Vaccinium as a comparative system for understanding of complex flavonoid accumulation profiles and regulation in fruit.

The genus Vaccinium L. (Ericaceae) contains premium berryfruit crops, including blueberry, cranberry, bilberry, and lingonberry. Consumption of Vaccinium berries is strongly associated with various potential health benefits, many of which are attributed to the relatively high concentrations of flavonoids, including the anthocyanins that provide the attractive red and blue berry colors. Because these phytochemicals are increasingly appealing to consumers, they have become a crop breeding target. There has been substantial recent progress in Vaccinium genomics and genetics together with new functional data on the transcriptional regulation of flavonoids. This is helping to unravel the developmental control of flavonoids and identify genetic regions and genes that can be selected for to further improve Vaccinium crops and advance our understanding of flavonoid regulation and biosynthesis across a broader range of fruit crops. In this update we consider the recent progress in understanding flavonoid regulation in fruit crops, using Vaccinium as an example and highlighting the significant gains in both genomic tools and functional analysis.

The New Cranberry Wisconsin Research Station: Renovation Priorities of a 'Stevens' Cranberry Marsh Based on Visual Mapping, Genetic Testing, and Yield Data.

Cultivar contamination is a common issue in commercial cranberry production. Unknown or unwanted cranberry genotypes are found in commercial cranberry beds that are intended to be a single uniform genotype. Identification of contamination and the impacts of contamination remain crucial issues to the cranberry industry to maintain long-term high productivity. To address this issue, tissue samples were taken from the former commercial beds of the new Wisconsin Cranberry Research Station (WCRS) for genetic fingerprinting analysis. The goals of this collection were to analyze the ten beds for genetic uniformity to determine if any should be maintained or replaced, and to assess the accuracy of visual perception of genetic contamination in the field. A total of 288 DNA samples were collected in the ten cranberry beds, and the 'Stevens' cultivar represented 180 samples, or 69% of the 261 samples expected to be 'Stevens'. Therefore, genotype contamination in the 'Stevens' beds was 31% overall. Overall, visual differentiation was accurate in distinguishing between genotypes and detecting large areas of contamination. A yield analysis was conducted along with the genotypic uniformity assessments, and a significant correlation was found between the 2017 yield of the beds and their level of genetic contamination. This study demonstrates the usefulness of genetic uniformity testing and mapping for cranberry bed management and renovation decision-making.

The Genetic Diversity of Cranberry Crop Wild Relatives, Vaccinium macrocarpon Aiton and V. oxycoccos L., in the US, with Special Emphasis on National Forests.

Knowledge of the genetic diversity in populations of crop wild relatives (CWR) can inform effective strategies for their conservation and facilitate utilization to solve agricultural challenges. Two crop wild relatives of the cultivated cranberry are widely distributed in the US. We studied 21 populations of Vaccinium macrocarpon Aiton and 24 populations of Vaccinium oxycoccos L. across much of their native ranges in the US using 32 simple sequence repeat (SSR) markers. We observed high levels of heterozygosity for both species across populations with private alleles ranging from 0 to 26. For V. macrocarpon, we found a total of 613 alleles and high levels of heterozygosity (HO = 0.99, HT = 0.75). We also observed high numbers of alleles (881) and levels of heterozygosity (HO = 0.71, HT = 0.80) in V. oxycoccos (4x). Our genetic analyses confirmed the field identification of a native population of V. macrocarpon on the Okanogan-Wenatchee National Forest in the state of Washington, far outside the previously reported range for the species. Our results will help to inform efforts of the United States Department of Agriculture Agricultural Research Service (USDA-ARS) and the United States Forest Service (USFS) to conserve the most diverse and unique wild cranberry populations through ex situ preservation of germplasm and in situ conservation in designated sites on National Forests.

Massive phenotyping of multiple cranberry populations reveals novel QTLs for fruit anthocyanin content and other important chemical traits.

Because of its known phytochemical activity and benefits for human health, American cranberry (Vaccinium macrocarpon L.) production and commercialization around the world has gained importance in recent years. Flavonoid compounds as well as the balance of sugars and acids are key quality characteristics of fresh and processed cranberry products. In this study, we identified novel QTL that influence total anthocyanin content (TAcy), titratable acidity (TA), proanthocyanidin content (PAC), Brix, and mean fruit weight (MFW) in cranberry fruits. Using repeated measurements over the fruit ripening period, different QTLs were identified at specific time points that coincide with known chemical changes during fruit development and maturation. Some genetic regions appear to be regulating more than one trait. In addition, we demonstrate the utility of digital imaging as a reliable, inexpensive and high-throughput strategy for the quantification of anthocyanin content in cranberry fruits. Using this imaging approach, we identified a set of QTLs across three different breeding populations which collocated with anthocyanin QTL identified using wet-lab approaches. We demonstrate the use of a high-throughput, reliable and highly accessible imaging strategy for predicting anthocyanin content based on cranberry fruit color, which could have a large impact for both industry and cranberry research.

RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization.

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus.

Love Me Not Meter: A Sensor Device for Detecting Petal Detachment Forces in Arabidopsis thaliana.

Abscission is the developmentally controlled loss of plant organs, providing diverse functions such as shedding senescent or diseased leaves, dispersing seeds, or dropping ripened fruit. This process is defined by the hydrolytic breakdown of the middle lamella in the abscission zone, allowing for organ detachment. While the model plant Arabidopsis thaliana does not undergo leaf abscission, it does have a predictable progression of floral organ abscission. To study abscission zone physical integrity in Arabidopsis, a breakstrength meter for Arabidopsis was developed to reliably measure the petal detachment forces.

The aspartate-less receiver (ALR) domains: distribution, structure and function.

Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from Streptococcus pneumoniae presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.

Are We on the Right Track: Can Our Understanding of Abscission in Model Systems Promote or Derail Making Improvements in Less Studied Crops?

As the world population grows and resources and climate conditions change, crop improvement continues to be one of the most important challenges for agriculturalists. The yield and quality of many crops is affected by abscission or shattering, and environmental stresses often hasten or alter the abscission process. Understanding this process can not only lead to genetic improvement, but also changes in cultural practices and management that will contribute to higher yields, improved quality and greater sustainability. As plant scientists, we have learned significant amounts about this process through the study of model plants such as Arabidopsis, tomato, rice, and maize. While these model systems have provided significant valuable information, we are sometimes challenged to use this knowledge effectively as variables including the economic value of the crop, the uniformity of the crop, ploidy levels, flowering and crossing mechanisms, ethylene responses, cultural requirements, responses to changes in environment, and cellular and tissue specific morphological differences can significantly influence outcomes. The value of genomic resources for lesser-studied crops such as cranberries and grapes and the orphan crop fonio will also be considered.

Symplastic intercellular connectivity regulates lateral root patterning.

Cell-to-cell communication coordinates the behavior of individual cells to establish organ patterning and development. Although mobile signals are known to be important in lateral root development, the role of plasmodesmata (PD)-mediated transport in this process has not been investigated. Here, we show that changes in symplastic connectivity accompany and regulate lateral root organogenesis in Arabidopsis. This connectivity is dependent upon callose deposition around PD affecting molecular flux through the channel. Two plasmodesmal-localized ß-1,3 glucanases (PdBGs) were identified that regulate callose accumulation and the number and distribution of lateral roots. The fundamental role of PD-associated callose in this process was illustrated by the induction of similar phenotypes in lines with altered callose turnover. Our results show that regulation of callose and cell-to-cell connectivity is critical in determining the pattern of lateral root formation, which influences root architecture and optimal plant performance.

LYM2-dependent chitin perception limits molecular flux via plasmodesmata.

Chitin acts as a pathogen-associated molecular pattern from fungal pathogens whose perception triggers a range of defense responses. We show that LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN 2 (LYM2), the Arabidopsis homolog of a rice chitin receptor-like protein, mediates a reduction in molecular flux via plasmodesmata in the presence of chitin. For this response, lym2-1 mutants are insensitive to the presence of chitin, but not to the flagellin derivative flg22. Surprisingly, the chitin-recognition receptor CHITIN ELCITOR RECEPTOR KINASE 1 (CERK1) is not required for chitin-induced changes to plasmodesmata flux, suggesting that there are at least two chitin-activated response pathways in Arabidopsis and that LYM2 is not required for CERK1-mediated chitin-triggered defense responses, indicating that these pathways are independent. In accordance with a role in the regulation of intercellular flux, LYM2 is resident at the plasma membrane and is enriched at plasmodesmata. Chitin-triggered regulation of molecular flux between cells is required for defense responses against the fungal pathogen Botrytis cinerea, and thus we conclude that the regulation of symplastic continuity and molecular flux between cells is a vital component of chitin-triggered immunity in Arabidopsis.

Callose deposition and symplastic connectivity are regulated prior to lateral root emergence.

Root growth is critical for the effective exploitation of the rhizosphere and productive plant growth. Our recent work(1) showed that root architecture was dependent upon the degree of symplastic connectivity between neighboring cells during the specification of lateral root primordia and was affected by genes regulating callose deposition at plasmodesmata (PD). Here we provide additional evidence that both symplastic connectivity and callose are also important during the later phase of lateral root development: emergence. Callose immunolocalization assays indicated that transient symplastic isolation of the primordium occur immediately prior to emergence through the overlaying tissues to produce the mature lateral root.(1) Here we could corroborate these results by analyzing the mobility of a symplastic tracer and the expression of PD genes in lateral roots and in response to auxins. Moreover, we show that altering callose deposition affects the number of emerged lateral roots suggesting that PD regulation is important for emergence.

Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function.

Herbivory results in an array of physiological changes in the host that are separable from the associated physical damage. We have made the surprising observation that an Arabidopsis line (pdko3) mutated in genes encoding plasmodesmal proteins is defective in some, but not all, of the typical plant responses to herbivory. We tested the responses of plasma transmembrane potential (Vm) depolarization, voltage gated K(+) channel activity, cytosolic calcium [Ca2+]cyt and reactive oxygen species (ROS) (H2 O2 and NO) release, shoot-to-root signaling, biosynthesis of the phytohormone jasmonic acid (JA) and the elicitation of volatile organic compounds (VOCs). Following herbivory and the release of factors present in insect oral secretions (including a putative ß-galactofuranose polysaccharide), both the pdko3 and wild type (WT) plants showed a increased accumulation of [Ca2+]cyt , NO and H2 O2 . In contrast, unlike WT plants, the mutant line showed an almost complete loss of voltage gated K(+) channel activity and Vm depolarization, a loss of shoot-induced root-Vm depolarization, a loss of activation and regulation of gene expression of the JA defense pathway, and a much diminished release and altered profile of VOCs. The mutations in genes for plasmodesmal proteins have provided valuable genetic tools for the dissection of the complex spectrum of responses to herbivory and shown us that the responses to herbivory can be separated into a calcium-activated oxidative response and a K(+) -dependent Vm-activated jasmonate response associated with the release of VOCs.

Plasmodesmata - membrane tunnels with attitude.

Plasmodesmata are doors in the rigid cell wall. In multicellular tissues, they allow the passage of molecules needed to create physiological gradients and, by closure, symplastic boundaries, which are necessary for the fundamental processes of plant growth, development and defence. Despite this central role in plant growth our knowledge of their contribution has been hindered by difficulties in biochemical and molecular characterisation. Recent advances in proteomic, biochemical, cell biological and genetic analysis of their structure and function is showing that plasmodesmata are plastic yet highly regulated structures. They require the perception of small molecule signals (such as reactive oxygen species) to activate local changes in the cell wall that place physical constraints on the channel. This article reviews recent evidence that highlights the roles of the membrane subcomponents both as structural elements and as environments for resident signalling molecules.

Arabidopsis plasmodesmal proteome.

The multicellular nature of plants requires that cells should communicate in order to coordinate essential functions. This is achieved in part by molecular flux through pores in the cell wall, called plasmodesmata. We describe the proteomic analysis of plasmodesmata purified from the walls of Arabidopsis suspension cells. Isolated plasmodesmata were seen as membrane-rich structures largely devoid of immunoreactive markers for the plasma membrane, endoplasmic reticulum and cytoplasmic components. Using nano-liquid chromatography and an Orbitrap ion-trap tandem mass spectrometer, 1341 proteins were identified. We refer to this list as the plasmodesmata- or PD-proteome. Relative to other cell wall proteomes, the PD-proteome is depleted in wall proteins and enriched for membrane proteins, but still has a significant number (35%) of putative cytoplasmic contaminants, probably reflecting the sensitivity of the proteomic detection system. To validate the PD-proteome we searched for known plasmodesmal proteins and used molecular and cell biological techniques to identify novel putative plasmodesmal proteins from a small subset of candidates. The PD-proteome contained known plasmodesmal proteins and some inferred plasmodesmal proteins, based upon sequence or functional homology with examples identified in different plant systems. Many of these had a membrane association reflecting the membranous nature of isolated structures. Exploiting this connection we analysed a sample of the abundant receptor-like class of membrane proteins and a small random selection of other membrane proteins for their ability to target plasmodesmata as fluorescently-tagged fusion proteins. From 15 candidates we identified three receptor-like kinases, a tetraspanin and a protein of unknown function as novel potential plasmodesmal proteins. Together with published work, these data suggest that the membranous elements in plasmodesmata may be rich in receptor-like functions, and they validate the content of the PD-proteome as a valuable resource for the further uncovering of the structure and function of plasmodesmata as key components in cell-to-cell communication in plants.

Structure-based mutational analysis of eIF4E in relation to sbm1 resistance to pea seed-borne mosaic virus in pea.

BACKGROUND: Pea encodes eukaryotic translation initiation factor eIF4E (eIF4E(S)), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4E(R)) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection. METHODOLOGY/PRINCIPAL FINDINGS: To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4E(S) bound to m(7)GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m(7)GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4E(R) and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m(7)GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation. CONCLUSIONS/SIGNIFICANCE: The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional domains within the eIF4E structure, we identified eIF4E alleles with the potential to convey novel virus resistance phenotypes.

A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

Plasmodesmata: gateways to local and systemic virus infection.

As channels that provide cell-to-cell connectivity, plasmodesmata are central to the local and systemic spread of viruses in plants. This review discusses the current state of knowledge of the structure and function of these channels and the ways in which viruses bring about functional changes that allow macromolecular trafficking to occur. Despite the passing of two decades since the first identification of a viral movement protein that mediates these changes, our understanding of the relevant molecular mechanisms remains in its infancy. However, viral movement proteins provide valuable tools for the modification of plasmodesmata and will continue to assist in the dissection of plasmodesmal properties in relation to their core roles in cell-to-cell communication.

Differing requirements for actin and myosin by plant viruses for sustained intercellular movement.

The actin cytoskeleton has been implicated in the intra- and intercellular movement of a growing number of plant and animal viruses. However, the range of viruses influenced by actin for movement and the mechanism of this transport are poorly understood. Here we determine the importance of microfilaments and myosins for the sustained intercellular movement of a group of RNA-based plant viruses. We demonstrate that the intercellular movement of viruses from different genera [tobacco mosaic virus (TMV), potato virus X (PVX), tomato bushy stunt virus (TBSV)], is inhibited by disruption of microfilaments. Surprisingly, turnip vein-clearing virus (TVCV), a virus from the same genus as TMV, did not require intact microfilaments for normal spread. To investigate the molecular basis for this difference we compared the subcellular location of GFP fusions to the 126-kDa protein and the homologous 125-kDa protein from TMV and TVCV, respectively. The 126-kDa protein formed numerous large cytoplasmic inclusions associated with microfilaments, whereas the 125-kDa protein formed few small possible inclusions, none associated with microfilaments. The dependence of TMV, PVX, and TBSV on intact microfilaments for intercellular movement led us to investigate the role of myosin motors in this process. Virus-induced gene silencing of the Nicotiana benthamiana myosin XI-2 gene, but not three other myosins, inhibited only TMV movement. These results indicate that RNA viruses have evolved differently in their requirements for microfilaments and the associated myosin motors, in a manner not correlated with predicted phylogeny.

Crystallization and preliminary X-ray analysis of eukaryotic initiation factor 4E from Pisum sativum.

Crystals of an N-terminally truncated 20 kDa fragment of Pisum sativum eIF4E (DeltaN-eIF4E) were grown by vapour diffusion. X-ray data were recorded to a resolution of 2.2 A from a single crystal in-house. Indexing was consistent with primitive monoclinic symmetry and solvent-content estimations suggested that between four and nine copies of the eIF4E fragment were possible per crystallographic asymmetric unit. eIF4E is an essential component of the eukaryotic translation machinery and recent studies have shown that point mutations of plant eIF4Es can confer resistance to potyvirus infection.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.