Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

AbstractNicotiana benthamianais an invaluable model plant and biotechnology platform. To further improve its usefulness and versatility, we have produced high quality chromosome level genome assemblies and multi-omic datasets for both the ubiquitously used LAB strain and a distantly related wild accession, QLD, as well as mapping their single nucleotide polymorphisms with two additional laboratory and four additional wild accessions. LAB and QLD have many genetic, functional, and metabolic differences. These coupled with their high inter-fertility and equally efficient transient and stable transformation and genome editing provide a powerful resource partnership. Their ∼3Gb allotetraploid genomes show advanced diploidisation with major chromosome loss and rearrangement, extensive homoeologous gene loss, and widespread segmental allopolyploidy. Recent bursts of Copia mobility, not seen in otherNicotianagenomes, have probably aidedN. benthamiana’s adaptation to a spectrum of Australian ecologies.

Nicotiana is found predominantly in the Americas and Australia, but also has representatives in Africa and the Pacific Islands. All native Australian Nicotiana species belong to section Suaveolentes. The number of species in this section is uncertain and subject to revision. An example of this uncertainty is the taxonomic status of a South Australian Nicotiana accession colloquially termed 'Corunna'. Here, we report sequences for nuclear and plastid markers for N. sp. Corunna (D.E.Symon 17088) and accessions of two other Australian species, N. burbidgeae and N. benthamiana. Phylogenetic comparison of these sequences with those of other members of Nicotiana places all three taxa in N. section Suaveolentes and shows that 'Corunna' represents a distinct phylogenetic lineage in a well supported clade along with N. goodspeedii, N. maritima, N. amplexicaulis and N. suaveolentes. Phenetic analysis of floral characters also supports recognition of N. sp. Corunna (D.E.Symon 17088) as a distinct species, which we describe here as Nicotiana paulineana Newbigin & P.M.Waterh., sp. nov. The enlarged molecular dataset described here contributes to a better understanding of taxonomic relationships within the section.

Ascorbate (vitamin C) is an essential multifunctional molecule for both plants and mammals. In plants, ascorbate is the most abundant water-soluble antioxidant that supports stress tolerance. In humans, ascorbate is an essential micronutrient and promotes iron (Fe) absorption in the gut. Engineering crops with increased ascorbate levels have the potential to improve both crop stress tolerance and human health. Here, rice (Oryza sativa L.) plants were engineered to constitutively overexpress the rice GDP-L-galactose phosphorylase coding sequence (35S-OsGGP), which encodes the rate-limiting enzymatic step of the L-galactose pathway. Ascorbate concentrations were negligible in both null segregant (NS) and 35S-OsGGP brown rice (BR, unpolished grain), but significantly increased in 35S-OsGGP germinated brown rice (GBR) relative to NS. Foliar ascorbate concentrations were significantly increased in 35S-OsGGP plants in the vegetative growth phase relative to NS, but significantly reduced at the reproductive growth phase and were associated with reduced OsGGP transcript levels. The 35S-OsGGP plants did not display altered salt tolerance at the vegetative growth phase despite having elevated ascorbate concentrations. Ascorbate concentrations were positively correlated with ferritin concentrations in Caco-2 cells – an accurate predictor of Fe bioavailability in human digestion – exposed to in vitro digests of NS and 35S-OsGGP BR and GBR samples.

Transgenes have become essential to modern biology, being an important tool in functional genomic studies and also in the development of biotechnological products. One of the major challenges in the generation of transgenic lines concerns the expression of transgenes, which, compared to endogenes, are particularly susceptible to silencing mediated by small RNAs (sRNAs). Several reasons have been put forward to explain why transgenes often trigger the production of sRNAs, such as the high level of expression induced by commonly used strong constitutive promoters, the lack of introns, and features resembling viral and other exogenous sequences. However, the relative contributions of the different genomic elements with respect to protecting genes from the silencing machinery and their molecular mechanisms remain unclear. Here, we present the results of a mutagenesis screen conceived to identify features involved in the protection of endogenes against becoming a template for the production of sRNAs. Interestingly, all of the recovered mutants had alterations in genes with proposed function in transcription termination, suggesting a central role of terminators in this process. Indeed, using a GFP reporter system, we show that, among different genetic elements tested, the terminator sequence had the greatest effect on transgene-derived sRNA accumulation and that a well-defined poly(A) site might be especially important. Finally, we describe an unexpected mechanism, where transgenes containing certain intron/terminator combinations lead to an increase in the production of sRNAs, which appears to interfere with splicing.

Background Ascorbate is a powerful antioxidant in plants and an essential micronutrient for humans. The GDP-L-galactose phosphorylase ( GGP ) gene encodes the rate-limiting enzyme of the L-galactose pathway—the dominant ascorbate biosynthetic pathway in plants—and is a promising gene candidate for increasing ascorbate in crops. In addition to transcriptional regulation, GGP production is regulated at the translational level through an upstream open reading frame (uORF) in the long 5′-untranslated region (5’UTR). The GGP genes have yet to be identified in bread wheat ( Triticum aestivum L.), one of the most important food grain sources for humans. Results Bread wheat chromosomal groups 4 and 5 were found to each contain three homoeologous TaGGP genes on the A, B, and D subgenomes ( TaGGP2-A / B / D and TaGGP1-A / B/D , respectively) and a highly conserved uORF was present in the long 5’UTR of all six genes. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium /Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. The two groups of TaGGP genes have distinct expression patterns with the TaGGP1 homoeologs broadly expressed across different tissues and developmental stages and the TaGGP2 homoeologs highly expressed in anthers. Transient transformation of the TaGGP coding sequences in Nicotiana benthamiana leaf tissue increased ascorbate concentrations more than five-fold, confirming their functional role in ascorbate biosynthesis in planta . Conclusions We have identified six TaGGP genes in the bread wheat genome, each with a highly conserved uORF. Phylogenetic and microsynteny analyses highlight that a transposable element may have been responsible for the duplication and specialized expression of GGP2 in anthers in the Brachypodium /Triticeae lineage. Transient transformation of the TaGGP coding sequences in N. benthamiana demonstrated their activity in planta . The six TaGGP genes and uORFs identified in this study provide a valuable genetic resource for increasing ascorbate concentrations in bread wheat.