Marc Jakoby

FRU (BHLH029) is required for induction of iron mobilization genes in Arabidopsis thaliana

Iron mobilization responses are induced by low iron supply at transcriptional level. In tomato, the basic helix‐loop‐helix gene FER is required for induction of iron mobilization. Using molecular‐genetic techniques, we analyzed the function of BHLH029, named FRU (FER‐like regulator of iron uptake), the Arabidopsis thaliana homolog of the tomato FER gene. The FRU gene was mainly expressed in roots in a cell‐specific pattern and induced by iron deficiency. FRU mutant plants were chlorotic, and the FRU gene was found necessary for induction of the essential iron mobilization genes FRO2 (ferric chelate reductase gene) and IRT1 (iron‐regulated transporter gene). Overexpression of FRU resulted in an increase of iron mobilization responses at low iron supply. Thus, the FRU gene is a mediator in induction of iron mobilization responses in Arabidopsis, indicating that regulation of iron uptake is conserved in dicot species.

A positive signal from the fertilization of the egg cell sets off endosperm proliferation in angiosperm embryogenesis

Double fertilization of the egg cell and the central cell by one sperm cell each produces the diploid embryo and the typically triploid endosperm and is one of the defining characteristics of flowering plants (angiosperms). Endosperm and embryo develop in parallel to form the mature seed, but little is known about the coordination between these two organisms. We characterized a mutation of the Arabidopsis thaliana Cdc2 homolog CDC2A (also called CDKA;1), which has a paternal effect. In cdc2a mutant pollen, only one sperm cell, instead of two, is produced. Mutant pollen is viable but can fertilize only one cell in the embryo sac, allowing for a genetic dissection of the double fertilization process. We observed exclusive fertilization of the egg cell by cdc2a sperm cells. Moreover, we found that unfertilized endosperm developed, suggesting that a previously unrecognized positive signal from the fertilization of the egg cell initiates proliferation of the central cell.

Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana

Basic helix-loop-helix (bHLH) transcription factors represent a family of proteins that contain a bHLH domain, a motif involved in binding DNA. Recently, two groups independently analyzed the BHLH gene family of Arabidopsis thaliana ([Heim et al., 2003][1]; [Toledo-Ortiz et al., 2003][2]). These

Novel Functions of Plant Cyclin-Dependent Kinase Inhibitors, ICK1/KRP1, Can Act Non-Cell-Autonomously and Inhibit Entry into Mitosis

In animals, cyclin-dependent kinase inhibitors (CKIs) are important regulators of cell cycle progression. Recently, putative CKIs were also identified in plants, and in previous studies, Arabidopsis thaliana plants misexpressing CKIs were found to have reduced endoreplication levels and decreased numbers of cells consistent with a function of CKIs in blocking the G1-S cell cycle transition. Here, we demonstrate that at least one inhibitor from Arabidopsis, ICK1/KRP1, can also block entry into mitosis but allows S-phase progression causing endoreplication. Our data suggest that plant CKIs act in a concentration-dependent manner and have an important function in cell proliferation as well as in cell cycle exit and in turning from a mitotic to an endoreplicating cell cycle mode. Endoreplication is usually associated with terminal differentiation; we observed, however, that cell fate specification proceeded independently from ICK1/KRP1-induced endoreplication. Strikingly, we found that endoreplicated cells were able to reenter mitosis, emphasizing the high degree of flexibility of plant cells during development. Moreover, we show that in contrast with animal CDK inhibitors, ICK1/KRP1 can move between cells. On the one hand, this challenges plant cell cycle control with keeping CKIs locally controlled, and on the other hand this provides a possibility of linking cell cycle control in single cells with the supracellular organization of a tissue or an organ.

Iron deficiency-mediated stress regulation of four subgroup Ib BHLH genes in Arabidopsis thaliana

Networks of transcription factors control physiological, developmental and environmental responses. Root iron acquisition responses are controlled by the essential bHLH protein FIT. Recently, two group Ib BHLH genes were reported to be iron deficiency-regulated. Here, we studied expression patterns of these two group Ib BHLH genes and of their two closest homologs to analyze whether their regulation would support a function in iron deficiency responses. We found that BHLH038, BHLH039, BHLH100 and BHLH101 (comprising a subgroup of BHLH Ib genes) were up regulated by iron deficiency in roots and leaves. Single insertion mutants had no visible phenotype and were capable of inducing root iron acquisition responses, presumably due to functional redundancy. Specific metal treatments like nickel, high zinc or high copper resulted in induction of the four BHLH Ib genes whereas high iron, low copper and low zinc repressed gene expression. Induction of the four BHLH Ib genes was also found in multiple iron acquisition mutants including fit. Ectopic activation of FIT did not suppress the four BHLH Ib genes. Split-root analyses using promoter-GUS lines showed that FIT and BHLH100 promoters were controlled by different local and systemic signals involved in their regulation by iron. These results indicated that the four BHLH Ib genes were induced independently from FIT by conditions causing iron deficiency. Taken together, BHLH038, BHLH039, BHLH100 and BHLH101 function differently from FIT and may be involved in mediating a signal related to iron deficiency-induced stress and/or internal iron homeostasis.

Transcriptional Profiling of Mature Arabidopsis Trichomes Reveals That NOECK Encodes the MIXTA-Like Transcriptional Regulator MYB106

Leaf hairs (trichomes) of Arabidopsis (Arabidopsis thaliana) have been extensively used as a model to address general questions in cell and developmental biology. Here, we lay the foundation for a systems-level understanding of the biology of this model cell type by performing genome-wide gene expression analyses. We have identified 3,231 genes that are up-regulated in mature trichomes relative to leaves without trichomes, and we compared wild-type trichomes with two mutants, glabra3 and triptychon, that affect trichome morphology and physiology in contrasting ways. We found that cell wall-related transcripts were particularly overrepresented in trichomes, consistent with their highly elaborated structure. In addition, trichome expression maps revealed high activities of anthocyanin, flavonoid, and glucosinolate pathways, indicative of the roles of trichomes in the biosynthesis of secondary compounds and defense. Interspecies comparisons revealed that Arabidopsis trichomes share many expressed genes with cotton (Gossypium hirsutum) fibers, making them an attractive model to study industrially important fibers. In addition to identifying physiological processes involved in the development of a specific cell type, we also demonstrated the utility of transcript profiling for identifying and analyzing regulatory gene function. One of the genes that are differentially expressed in fibers is the MYB transcription factor GhMYB25. A combination of transcript profiling and map-based cloning revealed that the NOECK gene of Arabidopsis encodes AtMYB106, a MIXTA-like transcription factor and homolog of cotton GhMYB25. However, in contrast to Antirrhinum, in which MIXTA promotes epidermal cell outgrowth, AtMYB106 appears to function as a repressor of cell outgrowth in Arabidopsis.

Construction and application of new Corynebacterium glutamicum vectors

The construction of new Corynebacterium glutamicum/Escherichia coli shuttle vectors with improved cloning properties and an increased chloramphenicol resistance (50 μg ml−1 MIC) is described. A modified glnB gene encoding a Strep-tag II domain modified PII protein was expressed in C. glutamicum and streptavidin affinity chromatography was used to purify this protein.

AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum

The uptake and assimilation of nitrogen sources is effectively regulated in bacteria. In the Gram‐negative enterobacterium Escherichia coli, the NtrB/C two‐component system is responsible for the activation of transcription of different enzymes and transporters, depending on the nitrogen status of the cell. In this study, we investigated regulation of ammonium uptake in Corynebacterium glutamicum, a Gram‐positive soil bacterium closely related to Mycobacterium tuberculosis. As shown by Northern blot hybridizations, regulation occurs on the level of transcription upon nitrogen starvation. In contrast to enterobacteria, a repressor protein is involved in regulation, as revealed by measurements of methylammonium uptake and β‐galactosidase activity in reporter strains. The repressor‐encoding gene, designated amtR, was isolated and sequenced. Deletion of amtR led to deregulation of transcription of amt coding for the C. glutamicum (methyl)ammonium uptake system. E. coli extracts from amtR‐expressing cells were applied in gel retardation experiments, and binding of AmtR to the amt upstream region was observed. By deletion analyses, a target motif for AmtR binding was identified, and binding of purified AmtR protein to this motif, ATCTATAGN1−4ATAG, was shown. Furthermore, the binding of AmtR to this sequence was proven in vivo using a yeast one‐hybrid system. Subsequent studies showed that AmtR not only regulates transcription of the amt gene but also of the amtB–glnK–glnD operon encoding an amt paralogue, the signal transduction protein PII and the uridylyltransferase/uridylyl‐removing enzyme, key components of the nitrogen regulatory cascade. In summary, regulation of ammonium uptake and assimilation in the high G+C content Gram‐positive bacterium C. glutamicum differs significantly from the mechanism found in the low G+C content Gram‐positive model organism Bacillus subtilis and from the paradigm of nitrogen control in the Gram‐negative enterobacteria.

Multiplicity of ammonium uptake systems in Corynebacterium glutamicum : role of Amt and AmtB

In Corynebacterium glutamicum, a Gram-positive soil bacterium widely used in the industrial production of amino acids, two genes encoding (putative) ammonium uptake carriers have been described. The isolation of amt was the first report of the sequence of a gene encoding a bacterial ammonium uptake system combined with the characterization of the corresponding protein. Recently, a second amt gene, amtB, with so far unknown function, was isolated. The isolation of this gene and the suggestion of a new concept for ammonium acquisition prompted the reinvestigation of ammonium transport in C. glutamicum. In this study it is shown that Amt mediates uptake of (methyl)ammonium into the cell with high affinity and strictly depending on the membrane potential. As shown by the determination of K:(m) at different pH values, ammonium/methylammonium, but not ammonia/methylamine, are substrates of Amt. AmtB exclusively accepts ammonium as a transport substrate. In addition, hints of another, until now unknown, low-affinity, ammonium-specific uptake system were found.

Analysis of the Subcellular Localization, Function, and Proteolytic Control of the Arabidopsis Cyclin-Dependent Kinase Inhibitor ICK1/KRP1

Recent studies have shown that cyclin-dependent kinase (CDK) inhibitors can have a tremendous impact on cell cycle progression in plants. In animals, CDK inhibitors are tightly regulated, especially by posttranslational mechanisms of which control of nuclear access and regulation of protein turnover are particularly important. Here we address the posttranslational regulation of INHIBITOR/INTERACTOR OF CDK 1 (ICK1)/KIP RELATED PROTEIN 1 (KRP1), an Arabidopsis (Arabidopsis thaliana) CDK inhibitor. We show that ICK1/KRP1 exerts its function in the nucleus and its presence in the nucleus is controlled by multiple nuclear localization signals as well as by nuclear export. In addition, we show that ICK1/KRP1 localizes to different subnuclear domains, i.e. in the nucleoplasm and to the chromocenters, hinting at specific actions within the nuclear compartment. Localization to the chromocenters is mediated by an N-terminal domain, in addition we find that this domain may be involved in cyclin binding. Further we demonstrate that ICK1/KRP1 is an unstable protein and degraded by the 26S proteasome in the nucleus. This degradation is mediated by at least two domains indicating the presence of at least two different pathways impinging on ICK1/KRP1 protein stability.