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NORMAL GENOMIC

• Zhou X et al.
• A maize jasmonate Zim-domain protein, ZmJAZ14, associates with the JA, ABA, and GA signaling pathways in transgenic Arabidopsis.
• PLoS One. 2015; 10: 121824-121824
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• Jasmonate (JA) is an important signaling molecule involved in the regulation of many physiological and stress-related processes in plants. Jasmonate ZIM-domain (JAZ) proteins have been implicated in regulating JA signaling pathways and the cross talk between various phytohormones. Maize is not only an important cereal crop, but also a model plant for monocotyledon studies. Although many JAZ proteins have been characterized in Arabidopsis and rice, few reports have examined the function of JAZ proteins in maize. In this report, we examined the phylogenetic relationship and expression pattern of JAZ family genes in maize. In addition, a tassel and endosperm-specific JAZ gene, ZmJAZ14, was identified using microarray data analysis and real-time RT-PCR, and its expression was induced by polyethylene glycol (PEG), jasmonate (JA), abscisic acid (ABA), and gibberellins (GAs). ZmJAZ14 was shown to be localized in the nucleus and possessed no transcriptional activating activity, suggesting that it functions as a transcriptional regulator. We found that overexpression of ZmJAZ14 in Arabidopsis enhanced plant tolerance to JA and ABA treatment, as well as PEG stress, while it promoted growth under GA stimulus. Moreover, ZmJAZ14 interacted with a subset of transcription factors in Arabidopsis, and the accumulation of several marker genes involved in JA, ABA, and GA signaling pathways were altered in the overexpression lines. These results suggest that ZmJAZ14 may serve as a hub for the cross talk among the JA, ABA, and GA signaling pathways. Our results can be used to further characterize the function of JAZ family proteins in maize, and the gene cloned in this study may serve as a candidate for drought tolerance and growth promotion regulation in maize.

• K MJ, Laxmi A
• DUF581 is plant specific FCS-like zinc finger involved in protein-protein interaction.
• PLoS One. 2014; 9: 99074-99074
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• Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ) domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.

• Brand LH et al.
• Screening for protein-DNA interactions by automatable DNA-protein interaction ELISA.
• PLoS One. 2013; 8: 75177-75177
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• DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding of gene regulation in any organism of choice.

• Sharma N, Bhalla PL, Singh MB
• Transcriptome-wide profiling and expression analysis of transcription factor families in a liverwort, Marchantia polymorpha.
• BMC Genomics. 2013; 14: 915-915
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• BACKGROUND: Transcription factors (TFs) are vital elements that regulate transcription and the spatio-temporal expression of genes, thereby ensuring the accurate development and functioning of an organism. The identification of TF-encoding genes in a liverwort, Marchantia polymorpha, offers insights into TF organization in the members of the most basal lineages of land plants (embryophytes). Therefore, a comparison of Marchantia TF genes with other land plants (monocots, dicots, bryophytes) and algae (chlorophytes, rhodophytes) provides the most comprehensive view of the rates of expansion or contraction of TF genes in plant evolution. RESULTS: In this study, we report the identification of TF-encoding transcripts in M. polymorpha for the first time, as evidenced by deep RNA sequencing data. In total, 3,471 putative TF encoding transcripts, distributed in 80 families, were identified, representing 7.4% of the generated Marchantia gametophytic transcriptome dataset. Overall, TF basic functions and distribution across families appear to be conserved when compared to other plant species. However, it is of interest to observe the genesis of novel sequences in 24 TF families and the apparent termination of 2 TF families with the emergence of Marchantia. Out of 24 TF families, 6 are known to be associated with plant reproductive development processes. We also examined the expression pattern of these TF-encoding transcripts in six male and female developmental stages in vegetative and reproductive gametophytic tissues of Marchantia. CONCLUSIONS: The analysis highlighted the importance of Marchantia, a model plant system, in an evolutionary context. The dataset generated here provides a scientific resource for TF gene discovery and other comparative evolutionary studies of land plants.

• Wu X, Cheng Y, Li T, Wang Z, Liu JY
• In vitro identification of DNA-binding motif for the new zinc finger protein AtYY1.
• Acta Biochim Biophys Sin (Shanghai). 2012; 44: 483-9
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• The functional characterization of novel transcription factors identified by systematic analysis remains a major challenge due to insufficient data to interpret their specific roles in signaling networks. Here we present a DNA-binding sequence discovery method to in vitro identify a G-rich, 11-bp DNA-binding motif of a novel potential transcription factor AtYY1, a zinc finger protein in Arabidopsis, by using polymerase chain reaction-assisted in vitro selection and surface plasmon resonance analysis. Further mutational analysis of the conserved G bases of the potential motif confirmed that AtYY1 specifically bound to these conserved G sites. Additionally, genome-wide target gene analysis revealed that AtYY1 was involved in diverse cellular pathways, including glucose metabolism, photosynthesis, phototropism, and stress response.

• Shyu C et al.
• JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis.
• Plant Cell. 2012; 24: 536-50
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• The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif-containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors.

• Ismail A, Riemann M, Nick P
• The jasmonate pathway mediates salt tolerance in grapevines.
• J Exp Bot. 2012; 63: 2127-39
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• Salt stress is a major constraint for many crop plants, such as the moderately salt-sensitive economically important fruit crop grapevine. Plants have evolved different strategies for protection against salinity and drought. Jasmonate signalling is a central element of both biotic and abiotic stress responses. To discriminate stress quality, there must be cross-talk with parallel signal chains. Using two grapevine cell lines differing in salt tolerance, the response of jasmonate ZIM/tify-domain (JAZ/TIFY) proteins (negative regulators of jasmonate signalling), a marker for salt adaptation Na(+)/H(+) EXCHANGER (NHX1), and markers for biotic defence STILBENE SYNTHASE (StSy) and RESVERATROL SYNTHASE (RS) were analysed. It is shown that salt stress signalling shares several events with biotic defence including activity of a gadolinium-sensitive calcium influx channel (monitored by apoplastic alkalinization) and transient induction of JAZ/TIFY transcripts. Exogenous jasmonate can rescue growth in the salt-sensitive cell line. Suppression of jasmonate signalling by phenidone or aspirin blocks the induction of JAZ/TIFY transcripts. The rapid induction of RS and StSy characteristic for biotic defence in grapevine is strongly delayed in response to salt stress. In the salt-tolerant line, NHX1 is induced and the formation of reactive oxygen species, monitored as stress markers in the sensitive cell line, is suppressed. The data are discussed in terms of a model where salt stress signalling acts as a default pathway whose readout is modulated by a parallel signal chain triggered by biotic factors downstream of jasmonate signalling.

• Shaikhali J et al.
• The CRYPTOCHROME1-dependent response to excess light is mediated through the transcriptional activators ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 and ZML2 in Arabidopsis.
• Plant Cell. 2012; 24: 3009-25
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• Exposure of plants to light intensities that exceed the electron utilization capacity of the chloroplast has a dramatic impact on nuclear gene expression. The photoreceptor Cryptochrome 1 (cry1) is essential to the induction of genes encoding photoprotective components in Arabidopsis thaliana. Bioinformatic analysis of the cry1 regulon revealed the putative cis-element CryR1 (GnTCKAG), and here we demonstrate an interaction between CryR1 and the zinc finger GATA-type transcription factors ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 (ZML1) and ZML2. The ZML proteins specifically bind to the CryR1 cis-element as demonstrated in vitro and in vivo, and TCTAG was shown to constitute the core sequence required for ZML2 binding. In addition, ZML2 activated transcription of the yellow fluorescent protein reporter gene driven by the CryR1 cis-element in Arabidopsis leaf protoplasts. T-DNA insertion lines for ZML2 and its homolog ZML1 demonstrated misregulation of several cry1-dependent genes in response to excess light. Furthermore, the zml1 and zml2 T-DNA insertion lines displayed a high irradiance-sensitive phenotype with significant photoinactivation of photosystem II (PSII), indicated by reduced maximum quantum efficiency of PSII, and severe photobleaching. Thus, we identified the ZML2 and ZML1 GATA transcription factors as two essential components of the cry1-mediated photoprotective response.

• Bowen AJ, Gonzalez D, Mullins JG, Bhatt AM, Martinez A, Conlan RS
• PAH-domain-specific interactions of the Arabidopsis transcription coregulator SIN3-LIKE1 (SNL1) with telomere-binding protein 1 and ALWAYS EARLY2 Myb-DNA binding factors.
• J Mol Biol. 2010; 395: 937-49
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• The eukaryotic SIN3 protein is the central component of the evolutionarily conserved multisubunit SIN3 complex that has roles in regulating gene expression and genome stability. Here we characterise the structure of the SIN3 protein in higher plants through the analysis of SNL1 (SIN3-LIKE1), SNL2, SNL3, SNL4, SNL5 and SNL6, a family of six SIN3 homologues in Arabidopsis thaliana. In an Arabidopsis-protoplast beta-glucuronidase reporter gene assay, as well as in a heterologous yeast repression assay, full-length SNL1 was shown to repress transcription in a histone-deacetylase-dependent manner, demonstrating the conserved nature of SIN3 function. Yeast two-hybrid screening identified a number of DNA binding proteins each containing a single Myb domain that included the Arabidopsis ALWAYS EARLY proteins AtALY2 and AtALY3, and two telomere binding proteins AtTBP1 and AtTRP2/TRFL1 as SNL1 partners, suggesting potential functions for SNL1 in development and telomere maintenance. The interaction with telomere-binding protein 1 was found to be mediated through the well-defined paired amphipathic helix domain PAH2. In contrast, the AtALY2 interaction was mediated through the PAH3 domain of SNL1, which is structurally distinct from PAH1 and PAH2, suggesting that evolution of this domain to a more novel structural motif has occurred. These findings support a diverse role of SNL1 in the regulation of transcription and genome stability.

• Grunewald W et al.
• Expression of the Arabidopsis jasmonate signalling repressor JAZ1/TIFY10A is stimulated by auxin.
• EMBO Rep. 2009; 10: 923-8
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• Plant hormones have pivotal roles in almost every aspect of plant development. Over the past decades, physiological and genetic studies have revealed that hormone action in plants is determined by complex interactions between hormonal signalling pathways. Evidence is accumulating for the existence of crosstalk between the auxin and jasmonate (JA) signalling pathways. Recently, the JASMONATE ZIM-domain (JAZ) proteins have been identified as the long-sought repressors of JA signalling. Here, we show that expression of JAZ1/TIFY10A is not solely inducible by JA, but that it is also an early auxin-responsive gene. Furthermore, we could show that the auxin-inducible expression of JAZ1/TIFY10A is independent of the JA signalling pathway but is controlled by the auxin/indole-3-acetic acid-auxin response transcription factor signalling pathway. Our results provide evidence for the existence of at least two different input signals regarding JAZ1/TIFY10A expression and thus support the idea of an intimate molecular interplay between auxin and JA signalling.

• Chung HS, Howe GA
• A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis.
• Plant Cell. 2009; 21: 131-45
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• JASMONATE ZIM-domain (JAZ) proteins act as repressors of jasmonate (JA) signaling. Perception of bioactive JAs by the F-box protein CORONATINE INSENSITIVE1 (COI1) causes degradation of JAZs via the ubiquitin-proteasome pathway, which in turn activates the expression of genes involved in plant growth, development, and defense. JAZ proteins contain two highly conserved sequence regions: the Jas domain that interacts with COI1 to destabilize the repressor and the ZIM domain of unknown function. Here, we show that the conserved TIFY motif (TIFF/YXG) within the ZIM domain mediates homo- and heteromeric interactions between most Arabidopsis thaliana JAZs. We have also identified an alternatively spliced form (JAZ10.4) of JAZ10 that lacks the Jas domain and, as a consequence, is highly resistant to JA-induced degradation. Strong JA-insensitive phenotypes conferred by overexpression of JAZ10.4 were suppressed by mutations in the TIFY motif that block JAZ10.4-JAZ interactions. We conclude that JAZ10.4 functions to attenuate signal output in the presence of JA and further suggest that the dominant-negative action of this splice variant involves protein-protein interaction through the ZIM/TIFY domain. The ability of JAZ10.4 to interact with MYC2 is consistent with a model in which a JAZ10.4-containing protein complex directly represses the activity of transcription factors that promote expression of JA response genes.

• Grabowska A, Wisniewska A, Tagashira N, Malepszy S, Filipecki M
• Characterization of CsSEF1 gene encoding putative CCCH-type zinc finger protein expressed during cucumber somatic embryogenesis.
• J Plant Physiol. 2009; 166: 310-23
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• Somatic embryos obtained in vitro are a form of vegetative reproduction that can be used in artificial seed technology, as well as a model to study the principles of plant development. In order to isolate the genes involved in somatic embryogenesis of the cucumber (Cucumis sativus L.), we utilized the suppression subtractive hybridization (SSH). One of the obtained sequences was the CsSEF1 clone (Cucumis sativus Somatic Embryogenesis Zinc Finger 1), with a level of expression that sharply increased with the induction of embryogenesis. The full length cDNA of CsSEF1 encodes the putative 307 amino acid long protein containing three zinc finger motifs, two with CCCH and one with the atypical CHCH pattern. The CsSEF1 protein shows significant similarity to other proteins from plants, in which the zinc fingers arrangement and patterns are very similar. Transcripts of CsSEF1 were localized in the apical part of somatic embryos, starting as early as the polarity was visible and in later developmental stages marking the cotyledon primordia and procambium tissues. As a result of transferring an antisense fragment of CsSEF1 into Arabidopsis thaliana abnormalities in zygotic embryos and also in cotyledons and root development were observed.

• Khanna R et al.
• The Arabidopsis B-box zinc finger family.
• Plant Cell. 2009; 21: 3416-20

• Yan Y et al.
• A downstream mediator in the growth repression limb of the jasmonate pathway.
• Plant Cell. 2007; 19: 2470-83
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• Wounding plant tissues initiates large-scale changes in transcription coupled to growth arrest, allowing resource diversion for defense. These processes are mediated in large part by the potent lipid regulator jasmonic acid (JA). Genes selected from a list of wound-inducible transcripts regulated by the jasmonate pathway were overexpressed in Arabidopsis thaliana, and the transgenic plants were then assayed for sensitivity to methyl jasmonate (MeJA). When grown in the presence of MeJA, the roots of plants overexpressing a gene of unknown function were longer than those of wild-type plants. When transcript levels for this gene, which we named JASMONATE-ASSOCIATED1 (JAS1), were reduced by RNA interference, the plants showed increased sensitivity to MeJA and growth was inhibited. These gain- and loss-of-function assays suggest that this gene acts as a repressor of JA-inhibited growth. An alternative transcript from the gene encoding a second protein isoform with a longer C terminus failed to repress jasmonate sensitivity. This identified a conserved C-terminal sequence in JAS1 and related genes, all of which also contain Zim motifs and many of which are jasmonate-regulated. Both forms of JAS1 were found to localize to the nucleus in transient expression assays. Physiological tests of growth responses after wounding were consistent with the fact that JAS1 is a repressor of JA-regulated growth retardation.

• Barakat A, Muller KF, Saenz-de-Miera LE
• Molecular evolutionary analyses of the Arabidopsis L7 ribosomal protein gene family.
• Gene. 2007; 403: 143-50
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• Cytoplasmic ribosomal protein (r-protein) genes in Arabidopsis thaliana are encoded by 80 multigene families that contain between two and seven members. Gene family members are typically similar at the protein sequence level, with the most divergent members of any gene family retaining 94% identity, on average. However, three Arabidopsis r-protein families - S15a, L7 and P2 - contain highly divergent family members. Here, we investigated the organization, structure, expression and molecular evolution of the L7 r-protein family. Phylogenetic analyses showed that L7 r-protein gene family members constitute two distinct phylogenetic groups. The first group including RPL7B, RPL7C and RPL7D has homologs in plants, animals and fungi. The second group represented by RPL7A is found in plants but has no orthologs from other fully-sequenced eukaryotic genomes. These two groups may have derived from a duplication event prior to the divergence of animals and plants. All four L7 r-protein genes are expressed and all exhibit a differential expression in inflorescence and flowers. RPL7A and RPL7B are less expressed than the other genes in all tissues analyzed. Molecular characterization of nucleic and protein sequences of L7 r-protein genes and analysis of their codon usage did not indicate any functional divergence. The probable evolution of an extra-ribosomal function of group 2 genes is discussed.

• Thines B et al.
• JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.
• Nature. 2007; 448: 661-5
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• Jasmonate and related signalling compounds have a crucial role in both host immunity and development in plants, but the molecular details of the signalling mechanism are poorly understood. Here we identify members of the jasmonate ZIM-domain (JAZ) protein family as key regulators of jasmonate signalling. JAZ1 protein acts to repress transcription of jasmonate-responsive genes. Jasmonate treatment causes JAZ1 degradation and this degradation is dependent on activities of the SCF(COI1) ubiquitin ligase and the 26S proteasome. Furthermore, the jasmonoyl-isoleucine (JA-Ile) conjugate, but not other jasmonate-derivatives such as jasmonate, 12-oxo-phytodienoic acid, or methyl-jasmonate, promotes physical interaction between COI1 and JAZ1 proteins in the absence of other plant proteins. Our results suggest a model in which jasmonate ligands promote the binding of the SCF(COI1) ubiquitin ligase to and subsequent degradation of the JAZ1 repressor protein, and implicate the SCF(COI1)-JAZ1 protein complex as a site of perception of the plant hormone JA-Ile.

• Stephenson TJ, McIntyre CL, Collet C, Xue GP
• Genome-wide identification and expression analysis of the NF-Y family of transcription factors in Triticum aestivum.
• Plant Mol Biol. 2007; 65: 77-92
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• Nuclear Factor Y (NF-Y) is a trimeric complex that binds to the CCAAT box, a ubiquitous eukaryotic promoter element. The three subunits NF-YA, NF-YB and NF-YC are represented by single genes in yeast and mammals. However, in model plant species (Arabidopsis and rice) multiple genes encode each subunit providing the impetus for the investigation of the NF-Y transcription factor family in wheat. A total of 37 NF-Y and Dr1 genes (10 NF-YA, 11 NF-YB, 14 NF-YC and 2 Dr1) in Triticum aestivum were identified in the global DNA databases by computational analysis in this study. Each of the wheat NF-Y subunit families could be further divided into 4-5 clades based on their conserved core region sequences. Several conserved motifs outside of the NF-Y core regions were also identified by comparison of NF-Y members from wheat, rice and Arabidopsis. Quantitative RT-PCR analysis revealed that some of the wheat NF-Y genes were expressed ubiquitously, while others were expressed in an organ-specific manner. In particular, each TaNF-Y subunit family had members that were expressed predominantly in the endosperm. The expression of nine NF-Y and two Dr1 genes in wheat leaves appeared to be responsive to drought stress. Three of these genes were up-regulated under drought conditions, indicating that these members of the NF-Y and Dr1 families are potentially involved in plant drought adaptation. The combined expression and phylogenetic analyses revealed that members within the same phylogenetic clade generally shared a similar expression profile. Organ-specific expression and differential response to drought indicate a plant-specific biological role for various members of this transcription factor family.

• Chini A et al.
• The JAZ family of repressors is the missing link in jasmonate signalling.
• Nature. 2007; 448: 666-71
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• Jasmonates are essential phytohormones for plant development and survival. However, the molecular details of their signalling pathway remain largely unknown. The identification more than a decade ago of COI1 as an F-box protein suggested the existence of a repressor of jasmonate responses that is targeted by the SCF(COI1) complex for proteasome degradation in response to jasmonate. Here we report the identification of JASMONATE-INSENSITIVE 3 (JAI3) and a family of related proteins named JAZ (jasmonate ZIM-domain), in Arabidopsis thaliana. Our results demonstrate that JAI3 and other JAZs are direct targets of the SCF(COI1) E3 ubiquitin ligase and jasmonate treatment induces their proteasome degradation. Moreover, JAI3 negatively regulates the key transcriptional activator of jasmonate responses, MYC2. The JAZ family therefore represents the molecular link between the two previously known steps in the jasmonate pathway. Furthermore, we demonstrate the existence of a regulatory feed-back loop involving MYC2 and JAZ proteins, which provides a mechanistic explanation for the pulsed response to jasmonate and the subsequent desensitization of the cell.

• Bi YM, Wang RL, Zhu T, Rothstein SJ
• Global transcription profiling reveals differential responses to chronic nitrogen stress and putative nitrogen regulatory components in Arabidopsis.
• BMC Genomics. 2007; 8: 281-281
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• BACKGROUND: A large quantity of nitrogen (N) fertilizer is used for crop production to achieve high yields at a significant economic and environmental cost. Efforts have been directed to understanding the molecular basis of plant responses to N and identifying N-responsive genes in order to manipulate their expression, thus enabling plants to use N more efficiently. No studies have yet delineated these responses at the transcriptional level when plants are grown under chronic N stress and the understanding of regulatory elements involved in N response is very limited. RESULTS: To further our understanding of the response of plants to varying N levels, a growth system was developed where N was the growth-limiting factor. An Arabidopsis whole genome microarray was used to evaluate global gene expression under different N conditions. Differentially expressed genes under mild or severe chronic N stress were identified. Mild N stress triggered only a small set of genes significantly different at the transcriptional level, which are largely involved in various stress responses. Plant responses were much more pronounced under severe N stress, involving a large number of genes in many different biological processes. Differentially expressed genes were also identified in response to short- and long-term N availability increases. Putative N regulatory elements were determined along with several previously known motifs involved in the responses to N and carbon availability as well as plant stress. CONCLUSION: Differentially expressed genes identified provide additional insights into the coordination of the complex N responses of plants and the components of the N response mechanism. Putative N regulatory elements were identified to reveal possible new components of the regulatory network for plant N responses. A better understanding of the complex regulatory network for plant N responses will help lead to strategies to improve N use efficiency.

• White DW
• PEAPOD regulates lamina size and curvature in Arabidopsis.
• Proc Natl Acad Sci U S A. 2006; 103: 13238-43
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• Although a complex pattern of interspersed cell proliferation and cell differentiation is known to occur during leaf blade development in eudicot plants, the genetic mechanisms coordinating this growth are unclear. In Arabidopsis, deletion of the PEAPOD (PPD) locus increases leaf lamina size and results in dome-shaped rather than flat leaves. Siliques are also altered in shape because of extra lamina growth. The curvature of a Deltappd leaf reflects the difference between excess growth of the lamina and a limitation to the extension capacity of its perimeter. Excess lamina growth in Deltappd plants is due to a prolonged phase of dispersed meristematic cell (DMC) proliferation (for example, the meristemoid and procambium cells that form stomatal stem cells and vascular cells, respectively) during blade development. The PPD locus is composed of two homologous genes, PPD1 and PPD2, which encode plant-specific putative DNA-binding proteins. Overexpression of PPD reduces lamina size by promoting the early arrest of DMC proliferation during leaf and silique development. Therefore, by regulating the arrest of DMC proliferation, the PPD genes coordinate tissue growth, modulate lamina size, and limit curvature of the leaf blade. I propose a revised model of leaf development with two cell-cycle arrest fronts progressing from the tip to the base: the known primary front, which determines arrest of general cell proliferation, followed by a secondary front that involves PPD and arrests DMC division.

• Sanchez JP, Ullman C, Moore M, Choo Y, Chua NH
• Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A ligase-1 expression by artificial zinc finger chimeras.
• Plant Biotechnol J. 2006; 4: 103-14
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• The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sanchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 1465-1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 (At4CL1) gene in A. thaliana. At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1.

• Yoshizumi T et al.
• Increased level of polyploidy1, a conserved repressor of CYCLINA2 transcription, controls endoreduplication in Arabidopsis.
• Plant Cell. 2006; 18: 2452-68
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• Endoreduplication is a type of cell cycle in which DNA replication continues without cell division. We have isolated several dominant mutants from Arabidopsis thaliana activation tagging lines by flow cytometry. One of the mutants, increased level of polyploidy1-1D (ilp1-1D), showed increased polyploidy in both light- and dark-grown hypocotyls. The corresponding gene of ilp1-1D encodes a protein homologous to the C-terminal region of mammalian GC binding factor. We demonstrate that this protein functions as a transcriptional repressor in vivo. The expression of all members of the CYCLINA2 (CYCA2) family was reduced in an ILP1 overexpressing line, and the mouse (Mus musculus) homolog of ILP1 repressed cyclin A2 expression in mouse NIH3T3 cells. T-DNA insertion mutants of ILP1 showed reduced polyploidy and upregulated all CYCA2 expression. Furthermore, loss of CYCA2;1 expression induces an increase in polyploidy in Arabidopsis. We demonstrate that this protein regulates endoreduplication through control of CYCA2 expression in Arabidopsis.

• Milla MA, Townsend J, Chang IF, Cushman JC
• The Arabidopsis AtDi19 gene family encodes a novel type of Cys2/His2 zinc-finger protein implicated in ABA-independent dehydration, high-salinity stress and light signaling pathways.
• Plant Mol Biol. 2006; 61: 13-30
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• The AtDi19 (drought-induced) gene family encodes seven hydrophilic proteins that contain two atypical Cys2/His2 (C2H2) zinc finger-like domains that are evolutionarily well-conserved within angiosperms suggesting a conserved and important function. Five of the seven Arabidopsis AtDi19-related:DsRed2 fusion proteins exhibited speckled patterns of localization within the nucleus as shown by transient expression analysis in Arabidopsis protoplasts. In contrast, AtDi19-2:DsRed2 was present in the nucleus and cytoplasm, whereas AtDi19-4:DsRed2 was localized to the nuclear periphery. mRNA expression studies showed that AtDi19 genes are ubiquitously expressed in Arabidopsis tissues, although some differences were observed. In seedlings, RT-PCR analyses showed that AtDi19-1 and AtDi19-3 steady-state transcript amounts were rapidly induced by dehydration, whereas transcript amounts for AtDi19-2 and AtDi19-4 increased in response to high-salt stress. In addition, the mRNA abundance of all the AtDi19-related gene family members was not regulated by ABA. These data, taken together, suggest that several AtDi19-related gene family members may function in ABA-independent, dehydration and salinity stress signaling pathways. However, they may also be regulated by other abiotic stimuli. AtDi19-7, for example, has been implicated in regulating light signaling and responses. Finally, we show that most AtDi19-related proteins are phosphorylated in vitro by calcium-dependent protein kinases suggesting that this post-translational modification may be important for regulating the function of this novel protein family.

• AbuQamar S et al.
• Expression profiling and mutant analysis reveals complex regulatory networks involved in Arabidopsis response to Botrytis infection.
• Plant J. 2006; 48: 28-44
• Display abstract

• The expression profiles of Botrytis-inoculated Arabidopsis plants were studied to determine the nature of the defense transcriptome and to identify genes involved in host responses to the pathogen. Normally resistant Arabidopsis wild-type plants were compared with coi1, ein2, and nahG plants that are defective in various defense responses and/or show increased susceptibility to Botrytis. In wild-type plants, the expression of 621 genes representing approximately 0.48% of the Arabidopsis transcriptome was induced greater than or equal to twofold after infection. Of these 621 Botrytis-induced genes (BIGs), 462 were induced at or before 36 h post-inoculation, and may be involved in resistance to the pathogen. The expression of 181 BIGs was dependent on a functional COI1 gene required for jasmonate signaling, whereas the expression of 63 and 80 BIGs were dependent on ethylene (ET) signaling or salicylic acid accumulation, respectively, based on results from ein2 and nahG plants. BIGs encode diverse regulatory and structural proteins implicated in pathogen defense and abiotic and oxidative-stress responses. Thirty BIGs encode putative DNA-binding proteins that belong to ET response, zinc-finger, MYB, WRKY, and HD-ZIP family transcription-factor proteins. Fourteen BIGs were studied in detail to determine their role in resistance to Botrytis. T-DNA insertion alleles of ZFAR1 (At2G40140), the gene encoding a putative zinc-finger protein with ankyrin-repeat domains, showed increased local susceptibility to Botrytis and sensitivity to germination in the presence of abscisic acid (ABA), supporting the role of ABA in mediating responses to Botrytis infection. In addition, two independent T-DNA insertion alleles in the WRKY70 gene showed increased susceptibility to Botrytis. The transcriptional activation of genes involved in plant hormone signaling and synthesis, removal of reactive oxygen species, and defense and abiotic-stress responses, coupled with the susceptibility of the wrky70 and zfar1 mutants, highlights the complex genetic network underlying defense responses to Botrytis in Arabidopsis.

• Hsieh MH, Goodman HM
• A novel gene family in Arabidopsis encoding putative heptahelical transmembrane proteins homologous to human adiponectin receptors and progestin receptors.
• J Exp Bot. 2005; 56: 3137-47
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• A novel seven-transmembrane receptor family, that is comprised of human adiponectin receptors (AdipoRs) and membrane progestin receptors (mPRs) that share little sequence homology with all known G protein-coupled receptors (GPCRs), has been identified recently. Although a fish mPR has been suggested to be a GPCR, human AdipoRs seem to be structurally and functionally distinct from all known GPCRs. The identification of a novel gene family, the heptahelical protein (HHP) gene family, encoding proteins in Arabidopsis predicted to have a heptahelical transmembrane topology is reported here. There are at least five HHP genes in Arabidopsis whose encoded amino acid sequences have significant similarities to human AdipoRs and mPRs. The expression and regulation of the Arabidopsis HHP gene family has been studied here. The expression of the HHP gene family is differentially regulated by plant hormones. Steady-state levels of HHP1 mRNA are increased by treatments with abscisic acid and gibberellic acid, whereas levels of HHP2 mRNA are increased by abscisic acid and benzyladenine treatments. In addition, the expression of the HHP gene family is up-regulated by the presence of sucrose in the medium. Temperature and salt stress treatments also differentially affect the expression of the HHP genes. These novel seven-transmembrane proteins previously described in yeast and animals, and now identified in plants, may represent a new class of receptors that are highly conserved across kingdoms.

• Phelps-Durr TL, Thomas J, Vahab P, Timmermans MC
• Maize rough sheath2 and its Arabidopsis orthologue ASYMMETRIC LEAVES1 interact with HIRA, a predicted histone chaperone, to maintain knox gene silencing and determinacy during organogenesis.
• Plant Cell. 2005; 17: 2886-98
• Display abstract

• Plant shoots are characterized by indeterminate growth resulting from the action of a population of stem cells in the shoot apical meristem (SAM). Indeterminacy within the SAM is specified in part by the class I knox homeobox genes. The myb domain proteins rough sheath2 (RS2) and ASYMMETRIC LEAVES1 (AS1) from maize (Zea mays) and Arabidopsis thaliana, respectively, are required to establish determinacy during leaf development. These proteins are part of a cellular memory system that in response to a stem cell-derived signal keeps knox genes in an off state during organogenesis. Here, we show that RS2/AS1 can form conserved protein complexes through interaction with the DNA binding factor ASYMMETRIC LEAVES2, a predicted RNA binding protein (RIK, for RS2-Interacting KH protein), and a homologue of the chromatin-remodeling protein HIRA. Partial loss of HIRA function in Arabidopsis results in developmental defects comparable to those of as1 and causes reactivation of knox genes in developing leaves, demonstrating a direct role for HIRA in knox gene repression and the establishment of determinacy during leaf formation. Our data suggest that RS2/AS1 and HIRA mediate the epigenetic silencing of knox genes, possibly by modulating chromatin structure. Components of this process are conserved in animals, suggesting the possibility that a similar epigenetic mechanism maintains determinacy during both plant and animal development.

• Wu KL, Guo ZJ, Wang HH, Li J
• The WRKY family of transcription factors in rice and Arabidopsis and their origins.
• DNA Res. 2005; 12: 9-26
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• WRKY transcription factors, originally isolated from plants contain one or two conserved WRKY domains, about 60 amino acid residues with the WRKYGQK sequence followed by a C2H2 or C2HC zinc finger motif. Evidence is accumulating to suggest that the WRKY proteins play significant roles in responses to biotic and abiotic stresses, and in development. In this research, we identified 102 putative WRKY genes from the rice genome and compared them with those from Arabidopsis. The WRKY genes from rice and Arabidopsis were divided into three groups with several subgroups on the basis of phylogenies and the basic structure of the WRKY domains (WDs). The phylogenetic trees generated from the WDs and the genes indicate that the WRKY gene family arose during evolution through duplication and that the dramatic amplification of rice WRKY genes in group III is due to tandem and segmental gene duplication compared with those of Arabidopsis. The result suggests that some of the rice WRKY genes in group III are evolutionarily more active than those in Arabidopsis, and may have specific roles in monocotyledonous plants. Further, it was possible to identify the presence of WRKY-like genes in protists (Giardia lamblia and Dictyostelium discoideum) and green algae Chlamydomonas reinhardtii through database research, demonstrating the ancient origin of the gene family. The results obtained by alignments of the WDs from different species and other analysis imply that domain gain and loss is a divergent force for expansion of the WRKY gene family, and that a rapid amplification of the WRKY genes predate the divergence of monocots and dicots. On the basis of these results, we believe that genes encoding a single WD may have been derived from the C-terminal WD of the genes harboring two WDs. The conserved intron splicing positions in the WDs of higher plants offer clues about WRKY gene evolution, annotation, and classification.

• Vogel JT, Zarka DG, Van Buskirk HA, Fowler SG, Thomashow MF
• Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis.
• Plant J. 2005; 41: 195-211
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• Summary The CBF cold response pathway has a prominent role in cold acclimation. The pathway includes action of three transcription factors, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), that are rapidly induced in response to low temperature followed by expression of the CBF-targeted genes (the CBF regulon) that act in concert to increase plant-freezing tolerance. The results of transcriptome profiling and mutagenesis experiments, however, indicate that additional cold response pathways exist and may have important roles in life at low temperature. To further understand the roles that the CBF proteins play in configuring the low temperature transcriptome and to identify additional transcription factors with roles in cold acclimation, we used the Affymetrix GeneChip containing probe sets for approximately 24,000 Arabidopsis genes to define a core set of cold-responsive genes and to determine which genes were targets of CBF2 and 6 other transcription factors that appeared to be coordinately regulated with CBF2. A total of 514 genes were placed in the core set of cold-responsive genes, 302 of which were upregulated and 212 downregulated. Hierarchical clustering and bioinformatic analysis indicated that the 514 cold-responsive transcripts could be assigned to one of seven distinct expression classes and identified multiple potential novel cis-acting cold-regulatory elements. Eighty-five cold-induced genes and eight cold-repressed genes were assigned to the CBF2 regulon. An additional nine cold-induced genes and 15 cold-repressed genes were assigned to a regulon controlled by ZAT12. Of the 25 core cold-induced genes that were most highly upregulated (induced over 15-fold), 19 genes (84%) were induced by CBF2 and another two genes (8%) were regulated by both CBF2 and ZAT12. Thus, the large majority (92%) of the most highly induced genes belong to the CBF and ZAT12 regulons. Constitutive expression of ZAT12 in Arabidopsis caused a small, but reproducible, increase in freezing tolerance, indicating a role for the ZAT12 regulon in cold acclimation. In addition, ZAT12 downregulated the expression of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that dampens expression of the CBF cold response pathway.

• Nole-Wilson S, Tranby TL, Krizek BA
• AINTEGUMENTA-like (AIL) genes are expressed in young tissues and may specify meristematic or division-competent states.
• Plant Mol Biol. 2005; 57: 613-28
• Display abstract

• Although several members of the AP2/ERF family of transcription factors are important developmental regulators in plants, many genes in this large protein family remain uncharacterized. Here, we present a phylogenetic analysis of the 18 genes that make up the AP2 subgroup of this family. We report expression analyses of seven Arabidopsis genes most closely related to the floral development gene AINTEGUMENTA (ANT) and show that all AINTEGUMENTA-like (AIL) genes are transcribed in multiple tissues during development. They are expressed primarily in young actively dividing tissues of a plant and not in mature leaves or stems. The spatial distribution of AIL5, AIL6, and AIL7 mRNA in inflorescences was characterized by in situ hybridization. Each of these genes is expressed in a spatially and temporally distinct pattern within inflorescence meristems and flowers. Ectopic expression of AIL5 resulted in a larger floral organ phenotype, similar to that resulting from ectopic expression of ANT. Our results are consistent with AIL genes having roles in specification of meristematic or division-competent states.

• Wang JY, Yin WL, Xia XL
• [Cloning and structure analysis of zinc finger protein gene in Populus euphratica Oliv].
• Yi Chuan. 2005; 27: 245-8
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• Zinc finger proteins belong to a family of nuclear transcription factors which function is to regulate gene expression in both prokaryotic and eukaryotic cells. A pair of primers was designed after analyzing the conservation of salt-tolerant zinc protein Alfin-1 in such diverse plants as alfalfa and Arabidopsis. The zinc finger protein gene is isolated from total RNA with RT-PCR in aquaculture leaves of Populus euphratica . Its full cDNA length is 924bp. Analysis of its amino acid sequence showed it has a typical Cys(2)/His(2) zinc finger structure and a G-rich promoter binding site GTGGGG, starting from position 556. Since transcrptional factors which have the same function show conservation in structure and amino acid sequence of DNA binding region, the structure analysis in this paper indicates the cloned zinc finger protein gene may have functional correlation to Alfin-1.

• Yamasaki K et al.
• A novel zinc-binding motif revealed by solution structures of DNA-binding domains of Arabidopsis SBP-family transcription factors.
• J Mol Biol. 2004; 337: 49-63
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• SQUAMOSA promoter binding proteins (SBPs) form a major family of plant-specific transcription factors related to flower development. Although SBPs are heterogeneous in primary structure, they share a highly conserved DNA-binding domain (DBD) that has been suggested to be zinc binding. Here we report the NMR solution structures of DBDs of two SBPs of Arabidopsis thaliana, SPL4 and SPL7. The two share essentially the same structural features. Each structure contains two zinc-binding sites consisting of eight Cys or His residues in a Cys3HisCys2HisCys or Cys6HisCys sequence motif in which the first four residues coordinate to one zinc and the last four coordinate to the other. These structures are dissimilar to other known zinc-binding structures, and thus represent a novel type of zinc-binding motif. The electrostatic profile on the surface suggested that a continuous region, including all the conserved basic residues, is involved in the DNA binding, the mode of which is likely to be novel as well.

• Narita NN et al.
• Overexpression of a novel small peptide ROTUNDIFOLIA4 decreases cell proliferation and alters leaf shape in Arabidopsis thaliana.
• Plant J. 2004; 38: 699-713
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• Leaf shape is determined by polar cell expansion and polar cell proliferation along the leaf axes. However, the genes controlling polar cell proliferation during leaf morphogenesis are largely unknown. We identified a dominant mutant of Arabidopsis thaliana, rotundifolia4-1D (rot4-1D), which possessed short leaves and floral organs. We showed that the altered leaf shape is caused by reduced cell proliferation, specifically in the longitudinal (proximal-distal) axis of the leaf, suggesting that the ROT4 gene controls polar cell proliferation in lateral organs. The ROT4 open-reading frame (ORF) encodes a novel small peptide that had not been identified in the Arabidopsis genome annotation. Overexpression of a ROT4-green fluorescence protein (GFP) fusion protein in transgenic plants recapitulated the rot4 phenotype, suggesting that ROT4 acts to restrict cell proliferation. The ROT4-GFP fusion protein localized to the plasma membrane when expressed in transgenic Arabidopsis plants. Phylogenetic analysis indicates that ROT4 defines a novel seed plant-specific family of small peptides with 22 members in Arabidopsis, ROT FOUR LIKE1-22 (RTFL1-22). All RTFL members share a conserved 29-amino acid domain, the RTF domain, and overexpression of the ROT4 RTF domain alone is sufficient to confer a rot4-1D phenotype. Loss-of-function mutations in several RTFL genes were aphenotypic, suggesting that there may be some functional redundancy between family members. Analyses by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that ROT4 is expressed in the shoot apex and young leaves of wild-type plants, consistent with a role for ROT4 in controlling polarity-dependent cell proliferation during wild-type leaf morphogenesis.

• Lin R, Wang H
• Arabidopsis FHY3/FAR1 gene family and distinct roles of its members in light control of Arabidopsis development.
• Plant Physiol. 2004; 136: 4010-22
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• FHY3 (far-red elongated hypocotyls 3) and FAR1 (far-red-impaired response) are two homologous proteins essential for phytochrome A controlled far-red responses in Arabidopsis (Arabidopsis thaliana). There are 12 additional FHY3/FAR1-related genes in the Arabidopsis genome. The predicted sizes of this family of proteins range from 531 amino acids to 851 amino acids, and they share 12.0% to 82.4% amino acid identities over their entire lengths. In addition, most FRS proteins contain one to three coiled-coil domains and one or two putative nuclear localization signals. Semiquantitative reverse transcription-polymerase chain reaction analyses revealed that all FRS genes except FRS10 are expressed in all tissues examined, including rosette leaves, cauline leaves, inflorescence stems, flowers, and siliques. Analyses of gene specific promoterGUS fusion reporter gene expression revealed that all FRS genes except FRS1 are expressed in hypocotyls, and their expression in hypocotyl is induced by far-red light treatment. Transient expression of green fluorescent protein tagged FRS fusion proteins in onion (Allium cepa) epidermal cells revealed that all FRS proteins are targeted into the nucleus. T-DNA knockout frs6 and frs8 mutants flowered early under both long-day and short-day conditions (with much more drastic effects under short-day conditions), suggesting that FRS6 and FRS8 regulate flowering time. In addition, FRS9 RNAi transgenic plants showed a specific hypersensitivity to red light inhibition of hypocotyl elongation and light-regulated gene expression, indicating that FRS9 is a specific negative regulator of phyB signaling mediating seedling deetiolation. In summary, our results support the notion that FRS family members play distinct roles in light control of Arabidopsis development, most likely by regulating nuclear gene expression.

• Jang S, Hur J, Kim SJ, Han MJ, Kim SR, An G
• Ectopic expression of OsYAB1 causes extra stamens and carpels in rice.
• Plant Mol Biol. 2004; 56: 133-43
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• Members in the YABBY gene family of proteins are plant-specific transcription factors that play critical roles in determining organ polarity. We have isolated a cDNA clone from rice that encodes a YABBY protein. This protein, OsYAB1, is similar to Arabidopsis YAB2 (50.3%) and YAB5 (47.6%). It carries a zinc-finger motif and a YABBY domain, as do those in Arabidopsis . A fusion protein between OsYAB1 and GFP is located in the nucleus. RNA gel-blot analysis showed that the OsYAB1 gene is preferentially expressed in flowers. In-situ hybridization experiments also indicated that the transcript accumulated in the stamen and carpel primordia. Unlike the Arabidopsis YABBY genes, however, the OsYAB1 gene does not show polar expression pattern in the tissues of floral organs. Our transgenic plants that ectopically expressed OsYAB1 were normal during the vegetative growth period, but then showed abnormalities in their floral structures. Spikelets contained supernumerary stamens and carpels compared with those of the wild types. These results suggest that OsYAB1 plays a major role in meristem development and maintenance of stamens and carpels, rather than in determining polarity.

• Feng Y, Liu QP, Xue QZ
• [Comparative phylogenetic analysis of the rice and Arabidopsis PHD-finger proteins].
• Yi Chuan Xue Bao. 2004; 31: 1284-93
• Display abstract

• PHD-finger (plant homeodomain finger), a unique Cys4-His-Cys3 pattern zinc-containing domain, is widely found in many transcriptional regulation proteins from plants or animals. This protein has many different family members distributing in plant or animal proteome,and plays key roles in the development. Here, we identified 44 different putative PHD-finger proteins in the rice (Oryza sativa) genome and performed a phylogenetic analysis with 45 Arabidopsis thaliana proteins. The analysis led to a classification of the 89 proteins into two major groups based on the number of PHD-fingers. Phylogentic analysis of these proteins indicated that there are common ancestors in both plant and human genomes, from which two or three PHD-finger containing proteins might duplicate.

• Jakoby M, Wang HY, Reidt W, Weisshaar B, Bauer P
• FRU (BHLH029) is required for induction of iron mobilization genes in Arabidopsis thaliana.
• FEBS Lett. 2004; 577: 528-34
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• 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.

• Reyes JC, Muro-Pastor MI, Florencio FJ
• The GATA family of transcription factors in Arabidopsis and rice.
• Plant Physiol. 2004; 134: 1718-32
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• GATA transcription factors are a group of DNA binding proteins broadly distributed in eukaryotes. The GATA factors DNA binding domain is a class IV zinc finger motif in the form CX(2)CX(17-20)CX(2)C followed by a basic region. In plants, GATA DNA motifs have been implicated in light-dependent and nitrate-dependent control of transcription. Herein, we show that the Arabidopsis and the rice (Oryza sativa) genomes present 29 and 28 loci, respectively, that encode for putative GATA factors. A phylogenetic analysis of the 57 GATA factors encoding genes, as well as the study of their intron-exon structure, indicates the existence of seven subfamilies of GATA genes. Some of these subfamilies are represented in both species but others are exclusive for one of them. In addition to the GATA zinc finger motif, polypeptides of the different subfamilies are characterized by the presence of additional domains such as an acidic domain, a CCT (CONSTANS, CO-like, and TOC1) domain, or a transposase-like domain also found in FAR1 and FHY3. Subfamily VI comprises genes that encode putative bi-zinc finger polypeptides, also found in metazoan and fungi, and a tri-zinc finger protein which has not been previously reported in eukaryotes. The phylogeny of the GATA zinc finger motif, excluding flanking regions, evidenced the existence of four classes of GATA zinc fingers, three of them containing 18 residues in the zinc finger loop and one containing a 20-residue loop. Our results support multiple models of evolution of the GATA gene family in plants including gene duplication and exon shuffling.

• Gutierrez RA, Green PJ, Keegstra K, Ohlrogge JB
• Phylogenetic profiling of the Arabidopsis thaliana proteome: what proteins distinguish plants from other organisms?
• Genome Biol. 2004; 5: 53-53
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• BACKGROUND: The availability of the complete genome sequence of Arabidopsis thaliana together with those of other organisms provides an opportunity to decipher the genetic factors that define plant form and function. To begin this task, we have classified the nuclear protein-coding genes of Arabidopsis thaliana on the basis of their pattern of sequence similarity to organisms across the three domains of life. RESULTS: We identified 3,848 Arabidopsis proteins that are likely to be found solely within the plant lineage. More than half of these plant-specific proteins are of unknown function, emphasizing the general lack of knowledge of processes unique to plants. Plant-specific proteins that are membrane-associated and/or targeted to the mitochondria or chloroplasts are the most poorly characterized. Analyses of microarray data indicate that genes coding for plant-specific proteins, but not evolutionarily conserved proteins, are more likely to be expressed in an organ-specific manner. A large proportion (13%) of plant-specific proteins are transcription factors, whereas other basic cellular processes are under-represented, suggesting that evolution of plant-specific control of gene expression contributed to making plants different from other eukaryotes. CONCLUSIONS: We identified and characterized the Arabidopsis proteins that are most likely to be plant-specific. Our results provide a genome-wide assessment that supports the hypothesis that evolution of higher plant complexity and diversity is related to the evolution of regulatory mechanisms. Because proteins that are unique to the green plant lineage will not be studied in other model systems, they should be attractive priorities for future studies.

• Shikata M et al.
• Characterization of Arabidopsis ZIM, a member of a novel plant-specific GATA factor gene family.
• J Exp Bot. 2004; 55: 631-9
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• The Arabidopsis gene ZIM encodes a putative transcription factor containing a novel GATA-type zinc-finger domain with a longer spacer between its two sets of conserved cysteine residues (C-X2-C-X20-C-X2-C). In Arabidopsis, ZIM and homologous proteins, ZML1 and ZML2, were identified as GATA factors containing the C-X2-C-X20-C-X2-C motif, a CCT domain, and an uncharacterized conserved domain. Proteins that possess this domain structure were found exclusively in plants, indicating that they belong to a novel family of plant-specific GATA-type transcription factors. When ZIM was overexpressed using a CaMV 35S promoter in Arabidopsis, hypocotyls and petioles were elongated. The elongation phenotype was observed under all wavelengths of light tested and even in the presence of biosynthetic inhibitors of either brassinosteroid or gibberellin. In ZIM-overexpressing plants, XTH33 which is predicted to function in cell wall modification was detected as an up-regulated gene by microarray analysis, and this could account for the elongation phenotype. Genes in ZIM-overexpressing plants were identified that were up-regulated in a tissue-specific manner, which suggests that transcriptional regulation by ZIM and its consequent effects are spatially controlled.

• Takeda S, Matsumoto N, Okada K
• RABBIT EARS, encoding a SUPERMAN-like zinc finger protein, regulates petal development in Arabidopsis thaliana.
• Development. 2004; 131: 425-34
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• Floral organs usually initiate at fixed positions in concentric whorls within a flower. Although it is understood that floral homeotic genes determine the identity of floral organs, the mechanisms of position determination and the development of each organ have not been clearly explained. We isolated a novel mutant, rabbit ears (rbe), with defects in petal development. In rbe, under-developed petals are formed at the correct position in a flower, and the initiation of petal primordia is altered. The rbe mutation affects the second whorl organ shapes independently of the organ identity. RBE encodes a SUPERMAN-like protein and is located in the nucleus, and thus may be a transcription factor. RBE transcripts are expressed in petal primordia and their precursor cells, and disappeared at later stages. When cells that express RBE are ablated genetically, no petal primordia arise. RBE is not expressed in ap1-1 and ptl-1 mutants, indicating that RBE acts downstream of AP1 and PTL genes. These characteristics suggest that RBE is required for the early development of the organ primordia of the second whorl.

• Wen J, Lease KA, Walker JC
• DVL, a novel class of small polypeptides: overexpression alters Arabidopsis development.
• Plant J. 2004; 37: 668-77
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• Small polypeptides can act as important regulatory molecules that coordinate cellular responses required for differentiation, growth, and development. In a gain-of-function genetic screen for genes that influence fruit development in Arabidopsis, we identified a novel gene -DEVIL1 (DVL1) - encoding a small protein. Overexpression of DVL1 results in pleiotropic phenotypes featured by shortened stature, rounder rosette leaves, clustered inflorescences, shortened pedicles, and siliques with pronged tips. cDNA analysis indicates that DVL1 has a 153-nucleotide (nt) open-reading frame (ORF) encoding a 51-amino acid polypeptide that shares no significant similarity to previously identified proteins. Sequence alignment shows that DVL1 belongs to a family of related genes that are limited to angiosperm plants. Ectopic overexpression of each of the five closely related Arabidopsis DVL genes causes similar phenotypic changes, suggesting overlapping function in the DVL gene family. Point mutations of conserved amino acids in the C-terminal region of the DVL1 polypeptide reveal that these conserved residues are required for DVL1-overexpression phenotypes. Our results show that the DVL family is a novel class of small polypeptides and the overexpression phenotypes suggest that these polypeptides may have a role in plant development.

• Belenghi B et al.
• AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death.
• Eur J Biochem. 2003; 270: 2593-604
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• In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (Kd = 4.0 x 10-2 micro m, at pH 7.0 and 25 degrees C), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana. In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.

• Nomura T, Bishop GJ, Kaneta T, Reid JB, Chory J, Yokota T
• The LKA gene is a BRASSINOSTEROID INSENSITIVE 1 homolog of pea.
• Plant J. 2003; 36: 291-300
• Display abstract

• Brassinosteroids (BRs) are growth-promoting plant steroid hormones, and in garden pea (Pisum sativum L.), the lka mutant is defective in BR perception. Here, we show that LKA encodes P. sativum BRI1 (PsBRI1), a homolog of BRI1, which is the Arabidopsis leucine-rich repeat receptor-like kinase/BR receptor. PsBRI1 was isolated by screening a pea cDNA library using Arabidopsis BRI1 cDNA as the probe. PsBRI1 is predicted to encode a 1188-amino-acid protein that has 78% similarity with Arabidopsis BRI1. Sequence analysis of PsBRI1 in the lka mutant led to the identification of a missense mutation that converts the highly conserved aspartic acid residue to asparagine, which is located in the leucine-rich repeat, just before the island domain that may bind BR or a BR-protein complex. The mutation identified in PsBRI1 co-segregated with the semi-erectoide lka phenotype. Transcript analysis of LKA/PsBRI1 indicates that it is ubiquitously expressed in pea and that the expression was downregulated by exogenous BR. The lka mutant was then utilized in further studies to analyze the independent actions of BR and gibberellin (GA) through the characterization of BR response on GA mutants and GA response on BR mutants.

• Yan W, Burns KH, Ma L, Matzuk MM
• Identification of Zfp393, a germ cell-specific gene encoding a novel zinc finger protein.
• Mech Dev. 2002; 118: 233-9
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• Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) which were unique to ovary, testis, and egg libraries. The full-length cDNA of this transcript was deduced and further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA encodes a novel protein of 341 amino acids with a nuclear localization signal. The carboxyl-terminus of the protein contains three C2H2 zinc fingers, and the NH(2)-terminus is proline and serine-rich. Based on the conserved zinc finger motifs, we have termed this novel protein as zinc finger protein 393 (ZFP393). Northern blot and RT-PCR analyses revealed that Zfp393 mRNA was exclusively expressed in testis and ovary. The expression sites were further localized by in situ hybridization to step 3-8 spermatids in testis and growing oocytes in ovary. The Zfp393 gene consists of three exons spanning approximately 8 kb on the distal part of mouse chromosome 4. The carboxyl-terminal zinc finger region is highly homologous to several zinc finger-containing proteins, but no proteins were found to share sequence similarity with the NH(2)-terminal region of ZFP393. Genomic database mining and Southern blot analysis indicate that Zfp393 is a single copy gene. We hypothesize that ZFP393 functions as a germ cell-specific transcription factor that plays important roles in spermatid differentiation and oocyte development.

• Aubert D et al.
• EMF1, a novel protein involved in the control of shoot architecture and flowering in Arabidopsis.
• Plant Cell. 2001; 13: 1865-75
• Display abstract

• Shoot architecture and flowering time in angiosperms depend on the balanced expression of a large number of flowering time and flower meristem identity genes. Loss-of-function mutations in the Arabidopsis EMBRYONIC FLOWER (EMF) genes cause Arabidopsis to eliminate rosette shoot growth and transform the apical meristem from indeterminate to determinate growth by producing a single terminal flower on all nodes. We have identified the EMF1 gene by positional cloning. The deduced polypeptide has no homology with any protein of known function except a putative protein in the rice genome with which EMF1 shares common motifs that include nuclear localization signals, P-loop, and LXXLL elements. Alteration of EMF1 expression in transgenic plants caused progressive changes in flowering time, shoot determinacy, and inflorescence architecture. EMF1 and its related sequence may belong to a new class of proteins that function as transcriptional regulators of phase transition during shoot development.

• Robson F et al.
• Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants.
• Plant J. 2001; 28: 619-31
• Display abstract

• CONSTANS promotes flowering of Arabidopsis in response to long-day conditions. We show that CONSTANS is a member of an Arabidopsis gene family that comprises 16 other members. The CO-Like proteins encoded by these genes contain two segments of homology: a zinc finger containing region near their amino terminus and a CCT (CO, CO-Like, TOC1) domain near their carboxy terminus. Analysis of seven classical co mutant alleles demonstrated that the mutations all occur within either the zinc finger region or the CCT domain, confirming that the two regions of homology are important for CO function. The zinc fingers are most similar to those of B-boxes, which act as protein-protein interaction domains in several transcription factors described in animals. Segments of CO protein containing the CCT domain localize GFP to the nucleus, but one mutation that affects the CCT domain delays flowering without affecting the nuclear localization function, suggesting that this domain has additional functions. All eight co alleles, including one recovered by pollen irradiation in which DNA encoding both B-boxes is deleted, are shown to be semidominant. This dominance appears to be largely due to a reduction in CO dosage in the heterozygous plants. However, some alleles may also actively delay flowering, because overexpression from the CaMV 35S promoter of the co-3 allele, that has a mutation in the second B-box, delayed flowering of wild-type plants. The significance of these observations for the role of CO in the control of flowering time is discussed.

• Conlon H, Zadra I, Haas H, Arst HN Jr, Jones MG, Caddick MX
• The Aspergillus nidulans GATA transcription factor gene areB encodes at least three proteins and features three classes of mutation.
• Mol Microbiol. 2001; 40: 361-75
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• In Aspergillus nidulans, the principal transcription factor regulating nitrogen metabolism, AREA, belongs to the GATA family of DNA-binding proteins. In seeking additional GATA factors, we have cloned areB, which was originally identified via a genetic screen for suppressors of areA loss-of-function mutations. Based on our analysis, areB is predicted to encode at least three distinct protein products. These arise from the use of two promoters, differential splicing and translation initiating at AUG and non-AUG start codons. All the putative products include a GATA domain and a putative Leu zipper. These regions show strong sequence similarity to regulatory proteins from Saccharomyces cerevisiae (Dal80p and Gzf3p), Penicillium chrysogenum (NREB) and Neurospora crassa (ASD4). We have characterized three classes of mutation in areB; the first are loss-of-function mutations that terminate the polypeptides within or before the GATA domain. The second class truncates the GATA factor either within or upstream of the putative Leu zipper but retains the GATA domain. The third class fuses novel gene sequences to areB with the potential to produce putative chimeric polypeptides. These novel gene fusions transform the putative negative-acting transcription factor into an activator that can partially replace areA.

• Lowry JA, Atchley WR
• Molecular evolution of the GATA family of transcription factors: conservation within the DNA-binding domain.
• J Mol Evol. 2000; 50: 103-15
• Display abstract

• The GATA-binding transcription factors comprise a protein family whose members contain either one or two highly conserved zinc finger DNA-binding domains. Members of this group have been identified in organisms ranging from cellular slime mold to vertebrates, including plants, fungi, nematodes, insects, and echinoderms. While much work has been done describing the expression patterns, functional aspects, and target genes for many of these proteins, an evolutionary analysis of the entire family has been lacking. Herein we show that only the C-terminal zinc finger (Cf) and basic domain, which together constitute the GATA-binding domain, are conserved throughout this protein family. Phylogenetic analyses of amino acid sequences demonstrate distinct evolutionary pathways. Analysis of GATA factors isolated from vertebrates suggests that the six distinct vertebrate GATAs are descended from a common ancestral sequence, while those isolated from nonvertebrates (with the exception of the fungal AREA orthologues and Arabidopsis paralogues) appear to be related only within the DNA-binding domain and otherwise provide little insight into their evolutionary history. These results suggest multiple modes of evolution, including gene duplication and modular evolution of GATA factors based upon inclusion of a class IV zinc finger motif. As such, GATA transcription factors represent a group of proteins related solely by their homologous DNA-binding domains. Further analysis of this domain examines the degree of conservation at each amino acid site using the Boltzmann entropy measure, thereby identifying residues critical to preservation of structure and function. Finally, we construct a predictive motif that can accurately identify potential GATA proteins.

• Mark C, Abrink M, Hellman L
• Comparative analysis of KRAB zinc finger proteins in rodents and man: evidence for several evolutionarily distinct subfamilies of KRAB zinc finger genes.
• DNA Cell Biol. 1999; 18: 381-96
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• Although the KRAB zinc finger proteins probably constitute the single largest class of transcription factors within the human genome, almost nothing is known about their biological function. To increase our knowledge about this interesting and relatively unexplored family of potent transcriptional repressors, we here present the cloning, structural analysis, and expression study of three novel mouse KRAB zinc finger proteins. In addition, we present an extensive comparative analysis of various members of this gene family based on the structure of the common KRAB A motif. At least three larger subfamilies of KRAB zinc finger proteins are identified: one carrying the classical KRAB A motif only, another holding both a classical KRAB A and a classical KRAB B motif, and a third holding a classical KRAB A and a highly divergent KRAB B domain, named b. A large variation both in size and in primary amino acid sequence was observed in the linker region between the KRAB domain and the C-terminally located zinc finger repeats. This variability indicates that this region is of minor importance for the biological function of KRAB-containing zinc finger proteins. The fact that in many zinc finger genes, the entire or almost the entire linker region is composed of degenerate finger motifs substantiates this conclusion. The absence of identifiable KRAB A and B motifs in the genome of yeast, Saccharomyces cerevisiae, indicates a relatively late appearance of the KRAB domain in evolution and may suggest that the biological functions are restricted to multicellular organisms. In addition, we show that the expression of individual members of one subfamily of KRAB zinc finger genes is restricted to specific hematopoietic cell lineages. This finding suggests that KRAB zinc finger proteins may play a role in lineage commitment, possibly silencing leakage transcription from nonlineage-expressed genes.

• van der Reijden BA, Erpelinck-Verschueren CA, Lowenberg B, Jansen JH
• TRIADs: a new class of proteins with a novel cysteine-rich signature.
• Protein Sci. 1999; 8: 1557-61
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• Triad1 was recently identified as a nuclear RING finger protein, which is up-regulated during retinoic acid induced granulocytic differentiation of acute leukemia cells. Here we show that a cysteine-rich domain (C6HC), present in Triad1, is conserved in at least 24 proteins encoded by various eukaryotes. The C6HC consensus pattern C-x(4)-C-x(14-30)-C-x(1-4)-C-x(4)-C-x(2)-C-x(4)-H-x(4)-C defines this structure as the fourth family member of the zinc-binding RING, LIM, and LAP/PHD fingers. Strikingly, in 22 of 24 proteins the C6HC domain is flanked by two RING finger structures. We have termed the novel C6HC motif DRIL (double RING finger linked). The strong conservation of the larger tripartite TRIAD (two RING fingers and DRIL) structure indicates that the three subdomains are functionally linked and identifies a novel class of proteins.

• Brzeski J, Podstolski W, Olczak K, Jerzmanowski A
• Identification and analysis of the Arabidopsis thaliana BSH gene, a member of the SNF5 gene family.
• Nucleic Acids Res. 1999; 27: 2393-9
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• The multiprotein complexes involved in active dis-ruption of chromatin structure, homologous to yeast SWI/SNF complex, have been described for human and Drosophila cells. In all SWI/SNF-class complexes characterised so far, one of the key components is the SNF5-type protein. Here we describe the isolation of a plant (Arabidopsis thaliana ) cDNA encoding a 27 kDa protein which we named BSH, with high homology to yeast SNF5p and its human (INI1) and Drosophila (SNR1) counterparts as well as to other putative SNF5-type proteins from Caenorhabditis elegans, fish and yeast. With 240 amino acids, the Arabidopsis BSH is the smallest SNF5-type protein so far identified. When expressed in Saccharomyces cerevisiae, the gene for BSH partially complements the snf5 mutation. BSH is, however, unable to activate transcription in yeast when tethered to DNA. The gene for BSH occurs in single copy in the Arabidopsis genome and is ubiquitously expressed in the plant. Analysis of the whole cell and nuclear protein extracts with antibodies against recombinant BSH indicates that the protein is localised in nuclei. Transgenic Arabidopsis plants with markedly decreased physiological level of the BSH mRNA, resulting from the expression of antisense messenger, are viable but exhibit a distinctive phenotype characterised by bushy growth and flowers that are unable to produce seeds.

• Henry J, Mather IH, McDermott MF, Pontarotti P
• B30.2-like domain proteins: update and new insights into a rapidly expanding family of proteins.
• Mol Biol Evol. 1998; 15: 1696-705
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• The B30.2 domain is a conserved region of around 170 amino acids associated with several different protein domains, including the immunoglobulin folds of butyrophilin and the RING finger domain of ret finger protein. We recently reported several novel members of this family as well as previously undescribed protein families possessing the B30.2 domain. Many proteins have subsequently been found to possess this domain, including pyrin/marenostrin and the midline 1 (MID1) protein. Mutations in the B30.2 domain of pyrin/marenostrin are implicated in familial Mediterranean fever, and partial loss of the B30.2 domain of MID1 is responsible for Opitz G/BBB syndrome, characterized by developmental midline defects. In this study, we scrutinized the available sequence data bases for the identification of novel B30.2 domain proteins using highly sensitive database-searching tools. In addition, we discuss the chromosomal localization of genes in the B30.2 family, since the encoded proteins are likely to be involved in other forms of periodic fever, autoimmune, and genetic diseases.

• Maia IG, Benedetti CE, Leite A, Turcinelli SR, Vercesi AE, Arruda P
• AtPUMP: an Arabidopsis gene encoding a plant uncoupling mitochondrial protein.
• FEBS Lett. 1998; 429: 403-6
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• A cDNA clone (AtPUMP) encoding a plant uncoupling mitochondrial protein was isolated from Arabidopsis thaliana. The cDNA contains an open reading frame of 921 nucleotides encoding 306 amino acids (predicted molecular weight 32,708). The predicted polypeptide is 81% identical and 89% similar to the potato UCP-like protein, and includes an energy transfer protein motif common to mitochondrial transporters. The AtPUMP gene exists as a single copy in the Arabidopsis genome. The corresponding transcript was expressed in all tissues and was strongly induced by cold treatment. We suggest that the putative AtPUMP protein may play a role in heat-requiring physiological events in Arabidopsis.

• Matsuda N, Nakano A
• RMA1, an Arabidopsis thaliana gene whose cDNA suppresses the yeast sec15 mutation, encodes a novel protein with a RING finger motif and a membrane anchor.
• Plant Cell Physiol. 1998; 39: 545-54
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• To identify molecules that function in the plant secretory pathway, we screened for Arabidopsis thaliana cDNA clones that complement the temperature-sensitive (ts), secretion-deficient sec15 mutation of yeast Saccharomyces cerevisiae. RMA1, one of the genes obtained in this screening, suppressed not only the ts growth of sec15 but also its secretory defect. RMA1 is not a structural homologue of SEC15 but encodes a novel 28 kDa protein with a RING finger motif and a C-terminal membrane-anchoring domain. Mutational analysis indicates that the RING finger motif of RMA1 is important for its suppression activity. In Arabidopsis plant, RMA1 is ubiquitously expressed. A search for homologous proteins in the database revealed that Arabidopsis, nematode, mouse and human possess close homologues of RMA1.

• Jensen RB, Jensen KL, Jespersen HM, Skriver K
• Widespread occurrence of a highly conserved RING-H2 zinc finger motif in the model plant Arabidopsis thaliana.
• FEBS Lett. 1998; 436: 283-7
• Display abstract

• Several novel Arabidopsis thaliana proteins containing a RING-H2 zinc finger motif were predicted after database searches. Alignment of 29 RING-H2 finger sequences shows that the motif is strikingly conserved in otherwise unrelated proteins. Only short, non-conserved polar/charged sequences distinguish these domains. The RING-H2 domain is most often present in multi-domain structures, a number of which are likely to contain a membrane-spanning region or an additional zinc finger. However, there are several small (126-200 residues) proteins consisting of an N-terminal domain, rich in aliphatic residues, and a C-terminal RING-H2 domain. Reverse-transcription PCR suggests that the RING-H2 genes are widely expressed at low levels.

• Dietrich RA, Richberg MH, Schmidt R, Dean C, Dangl JL
• A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death.
• Cell. 1997; 88: 685-94
• Display abstract

• Arabidopsis Isd1 mutants are hyperresponsive to cell death initiators and fail to limit the extent of cell death. Superoxide is a necessary and sufficient signal for cell death propagation. Thus, LSD1 monitors a superoxide-dependent signal and negatively regulates a plant cell death pathway. We isolated LSD1 via its map position. The predicted LSD1 protein contains three zinc finger domains, defined by CxxCxRxxLMYxxGASxVxCxxC. These domains are present in three additional Arabidopsis genes, suggesting that LSD1 defines a zinc finger protein subclass. LSD1 is constitutively expressed, consistent with the mutant phenotype. Alternate splicing gives rise to a low abundance mRNA encoding an extra five amino-terminal amino acids. We propose that LSD1 regulates transcription, via either repression of a prodeath pathway or activation of an antideath pathway, in response to signals emanating from cells undergoing pathogen-induced hypersensitive cell death.

• Cardon GH, Hohmann S, Nettesheim K, Saedler H, Huijser P
• Functional analysis of the Arabidopsis thaliana SBP-box gene SPL3: a novel gene involved in the floral transition.
• Plant J. 1997; 12: 367-77
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• The isolation and initial characterization of the Arabidopsis thaliana SPL3 gene are described. SPL3 belongs to a gene family encoding putative transcription factors characterized by a conserved DNA-binding domain, the SBP domain. SPL3 transcription is developmentally regulated and is localized mainly in vegetative and inflorescence apical meristems, floral meristems and in leaf and floral organ primordia. SPL3 recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene AP1. Similarly to AP1, constitutive expression of SPL3 results in early flowering. However, constitutive expression of SPL3 in an ap1 mutant background showed that AP1 is not required for the early flowering phenotype of the SPL3 transgenic plants. The function of SPL3 during flowering as well as its possible functional redundancy are discussed.

• Tague BW, Gallant P, Goodman HM
• Expression analysis of an Arabidopsis C2H2 zinc finger protein gene.
• Plant Mol Biol. 1996; 32: 785-96
• Display abstract

• C2H2 zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C2H2 zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the beta-glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promoter: beta-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.

• Abrink M, Aveskogh M, Hellman L
• Isolation of cDNA clones for 42 different Kruppel-related zinc finger proteins expressed in the human monoblast cell line U-937.
• DNA Cell Biol. 1995; 14: 125-36
• Display abstract

• To study the complexity and structural characteristics of zinc finger proteins expressed during human hematopoiesis and to isolate novel regulators of blood cell development, a degenerate oligonucleotide probe specific for a consensus zinc finger peptide domain was used to isolate 63 cDNA clones for Kruppel-related zinc finger genes from the human monoblast cell line U-937. By extensive nucleotide sequence and Northern blot analysis, these cDNA clones were found to originate from approximately 42 different genes (HZF 1-42) of which only 8 have previously been described. Northern blot analysis showed that a majority of these genes were expressed at comparable levels in U-937 and HeLa cells. The large number of individual genes represented among the 63 clones and their apparent non-cell-type-specific expression suggest that the majority of the Kruppel-related zinc finger genes are likely to be expressed in most human tissues. In contrast, some of the genes displayed a restricted expression pattern, indicating that they represent potential regulators of monocyte differentiation or proliferation. Detailed structural analysis of the first 12 cDNAs (HZF 1-10) and a partial characterization of HZF 11-42 revealed that a common feature of human Kruppel-related zinc finger proteins is the presence of tandem arrays of zinc fingers ranging in number from 3 to over 20 that are preferentially located in the carboxy-terminal regions of the proteins. In addition, several novel KRAB-containing zinc finger genes and a novel conserved sequence element were identified.

• Sakai H, Medrano LJ, Meyerowitz EM
• Role of SUPERMAN in maintaining Arabidopsis floral whorl boundaries.
• Nature. 1995; 378: 199-203
• Display abstract

• The Arabidopsis gene SUPERMAN (SUP) is necessary for the proper spatial development of reproductive floral tissues. Recessive mutations cause extra stamens to form interior to the normal third whorl stamens, at the expense of fourth whorl carpel development. The mutant phenotype is associated with the ectopic expression of the B function genes, AP3 and PI, in the altered floral region, closer to the centre of the flower than in the wild type, and ap3 sup and pi sup double mutants exhibit a phenotype similar to ap3 and pi single mutants. These findings led to SUP being interpreted as an upstream negative regulator of the B function organ-identity genes, acting in the fourth whorl, to establish a boundary between stamen and carpel whorls. Here we show, using molecular cloning and analysis, that it is expressed in the third whorl and acts to maintain this boundary in developing flowers. The putative SUPERMAN protein contains one zinc-finger and a region resembling a basic leucine zipper motif, suggesting a function in transcriptional regulation.

• Yanagisawa S
• A novel DNA-binding domain that may form a single zinc finger motif.
• Nucleic Acids Res. 1995; 23: 3403-10
• Display abstract

• MNB1a is a DNA-binding protein from maize that interacts with the 35S promoter of cauliflower mosaic virus. This protein did not show significant homologies with any other DNA-binding protein and MNB1a seemed to be a member of a multigene family. In this study, isolation of cDNAs from the gene family to which MNB1a belongs revealed a unique conserved domain, referred to herein as the Dof domain, that contains a novel cysteine-rich motif for a single putative zinc finger. The amino acid sequence of the Dof domain and the arrangement of cysteine residues in this domain differ from those of known zinc finger motifs. However, the Dof domain was shown to be a DNA-binding domain that required Zn2+ ions for activity. Mutations at cysteine residues eliminated the DNA-binding activity of MNB1a. Thus, the Dof domain may be classified as a novel zinc finger motif. In addition, Southern blot analysis and a survey of DNA databases suggested that proteins that include Dof domains might exist in other eukaryotes, at least in the plant kingdom.

• Daniel-Vedele F, Caboche M
• A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi.
• Mol Gen Genet. 1993; 240: 365-73
• Display abstract

• In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X2-C-X17-C-X2-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X2-C-X18-C-X2-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light.

• de Oliveira DE et al.
• Inflorescence-specific genes from Arabidopsis thaliana encoding glycine-rich proteins.
• Plant J. 1993; 3: 495-507
• Display abstract

• Genomic and cDNA clones for three inflorescence-specific genes from Arabidopsis thaliana were isolated and characterized. The genes are tandemly organized in the genome on a 10 kb fragment. The expression of these genes is coordinately regulated in a developmental and organ-specific pattern. They are expressed predominantly in anthers at the later stage of flower development. The primary structure of the encoded gene products exhibits comparable features consisting of a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Little homology is observed either between the glycine-rich domain of the three genes or with previously described glycine-rich proteins from other plant species.

• Baltz R, Domon C, Pillay DT, Steinmetz A
• Characterization of a pollen-specific cDNA from sunflower encoding a zinc finger protein.
• Plant J. 1992; 2: 713-21
• Display abstract

• We have isolated, via differential hybridization screening of a floral cDNA library from sunflower, a cDNA clone that hybridizes to a 1100 nucleotide-long mRNA found exclusively in mature pollen grains. The cDNA encodes a 219 amino acid-long polypeptide containing two potential zinc fingers alternating with two basic domains. A similar organization is found in the erythroid-specific transcription factor Eryf1 from chicken and its murine homolog GF-1. The C-terminus of the protein contains a sixfold repeat of the pentapeptide sequence (S,T,A)(E,D)TQN. These features suggest that the SF3 protein is a transcription factor required for the expression of late pollen genes. The SF3 gene is a member of a multicopy gene family. A genomic copy of the gene has been isolated and sequenced; it is split by four short, AT-rich introns.

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