Rice (Oryza sativa
L.) is an important food crop and a source of calories for billions of people [1
]. Many insects attack rice. Of these insects, the brown planthopper (BPH; Nilaparvata lugens
Stål), the small brown planthopper (SBPH; Laodelphax striatellus
Fallén) and the white-backed planthopper (WBPH; Sogatella furcifera
Horváth) are three major planthoppers attacking rice, causing significant loss of rice yield [2
]. Understanding the defense responses of rice to planthopper infestation is important for developing strategies for such insect control. Studies of the molecular responses of rice to these species of sucking insects have shown that rice responses to these species of sucking insects are regulated through a complex network of salicylic acid (SA)- and jasmonic acid (JA)/ethylene (ET)-dependent signaling pathways [3
]. Gene expression profiles and analyses of physiological responses suggested that the genes involved in several different pathways including cell defense, cellular transport, metabolism, signal transduction, macromolecular degradation and biogenesis, cellular communication and plant defenses were found to be significantly differentially regulated by BPH/SBPH feeding [3
]. Transcription factors (TFs) play a key role in regulation of transcriptional expression in biological processes [13
]. Microarray analysis suggested that rice TFs were involved in defense response to BPH attack [8
] and over-expression of rice TFs in transgenic rice plants increased resistance to WBPH [15
]. Although gene expression profiles resulting from planthopper infestation have been studied by several groups by using two contrasting rice genotypes, the defense mechanisms of rice against these species of sucking insects remain to be largely explored.
In attempt to gain insight into the molecular mechanisms of rice resistance to SBPH, in this study, two contrasting rice genotypes, an SBPH-resistant introgression line (Pf9279-4; R) and its SBPH-susceptible parent (02428; S), were assessed for rice plant responses to SBPH attack at the molecular level. Previously, two-dimensional fluorescence difference gel electrophoresis analysis of these two contrasting rice genotypes that sampled at different time-points after SBPH infestation showed that the differential protein expression between these two contrasting rice genotypes were obvious within 6 h of infestation by SBPH. We therefore examined the comparative transcriptional profiling of these two contrasting rice genotypes which infested with SBPH for 0 h and 6 h by using Affymetrix GeneChip analysis. Although different biochemical pathways including cell defense, cellular transport, metabolism, signal transduction, macromolecular degradation and biogenesis, cellular communication and plant defenses have been documented [3
], it is unclear which individual pathways are involved in the response to planthopper infestation in rice. The increasing number of sequenced plant genomes and advances in bioinformatics strategies offer immense opportunities to study biological processes related to biotic and/or abiotic stress at the cellular and whole-organism level by using a novel systems-level approach [16
]. Among the bioinformatics strategies, because of the unique ability to display the differentially regulated individual biochemical pathways with omics data painted onto the pathway, the Pathway Tools Omics Viewer (http://pathway.iplantcollaborative.org/
) was ultimately employed to visualize the expression patterns and further identify the individual pathways that are responsive to SBPH feeding in rice.
The Pathway Tools Omics Viewer, an integrated tool in the Gramene “Cyc” Pathways databases (http://www.gramene.org
), provides visual analysis of whole-organism datasets and maps gene expression data sets to the metabolic pathways [17
]. The ability to visualize the expression patterns facilitates the ability to pinpoint and interpret expression patterns of interest [16
]. Of the Gramene “Cyc” Pathways databases, the RiceCyc pathway database now features 306 metabolic pathways; 2103 enzymatic reactions, 87 transport reactions, 1543 compounds and metabolites and 6040 protein coding genes are mapped to these reactions and pathways (http://pathway.iplantcollaborative.org/
). In the present study, to understand the global gene expression profiles, the transcriptomic data was uploaded to visualize expression patterns by using the Pathway Tools Omics Viewer. To identify the SBPH resistance and susceptibility-related metabolic pathways, by using Pathway Tools Omics Viewer, the individual metabolic pathways that were differentially regulated were displayed with omics data painted onto the pathway; by combining Pathway Tools Omics Viewer with the web tool Venn (http://bioinfogp.cnb.csic.es/
), 21 and 6 metabolic pathways potentially related to SBPH resistance and susceptibility in rice were identified, respectively. This study presents an omics-based comparative transcriptional profiling of SBPH-resistant and SBPH-susceptible rice plants during early infestation by SBPH and aims to identify individual pathways that are responsive to early infestation by SBPH in rice. The identified biochemical pathways will provide insight into how rice plants respond to early infestation by SBPH and the results will help us understand the general defense responses of rice plants to insect infestation from the perspective of biochemical pathways.
In this study, we visualized the rice transcriptomic data of microarray expression profiling by using Pathway Tools Omics Viewer. By using the capabilities of Pathway Tools Omics Viewer for overlaying transcriptomic data onto the overview as a whole and animation of the comparative transcriptome analysis, our data suggested that the difference of change pattern between these two contrasting rice genotypes mostly lies in biosynthetic pathways when exposed to SBPH; while the obvious difference of change pattern between these two contrasting rice genotypes lies in energy metabolism pathways (Table S2
). By using the capabilities of Pathway Tools Omics Viewer for generation a table containing all individual pathways with omics data painted onto the pathway, 166 metabolic pathways that were differentially regulated when exposed to SBPH were identified; of these, 64 metabolic pathways displayed similar change pattern in both two contrasting rice genotypes when exposed to SBPH (Table S4
); in response to SBPH infestation, biosynthesis was the largest class of the differentially regulated metabolic pathways in both resistant and susceptible rice plants (R6_R0 and S6_S0; Table 1
). By combination Pathway Tools Omics Viewer and the web tool Venn, 21 and 6 metabolic pathways were considered to be potentially associated with SBPH resistance and susceptibility in rice; these 21 SBPH resistance-related metabolic pathways were related to cuticle, phytoalexin, amine and polyamine, alkaloid, hormone, amino acid, fermentation and C1 compounds utilization and assimilation; in response to SBPH infestation, the metabolic pathways derived from phenylalanine including betanidin degradation, flavonoid biosynthesis, pinobanksin biosynthesis, salicylate biosynthesis and phenylalanine degradation III were down-regulated in the resistant rice plants (Figure 2
); these six SBPH susceptibility-related metabolic pathways were related to amino acid, lipid, cofactors, prosthetic groups, electron carriers biosynthesis as well as amine and polyamine. Of the metabolic pathways that were considered to be potentially associated with SBPH resistance and susceptibility in rice, the change pattern of the phenylalanine degradation III and ureide biosynthesis in the resistant rice plants displayed opposing differential changes in the susceptible rice plants (Table 2
and Table 3
), indicating these two metabolic pathways are highly potentially responsive to SBPH infestation.
The plant epidermis is considered to have an important role in defense against insect attack [23
]. Observations implicated that rice plant epidermis plays a role in selection of food plant by BPH and that a high ratio of long to short carbon-chain compounds in BPH resistant rice varieties largely determined planthopper feeding responses [24
]. These observations provide support for the viewpoint that increased activity in response to chemical characteristics of epidermis wax may play an important role in field resistance [24
]. In this study, the epidermis wax related metabolic pathway, the very long chain fatty acid biosynthesis, was found to be up-regulated in the SBPH-resistant rice plants. The very long chain fatty acids are fatty acids of 20 to 36 carbons and are required for plant cuticle biosynthesis [25
]. Recently, the very long chain fatty acid biosynthesis pathway has been found to be associated with plant defense; well-organized cuticle layers constitute the outermost layer of epidermal cells and thereafter act as the first natural barrier when encountered by pathogens [27
]. Similarly, the very long chain fatty acid biosynthesis in the SBPH resistant rice plants was up-regulated when exposed to SBPH infestation.
It is reported that some phloem feeding insects induce the SA pathway while suppressing the plant’s JA response [28
]. In response to planthopper attack, these antagonistic phenomena of SA and JA have also been observed. However, these antagonistic phenomena associated with rice resistance to planthopper seem to be in a contrasting manner. In response to BPH infestation, JA activity was suppressed while levels of SA were much higher accumulated [28
]. In contrast, in this study, in the SBPH resistant rice plants, the JA biosynthesis was up-regulated while the SA biosynthesis and its branch pinobanksin biosynthesis were down-regulated. Growing evidence shows that plant defense against their different kinds of attackers is also in a hormones-dependent manner [29
]. Besides JA and SA, other plant hormones such as auxin and cytokinin (CK) also serve as modulators of the hormone signaling backbone [29
]. Auxin signaling is critical in regulation plant growth and development [30
]. SBPH infestation seems to significantly modify auxin metabolism to decrease plant growth capacity. In the SBPH resistant rice plants, the inactivation of IAA (IAA, indole-3-acetic acid; IAA conjugate biosynthesis I and II), showed the strongest response among the 21 metabolic pathways potentially related to SBPH resistance in rice and was significantly up-regulated after attack by SBPH (Table S5
). In rice-WBPH interactions, genes involved in auxin signaling such as auxin-responsive protein, auxin efflux carrier component 4, and auxin-responsive protein IAA14 were also strongly suppressed [31
]. It has been reported that CK mediates insect resistance and also has an important role in the activation of JA biosynthesis [32
]. For example, after wounding, the level of CK increased in transgenic tobacco (Nicotiana tabacum
cv. Xanthinc) plants over-expressing a small GTP-binding protein, which resulted in increased rates of JA production [33
]. In this study, we also observed that CK degradation was down-regulated and JA biosynthesis was up-regulated in the SBPH-resistant plant.
Betanidin is synthesized from tyrosine and is water-soluble nitrogen-containing pigment [34
]. Pigment accumulation has been reported to be induced in response to insect feeding and the corresponding biosynthetic genes maybe directed against herbivores [35
]. In the SBPH-resistant rice plants, betanidin degradation was down-regulated. It is therefore significant that tyrosine, a precursor of betanidin, showed the second most significant change in the SBPH-resistant rice plants, increasing about 300%. Phytoalexins are antimicrobial specialised compounds which are produced by plants as a response to pathogen attack [36
]. Momilactones from rice are recognized as the major diterpenoid phytoalexins and growth inhibitors [37
]. It has been reported that WBPH attack resulted in increased levels of momilactone A within 24 h [39
]. Of the 21 metabolic pathways potentially related to SBPH resistance in rice, momilactone biosynthesis was the second most significantly altered pathway after attack by SBPH and was up-regulated in the SBPH-resistant rice plants (Table S5
). In plants, flavonoids are thought to have many functions including attracting pollinating insects [40
] and have been reported to stimulate the probing of rice WBPH and SBPH [41
]. In accordance with the resistance, in the SBPH-resistant rice plants, flavonoid biosynthesis was down-regulated.
Amino acids and amino acid derivatives have been considered to have an important role in defense response [43
]. Glutamate was the most abundant amino acid in the resistant rice plants before and after attack by SBPH (Table 4
). Arginine is synthesized from glutamate and is the precursor of spermine and γ-amino-butyric acid [44
]. The level of glutamate and arginine increased in the resistant rice plants but decreased in the susceptible rice plants after attack by SBPH. The γ-amino-butyric acid was the amino acid showing the largest change in concentration in the resistant rice plants in response to SBPH infestation (an increase of about 900%). Several lines of evidence indicate that increased γ-amino-butyric acid level is related to plant defense [46
]. Spermine has also been implicated in plant defenses and its intermediate putrescine has been reported to directly affect insect growth and development and therefore could also act as a defense mechanism against some herbivorous insects [47
]. Thus, the coordinated and linked changes pattern of glutamate and arginine level maybe enhance spermine and γ-amino-butyric acid biosynthesis and act as part of the defense against SBPH infestation. Meanwhile, before attack by SBPH, the level of isoleucine was the second highest, whereas, after attack by SBPH, the second highest level of amino acid was replaced by arginine. The two most abundant amino acid accounts for nearly one half of the total amino acid content when exposed to SBPH. Furthermore, although the functional significance of the phosphorylated pathway of serine biosynthesis in plants is not yet known, it is clear that in the phosphorylated pathway of serine biosynthesisthe glutamate functions as the substrate for the transfer of an amino group from glutamate to 3-phosphoserine [48
]; in the resistant rice plants, the phosphorylated pathway of serine biosynthesiswas down-regulated in the resistant rice plants, meaning less glutamate used in the phosphorylated pathway of serine biosynthesis. In addition, the glutamate degradation III was down-regulated in the resistant rice plants. In the last step of synthesis of isoleucine, an amino group from glutamate was transferred to produce the isoleucine (http://pathway.iplantcollaborative.org
); in the resistant rice plants, isoleucine level which was the second most abundant before attack showed the third significant decreased after attack, meaning less glutamate used in synthesis of isoleucine. Collectively, the increased N-rich glutamate and arginine in the resistant rice plants means that the N-rich soluble molecules accumulated. The accumulated N-rich soluble molecules have been considered as a means of nitrogen (N) and the carbon (C) store after wounding, which helps to prevent N losses during the wounding response and could work as a source for new growth after the wound recovery phase [49
]. Additionally, C store was also modulated in the resistant rice plants during the SBPH response by down-regulation of the mixed acid fermentation in which the products are mixed acids, particularly equal amounts of carbon dioxide (CO2
] and by up-regulation of the reductive TCA cycle I in which two molecules of CO2
can be fixed [53
Threonine has been conferred as the amino acid that resistant to Hyaloperonospora. arabidopsidis
, presumably by altering the pathogen’s ability to grow under that condition; mutations in threonine biosynthesis gene displayed increased resistance to H. arabidopsidis
and increased accumulation of threonine [54
]. Similarly, the threonine level increased and its expression (aminopropanol biosynthesis, threonine degradation II and threonine degradation III (to methylglyoxal)) was down-regulated in the resistant rice plants when exposed to SBPH. Hopperburn partially results from the amides accumulation in infested rice leaf blade; the amides accumulate in the leaf-blade tissues because translocation systems of amides are not functioning [55
]. There are two major forms of organic nitrogen compounds that can be transported amides (such as asparagine and glutamine) and ureides (such as allantoin and allantoate) [56
]. In response to SBPH infestation, the ureide biosynthesis was down-regulated in the resistant rice plants but up-regulated in the susceptible rice plants, presumably indicating the amide transportation systems are not functioning in the susceptible rice plants.
Overall, more than one third of the 166 differentially regulated metabolic pathways displayed similar change pattern in both two contrasting rice genotypes when exposed to SBPH. Twenty-one differentially regulated metabolic pathways were associated with SBPH resistance in rice. Briefly, SBPH infestation modulated phytohormones such as SA, JA, auxin and cytokinin; particularly, SA and JA showed antagonistic phenomena; inactivation of auxin enhanced significantly and showed the strongest response, meaning a shift from growth and development to defense. Following SBPH infestation, the major diterpenoid phytoalexin momilactone A metabolism enhanced significantly and showed the second strongest response; the N-rich glutamate and arginine levels increased and were the most abundant in resistant rice plants, together with the down-regulation of mixed acid fermentation and up-regulation of the C1 compounds utilization and assimilation, suggesting a N and C store to prevent N and C losses and work as a source for new growth after the phase of wound recovery. In summary, SBPH infestation caused changes in the primary and secondary metabolism; rice plants protect themselves against SBPH at several levels. Of these, one is the well-organized cuticle layers as first barrier encountered by SBPH. The second is the biosynthesis of inducible chemical defences such as the major diterpenoid phytoalexin momilactone A. The third is a shift from growth and development to defense, enhancing the capacity of rice plants to main high fitness in the face of enemies. The fourth is the modulation of N and C store and work as a source for new growth after the phase of wound recovery. In infested resistant rice plants, metabolic processes presumably not only protect them against SBPH but also enhance the capacity of rice plants to repair the tissue that injured by SBPH attack.