Download Report - ClimMani

ClimMani
Short Term Scientific Mission REPORT
Applicant name:
Susanna Pollastri
Education:
PhD in Plant Biology
Affiliation:
Institute for Sustainable Plant Protection
National Research Council
Italy
Research interests (keywords):
Isoprene, temperature, plant stress
Host (name and institute):
Prof. Josep Peñuelas
Period for the mission:
From the 5th till 22nd of July
Career stage (e.g. years since PhD)
Researcher
Global Ecology Unit CREAF-CSIC-UAB,
Center for Ecological Research and Forestry
Applications (CREAF) - National Research Council
(CSIC)
Edifici C, Universitat Autonoma de Barcelona,
08193 Bellaterra, Barcelona
Title of project:
Using Atomic Force Microscopy to study the differences between thylakoid membranes of
isoprene emitting and non-emitting plants submitted to field experimental warming
Short description and motivation of mission:
Many studies have already demonstrated that thylakoid membranes of isoprene emitting plants are
more stable at high temperatures (Loreto and Schnitzler, 2010; Velikova et al., 2011). Different
hypotheses have been proposed to explain how isoprene is able to confer such stability, as enhancing
hydrophobic interactions within thylakoids molecules (Sharkey and Yeh, 2001, Siwko et al. 2007),
preventing the formation of water channel (Singsaas et al., 1997; Sharkey et al., 2001), and dissipating
heat (Pollastri et al., 2014). However, the mechanism of action has not yet been clarified.
Recent studies were conducted to investigate some physical and chemical aspects of thylakoid
membranes. Different biophysical technique, providing indirect measures, were used to determine
thylakoid stability and functionality in isoprene emitting and non-emitting plants at higher
temperatures (Velikova et al.2011), but no direct measurements was performed till now.
The aim of the mission is to study thylakoid membrane in isoprene emitting and non-emitting plants,
at different temperature with atomic force microscopy (AFM) present at the Universitat Autonoma
de Barcelona. AFM will allow us to measure viscosity, to image the composition of the thylakoid
membrane, the thylakoid superstructure, and changes in the protein arrangement in transgenic
isoprene emitting and non-emitting plants submitted to field experimental warming.
Relevance to overall ClimMani COST objectives and WG activities (see ClimMani website for details on WGs):
The mission fits very well the objectives of ClimMani COST since we will try to discern the
mechanisms involved in the increased isoprene emissions our ClimMani community been observing
in our warming and drought experimental ClimMani field sites.
Outcome:
During my stay in Barcelona I had the opportunity to study thylakoid membranes with Atomic Force
Microscopy (AFM). This is a new excellent technique which can allow to gain more knowledge about
chloroplasts.
It’s already known that plants who emits isoprene are better protected against hot temperature
stresses compared to the non-emitting plants. Furthermore isoprene emitting plants are able to keep
photosynthesis values higher during heat stress.
The idea was to investigate the structure of the thylakoid’s membranes of isoprene emitting and nonemitting species with AFM.
To perform this experiment we use wild type tobacco plants, who naturally do not emit isoprene, and
transgenic isoprene emitting tobacco plants.
We isolated de-enveloped chloroplasts from different plants. Once isolated, a layer of de-enveloped
chlroplasts was distribuited over a glass coverslip in order to be measured with Atomic Force
Microscope.
We performed the following measurements:
-the Young’s modulus, that is a measure of the stiffness, of the thylakoid’s membrane. Young's
modulus is the elastic modulus, is a number that measures an object or substance's resistance to
being deformed elastically (i.e., non-permanently) when a force is applied to it;
- the viscosity of the membranes.
All these measurements were perforemd at different temperature points. The temperature was
controlled thanks to a Peltier element that was located under the sample. Moreover a tiny
thermometer was used to measure directly the temperature of the sample.
We have also collected some sample to be imaged with SEM and TEM, in order to:
- show that chloroplasts are de-enveloped, and so we are measuring the properties of the thylakoid
membranes and not of the intact chlroplasts.
-to measure the lenght of grana’s stacks, which can vary between samples and maybe contribute to
the measuring parameters.
Our preliminary results showed that isoprene emitters chloroplasts have an higher Young's modulus
compared to the isoprene non-emitting ones as the temperature rises. This means that the thylakoid's
membrane of isoprene ones are more able to mantain their "stucture" as the temperature rise,
compared to the non-emitting ones.
These are preliminary results that must be confermed by further measurements with AFM, at
different temperature. These will allow us to better understand why isoprene emitting plants are
better protected against heat (and drought) stress.