The Rapid Methylation of T-DNAs Upon Agrobacterium Inoculation in Plant Leaves

Agrobacterium tumefaciens has been foundational in the development of transgenic plants for both agricultural biotechnology and plant molecular research. However, the transformation efficiency and level of transgene expression obtained for any given construct can be highly variable. These inefficiencies often require screening of many lines to find one with consistent and heritable transgene expression. Transcriptional gene silencing is known to affect transgene expression, and is associated with DNA methylation, especially of cytosines in symmetric CG and CHG contexts. While the specificity, heritability and silencing-associated effects of DNA methylation of transgene sequences have been analyzed in many stably transformed plants, the methylation status of transgene sequences in the T-DNA during the transformation process has not been well-studied. Here we used agro-infiltration of the eGFP reporter gene in Nicotiana benthamiana leaves driven by either an AtEF1α-A4 or a CaMV-35S promoter to study early T-DNA methylation patterns of these promoter sequences. The T-DNA was examined by amplicon sequencing following sodium bisulfite treatment using three different sequencing platforms: Sanger sequencing, Ion Torrent PGM, and the Illumina MiSeq. Rapid DNA methylation was detectable in each promoter region just 2–3 days post-infiltration and the levels continued to rapidly accumulate over the first week, then steadily up to 21 days later. Cytosines in an asymmetric context (CHH) were the most heavily and rapidly methylated. This suggests that early T-DNA methylation may be important in determining the epigenetic and transcriptional fate of integrated transgenes. The Illumina MiSeq platform was the most sensitive and robust way of detecting and following the methylation profiles of the T-DNA promoters. The utility of the methods was then used to show a subtle but significant difference in promoter methylation during intron-mediated enhancement. In addition, the method was able to detect an increase in promoter methylation when the eGFP reporter gene was targeted by siRNAs generated by co-infiltration of a hairpin RNAi construct

Effect of 5'UTR introns on gene expression in Arabidopsis thaliana.

BACKGROUND: The majority of introns in gene transcripts are found within the coding sequences (CDSs). A small but significant fraction of introns are also found to reside within the untranslated regions (5'UTRs and 3'UTRs) of expressed sequences. Alignment of the whole genome and expressed sequence tags (ESTs) of the model plant Arabidopsis thaliana has identified introns residing in both coding and non-coding regions of the genome. RESULTS: A bioinformatic analysis revealed some interesting observations: (1) the density of introns in 5'UTRs is similar to that in CDSs but much higher than that in 3'UTRs; (2) the 5'UTR introns are preferentially located close to the initiating ATG codon; (3) introns in the 5'UTRs are, on average, longer than introns in the CDSs and 3'UTRs; and (4) 5'UTR introns have a different nucleotide composition to that of CDS and 3'UTR introns. Furthermore, we show that the 5'UTR intron of the A. thaliana EF1alpha-A3 gene affects the gene expression and the size of the 5'UTR intron influences the level of gene expression. CONCLUSION: Introns within the 5'UTR show specific features that distinguish them from introns that reside within the coding sequence and the 3'UTR. In the EF1alpha-A3 gene, the presence of a long intron in the 5'UTR is sufficient to enhance gene expression in plants in a size dependent manner

The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit

The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-β) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-β), and epsilon carotene hydroxylase (CRH-ϵ) showed some variation in gene expression. The LCY-β gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-β gene

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

<p>Abstract</p> <p>Background</p> <p>The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called <it>MYBA </it>or <it>MYB1</it>, while the gene determining fruit flesh and foliage anthocyanin has been termed <it>MYB10</it>. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous <it>MYB </it>genes from all the commercially important rosaceous species.</p> <p>Results</p> <p>We use gene specific primers to show that the three MYB activators of apple anthocyanin (<it>MYB10/MYB1/MYBA) </it>are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these <it>MYB10 </it>genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins.</p> <p>Conclusions</p> <p>This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.</p

Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple

<p>Abstract</p> <p>Background</p> <p>Integrating plant genomics and classical breeding is a challenge for both plant breeders and molecular biologists. Marker-assisted selection (MAS) is a tool that can be used to accelerate the development of novel apple varieties such as cultivars that have fruit with anthocyanin through to the core. In addition, determining the inheritance of novel alleles, such as the one responsible for red flesh, adds to our understanding of allelic variation. Our goal was to map candidate anthocyanin biosynthetic and regulatory genes in a population segregating for the red flesh phenotypes.</p> <p>Results</p> <p>We have identified the <it>Rni </it>locus, a major genetic determinant of the red foliage and red colour in the core of apple fruit. In a population segregating for the red flesh and foliage phenotype we have determined the inheritance of the <it>Rni </it>locus and DNA polymorphisms of candidate anthocyanin biosynthetic and regulatory genes. Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs) in the candidate genes were also located on an apple genetic map. We have shown that the MdMYB10 gene co-segregates with the <it>Rni </it>locus and is on Linkage Group (LG) 09 of the apple genome.</p> <p>Conclusion</p> <p>We have performed candidate gene mapping in a fruit tree crop and have provided genetic evidence that red colouration in the fruit core as well as red foliage are both controlled by a single locus named <it>Rni</it>. We have shown that the transcription factor MdMYB10 may be the gene underlying <it>Rni </it>as there were no recombinants between the marker for this gene and the red phenotype in a population of 516 individuals. Associating markers derived from candidate genes with a desirable phenotypic trait has demonstrated the application of genomic tools in a breeding programme of a horticultural crop species.</p

Apple skin patterning is associated with differential expression of MYB10

Background: Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. ‘Royal Gala’) produce fruit with a defined fruit pigment pattern, but in the case of ‘Honeycrisp’ apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown. Results: Here we show that striped areas of ‘Honeycrisp’ and ‘Royal Gala’ are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in ‘Honeycrisp’ and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5’ of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in ‘Honeycrisp’ red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes. Conclusions: Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in ‘Honeycrisp’. We suggest that methylation in the MYB10 promoter is more variable in ‘Honeycrisp’ than in ‘Royal Gala’, leading to more variable color patterns in the peel of this cultivar.https://doi.org/10.1186/1471-2229-11-9

Identification of Mendel's White Flower Character

BACKGROUND: The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea. METHODOLOGY/PRINCIPAL FINDINGS: We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates. CONCLUSIONS/SIGNIFICANCE: We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex

Comprehensive transcriptome analysis of the highly complex Pisum sativum genome using next generation sequencing

<p>Abstract</p> <p>Background</p> <p>The garden pea, <it>Pisum sativum</it>, is among the best-investigated legume plants and of significant agro-commercial relevance. <it>Pisum sativum </it>has a large and complex genome and accordingly few comprehensive genomic resources exist.</p> <p>Results</p> <p>We analyzed the pea transcriptome at the highest possible amount of accuracy by current technology. We used next generation sequencing with the Roche/454 platform and evaluated and compared a variety of approaches, including diverse tissue libraries, normalization, alternative sequencing technologies, saturation estimation and diverse assembly strategies. We generated libraries from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings, comprising a total of 450 megabases. Libraries were assembled into 324,428 unigenes in a first pass assembly.</p> <p>A second pass assembly reduced the amount to 81,449 unigenes but caused a significant number of chimeras. Analyses of the assemblies identified the assembly step as a major possibility for improvement. By recording frequencies of Arabidopsis orthologs hit by randomly drawn reads and fitting parameters of the saturation curve we concluded that sequencing was exhaustive. For leaf libraries we found normalization allows partial recovery of expression strength aside the desired effect of increased coverage. Based on theoretical and biological considerations we concluded that the sequence reads in the database tagged the vast majority of transcripts in the aerial tissues. A pathway representation analysis showed the merits of sampling multiple aerial tissues to increase the number of tagged genes. All results have been made available as a fully annotated database in fasta format.</p> <p>Conclusions</p> <p>We conclude that the approach taken resulted in a high quality - dataset which serves well as a first comprehensive reference set for the model legume pea. We suggest future deep sequencing transcriptome projects of species lacking a genomics backbone will need to concentrate mainly on resolving the issues of redundancy and paralogy during transcriptome assembly.</p