The auronidin flavonoid pigments of the liverwort Marchantia polymorpha form polymers that modify cell wall properties.

Plant adaptation from aquatic to terrestrial environments required modifications to cell wall structure and function to provide tolerance to new abiotic and biotic stressors. Here, we investigate the nature and function of red auronidin pigment accumulation in the cell wall of the liverwort Marchantia polymorpha. Transgenic plants with auronidin production either constitutive or absent were analysed for their cell wall properties, including fractionation of polysaccharide and phenolic components. While small amounts of auronidin and other flavonoids were loosely associated with the cell wall, the majority of the pigments were tightly associated, similar to what is observed in angiosperms for polyphenolics such as lignin. No evidence of covalent binding to a polysaccharide component was found: we propose auronidin is present in the wall as a physically entrapped large molecular weight polymer. The results suggested auronidin is a dual function molecule that can both screen excess light and increase wall strength, hydrophobicity and resistance to enzymatic degradation by pathogens. Thus, liverworts have expanded the core phenylpropanoid toolkit that was present in the ancestor of all land plants, to deliver a lineage-specific solution to some of the environmental stresses faced from a terrestrial lifestyle.

The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum).

Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter-specific, and inter-ploidy admixture of its genome and lack of in depth genome-wide inheritance and comparative structural studies. Here we describe a new high-quality, phased, chromosome-scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high-density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species. Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr-6 and one of chr-10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter-crossable wild diploid species. These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this crop.

Stress, senescence, and specialized metabolites in bryophytes.

Life on land exposes plants to varied abiotic and biotic environmental stresses. These environmental drivers contributed to a large expansion of metabolic capabilities during land plant evolution and species diversification. In this review we summarize knowledge on how the specialized metabolite pathways of bryophytes may contribute to stress tolerance capabilities. Bryophytes are the non-tracheophyte land plant group (comprising the hornworts, liverworts, and mosses) and rapidly diversified following the colonization of land. Mosses and liverworts have as wide a distribution as flowering plants with regard to available environments, able to grow in polar regions through to hot desert landscapes. Yet in contrast to flowering plants, for which the biosynthetic pathways, transcriptional regulation, and compound function of stress tolerance-related metabolite pathways have been extensively characterized, it is only recently that similar data have become available for bryophytes. The bryophyte data are compared with those available for angiosperms, including examining how the differing plant forms of bryophytes and angiosperms may influence specialized metabolite diversity and function. The involvement of stress-induced specialized metabolites in senescence and nutrient response pathways is also discussed.

Evolution and function of red pigmentation in land plants.

BACKGROUND: Land plants commonly produce red pigmentation as a response to environmental stressors, both abiotic and biotic. The type of pigment produced varies among different land plant lineages. In the majority of species they are flavonoids, a large branch of the phenylpropanoid pathway. Flavonoids that can confer red colours include 3-hydroxyanthocyanins, 3-deoxyanthocyanins, sphagnorubins and auronidins, which are the predominant red pigments in flowering plants, ferns, mosses and liverworts, respectively. However, some flowering plants have lost the capacity for anthocyanin biosynthesis and produce nitrogen-containing betalain pigments instead. Some terrestrial algal species also produce red pigmentation as an abiotic stress response, and these include both carotenoid and phenolic pigments. SCOPE: In this review, we examine: which environmental triggers induce red pigmentation in non-reproductive tissues; theories on the functions of stress-induced pigmentation; the evolution of the biosynthetic pathways; and structure-function aspects of different pigment types. We also compare data on stress-induced pigmentation in land plants with those for terrestrial algae, and discuss possible explanations for the lack of red pigmentation in the hornwort lineage of land plants. CONCLUSIONS: The evidence suggests that pigment biosynthetic pathways have evolved numerous times in land plants to provide compounds that have red colour to screen damaging photosynthetically active radiation but that also have secondary functions that provide specific benefits to the particular land plant lineage.

Strigolactones regulate sepal senescence in Arabidopsis.

Flower sepals are critical for flower development and vary greatly in life span depending on their function post-pollination. Very little is known about what controls sepal longevity. Using a sepal senescence mutant screen, we identified two Arabidopsis mutants with delayed senescence directly connecting strigolactones with senescence regulation in a novel floral context that hitherto has not been explored. The mutations were in the strigolactone biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and in the strigolactone receptor gene DWARF14 (AtD14). The mutation in AtD14 changed the catalytic Ser97 to Phe in the enzyme active site, which is the first mutation of its kind in planta. The lesion in MAX1 was in the haem-iron ligand signature of the cytochrome P450 protein, converting the highly conserved Gly469 to Arg, which was shown in a transient expression assay to substantially inhibit the activity of MAX1. The two mutations highlighted the importance of strigolactone activity for driving to completion senescence initiated both developmentally and in response to carbon-limiting stress, as has been found for the more well-known senescence-associated regulators ethylene and abscisic acid. Analysis of transcript abundance in excised inflorescences during an extended night suggested an intricate relationship among sugar starvation, senescence, and strigolactone biosynthesis and signalling.

Staying green postharvest: how three mutations in the Arabidopsis chlorophyll b reductase gene NYC1 delay degreening by distinct mechanisms.

Stresses such as energy deprivation, wounding and water-supply disruption often contribute to rapid deterioration of harvested tissues. To uncover the genetic regulation behind such stresses, a simple assessment system was used to detect senescence mutants in conjunction with two rapid mapping techniques to identify the causal mutations. To demonstrate the power of this approach, immature inflorescences of Arabidopsis plants that contained ethyl methanesulfonate-induced lesions were detached and screened for altered timing of dark-induced senescence. Numerous mutant lines displaying accelerated or delayed timing of senescence relative to wild type were discovered. The underlying mutations in three of these were identified using High Resolution Melting analysis to map to a chromosomal arm followed by a whole-genome sequencing-based mapping method, termed 'Needle in the K-Stack', to identify the causal lesions. All three mutations were single base pair changes and occurred in the same gene, NON-YELLOW COLORING1 (NYC1), a chlorophyll b reductase of the short-chain dehydrogenase/reductase (SDR) superfamily. This was consistent with the mutants preferentially retaining chlorophyll b, although substantial amounts of chlorophyll b were still lost. The single base pair mutations disrupted NYC1 function by three distinct mechanisms, one by producing a termination codon, the second by interfering with correct intron splicing and the third by replacing a highly conserved proline with a non-equivalent serine residue. This non-synonymous amino acid change, which occurred in the NADPH binding domain of NYC1, is the first example of such a mutation in an SDR protein inhibiting a physiological response in plants.

DWARF27 and CAROTENOID CLEAVAGE DIOXYGENASE 7 genes regulate release, germination and growth of gemma in Marchantia polymorpha.

Strigolactones (SLs), a class of carotenoid-derived hormones, play a crucial role in flowering plants by regulating underground communication with symbiotic arbuscular mycorrhizal fungi (AM) and controlling shoot and root architecture. While the functions of core SL genes have been characterized in many plants, their roles in non-tracheophyte plants like liverworts require further investigation. In this study, we employed the model liverwort species Marchantia polymorpha, which lacks detectable SL production and orthologs of key SL biosynthetic genes, including CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) and MORE AXILLARY GROWTH 1 (MAX1). However, it retains some SL pathway components, including DWARF27 (D27) and CCD7. To help elucidate the function of these remaining components in M. polymorpha, knockout mutants were generated for MpD27-1, MpD27-2 and MpCCD7. Phenotypic comparisons of these mutants with the wild-type control revealed a novel role for these genes in regulating the release of gemmae from the gemma cup and the germination and growth of gemmae in the dark. Mpd27-1, Mpd27-2, and Mpccd7 mutants showed lower transcript abundance of genes involved in photosynthesis, such as EARLY LIGHT INDUCED (ELI), and stress responses such as LATE EMBRYOGENESIS ABUNDANT (LEA) but exhibited higher transcript levels of ETHYLENE RESPONSE FACTORS (ERFs) and SL and carotenoid related genes, such as TERPENE SYNTHASE (TS), CCD7 and LECITHIN-RETINAL ACYL TRANSFERASE (LRAT). Furthermore, the mutants of M. polymorpha in the SL pathway exhibited increased contents of carotenoid. This unveils a previously unrecognized role for MpD27-1, MpD27-2 and MpCCD7 in controlling release, germination, and growth of gemmae in response to varying light conditions. These discoveries enhance our comprehension of the regulatory functions of SL biosynthesis genes in non-flowering plants.

A population genomics analysis of the Aotearoa New Zealand endemic rewarewa tree (Knightia excelsa).

Rewarewa (Knightia excelsa, Proteaceae) is a tree species endemic to Aotearoa New Zealand, with a natural distribution spanning Te Ika-a-Maui (North Island) and the top of Te Waipounamu (South Island). We used the pseudo-chromosome genome assembly of rewarewa as a reference and whole genome pooled sequencing from 35 populations sampled across Aotearoa New Zealand, including trees growing on Maori-owned land, to identify 1,443,255 single nucleotide polymorphisms (SNPs). Four genetic clusters located in the northern North Island (NNI), eastern North Island (NIE), western and southern North Island (NIWS), and the South Island (SI) were identified. Gene flow was revealed between the SI and NIE genetic clusters, plus bottleneck and contraction events within the genetic clusters since the mid-late Pleistocene, with divergence between North and South Island clusters estimated to have occurred ~115,000-230,000 years ago. Genotype environment analysis (GEA) was used to identify loci and genes linked with altitude, soil pH, soil carbon, slope, soil size, annual mean temperature, mean diurnal range, isothermality, annual precipitation, and precipitation seasonality. The location of the SNPs associated with these environmental variables was compared with the position of 52,192 gene-coding sequences that were predicted in the rewarewa genome using RNA sequencing. This new understanding of the genetic variation present in rewarewa and insights into the genetic control of adaptive traits will inform efforts to incorporate the species in restoration plantings and for marketing rewarewa honey based on provenance.

Hormonal regulation of leaf senescence through integration of developmental and stress signals.

Leaf senescence is a genetically controlled dismantling programme that enables plants to efficiently remobilise nutrients to new growing sinks. It involves substantial metabolic reprogramming whose timing is affected by developmental and environmental signals. Plant hormones have long been known to affect the timing of leaf senescence, but they also affect plant development and stress responses. It has therefore been difficult to tease apart how the different hormones regulate the onset and progression of leaf senescence, i.e., whether they directly affect leaf senescence or affect it indirectly by altering the developmental programme or by altering plants' response to stress. Here we review research on hormonal regulation of leaf senescence and propose that hormones affect senescence through differential responses to developmental and environmental signals. We suggest that leaf senescence strictly depends on developmental changes, after which senescence can be induced, depending on the type of hormonal and environmental cues.

Carbon deprivation-driven transcriptome reprogramming in detached developmentally arresting Arabidopsis inflorescences.

Senescence is genetically controlled and activated in mature tissues during aging. However, immature plant tissues also display senescence-like symptoms when continuously exposed to adverse energy-depleting conditions. We used detached dark-held immature inflorescences of Arabidopsis (Arabidopsis thaliana) to understand the metabolic reprogramming occurring in immature tissues transitioning from rapid growth to precocious senescence. Macroscopic growth of the detached inflorescences rapidly ceased upon placement in water in the dark at 21°C. Inflorescences were completely degreened by 120 h of dark incubation and by 24 h had already lost 24% of their chlorophyll and 34% of their protein content. Comparative transcriptome profiling at 24 h revealed that inflorescence response at 24 h had a large carbon-deprivation component. Genes that positively regulate developmental senescence (ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN92) and shade-avoidance syndrome (PHYTOCHROME INTERACTING FACTOR4 [PIF4] and PIF5) were up-regulated within 24 h. Mutations in these genes delayed degreening of the inflorescences. Their up-regulation was suppressed in dark-held inflorescences by glucose treatment, which promoted macroscopic growth and development and inhibited degreening of the inflorescences. Detached inflorescences held in the dark for 4 d were still able to reinitiate development to produce siliques upon being brought out to the light, indicating that the transcriptional reprogramming at 24 h was adaptive and reversible. Our results suggest that the response of detached immature tissues to dark storage involves interactions between carbohydrate status sensing and light deprivation signaling and that the dark-adaptive response of the tissues appears to utilize some of the same key regulators as developmental senescence.

The Evolution of Flavonoid Biosynthesis: A Bryophyte Perspective.

The flavonoid pathway is one of the best characterized specialized metabolite pathways of plants. In angiosperms, the flavonoids have varied roles in assisting with tolerance to abiotic stress and are also key for signaling to pollinators and seed dispersal agents. The pathway is thought to be specific to land plants and to have arisen during the period of land colonization around 550-470 million years ago. In this review we consider current knowledge of the flavonoid pathway in the bryophytes, consisting of the liverworts, hornworts, and mosses. The pathway is less characterized for bryophytes than angiosperms, and the first genetic and molecular studies on bryophytes are finding both commonalities and significant differences in flavonoid biosynthesis and pathway regulation between angiosperms and bryophytes. This includes biosynthetic pathway branches specific to each plant group and the apparent complete absence of flavonoids from the hornworts.

Two Loci, RiAF3 and RiAF4, Contribute to the Annual-Fruiting Trait in Rubus.

Most Rubus species have a biennial cycle of flowering and fruiting with an intervening period of winter dormancy, in common with many perennial fruit crops. Annual-fruiting (AF) varieties of raspberry (Rubus idaeus and Rubus occidentalis L.) and blackberry (Rubus subgenus Rubus) are able to flower and fruit in one growing season, without the intervening dormant period normally required in biennial-fruiting (BF) varieties. We used a red raspberry (R. idaeus) population segregating for AF obtained from a cross between NC493 and 'Chilliwack' to identify genetic factors controlling AF. Genotyping by sequencing (GBS) was used to generate saturated linkage maps in both parents. Trait mapping in this population indicated that AF is controlled by two newly identified loci (RiAF3 and RiAF4) located on Rubus linkage groups (LGs) 3 and 4. The location of these loci was analyzed using single-nucleotide polymorphism (SNP) markers on independent red raspberry and blackberry populations segregating for the AF trait. This confirmed that AF in Rubus is regulated by loci on LG 3 and 4, in addition to a previously reported locus on LG 7. Comparative RNAseq analysis at the time of floral bud differentiation in an AF and a BF variety revealed candidate genes potentially regulating the trait.

Cold, antioxidant and osmotic pre-treatments maintain the structural integrity of meristematic cells and improve plant regeneration in cryopreserved kiwifruit shoot tips.

Cryopreservation is a reliable and cost-effective method for the long-term preservation of clonally propagated species. The number of vegetatively propagated species conserved by cryopreservation is increasing through development of vitrification-based methods; droplet vitrification in particular is becoming the preferred method for many species, as it ensures fast freezing and thawing rates. This research investigated if cold, antioxidant and osmotic pre-treatments could maintain the structural integrity of cells, thence aid in developing a droplet vitrification protocol for kiwifruit using Actinidia chinensis var. chinensis 'Hort16A' as a model. Cold acclimation of donor plantlets at 4 °C for 2 weeks followed by sucrose pre-culture of shoot tips and supplementation of ascorbic acid (0.4 mM) in all media throughout the procedure registered 40% regeneration after cryopreservation. Transmission electron microscope imaging of meristematic cells confirmed sucrose and ascorbic acid pre-treatment of shoot tips from cold acclimated plantlets following treatment in vitrification solution exhibited severe plasmolysis and some disruption of membrane and vacuoles. In contrast cells without cold acclimation or sucrose and ascorbic acid pre-treatments exhibited minimal change after exposure to vitrification solution. After cryopreservation and recovery, all cells of untreated shoot tips showed rupture of the plasma membrane, loss of cytoplasmic contents and organelle distortions. By comparison, most pre-treated shoot-tip cells from cold acclimated plantlets retained their structural integrity, showing that only those cells that have been dehydrated and plasmolysed can withstand cryopreservation by vitrification.

Identification of Postharvest Senescence Regulators Through Map-Based Cloning Using Detached Arabidopsis Inflorescences as a Model Tissue.

Postharvest deterioration of fruits and vegetables can be accelerated by biological, environmental, and physiological stresses. Fully understanding tissue response to harvest will provide new opportunities for limiting postharvest losses during handling and storage. The model plant Arabidopsis thaliana (Arabidopsis) has many attributes that make it excellent for studying the underlying control of postharvest responses. It is also one of the best resourced plants with numerous web-based bioinformatic programs and large numbers of mutant collections. Here we introduce a novel assay system called AIDA (the Arabidopsis Inflorescence Degreening Assay) that we developed for understanding postharvest response of immature tissues. We also demonstrate how the high-throughput screening capability of AIDA can be used with mapping technologies (high-resolution melting [HRM] and needle in the k-stack [NIKS]) to identify regulators of postharvest senescence in ethyl methanesulfonate (EMS) mutagenized plant populations. Whether it is best to use HRM or NIKS or both technologies will depend on your laboratory facilities and computing capabilities.

Chromosome-scale scaffolding of the black raspberry (Rubus occidentalis L.) genome based on chromatin interaction data.

Black raspberry (Rubus occidentalis L.) is a niche fruit crop valued for its flavor and potential health benefits. The improvement of fruit and cane characteristics via molecular breeding technologies has been hindered by the lack of a high-quality reference genome. The recently released draft genome for black raspberry (ORUS 4115-3) lacks assembly of scaffolds to chromosome scale. We used high-throughput chromatin conformation capture (Hi-C) and Proximity-Guided Assembly (PGA) to cluster and order 9650 out of 11,936 contigs of this draft genome assembly into seven pseudo-chromosomes. The seven pseudo-chromosomes cover ~97.2% of the total contig length (~223.8 Mb). Locating existing genetic markers on the physical map resolved multiple discrepancies in marker order on the genetic map. Centromeric regions were inferred from recombination frequencies of genetic markers, alignment of 303 bp centromeric sequence with the PGA, and heat map showing the physical contact matrix over the entire genome. We demonstrate a high degree of synteny between each of the seven chromosomes of black raspberry and a high-quality reference genome for strawberry (Fragaria vesca L.) assembled using only PacBio long-read sequences. We conclude that PGA is a cost-effective and rapid method of generating chromosome-scale assemblies from Illumina short-read sequencing data.

A near complete, chromosome-scale assembly of the black raspberry (Rubus occidentalis) genome.

Background: The fragmented nature of most draft plant genomes has hindered downstream gene discovery, trait mapping for breeding, and other functional genomics applications. There is a pressing need to improve or finish draft plant genome assemblies. Findings: Here, we present a chromosome-scale assembly of the black raspberry genome using single-molecule real-time Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated V3 assembly has a contig N50 of 5.1 Mb, representing an ∼200-fold improvement over the previous Illumina-based version. Each of the 235 contigs was anchored and oriented into seven chromosomes, correcting several major misassemblies. Black raspberry V3 contains 47 Mb of new sequences including large pericentromeric regions and thousands of previously unannotated protein-coding genes. Among the new genes are hundreds of expanded tandem gene arrays that were collapsed in the Illumina-based assembly. Detailed comparative genomics with the high-quality V4 woodland strawberry genome (Fragaria vesca) revealed near-perfect 1:1 synteny with dramatic divergence in tandem gene array composition. Lineage-specific tandem gene arrays in black raspberry are related to agronomic traits such as disease resistance and secondary metabolite biosynthesis. Conclusions: The improved resolution of tandem gene arrays highlights the need to reassemble these highly complex and biologically important regions in draft plant genomes. The updated, high-quality black raspberry reference genome will be useful for comparative genomics across the horticulturally important Rosaceae family and enable the development of marker assisted breeding in Rubus.

Arabidopsis AGAMOUS Regulates Sepal Senescence by Driving Jasmonate Production.

The signal that initiates the age-regulated senescence program in flowers is still unknown. Here we propose for the ephemeral Arabidopsis thaliana flower that it dies because of continued expression of the MADS-box transcription factor AGAMOUS (AG). AG is necessary for specifying the reproductive structures of the flower. Flowers of ag-1, which lack AG, exhibited delayed sepal senescence and abscission. The flowers also had reduced jasmonic acid (JA) content. Other anther-defective sterile mutants deficient in JA, defective in anther dehiscence 1 (dad1) and delayed dehiscence 2 (dde2), exhibited delayed sepal senescence and abscission as well. Manually pollinated dad1 flowers produced siliques but still had delayed senescence, demonstrating that absence of pollination does not cause delayed senescence. When ag-1, dad1 and dde2 flowers were sprayed with 100 µM methyl jasmonate, the sepal senescence and abscission phenotypes were rescued, suggesting that JA has a role in these processes. Our study uncovers a novel role for AG in determining the timing of death of the flower it helps develop and highlights a role for JA in sepal senescence.