Chromosome-level genomes of three key Allium crops and their trait evolution.

Allium crop breeding remains severely hindered due to the lack of high-quality reference genomes. Here we report high-quality chromosome-level genome assemblies for three key Allium crops (Welsh onion, garlic and onion), which are 11.17 Gb, 15.52 Gb and 15.78 Gb in size with the highest recorded contig N50 of 507.27 Mb, 109.82 Mb and 81.66 Mb, respectively. Beyond revealing the genome evolutionary process of Allium species, our pathogen infection experiments and comparative metabolomic and genomic analyses showed that genes encoding enzymes involved in the metabolic pathway of Allium-specific flavor compounds may have evolved from an ancient uncharacterized plant defense system widely existing in many plant lineages but extensively boosted in alliums. Using in situ hybridization and spatial RNA sequencing, we obtained an overview of cell-type categorization and gene expression changes associated with spongy mesophyll cell expansion during onion bulb formation, thus indicating the functional roles of bulb formation genes.

Comparative Transcriptomics of Multi-Stress Responses in Pachycladon cheesemanii and Arabidopsis thaliana.

The environment is seldom optimal for plant growth and changes in abiotic and biotic signals, including temperature, water availability, radiation and pests, induce plant responses to optimise survival. The New Zealand native plant species and close relative to Arabidopsis thaliana, Pachycladon cheesemanii, grows under environmental conditions that are unsustainable for many plant species. Here, we compare the responses of both species to different stressors (low temperature, salt and UV-B radiation) to help understand how P. cheesemanii can grow in such harsh environments. The stress transcriptomes were determined and comparative transcriptome and network analyses discovered similar and unique responses within species, and between the two plant species. A number of widely studied plant stress processes were highly conserved in A. thaliana and P. cheesemanii. However, in response to cold stress, Gene Ontology terms related to glycosinolate metabolism were only enriched in P. cheesemanii. Salt stress was associated with alteration of the cuticle and proline biosynthesis in A. thaliana and P. cheesemanii, respectively. Anthocyanin production may be a more important strategy to contribute to the UV-B radiation tolerance in P. cheesemanii. These results allowed us to define broad stress response pathways in A. thaliana and P. cheesemanii and suggested that regulation of glycosinolate, proline and anthocyanin metabolism are strategies that help mitigate environmental stress.

The 5'-3' mRNA Decay Pathway Modulates the Plant Circadian Network in Arabidopsis.

Circadian rhythms enable organisms to anticipate and adjust their physiology to periodic environmental changes. These rhythms are controlled by biological clocks that consist of a set of clock genes that regulate each other's expression. Circadian oscillations in messenger RNA (mRNA) levels require the regulation of mRNA production and degradation. While transcription factors controlling clock function have been well characterized from cyanobacteria to humans, the role of factors controlling mRNA decay is largely unknown. Here, we show that mutations in SM-LIKE PROTEIN 1 (LSM1) and exoribonucleases 4 (XRN4), components of the 5'-3' mRNA decay pathway, alter clock function in Arabidopsis. We found that lsm1 and xrn4 mutants display long-period phenotypes for clock gene expression. In xrn4, these circadian defects were associated with changes in circadian phases of expression, but not overall mRNA levels, of several core-clock genes. We then used noninvasive transcriptome-wide mRNA stability analysis to identify genes and pathways regulated by XRN4. Among genes affected in the xrn4 mutant at the transcriptional and posttranscriptional level, we found an enrichment in genes involved in auxin, ethylene and drought recovery. Large effects were not observed for canonical core-clock genes, although the mRNAs of several auxiliary clock genes that control the pace of the clock were stabilized in xrn4 mutants. Our results establish that the 5'-3' mRNA decay pathway constitutes a novel posttranscriptional regulatory layer of the circadian gene network, which probably acts through a combination of small effects on mRNA stability of several auxiliary and some core-clock genes.

A Point Mutation in Phytochromobilin synthase Alters the Circadian Clock and Photoperiodic Flowering of Medicago truncatula.

Plants use seasonal cues to initiate flowering at an appropriate time of year to ensure optimal reproductive success. The circadian clock integrates these daily and seasonal cues with internal cues to initiate flowering. The molecular pathways that control the sensitivity of flowering to photoperiods (daylengths) are well described in the model plant Arabidopsis. However, much less is known for crop species, such as legumes. Here, we performed a flowering time screen of a TILLING population of Medicago truncatula and found a line with late-flowering and altered light-sensing phenotypes. Using RNA sequencing, we identified a nonsense mutation in the Phytochromobilin synthase (MtPΦBS) gene, which encodes an enzyme that carries out the final step in the biosynthesis of the chromophore required for phytochrome (phy) activity. The analysis of the circadian clock in the MtpΦbs mutant revealed a shorter circadian period, which was shared with the MtphyA mutant. The MtpΦbs and MtphyA mutants showed downregulation of the FT floral regulators MtFTa1 and MtFTb1/b2 and a change in phase for morning and night core clock genes. Our findings show that phyA is necessary to synchronize the circadian clock and integration of light signalling to precisely control the timing of flowering.

Molecular control of the floral transition in the mast seeding plant Celmisia lyallii (Asteraceae).

Mast flowering (or masting) is synchronous, highly variable flowering among years in populations of perennial plants. Despite having widespread consequences for seed consumers, endangered fauna and human health, masting is hard to predict. While observational studies show links to various weather patterns in different plant species, the mechanism(s) underpinning the regulation of masting is still not fully explained. We studied floral induction in Celmisia lyallii (Asteraceae), a mast flowering herbaceous alpine perennial, comparing gene expression in flowering and nonflowering plants. We performed translocation experiments to induce the floral transition in C. lyallii plants followed by both global and targeted expression analysis of flowering-pathway genes. Differential expression analysis showed elevated expression of ClSOC1 and ClmiR172 (promoters of flowering) in leaves of plants that subsequently flowered, in contrast to elevated expression of ClAFT and ClTOE1 (repressors of flowering) in leaves of plants that did not flower. The warm summer conditions that promoted flowering led to differential regulation of age and hormonal pathway genes, including ClmiR172 and ClGA20ox2, known to repress the expression of floral repressors and permit flowering. Upregulated expression of epigenetic modifiers of floral promoters also suggests that plants may maintain a novel "summer memory" across years to induce flowering. These results provide a basic mechanistic understanding of floral induction in masting plants and evidence of their ability to imprint various environmental cues to synchronize flowering, allowing us to better predict masting events under climate change.

RNAi-mediated repression of dormancy-related genes results in evergrowing apple trees.

DORMANCY-ASSOCIATED MADS-box (DAM) and SHORT VEGETATIVE PHASE (SVP) genes have been implicated in the regulation of winter dormancy in perennials. Ectopic expression of apple (Malus × domestica Borkh. 'Royal Gala') DAM and SVP genes delays budbreak and constrains lateral shoot outgrowth. In this study, we used RNA interference (RNAi) to simultaneously target all apple DAM and SVP genes in order to study their role and mode of action in the regulation of bud dormancy, budbreak and flowering. A synthetic construct carrying a hairpin fragment assembled from sequences specific to coding regions of three DAM and two SVP genes was used to generate transgenic lines. Reduced expression of DAM/SVP genes resulted in delayed leaf senescence and abscission in autumn, failure to enter bud dormancy in winter and continual growth of new leaves regardless of the season for over 3 years. Precocious flowering but normal flower morphology, fertility and fruit development were observed. The non-dormant phenotype was associated with modified phytohormone composition. The content of gibberellins (GAs) and jasmonates (JAs) was significantly increased in terminal buds of RNAi lines compared with wildtype plants, accompanied by elevated expression of the key GA biosynthesis pathway gene GIBBERELLIN 20 OXIDASE-2 (MdGA20ox-2) along with the FLOWERING LOCUS T gene MdFT2. The key mediator of plasmodesmatal closure, MdCALLOSE SYNTHASE 1 (MdCALS1), was repressed in RNAi lines. This study provides functional evidence for the role of DAM/SVP genes in vegetative phenology of apple and paves the way for production of low-chill varieties suitable for growth in warming climates.

Identification of flowering-time genes in mast flowering plants using De Novo transcriptomic analysis.

Mast flowering is synchronised highly variable flowering by a population of perennial plants over a wide geographical area. High seeding years are seen as a threat to native and endangered species due to high predator density caused by the abundance of seed. An understanding of the molecular pathways that influence masting behaviour in plants could provide better prediction of a forthcoming masting season and enable conservation strategies to be deployed. The goal of this study was to identify candidate flowering genes that might be involved in regulating mast flowering. To achieve this, high-throughput large-scale RNA-sequencing was performed on two masting plant species, Celmisia lyallii (Asteraceae), and Chionochloa pallens (Poaceae) to develop a reference transcriptome for functional and molecular analysis. An average total of 33 million 150 base-paired reads, for both species, were assembled using the Trinity pipeline, resulting in 151,803 and 348,649 transcripts respectively for C. lyallii and C. pallens. For both species, about 56% of the unigenes were annotated with gene descriptions to known proteins followed by Gene Ontology analysis, categorising them on the basis of putative biological processes, molecular function, and cellular localization. A total of 543 transcripts from C. lyallii and 470 transcripts from C. pallens were also mapped to unique flowering-time proteins identified in Arabidopsis thaliana, suggesting the conservation of the flowering network in these wild alpine plants growing in natural field conditions. Expression analysis of several selected homologous flowering-pathway genes showed seasonal and photoperiodic variations. These genes can further be analysed to understand why seasonal cues, such as the increasing photoperiod in spring, that triggers the annual flowering of most plants, are insufficient to always trigger flowering in masting plants and to uncover the molecular basis of how additional cues (such as temperature during the previous growing seasons) then determines flowering in mast years.

Histone modification and activation by SOC1-like and drought stress-related transcription factors may regulate AcSVP2 expression during kiwifruit winter dormancy.

The SHORT VEGETATIVE PHASE (SVP)-like and DORMANCY ASSOCIATED MADS-BOX (DAM) genes have been shown to regulate winter dormancy in woody perennials. In kiwifruit, AcSVP2 affects the duration of dormancy in cultivars that require high chill for dormancy release. In this study, we used a low-chill kiwifruit Actinidia chinensis 'Hort16A' to further study the function and regulation of AcSVP2. Overexpression of AcSVP2 in transgenic A. chinensis delayed budbreak in spring. A reduction in the active trimethylation histone marks of the histone H3K4 and acetylation of histone H3 contributed to the reduction of AcSVP2 expression towards dormancy release, while the inactive histone marks of trimethylation of the histone H3K27 and H3K9 in AcSVP2 locus did not show significant enrichment at the end of winter dormancy. Analysis of expression in shoot buds showed that AcSVP2 transcript was elevated in dormant buds during winter months and declined prior to budbreak, which was coordinated with expression of some of kiwifruit SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1)-like genes. Screening of 101 transcription factors in an assay with a 2.3 kb promoter region of AcSVP2 found that kiwifruit SOC1-like genes are able to activate the AcSVP2 promoter. We further identified additional transcription factors associated with drought/osmotic stress and dormancy which may regulate AcSVP2 expression.

Comparative transcriptome analysis of the wild-type model apomict Hieracium praealtum and its loss of parthenogenesis (lop) mutant.

BACKGROUND: Asexual seed formation (apomixis) has been observed in diverse plant families but is rare in crop plants. The generation of apomictic crops would revolutionize agriculture, as clonal seed production provides a low cost and efficient way to produce hybrid seed. Hieracium (Asteraceae) is a model system for studying the molecular components of gametophytic apomixis (asexual seed reproduction). RESULTS: In this study, a reference transcriptome was produced from apomictic Hieracium undergoing the key apomictic events of apomeiosis, parthenogenesis and autonomous endosperm development. In addition, transcriptome sequences from pre-pollination and post-pollination stages were generated from a loss of parthenogenesis (lop) mutant accession that exhibits loss of parthenogenesis and autonomous endosperm development. The transcriptome is composed of 147,632 contigs, 50% of which were annotated with orthologous genes and their probable function. The transcriptome was used to identify transcripts differentially expressed during apomictic and pollination dependent (lop) seed development. Gene Ontology enrichment analysis of differentially expressed transcripts showed that an important difference between apomictic and pollination dependent seed development was the expression of genes relating to epigenetic gene regulation. Genes that mark key developmental stages, i.e. aposporous embryo sac development and seed development, were also identified through their enhanced expression at those stages. CONCLUSION: The production of a comprehensive floral reference transcriptome for Hieracium provides a valuable resource for research into the molecular basis of apomixis and the identification of the genes underlying the LOP locus.

Functional Genomics and Flowering Time in Medicago truncatula: An Overview.

Flowering time is an important trait that influences adaptation and yield in many crop legumes. Both the inherent earliness of flowering and the degree to which it is responsive to environmental factors determine both the eco-geographic range across which crops can be successfully grown and the seasonal cycles most suitable for production. This chapter will provide a brief review of studies investigating the genetic control of flowering time in Medicago truncatula.

Medicago truncatula SOC1 Genes Are Up-regulated by Environmental Cues That Promote Flowering.

Like Arabidopsis thaliana, the flowering of the legume Medicago truncatula is promoted by long day (LD) photoperiod and vernalization. However, there are differences in the molecular mechanisms involved, with orthologs of two key Arabidopsis thaliana regulators, FLOWERING LOCUS C (FLC) and CONSTANS (CO), being absent or not having a role in flowering time function in Medicago. In Arabidopsis, the MADS-box transcription factor gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (AtSOC1), plays a key role in integrating the photoperiodic and vernalization pathways. In this study, we set out to investigate whether the Medicago SOC1 genes play a role in regulating flowering time. Three Medicago SOC1 genes were identified and characterized (MtSOC1a-MtSOC1c). All three MtSOC1 genes, when heterologously expressed, were able to promote earlier flowering of the late-flowering Arabidopsis soc1-2 mutant. The three MtSOC1 genes have different patterns of expression. However, consistent with a potential role in flowering time regulation, all three MtSOC1 genes are expressed in the shoot apex and are up-regulated in the shoot apex of plants in response to LD photoperiods and vernalization. The up-regulation of MtSOC1 genes was reduced in Medicago fta1-1 mutants, indicating that they are downstream of MtFTa1. Insertion mutant alleles of Medicago soc1b do not flower late, suggestive of functional redundancy among Medicago SOC1 genes in promoting flowering.

Complete Genome Sequence of a New Zealand Isolate of the Bovine Pathogen Streptococcus uberis.

Streptococcus uberis forms part of the native microbiota of cattle and is able to opportunistically infect the mammary gland; as such, it is a leading cause of bovine mastitis globally. Here, we report the complete genome sequence of S. uberis NZ01, isolated in New Zealand from a cow with a clinical case of bovine mastitis.

Kiwifruit SVP2 controls developmental and drought-stress pathways.

KEY MESSAGE: Genome-wide targets of Actinidia chinensis SVP2 confirm roles in ABA- and dehydration-mediated growth repression and reveal a conservation in mechanism of action between SVP genes of taxonomically distant Arabidopsis and a woody perennial kiwifruit. The molecular mechanisms underlying growth and dormancy in woody perennials are largely unknown. In Arabidopsis, the MADS-box transcription factor SHORT VEGETATIVE PHASE (SVP) plays a key role in the progression from vegetative to floral development, and in woody perennials SVP-like genes are also proposed to be involved in controlling dormancy. During kiwifruit development SVP2 has a role in growth inhibition, with high-chill kiwifruit Actinidia deliciosa transgenic lines overexpressing SVP2 showing suppressed bud outgrowth. Transcriptomic analyses of these plants suggests that SVP2 mimics the well-documented abscisic acid (ABA) effect on the plant dehydration response. To corroborate the growth inhibition role of SVP2 in kiwifruit development at the molecular level, we analysed the genome-wide direct targets of SVP2 using chromatin immunoprecipitation followed by high-throughput sequencing in kiwifruit A. chinensis. SVP2 was found to bind to at least 297 target sites in the kiwifruit genome, and potentially modulates 252 genes that function in a range of biological processes, especially those involved in repressing meristem activity and ABA-mediated dehydration pathways. In addition, our ChIP-seq analysis reveals remarkable conservation in mechanism of action between SVP genes of taxonomically distant plant species.

The Chickpea Early Flowering 1 (Efl1) Locus Is an Ortholog of Arabidopsis ELF3.

In climates that experience short growing seasons due to drought, heat, or end-of-season frost, early flowering is a highly desirable trait for chickpea (Cicer arietinum). In this study, we mapped, sequenced, and characterized Early flowering3 (Efl3), an ortholog of Arabidopsis (Arabidopsis thaliana) EARLY FLOWERING3 (ELF3) that confers early flowering in chickpea. In a recombinant inbred line population derived from a cross between CDC Frontier and ICCV 96029, this gene was mapped to the site of a quantitative trait locus on Ca5 that explained 59% of flowering time variation under short days. Sequencing of ELF3 in ICCV 96029 revealed an 11-bp deletion in the first exon that was predicted to result in a premature stop codon. The effect of this mutation was tested by transgenic complementation in the Arabidopsis elf3-1 mutant, with the CDC Frontier form of CaELF3a partially complementing elf3-1 while the ICCV 96029 form had no effect on flowering time. While induction of FLOWERING LOCUS T homologs was very early in ICCV 96029, an analysis of circadian clock function failed to show any clear loss of rhythm in the expression of clock genes in ICCV 96029 grown under continuous light, suggesting redundancy with other ELF3 homologs or possibly an alternative mode of action for this gene in chickpea. The 11-bp deletion was associated with early flowering in global chickpea germplasm but was not widely distributed, indicating that this mutation arose relatively recently.

SVP-like MADS Box Genes Control Dormancy and Budbreak in Apple.

The annual growth cycle of trees is the result of seasonal cues. The onset of winter triggers an endodormant state preventing bud growth and, once a chilling requirement is satisfied, these buds enter an ecodormant state and resume growing. MADS-box genes with similarity to Arabidopsis SHORT VEGETATIVE PHASE (SVP) [the SVP-like and DORMANCY ASSOCIATED MADS-BOX (DAM) genes] have been implicated in regulating flowering and growth-dormancy cycles in perennials. Here, we identified and characterized three DAM-like (MdDAMs) and two SHORT VEGETATIVE PHASE-like (MdSVPs) genes from apple (Malus × domestica 'Royal Gala'). The expression of MdDAMa and MdDAMc indicated they may play a role in triggering autumn growth cessation. In contrast, the expression of MdDAMb, MdSVPa and MdSVPb suggested a role in maintaining bud dormancy. Consistent with this, ectopic expression of MdDAMb and MdSVPa in 'Royal Gala' apple plants resulted in delayed budbreak and architecture change due to constrained lateral shoot outgrowth, but normal flower and fruit development. The association of MdSVPa and MdSVPb expression with floral bud development in the low fruiting 'Off' trees of a biennial bearing cultivar 'Sciros' suggested the SVP genes might also play a role in floral meristem identity.

Kiwifruit SVP2 gene prevents premature budbreak during dormancy.

Overexpression of SVP2 in kiwifruit delays budbreak before sufficient winter chilling. SVP2-mediated vegetative growth restriction involves stress response pathways, and commonalities exist between Arabidopsis and kiwifruit SVP targets.

Increasing ascorbate levels in crops to enhance human nutrition and plant abiotic stress tolerance.

Ascorbate (or vitamin C) is an essential human micronutrient predominantly obtained from plants. In addition to preventing scurvy, it is now known to have broader roles in human health, for example as a cofactor for enzymes involved in epigenetic programming and as regulator of cellular iron uptake. Furthermore, ascorbate is the major antioxidant in plants and underpins many environmentally induced abiotic stress responses. Biotechnological approaches to enhance the ascorbate content of crops therefore have potential to improve both human health and abiotic stress tolerance of crops. Identifying the genetic basis of ascorbate variation between plant varieties and discovering how some 'super fruits' accumulate extremely high levels of ascorbate should reveal new ways to more effectively manipulate the production of ascorbate in crops.

Doubled Haploid 'CUDH2107' as a Reference for Bulb Onion (Allium cepa L.) Research: Development of a Transcriptome Catalogue and Identification of Transcripts Associated with Male Fertility.

Researchers working on model plants have derived great benefit from developing genomic and genetic resources using 'reference' genotypes. Onion has a large and highly heterozygous genome making the sharing of germplasm and analysis of sequencing data complicated. To simplify the discovery and analysis of genes underlying important onion traits, we are promoting the use of the homozygous double haploid line 'CUDH2107' by the onion research community. In the present investigation, we performed transcriptome sequencing on vegetative and reproductive tissues of CUDH2107 to develop a multi-organ reference transcriptome catalogue. A total of 396 million 100 base pair paired reads was assembled using the Trinity pipeline, resulting in 271,665 transcript contigs. This dataset was analysed for gene ontology and transcripts were classified on the basis of putative biological processes, molecular function and cellular localization. Significant differences were observed in transcript expression profiles between different tissues. To demonstrate the utility of our CUDH2107 transcriptome catalogue for understanding the genetic and molecular basis of various traits, we identified orthologues of rice genes involved in male fertility and flower development. These genes provide an excellent starting point for studying the molecular regulation, and the engineering of reproductive traits.

Infiltration-RNAseq: transcriptome profiling of Agrobacterium-mediated infiltration of transcription factors to discover gene function and expression networks in plants.

BACKGROUND: Transcription factors (TFs) coordinate precise gene expression patterns that give rise to distinct phenotypic outputs. The identification of genes and transcriptional networks regulated by a TF often requires stable transformation and expression changes in plant cells. However, the production of stable transformants can be slow and laborious with no guarantee of success. Furthermore, transgenic plants overexpressing a TF of interest can present pleiotropic phenotypes and/or result in a high number of indirect gene expression changes. Therefore, fast, efficient, high-throughput methods for assaying TF function are needed. RESULTS: Agroinfiltration is a simple plant biology method that allows transient gene expression. It is a rapid and powerful tool for the functional characterisation of TF genes in planta. High throughput RNA sequencing is now a widely used method for analysing gene expression profiles (transcriptomes). By coupling TF agroinfiltration with RNA sequencing (named here as Infiltration-RNAseq), gene expression networks and gene function can be identified within a few weeks rather than many months. As a proof of concept, we agroinfiltrated Medicago truncatula leaves with M. truncatula LEGUME ANTHOCYANIN PRODUCITION 1 (MtLAP1), a MYB transcription factor involved in the regulation of the anthocyanin pathway, and assessed the resulting transcriptome. Leaves infiltrated with MtLAP1 turned red indicating the production of anthocyanin pigment. Consistent with this, genes encoding enzymes in the anthocyanin biosynthetic pathway, and known transcriptional activators and repressors of the anthocyanin biosynthetic pathway, were upregulated. A novel observation was the induction of a R3-MYB transcriptional repressor that likely provides transcriptional feedback inhibition to prevent the deleterious effects of excess anthocyanins on photosynthesis. CONCLUSIONS: Infiltration-RNAseq is a fast and convenient method for profiling TF-mediated gene expression changes. We utilised this method to identify TF-mediated transcriptional changes and TF target genes in M. truncatula and Nicotiana benthamiana. This included the identification of target genes of a TF not normally expressed in leaves, and targets of TFs from other plant species. Infiltration-RNAseq can be easily adapted to other plant species where agroinfiltration protocols have been optimised. The ability to identify downstream genes, including positive and negative transcriptional regulators, will result in a greater understanding of TF function.