DELAY OF GERMINATION1 DOG1) Regulates both Seed … · Seed Germination Assays For lettuce seed...
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Supplementary Information (SI Appendix)
Huo et al. (2016) – www.pnas.org/cgi/doi/10.1073/pnas.1600558113
DELAY OF GERMINATION1 (DOG1) Regulates both Seed Dormancy and Flowering Time
through MicroRNA Pathways
Heqiang Huo, Shouhui Wei, Kent J. Bradford
Supplementary Methods
Supplementary Text - Screening for altered flowering pathways in lettuce LsDOG1-RNAi plants
Supplementary Figures
Figure S1. Temperature sensitivity of different lettuce genotypes and characterization of lettuce DOG1
expression and function.
Figure S2. Protein amino acid sequence alignment of Arabidopsis Col DOG1 (AtDOG1) and lettuce
Salinas DOG1 (SalDOG1).
Figure S3. Gibberellin (but not fluridone, an ABA biosynthesis inhibitor) alleviates primary dormancy of
Arabidopsis seeds caused by overexpression of LsDOG1.
Figure S4. Relative mRNA levels of genes in different pathways regulating flowering.
Figure S5. Potential interactions of the DOG1-miR156-miR172 module in regulating developmental
phase transitions.
Figure S6. Overexpression of LsMIR156 resulted in delayed flowering in lettuce.
Figure S7. Relative mRNA and miRNA levels in leaves and apical meristems of Arabidopsis at different
developmental stages.
Figure S8. Primary dormancy of Col-WT, dog1-3 and dog1-5 seeds.
Supplementary Tables
Table S1. DOG1 protein amino acid sequence similarity among four lettuce and two Arabidopsis
genotypes.
Table S2. DOG1 effect on number of leaves at flowering of Arabidopsis genotypes.
Table S3. Sequences of primers used in this study.
Supplementary Methods
Generation of dog1-3 × 35S:AtMIR156 and nced 9-1× 35S:AtMIR156 Lines
Because Col-35S:AtMIR156 displayed a strong late flowering phenotype (1) we used it as a pollen
donor to cross with dog1-3 and nced9-1. The F1 seedlings from multiple crossing events were screened
based on flowering phenotype. At least three seedlings that displayed the most severe flowering
phenotypes were genotyped to confirm the T-DNA insertion in NCED9 locus and advanced to the F3
generation.
Seed Germination Assays
For lettuce seed germination tests, seeds were placed on one paper blotter disc in a 4.7-cm Petri dish
that was moistened with 3 mL of deionized water. For Arabidopsis seed germination tests, seeds were
placed in wells containing 400 µL of deionized water or solutions in 24-well plates. The plates were
sealed with Parafilm. All deionized water or solutions used for seed germination tests were supplemented
with 0.2% Plant Preservative Mixture (PPM, Caisson Labs, Smithfield, UT, USA) to protect seeds from
fungal development. Three replications of 30 lettuce seeds or 50 Arabidopsis seeds were utilized in all
tests.
Measurements of Flowering Times
To measure lettuce flowering times, at least 20 plants of each transgenic line and the segregated
control lines were grown in the greenhouse. Lettuces were irrigated on a cycle of twice with deionized
water and once with liquid nutrient solution through computer-controlled drip. The flowering times for
lettuce were measured as days from the planting date to the first open flower.
To measure Arabidopsis flowering times, two or three genotypes of Arabidopsis plants were
randomly arranged in multiple trays. Col-WT, dog1-3 and dog1-5, Col-WT and need 9-1, Col-
35S:LsMIR156, dog1-3-35S:LsMIR156, nced9-1-35S:LsMIR156 and dog1-5-35S:LsMIR156, Col-
35S:AtMIR156, dog1-3 × 35S:AtMIR156 and nced9-1× 35S:AtMIR156, Ler-WT and dog1-1, Ler-35S:
LsMIR156 and dog1-1-35S:LsMIR156 were randomly distributed within the same flat trays with multiple
replications; water and nutrition were provided as needed. The flowering times were measured as days to
flower appearance and number of rosette leaves at flowering. Data were expressed as percentages of
plants flowering or as percentages of flowering plants having similar ranges of rosette leaf numbers at
different days after trays were transferred to the growth chamber following stratification.
Isolation of LsDOG1 and Vector Construction
The Arabidopsis DOG1 (AT5G45830) coding sequence was used to BLAST search the lettuce
Transcriptome Shotgun Assembly (http://blast.ncbi.nlm.nih.gov). The best-matched EST was used for
reciprocal BLAST against the Arabidopsis genome (www.arabidopsis.org) to further confirm identity.
Primers were subsequently designed for RACE amplification of DOG1 cDNA. The lettuce DOG1 cDNA
was TA-cloned into pGEMT-easy vector (Promega, Madison, WI, USA) using the primers listed in Table
S3. For suppressing DOG1 in lettuce, the CaMV35S promoter in binary vector pGSA1165 (ABRC stock
CD3-450) was replaced by a 2.6 kb LsNCED4 promoter through the BglII and SacI restriction sites. A
350 bp fragment was amplified from the Salinas DOG1 cDNA and introduced into the previous
pGSA1165-ProLsNCED4 RNAi vector with a two-step procedure through AscI/NcoI and BamHI/SpeI
digestions and ligations. The RNAi fragment was compared to the lettuce genome
(http://compgenomics.ucdavis.edu/) by BLASTN using low stringency to confirm no potential off-targets.
For ectopic expression of LsDOG1, the CaMV35S promoter of pGSA1165 vector was replaced with the
LsDOG1 promoter through digestion and ligation of BglII/SacI, and the GUS linker of the engineered
pGSA1165 was replaced with PI-LsDOG1 through NcoI/PacI to form the ProDOG1:PIDOG1 ectopic
expression vector. For overexpression of LsDOG1 in Arabidopsis, the GUSplus fragment of
pCAMBIA1305 was replaced with LsDOG1 coding regions from Sal, PI, UC and Saligna genotypes
through NcoI/PmlI digestion/ligation to form the Pro35S:LsDOG1 overexpression vectors. All cloned
fragments were error-proofed by sequencing.
Identification of LsMIR156 and LsMIR172
The mature sequences of Arabidopsis miR156 and miR172 obtained from the miRBase database
(www.miRBase.org) were used to BLAST against the lettuce Transcriptome Shotgun Assembly
(http://blast.ncbi.nlm.nih.gov). The candidate orthologous genes to the AtMIR156 genes were first used
for BLASTX against the Arabidopsis genome and NCBI protein database (http://www.ncbi.nlm.nih.gov/)
to rule out the protein-encoding genes such as LsSPLs. The rest of the candidate sequences were further
used for BLASTN against the Arabidopsis genome for confirmation. The EST contigs of these candidates
were used to search the lettuce genome browser (http://gviewer.gc.ucdavis.edu/cgi-
bin/gbrowse/alface_version_5_1/) to obtain the 5’ and 3’ extended sequences of miRNA binding sites if
needed. CENTROIDFOLD (http://www.ncrna.org/centroidfold) was used to define the potential stem-
loop precursors within the extended sequences of these candidates, and ViennaRNA Web Services
(RNAfold web server, http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) was used for the structure
prediction.
Vector Construction for Overexpression of LsMIR156 and LsMIR172
For overexpression of LsMIR156 in lettuce, sequences comprising LsMIR156 were amplified using
the primers listed in Table S3 and cloned into the Gateway cloning vector pDONR221 through BP
reaction. The cloned LsMIR156 was subsequently transferred to the Gateway binary expression vector
pGWB402Ω via LR reaction (2). For overexpression in Arabidopsis, LsMIR156 and LsMIR172 were
amplified using the primers listed in Table S3 and cloned into the Gateway cloning vector pDONR207
through BP reaction. These were subsequently transferred to the Gateway binary expression vector
pEarleyGate100 via LR reaction (3). All cloned fragments were error-proofed by sequencing.
Plant Transformation
For lettuce, the DOG1-RNAi and ProDOG1:SalDOG1 constructs were introduced into
Agrobacterium tumefasciens LBA4404 and the LsMIR156 construct was introduced into EHA105, all of
which were then used for Salinas or PI251246 transformation at the UC Davis Ralph M. Parsons
Foundation Plant Transformation Facility. For Arabidopsis, the Pro35S:LsDOG1, Pro35S:LsMIR156 and
Pro35S:LsMIR172 overexpression vectors were introduced into Agrobacterium GV3101 and used for
transformation through the floral dip method (4). The primers in Table S3 were used to genotype
Arabidopsis T-DNA mutants prior to plant transformation and to confirm the T1 and T3 transgenic plants.
mRNA and miRNA Analyses
Three lettuce leaves of similar developmental ages were pooled from three individual plants for each
line to form one biological sample. For lettuce apical meristems, one biological sample was pooled from 8
lettuce plants for each line. For lettuce, ~100 mg seeds were used for one biological sample. For
Arabidopsis leaves and apical meristems, one biological sample was pooled from ~20 individual plants.
For Arabidopsis seeds, ~50 mg seeds were used for one biological sample. In all cases, three biological
samples were assayed for each line.
Total RNA with enriched small RNA from leaves and apical meristems of lettuce and Arabidopsis
was isolated using the RNAzol RT kit as described in the instruction manual (Molecular Research Center,
Cincinnati, OH, USA). Total RNA with enriched small RNA from seeds of lettuce and Arabidopsis was
isolated using PureLink® Plant RNA Reagent following the instruction manual (Life
Technologies/Thermo Fisher Scientific, Carlsbad, CA, USA). All total RNAs were treated with TURBO
DNAase I (Life Technologies) prior to downstream analysis. For mRNA quantitation, 1 µg of total RNA
was used for cDNA synthesis using QuantiTect Reverse Transcription Kit (Qiagen, Redwood City, CA,
USA) and real-time PCR was performed as described previously (5). miRNAs were detected with
TaqMan® MicroRNA Assay (Life Technologies) with slight modifications. In brief, 300 ng of total RNA
was used for miRNA reverse transcription and 4 µL of 400x diluted miRNA RT was used for real-time
PCR in a 10 µL of reaction buffer that contained 5 µL of TaqMan Universal PCR master Mix II and 0.5
µL of TaqMan® Small RNA Assay probe. All real-time PCR assays were performed on a StepOnePlus
Real-Time PCR system (Life Technologies).
Supplementary Text
Screening for altered flowering pathways in lettuce LsDOG1-RNAi plants
Flowering of plants is dependent on developmental programs that are mediated by both
environmental and endogenous cues and are under tight genetic control. The primary environmental cues
for regulating flowering include the photoperiod, ambient temperature and chilling (vernalization), while
endogenous factors such as phytohormones and carbohydrate status regulate the so-called autonomous,
gibberellin (GA), nutrient, and aging pathways influencing flowering (6-10). In Arabidopsis (and other
plants), these components form a complex gene regulatory network in which both types of cues converge
at a set of integrator genes including FLOWERING LOCUS C (FLC), SHORT VEGETATIVE PHASE
(SVP), FLOWERING LOCUS M (FLM), FLOWERING LOCUS T (FT), etc. Like Arabidopsis, lettuce has
a winter annual flowering habit, with flowering being promoted by long days and chilling. To understand
how suppression of LsDOG1 expression could affect flowering time in lettuce, we tested expression of
several key integrator genes involved in different pathways.
FLOWERING LOCUS T (FT) is the most important integrator for regulating flowering time. FT is
mainly expressed in leaves and is induced by the transcriptional activator CONSTANS (CO) through its
binding to the FT promoter under long-day conditions (11, 12). The CO-FT module in flowering
regulation has been demonstrated to be conserved in other plant species, including sunflowers (Helianthus
annuus) in the Asteraceae with lettuce (7, 10, 13). To test whether LsDOG1 might influence FT
expression, we measured CO and FT mRNA abundances in leaves of Salinas and DOG1-RNAi plants. FT
mRNA was almost 25-fold higher, consistent with the early flowering phenotype, while CO mRNA was
only 75% higher in the LsDOG1-suppressed plants compared to the control (Fig. S4), suggesting that
other pathways might trigger the change in FT mRNA accumulation (Fig. S5).
In addition to photoperiod, temperature is another important environmental cue that greatly influences
plant flowering (Fig. S5). Arabidopsis plants flower earlier when grown at higher temperatures (23°C)
than at lower temperatures (16°C) (14). Lettuce also responds to high temperatures (particularly night
temperatures) with early flowering (15). At lower temperature (16°C), FLM and SVP proteins form a
complex to bind to the promoters of floral activator genes like FT and SOC1 to repress their transcription
(16, 17). This repression is released by increased temperatures (16-18). In contrast, the extended low
ambient temperature during the winter season can promote rapid flowering of winter annual plants in the
following spring season. The requirement for this chilling period (also called vernalization) prevents the
improper flowering of winter annual plants until after the winter season (9, 19). In Arabidopsis, chilling
represses the transcription of FLC through epigenetic silencing that is mediated by a conserved Polycomb
(PcG) mechanism. FLC is a MADS box transcription factor that represses the floral integrators FT, TSF
and SOC1 to inhibit flowering (19-23). FLC is also a core component in the autonomous flowering
pathway (10). Autonomous pathway mutants derived from rapid-flowering accessions are characterized
by delayed flowering irrespective of day length (9, 10). Genes involved in the autonomous pathway
regulate flowering by repressing FLC through epigenetic modification and/or RNA processing (10, 24).
With respect to FLC, FLM and SVP homolog genes in lettuce, we found either no effect of silencing
LsDOG1or that mRNA amounts were increased (Fig. S4). As FLC and SVP are repressors of flowering,
this makes it unlikely that DOG1 acts on flowering through modification of the vernalization or
autonomous pathways (Fig. S5), although mRNA abundance may not always reflect protein abundance or
activity.
Gibberellin can promote both seed germination and flowering (10, 25). Because enhanced seed
germination of atdog1 and lettuce DOG1-RNAi may be attributed to alteration in GA biosynthesis and
signaling (Fig. S3) (26, 27), we tested whether there are any changes in the key enzymes involved in
biosynthesis of bioactive GA. The biosynthesis of active GA is tightly regulated by the relative activities
of anabolic (GA20ox and GA3ox) and catabolic enzymes (GA2ox) under different environmental
conditions (Fig. S5) (28). Mutations that inhibit this biosynthetic pathway or increase the degradation of
GA can delay flowering, particularly under short-day conditions (10, 29-31). It has been proposed that
GA promotes SOC1 gene expression and its target LFY in apical meristems to accelerate flowering under
short days (32-34). The GA signaling pathway also more directly regulates flowering time (Fig. S5). GA
is perceived by the GA receptors, GIBBERELLIC ACID-INSENSITIVE DWARF 1 (GID1a, GID1b, and
GID1c) (35). The lower expression of FT and TSF in a triple mutant of gid1 caused a late-flowering
phenotype in long days (36). The reduction of expression of FT and TSF is attributed to the repression by
DELLA proteins of SPL3 expression in leaves and of SPL3, SPL4 and of SPL5 expression in the shoot
apex (36, 37). We measured the transcript abundances in lettuce of GA3ox2, GA20ox1, GA2ox2, GA2ox3,
GA2ox6 homologs and of DELLA homologs RGL1 and RGA. If silencing of LsDOG1were acting to
accelerate flowering by enhancing GA levels, we would expect expression of GA3ox2 and GA20ox1 to be
elevated and expression of the GA2ox genes and of RGL1/RGA to be reduced. However, the opposite was
the case, with expression of GA3ox2 and GA20ox1 being reduced while expression of the GA2ox genes
and of RGL1/RGA were either unaffected or increased (Fig. S4). Consistent with this data, we also
observed no obvious change in internode lengths between DOG1-RNAi and its control lines. These
results indicate that the early flowering in DOG1-RNAi is unlikely to be caused by an alteration in GA-
related flowering pathways (Fig. S5).
Regulation of flowering time by the maturity (phase change) or “aging” pathway prevents flowering
until the plant transitions from the juvenile to the adult phase, even under inductive environmental
conditions like long days and high temperature (10). miR156 and miR172, and their target genes, are
major components of the aging pathway and act sequentially to regulate flowering time (Fig. S5) (8).
miR156 expression is high in the embryo and early seedling stages and decreases in older plants, resulting
in a concomitant increase in expression of SPLs with aging, especially SPL3, 4, 5, 9, 10 and 15 (38). The
up-regulation of SPL3/4/5/9/10/15 caused an early-flowering phenotype through induction of FT and
floral meristem genes by binding the promoter elements of these genes (38-41). miR156 is not regulated
by the vernalization-, photoperiod-, and GA-dependent flowering pathways (40). Inactivation or
overexpression of the flowering regulators FLC, CO, FT, or SOC1 had no obvious effects on miR156
levels, and miR156 is also not responsive to exogenous application of GA, auxin and cytokinin (41).
However, miR156 is reported to respond to ambient temperatures. At 16ºC, the miR156 level is higher
than at 23ºC (42). Higher miR156 leads to a lower level of its target gene SPL3, resulting in lower
expression of FT (40). In contrast, the level of miR172 is lower at 16°C than at 23ºC, as SPL9 and 10
(targets of miR156) directly promote miR172 expression, which has an opposite effect to miR156 on
flowering time (1). Similarly, expression of the target genes of miR172, TARGET OF EAT1 (TOE1),
TOE2 and SCHLAFMüTZE (SMZ) were observed to increase at 16ºC and act as floral repressors (8, 42).
miR172 positively regulates flowering by silencing floral repressor genes: AP2, TOE1, TOE2, TOE3,
SMZ, and SCHNARCHZAPFEN (SNZ) (43). In addition, miR156 was also reported to crosstalk with the
sugar-signaling pathway in regulating flowering time. The enzyme TREHALOSE-6-PHOSPHATE
SYNTHASE 1 (encoded by TPS1) produces trehalose- 6-phosphate (T6P) which serves as a signal for
carbohydrate availability in plants; TPS1 regulates flowering time through mediating the expression of
SPL genes in the shoot apical meristem via a miR156-dependent mechanism (44). Two additional studies
provide supporting evidence that miR156 abundance is regulated by sugars such as glucose and sucrose to
control the vegetative to flowering phase transition (45, 46).
In the early flowering DOG1-RNAi line in lettuce, we found that expression of at least three SPL
homolog genes was up-regulated several fold compared to control plants (Fig. S4). These SPLs could be
regulated by either miR156 or GA. As discussed above, the up-regulation of SPL genes is less likely to be
caused by alterations in GA biosynthesis or degradation. Thus, gene expression assays indicated that the
early flowering phenotype in DOG1-RNAi lettuce is likely to be mainly attributed to the alteration in the
miR156-SPL pathway (Fig. S5).
Supplementary Figures
Fig. S1
Fig. S1. Temperature sensitivity of different lettuce genotypes and characterization of lettuce DOG1
expression and function. (A) Sensitivity to temperature of seed germination of four lettuce genotypes: cv.
Salinas (Sal, L. sativa), PI251246 (PI, L. sativa), US96UC23 (UC, L. serriola), and PI261653 (Saligna, L.
saligna). (B) Functional tests of the ability of LsDOG1 from four lettuce genotypes to complement the
dog1-3 mutant of Arabidopsis and restore seed thermoinhibition at 32°C. (C) Relative LsDOG1 mRNA
levels in Salinas roots, leaves and dry seeds. Root and leaf tissues were from 6-week-old plants. (D)
Relative LsDOG1 mRNA levels in dry seeds of Sal and PI that were matured at 18°C or 32°C. (E)
Overexpression of PI-DOG1 inhibited germination of nced6-1 nced9-1 (nced6/9) double mutant seeds at
32°C. (F) Germination of dog1-3, nced9-1, nced 6-1 nced9-1 (nced6/9), and dog1-3/nced9-1
(dog1/nced9) seeds at 35°C. In E and F, seeds were imbibed in the light for 5 d. In panel F, no dog1-3
seeds germinated. ** Denotes a significant difference at 0.01 level (Student’s t-test). Error bars = SE, n =
3. All Arabidopsis seeds were tested at approximately 3 weeks after harvest.
Fig. S2
Fig. S2. Protein amino acid sequence alignment of Arabidopsis Col DOG1 (AtDOG1) and lettuce Salinas
DOG1 (SalDOG1). The DOG1 domain is underlined in red.
Fig. S3
Fig. S3. Gibberellin, but not fluridone (an ABA biosynthesis inhibitor), alleviates primary dormancy of
Arabidopsis seeds caused by overexpression of LsDOG1. Seeds (3 weeks after harvest) of Col plants
overexpressing LsDOG1 were imbibed in water (H2O), 300 µM fluridone (FLU) or 300 µM gibberellin
4+7 (GA) for 120 h in the light at 20˚C. No seeds germinated in water.
Fig. S4
Fig. S4. Relative mRNA levels of genes in different pathways regulating flowering. The mRNA levels in
leaves of Sal (CTL) and DOG1-RNAi line at 6 weeks after planting were normalized by the geometric
mean of three reference genes: AP2M (Clathrin adaptor complexes medium subunit family), PDF1 and
ef1α; they are shown relative to the levels in CTL leaves for each gene. The left y-axis shows values for
all genes except FT, whose values are on the right y-axis. Error bars = SE, n = 3.
See Supplementary Text for further discussion of these data.
Fig. S5. Potential interactions of the DOG1-miR156-miR172 module in regulating developmental phase
transitions. Diagramed here are potential interactions of genes and pathways described in this work. The
central focus is on our hypothesis that at least one action of DOG1 is to promote the conversion of
MIR156 and MIR172 transcripts to miR156 and miR172. This is shown as acting through DOG1
promotion of expression of DICER complex components (see Fig. 4E-G), but additional mechanisms are
possible. miR156 targets SPL transcripts for degradation, which delays or prevents flowering (6, 8). When
DOG1 function is compromised, miR156 levels are reduced, SPL expression is enhanced, and early
flowering would result, especially when MIR156 is overexpressed (Figs. 2, 3). SPL3/4/5/9/15 can directly
promote expression of FT in leaves and of LEAFY (LFY), FRUITFULL (FUL), APETALA1 (AP1),
SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and AGAMOUS LIKE 24 (AGL24)
in apical meristems (6, 8). In addition, miR156 can repress SPL10 and 11 to prevent precocious
expression of genes during seed maturation phase (47), while FUSCA3 (FUS3) can promote MIR156
expression during seed development (48), enabling generational resetting of miR156 levels. SPL9/10/15
can upregulate miR172 through promoting MIR172 expression (38, 41); since miR156 inhibits SPL
expression, when miR156 levels go down, miR172 levels increase (Figs. 2D, 3B). The conversion of
primary MIR172 to miR172 is also proposed to be affected by DOG1’s influence on the DICER complex
(Fig. 3C, 4E-G). miR172 represses AP2-like genes including SCHNARCHZAPFEN (SNZ), which can
affect seedling development (49) and juvenile to adult transition (1, 38, 41). miR172 also can promote
seed germination (Fig. 4D), although the specific mechanism remains unknown. GA promotes both seed
germination and flowering (see Supplementary Text). Other flowering regulatory pathways in the
diagram are discussed in the Supplementary Text. ABA and DOG1 can act in parallel to regulate seed
dormancy (thermoinhibition) (Fig. 4C; S1E-F; S3) through regulating GA biosynthesis or signal
transduction or possibly through additional mechanisms (27, 50-52).
SPL10,11
SPL3,4,5
SPL9,10,15
FT
miR156
DOG1
Ju
ven
ile to
ad
ult
Germination
Em
bry
o to
seed
ling
Flowering
phase
Em
bry
og
en
esis
an
d s
eed
develo
pm
en
t tr
an
sit
ion
Dormancy to germination transition
miR172
AP2-like
LFY, FUL, AP1,
SOC1, AGL24
Autonomous
pathway
FLC
Vernalization
pathway
Ambient low
temperature
SVP/FLMFT
Photoperiod
CO
SNZ
Dormant
seeds
Juvenile
phase
Adult phase
High
temperature
ABA
GA
MIR156
DICER complex
(DCL1, HYL1, TGH, SE, CDC5)
GA20ox
GA pathway
GA3ox
RGL1,
RGAGA2ox
GA
MIR172FUS3
Adult to flowering transition
Fig. S6
Fig. S6. Overexpression of LsMIR156 resulted in delayed flowering in lettuce. Representative lettuce
plants of Sal-WT (left) and Sal overexpressing LsMIR156 (Sal-LsMIR56OX) (right) are shown at 97 days
after germination.
Fig. S7
Fig. S7. Relative mRNA and miRNA levels in leaves and apical meristems of Arabidopsis at different
developmental stages. Relative levels of (A) LsMIR156, (B) miR156, (C) miR172, (D) SPL3 and (E)
SPL9 in Col-35S:LsMIR156 (Col-LsMIR156) and dog1-3-35S:LsMIR156-I (dog1-LsMIR156) leaves at
15, 25 and 35 days after germination (F). Relative mRNA levels of AtMIR156, miR156 and miR172 in
apical meristems of 25-day-old Col-35S:AtMIR156 (Col-AtMIR156) plants and in apical meristems of
dog1-3 plants of the same age into which Col-35S:AtMIR156 had been crossed (dog1×AtMIR156).
Fig. S8. Primary dormancy of Col-WT, dog1-3 and dog1-5 seeds. Freshly-harvested seeds (5 days after
harvesting) were imbibed at 25°C for 5 days.
Supplemental Tables
Table S1. DOG1 protein amino acid sequence similarity (%) among four lettuce and two Arabidopsis
genotypes.
Salinas Saligna PI251246 US96UC23 At-Col At-Cvi
Salinas 100
Saligna 96.0 100
PI251246 99.3 95.2 100
US96UC23 96.7 98.2 95.2 100
At-Col 49.5 49.8 49.1 50.2 100
At-Cvi 50.4 49.7 50.4 50.3 90.4 100
Table S2. DOG1 effect on number of leaves at flowering of Arabidopsis genotypes.
Columns are categories of the number of rosette leaves present at the time of flowering. Values for
different genotypes or growth conditions indicate the percentage of the total number of plants that have
the indicated number of rosette leaves at flowering.
Experiment 1
(LD, 22°C,135 µmol m-2s-1) Hm/Hta PNb Percentage of Flowering Plants
Number of Rosette leaves 5-10 11-15 16-20 21-25 26-30 31-35 36-40 41-50 >51
Col Hm 72 79.2 20.8
Col-35S:LsMIR156-G Hm 65 4.6 6.2 43.1 46.1
Col-35S:LsMIR156-C Hm 53 9.4 24.5 62.3 3.8
Col-35S: AtMIR156 Hm 70 1.4 4.3 11.4 42.9 40.0
dog1-3 Hm 61 67.2 32.8
dog1-3-LsMIR156-I Hm 69 23.2 52.2 24.6
dog1-3-35S:LsMIR156-G Hm 68 1.5 58.8 39.7
dog1-3 × 35S:AtMIR156-A Hm 63 3.3 39.7 39.7 17.5
dog1-3 × 35S:AtMIR156-G Hm 65 4.6 32.3 33.8 18.5 7.7 3.1
nced9-1 Hm 48 62.5 37.5
nced9-1×AtMIR156#7 Hm 40 7.5 15.0 40.0 37.5
Experiment 2
(LD, 21°C,100 µmol m-2s-1) Hm/Hta PNb Percentage of Flowering Plants
Number of Rosette leaves 5-10 11-15 16-20 21-25 26-30 31-35 36-40 41-50 >51
Col Hm 48 83.3 16.7
Col-35S:LsMIR156 Ht 72 1.4 1.4 5.6 8.3 4.2 9.7 4.2 65.2
dog1-3 Hm 36 77.2 22.8
dog1-3-35S:LsMIR156 Ht 48 1.1 28.1 27.1 14.5 25.0 2.1 2.1
dog1-5 Hm 20 80 20
dog1-5-35S:LsMIR156 Ht 28 7.1 3.6 3.6 14.3 71.4
nced9-1 Hm 47 85.1 14.9
nced9-1-35S:LsMIR156 Ht 37 2.7 5.4 2.7 8.1 5.4 13.5 62.2
Experiment 3
(LD, 22°C,135 µmol m-2s-1) Hm/Hta PNb Percentage of Flowering Plants
Number of Rosette leaves 5-10 11-15 16-20 21-25 26-30 31-35 36-40 41-50 >51
Ler Hm 48 62.5 37.5
Ler-35S:LsMIR156 Ht 63 3.2 9.6 23.8 60.3 3.1
dog1-1 Hm 57 64.9 35.1
dog1-1-35S:LsMIR156 Ht 52 34.6 44.2 21.2
Experiment 4
(SD, 22°C,135 µmol m-2s-1) Hm/Hta PNb Percentage of Flowering Plants
Number of Rosette leaves 11-20 21-30 31-40 41-50 51-60 61-70 >71
Col Hm 37 29.7 64.9 5.4
Col-35S:LsMIR172 Ht 77 18.2 45.5 22.1 11.7 2.5
dog1-3 Hm 64 3.1 17.2 67.2 12.5
dog1-3-35S:LsMIR172 Ht 100 12 27 30 25 3 3 a Hm: Homozygous; Ht: Heterozygous
b PN: number of plants when homozygous; number of independent transgenic individuals when heterozygous.
Table S3. Sequences of primers used in this study.
PRIMER NAME SEQUENCE SEQUENCE
ID
PURPOSE
AtSPL4-qF TCAAGGGTAGAGATGACACTTCCTATGC AT1G53160 RT-PCR
AtSPL4-qR TCTCTCATCATAGCAAGTGATGGACCCTG
AtSPL5-qF CCAGACTCAAGAAAGAAACAGGGTAGACAG AT3G15270 RT-PCR
AtSPL5-qF TCCGTGTAGGATTTAATACCATGACC
AtSPL9-qF CAAGGTTCAGTTGGTGGAGGA AT2G42200 RT-PCR
AtSPL9-qR TGAAGAAGCTCGCCATGTATTG
AtSPL10-qF TCAGGAGGCCTCCATGAATCTCA AT1G27370 RT-PCR
AtSPL10-qR GGCCACGGGAGTGTGTTTGAT
LBa1 TGGTTCACGTAGTGGGCCATCG PROK2 GENOTYPING T-DNA
MUTANT
AtDOG1-3LP TTCCAGGAACGTTGTCGTATC AT5G45830 GENOTYPING OF
SALK_000867
AtDOG1-3RP AGTTTGTGACCCACACAAAGC GENOTYPING OF
SALK_000867
AtDOG1-5LP AAGTTGATCATGTTCATGGGG AT5G45830 GENOTYPING OF
SALK_022748C
AtDOG1-5RP TATGGTAGCAAGGTGCAATGC GENOTYPING OF
SALK_022748C
AtNCED9-LP ATTCCGCTTGATCAACCAAC AT1G78390 GENOTYPING OF
ATNCED9-1
AtNCED9-RP CACAGTTGGATCATTGGACACT GENOTYPING OF
ATNCED9-1
AtMIR159A-qF TCAGGAGCTTTAACTTGCCCTTT AT1G73687 RT-PCR
AtMIR159A-qR CACGCTAAACATTGCTTCGGAAT
AtMIR319B-qF AGCTTTCTTCGGTCCACTCATGG AT5G41663 RT-PCR
AtMIR319B-qR GAGCTCCCTTCAGTCCAAGCATA
AtMIR156-qF TGAGCACACAAAGGCAATTT AT2G25095 RT-PCR
AtMIR156-qR CAGTGAGCACGCAAGAGAAG
AtMIR172-qF ATCTGTTGATGGACGGTGGT AT2G28056 RT-PCR
AtMIR172-qR AATAGTCGTTGATTGCCGATG
AtACT8-qF CTCAGGTATTGCAGACCGTATGAG AT1G49240 RT-PCR
AtACT8-qR CTGGACCTGCTTCATCATACTCTG
AtACT2-qF CTTGCACCAAGCAGCATGAA AT3G18780 RT-PCR
AtACT2-qR CCGATCCAGACACTGTACTTCCTT
LsDOG1-RACE-F ATGGCCAAACAAATGAAACACCA KT337314 LsDOG1 CDNA
CLONING
GENERACER-
ADAPTER
GCTGTCAACGATACGCTACGTAACGGCATGAC
AGTGTTTTTTTTTTTTTTTTTTTTTTTT
LsDOG1 CDNA
CLONING
GENERACER 3’R GCTGTCAACGATACGCTACGTAACG LsDOG1 CDNA
CLONING
GENERACER
NESTED 3’R
CGCTACGTAACGGCATGACAGTG LsDOG1 CDNA
CLONING
LsDOG1-RACE-R1 TCTCTAAAATACCCTTCAGTGTACTCATCCTC LsDOG1 CDNA
CLONING
LsDOG1-RACE-
NESTEDR1
TCTGCTTCCATCAAAACATTATACATATCA LsDOG1 CDNA
CLONING
LsDOG1-Saligna-F CACCATGGCCAAACAAATGAAACACC KT337316 SALIGNA DOG1
CONSTRUCT
LsDOG1-Saligana-R TTCAGTGTCACGTCTCCGCTG GTGGTG
LsNCED4Pro-F TTAAAGATCTAACAGACAAAAGTCAACGGAGT
TAG
KC676791 SALIGNA DOG1
CONSTRUCT
LsNCED4Pro-R TTGAGCTCTGGAGGCGGTGGTAGTGATG
LsDOG1-UC-F CACCATGGCCAAAAAAATGAAACACC JO034198 UCDOG1
CONSTRUCT
LsDOG1-UC-R TTCAGTGTCACGTCTCCGGTGGTGGTG
LsDOG1-PI/SAL-F CACCATGGCCAAACAAATGAAACACC KT337314/
KT337315
FOR SALINAS AND PI
DOG1 CONSTRUCT
LsDOG1-PI/SAL-R TTCAGTGTCACGTCTCCGGTGGTGGGT
PRODOG1-F AGATCTTATGGGTCGAAGGGACCAAT KT290282 PI DOG1 ECTOPIC
EXPRESSION
CONSTRUCT
PRODOG1-R GAGCTCTGGTTTTTTTTGTAAGGGGTGACTC
LsMIR156-qF TGATGCTGCATGTCAACAGA JI599382 RT-PCR
LsMIR156-qR CTCTATCGCCCCCACAAGTA
LsMIR156-BPF GGGGACAAGTTTGTACAAAAAAGCAGGCTTCC
GGTTCTGTTCCGATATCC
JI599382 LsMIR156
CONSTRUCT
LsMIR156-BPR GGGGACCACTTTGTACAAGAAAGCTGGGTCTC
TAAATTGGGATTCAACAAATTC
LsMIR172-BPF GGGGACAAGTTTGTACAAAAAAGCAGGCTTCT
ACTCCTCATCTCTATCCTCTCCTTG
JI585366 LsMIR172
CONSTRUCT
LsMIR172-BPR GGGGACCACTTTGTACAAGAAAGCTGGGTCAG
TTTTTGTTATCTCAAGTTGTCTAATCC
SalDOGRNAi-F GCACTAGTCCATGGCAACAGCTCGATCTGGAC
GAATTA
KT337314 LsDOG1-RNAi
CONSTRUCT
SalDOGRNAi-R CGGGATCCGGCGCGCCGCATGAAGTTCATCTA
TTCTTTTGAGC
LsDOG1-qF CCAAAAAAATCGTCTCCCACTT KT337314 RT-PCR
LsDOG1-qR CAAAAAGGAAGGCCCATCGT RT-PCR
LsSPL3-qF TAGCCGGTTTCATGAGCTTTC JI599986 RT-PCR
LsSPL3-qR TCATTGTGCCCTGCTAAACG
LsSPL4-qF GTAGGCGTTTAGCTGGGCATA JI598022 RT-PCR
LsSPL4-qR CCGTCTCCATAAGTTTCAAAGGA
LsSPL9-qF TGCTCATTCGAAAACGGCTAA JI582339 RT-PCR
LsSPL9-qR TGGAACCTGCTGCACTGTTG
LsFT-qF GGACTCTCATAGCACACAATTTCTTG JI597116 RT-PCR
LsFT-qR CATTGGTTGGTGACCGATATACC
LsSVP-qF GGCATAGCCTGCATTCAAAAA JI583990 RT-PCR
LsSVP-qR GGCATAGTTGGCGTCTTCAAC
LsFLC-qF GCTCAGAATCTTGCTCATGCTTT JI587615 RT-PCR
LsFLC-qR CGTCGCTCTTTTCATCTTTTCC
LsFLM-qF TCCGCCCCATTGATATTGA JI603388 RT-PCR
LsFLM-qR ACAAAGAAAGTTAGGTCTAGCCACAAG
LsCO-qF GTGGCAACGCCGATAGTGT JI585578 RT-PCR
LsCO-qR CATATTCCATCCCCAACTGAAAC
LsCLARI-qF CTGCTTCCGCTATCTACTTCCTAAA JI580445 RT-PCR
LsCLARI-qR TTCCCCCGACGTCATCAC
LsPDF1-qF AAGCTTGGTGCTCTCTGCAT JI579444 RT-PCR
LsPDF1-qR GGGACCAAATTCCTCTGCGA
LsEF4α-qF TCGTCATCGGCCATGTTGAC JI582565 RT-PCR
LsEF4α-qR TCGTCATCGGCCATGTTGAC
LsACT7-qF GATCACGATTGGAGCTGAAAGA JI581679 RT-PCR
LsACT7-qR GCAGCTTCCATTCCAATCAAA
LsUBQ10-qF TGGTCGTACGCTTGCTGATT JI587827 RT-PCR
LsUBQ10-qR AAAAACACCCACCACGAAGC
AtDCL1-qF CGTTGTTATGCGTTTCGACCTTGC AT1G01040 RT-PCR
AtDCL1-qR AACGCTGCGTGAGATACATTTCCTC
AtHYL1-qF TTGCCTGGATTCTTCAATCGTAAGG AT1G09700 RT-PCR
AtHYL1-qR TAGGTTCTTGCATAATCCCGTTTCG
AtTGH-qF AGATCTCGCATGTCTGCCAA AT5G23080 RT-PCR
AtTGH-qR ACCCGGAGCAATCTTTCCTG
AtSE-qF CCACCGCCTCGTAGGGATTACA AT2G27100 RT-PCR
AtSE-qR CCACCATGGTCATACCCAAATCTTC
AtCDC5-qF GTTTCCGAGCACAGGCATTG AT1G09770 RT-PCR
AtCDC5-qR TCCTCTCCAGTGGCTAGCTT
LsTGH-qF AGCGGTTTGATTTTGTCACC JI574976 RT-PCR
LsTGH-qR CGAGGGAGTTGCGACTTTAG
LsSE-qF TGGATACCAAGGTGGTCCAT JI576033 RT-PCR
LsSE-qR ACCTGCGTTCAGCTTCTGAT
LsCDC5-qF GATTATGATGGAGGCCGAGA JI575030
RT-PCR
LsCDC5-qR GAAAAATCCGAAGGATGCAA
LsDCL1-qF TGGTTGCAACAGAGGTTGGA JI573836 RT-PCR
LsDCL1-qR TTGCTGGTGCCATCTTCTTG
LsHYL1-qF GCGATAAACCTCCCCAATTTT JI587263 RT-PCR
LsHYL1-qR GGAAACGGAGTCAGCGAACA
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