3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P40 SEASONAL ChANGES OF RubISCO
ACTIVITy AND ITS CONTENT IN NORwAy
SPRuCE ExPOSED TO AMbIENT AND
ELEVATED CO 2 CONCENTRATIONS
MIROSLAV HRSTKA a , LUCIE ZACHOVá a , OTMAR
URBAn b and MARTInA KOŠVAnCOVá b
a Department of Food Chemistry and Biotechnology, Faculty
of Chemistry, Brno University of Technology, Purkyňova 118,
612 00 Brno, Czech Republic,
b Institute of Systems Biology and Ecology AS CR, Poříčí 3b,
603 00 Brno, Czech Republic,
htka@fch.vutbr.cz
Introduction
Ribulose-1,5-bisphosphate carboxylase/oxygenase,
Rubisco (EC 4.1.1.39) is the most abundant protein on the
Earth. This enzyme catalyses carboxylation of D-ribulose-
1,5-bisphosphate (RuBP), the first step of the Calvin cycle
in competition with oxygenation of RuBP that leads to the
photorespiratory pathway. Rubisco is a key enzyme of photosynthesis
regulation1 . It must be reversibly activated with
CO2 and Mg2+ before catalysis can occur. The activation in
vivo must be facilitated by the presence of a second protein,
Rubisco activase2 .
Rubisco activity depends on the amount of enzyme and
the activation state of its active sites and changes within
several minutes depending on temperature, RuBP supply,
irradiance, CO2 concentration, inorganic phosphate content,
and presence of inhibitors in the active site3 . Rubisco content
varies over the time scale of hours and days in dependence on
specific saccharide contents (e.g. glucose, sucrose). Increased
contents of these sugars lead, via a hexokinase-related signal,
to the repression of Rubisco gene expression and subsequent
decrease in the content of Rubisco protein4,5 . Decrease
in Rubisco amount may be also the result of non-selective
decrease in leaf nitrogen content6,7 . Many authors8–10 present
that Rubisco content reaches a maximum soon after full
expansion of the leaf. Then Rubisco is gradually degraded
and its nitrogen is translocated into growing organs during
senescence.
Short-term exposure of higher plants to elevated CO2 concentrations usually increases photosynthetic CO2 uptake.
Long-term growth in elevated CO2 usually leads to decrease
of content and activity of Rubisco. This biochemical
adjustment, which is often termed acclimation or down-regulation,
reduces photosynthetic capacity11 .
In this work we measured seasonal changes of Rubisco
initial and total activities in vitro as well as Rubisco content
in norway spruce needles to answer following quesions:
• Is there a decrease of Rubisco activity and content in
norway spruce during the growing season?
• Is there a down-regulation of Rubisco activity and content
in norway spruce cultivated in elevated CO2 concentration?
s657
Experimental
M a t e r i a l s
The experiment was conducted in 2007 at the experimental
site Bílý Kříž in Beskydy Mts. Five-year old seedlings
of norway spruce (Picea abies [L.] Karst.) were grown
under ambient (A = 375 µmol (CO 2 ) mol –1 ) and elevated
(E = 700 µmol (CO 2 ) mol –1 ) CO 2 concentrations using the
glass domes with adjustable windows. Last year needles were
sampled between 11 a.m. and 3 p.m. at the following dates:
15th May, 23th July and 10th October. About 0.06 g of needles
were weighed and the projected area of these needles was
estimated according 12 . needles were inserted into microtube
Eppendorf and put into liquid nitrogen.
M e t h o d s
Rubisco activity assay. needles from one microtube
were homogenized in a chilled mortar with 0.02 g inert sand
and 5 ml extraction buffer composed of: 50 mM HEPES,
5 mM na 2 EDTA, 5 mM dithiothreitol (DTT), and 1 % insoluble
polyvinylpolypyrrolidone, all at pH 8.0. The homogenate
was centrifuged at 10,000 × g for 30 s and an aliquot of
the supernatant was used immediately for spectrophotometric
Rubisco activity assay, based on the continuous measurement
of 3-phosphoglycerate-dependent nADH oxidation in
a coupled enzyme system 13 . The initial Rubisco activity was
determined by adding 20 μl of extract to 100 μl of activation
solution which contained 25 mM KHCO 3 and 20 mM MgCl 2
and 850 μl of assay solution composed of: 50 mM HEPES
(pH 8.0), 25 mM KHCO 3 , 20 mM MgCl 2 , 5 mM na 2 EDTA,
5 mM DTT, 3.5 mM ATP, 0.35 mM nADH, 3.5 mM phosphocreatine,
80 nkat glyceraldehyde-3-phosphate dehydrogenase,
80 nkat 3-phosphoglyceric phosphokinase, and
80 nkat creatine phosphokinase. The reaction was started by
the addition 30 μl of ribose-5-phosphate (R5P) at final concentration
0.4 mM and the changes in absorbance at 340 nm
were immediately measured at 25 ºC using Helios γ (Spectronic
Unicam, UK) spectrophotometer.
The total activity was measured after 15 min incubating
20 μl of the extract with 100 μl of activation solution. 850 μl
of assay solution were added, the reaction was again started
by adding 30 μl of R5P at final concentration 0.4 mM and the
changes in absorbance at 340 nm were measured.
Rubisco content determination. needles from one
microtube were homogenized in a chilled mortar with 0.02 g
inert sand and 2 ml extraction buffer containing 62 mM Tris,
2% (w/v) sodium dodecyl sulphate (SDS), 65 mM DTT, and
10% (v/v) glycerol, all at pH 6.8. The homogenate was centrifuged
at 10,000 × g for 2 min, 0.5 ml of the supernatant
were added to 0.5 ml sample buffer composed of 3% (w/v)
Tris, 5% 2-mercaptoethanol, 10% (w/v) SDS, 20% (v/v) glycerol,
and 0.2% (w/v) bromophenol blue and the mixture was
incubated 5 min at 100 ºC.
Rubisco content was determined by SDS-PAGE with a
Mini-PROTEAn 3 system (Bio-Rad). Resolving gels contained
10 % (w/v) acrylamide, 0.27 % (w/v) n,n´-methy-