Effect of red LED light on steviol
glycoside accumulation and
transcriptional analysis of the
genes involved
Stijn Ceunen and Jan M.C. Geuns
Laboratory of Functional Biology
KU Leuven
Belgium
Contents
Introduction
 Aim of the research
 Material and methods
 Results

 Plant
development
 Spatio-temporal SVgly accumulation
 Transcriptional analysis
 Correlation dynamics
Summary
 Acknowledgments

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Introduction

State of the art
 Stevia
rebaudiana is a short-day plant (SD)
 SDs
(< 12 h photoperiod) cause rapid flowering, contrary
to long-days (LDs)
 During
SDs, LD conditions are simulated with red LED
light illumination during the night
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Introduction

State of the art
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Week 25
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Week 25
Week 32
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Introduction
SD
Leaf
dry
SD +
weight
5’ LED
SD
SVglys
W9
W25
W35
2.3 g
-
-
3.5 g
34.2 g
130.8 g
0.05 g
(2.0 %)
-
-
4.0 g
(11.8 %)
14.5 g
(11.0 %)
SD +
0.15 g
5’ LED (4.3 %)
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Introduction
SD
Leaf
dry
SD +
weight
5’ LED
SD
SVglys
W9
W25
W35
2.3 g
-
-
1 Year
13.3 g
15x
3.5 g
34.2 g
130.8 g
194.3 g
0.05 g
(2.0 %)
-
-
0.3 g
SD +
0.15 g
5’ LED (4.3 %)
4.0 g
(11.8 %)
14.5 g
(11.0 %)
74x
21.3 g
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Introduction

Practical application: simulating LDs on a field
 Belts
of red LED lights
 Only 5 min illumination is sufficient
 Advantages
Less harvests / year, thus less labour
 Low amounts of electricity needed (solar power!)
 LEDs have long lifespans (> 20,000 hours)
 Small investment per hectare (± 10,000 euro)
 Easier extraction due to greater percentage yield
 Temperate climate: at least two-month prolongation of
growth season

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Introduction

Biosynthesis of SVglys
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Introduction
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Aim
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Aim
1) Is there a physiological difference between
plants grown under normal LDs and those
under either LDs or SDs receiving red LED
light?
2) How is the phytochrome effect reflected
on the transcription of the genes involved in
SVgly biosynthesis?
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Material and methods

Experimental set-up: preliminary test

Mid-vegetative plants under LDs (16 h), with or without
red LED illumination during the night

Parameters: amount of flower buds and flowers,
flowering rate
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Material and methods

Experimental set-up: main experiment
 Plant
material: 180 in vitro propagated seedlings of similar
morphology
 Conditions: LD (16 h photoperiod), SD (8 h) and SD (8
h) +5’ LED
 Sampling: leaves at nodes 3-4, 7-8, 11-12, 15-16 at
different ontogenetic stages (determined by ontogeny
under SDs)
 Parameters: SVglys* (Reb A, ST, Reb F, Reb C, Dul A,
Reb G, Rub, Reb B, SB, SVE, SM), Reb A/ST, SV*,
proteins*, gene transcription
* Calculated per g dry matter
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Material and methods

Experimental set-up: main experiment
 Gene
transcription
RNA extraction (high purity only!) + cDNA synthesis
 Primers were based upon Mohamed et al. (2011)
 House-keeping genes: 18S rRNA and -actin
 qPCR analysis: GoTaq® qPCR Master Mix (Promega)
 Normalisation procedures according to 2−ΔΔCT method

First normalisation against 18S rRNA or -actin
 Second normalisation against t0 (leaves at nodes 7-8 (upper leaves) of
untreated, young vegetative plants)

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Results
Plant development
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Results

Preliminary test
AVERAGE
COUNTS
LD
Flower
Flowering
Flowers
rate
buds
Total in
3 plants
289
115
40 %
Per stem
32
13
40 %
Total in
3 plants
153
25
16 %
17
3
16 %
LD +
5’ LED Per stem
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Results

Main experiment
 SD

76 d from young vegetative stage to seed dispersal stage (12-13
nodes)
 LD
and SD+LED
After 76 d: still vegetative (17-18 nodes)
 No morphological differences

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Results
Spatio-temporal
SVgly accumulation
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Results
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Results
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Results
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Results
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Results
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Results
Transcriptional analysis
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Introduction
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Introduction
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Results
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Introduction
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Results
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Introduction
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Results
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Introduction
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Results
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Results
Correlation dynamics
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Results

Gene transcription – product accumulation
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Results
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Results

Gene 1 transcription – gene 2 transcription
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Results

Cultivar 1 – cultivar 2
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Results

Cultivar 1 – cultivar 2
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Results

Cultivar 1 – cultivar 2
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Summary

LD + LED causes further delay of flowering

Plant morphology under SD + LED ~ LD

% SVglys under SD + LED < LD (photosynthesis ↓)

SD: sharp decline of % SVglys during flowering
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Summary

Reb A/ST ratio is stable under LD and SD+LED, but
increases under SDs due to flowering (effect of ST ↓)

Transcript levels: UGT74G1 > UGT85C2 > ent-KAH >
UGT76G1 > ent-KS >> ent-KO

Transcription ↓ during generative development under SD
 Note:

transcription (HK) ↓, thus actual transcription (targets) ↓
LD and SD+LED: constant transcription
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Summary

Model
Time
Time
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Summary

Correlation dynamics
 No
significant correlations between gene transcription and product
accumulation
 Significant
correlation between transcript levels of the different
genes
 The
variation of Reb A/ST ratio in leaves with different % SVgly
content is cultivar-dependent, although the general profile remains:


Smallest ratio: large, mid-stem leaves
Greatest ratio: small, upper leaves (great % SVglys) or small, lower leaves
(small % SVglys)
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Acknowledgments

Prof. Dr. Jan Geuns for supervising the research

Shokoofeh Hajihashemi and Hilde Verlinden for the in vitro propagation
of the Stevia plants

Shokoofeh Hajihashemi and Sofie Deroover for help with the qPCR
equipment

Tom Struyf for helpful discussions
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Thank you for your attention!
Questions?
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