A signaling cascade mediating fruit trait development via phosphorylation-modulated nuclear accumulation of JAZ repressor.

It is generally accepted that jasmonate-ZIM domain (JAZ) repressors act to mediate jasmonate (JA) signaling via CORONATINE-INSENSITIVE1 (COI1)-mediated degradation. Here, we report a cryptic signaling cascade where a JAZ repressor, FvJAZ12, mediates multiple signaling inputs via phosphorylation-modulated subcellular translocation rather than the COI1-mediated degradation mechanism in strawberry (Fragaria vesca). FvJAZ12 acts to regulate flavor metabolism and defense response, and was found to be the target of FvMPK6, a mitogen-activated protein kinase that is capable of responding to multiple signal stimuli. FvMPK6 phosphorylates FvJAZ12 at the amino acid residues S179 and T183 adjacent to the PY residues, thereby attenuating its nuclear accumulation and relieving its repression for FvMYC2, which acts to control the expression of lipoxygenase 3 (FvLOX3), an important gene involved in JA biosynthesis and a diverse array of cellular metabolisms. Our data reveal a previously unreported mechanism for JA signaling and decipher a signaling cascade that links multiple signaling inputs with fruit trait development.

Expansin SlExp1 and endoglucanase SlCel2 synergistically promote fruit softening and cell wall disassembly in tomato.

Fruit softening, an irreversible process that occurs during fruit ripening, can lead to losses and waste during postharvest transportation and storage. Cell wall disassembly is the main factor leading to loss of fruit firmness, and several ripening-associated cell wall genes have been targeted for genetic modification, particularly pectin modifiers. However, individual knockdown of most cell wall-related genes has had minimal influence on cell wall integrity and fruit firmness, with the notable exception of pectate lyase. Compared to pectin disassembly, studies of the cell wall matrix, the xyloglucan-cellulose framework, and underlying mechanisms during fruit softening are limited. Here, a tomato (Solanum lycopersicum) fruit ripening-associated α-expansin (SlExpansin1/SlExp1) and an endoglucanase (SlCellulase2/SlCel2), which function in the cell wall matrix, were knocked out individually and together using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing. Simultaneous knockout of SlExp1 and SlCel2 enhanced fruit firmness, reduced depolymerization of homogalacturonan-type pectin and xyloglucan, and increased cell adhesion. In contrast, single knockouts of either SlExp1 or SlCel2 did not substantially change fruit firmness, while simultaneous overexpression of SlExp1 and SlCel2 promoted early fruit softening. Collectively, our results demonstrate that SlExp1 and SlCel2 synergistically regulate cell wall disassembly and fruit softening in tomato.

Cannabinoids function in defense against chewing herbivores in Cannabis sativa L.

In the decades since the first cannabinoids were identified by scientists, research has focused almost exclusively on the function and capacity of cannabinoids as medicines and intoxicants for humans and other vertebrates. Very little is known about the adaptive value of cannabinoid production, though several hypotheses have been proposed including protection from ultraviolet radiation, pathogens, and herbivores. To test the prediction that genotypes with greater concentrations of cannabinoids will have reduced herbivory, a segregating F2 population of Cannabis sativa was leveraged to conduct lab- and field-based bioassays investigating the function of cannabinoids in mediating interactions with chewing herbivores. In the field, foliar cannabinoid concentration was inversely correlated with chewing herbivore damage. On detached leaves, Trichoplusia ni larvae consumed less leaf area and grew less when feeding on leaves with greater concentrations of cannabinoids. Scanning electron and light microscopy were used to characterize variation in glandular trichome morphology. Cannabinoid-free genotypes had trichomes that appeared collapsed. To isolate cannabinoids from confounding factors, artificial insect diet was amended with cannabinoids in a range of physiologically relevant concentrations. Larvae grew less and had lower rates of survival as cannabinoid concentration increased. These results support the hypothesis that cannabinoids function in defense against chewing herbivores.

Cuscuta campestris fine-tunes gene expression during haustoriogenesis as an adaptation to different hosts.

The Cuscuta genus comprises obligate parasitic plants that have an unusually wide host range. Whether Cuscuta uses different infection strategies for different hosts or whether the infection strategy is mechanistically and enzymatically conserved remains unknown. To address this, we investigated molecular events during the interaction between field dodder (Cuscuta campestris) and two host species of the Solanum genus that are known to react differently to parasitic infection. We found that host gene induction, particularly of cell wall fortifying genes, coincided with a differential induction of genes for cell wall degradation in the parasite in the cultivated tomato (Solanum lycopersicum) but not in a wild relative (Solanum pennellii). This indicates that the parasite can adjust its gene expression in response to its host. This idea was supported by the increased expression of C. campestris genes encoding an endo-ß-1,4-mannanase in response to exposure of the parasite to purified mono- and polysaccharides in a host-independent infection system. Our results suggest multiple key roles of the host cell wall in determining the outcome of an infection attempt.

Correlations among morphological and biochemical traits in high-cannabidiol hemp (Cannabis sativa L.).

Cannabis sativa is cultivated for multiple uses including the production of cannabinoids. In developing improved production systems for high-cannabinoid cultivars, scientists and cultivators must consider the optimization of complex and interacting sets of morphological, phenological, and biochemical traits, which have historically been shaped by natural and anthropogenic selection. Determining factors that modulate cannabinoid variation within and among genotypes is fundamental to developing efficient production systems and understanding the ecological significance of cannabinoids. Thirty-two high-cannabinoid hemp cultivars were characterized for traits including flowering date and shoot-tip cannabinoid concentration. Additionally, a set of plant architecture traits, as well as wet, dry, and stripped inflorescence biomass were measured at harvest. One plant per plot was partitioned post-harvest to quantify intra-plant variation in inflorescence biomass production and cannabinoid concentration. Some cultivars showed intra-plant variation in cannabinoid concentration, while many had a consistent concentration regardless of canopy position. There was both intra- and inter-cultivar variation in architecture that correlated with intra-plant distribution of inflorescence biomass, and concentration of cannabinoids sampled from various positions within a plant. These relationships among morphological and biochemical traits will inform future decisions by cultivators, regulators, and plant breeders.

Abscisic acid mediated strawberry receptacle ripening involves the interplay of multiple phytohormone signaling networks.

As a canonical non-climacteric fruit, strawberry (Fragaria spp.) ripening is mainly mediated by abscisic acid (ABA), which involves multiple other phytohormone signalings. Many details of these complex associations are not well understood. We present an coexpression network, involving ABA and other phytohormone signalings, based on weighted gene coexpression network analysis of spatiotemporally resolved transcriptome data and phenotypic changes of strawberry receptacles during development and following various treatments. This coexpression network consists of 18,998 transcripts and includes transcripts related to phytohormone signaling pathways, MADS and NAC family transcription factors and biosynthetic pathways associated with fruit quality. Members of eight phytohormone signaling pathways are predicted to participate in ripening and fruit quality attributes mediated by ABA, of which 43 transcripts were screened to consist of the hub phytohormone signalings. In addition to using several genes reported from previous studies to verify the reliability and accuracy of this network, we explored the role of two hub signalings, small auxin up-regulated RNA 1 and 2 in receptacle ripening mediated by ABA, which are also predicted to contribute to fruit quality. These results and publicly accessible datasets provide a valuable resource to elucidate ripening and quality formation mediated by ABA and involves multiple other phytohormone signalings in strawberry receptacle and serve as a model for other non-climacteric fruits.

Spatiotemporal dynamics of the tomato fruit transcriptome under prolonged water stress.

Water availability influences all aspects of plant growth and development; however, most studies of plant responses to drought have focused on vegetative organs, notably roots and leaves. Far less is known about the molecular bases of drought acclimation responses in fruits, which are complex organs with distinct tissue types. To obtain a more comprehensive picture of the molecular mechanisms governing fruit development under drought, we profiled the transcriptomes of a spectrum of fruit tissues from tomato (Solanum lycopersicum), spanning early growth through ripening and collected from plants grown under varying intensities of water stress. In addition, we compared transcriptional changes in fruit with those in leaves to highlight different and conserved transcriptome signatures in vegetative and reproductive organs. We observed extensive and diverse genetic reprogramming in different fruit tissues and leaves, each associated with a unique response to drought acclimation. These included major transcriptional shifts in the placenta of growing fruit and in the seeds of ripe fruit related to cell growth and epigenetic regulation, respectively. Changes in metabolic and hormonal pathways, such as those related to starch, carotenoids, jasmonic acid, and ethylene metabolism, were associated with distinct fruit tissues and developmental stages. Gene coexpression network analysis provided further insights into the tissue-specific regulation of distinct responses to water stress. Our data highlight the spatiotemporal specificity of drought responses in tomato fruit and indicate known and unrevealed molecular regulatory mechanisms involved in drought acclimation, during both vegetative and reproductive stages of development.

A tomato LATERAL ORGAN BOUNDARIES transcription factor, SlLOB1, predominantly regulates cell wall and softening components of ripening.

Fruit softening is a key component of the irreversible ripening program, contributing to the palatability necessary for frugivore-mediated seed dispersal. The underlying textural changes are complex and result from cell wall remodeling and changes in both cell adhesion and turgor. While a number of transcription factors (TFs) that regulate ripening have been identified, these affect most canonical ripening-related physiological processes. Here, we show that a tomato fruit ripening-specific LATERAL ORGAN BOUNDRIES (LOB) TF, SlLOB1, up-regulates a suite of cell wall-associated genes during late maturation and ripening of locule and pericarp tissues. SlLOB1 repression in transgenic fruit impedes softening, while overexpression throughout the plant under the direction of the 35s promoter confers precocious induction of cell wall gene expression and premature softening. Transcript and protein levels of the wall-loosening protein EXPANSIN1 (EXP1) are strongly suppressed in SlLOB1 RNA interference lines, while EXP1 is induced in SlLOB1-overexpressing transgenic leaves and fruit. In contrast to the role of ethylene and previously characterized ripening TFs, which are comprehensive facilitators of ripening phenomena including softening, SlLOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening.

Apple Ripening Is Controlled by a NAC Transcription Factor.

Softening is a hallmark of ripening in fleshy fruits, and has both desirable and undesirable implications for texture and postharvest stability. Accordingly, the timing and extent of pre-harvest ripening and associated textural changes following harvest are key targets for improving fruit quality through breeding. Previously, we identified a large effect locus associated with harvest date and firmness in apple (Malus domestica) using genome-wide association studies (GWAS). Here, we present additional evidence that polymorphisms in or around a transcription factor gene, NAC18.1, may cause variation in these traits. First, we confirmed our previous findings with new phenotype and genotype data from ∼800 apple accessions. In this population, we compared a genetic marker within NAC18.1 to markers targeting three other firmness-related genes currently used by breeders (ACS1, ACO1, and PG1), and found that the NAC18.1 marker was the strongest predictor of both firmness at harvest and firmness after 3 months of cold storage. By sequencing NAC18.1 across 18 accessions, we revealed two predominant haplotypes containing the single nucleotide polymorphism (SNP) previously identified using GWAS, as well as dozens of additional SNPs and indels in both the coding and promoter sequences. NAC18.1 encodes a protein that is orthogolous to the NON-RIPENING (NOR) transcription factor, a regulator of ripening in tomato (Solanum lycopersicum). We introduced both NAC18.1 transgene haplotypes into the tomato nor mutant and showed that both haplotypes complement the nor ripening deficiency. Taken together, these results indicate that polymorphisms in NAC18.1 may underlie substantial variation in apple firmness through modulation of a conserved ripening program.

Morphometric relationships and their contribution to biomass and cannabinoid yield in hybrids of hemp (Cannabis sativa).

The breeding of hybrid cultivars of hemp (Cannabis sativa L.) is not well described, especially the segregation and inheritance of traits that are important for yield. A total of 23 families were produced from genetically diverse parents to investigate the inheritance of morphological traits and their association with biomass accumulation and cannabinoid yield. In addition, a novel classification method for canopy architecture was developed. The strong linear relationship between wet and dry biomass provided an accurate estimate of final dry stripped floral biomass. Of all field and aerial measurements, basal stem diameter was determined to be the single best selection criterion for final dry stripped floral biomass yield. Along with stem diameter, canopy architecture and stem growth predictors described the majority of the explainable variation of biomass yield. Within-family variance for morphological and cannabinoid measurements reflected the heterozygosity of the parents. While selfed populations suffered from inbreeding depression, hybrid development in hemp will require at least one inbred parent to achieve uniform growth and biomass yield. Nevertheless, floral phenology remains a confounding factor in selection because of its underlying influence on biomass production, highlighting the need to understand the genetic basis for flowering time in the breeding of uniform cultivars.

An HD-ZIP-MYB complex regulates glandular secretory trichome initiation in Artemisia annua.

Plant glandular secretory trichomes (GSTs) produce various specialized metabolites. Increasing GST density represents a strategy to enhance the yield of these chemicals; however, the gene regulatory network that controls GST initiation remains unclear. In a previous study of Artemisia annua L., we found that a HD-ZIP IV transcription factor, AaHD1, promotes GST initiation by directly regulating AaGSW2. Here, we identified two AaHD1-interacting transcription factors, namely AaMIXTA-like 2 (AaMYB16) and AaMYB5. Through the generation and characterization of transgenic plants, we found that AaMYB16 is a positive regulator of GST initiation, whereas AaMYB5 has the opposite effect. Notably, neither of them regulates GST formation independently. Rather, they act competitively, by interacting and modulating AaHD1 promoter binding activity. Additionally, the phytohormone jasmonic acid (JA) was shown to be associated with the AaHD1-AaMYB16/AaMYB5 regulatory network through transcriptional regulation via a JASMONATE-ZIM DOMAIN (JAZ) protein repressor. These results bring new insights into the mechanism of GST initiation through regulatory complexes, which appear to have similar functions in a range of vascular plant taxa.

Function of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE is evolutionarily conserved in embryophytes.

The plant phenylpropanoid pathway generates a major class of specialized metabolites and precursors of essential extracellular polymers that initially appeared upon plant terrestrialization. Despite its evolutionary significance, little is known about the complexity and function of this major metabolic pathway in extant bryophytes, which represent the non-vascular stage of embryophyte evolution. Here, we report that the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) gene, which plays a critical function in the phenylpropanoid pathway during seed plant development, is functionally conserved in Physcomitrium patens (Physcomitrella), in the moss lineage of bryophytes. Phylogenetic analysis indicates that bona fide HCT function emerged in the progenitor of embryophytes. In vitro enzyme assays, moss phenolic pathway reconstitution in yeast and in planta gene inactivation coupled to targeted metabolic profiling, collectively indicate that P. patens HCT (PpHCT), similar to tracheophyte HCT orthologs, uses shikimate as a native acyl acceptor to produce a p-coumaroyl-5-O-shikimate intermediate. Phenotypic and metabolic analyses of loss-of-function mutants show that PpHCT is necessary for the production of caffeate derivatives, including previously reported caffeoyl-threonate esters, and for the formation of an intact cuticle. Deep conservation of HCT function in embryophytes is further suggested by the ability of HCT genes from P. patens and the liverwort Marchantia polymorpha to complement an Arabidopsis thaliana CRISPR/Cas9 hct mutant, and by the presence of phenolic esters of shikimate in representative species of the three bryophyte lineages.

Trafficking Processes and Secretion Pathways Underlying the Formation of Plant Cuticles.

Cuticles are specialized cell wall structures that form at the surface of terrestrial plant organs. They are largely comprised lipidic compounds and are deposited in the apoplast, external to the polysaccharide-rich primary wall, creating a barrier to diffusion of water and solutes, as well as to environmental factors. The predominant cuticle component is cutin, a polyester that is assembled as a complex matrix, within and on the surface of which aliphatic and aromatic wax molecules accumulate, further modifying its properties. To reach the point of cuticle assembly the different acyl lipid-containing components are first exported from the cell across the plasma membrane and then traffic across the polysaccharide wall. The export of cutin precursors and waxes from the cell is known to involve plasma membrane-localized ATP-binding cassette (ABC) transporters; however, other secretion mechanisms may also contribute. Indeed, extracellular vesiculo-tubular structures have recently been reported in Arabidopsis thaliana (Arabidopsis) to be associated with the deposition of suberin, a polyester that is structurally closely related to cutin. Intriguingly, similar membranous structures have been observed in leaves and petals of Arabidopsis, although in lower numbers, but no close association with cutin formation has been identified. The possibility of multiple export mechanisms for cuticular components acting in parallel will be discussed, together with proposals for how cuticle precursors may traverse the polysaccharide cell wall before their assimilation into the cuticle macromolecular architecture.

The WRKY transcription factor AaGSW2 promotes glandular trichome initiation in Artemisia annua.

Glandular secreting trichomes (GSTs) synthesize and secrete large quantities of secondary metabolites, some of which have well-established commercial value. An example is the anti-malarial compound artemisinin, which is synthesized in the GSTs of Artemisia annua. Accordingly, there is considerable interest in understanding the processes that regulate GST density as a strategy to increase artemisinin production. In this study, we identified a GST-specific WRKY transcription factor from A. annua, AaGSW2, which is positively regulated by the direct binding of the homeodomain proteins AaHD1 and AaHD8 to the L1-box of the AaGSW2 promoter. Overexpression of AaGSW2 in A. annua significantly increased GST density, while AaGSW2 knockdown lines showed impaired GST initiation. Ectopic expression of AaGSW2 homologs from two mint cultivars, Mentha spicata and Mentha haplocalyx, in A. annua also induced GST formation. These results reveal a molecular mechanism involving homeodomain and WRKY proteins that controls glandular trichome initiation, at least part of which is shared by A. annua and mint.

Transpiration from Tomato Fruit Occurs Primarily via Trichome-Associated Transcuticular Polar Pores.

Nonstomatal water loss by transpiration through the hydrophobic cuticle is ubiquitous in land plants, but the pathways along which this occurs have not been identified. Tomato (Solanum lycopersicum) provides an excellent system in which to study this phenomenon, as its fruit are astomatous and a major target for desiccation resistance to enhance shelf life. We screened a tomato core collection of 398 accessions from around the world and selected seven cultivars that collectively exhibited the lowest and highest degrees of transpirational water loss for a more detailed study. The transpirational differences between these lines reflected the permeances of their isolated cuticles, but this did not correlate with various measures of cuticle abundance or composition. Rather, we found that fruit cuticle permeance has a strong dependence on the abundance of microscopic polar pores. We further observed that these transcuticular pores are associated with trichomes and are exposed when the trichomes are dislodged, revealing a previously unreported link between fruit trichome density and transpirational water loss. During postharvest storage, limited self-sealing of the pores was detected for certain cultivars, in contrast with the stem scar, which healed relatively rapidly. The abundance of trichome-associated pores, together with their self-sealing capacity, presents a promising target for breeding or engineering efforts to reduce fruit transpirational water loss.

Callose deposition is essential for the completion of cytokinesis in the unicellular alga Penium margaritaceum.

Cytokinesis in land plants involves the formation of a cell plate that develops into the new cell wall. Callose, a ß-1,3 glucan, accumulates at later stages of cell plate development, presumably to stabilize this delicate membrane network during expansion. Cytokinetic callose is considered specific to multicellular plant species, because it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte, Penium margaritaceum Callose deposition at the division plane of P. margaritaceum showed distinct, spatiotemporal patterns likely representing distinct roles of this polymer in cytokinesis. Pharmacological inhibition of callose deposition by endosidin 7 resulted in cytokinesis defects, consistent with the essential role for this polymer in P. margaritaceum cell division. Cell wall deposition at the isthmus zone was also affected by the absence of callose, demonstrating the dynamic nature of new wall assembly in P. margaritaceum The identification of candidate callose synthase genes provides molecular evidence for callose biosynthesis in P. margaritaceum The evolutionary implications of cytokinetic callose in this unicellular zygnematopycean alga is discussed in the context of the conquest of land by plants.This article has an associated First Person interview with the first author of the paper.

Comparative genomics of muskmelon reveals a potential role for retrotransposons in the modification of gene expression.

Melon exhibits substantial natural variation especially in fruit ripening physiology, including both climacteric (ethylene-producing) and non-climacteric types. However, genomic mechanisms underlying such variation are not yet fully understood. Here, we report an Oxford Nanopore-based high-grade genome reference in the semi-climacteric cultivar Harukei-3 (378 Mb + 33,829 protein-coding genes), with an update of tissue-wide RNA-seq atlas in the Melonet-DB database. Comparison between Harukei-3 and DHL92, the first published melon genome, enabled identification of 24,758 one-to-one orthologue gene pairs, whereas others were candidates of copy number variation or presence/absence polymorphisms (PAPs). Further comparison based on 10 melon genome assemblies identified genome-wide PAPs of 415 retrotransposon Gag-like sequences. Of these, 160 showed fruit ripening-inducible expression, with 59.4% of the neighboring genes showing similar expression patterns (r > 0.8). Our results suggest that retrotransposons contributed to the modification of gene expression during diversification of melon genomes, and may affect fruit ripening-inducible gene expression.

The Tomato Guanylate-Binding Protein SlGBP1 Enables Fruit Tissue Differentiation by Maintaining Endopolyploid Cells in a Non-Proliferative State.

Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato (Solanum lycopersicum), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein.

Experimental Manipulation of Pectin Architecture in the Cell Wall of the Unicellular Charophyte, Penium Margaritaceum.

Pectins represent one of the main components of the plant primary cell wall. These polymers have critical roles in cell expansion, cell-cell adhesion and response to biotic stress. We present a comprehensive screening of pectin architecture of the unicellular streptophyte, Penium margaritaceum. Penium possesses a distinct cell wall whose outer layer consists of a lattice of pectin-rich fibers and projections. In this study, cells were exposed to a variety of physical, chemical and enzymatic treatments that directly affect the cell wall, especially the pectin lattice. Correlative analyses of pectin lattice perturbation using field emission scanning electron microscopy, confocal laser scanning microscopy, and transmission electron microscopy demonstrate that pectin lattice microarchitecture is both highly sensitive and malleable.