All eukaryotes have three nuclear DNA-dependent RNA polymerases, namely, Pol I, II, and III. Interestingly, plants have catalytic subunits for a fourth nuclear polymerase, Pol IV. Genetic and biochemical evidence indicates that Pol IV does not functionally overlap with Pol I, II, or III and is nonessential for viability. However, disruption of the Pol IV catalytic subunit genes NRPD1 or NRPD2 inhibits heterochromatin association into chromocenters, coincident with losses in cytosine methylation at pericentromeric 5S gene clusters and AtSN1 retroelements. Loss of CG, CNG, and CNN methylation in Pol IV mutants implicates a partnership between Pol IV and the methyltransferase responsible for RNA-directed de novo methylation. Consistent with this hypothesis, 5S gene and AtSN1 siRNAs are essentially eliminated in Pol IV mutants. The data suggest that Pol IV helps produce siRNAs that target de novo cytosine methylation events required for facultative heterochromatin formation and higher-order heterochromatin associations.

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 transactivation 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.

The GCN4 motif, a cis-element that is highly conserved in the promoters of cereal seed storage protein genes, plays a central role in controlling endospermspecific expression. This motif is the recognition site for a basic leucine zipper transcriptional factor that belongs to the group of maize Opaque-2 (O2)-like proteins. Five different basic leucine zipper cDNA clones, designated RISBZ1-5, have been isolated from a rice seed cDNA library. The predicted gene products can be divided into two groups based on their amino acid sequences. Although all the RISBZ proteins are able to interact with the GCN4 motif, only RISBZ1 is capable of activating (more than 100-fold expression) the expression of a reporter gene under a minimal promoter fused to a pentamer of the GCN4 motif. Loss-of-function and gain-of-function experiments using the yeast GAL4 DNA binding domain revealed that the proline-rich N-terminal domain (27 amino acids in length) is responsible for transactivation. The RISBZ1 protein is capable of forming homodimers as well as heterodimers with other RISBZ subunit proteins. RISBZ1 gene expression is restricted to the seed, where it precedes the expression of storage protein genes. When the RISBZ1 promoter was transcriptionally fused to the ␤-glucuronidase reporter gene and the chimeric gene was introduced into rice, the ␤-glucuronidase gene is specifically expressed in aleurone and subaleurone layer of the developing endosperm. These findings suggest that the specific expression of transcriptional activator RISBZ1 gene may determine the endosperm specificity of the storage protein genes.Regulated gene expression is mediated by the combinatorial interactions of multiple cis-elements in the gene's promoter. Specific binding of transcriptional factors to the cognate ciselements constitute a crucial step in transcription initiation and, in turn, on the spatial and temporal expression of genes.Seed storage protein genes provide a model system for the study on the regulatory mechanisms of plant genes (1), since their expression is restricted to a specific tissue and stage during seed development. These specific temporal and spatial expression patterns may be explained as the result of regulatory assemblies of several transcriptional activators that recognize the cis-elements implicated in seed-specific expression. Therefore, to understand such molecular mechanisms, characterization of cis-elements and transcription factors has been performed on many storage protein genes of several crop plants (2,3). Despite numerous studies, the mechanism by which these genes are regulated are poorly understood, since many of the essential cis-elements have not been identified. This is especially true in the case of monocot plants, where many of the promoter analyses of cereal storage protein genes have carried out by transient assays using particle bombardment or heterologous transgenic tobacco system (4 -6). Dissection analyses of promoter using homologous stable transgenic plant have been carried out only on glutelin genes of ...

SummaryThe ±197 bp promoter of the rice seed storage protein gene, GluB-1, is capable of conferring endospermspeci®c gene expression. This proximal 5¢¯anking region contains four motifs, GCN4, AACA, ACGT and Prolamin-box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the ±46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three motifs are unable to direct expression in seeds as well as other tissues of transgenic rice plants. Mutational analysis of individual motifs in the ±197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determining endosperm-speci®c expression and that the AACA, ACGT and Prolaminbox are involved in quantitative regulation of the GluB-1 gene. A set of gain-of-function experiments using transgenic rice showed that either the Prolamin-box or AACA, although often coupled with GCN4 in many genes, is insuf®cient to form a functional promoter unit with GCN4, whereas a combination of GCN4, AACA and ACGT motifs was found suf®cient to confer a detectable level of endosperm expression. Taken together, our results provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the expression of seed storage protein genes.

The optical differential pathlength factor (DPF) is an important parameter for physiological measurement using near infrared spectroscopy, but for the human adult head it has been available only for the forehead. Here we report measured DPF results for the forehead, somatosensory motor and occipital regions from measurements on 11 adult volunteers using a time-resolved optical imaging system. The optode separation was about 30 mm and the wavelengths used were 759 nm, 799 nm and 834 nm. Measured DPFs were 7.25 for the central forehead and 6.25 for the temple region at 799 nm. For the central somatosensory and occipital areas (10 mm above the inion), DPFs at 799 nm are 7.5 and 8.75, respectively. Less than 10% decreases of DPF for all these regions were observed when the wavelength increased from 759 nm to 834 nm. To compare these DPF maps with the anatomical structure of the head, a Monte Carlo simulation was carried out to calculate DPF for these regions by using a two-layered semi-infinite model and assuming the thickness of the upper layer to be the sum of the thicknesses of scalp and skull, which was measured from MRI images of a subject's head. The DPF data will be useful for quantitative monitoring of the haemodynamic changes occurring in adult heads.

SummaryIn 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 CMSencoding 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.

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Unlike animals, whose gametes are direct products of meiosis, plant meiotic products undergo additional rounds of mitosis, developing into multicellular haploid gametophytes that produce egg or sperm cells. The complex development of gametophytes requires extensive expression of the genome, with DNAdependent RNA polymerases I, II, and III being the key enzymes for nuclear gene expression. We show that loss-of-function mutations in genes encoding key subunits of RNA polymerases I, II, or III are not transmitted maternally due to the failure of female megaspores to complete the three rounds of mitosis required for the development of mature gametophytes. However, male microspores bearing defective polymerase alleles develop into mature gametophytes (pollen) that germinate, grow pollen tubes, fertilize wild-type female gametophytes, and transmit the mutant genes to the next generation at moderate frequency. These results indicate that female gametophytes are autonomous with regard to gene expression, relying on transcription machinery encoded by their haploid nuclei. By contrast, male gametophytes make extensive use of transcription machinery that is synthesized by the diploid parent plant (sporophyte) and persists in mature pollen. As a result, the expected stringent selection against nonfunctional essential genes in the haploid state occurs in the female lineage but is relaxed in the male lineage.