Download Plant growth: the translational connection

Post-Transcriptional Regulation of Plant Gene Expression
Plant growth: the translational connection
C. Robaglia*1 , B. Menand†, Y. Lei*, R. Sormani*, M. Nicolaı̈*, C. Gery‡, E. Teoulé‡, D. Deprost§ and C. Meyer§
*Laboratoire de Génétique et Biophysique des Plantes, Département d’Ecophysiologie Végétale et Microbiologie, UMR 6191 CNRS-CEA-Université de la
Méditerranée, Luminy, Marseille, France, †Department of Cell and Molecular Biology, John Innes Center, Norwich, U.K., ‡Unité de Génétique, INRA,
Versailles, France, and §Laboratoire de Nutrition Azotée des Plantes, INRA, Versailles, France
Abstract
The TOR (target of rapamycin) pathway is a phylogenetically conserved transduction system in eukaryotes
linking the energy status of the cell to the protein synthesis apparatus and to cell growth. The TOR protein
is specifically inhibited by a rapamycin–FKBP12 complex (where FKBP stands for FK506-binding protein) in
yeast and animal cells. Whereas plants appear insensitive to rapamycin, Arabidopsis thaliana harbours a
single TOR gene, which is essential for embryonic development. It was found that the product of this gene
was capable of binding to rapamycin and yeast FKBP12. In-frame fusion with a GUS reporter gene shows
that the TOR protein is produced essentially in proliferating zones, whereas the TOR mRNA can be detected
in all organs suggesting a translational regulation of TOR. Phenotypic analysis of Arabidopsis TOR mutants
indicates that the plant TOR pathway fulfils the same role in controlling cell growth as its other eukaryotic
counterparts.
Introduction
The growth of an organism depends on the size and the
number of its cells. Increase in cell numbers depends on
the rate of cell division and on the number of proliferating
cells. Cell size can be variably adjusted during cell division
or during differentiation [1]. In plants, a large size can be
reached by the process of cell expansion, where cells can
enlarge post-mitotically up to 1000 times by taking up water
and synthesizing a cell wall: ‘liquid growth’ [2]. This kind of
cell differentiation is specific to plants; however, the provision
of new cells to constitute new organs still depends on cell
proliferation as in other multicellular organisms. To provide
the building blocks of new organs, cell proliferation must
be linked to cell growth, which accounts for the increase
in mass, which will be shared by the daughter cells. This
increase in mass is intimately linked to the process of protein
synthesis and to the nutritional input perceived by the cell. A
connection between nutrient perception, protein synthesis
and growth is certainly essential for all cells [3,4], and
genes that are supposed to be involved in this regulation
appear to be phylogenetically conserved among eukaryotes.
One of the pathways controlling growth in eukaryotes is
known as the TOR (target of rapamycin) pathway. TOR
is a very large protein kinase, initially discovered in yeast,
which is the target of the antiproliferative drug rapamycin. A
decade of research on TOR in yeast and animals has shown
that this protein is at the heart of a regulatory system linking
nutrient availability to the activity of the protein synthesis
Key words: Arabidopsis thaliana, cell growth, cell proliferation, meristem, target of rapamycin
(TOR), translation.
Abbreviations used: eIF, eukaryotic initiation factor; eIF4E-BP, eIF4E-binding protein; FKBP,
FK506-binding protein; TOR, target of rapamycin.
1
To whom correspondence should be addressed (email [email protected]).
machinery to regulate cell proliferation in unicellular organisms and body growth in multicellular organisms [5–7]. TOR
responds to various inputs linked to the energy available
to the cell such as amino acids, ATP status and stresses,
but also to mitosis-promoting hormones (insulin) in multicellular animals and integrates them to regulate several
effectors of cell proliferation and survival such as the protein
synthesis machinery, the cytoskeleton, the uptake of nutrients
and autophagy (Figure 1). TOR is supposed to integrate its
inputs by engaging in several large molecular mass complexes regulating its protein kinase activity towards various
substrates. The rapamycin inhibitor triggers the formation
of a complex with an FKBP12 (FK506-binding protein),
which in turn binds TOR and blocks its activity. One of
the main TOR targets is translation initiation mediated by the
eIF4F (where eIF stands for eukaryotic initiation factor)
translation–initiation complex. In animal cells, TOR activation releases an active eIF4E cap-binding protein by
phosphorylating the repressor eIF4E-BP (eIF4E-binding
protein, [8]). In yeast, a different eIF4E-BP named Eap1
is supposed to play a similar role and TOR was found to
regulate cell-cycle progression by activating the translation
of the mRNA for the G1 cyclin CLN3 [9,10]. In animal cells,
TOR also promotes the up-regulation of translation of a class
of mRNAs bearing 5 -terminal oligopyrimidine tracts (5 TOP mRNAs) encoding components of the translational apparatus, through the activation of the p70 ribosomal protein
S6 kinase (p70S6k ), thereby controlling the biogenesis of the
ribosome [11]. Although yeast is not known to possess
the equivalent of p70S6k , TOR also controls ribosome biogenesis through regulation of the translation of the ribosomal
protein mRNA as well as the transcription of rRNA and
tRNA by polymerase I and polymerase III and the structure
of the chromatin of rDNA genes [12].
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Figure 1 TOR cellular roles: summary of data from yeast,
Figure 2 AtTOR functions. AtTOR knockout phenotypical analysis
mammalian cells, Caenorhabditis elegans and Drosophila
melanogaster
and expression pattern are summarized
Grey areas symbolize the cytoplasm, white areas symbolize the vacuole.
Features of the AtTOR gene
In plants, cell proliferation occurs in meristems, dynamic
structures continuously traversed by a flux of cells that
divide and grow at different rates, and which are defined
during embryogenesis. Although the plants are autotrophic,
the meristems can be considered as heterotrophs and they
depend on the rest of the plant for their supply of nutrients
to sustain growth. As plant development and morphogenesis
are highly adaptive, cell proliferation in the meristems has to
be integrated all through a plant’s life with the various
environmental stimuli, including nutrient supply [13].
Investigation of the Arabidopsis genome reveals that plants
possess a probable orthologue of the TOR gene. This gene
is located on the lower arm of chromosome 1 and encodes
a 2481-amino-acid protein with all the attributes of a TOR
protein including the FKBP-rapamycin-binding domain, the
C-terminal kinase domain and the presence of HEAT repeats
[14].
Investigations of the plant-TOR pathway have been
hampered by the natural resistance of plants to rapamycin.
Identification of a potential FRB domain in the AtTOR
protein raises the question of the basis of this resistance, since
plants also possess orthologues of FKBP12 proteins. A yeast
three-hybrid analysis was therefore undertaken to determine
if the AtTOR FRB domain was capable of interacting with
plant orthologues of Saccharomyces cerevisiae FKBP12 in
the presence of rapamycin. This revealed that none of the
plant FKBPs tested (AtFKBP12, AtFKBP15-1, AtFKBP152 and AtFKBP62) could interact with the AtTOR FRB
domain in the presence of rapamycin (B. Menand and C.
Robaglia, unpublished work). However, a control with yeast
FKBP12 (ScFKBP12), revealed that complex formation could
occur [14]. This showed that the FRB domain of Arabidopsis
thaliana TOR was still functional for a ternary-complex
formation, despite the fact that none of the plant FKBPs
could engage in such a complex and suggests that this domain
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is involved in a TOR function conserved in plants, yeast and
animals.
Mutants of AtTOR were readily identified in T-DNA
insertion libraries and two mutants with insertions interrupting the gene near the FRB domain and upstream
of the kinase domain were analysed in depth [14]. The
siliques of those plants contained one quarter aborted seeds
suggesting that the mutation was lethal at the homozygous
stage, indeed the observation of embryos showed that
development was arrested at the globular stage. Interestingly,
those arrested embryos still occasionally contained cells
undertaking division. This shows that AtTOR inactivation
was not detrimental for the cell-division machinery. During
the early stages of Arabidopsis embryo development, there is
a small increase in the total embryo volume and the size of
cells decreases during divisions. The developmental arrest of
a tor mutant embryo at the globular stage correlates with the
change from divisions without growth to divisions coupled
with growth [15]. Therefore AtTOR is probably required
for premitotic cytoplasmic growth during cell proliferation.
This is supported by the absence of segregation distortion
in the transmission of the tor mutation showing that correct
divisions of the tor gametophytes can occur, leading to gamete
formation. Again, these divisions are the result of cleavages
within a pre-existing cell mass without net enlargement of the
cytoplasm (the alternance of generations where both haploid
and diploid forms can divide is specific to plants).
In-frame fusion with the GUS reporter further reveals
that the synthesis of the AtTOR–GUS fusion protein occurs
in embryo, endosperm, primary meristems and primordia
but not in differentiated organs such as fully expanded leaf
cells and root tissue (Figure 3, lower panel). During the
formation of secondary roots AtTOR expression occurs very
early, as soon as the first periclinal divisions of the lateral
root primordia and well before the meristem structure was
clearly established [14]. Remarkably, the precursors of the
stomatal guard cells, which return to a cycle of cell division
after differentiation and with a small increase in cytosol
mass, are not expressing the AtTOR-GUS marker, whereas
they express the CycB-GUS marker, further providing
an indication that AtTOR is essential for cytoplasmic
Post-Transcriptional Regulation of Plant Gene Expression
Figure 3 Post-transcriptional regulation of AtTOR
Upper panel: reverse transcriptase–PCR analysis of AtTOR expression in plant tissues. Lower panel: GUS staining of an
heterozygous TOR/tor-1 plant, the tor-1 allele is fused with the GUS gene at amino acid 1555 of AtTOR. Total RNA was
extracted from the indicated tissues and 1 µg was reverse transcribed using oligo(dT). After a 10× dilution, PCR analysis
was performed using AtTOR or actin 8 specific primers for the indicated number of cycles. PCR products were separated by
agarose gel electrophoresis, blotted on to nylon and hybridized with AtTOR or actin 8 32 P-labelled probes.
growth. The above observations and their interpretation are
summarized in Figure 2.
Post-transcriptional regulation of AtTOR
gene expression
As stated above, the production of the TOR protein, as
attested by the in-frame GUS fusion is limited to proliferating cells and tissues. However, reverse transcriptase–
PCR experiments designed to detect the AtTOR mRNA
reveal that it is expressed at nearly equal levels in all
plant tissues including differentiated cells at the tip of
the leaves and fully expanded leaves where TOR-GUS
protein is undetectable (Figure 3). This suggests that
AtTOR is regulated post-transcriptionally by translational
repression in differentiated cells. As translational regulation
is often linked to particular features of untranslated mRNA
sequences, the AtTOR 5 -leader sequence was obtained by
5 -RACE (where RACE stands for rapid amplification of
cDNA ends) analysis. Visual inspection of the resulting
230 bp sequence reveals the presence of a short microORF
(open reading frame) having the potential to encode for
a single amino acid, AUGUGUUGA. MicroORFs were
found to positively or negatively regulate translation of
downstream genes in many cases [16]. An indication of their
function is their conservation since sequence constraints are
generally not more rigid in untranslated regions than in
coding sequences. Indeed, sequencing the DNA upstream of
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the AtTOR coding region in 12 Arabidopsis ecotypes chosen
from a collection maximizing genetic diversity [17] shows
that the AtTOR leader region, including the uORF, is highly
conserved, thus supporting the maintenance of a functional
regulatory role (results not shown).
Concluding remarks
The lethal phenotype of knockout AtTOR mutants and the
insensitivity of plants to rapamycin currently limits the study
of the plant TOR pathway. Innovative methods to conditionally inactivate AtTOR or the isolation of hypomorphic
mutants are necessary. Post-transcriptional gene silencing
through the expression of double-stranded RNA is a way
to achieve this goal, since a range of phenotypes can in
principle be obtained [18]. Another strategy would be to
generate tissue sectors devoid of AtTOR by complementation
of the available mutants with the AtTOR gene bordered by
target sites for an inducible recombinase such as FLP [19].
Plants harbour orthologues of many genes linked to TOR
in other organisms such as those coding for ribosomal S6
kinase, PDK, eIF4E and TAP 42, but lack others such as
genes coding for PKB and eIF4E-BP [20]. Therefore the
precise architecture of the plant TOR pathway remains
to be determined. Since plants and animals independently
acquire multicellularity, it is probable that signals acting
upstream of TOR to orchestrate growth and morphogenesis
are different [21]. An important area of future research will
be the exploration of the integration of the plant TOR at the
level of the organism.
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Received 27 April 2004