Regulation of tomato epidermal morphology by
anthocyanin transcription factors
Xandra Schrama
MSc thesis MPB Plant Biotechnology
Supervisor: Jules Beekwilder
Examiner: Robert Hall
22-01-16
1
Abstract
Anthocyanin production is regulated by a MYB, bHLH and WDR transcription factor complex.
Overexpression of a MYB (Rosea1) and a bHLH (Delila) from Snapdragon in tomato induces ectopic
anthocyanin production in all plant parts except fruits. Surprisingly, also genes involved in epidermal
cell fate were upregulated upon overexpression of anthocyanin inducing Rosea1 and Delila
(Ros1&Del) transcription factors. One gene highly upregulated was the epidermal cell fate
transcription factor GLABRA2 (GL2). In Arabidopsis, GL2 is involved in root hair, trichome and
stomata formation. It is not clear yet what the relation between upregulation of anthocyanin related
genes and GL2 is. It is shown here that Ros1&Del induced tomato plants have deviating phenotypes
in root hairs, trichomes and stomata compared to control plants. In addition it is shown that
Ros1&Del directly activate the epithelial cell fate regulator GL2. Ros1&Del overexpressing tomato
plants were used for SEM analysis of trichomes and stomata abundance. Trichome type 7 was
downregulated on Ros1&Del induced tomato leaves. Stomata counts were identical on control and
induced plants, however on induced plants stomata had a wider aperture. Root hairs analysed on
Ros1&Del induced seedlings were 87% shorter than control root hairs. Ros1&Del overexpression thus
induced several differences in epidermal phenotypes compared to control plants. These effects
might be explained by GL2 overexpression but also by induction of auxin related genes. The
promoter of Glabra2 was fused to a luciferase reporter gene to use for trans-activation assays with
35S-Ros1 and 35S-Del. It was found that Ros1&Del directly activate Glabra2. CHS-RNAi also plays a
role in promoter Glabra2 induction, however the exact role it plays is not clear yet. 35S::GL2 tomato
plants were produced but no T1 generation is produced yet and phenotyping of these lines needs to
be done. In conclusion, the phenotypic deviations found in Ros1&Del overexpression tomato plants is
probably caused by direct activation of GL2 but 35S::GL2 T1 generation plants need to be examined
first in order to conclude this. Tomatoes overexpressing Ros1&Del under a fruit specific promoter are
probably soon to go commercial and are said to differ only in anthocyanin content compared to WT
fruits. We have shown with this study that Ros1&Del expression in tomato plants does not only
trigger anthocyanin biosynthesis genes but a range of other genes as well. Probably the effect of
Ros1&Del depends on promoter usage.
2
Introduction
Anthocyanins naturally occur in many plant species as red, blue and purple pigments. Examples are
fruits and vegetables (e.g. blueberries, red cabbage and eggplant), autumn leaves and flowers.
Anthocyanins are part of the large polyphenolic flavonoids group. Flavonoids belong to the group of
secondary plant compounds (Kong et al., 2003). Other compounds which belong to the flavonoids
group are flavonols, flavones, flavanones, flavan-3-ols and isoflavones (Lila, 2004).
Anthocyanins are frequently used in the food industry as natural colorants (Castañeda-Ovando et al.,
2009). Moreover, anthocyanins have several effects on the health status of the human body. In
general, anthocyanins have antimicrobial effects (Lacombe et al., 2010; Burdulis etal., 2008).
However, there are also more specialized effects known. Anthocyanins are antioxidants and are
therefore suggested to have anti-inflammatory effects, inhibit cancer development and help to
control obesity and diabetes (Reviewed in He and Giusti, 2010). Anthocyanins do not only play a role
in human health status, anthocyanins also play a role in plants themselves and might be critical to
plant survival in some environments. It is believed that anthocyanins are involved in attracting
pollinators and seed dispensers due to their ability to form different colour patterns (Zhang et al.,
2013). There are also several physiological processes in which anthocyanins play a role in plants.
Anthocyanins are involved in resistance to drought stress, photo-oxidative damage and heavy metals.
Furthermore, anthocyanins play a role in resistance to herbivory and pathogens (Reviewed in Gould,
2004).
Anthocyanin biochemistry
The basic anthocyanin structure is the anthocyanidin
(Figure 1), which consist of an aromatic ring (A), which
is bound to a heterocyclic ring containing an oxygen (C),
the C ring is bound by a carbon-carbon bond to another
aromatic
ring
(B)
(Castañeda-Ovando).
This
anthocyanidin structure is very unstable and is
therefore hardly found in this form in nature (Mazza
and Brouillard, 1987). Anthocyanidins are therefore
always glycosylated, this glycosylated form of Figure 1 | Anthocyanidins general structure. R2 to R7
anthocyanidin is called anthocyanin (Castañeda-Ovando are common modification positions. CastañedaOvando et al., 2009
et al., 2009).
Anthocyanin biosynthesis
Anthocyanin biosynthesis begins with a basic pathway (Figure 2); the shikimic acid pathway. The
amino acid phenylalanine is formed in this pathway. From phenylalanine, 4-coumaroyl-Coa is
produced by several enzymes (PAL1, CH4/CPR, 4CL). Naringenin chalcone is then formed by
combining one 4-coumaroyl-Coa with three malonyl-Coa molecules, and this is catalysed by the first
committed enzyme in flavonoid biosynthesis, chalcone synthase (CHS). The key intermediate
flavonoid naringenin is then formed, catalysed by chalcone isomerase (CHI)(Patra et al., 2013). From
naringenin, dihydrokaempferol is formed, catalysed by the enzyme flavanone-3-hydroxylase (F3H).
Dihydrokaempferol can be converted to dihydromyricetin by flavonoid-3’-hydroxylase (F3’H) and to
dihydroquercetin by flavonoid-3’5’-hydroxylase (F3’5’H) (Butelli et al., 2008). The dihydroxyflavonols
dihydromyricetin, dihydrokaempferol and dihydroquercetin can be converted to the anthocyanidins
delphinidin, pelargonidin and cyanidin respectively, catalysed by dihydroflavonol reductase (DFR) and
anthocyanidin synthase (ANS). After synthesis, delphinidin can be methylated on the hydroxylgroups
to form petunidin or malvidin. Cyanidin can be methylated to form peonidin. Together with
pelargonidin, these 6 anthocyanidins form the main anthocyanidins found in nature (Zhang et al.,
2014). These anthocyanidins can be processed into anthocyanins by several enzymes; flavonoid 3-Oglucosyltransferase (3-GT), flavonoid 3-O-glucoside-rhamnosyltransferase (RT), anthocyanin
acyltransferase (AAC) and flavonoid-5-glucosyltransferase (5GT) (Butelli et al., 2008). The
3
anthocyanidin delphinidin is purple, pelargonidin is pink and cyanidin is red. This diversity in colour is
caused by differences in B-ring hydroxylation of the anthocyanidin general structure (Zhang et al.,
2014). In addition also flavonols can be produced by this pathway. Dihydrokaempferol,
dihydromyricetin and dihydroquercetin can be converted to the flavonols kaempferol, myricetin and
quercetin respectively by flavonol synthase (FLS) (Butelli et al., 2008).
In nature more than 600 different anthocyanins occur (Zhang et al., 2014). This large collection of
anthocyanins can be explained by structural variations and these are caused by diversity in the
number of hydroxyl groups in the molecule, the amount of hydroxyl groups methylated, the number,
forms and position of sugar functional groups which are attached to the phenolic molecule and the
number and forms of aliphatic and aromatic acids attached to the sugars (Mazza and Miniati, 1993).
These side chain decorations affect colour, stability, interactions, bioavailability and possibly also
proposed health effects of anthocyanins (Zhang et al., 2014).
Figure 2 | Schematic visualisation of the anthocyanin biosynthesis pathway. Enzymes are shown in green boxes,
dihydroxyflavonols in orange box, flavonols in yellow boxes, anthocyanidins in red box and anthocyanins in purple box. CHS:
chalcone synthase, CHI: chalcone isomerase, F3H: flavanone-3-hydroxylase, F3’H: flavonoid-3’-hydroxylase, F3’5’H:
flavonoid-3’5’-hydroxylase, DFR: dihydroflavonol reductase, ANS: anthocyanidin synthase, 3-GT: flavonoid 3-Oglucosyltransferase, RT: flavonoid 3-O-glucoside-rhamnosyltransferase, AAC: anthocyanin acyltransferase, 5-GT: flavonoid5-glucosyltransferase (Katsumoto et al., 2007; Petroni and Tonelli, 2011; Butelli et al., 2008; Koopman et al., 2012).
4
Anthocyanin biosynthesis regulation
The anthocyanin biosynthesis pathway is under control of transcription factors of three protein
families; R2R3MYB transcription factors, bHLH transcription factors and WD Repeat Proteins (WDR)
(Zhang et al., 2014). Together, these three protein families interact to form a complex (MBW) and
this complex can activate anthocyanin production (Zhang et al., 2014; Ramsay and Glover, 2005).
bHLH and R2R3MYB belong to the largest families of transcription factors. Subgroups within the
bHLH and MYB TF exist and each group has distinct functions. Subgroups of R2R3MYBs are involved
in phenylpropanoid and glucosinolate biosynthesis, plant hormone and pathogen related stresses,
organ determination, control of cell shape and formation of root hairs and trichomes. bHLH
subgroups are involved in regulation of fruit splitting at maturity, carpel and anther formation,
epidermal cell development, phytochrome signalling, flavonoid biosynthesis, hormone signalling and
stress responses. It is not clear yet if R2R3MYBs and bHLHs predominantly work together or if they
also execute functions separately. Nevertheless, it is already known that, in a complex, bHLH-MYBs
are involved in the anthocyanin biosynthesis pathway, epidermal cell differentiation (trichomes, root
hairs and seed coat), phytochrome A signalling, cold stress tolerance and vacuolar acidification (Feller
et al., 2011). The function of WDR proteins is probably performing a stabilizing function in a protein
complex (eg. MBW), in addition the WDR domain is a site for protein interactions and WDR can be an
interaction domain of larger proteins (Van Nocker & Ludwig, 2003).
In Arabidopsis the MBW complex involved in anthocyanin production is composed of a R2R3MYB
(PAP1, PAP2, MYB113 or MYB114), a bHLH (TT8, GLABRA3 or EGL3) and a WDR protein (TTG1) (Wang
et al., 2015). The R2R3MYB transcription factor is probably the limiting factor in anthocyanin
production because it is thought that the WDR and bHLH proteins are constitutively expressed
(Zhang et al., 2014). This is confirmed when specific MYB proteins are expressed in plants, which
leads to anthocyanin accumulation. However, expression of both MYB and bHLH leads to higher
anthocyanin accumulation compared to a MYB alone, signifying the effect that bHLH can potentiate
the production of anthocyanins (Outchkourov et al., 2014)
Anthocyanins can be ectopically produced by expressing MBW complex transcription factors. For
example stable expression of transcription factors R2R3MYB, Rosea1 (Ros1) and bHLH, Delila (Del)
from Antirrhinum majus (Snapdragon) under a fruit specific promoter in tomato led to ripe tomato
fruits with purple coloration (Butelli et al., 2008). Ros1 and Del expression in tomato increased all
genes required for anthocyanin production (Butelli et al., 2008, Toghe et al., 2015). Anthocyanins are
not usually found in fruits of tomato plants, however, the pathway needed for anthocyanin
production (Figure 2) is present in the tomato genome, albeit only highly active in hypocotyls
(Gomez-Roldan et al., 2014). Also ectopic expression of MYB75/PAP1 from Arabidopsis led to purple
coloration in tomato plants (Zuluaga et al., 2008). Transient expression of Ros1 and Del in N.
benthamiana induced, as expected, purple coloration of leaves. HPLC profiling of the purple leaves
showed that delphinidin-3-rutinoside (D3R) was the only anthocyanin produced. However,
untargeted LCMS analysis showed that in addition to D3R, a spectrum of other phenolic and nonphenolic compounds was upregulated by Ros1&Del expression (Outchkourov et al., 2014).
In order to get more insight in gene upregulation upon Ros1 and Del expression, a dexamethasone
(DEX) inducible Ros1 and Del system was created in tomato (Outchkourov, unpublished). In these
Ros1&Del tomato plants, Ros1 and Del were both fused to a GAL4 promoter. Upon dexamethasone
treatment of the plants, the constitutively expressed GAL4 transcription factor is able to migrate to
the nucleus and activate Ros1 and Del production.
RNA of callus and roots of Ros1&Del induced tomato plants was compared to control plants in order
to achieve information about up-or downregulated genes by Ros1&Del activation. More than 400
genes were found to be up-or down regulated in the activated Ros1 and Del plant material. Genes
5
from the anthocyanin biosynthesis route were highly upregulated. Some of these genes were already
identified in tomato, such as F3’5’H and DFR. Others were known to be involved in anthocyanin
production based on homology, such as 3-GT, RT, AAC and 5GT. Among the surprising genes found in
this set were genes involved in epithelial cell fate specification and auxin accumulation. One gene
which was highly upregulated in the sequence database was a homeobox-leucine zipper protein 61%
identical to Arabidopsis GLABRA2 (solyc03g120620.2). In callus GLABRA2 was upregulated 6 fold, in
roots 30-40 fold.
GLABRA2
The GLABRA2 (GL2) gene encodes a homeobox leucine zipper protein (Rerie et al., 1994; Gao et al.,
2008), which is able to bind DNA and activates transcription. In Arabidopsis, GL2 is already identified
to regulate several processes. Trichome formation, stomata control and root hair pattern formation
are three processes in which GL2 plays a pivotal role (Johnson et al., 2002; Gao et al., 2008; Rerie et
al., 1994). GL2 positively influences trichome formation; overexpression of GL2 leads to more
trichomes (Ohashi et al., 2002). GL2 negatively regulates stomata formation (Qing and Aoyama,
2012) and hair development in differentiating hairless cells (Masucci et al., 1996). However, only very
few genes are known to be a downstream target of GL2 (Lin et al., 2015). Recently it was found that
GL2 also plays a role in controlling anthocyanin production in Arabidopsis (Wang et al., 2015).
In Arabidopsis, GL2 is activated by a R2R3-MYB (WER or MYB23), bHLH (GL3/EGL3) and a WD40 (TTG)
protein complex (MBW complex) (Schiefelbein et al., 2009, Qing and Aoyama, 2012). This MBW
complex is very much alike the MBW complex which can activate anthocyanin production. A R3-MYB
(CPC, TRY, ETC1, ETC2, ETC3/CPL3) transcription factor can inhibit GL2 regulation by competing with
the R2R3-MYB protein to form the MBW complex (Wang et al., 2015, Qing and Aoyama, 2012). In
tomato, GL2 was discovered when a high anthocyanin line was produced. (Mathews et al., 2003).
This anthocyanin overexpression was caused by activation tagging of a MYB transcription factor,
ANT1. ANT1 is closely related to other MYB transcription factors (e.g. PAP1, P1, C1) involved in
anthocyanin regulation. This indicates GL2 might be regulated by an anthocyanin regulating MYB
transcription factor in tomato. Due to these facts I think Ros1&Del can regulate GL2 expression in
tomato and GL2 might play a role in anthocyanin production in tomato.
Aim
First the phenotype of Ros1 and Del overexpressing Micro-Tom plants was characterized. As GL2
controls trichome-, stomata-, and root hair pattern formation in Arabidopsis, Ros1&Del, and thus
GL2, overexpressing plants were expected to show a deviating phenotype in these traits in tomato. A
previously performed root morphology experiment on transformed tomato plants with a DEX
inducible Ros1 and Del system showed more root branching and shorter and less root hairs than
wildtype plants (Matthijs Hölscher, MSc thesis). However, these root phenotypes were not
quantitatively assessed. In this study a larger, more quantitative experiment on root hair formation
was carried out. Moreover, trichomes and stomata were visualized by scanning electron microscopy
on Ros1 and Del overexpressing tomato plants.
In order to figure out if GL2 is directly activated by Ros1&Del, a trans-activation assay in N.
benthamiana was performed. GL2 promoter was fused to a luciferase reporter gene in order to
execute trans-activation assays. It was expected that Ros1 and Del regulate both anthocyanin
production and GL2 expression.
Lastly, tomato plants stably over-expressing GL2 were produced in order to do phenotypic and
metabolic characterization. Since GL2 has an effect on trichomes, stomata, root hairs and
anthocyanin formation in Arabidopsis the aim was to figure out whether these traits are similarly or
differently controlled by GL2 in tomato.
6
Materials and Methods
Phenotyping of Ros1&Del induced tomato plants
Ros1&Del tomato Micro-Tom plants
Ros1&Del tomato plants were used for phenotyping of trichomes, stomata and roots. Ros1&Del
Micro-Tom tomato plants were previously produced (Outchkourov et al., unpublished). This MicroTom line produces anthocyanins in all parts of the plant except the fruits upon treatment with DEX.
(Ros1) and Delila (Del) from snapdragon (Antirrhinum majus) have been placed under the control of a
GAL4 promoter. GAL4, an activation domain (ACT) and the hormone binding domain of a
glucocorticoid receptor (GR) were transformed into tomato plants under a constitutively active
promoter (UBQ10). The GAL4-ACT-GR fusion protein is retained in the cytosol by binding of heat
shock proteins and GAL4 is therefore not able to activate Ros1 and Del. Upon dexamethasone (DEX)
treatment of the plants, DEX binds to the GR receptor causing a conformational change and releases
heat shock proteins. This enables GAL4 transcription factor to migrate to the nucleus and active Ros1
and Del.
Scanning electron microscopy of Ros1&Del Micro-Tom trichomes and stomata
Seeds of Micro-Tom Ros1&Del plants were sown and plants were grown in soil. Plants were watered
for 20 days with water containing DEX (10µg/ml). Leaves of the wild type and Micro-Tom Ros1&Del
plants were cut off and prepared for SEM analysis (Tiny Franssen-Verheijen). Analysed SEM pictures
were 1200μmx800μm and had a magnitude of 250x. Total numbers of trichomes were counted. Hairs
and stomata were counted on a 500μmx500μm picture with a 250x magnitude. Counted numbers
were analysed using IPM SPSS Statistics 22.
Microscopy of Ros1&Del Micro-Tom root hairs
Micro-Tom Wild type and Ros1&Del seeds were sterilized in 1% bleach for 20 minutes in a 50 mL blue
cap. Thereafter seeds were washed 2 times 5 minutes in MQ water. Seeds were sown on 3 layers of
sterilized filter paper in Duchefa containers (12x6.5x7) moistened with 7 mL of sterilized water with
2.2g/L Duchefa MS with vitamins. On subsequent days 1mL of water with 2.2g/L Duchefa MS with
vitamins was added to the containers. After 7 days the seedlings (0.5-3 cm long) were transferred to
plates (11x11cm) with agar or agar containing DEX(2gr/L MS with vitamins, 13g/L agar, 10mL MES
buffer, pH 5.8). DEX was dissolved in DMSO (10µg/ml) and added after autoclaving of agar. Seedlings
were placed ± 2cm from the top of the plate in order to grown roots downwards, root length was
marked in order to know from which length the root grows in DEX. Plates were placed in an angle of
60°. Seeds were germinated and seedlings were grown in a tissue culture room (16hours light, 25°C).
Microscopy was performed 3 days after transferring the seedlings to the plates. From five roots per
treatment, two pictures were made. From each picture the length of 10 root hairs was measured
using ImageJ. So in total 20 root hairs per root and 100 root hairs per treatment were measured.
Trans-activation assays
Constructs (Construction of Expression Cassettes and Vector)
GL2 promoter (pGL2) was amplified from tomato genomic DNA using primers (Table 2). The length of
the promoter is 1938 bp.
Table 2 | Primers used to amplify the promoter of GL2 from tomato genomic DNA.
Gene
Forward primer
GL2 promoter AGA ATA AAT ATT CAA TGT TAT TG
Reverse primer
AGT AGT GAT TAT CCT GTT AAA GG
3’A overhangs were added to the amplified promoter of GL2 to use it for TOPO cloning into
pCR8/GW/TOPO-TA vector (Invitrogen). The sequence was verified (EZ-seq Macrogen) and pGL2 was
recombined by Gateway technology into pGKGWG (contains GFP reporter gene) and pGreen
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(contains luciferase reporter gene) vectors. The plasmid was transformed to Agrobacterium
tumefaciens strain AGL0 for plant infiltration.
AGL0 agrobacterium strain containing pBin and pGreen vectors containing 35S-Ros1 and 35S-Del
were previously produced by Nikolay. CHS-RNAi construct was obtained from Bo Wang.
Agrobacterium tumefaciens Transient Transformation Assay
Agrobacterium clones were grown for 24 hours at 28°C in LB medium (10g/L tryptone, 5g/L Yeast
extract, 10g/L NaCl) with antibiotics (kanamycin 50μg/ml or spectinomycin 100μg/ml and rifampicin
25μg/ml). Ten times diluted OD of the cultures was measured at 600nm and bacteria were
resuspended in infiltration media (10mM MES buffer, 10mM MgCL2, 100μm acetosyringone) to an
OD of 0.5. After 3 hours incubation on the roller bank (±20°C), leaves of 4-5 weeks old Nicotiana
benthamiana plants were infiltrated. Infiltration was done on the abaxial side of the leaves using a
1mL syringe. Nicotiana benthamiana plants were grown under 16 hours light and 8 hours dark
conditions, 18-22°C and a humidity of ±60%.
Sample homogenizing
Leaf disks (± 15 gram) were harvested by pressing the agro-infiltrated leaf between the lid of a 1,5 mL
Eppendorf tube, 3 days after agro-infiltration. Leaf disks were transferred to a 96 deep well plate
containing 2 metal beads (Ø 3mm) per tube and deep well plate was frozen in liquid nitrogen.
Samples were homogenized using a Tissue-Lyser II (Qiagen), 1 minute at 30r/s and stored at -80 until
further use.
Luminescence measurements with luminometer
Agro-infiltrated samples from -80 were defrosted by adding 100μl PLB buffer (Dual-Luciferase
Reporter Assay, Promega) per 30 gram of leaf material. Samples were vortexed for 5 seconds and
centrifuged for 5 minutes at 13000 rpm. 20μl of the supernatant was added to a 96-well
luminometer white plate. In method one LARII and Stop & Glo buffer (Dual-Luciferase Reporter
Assay, Promega) were used to measure luciferase and renilla activity in the samples. In method two
LAR buffer (Luciferase Assay System, Promega) was used to measure Luciferase activity. A
Luminometer (GloMax®-96 Microplate Luminometer from Promega) was used for measurements,
40μl of buffer was used per 20μl of supernatant. Delay time was 0.8 seconds, integration time of
buffers into supernatant was ten seconds. Obtained values using the Luminometer were direct
measurements of luminescence and are therefore proportional to the amount of luciferase
produced. Values were further analysed using IPM SPSS Statistics 22.
Luminescence pictures
Agro-infiltrated leaves were sprayed with luciferin (1mM) 3 days post agro-infiltration to inactivate
accumulated luciferase. 4 days post infiltration leaves were again sprayed and with luciferin (1mM). 5
minutes after luciferin spraying leaves were cut from the plant and measured with a cooled CCD
camera. Leaves were places on a plastic tray in a box coated with aluminium foil to reduce noise in
the pictures caused by cosmic rays. Measurements were done for 5 or 10 minutes. Emission of
luminescence has a maximum at 560nm therefore a filter in the camera is used to block most other
wavelengths. Analysis of the pictures was performed with ImageJ software. The measured intensity is
proportional to the amount of luciferase produced. The intensity of selected areas was measured
and values were processed using IPM SPSS Statistics 22.
LCMS analysis of agro-infiltrated leaves
Agro-infiltrated leaves were cut off from the plants and ground in liquid nitrogen. 200mg±10mg was
added into a 1.5mL Eppendorf tube. 1mL methanol and 1% formic acid was added. Samples were put
in an ultrasonic bath for 10 minutes. Samples were centrifuged for 5 minutes and after that filtered
8
using a Minisart SPR4 filter. Samples were analysed in positive mode. Analysis of the results was
performed with MassLynx and Thermo Xcalibur Qual Browser.
35S::GL2 tomato Micro-Tom plants
Plant transformation
Seeds of Micro-Tom were sterilized and grown on germination medium (2.2g/L MS salt with vitamins,
10g/L sucrose and 8g/L Daishin agar) for 9 days. Rectangular pieces were cut from the cotyledons
and transferred to plates with COM (4.8g/L MS salts minus vitamins, 112mg/L Gamborg B5 vitamins,
30g/L sucrose, 0.5g/L MES, 8g/L Daishin agar, 0.05mg/L 2.4D, 0.1mg/L IAA, 2mg/L Zeatin, 200μM
acetosyringone, pH 5.8) and incubated overnight. Agrobacterium cells containing 35S::GL2 and the
NPTII gene with an OD of 0.2 were resuspended in 20 mL LIM (4.8 mg/L MS salts minus vitamins,
112mg/L Gamborg B5 vitamins, 30g/L sucrose, 0.5g/L MES, pH 5.8) and 200μM acetosyringone and
incubated for 1 hour. Explants were cultured in agrobacterium & LIM mixture for 20 minutes and
cleaned in LIM twice afterwards. Explants were transferred to plates with COM. After culturing for
two days the explants were transferred to POM (4.8g/Lplates MS salts minus vitamins, 112mg/L
Gamborg B5 vitamins, 30g/L sucrose, 0.5g/L MES, 8g/L Daishin agar, 0.1 mg/L IAA, 2 mg/L Zeatin,
200mg/L cefotaxim, 50 mg/L vancomycin, pH 5.8). After 3 days of culturing on POM, explants were
transferred to SIM (4.8g/L MS salts minus vitamins, 112mg/L Gamborg B5 vitamins , 10g/L glucose,
0.5g/L MES, 8g/L daishin agar, IAA 0.1 mg/L, zeatin riboside 1mg/L, kanamycin 1mg/L, cefotaxim
1mg/L, vancomycin 0.5mg/L) plates. Every two weeks callus was transferred to freshly made SIM
plates. After shoots formed on the explants these were cut an put on RIM medium (4.4g/L MS salt
with vitamins, 15g/L sucrose, 4 gr/L daishin agar, 0.25mg/L IBA, 100mg/L cefotaxime, 100mg/L
vancomycin) and grown until roots formed. Rim medium was also made freshly every two weeks.
Plants with roots were transferred to soil, watered and put in a tray with a closed but transparent lid.
Lid was gradually shifted to get plants adapted to a less humid environment after at least 3 days of
growing in a closed container. Plants were moved to the greenhouse after a total of 10 days in the
container. Greenhouse conditions were 8 hours dark 18°C and 16 hours light 23°C with a humidity of
70%. Plants were grown until harvesting of the tomato’s.
Genotyping of GL2 overexpressing tomato plants: PCR
Small leaves of 27 transformed Micro-Tom plants were cut off, put in 2mL Eppendorf tubes and
frozen in liquid nitrogen. Leaf material was crushed in liquid nitrogen and 1mL CTAB was added
(0.01g/L CTAB, 0.1ml 1M tris (pH 8.0), 0.14mL 5M NaCl, 0.02mL 0.5M EDTA (pH 8.0). Samples were
incubated for 90 minutes at 65°C, shaking. After cooling the samples, 0.8 mL chloroform/isoamylalcohol (24:1) was added, samples were keeled 40 times. Samples were centrifuged for 10 min.
at 13000rpm. The aqueous layer was pipetted into a new tube and centrifuged for 10 min. at
13000rpm. The pellet was washed with 0.5mL 76% ethanol and DNA was dried in a centrifuge
3000rpm with open lid. DNA was dissolved in 50µl water. DNA was amplified with a forward and
reverse primer for the NPTII gene. If NPTII gene is amplified the plants are transgenic. 1µl DNA, 2.5µl
Taq buffer, 0.5µl 10mM dNTP, 0.25µl 10µM forward and backward primer, 0.5µl supertaq and 20µl
water were mixed and used for PCR.
Genotyping of GL2 overexpressing tomato plants: qPCR
Small leaves of 20 transformed, 2 non-transformed and 2 WT plants were harvested and frozen in
liquid nitrogen. RNA was isolated using Invitrap Spin plant RNA minikit. The protocol of the kit was
followed. After RNA was isolated samples were put for 30 minutes on ice. RNA was treated with a
DNase to remove DNA from the samples (Ambion® TURBO DNA-free™ DNase Treatment and
Removal Reagents). 25µl DNase and 25µl buffer was added to each sample and incubated 30 min. at
37°C. DNase was inactivated by adding DNase inactivating reagent and incubated for 5 minutes at
room temperature. RNA was measured in the NanoDrop for concentration and put on gel to check
whether RNA was pure. After RNA was checked, cDNA was synthesised (iScript™ cDNA Synthesis Kit ).
500ng rna, 2µl 5x iscript reaction mix, 0.5µl iscript reverse transcriptase was added and put 5
9
minutes at 25°C, 30 minutes at 42°C and 5 minutes at 85°C. 10µl SYBR green, 2mM forward and
reverse primer (Table 3), 4.5µl HPLC water and 50ng/µl cDNA was combined and used for qPCR.
Actine primers were used as a control. qPCR was performed using a BioRad MyIQ Single Color RTPCR. Program was one time 5.5min at 95°C, 40 times 15sec 95°C and 1min 50°C.
Table 3 | Primers used for qPCR analysis of GL2
Gene
Forward primer
Reverse primer
AAA CAT AGT GGA CTC TCT GTA CC TCA CAT TGC TGC TGA AGA GTA G
Glabra2
CT values were acquired using the qPCR measurement. Gene expression levels were obtained using
the following formula:
ΔΔCT=((Ct GL2 WT tomato – Ct actine WT tomato) – (Ct GL2 transformed plant – Ct actine
transformed plant))
Phenotyping of GL2 overexpressing tomato plants
Phenotypes of transformed tomato plants were captured using a Canon PowerShot G12. An excel file
was made with several observational phenotypic traits.
Seed storage of GL2 overexpressing tomato plants
Ripe tomatoes were harvested and seeds were collected. Seeds were washed in 1% acetic acid for 24
hours and dried on sterilized filter paper.
Statistical analyses
Statistical analyses were performed using IBM SPSS Statistics 23. For root hair analysis an ANOVA was
performed with genotype and DEX treatment in a model. The natural logarithm of the root hair
length was calculated to obtain a normal distribution and values were used in the ANOVA and LSD
test. For the luciferase assay background values were subtracted and log values were calculated from
remaining luminescence values to achieve a normal distribution. Values were used for a LSD test.
10
Results
Two transcription factors, Ros1 (MYB transcription factor) and Del (bHLH transcription factor), from
Snapdragon, were stably transformed in tomato cultivar Micro-Tom under an inducible promoter.
Ros1 and Del can induce anthocyanin production in tomato by upregulating all steps in the
anthocyanin pathway. Ros1&Del transcription could be activated upon dexamethasone (DEX)
treatment. This inducible system was created to study anthocyanin production in tomato plants,
however it was found that an epithelial cell fate determinant, GL2, was also highly upregulated by
the transcription factors Ros1&Del.
Less trichome type 7 and wider stomatal aperture observed on Ros1&Del induced plants
GL2 regulates trichome- and stomata formation in Arabidopsis (Qing and Aoyama, 2012). Therefore it
was studied whether Ros1&Del and thus GL2 induced tomato plants show a deviating phenotype in
trichomes and stomata from non-induced tomato plants. To investigate the effect of Ros1&Del
induction in tomato plants, DEX was applied to activate the Ros1&Del system. Plants were watered
20 days with or without DEX (10µg/L) before analysing newly grown leaves. In order to visualize
trichomes and stomata, a scanning electron microscope (SEM) was used. Leaves were magnified
250x in order to be able to visualize and count trichomes, hairs and stomata.
Trichome type 6
15
Ros1&Del non-induced
B
10
5
Trichome type 7
15
Ros1&Del induced
# trichomes counted
# trichomes counted
A
Ros1&Del non-induced
Ros1&Del induced
10
5
0
0
Downside of leaf
C Ros1&Del -
Downside of leaf
Upperside of leaf
Upperside of leaf
D Ros1&Del +
B
400 µm
Figure 1 | Counted numbers of trichomes type 6 (A) and type 7 (B) on a 1200μmx800μm piece of Ros1&Del
induced or non-induced tomato leave. Tomato plants were induced or not induced with DEX for 20 days prior
to leave examination. Newly formed leaf surface was observed under a scanning electron microscope. Error
bars indicate standard error (n=5); 5 pictures were made from 1 leaf. Representative examples of SEM pictures
of abaxial side of non-induced (C) and induced Ros1&Del tomato leaves (D) (1200μmx800μm). Squares indicate
type 6 trichomes, circles type 7 trichomes.
One induced leaf and one non-induced leaf of Ros1&Del plants were observed with SEM. Glandular
trichomes type 6 and 7 were identified based on their morphology (Glas et al., 2012), observed on
tomato leaves and counted (Figure 1) both on the abaxial and adaxial side of the leaf. For trichome
11
type 6 no differences could be observed between the wild type and Ros1&Del induced plant. Less
trichomes type 7 were present on the Ros1&Del induced leaf. However, there were not enough
replicates tested to make solid conclusions on the significance of this observation and extrapolate
this result to a population of induced Ros1&Del plants.
Trichome type 3 and 8
A
Ros1&Del non-induced
B
Ros1&Del induced
# counted hairs
50
40
30
20
10
0
Downside of leaf
Upperside of leaf
Figure 2 | (A) Total numbers of trichome type 3 and 8 counted on a 500μmx500μm piece of leave of induced or
non-induced Ros1&Del plant. (B) Aberrant rod shaped trichome on Ros1&Del induced leaf, indicated by arrow.
Also, non-glandular trichomes type 3 or 5 (from these pictures it was not possible to distinguish
them) and 8 were observed but were not quantified separately. The total amount of non-glandular
trichomes is the same on induced and non-induced tomato leaves (Figure 2A). One aberrant looking
trichome was observed on a Ros1&Del induced leaf (Figure 2B), which had a rod shaped top. All
other non-glandular trichomes had a pointy top.
12
Stomata were also observed and counted on SEM pictures (Figure 3A). No differences in amounts of
stomata could be observed between induced and non-induced Ros1&Del leaves. However, the
Ros1&Del induced leaf showed wide open stomata while the wild type leaf showed more stomata
which were closed or almost closed (Figure 3B,C &D). On Ros1&Del non-induced leaves, three times
as much stomata were open than closed. On Ros1&Del induced leaves there were 15 times more
open stomata than closed ones. In addition it is visible that stomata on Ros1&Del induced leaves
have a wider stomatal aperture (Figure 3D).
Stomata
A
Ros1&Del non-induced
Ros1&Del induced
100
50
Closed
100
# open and closed stomata
# counted stomata
150
Stomatal opening
B
Open
80
60
40
20
0
0
Downside of leaf
Upperside of leaf
C Ros1&Del -
Green
Down
Purple
Down
Green Up Purple Up
D Ros1&Del +
100 μm
100 μm
Figure 3 | Stomatal counting and aperture. (A) Total numbers of counted hairs. Error bars indicate standard
error (n=3). (B) Ratio between open and closed stomata counted on downside of induced and non-induced
tomato leaves. (A) and (B) were both counted on a 500μmx500μm piece of leave of induced or non-induced
Ros1&Del plant. Representative SEM picture of a 500μmx500μm piece of non-induced (C) and induced (D)
Ros1&Del leave. Scale bars are shown in pictures.
13
Shorter root hair length upon Ros1&Del expression
GL2 also regulates root hair growth by suppressing development of root hairs in non-hair cells. GL2
mutants or knockout plants grow more root hairs; extra formed root hairs show similarity in timing
and elongation compared to wild type root hairs (Masucci et al., 1996). Ros1&Del induced plants
show GL2 upregulation, therefore a reduction in amount of root hairs was expected in Ros1&Del
transformed plants. Wildtype and Ros1&Del tomato plants were grown to figure out whether
Ros1&Del induction influences root hair development. Wildtype and Ros1&Del tomato seeds were
germinated and after 8 days small seedlings (0.3-1.5 cm) were transferred to agar plates containing
DEX (10µg/L) or no DEX. Seedlings were grown for 3 days in an angle of ±60° before examining roots.
Roots from Ros1&Del plants became light purple to intense purple one day after transferring
seedlings to DEX containing growth medium. Ros1&Del induced plants formed shorter root hairs
(Figure 4). Furthermore it was observed that some Ros1&Del induced roots did not stay attached to
the agar and started to grow into the air.
A
B
Figure 4 | (A) Root pictures of Ros1&Del and Wildtype plants treated with DEX (right side) or without DEX.
Plants were germinated on filter paper and after 8 days transferred to plates with medium containing DEX
(10µg/L) or no DEX. Plants were grown 3 days on this medium, in an angle of ±60°, before examining. (B)
Detailed pictures of roots. For every root pictured in (A), two pictures were made with a higher magnification
and shown in (B). Scales are shown in pictures.
14
Root hair length
Length of root hairs (mm)
0.7
c
0.6
0.5
0.4
a
a
0.3
0.2
b
0.1
Figure 5 | Average root hair length of Ros1&Del and
WT plants grown with or without DEX. Root hair
length was measured on two positions per root and
on five roots per treatment (Figure 4). DEX was
applied 8 days post germination and seedlings were
grown 3 days with or without DEX on agar medium.
Error bar indicate standard error (n=5). Letters in
graph indicate statistically different from the other
groups LSD test (Table S1). RD-: Ros1&Del plants no
DEX, RD+: Ros1&Del plants with DEX, WT-: Wildtype
plants non DEX, WT+: Wildtype plants with DEX.
0
RD-
RD+
WT-
WT+
Root hair length of ten root hairs was measured on two places of each root using ImageJ (Figure 5).
Ros1&Del induced tomato roots grow shorter root hairs (p<0.001 LSD test, Supplementary table 1
(Table S1)) compared to non-induced roots. Also, WT plants treated with DEX show a decrease in
root hair length (p=0.046 LSD test, Table S1). The effect of DEX on Ros1&Del roots is however much
larger (p<0.001) than on WT roots (ANOVA, Table S2). Root hair length decrease of treated versus
non-treated plants is 86% and 27% for Ros1&Del and Wildtype plants respectively.
Roots grown on DEX containing agar became very light to intensely purple. The more purple a root
became induced by DEX treatment, the shorter root hairs were observed (Figure 6). Purple
coloration was not quantified.
Figure 6 | DEX induced Ros1&Del Micro-Tom
root. The more purple the root became the
shorter root hairs were observed.
Interesting genes up or down regulated in Ros1&Del induced callus and roots
Ros1&Del induced roots of tomato show a deviating phenotype from wild type roots. Especially
purple coloration and root hair length differ. A database was inspected in which was stated which
genes were up or down regulated in Ros1&Del induced callus and roots. Anthocyanin related genes
and GL2 were already found to be upregulated in both Ros1&Del induced callus and roots. In
addition was looked for genes which could explain the phenotypic differences found in Ros1&Del
plants. Genes involved in cell elongation were found, several expansins, extensins, P4Hs (prolyl 4hydroxylase) and RDH6 (root hair defective 6) were found in the database (Table 1). Hormones are
known to be involved in root hair elongation as well (Kapulnik et al., 2010), therefore also auxin,
ethylene and strigolactone related genes were searched in the database. Several auxin related genes
were down or up regulated (e.g. GH3-8). Furthermore was looked for genes involved in MBW
complex in order to figure out if more MBW complex genes were upregulated as a result of
Ros1&Del expression.
15
MBW related genes
Cell wall related genes
Table 1 | Genes up or down regulated in Ros1&Del induced callus and root tissue. Genes related to cell wall
functioning and genes involved in MBW complex were examined. For MYB113/114 more homologs were found
and were either down or up regulated.
Abbreviation
Solyc Number
GL2
Solyc03g120620
DF4R
GH3-8
EXPB2
Solyc02g085020
Solyc02g092820
Solyc03g093390
AT5G64100
Solyc11g007210
HRGP1
Solyc01g005880
RHD6/RSL4
P4H
EXT
Solyc02g091440
Solyc02g067530
Solyc01g005880
Solyc01g097680
Solyc12g100110
TT8
PAP
Solyc09g065100
Solyc07g008570
TTG1
Solyc03g097340
MYB113/11
4
Solyc07g008010
Solyc05g051550
Function
homeobox leucine zipper
protein
Anthocyanidin
biosynthesis
Auxin synthetase
Expansin
Peroxidase superfamily
protein
hydroxyproline-rich
glycoprotein
bHLH TF, Promotes postmitotic cell growth in roothair cells
Prolyl 4-hydroxylase
Extensin-like proteins
bHLH (regulates flavonoid
biosynthesis)
purple acid phosphatase
Anthocyanin
accumulation, trichome &
root hair regulation
MYB TF. Involved in
regulation of anthocyanin
biosynthesis
Fold change
Callus 3hr
Callus 24hr
Roots 3hr
Roots 24hr
6.4
5.92
39
30.2
14651
6.73
-54.11
19492.1
3.93
-7.71
4810.8
8.08
-33.26
5442.8
6.92
-19.83
25.20
1
-34.47
-67.24
1
1
-58.16
-58.16
4.77
2.03
1
1
1
3.28
-1.14
1
1
4.1
1.08
2.25
-58.16
-44.17
-179.63
-1.42
-1.01
-58.16
-44.17
-32.81
68.93
-5.12
1327.67
-1.86
224.24
-1.18
2558.87
-2.25
4.35
2.87
4.14
2.94
-3.45
-5.83
2.11
-5.83
3.59
-2.45
12.22
-6.89
16
Ros1&Del activate promoter of GLABRA2
GL2 is upregulated in tomato plants overexpressing Ros1&Del transcription factors (Table 1). In
addition, in above experiments using Ros1&Del induced plants, phenotypes were found (less
trichomes and shorter root hairs) which might be linked to overexpression of GL2. However, there is
no evidence yet if GL2 is regulated by Ros1&Del and if this activation is direct or whether it goes via
other activators. In tomato, GL2 was identified in ANT1 overexpressing plants. ANT1 is a MYB
transcription factor involved in anthocyanin production. ANT1 is closely related to Rosea1 (Mathews
et al., 2003) and this therefore suggests that GL2 is activated by a anthocyanin regulating
transcription factor.
In order to figure out if GL2 is regulated by Ros1&Del, a transactivation assay was performed. The
GL2 promoter was fused to a luciferase reporter gene in a GW-Greenluc-pGreen vector (pGL2),
enabling to visualize GL2 promoter activation. Transactivation assays were performed by agroinfiltration of N. benthamiana plants.
Three different methods were tested to measure luminescence. First, a dual luciferase reporter assay
system (Promega) was used. This system measures firefly luminescence (LUC) and Renilla
luminescence (REN) in one sample using two different reagents. This implies both pGL2-luciferase
and 35S-Renilla were infiltrated in the leaves. REN was measured as an internal control to correct for
infiltration differences. After measuring LUC and REN, normalized values were obtained by dividing
firefly over renilla luminescence to achieve normalized values.
Leaf disks from infiltrated N. benthamiana plants were harvested and snap frozen, samples were
homogenized in a 96 wells plate by a ball mill (Retsch) and thawed by adding lysis buffer. Samples
were centrifuged and 20µl supernatant was used for luminescence measurements. Substrates
needed to measure LUC and REN were added and luminescence was measured by a luminometer
(GloMax®-96 Microplate Luminometer, Promega) (Figure 7).
LUC
Luminescence (RLU) x1000
Luminescence (RLU) x 1000
2500
2000
1500
1000
500
0
Ren pGL2 pGL2 RD
+ Ren + RD +
Ren
RD +
Ren
C
LUC/REN
0.025
400000
350000
0.02
300000
Luciferase / Renilla
A
Luminescence 1st method
B
REN
250000
200000
150000
100000
50000
0
0.015
0.01
0.005
0
Ren pGL2 pGL2 RD
+ Ren + RD
+ Ren
RD +
Ren
Ren pGL2 +pGL2 + RD
Ren RD +
Ren
Figure 7 | First method to measure Luminescence. (A) Luminescence from firefly luciferase (B) Luminescence
from Renilla luciferase. (C) Normalized values by dividing LUC over REN. Error bars indicate standard error
(n=9). Ren: Renilla, pGL2: promoter-GL2-luciferase, RD: Ros1&Del.
Increased LUC signals were measured after co-infiltration of pGL2 with Ros1&Del (Figure 7A).
However, after correcting LUC values with REN for infiltration differences, no effect of Ros1&Del on
pGL2 induction was found (Figure 7C). Interestingly, REN values increased when co-infiltrated with
Ros1&Del, with or without presence of pGL2 (Figure 7B). This means more Renilla is expressed in
Ros1&Del infiltrated leaves or the renilla signal measured is influenced and gives higher values.
17
RD +
Ren
Therefore a new approach was tested in which only firefly luciferase was used (Luciferase Assay
Systems, Promega).
In this second approach leaf disks were harvested and homogenized using the same protocol as in
the first method. Differences with the previous method were the use of only one reagent, firefly
luciferase reagent and a different lysis buffer.
Luminescence 2nd method
B
A 1600
Luminescence (RLU) x1000
1400
1200
pBin
1000
800
577x
600
pGL2
11x
pGL2+
Ros1&Del
4x
400
pGL2
200
0
pGL2
pGL2 + RDpGL2 + RD
+ CHSrnai
pBin
RD
CHS rnai
pGL2+
Ros1&Del
pGL2+
Ros1&Del+
CHS-RNAi
Figure 8 |(A) Luminescence measured by luminometer (Promega). pGL2 was agro-infiltrated in leaves in
combination with Ros1&Del and RD+CHS-RNAi. pBIN, Ros1&Del, and CHS-RNAi were measured as controls.
Three leaves were infiltrated on separate plants; from each leave 3 leaf disks were taken. Error bar indicates
standard error (n=9). pGL2: promoter of GL2 fused to luciferin, RD: Ros1&Del. (B) Fold change in luminescence
measured between samples from values showed in (A).
The second approach gave in general 7-fold lower LUC signals, but the noise from background signals
was 40 times lower (Table S3). However, the increase of LUC signal after co-infiltration of pGL2 with
Ros1&Del compared to pGL2 alone was much higher in this new method (11x versus 3x) (Table S4).
This second approach had less background signal but this method still gave large standard errors. In
addition, this technique was very sensitive to oxidation of the leaf samples and therefore no valuable
replications could be performed.
A different technique for measuring luminescence is by making pictures with a highly sensitive,
cooled, CCD camera. This technique makes it possible to visualize luminescence and examine how
expressed proteins are located within leaves. Leaves were agro-infiltrated and sprayed 3 days post
infiltration (dpi) with 1mM Luciferin and measured 4 dpi after spraying or dipping of leaf disks for a
second time with 1mM luciferin. This luciferin pre-spraying on 3 dpi inactivates all luciferase
produced until then, thus on day 4 only the ongoing transcription and translation was measured.
Both leaf disks (Figure 9) and whole leaves (Figure S1) were measured. This third method differs from
the other two by inactivating the accumulated luciferase produced during day one to three. Now
only the luciferase produced during day four is measured. So this method shows the actual luciferase
production in 24 hours.
The pGL2 only construct shows very low luminescence, 1.05 x higher than background, compared to
577x higher than background in method 2. In addition this method nicely displays the measured
luminescence between different constructs (Figure 9A).
18
pGL2
pGL2
+RD
pGL2+
CHSRNAi
RD+CHSRNAi
Leaf 1
Leaf 2
Leaf 3
mean luciferase signal per pixel (RLU)
A
Luminescence 3rd method
B 1200
pGL2+
1000
800
600
400
200
0
pGL2
Figure 9 | (A) Leaf disks of agro-infiltrated leaves
incubated in 1mM luciferin. All constructs were spotinfiltrated on one leaf, in total three leaves were
infiltrated. Measurement was done for 5 minutes. (B)
Relative light units were measured by ImageJ,
background signal was subtracted from samples.
Standard error is visible in graph (n=3). (C) Fold change
in luminescence between different samples measured
in leaf disks.
pGL2+ RD
C
pGL2
8x
pGL2
6x
pGL2+ CHSrnai
pGL2+
RD+CHSrnai
pGL2+
Ros1&Del
3x
pGL2+
pGL2
pGL2+
+Ros1&Del CHS-RNAi Ros1&Del+
CHS-RNAi
The third method was used for calculations because it was decided as the most reliable method and
gives ongoing, not accumulated, luciferase activation values. ImageJ was used to measure the
intensity of different leaf disks. This measured intensity is proportional to the amount of
luminescence and thus luciferase produced, which is proportional to the amount of pGL2 activation.
Ros1&Del co-infiltrated with pGL2 gave higher luminescence values (p=0.003, Tukey’s HSD, Table S5)
compared to pGL2 alone. Besides leaf disks whole leaves were measured (Figure S1) and in whole
leaves the same significance was found for pGL2 induction by Ros1&Del (p=0.003, Tukey’s HSD, Table
S6).
CHS-RNAi increases pGL2 activation
The above results suggest that Ros1&Del directly activate pGL2. However, Ros1&Del expression
changed plant colour by inducing anthocyanin production. This means also anthocyanins could play a
role in the above found promoter of GL2 activation. To assess the effect of anthocyanins on pGL2
activation, CHS-RNAi was co-infiltrated with Ros1&Del and pGL2. CHS-RNAi decreases naringenin
chalcone and thus the whole flavonoid en anthocyanin pathway (Schijlen et al., 2007).
A 3-fold increase in luminescence was measured when Ros1&Del and pGL2 were co-infiltrated with
CHS-RNAi (Figure 9C). In whole leaves this increase was significant (p=0.017) in leaf disks just not
significant (p=0.071). Surprisingly pGL2 induction was 6-fold increased upon co-infiltration with CHSRNAi. This was not expected while no transcription factors were infiltrated in these leaves which
could have activated the promoter. Furthermore no anthocyanins were present in these leaves so no
anthocyanins could be down-regulated. Both in leaf disks (p=0.007) and whole leaves (p=0.014) this
effect of CHS-RNAi on pGL2 induction was significant.
19
Figure 10 | Infiltration of agrobacterium strains in N. benthamiana leaves. 1:control 2:pGL2+RD 3:pGL2+CHSRNAi 4:pGL2+RD+CHS-RNAi 5:pGL2+DFR 6:pGL2+RD+DFR. Pictures were made 5dpi.
Leaves which were agro-infiltrated and used for luminescence measurements were observed on
anthocyanin formation (Figure 10). All combinations with Ros1&Del gave purple coloration and thus
formed anthocyanins. The combination Ros1&Del with CHS-RNAi was expected to give a reduction in
anthocyanin formation and thus purple coloration. A decrease in anthocyanin production can be
observed on leaves infiltrated with Ros1&Del and CHS-RNAi. However, no big effects in purple
coloration could be observed on the infiltrated leaves. To obtain more quantitative results on
anthocyanin production, LCMS was performed on infiltrated N.benthamiana leaves.
To figure out whether CHS-RNAi causes a reduction in anthocyanin formation, leaves of N.
benthamiana were infiltrated with either RD, CHS-RNAi, RD+CHS-RNAi and after 5 days harvested.
Leaves were analysed using a LCMS to find differences in anthocyanin content or to find other
metabolites that can explain the higher pGL2 induction by CHS-RNAi co-infiltration. Metabolites were
analysed in positive mode. First, the only anthocyanin produced in N. benthamiana leaves,
delphinidin-3-rutinoside (D3R), was analysed (Figure 11).
A
Delphinidin 3 Rutinoside
M/Z 611 RT=8.9 min
3000000
2500000
B
Average D3R
2000000
1500000
1000000
500000
Mass Counts
2500000
0
Control
RD
CHS-RNAi RD- CHSRNAi
2000000
1500000
1000000
500000
0
Figure 11. LCMS analysis of D3R. Three control leaves and five leaves of other constructs were measured by
LCMS. (A) Amount of anthocyanins detected per leaf analysed (B) Average amount of anthocyanin D3R with
standard error visible in graph (n=5).
On average Ros1&Del and Ros1&Del+CHS-RNAi gave a small reduction in D3R production (Figure
11B), this effect is however not significant (p=0.113, independent samples t-test). There are large
20
variations found in anthocyanin production between leaves (Figure 11A). The effect of CHS-RNAi on
pGL2 activation is probably not caused by anthocyanin reduction. However, from these results no
conclusions can be drawn on involvement of anthocyanin on pGL2 activation.
The dataset of obtained masses was examined for compounds which had a large difference in
expression between Ros1&Del and Ros1&Del+CHS-RNAi. Several compounds were found which
ranged from 2x to 14x less expression upon CHS-RNAi co-infiltration (Figure S2). These compounds
were however not identified so further research into these compounds is needed.
35S::GL2 stable Micro-Tom production
To identify the functions of GL2 in tomato and to figure out whether the observed phenotypic
aberrations in Ros1&Del plants were induced by GL2, GL2 overexpressing plants were produced.
Micro-Tom leaf disks were transfected with agrobacterium containing 35S::GL2 and NPTII genes.
Shoots were formed 5 months after transfection and roots were formed at least 4 weeks after
transferring shoots to root inducing medium. Root producing shoots were transferred to soil and
viable plants were grown further in a greenhouse. To verify whether the transgene was present in
the Micro-Tom plants first a PCR was performed on the NPTII gene. Out of 25 tomato plants, 23
turned out to be transgenic. Plant numbers 4 and 8 were not transgenic (Figure 12).
To quantify the amount of GL2 expressed in the transformed tomato plants a qPCR on RNA was
performed. Pictures of transformed plants were combined with level of GL2 expression (Figure 12).
Several plants showed uncommon phenotypes which could be caused by GL2 overexpression but
also by tissue culture conditions. Phenotypic traits were recorded in a table (Table S7). No
correlations could be made between qPCR data and phenotypes. For example seedless plants were
found in the low, medium and high GL2 expressing lines. In addition, some plants remained very
small and did not elongate well, these phenotypes were also found in low, medium and high
expressing lines.
21
Figure 12 | 35S::GL2 expression data. qPCR was performed to identify GL2 expression levels in transformed plants. Plant numbers and pictures are shown on the x-axis.
Amount of GL2 expression compared to control is shown on the y-axis. One replicate per plant was taken. 35SGL2-4 and 35SGL2-8 did not contain transgene, WT1 and WT2
22
are Micro-Tom wild type plants grown from seeds.
Discussion
Tomato Micro-Tom plants transformed with a DEX-inducible Ros1&Del system were used in order to
study the phenotype of GL2 overexpression. In addition a trans-activation assay was performed using
a luciferase reporter system for examining if and how GL2 is regulated by Ros1&Del.
Phenotypic effects of Ros1 and Del, anthocyanins transcription factors, overexpression
An initial goal of the project was to identify phenotypic differences between Ros1&Del induced and
non-induced tomato plants.
Trichomes and stomata
Ros1&Del expression increased GL2 production and therefore it was expected that more or different
trichomes would be produced on Ros1&Del induced plants. This was expected because upon
transient expression of Ros1&Del in N. benthaminana more nornicotine conjugates were identified
than in WT plants (Outchkourov et al., 2014). Nornicotine conjugates are found in trichomes (Laue et
al., 2000). Furthermore GL2 overexpression increases trichome initiation in Arabidopsis; additive
pGL2::GL2 expression induced more trichomes (Ohashi et al., 2002). In order to figure out the effect
of Ros1&Del overexpression on trichomes of tomato plants, a scanning electron microscope (SEM)
was used. Trichome type 3/5, 6 and 7 and 8 were identified on both induced and non-induced leaves.
No differences in trichome type 6 abundance could be observed but less trichomes type 7 were
observed on Ros1&Del induced plants. Trichome type 3/5 and 8 were not evaluated separately while
too little details were available in the pictures. Trichome type 6 and 7 are both glandular trichomes,
these trichomes contain chemicals which might play a role in pest resistance. Trichome type 6 is the
most abundantly found trichome in S. lycopersicum. A large variety of compounds can be produced
by these trichomes (Bergau et al., 2015). For both type 6 and 7 not much is known yet about the
exact metabolites these trichomes produce and what function these chemicals have. Therefore it is
hard to speculate about the chemical or physiological consequences of trichome type 7. Probably this
was just a leaf effect because only one leaf was pictured for each treatment in this experiment. To
make any solid conclusions more replicates should be used in the follow up experiments. Also, one
abnormal looking trichome was found on the Ros1&Del induced leaf. This rod shaped phenotype is
also found in an odourless mutant (od-2) which is defect in terpene production (Kang et al., 2010). To
figure out if this phenotype is found more often by Ros1&Del induction, again, more replicates
should be taken into account.
On the same SEM pictures stomata abundance was counted. No difference in amount of stomata
was found. However, Ros1&Del induced leaves showed more open stomata than non-induced leaves.
This phenomenon could be explained by several points. Physiological differences generated by the
induction of Ros1&Del and the consecutive purple coloration of the leaves could be the cause.
Abscisic acid has an influence on stomatal opening, mutants in which stomata resist to close can be
treated with abscisic acid and revert to a wild type phenotype (Tal and Nevo, 1973). These abscisic
acid mutants wilt easily. The purple Ros1&Del tomato plant did not wilt easier than the non-induced
Ros1&Del tomato plant so this makes it less likely that a lack in abscisic acid is causing the open
stomata. Another explanation could be GH3-8 which was found to be highly upregulated in Ros1&Del
induced plant material (Table 1). In rice, GH3-2 overexpression, which has the same function as GH38, leads to stomata with a higher aperture (Du et al., 2012). The open stomata could also be caused
by treatment differences, but the leaf samples of Ros1&Del induced and non-induced plants were
embedded at the same time point. Furthermore, the plants got approximately the same amount of
soil, water and light. In order to know what caused the open stomata more leaves should be
visualized, probably lower-cost microscopes could be used. Also the stomatal conductance could be
measured in order to figure out if leave transpiration is higher in induced plants. This could easily be
23
measured with a porometer. ABA levels in tomato leaves could be measured as well, ABA stimulates
stomatal closure in order to minimize transpiration levels. ABA could be measured by LCMS in order
to figure out whether levels are changed in Ros1&Del induced plants (López-Carbonell & Jáuregui,
2005).
Root hairs
GL2 is known to have a supressing effect on root hair formation in Arabidopsis. In Arabidopsis the
root consists of hair and non-hair cell files. GL2 suppresses hair formation in the non-hair cells;
mutants defect in GL2 also form root hairs in non-hair cells (Lin et al., 2015). In Arabidopsis all root
hair experiments were based on knock-out or GL2 mutants, which led to more root hairs. In
Arabidopsis two reports were found on GL2 overexpression. One did not report anything about root
hairs (Ohashi et al., 2002), the other did not find any epidermal processes changed by overexpression
of GL2 (Wang et al., 2015).
In this study a clear effect of Ros1&Del over-expression was observed on root hair formation. On
induced plants shoots and roots became purple and root hairs were shorter and on some roots
hardly visible. DEX treatment of WT plants also caused slightly shorter root hairs. This could be due
to an effect of DEX on root hair growth in general. Surprisingly the root hairs on Ros1&Del induced
tomato plants became shorter while it was expected to get less hairy roots but no changes in length
(Masucci et al., 1996). This discrepancy could be attributed to the differences between Arabidopsis
and tomato root hair formation. Tomato plants have type 1 root hair patterning which means every
cell can produce a root hair whereas Arabidopsis has type 3 patterning which means hair cell files are
separated by one to three non-hair cell files (Pemberton et al., 2001). In Arabidopsis GL2 has an
effect on the non-hair cells but tomato plants do not have non-hair cells. It is possible, therefore, that
GL2 works by a different mechanism in tomato roots.
Another possible reason for the short root hairs is the effect of up-or down
regulated genes in Ros1&Del induced plants in addition to GL2 upregulation.
GH3-8 turned out to be also upregulated in callus and roots of Ros1&Del
induced tomato plants. GH3-8 is an IAA-amino synthetase and inhibits free
IAA accumulation by coupling IAA to amino acids and thus inactivating it (Ding
et al., 2008). In Arabidopsis the GH3 family is only highly expressed in the root
and especially the root tip (eFP browser). GH3-8 overexpression in rice led to
abnormal morphology including shorter plants and shorter and less roots. This
altered morphology in rice is probably caused by enhanced auxin inactivation
by overexpression of GH3-8. Thus higher GH3-8 levels leads to less auxin
availability. Auxin induces the expression of expansins, which are proteins
found in the plant cell wall (Ding et al., 2008). Expansins can regulate cell
enlargement by loosening cell wall proteins (Cho and Cosgrove, 2002). Two
expansin genes are identified as most important for root hair elongation in
Arabidopsis. GL2 is known to reduce expression of at least one of these
expansin genes (Cho and Cosgrove, 2002). In the tomato database with up or
down regulated genes in Ros1&Del induced callus and roots, several expansins
were found to be highly downregulated (Table 1). This can explain the shorter
root hairs while fewer expansins were produced in Ros1&Del induced roots
(Figure 5).
GH3-8 ↑
Auxin ↓
Expansin ↓
Root Hair length ↓
Figure 13 | Proposed
explanation shorter
root hair formation
by Ros1&Del
induction
Another gene which could be involved in root hair elongation is P4H. P4H (prolyl 4-hydroxylase) is
found to be the most important gene for root hair elongation in Arabidopsis (Velasquez et al., 2015).
Prolyl 4-hydroxylase is slightly down regulated in Ros1&Del induced plants, Prolyl 4-hydroxylase
alpha subunit like protein is slightly upregulated after 3 hours and slightly downregulated after 24
hours. This expression data makes it less likely that P4H is the main cause of shorter root hairs in
24
these tomato plants. However, P4H is important in proline hydroxylation of extensins. Extensins are
essential for cell extension, in Arabidopsis one extensin was identified required for root hair
elongation (Baumberger et al., 2001). In the database with gene alterations in Ros1&Del induced
plants, several extensin like proteins were highly (more than 50 fold) downregulated in root tissue
(Table 1). This could imply that less extensins could also have an effect on root hair elongation.
In Arabidopsis RDH6 was suggested as an important downstream gene of GL2 involved in root hair
initiation (Cho and Cosgrove, 2002). RHD6 positively regulates RSL4 expression (Lin et al., 2015). RSL4
controls root hair length by controlling genes involved in cell expansion (Yi et al., 2010). It is also
found that RHD6 downregulates two main expansins needed for root hair elongation (Cho and
Coscrove, 2002). RHD6 and RSL4 were blasted in tomato genome and both were corresponded to
Solyc02g091440. Solyc02g091440 is a BHLH transcription factor, in callus this TF is upregulated ± 4
times, in roots no effect is found. Probably in tomato homologs of RHD6 and/or RSL4 do have a
different function or these genes are not corresponded to the right Solyc number in the gene
database.
In conclusion Ros1&Del overexpression has influences on epidermal phenotype. Probably effects are
found in trichome and stomata and significantly shorter root hairs were found in Ros1&Del induced
plants. These effects could be caused by GL2 induction. However, the decrease in root hair length is
also likely to be caused by GH3-8 induction, which in turn down-regulates auxin, which leads to
downregulation of expansins. Moreover, extensins could also be in involved. It is possible GL2, GH38, expansins and extensins are all interacting with each other to initiate root hairs and elongate
them, probably with GL2 as an upstream transcription factor.
GLABRA2 induction by Ros1&Del
Another goal of this project was to figure out if GL2 is directly activated by RD or if there is an indirect
activation. In order to figure this out a trans-activation assay was performed. The GL2 promoter was
fused to a luciferase reporter gene. The amount of luminescence (LUC) measured gave information
about the activation of GL2 promoter. It was expected that Ros1&Del directly activate GL2.
Three different methods were used to measure LUC. First a dual reporter system was used in which
LUC and renilla luminescence (REN) were measured. A problem encountered with this test was a
measured increase the internal control REN values upon co-infiltration with RD (with or without GL2),
which was observed repeatedly. This effect was not expected to come from the a-specific promoter
activation of renilla while a 35S promoter was used. This effect might be caused by increased cell
proliferation or increased cell metabolism of RD infiltrated leaves. If cells thrive by RD infiltration the
transfection efficiency is affected and then probably more renilla is produced. The increased REN
signal might also be caused by a Ros1&Del or anthocyanin effect on REN production or by an effect
of anthocyanins on renilla perception. Because problems with renilla production could not be
avoided easily a second approach was tried. In the second method only LUC was measured by using a
different assay system. Compared to method one the controls were very stable and gave very low
signals, indicating less background signals. However, the standard deviations were still large. A
reason for the large standard deviation might be expression differences found within the leaf. Most
variation in agroinfiltration is caused by within leaf expression differences (Bashandy et al., 2015).
Only very small spots were taken from leaves (Ø1 cm) to use for this assay so probably larger areas
should have been taken to overcome the differences in expression. The first two methods measured
the accumulated luciferase production, which is not the real, ongoing, promoter activation. In the
third method, leaves were not ground but pictures of leaves were made with a cooled CCD camera.
In addition leaves were sprayed one day before measuring with luciferin to inactivate the
accumulated luciferase. This method gave lower standard deviation and less background signals in
controls therefore this method was chosen as the most reliable.
25
In all three methods could be seen that RD induced pGL2 activation. From method three could be
concluded that co-infiltration of RD with pGL2 gave 8 fold induction of luminescence. So these results
support the expectation that RD directly induces GL2 (Figure 14A). Surprisingly, co-infiltration of CHSRNAi with pGL2+Ros1&Del gave higher promoter induction. This result could imply that CHS-RNAi
induced anthocyanin downregulation leads to more pGL2 activation (Figure 14B). Remarkably, coinfiltration of pGL2 with CHS-RNAi gave six fold higher luminescence compared to pGL2 alone. This
GL2 promoter induction by CHS-RNAi was not expected because no transcription factors were coinfiltrated. CHS-RNAi downregulates flavonols and anthocyanins, therefore probably downregulation
of flavonols and/or anthocyanins could have increased pGL2 activation (Figure 14C). Due to the fact
that no anthocyanins were present when Ros1&Del were not infiltrated, CHS-RNAi could only down
regulate already present flavonols in the pGL2+CHS-RNAi infiltrated leaves. Besides that, LCMS
analysis of CHS-RNAi co-infiltration with RD did not show a significant downregulation of
anthocyanins. Therefore the effect found in increased pGL2 activation is possibly mainly caused by
flavonols. Flavonols are known to play roles in several developmental and physiological pathways in
Arabidopsis (Maloney et al., 2014). Wang et al (2015) found a negative correlation between GL2 and
anthocyanin; GL2 overexpression resulted in less anthocyanin production. So probably both flavonols
and anthocyanins have an effect on pGL2 activation. However, it is also possible that accumulated
compounds upstream of CHS caused this effect found.
Figure 14 | Proposed Ros1&Del and CHS-RNAi mechanism of action. Ros1&Del increase pGL2 activation. CHSRNAi decreases flavonols and anthocyanins. Upon downregulation of flavonols and anthocyanins, GL2
promoter induction goes up via unknown TFs.
It could also be possible that RNAi in general causes pGL2 upregulation. No control RNAi construct
was tested. A general off-target effect of RNAi is activation of non-specific genes. A scrambled or
non-targeting RNAi construct should have been tested (Echeverri et al., 2006). A RNAi construct
against GFP or luciferase could have been used as negative control, however these controls still do
not provide evidence whether the observed phenotype was specific. Also a construct with at least 3
mutations in the binding site of the RNAi construct could have been used as a control. This construct
fails to bind to the specific gene (CHS) but also the unspecific gene (GL2) and thus the effect on pGL2
should not be seen anymore (Reviewed in Cullen, 2006). These suggestions on negative controls
indicate no perfect controls are available for RNAi experiments.
Phenotyping of GLABRA2 overexpressing tomato plants
In order to get more insight in the phenotypic changes GL2 generates in tomato, GL2 overexpressing
lines were produced. In Arabidopsis no 35S::GL2 lines could be produced because this was lethal,
caused by heterodimerization of ectopically produced GL2 with endogenous GL2 (Ohashi et al.,
2002). However, they managed to get GL2 overexpression by introducing a pGL2::GL2 into
Arabidopsis and found increased trichome numbers in these plants. In addition, via activation tagging
a mutant in Arabidopsis was found which had overexpression of GL2 (Wang et al., 2015). This mutant
accumulated less anthocyanins but showed no epidermal differences with WT plants. In this study,
GL2 overexpression in Micro-Tom yielded living tomato plants which were able to produce seeds.
These 35S::GL2 Micro-Tom plants were very different in phenotype but no experiments can be done
on this T0 generation. These plants come from tissue culture conditions and variation seen between
26
plants can be caused by soma clonal variation. Therefore the next generation (T1) should be used for
phenotyping experiments. This should initially focus on root hairs growth in seedlings which should
examined under microscope and measured with ImageJ. Also leaf surface with focus on stomata and
trichomes should be examined.
The next generation of these plants should be grown in order to identify phenotypic aberrations
caused by elevated expression of GL2. Furthermore, these plants could be used in order to confirm
the phenotypes found in Ros1&Del induced plants were induced by overexpression of GL2.
Broader perspective
Ros1&Del form Snapdragon were initially introduce in tomato lines to produce healthier tomatoes
(Butelli et al., 2008). Within a year they would like to produce these tomatoes commercially in
Northern America (Shukman, 2014). Ros1&Del are placed under a fruit specific E8 promoter. WT and
Ros1&Del plants were physiologically identical. Fruits started to accumulate anthocyanins after the
mature green stage. In our research Ros1&Del were not placed under a fruit specific promoter but
under a ubiquitous promoter which was only not active in the fruits. We saw a lot of physiological
changes upon Ros1&Del expression in tomato plants. Even though a different promoter is used, still
these findings suggest not only anthocyanin production is affected by introduction of Ros1&Del. Do
the purple tomato fruits harvested from Ros1&Del under E8 promoter only differ in anthocyanin
content from WT fruits? If yes, probably the different effect of Ros1&Del on tomato physiology found
in our study is dependent on the promoter used.
Conclusion
This research showed that overexpression of Ros1&Del leads to shorter root hairs and possibly to
less trichome type 7 formation and stomata with a wider aperture. Furthermore is shown that
Ros1&Del directly activate the promoter of GL2. In Arabidopsis GL2 regulates root hair, trichome and
stomata formation. Therefore probably GL2 causes the deviating phenotypes found in Ros1&Del
induced tomato plants. In order to conclude GL2 causes the deviating phenotypes, the T1 generation
of 35S::GL2 Micro-Tom should be examined on root hair, trichome and stomata formation.
27
Acknowledgements
I would like to thank my supervisors Jules Beekwilder and Nikolay Outchkourov for helping me with
all the lab-and think work needed for this thesis. Also all the others of lab 1.062 for helping me with
all kind of problems. I would like to thank Robert Hall for providing feedback during meetings on my
practical work. I would like to thank Rumyana Karlova for helping me with my overexpressing GL2
plants, from feedback on the callus to qPCR. Sander van der Krol and Umidjon Shapulatov helped me
with the luciferase assay which led to very nice luminescence pictures. Ikram Blilou helped me a lot
with microscopy of the root hairs and with possible explanations why shorter root hairs were
formed.
28
Supplemental Data
Table S1 | Tukey HSD test performed in SPSS on root hair length measured in ImageJ. Genotype
wild type (WT) or Ros1&Del (RD), treatment Dex or no Dex application.
Multiple Comparisons
Dependent Variable: Length
95% Confidence Interval
Mean
Tukey HSD
(I) GxT
(J) GxT
Difference (I-J)
RD-
2,00
RD+
WT-
WT+
Std. Error
Sig.
Lower Bound
Upper Bound
1,9421
,18672
,000
1,4079
2,4763
3,00
-,3876
,18672
,203
-,9218
,1466
4,00
,0167
*
,18672
1,000
-,5175
,5509
,18672
,000
-2,4763
-1,4079
1,00
-1,9421
*
3,00
-2,3297
*
,18672
,000
-2,8639
-1,7955
4,00
-1,9254
*
,18672
,000
-2,4596
-1,3912
1,00
,3876
,18672
,203
-,1466
,9218
*
2,00
2,3297
,18672
,000
1,7955
2,8639
4,00
,4043
,18672
,175
-,1299
,9385
1,00
-,0167
,18672
1,000
-,5509
,5175
2,00
1,9254
,18672
,000
1,3912
2,4596
3,00
-,4043
,18672
,175
-,9385
,1299
*
Table S2 | Anova performed in SPSS on root hair length. Genotype wild type (WT) or Ros1&Del (RD), treatment
Dex or no Dex application.
Tests of Between-Subjects Effects
Dependent Variable: Length
Type III Sum of
Source
Squares
df
Mean Square
F
Sig.
Corrected Model
16,525
3
5,508
63,201
,000
Intercept
50,881
1
50,881
583,774
,000
Genotype
6,687
1
6,687
76,728
,000
Treatment
6,882
1
6,882
78,960
,000
Genotype * Treatment
2,956
1
2,956
33,916
,000
Error
1,395
16
,087
Total
68,801
20
Corrected Total
17,920
19
a
29
Table S3 |Differences in luciferase signal between approach 1 and 2 for pGL2-RD and pBin. Mean of
luminescence signal was taken from 9 replicates. Ratio was calculated by dividing approach 1 by approach 2.
Approach 1/approach 2
Mean luciferase signal
(Fold change)
pGl2-RD
approach 1
1891619
approach 2
259790
7.28x
pBin
approach 1
1609
approach 2
41
38.93x
Table S4 | Effect of RD co-infiltration with pGL2 in luminescence assay. Approach 1 and 2 are compared in how
much increased signal is shown by each method. Mean of luminescence signal was taken from 9 replicates.
approach 1
approach 2
pGL2
pGl2-RD
604301
1891619
23885
259790
ratio
3.130262075
10.87680671
pGL2
pGL2
Ros1&Del
pGL2
CHS-RNAi
pGL2
Ros1&Del
CHS-RNAi
Figure S1 | Agro-infiltrated leaves sprayed with 1 mM luciferin 3dpi and 4dpi. Pictures were made 4dpi for a
total of 10 minutes. From left to right pGL2, pGL2 + Ros1&Del, pGL2+ CHS-RNAi and pGL2+Ros1&Del+ CHSRNAi.
30
Table S5 | Tukey HSD performed in SPSS on luminescence values of leaf disks. Background values were
subtracted before analysing the data. 3 leaf disks per construct were measured.
Multiple Comparisons
Dependent Variable: Luminescence_ln_leafdisks
Mean
(I) constructs
Tukey HSD
pGL2
(J) constructs
pGL2_RD
pGL2_CHS
pGL2_RD_CHS
pGL2_RD
pGL2_CHS
(I-J)
Std. Error
Sig.
Lower Bound Upper Bound
-2,0794
*
,37816
,003
-3,2904
-,8684
-1,7464
*
,37816
,007
-2,9574
-,5354
-3,1955
*
,37816
,000
-4,4065
-1,9845
,37816
,003
,8684
3,2904
,37816
,815
-,8781
1,5439
*
pGL2
2,0794
pGL2_CHS
,3329
pGL2_RD_CHS
-1,1162
,37816
,071
-2,3272
,0948
*
,37816
,007
,5354
2,9574
,37816
,815
-1,5439
,8781
,37816
,021
-2,6601
-,2381
,37816
,000
1,9845
4,4065
,37816
,071
-,0948
2,3272
,37816
,021
,2381
2,6601
pGL2
1,7464
pGL2_RD
-,3329
pGL2_RD_CHS
pGL2_RD_CHS
95% Confidence Interval
Difference
-1,4491
pGL2
3,1955
pGL2_RD
1,1162
pGL2_CHS
1,4491
*
*
*
Table S6 | Tukey HSD performed in SPSS on whole leaf luminescence values. 3 leaves per construct were
measured and background values were subtracted before analysing the data.
Multiple Comparisons
Dependent Variable: Luminescence_ln_whole leaf
Mean
Tukey HSD
(I) Constructs
(J) Constructs
(I-J)
pGL2
pGL2+RD
-1,3952
pGL2+CHS
pGL2+RD+CHS
pGL2+RD
pGL2+CHS
pGL2+RD+CHS
95% Confidence Interval
Difference
Std. Error
Sig.
Lower Bound
Upper Bound
*
,25333
,003
-2,2065
-,5840
-1,0488
*
,25333
,014
-1,8601
-,2376
-2,3995
*
,25333
,000
-3,2107
-1,5882
,25333
,003
,5840
2,2065
,25333
,551
-,4648
1,1577
,25333
,017
-1,8155
-,1930
,25333
,014
,2376
1,8601
,25333
,551
-1,1577
,4648
pGL2
1,3952
pGL2+CHS
,3464
*
pGL2+RD+CHS
-1,0042
pGL2
1,0488
pGL2+RD
-,3464
pGL2+RD+CHS
-1,3507
*
*
*
,25333
,003
-2,1619
-,5394
,25333
,000
1,5882
3,2107
pGL2
2,3995
*
pGL2+RD
1,0042
*
,25333
,017
,1930
1,8155
1,3507
*
,25333
,003
,5394
2,1619
pGL2+CHS
31
1000
Fold over pBIN
800
600
400
Ratio RD
Ratio CHS
Ratio RD-CHS
200
0
-200
Figure S2 | LCMS data of agro-infiltrated leaves was analysed for compounds which differed in amount
between RD and RD+ CHS-RNAi. Hexanoyl-nornicotine, N’ Octanoylnornicotine and D3R could be identified by
previous obtained LCMS results (Outckourov et al., 2012). Several other compounds could not be identified.
Ratios are shown of values divided by control (pBin). Error bars indicate standard deviation (n=5).
32
Table S7 | Phenotypic and genotypic features of 35S::GL2 plants. Flat, Tall, Normal Looking and Curled Leaves were observed by eye. Transgenic was confirmed with a PCR,
35S-GL2 expression was analysed with qPCR, low is until 2,5-fold induction compared to WT, medium from 2,5 till 10, high 10-fold and higher. Seeds available (Y) or seedless
st
(N). Number of seeds were counted after harvesting tomatoes. Remarks are observations. Tomato plants are still growing and last update of this table was on January 21 .
Plant #
Flat
1
Curled leaves
Transgenic
x
x
x
low
2
x
x
x
3
x
x
x
4
5
Tall
“Normal looking”
Seeds?
# seeds
1.328686
Y
10
medium
6.364292
N
medium
4.228072
N
low
0.094732
Y
+
medium
3.160165
Y
+
low
0.528509
Y
+
x
high
10.26741
N
x
NA
NA
Y
x
x
8
x
x
x
Remark
Tomatoes with a pointy end
Flat tomatoes, strange flowers, hardly any leaves
‘Wind Tree’ form
13
x
x
14
x
x
16
x
x
x
low
1.580083
N
18
x
x
x
medium
4.438278
Y
1
19
x
x
x
NA
NA
Y
10
Looks like a small clump
x
medium
7.78124
Y
+
Lots of flowers, zigzag flowering
20
22
x
x
Grows wide instead of in height
++
Looks like a small clump
x
x
x
medium
9.12611
Y
2
23
x
x
x
NA
NA
Y
5
24
x
x
high
15.13692
Y
+
x
x
high
17.26765
N
x
x
low
2.313376
Y
3
x
NA
NA
Y
+++
25
x
x
35S-GL2 expression
x
x
26
x
27
x
x
Looks like a small clump
Lots of flowers, ‘Wind Tree’ form, zigzag flowering
Looks like a small clump
Looks like 23
28
x
x
x
NA
NA
29
x
x
x
low
1.693491
Y
1
30
x
x
NA
NA
Y
10
Flat but normal leaves
32
x
x
NA
NA
Y
5
Looks like 30
34
x
x
NA
NA
Dying
35
x
x
NA
NA
Small
x
medium
6.364292
36
x
x
x
Dying
Y
++
Chlorophyll deficiency in leaves, does not really elongate
33
References
Bashandy, H., Jalkanen, S., & Teeri, T. H. (2015). Within leaf variation is the largest source of variation in agroinfiltration of
Nicotiana benthamiana. Plant methods, 11(1), 1.
Baumberger, N., Ringli, C., & Keller, B. (2001). The chimeric leucine-rich repeat/extensin cell wall protein LRX1 is required
for root hair morphogenesis in Arabidopsis thaliana. Genes & Development, 15(9), 1128-1139.
Bergau, N., Bennewitz, S., Syrowatka, F., Hause, G., & Tissier, A. (2015). The development of type VI glandular trichomes in
the cultivated tomato Solanum lycopersicum and a related wild species S. habrochaites. BMC plant biology, 15(1), 1.
Bovy, A., de Vos, R., Kemper, M., Schijlen, E., Pertejo, M. A., Muir, S., Collins, G., Robinson, S., Verhoeyen, M., Hughes, S.,
Santos-Buelga, C., van Tunen, A. (2002). High-flavonol tomatoes resulting from the heterologous expression of the maize
transcription factor genes LC and C1. The Plant Cell Online, 14(10), 2509-2526.
Brouillard, R. (1988). Flavonoids and flower colour. In The flavonoids (pp. 525-538). Springer US.
Burdulis, D., Sarkinas, A., Jasutiene, I., Stackevicene, E., Nikolajevas, L., & Janulis, V. (2008). Comparative study of
anthocyanin composition, antimicrobial and antioxidant activity in bilberry (Vaccinium myrtillus L.) and blueberry
(Vaccinium corymbosum L.) fruits. Acta poloniae pharmaceutica, 66(4), 399-408.
Butelli, E., Titta, L., Giorgio, M., Mock, H.P., Matros, A., Peterek, S., Schijlen, E.G.W.M., Hall, R.D., Bovy, A.G., Luo, J., Martin,
C. (2008). Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors.
Nature biotechnology, 26(11), 1301-1308.
Castañeda-Ovando, A., de Lourdes Pacheco-Hernández, M., Páez-Hernández, M. E., Rodríguez, J. A., & Galán-Vidal, C. A.
(2009). Chemical studies of anthocyanins: A review. Food chemistry, 113(4), 859-871.
Cho, H. T., & Cosgrove, D. J. (2002). Regulation of root hair initiation and expansin gene expression in Arabidopsis. The Plant
Cell, 14(12), 3237-3253.
Cullen, B. R. (2006). Enhancing and confirming the specificity of RNAi experiments. Nature methods, 3(9), 677-681.
Ding, X., Cao, Y., Huang, L., Zhao, J., Xu, C., Li, X., & Wang, S. (2008). Activation of the indole-3-acetic acid–amido synthetase
GH3-8 suppresses expansin expression and promotes salicylate-and jasmonate-independent basal immunity in rice. The
Plant Cell, 20(1), 228-240.
Du, H., Wu, N., Fu, J., Wang, S., Li, X., Xiao, J., & Xiong, L. (2012). A GH3 family member, OsGH3-2, modulates auxin and
abscisic acid levels and differentially affects drought and cold tolerance in rice. Journal of experimental botany, 63(18),
6467-6480.
Echeverri C.J., Beachy P.A., Baum B., Boutros M., Buchholz F., Chanda S.K., et al. Minimizing the risk of reporting false
positives in large-scale RNAi screens. Nat Methods 2006;3:777-779
Feller, A., Machemer, K., Braun, E. L., & Grotewold, E. (2011). Evolutionary and comparative analysis of MYB and bHLH plant
transcription factors. The Plant Journal, 66(1), 94-116.
Gao, Y., Gong, X., Cao, W., Zhao, J., Fu, L., Wang, X., Schumaker, K.S., Guo, Y. (2008). SAD2 in Arabidopsis functions in
trichome initiation through mediating GL3 function and regulating GL1, TTG1 and GLABRA2 expression. Journal of
integrative plant biology, 50(7), 906-917.
Glas, J. J., Schimmel, B. C., Alba, J. M., Escobar-Bravo, R., Schuurink, R. C., & Kant, M. R. (2012). Plant glandular trichomes as
targets for breeding or engineering of resistance to herbivores. International journal of molecular sciences, 13(12), 1707717103.
Gomez-Roldan, M., Engel, B., Vos, R.H. et al. (2014) Metabolomics reveals organ-specific metabolic rearrangements during
early tomato seedling development. Metabolomics, 10, 958–974.
Gould, K. S. (2004). Nature's Swiss army knife: the diverse protective roles of anthocyanins in leaves. BioMed Research
International, 2004(5), 314-320.
Johnson, C. S., Kolevski, B., & Smyth, D. R. (2002). Transparent Testa Glabra2, a trichome and seed coat development gene
of Arabidopsis, encodes a WRKY transcription factor. The Plant Cell Online, 14(6), 1359-1375.
34
Kang, J. H., Liu, G., Shi, F., Jones, A. D., Beaudry, R. M., & Howe, G. A. (2010). The tomato odorless-2 mutant is defective in
trichome-based production of diverse specialized metabolites and broad-spectrum resistance to insect herbivores. Plant
physiology, 154(1), 262-272.
Kapulnik, Y., Resnick, N., Mayzlish-Gati, E., Kaplan, Y., Wininger, S., Hershenhorn, J., & Koltai, H. (2011). Strigolactones
interact with ethylene and auxin in regulating root-hair elongation in Arabidopsis. Arabidopsis. J. Exp. Bot. 62: 2915–24
Katsumoto, Y., Fukuchi-Mizutani, M., Fukui, Y., Brugliera, F., Holton, T. A., Karan, M., Nakamura, N., Yonekura-Sakakibara,
K., Togamii, J., Pigeaire, A., Tao, G., Nehra, N.S., Lu, C., Dyson, B.K., Tsuda, S., Ashikari, T., Kusumi, T., Mason, J.G., Tanaka, Y.
(2007). Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating
delphinidin. Plant and cell physiology, 48(11), 1589-1600.
Kong, J. M., Chia, L. S., Goh, N. K., Chia, T. F., & Brouillard, R. (2003). Analysis and biological activities of anthocyanins.
Phytochemistry, 64(5), 923-933.
Koopman, F., Beekwilder, J., Crimi, B., van Houwelingen, A., Hall, R. D., Bosch, D., Van Maris, A.J.A., Pronk, J.T., Daran, J. M.
(2012). De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microbial Cell Factories,
11, 155.
Lacombe, A., Wu, V. C., Tyler, S., & Edwards, K. (2010). Antimicrobial action of the American cranberry constituents;
phenolics, anthocyanins, and organic acids, against Escherichia coli O157: H7. International journal of food microbiology,
139(1), 102-107.
Laue, G., Preston, C. A., & Baldwin, I. T. (2000). Fast track to the trichome: induction of N-acyl nornicotines precedes
nicotine induction in Nicotiana repanda. Planta, 210(3), 510-514.
Lila, M. A. (2004). Anthocyanins and human health: an in vitro investigative approach. BioMed Research International,
2004(5), 306-313.
Lin, Q., Ohashi, Y., Kato, M., Tsuge, T., Gu, H., Qu, L. J., & Aoyama, T. (2015). GLABRA2 Directly Suppresses Basic Helix-LoopHelix Transcription Factor Genes with Diverse Functions in Root Hair Development. The Plant Cell, 27(10), 2894-2906.
López-Carbonell, M., & Jáuregui, O. (2005). A rapid method for analysis of abscisic acid (ABA) in crude extracts of water
stressed Arabidopsis thaliana plants by liquid chromatography—mass spectrometry in tandem mode. Plant Physiology and
Biochemistry, 43(4), 407-411.
Masucci, J. D., Rerie, W. G., Foreman, D. R., Zhang, M., Galway, M. E., Marks, M. D., & Schiefelbein, J. W. (1996). The
homeobox gene GLABRA2 is required for position-dependent cell differentiation in the root epidermis of Arabidopsis
thaliana. Development, 122(4), 1253-1260.
Mathews, H., Clendennen, S. K., Caldwell, C. G., Liu, X. L., Connors, K., Matheis, N., Schuster D.K., Menasco D.J., Wagoner
W., Wagner D.R. (2003). Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis,
modification, and transport. The Plant Cell Online, 15(8), 1689-1703.
Mazza, G., & Brouillard, R. (1987). Recent developments in the stabilization of anthocyanins in food products. Food
Chemistry, 25(3), 207-225.
Mazza, G., & Miniati, E. (1993). Anthocyanins in fruits, vegetables, and grains. Boca Raton, FL: CRC Press, 1993
Van Nocker, S., & Ludwig, P. (2003). The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in
structure and function. BMC genomics, 4(1), 50.
Ohashi, Y., Oka, A., Ruberti, I., Morelli, G., & Aoyama, T. (2002). Entopically additive expression of GLABRA2 alters the
frequency and spacing of trichome initiation. The Plant Journal, 29(3), 359-369.
Outchkourov, N. S., Carollo, C. A., Gomez-Roldan, V., de Vos, R. C., Bosch, D., Hall, R. D., & Beekwilder, J. (2014). Control of
anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana
benthamiana leaves. Frontiers in plant science, 5:519.
Patra, B., Schluttenhofer, C., Wu, Y., Pattaniak, S., Yuan, L., 2013. Transcriptional regulation of secondary metabolite
biosynthesis in plants. Biochimica et Biophysica Acta-Gene Regulatory Mechanisms, 1829, 1236-1247.
35
Pemberton, L. M., Tsai, S. L., Lovell, P. H., & Harris, P. J. (2001). Epidermal patterning in seedling roots of eudicotyledons.
Annals of Botany, 87(5), 649-654.
Petroni, K., & Tonelli, C. (2011). Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant
Science, 181(3), 219-229.
Povero, G., Gonzali, S., Bassolino, L., Mazzucato, A., & Perata, P. (2011). Transcriptional analysis in high-anthocyanin
tomatoes reveals synergistic effect of Aft and atv genes. Journal of plant physiology, 168(3), 270-279.
Qing, L., & Aoyama, T. (2012). Pathways for Epidermal Cell Differentiation via the Homeobox Gene GLABRA2: Update on the
Roles of the Classic Regulator. Journal of integrative plant biology, 54(10), 729-737.
Ramsay, N. A., & Glover, B. J. (2005). MYB–bHLH–WD40 protein complex and the evolution of cellular diversity. Trends in
plant science, 10(2), 63-70.
Rerie, W. G., Feldmann, K. A., & Marks, M. D. (1994). The GLABRA2 gene encodes a homeo-domain protein required for
normal trichome development in Arabidopsis. Genes & Development, 8(12), 1388-1399.
Schiefelbein, J., Kwak, S. H., Wieckowski, Y., Barron, C., & Bruex, A. (2009). The gene regulatory network for root epidermal
cell-type pattern formation in Arabidopsis. Journal of experimental botany, 60(5), 1515-1521.
Schijlen, E. G., de Vos, C. R., Martens, S., Jonker, H. H., Rosin, F. M., Molthoff, J. W., Tikunov, Y.M., Angenent, G.C., Van
Tunen, A.J. Bovy, A. G. (2007). RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis
pathway, leads to parthenocarpic tomato fruits. Plant Physiology, 144(3), 1520-1530.
Shukman, D., (2014, January 24) Genetically-modified purple tomatoes heading for shops. BBC news. Retrieved from
http://www.bbc.com/news
Tal, M., & Nevo, Y. (1973). Abnormal stomatal behavior and root resistance, and hormonal imbalance in three wilty mutants
of tomato. Biochemical genetics, 8(3), 291-300.
Velasquez, S. M., Ricardi, M. M., Poulsen, C. P., Oikawa, A., Dilokpimol, A., Halim, A., et al. (2015). Complex regulation of
prolyl-4-hydroxylases impacts root hair expansion. Molecular plant, 8(5), 734-746.
Wang, X., Wang, X., Hu, Q., Dai, X., Tian, H., Zheng, K., Wang, X., Mao, T., Chen, J., Wang, S. (2015). Characterization of an
activation‐tagged mutant uncovers a role of GLABRA2 in anthocyanin biosynthesis in Arabidopsis. The Plant Journal. 83 (2),
300-311
Yi, K., Menand, B., Bell, E., & Dolan, L. (2010). A basic helix-loop-helix transcription factor controls cell growth and size in
root hairs. Nature genetics, 42(3), 264-267.
Zhang, Y., Butelli, E., De Stefano, R., Schoonbeek, H. J., Magusin, A., Pagliarani, C., Wellner, N., Hill, L., Orzaez, D., Granell, A.,
Jones, J.D.G., Martin, C. (2013). Anthocyanins double the shelf life of tomatoes by delaying over ripening and reducing
susceptibility to gray mold. Current Biology, 23(12), 1094-1100.
Zhang, Y., Butelli, E., & Martin, C. (2014). Engineering anthocyanin biosynthesis in plants. Current opinion in plant biology,
19, 81-90.
Zimmermann, I. M., Heim, M. A., Weisshaar, B., & Uhrig, J. F. (2004). Comprehensive identification of Arabidopsis thaliana
MYB transcription factors interacting with R/B‐like BHLH proteins. The Plant Journal, 40(1), 22-34.
Zuluaga, D. L., Gonzali, S., Loreti, E., Pucciariello, C., Degl'Innocenti, E., Guidi, L., Alpi, A., Perata, P. (2008). Arabidopsis
thaliana MYB75/PAP1 transcription factor induces anthocyanin production in transgenic tomato plants. Functional Plant
Biology, 35(7), 606-618.
36