Wolfgang Busch

WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators

Plants continuously maintain pools of totipotent stem cells in their apical meristems from which elaborate root and shoot systems are produced. In Arabidopsis thaliana, stem cell fate in the shoot apical meristem is controlled by a regulatory network that includes the CLAVATA (CLV) ligand–receptor system and the homeodomain protein WUSCHEL (WUS). Phytohormones such as auxin and cytokinin are also important for meristem regulation. Here we show a mechanistic link between the CLV/WUS network and hormonal control. WUS, a positive regulator of stem cells, directly represses the transcription of several two-component ARABIDOPSIS RESPONSE REGULATOR genes (ARR5, ARR6, ARR7 and ARR15), which act in the negative-feedback loop of cytokinin signalling. These data indicate that ARR genes might negatively influence meristem size and that their repression by WUS might be necessary for proper meristem function. Consistent with this hypothesis is our observation that a mutant ARR7 allele, which mimics the active, phosphorylated form, causes the formation of aberrant shoot apical meristems. Conversely, a loss-of-function mutation in a maize ARR homologue was recently shown to cause enlarged meristems.

Transcriptional Regulation of ROS Controls Transition from Proliferation to Differentiation in the Root

The balance between cellular proliferation and differentiation is a key aspect of development in multicellular organisms. Using high-resolution expression data from the Arabidopsis root, we identified a transcription factor, UPBEAT1 (UPB1), that regulates this balance. Genomewide expression profiling coupled with ChIP-chip analysis revealed that UPB1 directly regulates the expression of a set of peroxidases that modulate the balance of reactive oxygen species (ROS) between the zones of cell proliferation and the zone of cell elongation where differentiation begins. Disruption of UPB1 activity alters this ROS balance, leading to a delay in the onset of differentiation. Modulation of either ROS balance or peroxidase activity through chemical reagents affects the onset of differentiation in a manner consistent with the postulated UPB1 function. This pathway functions independently of auxin and cytokinin plant hormonal signaling. Comparison to ROS-regulated growth control in animals suggests that a similar mechanism is used in plants and animals.

Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana

The processing of Arabidopsis thaliana microRNAs (miRNAs) from longer primary transcripts (pri-miRNAs) requires the activity of several proteins, including DICER-LIKE1 (DCL1), the double-stranded RNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). It has been noted before that the morphological appearance of weak se mutants is reminiscent of plants with mutations in ABH1/CBP80 and CBP20, which encode the two subunits of the nuclear cap-binding complex. We report that, like SE, the cap-binding complex is necessary for proper processing of pri-miRNAs. Inactivation of either ABH1/CBP80 or CBP20 results in decreased levels of mature miRNAs accompanied by apparent stabilization of pri-miRNAs. Whole-genome tiling array analyses reveal that se, abh1/cbp80, and cbp20 mutants also share similar splicing defects, leading to the accumulation of many partially spliced transcripts. This is unlikely to be an indirect consequence of improper miRNA processing or other mRNA turnover pathways, because introns retained in se, abh1/cbp80, and cbp20 mutants are not affected by mutations in other genes required for miRNA processing or for nonsense-mediated mRNA decay. Taken together, our results uncover dual roles in splicing and miRNA processing that distinguish SE and the cap-binding complex from specialized miRNA processing factors such as DCL1 and HYL1.

The bHLH Transcription Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots

Iron deficiency induces a range of physiological responses that are controlled by transcriptional alterations concentrated in the root pericycle. The transcriptional regulator POPEYE regulates many of these responses possibly through interaction with iron deficiency response protein ILR3 and the putative E3 ligase protein BRUTUS. Global population increases and climate change underscore the need for better comprehension of how plants acquire and process nutrients such as iron. Using cell type–specific transcriptional profiling, we identified a pericycle-specific iron deficiency response and a bHLH transcription factor, POPEYE (PYE), that may play an important role in this response. Functional analysis of PYE suggests that it positively regulates growth and development under iron-deficient conditions. Chromatin immunoprecipitation-on-chip analysis and transcriptional profiling reveal that PYE helps maintain iron homeostasis by regulating the expression of known iron homeostasis genes and other genes involved in transcription, development, and stress response. PYE interacts with PYE homologs, including IAA–Leu Resistant3 (ILR3), another bHLH transcription factor that is involved in metal ion homeostasis. Moreover, ILR3 interacts with a third protein, BRUTUS (BTS), a putative E3 ligase protein, with metal ion binding and DNA binding domains, which negatively regulates the response to iron deficiency. PYE and BTS expression is also tightly coregulated. We propose that interactions among PYE, PYE homologs, and BTS are important for maintaining iron homeostasis under low iron conditions.

Spatiotemporal regulation of cell-cycle genes by SHORTROOT links patterning and growth.

The development of multicellular organisms relies on the coordinated control of cell divisions leading to proper patterning and growth. The molecular mechanisms underlying pattern formation, particularly the regulation of formative cell divisions, remain poorly understood. In Arabidopsis, formative divisions generating the root ground tissue are controlled by SHORTROOT (SHR) and SCARECROW (SCR). Here we show, using cell-type-specific transcriptional effects of SHR and SCR combined with data from chromatin immunoprecipitation-based microarray experiments, that SHR regulates the spatiotemporal activation of specific genes involved in cell division. Coincident with the onset of a specific formative division, SHR and SCR directly activate a D-type cyclin; furthermore, altering the expression of this cyclin resulted in formative division defects. Our results indicate that proper pattern formation is achieved through transcriptional regulation of specific cell-cycle genes in a cell-type- and developmental-stage-specific context. Taken together, we provide evidence for a direct link between developmental regulators, specific components of the cell-cycle machinery and organ patterning.

The Transporter Classification (TC) System, 2002

The Transporter Classification (TC) system is a functional/phylogenetic system designed for the classification of all transmembrane transport proteins found in living organisms on Earth. It parallels but differs from the strictly functional EC system developed decades ago by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB) for the classification of enzymes. Recently, the TC system has been adopted by the IUBMB as the internationally acclaimed system for the classification of transporters. Here we present the characteristics of the nearly 400 families of transport systems included in the TC system and provide statistical analyses of these families and their constituent proteins. Specifically, we analyze the transporter types for size and topological differences and analyze the families for the numbers and organismal sources of their constituent members. We show that channels and carriers exhibit distinctive structural and topological features. Bacterial-specific families outnumber eukaryotic-specific families about 2 to 1, while ubiquitous families, found in all three domains of life, are about half as numerous as eukaryotic-specific families. The results argue against appreciable horizontal transfer of genes encoding transporters between the three domains of life over the last 2 billion years.

Two Families of Mechanosensitive Channel Proteins

SUMMARY Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 α-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.

Transcriptional Control of a Plant Stem Cell Niche

Despite the independent evolution of multicellularity in plants and animals, the basic organization of their stem cell niches is remarkably similar. Here, we report the genome-wide regulatory potential of WUSCHEL, the key transcription factor for stem cell maintenance in the shoot apical meristem of the reference plant Arabidopsis thaliana. WUSCHEL acts by directly binding to at least two distinct DNA motifs in more than 100 target promoters and preferentially affects the expression of genes with roles in hormone signaling, metabolism, and development. Striking examples are the direct transcriptional repression of CLAVATA1, which is part of a negative feedback regulation of WUSCHEL, and the immediate regulation of transcriptional repressors of the TOPLESS family, which are involved in auxin signaling. Our results shed light on the complex transcriptional programs required for the maintenance of a dynamic and essential stem cell niche.

Requirement of B2-Type Cyclin-Dependent Kinases for Meristem Integrity in Arabidopsis thaliana

To maintain proper meristem function, cell division and differentiation must be coordinately regulated in distinct subdomains of the meristem. Although a number of regulators necessary for the correct organization of the shoot apical meristem (SAM) have been identified, it is still largely unknown how their function is integrated with the cell cycle machinery to translate domain identity into correct cellular behavior. We show here that the cyclin-dependent kinases CDKB2;1 and CDKB2;2 are required both for normal cell cycle progression and for meristem organization. Consistently, the CDKB2 genes are highly expressed in the SAM in a cell cycle–dependent fashion, and disruption of CDKB2 function leads to severe meristematic defects. In addition, strong alterations in hormone signaling both at the level of active hormones and with respect to transcriptional and physiological outputs were observed in plants with disturbed CDKB2 activity.

Cell Identity Regulators Link Development and Stress Responses in the Arabidopsis Root

Stress responses in plants are tightly coordinated with developmental processes, but interaction of these pathways is poorly understood. We used genome-wide assays at high spatiotemporal resolution to understand the processes that link development and stress in the Arabidopsis root. Our meta-analysis finds little evidence for a universal stress response. However, common stress responses appear to exist with many showing cell type specificity. Common stress responses may be mediated by cell identity regulators because mutations in these genes resulted in altered responses to stress. Evidence for a direct role for cell identity regulators came from genome-wide binding profiling of the key regulator SCARECROW, which showed binding to regulatory regions of stress-responsive genes. Coexpression in response to stress was used to identify genes involved in specific developmental processes. These results reveal surprising linkages between stress and development at cellular resolution, and show the power of multiple genome-wide data sets to elucidate biological processes.