SUMMARYThe LOS2 gene in Arabidopsis encodes an enolase with 72% amino acid sequence identity with human ENO1. In mammalian cells, the a-enolase (ENO1) gene encodes both a 48 kDa glycolytic enzyme and a 37 kDa transcriptional suppressor protein that are targeted to different cellular compartments. The tumor suppressor c-myc binding protein (MBP-1), which is alternatively translated from the second start codon of ENO1 transcripts, is preferentially localized in nuclei while a-enolase is found in the cytoplasm. We report here that an Arabidopsis MBP-1-like protein (AtMBP-1) is alternatively translated from full-length LOS2 transcripts using a second start codon. Like mammalian MBP-1, this truncated form of LOS2 has little, if any, enolase activity, indicating that an intact N-terminal region of LOS2 is critical for catalysis. AtMBP-1 has a short half-life in vivo and is stabilized by the proteasome inhibitor MG132, indicating that it is degraded via the ubiquitin-dependent proteasome pathway. Arabidopsis plants that over-express AtMBP-1 are hypersensitive to abscisic acid (ABA) during seed germination and show defects in vegetative growth and lateral stem development. AtMBP-1 interacts directly with the E3 ubiquitin ligase AtSAP5 and co-expression of these proteins resulted in destabilization of AtMBP-1 in vivo and abolished the developmental defects associated with AtMBP-1 over-expression. Thus, AtMBP-1 is as a bona fide alternative translation product of LOS2. Accumulation of this factor is limited by ubiquitin-dependent destabilization, apparently mediated by AtSAP5.
PAL (L-phenylalanine ammonia-lyase), the first enzyme of phenylpropanoid biosynthesis, is often encoded by multigene families in plants. A PCR-based approach was used to isolate cDNA clones corresponding to the four PAL genes of tobacco (Nicotiana tabacum). By careful comparison of cDNA and genomic clones, a new PAL gene (PAL4) was defined. PCR amplification of PAL sequences from cDNA led to the generation of chimaeric clones between PAL1 and PAL4, and incorrect annotation of PAL4 ESTs (expressed sequence tags) as PAL1 in the EST database has given rise to a randomly shuffled tentative consensus sequence. The PAL2 previously described in the literature was shown, by domain swapping experiments with PAL1, to possess a single nucleotide substitution leading to an inactive enzyme. The altered amino acid resulting from this substitution maps to the base of the active site pocket in the three-dimensional structure of PAL. The inactive PAL2 allele could not be recovered from 13 different tobacco cultivars examined. PALs 1-4 were co-expressed in multiple plant organs, and were also co-induced following exposure of cell cultures to yeast elicitor or methyl jasmonate. All four tobacco PAL proteins expressed in Escherichia coli displayed normal Michaelis-Menten kinetics, with Km values between 36 and 60 muM. Co-expression of different PAL proteins in E. coli resulted in formation of heterotetramers, which possessed kinetic properties within the same range as those of the individual homotetramers. The potential physiological function of heterotetrameric PAL forms is discussed.
Arabidopsis thaliana Stress Associated Protein 9 (AtSAP9) is a member of the A20/AN1 zinc finger protein family known to play important roles in plant stress responses and in the mammalian immune response. Although SAPs of several plant species were shown to be involved in abiotic stress responses, the underlying molecular mechanisms are largely unknown, and little is known about the involvement of SAPs in plant disease responses. Expression of SAP9 in Arabidopsis is up-regulated in response to dehydration, cold, salinity and abscisic acid (ABA), as well as pathogen infection. Constitutive expression of AtSAP9 in Arabidopsis leads to increased sensitivity to ABA and osmotic stress during germination and post-germinative development. Plants that overexpress AtSAP9 also showed increased susceptibility to infection by non-host pathogen Pseudomonas syringae pv. phaseolicola, indicating a potential role of AtSAP9 in disease resistance. AtSAP9 was found to interact with RADIATION SENSITIVE23d (Rad23d), a shuttle factor for the transport of ubiquitinated substrates to the proteasome, and it is co-localized with Rad23d in the nucleus. Thus, AtSAP9 may promote the protein degradation process by mediating the interaction of ubiquitinated targets with Rad23d. Taken together, these results indicate that AtSAP9 regulates abiotic and biotic stress responses, possibly via the ubiquitination/proteasome pathway.
BackgroundThe novel mutant allele hsi2-4 was isolated in a genetic screen to identify Arabidopsis mutants with constitutively elevated expression of a glutathione S-transferase F8::luciferase (GSTF8::LUC) reporter gene in Arabidopsis. The hsi2-4 mutant harbors a point mutation that affects the plant homeodomain (PHD)-like domain in HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2 (HSI2)/VIVIPAROUS1/ABI3-LIKE1 (VAL1). In hsi2-4 seedlings, expression of this LUC transgene and certain endogenous seed-maturation genes is constitutively enhanced. The parental reporter line (WTLUC) that was used for mutagenesis harbors two independent transgene loci, KanR and KanS. Both loci express luciferase whereas only the KanR locus confers resistance to kanamycin.ResultsHere we show that both transgene loci harbor multiple tandem insertions at single sites. Luciferase expression from these sites is regulated by the HSI2 PHD-like domain, which is required for the deposition of repressive histone methylation marks (H3K27me3) at both KanR and KanS loci. Expression of LUC and Neomycin Phosphotransferase II transgenes is associated with dynamic changes in H3K27me3 levels, and the activation marks H3K4me3 and H3K36me3 but does not appear to involve repressive H3K9me2 marks, DNA methylation or histone deacetylation. However, hsi2-2 and hsi2-4 mutants are partially resistant to growth inhibition associated with exposure to the DNA methylation inhibitor 5-aza-2′-deoxycytidine. HSI2 is also required for the repression of a subset of regulatory and structural seed maturation genes in vegetative tissues and H3K27me3 marks associated with most of these genes are also HSI2-dependent.ConclusionsThese data implicate HSI2 PHD-like domain in the regulation of gene expression involving histone modifications and DNA methylation-mediated epigenetic mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0293-4) contains supplementary material, which is available to authorized users.
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