fe nutrition levels. Meanwhile, ethylene-responsive late embryogenesis-like protein (spot 63), peroxidase (spot 64), 14-3-3-like protein (spots 60 and 61), and other stress-related proteins are known to be upregulated under various stress conditions [59–61], and were present at higher levels in monocropped maize roots (Table 4). Enhanced JA levels may result in a high ascorbate content [62]. Consistent with the increased ascorbate peroxidase level in intercropped maize (Table 3), two important enzymes involved in JA biosynthesis, allene oxide synthase (spot 59) and 12-oxo-phytodienoic acid reductase (spot 58), displayed increased activity during intercropping with peanuts (Table 4). Furthermore, chitinase (spot 62), also induced by phytohormones such as JA, plays a role in the defense against fungal infection [63–65], and its levels increased in intercropped maize roots (Table 4), indicating maize intercropping with peanuts may result in an improved ability to resist stress conditions. The higher levels of stress-responsive proteins in both monocropped peanuts and maize indicate that they are suffering more stress than intercropped plants. In turn, it has been suggested that peanut and maize which are intercropped have stronger stress-resistance compared to monocropped

plants through the positive interactions that exist within a mixed culture. It has also been suggested that intercropping of maize and peanuts results in a significant ecological advantage over monocropped systems to enable the plants to adapt to stressful environments in calcareous soils.

3.6. Jasmonate may act as a signaling molecule for stress resistance during intercropping
In this study, two important enzymes involved in JA biosynthesis (spot 58 and 59) accumulated in maize roots when intercropped with peanuts (Table 4). JA is a ubiquitous signaling molecule found in more than 160 plant families, including angiosperms and gymnosperms, as well as algae [66]. Defects in JA response or disruption of the JA biosynthetic pathway results in susceptibility to various pathogens and pests [67–72]. Perhaps the most significant role of JA is that of plant defense, due to its ability to inhibit the expression of genes involved in photosynthesis and ribosome-inactivating proteins following pathogen invasion. This leads to localized cell death, blocking further pathogen invasion [73]. Intercropping [74,75] and mixed cultivars [76,77] have been successfully used to reduce the levels of both pests and disease. Methyl JA (MeJA), involved in mediation of the interplant defense response, volatilized from Artemesia, has been shown to trigger gene expression and defense responses in adjacent tomato plants [67]. In this study, JA biosynthesis proteins were detected at high levels in the roots of intercropped maize. Moreover, from the abundant variation in stress-response proteins, it is speculated that both maize and peanut plants that are intercropped possess a stronger resistance to biotic or abiotic stressors than those that are monocropped. Therefore, the higher levels of JA biosynthesis proteins indicate that JA signaling may play an important role in the self-protection responses against opportunistic damage in intercropping maize, and increase stress resistance ability in intercropped peanuts through rhizosphere interactions.

3.7.

Proteins involved in C and N metabolism

Most of the proteins involved in the TCA cycle and glycolytic processes were found at higher levels in monocropped maize compared to intercropped plants (Tables 3 and 4). Two protein spots (spot 54 and 55), identified as putative aconitate hydratase 1, showed an increased relative signal intensity in monocropped maize roots. Aconitate hydratase is the key enzyme responsible for the conversion of citrate into isocitrate in the TCA cycle. 3-isopropylmalate dehydrogenase (spot 52), the enzyme used in the conversion of isocitrate into 2-ketoglutarate in TCA cycle, was present at higher levels in monocropped maize roots. With regards to glycolysis-related proteins, two proteins (spots 50 and 53), identified as enolase 1, were only found in monocropped maize roots. Enolase is the enzyme responsible for catalyzing the reversible dehydration of 2-phospho-D-glycerate into phosphoenolpyruvate as part of the glycolytic and gluconeogenesis pathways. Furthermore, phosphoenolpyruvate carboxykinase (PEPCK, spot 36), a key enzyme for converting oxaloacetate into phosphoenolpyruvate, was only detected in monocropped maize leaves.

456

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Table 4 – Maize root proteins differentially expressed in mono- and inter-cropping systems as identified by MALDI-TOF/TOF. SP a Accession no. b Homologous protein name Species MW/pI exp c MW/pI theo d MOWSE score e Cv. f Fold change g IM/M
Lost 2.0 0.4 Lost 0.4 0.5

Carbon metabolism 50 gi|162458207 51 gi|34501455 52 gi|194689986 53 gi|162458207 54 gi|92429669 55 gi|92429669

Enolase 1 NADH dehydrogenase subunit I 3-Isopropylmalate dehydrogenase Enolase 1 Putative aconitate hydratase 1 Putative aconitate hydratase 1

Zea mays Physcomitrella patens Oryza sativa Zea mays Sorghum bicolor Sorghum bicolor

29.0/5.5 40.9/5.7 42.9/6.3 61.9/5.0 118.0/6.3 120.6/6.3

48.3/5.2 21.4/6.4 40.2/6.5 48.3/5.2 107.5/6.6 107.5/6.6

75 73 124 90 138 91

32% 32% 48% 22% 27% 33%

Amino acid metabolism 56 gi|162462528 Glutamine synthetase AMP Biosynthesis 57 gi|162463092 Jasmonate biosynthesis 58 gi|162462789 59 gi|162460508 Defense/interaction with 60 gi|1345587 61 gi|110278805 62 gi|48093272 63 gi|1684830 64 gi|45685281

Zea mays

42.4/5.5

39.4/5.6

159

59%

2.1

Adenylosuccinate synthetase

Zea mays

59.5/6.5

53.3/6.9

83

50%

New

12-oxo-phytodienoic acid reductase Allene oxide synthase environment 14-3-3-like protein GF14-6 14-3-3-like protein GF14-D Chitinase Ethylene-responsive late embryogenesis-like protein Peroxidase

Zea mays Zea mays

51.9/5.6 59.7/6.7

42.4/5.5 53.3/6.5

211 209

49% 51%

2.1 2.3

Zea mays Oryza sativa Zea mays Solanum lycopersicum Zea mays

33.7/4.5 33.8/4.6 36.1/4.3 38.3/4.7 61.8/5.7

29.8/4.8 29.6/4.8 34.4/4.9 17.7/4.6 38.9/6.5

175 120 75 71 122

65% 74% 36% 53% 35%

Lost Lost 3.1 0.5 0.3

Protein synthesis 65 gi|162458395 66 gi|162460883 Protein fate 67 gi|162463728 Unknown 68 gi|194697160 69 gi|194701874 70 gi|168023539 71 gi|194693872 72 gi|168011677
a

Translational initiation factor eIF-4A Translational initiation factor eIF-4A

Zea mays Zea mays

54.5/5.4 56.2/5.3

47.2/5.4 46.8/5.4

208 99

63% 37%

2.3 0.3

Maize 20S proteasome alpha SU

Zea mays

29.6/6.3

27.6/6.1

162

65%

2.0

Unknown Unknown Predicted protein Unknown Predicted protein

Zea mays Zea mays Physcomitrella patens Zea mays Physcomitrella patens

39.9/6.4 40.2/6.7 44.4/4.2 61.6/5.4 131.9/5.3

36.7/6.1 35.4/6.2 35.1/6.1 25.8/5.5 25.6/5.7

121 114 78 147 83

77% 55% 27% 54% 24%

Lost 3.9 Lost 0.3 2.0

Spot number in 2-DE gel as shown in Fig. 2. Accession number of Uniport and/or NCBI database. c MW/pI exp: Experimental molecular weight and pI. d MW/pI theo: Theoretical molecular weight and pI. e MOWSE score: statistical probability of true positive identification of predicted proteins calculated by MASCOT (http://www.matrixscience.com) with 150 ppm masses tolerance and one allowed missed cleavage (MOWSE score≥70). f Cv.: sequence coverage. g Fold change IM/M denotes the ratio of accumulation of a particular protein from leaves of maize plants under intercropping versus monocropping condition. IM: intercropped maize, M: monocropped maize. New means the protein only detected in intercropping. Lost indicates the protein only detected in monocropping.
b

Several researchers have reported that Fe deficiency induces the activation of the TCA cycle and glycolysis due to its important role in providing organic acids, protons, and energy production during Fe depletion [78–81]. In particular, the activity of PEPCK has been found to increase markedly in plants suffering from Fe deficiency [78,80–82]. Moreover, proteomic studies have also reported an increase in proteins involved in the TCA cycle and glycolytic pathways in tomato (L. esculentum)

[17], cucumber (C. sativus) [18], sugar beet (B. vulgaris) [19], and M. truncatula [20] with Fe deprivation. The induction of proteins belonging to the TCA cycle and glycolysis in monocropped young maize leaves or roots indicates that maize may suffer from lower Fe conditions compared to when it is intercropped. Importantly, in accordance with the proteomic changes, physiological evidence also suggests that the active Fe content from the rhizosphere soil of intercropped maize is significantly

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higher than that from monocropped maize. The active Fe content of rhizosphere soil in intercropping is 7.4 ± 0.28 mg kg?1, whereas it is only 5.9 ± 0.14 mg kg?1 in monocropped maize. For peanut, NADP-dependent malic enzyme (NADP-ME, spot 19) increased in monocropping systems. NADP-ME catalyzes the oxidative decarboxylation of malate into pyruvate and links the glycolysis and TCA cycles [83]. The activity of NADP-ME decreases following the addition of Fe [84], and NADP-ME also plays a role in the response to plant defenses [85]. Meanwhile, amino acid metabolism proteins were found at lower levels in monocropping systems. Aspartate aminotransferase (spot 41), facilitating the conversion of aspartate into glutamate and oxaloacetate, was only detected in intercropped maize leaves. Glutamine synthetase, an essential enzyme of N metabolism for catalyzing glutamate and ammonia into glutamine, had increased expression not only in intercropped maize leaves (spot 42) but also in the roots (spot 56). Previous studies have suggested that Fe shortage also causes abundant changes in amino acid metabolism-related proteins, as well as proteins involved in the TCA cycle and glycolysis [17,19]. Taken together, the differences in the levels of Fe available in the rhizosphere soil between intercropped and monocropped maize results in changes to C and N metabolism. This provides further evidence that peanut/maize intercropping has an ecological advantage, with high Fe being available in the rhizosphere soil, leading to variation in the proteins related to C and N metabolism.

3.9. Transcript patterns related to stress responses in maize roots by microarray analysis
To further illustrate the ecological significance of intercropping of maize and peanut at the transcript and protein levels, microarray analysis was performed to identify the transcript patterns of the differentially expressed proteins in maize root under monocropping and intercropping treatments. Based on the proteins identified under each treatment, only a few genes related to stress responses underwent changes in transcription levels (Table 5). Three chitin-related genes were induced in the monocropping system. In contrast, six peroxidases were highly abundant in monocropped maize roots, showing a similar expression pattern to the proteomic data. Interestingly, in accordance with the high abundance of proteins involved in JA biosynthesis in the intercropped maize roots, jasmonateinduced protein levels increased at the transcription level during intercropping, as assessed by microarray analysis. This further supports the hypothesis that JA may play an important role in the facilitation of the interaction between the two plant species during intercropping. Further studies are now required to clarify the contribution of JA to the Fe acquisition of peanuts and its ecological significance for plants in the intercropping system.

3.10. Theoretical model of the ecological significance of peanut/maize intercropping illustrated by proteomics profiles
In agreement with the observed improved Fe nutrition in the intercropped peanuts, proteins related to photosynthesis generally displayed a high abundance in young peanut leaves during intercropping, suggesting that intercropping enhanced the efficiency of peanut photosynthesis. The low levels of stress-related proteins in both plants during intercropping

3.8. Transcript response of iron uptake-related genes in peanut roots
AhFRO1, which encodes Fe(III)–chelate reductase, and AhIRT1, responsible for the absorption of Fe(II) into root cells, are key genes for peanut Fe acquisition [86,87]. When AhFRO1 and AhIRT1 transcript levels in intercropped and monocropped peanut roots were compared, the expression of both AhFRO1 and AhIRT1 were significantly upregulated by monocropping (Fig. 4), suggesting that monocropped peanuts suffer from lower Fe availability and thus upregulate Fe-uptake genes.

Relative expression level (X10000)

300 250 200 150

*

Mono Inter

Table 5 – Microarray transcription comparison of the stress-related genes showing changes in protein abundance between intercropped and monocropped maize roots. Genes showing at least a two-fold change in transcription levels are listed. The values indicate the relative expression ratios of intercropped maize (IM) to monocropped maize (M). Affymetrix probe ID
Zm.550.1.A1_at

Fold change (IM/M)
2.0 2.8 2.0 0.3 0.3 0.4 0.2 0.3 0.4 0.4 0.4 0.3 0.5

Description
Glutathione S-transferase GST 22 Glutathione transferase19 Jasmonate-induced protein Peroxidase 12 Putative peroxidase Putative peroxidase Anionic peroxidase H Peroxidase R15 Peroxidase 12 Acidic class I chitinase Chitinase 2 Chitin binding Glutathione transferase30

*
100 50 0 AhFRO1 AhIRT1
Zm.2136.1.S1_at Zm.16176.1.A1_at Zm.16176.1.A1_s_at Zm.404.1.S1_at Zm.13915.1.S1_at Zm.11803.1.A1_at Zm.847.1.S1_at Zm.9675.1.A1_at Zm.2227.1.A1_at Zm.864.1.S1_at Zm.548.1.S1_at Zm.3634.1.A1_at

Fig. 4 – Transcription levels of AhFRO1 and AhIRT1 in intercropped and monocropped peanut roots. Inter, intercropping; Mono, monocropping. Vertical bars indicate expression levels relative to the control 18S ribosomal RNA gene. Error bars represent SD from three replicates. Asterisks indicate P < 0.05 by t-test.

458
Maize

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Improved ecological adaptation of intercropping peanut and maize
Stress-related proteins Stress-related proteins Glutathione SGlutathione StransferaseparA transferaseparA IN2-1 protein protein Lactoylglutathionelyase Lactoylglutathionelyase Stress-related proteins Stress-related proteins Ethylene-responsive late Ethylene-responsive late embryogenesis-like protein embryogenesis-like protein 14-3-3-like protein GF14-6 14-3-3-like protein GF14-6 14-3-3-like protein GF14-D 14-3-3-like protein GF14-D JA biosynthesis biosynthesis Alleneoxide synthase Alleneoxide synthase 12-oxo-phytodienoic 12-oxo-phytodienoic acid reductase reductase JA-induced protein (mRNA) JA-induced protein (mRNA) Photosynthesis Photosynthesis Rubisco-related proteins Rubisco-related proteins Chlorophyll a/b-binding Chlorophyll a/b-binding protein Transketolase1 Transketolase1
Stress-related proteins Stress-related proteins Pathogenesis-related Pathogenesis-related class 10 protein protein Ara h 8 allergen isoform allergen isoform Putative lectinprecursor lectinprecursor Putative heat shock protein heat shock protein Fe acquisition acquisition genes (mRNA) (mRNA) AhIRT1 AhFRO1

Peanut

Higher stressresistant ability

Rhizosphere Rhizosphere interactions interactions

Fig. 5 – Improved ecological adaptation of intercropping peanut and maize. Red arrows represent proteins (dots) or mRNA (squares) expressed at higher levels during intercropping compared to monocropping. Blue arrows indicate proteins or mRNA present at lower levels during intercropping. The proteins shown in the blue and yellow boxes are from young leaves and roots, respectively. As intercropping improved the Fe nutrition of peanuts, photosynthesic proteins displayed high expression levels in the young leaves, but Fe acquisition genes were downregulated in the peanut roots during intercropping. Rhizosphere interactions gave intercropping plants a high stress-resistance, therefore stress-related proteins were not induced during intercropping. Key proteins in JA biosynthesis accumulated in the intercropped maize roots, indicating that JA may contribute to high stress resistance in intercropping via rhizosphere interactions. Finally, the interactions between maize and peanut during intercropping improved the ecological adaptation of the plants to environmental stress.

resulted from the high stress-resistance during intercropping, through interspecific above ground and below ground interactions. The upregulation of allene oxide synthase and 12-oxophytodienoic acid reductase, and the increased induction of JA-induced protein, imply that JA acts as a signaling molecule through rhizosphere interactions, and thus improves the stress-resistance capability of both plants in the intercropping system. Taken together, the advantages of the interaction between peanut and maize in intercropping systems may improve the ecological adaptation of the plants to calcareous soils (Fig. 5). It is suggested that fine-tuning cropping patterns based on a better understanding of the molecular, physiological and ecological processes and interactions, at the individual level and right up to the agroecosystem level, is vital for a sustainable agriculture with high yields, high nutrient-use efficiency and environmental health. Substantial efforts are being made to improve plant nutrition efficiency and stress-resistance during intercropping at the molecular, cellular, whole-plant and ecological population levels. Intercropping strategies of dicotyledonous and gramineous plants could contribute to micronutrient biofortification in developing countries and food security in a more practical, effective, and sustainable manner.

China (Grant No. 20100008110001) and the Innovative Group Grant of the National Science Foundation of China (Grant No. 31121062).

Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2012.10.013.

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We thank Dr. Frank Hochholdinger (University of Bonn Katzenburgweg) for the helpful discussion of the manuscript. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31071840 and 31272223), the Ph.D. Programs Foundation of the Ministry of Education of

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