AUXIN TRANSPORT AND RIBOSOME BIOGENESIS MUTANT / REPORTER LINES TO STUDY PLANT CELL GROWTH AND PROLIFERATION UNDER ALTERED GRAVITY Miguel A Valbuena1, Ana I Manzano1, Jack JWA van Loon2, Julio Sáez-Vásquez3, Eugénie Carnero-Díaz4, Raúl Herranz1 and F Javier Medina1 1
Centro de Investigaciones Biológicas (CSIC), C/Ramiro de Maeztu 9, E-28040, Madrid, Spain;
[email protected];
[email protected];
[email protected];
[email protected]. 2 Dutch Experiment Support Center (DESC) @ ACTA, University of Amsterdam & VU University Amsterdam, Dept. Oral Cell Biology, Research Institute MOVE, Amsterdam, The Netherlands / European Space Agency (ESA), TEC-MMG, ESTEC, Noordwijk, The Netherlands;
[email protected]. 3 Lab. Génome et Dévelopment des Plants (CNRS), Univ. Perpignan via Domitia, Perpignan, France;
[email protected] 4 Équipe Régéneration (CNRS), Université Pierre et Marie Curie, Paris, France.
[email protected] ABSTRACT We tested different Arabidopsis thaliana strains to check their availability for space use in the International Space Station (ISS). We used mutants and reporter gene strains affecting factors of cell proliferation and cell growth, to check variations induced by an altered gravity vector. Seedlings were grown either in a Random Positioning Machine (RPM), under simulated microgravity (µg), or in a Large Diameter Centrifuge (LDC), under hypergravity (2g). A combination of the two devices (µgRPM+LDC) was also used. Under all gravity alterations, seedling roots were longer than in control 1g conditions, while the levels of the nucleolar protein nucleolin were depleted. Alterations in the pattern of expression of PIN2, an auxin transporter, and of cyclin B1, a cell cycle regulator, were shown. All these alterations are compatible with previous space data, so the use of these strains will be useful in the next experiments in ISS, under real microgravity. 1. INTRODUCTION In a weightless environment, e.g. that existing on board the ISS, plants undergo alterations in fundamental processes, like cell proliferation and cell growth [1], that could seriously reduce their viability and compromise their usefulness in life support systems as food and oxygen suppliers. Since the access to ISS to perform real microgravity research is necessarily limited, some ground-based facilities (GBFs) have been developed to compensate the Earth gravity vector, simulating the conditions that may occur in space (review in [2]). New, fast and reliable tools for in situ analyses should be first explored in GBFs and preliminary data should be obtained in them, prior to their implementation in space experiments. In this study, newly genetically engineered reporter gene constructions (GFP) and well established Arabidopsis thaliana mutant lines have been exposed to altered gravity environments on ground with the purpose of monitoring plant cell growth and proliferation processes.
2. MATERIAL AND METHODS 2.1 Construction of new GFP reporter lines Transgenic lines expressing the GFP reporter gene were produced by Agrobacterium-mediated transformation of Arabidopsis thaliana ecotype Col-0 plants with the following constructions: Nucleolin (ATNUC-L1:GFP) strain: to be used as a marker of ribosome biogenesis and cell growth. We expressed the mGFP4 gene product under the control of a 1197bp sequence immediately upstream the +1 position of the nucleolin gene (AT1G48920,1) promoter. Cyclin B1 (CYCB1:GFP) strain: to be used as a marker of cell proliferation and cell cycle progression. We expressed the mGFP4 gene product under the control of a 1128bp sequence immediately upstream the +1 position of the cyclin B1 gene (AT4G37490,1) promoter. We have also used a fusion PIN2:PIN2-GFP strain for auxin transport studies [3]. 2.2 Arabidopsis mutants atnuc-L1 mutant (SALK-053590) was used to further analyze cell growth function [4]. eir1.1 mutant (AT5G57090) was used to complement our studies about auxin efflux transport regulation [5]. 2.3 Testing materials in altered gravity GBF To cover a wide range of gravitational levels we have used a microgravity simulator (RPM) and a hypergravity dedicated facility (LDC), used either independently or in combination. Consequently, 3 environmental conditions of altered gravity were obtained and labeled as µgRPM, µgRPM+LDC, and 2gLDC; the µgRPM+LDC condition was obtained by placing a desktop RPM into a 2g environment (LDC). In addition, the respective controls, 1gRPM and 1gLDC, have been analyzed.
—————————————————————————– Proceedings of the Joint Life Science Symposium, Aberdeen, United Kingdom, 18-22 June 2012.
2.4 Evaluated parameters The root length was measured in wild type, eir1.1 and atnuc-L1 strains from pictures taken immediately after the 4 day-long altered gravity treatment. Nucleolar size of root meristematic cells was estimated by immunofluorescence, using anti-AtNUC polyclonal antibody. By confocal microscopy, GFP signal driven by the nucleolin or cyclin B1 promoters were used to estimate cell growth and proliferation in the previous 24h (estimated half-life of the mGFP protein used in transgenic plants expression, [6]). Fusion PIN2-GFP protein was used as an indicator of auxin transport. 3. RESULTS AND DISCUSSION Roots were longer in seedlings exposed to the RPM, as described in previous real or simulated microgravity experiments [1]. However, the longest growth was detected in the case of µgRPM+LDC (Table 1). It is important to point out that the environmental gravity in which the RPM was placed in this case, was different from the case of the RPM alone (2g vs. 1g). This means that the differential g force between effective gravity and environmental gravity was different in the two cases. Strain Root length Nucleolar size
A
B
C
D
RPM+LDC 2gLDC µgRPM µg RPM vs 1g vs 1gLDC
Wt
+
+*
+*
eir1.1
+*
+*
+
atnuc-L1
+
+*
+
Wt
-*
-
-*
eir1.1
-*
+
+
PIN2:GFP Protein expression ATNUC-L1:GFP pattern CYCB1:GFP
gravitropic signaling (see table 1 - Protein expression pattern). In the other two cases, ATNUC-L1:GFP and CYCB1:GFP, subcellular localization was not preserved but GFP signal can be used to map the meristematic areas of expression of these genes. The respective patterns appeared affected in different degrees in all cases of modified gravity, and even in 1gLDC due to the effect of centrifugal forces. Signals were especially weak, in the 2g condition, and in the case of CYCB1:GFP under microgravity (Fig. 1).
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Table 1. Summary of results comparing the three different treatments and the different strains used. (+) means that the value is higher compared with the control. (-) means that the value is lower compared with the control (two or more symbols indicate greater differences).(*) indicates that differences are statistically significant (p
