Evaluation of various proteomic techniques to identify proteins involved in cereal stress responses to aphid infestation
- Authors: Nqumla, Ntombekhaya
- Date: 2012
- Subjects: Aphids , Wheat , Plant proteomics , Rice
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11270 , http://hdl.handle.net/10353/d1004572 , Aphids , Wheat , Plant proteomics , Rice
- Description: All plants are exposed to abiotic and biotic stresses and have developed intricate signalling responses to survive. They respond to cell-structure disruption caused by herbivore probing and feeding by the formation of callose. Callose is a linear homopolymer made up of β-1,3-linked glucose residues with some β-1,6-branches. Plant responses to abiotic or biotic stress share events such as phosphorylation, membrane depolarization, calcium influx and the release of reactive oxygen species such as hydrogen peroxide. These events lead to the up-regulation of several pathways leading to biosynthesis of signalling molecules such as salicylic acid, jasmonate, abscisic acid and ethylene pathways. The aim of this study was to determine the most suitable proteomic approach for identifying proteins and signalling pathways involved in cereal response to aphid infestation. An in silico approach was first evaluated in which the 5ʹ upstream regulatory region of proteins belonging to the family of callose synthases was scanned for cis-regulatory elements in order to identify which callose synthases are possibly expressed in plants during biotic or abiotic stresses. Bioinformatics tools were used in the identification of twelve Arabidopsis and ten rice callose synthase coding regions. Genome sequences for rice and Arabidopsis were scanned for the 2000 bp 5ʹ region upstream of the start codon of each callose synthase coding region. PlantCare, PLACE and Athena software were used to identify putative cis-regulatory elements present in the 2000 bp 5ʹ upstream sequences. The majority of cis-acting elements identified were involved in drought and high temperature responses and only one cis-acting element was involved in wound stress. These results therefore indicated a probable role for plant callose synthases in drought stress responses rather than in biotic stress responses. Genevestigator analysis of Arabidopsis results of micro-array experiments indicated that AtGSL10 is highly up-regulated, with AtGSL1, 3, 5, 6, 7, 8, 11 and 12 showing medium up-regulation and AtGSL2, 4 and 9 no up-regulation during aphid infestation of Arabidopsis plants, implicating a possible role for AtGSL10 in the plant response to aphid infestation. An LC/MS/MS approach was used to identify specific signalling pathways involved in wheat resistance or stress response to aphid infestation. Eight proteins were identified as being up-regulated during aphid feeding in wheat, and 11 proteins were identified as possibly involved in the wheat resistance mechanism against aphid infestation. Several proteins were also identified as constitutively expressed proteins, during normal conditions and aphid infestation. Most pathways identified with proteins up-regulated in the resistance mechanisms of TugelaDN plants, were related to energy metabolism and located in the chloroplast. Evaluation of two dimensional gel electrophoresis to identify phosphoproteins differentially regulated in wheat during aphid infestation, revealed the up-regulation of three proteins namely photosystem II oxygen-evolving complex protein 2, HVUNKNOWN from Hordeum vulgare subsp vulgare and HSKERAT9 NID from Homo sapiens. Additional 57 proteins were partially identified as involved in the stress response but due to low protein levels, the percentage of matching peptides to these proteins was below the required confidence level. Although these protein identifications were below the confidence level, it is interesting to note that several of the proteins are known stress response proteins, and therefore could serve as potential targets for future investigations. In conclusion, the down and up-regulation of wheat proteins after aphid feeding reported in this study suggest that several signalling pathways are involved in the cereal stress response to aphid feeding. In silico approaches require knowledge or identification of potential proteins whereas 2D and LC/MS can identify numerous proteins still unknown to be involved in specific stress responses. The 2D approach is also limited in that the proteins of interest may be in low abundance and therefore not detected in the gels due to the presence of high abundant proteins. Therefore the best approach to identify proteins and signalling pathways involved in the stress response of wheat to aphid infestation, is the LC/MS/MS approach, as this proved to be the most sensitive and robust, identifying the most proteins with a high degree of confidence.
- Full Text:
- Date Issued: 2012
- Authors: Nqumla, Ntombekhaya
- Date: 2012
- Subjects: Aphids , Wheat , Plant proteomics , Rice
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11270 , http://hdl.handle.net/10353/d1004572 , Aphids , Wheat , Plant proteomics , Rice
- Description: All plants are exposed to abiotic and biotic stresses and have developed intricate signalling responses to survive. They respond to cell-structure disruption caused by herbivore probing and feeding by the formation of callose. Callose is a linear homopolymer made up of β-1,3-linked glucose residues with some β-1,6-branches. Plant responses to abiotic or biotic stress share events such as phosphorylation, membrane depolarization, calcium influx and the release of reactive oxygen species such as hydrogen peroxide. These events lead to the up-regulation of several pathways leading to biosynthesis of signalling molecules such as salicylic acid, jasmonate, abscisic acid and ethylene pathways. The aim of this study was to determine the most suitable proteomic approach for identifying proteins and signalling pathways involved in cereal response to aphid infestation. An in silico approach was first evaluated in which the 5ʹ upstream regulatory region of proteins belonging to the family of callose synthases was scanned for cis-regulatory elements in order to identify which callose synthases are possibly expressed in plants during biotic or abiotic stresses. Bioinformatics tools were used in the identification of twelve Arabidopsis and ten rice callose synthase coding regions. Genome sequences for rice and Arabidopsis were scanned for the 2000 bp 5ʹ region upstream of the start codon of each callose synthase coding region. PlantCare, PLACE and Athena software were used to identify putative cis-regulatory elements present in the 2000 bp 5ʹ upstream sequences. The majority of cis-acting elements identified were involved in drought and high temperature responses and only one cis-acting element was involved in wound stress. These results therefore indicated a probable role for plant callose synthases in drought stress responses rather than in biotic stress responses. Genevestigator analysis of Arabidopsis results of micro-array experiments indicated that AtGSL10 is highly up-regulated, with AtGSL1, 3, 5, 6, 7, 8, 11 and 12 showing medium up-regulation and AtGSL2, 4 and 9 no up-regulation during aphid infestation of Arabidopsis plants, implicating a possible role for AtGSL10 in the plant response to aphid infestation. An LC/MS/MS approach was used to identify specific signalling pathways involved in wheat resistance or stress response to aphid infestation. Eight proteins were identified as being up-regulated during aphid feeding in wheat, and 11 proteins were identified as possibly involved in the wheat resistance mechanism against aphid infestation. Several proteins were also identified as constitutively expressed proteins, during normal conditions and aphid infestation. Most pathways identified with proteins up-regulated in the resistance mechanisms of TugelaDN plants, were related to energy metabolism and located in the chloroplast. Evaluation of two dimensional gel electrophoresis to identify phosphoproteins differentially regulated in wheat during aphid infestation, revealed the up-regulation of three proteins namely photosystem II oxygen-evolving complex protein 2, HVUNKNOWN from Hordeum vulgare subsp vulgare and HSKERAT9 NID from Homo sapiens. Additional 57 proteins were partially identified as involved in the stress response but due to low protein levels, the percentage of matching peptides to these proteins was below the required confidence level. Although these protein identifications were below the confidence level, it is interesting to note that several of the proteins are known stress response proteins, and therefore could serve as potential targets for future investigations. In conclusion, the down and up-regulation of wheat proteins after aphid feeding reported in this study suggest that several signalling pathways are involved in the cereal stress response to aphid feeding. In silico approaches require knowledge or identification of potential proteins whereas 2D and LC/MS can identify numerous proteins still unknown to be involved in specific stress responses. The 2D approach is also limited in that the proteins of interest may be in low abundance and therefore not detected in the gels due to the presence of high abundant proteins. Therefore the best approach to identify proteins and signalling pathways involved in the stress response of wheat to aphid infestation, is the LC/MS/MS approach, as this proved to be the most sensitive and robust, identifying the most proteins with a high degree of confidence.
- Full Text:
- Date Issued: 2012
Observations of selective feeding of the aphid, Sitobion yakini (eastop) on leaf blades of barley (Hordeum vulgare L)
- Authors: Matsiliza, Babalwa
- Date: 2000
- Subjects: Aphids , Phloem , Barley
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4212 , http://hdl.handle.net/10962/d1003781 , Aphids , Phloem , Barley
- Description: Penetration of leaves of barley (Hordeum vulgare L.) plants grown under normal conditions and those exposed to physiological stress, by the aphid, Sitobion yakini was investigated using light and electron microscope techniques. This was carried out to determine if there was evidence of preferential feeding on either thin- or thick-walled sieve tubes in the barley leaf. Under both stress and non-stress conditions, preliminary results of an electron microscope investigation showed that penetration of the epidermis and mesophyll was largely intercellular, becoming partly intercellular and in part intracellular inside the vascular bundle. A total of 317 stylets and stylet tracks were encountered during the examination of 2000 serial sections. In non-stressed plant material, 293 (92%) terminated in thin-walled sieve tubes and only 24 (8%) in thick-walled sieve tubes. Investigation of 1000 serial sections using stressed plant material showed, that 84 of 89 (94%) stylets and stylet tracks encountered terminated in thin-walled sieve tubes. Furthermore, 90 of 94 (96%) stylets and stylet tracks encountered in 1000 serial sections from the second experiment of control non-stressed plant material (control) terminated in thin-walled sieve tubes. The thin-walled sieve tubes were significantly more visited (probed) by the aphid than the thick-walled sieve tubes. Under stress conditions, 50 of 89 (56%) stylets and stylet tracks which terminated in thin-walled sieve tubes were associated with the small longitudinal bundles. Under normal conditions, 65 of 94 (69%) stylets and stylet tracks which terminated in thin-walled sieve tubes were associated with the small longitudinal bundles. There were no significant differences on the number of probes of sieve elements between the two treatments. These data suggest that the aphid S. yakini feeds preferentially on the thin-walled sieve tubes of the small longitudinal vascular bundles in plants grown under normal conditions and those exposed to physiological stress, such as water stress. This further suggests that the thin-walled sieve tubes in barley leaf blades are more attractive to the aphid and are probably most functional in terms of phloem loading and transport.
- Full Text:
- Date Issued: 2000
- Authors: Matsiliza, Babalwa
- Date: 2000
- Subjects: Aphids , Phloem , Barley
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4212 , http://hdl.handle.net/10962/d1003781 , Aphids , Phloem , Barley
- Description: Penetration of leaves of barley (Hordeum vulgare L.) plants grown under normal conditions and those exposed to physiological stress, by the aphid, Sitobion yakini was investigated using light and electron microscope techniques. This was carried out to determine if there was evidence of preferential feeding on either thin- or thick-walled sieve tubes in the barley leaf. Under both stress and non-stress conditions, preliminary results of an electron microscope investigation showed that penetration of the epidermis and mesophyll was largely intercellular, becoming partly intercellular and in part intracellular inside the vascular bundle. A total of 317 stylets and stylet tracks were encountered during the examination of 2000 serial sections. In non-stressed plant material, 293 (92%) terminated in thin-walled sieve tubes and only 24 (8%) in thick-walled sieve tubes. Investigation of 1000 serial sections using stressed plant material showed, that 84 of 89 (94%) stylets and stylet tracks encountered terminated in thin-walled sieve tubes. Furthermore, 90 of 94 (96%) stylets and stylet tracks encountered in 1000 serial sections from the second experiment of control non-stressed plant material (control) terminated in thin-walled sieve tubes. The thin-walled sieve tubes were significantly more visited (probed) by the aphid than the thick-walled sieve tubes. Under stress conditions, 50 of 89 (56%) stylets and stylet tracks which terminated in thin-walled sieve tubes were associated with the small longitudinal bundles. Under normal conditions, 65 of 94 (69%) stylets and stylet tracks which terminated in thin-walled sieve tubes were associated with the small longitudinal bundles. There were no significant differences on the number of probes of sieve elements between the two treatments. These data suggest that the aphid S. yakini feeds preferentially on the thin-walled sieve tubes of the small longitudinal vascular bundles in plants grown under normal conditions and those exposed to physiological stress, such as water stress. This further suggests that the thin-walled sieve tubes in barley leaf blades are more attractive to the aphid and are probably most functional in terms of phloem loading and transport.
- Full Text:
- Date Issued: 2000
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