Andreas Czihal

Iron assimilation and transcription factor controlled synthesis of riboflavin in plants

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.

Members of the RKD transcription factor family induce an egg cell‐like gene expression program

In 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.

Gene Farming in Plants: Expression of a Heatstable Bacillus Amylase in Transgenic Legume Seeds

Summary Seeds of crop plants are promising expression and storage systems for gene farming purposes. We report the generation of transgenic narbon beans (Vicia narbonensis L.) containing the Bacillus licheniformis (α-amylase gene. The transgene is controlled by the seed specific USP gene promoter and encodes a heatstable enzyme. The transgene product accumulates in the storage vacuoles of cotyledon cells and retains its thermostability and starch degradation specificity. The enzyme is able to liquefy the endogenous seed starch reserves of this grain legume.

Regulation and evolution of seed globulin genes

Summary The expression of seed protein genes is precisely regulated in a tissue-specific and development dependent manner mainly as the result of specific interactions between trans-acting factors and corresponding cis-elements. The RY element CATGCATG is one of the best known df-motifs found in the promoters of many seed specific genes. The expression analysis of in vitro generated promoter mutants suggests that mainly the actual nucleotide sequence and not the RY character itself is essential for the function of the motif as a positively acting element of the legumin gene promoter. Southwestern screening of a cotyledon specific expression library resulted in the isolation of several seed promoter binding factors representing new members of the Zn-finger-, bZIP-, RING- and HMG-families. One factor isolated both from Vicia faba and Arabidopsis thaliana contains a typical repeat pattern of amino acids and might represent a novel class of DNA binding factors. Embryos of the conditionally embryo lethal fus3 mutant of Arabidopsis thaliana fail to acquire dormancy and desiccation tolerance. The activity of several seed specific gene promoters depends on an intact FUS 3 gene product. Using transient expression assays in isolated protoplasts we demonstrate that the FUS 3 gene product strongly induces the activity of seed gene promoters. The destruction of the RY motif reduces this inducibility, suggesting that the RY motif is an essential target of the FUS 3 pathway. Based on extensive comparisons of globulin genes we suggest that seed globulins of the legumin and vicilin type are members of a large superfamily of structurally related, functionally distinct proteins and have been recruited from a group of ancient proteins functional in basic cellular desiccation/hydration processes.

Transcriptional regulator AtET2 is required for the induction of dormancy during late seed development

During the late stages of seed development, the embryo patterning program is completed and maturation is initiated. One of the main events during the maturation phase is the acquisition of dormancy, characterized by the failure of a normally developed seed to germinate precociously. Dormancy is controlled by a complex regulatory mechanism that involves the phytohormone gibberellin (GA) and the transcription factor FUSCA3 (FUS3). Here, we demonstrate the importance of the previously characterized GA regulator EFFECTOR OF TRANSCRIPTION2 (AtET2) for correct seed development. We show that entering the maturation phase, seeds of the et2-1 mutant, which contain a non-functional AtET2 gene, fail to induce dormancy. This correlates well with the observed activity pattern of the AtET2 promoter, which is active in the maturing embryo. AtET2 action during seed development is dependent on a complex interaction with GA and the FUS3 gene, the latter evidenced by the phenotypes of the et2-1 fus3-T double mutant. We show that in vitro expressed AtET2 protein can bind to both linear and supercoiled DNA without any obvious sequence preference. This suggests that, within a larger protein complex, AtET2 might be required for the correct positioning upon the DNA.

EFFECTOR OF TRANSCRIPTION factors are novel plant-specific regulators associated with genomic DNA methylation in Arabidopsis

Summary Plant‐specific EFFECTORS OF TRANSCRIPTION (ET) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ETs share a GIY‐YIG domain, involved in DNA nicking activity. It was hypothesised that ETs might act as epigenetic regulators. Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana. Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET‐specific differentially expressed genes and mostly independently characteristic, ET‐specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects. The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair.