A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

The recalcitrant plant species, Castanospermum australe and Trichilia dregeana, differ in their ability to produce dehydrin-related polypeptides during seed maturation and in response to ABA or water-deficit-related stresses

In constrast to seeds of orthodox species, those of recalcitrantspecies do not acquire desiccation tolerance during their developmentand are shed from the parent plant at high water contents. Dehydrinproduction in seeds of recalcitrant species was examined duringdevelopment and germination, in response to abscisic acid (ABA),and following the imposition of various water-deficit-relatedstresses, including desiccation, water stress, high salt, highosmolarity, and low temperature. Two tropical species exhibiteda differential capacity to produce dehydrin-related proteinsduring seed maturation. Dehydrins were present in axes and cotyledonsof Castanospermum australe seeds during mid-maturation and atmaturity. In Trichilia dregeana, no dehydrin-related polypeptideswere detected in the mature seed. During the development ofC. australe seeds, the nature of the dehydrin related polypeptidesaccumulated in the cotyledons and axis changed and new polypeptideswere detected in the mature seeds that were not present duringmid-maturation. The dehydrins present in cotyledons of matureseeds (31, 37 and 40 kDa) were still detectable after germination(i.e. in untreated seedlings). These dehydrins became less abundantin the cotyledons of C. australe seedlings following ABA andall stress treatments except cold, although most of the dehydrinswere still detectable. An exception was the desiccation-treatedseedlings, in which no dehydrins were detected. In the rootsof C. australe seedlings, no dehydrins were found after germinationnor were they induced in the root by ABA or any of the stresstreatments imposed on seedlings. Seedlings of Trichilia dregeanadid not produce dehydrins in the roots or cotyledons when exposedto ABA or water-deficit-related stresses. Key words: Dehydrin, ABA, desiccation, recalcitrant, seed     

A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn

Several cDNAs related to an ABA-induced cDNA from barley aleurone were isolated from barley and corn seedlings that were undergoing dehydration. Four different barley polypeptides with sizes of 22.6, 16.2, 14.4 and 14.2 kDa and a single corn polypeptide with a size of 17.0 kDa were predicted from the nucleotide sequences of the cDNAs. These dehydration-induced proteins (dehydrins) are very similar to each other and to a previously identified rice protein induced by ABA and salt, and have at least some similarity to a previously identified cotton embryo protein. Each dehydrin is extremely hydrophilic, glycine-rich, cysteine- and tryptophan-free and contains repeated units in a conserved linear order. A lysine-rich repeating unit occurs twice in each protein, once at the carboxy terminus and once partway through the polypeptide, adjacent to a succession of serines. This repeating unit and the adjacent flanking run of serines are conserved with minimal variation among all dehydrins. Another repeating unit is flanked by the two copies of the lysine-rich unit, and varies in number from one to five copies. This latter repeating unit is less conserved than the former, varying even within a singly dehydrin. The messenger RNAs corresponding to each cDNA are abundant in dehydrating, but not in well-watered seedlings. The amino acid sequence of tryptic peptides from purified dehydration-induced proteins of corn established that the corn cDNAs correspond to a protein that is produced in abundance during the response of corn seedlings to dehydration.Abbreviations ABA abscisic acid - GA gibberellin - SDS sodium dodecyl sulfate - EDTA ethylenediaminetetraacetic acid - SSC 0.15 M NaCl, 0.015 M Na₃ citrate, pH 7.0 - ddH₂O double-distilled water - TES N-tris [hydroxymethyl]-methyl-2-aminoethanesulfonic acid     

Molecular Cloning and Characterization of a Novel Dehydrin Gene from Ginkgo biloba

A full-length cDNA encoding a dehydrin was cloned from the living fossil plant Ginkgo biloba by rapid amplification of cDNA ends (RACE). The cDNA, designated as GbDHN, was 813 bp long containing an open reading frame of 489 bp. The deduced GbDHN protein had 163 amino acid residues, which formed a 17 kDa polypeptide with a predicted isoelectric point (pI) of 5.75. GbDHN had an S-segment and a K-segment, indicative of dehydrins, but no Y-segments. Homology analysis indicated that the S-segment and K-segment of GbDHN shared identity with those of other reported dehydrins, indicating that GbDHN belonged to dehydrin superfamily. Genomic sequence of GbDHN was also cloned using genomic walker technology. By comparing genomic DNA with the cDNA, it was found that there was a 257-bp intron in this gene. Promoter analysis indicated that it contained six CAAT boxes, one TATA box, one ABRE box and one GC-motif in the 5′-flanking region. Southern blot analysis revealed that GbDHN belonged to a single copy gene family. RT-PCR analysis revealed that GbDHN constitutively expressed in stems and roots. The increased expression of GbDHN was detected when G. biloba seedlings were treated with exogenous abscisic acid (ABA), salt stress and drought stress. These results indicate that the GbDHN has the potential to play a role in response to ABA and environmental stresses that can cause plant dehydration.     

Identification and characterization of dehydrins in horse chestnut recalcitrant seeds

The fraction of heat-stable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stress-induced dehydrin-like polypeptides. In our experiments, in tissues of dormant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Low-molecular-weight heat-stable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not cross-react with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissue-specificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heat-stable proteins during the entire period of stratification and also radicle emersion. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cycloheximide, or α-amanitin, dehydrins did not disappeared from the fraction of heat-stable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could cross-react with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50-kD heat-stable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heat-sensitive proteins and was named as a dehydrin-like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heat-stable cytosolic proteins. The role and function of major heat-stable proteins in horse chestnut seeds are yet to be studied.     

GTP Promotes the Formation of Early-Import Intermediates But Is Not Required during the Translocation Step of Protein Import into Chloroplasts

Dehydrins are a family of proteins (LEA [late-embryogenesis abundant] D11) commonly induced by environmental stresses associated with low temperature or dehydration and during seed maturation drying. Our previous genetic studies suggested an association of an approximately 35-kD protein (by immunological evidence a dehydrin) with chilling tolerance during emergence of seedlings of cowpea (Vigna unguiculata) line 1393-2-11. In the present study we found that the accumulation of this protein in developing cowpea seeds is coordinated with the start of the dehydration phase of embryo development. We purified this protein from dry seeds of cowpea line 1393-2-11 by using the characteristic high-temperature solubility of dehydrins as an initial enrichment step, which was followed by three chromatography steps involving cation exchange, hydrophobic interaction, and anion exchange. Various characteristics of this protein confirmed that indeed it is a dehydrin, including total amino acid composition, partial amino acid sequencing, and the adoption of alpha-helical structure in the presence of sodium dodecyl sulfate. The propensity of dehydrins to adopt alpha-helical structure in the presence of sodium dodecyl sulfate, together with the apparent polypeptide adhesion property of this cowpea dehydrin, suggests a role in stabilizing other proteins or membranes. Taken together, the genetic, physiological, and physicochemical data are at this stage consistent with a cause-and-effect relationship between the presence in mature seeds of the approximately 35-kD dehydrin, which is the product of a single member of a multigene family, and an increment of chilling tolerance during emergence of cowpea seedlings.     

Proteins arising during the late stages of embryogenesis in Pisum sativum L.

Two abscisic acid (ABA)-responsive seed proteins, ABR17 and ABR18 (ABA-responsive 17000-M_r and 18000-M_r, respectively), previously found to be induced in cultured embryos of pea (Pisum sativum L.) are major components synthesised during normal seed desiccation. The ABR17 and ABR18 proteins showed different patterns of accumulation. The ABR18 protein was abundant in the testa during early seed development but in desiccating seed it was synthesised in the embryo, indicating spacial as well as temporal regulation of expression. The ABR18 protein was undetectable soon after germination but reappeared after adding ABA. The ABR17 protein was not detected in the testa but appeared in the embryo just prior to maximum fresh weight. The ABR17 protein continued to be synthesised during germination and was also present in non-stressed leaves. A high level of endogenous ABA or added ABA increased levels of translatable ABR17 mRNA. The ABR17 and ABR18 proteins were further characterised so as to help determine their structure and function. Neither protein appeared to contain a signal peptide but both proteins appeared to be glycosylated. The proteins had similar amino-acid compositions and limited Nterminal analysis showed 56% sequence identity. Neither protein had any significant N-terminal sequence homology to any of the late embryogenesis-abundant (LEA) proteins or dehydrins. Both proteins, however, show striking homology with a pea disease-resistance-response protein and the major birch pollen allergen, indicating that the ABR17 and ABR18 proteins may be members of a distinct group of stress-induced proteins.Abbreviations ABA (±) cis,trans-abscisic acid - ABR17 M_r-17200 ABA-responsive protein - ABR18 M_r-18 100 ABA-responsive protein - FW fresh weight - I_gG immunoglobulin G - LEA late embryogenesis-abundant - M_r apparent molecularmass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station.     

Expression of KS-type dehydrins is primarily regulated by factors related to organ type and leaf developmental stage during vegetative growth

The expression of a gene, designated as DHN10, was analyzed at the protein level in two Solanum species. The DHN10 protein displays some consensus amino acid sequences of dehydrins, termed K- and S-segments. Unlike most dehydrins, both segments occur only in single copies in the DHN10 sequence and the S-segment is at a C-terminal position. Database searches revealed that KS-type dehydrins constitute a specific subclass distributed in dicotyledons and monocotyledons. In Solanum tuberosum L. plants, a high DHN10 abundance was observed under control conditions, particularly in flowers, stems, tubers and young developing leaves. In other Solanaceae and in barley (Hordeum vulgare L.), the amount of DHN10 was much more elevated in young leaves than in old leaves. DHN10 abundance was investigated in two Solanum species subjected to low temperature or to drought. Under stress conditions, we observed substantially higher protein levels only in mature expanded leaves. These findings clearly indicate that KS-type dehydrins are present at a high level in the absence of stress during vegetative growth and that their expression is primarily regulated by factors related to organ type and to leaf development stage. A potential role for the DHN10 dehydrin during plant development and in tolerance to environmental stress is discussed.Abbreviations DHN10 Dehydrin protein of 10 kDa - His Histidine - KS-type dehydrin Dehydrin containing a single K-segment followed by a single S-segment - LEA Late embryogenesis abundant - NTS Nuclear targeting signal     

A shoot-specific mRNA from pea: nucleotide sequence and regulation as compared to light-induced mRNAs

The regulation of a mRNA encoding a shoot-specific polypeptide from developing pea seedlings was studied and compared to the regulation of mRNAs encoding two major light-induced nuclear-encoded polypeptides, the small subunit of the ribulose 1,5 biphosphate carboxylase (ssRuBPCase) and a polypeptide of the light-harvesting chlorophyll a/b complex (LHCP). By using cDNA clones as probes in Northern blottings of total cellular RNA it was found that both ssRuBPCase and LHCP mRNA could be induced in shoots by white and red light but to lower levels in roots and cotyledons. In contrast, the mRNA for the shoot-specific polypeptide was only found in shoots, and was present approximately two days after the start of germination. The shoot-specific mRNA sequence was predominantly found in stem tissue, irrespective of illumination, both in the young seedlings and adult plants. Only very low amounts could be detected in plumule and leaf. The shoot-specific sequence could also be detected in RNA isolated from developing shoots of another pea cultivar but not in those of other legumes and of cereals. The primary sequence of the complete coding portion and the deduced amino acid sequence of the mRNA encoding the shoot-specific polypeptide was determined. The observed codon usage is non-random and is consistent with data from other high plant genes. Possible polyadenylation signal sequences (AATAAG and AATAAT) were present at 55 and 124 bases 5′ of the poly(A) tail. The polypeptide encoded by the shoot-specific mRNA consists of 196 amino acids with a calculated molecular weight of 21 898. It contains a four times reiterated highly conserved unit of 26 amino acids. The NH₂-terminal end is highly hydrophobic and resembles a signal polypeptide.     

Development of Desiccation Tolerance during Embryogenesis in Rice (Oryza sativa) and Wild Rice (Zizania palustris) (Dehydrin Expression,Abscisic Acid Content,and Sucrose Accumulation)

The ability of seeds to withstand desiccation develops during embryogenesis and differs considerably among species. Paddy rice (Oryza sativa L.) grains readily survive dehydration to as low as 2% water content, whereas North American wild rice (Zizania palustris var interior [Fasset] Dore) grains are not tolerant of water contents below 6% and are sensitive to drying and imbibition conditions. During embryogenesis, dehydrin proteins, abscisic acid (ABA), and saccharides are synthesized, and all have been implicated in the development of desiccation tolerance. We examined the accumulation patterns of dehydrin protein, ABA, and soluble saccharides (sucrose and oligosaccharides) of rice embryos and wild rice axes in relation to the development of desiccation tolerance during embryogenesis. Dehydrin protein was detected immunologically with an antibody raised against a conserved dehydrin amino acid sequence. Both rice and wild rice embryos accumulated a 21-kD dehydrin protein during development, and an immunologically related 38-kD protein accumulated similarly in rice. Dehydrin protein synthesis was detected before desiccation tolerance had developed in both rice embryos and wild rice axes. However, the major accumulation of dehydrin occurred after most seeds of both species had become desiccation tolerant. ABA accumulated in wild rice axes to about twice the amount present in rice embryos. There were no obvious relationships between ABA and the temporal expression patterns of dehydrin protein in either rice or wild rice. Wild rice axes accumulated about twice as much sucrose as rice embryos. Oligosaccharides were present at only about one-tenth of the maximum sucrose concentrations in both rice and wild rice. We conclude that the desiccation sensitivity displayed by wild rice grains is not due to an inability to synthesize dehydrin proteins, ABA, or soluble carbohydrates.     

A comparative study of water distribution and dehydrin protein localization in maturing pea seeds

In this study, the distribution of water in pea seeds after harvesting at different seed stages was traced by magnetic resonance imaging (MRI). MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside seeds. The intensity of the signal coming from water declined from the inner to the outer part of cotyledon tissue. This spatial inhomogeneity of water signals inside cotyledons may be correlated with the gradient of storage substances accumulation within cotyledons. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of protovascular tissue of both the embryo axis and cotyledons. The temporal accumulation of two dehydrin proteins with molecular masses of 30 and 35kDa correlated well with seed desiccation. The pattern of dehydrin localization reflected the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles.     

Genetic variation in pea (Pisum) dehydrins: sequence elements responsible for length differences between dehydrin alleles and between dehydrin loci in Pisum sativum L.

 The electrophoretic patterns of dehydrins extracted from mature seeds of a range of pea (Pisum) species revealed extensive variation in dehydrin polypeptide mobility. Variation was also observed among lines of P. sativum. Crosses between lines with different dehydrin electrophoretic patterns produced F₁ seeds with additive patterns, and segregation in the F₂ generation was consistent with a 1 : 2 : 1 ratio, indicating allelic variation at each of two dehydrin loci (Dhn2, Dhn3). Genetic linkage was observed between Dhn2 and Dhn3, and the segregation ratios indicated preferential transmission of one allele at the Dhn3 locus. Dehydrin cDNA clones were characterised that encoded the allelic variants at Dhn2 and Dhn3. Their deduced amino-acid sequences were very similar to each other as well as to the product of the Dhn1 locus reported previously. Comparisons were made between the sequences of allelic variants at a single locus, and between the products of different loci. Differences in the electrophoretic mobilities between allelic variants at Dhn2 and Dhn3 were associated with differences in polypeptide length resulting principally from tandem duplications of 21 (Dhn2) or 24 (Dhn3) amino-acid residues. These duplications accounted for much of the difference in length between dehydrins encoded by the different loci. The conserved core of one of the duplicated regions varied in copy number, and small insertions/deletions of amino acids near this core also contributed to length variation both between allelic forms and between loci. Dehydrins possess characteristic highly conserved amino-acid sequence motifs, yet vary considerably in length. Mechanisms involving sequence duplication appear to be responsible for generating the length differences observed between allelic variants as well as between the products of different loci. Received: 12 June 1997 / Accepted: 29 October 1997     

Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development

We have analyzed the nucleotide sequence and accumulation of an mRNA which is prevalent in seeds of Brassica napus L. During normal development, the mRNA begins to accumulate during late embryogeny, is stored in dry seeds, and becomes undetectable in seedlings within 24 hours after imbibition. Moreover, abscisic acid treatment of embryos precociously induces or enhances accumulation of the mRNA. Nucleotide sequencing studies show that the deduced 30 kDa polypeptide has an unusual primary structure; the polypeptide possesses direct amino acid sequence repeats and is virtually entirely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Based upon the expression pattern and predicted polypeptide sequence, we conclude that the mRNA is encoded by a late embryogenesis-abundant (Lea) gene in B. napus.Abbreviations ABA abscisic acid - bp base pairs - DAF days after flowering - HAI hours after the start of imbibition - kb kilobase (pairs)     

Water Deficit in Pea Root Tips: Effects on the Cell Cycle and on the Production of Dehydrin-like Proteins

Dehydrin-like proteins have been detected in nuclei and cytoplasmof meristematic root tip cells from pea seedlings subjectedto slow dehydration at 90% relative humidity for 48 h or more.Evidence was gained from Western blotting and immunocytochemicalexperiments using an antibody raised against the conserved domainof dehydrin proteins. Flow cytometer analysis has shown thatcycling cells of root tip meristems from dehydrated seedlingsare mostly arrested in G2 phase. Other stress treatments thoughtto involve water depletion (osmotic stress, cold treatment)or to modulate cell response to water deficit (abscisic acid)gave less clear-cut results with all treatments lowering theproportion of cells entering the S phase, but without a definiteand persistent arrest in any preferential phase of the cycle.Possible interrelationships between G2 arrest and dehydrin productionare discussed. Cell cycle; dehydrins; flow cytometry; nuclei; pea; Pisum sativum L.; water stress     

Dehydrin-like proteins in castor bean seeds and seedlings are differentially produced in response to ABA and water-deficit-related stresses

The stress inducibility of dehydrin protein production in seedlingsof castor bean was analysed by subjecting them to ABA and variouswater-deficit-related treatments including desiccation, waterstress, high salt, high osmolarity, and low temperature. A furthergoal was to determine whether the immature seed (at stages priorto major dehydrin synthesis) would respond in a similar mannerto these stresses. A number of dehydrin-like proteins increasedin seedlings subjected to the various stress treatments. Inthe endosperm, these appear to be different from the dehydrin-relatedpolypeptides that are induced during late seed development andwhich persist following germination/growth of mature seeds.In the endosperm of seedlings, ABA, water stress and desiccationinduced the same dehydrin polypeptides, while high osmolarity,high salt and low temperature induced a different set. Stress-specificdifferences in dehydrin synthesis were also found in the cotyledonsand radicle of castor bean seedlings; however, dehydrins indu-cibleby exogenous ABA were consistently produced. Immature seedstreated with ABA or subjected to stress responded by producingdehydrin-like proteins associated with late development; however,the same proteins were induced following detachment of immatureseeds from the parent plant and maintenance on water. When seedlingswere exposed simultaneously to GA and either ABA, high salt,or low temperature, dehydrin production was suppressed. It isconcluded that dehydrin production in castor bean is tissue-specificand is dependent upon the physiological stage of the seed. Inthe endosperm, the response to different stresses may rely uponmore than one signal trans-duction pathway. Key words: Dehydrin, castor bean, ABA, desiccation     

High salinity induces dehydrin accumulation in Chenopodium quinoa Willd. cv. Hualhuas embryos

Background and Aims

Chenopodium quinoa can grow at altitudes of 3,600–4,000 masl and is adapted to the highly arid conditions typical of the salty soils in the South American Altiplano, with less than 250?mm of annual rain and temperatures below 0°C. The aim of the study was to investigate the effect of salinity on the dehydrin content of mature embryos harvested from salt-stressed Chenopodium quinoa cv. Hualhuas plants grown at 100 to 500?mM NaCl. To date, no studies exist on the dehydrins of seeds from salt-stressed plants, although dehydrins in the root, stems and leaves have been reported as an adaptation to water deficit produced by salinity.

Methods

Dehydrin-like protein detection was carried out with an antiserum raised against a highly-conserved lysine-rich 15-amino acid sequence known as the K-segment, which is capable of recognizing proteins immunologically related to the dehydrin family.

Results

Dehydrins were analyzed in embryos by both western blot and in situ immunolocalization. Western blot analysis detected at least four dehydrins (55, 50, 34, and 30?kDa) in seeds harvested from quinoa salt-stressed plants treated under a wide range of salinities. The 30?kDa dehydrin increased its accumulation in both 300 and 500?mM NaCl growth conditions as revealed by densitometric analyses. Dehydrin subcellular localization was mostly nuclear at 500?mM of NaCl. A phosphatase treatment of protein extracts caused a mobility shift of the 34 and 30?kDa dehydrin bands suggesting a putative modulation mechanism based on protein phosphorylation.

Conclusions

We propose that these novel observations regarding dehydrin accumulation, subcellular localization and phosphorylation state are related to the high salt stress tolerant phenotype previously reported on this cultivar.     

Sequence of a plant cDNA from Vicia faba encoding a novel Ran-related GTP-binding protein

A clone obtained from a broad bean (Vicia faba) developing cotyledon cDNA library contained the complete coding sequence of a polypeptide with very high homology to the small GTP-binding proteins Ran from human cells and Spi1 from yeast. These proteins belong to the ras superfamily of proteins involved in different basic cellular processes. The Ran/Spi1 proteins interact with a protein bound to DNA (RCC1) and are thought to function in the regulation of the cell cycle. The amino acid sequence of the obtained plant Ran-homologue, designated Vfa-ran, is 74% and 76% identical to Ran and Spi1, respectively. The five functional, conserved domains of ras-related proteins are present in the Vfa-ran sequence. However, as in Ran/Spi1 the C-terminus of Vfa-ran is very acidic and lacks the Cys motif for isoprenylation.Northern blotting revealed a corresponding mRNA expression in broad bean roots, leaves, and cotyledons with the highest level in roots.