SummaryThe Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-speci®c seed proteins. To investigate the interaction between the B3-domain-containing transcription factors FUS3 and ABI3 with the RY cis-motif, conserved in many seed-speci®c promoters, a promoter analysis as well as band-shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY ciselement. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis-motif is a target for both transcription factors. For FUS3, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development.

SUMMARYIn contrast to animals, the life cycle of higher plants alternates between a gamete-producing (gametophyte) and a spore-producing (sporophyte) generation. The female gametophyte of angiosperms consists of four distinct cell types, including two gametes, the egg and the central cell, which give rise to embryo and endosperm, respectively. Based on a combined subtractive hybridization and virtual subtraction approach in wheat (Triticum aestivum L.), we have isolated a class of transcription factors not found in animal genomes, the RKD (RWP-RK domain-containing) factors, which share a highly conserved RWP-RK domain. Single-cell RT-PCR revealed that the genes TaRKD1 and TaRKD2 are preferentially expressed in the egg cell of wheat. The Arabidopsis genome contains five RKD genes, at least two of them, AtRKD1 and AtRKD2, are preferentially expressed in the egg cell of Arabidopsis. Ectopic expression of the AtRKD1 and AtRKD2 genes induces cell proliferation and the expression of an egg cell marker. Analyses of RKD-induced proliferating cells exhibit a shift of gene expression towards an egg cell-like transcriptome. Promoters of selected RKD-induced genes were shown to be predominantly active in the egg cell and can be activated by RKD in a transient protoplast expression assay. The data show that egg cell-specific RKD factors control a transcriptional program, which is characteristic for plant egg cells.

Basic cellular processes such as electron transport in photosynthesis and respiration require the precise control of iron homeostasis. To mobilize iron, plants have evolved at least two different strategies. The nonproteinogenous amino acid nicotianamine which is synthesized from three molecules of S-adenosyl-l-methionine, is an essential component of both pathways. This compound is missing in the tomato mutant chloronerva, which exhibits severe defects in the regulation of iron metabolism. We report the purification and partial characterization of the nicotianamine synthase from barley roots as well as the cloning of two corresponding gene sequences. The function of the gene sequence has been verified by overexpression in Escherichia coli. Further confirmation comes from reduction of the nicotianamine content and the exhibition of a chloronerva-like phenotype due to the expression of heterologous antisense constructs in transgenic tobacco plants. The native enzyme with an apparent M r of < 105 000 probably represents a trimer of S-adenosyl-l-methionine-binding subunits. A comparison with the recently cloned chloronerva gene of tomato reveals striking sequence homology, providing support for the suggestion that the destruction of the nicotianamine synthase encoding gene is the molecular basis of the tomato mutation.Keywords: antisense constructs; chloronerva mutation; gene isolation; Hordeum vulgare; iron metabolism.Iron is essential for fundamental cellular processes such as electron transfer in photosynthesis, respiration, nitrogen fixation as well as DNA synthesis [1]. Excessive accumulation causes severe damage to cellular components due to the formation of highly reactive hydroxyl radicals by the Fenton reaction [2]. Thus, the precise control of iron homeostasis is a basic prerequisite for cellular function. According to WHO data the health of more than three billion people worldwide is affected by iron deficient diet. Crop plants with a higher iron content, for example in the endosperm of cereals, could contribute to the improvement of this situation. In soil iron is mainly found as stable Fe(III) compounds with low solubility at neutral pH [1,3]. Therefore, plants have evolved special mechanisms of iron acquisition, classified into two strategies [4]. Strategy I plants, including dicots and nongraminaceous monocots, facilitate iron uptake mainly by increased acidification of the rhizosphere due to enhanced proton extrusion and the reduction of Fe(III) to Fe(II) by an inducible plasma membrane-bound reductase. In contrast, graminaceous monocots (strategy II plants) release phytosiderophores of the mugineic acid family into the rhizosphere. These compounds act as chelators of ferric ions and are taken up by root cells as Fe(III)-phytosiderophore complexes.The nonproteinogenous amino acid nicotianamine (NA) is found in all multicellular plants [5] and is considered to be a key component for both strategies of iron acquisition (Fig.1). In strategy I plants NA might function as a chelator of iron in symplastic...

Iron homeostasis is vital for many cellular processes and requires a precise regulation. Several iron efficient plants respond to iron starvation with the excretion of riboflavin and other flavins. Basic helix-loop-helix transcription factors (TF) are involved in the regulation of many developmental processes, including iron assimilation. Here we describe the isolation and characterisation of two Arabidopsis bHLH TF genes, which are strongly induced under iron starvation. Their heterologous ectopic expression causes constitutive, iron starvation independent excretion of riboflavin. The results show that both bHLH TFs represent an essential component of the regulatory pathway connecting iron deficiency perception and riboflavin excretion and might act as integrators of various stress reactions.

Seed storage globulins of the 7S and 11S type are synthesized in the seeds of angiosperms and gymnosperms. We have isolated and characterized a vicilin-like gene expressed in the cycad Zamia furfuraceae. Sequence comparisons reveal clear similarities to a sucrose-binding protein isolated from soybean. We suggest the existence of a superfamily of related genes including both vicilin-like and legumin-like seed globulin genes as well as genes coding for spherulins, germins and sucrose-binding-proteins.