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The Plant Cell, Vol. 24: 4850–4874, December 2012, www.plantcell.org ã 2012 NRC Canada.
Target of Rapamycin Signaling Regulates Metabolism,
Growth, and Life Span in Arabidopsis
W OA
Maozhi Ren,a Prakash Venglat,a Shuqing Qiu,a Li Feng,a Yongguo Cao,a Edwin Wang,b Daoquan Xiang,a
Jinghe Wang,a Danny Alexander,c Subbaiah Chalivendra,d David Logan,e Autar Mattoo,f Gopalan Selvaraj,a
and Raju Datlaa,1
a Plant
Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan S7N 0W9, Canada
Chemistry and Bioinformatics Group, Biotechnology Research Institute, National Research Council of Canada,
Montreal, Quebec H4P 2R2, Canada
c Metabolon, Durham, North Carolina 27713
d Valent BioSciences Corporation, Long Grove, Illinois 60047
e Université d’Angers, Institut de Recherche en Horticulture et Semences, SFR 4207 QUASAV, LUNAM Université, Angers cedex 1,
France
f Sustainable Agricultural Systems Laboratory, U.S. Department of Agriculture–Agricultural Research Service, Beltsville Agricultural
Research Center, Beltsville, Maryland 20705-2350
b Computational
Target of Rapamycin (TOR) is a major nutrition and energy sensor that regulates growth and life span in yeast and animals. In
plants, growth and life span are intertwined not only with nutrient acquisition from the soil and nutrition generation via
photosynthesis but also with their unique modes of development and differentiation. How TOR functions in these processes
has not yet been determined. To gain further insights, rapamycin-sensitive transgenic Arabidopsis thaliana lines (BP12)
expressing yeast FK506 Binding Protein12 were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower
overall root, leaf, and shoot growth and development leading to poor nutrient uptake and light energy utilization. Experimental
limitation of nutrient availability and light energy supply in wild-type Arabidopsis produced phenotypes observed with TOR
knockdown plants, indicating a link between TOR signaling and nutrition/light energy status. Genetic and physiological studies
together with RNA sequencing and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development
and life span in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, and rRNA and protein
synthesis. Gain- and loss-of-function Ribosomal Protein S6 (RPS6) mutants additionally show that TOR function involves RPS6mediated nutrition and light-dependent growth and life span in Arabidopsis.
INTRODUCTION
Among all extant organisms, many of the longest living species
are plants. For example, a creosote bush (Larrea tridentata)
called King Clone, which has lived for over 10,000 years, was
found in the Mojave Desert (Vasek, 1980). The giant redwood
trees in California (Sequoia sempervirens) live for well over 2000
years (Scheres, 2007) and several other tree species have a long
life span. However, the mechanisms that underpin longevity
in plants are not known. Dissecting the control mechanisms
of growth and life span in plants has many implications. It will
provide a framework for addressing the key components and
regulators of life span in plants. Engineering life span in plants
has multiple applications, including early maturation for short
seasons, long-lasting horticultural plants, and trees of desirable
1 Address
correspondence to [email protected].
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: Raju Datla (raju.datla@
nrc-cnrc.gc.ca).
W
Online version contains Web-only data.
OA
Open access articles can be viewed online without a subscription.
www.plantcell.org/cgi/doi/10.1105/tpc.112.107144
life span in silviculture (McCouch, 2004; Neale, 2007; Takeda
and Matsuoka, 2008; Sonah et al., 2011). Recent work identified
genetic factors that can be modified via breeding techniques to
improve crop yield through modulating the growth phases (Moose
and Mumm, 2008). Uauy et al. (2006) showed that a NAC transcription factor–mediated acceleration of senescence impacted
nutrient remobilization in wheat (Triticum aestivum), resulting in
significant increase in protein content and micronutrients in the
grains (Uauy et al., 2006). Thus, life span alteration can have
several beneficial outcomes.
Plants are distinct from most other multicellular eukaryotes in
having a modular body plan with immortal totipotent stem cells,
sessile but autotrophic lifestyle, and very extensive biosynthetic
capabilities and adaptation to various stress factors. Therefore,
the determinants of growth habit and life span are expected
to be different in plants and animals (Thomas, 2002). Nevertheless, it is important to determine if plants and animals share
any common regulators or mediators of the processes involved.
In this regard, the Target of Rapamycin (TOR) pathway is a fundamental commonality. In other eukaryotes, the TOR signaling
pathway is considered to have a central role in sensing nutrient
and energy status. It plays a critical role in regulating cellular
metabolism (Fontana et al., 2010; Laplante and Sabatini, 2012)
TOR Regulates Life Span
and anabolic functions through alterations in ribosome biogenesis, mRNA translation, and polysome integrity (Proud, 2004;
Tavernarakis, 2008). The essential role of TOR in plants became
evident as research showed that mutations in TOR caused
embryo lethality (Menand et al., 2002). Although the roles of
TOR in postembryonic plant growth and life span are largely
undetermined, experimental findings suggest TOR involvement
in the regulatory program that coordinates cell wall components
with cell elongation as well as growth and development in plants
(Leiber et al., 2010). Plant cell growth is tightly linked to the
physico-chemical aspects of the cell wall. The components and
processes associated with cell wall extension are critical for
cell enlargement and organ growth (Carpita and Gibeaut, 1993;
Martin et al., 2001). Mutation in Lethal with Sec Thirteen8 (LST8),
a member of the TOR complex, leads to cell wall alteration and
reduction of vegetative growth in Arabidopsis thaliana (Moreau
et al., 2012). TAP46, another factor that functions as a positive
downstream effector of TOR, regulates the growth and metabolism of plants via modulation of polysome abundance and
global translation activities in response to the nutrient availability
(Ahn et al., 2011).
Ribosome biogenesis and protein synthesis command a major portion of available nutrients and energy in growing cells of all
eukaryotic species and affect the growth rate and life span
(Byrne, 2009; Lempiäinen and Shore, 2009). The TOR signaling
pathway tightly couples energy and nutrient availability to protein
and ribosome production (Wullschleger et al., 2006). Inhibition
of the TOR signaling pathway by nutrient starvation, rapamycin
treatment, or genetic mutation results in rapid downregulation
of protein synthesis and ribosome biogenesis in diverse eukaryotes (Li et al., 2006). Mechanistically, TOR acts as a protein
kinase to change the phosphorylation state of many target
proteins. Ribosomal protein S6 (RPS6) is a key component of
the 40S ribosomal subunit, integrating t-RNA recruitment to
mRNA and translation initiation factors, and thus regulating
translation of mRNAs (Ruvinsky and Meyuhas, 2006). In mammals, RPS6 was the first identified ribosomal protein target of
inducible phosphorylation by growth factors and also by ribosome protein S6 protein kinase (S6K) (Krieg et al., 1988; Franco
and Rosenfeld, 1990; Ruvinsky and Meyuhas, 2006). S6K is
a known target of mammalian TOR, participating in the TOR signaling pathway to regulate growth and life span in mice (Pende
et al., 2004; Holz et al., 2005; Selman et al., 2009). Additionally,
in yeast, RPS6 regulates cell size, growth, and life span (Fabrizio
et al., 2001; Chiocchetti et al., 2007), suggesting a coordination
with TOR.
In Arabidopsis, a connection of the TOR signaling pathway to
growth was apparent when embryo lethality was observed in
TOR T-DNA insertion mutants (Menand et al., 2002; Ren et al.,
2011). Also, mutants lacking TOR pathway components, including S6K, LST8, TAP46, and RPS6, were lethal at an early
stage (Creff et al., 2010; Henriques et al., 2010; Ahn et al., 2011;
Moreau et al., 2012). Rapamycin, an immunosuppressant drug
that was first discovered as an antifungal compound, is a valuable tool for dissecting the functions of the TOR pathway in
mammalian and other systems (e.g., yeast) (Loewith and Hall,
2011). Rapamycin specifically binds to FKBP12 (for FK506
binding protein 12), which interacts with the FRB domain of
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TOR, and the resultant rapamycin-FKBP12-TOR inhibitory complex
renders the TOR pathway inactive (Heitman et al., 1991; Stan
et al., 1994; Zheng et al., 1995; Loewith et al., 2002). Studies
with Arabidopsis have shown that rapamycin treatment does not
produce phenotypes associated with inhibition of TOR, and this
is attributed to structural differences of the target Arabidopsis
FKBP12 proteins, which are unlike their yeast or mammalian
counterparts (Xu et al., 1998; Menand et al., 2002; Sormani et al.,
2007). This is further supported by studies that showed that
transgenic Arabidopsis plants that expressed yeast or human
FKBP12 proteins are rapamycin sensitive (Sormani et al., 2007;
Leiber et al., 2010; Xiong and Sheen, 2012).
This study was designed to gain further insights into the postembryonic plant growth and life span roles of the TOR signaling
pathway and its downstream effectors in plants through analysis
of yeast FKBP12-based rapamycin-sensitive Arabidopsis transgenic lines (BP12) as well as in conjunction with rsp6 mutants.
Independent BP12 lines as well as the rps6 mutants produced
phenotypes of reduced root and leaf growth but an extended
life span. Furthermore, we report that these phenotypes were
associated with increased catabolism, as evident from metabolite and transcript profiles. These studies demonstrate
that TOR signaling in concert with RPS6 as a downstream
component couples metabolic processes associated with
growth and life span to nutritional and light energy status in
Arabidopsis.
RESULTS
Establishment of Rapamycin-Sensitive Arabidopsis Lines
Expressing Yeast FKBP12
We generated 81 independent transgenic Arabidopsis lines
(BP12 lines) by expressing the yeast FKBP12 gene under the
control of a constitutive (cauliflower mosaic virus 35S) promoter.
None of these lines showed any detectable or visible developmental or growth defects. PCR identification of 29 T1 lines
confirmed the presence of FKBP12 in all the lines except line
24 (Figure 1A). From these 28 T1 independent lines, 12 T2 BP12
lines (lines 1 to 12) were selected for rapamycin sensitivity
and segregation analysis in the T2 generation. Compared with
wild-type plants, all 12 independent BP12 lines were responsive
to rapamycin treatment, causing phenotypes including slower
plant growth with shorter shoots and roots (see Supplemental
Figure 1A online). Copy number for FKBP12 in these 12 lines
was verified by DNA gel blots: The BP12-2 line contained a
single copy, BP12-1 two copies, BP12-4 and BP12-6 three
copies each, and the rest of the transformed lines had multiple
copies (Figure 1B). We selected five independent T3 generation
lines, BP12-1 to BP12-5, and confirmed the expression of the
FKBP12 transcript and corresponding protein in all of them
(Figure 1C). Then, seedling growth of BP12-1, BP12-2, BP12-4,
and BP12-5 lines was examined in the same plate at incremental
rapamycin concentrations, 0.5 to 20 µg/mL in the medium, using
0.53 Murashige and Skoog (MS) containing 1 µg/mL DMSO as
controls (Figure 1D). On 0.53 MS + DMSO medium, the seedling
growth of these four BP12 lines was similar to wild-type plants
Figure 1. Molecular and Functional Analyses of Transgenic Rapamycin-Sensitive Arabidopsis BP12 Lines.
(A) The presence of yeast FKBP12 gene was confirmed by PCR in 28 of the 29 P35S:FKBP12-Myc transgenic lines (BP12), except line 24, using 35SF1
and FKBP12R primers. M, DNA ladder; WT, the wild type.
(B) DNA gel blot analysis of BP12 lines (lanes 2 to 11) showed that BP12-2 (lane 2) contained a single copy of yeast FKBP12 transgene and in the others
two or more copies.
(C) Expression of FKBP12 in BP12 lines 1 to 5 confirmed by RT-PCR analysis (top panel); Actin 2 was used as internal control (middle panel) (1).
Immunoblot analysis using Myc epitope tag-based antibody showed the expression of yeast FKBP12 protein (bottom panel) (2).
(D) Rapamycin (RAP) sensitivity of wild-type and four BP12 seedlings grown on 0.53 MS plates containing incremental concentrations of rapamycin
(0.5 to 20 µg/mL) at 7 DAG. The growth of wild-type seedlings in all the rapamycin-containing medium is similar to the control without rapamycin,
whereas the four BP12-2, BP12-5, BP12-1, and BP12-4 lines showed reduced root and shoot growth in the presence of rapamycin.
(E) and (F) BP12-2 and BP12-5 seedlings at 7 DAG with rapamycin (10 µg/mL) showed reduced seedling length (E) and fresh weight (F) compared with
the wild type.
(G) Both BP12 lines displayed reduced growth rate compared with the wild type as measured by fresh weight at 7, 15, 25, 35, and 45 DAG. Error bars
indicate SD for triplicates.
TOR Regulates Life Span
grown on 0.53 MS medium (see Supplemental Figure 1B online), confirming that overexpression of FKBP12 does not alter
plant growth or development. The growth of wild-type seedlings
was no different at 0 or 20 µg/mL rapamycin but clear phenotypes affecting primary root growth, lateral root initiation, cotyledon growth, rosette leaf initiation and expansion, and shoot
growth were observed in the four independent transgenic BP12
lines (Figure 1D). The severity of these phenotypes increased
with increasing concentrations of rapamycin, with even a dose
of 0.5 µg/mL rapamycin being sufficient to observe effects on
seedling growth in all the four BP12 lines (Figure 1D). These
results clearly indicate that Arabidopsis seedling growth and the
observed root and shoot phenotypes are dependent on both
the FKBP12 transgene expression and rapamycin, consistent
with previous observations (Mahfouz et al., 2006; Sormani et al.,
2007; Leiber et al., 2010). We would like to note here that compared
with the findings with transgenic Arabidopsis lines, the wild-type
seedling growth was normal even at the highest concentration
of rapamycin tested (i.e., 20 µg/mL medium) (Figure 1D). We
used rapamycin at 10 µg/mL concentration in the remainder
of the experiments because this level allowed BP12 lines to
complete their life cycle in spite of its effect on growth, and the
effects of TOR signaling at different stages of postembryonic
development could be assessed.
To address subcellular localization of FKBP12, we generated
81 P35S:FKBP12-GFP (for green fluorescent protein) transgenic
Arabidopsis lines, out of which 17 selected T1 lines were analyzed by PCR to confirm the presence of the transgene with GFP
fusion. All the selected lines except line 13 were positive (see
Supplemental Figure 1C online). We also confirmed rapamycin
sensitivity in lines 1 to 9 (see Supplemental Figure 1D online),
suggesting that C-terminal fusion of GFP did not significantly
affect the rapamycin sensitivity of ScFKBP12 in the TOR complex. The FKBP12 protein was localized to two compartments,
the nucleus and the cytoplasm (see Supplemental Figures 1E to
1G online), which is similar to the subcellular localization pattern
previously observed in TOR-GFP lines (Ren et al., 2011). The
colocalization of FKBP12 with TOR protein in the nucleus and
cytoplasm may be an indication of a functional interaction between these proteins in the presence of rapamycin, similar to that
shown in yeast and animal systems (Li et al., 2006; Wullschleger
et al., 2006).
Rapamycin Affects TOR Kinase in BP12-2
TOR kinase function involves phosphorylation of its downstream
target proteins, such as S6K, 4E-BP1, AKT, and TAP46. Phosphorylation of S6K at Thr-389 residue (pT389) has been used
to monitor TOR activity in mammals (Wullschleger et al., 2006).
Since the human S6K (Hs-S6K) and Arabidopsis S6K (At-S6K)
homologs share a similar consensus phosphorylation motif for
TOR (Turck et al., 1998), it is possible to detect the phosphorylation status of human S6K (Hs-S6K) as substrate for Arabidopsis TOR kinase using an Hs-S6K (p70-S6K-pT389) specific
antibody. Therefore, we explored this possibility in BP12 lines
with the Hs-S6K antibody by creating double transgenics expressing both FKBP12 and Hs-S6K transgenes. We introduced
the human p70-S6K construct P35S:Hs-S6K (with kanamycin
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selection marker) in the single-copy FKBP12 homozygous
BP12-2 line (with basta selection marker). T0 generation seeds
were screened on 0.53 MS medium containing both Basta and
kanamycin. Nine independent lines containing both P35S:HsS6K and P35S:FKBP12 were identified by PCR and RT-PCR
(see Supplemental Figures 2A and 2B online). The S6K/BP12-2
line that showed the highest expression level of HsS6K was used to
determine At-TOR kinase activity. The ELISA data showed that,
in S6K/BP12-C plants, 76.01% of S6K proteins were phosphorylated at Thr-389 by TOR kinase protein in the control,
whereas in the presence of rapamycin, S6K protein phosphorylation was reduced to 24.05%, indicating that rapamycin significantly inhibited TOR kinase activity (see Supplemental Figure
2C online). A recent study also used Hs-S6K (p70-S6K-pT389)
specific antibody to monitor TOR activity in Arabidopsis (Xiong
and Sheen, 2012).
Inhibition of TOR Signaling Suppresses Plant Growth
and Development
TOR has been shown to promote anabolic activity and cell
growth in yeast and animals (Topisirovic and Sonenberg, 2010).
To explore the possible links between TOR and plant growth, we
selected BP12-2, with a single FKBP12 transgene copy, and
BP12-5, with multiple copies, for further study. In the presence
of rapamycin, both BP12-2 and BP12-5 displayed a consistent
reduction in growth and uniform rapamycin-sensitive phenotypes. The wild type did not have any discernible phenotypes.
The transgenic lines showed a significant reduction in seedling
length (Figure 1E) and fresh weight (Figure 1F) compared with
the wild type at 7 d after germination (DAG). The slow growth
was more pronounced (12-fold) by 45 DAG (Figure 1G). Also, the
growth of primary and lateral roots was significantly inhibited in
BP12-2 and BP12-5 plants (Figure 2A). Closer examination of
the roots showed that the root hair growth was severely inhibited by rapamycin in BP12-2 and BP12-5 lines compared
with the wild type (Figures 2B and 2E). Since the root apical
meristem (RAM) size is defined by the proliferation of the transit
amplifying cells (TACs), the daughter cells of the stem cells
(Scheres, 2007), we compared the RAM region of BP12-2 and
the wild type. BP12-2 RAM showed a significant reduction in the
TAC zone resulting in a smaller RAM (Figures 2E and 2F). This is
likely due to decreased cell division rate in the meristem region
and in the TAC zone. The smaller RAM in BP12-2 was associated with differentiation of the xylem tracheary elements closer
to the RAM region compared with the wild type (Figures 2F and
2G).
To investigate the effects of rapamycin treatment on cell
elongation, we focused on the hypocotyl region of seedlings,
which shows rapid cell elongation in the dark. Upon rapamycin
treatment, both BP12-2 and BP12-5 hypocotyls elongated to
only half the length of wild-type hypocotyls (Figure 2C). Elongation of cells from the mid-region of the hypocotyl was reduced
by 50% in BP12-2 and BP12-5 compared with the wild type
(Figure 2D). We also examined cell size in epidermal, palisade,
and spongy mesophyll cells of the leaves treated with rapamycin. Significantly smaller cells (3 6 0.2-fold, n = 50) were observed in rapamycin-treated BP12-2 line (Figures 2H to 2K) but
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Figure 2. Phenotypic Analysis of Rapamycin-Treated BP12-2 and BP12-5 Lines.
(A) BP12-2 and BP12-5 lines showed significant reduction in root and shoot growth compared with the wild type (WT) when grown on 0.53 MS +
rapamycin (10 µg/mL) (right panel) versus 0.53 MS + DMSO control (left panel) at 30 DAG.
(B) Rapamycin treatment reduced root hair elongation in BP12-2 and BP12-5 seedlings, whereas normal growth was observed in the wild type.
(C) and (D) Rapamycin treatment significantly reduced the elongation of hypocotyl in dark-grown BP12-2 and BP12-5 3-DAG seedlings compared with
the wild type and also showed ;50% reduction in hypocotyl cell length in BP12 plants compared with the wild type (D). Bracketed regions indicate
single cell length.
(E) to (G) Rapamycin treatment resulted in significant reduction in the size of the RAM in BP12-2 compared with the wild type ([E]; bracketed region in
[F]). In BP12-2, root hair initiation was inhibited (red star) compared with the wild type (E). Red stars in (F) and (G) indicate differentiation of xylem
tracheary elements with reference to the RAM.
(H) and (I) Scanning electron microscopy analysis of leaf and leaf epidermal cells of 2-week-old seedlings showed smaller cells and overall reduction in
leaf size in rapamycin treated BP12-2 line compared with wild-type controls.
(J) and (K) The analysis of confocal images of palisade (J) and spongy (K) mesophyll cells of leaves at the same stage showed similar reduction in cell
size in BP12-2 line compared with the wild type.
Bars = 1 mm in (B), (C), and (H), 100 µm in (D) to (G) and (I), and 50 µm in (J) and (K).
TOR Regulates Life Span
not the wild type. Together, these results indicate that TOR
activity is required for normal cell elongation and expansion in
Arabidopsis.
Suppression of TOR Activity Influences
Metabolism in Arabidopsis
Changes in cellular metabolism, particularly the accumulation of
amino acids in response to suppression of TOR signaling, have
been previously observed in yeast, animals, and Arabidopsis
(Chen and Kaiser, 2003; Laplante and Sabatini, 2012; Moreau
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et al., 2012). To ascertain which metabolic changes accompany
the growth pattern of TOR suppression lines, we analyzed metabolite profiles in the BP12-2 and BP12-5 lines and in the wild
type in the presence or absence of rapamycin. Both the transgenic lines showed robust rapamycin-dependent responses,
but the wild type exhibited few changes (Figures 3A and 3B;
see Supplemental Figure 3 online). Quite remarkably, rapamycin
significantly affected primary metabolism, with increases in the
glycolytic and tricarboxylic acid (TCA) cycle intermediates and
a large number of amino acids (Figures 3B and 3C, I and II; see
Supplemental Figures 3A and 3B online); there were also notable
Figure 3. Suppression of TOR Activity in BP12-2 and BP12-5 Lines Affects Metabolite Levels.
(A) Principal component analysis of the metabolite data illustrating the clear separation of rapamycin-treated BP12 lines from the untreated BP12 and
treated and untreated wild type (WT) based on the principal component 1 (x axis). In this analysis, the rapamycin treated wild-type line behaved similar
to its untreated group, while both BP12 lines showed a strong rapamycin-dependent response.
(B) Heat map showing metabolite profiles of rapamycin-treated BP12 lines compared with the control. Carbo, carbohydrate; Cof, cofactor; Lip, lipid;
Nucl, nucleotide; SM, secondary metabolite/xenobiotic pools. The star indicates rapamycin. Supplemental Figures 3A to 3F online list the compound
names and their mean values for each pathway group. Four lanes in the heat map for each treatment correspond to four biological replicates.
(C) Rapamycin-treated BP12 lines showed increase in carbohydrates (I) and amino acids (II) as well as decrease in peptides (III) compared with control.
1,3-DHA, 1,3-dihydroxyacetone; F-6-P, Fru-6-phosphate; G-6-P, Glc-6-phosphate; NO, no rapamycin treatment; RAP, rapamycin treatment. Error bars
indicate SD for four biological replicates. The relative value of each metabolite shown is the ratio between rapamycin-treated wild type, BP12-2 and
BP12-5, and nontreated wild type, BP12-2, and BP12-5 seedlings. All values indicated are statistically significant (P < 0.05) compared with the control.
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increases in some components of the secondary metabolism,
such as the polyamine pathway, antioxidative pathway, and
phenylpropanoid pathway, in the two BP12 lines analyzed
(Figure 3B; see Supplemental Figures 3D and 3E online). There
was an increase in methanol-extractable D-galacturonate (see
Supplemental Figure 3A online). As noted above, TOR inhibition
results in a reduction of hypocotyl elongation and smaller cells in
the leaves (Figures 2C and 2H). As cell expansion and tissue growth
require incorporation of D-galacturonate into polymeric form in cell
wall pectin, we attribute the increase in D-galacturonate to tempered
cell wall assembly upon TOR inhibition. 3-Deoxyoctulosonate,
another substrate for pectin synthesis, had also increased (see
Supplemental Figure 3A online). Interestingly, there was a reduction in the myo-inositol level (see Supplemental Figure 3A
online). Myo-inositol is a precursor of D-glucuronate that in turn
can be metabolized to D-galacturonate (Reiter and Vanzin,
2001). The TCA cycle intermediates, citrate, fumarate, malate,
and succinate registered significant increases. Since photosynthesis is diminished in TOR-inhibited lines (noted below),
the TCA intermediates required for cellular respiration and maintenance of growth likely arise from reorganized metabolism. Since
the nitrogen supply in the growth medium was the same, regardless of rapamycin inclusion, reduced carbon fixation would
be expected to distort carbon-nitrogen balance in the cells.
The interrelationship in carbon-nitrogen metabolism is complex
(Nunes-Nesi et al., 2010).
Like the increase in organic acids, prominent increases in the
amino acids and polyamines (Kausch et al., 2012) were apparent
in rapamycin-treated BP-12 transgenic lines (Figures 3B and
3C, II; see Supplemental Figure 3B online). The increased level
of several amino acids is attributed to protein degradation as
a source for energy metabolism. The reduction in dipeptides is
presumably an adaptive measure of TOR-inhibited cells to make
amino acids to support respiration and limited growth (Figures
3B and 3C, III; see Supplemental Figure 3C online). The decrease in Arg levels parallels a decrease in N-acetylglutamate,
N-acetylornithine, Orn, and citrulline, the precursors of Arg biosynthesis (see Supplemental Figure 3B online). This decrease in
Arg levels and a concomitant increase in dimethylarginine
under rapamycin treatment is an indication of methylation of
proteinaceous Arg. Arg dimethylation acts as an antiaging modification in Caenorhabditis elegans and increased its life span
(Takahashi et al., 2011). Decreased levels of the polyamine precursors, Arg and Orn, were noted alongside increased levels of
the biogenic amines, agmatine and putrescine, and the product
of their catabolism, 5-methylthioadenosine (see Supplemental
Figure 3D online). These results suggest that a perceived signal
generated by TOR suppression redirects carbon flow and metabolic shift to other pathways to support the growth of rapamycintreated BP12 plants.
Other metabolic alterations arising from rapamycin treatment
included some secondary metabolites. The phenylpropanoid
pathway generates among precursors for other products substrates for flavonoids that exist in relatively simple forms and
more complex forms that are recalcitrant to methanol extraction.
This pathway generates hydroxycinnamic acid (HCA)–derived
monolignols that when polymerized appropriately become the
lignin component of the cell wall (Vogt, 2010). Rapamycin
suppression of TOR activity led to increases in methanolextractable HCA (ferulate and sinapate) and monolignols (coniferyl alcohol) (see Supplemental Figure 3E online), supporting
the inference noted above with reference to tempered cell wall
assembly. Tyramine, derived from L-Tyr, can condense with
hydroxycinnamoyl CoA esters to form amides that can then be
incorporated in cell walls (Facchini et al., 2002). The levels of
tyramine were also lower when TOR was inhibited even though
the HCA content was elevated (see Supplemental Figure 3E
online).
Also, members of anti-reactive oxygen species (ROS), including 5-oxoproline, and both oxidized and reduced forms of
glutathione, all increased in rapamycin-treated BP12 plants (see
Supplemental Figure 3B online). The ratio of GSH/GSSG is
significantly reduced in BP12 lines (GSH/GSSG = 0.73, P < 0.05)
compared with the wild-type control (GSH/GSSG = 1.32, P <
0.05) indicative of active ROS quenching. In C. elegans, genetic
or rapamycin inhibition of TOR activates a stress-protective
response that includes glutathione S-transferase and genes
involved in glutathione among others. This, while reducing
growth rate, enhances life span of the worm (Robida-Stubbs
et al., 2012) similar to that observed in BP12 plants treated with
rapamycin. Purine and pyrimidine metabolism and also the
lipid metabolism were notably the least affected by rapamycinmediated TOR signaling suppression (see Supplemental Figure
3F online). An active protein metabolism is apparent from an
increase in dimethylarginine, which is posttranslationally formed
in proteins (see Supplemental Figure 3B online). These changes
in metabolic profiles of rapamycin-treated BP12 plants compared with the wild type indicate redirection of specific carbon
and nitrogen metabolites involving both primary and secondary
metabolism, suggesting that TOR is associated with coordination of several primary and secondary metabolic activities during
growth and development of Arabidopsis.
Inhibition of TOR Activity Is Associated with Altered
Gene Expression
We performed global RNA sequencing (RNA-seq) analysis
in BP12-2 to obtain a perspective of gene expression under
conditions of TOR inhibition. Comparative transcriptomes of
rapamycin-treated BP12-2 line and the wild type identified a large
number of differentially expressed genes (see Supplemental
Data Sets 1 and 2 online). Among these, the most prominent
downregulated genes under TOR inhibition in rapamycin-treated
BP12-2 line were associated with major anabolic pathways, including cell wall biogenesis, photosynthesis, and inorganic nutrient transporters (see Supplemental Data Sets 2 and 3 online).
By contrast, a large number of genes associated with catabolic
pathways involving carbohydrates and proteins were upregulated in rapamycin-treated BP12-2 plants (see Supplemental
Data Sets 2 and 3 online). In the category of cell wall biogenesis,
genes downregulated in rapamycin-treated BP12-2 included
structural proteins extensins, expansins, Pro-rich proteins, and
arabinogalactan- and Hyp-rich glycoproteins (see Supplemental
Figure 4 and Supplemental Data Set 3 online). Extensins are
involved in cell wall architecture, whereas expansins function to
enable plant cell expansion (Martin et al., 2001; Velasquez et al.,
TOR Regulates Life Span
2011; Bruex et al., 2012). Since cell wall development governs
cell size, shape, and function (Burton et al., 2010) together with
the fact that genes that enable restructuring of cell wall during
growth were affected in rapamycin-treated BP12 plants, we
quantified these genes and confirmed the transcriptome data
using real-time PCR. All of the 16 extensin genes, seven of the
nine expansin genes, and Pro- and glyco-rich cell wall protein
genes tested were significantly downregulated in rapamycintreated BP12-2 compared with the wild type (see Supplemental
Figure 4 and Supplemental Data Set 3 online). Also, galactinol
synthase 3, involved in the synthesis of raffinose family of oligosaccharides (RFOs), was significantly downregulated. This is
consistent with a decreased level of galactinol and raffinose in
TOR knockdown plants. Biosynthesis of RFOs is catalyzed via
the formation of galactinol from myo-inositol and UDP-Gal by
galactinol synthase. Sequential transfer of Gal units from galactinol to Suc leads to the formation of raffinose and higher
order RFOs (Peterbauer and Richter, 2001). Both galactinol and
raffinose are proposed to protect plant cells from oxidative damage by scavenging hydroxyl radicals when challenged by strong
oxidizing agents, such as methyl viologen or chilling stress (Taji
et al., 2002; Nishizawa et al., 2008). The low level of RFOs in
rapamycin-treated plants is consistent with a lowered oxidative
challenge in these plants. Taken together, these transcriptome
data are consistent with and complement the carbohydrate
metabolite data and suggest that TOR controls cell expansion
and elongation likely via regulating the expression of the cell wall
structural proteins.
Inorganic nutrition transporters that are essential for anabolic
processes in plants were also downregulated in rapamycintreated BP12. This is reflected by the poor growth of rapamycintreated BP12-2 plants even when grown under nonlimiting
supply of inorganic nutrients in the medium. Defensin genes
that are part of the stress response pathway in Arabidopsis
are upregulated (see Supplemental Figure 4 and Supplemental
Data Set 1 online). In parallel with these observations is downregulation of genes encoding ribulose-1,5-bis-phosphate carboxylase/oxygenase small subunit (Izumi et al., 2012) and NDP
kinase in rapamycin-treated BP12-2 plants (Choi et al., 1999),
which are important for carbon fixation and the light signaling
pathway, respectively, in the chloroplast (Izumi et al., 2012).
Thus, downregulation of photosynthesis, a major anabolic process in plants, can contribute to growth defects in rapamycintreated BP12-2 plants. Similarly, defects in the development
of root hairs and elongation of lateral and primary roots in
rapamycin-treated BP12-2 plants are consistent with the transcriptome data showing differential expression of 40 rootrelated genes in these plants (see Supplemental Data Set 3
online). Thirty-three of these were found to be significantly
downregulated (see Supplemental Data Set 3 online), including
MORPHOGENESIS OF ROOT HAIR6 (MRH6), which affects root
hair development (Jones et al., 2006).
Metabolite data clearly indicated increased polyamine biosynthesis in rapamycin-treated BP12-2 plants. A limiting reaction in this pathway is catalyzed by S-adenosylmethionine
decarboxylase (Mehta et al., 2002). S-adenosylmethionine decarboxylase was one of the upregulated genes featured in
rapamycin-treated BP12-2 plants whose expression was also
4857
quantified and verified (see Supplemental Figure 4 online). Thus,
the transcriptome and metabolite data are congruous and support
the finding that inhibition of TOR activity affects metabolic processes linked to important plant growth processes.
TOR Modulates Plant Growth in
a Nutrient-Dependent Manner
The above-described data bring to the fore the nutrition relatedness of the plant TOR pathway and the question arose
whether, similar to the role of TOR as a nutrition sensor in yeast
and animals, TOR regulation of Arabidopsis development and
metabolism are influenced by nutrition. Therefore, wild-type
and BP12-2 seedling growth was followed on different concentrations of MS medium (see Supplemental Table 1 online). Wildtype plants grown on nutrient poor medium (0.0013 MS)
resembled the BP12-2 plant grown on 500-fold richer medium
(0.53 MS) but containing rapamycin (Figure 4A), indicating both
nutrient starvation and TOR inhibition by rapamycin produce
similar phenotypes. A gradual reduction of fresh weight in wildtype plants was observed on decreasing MS concentration in
the medium (Figure 4B). In 0.0013 MS + rapamycin medium that
provides a condition of nutrient starvation and TOR inhibition,
both wild-type and BP12-2 plants displayed similar growth rates
(Figures 4C and 4D). Under these conditions, increasing the
nutrient supply up to 13 MS while maintaining the rapamycin
concentration permitted a ninefold growth increase in the wild
type, but not in BP12-2, whose growth remained close to that
observed in 0.0013 MS + rapamycin (Figures 4C and 4D). These
results indicated that BP12-2 plants could not respond positively to increasing nutrition levels when TOR is inhibited. Under
hypernutritive conditions (23 MS + rapamycin), which also
represent higher osmotic potential, growth of the wild type and
BP12-2 line decreased relative to 13 MS + rapamycin (Figures
4C and 4D). Increasing the concentrations of nitrogen and carbon (Suc) significantly increased the growth rates of wild-type
seedlings, whereas the BP12-2 seedlings with rapamycin remained insensitive to this stimulation (Figures 4E and 4F). We
note here that S6K phosphorylation was reduced to 25.18%
when nutrition was limited (see Supplemental Figure 2C online),
and this is similar to the reduced level of phosphorylation (24%)
when TOR is inhibited by rapamycin. Thus, nutrition starvation
phenocopies the suppression of TOR kinase activity. Together,
these results suggest that TOR signaling senses nutritional status
and may thereby control cell growth and development.
TOR Is Required for Light Energy–Dependent
Plant Growth
Plants respond positively to photosynthetic growth when light
intensity is increased from suboptimal levels. This was observed
with wild-type plants (Figures 5A and 5B). By contrast, rapamycintreated BP12-2 line failed to respond to increasing light intensities
(Figure 5A), resulting in a growth rate that was >10-fold lower than
the wild-type control (Figure 5B). To test the effect of combined
light and nutrition conditions on plant growth, fresh weight ratio
between wild-type and rapamycin-treated BP12-2 plants under
various light intensities and nutrient concentrations (MS media)
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Figure 4. TOR Promotes Plant Growth in a Nutrient-Dependent Manner.
(A) Phenotypes of BP12-2 plants grown on 0.53 MS with rapamycin (Rap) at 20 DAG resemble 20-DAG wild-type (WT) plants grown on 0.0013 MS that
represents a nutrition starvation condition. At 20 DAG, clear similarities in phenotypes were observed between TOR suppression in BP12-2 and
nutritional starvation in the wild type.
(B) The wild-type seedlings grown on decreasing concentrations of MS showed a consistent decrease in fresh weight (FW); BP12-2 seedlings grown on
0.53 MS with rapamycin showed similar fresh weight as the wild type grown on 0.0013 MS, whereas without rapamycin treatment in BP12-2 seedling
the fresh weight was similar to the wild type grown on 0.53 MS.
(C) Phenotypes of 9-DAG seedlings of the wild type and BP12-2 grown on different concentrations of MS media containing rapamycin showed that
increasing concentrations of MS did not increase the growth rate of BP12-2 seedlings compared with the wild type in the presence of rapamycin. At 9
DAG, clear growth differences become apparent between the wild type and BP12-2 under rapamycin treatment.
(D) to (F) Growth rates (measured in fresh weight) of 9-DAG wild-type and BP12-2 seedlings grown on increasing concentrations of MS media (D) and
increasing concentrations of nitrogen (E) and Suc (F) in MS with rapamycin. FW, fresh weight; RAP, rapamycin. Error bars indicate SD for triplicates.
was determined (Figure 5C). The largest fresh weight ratio
value, indicating a significant difference between wild-type and
rapamycin-treated BP12-2 plants, was observed at 100 µmol
m22 s21 light intensity (Figure 5C). This ratio peaked at 0.53 MS
and was significantly lower under weak light (10 µmol m22 s21)
and dark conditions (Figure 5C). Consistent with these observations, upon light energy limitation, TOR kinase activity
was significantly suppressed similar to that observed in BP12-2
plants treated with rapamycin (see Supplemental Figure 2C
online).
We next determined whether the reduction of growth and
biomass of TOR suppression lines is associated with ROS and
NAD(P)H reduction (Apel and Hirt, 2004). ROS signals in the root
hair of wild-type plants treated with rapamycin were similar to
the control plants; however, significant reduction of ROS signals
was observed in rapamycin-treated BP12-2 plants compared
with the control (Figure 5D), as was also the case with NAD(P)H
levels (Figure 5E). Interestingly, the ratios of NADH/NAD and
NADPH/NADP in BP12 lines did not differ from the wild type in
response to rapamycin treatment, indicating that the homeostasis of NAD(P)H/NAD(P) is maintained in BP12 lines but the
relative levels of NAD(P)H were lower compared with the wild
type. Glutathione reductase uses NADPH to reduce the oxidized
form of glutathione (GSSG) to the reduced form (GSH). The
TOR Regulates Life Span
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Figure 5. Influence of Light-Dependent Functions of TOR on Plant Growth.
(A) Phenotypes of 11-DAG seedlings grown on 0.53 MS media with rapamycin (RAP) under different light intensities. Strong light (SL; 100 µmol m22 s21)
severely inhibited seedling growth in the BP12-2 line compared with the wild type (WT), whereas in weak light (WL; 30 µmol m22 s21) and dark conditions,
growth of BP12-2 seedlings was less inhibited.
(B) The 11-DAG wild type showed increased growth rates on 0.53 MS with rapamycin at increasing light intensities, whereas BP12-2 seedlings failed to
respond to these favorable conditions. FW, fresh weight.
(C) Fresh weight ratios of wild-type/BP12-2 seedlings peaked under strong light conditions and remained neutral under weak light and dark conditions
when grown under different light conditions (100 µmol m22 s21, 30 µmol m22 s21, dark) and at various MS concentrations (0.01, 0.1, 0.5, 1.0, and 2.03)
with rapamycin. Fresh weight ratio quantified the difference in response of wild-type and BP12-2 seedlings on the combined effect of light intensity and
nutrition levels.
(D) ROS levels as detected by H2DCFDA fluorescence in the root hairs of 11-DAG wild-type and BP12-2 plants grown in the presence and absence of
rapamycin. Weak fluorescence signal in rapamycin-treated BP12-2 plants indicate reduced ROS levels in BP12-2 compared with the wild type.
(E) NADH and NADPH levels were reduced in 11-DAG BP12-2 seedlings compared with the wild type in the presence of rapamycin. Error bars indicate
SD for triplicates.
reduction of NADPH is highly consistent with the ratio of GSH/
GSSG significantly decreasing in rapamycin-treated BP12 lines
compared with the wild type control observed in metabolite
profile. These results suggest a mechanistic link between light
energy-TOR-ROS/NAD(P)H and plant growth.
Nutrition and Energy-Dependent TOR Function
Regulates Production of rRNAs
It is estimated that cells dedicate ;80% of total transcriptional
activity to the synthesis of rRNAs and proteins for ribosome biogenesis (Warner, 1999), making ribosome biogenesis a major nutrient and energy-consuming process in growing cells (Lempiäinen
and Shore, 2009). The TOR regulation of energy and nutrient
availability is known to be mediated by alterations in protein and
ribosome production (Wullschleger et al., 2006). We therefore
quantified total RNA production of wild-type and BP12-2 plants
with and without rapamycin. Total RNA was significantly reduced in rapamycin-treated BP12-2 plants (Figure 6A and inset),
as also observed under light-limiting conditions (Figure 6B and
inset), nutrition starvation (Figure 6C and inset), and carbon
(Suc) or nitrogen limitation (Figure 6D and inset). Under nutrient
limitation (0.0013 MS) or when kept in the dark, no further reduction in total RNA was found in rapamycin-treated BP12-2
plants (Figure 6E and inset). Thus, TOR requires both light
and nutrition signals to regulate total RNA expression in Arabidopsis. Previously, TOR modulation of rRNA expression has
been shown (Ren et al., 2011). We quantified rRNA content in
BP12-2 plants using a GFP reporter construct (45S rRNA promoter fused with GFP; Pr-RNA:GFP) previously described (Ren
et al., 2011). The GFP transcripts quantified by real time-PCR
(see Supplemental Table 4 online) essentially mirrored the trends
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Figure 6. TOR Couples Light and Nutrition Signals to Regulate Total RNA Production in Arabidopsis.
Relative total RNA levels in BP12-2 and wild-type (WT) seedlings grown under different nutrient and light with or without rapamycin (RAP). RNA
denatured gels are shown as insets in all panels representing RNA from samples shown in the histogram from left to right. Total RNA was isolated from
the same amount (0.9 g) of seedling tissues. MS = 0.53 MS; error bars indicate SD for triplicates.
(A) Decreased RNA levels in BP12-2 treated with rapamycin for 24 h compared with the wild type.
(B) Decreased RNA levels in both the wild type and BP12-2 under both dark and nutrition starvation (0.0013 MS) conditions.
(C) Decreased RNA levels in the wild type and BP12-2 under nutrition starvation, which is similar to that observed in rapamycin-treated BP12-2
seedlings.
(D) Decreased RNA levels in the wild type and BP12-2 under carbon or nitrogen starvation. -N, without nitrogen (see Supplemental Table 2 online);
-C, without Suc (see Supplemental Table 3 online).
(E) RNA levels are not further reduced in rapamycin-treated BP12-2 seedlings grown under dark or nutrition starvation conditions.
observed for the total RNA levels in the wild type and BP12-2
with analogous treatments (Figure 6).
TOR Suppression Significantly Extends Life Span
Irrespective of Negative Effect on Biomass in Arabidopsis
In order to delineate effects of TOR suppression on growth of
Arabidopsis, we examined the kinetics of phase change, flowering time, chlorophyll content, cell death rate, and the senescence-associated gene (SAG) expression in BP12-2 lines in
0.53 MS + rapamycin medium at 100 µmol m22 s21 fluence
(Figure 7). The BP12-2 plants characteristically had smaller
rosette leaves, slower phase change, delayed flowering, and
shorter roots compared with the wild type (Figures 7A to 7C; see
Supplemental Table 5 online). By 70 DAG, the wild-type plants
had senesced, whereas the BP12-2 rosettes were still green and
their roots had lengthened, indicating sustained growth and
longer life span (Figure 7D; see Supplemental Table 5 online).
Rapamycin application to BP12-2 plants even at an advanced
growth stage effectively delayed their flowering time and further
aging (see Supplemental Figure 5 online).
The delayed senescence in rapamycin-treated BP12-2 plants
prompted us to further examine aging in the first rosette leaf pair
in Arabidopsis using known assays and markers: chlorophyll
TOR Regulates Life Span
4861
Figure 7. Reduced TOR Activity Delays the Growth and Phase Changes in Arabidopsis.
(A) to (D) BP12-2 plants grown on 0.53 MS + rapamycin displayed fewer leaves and delayed flowering at 30 DAG (A), smaller and rounded leaves with
shorter petioles (B), slower growth and delayed bolting at 40 DAG (C), and delayed senescence in 70 DAG (D) compared with the wild type (WT).
(E) to (H) Slower growth, delayed phase change, later flowering, and delayed senescence in BP12-2 plants compared with the wild type were defined by
increased chlorophyll retention (E), decreased cell death rate (F), delayed flowering time (G), and delayed SAG12 expression (H). Error bars indicate SD
for triplicates.
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content, cell death rate, and SAG12 expression levels (Noh and
Amasino, 1999; Buchanan-Wollaston et al., 2005; Schippers
et al., 2008). At 32 DAG, total chlorophyll content in the leaves
had only reduced by 11% in BP12-2 line in contrast with 83% in
the wild type (Figure 7E). Cell death (measured by Evans blue
staining; Guo and Crawford, 2005) was only 19% in BP12-2
compared with 94% in the wild type (Figure 7F). The flowering
time of BP12-2 plants was delayed by 13 d compared with the
wild type (Figure 7G). Senescence rate was also measured by
quantifying SAG12 marker expression in the first leaf pair of the
rosette treated with rapamycin. It should be noted that only
SAG12 was used because both SAG12 and SAG13 had shown
similar trends in this study. SAG12 expression in the wild type
was clearly higher, and in comparison it was initiated 4 d later in
the BP12-2 line but continued to gradually increase to the wildtype levels by day 44 (Figure 7H). Rapamycin treatment of
BP12-2 consistently delayed the emergence of hypocotyl, establishment of rosette leaves, bolting, opening of the first flower,
and whole-plant senescence relative to that in the wild type.
Consequently, BP12-2 plants lived 17 d longer than the wild
type (see Supplemental Table 5 online).
Does TOR Signaling Recruit RPS6 to Regulate Growth
and Life Span of Arabidopsis?
A previous study suggested involvement of RPS6 in regulating
growth processes in Arabidopsis (Creff et al., 2010). We further
pursued this observation and found that rps6 mutant lines show
growth phenotypes similar to rapamycin-treated BP12-2 plants.
Independent T-DNA knockout lines representing loss of function
of two RPS6 homologs, rps6a and rps6b (see Supplemental
Figures 6A and 6B online and Supplemental Methods 1 online),
had smaller leaves, slower root growth rate, delayed phase
change, delayed flowering, and longer life span in comparison to
the wild type (Figures 8A and 8B; see Supplemental Tables 5 and
6 online). Our attempts to construct double mutants of rps6a and
rps6b were not successful due to lethality in the double homozygous seeds (Supplemental Methods 1 online). Interestingly, all
the 54 F1 generation plants (rps6a/+ rps6b/+) confirmed by PCRbased genotyping, had the same phenotype as the rps6a or rps6b
single mutants (Figure 8C). This further confirmed that heterozygosity of the two RPS6 genes results in dosage effect or haploinsufficiency as also previously noted (Creff et al., 2010).
Because stronger developmental defects were observed in
our studies with rps6b compared with the rps6a, rps6b plants
were used for additional experiments. The smaller leaves of
rps6b mutants had fewer secondary veins and the epidermal
pavement cells were two-thirds the size of comparable cells in
the wild type (Figure 8D), while the TAC zone in rps6b mutant
roots was much smaller than the wild type (Figure 8E), similar to
observations shown above in rapamycin-treated BP12-2 RAM.
Complementation tests confirmed the respective mutant alleles
of RPS6A and RPS6B genes (Figure 8F; see Supplemental
Methods online). Furthermore, growth rates of rps6a and rps6b
single mutants in different nutrition conditions and two light intensities were examined (see Supplemental Figure 7A online). In
normal to rich nutrition medium or high light conditions, the
growth rate of the mutant seedlings was slower than that of the
wild type but slightly better than wild-type controls under poor
nutrition or low light conditions (see Supplemental Figures 7B
and 7C online). The observations of slow growth of rps6 single
mutants and the lethality of the rps6a-rps6b double mutants are
reminiscent of rapamycin-treated BP12-2 plants and the tor
mutants, respectively (Menand et al., 2002; Ren et al., 2011).
Thus, both TOR and RPS6 are limiting factors for normal plant
growth.
These findings prompted us to test if RPS6 functions as
a downstream mediator (component) of TOR signaling as seem
to be the case in yeast and animals. We therefore tested genetic
interactions between TOR and RPS6. TOR was suppressed in
the rps6 mutant by crossing BP12-2 line with T3 generation
homozygous lines of rps6 carrying P35S:FKBP12. In both the
rps6a and rps6b mutants carrying P35S:FKBP12, rapamycin
treatment did not significantly decrease the growth rate to the
levels observed in the rapamycin-treated BP12-2 line (Figures
9A and 9B). This indicated that TOR inhibition phenotype to
manifest fully requires functional and homozygous copies of
both RPS6A and RPS6B. However, the chlorophyll content
(Figure 9C), cell death rate (Figure 9D), flowering time (Figure
9E), and SAG12 expression level (Figure 9F) in the rapamycintreated rps6b carrying P35S:FKBP12 followed the trend observed in rapamycin-treated BP12-2 plants and rps6b single
mutants. The values were consistently intermediate between
those observed in rps6b and BP12-2 lines. Rapamycin treatment produced significant increase in the life span of the rps6b
mutants (Figure 9; see Supplemental Table 6 online) but not to
the same extent as seen in the rapamycin-treated BP12-2 line
(Figure 9). These observations suggest that both RPS6A and
RPS6B are required for TOR suppression-dependent increase in
life span observed in BP12-2 plants.
Overexpression of TOR and RPS6 Leads to Accelerated
Phase Change, Earlier Ripening, and Shorter Life Span
Since TOR-inhibited plants and rps6 mutants displayed delayed
phase change and late flowering, we used P35S:TOR transgenic
plants (Ren et al., 2011) and RPS6A and RPS6B overexpression
lines (P35S:RPS6A and P35S:RPS6B) generated in this study
(see Supplemental Figure 8 online; Figures 10A and 10C) to
obtain additional insights into the TOR-RPS6 connection. The
TOR overexpression line (TOR-OE1) flowers and senesces earlier compared with the wild type (Figure 10A; see Supplemental
Table 6 online). Although the first five leaves of the TOR-OE1
rosette retain a shape similar to the wild type, the latter formed
more rounded leaves with short petioles (Figure 10A). TOR-OE1
siliques ripened earlier than the wild type (Figure 10B; see
Supplemental Table 6 online). Both the P35S:RPS6A (RPS6AOE1) and P35S:RPS6B (RPS6B-OE1) lines showed the following
phenotypes: early flowering (see Supplemental Table 6 online),
early onset of senescence in rosette leaves and siliques (Figures
10C and 10D; see Supplemental Table 6 online), and shorter
petioles and bent siliques (Figures 10C and 10D). In this regard,
these plants resemble the TOR kinase domain overexpression
lines (Ren et al., 2011).
The results of reduced life span in overexpressors of TOR
and RPS6 (see Supplemental Table 6 online) together with the
TOR Regulates Life Span
4863
Figure 8. rps6 Mutants Resemble TOR Knockdown Lines, Exhibiting Slower Growth, Longer Life Span, and Defects in Leaf Shape, Cell Size, and Root.
(A) Slower growth with reduced leaf size and delayed flowering time in rps6a (panels 1 and 2) and rps6b (panels 3 and 4) mutants compared with the wild
type (WT). rps6b mutant showed stronger phenotypes compared with rps6a.
(B) Delayed flowering of rps6a and rps6b seedlings compared with the wild type was also observed when grown on 0.53 MS medium.
(C) F1 heterozygous seedlings of rps6a/+/rps6b/+ double mutant display rps6a or rps6b single mutant phenotypes.
(D) Cleared rps6b mature leaves display defective vascular patterning as shown by reduction in the number of secondary branching veins compared
with the wild type (left); scanning electron microscopy images of leaf abaxial surface showed that epidermal pavement cells are ;1.5 times smaller in
rps6b mutant compared with the wild type (right).
(E) Nomarski images of cleared rps6b roots show reduced TAC zone compared with the wild type; the meristem/root cap region is bracketed in orange,
and the TAC zone is bracketed in yellow.
(F) Complementation assays of rps6a and rps6b mutants: (1) PRPS6A:RPS6A genomic DNA rescued the rps6a to normal growth. (2) rps6b is completely restored by RPS6B genomic DNA. (3) and (4) rps6a and rps6b can be recovered by RPS6B and RPS6A, respectively, indicating functional
equivalency of RPS6A and RPS6B in Arabidopsis.
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Figure 9. rps6 Mutants Partially Recover Rapamycin-Treated BP12-2 Phenotypes.
(A) and (B) Rapamycin-treated rps6a/BP12-2 and rps6b/BP12-2 plants display partially suppressed slower growth and delayed bolting phenotypes
observed in rapamycin-treated BP12-2 at 35 DAG (A) and 45 DAG (B).
(C) to (F) The partial suppression of slower growth and delayed bolting phenotypes observed in rapamycin-treated rps6a/BP12-2 and rps6b/BP12-2
plants compared with rapamycin-treated BP12-2 were further defined by suppression of delayed chlorophyll degradation (C), cell death rate (D),
flowering time (E), and SAG12 expression (F). For SAG12 expression, wild-type (WT) values were set as 1. Error bars indicate SD for triplicates.
finding that the rps6b mutation mitigates early flowering and
senescence phenotypes of TOR-OE1 (Figure 10F) in the rps6b/
TOR-OE1 plants (Figure 10F) suggest that the TOR-mediated
life span phenotypes require RPS6 function. We also verified
the ability of rapamycin to specifically inhibit TOR using the
TOR-OE1xBP12-2 line. T3 generation TOR-OE1xBP12-2 plants
showed early senescence and short life span as observed in
TOR-OE1 in the absence of rapamycin, but these phenotypes
were suppressed in the presence of rapamycin (Figure 10G; see
Supplemental Table 5 online). Notably, the TOR overexpression
phenotypes were suppressed by rapamycin only in lines with the
yeast FKBP12 background, further confirming that rapamycin
specifically inhibits TOR activity.
Next, we used TOR-OE1, RPS6A-OE1, and RPS6B-OE1 lines
that have high expression of the respective transgenes (see
Supplemental Table 7 online) to determine the effect of TOR and
RPS6 on life span in Arabidopsis. Both in the soil and on MS
media, the rosette leaves of all these transgenic lines showed an
early onset of senescence (see Supplemental Figures 9A to 9C
online) with rapid decrease in chlorophyll content (at 24 DAG,
TOR-OE1 [56%], RPS6A-OE1 [30%], and RPS6B-OE1 [25%] in
contrast with 6% in the wild type) (see Supplemental Figure 9D
online) and faster cell death rate (see Supplemental Figure 9E
online). Thus, TOR and RPS6 overexpression contribute to accelerated senescence and flowering time. These observations
are consistent with SAG12 and SAG13 expression, detected in
the wild type at or after 24 DAG but expressed as early as 16
DAG in TOR and RPS6 overexpression lines (see Supplemental
Figures 9F and 9G online). At 36 DAG, SAG13 expression in
TOR and RPS6 overexpression lines was ;200 and ;1000-fold
higher, respectively, relative to the wild type (see Supplemental
Figure 9G online). Collectively, these results demonstrated that
the high TOR and RPS6 expression levels (see Supplemental
Table 7 online) confer faster growth cycle and decreased life
span in Arabidopsis.
RPS6 Expression and Localization Overlaps with TOR
We hypothesized that if TOR and RPS6 products have overlapping functions, they should colocalize to same cellular
TOR Regulates Life Span
4865
Figure 10. RPS6 Is Required for TOR Function in Arabidopsis, and Both Genes Show Overlapping Expression Patterns and Subcellular Localization.
(A) to (H) The TOR overexpression line (TOR-OE1) showed early senescence, rounded leaves with short petioles (A), and early ripening siliques (B).
Overexpression of RPS6A (RPS6A-OE1) and RPS6B (RPS6B-OE1) also produced rounded and short petiole leaves that display early senescence (C)
and early ripening siliques ([D] and [E]). The early senescence phenotype of TOR-OE1 is partially suppressed by rps6b mutant (F) and rapamycintreated BP12-2 (G). Seedlings and inflorescences of PTOR:GUS, PRPS6A:GUS, and PRPS6B:GUS lines showed similar patterns of GUS reporter
expression (H). WT, the wild type.
(I) and (J) Nuclear and cytoplasmic localization of transiently expressed RPS6A-GFP (I) and RPS6B-GFP (J) proteins in onion epidermal cells; left,
middle, and right panels show bright-field, GFP, and 49,6-diamidino-2-phenylindole, respectively.
location(s). Therefore, subcellular localization of RPS6A and RPS6B
was determined by transient expression of P35S:RPS6A-GFP and
P35S:RPS6B-GFP fusion constructs in onion epidermal cells.
The GFP signal of RPS6A and RPS6B was found localized both
in the nucleus and cytoplasm (Figures 10I and 10J), which is
similar to previously observed localization of TOR (Ren et al.,
2011). In addition to their colocalization, we also determined
that they share overlapping expression patterns. Both PRPS6A:
GUS (for b-glucuronidase) and PRPS6B:GUS were found to
be ubiquitously expressed in the seedling and inflorescence
tissues and closely resembled the expression pattern observed
with PTOR:GUS (Figure 10H). These overlapping expression
and subcellular localization patterns for TOR, RPS6A, and
RPS6B along with similar phenotypes in their loss- and gainof-function lines support our contention that these genes
perform coordinated functions in the TOR signaling pathway
in Arabidopsis.
TOR and RPS6 Modulate Protein Synthesis
Protein synthesis is integral to active growth of organisms
(Tavernarakis, 2008). We surmised that TOR- and RPS6-dependent
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pathways in Arabidopsis would also be associated with active
protein synthesis. Changes in the rate of protein synthesis was
followed by quantifying GUS activity using 4-methylumbelliferylbeta-D-glucuronide fluorometric enzymatic assays in the leaves
of the wild type and TOR-OE1, RPS6A-OE1, and RPS6B-OE1,
BP12-2, rps6a, and rps6b transgenic Arabidopsis lines carrying
a single copy of the P35S:GUS construct. The GUS activity in the
leaves of TOR and RPS6 overexpression lines was four and two
times, respectively, that of the wild-type leaves (see Supplemental
Figure 10 online), and a significant reduction was seen in rps6a
(27%) and rps6b (31%) leaves (see Supplemental Figure 9 online).
In the presence of rapamycin, GUS activity was reduced by 61%
in BP12-2 leaves compared with the wild type (see Supplemental
Figure 10 online). Thus, we inferred from this reporter study that
protein synthesis was significantly upregulated in lines overexpressing TOR or RPS6 and downregulated in TOR or RPS6
suppressors. Taken together, these data show that the role(s) of
TOR and RPS6 in growth rate and life span in Arabidopsis are
linked with rRNA, total RNA production, and protein synthesis.
DISCUSSION
Organismal life span is a fundamental and intriguing aspect of
biology. New insights into basic regulators controlling growth
and life span in yeast and animals have brought to the fore the
TOR signaling pathway as a central component of this network
(Fontana et al., 2010; Zoncu et al., 2011). As autotrophic organisms with a fundamentally different growth, development,
and differentiation regime, plants present a paradigm of their
own in this regard. In this respect, our knowledge of the involvement of TOR in these processes is rudimentary. Major
advances in the current understanding of the functions of TOR
signaling in yeast and animal systems were made possible
by the development of several key molecular, biochemical, and
genetic tools, including rapamycin-dependent conditional TOR
repression systems (Harrison et al., 2009; Bjedov et al., 2010;
Loewith and Hall, 2011). By contrast, such tools are either
lacking or limiting for addressing TOR signaling in plants. Even
though conditional rapamycin-sensitive systems using yeast
and human FKBP12 transgenes have been reported, they were
not explored extensively to dissect the TOR functions in plant
systems, especially involving mid to late phases of development (Sormani et al., 2007; Leiber et al., 2010). In this study, we
took advantage of the relevant previous findings and established
rapamycin-sensitive BP12 lines in Arabidopsis carrying the yeast
FKBP12. Our results show that rapamycin treatment caused
growth inhibition in all independent BP12 transgenic lines. This
effect was specific such that this treatment caused no detectable phenotypic differences in the growth or phenology of wildtype plants (treated or untreated) and untreated BP12 plants.
These results are consistent with earlier observations of wildtype Arabidopsis growth and development not being sensitive
to rapamycin (Xu et al., 1998; Menand et al., 2002; Mahfouz
et al., 2006; Sormani et al., 2007; Leiber et al., 2010). BP12-2 is
a single-copy line that represented the rapamycin-sensitive
lines we had generated. With a derivative of this line carrying
human S6K, we showed that rapamycin reduced T389-S6K
phosphorylation by TOR, thus establishing that TOR kinase is
inhibited by rapamycin in BP12-2. In contrast with these observations and those mentioned above, a recent report has
indicated that rapamycin affects TOR activity in wild-type
Arabidopsis (Xiong and Sheen, 2012). In that study, the concentration of rapamycin that inhibited seedling growth was
10 µM (equivalent to 9.14 mg/mL), but no phenotypic changes
were apparent at 1 and 5 µM rapamycin. Also, they used Glc in
the liquid medium where the effective rapamycin dose experienced by the seedlings is likely higher, but studies including
ours and previous reports employed solid medium and Suc. The
presence of Glc in the medium may have also contributed to the
altered responses in their study (Xiong and Sheen, 2012). In
numerous trials in experiments, we have not noticed any phenotypic change in wild-type Arabidopsis seedlings even at 21.8
mM (20 mg/mL) rapamycin. Our results clearly show that transgenic Arabidopsis lines expressing yeast FKBP12 are conditionally sensitive to rapamycin and that rapamycin specifically
targets TOR protein. In our metabolite analysis, both BP12 lines
displayed rapamycin-dependent response, and the TOR overexpression line in the presence of the FKBP12 transgene showed
rapamycin-dependent suppression of the overexpression phenotypes indicative of both specificity and function. We demonstrated
that TOR signaling is important in plant growth and longevity,
particularly in the development of roots, root hairs, leaf, and shoot.
We have also shown that TOR, in concert with RPS6, regulates
cell size, growth, flowering time, senescence, and life span in
Arabidopsis.
Inhibition of TOR Rebalances the Nutrition
and Energy Homeostasis
The roots, including the root hairs, function as efficient sensing
systems that rapidly signal rhizosphere concentrations of nutrients
and thus monitor the fluctuations in both time and space. The
elongation of roots and root hairs is essential for uptake of
nutrients from the soil/medium. In this study, we showed that
inhibition of TOR activity in Arabidopsis significantly suppressed
primary and lateral root growth by reducing TAC production,
resulting in a smaller RAM, thus also linking TOR functions to
root meristem. Furthermore, the growth of root hairs was also
significantly inhibited, consequently reducing the rate of nutrient
uptake. Consistent with this, the global transcriptome analysis
further showed that inhibition of TOR activity evokes downregulation of root hair–related genes, viz., MRH6 and LRX1. It is
interesting to note that ROL5, a putative downstream component
of the TOR pathway, was previously implicated in root development in Arabidopsis (Leiber et al., 2010). Furthermore, we
observed a reduced level of ROS signal in root hairs upon TOR
inhibition. This is consistent with an earlier study that showed
ROS accumulation in developing root hair cells was essential for
their elongation (Foreman et al., 2003). It has also been shown
that ROS induction is critical for plant development (Mittler et al.,
2011). For example, mutations in genes involved in ROS generation (e.g., respiratory burst oxidase homolog [rboh] genes
in Arabidopsis) compromise growth and development; plant
size is affected in rbohF mutants, whereas root and root hair
TOR Regulates Life Span
elongation is suppressed in rbohC (rhd2) mutants (Torres
et al., 2002; Foreman et al., 2003). Placing these root phenotypes in the context of extended life span in TOR knockdown
plants suggests that TOR likely acts as a key nutrition sensor in
plants by regulating the development of the root system and its
associated functions.
Leaves are the major photosynthetic organs that supply
carbon for plant growth. Inhibition of TOR activity significantly
reduced leaf size (Figures 2 and 7) and failed to increase growth
rate and biomass in response to increasing light intensities
(Figure 5). In addition, TOR inhibition caused downregulation of
biochemical and genetic markers of carbon fixation: ribulose as
a precursor of ribulose-1,5-bisphosphate for the Calvin cycle;
the small subunit of ribulose-1,5-bis-phosphate carboxylase/
oxygenase (which catalyzes carbon fixation), and NAD(P)H (the
reducing power) (Figure 5; see Supplemental Figures 3A and 4
online). These results suggest that TOR functions mediate nutrient uptake and light energy utilization in Arabidopsis and likely
in other plants.
TOR Inhibition Is Associated with Restructured
Metabolism, Transcriptome, and Life Span
TOR functions as a major growth regulator by integrating
the nutrient and energy requirements to promote anabolism
while inhibiting the catabolism in yeast and animals (Laplante
and Sabatini, 2012). In plants, previous studies showed that
suppression of TOR either by ethanol-induced TOR RNA interference (RNAi) or by mutation of LST8, a partner of TOR, causes
growth and developmental arrest and evokes restructuring of
metabolite/transcript profiles, including accumulation of soluble
sugars and amino acids, downregulated cell wall–associated
gene expression, hypersensitivity to high sugar concentration,
and decreased polysome/ribosome content (Deprost et al.,
2007; Moreau et al., 2012). Consistent with this, in this study,
rapamycin-treated BP12 plants resembled the wild-type plants
undergoing nutrient starvation and light energy limitation, and
this was linked to a threefold reduction in TOR kinase activity.
This translated into a reduction in growth, cell size, fresh weight,
rRNA, and protein to ;25% that of control, respectively. Importantly, when TOR is inhibited by rapamycin, BP12 plants
failed to respond to the increasing levels of nutrient and light
energy, implicating nutrition and energy sensing functions
of TOR for plant normal growth and development. To adapt
to these conditions of nutrient and energy limitation, BP12
plants likely restructured their metabolic balance by upregulating the glycolysis, amino acid, and TCA pathway, resulting
in the accumulation of their intermediates, which likely contribute as an alternate nutrient and energy source for survival.
TOR negatively regulates autophagy and inhibition of TOR
likely results in selective autophagy, which enables recycling
of cellular nutrients (Liu and Bassham, 2012). Consistent with
this, rapamycin-treated BP12 plants displayed accumulation
of the intermediates in glycolysis, amino acid, and TCA pathway. There was also a lower abundance of dipeptides paralleling the accumulation of amino acids in rapamycin-treated
BP12, implicating TOR inhibition in suppression of anabolic
processes in Arabidopsis.
4867
TOR inhibition by rapamycin treatment in BP12 plants highly
mimics the metabolite and transcriptome profiles observed in
TOR RNAi and lst8 mutant plants (Deprost et al., 2007; Moreau
et al., 2012). However, TOR RNAi caused etiolation and lethality,
whereas rapamycin inhibition in BP12 plants did not abolish the
essential functions but slowed plant growth and development.
The lethal phenotypes of the inducible TOR RNAi lines is likely
due to the inhibition of both TORC1 and TORC2 complexes
(Deprost et al., 2007), whereas the TOR repression observed in
BP12 is likely mediated through TORC1. These observations indicate the tight connectivity among TOR kinase activity, nutrient/
energy status, metabolism, and growth in plants. Taken together,
these data suggest that TOR plays several critical roles to control
nutrient and light energy–dependent growth by modulating metabolic processes.
ROS and NADPH are key metabolites involved in various
important cellular functions. In yeast and animals, mitochondria
are the major source of ROS and NAD(P)H. Therefore, increased
TCA flux would lead to an increase in ROS and NAD(P)H. In
plants, because of photosynthesis, chloroplasts are a major
source of ROS and NAD(P)H (Dietz and Pfannschmidt, 2011).
In TOR knockdown plants, severely retarded leaf development
likely resulted in lower photosynthetic efficiency, which may
account for the reduced levels of total ROS and NAD(P)H
levels compared with the wild type (Figures 5D and 5E). Glutathione reductase uses NADPH to reduce the oxidized form
of glutathione (GSSG) to the reduced form (GSH). Although
the glutathione content is increased in the metabolite profile
of rapamycin-treated BP12-2 plants, the ratio of GSH/GSSG
was significantly decreased in rapamycin-treated BP12 lines
compared with the wild-type control, indicating that BP12
plants actively dampen ROS levels at the cellular level as
part of the restructured metabolism for sustainable growth
and development in the presence of rapamycin. Decreased
levels of NADPH without a change in the ascorbate levels or
ascorbate/dehydroascorbate ratio may also explain the elevated levels of total glutathione but a lower GSH/GSSG ratio in
rapamycin-treated plants (see Supplemental Figure 11 online).
One of the specific functions of glutathione in plants is to
maintain the levels of ascorbic acid by the nonenzymatic reduction of dehydroascorbate. Ascorbic acid acts as an electron donor in the reduction and detoxification of hydrogen
peroxide and dehydroascorbate formed in the process must
be rereduced in order to prevent a drop in the total ascorbate
pool (Noctor et al., 1998). An accumulation of oxidized glutathione (GSSG) levels is known to trigger Glu-Cys ligase, the
rate-limiting enzyme in glutathione biosynthesis (Hicks et al.,
2007), which is consistent with the elevated levels of total
glutathione in rapamycin-treated plants that we have observed
(see Supplemental Figure 11 online). Recently, overexpression
of a NAC transcription factor, JUNGBRUNNEN1 (fountain of
youth), of Arabidopsis was shown to reduce ROS levels, delay
senescence, and increase longevity (Wu et al., 2012), which
agrees with our observations of lower ROS levels and increased
longevity in TOR-suppressed BP12 lines.
The polyamine pathway was upregulated, diamine putrescine and its precursors, together with its rate-limiting gene, Sadenosylmethionine decarboxylase (Mehta et al., 2002), featuring
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Figure 11. Model Explaining Potential Link between TOR Pathway and Life Span in Arabidopsis.
(A) High TOR-RPS6 expression levels confer early flowering and early onset of leaf senescence indicative of shorter life span, whereas low TOR-RPS6
expression leads to slower growth as a result of restructured metabolism, late flowering, and delayed onset of leaf senescence, which are indicative of
longer life span during postembryonic development. Loss of TOR and RPS6 functions leads to early embryo lethality.
(B) Components of the TOR signaling pathway regulate growth and life span likely through ribosome biogenesis and protein translation that influences
the overall growth rate and life span. Suppression of TOR signaling influences the overall metabolic rate via increased catabolism and decreased
anabolism, which results in delayed growth rate and extended life span. Furthermore, light energy and nutrition status have a major influence on the
TOR pathway, and characterization of other players in this process will aid to advance our understanding of life span regulation in Arabidopsis and other
important plant species.
characteristically with TOR inhibition. Interestingly, the noted
redirection of carbon (C) and nitrogen (N) metabolism in TORinhibited plants may well be connected to polyamines since
such a connection of polyamines with anabolic processes and
C:N signaling has been previously reported (Mattoo et al., 2006;
Handa and Mattoo, 2010). C:N metabolism is intimately connected with plant growth and development (Nunes-Nesi et al.,
2010). A polyamine connection with increased longevity has also
been indicated in studies with yeast and animal models (Eisenberg
et al., 2009) and delayed ripening and extended vegetative phase
of growth in transgenic tomato (Solanum lycopersicum) plants
expressing yeast spermidine synthase (Nambeesan et al., 2010).
Thus, several other cellular signals may be in tune with TOR to
integrate the nutrient and energy requirements with anabolism in
plants.
Cell wall remodeling is a limiting factor for growth. Cell growth
depends on cell elongation and expansion that involves cell wall
loosening and addition of cellulosic microfibrils (Martin et al.,
2001). The expansins and extensins are the major structural
proteins of cell wall and play an important role in restructuring
cell growth. The results from our global transcriptome analysis
showed that a significant downregulation of the extensins and
expansins occurred in TOR suppression plants, indicating that
TOR is a crucial regulator of cell wall biogenesis, consistent with
a recent study (Moreau et al., 2012).
Plants attempt to complete their lifecycle as an avoidance
mechanism against environmental stresses, such as drought
and salinity, and the life span is therefore shortened. Although
certain gene expression signatures resembling stress responses
were seen in TOR suppressed lines, these responses did not
result in hastened lifecycle. The extended life span suggests that
the TOR suppression resembles the calorie restriction–mediated
programs observed in yeast and animal model systems (Fontana
et al., 2010). The 17-d extension of life span in BP12-2 recorded in our study resembles the 25% extension of life span
in rapamycin-fed mouse (Harrison et al., 2009). These parallels
suggest conservation of several key functions of TOR, including control of life span in plants as observed in other diverse eukaryotes.
RPS6 Is an Important Mediator of TOR Signaling
in Arabidopsis
In Arabidopsis, the sequential phosphorylation cascade of TOR,
S6K, and RPS6 is suggested to be conserved (Mahfouz et al.,
2006). Notably, when Arabidopsis S6K is expressed in animal
cells, it can substitute the animal S6K to phosphorylate RPS6
protein (Turck et al., 1998). An S6K double mutant displays an
embryo lethal phenotype similar to tor knockout mutant lines in
Arabidopsis (Henriques et al., 2010). Notwithstanding this conservation, TOR-independent functions of S6K and RPS6 in
plants have also been observed (Mahfouz et al., 2006; Creff
et al., 2010; Henriques et al., 2010). Overexpression of S6K,
unlike that of TOR or RPS6, does not advance flowering or senescence in Arabidopsis (Mahfouz et al., 2006; Henriques et al.,
2010). This suggests that other upstream kinases, like pyruvate
dehydrogenase kinase and signaling factors of hormonal pathways, may be involved in the phosphorylation of S6K (Turck
et al., 2004; Mahfouz et al., 2006). The observation that rps6
mutations retard growth to a lesser extent than low light or
nutrition (in the wild-type background) and in combination with
the more severe growth retardation by TOR inhibition (in BP122) indicates that RPS6 also has some functions that are independent of TOR signaling in Arabidopsis. Indeed, another
study has shown that RPS6 as the key phosphoprotein of
the 40S ribosome is targeted by various upstream kinases in
TOR Regulates Life Span
addition to TOR and S6K kinases to regulate many biological
processes in response to various growth and stress signals (Turck
et al., 2004). Interestingly, mutations in many ribosome protein
genes of plants also display growth-related phenotypes similar
to RPS6 (Byrne, 2009), indicating that they perform similar essential functions as key components of translation machinery. It
is possible that some or majority of these ribosomal proteins are
likely regulated at least in part by TOR.
Evidence presented here favors overlapping functions of TOR
with RPS6. Unlike TOR, which is a single-copy gene in the
Arabidopsis genome, RPS6 is present as two closely related
genes (RPS6A and RPS6B). As with TOR, RPS6 function is
essential for embryo viability. Interestingly, our results show that
loss of function mutation in any one of the two RPS6 genes
results in slower growth and developmental phenotypes, such
as smaller leaves, shorter roots, and smaller RAM, similar to that
observed in rapamycin-treated BP12-2 plants. Furthermore, the
functions of RPS6 are regulated in a dose-dependent manner as
shown by the heterozygous double mutants of rps6a and rps6b
that resemble the single mutant and homozygous double mutants that are lethal like the tor mutants. Consistent with the
loss-of-function studies, overexpression of RPS6A or RPS6B
produced phenotypes that resembled TOR overexpression lines
in that they showed an opposite effect to that seen with inhibition of TOR. We therefore propose that RPS6 is a downstream component of the TOR signaling pathway in Arabidopsis.
The advancement of phase change during the vegetative development, early flowering, and onset of leaf senescence in the
RPS6 overexpression lines also show that RPS6 is a downstream component in the TOR pathway in plants. With reference
to the senescence of leaves, SAG12 and SAG13 marker gene
expression (Quirino et al., 2000) also correlated well with chlorophyll loss and greater cell death when TOR or RPS6 was
overexpressed (see Supplemental Figure 9 online). Consistent
with these observations, the old5 mutant of Arabidopsis that has
a shorter life span than the wild type undergoes accelerated
aging as shown by early senescing leaves and early SAG expression (Schippers et al., 2008). Furthermore, loss of chlorophyll in TOR or RPS6 overexpression lines likely involves internal
reallocation of storage reserves from the old leaves to the developing flowers and fruits to accelerate the phase change and
to shorten the life span as previously implied for loss of chlorophyll (Lim et al., 2007).
In summary, our results show that growth rate, phase change,
and life span of Arabidopsis are regulated through modulation
of TOR-RPS6–dependent pathways that link nutrition and light
signals. Based on our studies, we present a working model
highlighting the role of TOR pathway in plants (Figure 11). In this
model, loss of TOR or RPS6 activity results in lethality since both
are critical for cell survival (Menand et al., 2002; Creff et al.,
2010; Ren et al., 2011). However, by modulating the levels of
TOR or RPS6 expression, the growth, flowering time, and senescence can be altered to produce a longer or shorter life span
in Arabidopsis. Also, ribosome biogenesis and protein synthesis
are important players in this regard as observed in other eukaryotes (Ruvinsky and Meyuhas, 2006). As TOR couples light
and nutritional signals with ribosome biogenesis, the processes
associated with specific cellular metabolism are also implicated
4869
in the regulation of life span in Arabidopsis. Plants have a remarkable ability to survive and grow under extreme conditions,
such as very low light and nutrition deprivation, and display
a remarkable plasticity in growth and life span. It is conceivable
that TOR and its associated signaling components contribute to
making plants resilient under unfavorable environments. Future
research in plant TOR signaling should help to advance the critical
understanding and provide new insights in this important field.
METHODS
Arabidopsis thaliana Growth and Transformation
In this study, the wild-type Arabidopsis Columbia (Columbia-0) ecotype was
used. Arabidopsis seedlings and plants were grown in growth chambers
with 22°C and 16-h/8-h light/dark cycle settings unless indicated otherwise. Transgenic plants were generated by the floral dipping method
(Zhang et al., 2006). Arabidopsis growth, transformation, and screening of
primary transformants were performed according to the published protocols (Zhang et al., 2006).
Isolation and Characterization of T-DNA Insertion Lines
The mutants rps6a (SALK_012147) and rps6b (SALK_061539) in Columbia
background were obtained from the ABRC. Detailed information describing mutant lines is listed in Supplemental Table 8 online. The
knockout lines were identified and confirmed by PCR using primers
designed using the T-DNA Primer Design website, http://signal.salk.edu/
tdnaprimers.2.html. The details of these primers are listed in Supplemental
Table 9 online.
Growth Rate Measurement
To measure the rate of plant growth, sterilized seeds were planted on the
nutrient-solidified agar plates. Plates were sealed with Parafilm and held
vertically in the growth chamber under the indicated light and temperature
conditions. The seedling samples were harvested, and the fresh weight
was examined 11 DAG. For rapamycin treatments, exposure of BP12
plants to different concentrations (0.5, 1, 5, 10, and 20 µg/mL) of rapamycin showed that 10 mg/mL rapamycin was the optimal concentration to
inhibit TOR as also documented in the previous study (Sormani et al.,
2007). In nutrition limitation or light limitation conditions, the wild-type
control and BP12-2 transgenic plants or rps6 mutants were germinated on
the same plate containing 10 µg/mL rapamycin (Bioshop Canada) or the
same nitrogen/carbon concentration unless indicated otherwise. Rapamycin was dissolved in DMSO (Fisher ChemAlert). It was noted that MS
plates containing 1 µL/mL DMSO had no effects on the growth of wildtype, BP12-1, BP12-2, BP12-4, and BP12-5 plants compared with MS
plates lacking DMSO (see Supplemental Figure 1B online). Therefore, MS/
DMSO plates are referred as MS plates in all the rapamycin treatment
assays. Each fresh weight data point represents the average of three
independent experiments.
Generation of Overexpression Constructs
The full-length coding sequence of RPS6A (750 bp) and RPS6B (753 bp)
was amplified by RT-PCR using the Advantage 2 Polymerase Mix kit
(Clontech) following the manufacturer’s instructions. RT-PCR primer pairs
for amplification of the full-length genes from Arabidopsis were designed
based on the cDNA sequence (http://www.Arabidopsis.org). These primers introduced a NotI site at the 59 end of the respective forward primer
and an XmalI site in the 39 end of reverse primer. The PCR products, with
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The Plant Cell
the 59 NotI site and 39 XmalI site, were cloned into the TA cloning vector
pCR2.1-TOPO (Invitrogen). The recombinant clones were verified by DNA
sequencing and cloned into p8GWN (Ren et al., 2011) to generate the
Gateway system–based Entry vector (Earley et al., 2006). Similarly, the
coding sequence of full-length yeast FKBP12 (345 bp) containing NotIXmaI restriction sites was PCR amplified from yeast genomic DNA and
cloned into the NotI-XmaI sites of LR reaction gateway entry vector
p8GWN (Ren et al., 2011) to generate Gateway entry vector constructs.
The Gateway system (Invitrogen) was employed for creating different
overexpression constructs, which were developed by transferring target
genes from p8GWN into the appropriate pEarleyGate vectors (pEarleyGate 201 containing HA tag; pEarleyGate 201 containing cMyc tag and
pEarleyGate 103 containing GFP) through LR recombination reactions in
Escherichia coli (Earley et al., 2006) to generate RPS6A-OE(P35S:HARPS6A), RPS6B-OE(P35S:cMyc-RPS6B), P35S:RPS6A-GFP, and P35S:
RPS6B-GFP. The lines of TOR-OE (P35S:TOR) were described previously
(Ren et al., 2011). The resulting plasmids were used to transform wild-type
Arabidopsis plants (Columbia-0) for developmental and phenotypic
analyses or onion (Allium cepa) cells for cellular localization studies.
Total protein was extracted from 100 mg of fresh leaf tissue obtained
from 3-week-old plants with 300 mL extraction buffer (0.1 M Tris-HCl, pH
8.0, 0.01 M MgCl2, 18% [w/v] Suc, and 40 mM 2-mercaptoethanol). SDS
(2%, w/v) was added to extraction buffer to solubilize membrane and
other insoluble proteins. Protein samples were subjected to SDS-PAGE
and transferred to Hybond-P polyvinylidene difluoride transfer membrane
(Amersham). Membranes were blocked with 5% ECL blocking agent
(dissolved with 13 TBST) and then incubated with 1:2000 diluted primary
antibody (mouse anti-c-Myc). After three washings with 13 TBST, the blot
was incubated with 1:5000 diluted horseradish peroxidase–conjugated
goat anti-mouse IgG antibodies. Supersignal west pico chemiluminescent substrate (Thermo Scientific) was used for signal detection.
Quantitative Real-Time PCR
Real-time PCR was performed as described previously using the Step
One Real-Time PCR system from Applied Biosystems (Ren et al., 2011).
The primers used in this study are listed in Supplemental Table 11 online.
Biochemical Assays
Generation of Promoter-GUS and Complementation Constructs
Based on our previous PrRNA:GFP construct (Ren et al., 2011), the PrRNA
was replaced by the promoters of TOR (2.7 kb), RPS6A (1.2 kb), and
RPS6B (2.1 kb) through AsisI and NotI restriction sites, respectively.
Furthermore, the GFP was replaced by 1.8-kb GUS marker gene containing NotI-XmaI restriction sites and PCR amplified using forward primer
GUSF and reverse primer GUSR (see Supplemental Table 10 online). The
constructs of PTOR:GUS, PRPS6A:GUS, and PRPS6B:GUS were transferred into destination vector pEarleyGate303 through LR recombination
reactions. Based on PRPS6A:GUS and PRPS6B:GUS constructs, the GUS
gene was replaced by 2632-bp genome fragment containing the RPS6A
and 1406-bp genome fragment of RPS6B coding sequence to generate
PRPS6A:RPS6A and PRPS6B:RPS6B complementation vectors. The
corresponding primers are listed in Supplemental Table 10 online. The
resulting plasmids were used to transform wild-type Arabidopsis plants
(Columbia-0) for expression analysis and rps6a or rps6b mutants background for complementation studies.
GFP Transient Expression and Particle Bombardment
Particle bombardment was performed as described (Ren et al., 2011).
For the GUS assay, the GUS activity and staining were performed as
described previously (Jefferson et al., 1987; Jefferson, 1989).
For the NAD/NADH and NADP/NADPH assay, 11-DAG wild-type and
BP12-2 seedlings were collected in the presence of rapamycin, ground in
liquid nitrogen, and assayed as described in the e.ENZYME LLC kit instructions. Three biological replicates were used for each assay.
For the p70-S6K [pT389] phosphorylation assay, S6K/BP12-C seedlings
at 15 DAG were treated with rapamycin, dark and nutrient starvation for
3 d by transferring the seedlings from half-strength MS medium to halfstrength MS + 10 mg/mL rapamycin, half-strength MS + dark, and 0.0013
MS, respectively, along with control (half-strength MS + 1 mL/mL DMSO)
were used. The ELISA assay was performed on seedling extracts made
following the kit manual (p70-S6K [pT389] human ELISA kit; Invitrogen).
Three biological replicates were used for each assay.
ROS Detection
The probe 29,79-dichlorofluorescin diacetate (H2DCFDA) (Molecular Probes/
Invitrogen) was used at 20 mM to detect ROS. The roots were treated with the
H2DCFDA for 45 min at 4°C and washed twice in the same buffer, and then
images were captured with Leica SP5 confocal microscope using 488-nm/
522-nm excitation/emission.
Measurements of Chlorophyll Content and Cell Death Rate
The relative chlorophyll content, cell death rate, and SAG12/SAG13
expression level in the first leaf pair was measured every fourth day
starting from 20 to 44 DAG. Chlorophyll was extracted from the first leaf
pair and quantified (Weaver and Amasino, 2001). The chlorophyll content
at 20 DAG was used as the reference (values set as 100%), to which the
relative chlorophyll contents in experimental samples were compared.
The cell death rate was determined after spectrophotometric measurement of absorbance at 600 nm (Guo and Crawford, 2005). The cell death
rate at 44 DAG was used as the reference control (values set as 100%) for
the relative cell death rates shown in the figures. Each data point represents the average of three pools of the first leaf pair taken from separate
plants at each sampling time.
DNA Gel Blotting and Immunoblotting
The genomic DNA samples were isolated from the leaves at 20 DAG of
BP12 lines. DNA gel blotting was performed using DIG-High prime DNA
labeling and detection starter kit II (Roche) following the protocol provided
with the kit.
Microscopy
Cellular details were observed with a Leica DMR microscope after clearing
the tissues in a chloral hydrate solution (8:1:2 chloral hydrate/glycerol/
water [w/v/v]), and the images were captured using a Microfire camera
(Optronics). Scanning electron microscopy was performed as described
previously (Venglat et al., 2002). Confocal images of the palisade and
spongy mesophyll cells were captured with the Leica SP5 confocal microscope after staining the tissues with propidium iodide.
Metabolite Analysis
Wild-type, BP12-2, and BP12-5 seedlings (incubated in growth chambers
with the following settings: 22°C, 16-h-light [7 AM to 11 PM]/ 8-h-dark cycle,
and 100 µmol m22 s21 light intensity) at 15 DAG were treated with rapamycin for 3 d by transferring from 0.53 MS medium to 0.53 MS +
10 mg/mL rapamycin along with control (0.5 MS + 1 mL/mL DMSO).
Samples collected between 1 and 2 PM were ground in liquid nitrogen and
freeze dried for 6 d under vacuum. Four biological replicates for each of
TOR Regulates Life Span
these samples were further processed and analyzed by Metabolon for global
unbiased metabolite profiling involving a combination of three platforms:
ultra-HPLC–tandem mass spectrometry optimized for basic species,
ultra-HPLC/tandem mass spectrometry optimized for acidic species, and
gas chromatography–mass spectrometry. Methods used were as described previously (Evans et al., 2009; Oliver et al., 2011).
Metabolite Heat Map Generation
Wild-type, BP12-2, and BP12-5 seedlings at 15 DAG were treated with
rapamycin for 3 d by transferring from 0.5 MS medium to 0.5 MS + 10 µg/mL
rapamycin along with control (0.5 MS + 1 µL/mL DMSO). Four lanes in the
heat map for each treatment correspond to four biological replicates. Raw
data for each compound from the entire metabolic profiling data set were
scaled to the median of the detected values, and null values were imputed
with the minimum value detected for the compound. A log2 transformation
was then performed, which rendered the median values as zero. False color
was applied as indicated in the legend scale, in both cases with color
saturation at two log2 units above or below the median, with yellow representing values higher than the median and blue representing values lower
than the median. Compounds in the figure were blocked by general
pathway group as indicated. Supplemental Figures 3A to 3E online list
the respective compound names and their mean values for each
pathway group. Rapamycin, indicated by red arrow, was at detectable
levels in all treated plants, but not in any of the untreated plants, as
expected.
RNA-Seq Data Analysis
Paired-end alignments were obtained through aligning short reads onto
the reference Arabidopsis genome (TAIR9) using Bowtie (http://bowtie-bio.
sourceforge.net/index.shtml). Approximately 70% of the reads mapped to
at least one genomic location on either of the two strands. For those reads
that did not align to the genome, a second-round alignment was performed
in which we recursively trimmed one base at a time (up to 21-mers) at either
the 59 or 39 end of the reads until we found a (perfect) match to the genome.
Finally, more than 80% of the reads mapped onto the genome. Htseq-count
(http://www-huber.embl.de/users/anders/HTSeq/doc/count.html) was used
to count the reads from the Bowtie derived output files. Differentially
expressed genes were identified using edgeR (http://www.bioconductor.
org/packages/release/bioc/html/edgeR.html). The false discovery rate–
corrected P value for differential expression was set to be #0.05.
For Gene Ontology (GO) analysis, GO terms were obtained from The
Arabidopsis Information Resource. GO enrichment analysis was conducted as described previously (Fu et al., 2009; Paliouras et al., 2011).
Specifically, to identify differentially expressed GO terms, we considered
the gene expression values for the GO analysis using parametric analysis
of gene set enrichment (Kim and Volsky, 2005).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: TOR, At1g50030; yeast FKBP(12), YNL135C; Arabidopsis S6K,
At3g08730; human S6K, NM_003161; RPS6A, At4g31700; and RPS6B,
At5g10360. The RNA-seq data have been uploaded to the Gene Expression Omnibus under accession number GSE42968.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Functional Analysis of BP12 Lines and
Subcellular Localization of GFP-Fused FKBP12 Protein in Arabidopsis.
4871
Supplemental Figure 2. Molecular Identification of Transgenics and
Analysis of Human S6K1 Phosphorylation Status in Arabidopsis.
Supplemental Figure 3A. Heat Map of Biochemicals in the Carbohydrate Pathway.
Supplemental Figure 3B. Heat Map of Biochemicals in the Amino
Acid Pathway and Polyamine Subpathway.
Supplemental Figure 3C. Heat Map of Biochemicals in the Peptide
Pathway.
Supplemental Figure 3D. Polyamine Pathway Showing Metabolites in
Red and Blue That Are Significantly Up- and Downregulated
Supplemental Figure 3E. Heat Map of Biochemicals in the Secondary
Metabolism Pathway.
Supplemental Figure 3F. Heat Map of Biochemicals in the Lipid,
Cofactors, and Nucleotide Pathway.
Supplemental Figure 4. Gene Expression Levels in RapamycinTreated BP12-2 Plants Compared with the Wild Type.
Supplemental Figure 5. Rapamycin Treatment at Preflowering Stage
Delayed the Growth Rate and Phase Change in BP12-2 Plants.
Supplemental Figure 6. Molecular Identification of rps6a and rps6b
Mutants.
Supplemental Figure 7. rps6 Mutants Show Nutrition- and LightDependent Growth Performance.
Supplemental Figure 8. Molecular Identification of RPS6A-OE1 and
RPS6B-OE1 Transgenic Plants by DNA PCR, RT-PCR, and Immunoblot Analyses.
Supplemental Figure 9. TOR, RPS6A, and RPS6B Overexpression
Result in Early Onset of Senescence.
Supplemental Figure 10. Levels of TOR and RPS6 Determine the
Rate of Protein Synthesis as Measured by GUS Reporter Expression in
Arabidopsis.
Supplemental Figure 11. Proposed Mechanism of Rapamycin-Induced
Redox and Metabolite Alterations in BP-12 Plants.
Supplemental Table 1. Different Concentrations of MS Medium Used
in This Study.
Supplemental Table 2. Different Nitrogen Concentration Used in MS
Medium.
Supplemental Table 3. Different Suc Concentration Used in the MS
Medium.
Supplemental Table 4. Analysis of GFP Expression Levels of PrRNA:
GFP in the Wild Type and BP12-2 Background under Different Nutrition
and Rapamycin Treatment Conditions.
Supplemental Table 5. Growth Stages and Leaf Numbers of BP12-2,
rps6a, rps6b, rps6b/BP12-2, TOR-OX1, and TOR-OX1/BP12-2 Grown
on MS + Rapamycin Plates compared with Wild-Type Control.
Supplemental Table 6. Growth Stages and Leaf Numbers of rps6a,
rps6b, TOR-OE1, RPS6A-OE1, and RPS6B-OE1 and Wild-Type Lines
Grown in Soil.
Supplemental Table 7. Expression Levels of Transgenes in TOR-OE1,
RPS6A-OE1, and RPS6B-OE1 Arabidopsis Lines.
Supplemental Table 8. Characterization of T-DNA Insertion Lines and
Their Phenotypes.
Supplemental Table 9. PCR Primers Used for Identification of SALK
Knockout Lines.
Supplemental Table 10. PCR Primers Used for Cloning.
4872
The Plant Cell
Supplemental Table 11. Primers Used for Real-Time PCR.
Supplemental Methods 1. Identification of RPS6 Single and Double
Mutants in Arabidopsis.
Supplemental Data Set 1. List of genes from the RNA Seq data that
are differentially expressed in BP12-2 seedlings treated with rapamycin (P-Value < 0.05).
Supplemental Data Set 2. GO analysis of functional categories of
genes from the RNA Seq data that are differentially expressed in
BP12-2 seedlings treated with rapamycin (P-Value < 0.05).
Supplemental Data Set 3. Functional categories of genes from the
RNA Seq data that are differentially expressed in BP12-2 seedlings
treated with rapamycin (P-Value < 0.05). A- root; B- cell wall; Ctransporter and D- defense response.
ACKNOWLEDGMENTS
This work was supported by the National Research Council of Canada
Genomics and Health Initiative, Genome Canada, and Genome Prairie.
We thank Paul Arnison, Faouzi Bekkaoui, and Adrian Cutler for critical
reading and comments on the article. This is National Research Council
of Canada publication number 50192. Mention of trade names or
commercial products in this publication is solely for the purpose of
providing specific information and does not imply recommendation or
endorsement by the USDA.
AUTHOR CONTRIBUTIONS
M.R. and R.D. designed the experiments. P.V., M.R., and S.Q. performed
microscopy and developmental analyses. M.R., L.F., and J.W. made the
constructs, produced the transgenic lines, and performed the growth
and physiological assays. D.A, M.R., P.V., G.S., S.C., A.M., and R.D.
performed and analyzed metabolite data sets. E.W., M.R., P.V., Y.C.,
A.M., G.S., and R.D. performed and analyzed transcriptome data sets.
Y.C. and M.R. performed DNA gel blot analysis. S.Q., L.F., and M.R.
performed immunoblots and real-time PCR. D.X., D.L., and S.C. provided
valuable advice and new reagents. M.R., P.V., and R.D. prepared the
article with significant contributions from G.S., S.C., and A.M.
Received November 5, 2012; revised November 5, 2012; accepted
December 8, 2012; published December 28, 2012.
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Plant Cell 2012;24;4850-4874; originally published online December 28, 2012;
DOI 10.1105/tpc.112.107144
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