Transcriptional Regulation of Pine Male and Female Cone Initiation and Development: Key Players Identified Through Comparative Transcriptomics.

With long reproductive timescales, large complex genomes, and a lack of reliable reference genomes, understanding gene function in conifers is extremely challenging. Consequently, our understanding of which genetic factors influence the development of reproductive structures (cones) in monoecious conifers remains limited. Genes with inferred roles in conifer reproduction have mostly been identified through homology and phylogenetic reconstruction with their angiosperm counterparts. We used RNA-sequencing to generate transcriptomes of the early morphological stages of cone development in the conifer species Pinus densiflora and used these to gain a deeper insight into the transcriptional changes during male and female cone development. Paired-end Illumina sequencing was used to generate transcriptomes from non-reproductive tissue and male and female cones at four time points with a total of 382.82 Gbp of data generated. After assembly and stringent filtering, a total of 37,164 transcripts were retrieved, of which a third were functionally annotated using the Mercator plant pipeline. Differentially expressed gene (DEG) analysis resulted in the identification of 172,092 DEGs in the nine tissue types. This, alongside GO gene enrichment analyses, pinpointed transcripts putatively involved in conifer reproductive structure development, including co-orthologs of several angiosperm flowering genes and several that have not been previously reported in conifers. This study provides a comprehensive transcriptome resource for male and early female cone development in the gymnosperm species Pinus densiflora. Characterisation of this resource has allowed the identification of potential key players and thus provides valuable insights into the molecular regulation of reproductive structure development in monoecious conifers.

A role for SPEECHLESS in the integration of leaf stomatal patterning with the growth vs disease trade-off in poplar.

Occurrence of stomata on both leaf surfaces (amphistomaty) promotes higher stomatal conductance and photosynthesis while simultaneously increasing exposure to potential disease agents in black cottonwood (Populus trichocarpa). A genome-wide association study (GWAS) with 2.2M single nucleotide polymorphisms generated through whole-genome sequencing found 280 loci associated with variation in adaxial stomatal traits, implicating genes regulating stomatal development and behavior. Strikingly, numerous loci regulating plant growth and response to biotic and abiotic stresses were also identified. The most significant locus was a poplar homologue of SPEECHLESS (PtSPCH1). Individuals possessing PtSPCH1 alleles associated with greater adaxial stomatal density originated primarily from environments with shorter growing seasons (e.g. northern latitudes, high elevations) or with less precipitation. PtSPCH1 was expressed in developing leaves but not developing stem xylem. In developing leaves, RNA sequencing showed patterns of coordinated expression between PtSPCH1 and other GWAS-identified genes. The breadth of our GWAS results suggests that the evolution of amphistomaty is part of a larger, complex response in plants. Suites of genes underpin this response, retrieved through genetic association to adaxial stomata, and show coordinated expression during development. We propose that the occurrence of amphistomaty in P. trichocarpa involves PtSPCH1 and reflects selection for supporting rapid growth over investment in immunity.

Strategies for Engineering Reproductive Sterility in Plantation Forests.

A considerable body of research exists concerning the development of technologies to engineer sterility in forest trees. The primary driver for this work has been to mitigate concerns arising from gene flow from commercial plantings of genetically engineered (GE) trees to non-GE plantations, or to wild or feral relatives. More recently, there has been interest in the use of sterility technologies as a means to mitigate the global environmental and socio-economic damage caused by the escape of non-native invasive tree species from planted forests. The current sophisticated understanding of the molecular processes underpinning sexual reproduction in angiosperms has facilitated the successful demonstration of a number of control strategies in hardwood tree species, particularly in the model hardwood tree Poplar. Despite gymnosperm softwood trees, such as pines, making up the majority of the global planted forest estate, only pollen sterility, via cell ablation, has been demonstrated in softwoods. Progress has been limited by the lack of an endogenous model system, long timescales required for testing, and key differences between softwood reproductive pathways and those of well characterized angiosperm model systems. The availability of comprehensive genome and transcriptome resources has allowed unprecedented insights into the reproductive processes of both hardwood and softwood tree species. This increased fundamental knowledge together with the implementation of new breeding technologies, such as gene editing, which potentially face a less oppressive regulatory regime, is making the implementation of engineered sterility into commercial forestry a realistic possibility.

A New Zealand Perspective on the Application and Regulation of Gene Editing.

New Zealand (NZ) is a small country with an export-led economy with above 90% of primary production exported. Plant-based primary commodities derived from the pastoral, horticultural and forestry sectors account for around half of the export earnings. Productivity is characterized by a history of innovation and the early adoption of advanced technologies. Gene editing has the potential to revolutionize breeding programmes, particularly in NZ. Here, perennials such as tree crops and forestry species are key components of the primary production value chain but are challenging for conventional breeding and only recently domesticated. Uncertainty over the global regulatory status of gene editing products is a barrier to invest in and apply editing techniques in plant breeding. NZs major trading partners including Europe, Asia and Australia are currently evaluating the regulatory status of these technologies and have not made definitive decisions. NZ is one of the few countries where the regulatory status of gene editing has been clarified. In 2014, the NZ Environmental Protection Authority ruled that plants produced via gene editing methods, where no foreign DNA remained in the edited plant, would not be regulated as GMOs. However, following a challenge in the High Court, this decision was overturned such that NZ currently controls all products of gene editing as GMOs. Here, we illustrate the potential benefits of integrating gene editing into plant breeding programmes using targets and traits with application in NZ. The regulatory process which led to gene editing's current GMO classification in NZ is described and the importance of globally harmonized regulations, particularly to small export-driven nations is discussed.

Recent Advances in our Understanding of Tocopherol Biosynthesis in Plants: An Overview of Key Genes, Functions, and Breeding of Vitamin E Improved Crops.

Tocopherols, together with tocotrienols and plastochromanols belong to a group of lipophilic compounds also called tocochromanols or vitamin E. Considered to be one of the most powerful antioxidants, tocochromanols are solely synthesized by photosynthetic organisms including plants, algae, and cyanobacteria and, therefore, are an essential component in the human diet. Tocochromanols potent antioxidative properties are due to their ability to interact with polyunsaturated acyl groups and scavenge lipid peroxyl radicals and quench reactive oxygen species (ROS), thus protecting fatty acids from lipid peroxidation. In the plant model species Arabidopsis thaliana, the required genes for tocopherol biosynthesis and functional roles of tocopherols were elucidated in mutant and transgenic plants. Recent research efforts have led to new outcomes for the vitamin E biosynthetic and related pathways, and new possible alternatives for the biofortification of important crops have been suggested. Here, we review 30 years of research on tocopherols in model and crop species, with emphasis on the improvement of vitamin E content using transgenic approaches and classical breeding. We will discuss future prospects to further improve the nutritional value of our food.

Genetic analysis of health-related secondary metabolites in a Brassica rapa recombinant inbred line population.

The genetic basis of the wide variation for nutritional traits in Brassica rapa is largely unknown. A new Recombinant Inbred Line (RIL) population was profiled using High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR) analysis to detect quantitative trait loci (QTLs) controlling seed tocopherol and seedling metabolite concentrations. RIL population parent L58 had a higher level of glucosinolates and phenylpropanoids, whereas levels of sucrose, glucose and glutamate were higher in the other RIL population parent, R-o-18. QTL related to seed tocopherol (α-, ß-, γ-, δ-, α-/γ- and total tocopherol) concentrations were detected on chromosomes A3, A6, A9 and A10, explaining 11%-35% of the respective variation. The locus on A3 co-locates with the BrVTE1gene, encoding tocopherol cyclase. NMR spectroscopy identified the presence of organic/amino acid, sugar/glucosinolate and aromatic compounds in seedlings. QTL positions were obtained for most of the identified compounds. Compared to previous studies, novel loci were found for glucosinolate concentrations. This work can be used to design markers for marker-assisted selection of nutritional compounds in B. rapa.

Unraveling the genetic basis of seed tocopherol content and composition in rapeseed (Brassica napus L.).

BACKGROUND: Tocopherols are important antioxidants in vegetable oils; when present as vitamin E, tocopherols are an essential nutrient for humans and livestock. Rapeseed (Brassica napus L, AACC, 2 n = 38) is one of the most important oil crops and a major source of tocopherols. Although the tocopherol biosynthetic pathway has been well elucidated in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803, knowledge about the genetic basis of tocopherol biosynthesis in seeds of rapeseed is scant. This project was carried out to dissect the genetic basis of seed tocopherol content and composition in rapeseed through quantitative trait loci (QTL) detection, genome-wide association analysis, and homologous gene mapping. METHODOLOGY/PRINCIPAL FINDINGS: We used a segregating Tapidor × Ningyou7 doubled haploid (TNDH) population, its reconstructed F(2) (RC-F(2)) population, and a panel of 142 rapeseed accessions (association panel). Genetic effects mainly contributed to phenotypic variations in tocopherol content and composition; environmental effects were also identified. Thirty-three unique QTL were detected for tocopherol content and composition in TNDH and RC-F(2) populations. Of these, seven QTL co-localized with candidate sequences associated with tocopherol biosynthesis through in silico and linkage mapping. Several near-isogenic lines carrying introgressions from the parent with higher tocopherol content showed highly increased tocopherol content compared with the recurrent parent. Genome-wide association analysis was performed with 142 B. napus accessions. Sixty-one loci were significantly associated with tocopherol content and composition, 11 of which were localized within the confidence intervals of tocopherol QTL. CONCLUSIONS/SIGNIFICANCE: This joint QTL, candidate gene, and association mapping study sheds light on the genetic basis of seed tocopherol biosynthesis in rapeseed. The sequences presented here may be used for marker-assisted selection of oilseed rape lines with superior tocopherol content and composition.

A candidate gene-based association study of tocopherol content and composition in rapeseed (Brassica napus).

Rapeseed (Brassica napus L.) is the most important oil crop of temperate climates. Rapeseed oil contains tocopherols, also known as vitamin E, which is an indispensable nutrient for humans and animals due to its antioxidant and radical scavenging abilities. Moreover, tocopherols are also important for the oxidative stability of vegetable oils. Therefore, seed oil with increased tocopherol content or altered tocopherol composition is a target for breeding. We investigated the role of nucleotide variations within candidate genes from the tocopherol biosynthesis pathway. Field trials were carried out with 229 accessions from a worldwide B. napus collection which was divided into two panels of 96 and 133 accessions. Seed tocopherol content and composition were measured by HPLC. High heritabilities were found for both traits, ranging from 0.62 to 0.94. We identified polymorphisms by sequencing selected regions of the tocopherol genes from the 96 accession panel. Subsequently, we determined the population structure (Q) and relative kinship (K) as detected by genotyping with genome-wide distributed SSR markers. Association studies were performed using two models, the structure-based GLM + Q and the PK-mixed model. Between 26 and 12 polymorphisms within two genes (BnaX.VTE3.a, BnaA.PDS1.c) were significantly associated with tocopherol traits. The SNPs explained up to 16.93% of the genetic variance for tocopherol composition and up to 10.48% for total tocopherol content. Based on the sequence information we designed CAPS markers for genotyping the 133 accessions from the second panel. Significant associations with various tocopherol traits confirmed the results from the first experiment. We demonstrate that the polymorphisms within the tocopherol genes clearly impact tocopherol content and composition in B. napus seeds. We suggest that these nucleotide variations may be used as selectable markers for breeding rapeseed with enhanced tocopherol quality.

Piceid (resveratrol glucoside) synthesis in stilbene synthase transgenic apple fruit.

A stilbene synthase gene along with the selectable marker gene bar for herbicide resistance was transferred via Agrobacterium tumefaciens mediated transformation into apple (Malus domesticaBorkh.) cvs. 'Elstar' and 'Holsteiner Cox'. The stilbene synthase catalyzes the conversion of 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA into 3,4',5-trihydroxystilbene, commonly known as resveratrol. This phytoalexin has implications in both phytopathology and human health. Greenhouse-grown transgenic and nontransformed control plants were grafted onto dwarfing rootstock M27. Flowering and fruiting occurred within the following years, offering the opportunity to analyze transgenic apple fruit and fertility of transgenic plants as well as inheritance of the transgenes into the seedling progeny. Molecular analysis revealed that the stilbene synthase is expressed in transgenic plants and in the skin and flesh of transgenic apple fruit. After formation, resveratrol is modified by the addition of a hexose sugar. The resulting component was characterized as piceid. With the aim of characterizing the influence of the novel biosynthetic pathway on the accumulation of other phenolic compounds naturally present in apple fruit, the amounts of flavanols, flavonols, phloretin derivatives and hydroxycinnamic acids in wild type and transgenic fruit were determined by HPLC. In all investigated transformed lines that accumulated piceid, no negative correlation between levels of piceid and the above-mentioned compounds was observed, except for the flavonol contents, which slightly decreased. Inheritance of the transgenes was confirmed in the seedling progeny, which were obtained after pollination of transgenic plants with nontransgenic pollen and vice versa after pollination of nontransgenic plants with pollen obtained from transgenic plants. The fertility of stilbene synthase transgenic plants was demonstrated. To the authors' knowledge this is the first time that data are available on piceid synthesis in transgenic apple fruit and the effects of its accumulation on levels of other phenolic compounds present in the fruit.