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2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage.

Simova-Stoilova LP, Romero-Rodríguez MC, Sánchez-Lucas R, Navarro-Cerrillo RM, Medina-Aunon JA, Jorrín-Novo JV - Front Plant Sci (2015)

Bottom Line: Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS).Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased.Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress.

View Article: PubMed Central - PubMed

Affiliation: Agricultural and Plant Biochemistry and Proteomics Research Group, Department of Biochemistry and Molecular Biology, University of Cordoba Cordoba, Spain.

ABSTRACT
Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress.

No MeSH data available.


Related in: MedlinePlus

Summary diagram of molecular mechanisms involved in Quercus ilex root response to water limitation.
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Figure 5: Summary diagram of molecular mechanisms involved in Quercus ilex root response to water limitation.

Mentions: Selected well defined spots, presenting significant changes under stress (at least 1.5-fold change compared to controls) and consistent in the three biological replicas, were subjected to MALDI-TOF PMF and MS/MS. For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Detailed tables with identified protein species and their most probable subcellular location are presented in the Supplementary Material (Supplementary Tables S1, S2). There were five cases of low score unreliable identification and three cases of 2–3 different proteins identified in the same spot, which were excluded from these tables. In spite of the fact that Quercus ilex is an orphan species and identification was mainly based on homology, the majority of reliable hits for one given spot were for the same protein in different plant species. A few of the proteins were detected in more than one spot, like actin 2, enolase (EC 4.2.1.11), betaine aldehyde dehydrogenase (EC 1.2.1.8), cysteine synthase (EC 2.5.1.47), pyruvate decarboxylase (EC 4.1.1.1), but with some exceptions they presented similar trends of changes. Differences in protein abundance are clearly distinguished in the heat map built on the basis of normalized spot abundancy ratios of treatment to the respective age control (Figure 4). Different patterns of changes were found when looking at the identification results; however, the majority of spot abundance changes presented the same trend at drought stress irrespective treatment duration—D10 or D20. The following types of dynamic changes were observed—changes in abundance under stress unrelated to how long was the treatment (43 protein spots—24 up and 19 down), increase or decrease in abundance only at D10 (14 protein spots—8 up and 6 down in abundance), decrease only at D20 (2 protein spots), spot abundance changes in different directions comparing D10 and D20 (4 protein spots), prominent abundance changes in recovery, mainly diminution (8 spots), and changes beyond detection level, so-called qualitative changes (11 protein spots). Among the identified proteins with earlier and reversible increase in abundance under drought followed by recovery were found many enzymes related to secondary metabolism such as: caffeoyl CoA 3-O-methyl transferase (EC 2.1.1.104), chalcone synthase (2 spots, EC 2.3.1.74), shikimate dehydrogenase (EC 1.1.1.25), quinone oxidoreductase (EC 1.6.5.5), as well as ubiquitin activating enzyme E1 (EC 6.3.2.19), DEAD box RNA helicase (EC 3.6.4.13), betaine aldehyde dehydrogenase (EC 1.2.1.8). Down-accumulated in D10 were some inducible and hormone responsive proteins, glycyl-tRNA synthetase (EC 6.1.1.14), D-3-phosphoglycerate dehydrogenase (L-serine biosynthesis, (EC 1.1.1.95). There were not up-accumulated proteins typical only for prolonged drought treatment but 2 spots presented decrease in abundancy only at D20—methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and methylene tetrahydrofolate reductase (EC 1.5.1.20), a cytoplasmic enzyme member of one-carbon metabolism. A few proteins had opposite trends of changes comparing D10 and D20—peroxidase (EC 1.11.1.7)—D10 decrease, D20 increase, S-adenosylmethionine synthase (one-carbon metabolism, 2 spots, (EC 2.5.1.6)—D10 increase, D20 decrease, and the mitochondrial NADH-ubiquinone oxidoreductase (EC 1.6.5.3)—D10 increase, D20 decrease. The following proteins presented prominent changes in abundance after recovery, mainly diminution: Pyruvate dehydrogenase E1 (EC 1.2.4.1) subunit beta, component 3 of pyruvate dehydrogenase complex, enolase (EC 4.2.1.11), protein disulfide-isomerase (EC 5.3.4.1), STI like HSP (2 spots). The only protein spot with increase in recovery was proteasome subunit beta (EC 3.4.25.1). The most interesting qualitative differences were in one cysteine synthase isoform (EC 2.5.1.47), which spot was below detection under drought; however, there was another spot identified also as cysteine synthase with increased abundance under drought. Identified proteins were classified into functional groups according to Bevan et al. (1998): primary and secondary metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. In Table 3 are summarized the main functional groups and subgroups and the tendencies of changes in individual proteins within groups. It is seen that several enzymes of the carbohydrate metabolism were decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism were increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract stress. A summary diagram of molecular mechanisms involved in Quercus ilex root response to water limitation is presented in Figure 5.


2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage.

Simova-Stoilova LP, Romero-Rodríguez MC, Sánchez-Lucas R, Navarro-Cerrillo RM, Medina-Aunon JA, Jorrín-Novo JV - Front Plant Sci (2015)

Summary diagram of molecular mechanisms involved in Quercus ilex root response to water limitation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4536546&req=5

Figure 5: Summary diagram of molecular mechanisms involved in Quercus ilex root response to water limitation.
Mentions: Selected well defined spots, presenting significant changes under stress (at least 1.5-fold change compared to controls) and consistent in the three biological replicas, were subjected to MALDI-TOF PMF and MS/MS. For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Detailed tables with identified protein species and their most probable subcellular location are presented in the Supplementary Material (Supplementary Tables S1, S2). There were five cases of low score unreliable identification and three cases of 2–3 different proteins identified in the same spot, which were excluded from these tables. In spite of the fact that Quercus ilex is an orphan species and identification was mainly based on homology, the majority of reliable hits for one given spot were for the same protein in different plant species. A few of the proteins were detected in more than one spot, like actin 2, enolase (EC 4.2.1.11), betaine aldehyde dehydrogenase (EC 1.2.1.8), cysteine synthase (EC 2.5.1.47), pyruvate decarboxylase (EC 4.1.1.1), but with some exceptions they presented similar trends of changes. Differences in protein abundance are clearly distinguished in the heat map built on the basis of normalized spot abundancy ratios of treatment to the respective age control (Figure 4). Different patterns of changes were found when looking at the identification results; however, the majority of spot abundance changes presented the same trend at drought stress irrespective treatment duration—D10 or D20. The following types of dynamic changes were observed—changes in abundance under stress unrelated to how long was the treatment (43 protein spots—24 up and 19 down), increase or decrease in abundance only at D10 (14 protein spots—8 up and 6 down in abundance), decrease only at D20 (2 protein spots), spot abundance changes in different directions comparing D10 and D20 (4 protein spots), prominent abundance changes in recovery, mainly diminution (8 spots), and changes beyond detection level, so-called qualitative changes (11 protein spots). Among the identified proteins with earlier and reversible increase in abundance under drought followed by recovery were found many enzymes related to secondary metabolism such as: caffeoyl CoA 3-O-methyl transferase (EC 2.1.1.104), chalcone synthase (2 spots, EC 2.3.1.74), shikimate dehydrogenase (EC 1.1.1.25), quinone oxidoreductase (EC 1.6.5.5), as well as ubiquitin activating enzyme E1 (EC 6.3.2.19), DEAD box RNA helicase (EC 3.6.4.13), betaine aldehyde dehydrogenase (EC 1.2.1.8). Down-accumulated in D10 were some inducible and hormone responsive proteins, glycyl-tRNA synthetase (EC 6.1.1.14), D-3-phosphoglycerate dehydrogenase (L-serine biosynthesis, (EC 1.1.1.95). There were not up-accumulated proteins typical only for prolonged drought treatment but 2 spots presented decrease in abundancy only at D20—methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and methylene tetrahydrofolate reductase (EC 1.5.1.20), a cytoplasmic enzyme member of one-carbon metabolism. A few proteins had opposite trends of changes comparing D10 and D20—peroxidase (EC 1.11.1.7)—D10 decrease, D20 increase, S-adenosylmethionine synthase (one-carbon metabolism, 2 spots, (EC 2.5.1.6)—D10 increase, D20 decrease, and the mitochondrial NADH-ubiquinone oxidoreductase (EC 1.6.5.3)—D10 increase, D20 decrease. The following proteins presented prominent changes in abundance after recovery, mainly diminution: Pyruvate dehydrogenase E1 (EC 1.2.4.1) subunit beta, component 3 of pyruvate dehydrogenase complex, enolase (EC 4.2.1.11), protein disulfide-isomerase (EC 5.3.4.1), STI like HSP (2 spots). The only protein spot with increase in recovery was proteasome subunit beta (EC 3.4.25.1). The most interesting qualitative differences were in one cysteine synthase isoform (EC 2.5.1.47), which spot was below detection under drought; however, there was another spot identified also as cysteine synthase with increased abundance under drought. Identified proteins were classified into functional groups according to Bevan et al. (1998): primary and secondary metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. In Table 3 are summarized the main functional groups and subgroups and the tendencies of changes in individual proteins within groups. It is seen that several enzymes of the carbohydrate metabolism were decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism were increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract stress. A summary diagram of molecular mechanisms involved in Quercus ilex root response to water limitation is presented in Figure 5.

Bottom Line: Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS).Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased.Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress.

View Article: PubMed Central - PubMed

Affiliation: Agricultural and Plant Biochemistry and Proteomics Research Group, Department of Biochemistry and Molecular Biology, University of Cordoba Cordoba, Spain.

ABSTRACT
Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress.

No MeSH data available.


Related in: MedlinePlus