Regulatory frameworks involved in the floral induction, formation and developmental programming of woody horticultural plants: a case study on blueberries.

Flowering represents a crucial stage in the life cycles of plants. Ensuring strong and consistent flowering is vital for maintaining crop production amidst the challenges presented by climate change. In this review, we summarized key recent efforts aimed at unraveling the complexities of plant flowering through genetic, genomic, physiological, and biochemical studies in woody species, with a special focus on the genetic control of floral initiation and activation in woody horticultural species. Key topics covered in the review include major flowering pathway genes in deciduous woody plants, regulation of the phase transition from juvenile to adult stage, the roles of CONSTANS (CO) and CO-like gene and FLOWERING LOCUS T genes in flower induction, the floral regulatory role of GA-DELLA pathway, and the multifunctional roles of MADS-box genes in flowering and dormancy release triggered by chilling. Based on our own research work in blueberries, we highlighted the central roles played by two key flowering pathway genes, FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, which regulate floral initiation and activation (dormancy release), respectively. Collectively, our survey shows both the conserved and diverse aspects of the flowering pathway in annual and woody plants, providing insights into the potential molecular mechanisms governing woody plants. This paves the way for enhancing the resilience and productivity of fruit-bearing crops in the face of changing climatic conditions, all through the perspective of genetic interventions.

Uncovering genetic and metabolite markers associated with resistance against anthracnose fruit rot in northern highbush blueberry.

Anthracnose fruit rot (AFR), caused by the fungal pathogen Colletotrichum fioriniae, is among the most destructive and widespread fruit disease of blueberry, impacting both yield and overall fruit quality. Blueberry cultivars have highly variable resistance against AFR. To date, this pathogen is largely controlled by applying various fungicides; thus, a more cost-effective and environmentally conscious solution for AFR is needed. Here we report three quantitative trait loci associated with AFR resistance in northern highbush blueberry (Vaccinium corymbosum). Candidate genes within these genomic regions are associated with the biosynthesis of flavonoids (e.g. anthocyanins) and resistance against pathogens. Furthermore, we examined gene expression changes in fruits following inoculation with Colletotrichum in a resistant cultivar, which revealed an enrichment of significantly differentially expressed genes associated with certain specialized metabolic pathways (e.g. flavonol biosynthesis) and pathogen resistance. Using non-targeted metabolite profiling, we identified a flavonol glycoside with properties consistent with a quercetin rhamnoside as a compound exhibiting significant abundance differences among the most resistant and susceptible individuals from the genetic mapping population. Further analysis revealed that this compound exhibits significant abundance differences among the most resistant and susceptible individuals when analyzed as two groups. However, individuals within each group displayed considerable overlapping variation in this compound, suggesting that its abundance may only be partially associated with resistance against C. fioriniae. These findings should serve as a powerful resource that will enable breeding programs to more easily develop new cultivars with superior resistance to AFR and as the basis of future research studies.

Multiple transcriptome comparisons reveal the essential roles of FLOWERING LOCUS T in floral initiation and SOC1 and SVP in floral activation in blueberry.

The flowering mechanisms, especially chilling requirement-regulated flowering, in deciduous woody crops remain to be elucidated. Flower buds of northern highbush blueberry cultivar Aurora require approximately 1,000 chilling hours to bloom. Overexpression of a blueberry FLOWERING LOCUS T (VcFT) enabled precocious flowering of transgenic "Aurora" mainly in non-terminated apical buds during flower bud formation, meanwhile, most of the mature flower buds could not break until they received enough chilling hours. In this study, we highlighted two groups of differentially expressed genes (DEGs) in flower buds caused by VcFT overexpression (VcFT-OX) and full chilling. We compared the two groups of DEGs with a focus on flowering pathway genes. We found: 1) In non-chilled flower buds, VcFT-OX drove a high VcFT expression and repressed expression of a major MADS-box gene, blueberry SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (VcSOC1) resulting an increased VcFT/VcSOC1 expression ratio; 2) In fully chilled flower buds that are ready to break, the chilling upregulated VcSOC1 expression in non-transgenic "Aurora" and repressed VcFT expression in VcFT-OX "Aurora", and each resulted in a decreased ratio of VcFT to VcSOC1; additionally, expression of a blueberry SHORT VEGETATIVE PHASE (VcSVP) was upregulated in chilled flower buds of both transgenic and non-transgenic' "Aurora". Together with additional analysis of VcFT and VcSOC1 in the transcriptome data of other genotypes and tissues, we provide evidence to support that VcFT expression plays a significant role in promoting floral initiation and that VcSOC1 expression is a key floral activator. We thus propose a new hypothesis on blueberry flowering mechanism, of which the ratios of VcFT-to-VcSOC1 at transcript levels in the flowering pathways determine flower bud formation and bud breaking. Generally, an increased VcFT/VcSOC1 ratio or increased VcSOC1 in leaf promotes precocious flowering and flower bud formation, and a decreased VcFT/VcSOC1 ratio with increased VcSOC1 in fully chilled flower buds contributes to flower bud breaking.

Gene Editing Profiles in 94 CRISPR-Cas9 Expressing T0 Transgenic Tobacco Lines Reveal High Frequencies of Chimeric Editing of the Target Gene.

Chimeric editing is often reported in gene editing. To assess how the general chimeric editing is, we created a transgenic tobacco line carrying a marker, beta-glucuronidase gene (gusA), introduced a CRISPR-Cas9 editing vector into the transgenic tobacco line for knocking out gusA, and then investigated the gusA editing efficiencies in T0 and subsequent generations. The editing vector carried a Cas9 gene, which was driven by the cauliflower mosaic virus 35S promoter, and two guide RNAs, gRNA1 and gRNA2, which were driven by Arabidopsis U6 (AtU6) and U3 (AtU3) promoter, respectively. The two gRNAs were designed to knock out a 42-nucleotide fragment of the coding region of gusA. The editing vector was transformed into gusA-containing tobacco leaves using Agrobacterium tumefaciens-mediated transformation and hygromycin selection. Hygromycin-resistant, independent T0 transgenic lines were used to evaluate gusA-editing efficiencies through histochemical GUS assays, polymerase chain reactions (PCR), and next-generation sequencing of PCR amplicons. Profiles of targeted sequences of 94 T0 transgenic lines revealed that these lines were regenerated from non-edited cells where subsequent editing occurred and created chimeric-edited cells in these lines during or after regeneration. Two of them had the target fragment of 42 bp pairs of nucleotides removed. Detail analysis showed that on-target mutations at the AtU6-gRNA1 site and the AtU3-gRNA2 site were found in 4.3% and 77.7% of T0 transgenic lines, respectively. To overcome the issue of extremely low editing efficiencies in T0 lines, we conducted a second round of shoot induction from the chimeric line(s) to enhance the success of obtaining lines with all or most cells edited. The mutation profiles in T0 transgenic lines provide valuable information to understand gene editing in plant cells with constitutively expressed CRISPR-Cas9 and gRNAs.

Removal of a 10-kb Gret1 transposon from VvMybA1 of Vitis vinifera cv. Chardonnay.

Many white grape cultivars have a nonfunctional VvMybA1 gene due to the presence of a 10-kb Gret1 transposon in its promoter. In this study, we successfully demonstrated removal of the 10-kb Gret1 transposon and functional restoration of a VvMybA1 allele in Vitis vinifera cv. Chardonnay through transgenic expression of Cas9 and two gRNAs simultaneously targeting two junction sequences between Gret1 LTRs and VvMybA1. We generated 67 and 24 Cas9-positive vines via Agrobacterium-mediated and biolistic bombardment transformation, respectively. While the editing efficiencies were as high as 17% for the 5' target site and 65% for the 3' target site, simultaneous editing of both 5' and 3' target sites resulting in the removal of Gret1 transposon from the VvMybA1 promoter was 0.5% or less in most transgenic calli, suggesting that these calli had very limited numbers of cells with the Gret1 removed. Nevertheless, two bombardment-transformed vines, which shared the same unique editing features and were likely derived from a singly edited event, were found to have the Gret1 successfully edited out from one of their two VvMybA1 alleles. The edited allele was functionally restored based on the detection of its expression and a positive coloring assay result in leaves. Precise removal of more than a 10-kb DNA fragment from a gene locus in grape broadens the possibilities of using gene editing technologies to modify various trait genes in grapes and other plants.

There and back again; historical perspective and future directions for Vaccinium breeding and research studies.

The genus Vaccinium L. (Ericaceae) contains a wide diversity of culturally and economically important berry crop species. Consumer demand and scientific research in blueberry (Vaccinium spp.) and cranberry (Vaccinium macrocarpon) have increased worldwide over the crops' relatively short domestication history (~100 years). Other species, including bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and ohelo berry (Vaccinium reticulatum) are largely still harvested from the wild but with crop improvement efforts underway. Here, we present a review article on these Vaccinium berry crops on topics that span taxonomy to genetics and genomics to breeding. We highlight the accomplishments made thus far for each of these crops, along their journey from the wild, and propose research areas and questions that will require investments by the community over the coming decades to guide future crop improvement efforts. New tools and resources are needed to underpin the development of superior cultivars that are not only more resilient to various environmental stresses and higher yielding, but also produce fruit that continue to meet a variety of consumer preferences, including fruit quality and health related traits.

Expression of a maize SOC1 gene enhances soybean yield potential through modulating plant growth and flowering.

Yield enhancement is a top priority for soybean (Glycine max Merr.) breeding. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) is a major integrator in flowering pathway, and it is anticipated to be capable of regulating soybean reproductive stages through its interactions with other MADS-box genes. Thus, we produced transgenic soybean for a constitutive expression of a maize SOC1 (ZmSOC1). T1 transgenic plants, in comparison with the nontransgenic plants, showed early flowering, reduced height of mature plants, and no significant impact on grain quality. The transgenic plants also had a 13.5-23.2% of higher grain weight per plant than the nontransgenic plants in two experiments. Transcriptome analysis in the leaves of 34-day old plants revealed 58 differentially expressed genes (DEGs) responding to the expression of the ZmSOC1, of which the upregulated FRUITFULL MADS-box gene, as well as the transcription factor VASCULAR PLANT ONE-ZINC FINGER1, contributed to the promoted flowering. The downregulated gibberellin receptor GID1B could play a major role in reducing the plant height. The remaining DEGs suggested broader effects on the other unmeasured traits (e.g., photosynthesis efficiency and abiotic tolerance), which could contribute to yield increase. Overall, modulating expression of SOC1 in soybean provides a novel and promising approach to regulate plant growth and reproductive development and thus has a potential either to enhance grain yield or to change plant adaptability.

Utilizing MIKC-type MADS-box protein SOC1 for yield potential enhancement in maize.

KEY MESSAGE: Overexpression of Zea mays SOC gene promotes flowering, reduces plant height, and leads to no reduction in grain production per plant, suggesting enhanced yield potential, at least, through increasing planting density. MIKC-type MADS-box gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) is an integrator conserved in the plant flowering pathway. In this study, the maize SOC1 (ZmSOC1) gene was cloned and overexpressed in transgenic maize Hi-II genotype. The T0 plants were backcrossed with nontransgenic inbred B73 to produce first generation backcross (BC1) seeds. Phenotyping of both transgenic and null segregant (NT) BC1 plants was conducted in three independent experiments. The BC1 transgenic plants showed new attributes such as increased vegetative growth, accelerated flowering time, reduced overall plant height, and increased grain weight. Second generation backcross (BC2) plants were evaluated in the field using two planting densities. Compared to BC2 NT plants, BC2 transgenic plants, were 12-18% shorter, flowered 5 days earlier, and showed no reduction in grain production per plant and an increase in fat, starch, and simple sugars in the grain. Transcriptome comparison in young leaves of 56-day-old BC1 plants revealed that the overexpressed ZmSOC1 resulted in 107 differentially expressed genes. The upregulated transcription factor DNA BINDING WITH ONE FINGER 5.4 (DOF5.4) was among the genes responsible for the reduced plant height. Modulating expression of SOC1 opens a new and effective approach to promote flowering and reduce plant height, which may have potential to enhance crop yield and improve grain quality.

K-Domain Technology: Constitutive Expression of a Blueberry Keratin-Like Domain Mimics Expression of Multiple MADS-Box Genes in Enhancing Maize Grain Yield.

MADS-box genes are considered as the foundation of all agronomic traits because they play essential roles in almost every aspect of plant reproductive development. Keratin-like (K) domain is a conserved protein domain of tens of MIKC-type MADS-box genes in plants. K-domain technology constitutively expresses a K-domain to mimic expression of the K-domains of other MADS-box genes simultaneously and thus to generate new opportunities for yield enhancement, because the increased K-domains can likely prevent MADS-domain proteins from binding to target DNA. In this study, we evaluated utilizing the K-domain technology to increase maize yield. The K-domain of a blueberry's SUPPRESSOR of CONSTITUTIVE EXPRESSION OF CONSTANS 1 (VcSOC1K) has similarities to five MADS-box genes in maize. Transgenic maize plants expressing the VcSOC1K showed 13-100% of more grain per plant than the nontransgenic plants in all five experiments conducted under different experimental conditions. Transcriptome comparisons revealed 982 differentially expressed genes (DEGs) in the leaves from 83-day old plants, supporting that the K-domain technology were powerful and multiple functional. The results demonstrated that constitutive expression of the VcSOC1K was very effective to enhance maize grain production. With the potential of mimicking the K-domains of multiple MADS-box genes, the K-domain technology opens a new approach to increase crop yield.

Transfer of endogenous small RNAs between branches of scions and rootstocks in grafted sweet cherry trees.

Grafting is a well-established agricultural practice in cherry production for clonal propagation, altered plant vigor and architecture, increased tolerance to biotic and abiotic stresses, precocity, and higher yield. Mobile molecules, such as water, hormones, nutrients, DNAs, RNAs, and proteins play essential roles in rootstock-scion interactions. Small RNAs (sRNAs) are 19 to 30-nucleotides (nt) RNA molecules that are a group of mobile signals in plants. Rootstock-to-scion transfer of transgene-derived small interfering RNAs enabled virus resistance in nontransgenic sweet cherry scion. To determine whether there was long-distance scion-to-rootstock transfer of endogenous sRNAs, we compared sRNAs profiles in bud tissues of an ungrafted 'Gisela 6' rootstock, two sweet cherry 'Emperor Francis' scions as well as their 'Gisela 6' rootstocks. Over two million sRNAs were detected in each sweet cherry scion, where 21-nt sRNA (56.1% and 55.8%) being the most abundant, followed by 24-nt sRNAs (13.1% and 12.5%). Furthermore, we identified over three thousand sRNAs that were potentially transferred from the sweet cherry scions to their corresponding rootstocks. In contrast to the sRNAs in scions, among the transferred sRNAs in rootstocks, the most abundant were 24-nt sRNAs (46.3% and 34.8%) followed by 21-nt sRNAs (14.6% and 19.3%). In other words, 21-nt sRNAs had the least transferred proportion out of the total sRNAs in sources (scions) while 24-nt had the largest proportion. The transferred sRNAs were from 574 cherry transcripts, of which 350 had a match from the Arabidopsis thaliana standard protein set. The finding that "DNA or RNA binding activity" was enriched in the transcripts producing transferred sRNAs indicated that they may affect the biological processes of the rootstocks at different regulatory levels. Overall, the profiles of the transported sRNAs and their annotations revealed in this study facilitate a better understanding of the role of the long-distance transported sRNAs in sweet cherry rootstock-scion interactions as well as in branch-to-branch interactions in a tree.

Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.).

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD600 of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L-1 glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm.

VcRR2 regulates chilling-mediated flowering through expression of hormone genes in a transgenic blueberry mutant.

The molecular mechanism underlying dormancy release and the induction of flowering remains poorly understood in woody plants. Mu-legacy is a valuable blueberry mutant, in which a transgene insertion caused increased expression of a RESPONSE REGULATOR 2-like gene (VcRR2). Mu-legacy plants, compared with nontransgenic 'Legacy' plants, show dwarfing, promotion of flower bud formation, and can flower under nonchilling conditions. We conducted transcriptomic comparisons in leaves, chilled and nonchilled flowering buds, and late-pink buds, and analyzed a total of 41 metabolites of six groups of hormones in leaf tissues of both Mu-legacy and 'Legacy' plants. These analyses uncovered that increased VcRR2 expression promotes the expression of a homolog of Arabidopsis thaliana ENT-COPALYL DIPHOSPHATE SYNTHETASE 1 (VcGA1), which induces new homeostasis of hormones, including increased gibberellin 4 (GA4) levels in Mu-legacy leaves. Consequently, increased expression of VcRR2 and VcGA1, which function in cytokinin responses and gibberellin synthesis, respectively, initiated the reduction in plant height and the enhancement of flower bud formation of the Mu-legacy plants through interactions of multiple approaches. In nonchilled flower buds, 29 differentially expressed transcripts of 17 genes of five groups of hormones were identified in transcriptome comparisons between Mu-legacy and 'Legacy' plants, of which 22 were chilling responsive. Thus, these analyses suggest that increased expression of VcRR2 was collectively responsible for promoting flower bud formation in highbush blueberry under nonchilling conditions. We report here for the first time the importance of VcRR2 to induce a suite of downstream hormones that promote flowering in woody plants.

VcFT-induced mobile florigenic signals in transgenic and transgrafted blueberries.

FLOWERING LOCUS T (FT) can promote early flowering in annual species, but such role has not been well demonstrated in woody species. We produced self and reciprocal grafts involving non-transgenic blueberry (NT) and transgenic blueberry (T) carrying a 35S-driven blueberry FT (VcFT-OX). We demonstrated that the transgenic VcFT-OX rootstock promoted flowering of non-transgenic blueberry scions in the NT (scion):T (rootstock) grafts. We further analyzed RNA-Seq profiles and six groups of phytohormones in both NT:T and NT:NT plants. We observed content changes of several hormone metabolites, in a descending order, in the transgenic NT:T, non-transgenic NT:T, and non-transgenic NT:NT leaves. By comparing differential expression transcripts (DETs) of these tissues in relative to their control, we found that the non-transgenic NT:T leaves had many DETs shared with the transgenic NT:T leaves, but very few with the transgenic NT:T roots. Interestingly, a number of these shared DETs belong to hormone pathway genes, concurring with the content changes of hormone metabolites in both transgenic and non-transgenic leaves of the NT:T plants. These results suggest that phytohormones induced by VcFT-OX in the transgenic leaves might serve as part of the signals that resulted in early flowering in both transgenic plants and the non-transgenic NT:T scions.

Interfering cellular lactate homeostasis overcomes Taxol resistance of breast cancer cells through the microRNA-124-mediated lactate transporter (MCT1) inhibition.

BACKGROUND: Breast cancer, the most common invasive cancer of women, is a malignant neoplasm and the second main cause of cancer death. Resistance to paclitaxel (Taxol), one of the frequently used chemotherapy agents for breast cancer, presents a major clinical challenge. Recent studies revealed that metabolic alterations of cancer cells play important roles in chemo-resistance. MATERIALS AND METHODS: In this study, Human breast cancer cells, BT474, SKBR3 and MCF7 were used to study the causal relationship between the lactate exporter, MCT1 (SLC16A1)-modulated glucose metabolism and Taxol resistance of breast cancer cells. Taxol resistant breast cancer cells were established. The intracellular lactate and extracellular lactate levels as well glucose uptake and oxygen consumption were measured. MicroRNA-124 expressions were detected by qRT-PCR from both breast cancer patient samples and breast cancer cells. Target of miR-124 was predicted and verified by Western blot and luciferase assay. An xenograft mice model was established and evaluated for the in vivo tumor therapeutic effects of MCT1 inhibitor plus microRNA-124 treatments. RESULTS: Low toxic Taxol treatments promoted cellular glucose metabolism and intracellular lactate accumulation with upregulated lactate dehydrogenase-A (LDHA) and MCT1 expressions. By establishing Taxol resistant breast cancer cell line, we found Taxol resistant cells exhibit upregulated LDHA and MCT1 expressions. Furthermore, glucose consumption, lactate production and intracellular ATP were elevated in Taxol resistant MCF7 cells compared with their parental cells. The miR-124, a tumor suppressive miRNA, was significantly downregulated in Taxol resistant cells. Luciferase assay and q-RT-PCR showed MCT1 is a direct target of miR-124 in both breast cancer cell lines and patient specimens. Moreover, co-treatment of breast cancer cells with either MCT1 inhibitor or miR-124 plus Taxol led to synergistically cytotoxic effects. Importantly, based on in vitro and in vivo results, inhibition of MCT1 significantly sensitized Taxol resistant cells. Finally, rescue experiments showed restoration of MCT1 in miR-124 overexpressing cells promoted Taxol resistance. CONCLUSIONS: This study reveals a possible role of miRNA-214-mediated Taxol resistance, contributing to identify novel therapeutic targets against chemoresistant breast cancers.

Constitutive Expression of an Apple FLC3-like Gene Promotes Flowering in Transgenic Blueberry under Nonchilling Conditions.

MADS-box transcription factors FLOWERING LOCUS C (FLC) and APETALA1 (AP1)/CAULIFLOWER (CAL) have an opposite effect in vernalization-regulated flowering in Arabidopsis. In woody plants, a functional FLC-like gene has not been verified through reverse genetics. To reveal chilling-regulated flowering mechanisms in woody fruit crops, we conducted phylogenetic analysis of the annotated FLC-like proteins of apple and found that these proteins are grouped more closely to Arabidopsis AP1 than the FLC group. An FLC3-like MADS-box gene from columnar apple trees (Malus domestica) (MdFLC3-like) was cloned for functional analysis through a constitutive transgenic expression. The MdFLC3-like shows 88% identity to pear's FLC-like genes and 82% identity to blueberry's CAL1 gene (VcCAL1). When constitutively expressed in a highbush blueberry (Vaccinium corymbosum L.) cultivar 'Legacy', the MdFLC3-like induced expressions of orthologues of three MADS-box genes, including APETALA1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, and CAL1. As a consequence, in contrast to the anticipated late flowering associated with an overexpressed FLC-like, the MdFLC3-like promoted flowering of transgenic blueberry plants under nonchilling conditions where nontransgenic 'Legacy' plants could not flower. Thus, the constitutively expressed MdFLC3-like in transgenic blueberries functioned likely as a blueberry's VcCAL1. The results are anticipated to facilitate future studies for revealing chilling-mediated flowering mechanisms in woody plants.

Agrobacterium-Mediated Transformation of Tree Fruit Crops: Methods, Progress, and Challenges.

Genetic engineering based on Agrobacterium-mediated transformation has been a desirable tool to manipulate single or multiple genes of existing genotypes of woody fruit crops, for which conventional breeding is a difficult and lengthy process due to heterozygosity, sexual incompatibility, juvenility, or a lack of natural sources. To date, successful transformation has been reported for many fruit crops. We review the major progress in genetic transformation of these fruit crops made in the past 5 years, emphasizing reproducible transformation protocols as well as the strategies that have been tested in fruit crops. While direct transformation of scion cultivars was mostly used for fruit quality improvement, biotic and abiotic tolerance, and functional gene analysis, transgrafting on genetically modified (GM) rootstocks showed a potential to produce non-GM fruit products. More recently, genome editing technology has demonstrated a potential for gene(s) manipulation of several fruit crops. However, substantial efforts are still needed to produce plants from gene-edited cells, for which tremendous challenge remains in the context of either cell's recalcitrance to regeneration or inefficient gene-editing due to their polyploidy. We propose that effective transient transformation and efficient regeneration are the key for future utilization of genome editing technologies for improvement of fruit crops.

Chemical synthesis, crystal structure, versatile evaluation of their biological activities and molecular simulations of novel pyrithiobac derivatives.

Since pyrithiobac (PTB) is a successful commercial herbicide with very low toxicity against mammals, it is worth exploring its derivatives for an extensive study. Herein, a total of 35 novel compounds were chemically synthesized and single crystal of 6-6 was obtained to confirm the molecular structure of this family of compounds. The novel PTB derivatives were fully evaluated against various biological platforms. From the bioassay results, the best AHAS inhibitor 6-22 displayed weaker herbicidal activity but stronger anti-Candida activity than PTB did. For plant pathogenic fungi, 6-26 showed excellent activity at 50 mg/L dosage. Preliminary insecticidal activity and antiviral activity were also observed for some title compounds. Strikingly, 6-5 exhibited a promising inhibitory activity against SARS-CoV Mpro with IC50 of 4.471 µM and a low cellular cytotoxicity against mammalian 293 T cells. Based on the results of molecular modeling, HOMO-1 was considered to be a factor that affects AHAS inhibition and a possible binding mode of 6-5 with SARS-CoV Mpro was predicted. This is the first time that PTB derivatives have been studied as biological agents other than herbicides. The present research hence has suggested that more attentions should be paid to compounds belonging to this family to develop novel agrochemicals or medicines.

Haplotype-phased genome and evolution of phytonutrient pathways of tetraploid blueberry.

BACKGROUND: Highbush blueberry (Vaccinium corymbosum) has long been consumed for its unique flavor and composition of health-promoting phytonutrients. However, breeding efforts to improve fruit quality in blueberry have been greatly hampered by the lack of adequate genomic resources and a limited understanding of the underlying genetics encoding key traits. The genome of highbush blueberry has been particularly challenging to assemble due, in large part, to its polyploid nature and genome size. FINDINGS: Here, we present a chromosome-scale and haplotype-phased genome assembly of the cultivar "Draper," which has the highest antioxidant levels among a diversity panel of 71 cultivars and 13 wild Vaccinium species. We leveraged this genome, combined with gene expression and metabolite data measured across fruit development, to identify candidate genes involved in the biosynthesis of important phytonutrients among other metabolites associated with superior fruit quality. Genome-wide analyses revealed that both polyploidy and tandem gene duplications modified various pathways involved in the biosynthesis of key phytonutrients. Furthermore, gene expression analyses hint at the presence of a spatial-temporal specific dominantly expressed subgenome including during fruit development. CONCLUSIONS: These findings and the reference genome will serve as a valuable resource to guide future genome-enabled breeding of important agronomic traits in highbush blueberry.