Romanian Biotechnological Letters
Copyright © 2009 Bucharest University
Romanian Society of Biological Sciences
Vol. 14, No. 6, 2009, pp. 4786-4791
Printed in Romania. All rights reserved
ORIGINAL PAPER
Expression of TRL1 gene in floral organs of tomato
(Solanum lycopersicum)
Received for publication, April 20, 2009
Accepted, November 5, 2009
VLADIMIR S. BONDARENCO
Institute of Genetics and Plant Physiology, Academy of Sciences of Moldova
For Correspondence:
Academy of Sciences of Moldova
Institute of Genetics and Plant Physiology, Laboratory of Molecular Genetics
20, Padurii str., MD 2002, Chisinau, the Republic of Moldova
Tel: (37322) 55 61 19, Fax: (37322) 55 61 80, e-mail: [email protected]
Abstract
The article describes the expression patterns of a RAD16-like TRL1 (tomato RAD16-like 1)
gene in the floral organs of tomato during flower development. The dependence between the organ’s
type and developmental stage and TRL1 expression is shown. The data on the induction of the TRL1
expression as a result of γ-irradiation are also presented.
Introduction
Floral organs are of a major interest as a model system for the investigation of gene
expression. This is owing to the fact that, in a short period of time, flower organs overtake
different stages of development, tissue- and organogenesis (Brukhin, 2003), which is reflected
on differential expression of the genes (Cnudde et al, 2006). Flower gene expression is
influenced by both internal and external factors. Numerous environmental mutagenic agents
(ex: UV rays, ionizing radiation, chemical mutagens, etc.) affect plant development and can
cause damage to DNA. Anthropogenic-related pressure, intensified during the last years, led
to the increase of the background ultraviolet radiation, having a negative impact over the
planet (Rousseeaux et al, 1999).
A TRL1 (tomato RAD16-like 1) gene (Accession Number EU434821) was identified in
the laboratory of Molecular Genetics, Institute of Genetics and Plant Physiology, Academy of
Sciences of Moldova. This gene presented structural homology to the yeast RAD16 gene
responsible for nucleotide excision repair (NER) of DNA (Guzder et al 1997, Blastyak et al
2007). In order to investigate the functional homology of the TRL1 with the RAD16, the
specifics of its expression in tomato floral organs at different stages of flower development
under background radiation was studied. The influence of γ- radiation on TRL1 expression in
floral organs was also investigated.
Materials and methods
Plant materials
Tomato (Solanum lycopersicum) plants, Gloria variety, were used in the research. The
stages of anther meiosis were determined according to the length of anthers (Deaghileva,
1999).
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Expression of TRL1 gene in floral organs of tomato
(Solanum lycopersicum)
Irradiation and sample preparation
Field-grown plants were exposed to γ-radiation, dose 50 Gy. After irradiation, the plants
were kept under normal light conditions. The samples were harvested and stored in liquid
nitrogen 2 hours after irradiation. The floral organs were isolated at different developmental
stages with anthers taken as standard. For each point, approximately 10-15 buds were used.
RNA isolation and reverse transcription
RNA was isolated using Qiagen RN-easy Plant Mini Kit, according to protocol. DNase
treatment was performed during RNA isolation employing Qiagen RN-ase Free DNase Set.
Reverse transcription was carried out with Reverse Transcription Kit “REVERTA”
(Inter Lab Service) using Rand-oligo, as indicated by the manufacture’s protocol.
Real Time PCR
The reactions were carried out on a Bio-Rad iQ5 real-time PCR detection system.
SYBR Green was used as fluorescent dye. The following sequences served as primers: P1 CCCAGGAACTCCCAACAGTA, P2 – TTCACCAACACTGACCGAAT, flanking a 290
nucleotide segment of the TRL1 second exon. Tomato actin gene (Tom52, Accession Number
SLU60482) was used as reference. The following primers were designed for amplifying actin:
ASL – TTCCCTCTATGCCAGTGGAC and ASR – CATCTCCAGAGTCCAGCACA,
flanking a 271 nucleotide fragment. The reaction conditions were: denaturation 95˚C - 30 sec,
annealing 62˚C - 30 sec, elongation 70˚C – 30 sec. Detection was performed during
elongation. No amplification of unspecific products was observed. The reactions with each
template were made in triplicate.
Expression levels (2-∆Ct) were calculated according to Livak and Schmittgen (2001) and
are relative to the level of actin expression. The 2-∆Ct values were consequently used for
graphics construction.
Results and discussion
TRL1 Expression under Background Radiation
The buds and mature flowers of field-grown plants were used for the investigation of the
TRL1 expression pattern in floral organs.
As figure 1 shows, the expression patterns vary in different flower organs. The common
feature for all the 4 organ types is the low transcript level of TRL1 at the premeiosis stage.
The highest transcript level of TRL1 was observed in sepals at all the investigated stages
of flower development. After the meiosis in anthers starts and till the maturing of the flower,
the transcript level is increased in sepals (being approximately 3 times higher than the
premeiosis stage value).
The lowest level of TRL1 expression was observed in petals. The situation persists
during the whole prophase I and tetrad stage in anthers. The enhancement of transcript level is
observed only at the stage of mature flower (becoming 5 – 15 times higher).
In anthers, an increase of the TRL1 transcript level is detected at the beginning of
prophase I (leptotene and zygotene stages), becoming 13 times higher than during premeiosis.
The transcript amplification persists for the period of pachytene and diplotene, although it is
less considerable. A 2.5 times decrease of the TRL1 expression is noticed for diakinesis
compared with pachytene-diplotene stage. At the phase of microspore tetrads, transcript level
Rom. Biotechnol. Lett., Vol. 14, No. 6, 4786-4791 (2009)
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VLADIMIR S. BONDARENCO
becomes 5 times higher than the one of diakinesis. An insignificant decrease of the expression
level was observed in anthers at the mature flower stage.
Figure 1. Relative expression of TRL1 in tomato floral organs under background radiation.
PM-premeiosis, L-Z – leptotene and zygotene, P-D – pachytene and diplotene, Dk – diakinesis, T – tetrad,
F – mature flower. The 2-∆Ct values were used for graphics construction.
In pistils, the TRL1 transcript level is approximately 12 times higher during leptotene
and zygotene in comparison with premeiosis phase. It is reduced for the duration of
pachytene-diplotene (approximately 2.5 times). During the next investigated stages, transcript
level increases, becoming the highest in mature flowers.
Thus, the lowest transcript level of TRL1 was detected at the premeiosis stage in all the
floral organs. In sepals, petals and pistils the highest transcript concentration was noticed in
mature flowers. The largest range of changes was noticed for the given gene expression levels
in pistils: the transcript level in mature flowers is 130 times higher than during premeiosis
stage.
The TRL1 expression patterns differences, revealed in floral organs, might serve as a
proof of the presupposition of the involvement of the given gene in DNA repair processes.
The observation that a stably high expression level of the investigated gene is observed in
sepals during all the stages of flower development is very important. It could be explained by
the fact that, during almost the whole period of flower system development, the buds are
closed and, therefore, the effects of natural solar radiation might act mostly on sepals. That is
probably why the highest level of TRL1 expression was detected in sepals. A different
expression pattern was observed in mature flowers. At this stage all floral organs are equally
exposed to background UV-radiation. A significant increase of transcript level is noticed in
both petals and pistils at the stage of mature flower. At the same stage, in anthers, the TRL1
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Rom. Biotechnol. Lett., Vol. 14, No. 6, 4786-4791 (2009)
Expression of TRL1 gene in floral organs of tomato
(Solanum lycopersicum)
transcript level is lower than during tetrads phase, although it is higher than through meiosis.
This circumstance might be connected with the significant decrease of the proliferating and/or
meiotic activity levels of anthers at the stage of mature flower as a consequence of the end of
fertile pollen formation processes (Brukhin et al, 2003). As regards the reduction of the TRL1
expression in anthers during diakinesis, the situation is probably caused by the high level of
DNA compactization, characteristic for the mentioned phase of meiosis prophase I, and
transcriptional activity is therefore reduced (Hong Ma, 2005).
Expression of TRL1 after γ-irradiation.
In order to investigate the involvement of TRL1 gene in DNA repair in tomato, fieldgrown plants were irradiated with γ-rays. The exposure to γ-radiation induces different
damages within DNA molecules (O’Neill P. and Fielden E.M., 1993) and, in case of
participation of the investigated gene in DNA damage removal, should enhance the
expression of TRL1.
Field-grown plants were γ-irradiated at a dose of 50 Gy. After the irradiation, plants
were kept under light during 2 hours, floral organs fixed afterwards in liquid nitrogen. Nonirradiated field-grown plants were used as control.
The data of the TRL1 gene expression in floral organs at the anther meiosis stage and
mature flower stage is presented in figure 2. The induction of TRL1 expression as response to
γ-irradiation is observed in all floral organs.
Figure 2. Relative expression of TRL1 in tomato floral organs before and after γ-irradiation
-∆Ct
The 2
values were used for graphics construction.
Table 1 includes the multiplicity values of TRL1 transcript level enhancement in tomato
floral organs. As the data illustrate, after γ- irradiation, an approximately the same multiplicity
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VLADIMIR S. BONDARENCO
value is observed in sepals during both the anther meiosis stage and mature flower in
comparison with the control. In petals and, especially, in anthers, this value is significantly
higher through meiosis than in mature flowers. Furthermore, after γ-irradiation in petals, at
the stage of mature flower, the TRL1 transcript level is higher than in anthers (Fig.2).
Apparently, this situation is caused by a sudden decrease of the anther proliferating and/or
meiotic activity at the given developmental phase of the flower development (Brukhin et al,
2003).
Table 1. Increase of TRL1 transcript concentration after γ-irradiation in reference to control
2-∆Ct irradiated/2-∆Ct control (×)
Anther Meiosis Stage
Mature flower stage
Sepals
3,9
4,6
Petals
Anthers
Pistils
22,3
16,4
5,4
9,9
2,5
8,3
As fig. 2 shows, following γ-irradiation, in pistils, at the mature flower stage, the
transcript level of the investigated gene is the highest amongst all floral organs. This might be
the consequence of the fact that, at this developmental stage, the pistil is an actively
proliferating organ, where the processes of double fertilization begin, while the other organs
have reached the apogee of their development (Brukhin et al, 2003).
Conclusions
1.
2.
3.
A correlation may exist between the expression of TRL1 gene and distinct flower organ
physiologies, as well as the amount of solar radiation received by the organ;
γ-irradiation enhances the expression of the gene under study;
The results of TRL1 expression suggest that the given gene might be involved in DNA
repair processes in tomato.
Acknowledgements
I am very grateful to my scientific advisor Dr. hab. Nicolae Barbacar of the Institute of
Genetics and Plant Physiology, ASM for helpful discussions. I also express my appreciation
to Dr. Dmitriy Ivanov and Dr. Elena Goncharova of the “DNA-Technology” ScientificProduction Firm, Moscow, Russian Federation for providing me with real time PCR detection
system for the given investigations and for technical advice. This research was supported by
Award № MTFP-013/05 of the Moldovan Research and Development Association (MRDA)
under funding from the U.S. Civilian Research & Development Foundation (CRDF).
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