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
Optimisation of expression of a rice (Oryza sativa L.cv Nipponbare) plant natriuretic peptide (OsPNP-B) and its functional characterisation
- Authors: Affun, Ogheneochuko Janet
- Date: 2012
- Subjects: Rice , Plant hormones , Water-electrolyte imbalances
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/24354 , vital:62644
- Description: Maintenance of water and solute homeostasis is a key requirement for living systems, and in vertebrates, homeostasis is in part achieved by natriuretic peptides (NP), a family of peptide hormones. A related family of peptide hormones have also been found in the plant kingdom. Plant natriuretic peptides (PNP) are a novel class of plant proteins with two closely related homologous genes (AtPNP-A and AtPNP-B) identified in the model plant Arabidopsis thaliana. AtPNP-A has been extensively studied and evidence obtained points to a role in plant water homeostasis. No research has been conducted on the function of PNP-B proteins. In this study, we focus on the bioinformatic analysis of the PNP-B gene in various plants, as well investigating whether PNP-B plays a role in water homeostasis in rice plants exposed to drought stress. Basic local alignment search tool (BLAST) queries of the ―The Gene Index‖, EST and available plant genome databases revealed the presence of the PNP-B mRNA in rice, oil seed rape, oak, leafy spurge, poplar, sugarcane, pepper, cotton, apple and maize. All the identified genome sequences contained a predicted intron/insert, which was not present in the related mRNA sequences. However, RT-PCR analysis revealed the presence of the 101 bp insert in the 976 bp amplified rice PNP-B (OsPNP-B) cDNA and therefore expression was optimized for the protein encoded by only the exon2 sequence as this contained the predicted active site region of PNP-B. OsPNP-B was shown to be translated to a protein of 14 kDa with a sequence similarity to AtPNP-B (54percent), AtPNP-A (37percent) and CjBAp12 (55percent), suggesting two possible functions for PNP-B viz water homeostasis and/or pathogenesis defence. To determine whether PNP-B is involved in water homeostasis, total protein extracted from 4 weeks old (4 leaves stage) rice plants subjected to drought treatment for a period of 24, 48, 72, 120, 168 and 240hrs were resolved by 17percent SDS-PAGE and analysed by western blot analysis. The PNP-B protein was found to be down-regulated during drought stress, implying that PNP-B may play a role in water homeostasis through the release of water from cells rather than the up-take of water as seen At-PNP-A. PNP-B could therefore also be involved in plant defence mechanisms to pathogens where plants induce desiccation of infected leaves, thereby ridding the plant of the relevant pathogen. , Thesis (MSc) -- Faculty of Science and Agriculture, 2012
- Full Text:
- Date Issued: 2012
- Authors: Affun, Ogheneochuko Janet
- Date: 2012
- Subjects: Rice , Plant hormones , Water-electrolyte imbalances
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/24354 , vital:62644
- Description: Maintenance of water and solute homeostasis is a key requirement for living systems, and in vertebrates, homeostasis is in part achieved by natriuretic peptides (NP), a family of peptide hormones. A related family of peptide hormones have also been found in the plant kingdom. Plant natriuretic peptides (PNP) are a novel class of plant proteins with two closely related homologous genes (AtPNP-A and AtPNP-B) identified in the model plant Arabidopsis thaliana. AtPNP-A has been extensively studied and evidence obtained points to a role in plant water homeostasis. No research has been conducted on the function of PNP-B proteins. In this study, we focus on the bioinformatic analysis of the PNP-B gene in various plants, as well investigating whether PNP-B plays a role in water homeostasis in rice plants exposed to drought stress. Basic local alignment search tool (BLAST) queries of the ―The Gene Index‖, EST and available plant genome databases revealed the presence of the PNP-B mRNA in rice, oil seed rape, oak, leafy spurge, poplar, sugarcane, pepper, cotton, apple and maize. All the identified genome sequences contained a predicted intron/insert, which was not present in the related mRNA sequences. However, RT-PCR analysis revealed the presence of the 101 bp insert in the 976 bp amplified rice PNP-B (OsPNP-B) cDNA and therefore expression was optimized for the protein encoded by only the exon2 sequence as this contained the predicted active site region of PNP-B. OsPNP-B was shown to be translated to a protein of 14 kDa with a sequence similarity to AtPNP-B (54percent), AtPNP-A (37percent) and CjBAp12 (55percent), suggesting two possible functions for PNP-B viz water homeostasis and/or pathogenesis defence. To determine whether PNP-B is involved in water homeostasis, total protein extracted from 4 weeks old (4 leaves stage) rice plants subjected to drought treatment for a period of 24, 48, 72, 120, 168 and 240hrs were resolved by 17percent SDS-PAGE and analysed by western blot analysis. The PNP-B protein was found to be down-regulated during drought stress, implying that PNP-B may play a role in water homeostasis through the release of water from cells rather than the up-take of water as seen At-PNP-A. PNP-B could therefore also be involved in plant defence mechanisms to pathogens where plants induce desiccation of infected leaves, thereby ridding the plant of the relevant pathogen. , Thesis (MSc) -- Faculty of Science and Agriculture, 2012
- Full Text:
- Date Issued: 2012
Differential expression and regulation of sucrose transporters in rice (Orzya sativa L, cv Nipponbare) during environmental stress conditions
- Authors: Ibraheem, Omodele
- Date: 2011
- Subjects: Crops -- Effect of stress on , Plant molecular genetics , Gene expression , Sucrose , Rice
- Language: English
- Type: Thesis , Doctoral , PhD (Biochemistry)
- Identifier: vital:11249 , http://hdl.handle.net/10353/330 , Crops -- Effect of stress on , Plant molecular genetics , Gene expression , Sucrose , Rice
- Description: Plant productivity is greatly affected by environmental stresses such as drought, salinity and insect herbivory. Plants respond and adapt to these stresses by exhibiting physiological as well as biochemical changes at the cellular and molecular levels in order to survive. Expression of a variety of genes which encode numerous membrane transporters have been demonstrated to be induced by these stresses in a variety of plants. The nutritional status of plants is controlled by these transporters, which are regulated by the transcription of the corresponding genes. In spite of these adverse stress effects on agricultural yield, only a few studies have focused on gene transcriptional and translational regulation of membrane transporters during environmental stress situations. Rice, like other plants, contains a number of sucrose transporters encoded by a family of genes. However, detailed knowledge of their roles, localization and regulation during environmental stress conditions is lacking. Bioinformatic tools were used to identify putative cis-acting regulatory elements that may be involved in the regulation of rice and Arabidopsis thaliana sucrose transporters. The possible cis-acting regulatory elements were predicted by scanning genomic sequences 1.5 kbp upstream of the sucrose transporter genes translational start sites, using Plant CARE, PLACE and Genomatix Matinspector professional data bases. Several cis-acting regulatory elements that are associated with plant development, plant hormonal regulation and stress response were identified, and were present in varying frequencies within the 1.5 kbp of 5′ regulatory region. The putative cis-acting regulatory elements that possibly are involved in the expression and regulation of sucrose transporter gene families in rice and Arabidopsis thaliana during cellular development or environmental stress conditions were identified as: A-box, RY, CAT, Pyrimidine-box, Sucrose-box, ABRE, ARF, ERE, GARE, Me-JA, ARE, DRE, GA-motif, GATA, GT-1, MYC, MYB, W-box, and I-box. Expression analysis was used to elucidate the role of rice (Oryza sativa L. cv Nipponbare) sucrose transporter (OsSUT) genes during drought and salinity treatments of three week old rice plants ( at four leaf stage) over a 10 days. Among the five rice OsSUT genes identified, only OsSUT2 was observed to be progressively up-regulated during drought and salinity treatments, while OsSUT1, OsSUT4 and OsSUT5 were expressed at low levels, and OsSUT3 showed no detectable transcript expression. Sucrose transport will be essential to meet the cellular energy demands and also for osmoprotectant activities during drought and salinity stresses. It therefore indicates that OsSUT2 which facilitates transport of sucrose from photosynthetic cells will be III essential for rice plants to cope with drought and salinity stresses, and cultivars with a higher OsSUT2 expression should be able to tolerate these environmental stresses better. The role of OsSUT in assimilate transport during rusty plum aphids (Hysteroneura setariae; Thomas) infestation on the leaves of three week old rice (Orzya sativa L. cv Nipponbare) cultivar plants, over a time-course of 1 to 10 days of treatments, was also examined by combination of gene expression and β-glucuronidase (GUS) reporter gene analysis. Real Time PCR analysis of the five OsSUT genes revealed that the expression of OsSUT1 was progressively up-regulated during the course of aphid infestation. OsSUT2 and OsSUT4 expression were comparatively low in both the control and treated plants. OsSUT5 showed no clear difference in transcript expression in both control and treated plants, while no detectable transcript expression of OsSUT3 could be found. The up-regulation of OsSUT1 gene was verified at protein level by western blot analysis in both the control and treated plants. OsSUT1 protein expression was found to increase with time during aphid infestation. A similar trend was noticeable in the control plants, however at a lower expression level. These demonstrate that the cellular expression of OsSUT1is regulated by both developmental and environmental factors. OsSUT1-promoter:::GUS reporter gene expression was observed within the vascular parenchyma and/or companion cells associated with phloem sieve elements of the large and small bundles in the phloem tissues of the flag leaf blade regions where feeding aphids were confined, which progressively increased with time of infestation. It is suggested that OsSUT1 may primarily play an essential role in phloem transport of assimilate to wounded tissues from adjacent health tissues or may be involved in the retrieval of assimilate back into the phloem to minimize loss caused by the infestation. Some OsSUT1-promoter:::GUS expression was also found in the metaxylem at 10 days after infestation, which could signify a recovery system in which sucrose lost into the xylem as a result of aphids feeding are retrieved back into the phloem through the vascular parenchyma. This was supported by the exposure of cut ends of matured OsSUT1-promoter:::GUS rice plant leaf to 2% sucrose solution. OsSUT1-promoter:::GUS expression was observed within the protoxylem, xylem and phloem parenchyma tissues. This indicates that sucrose translocating within the xylem tissues are retrieved into the phloem via the OsSUT1 localized within the parenchyma tissues. In conclusion, the differential expression and regulation of rice (Orzya sativa L. cv Nipponbare) sucrose transporters as reported here suggest that OsSUT2 and OsSUT1 were constitutively expressed compared to other rice sucrose transporters during drought and salinity, and rusty plum aphids (Hysteroneura setariae; Thomas) infestation stresses respectively. Thus, the expression and regulation of the sucrose transporters could be related to the physiological and nutritional requirements of the cells during plant developmental or environmental stress state that allows their differential expression.
- Full Text:
- Date Issued: 2011
- Authors: Ibraheem, Omodele
- Date: 2011
- Subjects: Crops -- Effect of stress on , Plant molecular genetics , Gene expression , Sucrose , Rice
- Language: English
- Type: Thesis , Doctoral , PhD (Biochemistry)
- Identifier: vital:11249 , http://hdl.handle.net/10353/330 , Crops -- Effect of stress on , Plant molecular genetics , Gene expression , Sucrose , Rice
- Description: Plant productivity is greatly affected by environmental stresses such as drought, salinity and insect herbivory. Plants respond and adapt to these stresses by exhibiting physiological as well as biochemical changes at the cellular and molecular levels in order to survive. Expression of a variety of genes which encode numerous membrane transporters have been demonstrated to be induced by these stresses in a variety of plants. The nutritional status of plants is controlled by these transporters, which are regulated by the transcription of the corresponding genes. In spite of these adverse stress effects on agricultural yield, only a few studies have focused on gene transcriptional and translational regulation of membrane transporters during environmental stress situations. Rice, like other plants, contains a number of sucrose transporters encoded by a family of genes. However, detailed knowledge of their roles, localization and regulation during environmental stress conditions is lacking. Bioinformatic tools were used to identify putative cis-acting regulatory elements that may be involved in the regulation of rice and Arabidopsis thaliana sucrose transporters. The possible cis-acting regulatory elements were predicted by scanning genomic sequences 1.5 kbp upstream of the sucrose transporter genes translational start sites, using Plant CARE, PLACE and Genomatix Matinspector professional data bases. Several cis-acting regulatory elements that are associated with plant development, plant hormonal regulation and stress response were identified, and were present in varying frequencies within the 1.5 kbp of 5′ regulatory region. The putative cis-acting regulatory elements that possibly are involved in the expression and regulation of sucrose transporter gene families in rice and Arabidopsis thaliana during cellular development or environmental stress conditions were identified as: A-box, RY, CAT, Pyrimidine-box, Sucrose-box, ABRE, ARF, ERE, GARE, Me-JA, ARE, DRE, GA-motif, GATA, GT-1, MYC, MYB, W-box, and I-box. Expression analysis was used to elucidate the role of rice (Oryza sativa L. cv Nipponbare) sucrose transporter (OsSUT) genes during drought and salinity treatments of three week old rice plants ( at four leaf stage) over a 10 days. Among the five rice OsSUT genes identified, only OsSUT2 was observed to be progressively up-regulated during drought and salinity treatments, while OsSUT1, OsSUT4 and OsSUT5 were expressed at low levels, and OsSUT3 showed no detectable transcript expression. Sucrose transport will be essential to meet the cellular energy demands and also for osmoprotectant activities during drought and salinity stresses. It therefore indicates that OsSUT2 which facilitates transport of sucrose from photosynthetic cells will be III essential for rice plants to cope with drought and salinity stresses, and cultivars with a higher OsSUT2 expression should be able to tolerate these environmental stresses better. The role of OsSUT in assimilate transport during rusty plum aphids (Hysteroneura setariae; Thomas) infestation on the leaves of three week old rice (Orzya sativa L. cv Nipponbare) cultivar plants, over a time-course of 1 to 10 days of treatments, was also examined by combination of gene expression and β-glucuronidase (GUS) reporter gene analysis. Real Time PCR analysis of the five OsSUT genes revealed that the expression of OsSUT1 was progressively up-regulated during the course of aphid infestation. OsSUT2 and OsSUT4 expression were comparatively low in both the control and treated plants. OsSUT5 showed no clear difference in transcript expression in both control and treated plants, while no detectable transcript expression of OsSUT3 could be found. The up-regulation of OsSUT1 gene was verified at protein level by western blot analysis in both the control and treated plants. OsSUT1 protein expression was found to increase with time during aphid infestation. A similar trend was noticeable in the control plants, however at a lower expression level. These demonstrate that the cellular expression of OsSUT1is regulated by both developmental and environmental factors. OsSUT1-promoter:::GUS reporter gene expression was observed within the vascular parenchyma and/or companion cells associated with phloem sieve elements of the large and small bundles in the phloem tissues of the flag leaf blade regions where feeding aphids were confined, which progressively increased with time of infestation. It is suggested that OsSUT1 may primarily play an essential role in phloem transport of assimilate to wounded tissues from adjacent health tissues or may be involved in the retrieval of assimilate back into the phloem to minimize loss caused by the infestation. Some OsSUT1-promoter:::GUS expression was also found in the metaxylem at 10 days after infestation, which could signify a recovery system in which sucrose lost into the xylem as a result of aphids feeding are retrieved back into the phloem through the vascular parenchyma. This was supported by the exposure of cut ends of matured OsSUT1-promoter:::GUS rice plant leaf to 2% sucrose solution. OsSUT1-promoter:::GUS expression was observed within the protoxylem, xylem and phloem parenchyma tissues. This indicates that sucrose translocating within the xylem tissues are retrieved into the phloem via the OsSUT1 localized within the parenchyma tissues. In conclusion, the differential expression and regulation of rice (Orzya sativa L. cv Nipponbare) sucrose transporters as reported here suggest that OsSUT2 and OsSUT1 were constitutively expressed compared to other rice sucrose transporters during drought and salinity, and rusty plum aphids (Hysteroneura setariae; Thomas) infestation stresses respectively. Thus, the expression and regulation of the sucrose transporters could be related to the physiological and nutritional requirements of the cells during plant developmental or environmental stress state that allows their differential expression.
- Full Text:
- Date Issued: 2011
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