Joint research project

MOXIVOL: Molecular characterization of the antioxidant system and volatolome in pomegranate (Punica granatum) under drought stress.

Project leaders
Biancaelena Maserti, Myrza Musayev
AZERBAIJAN - ANAS - Azerbaijan National Academy of Sciences
CNR/ANAS 2014-2015
Biology, agriculture and food sciences
Thematic area
Biology, agriculture and food sciences
Status of the project

Research proposal

1. Background: Pomegranate (Punica granatum L Pomegranate is a very interesting fruit plant species from a commercial point of view due to its adaptation to a wide range of climates and soil conditions. However, pomegranate has frequently been considered a minor crop, although the growing interest in the consumption of its fruits due to their organoleptic characteristics and their perceived health benefits is beginning to change the way it is regarded. Indeed pomegranate has a high content of vitamins, minerals and the polyphenol antioxidants content of pomegranate juice is among the highest of any foods (Guo et al., 2003) and it has been suggested that it may lower risk of heart disease (Aviram et al., 2004) and may slow cancer progress (Adams et al., 2006). Despite the increasing importance of pomegranate and the ever more frequent water shortages experienced in the growing regions to the best of our knowledge, no information exists on the molecular mechanisms developed by pomegranate to face to environmental changes, such as drought stress that may affect their therapeutic contents. Pomegranate is considered to be a drought-resistant crop because it copes heat and can thrives well in arid and semiarid areas, even under desert conditions (Aseri et al., 2008). Although water deficit during the final stage of fruit growth in fruit tree produces a decrease in final fruit size, beneficial aspects include increased total fruit soluble solids levels and earlier fruit ripening (Besset et al., 2001). Abiotic stresses such as drought lead to the overproduction of reactive oxygen species (ROS) in plants which are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates and DNA which ultimately results in oxidative stress (Gill and Tuteja (2010). The observation that VOC are often induced by stresses, among them drought and salt suggests that VOC might play a role as antioxidant compounds contributing to detoxify them (Loreto and Velikova, 2001) . Additionally, it has been demonstrated that VOC emissions modulate plant tolerance to heat, pollutant and oxidative stress (Centritto et al. 2011)A low VOC emission in pomegranate was measured by Rasmussen (1978), but the result has not been recently confirmed with more sensible instrumentations.
2. Methodologies/experiments (and deliverables Dxx) WP1- Physiological, metabolic and proteomics analysis (coordinated by CNR-IPP) CNR-1 Assessment of membrane stability upon exposure to drought stress: Membrane integrity will be experimentally assessed through measurements of biochemical metabolites. Membrane damage will be based on measurements of volatiles molecules, the C-6 compounds that are formed from the breakdown of membrane lipids, by PTR-MS (Loreto et al. 2006). Results of this analysis will be used as a markers of drought stress (D1). CNR-2 Physiological analysis (D2): characterization of gas exchange (particularly photosynthesis) and water relations in control and drought stressed plants will be achieved. Instantaneous exchanges and curves of assimilation will be quantified by using a Li-Cor 6400 analyzer. ). CNR-3 Biochemical analysis: Soluble carbohydrates will be identified and quantified using HPLC-RI analysis, starch will be analyzed by enzymatic digestion (D3); pigment analysis: individual carotenoids will be identified and quantified using HPLC-DAD analysis.. (D4); polyphenols: flavonoids will be identified using HPLC-DAD (based on their retention times and UV-spectral characteristics as compared with authentic standards) and HPLC-MS analysis. The quantification will be done using authentic standards (D5); antioxidant enzyme activities, H2O2, SOD, CAT , APX and POX will be estimated photometrically; the concentration of H2O2 will be determined following the potassium titanium oxalate method (D6). CNR-4 In-gel activity of SOD , CAT , APX and POX: The activities will be estimated by using in-gel activity protocols available in the literature. This technology is based on the enzyme separation on native-PAGE gels and will allow detecting the activities of specific isoforms of the antioxidant enzymes during drought stress. Computerized analysis with dedicated software will allow identifying enzyme isoforms whose activities changed as a function of drought stress. The activity bands of interest will excised from the gel and subjected to mass spectrometry analysis (LC-MS/MS) to identify their peptide sequences. CNR-5 RT-PCR analysis: primers will be designed on the antioxidant enzyme peptide sequences found by MS analysis. Total RNA will be extracted on leaf and fruit samples and retro-transcribed and RT-PCR analysis to compare cDNA levels in control and drought stressed plants will be performed. Bands will be excised and sequenced. CNR-4 and 5 analysis will provide the identification of antioxidant enzyme encoding genes involved in drought stress response of pomegranate trees (D7). CNR-6 Population genetic analysis for describing the genetic diversity of wild pomegranate Azeri populations. Nuclear genetic markers developed by Hasnaoui et al. (2010) and by Ebrahimi et al. (2010) will be applied for quantifying neutral genetic diversity within and among natural populations, and to compare Azeri populations with those already studied in other countries of the range (Norouzi et al. 2012) (D8). WP2 - Exploration and mapping of Azer pomegranate resources (coordinated by GRI-ANAS) GRI-ANAS-1 Expedition organization. Organization of expedition within the natural habitats of the wild populations of pomegranate in Azerbaijan and collection of samples of wild forms for drought-resistance studies (D9). GRI-ANAS-2 Drought stress experiments: Study of drought resistance of about 20 local pomegranate varieties - landraces (Girmizi gabig, Shirin girmizi, Nazik gabig, Veles, Shelli melesi, Guleyshe, Eflani, Shah nar, Bala Mursel etc.) and wild type accession in the field and laboratory conditions (D10). GRI-ANAS-3 Mapping pomegranate phenotypes in the field: Mapping the location of landrace phenotypes which are currently used by farmers and wild forms of pomegranate by using geographic information systems (GIS)and preparation of related distribution maps (Maps of pomegranate landraces and wild type in Azerbaijan, D11).
3.Gains and benefices for the two research institutions in carrying out MOXIVOL activities. - Development of a comparative study of Azeri pomegranate accessions under control and drought stress with - Access to plant material not available in EU - Access to high performance instrumentations and innovative technologies provided by CNR-IPP researchers - Beginning of a relationship between CNR-IPP and GRI-ANAS and enlarging the collaboration between Europe and Caucasian countries.

Research goals

Two laboratories which a large experience in performing researches on plant response to environmental stress are partners in the MOXIVOL project . These partners will integrate competences in ecology, physiology, biochemistry, molecular biology, functional genomics and bioinformatics to improve knowledge about the molecular and metabolic bases of antioxidant system and biogenic volatile organic compounds (BVOC) emission and their functions in plant protection during drought stress.
The rationale of the project is that an interdisciplinary approach providing a large set of knowledge on antioxidant system and VOC emission will allow a better understanding of pomegranate capacity to cope with drought stress. However, delivered results will go far beyond the scope of MOXIVOL, because the candidate genes for drought tolerance identified in pomegranate could be also used to improve the tolerance of other economical fruit trees by using breeding techniques.

2.1 Specific objectives to be achieved in control and drought-stressed samples are:
Measurements of the photosynthesis performance
Polyphenol compound levels
Evaluation of the Volatolome of pomegranate leaves and fruits and its involvment in drought response.
Assessment of antioxidant enzymes activities such as SOD, APX, CAT, GR by spectrophotometric analysis and in-gel activity staining
Identification of the genes encoding the antioxidant enzymes specifically involved in drought-stress response.

Last update: 27/11/2021