72575 - Stress Physiology and Redox Dynamics in Plants

Course Unit Page

Academic Year 2018/2019

Learning outcomes

At the end of the course, the student will have in-depth knowledge of modern developments in specific areas of plant physiology and the main methodologies targeting the expression of recombinant proteins in plants and their characterization. In particular, the student is able to: - analyze and discuss topics of genomics, redox biology, physiology of abiotic and biotic stress in higher plants; - critically evaluate the scientific literature on the topics covered; - analyze fragments of DNA and transform bacterial cells; - purify recombinant proteins by chromatographic techniques; - conduct analysis of biochemical and molecular characterization of the protein product.

Course contents

The course consists of Module 1 (lectures, 4 credits, Prof. Trost) and Module 2 (individual experimental laboratory, 3 credits, Dr. Sparla).  
Module 1 (Prof. Trost)
Arabidopsis thaliana as a model species in plant biology. Nuclear and plastid genome. Endosymbiotic gene transfer. 

Oxidative stess and redox signaling. Production and detoxification of ROS in plants. Photosynthesis and photoinhibition. Mitochondria. NADPH-oxidase. Thiol-based redox signaling. 

Abiotic stress. Water stress: osmotic adjustment and abscisic acid.

Biotic stress. Classes of plant pathogens and pathogenesis. Constitutive defenses of plants. Basal immunity and vertical resistance (guard hypothesis and gene for gene model). Oxidative burst, nitric oxide and hypersensitive response. Systemic responses.

Module 2 (Dr. Sparla):

Day 1: extraction and analysis of plasmid DNA by restriction endonuclesi. Calculations and discussion.

Day 2: transformation of E. coli BL21 (DE3) and selection of transformants on medium with kanamycin. NBT-staining to verify the production of ROS in the wound response of A. thaliana leaves.

Day 3: liquid culture of a colony grown on plate. Separation in SDS-PAGE and subsequent Western blot analysis of plant proteins.

Day 4: induction of the expression of the recombinant protein by IPTG; bacterial growth curve. Incubation with secondary antibody conjugated to alkaline phosphatase and colorimetric detection. Calculations and discussion.

Day 5: cell lysis and recombinant protein purification by metal affinity chromatography; desalting of the protein sample.

Day 6: denaturing gel electrophoresis of the purified recombinant protein; measurement of protein concentration. Calculations and discussion.

Day 7: kinetic analysis of the recombinant enzyme. Calculations and discussion.

Day 8: absorption spectra of the recombinant protein; assessment of the nature of the flavin cofactor. Calculations and conclusions.

Readings/Bibliography

- Rascio et al., Elementi di Fiologia vegetale, Edises, second edition 
- Taiz Zeiger, Plant physiology, fifth edition, Sinauer Associates
- Updated reviews on key issues (provided by the teacher)
- Power point files 
- Detailed protocol of the laboratory module.

Teaching methods

Module 1: Lectures  Module 2 (laboratory): Each student is required to carry out the experimental laboratory activities individually. The teacher will give a theoretical introduction to the lab and continuous technical assistance. The results obtained will be critically discussed.

Assessment methods

At the end of the laboratory Module 2 the students will have to pass a written test of verification of the experimental activity carried out. The students who pass the written test, will have access to the oral examination on the topics of Module 1.

Teaching tools

The course will take place in classrooms with PC projection. All lectures will be given with power point presentations. The files of power point presentations will be made ​​available to students at the end of the course (downloadable files from AMSCAMPUS). The lab course will take place in a didactic laboratory with single workstations, equipped with centrifuges, spectrophotometers and electrophoretic cells.

Office hours

See the website of Paolo Bernardo Trost

See the website of Francesca Sparla