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Digital archive of theses discussed at the University of Pisa


Thesis etd-03022011-191039

Thesis type
Tesi di dottorato di ricerca
Thesis title
Proteome change in citrus species challenged with salinity or arthropod attack
Academic discipline
Course of study
tutor Prof. Cercignani, Giovanni
tutor Dott.ssa Maserti, Biancaelena
  • citrus
  • proteomics
  • salt stress
Graduation session start date

Citrus plants grown in the Mediterranean area are currently facing biotic stress, and abiotic stress such as soil salinity. In order to cope with such a stress and improve fruit quality, citrus varieties are grafted on rootstocks with different tolerance to abiotic and biotic stresses.
Salinity stress is an increasing environmental problem for crop production worldwide. Citrus is one of the most important fruit crops globally and is considered as being salt sensitive.
In this work, the leaf proteome expressed during moderate salt stress in the leaves and roots of citrus rootstocks showing different salt tolerance were studied. Protein changes in both control and salt-treated leaves and roots were revealed using two-dimensional gel electrophoresis (2-DE), coupled with mass spectrometry and data base search.
About 900 spots were reproducibly detected in each gel for treated leaves. Twenty-five spots changed their expression level after salinity stress in the leaves of salt stressed citrus plants and were identified by liquid chromatography-tandem mass spectrometry. The majority of these proteins were related to photosynthesis (25%), energy production (16%), and defense activity (25%). The potential roles of these salt responsive proteins are discussed.
Moreover, salt stress negatively affects many physiological processes in plants. Some of these effects may involve the oxidative damage of cellular components, which can be promoted by reactive oxygen species (ROS) and prevented by antioxidants. In this study, using optimized protocols for in-gel activity staining on native PAGE, the isozyme pattern of two antioxidant enzymes, ascorbate peroxidase (APX) and superoxide dismutase (SOD), is characterized in the salt-treated leaves of citrus rootstocks showing different tolerance to salt stress. Diploid and tetraploid clones are used, because it seems that tetraploidy is to be a factor improving abiotic stress. Furthermore identification of Fe-SOD, after two dimensional native gel electrophoresis on immobilized gradient IPG strip and LC- MS/MS, is also reported. Data show involvement of APX in the response mechanisms to salt stress in citrus leaves. This study reports the isozyme patterns using in-gel activity staining of SOD and APX and their involvement in salt stress response in citrus. Twenty-five proteins were differentially expressed by 50 mM Na Cl exposure for three weeks in two different citrus rootstocks, the salt-tolerant C. reshni and the salt-sensitive Carrizo citrange. According to the biological processes in which the protein participates, the majority of identified proteins were correlated with oxidative stress (32%), metabolic processes (16%) and ATP synthesis (8%) and 16% of proteins have unknown function.
To characterize differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was carried out using two-dimensional gel electrophoresis. Different expression of mite responsive proteins were compared to expression pattern under MeJA treatment. MeJA is a compound recognized as playing a dominant role in promoting plant defense/response to many arthropod herbivores. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography–mass spectrometry–tandem mass spectrometry. Most proteins of the differentially expressed proteins were involves in photosynthesis and metabolism pathway. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Defence-related proteins were seven such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein.
In particular, analysis have been show the defence-related acidic chitinase II to be specifically induced in Citrus clementina leaves infested by the two-spotted spider mite Tetranychus urticae or treated with MeJA. Chitinases are considered pathogenesis-related proteins involved in viral infections, fungal and bacterial cell wall components, and also by more general sources of stress such as wounding, salicylic acid, ethylene, auxins and cytokinins.
In the present study, changes in the mRNA profiles of two partially homologous chitinase forms were shown by RT-PCR. In particular, the appearance of an additional cDNA chitinase fragment in T. urticae-infested and MeJA-treated leaves was observed. This finding may indicate a specific regulatory mechanism of chitinase expression.
Results report evidence for alternative splicing in T. urticae-infested C. clementina, where a premature stop codon after the first 135 amino acids was introduced. We observed inducible chitinase activity after MeJA treatment, indicative of a rapid plant response to infestation.
Finally a protocol of in-gel activity staining of antioxidant enzyme was optimized for bidimensional gel electrophoresis. The use of this approach allowed to identify a Fe-SOD from activity-stained gel, by mass spectrometry. Results are not discussed, but only presented as future perspective for a post-doctoral research.