The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon–intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Heading time in bread wheat (Triticum aestivum L.) is determined by three characters: vernalization requirement, photoperiodic sensitivity and narrow-sense earliness, which are involved in the phase transition from vegetative to reproductive growth. We identified and characterized the APETALA1 (AP1)-like MADS box gene in wheat (WAP1) as an activator of phase transition. Its expression starts just before the phase transition and is maintained during the reproductive phase. Inhibition of WAP1 expression in the transgenic plants by co-suppression affected neither vernalization requirement nor photoperiodic sensitivity, but resulted in delayed narrow-sense earliness, indicating that WAP1 accelerates autonomous phase transition. Analyses of the WAP1 expression in the near-isogenic lines (NILs) for spring habit genes (Vrn) revealed that WAP1 transcripts were induced by vernalization strongly in the NILs with Vrn dominant alleles and weakly with the recessive alleles. Furthermore, WAP1 expression was up-regulated by a long photoperiod in both NILs with and those without a photoperiod-insensitive gene (Ppd). These results suggest that WAP1 is a key gene in the regulatory pathway controlling the phase transition from vegetative to reproductive growth in wheat.

Photosystem I (PSI) photoinhibition suppresses plant photosynthesis and growth. However, the mechanism underlying PSI photoinhibition has not been fully clarified. In this study, in order to investigate the mechanism of PSI photoinhibition in higher plants, we applied repetitive short-pulse (rSP) illumination, which causes PSI-specific photoinhibition in chloroplasts isolated from spinach leaves. We found that rSP treatment caused PSI photoinhibition, but not PSII photoinhibition in isolated chloroplasts in the presence of O 2 . However, chloroplastic superoxide dismutase and ascorbate peroxidase activities failed to protect PSI from its photoinhibition. Importantly, PSI photoinhibition was largely alleviated in the presence of methyl viologen, which stimulates the production of reactive oxygen species (ROS) at the stromal region by accepting electrons from PSI, even under the conditions where CuZn-superoxide dismutase and ascorbate peroxidase activities were inactivated by KCN. These results suggest that the ROS production site, but not the ROS production rate, is critical for PSI photoinhibition. Furthermore, we found that not only superoxide (O 2 2 ) but also singlet oxygen ( 1 O 2 ) is involved in PSI photoinhibition induced by rSP treatment. From these results, we suggest that PSI photoinhibition is caused by both O 2 2 and 1 O 2 produced within the thylakoid membranes when electron carriers in PSI become highly reduced. Here, we show, to our knowledge, new insight into the PSI photoinhibition in higher plants.

Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in a cytoplasmic substitution (alloplasmic) line of wheat (Triticum aestivum L.) cv. Norin 26, which has the cytoplasm of a wild relative species, Aegilops crassa L. On the other hand, an alloplasmic line of wheat cv. Chinese Spring (CS) with Ae. crassa cytoplasm has normal¯owers. This is due to the presence in the CS nucleus of a fertility-restoring gene, Rfd1. Deletion mapping analysis revealed that Rfd1 is located on the middle part of the long arm of chromosome 7B. To investigate the function of the Rfd1 gene by a loss-of-function strategy, we produced alloplasmic lines of CS ditelosomic 7BS [(cr)-CSdt7BS] and CS monotelodisomic 7BS [(cr)-CSmd7BS] with the Ae. crassa cytoplasm, and characterized their phenotypes. The line (cr)-CSdt7BS without Rfd1 exhibited pistillody in all¯orets, and also female sterility. Scanning electron microscopy of the young spikes revealed that the pistillody was induced at an early stage of stamen development. The pistillate stamens often developed incomplete ovule-like structures with integuments instead of tapetum and pollen grains. It is possible that MADS box genes are associated with the induction of pistillody, because the expression of wheat APETALA3 homologue (WAP3) was reduced in the young spikes of (cr)-CSdt7BS. In addition, a histological study indicated that the female sterility in (cr)-CSdt7BS is due to the abnormality of the ovule, which fails to form an inner epidermis and integuments in the chalaza region. The line (cr)-CSmd7BS, hemizygous for Rfd1, showed partial pistillody (51%) and restored female fertility up to 72%. These results suggest that the induction of both pistillody and ovule de®ciency caused by the Ae. crassa cytoplasm is inhibited by the Rfd1 gene in a dose-dependent manner.

A number of cold responsive ( Cor )/late embryogenesis abundant ( Lea ) genes are induced by both low temperature (LT) and dehydration. To understand the molecular basis of cold acclimation and its relationship with drought stress response in wheat seedlings, we isolated a DREB2 homolog Wdreb2 , which is the candidate gene for a transcription factor of the Cor / Lea genes. The Wdreb2 expression was activated by cold, drought, salt and exogenous ABA treatment. Detailed expression studies of Wdreb2 indicated the involvement of two distinct pathways of its activation, a drought and salt stress-responsive pathway and a cold-responsive pathway. The transient expression analysis showed that the Wrab19 expression was directly activated by the WDREB2 transcription factor in wheat cells. Three transcript forms of Wdreb2 ( Wdreb2 α , Wdreb2 β and Wdreb2 γ ) were produced through alternative splicing. Under drought and salt stress conditions, the amount of the Wdreb2 β form remained fairly constant during 24-hour treatment, while those of the Wdreb2 α and Wdreb2 γ forms showed transient increases. On the other hand, the LT treatment resulted in increased transcript levels of all three forms of Wdreb2 . Thus, under the LT and drought/salt stress conditions the amount of the WDREB2 transcription factors in wheat is differentially controlled by the level of transcription and alternative splicing.