Research articleVipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes
Introduction
Oxygenic photosynthesis is a specific feature of cyanobacteria and chloroplasts of plants and algae. Characteristic for oxygenic photosynthesis is a specialized membrane system, the thylakoids, on which the components of the photosynthetic machinery are located [20]. Despite its importance in the process of oxygenic photosynthesis, very little is known about the origin of the thylakoid membrane in the ancestor of today's cyanobacteria. Furthermore, proteins and other factors that are involved in the formation and maintenance of the thylakoid membrane in either cyanobacteria or chloroplasts are not well defined.
We have shown recently that in Arabidopsis as well as in Synechocystis the vesicle inducing protein in plastids 1 (Vipp1) is essential for thylakoid biogenesis [3], [11], [21]. Δvipp1 mutant plants, in which the expression of the vipp1 gene is greatly reduced, have lost their ability to build up a proper thylakoid membrane system and consequently to perform oxygenic photosynthesis. Disruption of the vipp1 gene in Arabidopsis also abolished chloroplast vesicle transport, which has an alleged function in thylakoid formation in the chloroplasts of higher plants [22], [23]. Vipp1 apparently derived from a bacterial ancestor, the phage shock protein A (PspA), which is expressed in many bacteria under distinct stress conditions that affect the integrity of the plasma membrane [4], [5], [7], [9]. PspA and Vipp1 are both able to form homo-multimeric high-molecular-weight complexes but the significance of oligomerization for the function of these proteins is not clear [3], [8]. The C-terminal domain that distinguishes all Vipp1 proteins from PspA is not involved in oligomerization [3] and is therefore likely to have a specific function in thylakoid biogenesis. Since Δvipp1 mutant plants exhibit a nearly complete loss of thylakoid membrane formation, they are only capable of heterotrophic growth and develop an albinotic phenotype at a very early stage [11]. This leads to a number of secondary effects that cannot be correlated directly with the function of Vipp1. Therefore the primary function of Vipp1 in thylakoid membrane formation is difficult to assess from these plants.
In this work we made use of an Arabidopsis line that expresses a fusion protein of Vipp1 and ProteinA from Staphylococcus aureus in the Δvipp1 background. The Vipp1–ProteinA fusion protein is able to reinstate autotrophic growth. Plants which are homozygous with regard to the VIPP1–PROTEINA insertion possess a Vipp1–ProteinA content similar to the Vipp1 content of wild type and accordingly re-establish the wild-type phenotype. Nevertheless, plants heterozygous for the insertion have only about half the amount of Vipp1–ProteinA and do not reach a full recovery under normal growth conditions. Thus, these plants have enabled us to more clearly determine the primary physiological effects exerted by a deficiency in Vipp1.
Section snippets
Reduction in Vipp1 content affects plants differently under different light conditions
We have shown earlier that a T-DNA insertion into the promoter region of the At1g65260 gene (VIPP1) gives rise to mutants with a limited amount of Vipp1 (about 20% of wild type content) and a disability to form thylakoid membranes [11]. Mutant plants are no longer capable of photoautotrophic growth and develop an albinotic phenotype at an early stage. This phenotype could be rescued by production of a Vipp1–ProteinA fusion protein in the Δvipp1 mutant background. Transformants were initially
Discussion
We had shown previously that a severe reduction in Vipp1 content leads to a loss of thylakoid formation as well as photosynthetic capacity both in chloroplasts and cyanobacteria [11], [21]. Nevertheless, due to its albinotic phenotype the original Δvipp1 mutant suffered from a number of secondary defects not directly connected to the loss of Vipp1. This made it difficult to discern the exact function of Vipp1 in thylakoid development. It was suggested that Vipp1 is involved in the formation of
Plant material, growth conditions, and mutant selection
For most experiments seedlings of Arabidopsis thaliana (var. Columbia Col-0) were grown at 22 °C under a 16 h light and 8 h dark cycle on MS-plates supplemented with 1% (w/v) sucrose as described before [11]. Propagation of both Δvipp1 and VIPP1–PROTEINA plants occurred via heterozygous offspring. All comparisons between mutant and wild type plants were carried out with leaf material of the same developmental stage. To obtain the VIPP1–PROTEINA plants, the entire VIPP1 reading frame was
Acknowledgements
The skilful technical assistance of Ms Silvia Dobler in preparing the electron micrographs and of Ms Daniela Duy in performing real-time PCR is gratefully acknowledged. The original pea vipp1 clone was a gift from Dr K. Keegstra. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB-TR1).
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