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JXB Advance Access published online on February 3, 2009
Journal of Experimental Botany, doi:10.1093/jxb/ern349
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Characterization of pullulanase (PUL)-deficient mutants of rice (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm

Naoko Fujita1 *,{dagger}, Yoshiko Toyosawa *2, Yoshinori Utsumi1, Toshiyuki Higuchi3, Isao Hanashiro3, Akira Ikegami4, Sayuri Akuzawa4, Mayumi Yoshida1, Akiko Mori1, Kotaro Inomata1, Rumiko Itoh1, Akio Miyao5, Hirohiko Hirochika5, Hikaru Satoh2 and Yasunori Nakamura1

1Department of Biological Production, Akita Prefectural University, Akita City, Akita, 010-0195 Japan
2Institute of Genetic Resources, Faculty of Agriculture, Kyusyu University, Hakozaki, Fukuoka, 812-8581 Japan
3Department of Biochemical Science and Technology, Kagoshima University, 1-21-24, Korimoto, Kagoshima, 890-0065 Japan
4Department of Nutritional Science, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagayaku, Tokyo, 156-8502 Japan
5NIAS, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602 Japan

{dagger} To whom correspondence should be addressed: E-mail: naokof{at}akita-pu.ac.jp

Rice (Oryza sativa) allelic sugary1 (sug1) mutants defective in isoamylase 1 (ISA1) accumulate varying levels of starch and phytoglycogen in their endosperm, and the activity of a pullulanase-type of a debranching enzyme (PUL) was found to correlate closely with the severity of the sug1 phenotype. Thus, three PUL-deficient mutants were generated to investigate the function of PUL in starch biosynthesis. The reduction of PUL activity had no pleiotropic effects on the other enzymes involved in starch biosynthesis. The short chains (DP ≤13) of amylopectin in PUL mutants were increased compared with that of the wild type, but the extent of the changes was much smaller than that of sug1 mutants. The {alpha}-glucan composition [amylose, amylopectin, water-soluble polysaccharide (WSP)] and the structure of the starch components (amylose and amylopectin) of the PUL mutants were essentially the same, although the average chain length of the B2-3 chains of amylopectin in the PUL mutant was ~3 residues longer than that of the wild type. The double mutants between the PUL-null and mild sug1 mutants still retained starch in the outer layer of endosperm tissue, while the amounts of WSP and short chains (DP ≤7) of amylopectin were higher than those of the sug1 mutant; this indicates that the PUL function partially overlaps with that of ISA1 and its deficiency has a much smaller effect on the synthesis of amylopectin than ISA1 deficiency and the variation of the sug1 phenotype is not significantly dependent on the PUL activities.

Key words: Amylopectin, debranching enzyme, isoamylase, mutant, pullulanase, rice endosperm, starch biosynthesis

* These author contributed equally to the paper.

Received 29 September 2008; Revised 8 December 2008 Accepted 8 December 2008


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