Rosaceae fruit transcriptome database(ROFT)-a useful genomic resource for comparing fruits of apple,peach,strawberry,and raspberry

Rosaceae is a large plant family consisting of many economically important fruit crops including peach,apple,pear,strawberry,raspberry,plum,and others.Investigations into their growth and development will promote both basic understanding and progress toward increasing fruit yield and quality.With the ever-increasing high-throughput sequencing data of Rosaceae,comparative studies are hindered by inconsistency of sample collection with regard to tissue,stage,growth conditions,and by vastly different handling of the data.Therefore,databases that enable easy access and effective utilization of directly comparable transcript data are highly desirable.Here,we describe a database for comparative analysis,ROsaceae Fruit Transcriptome database(ROFT),based on RNA-seq data generated from the same laboratory using similarly dissected and staged fruit tissues of four important Rosaceae fruit crops:apple,peach,strawberry,and red raspberry.Hence,the database is unique in allowing easy and robust comparisons among fruit gene expression across the four species.ROFT enables researchers to query orthologous genes and their expression patterns during different fruit developmental stages in the four species,identify tissue-specific and tissue-/stage-specific genes,visualize and compare ortholog expression in different fruit types,explore consensus co-expression networks,and download different data types.The database provides users access to vast amounts of RNA-seq data across the four economically important fruits,enables investigations of fruit type specification and evolution,and facilitates the selection of genes with critical roles in fruit development for further studies.

Morphological and molecular characterization of culturable leaf endophytic fungi from Malabar Spinach:The first report

To isolate and identify the cultivable leaf endophytic fungi of Basella alba L.and B.rubra L.,healthy and fresh leaves were chosen and collected from Sri Devaraj Urs Academy of Higher Education and Research college botanical garden.Authenticated materials were made into small bits,surface sterilized by applying the standard protocol and cultured on a potato dextrose agar plate supplemented with appropriate antibiotics,incubated for 7 to10 days at 28˚C.Seed borne fungal endophytes were also identified by keeping scarified seeds on sterilized agarose.A phylogenetic tree was constructed for the fungi of both the plants.A total of 26 and 19 endophytic cultivable fungi were isolated from B.alba and B.rubra respectively.Majority of the endophytes belongs to the genus Alternaria and several genera in class Dothideomycetes.Our preliminary observation indicated that the vertically transmitted endophytic fungi are the dominant endophytes of the selected plants.We conclude that Alternaria is predominant in both the plants.The mechanism of transition of the microbe from being non-pathogenic in its host to being pathogenic in other plants needs to be studied and elucidated.

Assembly and annotation of Fragaria vesca‘Yellow Wonder'genome,a model diploid strawberry for molecular genetic research

Fragaria vesca,a wild diploid strawberry,serves as a fundamental research model for cultivated strawberry.The current reference genomes available are limited to two closely-related accessions,Hawaii 4 and CFRA2339.The widely-used model accession‘Yellow Wonder'does not yet have its reference genome.In this study,the genome of a 7^(th) generation inbred'Yellow Wonder'was assembled using a combination of Oxford Nanopore long reads and Illumina short reads.The de novo chromosome-scale assembly of this 220 megabase genome possesses 34,007 genes which were annotated through lift over from the Hawaii 4 genome annotation.Genome comparisons show that the‘Yellow Wonder'genome is relatively distinct from the two previously published F.vesca accessions,Hawaii 4 and CFRA2339.The availability of a‘Yellow Wonder'reference genome adds another important genomic resource to Fragaria vesca and enables rapid research progress in strawberry.

Integrated analysis of high-throughput sequencing data shows abscisic acidresponsive genes and miRNAs in strawberry receptacle fruit ripening

The perception and signal transduction of the plant hormone abscisic acid(ABA)are crucial for strawberry fruit ripening,but the underlying mechanism of how ABA regulates ripening-related genes has not been well understood.By employing high-throughput sequencing technology,we comprehensively analyzed transcriptomic and miRNA expression profiles simultaneously in ABA-and nordihydroguaiaretic acid(NDGA,an ABA biosynthesis blocker)-treated strawberry fruits with temporal resolution.The results revealed that ABA regulated many genes in different pathways,including hormone signal transduction and the biosynthesis of secondary metabolites.Transcription factor genes belonging to WRKY and heat shock factor(HSF)families might play key roles in regulating the expression of ABA inducible genes,whereas the KNOTTED1-like homeobox protein and Squamosa Promoter-Binding-like protein 18 might be responsible for ABA-downregulated genes.Additionally,20 known and six novel differentially expressed miRNAs might be important regulators that assist ABA in regulating target genes that are involved in versatile physiological processes,such as hormone balance regulation,pigments formation and cell wall degradation.Furthermore,degradome analysis showed that one novel miRNA,Fa_novel6,could degrade its target gene HERCULES1,which likely contributed to fruit size determination during strawberry ripening.These results expanded our understanding of how ABA drives the strawberry fruit ripening process as well as the role of miRNAs in this process.

Reannotation of the cultivated strawberry genome and establishment of a strawberry genome database

Cultivated strawberry(Fragaria×ananassa)is an important fruit crop species whose fruits are enjoyed by many worldwide.An octoploid of hybrid origin,the complex genome of this species was recently sequenced,serving as a key reference genome for cultivated strawberry and related species of the Rosaceae family.The current annotation of the F.ananassa genome mainly relies on ab initio predictions and,to a lesser extent,transcriptome data.Here,we present the structure and functional reannotation of the F.ananassa genome based on one PacBio full-length RNA library and ninety-two Illumina RNA-Seq libraries.This improved annotation of the F.ananassa genome,v1.0.a2,comprises a total of 108,447 gene models,with 97.85%complete BUSCOs.The models of 19,174 genes were modified,360 new genes were identified,and 11,044 genes were found to have alternatively spliced isoforms.Additionally,we constructed a strawberry genome database(SGD)for strawberry gene homolog searching and annotation downloading.Finally,the transcriptome of the receptacles and achenes of F.ananassa at four developmental stages were reanalyzed and qualified,and the expression profiles of all the genes in this annotation are also provided.Together,this study provides an updated annotation of the F.ananassa genome,which will facilitate genomic analyses across the Rosaceae family and gene functional studies in cultivated strawberry.

Identification of genes preferentially expressed in wild strawberry receptacle fruit and demonstration of their promoter activities

Fragaria vesca(F.vesca),the wild strawberry,is a diploid model for the commercial,octoploid strawberry as well as other members of the economically relevant Rosaceae family.Unlike the fruits of tomato and Arabidopsis,the fleshy fruit of strawberry is unique in that it is derived from the floral receptacle and has an external seed configuration.Thus,identification and subsequent characterization of receptacle-expressed genes may shed light on novel developmental processes or provide insight into how developmental regulation differs between receptacle-derived and ovary-derived fruits.Further,since fruit and flower tissues are the last organs to form on a plant,the development of receptacle fruitspecific promoters may provide useful molecular tools for research and application.In this work,we mined previously generated RNA-Seq datasets and identified 589 genes preferentially expressed in the strawberry receptacle versus all other profiled tissues.Promoters of a select subset of the 589 genes were isolated and their activities tested using a GUS transcriptional reporter.These promoters may now be used by the F.vesca research community for a variety of purposes,including driving expression of tissue-specific reporters,RNAi constructs,or specific genes to manipulate fruit development.Further,identified genes with receptacle-specific expression patterns,including MADS-Box and KNOX family transcription factors,are potential key regulators of fleshy fruit development and attractive candidates for functional characterization.

The red/far-red light photoreceptor FvePhyB regulates tissue elongation and anthocyanin accumulation in woodland strawberry

Light is an important environmental signal that influences plant growth and development.Among the photoreceptors,phytochromes can sense red/far-red light to coordinate various biological processes.However,their functions in strawberry are not yet known.In this study,we identified an EMS mutant,named P8,in woodland strawberry(Fragaria vesca)that showed greatly increased plant height and reduced anthocyanin content.Mapping-by-sequencing revealed that the causal mutation in FvePhyB leads to premature termination of translation.The light treatment assay revealed that FvePhyB is a bona fide red/far-red light photoreceptor,as it specifically inhibits hypocotyl length under red light.Transcriptome analysis showed that the FvePhyB mutation affects the expression levels of genes involved in hormone synthesis and signaling and anthocyanin biosynthesis in petioles and fruits.The srl mutant with a longer internode is caused by a mutation in the DELLA gene FveRGA1(Repressor of GA1)in the gibberellin pathway.We found that the P8 srl double mutant has much longer internodes than srl,suggesting a synergistic role of FvePhyB and FveRGA1 in this process.Taken together,these results demonstrate the important role of FvePhyB in regulating plant architecture and anthocyanin content in woodland strawberry.

Updated annotation of the wild strawberry Fragaria vesca V4 genome

The diploid strawberry Fragaria vesca serves as an ideal model plant for cultivated strawberry(Fragaria×ananassa,8x)and the Rosaceae family.The F.vesca genome was initially published in 2011 using older technologies.Recently,a new and greatly improved F.vesca genome,designated V4,was published.However,the number of annotated genes is remarkably reduced in V4(28,588 genes)compared to the prior annotations(32,831 to 33,673 genes).Additionally,the annotation of V4(v4.0.a1)implements a new nomenclature for gene IDs(FvH4_XgXXXXX),rather than the previous nomenclature(geneXXXXX).Hence,further improvement of the V4 genome annotation and assigning gene expression levels under the new gene IDs with existing transcriptome data are necessary to facilitate the utility of this high-quality F.vesca genome V4.Here,we built a new and improved annotation,v4.0.a2,for F.vesca genome V4.The new annotation has a total of 34,007 gene models with 98.1%complete Benchmarking Universal Single-Copy Orthologs(BUSCOs).In this v4.0.a2 annotation,gene models of 8,342 existing genes are modified,9,029 new genes are added,and 10,176 genes possess alternatively spliced isoforms with an average of 1.90 transcripts per locus.Transcription factors/regulators and protein kinases are globally identified.Interestingly,the transcription factor family FAr-red-impaired Response 1(FAR1)contains 82 genes in v4.0.a2 but only two members in v4.0.a1.Additionally,the expression levels of all genes in the new annotation across a total of 46 different tissues and stages are provided.Finally,miRNAs and their targets are reanalyzed and presented.Altogether,this work provides an updated genome annotation of the F.vesca V4 genome as well as a comprehensive gene expression atlas with the new gene ID nomenclature,which will greatly facilitate gene functional studies in strawberry and other evolutionarily related plant species.

An eFP browser for visualizing strawberry fruit and flower transcriptomes

Wild strawberry Fragaria vesca is emerging as an important model system for the cultivated strawberry due to its diploid genome and availability of extensive transcriptome data and a range of molecular genetic tools.Being able to better utilize these tools,especially the transcriptome data,will greatly facilitate research progress in strawberry and other Rosaceae fruit crops.The electronic fluorescent pictograph(eFP)software is a useful and popular tool to display transcriptome data visually,and is widely used in other model organisms including Arabidopsis and mouse.Here we applied eFP to display wild strawberry RNA sequencing(RNA-seq)data from 42 different tissues and stages,including various flower and fruit developmental stages.In addition,we generated eight additional RNA-seq data sets to represent tissues from ripening-stage receptacle fruit from yellow-colored and red-colored wild strawberry varieties.Differential gene expression analysis between these eight data sets provides additional information for understanding fruit-quality traits.Together,this work greatly facilitates the utility of the extensive transcriptome data for investigating strawberry flower and fruit development as well as fruit-quality traits.

Author Correction:Updated annotation of the wild strawberry Fragaria vesca V4 genome

Since the publication of this article,the authors have noticed that the GeneIDs from new and original genome annotations don’t match in Table S6,the correct Table S6 is given here.The authors would like to apologize for this error.

The MADS-box gene FveSEP3 plays essential roles in flower organogenesis and fruit development in woodland strawberry

Flower and fruit development are two key steps for plant reproduction.The ABCE model for flower development has been well established in model plant species;however,the functions of ABCE genes in fruit crops are less understood.In this work,we identi fied an EMS mutant named R27 in woodland strawberry(Fragaria vesca),showing the conversion of petals,stamens,and carpels to sepaloid organs in a semidominant inheritance fashion.Mapping by sequencing revealed that the class E gene homolog FveSEP3(FvH4_4g23530)possessed the causative mutation in R27 due to a G to E amino acid change in the conserved MADS domain.Additional fvesep3^(CR) mutants generated by CRISPR/Cas9 displayed similar phenotypes to fvesep3-R27.Overexpressing wild-type or mutated FveSEP3 in Arabidopsis suggested that the mutation in R27 might cause a dominant-negative effect.Further analyses indicated that FveSEP3 physically interacted with each of the ABCE proteins in strawberry.Moreover,both R27 and fvesep3^(CR) mutants exhibited parthenocarpic fruit growth and delayed fruit ripening.Transcriptome analysis revealed that both common and specific differentially expressed genes were identi fied in young fruit at 6-7 days post anthesis(DPA)of fvesep3 and pollinated wild type when compared to unpollinated wild type,especially those in the auxin pathway,a key hormone regulating fruit set in strawberry.Together,we provided compelling evidence that FveSEP3 plays predominant E functions compared to other E gene homologs in flower development and that FveSEP3 represses fruit growth in the absence of pollination and promotes fruit ripening in strawberry.

Gibberellic acid induced parthenocarpic‘Honeycrisp’apples(Malus domestica)exhibit reduced ovary width and lower acidity

Fruit set and development are dependent on auxin,gibberellin,and cytokinin,which cause parthenocarpic development in many species when applied ectopically.Commercial sprays containing these hormones are used to improve apple fruit set,size,and shape,but have been implicated negatively in other aspects of fruit quality.We applied gibberellic acid(GA_(3)),synthetic auxin(NAA),and the auxin-transport inhibitor NPA to‘Honeycrisp’apple flowers.Fruit retention and size were quantified throughout development,and seed number and fruit quality parameters were measured at maturity.GA_(3)alone caused the development of seedless parthenocarpic apples.At maturity,GA_(3)-treated apples were narrower due to reduced ovary width,indicating that GA_(3)induced normal growth of the hypanthium,but not the ovary.GA_(3)-treated fruits were also less acidic than hand-pollinated controls,but had similar firmness,starch,and sugar content.To further understand the regulation of parthenocarpy,we performed tissue-specific transcriptome analysis on GA_(3)-treated,NAA-treated,and control fruits,at 18 days after treatment and again at maturity.Overall,transcriptome analysis showed GA_(3)-treated and hand-pollinated fruits were highly similar in RNA expression profiles.Early expression differences in putative cell division,cytokinin degradation,and cell wall modification genes in GA_(3)-treated ovaries correlated with the observed shape differences,while early expression differences in the acidity gene Ma1 may be responsible for the changes in pH.Taken together,our results indicate that GA_(3)triggers the development of parthenocarpic apple fruit with morphological deviations that correlate with a number of candidate gene expression differences.

Impact of X-radiation in the management of COVID-19 disease

Coronaviruses are a diverse group of viruses that infect both animals and humans.Even though the existence of coronavirus and its infection to humans is not new,the 2019-novel coronavirus(nCoV)caused a major burden to individuals and society i.e.,anxiety,fear of infection,extreme competition for hospitalization,and more importantly financial liability.The nCoV infection/disease diagnosis was based on non-specific signs and symptoms,biochemical parameters,detection of the virus using reverse-transcription polymerase chain reaction(RTPCR),and X-ray-based imaging.This review focuses on the consolidation of potentials of X-ray-based imaging modality[chest-X radiography(CXR)and chest computed tomography(CT)]and low-dose radiation therapy(LDRT)for screening,severity,and management of COVID-19 disease.Reported studies suggest that CXR contributed significantly toward initial rapid screening/diagnosis and CT-imaging to monitor the disease severity.The chest CT has high sensitivity up to 98%and low specificity for diagnosis and severity of COVID-19 disease compared to RT-PCR.Similarly,LDRT compliments drug therapy in the early recovery/Less hospital stays by maintaining the physiological parameters better than the drug therapy alone.All the results undoubtedly demonstrated the evidence that X-ray-based technology continues to evolve and play a significant role in human health care even during the pandemic.

Macrophages and the maintenance of homeostasis

There have been many chapters written about macrophage polarization.These chapters generally focus on the role of macrophages in orchestrating immune responses by highlighting the T-cell-derived cytokines that shape these polarizing responses.This bias toward immunity is understandable,given the importance of macrophages to host defense.However,macrophages are ubiquitous and are involved in many different cellular processes,and describing them as immune cells is undoubtedly an oversimplification.It disregards their important roles in development,tissue remodeling,wound healing,angiogenesis,and metabolism,to name just a few processes.In this chapter,we propose that macrophages function as transducers in the body.According to Wikipedia,WA transducer is a device that converts energy from one form to another.^The word transducer is a term used to describe both the"sensor,which can interpret a wide range of energy forms,and the"actuator,which can switch voltages or currents to affect the environment.Macrophages are able to sense a seemingly endless variety of inputs from their environment and transduce these inputs into a variety of different response outcomes.Thus,rather than functioning as immune cells,they should be considered more broadly as cellular transducers that interpret microenvironmental changes and actuate vital tissue responses.In this chapter,we will describe some of the sensory stimuli that macrophages perceive and the responses they make to these stimuli to achieve their prime directive,which is the maintenance of homeostasis.

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

Fruit crops, including apple, orange, grape,banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops,including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including d Cas9-base-editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene-free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumerfriendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.

K＋ Transporter AtCHX17 with Its Hydrophilic C Tail Localizes to Membranes of the Secretory/Endocytic System： Role in Reproduction and Seed Set

The importance of sorting proteins and wall materials to their destination is critical for plant growth and development, though the machinery orchestrating membrane trafficking is poorly understood. Transporters that alter the environment across endomembrane compartments are thought to be important players. Using Escherichia coli and yeast, we previously showed that several Arabidopsis Cation/H＋ eXchanger （AtCHX） members were K＋ transporters with a role in pH homeostasis, though their subcellular location and biological roles in plants are unclear. Co-expression of markers with CHX16, CHX17, CHX18, or CHX19 tagged with a fluorescent protein indicated these transporters associated with plasma membrane （PM） and post-Golgi compartments. Under its native promoter, AtCHX17（l_820）-GFP localized to prevacuolar compartment （PVC） and to PM in roots. Brefeldin A diminished AtCHX17- GFP fluorescence at PM, whereas wortmannin caused formation of GFP-labeled ring-like structures, suggesting AtCHX17 trafficked among PVC, vacuole and PM. AtCHX17（1-472） lacking its carboxylic tail did not associate with PVC or PM in plant cells. Single chx17 mutant or higher-order mutants showed normal root growth and vegetative devel- opment. However, quadruple （chx16chx17chxlSchx19） mutants were reduced in frequency and produced 50%-70% fewer seeds, indicating overlapping roles of several AtCHX17-related transporters in reproduction and/or seed devel- opment. Together, our results suggest that successful reproduction and seed development depend on the ability to regulate cation and pH homeostasis by AtCHX17-1ike transporters on membranes that traffic in the endocytic and/or secretory pathways.

A Transcriptome Atlas of Physcomitrella patens Provides Insights into the Evolution and Development of Land Plants

Identifying the genetic mechanisms that underpin the evolution of new organ and tissue systems is an aim of evolutionary developmental biology. Comparative functional genetic studies between angiosperms and bryophytes can define those genetic changes that were responsible for developmental innovations. Here, we report the generation of a transcriptome atlas covering most phases in the life cycle of the model bryo- phyte Physcomitrella patens, including detailed sporophyte developmental progression. We identified a comprehensive set of sporophyte-specific transcription factors, and found that many of these genes have homologs in angiosperms that function in developmental processes such as flowering and shoot branching. Deletion of the PpTCP5 transcription factor results in development of supernumerary sporangia attached to a single seta, suggesting that it negatively regulates branching in the moss sporophyte. Given that TCP genes repress branching in angiosperms, we suggest that this activity is ancient. Finally, compar- ison of P. patens and Arabidopsis thaliana transcriptomes led us to the identification of a conserved core of transcription factors expressed in tip-growing cells. We identified modifications in the expression patterns of these genes that could account for developmental differences between P. patens tip-growing cells and A. thaliana pollen tubes and root hairs.

Subcellular Redistribution of Root Aquaporins nduced by Hydrogen Peroxide

Aquaporins are water channel proteins that mediate the fine-tuning of cell membrane water permeability during development or in response to environmental stresses. The present work focuses on the oxidative stress-induced redistribution of plasma membrane intrinsic protein （PIP） aquaporins from the plasma membrane （PM） to intracellular membranes. This process was investigated in the Arabidopsis root. Su- crose density gradient centrifugation showed that exposure of roots to 0.5 mM H2O2 induces significant depletion in PM fractions of several abundant PIP homologs after 15 rain. Analyses by single-particle tracking and fluorescence correlative spectroscopy showed that, in the PM of epidermal cells, H2O2 treat- ment induces an increase in lateral motion and a reduction in the density of a fluorescently tagged form of the prototypal AtPIP2;1 isoform, respectively. Co-expression analyses of AtPIP2;1 with endomembrane markers revealed that H2O2 triggers AtPIP2;1 accumulation in the late endosomal compartments. Life- time analyses established that the high stability of PIPs was maintained under oxidative stress conditions, suggesting that H2O2 triggers a mechanism for intracellular sequestration of PM aquaporins without further degradation. In addition to information on cellular regulation of aquaporins, this study provides novel and complementary insights into the dynamic remodeling of plant internal membranes during oxida- tive stress responses.

Disruption of Arabidopsis CHY1 Reveals an Important Role of Metabolic Status in Plant Cold Stress Signaling

To study cold signaling, we screened for Arabidopsis mutants with altered cold-induced transcription of a firefly luciferase reporter gene driven by the CBF3 promoter （CBF3-LUC）. One mutant, chy1-10, displayed reduced cold-induction of CBF3-LUC luminescence. RNA gel blot analysis revealed that expression of endogenous CBFs also was reduced in the chyl mutant, chy1-10 mutant plants are more sensitive to freezing treatment than wild-type after cold acclimation. Both the wild-type and chyl mutant plants are sensitive to darkness-induced starvation at warm temperatures, although chyl plants are slightly more sensitive. This dark-sensitivity is suppressed by cold temperature in the wildtype but not in chyl. Constitutive CBF3 expression partially rescues the sensitivity of chyl-10 plants to dark treatment in the cold. The chyl mutant accumulates higher levels of reactive oxygen species, and application of hydrogen peroxide can reduce cold-induction of CBF3-LUC in wild-type. Map-based cloning of the gene defective in the mutant revealed a nonsense mutation in CHY1, which encodes a peroxisomal β-hydroxyisobutyryl （HIBYL）-CoA hydrolase needed for valine catabolism and fatty acid β-oxidation. Our results suggest a role for peroxisomal metabolism in cold stress signaling, and plant tolerance to cold stress and darkness-induced starvation.

Illumina Sequencing Technology as a Method of Identifying T-DNA Insertion Loci in Activation- Tagged Arabidopsis thaliana Plants

Dear Editor, Forward genetic screens are commonly used as unbiased tools to isolate genes responsible for a phenotype of interest. In Arabidopsis thaliana, especially T-DNA activation tagging pop- ulations are frequently employed. These populations are gener- ated using vectors containing multiple copies of the constitutive 35S promoters derived from cau and often result in isolation i flower mosaic virus （35S CaMV） of dominant gain-of-function alleles （Weigel et al., 2000; Nakazawa et al., 2003）. This allows the study of members of large gene families that are often func- tionally redundant and, therefore, hard to identify in loss-of- function screens. Moreover, due to the dominant nature,