Advance Access published November 28, 2003

Journal of Experimental Botany, Page 1 of 2

DOI: 10.1093/jxb/erh029

GENE NOTE

The rice pyruvate decarboxylase 3 gene, which lacks introns, is transcribed

in mature pollen*
Yuhua Li1,2,, Kazuhiro Ohtsu1,, Keisuke Nemoto3, Nobuhiro Tsutsumi1, Atsushi Hirai1, and Mikio Nakazono1,
1
Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo,
Tokyo 113-8657, Japan
2
Research Institute of Flower Biotechnology, Northeast Forestry University, Harbin 150040, China
3
Asian Natural Environmental Science Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan

Received 17 July 2003; Accepted 7 October 2003

Abstract

Key words: Alcoholic fermentation, intron-less gene, Oryza

sativa, pollen, pyruvate decarboxylase.

Alcoholic fermentation consists of two reactions, in which pyruvate

is rst decarboxylated to acetaldehyde by pyruvate decarboxylase
(PDC), and then acetaldehyde is converted to ethanol by alcohol
dehydrogenase (ADH). In plants, this pathway occurs at low levels
under aerobic conditions, and is strongly induced under anaerobic
conditions (Peschke and Sachs, 1993; Sachs et al., 1996; Vartapetian
and Jackson, 1997). An exception is alcoholic fermentation during
pollen development, in which the pathway increases even under
aerobic conditions (Bucher et al., 1995; Tadege and Kuhlemeier,
1997). The products of aerobic fermentation (acetaldehyde and
ethanol) are reported to be metabolized in a pathway that bypasses
mitochondrial pyruvate dehydrogenase (Tadege et al., 1999). The
bypass contributes to respiration and lipid biosynthesis in tobacco
pollen (Tadege et al., 1999; Mellema et al., 2002).
Rice PDC is encoded by at least four PDC genes (Hossain et al.,
1996; Rivoal et al., 1997). Among the four PDC genes that have
been identied (PDC1PDC4), cDNA clones encoding PDC1,
PDC2 and PDC4 have been isolated and their expressions of the
three PDC genes have been shown to be induced under anaerobic
conditions (Hossain et al., 1994a; Huq et al., 1995; Rivoal et al.,
1997). A genomic clone of PDC3 was isolated from rice (cv. IR54)
and its nucleotide sequence was determined (Hossain et al., 1994b).
The rice PDC1 and PDC2 genes consist of six exons and ve introns

(Hossain et al., 1996; Huq et al., 1999), whereas the rice PDC3 gene
lacks introns (Hossain et al., 1994b). Furthermore, no transcript of
PDC3 was found even under anaerobic conditions by northern
hybridization. Therefore, PDC3 was suggested to be a pseudogene
(Hossain et al., 1994b). However, organ-specic expression of rice
PDC3, which would be a more sensitive test, was not examined. In
the present study, PDC3 transcripts were sought in various organs of
rice (cv. Nipponbare) using northern hybridization and were
detected in panicles after heading, but not in other organs or tissues
(Fig. 1A). On the other hand, PDC1 mRNA was found in roots of
light-grown seedlings, and PDC2 mRNA was observed in all the
organs examined, although its expression was highest in young
panicles (Fig. 1A).
Anthers in panicles after heading contain mature pollen. Because
mature pollen is a site of alcoholic fermentation (Tadege and
Kuhlemeier, 1997; Mellema et al., 2002), it may accumulate PDC3
mRNA. To examine this possibility, in situ hybridization of PDC3
transcripts was performed using anthers obtained from panicles after
heading, according to the method of Ishiwatari et al. (2000) except
that FAA (5% formalin, 45% ethanol and 5% acetic acid) was used
as a xative. As expected, the antisense PDC3 probe, but not the
sense probe, specically hybridized to mature pollen (Fig. 1B).
A search for PDC3 homologues in the rice EST clone database
found one clone, E3273, which was assumed to encode PDC3. The
EST clone was constructed from mRNA extracted from panicles at
the owering stage of rice (cv. Nipponbare). This stage perfectly
corresponds to the stage at which PDC3 mRNA was detected by
northern hybridization (Fig. 1A). The 1967 bp insert of the E3273
clone was completely sequenced (accession number AB111050).
The clone contained a complete open reading frame encoding a
polypeptide of 587 amino acid residues. The predicted amino acid
sequence was 94% identical to that of PDC3 reported previously
(Hossain et al., 1994b; data not shown). It was concluded that this
PDC gene is PDC3 because it lacks introns and its chromosomal
location corresponds to the location of PDC3 mapped by Huq et al.
(1999) (data not shown). The difference in the deduced amino acid
sequences is probably due to a difference in cultivars [Japonica rice

* The nucleotide sequence data reported here were deposited in DDBJ under the accession number AB111050.

These authors contributed equally to this work.

Present address: School of Agriculture, Meijo University, 1-501 Shiogamaguchi, Tenpaku-ku, Nagoya, Aichi 468-8502, Japan.

To whom correspondence should be addressed. Fax: +81 3 5841 5183. E-mail: anakazo@mail.ecc.u-tokyo.ac.jp

Journal of Experimental Botany, Society for Experimental Biology 2004; all rights reserved

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The rice pyruvate decarboxylase 3 gene (PDC3), which has no

introns, was previously postulated to be a pseudogene because
no PDC3 mRNA had been detected, even under anaerobic conditions. However, in this study, it was found that rice PDC3 transcripts accumulated in panicles after heading. Within anthers
obtained from the panicles, PDC3 was shown to be transcribed
in mature pollen by in situ hybridization. These results suggest
that the rice PDC3 is a functional gene. Its product may play a
role in aerobic alcoholic fermentation in mature pollen.

2 of 2 Li et al.
cv. Nipponbare (this study) and Indica rice cv. IR54 (Hossain et al.,
1994b)].
These results indicate that the rice PDC3 gene is not a pseudogene
but a functional gene and that PDC3 may play a role in alcoholic
fermentation in mature pollen.
Acknowledgements
The authors express their appreciation to Hiroyuki Tsuji for his technical
assistance. This work was partly supported by grants-in-aid from the Ministry
of Education, Culture, Sports, Science and Technology of Japan.

References

Fig. 1. (A) Northern hybridization of transcripts of the PDC3, PDC1 and

PDC2 genes in various organs. Each lane was loaded with 5 mg total RNA.
Gene-specic probes were prepared with the PCR products, which were
amplied from the 3-untranslated regions. Equal loadings of total RNA
were checked by ethidium bromide (EtBr) staining. (B) In situ
hybridization of PDC3 transcripts in horizontal sections of rice anthers
obtained from panicles after heading. These sections were hybridized with
an antisense or sense rice PDC3 probe. In the left panel, walls of two
anther loculi were dehisced. Scale bars represent 100 mm.

Downloaded from http://jxb.oxfordjournals.org/ by guest on September 9, 2014

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