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Work Package 2: Genetic and genomics strategies for protection of rice from major abiotic stresses (water shortage, saline stress, thermal stress)

Leaders: G.A.Sacchi e M.Kater (P3). Participants: P3-P7-P12-P11-P10-P6-P1-P2
WP2 deals with the identification of traits involved in tolerance mechanisms which may be profitably introduced into new rice genotypes, in order to overcome the main causes of abiotic/environmental stresses characterizing Italian areas. WP2 will develop on a common panel of rice genotypes chosen among the ITALORYZA collection for the specific research area.

WP2.1 Tolerance to thermal stress (P3, P10).
Transcriptome analysis will be used to identify specific response pathways determining cold tolerance. Analysis of transcripts will be carried out by means of massive sequencing, characterized by high efficiency and reliability to obtain informations on structure and function of differently expressed transcripts.
WP2.1a) Analysis of cold stress response in rice gametophyte (P3)
Cold susceptible and tolerant rice varieties will be exposed to cold stress and germination and male/female fertility will be assayed by means of in vitro and in vivo pollen tube growth assays and in vivo fertilization assays. Male and female developmental defects caused by the applied cold stresses will be histologically described. Most representative sample tissues will be used for transcriptome analysis using a deep sequencing approach and bioinformatic. The expression of candidate genes will be reanalysed during different cold stress treatments by quantitative real-time PCR using a group of different varieties with different levels of cold susceptibility: focus will be place on differentially expressed key candidates. Screen for mutants in selected candidate genes to access their importance will be performed on the Volano Tilling population (WP4.2) and other mutant collections available.
WP2.1b) Analysis of cold stress response in rice plants (seedlings and panicle exertion stages) (P10).
In a first step, the response to low temperature will be investigated on two contrasting rice cultivars selected from the panel of genotypes. Plants grown in controlled conditions will be characterized for their response to chilling using PSII photochemistry (Fv/Fm) and electrolyte leakage (EL) tests at both the seedling (three-leaf) and the heading (panicle exertion) stages. The collaboration with the consultant SSSUP will be focused to identify thresholds, timings, and other physiological parameters during the rice cycle necessary to plan and execute the transcription profiling experiment, and analyses of the obtained genomics results. Changes in rice transcriptome after chilling exposure will be focused to identify and map regulatory genes early expressed (initial 12/24 h of thermal stress). Quantitative studies of gene expression on treated and untreated (control) plants will be carried out on a genome scale by 454 deep-sequencing.

WP2.2 Tolerance to water stress (P3,P1,P2,P6,P11,P12).
Tolerance to water limiting conditions will be analyzed with particular concern on the early stages of plant development and on the effects on the grain composition, and complement with results obtained in WP2.1.b, WP2.2b and WP4.2, and from data made available by P1 from the previous Eu-funded project CEDROME.
WP2.2a Transcriptomic maps for the identification of genes involved in the response to water shortage at seedling level and identification of the associated polymorphisms (P6, P2).
This research line is expected to provide a characterization of Italian rice cultivars for the expression profile of water responsive transcription factors (TFs) by using the Real Time PCR platform for rice TFs established at Postdam University. Bioinformatic analysis will be performed to identify genes differentially expressed between the two cultivars under water stress. The analysis of the most promising genes will be extended to the previously characterized rice cultivars. Correlations between the expression profile of selected genes and the stress response phenotype (tolerance/susceptibility) will be analysed. Finally, the identification of polymorphisms in the differentially expressed genes will represent the basis for the development of molecular markers useful in breeding for water stress tolerance. Data will complement phenotyping data in the field obtained by P1, proteomic analyses of WP2.3a, and the analysis of the Volano TILLING population in WP4.2.
WP2.2b Association mapping for drought tolerance in the ITALORYZA working collection, the Volano TILLING population and the EcoTILLING collection (P1, P2)
Previous studies made available by P1 and P2 allow integrative evaluation in field condition for drought tolerance on three genetic resources created: a) the ITALORYZA working collection; b) the Volano TILLING population of about 2000 lines; c) the EcoTILLING collection of 100 genotypes. Data on yield and pheno-morphological traits will allow studies of association to validate and complement the research line of WP2.2a.

WP2.3 Tolerance to saline stress (P7, P11)
This area of research is targeted to the application of biochemical/molecular physiology to understand the key mechanisms conferring salt tolerance. Results will complement the ones obtained in WP2.2.
WP2.3a) Analysis of the ion transport system and study of the components of signalling pathways in response to saline stress (P7)
The aim of this action is the analysis of the transcriptional profile and the cellular localization of channels and transporters in different rice cultivars, in presence or absence of high concentrations of salt. A group of genes will be studied by RealTime-PCR on 2 clearly distinct cultivars (high- and low-salt sensitivity) chosen among genotypes already evaluated by P1. The study of the tissue and subcellular localization of the selected proteins by both RT-PCR and confocal microscopy will help us to discriminate between transport protein involved in Na transport and accumulation. Since nitric oxide (NO) is a regulator of Na/H antiporters and K+ channels, electrophysiological studies will be carried out to investigate the role of NO in the signalling pathway of salt stress. Finally, the selected genes will be tested as salt tolerance markers by screening about 20 Italian rice cultivars.
WP2.3b) Biochemical analysis of key enzymes controlling salt stress response (P11)
Three to five Italian rice cultivars with a contrasting capability to cope with salt stress conditions during the vegetative and reproductive stages will be selected. The levels and the properties of selected enzymes playing a pivotal role in antioxidant defense (namely superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) and in proline metabolism (namely P5C synthetase, P5C reductase, proline dehydrogenase and P5C dehydrogenase) will be studied in these genotypes, as well as the resulting levels of ROS, reduced ascorbate/GSH and free prolin level in tissues of osmotically-stressed seedlings. Changes in enzymatic activities or substrate affinities could provide basis for higher salt tolerance through increased ROS scavenging and/or osmolyte accumulation. The results will complement activities in WP2.3a.

WP2.4. Diagnostic Bio-markers for stress responses: ionomic, metabolomic and proteomic maps (P3, P12)
The research line will develop ionomic, metabolomic (P3), and proteomic maps (P12) by analyzing plant tissues and grains from plants exposed to stress, in order to identify changes in nutritional quality of the grain and/or bio-indicators for stress response detection.
WP2.4a) Ionomic and metabolomic maps for rice quality in different water availability conditions (P3, P1)
Ionomic maps will be developed for grain fractions (bran and white rice) from samples from 5 genotypes from two conditions (flooding and aerobic soil) by inductively coupled plasma-mass spectroscopy (ICP-MS) and/or combined HPLC-ICP-MS techniques. The maps will evaluate the mineral micronutrient (Fe, Zn, Mn, Cu, Se), eventual presence of toxic elements (Cd, Pb and As), metabolites with nutraceutical value (i.e., vitamins, phenols, ?.orizanol, ecc.) and antinutritional compounds (i.e. phytic acid). The maps will be intersected with transcriptomics (WP2.2a) and proteomic maps (WP2.4b), soil characteristics (P3) with the aims to define agro-techniques conserving qualitative aspects of produced grains in different water availabilities.
WP2.4b) Proteomic maps and identifications of quality bio-markers (P12)
Five Italian rice cultivars with a divergent capability to cope with drought and salt stress will be analyzed. Proteomic maps obtained by 2D- gels and 2D-LC HT techniques from leaf samples of different phenological stages of drought and salt-stressed rice plants will be utilised to identify markers linked to stress conditions that can be correlated to alterations in grain protein composition. ESI/MS and MALDI-TOF/MS will be performed on the crude protein extracts of grains to evidence quantitative and qualitative alterations in rice seed storage proteins. Modifications of the structure of grains and the distribution of storage proteins and starch granules in grains of the different cultivars and conditions will be analysed by Environmental Scanning Electron Microscope (ESEM) a versatile microscope with 2 nm ultimate resolution. The identification in grains of mineral elements improving quality will be also performed by EDX techniques and results complemented with WP2.4.a.