The study characterized the coding sequences of two sirtuin genes (VvSRT1 and VvSRT2) in Vitis vinifera (grapevine) and analyzed their expression levels in various leaf and berry developmental stages. VvSRT1 is similar to the human SIRT6 gene and VvSRT2 is similar to SIRT4. Both genes showed varying expression levels across tissues and developmental stages, with VvSRT2 exhibiting more variation. VvSRT2 generally showed higher expression than VvSRT1 in leaves. The differential expression of the genes suggests regulation during plant development and they may act in different tissues, stages, organs, and processes. This work provides a

1. CHARACTERIZATION AND STUDY OF VITIS VINIFERA L. SIRTUIN GENES
BUSCONI M.*, CUCURACHI M.**, MORREALE G.***, ZANETTI A.**, BAVARESCO L.***, FOGHER C.*
•*: Institute of Agronomy, Plant Genetics, and Field Crops, Università Cattolica del Sacro Cuore, Via Emilia Parmense, 84, 29122 Piacenza (Italy)
•**:PLANTECHNO SRL, Via Staffolo 60, 26041 Casalmaggiore Cremona (Italy)
•***: CRA Centro di ricerca per la viticoltura, viale XXVIII Aprile 26, 31015 – Conegliano, Treviso, Italy.
Aim
The aims of this study has been both the experimental characterization of the coding sequence of Vitis vinifera sirtuin genes and the analysis of their expression levels in several leaf and berry developmental stages.
Introduction
The sirtuin/Sir2 (Silent information regulator 2) family of NAD+-dependent deacetylases and mono-ADP-
ribosyltransferases plays an important role in several cellular processes including gene silencing, cell
cycle regulation and life span extension in yeast and animals. The functions of sirtuins have been
investigated mainly in fungi and vertebrates where they resulted to be connected with several processes
such as transcriptional silencing and changes in gene expression levels, counter aging, metabolic
regulation and pathogenesis (North and Verdin, 2004). Nevertheless there are still many substantial gaps to
the full understanding of the sirtuin functions(Greiss and Gartner, 2009). Despite the recent explosion in
the number of reports on sirtuins in fungi and animals, just few works dealing with plant sirtuins can be
retrieved from literature and few is known about their function in plants. Compared to other eukaryotes,
plants have relatively fewer SIR2 related genes and just two putative SIR2 family proteins can usually be
found in plant genomes.
It has recently been shown the presence of sirtuins in Vitis vinifera L. (Busconi et al., 2009). Vitis vinifera,
whose genome has been completely sequenced has been chosen also because it can be considered as a
model species for perennial woody plants. Considering that the function of Sir2 family in plants are poorly
understood, the aims of this study was both the experimental characterization of the coding sequence for
the two genes and the analysis of their expression levels in several leaf and berry developmental stages.
Material and methods
Leaves and berries were detached from Vitis vinifera L. ‘Merlot’ and the RNA was extracted according to standard
methods. The primers used to amplify the sirtuin gene coding sequences are reported (table 1). The experimental coding
sequences have been submitted to NCBI with the following accession numbers: VvSRT1 – JN252254; VvSRT2 –
JN252255. The variations in gene expression during the different developmental stages were evaluated by real time PCR
(RT-qPCR). The gene-specific primers were designed on the experimental coding sequences, obtained in this work,
within consecutive exons separated by an intron. Relative expression of each gene has been calculated according to the
2-∆∆Ct method using Glyceraldehyde-3-phosphate dehydrogenase for normalization.
Name Sequence Application Gene
IGP1094-RT1F 5’-ATGTCTCTGGGCTACGCA-3’ RT-PCR* VvSRT1
IGP1097-RT1R 5’-CTATGATCTAGGCTTCCTC-3’ RT-PCR VvSRT1
IGP1235-RT2F 5’-ATGACGGTGTCCTCCACC-3’ RT-PCR VvSRT2
IGP1101-RT2R 5’- CTAGATCACAGGTATGCTTAG-3’ RT-PCR VvSRT2
IGP1206 5’- GGGTGGTGCTAAGAAGGTTGTC-3’ Real Time-PCR GAPDH
IGP1207 5’- GGGTGGTGCTAAGAAGGTTGTC-3’ Real Time-PCR GAPDH
IGP1208 5’-CAGAATGGCTGATGTTGTATTATGC-3’ Real Time-PCR VvSRT2
IGP1209 5’ -CTAAAATCCCTCCGTGGTGG-3’ Real Time-PCR VvSRT2
IGP1210 5’-GATGTGATGCTTTACTGGTAGTTGGA -3’ Real Time-PCR VvSRT1
IGP1211 5’-TTCATGAGCAGCCCTGACAA-3’ Real Time-PCR VvSRT1
IGP1212 5’- CAGATCACTCCTGCATGCAACCTCCC -3’ Real Time-PCR VvSRT2
IGP1213 5’- CATCTCTGATGACCATGTCTGCTTTCCGA -3’ Real Time-PCR VvSRT1
IGP1214 5’-TCTCAGCCCCAAGCAAAGATGCTCC-3’ Real Time-PCR GAPDH
Results and discussion
In order to obtain a real coding sequence we considered both the putative sequences available online and the data
retrievable from the grape RNA-Seq database. Short regions, from 15 to 20 bases in length, of the putative coding
sequences were screened against the RNA-Seq database in search for proofs of expression. Several positive reads were
found and considered for the development of primers useful to amplify the experimental coding sequence. The main
characteristics of the encoded sirtuins are reported (Figure 1). VvSRT1 is analog to the human sirtuin SIRT6 while VvSRT2
is analog to the human sirtuin SIRT4.
Several significant expression level variations have been put in correlation with specific developmental stages, organ, and
age of the leaf (Figure 2). VvSRT2 is characterized by a greater expression variation in the different samples considered
than VvSRT1, whose expression remains generally low, without significant variations with the exception of Flowering
onset in berries.
VvSRT2 is generally more expressed than VvSRT1 in leaves, both in the 4th and 8th leaves. The presence of significant
variations, mainly for VvSRT2, suggests that the expression is regulated during plant development.
This work represents a first step in order to understand the role of sirtuins in grape. We succeeded in obtain two really
expressed coding sequences from start to stop codons. We can not exclude the presence of alternative spliced form, as
reported for other plants, but actually we have recovered only a single coding sequence for each gene. Then we obtained
a first characterization of their expression level changes in different plant organ (leaves and berries), in different
developmental stages, and in leaves of different ages. The two genes exhibit very different expression profiles and may
act in different tissues, stages, organs, and processes.
*: Reverse Trascriptase PCR
VvSRT1
VvSRT2
Figure 1. Schematic representation of grape sirtuins VvSRT1 and VvSRT2. The main predicted features are
reported: sirtuin conserved domains, secondary structure elements, binding sites, localization signals, and post-
translational modification sites.
Figure 2. Comparison of the expression profiles of VvSRT1 (fig 2a) and VvSRT2 (fig. 2b) obtained in leaves and berries. Figure 2a report the
expression levels of VvSRT1 during the different growing stages in different plant organ: berries (black boxes), 4th leaves (dark-grey boxes)
and 8th leaves (black/white striped boxes). Figure 2b report the expression level of VvSRT2 in the different stages and different plant organs
(i.e., berries (shaded grey boxes), 4th leaves (white boxes) and 8th leaves (light-grey boxes)). Error bar indicates +/- SD. (*) P < 0,05 (Post-Hoc
with Bonferroni correction).
2a - VvSRT1 2b - VvSRT2
References
Busconi, M., Reggi, S., Fogher, C., and Bavaresco, L. (2009) Evidence of a sirtuin gene family in grapevine (Vitis vinifera L.). Plant Phisiology and Biochemistry 47: 650-652.
Greiss, S., and Gartner, A. (2009) Sirtuin/Sir2 phylogeny, evolutionary considerations and structural conservation. Molecules and Cells (Mol. Cells) 28, 407-415.
North, B.J., and Verdin, E. (2004) Sirtuins: Sir2-related NAD dependent protein deacetylases. Genome Biology 5, 224-232.