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Energy homeostasis, Growth, Stress response, SurvivalDevelopment, Reproduction, Senescence
GlucoseSucrose
KIN10/11
Gen
e ex
pres
sion
PPs
Post-translational
Supplementary Figure 1 | KIN10/11 are central integrators of sugar, metabolic,stress, and developmental signals (see next page for Figure Legend)
TFs
AMP
SUPPLEMENTARY INFORMATION
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Supplementary Figure 1 | KIN10/11 are central integrators of sugar, metabolic,stress, and developmental signals. Plants are constantly challenged by multiple types ofstress that ultimately converge as an energy-deficiency signal in the cell, triggering the activationof KIN10/11. Conversely, sugars have a repressive effect, and at least in autotrophic tissues bothglucose and sucrose inhibit KIN10/11 action even when the stress factors persist. Upstreamprotein kinases (PKs), protein phosphatases1(PPs), and additional regulatory subunits2-6 maycontribute to the fine-tuning of the system and possibly confer tissue and cell-type specificity.Activated KIN10/11 initiates an energy-saving program at several levels, aiming at repressingbiosynthetic pathways and at promoting catabolic processes and photosynthesis to increase ATPgeneration. KIN10/11 directly and indirectly regulates key metabolic enzymes to inhibit specificassimilation pathways7-9. Remarkably, KIN10/11 regulation involves massive transcriptionalreprogramming that affects a wide range of both plant-specific and evolutionarily conservedpathways. This is partly mediated by the S-class of bZIP transcription factors. In addition tocontributing to the maintenance of cellular energy homeostasis and tolerance to (nutrient) stress,KIN10/11 have profound effects at the whole organism level influencing growth, viability,reproduction and senescence. KIN10/11 seem to be the convergence point linking multiplemetabolic, stress and developmental signals, thus playing a fundamental and highly conserved rolein vegetative and reproductive growth and survival. NR, nitrate reductase; SPS, sucrose phosphatesynthase; HMG-CoAR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
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aLUC/GUS
hypoxia + glucose
hypoxiacontro
l5 mM
10 mM25 mM
50 mM100 mM
50 mM
sucrose
048
121620
DIN6-LUCb
control treatmentD
IN1
Supplementary Figure 2 | Regulation of DIN6 expression. a, DIN1 and DIN6 are repressedby light and sugar but induced by multiple types of stress10-19. The data were obtained by using themicroarray database mining tool (Meta-Analyzer) provided by Genevestigator20. b, DIN6-LUCinduction by hypoxia is repressed by glucose and sucrose. Error bars indicate standard deviationsfrom three independent experiments. c, The PK inhibitor K252a (K) inhibits the induction of DINgene expression by darkness and stress. D, dark; H, hypoxia. Control, DCMU and hypoxia treatmentswere performed under light.
DIN
6
c
Contro
l
D+KD H H+KDCMUDCMU+K
Contro
l
D+K
D H H+KDCMUDCMU+K
UBQ4
DIN6
DIN1
Leaves Protoplasts
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Supplementary Figure 3 | Expression patterns of Arabidopsis genes encoding theputative catalytic and regulatory subunits of SnRK1. a, The Arabidopsis SnRKsuperfamily comprises 3 groups. The SnRK1s are most homologous to yeast Snf1 and mammalianAMPK. b, RT-PCR analyses. KIN10 and KIN11, but not KIN12 are expressed in Arabidopsisseedlings and mesophyll protoplasts. c, Expression of the catalytic subunit genes, KIN10, KIN11,KIN12 and the genes encoding putative regulatory subunits (α, β, γ, βγ)2-5 in the indicated plantmaterials and treatments. Without co-expression of β and γ regulatory subunits, an overexpressedmammalian AMPK catalytic α subunit was inactive and degraded21. Possibly, plant-specificregulation22,23 or/and sufficient endogenous regulatory β and γ proteins were present to supporttheir activities when KIN10/11 were overexpressed in leaf cells. The data were obtained from thegene expression search program provided by the Arabidopsis Membrane Protein Library (AMPL,http://www.cbs.umn.edu/arabidopsis/). 1, whole plant; 2, whole plant, ozone-treated; 3, wholeplant, constant light; 4, whole plant, constant light, water treated; 5, whole plant, constant light,glc treated; 6, primary roots, 1% suc; 7, lateral roots; 8, lateral roots, nematode infection; 9,shoots; 10, shoots, treated at 4ºC; 11, petioles; 12, active axillary buds; 13, dormant axillary buds;14, mature pollen; 15, suspension cells.
KIN11
KINβ1
KINγ
KINβ2
KINβ3
a1 2 3 4 5 6 7 8 9 10 1112 13 14 15
c
KIN10(At3g01090)
(At3g29160)
KIN12(At5g39440)
(At5g21170 )
(At4g16360)
(At2g28060)
KINβγ(At1g09020)
(At3g48530)
b
SnRK3
SnRK1Snf1SnRK1.3/KIN12SnRK1.1/KIN10SnRK1.2/KIN11
AMPKα1AMPKα2
(yeast)
(mammals)SnRK2
KIN1
0KI
N11
KIN1
2
KIN1
0KI
N11
KIN1
2
KIN1
0KI
N11
KIN1
2
genomic DNA
cDNA (RNA)seedlings
cDNA (RNA)protoplasts
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anti-HA
CKIN10
K48MKIN11
K49MPK
act
ivity
04812 SPS peptide
RBCL
anti-P-AMPK
anti-HA
0
4
8
PK a
ctiv
ity
CKIN10
K48MT175A
RBCL
SPS peptide
KIN10-HA
P-KIN10P-KIN11
P-KIN10-HA
Supplementary Figure 4 | KIN10 and KIN11 are active kinases. a, KIN10 andKIN11 expressed in protoplasts displayed the characteristic and conserved ability of SnRK1 tophosphorylate a specific peptide substrate (RDHMPRIRSEMQIWSED) derived from sucrosephosphate synthase (SPS)24,25. K48M and K49M are inactive KIN10 and KIN11 mutantcontrols, respectively. b, Phosphorylation of T175 is essential for KIN10 activity. KIN10 andKIN11 phosphorylated at the conserved T17526 are recognised by an antibody againstphosphorylated and activated mammalian AMPK on T172 (anti-P-AMPK). KIN10 is probablyphosphorylated by endogenous upstream kinase(s)21, but reduced phosphorylation levels ofK48M suggest also autophosphorylation. Total recombinant KIN10 and KIN11 were detectedwith an anti-HA antibody. RBCL coomassie staining served as endogenous protein control. P-KIN10, KIN10 phosphorylated at T175; C, control DNA. Error bars indicate standarddeviations (n=3).
a
b
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DIN6 PROMOTER SEQUENCE (-624) TGTATTCACTTTCTGATAAAATGCTAATCCTACAATCAAATGCAAGTGGTTCC T T ACCATTGTCGTGATAACACGTGTACGGCTCTAAAGCAATCAGAACAATCATT G2 D3 T GGACAGTTTTTACACCGTCAGATAAGTACCTATCCACTTGCTGACTCAGCCG T GATAAACCCTAAACCGGAAGTTTGCCCCACCGTCAAAATTGGAAGAAACCG T T A GACAAAAGAGAATGTAAAGACTAAGAAGTAAGAACCCATCGGACGTCGTAA D2 D1 C T A GAAGGTTAATTAACACGTGGAAACAGCTGGTCAGAGTTATCCGGTAACTTAT G1 CCGGTTACAAGTAAAAAAATAATTTGTTCCCATACACGACTCCTTCAGAACC AAACGCGACATCACGGCGCCGTTTAGTGTCTATAAATAGAGCAATCGGTCG TAGAAAACCAAGACATCAAAAACACGAATATCGATAGTACACTTCTACGTGC AATTTTCTCCTTTCTCTTCCTGGACATCTGTCTGTTTATTACATTTTCTTGTAA TCTCTTTTTGGGGTTTTACAATATCTATCCCCTAAAGTTTCGGAAAATTCTGT TTTTCTGTTCTCATTCTTCGTGATCTTTTTCACTTTCTTCAAAAAAAAAAC ATG
Supplementary Figure 5 | Regulation of DIN6 expression. DIN6 promoter sequences and selected putative regulatory cis-elements (in red and underlined)27. Mutated nucleotides are in bold. G1 and G2 represent G-box elements (CACGTG); C a C-box element (GACGTC). Both are bound by bZIP TFs28-30. D1, D2 and D3 indicate DOF TF binding sites (A/TAAAG). Plant-specific DOF TFs mediate stress, light and hormone signaling and control carbon and nitrogen metabolism31,32. T indicates the TATCCA element, together with the G-box isolated as an essential element of a sugar response sequence (SRS) required for expression of a rice α-amylase gene under starvation conditions33. The TATA-box and start codon are in bold, the leader sequence in blue.
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Transient KIN10 expression in protoplasts
RNA isolation-hybridization to ATH1 22K GeneChip
Data import, quality control,and preliminary filtering through GCOS (1822 genes)
Selection of 25 potential marker genes
Confirmation of marker gene expression using biological replicates and qRT-PCR
List A
Data import through rma
RankProd analysis
Further filtering of GCOS data based on marker gene expression
Data filtering based on marker gene expression
List B List C
Data import through gcrma
KIN10 target genes (1021)
A
B CD
Step 1
Step 2 KIN10-induced marker genes
Screen public databases and literature for matching expression patterns
four starvation-related datasets three sugar- or CO2- treatment datasetsGroup 1-positive correlation Group 2-negative correlation
filter
If fold-change of gene X > 2 in KIN10 gene listit should be > 0 in all datasets of Group 1 and < 0 in all datasets of Group 2
If fold-change of gene X < -2 in KIN10 gene listit should be < 0 in all datasets of Group 1 and > 0 in all datasets of Group 2
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GBF5 targets
DIN6 UDG
PPDK AXP
SEN5 LIP
DIN10
PGPD14
DIN1 INV
TPS8
KIN
10
sen
star
v
ext n
ight
suc
star
v
sen
dark
sucr
ose
gluc
ose
hypo
xia
DIN6UDGPPDKAXPSEN5LIPDIN10PGPD14DIN1INVTPS8
CO
2 fix
atio
n
2.0-2.0 0.0
log2 ratioa
6.1±1.2
9.2±2.6
6.5±1.1
2.0±1.6
6.1±1.9AXPTPS819.7±3.7SEN5
36.8±2.3DIN1
PGPD148±1.2UDG
17.1±1.9DIN6
fold changeGenefold changeGene
Induction byhypoxia treatment (n=3) qRT-PCR
4.9±1.3LIP
5.7±1.6PPDK
INV
10.6±1.6DIN10
CONTROL
b
f
Supplementary Figure 7 | Global gene expression regulation by KIN10. a,Selected KIN10 marker genes are activated by hypoxia and other types of cellular energystress. b, Analysis of hypoxia induction of KIN10 marker genes (Supplementary Table 2a) byqRT-PCR. c-e, Transient KIN10 expression in protoplasts results in the induction (c) andrepression (d, e) of genes involved in a wide variety of cellular processes and metabolism. f,GBF5 induces expression of endogenous KIN10 target genes. ext: extended; sen: senescence;starv: starvation; suc: sucrose, PK: protein kinase; PP: protein phosphatase. Error bars indicatestandard deviations (n=3).
KIN10repression
hormone response/metabolism
c-2.0 2.00.0
log2 ratio
amino acid degradation
transcriptionregulation
trehalose
lipid degradation
autophagy
KIN10activation
cell wall
DNA/chromatin
PKs/PPs
KIN
10
sen
star
vex
t nig
ht
suc
star
v
sen
dark
sucr
ose
CO
2 fix
atio
ngl
ucos
e
-2.0 2.00.0
log2 ratiod
protein synthesis
KIN
10
sen
star
vex
t nig
ht
suc
star
v
sen
dark
sucr
ose
CO
2 fix
atio
ngl
ucos
e
e
amino acid synthesis
nucleotidemetabolism
DNA/chromatin
cell wall
transcriptionregulation
PKs/PPs
KIN
10
sen
star
vex
t nig
ht
suc
star
v
sen
dark
sucr
ose
CO
2 fix
atio
ngl
ucos
e
-2.0 2.00.0
log2 ratio
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WTO1 10
-1 WTO1 10
-1WT
O1 10-1
anth
ocya
nin
O1 O210
-110
-2WT0
1
2
3
KIN10
KIN11
UBQ4
WTO1
10-1
mRNA
KIN10-HAKIN10
anti-KIN10
anti-P-AMPK
RBCL
P-KIN10-HAP-KIN10P-KIN11
Supplementary Figure 8 | Overexpression of KIN10 affects plant development,senescence and starvation responses. a, KIN10 overexpression (O1) and silencing(10-1) in transgenic plants were examined by RT-PCR and immunoblotting with anti-KIN10and anti-P-AMPK antibodies. Epitope-tagged transgenic KIN10-HA and endogenous KIN10protein levels were detected with a KIN10-specific antibody. P-KIN10-HA represented themajor phosphorylated form in the overexpression lines. KIN10 overexpression stimulatesgrowth in the absence of exogenous sugar (b, e), but reduces growth promotion by 1-3%sucrose (c, d, e). KIN10 overexpression prevents sucrose-induced anthocyanin accumulation(e) Error bars indicate standard deviations (e, n>60; f, n=3). Asterisks indicate statisticallysignificant differences (p<0.05).
a b c d
e
1% suc
3% suc
no suc
root
leng
th (c
m) WT O1 10-1
WT O1 10-1
WT O1 10-1
f
1% suc
3% suc
no suc
0.51.5
2.53.5
0.5
1.5
2.5
0.1
0.30.5
0.7
*
*
*
*
*
*
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PGPD14
0
10
20
30SEN5
0
20
40
60
CONTROL
0
1
2
3
O110
-210
-1WT O2d-2d-111
DIN10
0
10
20
30
40
10-2
10-1WT O2O1
d-2d-111
DIN10
0
20
40CONTROL
0
1
2
dark DCMUWT
WT d-1 d-1
SEN5
0
400
800
1200
dark DCMUWT
WT d-1 d-1
0
20
40
60 PGPD14
P-KIN10-HA
KIN10-HAKIN10
P-KIN10P-KIN11RBCL
10-2
10-1WT O2O1d-2d-111
anti-P-AMPK
anti-KIN10
Petiole length (mm) – leaves 11-12 (n=5)WT
130±2011
130±1210-1
152±14d-1
28±1110-2
170±23d-2
26±15O1
126±15O2
116±11
a b
c d
Supplementary Figure 9 | KIN10/11 silencing results in altered morphology anddisrupts transcriptional activation by stress. a, WT, KIN10 overexpression (O1, O2), kin10RNAi (10-1, 10-2), kin11 VIGS (11) and the kin10 kin11 VIGS double mutant (d-1, d-2) plants weresimilarly infiltrated with a control GFP or a KIN11 construct in the viral vector and examined after 3weeks using anti-KIN10 and anti-P-AMPK antibodies. b, The double mutants exhibit a dramaticreduction in petiole length. c-d, The KIN10 target gene response to various stresses is abolished inthe kin10 kin11 double mutants. Gene expression was measured by qRT-PCR (see SupplementaryTable 2a for gene annotation details and Supplementary Table 6 for other genes and numeric values)from hypoxia-treated mesophyll cells (c) and dark- or DCMU-treated leaves (d). Control, DCMU,and hypoxia treatments were performed under light. Error bars indicate standard deviations (c, n=3;d, n=2).
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SUPPLEMENTARY TABLES
Supplementary Table 1 (Microsoft Excel file). Global gene expression regulation
by KIN10 and hypoxic conditions. Raw data. Gene expression changes induced by 6h
of transient KIN10 expression or hypoxia treatment in Arabidopsis protoplasts. Eight µg
of total RNA was used for labeling and hybridization on 22K ATH1 Arabidopsis
GeneChips®. The data were compiled and normalized using the GCOS v. 1.0 software.
The document contains an Excel file with original unfiltered data from two biologically
independent replicates of the KIN10 experiment and one hypoxia experiment.
Supplementary Table 2. Experimental validation of selected KIN10 marker genes
by quantitative real time PCR (qRT-PCR). Twenty five putative KIN10 marker genes
covering diverse functional categories were selected based on their high (100-fold) or
moderate (3-fold) transcriptional changes upon KIN10 expression, as revealed in the
GeneChip® experiments described in Supplementary Table 1 (see Supplementary
Methods for details). Their expression changes were confirmed by two independent
protoplast transfection experiments and qRT-PCR analysis.
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Supplementary Table 2 (cont.) a, Induced KIN10 marker genes