Masayuki P. Yamamoto

Synergism between RPBF Dof and RISBZ1 bZIP Activators in the Regulation of Rice Seed Expression Genes

The Dof (DNA binding with one finger) transcriptional activator rice (Oryza sativa) prolamin box binding factor (RPBF), which is involved in gene regulation of rice seed storage proteins, has been isolated from rice cDNA expressed sequence tag clones containing the conserved Dof. RPBF is found as a single gene per haploid genome. Comparison of RPBF genomic and cDNA sequences revealed that the genomic copy is interrupted by one long intron of 1,892 bp in the 5′ noncoding region. We demonstrated by transient expression in rice callus protoplasts that the isolated RPBF trans-activated several storage protein genes via an AAAG target sequence located within their promoters, and with methylation interference experiments the additional AAAG-like sequences in promoters of genes expressed in maturing seeds were recognized by the RPBF protein. Binding was sequence specific, since mutation of the AAAG motif or its derivatives decreased both binding and trans-activation by RPBF. Synergism between RPBF and RISBZ1 recognizing the GCN4 motif [TGA(G/C)TCA] was observed in the expression of many storage protein genes. Overexpression of both transcription factors gave rise to much higher levels of expression than the sum of individual activities elicited by either RPBF or RISBZ1 alone. Furthermore, mutation of recognition sites suppressed reciprocal trans-activation ability, indicating that there are mutual interactions between RISBZ1 and RPBF. The RPBF gene is predominantly expressed in maturing endosperm and coordinately expressed with seed storage protein genes, and is involved in the quantitative regulation of genes expressed in the endosperm in cooperation with RISBZ1.

Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm

A new glutelin gene, designated GluD-1, has been discovered by comparing the seed storage proteins from 48 japonica and indica rice cultivars on SDS-PAGE gels. Evidence that GluD-1 is a member of the glutelin family was provided by Western blots using anti-glutelin antiserum and by mapping the gene to the chromosomal glutelin gene cluster. The limited GluD-1 size polymorphism among the rice varieties is due to amino acid substitutions rather than to post-transcriptional modification. GluD-1 is maximally expressed in the starchy endosperm starting at 5 d after flowering (DAF) and increasing through 30 DAF, a major difference from the other glutelins which are primarily expressed in the subaleurone from 10–16 DAF. Only about 0.2 kb of the GluD-1 promoter was sufficient to confer inner starchy endosperm-specific expression. The 0.2 kb truncated GluD-1 promoter contains a bifactorial endosperm box consisting of a truncated GCN4 motif (TGA(G/C)TCA) and AAAG Prolamin box (P box), and ACGT and AACA motifs as cis-regulatory elements. Gel retardation assays and trans-activation experiments indicated that the truncated GCN4 and P box are specifically recognized by RISBZ1 b-ZIP and RPBF Dof activators in vitro, respectively, and are synergistically transactivated, indicating that combinatorial interactions of these motifs are involved in essential endosperm-specific regulation. Furthermore, deviation from the cognate GCN4 motif alters tissue-specific expression in the inner starchy endosperm to include other endosperm tissues.

Compensation and interaction between RISBZ1 and RPBF during grain filling in rice

The rice (Oryza sativa L.) basic leucine Zipper factor RISBZ1 and rice prolamin box binding factor (RPBF) are transcriptional activators of rice seed storage protein (SSP) genes in vivo. To ascertain the functions of these trans-activators in seed development, knock-down (KD) transgenic rice plants were generated in which the accumulation of RISBZ1 and RPBF was reduced in an endosperm-specific manner by co-suppression (KD-RISBZ1 and KD-RPBF). The accumulation of most SSPs changed little between individual KD mutants and wild-type plants, whereas a double KD mutant (KD-RISBZ1/KD-RPBF) resulted in a significant reduction of most SSP gene expression and accumulation. The reduction of both trans-activators also caused a greater reduction in seed starch accumulation than individual KD mutants. Storage lipids were accumulated at reduced levels in KD-RISBZ1 and KD-RISBZ1/KD-RPBF seeds. KD-RPBF and KD-RISBZ1/KD-RPBF seeds exhibited multi-layered aleurone cells. Gene expression of DEFECTIVE KERNEL1 (OsDEK1), CRINKLY4 (OsCR4) and SUPERNUMERARY ALEURONE LAYER 1 (OsSAL1) rice homologues was decreased in the KD mutants, suggesting that these genes are regulated by RISBZ1 and RPBF. These phenotypes suggest that combinatorial interactions between RISBZ1 and RPBF play an essential role during grain filling. The functional redundancy and compensation between RISBZ1 and RPBF possibly account for weak effects on the SSP levels in single KD mutants, and help maintain various processes during seed development in rice. Physical interaction between RISBZ1 and RPBF may ensure that these processes are carried out properly.

The 5′-leader sequence of sugar beet mitochondrial atp6 encodes a novel polypeptide that is characteristic of Owen cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is a mitochondrially encoded trait, which is characterized by a failure of plants to produce viable pollen. We have investigated the protein profile of mitochondria from sugar beet plants with normal (fertile) or CMS cytoplasm, and observed that a 35-kDa polypeptide is expressed in Owen CMS plants but not in normal plants. The variant 35-kDa polypeptide was found in CMS mitochondria placed in five different nuclear backgrounds. Interestingly, this polypeptide proved to be antigenically related to a 387-codon ORF (preSatp6) that is fused in-frame with the downstream atp6. The presequence extension of the atp6 ORF is commonly found in higher plants, but whether or not it is normally expressed has hitherto remained unclear. Our study is thus the first to demonstrate that the atp6 presequence is actually translated in mitochondria. We also observed that preSATP6 is a mitochondrial membrane protein that assembles into a homogeneous 200-kDa protein complex. In organello translation experiments in the presence of protease inhibitors showed a reduction in the abundance of mature preSATP6 with time, suggesting that the mature preSATP6 may be derived by proteolytic processing of a translation product of the preSatp6/Satp6 ORF.

A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants.

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST-ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.

Heterogeneity of the atp6 Presequences in Normal and Different Sources of Male-Sterile Cytoplasms of Sugar Beet*

Summary We have characterized the mitochondrial atp6 loci from male-fertile (TK81-O), Owen CMS (TK81-MS), and wild beet-derived CMS (I-12CMS(3)) cytoplasms of sugar beet ( Beta vulgaris L.). The TK81-O atp6lacks a presequence characteristic of the atp6 genes from a variety of organisms. Interestingly, RNA editing was found to create an initiation codon in TK81-O atp6 mRNA, resulting in a 5 amino acid presequence. On the other hand, TK81-MS atp6 and I-12CMS(3) atp6have presequences of 387 and 389 residues, respectively. Western blot analysis failed to reveal polymorphisms in size and abundance of the accumulated ATP6 polypeptide among the three genotypes. Our results thus favour the view that the presequence is proteolytically discarded upon maturation of the gene product and that atp6 itself is not the cause of the sterility phenotype.

Promoter shuffling at a nuclear gene for mitochondrial RPL27. Involvement of interchromosome and subsequent intrachromosome recombinations

The Reclinomonas americana mitochondrial genome contains a mitochondrial ribosomal protein L27 (rpl27) gene, whereas the rpl27 gene is absent from all plant mitochondrial genomes examined to date. This suggests that plant mitochondrial rpl27 genes have been transferred previously from the mitochondrial genome to the nuclear genome. A nuclear cDNA encoding mitochondrial RPL27 was identified in rice (Oryza sativa). Three similar sequences were identified: rpl27-1 and rpl27-2 on chromosome 8 and rpl27-3 on chromosome 4. Harr plot analysis suggests that they were generated by inter- and intrachromosomal duplications. Interestingly, the transcribed rpl27 gene (rpl27-1) acquired a promoter sequence that was derived from the rice spt16 (Osspt16) gene, the homolog of a global transcription factor in yeast (Saccharomyces cerevisiae) located downstream from the rpl27-3 sequence on chromosome 4, after inter- and intrachromosomal recombination. Reverse transcription-PCR and promoter assay revealed that the rpl27 mRNAs were mainly transcribed from rpl27-1. A repeat of seven nucleotides (AATAGTT) was identified at the junction of rpl27-1 and rpl27-2 on chromosome 8, and the same repeat was also identified at the 5′ end of rpl27-2 and the 3′ end of rpl27-1. This repeat (AATAGTT) contains the hot-spot sequence AGTT, which is preferentially recognized by topoisomerase I in wheat (Triticum aestivum) germ, suggesting the involvement of topoisomerase I in this recombination. We here report the example of promoter shuffling and show that this promoter shuffling resulted from a recent segmental duplication through inter- and intrachromosomal recombination events.

The nad4L-orf25 gene cluster is conserved and expressed in sugar beet mitochondria

Abstract We have found that a gene coding for NADH dehydrogenase subunit 4L and a presumed gene, orf25, are linked and co-transcribed with each other in sugar beet mitochondria. Ten and twelve C-to-U editing events were observed in the mRNAs of nad4L and orf25, respectively; the amino-acid sequence specified after editing is better-conserved in comparison with the homologues of other organisms. It is interesting to note that the translation initiation codon of nad4L is created by editing. The conservation of the nad4L-orf25 linkage was examined by PCR-amplification of the intergenic region. We obtained successful PCR products from five dicots (spinach, apple, snapdragon, petunia and tobacco) and two monocots (tulip and pineapple), but not in two poaceous plants, rice and maize. The intergenic region, when present, was found to be well-conserved in its sequence, suggesting a monophyletic origin of this linkage. Our result, together with previous reports of Arabidopsis and four poaceous species, favour the argument that the nad4L-orf25 linkage is conserved throughout angiosperms except in the Poaceae.

The rps4 gene in sugar beet mitochondria: insertion/deletion mutations occur within the gene but do not disrupt the reading frame

Summary We have characterized a sugar beet mitochondrial gene, rps4, which is co-transcribed with the downstream gene nad6 . The sugar beet RPS4 polypeptide has two deletions of 8 and 49 amino acid residues and a duplication of 14 residues in the middle of the reading frame, relative to the rapeseed and rice homologues. Nevertheless, transcripts of the rps4 were observed to be edited at 10 nucleotides, suggesting that the sugar beet rps4 is functional. Western blot analysis identified the translation product of 33 kDa. The extensive rearrangements in rps4 were also found in spinach but not in pea, green pepper and rose.

Molecular basis of cytoplasmic male sterility in beets: an overview

Sugarbeet cultivars are almost exclusively hybrids, which are produced using the sole source of cytoplasmic male sterility (CMS), the so-called Owen CMS. Several alternative sources of CMS have been described. One of these, I-12CMS(3), was derived from wild beets collected in Pakistan, and another CMS source, GCMS, has a cytoplasmic origin in wild sea beets from France. During the past decade, male sterility-associated mitochondrial genes have been identified in these three CMS systems. Moreover, the recent development of a variety of DNA markers has permitted the genetic mapping of nuclear restorer-of-fertility genes for both Owen and GCMS. This review focuses on the mechanism of CMS in beets.