Differences in meristem size and expression of branching genes are associated with variation in panicle phenotype in wild and domesticated African rice.

BACKGROUND: The African rice Oryza glaberrima was domesticated from its wild relative Oryza barthii about 3000 years ago. During the domestication process, panicle complexity changed from a panicle with low complexity in O. barthii, to a highly branched panicle carrying more seeds in O. glaberrima. To understand the basis of this differential panicle development between the two species, we conducted morphological and molecular analyses of early panicle development. RESULTS: Using X-ray tomography, we analyzed the morphological basis of early developmental stages of panicle development. We uncovered evidence for a wider rachis meristem in O. glaberrima than in O. barthii. At the molecular level, spatial and temporal expression profiles of orthologs of O. sativa genes related to meristem activity and meristem fate control were obtained using in situ hybridization and qRT-PCR. Despite highly conserved spatial expression patterns between O. glaberrima and O. barthii, differences in the expression levels of these early acting genes were detected. CONCLUSION: The higher complexity of the O. glaberrima panicle compared to that of its wild relative O. barthii is associated with a wider rachis meristem and a modification of expression of branching-related genes. Our study indicates that the expression of genes in the miR156/miR529/SPL and TAW1 pathways, along with that of their target genes, is altered from the unbranched stage of development. This suggests that differences in panicle complexity between the two African rice species result from early alterations to gene expression during reproductive development.

Characterization of two members of the Arabidopsis thaliana gene family, At beta fruct3 and At beta fruct4, coding for vacuolar invertases.

We have isolated and characterized two Arabidopsis thaliana cDNAs and their cognate genes, At beta fruct3 and At beta fruct4, encoding vacuolar forms of invertase. Our sequencing results showed that the gene At beta fruct3 is located downstream of the 3-ketoacyl-acyl carrier protein synthase III gene (AtKasIII). At beta fruct3 and 4 are functional and organized into seven exons and six introns with an identical organization. The At beta fruct3 and At beta fruct4 genes encode, respectively, polypeptides of 648 and 664 residues that contain all the characteristic hallmarks of vacuolar invertases. A. thaliana is the first plant of which both cell-wall (At beta fruct1 and At beta fruct2) and vacuolar (At beta fruct3 and At beta fruct4) genes are characterized. The same number of exons and introns is seen in the genes At beta fruct1, At beta fruct3 and At beta fruct4 as well as in all other invertase genes described to date. However, the position of the third intron is different in At beta fruct3 and At beta fruct4. At beta fruct2 shows a different organization. A neighbour-joining distance tree shows that the A. thaliana vacuolar invertases described here are, as expected, more closely related to vacuolar invertases from other plant species (e.g., carrot) than to the A. thaliana cell-wall invertases. The evolution of plant invertase genes from a common ancestral gene is discussed. Our results demonstrate that in A. thaliana, at least two genes encoding vacuolar invertases are expressed during the development of the plant. Southern blot hybridization experiments suggest the presence of one copy of, respectively, At beta fruct3 and At beta fruct4 per haploid genome, and Northern blot analysis demonstrates that vacuolar invertase genes are highly expressed in stems, roots, flowers and at very low levels in mature leaves.

Characterisation of novel plant genes encoding MEKK/STE11 and RAF-related protein kinases.

Various elements of the MAP kinase module have been isolated in plants. We describe here the characterisation of 14 new plant cDNAs and genes encoding putative MAP kinase kinase kinases (MAP3Ks) related to the MEKK/STE11 and RAF protein kinases. Plant MAP3Ks are characterised by a variety of primary structures conserved within closely related proteins. Southern blot analysis suggests that plant MAP3Ks are heterogenous in their genomic structure, existing either as single copy genes or as small gene families. An RT-PCR analysis showed that in Arabidopsis thaliana, all organs studied contain detectable levels of transcripts of each of the MAP3K genes identified; however, signals obtained with mature pollen were weak or non-existent except for AtMAP3Kgamma. None of the reported genes share a cell-cycle or a cold stress regulated expression.

Plant MAP kinase kinase kinases structure, classification and evolution.

The increasing number of reports describing plant MAP kinase signalling components reflects the cardinal role that MAP kinase pathways are likely to play during plant growth and development. Relationship and structural analyses of plant MAP kinase kinase kinase related cDNAs and genes established, on one hand, the PMEKKs, which may be distinguished into the alpha, beta, gamma, and zeta groups, and, on the other hand, the PRAFs that consist of the delta, eta and theta groups. Plant MAP3Ks are characterized by different primary structures, but conserved within a single group. A relationship analysis, which included animal, fungal and plant MAP3Ks, revealed a high degree of diversity among this biochemically established set of proteins, thus suggesting a range of biological functions. Four major families emerged, namely the MEKK/STE11, including the PMEKKs, the RAF, including the PRAFs, as well as the MLK and CDC7 families. These four families showed phylum-dependent distributions. Signature sequences characterizing the RAF family and the RAF subfamilies have been evidenced. However, no equivalent sequence motifs were identified for the MEKK/STE11 family, which is highly heterogeneous.

EgAP2-1, an AINTEGUMENTA-like (AIL) gene expressed in meristematic and proliferating tissues of embryos in oil palm.

In order to better understand the developmental processes that govern the formation of somatic embryos in oil palm (Elaeis guineensis Jacq.), we investigated the transcription factor genes expressed during embryogenesis in this species. The AP2/EREBP transcription factor family includes the AP2 subgroup, which contains several proteins that play important roles in plant development. We identified and characterized EgAP2-1, which codes for a protein that contains two AP2 domains similar to those of the transcription factor BABYBOOM (BBM) and more generally AINTEGUMENTA-like (AIL) proteins of the AP2 subgroup. In a similar way to related genes from eudicots, ectopic expression of EgAP2-1 in transgenic Arabidopsis plants alters leaf morphology and enhances regeneration capacity. In oil palm, EgAP2-1 transcripts accumulate to the greatest extent in zygotic embryos. This expression pattern was investigated in more detail by in-situ hybridization, revealing that in both zygotic and somatic embryos, EgAP2-1 expression is concentrated in proliferating tissues associated with the early development of leaf primordia, root initials and provascular tissues.

The lectin gene family of Ricinus communis: cloning of a functional ricin gene and three lectin pseudogenes.

Molecular hybridisation using a ricin cDNA probe has revealed that the ricin/Ricinus communis agglutinin (RCA) multigene family is composed of approximately eight members. Several genomic clones containing preproricin and preproricin-like sequences have been isolated. Partial analysis of three different genomic clones by DNA sequencing and ribonuclease protection has indicated that at least three members of the lectin gene family are non-functional. None of the original seventeen positive clones isolated appears to contain a Ricinus communis agglutinin (RCA) gene. One gene member analysed (pCBG3H1) represents a functional ricin gene similar in coding sequence to the published cDNA sequence and possesses typical eukaryotic consensus sequences and seed-specific elements within the flanking sequences. Investigation at the transcriptional level of the expression pattern of this gene revealed that mRNA accumulates during the post-testa stages of seed development. The pattern of accumulation of steady-state transcripts correlates closely with that previously observed at the protein and translatable RNA levels.

Localisation of cis elements in the promoter of a wheat alpha-Amy2 gene.

A functional analysis of the promoter from the wheat alpha-amylase gene alpha-Amy2/54 is described. Mutant alpha-Amy2/54 promoters containing replacements or deletions were constructed and their ability to direct expression of the reporter gene beta-glucuronidase (GUS) in gibberellin-responsive oat aleurone protoplasts analysed. Chimaeric promoters using regions of the cauliflower mosaic virus (CaMV) 35S and alpha-Amy2/54 promoters were also analysed. The results suggest that at least three regions within the alpha-Amy2/54 promoter contain cis elements that are necessary for high-level gibberellin-regulated transcription. Fusion of 1.8 kb of promoter sequence upstream from -117 bp to a minimal (-55 CaMV 35S) promoter gave rise to hormone-independent expression implying that the region 3' to -117 bp contains an element which represses transcription in the absence of gibberellin or presence of abscisic acid.

Nuclear control of mitochondrial genome reorganization characterized using cultured cells of ditelosomic and nullisomic-tetrasomic wheat lines.

In vitro-mediated reorganization of the mitochondrial genome is governed by information contained in the nuclear genome. Here we show, from the study of tissue cultures initiated from an almost complete collection of ditelosomic and nullisomic-tetrasomic wheat lines, that nuclear control of the mitochondrial genome structure is a highly complex process. Whereas information responsible for the amplification of defined molecular configurations has been found to be located in either a single or a few chromosomal arms, other rearrangements such as changes in the relative amounts of interconverting subgenomic structures are governed by a number of genes scattered over most of the wheat chromosomes.

Functional analysis of linker insertions and point mutations in the alpha-Amy2/54 GA-regulated promoter.

Functional analysis of a gibberellin-regulated wheat alpha-amylase promoter, alpha-Amy2/54, has indicated that three regions were essential for expression. By studying the ability of mutant promoters, containing a randomly inserted 22 bp excision linker, to direct expression in oat aleurone protoplasts we have refined the positions and extents of these three cis elements and also demonstrated the presence of two additional elements. By converting the linker insertions to either single base point mutations or deletions using the class IIS restriction endonuclease BsmI we have shown that nucleotides -119 and -109 within the GARE -121GTAACAGAGTCTGG-108 and nucleotide -152 within the proposed element -156GATTGACTTGACC-144 are essential for high level expression from this promoter.

Molecular cloning of ricin.

A variety of strategies have been used to obtain cDNA and genomic clones encoding ricin. Since their isolation these sequences have been manipulated to allow expression of A chain (19) and A chain mutants (15,20,34), B chain (14,21-23) and proricin (24). Utilizing structural information (35), precise changes have been introduced into both A and B chains with the aim of probing catalytic and sugar-binding residues, respectively. In the longer term, such manipulations, coupled with successful expression and purification schemes, will allow the delineation of functional residues and domains, ensuring that ricin remains the prototype plant toxin with which to study cellular intoxication and ribosome inactivation and to utilize in pharmaceutical product development.

Cell cycle regulation by plant growth regulators: involvement of auxin and cytokinin in the re-entry of Petunia protoplasts into the cell cycle.

In order to understand the mode of action of auxins and cytokinins in the induction of cell division, the effects of the two plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-benzyladenine (BA) were investigated using mesophyll protoplasts of Petunia hybrida, cultivated in either complete medium or in medium deficient in cytokinin, auxin or both. Firstly we studied DNA synthesis, using 5-bromodeoxyuridine/bisbenzimide Hoechst/propidium iodide flow cytometry analyses and by the monitoring of histone H4 transcript levels. Roscovitine, a cyclin-dependent kinase (CDK) inhibitor, was found to block the cell cycle prior to entry into the S and M phases in the cultured P. hybrida protoplasts. This suggests that in Petunia cell there is a requirement for CDK activity in order to complete the G1 and G2 phases. Further experiments using roscovitine showed that neither 2,4-D nor BA were individually able to induce cell cycle progression beyond the roscovitine G1 arrest. We also monitored the phytohormonal induction of S phase by studying variations in transcript levels of the gene for mitogenactivated protein kinase (PMEK1) and transcript levels of the cell division cycle gene cdc2Pet. Only 2,4-D, and not BA, was able to stimulate PMEK1 gene transcription; thus, the more rapid S-phase induction in 2,4-D-treated protoplasts may be attributable to the activation of this transduction pathway. In contrast, both plant growth regulators were required to induce the appearance of cdc2Pet mRNA transcripts prior to S-phase engagement.

The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S. pombe cdc7p and may be involved in cell division.

We identified an Arabidopsis thaliana gene, AtMAP3Kepsilon1, and a Brassica napus cDNA, BnMAP3Kepsilon1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT-PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kepsilon1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.