Leader Intron Research Articles

Waxy gene haplotypes: Associations with apparent amylose content and the effect by the environment in an international rice germplasm collection

Apparent amylose content (AAC), the key determinant of rice end-use quality attributes, is primarily controlled by the Waxy gene which codes for granule bound starch synthase (GBSS). We examined the combination of sequence variation in the Waxy gene and environmental effects, and their associations with AAC using 171 rice accessions originating from 43 countries. The combination of two single-nucleotide-polymorphism (SNP) markers in the Waxy gene allows for the identification of three marker haplotypes in this gene. The first SNP is at the leader intron splice site (In1 SNP), and the second polymorphism is in exon 6. The haplotypes explained 86.7% of the variation in AAC and discriminated the three market classes of low, intermediate and high AAC rice from each other. The environment affected the AAC of all haplotypes. Higher air temperature during grain development associated with a decrease in AAC of low and intermediate AAC-types, but with an increase in AAC of high AAC-type. The association of AAC with several Waxy RM190 microsatellite-(CT n ) alleles in combination with the In1 SNP was also examined. In conclusion, the Waxy haplotypes studied appear to be useful markers for selecting the AAC of breeding lines developed from the world's rice germplasm.

The intron of the Arabidopsis thaliana COX5c gene is able to improve the drought tolerance conferred by the sunflower Hahb-4 transcription factor

Hahb-4 is a member of Helianthus annuus (sunflower) subfamily I of HD-Zip proteins. Transgenic Arabidopsis thaliana plants constitutively expressing this gene exhibit a strong tolerance of water stress in concert with morphological defects and a delay in development. In order to obtain a drought-tolerant phenotype without morphological associated phenotype, several stress inducible promoters were isolated and transgenic plants expressing Hahb-4 controlled by them were obtained and analyzed. These plants showed unchanged morphology in normal growth conditions and enhanced drought tolerance compared with non-transformed plants, but no as high as the one exhibited by the constitutively transformed genotype. A chimerical construction between the Hahb-4 promoter and the leader intron of the Arabidopsis Cox5c gene was made either directing gus or Hahb-4 expression. GUS activity increased in transgenic plants after induction, showing the same distribution pattern as in plants transformed with a construction lacking the intron. Transgenic plants, bearing the chimerical construct, are indistinguishable from wild type plants in normal growth conditions whereas the water stress tolerance achieved was as strong as the one shown by the constitutive genotype. This enhanced stress tolerance seemed to be due to a combination of an increase in transcription and translation rates in comparison to those of plants transformed with the Hahb-4 promoter. Similar strategies could be applied in the future for the obtaining of suitable promoters responsive to other external agents.

The M flax rust resistance pre-mRNA is alternatively spliced and contains a complex upstream untranslated region

Alternative splicing is an important step in controlling gene expression and has been shown to occur for a number of plant disease resistance (R) genes. The specific biological role of alternatively spliced transcripts from most R genes is unknown, yet in two cases it is clear that functional disease resistance cannot be activated without them. We report 12 splice isoforms of the M flax rust resistance gene, a TIR-NBS-LRR class of R gene. Collectively, these isoforms are predicted to encode at least nine different polypeptide products, only one of which is a full length peptide believed to confer functional M gene-specific disease resistance. An additional intron to that previously described was found in the 5' untranslated region. Splicing of this leader intron removes an upstream ORF (muORF) sequence. In some transcripts the leader intron is retained and in this case we predict negligible translation initiation of the full length M gene-encoding ORF. The majority of the alternatively spliced isoforms of M would encode truncated TIR and TIR-NBS containing proteins. Although the role of alternative splicing and the existence and function of the products they encode is still unclear, the complexities of the splicing profile, and the 5' UTR of the M gene, are likely to serve in mechanisms to regulate R protein levels.

Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L.

Cardosin A and cardosin B are two aspartic proteases mainly found in the pistils of cardoon Cynara cardunculus L., whose flowers are traditionally used in several Mediterranean countries in the manufacture of ewe's cheese. We have been characterizing cardosins at the biochemical, structural and molecular levels. In this study, we show that the cardoon aspartic proteases are encoded by a multigene family. The genes for cardosin A and cardosin B, as well as those for two new cardoon aspartic proteases, designated cardosin C and cardosin D, were characterized, and their expression in C. cardunculus L. was analyzed by RT-PCR. Together with cardosins, a partial clone of the cyprosin B gene was isolated, revealing that cardosin and cyprosin genes coexist in the genome of the same plant. As a first approach to understanding what dictates the flower-specific pattern of cardosin genes, the respective gene 5' regulatory sequences were fused with the reporter beta-glucuronidase and introduced into Arabidopsis thaliana. A subsequent deletion analysis of the promoter region of the cardosin A gene allowed the identification of a region of approximately 500 bp essential for gene expression in transgenic flowers. Additionally, the relevance of the leader intron of the cardosin A and B genes for gene expression was evaluated. Our data showed that the leader intron is essential for cardosin B gene expression in A. thaliana. In silico analysis revealed the presence of potential regulatory motifs that lay within the aforementioned regions and therefore might be important in the regulation of cardosin expression.

Overlapping expression patterns among the genes encoding Arabidopsis chromosomal high mobility group (HMG) proteins

High mobility group (HMG) proteins are usually considered ubiquitous components of the eukaryotic chromatin. Using HMG gene promoter- GUS reporter gene fusions we have examined the expression of the reporter gene in transgenic Arabidopsis plants. These experiments have revealed that the different HMGA and HMGB promoters display overlapping patterns of activity, but they also show tissue- and developmental stage-specific differences. Moreover, leader introns that are present in some of the HMGB genes can modulate reporter gene expression. The differential HMG gene expression supports the view that the various HMG proteins serve partially different architectural functions in plant chromatin.

An ACT-like Domain Participates in the Dimerization of Several Plant Basic-helix-loop-helix Transcription Factors

The maize basic-helix-loop-helix (bHLH) factor R belongs to a group of proteins with important functions in the regulation of metabolism and development through the cooperation with R2R3-MYB transcription factors. Here we show that in addition to the bHLH and the R2R3-MYB-interacting domains, R contains a dimerization region located C-terminal to the bHLH motif. This protein-protein interaction domain is important for the regulation of anthocyanin pigment biosynthesis by contributing to the recruitment of the C1 R2R3-MYB factor to the C1 binding sites present in the promoters of flavonoid biosynthetic genes. The R dimerization region bares structural similarity to the ACT domain present in several metabolic enzymes. Protein fold recognition analyses resulted in the identification of similar ACT-like domains in several other plant bHLH proteins. We show that at least one of these related motifs is capable of mediating homodimer formation. These findings underscore the function of R as a docking site for multiple protein-protein interactions and provide evidence for the presence of a novel dimerization domain in multiple plant bHLH proteins.

AtMHX is an auxin and ABA-regulated transporter whose expression pattern suggests a role in metal homeostasis in tissues with photosynthetic potential.

AtMHX is a vacuolar transporter encoded by a single gene in Arabidopsis thaliana (L.) Heynh. It exchanges protons with Mg2+, Zn2+, and Fe2+ ions. Proper homeostasis of these metals is essential for photosynthesis and numerous enzymatic reactions. In particular, very little is known about mechanisms involved in Mg2+ homeostasis in plants. Expression analysis using reporter-gene constructs suggested that AtMHX functions in metal homeostasis mainly in tissues with photosynthetic potential. This balancing is conducted by expression in the vascular region, the cortex of stems, trichomes, and hydathodes. Expression in stems is developmentally regulated, suggesting that minerals are accumulated in the upper regions of young stems, and are released during silique development. Mineral content in different stem parts was consistent with this possibility. Expression was induced by auxin and ABA, but not by the metal content of the growth medium, suggesting that expression is mainly regulated by endogenous developmental programs. AtMHX exhibits two distinguished regulatory properties. Its leader intron is absolutely essential for expression, and mediates an 86-fold enhancement of expression. This enhancement is the highest reported thus far for any dicot intron. Another remarkable feature is that a repetitive genomic element of 530 bp (or part of it) functions as an enhancer.

Functional analysis of DNA sequences controlling the expression of the rice OsCDPK2 gene

Plant calcium-dependent protein kinases (CDPKs) are involved in calcium-mediated signal transduction pathways. Their expression is finely tuned in different tissues and in response to specific signals, but the mechanism of such a regulation is still largely unknown. OsCDPK2 gene expression is modulated in vivo during rice (Oryza sativa L.) flower development and is downregulated by white light in leaves. In order to identify OsCDPK2 regulatory sequences, we amplified and cloned both the 5' and 3'-flanking regions of the gene. Sequence analysis revealed that the leader sequence is interrupted by an intron, whose regulatory role was investigated. Different ss-gucuronidase (GUS) expression vectors, carrying combinations of the putative OsCDPK2 regulatory regions, were generated and GUS expression was analyzed both in transient assays and in transgenic rice plants. The whole 5'-flanking sequence was able to drive GUS expression in rice calli and leaves transiently transformed with the biolistic technique. Analysis of the GUS expression pattern in transgenic plants revealed strong activity in root tips, leaf veins and mesophyll cells, in flower reproductive organs and in mature pollen grains. Expression was also shown to be subject to an intron-mediated enhancement (IME) mechanism, since the deletion of the leader intron sequence from chimeric OsCDPK2::GUS plasmids almost completely abolished GUS activity. Furthermore, in transiently transformed leaves, GUS expression driven by the OsCDPK2 promoter-leader region was constitutively observed regardless of light or dark exposure. Light-regulated expression was restored by inserting the OsCDPK2 3' untranslated region (3'UTR) downstream of the chimeric OsCDPK2::GUS transcription unit, suggesting that light down-regulation is mediated by a mechanism driven by the 3'UTR.

The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promotes high-level expression by increasing transcript abundance and translation efficiency

The involvement of regions located upstream of the translation start site in the expression of two Arabidopsis thaliana nuclear COX5c genes encoding subunit 5c of mitochondrial cytochrome c oxidase has been analysed. It was observed that these regions, which include a leader intron, direct the tissue-specific expression of the gus reporter gene, mainly in root and shoot meristems, actively growing tissues and vascular strands. Expression was also observed in flowers, specifically localized in anthers, stigma, and the receptacle, and in developing seeds. GUS activity measurements in protein extracts from transformed plants indicated that expression levels are higher than those observed with the constitutive CaMV 35S promoter. Removal of the leader intron produced a significant decrease in expression to values only slightly higher than those observed with a promoterless gus gene. Histochemical staining of plants transformed with the intronless construct revealed expression only in pollen, suggesting that regulatory elements capable of directing pollen-specific expression are present upstream of the intron. The COX5c-2 intron also increased GUS expression levels when fused in the correct orientation with the promoter of the unrelated COX5b-1 gene. Comparison of GUS activity values with the transcript levels suggests that the intron also increases translation efficiency of the corresponding mRNA. The results obtained point to an essential role of the intron present in the 5'-non-coding region of all known COX5c genes in directing the expression of these genes in plants.

A leader intron and 115-bp promoter region necessary for expression of the carnation S-adenosylmethionine decarboxylase gene in the pollen of transgenic tobacco

The expression of CSDC9 encoding S-adenosylmethionine decarboxylase (SAMDC) is developmentally and spatially regulated in carnation. To examine the regulation of the SAMDC gene, we analyzed the spatial expression of CSDC9 with a 5′-flanking β-glucuronidase fusion in transgenic tobacco plants. GUS was strongly expressed in flower, pollen, stem and vein of cotyledons. Expression in both anther and stigma was under developmental control; analysis of a series of mutants with deletions of the 5′-flanking region demonstrated differential activation in petal, anther, stigma and pollen grains. All the major cis-regulatory elements required for pollen-specific transcription were located in the upstream region between −273 and −158. This region contains four putative elements related to gibberellin induction (pyrimidine boxes, TTTTTTCC and CCTTTT) and pollen-specific expression (GTGA and AGAAA). In addition, the first 5′-leader intron was necessary for tissue-specific expression.

Differential Expression of Carotenoid Biosynthesis Genes among Different Colored Flesh in Watermelons [Citrullus lanatus (Thunb)

Carotenoids are plant compounds that serve a variety of essential functions in the plant and have also been found to have several health-promoting activities in humans. Carotenoids found in watermelon (Citrullus lanatus) flesh are responsible for the various colors such as red, yellow and orange. Previous inheritance studies of flesh color revealed that six genes were involved in color determination. The relationship and interaction of these genes suggests that some color-determining genes may be the result of mutations on the structural genes encoding enzymes in the carotenoid biosynthesis pathway. In this study we were able to isolate and sequence six genes encoding enzymes involved in the carotenoid biosynthetic pathway, and determine their expression in different colored watermelon fruit. The cDNA was synthesized from total RNA using RACE (Rapid Amplification of cDNA ends) kit (SMART RACE cDNA Amplification Kit; Clontech, Palo Alto, Calif.). Degenerate primers were designed based on published homologous genes from other species and were used to isolate gene fragments and full-length cDNAs of phytoene synthase, phytoene desaturase, _-carotene desaturase, β-cyclase, β-carotene hydroxylase and zeaxanthin expoxidase. RT-PCR was carried out to examine any differential expression of cloned genes in white, yellow, orange and red-fleshed watermelon. All cloned enzyme-encoding genes were expressed regardless of flesh colors. These results indicate that carotenoid biosynthesis may be regulated at the post-transcriptional level. One interesting feature supports this hypothesis. In case of β-cyclase, a 229-bp leader intron was identified, and an unspliced mRNA with this leader intron existed dominantly in cDNA pool of all samples.

Additional introns inserted within the luciferase reporter gene stabilise transgene expression in wheat

As part of a study to understand factors affecting transgene expression, constructs containing maize introns within the luciferase reporter gene coding region, in addition to a 5′ untranslated leader intron, were transformed into wheat plants. Adding a single intron at one particular position increased transgene activity by 1.5- to 2-fold, however the additional presence of a second intron reduced expression levels in both T 1 and T 2 populations. These results are in agreement with the transient assay results that we have previously reported. More importantly, we showed that both intron combinations led to a stabilisation of luciferase expression between generations in transgenic wheat plants.

The boundaries of the distribution of somatic hypermutation of rearranged immunoglobulin variable genes.

Available evidence about the mechanisms and distribution of somatic hypermutation (SHM) of rearranged immunoglobulin (IgV) genes is reviewed with particular emphasis on the 5' boundary. In heavy (H) chain genes, the 5' boundary of SHM is the transcription start site; in contrast to kappa light (L) chain genes, it is located in the leader (L) intron. DNA-based models of SHM cannot account for this difference. However, an updated reverse transcriptase (RT)-based model invoking error-prone RT activity of DNA polymerase eta copying IgV pre-mRNA templates to produce cDNA of the transcribed strand (TS) of IgV DNA, which then replaces the corresponding section of the original TS, can explain the difference. This explanation incorporates recent knowledge of pre-mRNA processing, in particular, binding of the splicing-associated protein termed U2AF to a pyrimidine-rich tract in the L intron of pre-mRNA of kappa L chains that may block RT progression further upstream to the end of the pre-mRNA template (transcription start site). Reasons why this block may not occur in H chains and other aspects of the updated RT-model are discussed.

Genes coding for starch branching enzymes are major contributors to starch viscosity characteristics in waxy rice (Oryza sativa L.)

Granule-bound starch synthase (GBSS) encoded by the Waxy (Wx) gene determines the synthesis of amylose; while, starch branching enzymes encoded by Sbe genes are involved in the formation of amylopectin. Both amylose and amylopectin form starch in the endosperm of seeds. Here, we report on the effects of the wx gene and the two Sbe genes, Sbe1 and Sbe3, on starch paste viscosity in waxy rice. Molecular markers for both Sbe1 and Sbe3 have been developed based on sequence diversity between the rice subspecies indica and japonica. All Sbe- and Wx- linked markers have been used to investigate 40 waxy rice cultivars. For the wx locus (null Wx locus in waxy rice), four classes of (CT)n repeats, including (CT)16, (CT)17, (CT)18, and (CT)19, were detected in the putative leader sequence. Moreover, a unique sequence, AGTTATA, is also present at the 5′ splice junction site of the putative leader intron. Overall, the wx locus has no effect on starch paste viscosity characteristics. Among all 40 waxy rice cultivars, five genotypes are observed at the Sbe1 and Sbe3 loci. Polymorphisms in both Sbe1 and Sbe3 loci account for ∼70% of the observed variations in both hot and cool viscosities, and for 40% of the observed variations in both peak viscosity and consistency.

The 5' leader of plant PgiC has an intron: the leader shows both the loss and maintenance of constraints compared with introns and exons in the coding region.

PgiC, a complex gene with 23 coding exons and 22 intervening introns, encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9) in higher plants. Here, we report RNA ligase-mediated rapid amplification of cDNA ends experiments that showed that PgiC in Clarkia (Onagraceae) and Arabidopsis thaliana has an intron in the 5' leader. Comparison of the EMBL accessions of the cDNA and genomic sequences showed that this is also the case in rice (Oryza sativa), suggesting that a leader intron is generally present in higher plant PgiC. The intron is bounded by consensus 5'-GT and AG-3' splice sites but showed alternative splicing in Clarkia, resulting in mature transcripts that differ by 8-19 nt in length. The intron is located 18 or 10 nt upstream of the start codon in Clarkia, 2 nt upstream in Arabidopsis, and 9 nt in rice. PgiC in Clarkia was duplicated before the divergence of the extant species, many of which have two expressed genes PgiC1 and PgiC2. Full-length transcripts of both genes identified the transcription start and made it possible to identify the leader intron and leader exon (between the transcription start and leader intron) from previously obtained genomic sequences of both genes in other Clarkia species. These data permit the comparison of evolution in the leader exon and intron with the exons and introns of the coding region, a topic that has not been studied previously. Both the leader exon and the leader intron resemble introns of the coding region in base substitution rate and accumulation of gaps. But the leader intron splice junctions are not strictly conserved in position as are those of the coding region introns. Also, in base composition, the leader intron resembles the other introns, whereas the leader exon more nearly resembles the coding exons. A difference in base composition between coding exons and flanking introns is known to be important for the recognition of splice sites. Thus, the marked difference in base composition between the leader exon and leader intron is probably maintained by selection despite a high rate of sequence divergence.

Molecular characterization of the Pri3 gene encoding a cysteine-rich protein, specifically expressed during fruiting initiation within the Agrocybe aegerita complex.

Coding and regulating sequences of Pri3 (a novel gene specifically expressed in both nucleus types of dikaryons during fruiting initiation) were compared within nine strains of the Agrocybe aegerita complex originating from Europe, Asia and Latin America. Highly homologous coding sequences were found; and the 95-amino-acid-long PRI3 deduced proteins all shared eight cysteines and eight glycines, a putative signal peptide suggesting an extra-cellular localization, a protein kinase C site (T(70)) and a cAMP-dependent kinase site (S(74)). The PRI3 proteins presented no significant homologies with already known proteins and constitute a new class of small cysteine-rich proteins. Within the 5' uncoding regions, two leader introns and a putative upstream regulating sequence resembling the QA1-F activator site were highly conserved. Comparison of the gene sequences within the A. aegerita complex suggested a divergent evolution of the European and Asian/Latin American groups of genes.

Vacuolar membrane localization of the Arabidopsis‘two‐pore’ K+ channel KCO1

Potassium (K+) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K+ channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K+ channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus-infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5' region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying beta-glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5' leader intron. The same overall structure was identified in two genes encoding AtKCO1-like K+ channels from Solanum tuberosum (StKCO1alpha and StKCO1beta). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green fluorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co-fractionate with tonoplast markers (Nt-TIPa, vATPase). In fluorescence images from transgenic AtKCO1-GFP BY2 cells fluorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the first cloned K+ channel demonstrated to be a vacuolar K+ channel.

Analysis of a single nucleotide polymorphism that controls the cooking quality of rice using a non-gel based assay

The waxy gene encoding granule-bound starch synthase (GBSS) is responsible for the synthesis of amylose in developing grain. Recent work has shown that a G-T polymorphism in the leader intron 5' splice site of GBSS plays a key role in determining the cooking and processing quality of rice. Cultivars with sequence AGGTATA at this location splice GBSS pre-mRNA efficiently and produce relatively large amounts of amylose. These varieties generally a have firm texture when cooked and the grains remain separate. In contrast, GBSS pre-mRNA splicing is temperature sensitive and generally less efficient in cultivars with the sequence AGTTATA. As a result, these cultivars generally have lower amylose content and produce soft and sticky cooked rice. We have used the READITTM assay, anon-gel based assay that uses the ability of DNA polymerase to perform pyrophosphoralysis, the reverse of DNA polymerization, to screen the critical G-T polymorphism in more than 750 samples from U.S. and Asian germplasm. We observed complete concordance between the results obtained using DNA sequencing or restriction enzyme digestion and the READITTM assay. It also gave accurate results with both heterozygous plants and with complex mixtures as might result when grain from advanced generation plants is pooled to obtain larger samples.

A long leader intron of the Ostub16 rice beta-tubulin gene is required for high-level gene expression and can autonomously promote transcription both in vivo and in vitro.

A 2 kb DNA fragment, upstream of the rice beta-tubulin isotype 16 (Ostub16) coding sequence, was isolated using inverse PCR and screening of a tubulin-enriched lambda library. An intron (863 bp) present in the 5' untranslated region (5' UTR) is spliced out to produce the most abundant mRNA species which corresponds to the previously cloned Ostub16 cDNA. Transient expression assays performed on rice embryogenic calluses with chimeric Ostub16::GUS constructs demonstrated that the entire 2 kb upstream sequence has a strong promoter activity, and that the 863 bp intron is required for high-level GUS expression. In addition, the intron sequence is capable per se of sustaining a weak but consistent GUS expression. Two rare Ostub16 transcripts, with a start site mapping within this intron sequence, were detected in rice coleoptile cells. The transcription start site mapped at position -290 with respect to the ATG codon, and the shorter molecule originated from splicing of the same precursor mRNA. Therefore transcriptional expression of rice beta-tubulin isotype 16 results in the synthesis of two premRNA molecules (I and II) encoding for three different mRNA species. We discuss these findings in terms of function and molecular evolution of the mechanisms that control plant beta-tubulin gene expression.

Introns and their positions affect the translational activity of mRNA in plant cells.

In an attempt to further increase transgene expression levels in plants over and above the enhancement obtained with a 5' untranslated leader intron, three different maize introns were inserted at three different positions within the coding sequence of the luciferase reporter gene. Constructs were transformed into maize (Black Mexican Sweet) cells and protoplasts, and their activity determined. Although all introns tested were correctly spliced, only one of them in a particular position was able to enhance gene expression. Correct splicing sites were used for intron removal and the quantity of luciferase mRNA produced did not differ significantly. These data indicate that both the position and the sequence of an intron have marked effects on expression levels, suggesting that nuclear processing of the pre-mRNA determines final expression levels through the structure of the mRNP.