GUS Fusion Research Articles

Molecular characterization of a light-responsive gene, breast basic conserved protein 1 ( OsiBBC1), encoding nuclear-localized protein homologous to ribosomal protein L13 from Oryza sativa indica

Rice ( Oryza sativa L. subsp. indica) cDNA for the gene OsiBBC1, encoding homologue of the breast basic conserved protein 1 (BBC1), similar to ribosomal protein L13, has been identified and characterized. OsiBBC1 codes for a 24 kDa highly basic protein with two potential bipartite nuclear localization signals (NLS) and a transcriptional activation domain (TAD). The structural part of the gene is interrupted by four introns. The OsiBBC1 gene is represented by two copies in the rice genome and both of them are expressed. Northern analysis showed that OsiBBC1 is expressed more in the young root, post-fertilized inflorescence, leaf base and callus tissue, which are comprised of actively dividing cells, indicating its role in cell division. The OsiBBC1 transcript accumulated more in the root of light-grown seedlings as compared to the shoot while its levels were higher in the shoot as compared to root of the etiolated seedlings, indicating its down-regulation by light. The western analysis, carried out using antibodies raised against a recombinant fusion protein, 6xHis–OsiBBC1, corroborated its tissue-specific expression profile observed by northern analysis. In addition, OsiBBC1/RPL13 protein could be targetted to the nucleus by particle bombardment of OsiBBC1::GUS fusion construct in the onion epidermal cells.

Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis

Pectin methylesterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell wall. Previous work indicated that plant PMEs may be subject to post-translational regulation. Here, we report the analysis of two proteinaceous inhibitors of PME in Arabidopsis thaliana (AtPMEI1 and 2). The functional analysis of recombinant AtPMEI1 and 2 proteins revealed that both proteins are able to inhibit PME activity from flowers and siliques. Quantitative RT-PCR analysis indicated that expression of AtPMEI1 and 2 mRNAs is tightly regulated during plant development with highest mRNA levels in flowers. Promotor::GUS fusions demonstrated that expression is mostly restricted to pollen.

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA(3) treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.

Cytokinin-Deficient Transgenic Arabidopsis Plants Show Multiple Developmental Alterations Indicating Opposite Functions of Cytokinins in the Regulation of Shoot and Root Meristem Activity

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.

Developmental regulation and significance of KNOX protein trafficking in Arabidopsis.

Intercellular communication delivers critical information for position-dependent specification of cell fate. In plants, a novel mechanism for cell-to-cell communication involves the intercellular trafficking of regulatory proteins and mRNAs. The maize KNOTTED1 (KN1) gene acts non cell-autonomously in the maize leaf, and KN1 was the first plant protein shown to traffic cell-to-cell, presumably through plasmodesmata. We have compared the intercellular trafficking of green fluorescent protein (GFP) fusions of KN1 and Arabidopsis KN1-related homeobox proteins to that of the viral movement protein from turnip vein clearing tobamovirus. We show that there is specific developmental regulation of GFP approximately KN1 trafficking. GFP -- KN1 was able to traffic from the inner layers of the leaf to the epidermis, but not in the opposite direction, from epidermis to mesophyll. However, GFP or the GFP -- movement protein fusion moved readily out of the epidermis. GFP -- KN1 was however able to traffic out of the epidermal (L1) layer in the shoot apical meristem, indicating that KN1 movement out of the L1 was developmentally regulated. GFP -- KNAT1/BREVIPEDICELLUS and GFP -- SHOOTMERISTEMLESS fusions could also traffic from the L1 to the L2/L3 layers of the meristem. In a test for the functional significance of trafficking, we showed that L1-specific expression of KN1 or of KNAT1 was able to partially complement the strong shootmeristemless-11 (stm-11) mutant. However, a cell-autonomous GUS fusion to KN1 showed neither trafficking ability nor complementation of stm-11 when expressed in the L1. These results suggest that the activity of KN1 and related homeobox proteins is maintained following intercellular trafficking, and that trafficking may be required for their normal developmental function.

Molecular characteristics and differential expression of two nuclear factors containing the FHA domain in Arabidopsis

The forkhead-associated (FHA) domain identified in a wide variety of proteins is a small module that recognizes phosphothreonine epitopes on proteins. AtFHA1 and AtFHA2 of Arabidopsis are homologs of tobacco NtFHA1 encoding a FHA domain-containing transcription activator. Previously, we showed that the FHA domain of NtFHA1 functionally substitutes for that of the Fhl1 forkhead transcription factor involved in rRNA processing in yeast, despite significant differences in their protein structures. In this study, we characterized AtFHA1 and AtFHA2 of Arabidopsis and analyzed their expression patterns. AtFHA1 and AtFHA2 contain an N-terminal FHA domain and a C-terminal acidic region, but lack any known DNA-binding motifs. Both proteins were targeted to the nucleus. GUS staining of transgenic Arabidopsis plants containing the promoter- GUS fusion gene demonstrated high expression of AtFHA2 in whole tissues throughout plant development. In contrast, AtFHA1 was temporally expressed at low levels in roots and vascular tissues of stems. Interestingly, AtFHA1 expression was significantly induced in whole plants in response to external stimuli. The observed differential expression of AtFHA1 and AtFHA2 implies that these two genes have distinct regulatory roles in Arabidopsis.

Endophytic Herbaspirillum seropedicae expresses nif genes in gramineous plants

The interactions between maize, sorghum, wheat and rice plants and Herbaspirillum seropedicae were examined microscopically following inoculation with the H. seropedicae LR15 strain, a Nif + (P nif:: gusA) mutant obtained by the insertion of a gusA-kanamycin cassette into the nifH gene of the H. seropedicae wild-type strain. The expression of the P nif:: gusA fusion was followed during the association of the diazotroph with the gramineous species. Histochemical analysis of seedlings of maize, sorghum, wheat and rice grown in vermiculite showed that strain LR15 colonized root surfaces and inner tissues. In early steps of the endophytic association, H. seropedicae colonized root exudation sites, such as axils of secondary roots and intercellular spaces of the root cortex; it then occupied the vascular tissue and there expressed nif genes. The expression of nif genes occurred in roots, stems and leaves as detected by the GUS reporter system. The expression of nif genes was also observed in bacterial colonies located in the external mucilaginous root material, 8 days after inoculation. Moreover, the colonization of plant tissue by H. seropedicae did not depend on the nitrogen-fixing ability, since similar numbers of cells were isolated from roots or shoots of the plants inoculated with Nif + or Nif − strains.

The unusual Arabidopsis extensin gene atExt1 is expressed throughout plant development and is induced by a variety of biotic and abiotic stresses.

We detail the expression of the Arabidopsis thaliana (L.) Heynh. atExt1 extensin gene. atExt1 is normally expressed in roots and inflorescences, and is induced by wounding, exogenously supplied salicylic acid, methyl jasmonate, auxins and brassinosteroids. Northern assays and histochemical analysis of transgenics expressing an atExt1:: gus fusion show that this gene is also induced by the brassica pathogen Xanthomonas campestris pv. campestris and that this induction is restricted to tissues close to the site of infection. Expression at regions of abscission and senescence also implicates atExt1 in these important developmental processes.

Transcriptional activation of phosphoenolpyruvate carboxylase by phosphorus deficiency in tobacco.

Phosphoenolpyruvate carboxylase (PEPC), which catalyses the carboxylation of phosphoenolpyruvate using HCO(3)(-) to generate oxaloacetic acid, is an important enzyme in the primary metabolism of plants. Although the PEPC genes (ppc) comprise only a small gene family, the function of each gene is not clear, except for roles in C(4) photosynthesis and CAM. Three PEPC genes (Nsppc1-3) from the C(3) plant Nicotiana sylvestris were used to investigate their roles and regulation in a C(3) plant, and their regulation by phosphorus depletion in particular. First, the induction of PEPC by phosphorus depletion was confirmed. Next, Nsppc1 was determined to be mainly responsive to phosphorus deficiency at the transcriptional level. Further studies using transgenic tobacco harbouring a chimeric gene consisting of the 2.0 kb promoter region of Nsppc1 and the beta-glucuronidase (GUS) reporter showed that PEPC is transcriptionally induced. It was also found that sucrose had a synergistic effect on the induction of PEPC by phosphorus deficiency. A series of transgenic tobacco containing 5'-deletion mutants of Nsppc1 promoter::GUS fusion revealed that the -539 to -442 bp Nsppc1 promoter region, relative to the translation start site, was necessary for the response to phosphorus deficiency. Gain-of-function analysis using a construct containing three tandem repeats of the -539 to -442 bp region confirmed that this region was sufficient to induce the phosphorus-deficiency response in tobacco.

The mid genes of Rhizobium sp. strain TAL1145 are required for degradation of mimosine into 3-hydroxy-4-pyridone and are inducible by mimosine

Mimosine is a toxin present in the tree-legume leucaena (Leucaena leucocephala), including its root nodules and the root exudates. The leucaena-nodulating Rhizobium sp. strain TAL1145 degrades mimosine (Mid(+)) and utilizes it as a source of carbon and nitrogen. Twelve TAL1145 mutants defective in mimosine degradation (Mid(-)) were made through Tn3Hogus, TnphoA or kanamycin-resistance-cassette insertions. A 5.0 kb PstI fragment of TAL1145, subcloned from a cosmid clone containing mid genes for mimosine degradation, complemented most of the Mid(-) mutants. Sequencing this fragment and the adjacent 0.9 kb PstI fragment identified five genes, midA, midB, midC, midD and midR, of which the first three genes encode ABC transporter proteins involved in mimosine uptake, while midD encodes an aminotransferase required for degrading mimosine into 3-hydroxy-4-pyridone, and midR is a regulatory gene encoding a LysR-type transcriptional activator. The location of MidA in the periplasm was shown by making two midA : : phoA fusions, which made active alkaline phosphatase in the periplasm. The various mid : : gus and midA : : phoA fusions were inducible by mimosine, and a midD : : gus fusion mutant showed beta-glucuronidase activity in the leucaena nodules, indicating that midD is expressed in the nodules. Similarly, a midA : : phoA fusion expressed alkaline phosphatase activity in the leucaena nodules, indicating that mimosine induces midA transcription in the bacteroids. mid genes are specific for the Mid(+) strains of leucaena Rhizobium and are absent in strains of other Rhizobium, Sinorhizobium and Bradyrhizobium spp.

The Promoter Region of Arabidopsis Metallothionein MT2a Gene is Strongly Induced by Treatment with Cu II, ZNII and CD II Ions in Transgenic Nicotiana Benthamiana Plants

ABSTRACTArabidopsis possess a small family of five metallothionein genes, as the expression of one of them (MT2a), was reported to be strongly induced by Cu II, ZnII and Cd II ions, (15). In present research, the expression of MT2a promoter region was studied in heterologous genetic background, through analysis of GUS activity of transgenic Nicotiana benthamiana plants carrying MT2a:GUS fusion gene. The fluorimetric GUS assay showed relatively low level of expression of MT2a promoter in the leaves of transgenic plants under non-induced conditions. Strong induction of MT2a:GUS gene, increasing of 103-, 84- and 59- times of the GUS activity, was detected 72 hours after treatment of the transgenic plants with respectively Cu II, ZnII and Cd II ions. The obtained results demonstrate a similarity between the heavy metal induction pattern of MT2a gene in Arabidopsis and MT2a promoter region in N. benthamiana. This suggests conservation of the signal transduction pathway conferring the transcriptional induction of MT2a metallothionein gene by the Cu II, ZnII and Cd II ions, in the both species. The possibilities of the practical utilization of the MT2a promoter region are discussed

Expression of the Arabidopsis MCM Gene PROLIFERA During Root-Knot and Cyst Nematode Infection

ABSTRACT Expression of the Arabidopsis thaliana gene PROLIFERA (PRL) was examined during development of root-knot and cyst nematode feeding sites. These obligate plant parasites establish specialized feeding structures in roots that allow them to withdraw nutrients from the host. In the process of establishing feeding sites, nematodes alter cell cycle regulation. PRL is normally expressed specifically in dividing cells at all stages of plant development and was used here as a marker for cell division. PRL expression, reported from a PRL::GUS fusion protein, was detected in nematode feeding sites of both root-knot and cyst nematodes from the earliest stages of infection in both giant cells and syncytia. However, unlike other cell cycle genes, expression of PRL was detected only occasionally in cells surrounding the feeding sites. PRL::GUS activity persisted until late in the infection cycle, past the time when other cell cycle genes are expressed. These data indicate that some aspects of the PRL expression pattern during nematode infection differ from that of other cell cycle genes.

Expression of Xanthomonas oryzae pv. oryzae hrp Genes in XOM2, a Novel Synthetic Medium

To analyze the regulation of hrp expression and to detect and identify hrp-dependent secretion proteins of plant-pathogenic bacteria, an appropriate hrp-inducing medium is indispensable. In this study, two efficient hrp-inducing media for Xanthomonas oryzae pv. oryzae were designed by assaying the expression of a hrcU (the first gene of the hrpC operon) and a gus (β-glucuronidase) fusion gene. We modified XVM2, which is a hrp-inducing medium for X. campestris pv. vesicatoria, by adding 0.01% xylose in place of fructose and sucrose (0.18 and 0.34%, respectively) as a sugar source. The resulting medium induced approximately 15-fold more GUS activity from transformants containing a hrcU::gus gene than did XVM2. Moreover, a methionine-containing synthetic medium with 0.18% xylose as a sugar source was able to induce much stronger expression of HrcU::GUS, with GUS activity approximately 100-fold greater than that in XVM2. Induction depended on a regulator, HrpXo, and the PIP (plant-inducible-promoter) box, suggesting that HrcU::GUS was expressed in a hrp-dependent manner. The induction of operons hrpA to hrpF in XOM2 was also confirmed. These results suggest that both media, especially XOM2, are highly efficient hrp-inducing media for X. oryzae pv. oryzae.

Embryo and endosperm development is disrupted in the female gametophytic capulet mutants of Arabidopsis.

The female gametophyte of higher plants gives rise, by double fertilization, to the diploid embryo and triploid endosperm, which develop in concert to produce the mature seed. What roles gametophytic maternal factors play in this process is not clear. The female-gametophytic effects on embryo and endosperm development in the Arabidopsis mea, fis, and fie mutants appear to be due to gametic imprinting that can be suppressed by METHYL TRANSFERASE1 antisense (MET1 a/s) transgene expression or by mutation of the DECREASE IN DNA METHYLATION1 (DDM1) gene. Here we describe two novel gametophytic maternal-effect mutants, capulet1 (cap1) and capulet2 (cap2). In the cap1 mutant, both embryo and endosperm development are arrested at early stages. In the cap2 mutant, endosperm development is blocked at very early stages, whereas embryos can develop to the early heart stage. The cap mutant phenotypes were not rescued by wild-type pollen nor by pollen from tetraploid plants. Furthermore, removal of silencing barriers from the paternal genome by MET1 a/s transgene expression or by the ddm1 mutation also failed to restore seed development in the cap mutants. Neither cap1 nor cap2 displayed autonomous seed development, in contrast to mea, fis, and fie mutants. In addition, cap2 was epistatic to fis1 in both autonomous endosperm and sexual development. Finally, both cap1 and cap2 mutant endosperms, like wild-type endosperms, expressed the paternally inactive endosperm-specific FIS2 promoter GUS fusion transgene only when the transgene was introduced via the embryo sac, indicating that imprinting was not affected. Our results suggest that the CAP genes represent novel maternal functions supplied by the female gametophyte that are required for embryo and endosperm development.

Identification of a NifL-like protein in a diazotroph of the beta-subgroup of the Proteobacteria, Azoarcus sp. strain BH72.

NifA, the transcriptional activator of nitrogenase (nif) genes, has up to now been described to be regulated in its activity via the sensor NifL only for members of the gamma-subgroup of the PROTEOBACTERIA: This paper reports a functionally similar NifL-like protein outside this group in Azoarcus sp. strain BH72, a diazotrophic grass endophyte belonging to the beta-subgroup of the PROTEOBACTERIA: Its structural genes for nitrogenase (nifHDK) are regulated in response to combined nitrogen and O(2) and expressed endophytically inside rice roots. In order to characterize nitrogen-regulatory genes, an Azoarcus sp. BH72 genomic library was used to select cosmids that complemented a nifA mutation in Azotobacter vinelandii. Sequence analysis of the 3.4 kb genomic region complementing nifA showed two ORFs with sequence identities of 44% to NifL and 61% to NifA of Azotobacter vinelandii. According to Northern blot and reverse transcriptase PCR analysis, the nifLA transcript was more abundant at low combined nitrogen and O(2) levels, results which were corroborated by GUS (beta-glucuronidase) assays using a transcriptional nifL::gusA fusion. N(2) fixation was abolished in a NifLA(-) and a NifA(-) mutant, wild-type fixation being restored by nifLA in trans. The NifLA(-) mutant also failed to activate nifH::gus expression, indicating that NifA is the obligate transcriptional activator for nifHDK. A nifL mutant was diazotrophic and did not show repression of nifH::gusA by ammonium or O(2), suggesting that NifL of Azoarcus sp. strain BH72 has a similar role in inactivating NifA in response to O(2) and combined nitrogen as NifL in bacteria of the gamma-PROTEOBACTERIA:

A novel cold-inducible gene from Arabidopsis, RCI3, encodes a peroxidase that constitutes a component for stress tolerance.

A cDNA from Arabidopsis corresponding to a new cold-inducible gene, RCI3 (for Rare Cold Inducible gene 3), was isolated. Isoelectric focusing electrophoresis and staining of peroxidase activity demonstrated that RCI3 encodes an active cationic peroxidase. RNA-blot analysis revealed that RCI3 expression in response to low temperature is negatively regulated by light, as RCI3 transcripts were exclusively detected in etiolated seedlings and roots of adult plants. RCI3 expression was also induced in etiolated seedlings, but not in roots, exposed to dehydration, salt stress or ABA, indicating that it is subjected to a complex regulation through different signaling pathways. Analysis of transgenic plants containing RCI3::GUS fusions established that this regulation occurs at the transcriptional level during plant development, and that cold-induced RCI3 expression in roots is mainly restricted to the endodermis. Plants overexpressing RCI3 showed an increase in dehydration and salt tolerance, while antisense suppression of RCI3 expression gave dehydration- and salt-sensitive phenotypes. These results indicate that RCI3 is involved in the tolerance to both stresses in Arabidopsis, and illustrate that manipulation of RCI3 has a potential with regard to plant improvement of stress tolerance.

Structural features of the lac promoter affecting gusA expression in Lactobacillus casei.

With the aim of designing efficient expression systems for Lactobacillus casei, different factors affecting gene expression from the strong and highly modulated lac promoter have been systematically analyzed, by using the Escherichia coli beta-glucuronidase gene (gusA) as reporter. The activity of this enzyme (GUS) was quantified when plac:: gusA fusions were cloned in plasmids with different copy number or when gusA was inserted in the chromosomal lactose operon (single copy). Results showed a clear gene dosage effect and a positive influence of the native lac operon transcription and translation signals on GUS expression.

The multigenic family of thioredoxin h in Arabidopsis thaliana: specific expression and stress response

In the model plant Arabidopsis thaliana, cytosolic thioredoxins h (TRXh) are encoded by a multigenic family of eight genes. Genomic studies have revealed that a number of these genes are duplicated genes originating from a common ancestor. This multiplicity of thioredoxin h genes raises questions of the specificity of plant thioredoxins and the function of such a large multigenic family in plant. The results from studies using northern blots, semi-quantitative RT-PCR and transgenic promoter– GUS fusions provide strong evidence that the members of the AtTRXh gene family show expression levels that vary among different plant organs. Moreover, distinct AtTRXh genes are induced in response to pathogenic elicitors. Together, our data suggest that the members of the multigenic family of AtTRXh may not have redundant functions.

LasX, a transcriptional regulator of the lactocin S biosynthetic genes in Lactobacillus sakei L45, acts both as an activator and a repressor

The 11 kb las locus, present on the 50 kb plasmid pCIM1, specifies the production of the lantibiotic lactocin S in Lactobacillus sakei L45. The gene cluster is organized into two oppositely orientated operons, lasAMNTUVPJW ( lasA–W) and lasXY, the former of which contains the biosynthetic, immunity and transport genes. We have previously shown that inactivation of lasX abolishes lactocin S production and causes a drastic reduction in lasA-specific transcripts (encoding pre-lactocin S). The aim of this study was to determine whether or not the product of lasX, which is significantly similar to Rgg-like regulators, was directly involved in transcriptional regulation of the lactocin S biosynthetic genes. The divergently orientated and overlapping promoters, P lasA – W and P lasXY , were transcriptionally fused to the Escherichia coli gusA gene, and the activity of the fusions was assayed in the presence and absence of lasX, which was expressed on a separate plasmid. A significant stimulation of expression (5–6-fold) of the P lasA-W – gusA fusion was observed in the presence of lasX, whereas expression of the P lasXY –gusA construct was reduced 1.5–2-fold. Our results strongly suggest that LasX is a bifunctional regulatory protein, acting both as an activator of lasA– W transcription and as a repressor of lasXY transcription. While a transcription stimulation activity has been described for several of the Rgg-like proteins, the present study is the first to report an autorepressor function for a member of this protein group.

Regulation of Arabidopsis thaliana Em genes: role of ABI5.

In order to identify new factors involved in Em (a class I Late Embryogenesis Abundant protein) gene expression, Arabidopsis mutants with an altered expression of an Em promoter GUS fusion construct and a modified accumulation of Em transcripts and proteins were isolated. Germination tests on ABA showed that the most affected mutant had a weak abi phenotype. Complementation tests further revealed this mutant to be a new abi5 allele, consequently named abi5-5. In addition to reducing the final level of Em transcripts in the dry seed, the abi5-5 mutation causes a delay in the accumulation of AtEm1 during seed development. An additional characteristic of the abi5-5 mutant, is the ability of its seeds to germinate at high concentrations of salt and mannitol. The abi5-5 mutation was characterized at the molecular level and was shown to result from a two base pair deletion in the coding sequence of the ABI 5 gene. The wild type and mutant recombinant proteins were produced in E. coli and were assayed for DNA-binding activity on their target promoters by electrophoretic mobility shift assay (EMSA). The ABI5 recombinant protein binds the ABRE sequence in the AtEm6 promoter as shown by Dnase footprinting. Among the ABRE-type sequences selected on both Em promoters, the G-box type AGACACGTGGCATGT element of the AtEm6 promoter shows the strongest binding by EMSA quantification.