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.

Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don).

BACKGROUND: To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. RESULTS: We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. CONCLUSIONS: We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.

Two conifer GUX clades are responsible for distinct glucuronic acid patterns on xylan.

Wood of coniferous trees (softwood), is a globally significant carbon sink and an important source of biomass. Despite that, little is known about the genetic basis of softwood cell wall biosynthesis. Branching of xylan, one of the main hemicelluloses in softwood secondary cell walls, with glucuronic acid (GlcA) is critical for biomass recalcitrance. Here, we investigate the decoration patterns of xylan by conifer GlucUronic acid substitution of Xylan (GUX) enzymes. Through molecular phylogenetics we identify two distinct conifer GUX clades. Using transcriptional profiling we show that the genes are preferentially expressed in secondary cell wall forming tissues. With in vitro and in planta assays we demonstrate that conifer GUX enzymes from both clades are active glucuronyltransferases. Conifer GUX enzymes from each clade have different specific activities. While members of clade one add evenly spaced GlcA branches, the members of clade two are also capable of glucuronidating two consecutive xyloses. Importantly, these types of xylan patterning are present in softwood. As xylan patterning might modulate xylan-cellulose and xylan-lignin interactions, our results further the understanding of softwood cell wall biosynthesis and provide breeding or genetic engineering targets that can be used to modify softwood properties.

MUR1-mediated cell-wall fucosylation is required for freezing tolerance in Arabidopsis thaliana.

Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE-TO-FREEZING8, in Arabidopsis thaliana. We identified SFR8 using recombination-based mapping and whole-genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC-MS and polyacrylamide gel electrophoresis to measure cell-wall fucose and boron (B)-dependent dimerization of the cell-wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate-bridging of RGII, we assessed freeze-induced damage in wild-type and sfr8 plants by measuring electrolyte leakage from freeze-thawed leaf discs. We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP-d-mannose-4,6-dehydratase. sfr8 cell walls exhibited low cell-wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing-sensitive. Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize.

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.

The sensitive to freezing3 mutation of Arabidopsis thaliana is a cold-sensitive allele of homomeric acetyl-CoA carboxylase that results in cold-induced cuticle deficiencies.

The sfr3 mutation causes freezing sensitivity in Arabidopsis thaliana. Mapping, sequencing, and transgenic complementation showed sfr3 to be a missense mutation in ACC1, an essential gene encoding homomeric (multifunctional) acetyl-CoA carboxylase. Cuticle permeability was compromised in the sfr3 mutant when plants were grown in the cold but not in the warm. Wax deposition on the inflorescence stem of cold-grown sfr3 plants was inhibited and the long-chain components of their leaf cuticular wax were reduced compared with wild-type plants. Thus, freezing sensitivity of sfr3 appears, from these results, to be due to cuticular deficiencies that develop during cold acclimation. These observations demonstrated the essential role of the cuticle in tolerance to freezing and drought.

Identification of SFR6, a key component in cold acclimation acting post-translationally on CBF function.

The sfr6-1 mutant of Arabidopsis thaliana was identified previously on the basis of its failure to undergo acclimation to freezing temperatures following exposure to low positive temperatures. This failure is attributed to a defect in the pathway leading to cold on-regulated (COR) gene expression via CBF (C-box binding factor) transcription factors. We identified a region of chromosome 4 containing SFR6 by positional mapping. Fine mapping of the sfr6-1 mutation proved impossible as the locus resides very close to the centromere. Therefore, we screened 380 T-DNA lines with insertions in genes within the large region to which sfr6-1 mapped. This resulted in the identification of two further mutant alleles of SFR6 (sfr6-2 and sfr6-3); like the original sfr6-1 mutation, these disrupt freezing tolerance and COR gene expression. To determine the protein sequence, we cloned an SFR6 cDNA based on the predicted coding sequence, but this offered no indication as to the mechanism by which SFR6 acts. The SFR6 gene itself is not strongly regulated by cold, thus discounting regulation of SFR6 activity at the transcriptional level. We show that over-expression of CBF1 or CBF2 transcription factors, which constitutively activate COR genes in the wild-type, cannot do so in sfr6-1. We demonstrate that CBF protein accumulates to wild-type levels in response to cold in sfr6-1. These results indicate a role for the SFR6 protein in the CBF pathway -downstream of CBF translation. The fact that the SFR6 protein is targeted to the nucleus may suggest a direct role in modulating gene expression.

A role for SENSITIVE TO FREEZING2 in protecting chloroplasts against freeze-induced damage in Arabidopsis.

SUMMARY: The sensitive to freezing2 (SFR2) gene has an important role in freezing tolerance in Arabidopsis thaliana. We show that homologous genes are present, and expressed, in a wide range of terrestrial plants, including species not able to tolerate freezing. Expression constructs derived from the cDNAs of a number of different plant species, including examples not tolerant to freezing, are able to complement the freezing sensitivity of the Arabidopsis sfr2 mutant. In Arabidopsis the SFR2 protein is localized to the chloroplast outer envelope membrane, as revealed by the analysis of transgenic plants expressing SFR2 fusions to GFP, by confocal microscopy, and by the immunological analysis of isolated chloroplasts treated with thermolysin protease. Moreover, the chloroplasts of the sfr2 mutant show clear evidence of rapid damage after a freezing episode, suggesting a role for SFR2 in the protection of the chloroplast.

crinkled leaves 8--a mutation in the large subunit of ribonucleotide reductase--leads to defects in leaf development and chloroplast division in Arabidopsis thaliana.

The crinkled leaves8 (cls8) mutant of Arabidopsis thaliana displays a developmental phenotype of abnormal leaf and flower morphology, reduced root growth and bleached leaf sections. Map-based cloning identified the mutation as being within the gene encoding the large subunit of ribonucleotide reductase (RNR1), the enzyme that catalyses the rate-limiting step in the production of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Levels of dTTP and dATP were significantly reduced in cls8. Two further mutant cls8 alleles and cls8::RNAi plants show similar or more severe phenotypes. The cls8-1 mutant has fewer copies of the chloroplast genome, and fewer, larger chloroplasts than wild-type plants. The ultrastructure of the chloroplast, however, appears normal in cls8-1 leaves. We present evidence that, under conditions of limited dNTP supply, the inhibition of chloroplast DNA replication may be the primary factor in inducing aberrant growth.

Nucleotide depletion and chloroplast division.

We have described the identification of crinkled leaves 8 (cls8) which contains a mutation within the gene encoding the large subunit of ribonucleotide reductase (RNR), the enzyme that catalyses the rate limiting step in the synthesis of deoxyribonucleotide triphosphates (dNTPs) for DNA synthesis and repair. The mutation resulted in plants with altered leaf and flower morphology, reduced root growth, bleached leaf sectors and reduced levels of dNTPs. An interesting consequence of the mutation was its effect on chloroplast division. Mutant plants had fewer, larger chloroplasts and a reduced number of chloroplast genomes compared to wild type plants. The morphological phenotype may be a consequence of altered chloroplast replication.

The SENSITIVE TO FREEZING2 gene, required for freezing tolerance in Arabidopsis thaliana, encodes a beta-glucosidase.

The sensitive to freezing2-1 (sfr2-1) mutation causes freezing sensitivity in Arabidopsis thaliana. By mapping, transgenic complementation, and sequencing, sfr2-1 was revealed to be a mutation in gene At3g06510. A new knockout allele was obtained, and its identical freezing-sensitive phenotype confirmed that the SFR2 gene product is essential for freezing tolerance. Transcription of SFR2 was observed to be constitutive rather than stress inducible and was distributed throughout most aerial tissues. SFR2 encodes a protein homologous to family 1 glycosyl hydrolases (beta-glycosidases), but the predicted AtSFR2 protein is divergent from all other family 1 beta-glycosidases of Arabidopsis, showing closer homology to the sequences of several beta-glycosidases from thermophilic archea and bacteria. After purification from a heterologous expression system, AtSFR2 displayed a specific hydrolytic activity against beta-d-glucosides.