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Identification of transcription factors regulated by LEC2 in
triacylglycerol biosynthesis

Hyung Ju Do and Hyun Uk Kim*
Department of Bioindustry and Bioresource Engineering, Sejong University, Seoul, 05006 Republic of Korea

Abstract

During the seed development, triacylglycerol (TAG) is accumulated in the cotyledon. The TAG is
used as an energy source for seedling development in germinated seeds. The complex network of tr
anscription factors (TFs) regulates genes involved in TAG biosynthesis during seed development. L
EAFY COTYLEDON 2 (LEC2) is the master regulator for maturation and TAG biosynthesis in see
d development. Currently, LEC2-downstream TF networks in the process of seed TAG biosynthesi
s have not been studied well. In this study, we identified 25 seed-specific TFs upregulated by LEC2
. To identify novel transcription factors that regulate TAG biosynthesis in the LEC2 regulatory net
work, each of 25 seed-specific candidate TFs has been transforming to overexpress in leaves of Nic
otiana benthamiana using Agrobacterium infiltration. Qualitative and quantitative analysis of TAG
content in transformed leaves is progressed using thin-layer chromatography (TLC) and gas chrom
atography (GC) to find TFs induce TAG accumulation. The discovery of novel TFs that regulate th
e biosynthesis of TAG can be usefully used to enhance the oil content of plants. Figure 4. Agrobacterium infiltration list of LEC2-induced transcription factors
Three independent experiments that infiltrate Agrobacterium into N. benthamiana leaves were
conducted. In these experiments, Agrobacterium suspension of negative control (EV), positive control
Introduction 1 (LEC2), and positive control 2 (WRI1) has infiltrated in the three leaves of different N. benthamiana
plants. Also, the suspension of TF-1 to TF-25 has infiltrated in the three leaves of different N.
benthamiana plants over four different experiments indicated lists in this figure. 5 days after infiltration
To find novel TFs that regulate TAG or fatty acid biosynthesis in the LEC2 regulatory network, we of the Agrobacterium suspension of those vectors, transformed leaves were harvested and used for
used Agrobacterium infiltration in N. benthamiana leaves and analyzed TAG content of the analysis. EV, Empty vector; LEC2, LEAFY COTYLEDON2; WRI1, WRINKLED1; TFs, Transcription
transformed leaves using TLC and GC.
factors
Figure 1. A model of transcription
factors regulated by LEC2 in TAG
biosynthesis
LEAFY COTYLEDON2 (LEC2), a
master regulator for seed development,
regulates several transcription factors in
triacylglycerol (TAG) biosynthesis.
WRINKLED1 (WRI1) is known as a
transcription factor regulated
transcriptionally by LEC2 in accumulating
TAG in the seed. Since many transcription
factors induced by LEC2 have been
identified among seed maturation and
TAG biosynthesis, unknown downstream
transcription factors networks in TAG
biosynthesis may present.

Figure 5. TAG content in transformed N. benthamiana leaves
Figure 2 . Experimental design to identify LEC2-regulated transcription factors TAG content of transformed leaves transiently expressing 25 transcription factors in Figure 4 is
analyzed by thin-layer chromatography (A) and gas chromatography (B). (A) The red letters indicate
To find transcription factors (TFs) involved in TAG biosynthesis among 25 TFs, empty vector (negative
control), LEC2, WRI1 (positive control 1 and 2), and 25 LEC2-regulated TFs are infiltrated in leaves of TFs showed high TAG content. Arabidopsis Col-0 seed and 15:0-TAG standard marker indicate as
N. benthamiana. Transformed leaves at 5 days after infiltration (DAI) are analyzed by TLC and GC to TAG. (B) Three measurements are averaged. Student’s t-test is used as statistical analysis (** p<0.01,*
see TAG accumulation and fatty acid composition. Also, the analysis of Nile-red to find TFs make p<0.05). We found high TAG content in N. benthamiana leaves transformed by TF-7, TF-9, TF-12,
changes of oil-body, iodine staining to find TFs make changes of starch content and RNA seq to find TF-19, and TF-22 compared with NC and comparable content with PC1 and PC2. These results
make changes of RNA expression, etc. We expect to find changes in TF candidates’ phenotype showed the TF-7, TF-9, TF-12, TF-19, and TF-22 may regulate or involve in TAG biosynthesis
compared with NC and PCs. EV, Empty vector; LEC2, LEAFY COTYLEDON2; WRI1, WRINKLED1; directly or indirectly by LEC2. The error bars indicate the SEM. TAG, Triacylglycerol; NC, Negative
TFs, Transcription factors; TLC, Thin layer chromatography; GC, Gas chromatography control (EV, Empty vector); PC1, Positive control (WRINKELD1); PC2, Positive control 2 (LEAFY
COTYLEDON2)
Results Summary and Ongoing study
❖Qualitative and quantitative analysis of triacylglycerol (TAG) in N. benthamiana leaves transformed
with negative control (empty vector), positive controls (LEC2, WRI1), unknown TF-1, TF-2, TF-3,
TF-4, TF-5, TF-6, TF-7, TF-8, TF-9, TF-10, TF-11, TF-12, TF-13, TF-14, TF-15, TF-16, TF-17, TF-
18, TF-19, TF-20, TF-21, TF-22, TF-23, TF-24, and TF-25 were underway by thin layer
chromatography (TLC) and gas chromatography (GC) over three independent experiments.
❖ TLC and GC analysis of the N. benthamiana leaves transformed with TF-7, TF-9, TF-12, TF-19, and
TF-22 showed increased TAG amount compared with N. benthamiana leaves transformed with
negative control and similar amount with two positive controls.
❖The novel TF-7, TF-9, TF-12, TF-19, and TF-22 may regulate the synthesis of TAG, which can be
usefully used to enhance the oil content of plants.
❖Oil-body in transformed N. benthamiana leaves with TF-7, TF-9, TF-12, TF-19, and TF-22 will be
observed by Nile-red.
❖Starch content of transformed N. benthamiana leaves with TF-7, TF-9, TF-12, TF-19, and TF-22 will
be analyzed by iodine staining.
❖RNA expression of genes involved in TAG biosynthesis will be analyzed by RNA seq in transformed
N. benthamiana leaves with TF-7, TF-9, TF-12, TF-19, and TF-22.
Figure 3. Identify 25 seed-specific transcription factors’ vectors for screening LEC2-induced
transcription factor candidates by Agrobacterium colony PCR References
25 LEC2-induced TFs, LEC2, WRI1, and EV are identified by Agrobacterium colony PCR using
vector-specific primer. EV, Empty vector; LEC2, LEAFY COTYLEDON2; WRI1, WRINKLED1; TFs, Kim HU et al.(2015) Senescence-inducible LEC2 enhances triacylglycerol accumulation in leaves
Transcription factors without negatively affecting plant growth. Plant Biotechnol J. 13(9): 1346–1359
Sejong University, Seoul Korea