Storage Protein Gene Expression Research Articles

Cloning and computer analysis of the promoter region of the legumin-like storage protein gene from buckwheat, Fagopyrum esculentum Moench

Using the modified 5?-RACE approach, a fragment containing the 955 bp long 5?- regulatory region of the buckwheat storage globulin gene (FeLEG1) has been amplified from the genomic DNA of buckwheat. The entire fragment was sequenced and the sequence analyzed by computer prediction of cis-regulatory elements possibly involved in tissue specific and developmentally controlled seed storage protein gene expression. The promoter obtained might be interesting not only for fundamental research, but also as a useful tool for biotechnological application.

The 5 UTR negatively regulates quantitative and spatial expression from the ABI3 promoter

The involvement of transcription factors Arabidopsis abscisic acid-insensitive3 (ABI3), maize viviparous1 (VP1) and Phaseolus vulgaris ABI3-like factor (PvALF) in the spatial control of storage protein gene expression is well established. However, little insight exists as to how they are themselves regulated. To address this, a 5.15 kb ABI3 upstream sequence including a 4.6 kb full-length promoter and 519 bp of 5'-untranslated region (UTR) was used to drive either beta-glucuronidase (GUS) or green fluorescent protein (GFP) expression in Arabidopsis. Expression from the full-length (- 4630/ + 519ABI3 ) and various 5'-truncated promoters was detected during embryogenesis in all lines, except those transgenic for promoter elements shorter than 364 bp. Two upstream activating regions, -3600 to -2033 and -2033 to -882, enhanced GUS expression in seeds. The -882 to -364 region was sufficient to confer seed-specific expression of GUS when fused to a - 64/ + 6CaMV 35S minimal promoter. Expression from the ABI3 promoter constructs was seed-specific, except in the presence of exogenous abscisic acid (ABA) (>0.3 microM), when GUS expression was detected in seedling roots. Excision of a 405 bp region containing three upstream open reading frames (uORFs) from the 5'-UTR dramatically increased GUS expression and debilitated constraint of reporter expression in roots. Negative regulation of ABI3 expression by the 5'-UTR may involve a post-transcriptional mechanism analogous to that of tumor suppressor genes which also bear long, uORF-containing, 5'-UTRs, or through interactions with RNA-binding proteins.

Regulation of storage protein gene expression in Arabidopsis.

The expression of seed storage proteins is under tight developmental regulation and represents a powerful model system to study the regulation of gene expression during plant development. In this study, we show that three homologous B3 type transcription factors regulate the model storage protein gene, At2S3, via two distinct mechanisms: FUSCA3 (FUS3) and LEAFY COTYLEDON2 (LEC2) activate the At2S3 promoter in yeast suggesting that they regulate At2S3 by directly binding its promoter; ABSCISIC ACID INSENSITIVE3 (ABI3), however, appears to act more indirectly on At2S3, possibly as a cofactor in an activation complex. In accordance with this, FUS3 and LEC2 were found to act in a partially redundant manner and differently from ABI3 in planta: At2S3 expression is reduced to variable and sometimes only moderate extent in fus3 and lec2 single mutants but is completely abolished in the lec2 fus3 double mutant. In addition, we found that FUS3 and LEC2 expression patterns, together with an unsuspected regulation of FUS3 by LEC2, enable us to explain the intriguing expression pattern of At2S3 in lec2 or fus3 single mutants. Based on these results, we present a model of At2S3 regulation and discuss its implications for other aspects of seed maturation.

Synergistic Activation of Seed Storage Protein Gene Expression in Arabidopsis by ABI3 and Two bZIPs Related to OPAQUE2

The expression of many seed storage protein genes in cereals relies on transcription factors of the bZIP class, belonging to the maize OPAQUE2 family. Here, we describe a survey of such factors in the genome of Arabidopsis thaliana, and the characterization of two of them, AtbZIP10 and AtbZIP25. Expression analysis by in situ hybridization shows that the occurrence of their mRNAs in the seed starts from early stages of development, peaks at maturation, and declines later in seed development, matching temporally and spatially those of the seed storage protein genes encoding 2S albumins and cruciferins. Gel mobility shift assays showed that AtbZIP10 and AtbZIP25 bind the ACGT boxes present in At2S and CRU3 promoters. Moreover, using the yeast two-hybrid system we show that AtbZIP10 and AtbZIP25 can interact in vivo with ABI3, an important regulator of gene expression in the seed of Arabidopsis. Transient expression analyses of a reporter gene under the control of the At2S1 promoter in transgenic plants overexpressing ectopically AtbZIP10, AtbZIP25, and ABI3 reveal that none of these factors could activate significantly the reporter gene when expressed individually. However, co-expression of AtbZIP10/25 with ABI3 resulted in a remarkable increase in the activation capacity over the At2S1 promoter, suggesting that they are part of a regulatory complex involved in seed-specific expression. This study shows a common mechanism of ABI3 in regulating different seed-specific genes through combinatorial interactions with particular bZIP proteins and a conserved role of O2-like bZIPs in monocot and dicot species.

Storage proteins.

Plants accumulate storage substances such as starch, lipids and proteins in certain phases of development. Storage proteins accumulate in both vegetative and reproductive tissues and serve as a reservoir to be used in later stages of plant development. The accumulation of storage protein is thus beneficial for the survival of plants. Storage proteins are also an important source of dietary plant proteins. Here, we summarize the genome organization and regulation of gene expression of storage protein genes in Arabidopsis.

QTL analysis of proteome and transcriptome variations for dissecting the genetic architecture of complex traits in maize.

In this review, we present some studies on genetic analysis of proteome and transcriptome variations, which exemplify new strategies for a better understanding of the molecular and genetic bases of complex traits. A large genetic variability was revealed at the proteome expression level, which raised the possibility to predict phenotypical performance on the basis of gene product variability. This approach yielded limited results, but could be re-newed by extensive identification of proteins now allowed by mass spectrometry. The dissection of the genetic basis of the variation of individual protein amounts proves very powerful to select 'candidate' proteins, physiologically relevant for a given phenotypical trait, as shown by a study on the effect of water stress in maize. In order to investigate factors of grain quality in maize, we selected a regulatory locus known to control the expression of several storage protein genes, Opaque-2, and investigated the relationships between variability in zein amount and composition and the molecular polymorphism at this locus. Moreover, a QTL analysis revealed that the variability in Opaque-2 transcript abundance was controlled by several polymorphic trans-acting regulators unlinked to the Opaque-2 structural gene. Such genetic approaches should represent additional tools for physiological analysis of the huge amounts of data generated by transcritome and proteome projects.

Ecotypic and genetic variation in poplar bark storage protein gene expression and accumulation.

Bark storage proteins (BSP) store nitrogen (N) translocated from senescing leaves in autumn, and supply reduced N for spring growth. Expression of bsp and BSP accumulation are associated with short day photoperiod. To determine if photoperiod-associated bsp expression varies among poplars native to different latitudes, Populus deltoides Bartr. clones originating from six latitudes were grown under natural conditions at a common location. Relative amounts of BSP mRNA in these clones were measured at 2-week intervals from August 7 to October 16. The date of maximum BSP mRNA accumulation was correlated with latitude of origin, and maximum accumulation of BSP mRNA occurred earlier in clones native to northern latitudes than in clones native to southern latitudes. This pattern of variation is consistent with photoperiodic responses of plants native to temperate climates. Genotypic variations in BSP accumulation, bark protein concentration and bark N concentration were compared among clones of six hybrid poplar (Populus trichocarpa Torr. and Gray x P. deltoides) full-sib families (three F(2) families, two F(1) families and one BC(1) family) after 6 weeks in a short day photoperiod and at midwinter. Significant differences in BSP accumulation occurred among clones within four of the six full-sib families after 6 weeks in a short day photoperiod and also at midwinter for outdoor-grown plants. Bark protein and bark N concentrations also varied significantly among clones within certain families. In general, the greatest variation was found in F(2) and BC(1) families. Within several families, relative BSP amounts were positively correlated with bark protein concentration and total bark N concentration. These results indicate a role of photoperiod in regulating bsp expression and demonstrate a genetic component underlying seasonal BSP accumulation. The results could have significance in selecting for clones with improved N storage capacity and N-use efficiency.

A Rice Functional Transcriptional Activator, RISBZ1, Responsible for Endosperm-specific Expression of Storage Protein Genes through GCN4 Motif

The GCN4 motif, a cis-element that is highly conserved in the promoters of cereal seed storage protein genes, plays a central role in controlling endosperm-specific expression. This motif is the recognition site for a basic leucine zipper transcriptional factor that belongs to the group of maize Opaque-2 (O2)-like proteins. Five different basic leucine zipper cDNA clones, designated RISBZ1-5, have been isolated from a rice seed cDNA library. The predicted gene products can be divided into two groups based on their amino acid sequences. Although all the RISBZ proteins are able to interact with the GCN4 motif, only RISBZ1 is capable of activating (more than 100-fold expression) the expression of a reporter gene under a minimal promoter fused to a pentamer of the GCN4 motif. Loss-of-function and gain-of-function experiments using the yeast GAL4 DNA binding domain revealed that the proline-rich N-terminal domain (27 amino acids in length) is responsible for transactivation. The RISBZ1 protein is capable of forming homodimers as well as heterodimers with other RISBZ subunit proteins. RISBZ1 gene expression is restricted to the seed, where it precedes the expression of storage protein genes. When the RISBZ1 promoter was transcriptionally fused to the beta-glucuronidase reporter gene and the chimeric gene was introduced into rice, the beta-glucuronidase gene is specifically expressed in aleurone and subaleurone layer of the developing endosperm. These findings suggest that the specific expression of transcriptional activator RISBZ1 gene may determine the endosperm specificity of the storage protein genes.

Differences in sucrose-to-starch metabolism of Solanum tuberosum and Solanum brevidens

Sucrose-to-starch metabolism of the tuberising species Solanum tuberosum and that of the non-tuberising Solanum brevidens was studied using in vitro stem cuttings cultured under tuber inducing conditions. The shoots growing from axillary buds of S. brevidens and the in vitro induced stolons and tubers of S. tuberosum were characterised with respect to their carbohydrate composition by measuring the glucose, fructose, sucrose and starch contents. Expression of the genes encoding vacuolar- and cell-wall-bound invertases, vascular- and tuber-specific sucrose synthases was studied by Northern blot hybridisation. Enzyme activity patterns of alkaline and acid invertases, sucrose synthase, hexokinase and ADP-glucose pyrophosphorylase were determined in both species. Major differences between S. brevidens and S. tuberosum were detected in the starch content, in the expression of cell-wall-bound invertase (cwInv), tuber-specific sucrose synthase (SusyI) and in the activities of sucrose synthase and ADP-glucose pyrophosphorylase that were low in S. brevidens. Genes homologous to the cwInv and SusyI were detected in S. brevidens, however, expression of the SusyI, unlike in S. tuberosum, was not sucrose-inducible in the non-tuberising species. These data together with previous findings (Z. Bánfalvi, A. Molnár, G. Molnár, L. Lakatos, L. Szabó, FEBS Lett. 383 (1996) 159–164) further support the idea that cell-wall-bound invertase, tuber-specific sucrose synthase and ADP-glucose pyrophosphorylase are important elements of regulatory and biosynthetic pathways resulting in storage starch synthesis and in the expression of tuber storage protein genes. The cwInv mRNA could be detected only in developing stolons and, thus, it can be a good molecular marker of stolon growth.

The effect of modifying carbohydrate metabolism on seed protein gene expression in peas

Mutations at the r locus in peas (Pisum sativum L.) affect starch content and composition by altering the activity of a specific isoform (SBEI) of starch-branching enzyme. There are several pleiotropic consequences of homozygosity for a mutant allele at the r locus, including seed wrinkling, and increased lipid and sugar concentrations that are thought to be, at least in part, a consequence of the re-partitioning of carbon that is incorporated into starch in the wild-type line. Mutant alleles at the r locus also have an effect on storage protein gene expression, apparently through the specific destabilization of legumin mRNAs in the high-sugar environment of the developing rr seed; the amounts of vicilin and convicilin mRNAs are unaffected. Other wrinkled-seeded mutants have been identified that perturb starch synthesis and result in elevated sugar levels in developing seeds; these include rb, a mutation in one of the structural genes for ADPglu-cose pyrophosphorylase. The recent production of a series of near-isogenic mutant and double mutant backcross Unes for r and rb, and of near-isogenic lines containing four additional mutations that influence starch synthesis (lam,rug-3, rug-4 and rug-5, defective in a granule-bound starch synthase, plastidial phosphoglucomutase, sucrose synthase and a soluble starch synthase, respectively) has provided the opportunity to examine the expression of legumin and vicilin genes in a range of different near-isogenic lines with different patterns of carbohydrate metabolism. We report and discuss here analyses of legumin and vicilin deposition in pea genotypes RRRbRb (wild-type round-seeded), rrRbRb, RRrbrb, rrrbrb (all wrinkled-seeded), the homozygous wrinkled-seeded mutants rug-3, rug-4 and rug-5 and the round-seeded low-amylose mutant lam.

Analysis of the Mechanisms Underlying Regulation of the Soybean Seed Storage Protein Gene Expression in Response to Sulfur Nutritional Conditions

Analysis of the Mechanisms Underlying Regulation of the Soybean Seed Storage Protein Gene Expression in Response to Sulfur Nutritional Conditions

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2.

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5'-TGTAAAG-3') found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

The same nuclear proteins bind to the 5'-flanking regions of genes for the rice seed storage protein: 16 kDa albumin, 13 kDa prolamin and type II glutelin.

Expression of rice seed storage-protein genes is dramatically regulated over a short period of seed maturation. To characterize the expression mechanism of the rice seed storage protein genes, their expression of major storage protein genes (16 kDa albumin, 13 kDa prolamin and type II glutelin) were compared by RNA blot analysis. Their coordinate expression suggests that the transcriptional regulatory machinery is shared among the glutelin, prolamin and albumin-genes. We isolated two novel genomic genes for prolamins (PG5a and PG5b) and obtained the promoter region of the glutelin gene by PCR. The 5'-flanking regions of these three rice seed storage protein genes were found to contain some similar conserved sequences. Nuclear extract partially purified from maturing rice seeds was used for the gel shift assay of the 5' region of the RA gene. We identified two DNA sequences of RA gene which were recognized by independent DNA-binding proteins. The complexes of these DNA sequences and DNA-binding proteins were inhibited by the fragments containing the 5' regions of the prolamin and glutelin genes, suggesting that these three genes share transcription factors.

The maize transcription factor Opaque-2 activates a wheat glutenin promoter in plant and yeast cells

The promoter of the wheat low-molecular-weight glutenin (LMWG1D1) gene contains a cis element called the GCN4 like motif (GLM) which has low homology to one class of binding site for the maize endosperm-specific b-ZIP transcription factor Opaque-2 (O2). Previous work has shown that the GLM element interacts with the nuclear factor ESBFII during wheat endosperm development at the time of maximum transcription of the LMWG1D1 gene. In this paper we demonstrate that O2 binds to the GLM element and can activate high levels of transcription from the wheat GLM in transient assays in plant protoplasts and in yeast cells. Lower levels of O2 activation through the GLM element in yeast containing a defective GCN4 gene showed that GCN4 was necessary for high levels of O2 transcriptional activation, indicating that O2 may need to heterodimerise with GCN4 to activate transcription in yeast. These observations provide evidence that the GLM represents a new type of O2 DNA-binding site, and support a postulate that an O2 homologue may activate endosperm-specific expression of wheat storage protein genes.

Transient expression of storage-protein genes during early embryogenesis of Vicia faba: synthesis and metabolization of vicilin and legumin in the embryo, suspensor and endosperm

Seed storage proteins are thought to be accumulated exclusively in the cell-expansion phase of embryogenesis and metabolized during germination and seedling growth. Here we show by a sensitive immunohistological technique that the two Vicia faba L. storage proteins vicilin and legumin are accumulated in substantial amounts in the suspensor and coenocytic endosperm and to a lesser extent in the mid-globular embryo. Both proteins appear and disappear at precise stages specific for each tissue. In the endosperm the accumulation starts around 12 d after pollination (DAP). After a maximum attained at 14–15 DAP, storage proteins are degraded within about 4 d. Accumulation is restricted to that part of the endosperm which covers the embryo and displays the highest levels of endoploidy (maximum 96n). In all other parts of the endosperm, storage proteins do not appear to accumulate, although storage-protein-specific mRNA synthesis takes place. In the suspensor, storage proteins are already observed at 6 DAP and disappear very quickly at approximately 10 DAP. Low amounts of legumin and vicilin are also detectable in the mid-globular embryo, but disappear completely as the embryo enters the heart stage. We conclude that storage proteins of Vicia faba accumulated transiently during early seed development are used as nutritive reserves for the growing embryo.

Isolation and characterisation of cDNA clones representing the genes encoding the major tuber storage protein (dioscorin) of yam (Dioscorea cayenensis Lam.)

cDNA clones encoding dioscorins, the major tuber storage proteins (M(r) 32,000) of yam (Dioscorea cayenesis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of M(r) 30,015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.

Effect of abscisic acid, osmoticum and desiccation on 11S storage protein gene expression in somatic embryos of white spruce

The effect of ABA, non-permeating osmoticum and late stage maturation drying on 11S storage protein gene expression in white spruce somatic embryos was investigated. A partial cDNA clone encoding the β-polypeptide of white spruce 11S legumin-like seed storage protein (named β-coniferin) was employed as a probe. Northern blot analyses demonstrated that in somatic embryos matured with 16 μM ABA and 7.5% PEG, the levels and patterns of accumulation of transcripts during early and mid-stages of embryogenesis were comparable to those observed in zygotic embryos at a similar morphological stage. Steady-state levels of coniferin transcripts declined when late stage somatic embryos were exposed to a desiccation treatment. Levels of coniferin transcripts in somatic embryos matured with 16 μM ABA and low osmoticum were substantially lower than those of somatic embryos matured with 7.5% PEG and 16 μM ABA. Increasing concentrations of ABA resulted in higher levels of accumulation of coniferin transcripts, however, the levels in somatic embryos matured with 32 μM ABA and low osmoticum remained lower than those of somatic embryos matured on 16 μM and 7.5% PEG indicating that the latter treatment is optimum for induction and accumulation of coniferin transcripts. The similarity of 11S storage protein gene expression in somatic embryos with zygotic embryos, along with their sensitivity to ABA and osmoticum indicates that somatic embryos may provide a useful system to facilitate studies of gene regulation in conifers.

Control of Seed Storage Protein Gene Expression: New Aspects on an Old Problem

Legume storage proteins are thought to be synthesized embryo-specifically during the cell-expansion phase of development and stored and metabolized not before seed germination. Instead we have shown that Vicia faba legumin and vicilin storage proteins are synthesized early in development in the suspensor, the endosperm, the globular stage embryo and even in the thin-walled parenchyma cells of the maternal seed coat. The proteins appear and disappear at precise developmental stages specific for each tissue before they again and finally accumulate in cotyledon-stage embryos. The final expression level of the V. faba legumin B4 gene is influenced by cis-regulatory elements upstream and downstream of the transcription initiation site acting at both the transcriptional and posttranscriptional/translational level. Studies in transgenic tobacco seeds revealed complex interactions between homologous transgene sequences resulting in both trans-suppression and trans-stimulation of legumin B4 target transgen expression. Seed protein gene expression is not only developmentally but also metabolically regulated. In Vicia faba cotyledons incubated ex planta for 2 to 4 days osmotic pressure and soluble sugar concentrations differentially influence storage protein mRNA levels. Legumin B mRNA levels are dramatically reduced by high hexose concentrations but only little influenced by osmotic values whereas vicilin mRNA levels strongly react to the osmotic value of the medium but much less to sugar concentrations. Particle bombardment experiments with legumin B4 promoter/GUS constructs suggest that 5'-flanking, cis-regulatory elements are involved in the response. The complexity of the regulatory network is underlined by the fact that in Arabidopsis thaliana the FUS3 locus specifically regulates storage protein synthesis but also induces changes in lipid and carbohydrate metabolism seemingly by partially omitting the dessication phase of development

Conifer zygotic embryogenesis, somatic embryogenesis, and seed germination: Biochemical and molecular advances

AbstractThe development of techniques for somatic embryogenesis in conifers has led to rapid advances in the ability to culture conifer tissuein vitro.Somatic embryogenesis now provides a means to clonally propagate commercially valuable conifers of several species and offers anin vitroexperimental system for studying embryogenesis. As a result of these developments, the conifer zygotic and somatic embryo system has recently attracted the attention not only of the cell biologist but also of biochemists and molecular biologists. In this review I have attempted to consolidate the recent information on ultrastructure, biochemical and molecular aspects of conifer zygotic and somatic embryogenesis. In the first section, salient features of zygotic embryogenesis are highlighted followed by biochemical and ultrastructural characterization of development with reference to storage reserve deposition. In the second section, detailed characterization of seed storage proteins, changes in gene expression with emphasis on storage protein genes and Lea genes during zygotic and somatic embryogenesis are described. Effect of culture treatments such as abscisic acid, osmoticum and desiccation on storage protein gene expression in somatic embryos is discussed. Based on these studies, comparisons are presented between angiosperm and gymnosperm embryogeny regarding the unique morphological and developmental patterns of conifer embryogenesis. Finally, a brief discussion of recent progress in biochemical and molecular characterization of conifer seed germination is presented.

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.