Mutations in the Arabidopsis Homolog of LST8/GbL, a Partner
of the Target of Rapamycin Kinase, Impair Plant Growth,
Flowering, and Metabolic Adaptation to Long Days
Manon Moreau,a,bMarianne Azzopardi,aGilles Cle ´ment,aThomas Dobrenel,aChloe ´ Marchive,a
Charlotte Renne,aMarie-Laure Martin-Magniette,c,dLudivine Taconnat,dJean-Pierre Renou,d
Christophe Robaglia,band Christian Meyera,1
aInstitut Jean-Pierre Bourgin, Unite ´ Mixte de Recherche 1318, Institut National de la Recherche Agronomique AgroParisTech,
78026 Versailles cedex, France
bLaboratoire de Ge ´ne ´tique et Biophysique des Plantes, Unite ´ Mixte de Recherche 7225, Commissariat a ` l’Energie Atomique–
Institut de Biologie Environnementale et Biotechnologie–Service de Biologie Ve ´ge ´tale et de Microbiologie Environnementales,
Centre National de la Recherche Scientifique, Universite ´ Aix Marseille, Faculte ´ des Sciences de Luminy, 13009 Marseille, France
cUnite ´ Mixte de Recherche 518, Institut National de la Recherche Agronomique AgroParisTech, 75005 Paris, France
dUnite ´ de Recherche en Ge ´nomique Ve ´ge ´tale, Unite ´ Mixte de Recherche 1165, Institut National de la Recherche Agronomique,
Universite ´ Evry Val d’Essonne, Centre National de la Recherche Scientifique, 91057 Evry cedex, France
The conserved Target of Rapamycin (TOR) kinase forms high molecular mass complexes and is a major regulator of cellular
adaptations to environmental cues. The Lethal with Sec Thirteen 8/G protein b subunit-like (LST8/GbL) protein is a member
of the TOR complexes, and two putative LST8 genes are present in Arabidopsis thaliana, of which only one (LST8-1) is
significantly expressed. The Arabidopsis LST8-1 protein is able to complement yeast lst8 mutations and interacts with the
TOR kinase. Mutations in the LST8-1 gene resulted in reduced vegetative growth and apical dominance with abnormal
development of flowers. Mutant plants were also highly sensitive to long days and accumulated, like TOR RNA interference
lines, higher amounts of starch and amino acids, including proline and glutamine, while showing reduced concentrations of
inositol and raffinose. Accordingly, transcriptomic and enzymatic analyses revealed a higher expression of genes involved
in nitrate assimilation when lst8-1 mutants were shifted to long days. The transcriptome of lst8-1 mutants in long days was
found to share similarities with that of a myo-inositol 1 phosphate synthase mutant that is also sensitive to the extension of
the light period. It thus appears that the LST8-1 protein has an important role in regulating amino acid accumulation and the
synthesis of myo-inositol and raffinose during plant adaptation to long days.
Cell growth is a fundamental energy-consuming process that
environmental stimuli to preservecellularhomeostasis. Inplants,
identification of central key regulators that integrate exogenous
signals, such as light, water, stress, or the presence of nutrient,
and adjust plant metabolism and morphogenesis to optimize
development and enhance survival is a challenging goal. The
Target of Rapamycin (TOR) pathway, which is conserved among
all eukaryotes, is now well known in animals and yeast as a
central regulator of growth in response to environmental cues
2009; Moreau et al., 2010). Indeed, this signaling pathway
integrates hormonal and nutritional information to translate
them into growth, developmental, and metabolic decisions.
The TOR kinase is a large protein (molecular mass of ;250 kD)
that belongs to the phosphatidyl inositol kinase-related kinase
family and is essential in all eukaryotic organisms. TOR associ-
ates in complexes with other protein partners to regulate, in
response to environmental signals(nutrient availability, stress,or
growth factors), various cellular processes like translation, tran-
scription, ribosome biogenesis, autophagy, actin organization,
animals, there are two TOR complexes (TORC1 and TORC2)
influencing different functions in cells. TORC1 is composed of
three major proteins: TOR, KOG1/RAPTOR, and LST8/GbL (for
Lethal with Sec Thirteen 8/G protein b subunit-like), whereas
TORC2 is composed of TOR, LST8/GbL, and AVO3/RICTOR
(Hara et al., 2002; Kim et al., 2002; Loewith et al., 2002). Other
than TOR, the small 34-kD LST8/GbL protein is the only one that
is common to both complexes.
LST8 was first identified in yeast through a screen for muta-
tions that show synthetic lethality with alleles of sec13, a gene
involved in endoplasmic reticulum–to–Golgi transport and also
required for the regulated transport from the Golgi to the plasma
membrane of Gap1, a general amino acid permease (Roberg
1Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Christian Meyer
CSome figures in this article are displayed in color online but in black
and white in the print edition.
WOnline version contains Web-only data.
The Plant Cell, Vol. 24: 463–481, February 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.
et al., 1997). The LST8 protein contains seven WD 40 repeats,
which are implicated in a wide range of functions like signal
transduction and vesicular trafficking (Neer et al., 1994), and its
protein b-subunits (Kim et al., 2003). These WD repeats result in
the formation of a stable, propeller-like platform allowing inter-
actions with several protein partners (Smith et al., 1999). At the
same time, LST8 was identified in fission yeast as Wat1p, a pro-
factor (Kemp et al., 1997). Mutations in Wat1p caused diploid-
ization, genome instability, and decreased level of a-tubulin
transcripts (Ochotorena et al., 2001). In yeast, LST8 is a negative
regulator of the retrograde (RTG) signaling pathway that medi-
of poor nitrogen sources by readjusting carbon and nitrogen
metabolism through nuclear translocation of the heterodimeric
transcription factors Rtg1/3 (Liu et al., 2001). Interestingly, the
inactivation of TOR activity by rapamycin also resulted in
nuclear accumulation of Rtg1/3 (Komeili et al., 2000). More
recently the LST8 protein was involved in connecting the TOR
and RTG signaling pathways, and some aspects of the LST8-
dependent RTG responses could be separated from TOR
activity (Giannattasio et al., 2005). In yeast, some of the effects
of lst8 mutations were ascribed to an intracellular accumulation
of amino acids and to a partial inhibition of the TOR signaling
pathway (Chen and Kaiser, 2003). Indeed, lst8 mutants were
found to be hypersensitive to rapamycin, an inhibitor of TORC1
functions, and had cell wall defects. In animal cells, the mLST8
protein binds and activates the TOR kinase domain and seems
to be required to maintain the TOR–RAPTOR interaction of the
TORC1 complex in a nutrient-dependent manner (Kim et al.,
2003; Adami et al., 2007). Recently, it has also been shown that
mLST8 interacts with IkappaB kinase and that it inhibits the
phosphorylation of this kinase by recruiting PP2A and PP6
phosphatases (You et al., 2010). So far, little is known about the
impact of LST8 mutations in multicellular organisms. In mouse,
it has been demonstrated that embryos devoid of mLST8 (GbL)
expression survive for some time and die at a period corre-
sponding to anincrease in the vasculature (Guertinet al., 2006).
These embryos resembled RICTOR-deficient ones, and the
lack of mLST8 protein appeared to affect only the functions of
the TORC2 complex without any visible effects on the readouts
of TORC1 activity.
In plants there are no clear homologs of the TOR complex
2 (TORC2)–specific components like AVO1/hSIN1 or AVO3/
RICTOR. Conversely, there is strong evidence for the existence
of a TORC1 complex in both land plants and algae (Mahfouz
et al., 2006; Moreau et al., 2010). In Chlamydomonas reinhardtii,
the LST8 protein (Cr-LST8), as well as Cr-TOR, were found in
high molecular masscomplexes thatareassociated withinternal
membranes (Dı ´az-Troya et al., 2008). The same report showed
that Cr-LST8 was also localized around the nucleus and near
peribasal bodies, close to the flagella. The fact that the expres-
sion of the Cr-LST8 coding sequence in yeast complemented
lst8 mutations suggests that Cr-LST8 is a functional homolog of
the yeast LST8 protein. In C. reinhardtii, the Cr-LST8 protein was
shown to copurify with the Cr-TOR protein and to interact, as in
Taken together, these data all support the assumption that a
conserved TORC1protein complex isformed in plants and algae
with the TOR, RAPTOR, and LST8 partners.
The loss of TOR or RAPTOR expression is embryo lethal in
plants (Menand et al., 2002; Deprost et al., 2005) even if the
penetrance of raptor mutation is not complete, allowing the re-
et al., 2005). By studying Arabidopsis thaliana lines either over-
or underexpressing the Arabidopsis TOR gene, we previously
showed that the level of TOR expression was very well corre-
lated with the size of the plants, the amount of seed produced,
and the abundance of polysomes (Deprost et al., 2007). Ethanol-
inducible RNA interference (RNAi) lines were also obtained that
allow a conditional silencing of TOR (Deprost et al., 2007). When
the expression of TOR was abolished by ethanol induction,
plants growth was arrested and senescence-linked markers
(genes and metabolites) became upregulated. As for RAPTOR,
there are two genes in Arabidopsis coding for proteins showing
high sequence identities when compared with the LST8 protein
sequences from other organisms (At3g18140 and At2g22040 for
LST8-1 and LST8-2 genes, respectively).
In this article, we investigated the role and properties of the
LST8 proteins in Arabidopsis. We showed that the LST8 protein
interacts with the TOR kinase domain and that mutations in the
most highlyexpressed LST8 geneaffect Arabidopsis growth and
development while impeding plant transcriptomic and metabolic
adaptations to long-day (LD) conditions. Our results provide
insight into the important role of LST8 in adapting plant metab-
olism and development to external conditions.
Only One of the Two Arabidopsis LST8 Genes Is
Asimilarity searchof the Arabidopsis translated genome withthe
yeast LST8 protein sequence revealed that it contains two genes
potentially coding for homologs of yeast and animal LST8
proteins, as described earlier (Mahfouz et al., 2006). An LST8-
hirsutum; Duan et al., 2006). These genes correspond to the
predicted At3g18140 and At2g22040 loci in Arabidopsis, which
we will name throughout this study LST8-1 and LST8-2, respec-
tively. Both LST8-1 and LST8-2 genes are ;2 kb long and are
composed of 10 exons and nine introns. The LST8-1 gene
encodes a protein of 305 amino acids witha predicted molecular
mass of 34 kD, whereas LST8-2 encodes a 35-kD protein of 312
amino acids. The two proteins are slightly more similar to yeast
LST8 (51% sequence identity) than to animal LST8 protein
sequences (45% sequence identity with mouse LST8). As in
other organisms, both Arabidopsis LST8 proteins contain seven
predicted WD 40 repeats, which can form a propeller-like plat-
form structure composed of b-strands (see Supplemental Figure
1 online; Dı ´az-Troya et al., 2008). The two Arabidopsis LST8
proteins have a high percentage of sequence identity to each
other (75%), but, interestingly, other angiosperm LST8 protein
sequences are more similar to LST8-1 than is LST8-2 (Figure 1).
464The Plant Cell
For example, the rice (Oryza sativa) LST8 protein sequence
only 77% identical. It seems that the LST8 gene was duplicated
in the ancestor of Arabidopsis and Arabidopsis lyrata since a
sequence closely related to LST8-2 was found only in A. lyrata
(Figure 1). The protein corresponding to LST8-2 in A. lyrata
seems to also have diverged from other plant LST8 sequences.
Numerous ESTs corresponding to the LST8-1 gene are available
in databases,butwehavenotbeenableto identifyspecific ESTs
the last exon of the LST8-2 gene has been labeled as non-
confirmed in the TAIR 10 Arabidopsis genome release (www.
Arabidopsis.org). Affymetrix microarrays contain only one oligo-
nucleotide set for both LST8 genes, which makes it difficult to
assess their expression levels. Conversely, CATMA microarrays
(Crowe et al., 2003) carry tags that are specific for each LST8
gene. A survey of all publicly available CATMA chip results
(CatDB; Gagnot et al., 2008) did not reveal any detectable
expression of LST8-2. It is thus likely, given the divergence of
the LST8-2 from other plant LST8 proteins and the lack of
detectable expression of its coding sequence, that LST8-2 is a
nonfunctional gene. This implies that expression data from
Affymetrix microarrays probably mainly reflect the level of
LST8-1–derived transcripts. Data from the Genevestigator web-
site (www.genevestigator.com) reveal that LST8-1 is expressed
throughout plant development and in all plant organs with a
higher level in micropylar and chalazal endosperm (see Supple-
mental Figure 2 online). Furthermore, LST8-1 expression seems
to be higher in aerial parts than in roots.
Subcellular Localization and Expression Pattern of the
First, we investigated the subcellular localization of the LST8-
1 protein by transiently expressing a 35S:LST8-1-green fluores-
cent protein (GFP) construct in Arabidopsis cotyledons. The
fusion protein was mainly detected in mobile dots (Figure 2). To
establish the nature of the mobile fluorescent dots, the 35S:
LST8-1-GFP construct was introduced in cotyledons of
Arabidopsis seedlings expressing a RabC1-red fluorescent
protein (RFP) fusion that specifically label the endosomes
(Rutherford and Moore, 2002). In most of the cases, the GFP-
and RFP-labeled dots were colocalized or at least very close
with endosomes. Next, the LST8-1 expression pattern in Arabi-
dopsis organs was investigated using plants expressing the
b-glucuronidase (GUS) reporter gene driven by the LST8-1 pro-
moter and 59-untranslated region (UTR; 1 kb upstream of ATG).
Staining was detected in plantlets at the level of the root central
cotyledon vasculature (Figures3Aand 3D),and instomata (Figure
3E). Promoter activity was also observed in leaf stipules (Figure
3F). In flowers, LST8-1 seemed to be also strongly expressed in
anthers, in pollen, and in the filament (Figures 3G and 3H; see
Supplemental Figure 3 online), as well as in the vasculature of
petals and sepals (Figure 3H).
The Arabidopsis LST8-1 Gene Can Complement Yeast
To test the conservation of LST8 function between yeast and
Arabidopsis, we tried to complement a yeast lst8 mutant strain.
Since LST8 is an essential gene in yeast, the mutant strain we
used expressed the yeast LST8 gene under the control of a Gal-
Figure 1. Distances between LST8 Protein Sequences.
Tree showing the average distances based on sequence identities
between plant and algae LST8 protein sequences using the COBALT
multiple alignment tool (Papadopoulos and Agarwala, 2007). The align-
ment is available as Supplemental Data Set 1 online.
Figure 2. Localization of a LST8-GFP Fusion Protein after Transient
Transformation of Cotyledons from a RabC1-RFP–Expressing Arabidop-
Each row is derived from a time-lapse series (5-s intervals). The arrows
indicate mobile dots. Scale bar = 10 mm.
(A) RFP-specific fluorescence from the RabC1-RFP construct that labels
(B) GFP-specific fluorescence from the 35S:LST8-GFP construct.
(C) RFP and GFP signals were merged.
Roles of the Arabidopsis LST8 Homolog465
inducible promoter (Loewith et al., 2002). This strain can grow
only on a Gal-containing medium, and, on Glc, a rapid growth
arrest of the lst8 mutant is observed. We expressed the Arabi-
a constitutive promoter (see Methods for details) and tested the
ability of transformants to grow on Glc as well as on the Gal
control medium (Figure 4). Yeast expressing the LST8-1 coding
sequence was able to grow normally on Glc medium, whereas
yeast transformed with the empty expression vector alone grew
only on Gal medium. This demonstrates that the Arabidopsis
perform the same functions as the yeast protein. This is of
Arabidopsis LST8-1 Interacts with the TOR FRB and
The yeast two-hybrid system was first used to test whether
Arabidopsis LST8-1 can interact with the TOR FRB and kinase
domain (FK domain) as described in animal cells (Kim et al.,
2003). A yeast strain expressing the Arabidopsis LST8-1 and
TOR FK proteins as prey and bait, respectively, was able to grow
expressing either the bait or the prey protein together with the
corresponding emptyvector failedtogrowonthesame medium.
To confirm this interaction in planta, we used a split-luciferase
system in which the two proteins to be tested are fused to either
the N- or C-terminal parts of firefly luciferase as we described
previously (Van Leene et al., 2010). After transient expression in
Arabidopsis cotyledons of the two fusion proteins, we measured
light emissions from 6 to 10 plantlets grown in vitro in multiwell
plates (Figure 5B). Although light levels were quite weak, a
reproducible signal was observed when the LST8-1 protein was
fused to luciferase N- or C-terminal parts and expressed to-
gether with the TOR FK domain. Negative controls expressing
the LST8-1 or TOR FK proteins together with GFP gave much
lower light signals (Figure 5B). Collectively, these results strongly
suggest that the LST8-1 protein interacts with the TOR FK
domain in Arabidopsis.
Disruption of the LST8-1 Gene Affects Plant Growth
Next, we investigated further the role of Arabidopsis LST8-
1 by studying the consequences of mutations in the correspond-
ing gene. We isolated two LST8-1 homozygous mutants named
lst8-1-1 (Salk collection; Alonso et al., 2003) and lst8-1-2 (SAIL
Figure 3. GUS Staining of Transformed Arabidopsis Plants Carrying a pLst8:GUS Construct Containing 1 kb of LST8-1 Promoter.
Plantlet (A), primary root tip (B), emerging secondary root (C), aerial part (D), close-up on a leaf showing staining of stomatal guard cells (E), emerging
leaves and stipules ([F], indicated by arrows), and flowers ([G] and [H]).
466The Plant Cell
collection) with T-DNA insertions in the 4thexon and 5th intron of
the gene, respectively (see Supplemental Figure 4 online). No
full-length LST8-1 transcripts were detected in these mutants by
for the LST8-2 gene, which displayed no visible phenotype.
Expression of the LST8-2 gene was not detected by RT-PCR in
the wild type or in the mutant lst8-1 background (Figure 6A). This
confirms that LST8-2 is not, or is only very weakly, expressed in
Under controlled short-day (SD) conditions (8 h of light), lst8-
1-1 and lst8-1-2 homozygous mutants showed a reduction in
growth compared with the wild type and did not flower (Figure
7A). By contrast, the mutant plants did not display defects in
rosette development. When plants were transferred to LD
conditions (16 h of light), growth of the mutants was much
more retarded compared with the wild type than under control
SD conditions both in vitro (Figure 7B) and when grown in soil
(Figures 7C and 7D). In the most severe cases, lst8-1 mutants
stopped growing and started to yellow and died after 2 weeks,
without producing flowers (Figure 7D). When the plants sur-
vived, they became bushy and developed multiple apical mer-
istems (Figures 7E and 7F). The severity of the phenotype
depended on the developmental stage of the plant when
transferred to LD conditions and on the quantity of light re-
ceived by the plants. Indeed, an early transfer of SD-grown
plants resulted in decreased survival and a stronger phenotype
in LDs. Both lst8-1 mutants exhibited the same growth defects
under LD conditions. In the growth chamber, or in the green-
house in summer or spring with high levels of outside light,
mutant plants were particularly affected by LD periods. In
winter, even if the LD conditions were maintained with artificial
light in the greenhouse, lst8-1 mutants showed a milder re-
sponse to extended light periods. Indeed, mutant plants
displayed defects in leaf development but continued to grow,
and after a few weeks under permissive LD conditions, lst8-
1 mutants became bushy and developed multiple apical mer-
istems (Figures 7E and 7F). Under these growth conditions, half
of the plants stayed at the vegetative stage for several months
without developing inflorescences. The other half of the mutant
plants started to flower while producing a higher number of
stems from the rosette than the control wild type (Figure 7).
However, the organization of the stem was affected (revealing a
type ones (see Supplemental Figure 5 online). The emerging
flower buds also presented an abnormal development (see
Supplemental Figure 5 online). Indeed, the flowers remained
closed, with no emergence of petals as the carpel started to
elongate. Afterwards, the flowers often generated very small
siliques containing aborted seeds. In one case, we were able to
obtain viable seeds from one lst8-1-1 mutant plant grown in a
growth chamber. These seeds germinated normally and pro-
duced plants similar to the parent homozygous mutant. It thus
Figure 5. Arabidopsis LST8-1 Interacts with the C-Terminal FRB-Kinase
Domain of TOR.
(A) Yeast two-hybrid assay with the TOR FRB-kinase domain as bait and
the LST8-1 protein as prey. C1-, pADH::GAL4BD pADH::GAL4AD-LST8;
C2-, pADH::GAL4BD-FRBK pADH::GAL4AD; C+, pADH::GAL4BD-
HSD1pADH::GAL4AD-GAPC2; 1, 2, 3, independent double yeast trans-
formants with pADH:GAL4BD-TOR/FRB and pADH:GAL4AD-LST8-1.
(B) Split-luciferase assay in Arabidopsis cotyledons after transient ex-
pression. Relative light emission of the different split-luciferase protein
pairs. Luciferase (LUC) activity was monitored with at least two inde-
pendent infiltration experiments per tested interactions. The mean of the
experiments is shown together with the corresponding SD values.
Figure 4. Complementation of a Yeast lst8 Mutant with the Arabidopsis
A yeast lst8 mutant strain expressing the Saccharomyces cerevisiae
LST8 cDNA under the control of an inducible Gal promoter was used for
complementation studies. On a permissive, Gal-containing medium, the
yeast lst8 mutant strain is able to grow (A), but on selective, Glc-
containing medium, the yeast lst8 mutant strain containing an empty
transformation vector fails to grow ([B], bottom part). The expression of
the Arabidopsis LST8-1 cDNA in the yeast lst8 mutant strain fully restores
the ability to grow on Glc medium ([B], top part).
[See online article for color version of this figure.]
Roles of the Arabidopsis LST8 Homolog467
seems that growth conditions are important for seed produc-
tion in the lst8-1 mutants.
The lst8-1-1 and lst8-1-2 mutants were transformed with a
driven by 1 kb of its own promoter. The wild-type phenotype
could be restored in the obtained transformants, and this con-
struct complemented the mutant phenotype under both SD and
LD conditions (see Supplemental Figure 6 online).
We then investigated in more detail the cause of the lst8-
1 mutants’ bushy phenotype. lst8-1 meristems were embedded
in resin or paraffin, cut in sections, and observed by microscopy.
This revealed that lst8-1 mutants presented not one but multiple
apical meristems that appeared correctly organized and func-
tional (Figure 8). These meristematic structures derive most
probably from activated axillary meristems, which could explain
the production of multiple stems and the bushy phenotype that
was observed in these mutants.
Metabolite and Enzyme Analysis of lst8-1-1 and
lst8-1-2 Mutants Reveals an Impaired Adaptation
to LD Conditions
The LST8 and TOR proteins have been implicated in the regu-
lation of several metabolic pathways. In yeast, it has been
demonstrated that under nitrogen-rich conditions, LST8 re-
presses transcription factors that promote the expression of
genes implicated in amino acid synthesis. Accordingly, these
genes are constitutively expressed in yeast lst8 mutants, result-
ing in an accumulation of amino acids (Liu et al., 2001; Chen and
This prompted us to investigate the nitrate assimilation path-
way in lst8-1 mutants under LD conditions. After transfer from
SDs to LDs, a decrease in nitrate contents was observed in the
leaves of wild-type plants with almost no nitrate left after 12 d of
growth in LDs (Figure 9A). By contrast, the amount of nitrate
stored in lst8-1 mutants was already higher in SDs and declined
only slightly in LDs. Enzymatic activities of the two first enzymes
involved in nitrate assimilation, namely, nitrate and nitrite reduc-
tases (NR and NiR, respectively), were always higher in leaves of
the mutant plants when compared with the wild type (Figures 9B
and 9C). NR activity showed a transient increase just after
transfer to LD conditions and decreased afterwards, whereas
NiR activity was more constant, except for in the mutant plants
12 d after transfer, where NiR activity was increased. Interest-
ingly, Gln synthetase activity diminished in the mutant plants,
whereas it increased in wild-type leaves (Figure 9D).
Sugar concentrations were shown to vary after exposure of
Arabidopsis to LDs (Corbesier et al., 1998). Therefore, we mea-
sured concentrations of soluble sugars and starch at the begin-
ning and at the end of the day during an SD-to-LD transition. Glc
and Fru concentrations in leaves did not show major changes
after transfer to LDs but were significantly higher in the lst8-1-2
mutant at the end of the day (Figure 10). Conversely, Suc was
less abundant in the mutant at the end of SDs or at the beginning
of LDs. Suc concentrations increased in LDs, but the difference
between the mutant and the wild type was unchanged (Figure
10). Although the amount of starch was similar in wild-type and
mutant lines at the end of a SD, it significantly increased in the
lst8-1-2 mutant under LD conditions. Similar results were ob-
tained with the lst8-1-1 mutant (see Supplemental Figure 7
sugar and starch metabolism properly in response to an abrupt
transition to LDs.
lst8-1 Mutants Show Faster Movements of
a Phloem Tracer
Sugar concentration in leaves is influenced by sugar export
through the phloem to sink organs. Moreover, it has been shown
previously that phloem transport is affected by transitions to LDs
(Gisel etal.,1999,2002).Several fluorescenttracerscan beused
Figure 6. Impact of T-DNA Insertions on Transcription of the LST8 Genes.
(A) Analysis of LST8-1 and LST8-2 expression levels in lst8-1-1 and lst8-1-2 mutants by RT-PCR. The reference constitutive gene is EF1a (Elongation
factor 1a). See Methods for details. WT, wild type.
(B) Analysis of LST8-1 expression level in lst8-1-1 and lst8-1-2 mutants by quantitative real-time RT-PCR. Arbitrary units are calculated relative to the
EF1a expression level. Values are the mean of at least three independent repetitions 6 SD.
468 The Plant Cell
to monitor phloem fluxes. For instance 6(5)-carboxyfluorescein
(CF) diacetate can be loaded into leaf cells and the impermeant
CF moiety can move only after loading into the phloem (Roberts
et al.,1997). Loading of onecotyledon from in vitro–grown plants
with CF allowed us to follow phloem flux and tracer unloading in
roots (Figure 11). We used a segregating population from lst8-
1 heterozygous mutants to compare the appearance of CF
fluorescence in the phloem of connected organs. The genotype
of the plants was determined by PCR analysis. A kinetic exper-
iment after CF labeling revealed that fluorescence appeared
morerapidly intheroots andcotyledons of lst8-1mutantsthanin
the wild type (Figure 11). This suggests that phloem loading and/
or transport is more active in lst8-1 mutants.
Global Metabolite Profiling of lst8-1 Mutants
To explore more globally the impact of lst8-1 mutation on
metabolism, we performed an analysis of metabolites by gas
chromatography coupled to mass spectrometry (GC-MS) using
leaves from the lst8-1-1 and lst8-1-2 mutant plants as well as
wild-type plants grown under SD or LD conditions. Under SD
growth conditions, lst8-1-1 and lst8-1-2 presented a higher level
of several amino acids, such as Gln, Pro, and Ala (see Supple-
mental Figure 8A online). The quantity of soluble protein was
found to be similar between lst8-1 mutants and wild-type plants
(11.8 and 12 mg·mg21fresh weight, respectively). By contrast, it
was found that lst8-1 mutants accumulated almost twofold more
ammonium than wild-type plants (2.8 versus 1.6 mmol·mg21
fresh weight, respectively).
Metabolite profiling analysis also showed that lst8-1-1 and
lst8-1-2 mutants underwent profound metabolic perturbations
when plants were transferred from SD to LD conditions. The
increase in amino acid content that was detected in mutants
when grown under SD conditions was dramatically amplified
under LD growth conditions in lst8-1-1 and lst8-1-2 compared
with the wild type (see Supplemental Figures 8B, 9, and 10
Figure 7. Phenotype of the lst8-1 Insertion Mutants.
(A) to (D) The control wild-type plants are on the left, and the lst8-1-1 mutant plants are on the right.
(A) Plants cultivated in growth chambers for 4 weeks under SD conditions.
(B) Plants grown in vitro for 7 d under LD conditions.
(C) Plants cultivated in the greenhouse for 6 weeks under LD conditions (winter).
(D) Plants cultivated for 4 weeks under SD conditions as in (A) followed by 1 week under LD conditions.
(E) and (F) lst8-1-1 mutant grown in the greenhouse under LD conditions. lst8-1 mutants develop multiple meristems ([E], indicated by red arrows),
become bushy, and produce several stems (F).
Roles of the Arabidopsis LST8 Homolog469
online). In particular, Pro, g amino-butyric acid, and Gln levels
the amount measured in wild-type plants, respectively (Figure
12; see Supplemental Figure 9 online). There was also an
increase in Gln and other amino acid levels when we compared
lst8-1 mutants grown in LDs to mutants grown in SDs (Figure 12;
see Supplemental Figure 10 online). Wild-type plants presented
a smaller increase in amino acids (see Supplemental Figure 11
online). For example, there was a transient increase in Pro after
transfer to LDs, but the Pro concentration later returned to the
level found in SDs. Conversely, lst8-1 mutants seem unable to
control this accumulation of Pro under LD conditions, which
continued progressively as most mutant plants were starting to
senesce (Figure 12). Similar accumulations of Gln, Pro, and
g amino-butyric acid but also of Leu and Ala were observed in
et al., 2007; see Supplemental Figure 12 online), which supports
the role of LST8 in regulating TOR kinase activity.
Concerning organic acids, there was also a strong increase in
the levels of malate, succinate (see Supplemental Figure 9
Figure 8. Development of Multiple Meristems in lst8-1-1 Mutant Plants.
Sections of the apical meristem zone were performed and observed after resin embedding and NBB staining (A) or paraffin embedding and Schiff
reagent staining (B). Arrows indicate multiple meristems.
[See online article for color version of this figure.]
Figure 9. Influence of Long-Day Conditions on Nitrogen Assimilation in lst8 Mutants.
Nitrate content (A), NiR (B), total NR (C), and Gln synthetase (GS; [D]) activities after transfer to LD conditions of wild-type and lst8-1-2 mutant plants.
Plants were grown under controlled conditions. Values are the mean of at least three independent repetitions 6 SD. The values for nitrate concentrations
(A) have been fitted to a regression line, and the corresponding slope is indicated. Statistically different values between the wild type and lst8-1-2 are
indicated by a star (Student’s t test). FW, fresh weight; LD+n, number of days under LD conditions.
470 The Plant Cell
online), and 2-oxoglutarate (Figure 12) in lst8-1 mutants and in
TOR RNAi lines (see Supplemental Figure 12 online). The higher
level of 2-oxoglutarate, the C-skeleton used for N assimilation, is
consistent withanincreased amount ofGln. Finally, after12 LDs,
nearly all metabolites were increased in lst8-1 mutant, which is
often indicative of cell death.
Whenwild-type plants were transferred to LDs, we observed a
dramatic increase in the amounts of trehalose, galactinol, and
raffinose stored in the leaves (Figure 12; see Supplemental
Figure 11 online). There was also an increase in myo-inositol but
to a lower extent (see Supplemental Figure 11 online). Galactinol
is made from UDP-Gal by addition of myo-inositol. Raffinose is a
trisaccharide synthesized by the addition of a Gal moiety do-
nated by galactinol to Suc. Raffinose and galactinol accumulate
in response to various stresses, including high light irradiance
(Nishizawa etal.,2008).Metabolite profiling clearlyreveals a lack
of myo-inositol, galactinol, and raffinose accumulation in lst8-
1 mutants when shifted to LD conditions (see Supplemental
level in wild-type plants was already 3 to 4 times higher than
those in lst8-1-1 and lst8-1-2 mutants (see Supplemental Figure
(see Supplemental Figures 8B, 9, and 10 online), galactinol, and
raffinose (Figure 12) remained very low in the lst8-1 mutants,
whereas it strongly increased in wild-type plants. In LDs, wild-
type plants also accumulated minor sugars like trehalose,
whereas lst8-1 mutants did not (Figure 12). Interestingly, dark-
treated seedlings of the TOR-inducible RNAi lines also show a
reduced level of both galactinol and raffinose (see Supplemen-
tal Figure 12 online). It thus appears that in the absence of
LST8-1, Arabidopsis plants exposed to LDs are unable to
induce the raffinose biosynthetic pathway and the production
of myo-inositol, which is needed for important signaling and
Transcriptomic Analyses of lst8-1 Mutants in SD and
To analyze variations in global gene expressions in the lst8-
1 mutants upon transfer to selective LD conditions, both lst8-1-
1 and lst8-1-2 mutant as well as wild-type plants were grown in
SD conditions until they reached the seven- to eight-leaf stage.
Subsequently, some plants were transferred to LD conditions for
2d,andthreeplantswere harvestedforeachcondition andeach
genotype to compare transcriptome variations. Global expres-
sion profiles were determined using RNA isolated from either SD
or LD conditions, and each lst8-1 mutant was compared with the
used as a reference for the lst8-1-1 mutant (Salk library) and
Col-0 for lst8-1-2 (SAIL library). mRNAs from each sample were
Figure 10. Diurnal Variations in Soluble Sugar and Starch Content during the Transition from SDs to LDs in the Wild Type and the lst8-1-2 Mutant.
Plants were first grown under controlled SD conditions and harvested at the beginning (morning [m]) and end (night [n]) of the day preceding the shift to
LD. Plants were again harvested at day 2 after the start of LD conditions. Results are mean of at least three different samples 6 SD. Statistically different
values between the wild type (WT) and lst8-1-2 are indicated by a star (Student’s t test). FW, fresh weight.
Roles of the Arabidopsis LST8 Homolog 471
extracted, amplified, and hybridized on CATMA microarrays
(Lurin etal.,2004; seeMethodsfor details). Therewere fewerup-
and downregulated genes in mutant plants grown in SDs than
there were under LD conditions (Figure 13A). The genes that are
downregulated in the mutants grown in SDs were mainly cate-
gorized in abiotic and biotic stress responses (see Supplemental
Figure 13 online). The two lst8-1 mutant lines together with the
corresponding wild type were grown in independent experi-
ments. Transcriptome comparisons between the two wild-type
ecotypes cultivated in either SDs or LDs reveals a good match
between the two experiments; indeed, ;70% of differentially
expressed genes were in common between the two wild-type
13A). Similarly, 50% of the differentially regulated genes in the
lst8-1-1 mutant grown in SDs were also detected in the other
mutant line. After transfer to LDs, only 25% of the differentially
expressed genes were conserved between the two mutant lines,
which probably reflects the wide range of phenotypes displayed
by the mutants in these conditions and possibly the different
genetic background of the two mutant lines. Therefore, we
decided to focus our analysis on genes that were found to be
statistically differentially expressed in both lst8-1 mutant lines.
confirm the microarrays data (see Supplemental Figure 14 on-
line). Furthermore, ;20% of differentially expressed genes in
LD-grown lst8-1 mutants were also differentially expressed after
induction of TOR silencing (Figure 13B). Genes that were found
to be downregulated in lst8-1 mutants transferred to LDs mainly
belong to stress response genes (see Supplemental Figure 15
online). Accordingly, we found that the lst8-1-1 mutant showed a
higher sensitivity to osmotic stress, similar to the TOR RNAi line
(Deprost et al., 2007; see Supplemental Figure 6 online). By
contrast, there was no significant effect of moderate salt stress.
Upregulated genes corresponded to genes involved in energy
generating pathways or coding for ribosomal proteins (see Sup-
plemental Figure 16 online).
Mutants affected in the MYO-INOSITOL1 PHOSPHATE SYN-
THASE (MIPS1) gene were also previously described as lacking
galactinol accumulation and being sensitive to LD conditions
(Meng et al., 2009). This prompted us to compare the tran-
scriptome of mips1 and lst8-1 mutants. Interestingly, out of 317
genes differentially expressed in the lst8-1-2 mutant grown in
LDs, 217 (68%) were also found to be either down- or upregu-
lated in the mips1 mutant in LDs when compared with wild-type
plants (Figure 13B). This indicates that a large proportion of the
impact of lst8-1 mutations on the transcriptome of LD-grown
plants can be explained by a default in MIPS1 activity. This gene
has been identified as being closely correlated with biomass
accumulation and carbon perturbations (Sulpice et al., 2009).
Next, we investigated the genes that are either up- or down-
regulated in both lst8-1 mutants after transfer to LDs. To better
explain the increased penetrance of the lst8-1 mutation in LD
conditions, we focused our analysis on differentially expressed
genes that show different trends in SDs or LDs (Figure 14).
Among genesthat were downregulated in the mutants, wefound
several genes involved in cell wall formation, such as expansin
genes and CELLULOSE SYNTHASE-LIKE G3. The gene encod-
ing phospholipase D, an enzyme involved in the synthesis of
phosphatidic acid, was also downregulated in lst8-1 mutants
phospholipase Da3 resulted in increased TOR activity in Arabi-
dopsis (Hong et al., 2008). As expected, MIPS1 and MIPS2 were
specifically repressed in the mutants grown in LDs, whereas
GALACTINOL SYNTHASE1, 2, and 3 were downregulated in
lst8-1 mutants in SDs and LDs. These results are in agreement
with the observed lack of galactinol and raffinose accumulation
in mutant leaves. The downregulation of the PIF4, a phyto-
regulations, and PIN4 proteins is also noteworthy (Figure 14).
Interestingly, these genes were also repressed in the mips1
mutant and in inducible TOR RNAi lines but were found to be
upregulated when wild-type plants were transferred to LDs,
which suggests that they may be involved in the adaptation to
these conditions (Figure 14).
A substantial number of genes that were upregulated in the
mutants compared with the wild type after transfer to LDs were
involved in either nitrate or sulfur assimilation (Figure 14). These
genes include adenosine 59-phosphosulfate kinase and reduc-
tase 2, NR, NiR, the nitrate-specific transcription factor LOB39,
ASPARAGINE SYNTHETASE2, and uroporphyrin methylases
(UPM1), which are involved in biosynthesis of siroheme, a
cofactor found in nitrite and sulfite reductases. The P5CS2
gene involved in Pro biosynthesis was also upregulated. In
addition, the expression of PEPC2 and IDH, which are involved
in thesynthesis oforganic acidsand ofcarbon skeletons needed
for the production of organic nitrogen, was also clearly induced
(Figure 14). This transcriptome analysis is in line with the ob-
served accumulation of amino acids, especially Gln and Pro,
after transfer to LDs and with the higher levels of NR and NiR
activities that were observed in lst8-1 mutants. Most of these
genes were foundto be repressedin wild-type plants transferred
Figure 11. Kinetic Analysis of Phloem Labeling Using CF Diacetate.
CF was applied on cotyledons of plants grown in vitro under LDs.
Fluorescence was recorded every 3 s. Magnification shows the labeling
of conductive tissues inside the root. WT, wild type.
472 The Plant Cell
to LDs, which is in agreement with the observed decrease in
nitrate assimilation and N metabolism. We also observed a
specific increase in the mRNA level of the BTB-TAZ2 (for
ADAPTOR ZINC FINGER2) and BTB-TAZ4 genes, which were
previously shown to be induced by nitrate but repressed by
sugars (Mandadi et al., 2009). The inhibition of the TOR kinase
results in the induction of autophagy (Wullschleger et al.,
2006). Interestingly, the expression of a known marker for the
formation of autophagic vesicles (ATG8) is induced in the lst8-
1 mutant, in the TOR RNAi lines, and in the mips1 mutant.
It is known that LDs induce flowering in Arabidopsis (Bernier
and Pe ´rilleux, 2005). Indeed, expression of the flowering inducer
gene Flowering Locus T (FT) is augmented after exposure of
Arabidopsis plants to LD growth conditions. As expected, FT
expression increased fourfold in wild-type plants after transfer
from SD to LD conditions but much less in lst8-1 mutants (log2
ratio of 0.3 and 0.1 for, respectively, lst8-1-1 and lst8-1-2
mutants as determined by quantitative RT-PCR). Accordingly,
Flowering Locus C, which represses flowering by regulating FT
(Greenup et al., 2009), shows the opposite trend. These results
could contribute to the delayed onset of flowering in LD-grown
In this article, we show that Arabidopsis LST8 plays an important
role in growth and organ development as well as metabolic
regulation and flowering in response to LDs. This protein is
known to be a component of the two TOR complexes in animals
and yeast (Wullschleger et al., 2006). In C. reinhardtii, it was also
demonstrated that LST8 interacts with the TOR kinase domain
(Dı ´az-Troya et al., 2008). In Arabidopsis, there are two genes
Figure 12. Leaf Metabolite Contents after Transfer to LD Conditions of Wild-Type and lst8-1-2 Mutant Plants.
Values are derived from normalized areas of specific peaks after GC-MS experiments (see Methods for details). Plants were grown in controlled growth
chambers. Values are the means of three independent repetitions 6 SD. Darker bars correspond to the mutant plants. LDn, number of days under LD
conditions (16 h light); SD, 8 h light.
Roles of the Arabidopsis LST8 Homolog 473
potentially coding for homologs of the LST8 proteins, and only
one of them (LST8-1, At3g18140) seems to be expressed at
significant levels.Interrogation ofpublicEST,transcriptome,and
protein databases gave no indication of expression of LST8-2
(At2g22040). In addition, the LST8-2 sequence seems to have
diverged from other plant LST8 sequences (Figure 1). It could
thus be the result of a recent duplication that is no longer
expressed and is therefore relieved from evolutionary pressure.
Moreover, we were not able to amplify a cDNA corresponding to
LST8-2 by RT-PCR in all tested organs and conditions (Figure 6).
Therefore, the vast majority of LST8 activity in Arabidopsis is
likely to derive from the expression of the sole LST8-1 gene.
seven conserved WD 40 repeats that can form the typical
200 proteins in Arabidopsis contain WD 40 repeats, which are
among these are several important signaling components like
regulator FY (van Nocker and Ludwig, 2003).
The localization of a LST8-GFP fusion protein indicates that it
is associated with endosomes and mobile vesicles. In yeast, the
LST8 protein, together with the TOR kinase, was also found to
copurify with endosomes and Golgi particles (Chen and Kaiser,
2003). Similarly, in C. reinhardtii, the LST8 protein was associ-
ated with microsomes (Dı ´az-Troya et al., 2008). Therefore, it
seems that the association of LST8 with intracellular vesicles is a
common trend in eukaryotes and could be linked to the regula-
tion of trafficking by the TOR kinase (Shaw, 2008).
The analysis of transformed Arabidopsis lines harboring a
translational fusion of the LST8-1 promoter and 59-UTR to the
GUS reporter gene shows that LST8-1 is mainly expressed in
root meristems and vascular tissues as well as in developing
organs like emerging root or leaf primordia. There was also a
strong GUS activity in flowers. This is similar to the GUS staining
pattern resulting from the expression of a TOR-GUS fusion in
have some overlapping domains of expression. Interestingly,
strong GUS staining was also detected in stomatal guard cells,
which are known to be central regulators of gas exchange
processes and were submitted to many metabolic and environ-
mental regulations (Casson and Gray, 2008). Like the C. rein-
hardtii LST8 gene (Dı ´az-Troya et al., 2008), the Arabidopsis
LST8-1 coding sequence can fully replace the lethal depletion of
is functionally conserved between Arabidopsis and yeast. More-
over, we have shown that, like in animal cells (Kim et al., 2002),
the LST8-1 protein interacts with the Arabidopsis TOR FRB and
kinase domains both in yeast and in planta (Figure 5). Unlike
yeast (Loewith et al., 2002) and mouse (Guertin et al., 2006),
mutations in the LST8-1 gene are not lethal in Arabidopsis. A
similar result has been observed in fission yeast (Kemp et al.,
1997). Nevertheless, the growth and development of lst8-1 mu-
tants was severely affected with a delay in plant growth, sterility
of most flowers, and high sensitivity to LDs. Interestingly, defi-
ciency in the RAPTOR protein, the other component of the
TORC1 complex, is also lethal in yeast and mice (Guertin et al.,
2006), whereas viable raptor mutants can be obtained in Arabi-
dopsis (Anderson et al., 2005; Moreau et al., 2010). The lethality
of lst8 mutations in other eukaryotes could be due to TOR-
effect of partial silencing of the expression of Arabidopsis TOR
(Deprost et al., 2007). The lst8-1 mutants have altered flower
development, and a recent report linked the overexpression of
the RIBOSOMAL S6 KINASE gene, a major readout of TOR
kinase activity, to abnormal flower development (Tzeng et al.,
2009). The bushy phenotype and the development of multiple
meristems in lst8-1 mutants could indicate a role for LST8 in the
regulation of apical dominance and meristem cell proliferation. It
is noteworthy that a knockdown of the ribosomal protein gene
RPL23aA in tobacco (Nicotiana tabacum) produced a pleiotropic
Figure 13. Differentially Expressed Genes in the Transcriptomic Analy-
sis of lst8-1 Mutants Using CATMA Arrays.
For each condition, gene expression in the mutant samples was com-
pared with that in wild-type samples grown under the same light regimes
as references. 1, lst8-1-2 to wild type in SD; 2, lst8-1-2 to wild type in LD
for 2 d; 3, wild type in LD to wild type in SD (reference); 4, lst8-1-2 in LD to
lst8-1-2 in SD; 5 to 8, same as 1 to 4, except with the lst8-1-1 mutant.
(A) Differentially expressed genes were ordered from the lowest to the
highest ratio with the wild type LDs (LD after 2 d) to SDs comparison as
reference (see Methods for the definition of differentially expressed
(B) Differentially expressed genes in the comparison between lst8-1-2
and wild-type grown under LD conditions, which were also differentially
expressed in a mips1 mutant compared with wild-type plants grown in
LD and in TOR ethanol-inducible RNAi lines induced by ethanol for 24 h
(see Methods for details). Only genes that are found in common between
at least two comparisons were retained for this analysis. Data were
obtained from the CatDB database and from Meng et al. (2009).
474 The Plant Cell
Figure 14. Differentially Expressed Genes in lst8-1 Mutants.
Transcriptome comparisons were performed between leaves of lst8-1 mutants and wild-type plants grown either in SDs (8 h) or transferred to LDs (16 h)
between wild-type plants grown in SDs and transferred to LDs and between mips1 mutants and the corresponding wild type in LDs, and between the
TOR ethanol-inducible RNAi lines and the corresponding control line (mean of 6-3 and 5-2 RNAi lines induced for 24 h with ethanol). Genes showing
opposite variations when compared with the wild type in SDs or LDs, or of special interest, were selected among differentially expressed transcripts in
the lst8-1 mutants. The results are the mean of the intensity ratios for lst8-1-1 and lst8-1-2 mutants and are presented as log2 ratios. Experiments were
run in duplicate. A color code was used to visualize the data.
Roles of the Arabidopsis LST8 Homolog475
phenotype characterized by altered and retarded growth, ab-
normal phyllotaxy, and loss of apical dominance (Degenhardt
and Bonham-Smith, 2008). Indeed, the TORC1 complex has a
central regulatory role in ribosome biogenesis and mRNA trans-
lation (Wullschleger et al., 2006).
The LD sensitivity and the delay in flowering in lst8-1 mutants
of the light period. One well-documented signal transduction
pathway responding to light level is the so-called RTG signaling
from chloroplast to nucleus (Leister, 2005; Queval et al., 2007;
Ferna ´ndez and Strand, 2008). Arabidopsis plants normally re-
spond to extension of the light period by adjusting the metabolic
and energetic status of the cell and also by inducing flowering
(Corbesier et al., 2002; Bernier and Pe ´rilleux, 2005; Greenup
et al., 2009). In yeast, LST8 has been implicated in vesicular
trafficking and in RTG responses linking mitochondrial dysfunc-
tion to nuclear gene transcription (Liu et al., 2001; Chen and
Kaiser, 2003). In response to upstream signals, LST8 negatively
regulates the transcription factors Rtg1/3 responsible for the
activation of amino acid synthesis genes in response to environ-
mental conditions. Indeed, yeast lst8 mutants show a strong
accumulation of amino acids due to an abolished repression of
RTG1/3 transcription factors by Glu (Liu et al., 2001). It was
and that this signal is conveyed to RTG1/3 through the action of
TOR and LST8. Interestingly, we observed the same accumula-
tion of amino acids, mainly Gln, in LD-grown lst8-1 mutant plants
but also in Arabidopsis plants silenced for TOR expression
(Figure 12; see Supplemental Figure 9 online). This accumulation
of amino acids could be the result of a stimulation of nitrate
assimilation and amino acid synthesis. Indeed, it is known that
overexpression of NR causes ammonium and Gln accumulation
like that observed in lst8-1 mutants (Lea et al., 2006). Accord-
ingly, transcriptome analysisof lst8-1 mutants transferred to LDs
assimilation pathway, including NR and NiR as well as Asn
synthetase (Meyer and Stitt, 2001; Lillo, 2008; Figure 14). In
Arabidopsis, silencing of the genes encoding either TOR or
TAP46, a target of TOR interacting with PP2A phosphatase,
resulted in a decreased NR activity (Deprost et al., 2007; Ahn
et al., 2011), whereas there was an accumulation of Gln. It thus
seems that inactivation of LST8-1 has the opposite effect on NR
to LD conditions causes in Arabidopsis a substantial decrease in
nitrate concentration and in the expression of genes involved in
its assimilation (Corbesier et al., 1998, 2002). Thus, it appears
that lst8-1 mutants do not sense properly the shift to LD condi-
tions and do not adjust their nitrogen metabolism accordingly.
Furthermore, the induction of the flowering locus FT did not
occur in lst8-1 mutants when transferred to LDs. FT is regulated
bytheexpression ofthe CONSTANS geneand representsone of
the most potent activators of flowering, integrating several
signaling pathways (Searle and Coupland, 2004). Taken to-
plants show a delay in the onset of flowering and appear to be
impaired in the perception of the extension of light period.
Arabidopsis LST8 also seems to be implicated in flower devel-
opment. Indeed, lst8-1 mutants displayed abnormal and often
sterile flowers, with a lack of petal and anther extension and
altered flower insertion on the stems (see Supplemental Figure 5
online). Moreover, we showed that LST8-1 is expressed in petal
and sepal conducting tissues, in pollen, and in stamen filaments.
It was previously described that a decrease in leaf-to-shoot
transition to flowering in Arabidopsis (Gisel et al., 1999, 2002).
The higher phloemloading and/or flux detected in lst8-1 mutants
of signaling molecules.
When wild-type plants were transferred to LD conditions, we
noticed an accumulation of Suc and of osmoprotectants like
raffinose and galactinol, which did not occur at all in lst8-1-1 and
lst8-1-2 mutants (Figure 12). Raffinose and galactinol have been
shown to be involved in resistance to various stresses, including
high light conditions, and to protect plants by detoxifying reac-
tive oxygen species (Taji et al., 2002; Nishizawa et al., 2008;
Usadel et al., 2008). When shifted to LDs, wild-type plants
displayed a transient increase in Pro concentration, followed by
raffinose and galactinol accumulation. It is possible that these
sugars replace Pro as osmoprotectants during adaptation to
LDs. By contrast, lst8-1 mutants fail to synthesize galactinol and
raffinose while showing a continuous increase in Pro. Interest-
ingly, TOR-silenced lines also showed a decrease in the accu-
mulation of galactinol and raffinose when germinated in the dark
(see Supplemental Figure 12 online). Therefore, the sensitivity of
lst8-1 mutants to LDs may be partly due to the absence of these
molecules. The low concentration of myo-inositol found in lst8-
1 mutants can explain the lack of galactinol and raffinose
production because myo-inositol is needed for the synthesis of
these compounds from UDP-Gal (Nishizawa et al., 2008). Inter-
estingly, Arabidopsis mutants affected in the MIPS1 gene had
lower levels of myo-inositol and galactinol and showed lesion
formation in LD conditions, like lst8-1 mutants (Meng et al.,
2009). Furthermore, we found a remarkable overlap between
the transcriptome of lst8-1 and mips1 mutants (Figure 13),
which suggests that a significant part of the consequences of
mutations intheLST8-1genecan beexplained byalackofmyo-
inositol synthesis. This is in agreement with the previous iden-
tification of MIPS1 as a candidate gene highly correlated with
carbon perturbations and growth (Sulpice et al., 2009). Apart
from the production of galactinol and raffinose, the lack of myo-
inositol also affects signal transduction via a decrease in phos-
phorylated forms of inositol like inositol 1,4,5-trisphosphate (IP3)
2005). lst8-1 mutants share several aspects of their phenotype,
like accumulation of amino acid or sensitivity to osmotic stress,
with TOR RNAi lines. Moreover, the absence of TOR or LST8
activity results in the differential expression of a common set of
genes (Figure 13B). Collectively, these results suggest that the
LST8-1 protein, probably by interacting with the TOR kinase
external cues and the regulation of growth and carbon metab-
olism by affecting the expression of MIPS1.
In conclusion, we have shown that Arabidopsis LST8 is im-
portant for growth and developmental processes linked to
changes in light conditions probably by influencing the activity
of the TOR complex. LST8-1 is needed for the adaptation of
476The Plant Cell
primary metabolism to changes in daylength by inducing the
synthesis of myo-inositol and of the osmoprotectants galactinol
and raffinose and by restraining nitrate assimilation and amino
acid accumulation that would exhaust cellular energy stores.
This is similar to the role of yeast LST8, the function of which is to
lower amino acid production under stress conditions by modu-
lating the TOR kinase activity. Our goal is now to identify the
targets and protein partners of LST8 in plants.
The lst8-1-1 (SALK_02459) and lst8-1-2 (SAIL_641D10) mutants were
obtained from the Nottingham Arabidopsis Stock Centre in a T-DNA–
mutagenized population of the Col-8 Arabidopsis thaliana ecotype and
population in the Col-0 Arabidopsis ecotype, respectively. Homozygous
mutant plants were identified by PCR using the primers listed in Supple-
mental Table 1 online. A DNA fragment comprising 1 kb of LST8-1
promoter and the 59-UTR (1 kb upstream of the gene initiation codon)
were amplified with forward and reverse primers containing EcoRI and
NotI restriction sites, respectively (primers are listed in Supplemental
Table 1 online). The resulting PCR product was cloned into the pENTR4
(Invitrogen) vector after digestion with the same restriction enzymes. The
pLst8:GUS construct was then obtained after cloning into Gateway
technology–compatible (Invitrogen) pGWB3 binary plasmid. Transgenic
plants were obtained by floral dipping (Clough and Bent, 1998) of Col-0
Arabidopsis plants. To complement the lst8-1 mutant, a genomic DNA
fragment containing the LST8-1 gene encompassing 1 kb of promoter
sequence was amplified by PCR with forward and reverse primers
containing a BamHI and a NotI restriction site, respectively (primers are
listed in Supplemental Table 1 online) and checked by sequencing. The
resulting PCR product was cloned into the pENTR4 vector after digestion
with the same restriction enzymes. The pLst8:LST8 construct was then
obtained after cloning into Gateway technology–compatible (Invitrogen)
of lst8-1-1 and lst8-1-2 heterozygous mutant plants.Theethanol-inducible
TOR RNAi lines 5-2 and 6-3, the control alcA:GUS line, as well as the
constitutive TOR RNAi line 35-7 were previously described by Deprost
et al. (2007). Plants used for either global metabolite profiling or tran-
scriptome analysis were harvested at the beginning of the light period.
Plant Growth Conditions
Seeds were sown in vitro on half-strength Murashige and Skoog medium
containing1% Sucand transferredtosoil7 d aftergermination.SD and LD
conditions were 8 h light/16 h night and 16 h light/8 h night, respectively, in
controlled growth chambers (70% relative humidity) with fluorescent tubes
and a light intensity of respectively 150 mmol·m22·s21and 130
mmol·m22·s21. The greenhouse was used for LDs with natural light,
supplemented, according to the season, with artificial light bulbs. For
in vitro studies, surface-sterilized seeds were sown on half-strength
Murashige and Skoog medium containing 1% Suc. Ethanol-inducible
TOR RNAi lines were induced either by direct sowing on half-strength
Murashige and Skoog medium containing 0.3% Suc and 50 mM ethanol
or by adding an Eppendorf cap containing 50% ethanol for 1 or 2 d on the
solid medium. Other details were as described by Deprost et al. (2007).
The LST8-GFP fusion protein was obtained and expressed transiently in
Arabidopsis cotyledons as described earlier (Marion et al., 2008). Con-
focal microscopy was performed on a Zeiss Lsm710 spectral laser
scanning microscope. GFP and mRFP1 fluorescence were detected with
argon laser line at 488 nm and a HeNe laser at 594 nm, respectively.
Transgenic plants carrying a pLST8-1:GUS construct were grown on
horizontal plates for 10 dat 258C underLD conditions (16 hlight/8 hdark).
Some of the plants were transferred to soil in growth chamber under LD
X-Gluc was performed as described (Menand et al., 2002) with 2 mM
ferricyanure (potassium hexacyanoferrate III) and 2 mM ferrocyanure
(potassium hexacyanoferrate II).
The LST8-1coding sequence was amplified byRT-PCR with forward and
reverse primers containing SalI and NotI restriction sites, respectively
(primers are listed in Supplemental Table 1 online) and checked by
sequencing. The resulting PCR product was cloned into the p424GPD
yeast expressionvector under control of the constitutive glyceraldehyde-
mutant yeast strain was then transformed with the empty p424GPD
vector or with the p424GPD-LST8-1 construct. Transformants were
plated on SD Glc-Trp medium or SD Gal-Trp medium and scored for
growth. Plates were incubated at 308C for 3 d.
Two-Hybrid Experiments in Yeast and Split-Luciferase
Assays in Arabidopsis Cotyledons
The Lst8-1 full-length cDNA was amplified by PCR as described above.
The resulting cDNA was digested with these enzymes and cloned by
ligation in a SalI- and NotI-digested pEXPAD-502 plasmid (Invitrogen)
containing a GAL4 activation domain. A 2.5-kb DNA fragment containing
the TOR FRB and kinase domains was amplified by PCR as described
previously (Deprost et al., 2007) and introduced in the pENTR4 plasmid
after digestion with EcoRI and NotI. This partial cDNA was then inserted
by recombination in the two-hybrid pDEST32 vector (Invitrogen) that
contains the GAL4 DNA binding domain. Two-hybrid experiments were
performed in yeast using the AH109 strain, and interaction between
LST8-1 and TOR proteinswas tested on a complete medium lacking Leu,
Trp, and His with increasing concentrations of 3-amino triazole as
described by the supplier (Matchmaker; Clontech).
Split-luciferase experiments were performed with the same LST8-1
and TOR proteins after cloning of the corresponding DNA fragmentsin
Gateway vectors carrying either the N- or C-terminal parts of firefly
luciferase as previously described (Van Leene et al., 2010). This way,
we obtained four different vector combinations. Transient transfor-
mation of Landsberg erecta Arabidopsis seedlings and detection of
luciferase activitywere performed as previouslydescribed (Van Leene
etal., 2010)except thatluciferaseemissionwasnormalizedaccording
to the number of infiltrated plants. Levels of light emissions were
measured with an ultra-amplified charge-coupled device camera
(Photonic Science) and obtained after integrating 2000 images (Pho-
tolite 32 software).
Paraffin or Resin Embedding and Chloro-Naphtol Staining
8 mm for paraffin-embedded samples and at 4 mm for resin-embedded
Roles of the Arabidopsis LST8 Homolog477
Rosettes of three plants of each genotype were harvested for each time
point. NiR, NR, and Gln synthetase activities were measured on leaf
tissue as previously described by Lea et al. (2006) and Deprost et al.
To image phloem transport, the adaxial surface of cotyledons was gently
applied to the cotyledon surface following the protocol of Roberts et al.
labeling using a Nikon SMZ 1500 binocular equipped with a fluorescence
excitation and detection module.
Total RNAs were prepared from shoots and roots using the Trizol reagent
(Invitrogen) following the manufacturer’s protocol.
Total RNA (1 mg) was used as a template to perform RT reactions using
Moloney murine leukemia virus reverse transcriptase (Invitrogen) accord-
ing to the manufacturer’s instructions. Quantitative RT-PCR reactions
were achieved using 23 Mesa Fast qPCR MasterMix Plus for SYBR
assay (Eurogentec) following the manufacturer’s protocols. The expres-
sion of the EF1a gene (At5g60390) was used as a constitutive reference
with primers described previously (Deprost et al., 2007).
Microarray analysis was performed at the Unite ´ de Recherche en
Ge ´nomique Ve ´ge ´tale (Evry, France) using the CATMA arrays containing
24,576 GSTs corresponding to 22,089 genes from Arabidopsis (Crowe
et al., 2003; Hilson et al., 2004). Two independent biological replicates
were produced. For each biological repetition and each point, RNA
collected from plants at the rosette 3.1 developmental growth stage
(Boyes et al., 2001) and cultivated in growth chamber conditions. Total
RNA was extracted using leaves according to the supplier’s instruc-
tions. For each comparison, one technical replicate with fluorochrome
reversal was performed for each biological replicate (i.e., four hybrid-
izationspercomparison).The labeling of cRNAswith Cy3-dUTPorCy5-
dUTP (Perkin-Elmer-NEN Life Science Products), the hybridization to
the slides, and the scanning were performed as described by Lurin et al.
Statistical Analysis of Microarray Data
four arrays, each containing 24,576 GSTs and 384 controls) and followed
the analysis described by Gagnot et al. (2008). For each array, the raw
data comprised the logarithm of median feature pixel intensity at wave-
lengths 635 nm (red) and 532 nm (green), and no background was
subtracted. An array-by-array normalization was performed to remove
systematic biases. First, spots considered as badly formed features were
excluded. Then, a global intensity-dependent normalization using the
loess procedure was performed to correct the dye bias. Finally, for each
block, the log ratio median calculated over the values for the entire block
was subtracted from each individual log ratio value to correct print tip
effects. To determine differentially expressed genes, we performed a
paired t test on the log ratios averaged on the dye swap. A trimmed
variance was then calculated from spots that did not display extreme
variance. The spots that were excluded were those with a specific
variance/common variance ratio smaller than the a-quantile of a x2
adjusted by the Bonferroni method, which controls the family-wise
error rate to keep a strong control of the false positives in a multiple-
comparison context. We considered asbeing differentially expressed the
probes with a Bonferroni P value # 0.05, as described by Gagnot et al.
(2008). The P values corresponding to the differentially expressed genes
Metabolite Profiling and Analysis
Rosettes of three plants (seven- to eight-leaf stage) of each genotype in
each growth condition were harvested, and 20 mg of powder of each
sample was used for extraction. Nitrate concentrations were measured
as described previously by Lea et al. (2006). Extraction, derivatization,
analysis, and data processing were performed according to Fiehn
(2006). Metabolites were analyzed by GC-MS 3 h and 20 min after
derivatization. One microliter of the derivatized samples was injected in
splitless mode on an Agilent 7890A gas chromatograph coupled to an
Restek (30 m with 10 m Integraguard column). The liner (Restek 20994)
was changed before each series of analysis and 10 cm of column was
cut. Oven temperature ramp was 708C for 7 min then 108C/min to 3258C
for 4 min (run length 36.5 min). Helium constant flow was 1.5231 mL/
source: 2508C; and quadripole, 1508C. Samples and blanks were
randomized. Amino acid standards were injected at the beginning
and end of the analysis for monitoring of the derivatization stability. An
alkane mix (C10, C12, C15, C19, C22, C28, C32, and C36) was injected
in the middle of the queue for external calibration. Five scans per
second were acquired.
Raw Agilent data files were converted in NetCDF format and analyzed
with AMDIS (http://chemdata.nist.gov/mass-spc/amdis/). An in-house
retention indices/mass spectra library built from the National Institute of
Standards and Technology, Golm, and Fiehn databases and standard
determined using the Quanlynx software (Waters) after conversion of the
NetCDF file in Masslynx format. Statistical analysis was made with TMEV
(http://www.tm4.org/mev.html): Univariate analysis by permutation (one-
way and two-way analysis of variance) was first used to select the
significant metabolites. Multivariate analysis (hierarchical clustering and
principal component analysis) was then performed on them. MapMan
(http://www.gabipd.org/projects/MapMan/) was used for graphical rep-
resentation of the metabolic changes after log2 transformation of the
mean of the three replicates.
Gene expression data were analyzed using the Genevestigator (www.
genevestigator.com), CatDB (Gagnot et al., 2008; urgv.evry.inra.fr/
CATdb), or BAR (bar.utoronto.ca) website. Metabolomic data were
analyzed using MapMan (Usadel et al., 2005; mapman.mpimp-golm.
Multiple alignment of protein sequences was performed using the
COBALT tool (Papadopoulos and Agarwala, 2007) with default settings.
This method uses progressive multiple alignment to combine pairwise
based on sequence identities and not on evolution rates. The resulting
alignment is available as Supplemental Data Set 1 online.
478 The Plant Cell
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or Genbank/EMBL databases under the following acces-
sion numbers: LST8-1 (At3g18140), LST8-2 (At2g22040), lst8-1-1
(SALK_02459), and lst8-1-2 (SAIL_641D10) mutants. The following pro-
tein sequences were used for multiple alignments: XP_003542977 and
XP_002880414 (Arabidopsis lyrata); 012143m (Manihot esculenta);
XP_002523640 (Ricinus communis); Gm15g09170 and Gm13g29940
sativa]); XP_003565062 (Brachypodium distachyon); PP00138G00630
(Physcomitrella patens); VC00031G01430 (Volvox carteri); CR17G03790
(Chlamydomonas reinhardtii); NP_001186102 (Homo sapiens); and P41318
(Saccharomyces cerevisiae).Microarraynormalizeddata are availableinthe
Gene Expression Omnibus database under accession numbers GSE25731
and GSE25721 for the analysis of lst8-1 mutants and TOR RNAi lines,
The following materials are available in the online version of this article.
Supplemental Figure 1. Alignment of Plants, Yeast, and Human
LST8 Protein Sequences.
Supplemental Figure 2. Expression of the Arabidopsis LST8 Genes
in Various Organs.
Supplemental Figure 3. GUS Staining of Transformed Arabidopsis
Plants Carrying a pLst8:GUS Construct.
Supplemental Figure 4. Position of the T-DNA Insertions in the lst8-
Supplemental Figure 5. Mutations in LST8-1 Gene Affect Stem and
Flower Bud Morphology and Organization.
Supplemental Figure 6. Germinating Plantlets from the lst8-1 Mutant
Line Display the Same Sensitivity to High Sugar Concentrations as a
TOR RNAi Line.
Supplemental Figure 7. Diurnal Variations in Sugar and Starch
Content during the Transition from Short Days to Long Days in the
Wild Type and in the lst8-1-1 Mutant.
Supplemental Figure 8. MapMan Representations of Metabolite
Variations in lst8-1-2 Mutants Using Global GC-MS Analysis.
Supplemental Figure 9. Variations in Metabolite Levels in lst8-1-2
Mutants and Wild-Type Plants Exposed to Long Days.
Supplemental Figure 10. MapMan Representations of Global Me-
tabolite Analysis Using GC-MS in lst8-1 Mutants.
Supplemental Figure 11. MapMan Representations of Global Me-
tabolite Analysis Using GC-MS in Wild-Type Plants.
Supplemental Figure 12. Changes in Metabolite Accumulations
Determined by GC-MS in the lst8-1-2 Mutant and in TOR-Inducible
Supplemental Figure 13. Frequency of Functional Classes in the
Genes Downregulated in lst8-1 Mutants Grown in Short-Day Condi-
tions and Compared with Wild-Type Plants Grown in Short Days.
Supplemental Figure 14. Changes in Transcript Abundance Deter-
mined by Either Quantitative Real-Time RT-PCR or Microarray Hy-
Supplemental Figure 15. Frequency of Functional Classes in the
Genes Downregulated in lst8-1 Mutants after 2 d under Long-Day
Supplemental Figure 16. Frequency of Functional Classes in the
Genes Upregulated in lst8-1 Mutants.
Supplemental Table 1. Primer Sequences.
Supplemental Data Set 1. Text File of the Alignment Corresponding
to the Tree in Figure 1.
We thank Halima Morin from the Institut Jean-Pierre Bourgin Plant
Imaging Platform (Unite ´ Mixte de Recherche 1318, Institut National de la
Recherche Agronomique, AgroParisTech, Versailles, France) for help
producing and imaging meristem sections, Lionel Gissot for help with
confocal microscopy and for providing the RabC1-RFP construct, Sylvie
Dinant for help with phloem labeling, Magali Bedu for performing
enzyme activity measurements, Jessica Marion for help with transient
transformation of Arabidopsis, and Michael Hall (Biozentrum, University
of Basel, Switzerland) for providing the yeast lst8 mutants. We thank
Joe ¨l Talbotec, Franc ¸ois Gosse, and Philippe Mare ´chal for taking care of
the plants and Jean-Christophe Palauqui, Patrick Laufs, Jean-Denis
Faure, and Hoai-Nam Truong for helpful discussions. This work was
partly supported by an Agence Nationale de la Recherche grant (ANR
Blanc06-3-135436) to J.-P.R., C.M., and C.R. and by a European Union
6th framework project (Agronomics) to C.M. M.M. was supported by a
joint PhD grant from Institut National de la Recherche Agronomique
(Plant Biology Department) and Direction des Sciences du Vivant-
Commissariat a ` l’Energie Atomique.
M.M. designed and performed research, analyzed data, and cowrote the
article. M.A., T.D., C. Renne, L.T., and C. Marchive designed and
performed research.G.C.designedand performedresearch,contributed
new analytic/computational tools, and analyzed data. J.-P.R. and M.-L.
M.-M. analyzed data and contributed new analytic/computational tools.
C. Robaglia and C. Meyer designed research, analyzed data, and
cowrote the article.
15, 2012; published February 3, 2012.
Adami, A., Garcı ´a-Alvarez, B., Arias-Palomo, E., Barford, D., and
Llorca, O. (2007). Structure of TOR and its complex with KOG1. Mol.
Cell 27: 509–516.
Ahn, C.S., Han, J.A., Lee, H.S., Lee, S., and Pai, H.S. (2011). The PP2A
regulatory subunit Tap46, a component of the TOR signaling pathway,
modulates growth and metabolism in plants. Plant Cell 23: 185–209.
Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of
Arabidopsis thaliana. Science 301: 653–657.
Anderson, G.H., Veit, B., and Hanson, M.R. (2005). The Arabidopsis
AtRaptor genes are essential for post-embryonic plant growth. BMC
Biol. 3: 12.
Bernier, G., and Pe ´rilleux, C. (2005). A physiological overview of the
genetics of flowering time control. Plant Biotechnol. J. 3: 3–16.
Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman,
N.E., Davis, K.R., and Go ¨rlach, J. (2001). Growth stage-based
phenotypic analysis of Arabidopsis: A model for high throughput func-
tional genomics in plants. Plant Cell 13: 1499–1510.
Roles of the Arabidopsis LST8 Homolog 479
Casson, S., and Gray, J.E. (2008). Influence of environmental factors on
stomatal development. New Phytol. 178: 9–23.
Chen, E.J., and Kaiser, C.A. (2003). LST8 negatively regulates amino
acid biosynthesis as a component of the TOR pathway. J. Cell Biol.
Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
J. 16: 735–743.
Corbesier, L., Bernier, G., and Pe ´rilleux, C. (2002). C:N ratio increases
in the phloem sap during floral transition of the long-day plants
Sinapis alba and Arabidopsis thaliana. Plant Cell Physiol. 43: 684–688.
Corbesier, L., Lejeune, P., and Bernier, G. (1998). The role of carbo-
hydrates in the induction of flowering in Arabidopsis thaliana: Com-
parison between the wild type and a starchless mutant. Planta 206:
Crowe, M.L., et al. (2003). CATMA: A complete Arabidopsis GST
database. Nucleic Acids Res. 31: 156–158.
Degenhardt, R.F., and Bonham-Smith, P.C. (2008). Arabidopsis ribo-
somal proteins RPL23aA and RPL23aB are differentially targeted to
the nucleolus and are disparately required for normal development.
Plant Physiol. 147: 128–142.
Deprost, D., Truong, H.N., Robaglia, C., and Meyer, C. (2005). An
Arabidopsis homolog of RAPTOR/KOG1 is essential for early embryo
development. Biochem. Biophys. Res. Commun. 326: 844–850.
Deprost, D., Yao, L., Sormani, R., Moreau, M., Leterreux, G., Nicolaı ¨,
M., Bedu, M., Robaglia, C., and Meyer, C. (2007). The Arabidopsis
TOR kinase links plant growth, yield, stress resistance and mRNA
translation. EMBO Rep. 8: 864–870.
Duan, H.Y., Li, F.G., Wu, X.D., Ma, D.M., Wang, M., and Hou, Y.X.
(2006). The cloning and sequencing of a cDNA encoding a WD repeat
protein in cotton (Gossypium hirsutum L.). DNA Seq. 17: 49–55.
Dı ´az-Troya, S., Florencio, F.J., and Crespo, J.L. (2008). Target of
rapamycin and LST8 proteins associate with membranes from the
endoplasmic reticulum in the unicellular green alga Chlamydomonas
reinhardtii. Eukaryot. Cell 7: 212–222.
Ferna ´ndez, A.P., and Strand, A. (2008). Retrograde signaling and plant
stress: Plastid signals initiate cellular stress responses. Curr. Opin.
Plant Biol. 11: 509–513.
Fiehn, O. (2006). Metabolite profiling in Arabidopsis. Methods Mol. Biol.
Gagnot, S., Tamby, J.P., Martin-Magniette, M.L., Bitton, F., Taconnat,
L., Balzergue, S., Aubourg, S., Renou, J.P., Lecharny, A., and
Brunaud, V. (2008). CATdb: A public access to Arabidopsis tran-
scriptome data from the URGV-CATMA platform. Nucleic Acids Res. 36
(Database issue): D986–D990.
Giannattasio, S., Liu, Z., Thornton, J., and Butow, R.A. (2005).
Retrograde response to mitochondrial dysfunction is separable from
TOR1/2 regulation of retrograde gene expression. J. Biol. Chem. 280:
Gisel, A., Barella, S., Hempel, F.D., and Zambryski, P.C. (1999).
Temporal and spatial regulation of symplastic trafficking during de-
velopment in Arabidopsis thaliana apices. Development 126: 1879–
Gisel, A., Hempel, F.D., Barella, S., and Zambryski, P. (2002). Leaf-to-
shoot apex movement of symplastic tracer is restricted coincident
with flowering in Arabidopsis. Proc. Natl. Acad. Sci. USA 99: 1713–
Greenup, A., Peacock, W.J., Dennis, E.S., and Trevaskis, B. (2009).
The molecular biology of seasonal flowering-responses in Arabidopsis
and the cereals. Ann. Bot. (Lond.) 103: 1165–1172.
Guertin, D.A., Stevens, D.M., Thoreen, C.C., Burds, A.A., Kalaany,
N.Y., Moffat, J., Brown, M., Fitzgerald, K.J., and Sabatini, D.M.
(2006). Ablation in mice of the mTORC components raptor, rictor, or
mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO
and PKCalpha, but not S6K1. Dev. Cell 11: 859–871.
Hara, K., Maruki, Y., Long, X., Yoshino, K., Oshiro, N., Hidayat, S.,
Tokunaga, C., Avruch, J., and Yonezawa, K. (2002). Raptor, a
binding partner of target of rapamycin (TOR), mediates TOR action.
Cell 110: 177–189.
Hilson, P., et al. (2004). Versatile gene-specific sequence tags for
Arabidopsis functional genomics: transcript profiling and reverse
genetics applications. Genome Res. 14: 2176–2189.
Hong, Y., Pan, X., Welti, R., and Wang, X. (2008). Phospholipase
Dalpha3 is involved in the hyperosmotic response in Arabidopsis.
Plant Cell 20: 803–816.
Kemp, J.T., Balasubramanian, M.K., and Gould, K.L. (1997). A wat1
mutant of fission yeast is defective in cell morphology. Mol. Gen.
Genet. 254: 127–138.
Kim, D.H., Sarbassov, D.D., Ali, S.M., King, J.E., Latek, R.R., Erdjument-
Bromage, H., Tempst, P., and Sabatini, D.M. (2002). mTOR interacts
with raptor to form a nutrient-sensitive complex that signals to the cell
growth machinery. Cell 110: 163–175.
Kim, D.H., Sarbassov, D.D., Ali, S.M., Latek, R.R., Guntur, K.V.,
Erdjument-Bromage, H., Tempst, P., and Sabatini, D.M. (2003).
GbetaL, a positive regulator of the rapamycin-sensitive pathway
required for the nutrient-sensitive interaction between raptor and
mTOR. Mol. Cell 11: 895–904.
Komeili, A., Wedaman, K.P., O’Shea, E.K., and Powers, T. (2000).
Mechanism of metabolic control. Target of rapamycin signaling links
nitrogen quality to the activity of the Rtg1 and Rtg3 transcription
factors. J. Cell Biol. 151: 863–878.
Lea, U.S., Leydecker, M.T., Quillere ´, I., Meyer, C., and Lillo, C. (2006).
Posttranslational regulation of nitrate reductase strongly affects the
levels of free amino acids and nitrate, whereas transcriptional regu-
lation has only minor influence. Plant Physiol. 140: 1085–1094.
Leister, D. (2005). Genomics-based dissection of the cross-talk of
chloroplasts with the nucleus and mitochondria in Arabidopsis. Gene
Lillo, C. (2008). Signalling cascades integrating light-enhanced nitrate
metabolism. Biochem. J. 415: 11–19.
Liu, Z., Sekito, T., Epstein, C.B., and Butow, R.A. (2001). RTG-
dependent mitochondria to nucleus signaling is negatively regulated
by the seven WD-repeat protein Lst8p. EMBO J. 20: 7209–7219.
Loewith, R., Jacinto, E., Wullschleger, S., Lorberg, A., Crespo, J.L.,
Bonenfant, D., Oppliger, W., Jenoe, P., and Hall, M.N. (2002). Two
TOR complexes, only one of which is rapamycin sensitive, have
distinct roles in cell growth control. Mol. Cell 10: 457–468.
Lurin, C., et al. (2004). Genome-wide analysis of Arabidopsis penta-
tricopeptide repeat proteins reveals their essential role in organelle
biogenesis. Plant Cell 16: 2089–2103.
Macquet, A., Ralet, M.C., Loudet, O., Kronenberger, J., Mouille, G.,
Marion-Poll, A., and North, H.M. (2007). A naturally occurring
mutation in an Arabidopsis accession affects a beta-D-galactosidase
that increases the hydrophilic potential of rhamnogalacturonan I in
seed mucilage. Plant Cell 19: 3990–4006.
Mahfouz, M.M., Kim, S., Delauney, A.J., and Verma, D.P. (2006).
Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which
regulates the activity of S6 kinase in response to osmotic stress
signals. Plant Cell 18: 477–490.
Mandadi, K.K., Misra, A., Ren, S., and McKnight, T.D. (2009). BT2, a
BTB protein, mediates multiple responses to nutrients, stresses, and
hormones in Arabidopsis. Plant Physiol. 150: 1930–1939.
Marion, J., Bach, L., Bellec, Y., Meyer, C., Gissot, L., and Faure, J.D.
(2008). Systematic analysis of protein subcellular localization and
interaction using high-throughput transient transformation of Arabi-
dopsis seedlings. Plant J. 56: 169–179.
480 The Plant Cell
Menand, B., Desnos, T., Nussaume, L., Berger, F., Bouchez, D., Download full-text
Meyer, C., and Robaglia, C. (2002). Expression and disruption of the
Arabidopsis TOR (target of rapamycin) gene. Proc. Natl. Acad. Sci.
USA 99: 6422–6427.
Meng, P.H., Raynaud, C., Tcherkez, G., Blanchet, S., Massoud, K.,
Domenichini, S., Henry, Y., Soubigou-Taconnat, L., Lelarge-
Trouverie, C., Saindrenan, P., Renou, J.P., and Bergounioux, C.
(2009). Crosstalks between myo-inositol metabolism, programmed
cell death and basal immunity in Arabidopsis. PLoS ONE 4: e7364.
Meyer, C., and Stitt, M. (2001). Nitrate reduction and signalling. In Plant
Nitrogen, P.J. Lea and J.-F. Morot-Gaudry, eds (Berlin: Springer), pp,
Moreau, M., Sormani, R., Menand, B., Veit, B., Robaglia, C., and
Meyer, C. (2010). The TOR complex and signaling pathway in plants.
In The TOR Complexes, Enzyme Series 27, M. Hall and F. Tamanoi,
eds (Oxford, UK: Academic Press/Elsevier), pp. 285–302.
Mumberg, D., Mu ¨ller, R., and Funk, M. (1995). Yeast vectors for the
controlled expression of heterologous proteins in different genetic
backgrounds. Gene 156: 119–122.
Neer, E.J., Schmidt, C.J., Nambudripad, R., and Smith, T.F. (1994).
The ancient regulatory-protein family of WD-repeat proteins. Nature
Nishizawa, A., Yabuta, Y., and Shigeoka, S. (2008). Galactinol and
raffinose constitute a novel function to protect plants from oxidative
damage. Plant Physiol. 147: 1251–1263.
Ochotorena, I.L., Hirata, D., Kominami, K., Potashkin, J., Sahin, F.,
Wentz-Hunter, K., Gould, K.L., Sato, K., Yoshida, Y., Vardy, L., and
Toda, T. (2001). Conserved Wat1/Pop3 WD-repeat protein of fission
yeast secures genome stability through microtubule integrity and may
be involved in mRNA maturation. J. Cell Sci. 114: 2911–2920.
Papadopoulos, J.S., and Agarwala, R. (2007). COBALT: Constraint-
based alignment tool for multiple protein sequences. Bioinformatics
Queval, G., Issakidis-Bourguet, E., Hoeberichts, F.A., Vandorpe, M.,
Gakie `re, B., Vanacker, H., Miginiac-Maslow, M., Van Breusegem,
F., and Noctor, G. (2007). Conditional oxidative stress responses in
the Arabidopsis photorespiratory mutant cat2 demonstrate that redox
state is a key modulator of daylength-dependent gene expression,
and define photoperiod as a crucial factor in the regulation of H2O2-
induced cell death. Plant J. 52: 640–657.
Roberg, K.J., Bickel, S., Rowley, N., and Kaiser, C.A. (1997). Control
of amino acid permease sorting in the late secretory pathway of
Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST8. Genet-
ics 147: 1569–1584.
Roberts, A.G., Cruz, S.S., Roberts, I.M., Prior, D., Turgeon, R., and
Oparka, K.J. (1997). Phloem unloading in sink leaves of Nicotiana
benthamiana: Comparison of a fluorescent solute with a fluorescent
virus. Plant Cell 9: 1381–1396.
Rutherford, S., and Moore, I. (2002). The Arabidopsis Rab GTPase
family: Another enigma variation. Curr. Opin. Plant Biol. 5: 518–528.
Searle, I., and Coupland, G. (2004). Induction of flowering by seasonal
changes in photoperiod. EMBO J. 23: 1217–1222.
Shaw, R.J. (2008). mTOR signaling: RAG GTPases transmit the amino
acid signal. Trends Biochem. Sci. 33: 565–568.
Smith, T.F., Gaitatzes, C., Saxena, K., and Neer, E.J. (1999). The WD
repeat: A common architecture for diverse functions. Trends Bio-
chem. Sci. 24: 181–185.
Soulard, A., Cohen, A., and Hall, M.N. (2009). TOR signaling in
invertebrates. Curr. Opin. Cell Biol. 21: 825–836.
Sulpice, R., et al. (2009). Starch as a major integrator in the regulation of
plant growth. Proc. Natl. Acad. Sci. USA 106: 10348–10353.
Taji, T., Ohsumi, C., Iuchi, S., Seki, M., Kasuga, M., Kobayashi, M.,
Yamaguchi-Shinozaki, K., and Shinozaki, K. (2002). Important roles
of drought- and cold-inducible genes for galactinol synthase in stress
tolerance in Arabidopsis thaliana. Plant J. 29: 417–426.
Tzeng, T.Y., Kong, L.R., Chen, C.H., Shaw, C.C., and Yang, C.H.
(2009). Overexpression of the lily p70(s6k) gene in Arabidopsis
affects elongation of flower organs and indicates TOR-dependent
regulation of AP3, PI and SUP translation. Plant Cell Physiol. 50:
Usadel, B., Bla ¨sing, O.E., Gibon, Y., Poree, F., Ho ¨hne, M., Gu ¨nter,
M., Trethewey, R., Kamlage, B., Poorter, H., and Stitt, M. (2008).
Multilevel genomic analysis of the response of transcripts, enzyme
activities and metabolites in Arabidopsis rosettes to a progressive
decrease of temperature in the non-freezing range. Plant Cell Environ.
Usadel, B., et al. (2005). Extension of the visualization tool MapMan to
allow statistical analysis of arrays, display of corresponding genes,
and comparison with known responses. Plant Physiol. 138: 1195–
Van Leene, J., et al. (2010). Targeted interactomics reveals a
complex core cell cycle machinery in Arabidopsis thaliana. Mol.
Syst. Biol. 6: 397.
van Nocker, S., and Ludwig, P. (2003). The WD-repeat protein super-
family in Arabidopsis: conservation and divergence in structure and
function. BMC Genomics 4: 50.
Wullschleger, S., Loewith, R., and Hall, M.N. (2006). TOR signaling in
growth and metabolism. Cell 124: 471–484.
Xu, J., Brearley, C.A., Lin, W.H., Wang, Y., Ye, R., Mueller-Roeber,
B., Xu, Z.H., and Xue, H.W. (2005). A role of Arabidopsis inositol
polyphosphate kinase, AtIPK2alpha, in pollen germination and root
growth. Plant Physiol. 137: 94–103.
You, D.J., Kim, Y.L., Park, C.R., Kim, D.K., Yeom, J., Lee, C., Ahn, C.,
Seong, J.Y., and Hwang, J.I. (2010). Regulation of IkB kinase by
GbL through recruitment of the protein phosphatases. Mol. Cells 30:
Roles of the Arabidopsis LST8 Homolog 481