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.

RNA-Seq reveals new DELLA targets and regulation in transgenic GA-insensitive grapevines.

BACKGROUND: Gibberellins (GAs) and their regulator DELLA are involved in many aspects of plant growth and development and most of our current knowledge in the DELLA-facilitated GA signaling was obtained from the studies of annual species. To understand GA-DELLA signaling in perennial species, we created ten GA-insensitive transgenic grapevines carrying a DELLA mutant allele (Vvgai1) in the background of Vitis vinifera 'Thompson Seedless' and conducted comprehensive analysis of their RNA expression profiles in the shoot, leaf and root tissues. RESULTS: The transgenic lines showed varying degrees of dwarf stature and other typical DELLA mutant phenotypes tightly correlated with the levels of Vvgai1 expression. A large number of differentially expressed genes (DEGs) were identified in the shoot, leaf and root tissues of the transgenic lines and these DEGs were involved in diverse biological processes; many of the DEGs showed strong tissue specificity and about 30% them carried a DELLA motif. We further discovered unexpected expression patterns of several key flowering induction genes VvCO, VvCOL1 and VvTFL1. CONCLUSIONS: Our results not only confirmed many previous DELLA study findings in annual species, but also revealed new DELLA targets and responses in grapevine, including the roles of homeodomain transcription factors as potential co-regulators with DELLA in controlling the development of grapevine which uniquely possess both vegetative and reproductive meristems at the same time. The contrasting responses of some key flowering induction pathway genes provides new insights into the divergence of GA-DELLA regulations between annual and perennial species in GA-DELLA signaling.

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.

Comparative transcriptome analysis of nonchilled, chilled, and late-pink bud reveals flowering pathway genes involved in chilling-mediated flowering in blueberry.

BACKGROUND: Blueberry cultivars require a fixed quantity of chilling hours during winter endo-dormancy for vernalization. In this study, transcriptome analysis using RNA sequencing data from nonchilled, chilled, and late pink buds of southern highbush blueberry 'Legacy' was performed to reveal genes associated with chilling accumulation and bud break. RESULTS: Fully chilled 'Legacy' plants flowered normally whereas nonchilled plants could not flower. Compared to nonchilled flower buds, chilled flower buds showed differential expression of 89% of flowering pathway genes, 86% of MADS-box genes, and 84% of cold-regulated genes. Blueberry orthologues of FLOWERING LOCUS T (FT) did not show a differential expression in chilled flower buds (compared to nonchilled flower bud) but were up-regulated in late-pink buds (compared to chilled flower bud). Orthologoues of major MADS-box genes were significantly up-regulated in chilled flower buds and down-regulated in late-pink buds. Functional orthologues of FLOWERING LOCUS C (FLC) were not found in blueberry. Orthologues of Protein FD (FD), TERMINAL FLOWER 1 (TFL1), and LEAFY (LFY) were down-regulated in chilled flower buds and in late-pink buds compared to nonchilled flower bud. CONCLUSIONS: The changes from nonchilled to chilled and chilled to late-pink buds are associated with transcriptional changes in a large number of differentially expressed (DE) phytohormone-related genes and DE flowering pathway genes. The profile of DE genes suggests that orthologues of FT, FD, TFL1, LFY, and MADS-box genes are the major genes involved in chilling-mediated blueberry bud-break. The results contribute to the comprehensive investigation of the vernalization-mediated flowering mechanism in woody plants.

An invaluable transgenic blueberry for studying chilling-induced flowering in woody plants.

BACKGROUND: Many deciduous woody crops require a minimum level of chilling to break dormancy and allow the seasonal growth of vegetative and floral buds. In this study, we report the discovery of an invaluable transgenic event of the blueberry cultivar 'Legacy' (hereafter, Mu-Legacy) for studying chilling-induced flowering in woody plants. Mu-legacy and its progeny provide a unique material to study the unknown mechanism of chilling-mediated flowering in woody plants. RESULTS: Unlike nontransgenic 'Legacy' and plants of 48 other transgenic events, Mu-Legacy plants were able to flower under nonchilling conditions and had early flower bud formation, reduced plant size, and reduced chilling requirement for normal flowering. These characteristics were heritable and also observed in self-pollinated, transgenic T1 progenies of Mu-Legacy. A 47-Kbp genomic sequence surrounding the transgene insertion position was identified. RNA-sequencing data showed increased expression of a RESPONSE REGULATOR 2-like gene (VcRR2), located adjacent to the insertion position in Mu-Legacy and likely driven by the CaMV 35S promoter of the transgene. The Mu-Legacy showed 209 differentially expressed genes (DEGs) in nonchilled flower buds (compared to nontransgenic 'Legacy'), of which only four DEGs were in the flowering pathway. This suggests altered expression of these few genes, VcRR2 and four flowering DEGs, is sufficient to significantly change flowering behavior in Mu-Legacy. CONCLUSIONS: The significance of VcRR2 in Mu-Legacy suggests that the VcRR2-involved cytokinin pathway likely contributes to the major differences in chilling-mediated flowering between woody and herbaceous plants. More importantly, Mu-Legacy shows increased yield potential, a decreased chilling requirement, and better winter hardiness than many low-chilling cultivars growing in southern warm winter conditions.

MAC30 knockdown involved in the activation of the Hippo signaling pathway in breast cancer cells.

Down-regulation of the meningioma-associated protein (MAC30) gene has been found in many solid cancers. This study was carried out to determine the roles and the mechanisms of MAC30 in breast cancer. We used our own data and a public database to analyze the MAC30 mRNA and protein levels in breast cancer tissues. In addition, we established MAC30 knockdown breast cancer cells using MAC30 siRNA. The roles of MAC30 were detected by using the Soft agar assay, Annexin-V-FITC/PI double staining and the Transwell assay. Western blotting was used to analyze the potential mechanism(s) of MAC30 in these cells. We found that MAC30 mRNA and protein were higher in the cancer tissues compared to the matched normal tissues. MAC30 expression was associated with tumor size, tumor differentiation and estrogen receptor (ER) status. Overall survival rate of the patients with low MAC30 expression was obviously higher than the ones with high expression. The apoptotic ratio was lower in MDA-MB-231 and MDA-MB-157 cells with MAC30 expression. By Western blot analysis, we found that increased levels of phosphorylated YAP1, MST1 and LATS1 after MAC30 siRNA transfection in these two cells. In summary, we demonstrate that MAC30 knockdown is involved in the activation of the Hippo signaling pathway.

The Cold-Regulated Genes of Blueberry and Their Response to Overexpression of VcDDF1 in Several Tissues.

Expression of blueberry cold-regulated genes (VcCORs) could play a role in the variable cold hardiness of blueberry tissues. In this study, transcriptome comparisons were conducted to reveal expression of VcCORs in non-acclimated leaves, flower buds, and flowers of both non-transgenic and transgenic blueberries containing an overexpressed blueberry DWARF AND DELAYED FLOWERING gene (VcDDF1) as well as in fully chilled flower buds of non-transgenic blueberry. In non-transgenic blueberries, 57.5% of VcCOR genes showed differential expression in at least one of the three pairwise comparisons between non-acclimated leaves, flower buds, and flowers, and six out of nine dehydration-responsive element-binding factors showed differential expression. In addition, expression of VcDDF1 was not cold-inducible in non-transgenic blueberries and had higher expression in flowers than in leaves or non-acclimated flower buds. In transgenic blueberries, overexpression of VcDDF1 resulted in higher VcDDF1 expression in leaves than in flower buds and flowers. VcDDF1 overexpression enhanced expression of blueberry CBF1 and CBF3 in leaves and repressed expression of CBF3 in both flower buds and flowers. Overall, the results revealed tissue-specific expression patterns of VcCORs. The responses of VcCORs to overexpression of VcDDF1 suggest that it is possible to increase plant cold hardiness through overexpression of a non-cold-inducible gene.

Transcriptomic changes reveal gene networks responding to the overexpression of a blueberry DWARF AND DELAYED FLOWERING 1 gene in transgenic blueberry plants.

BACKGROUND: Constitutive expression of the CBF/DREB1 for increasing freezing tolerance in woody plants is often associated with other phenotypic changes including dwarf plant and delayed flowering. These phenotypic changes have been observed when Arabidopsis DWARF AND DELAYED FLOWERING 1 (DDF1) was overexpressed in A. thaliana plants. To date, the DDF1 orthologues have not been studied in woody plants. The aim of this study is to investigate transcriptomic responses to the overexpression of blueberry (Vaccinium corymbosum) DDF1 (herein, VcDDF1-OX). RESULTS: The VcDDF1-OX resulted in enhanced freezing tolerance in tetraploid blueberry plants and did not result in significant changes in plant size, chilling requirement, and flowering time. Comparative transcriptome analysis of transgenic 'Legacy-VcDDF1-OX' plants containing an overexpressed VcDDF1 with non-transgenic highbush blueberry 'Legacy' plants revealed the VcDDF1-OX derived differentially expressed (DE) genes and transcripts in the pathways of cold-response, plant flowering, DELLA proteins, and plant phytohormones. The increase in freezing tolerance was associated to the expression of cold-regulated genes (CORs) and the ethylene pathway genes. The unchanged plant size, dormancy and flowering were due to the minimal effect of the VcDDF1-OX on the expression of DELLA proteins, flowering pathway genes, and the other phytohormone genes related to plant growth and development. The DE genes in auxin and cytokinin pathways suggest that the VcDDF1-OX has also altered plant tolerance to drought and high salinity. CONCLUSION: A DDF1 orthologue in blueberry functioned differently from the DDF1 reported in Arabidopsis. The overexpression of VcDDF1 or its orthologues is a new approach to increase freezing tolerance of deciduous woody plant species with no obvious effect on plant size and plant flowering time.

MicroRNA-211, a direct negative regulator of CDC25B expression, inhibits triple-negative breast cancer cells' growth and migration.

The non-coding microRNAs (miRNAs) have tissue- and disease-specific expression patterns. Dysregulation of miRNAs has been associated with initiation and progression of oncogenesis in humans. The abnormal expression of CDC25B phosphatases detected in a number of tumors implies that their dysregulation is involved in malignant transformation. Using miRNA target prediction software, we found that miR-211 could target the 3'UTR sequence of CDC25B. To shed light on their roles of miR-211 in breast cancer, the expression of miR-211 was examined by real-time RT-PCR in breast cancer and normal tissues. MiR-211 is significantly downregulated in breast cancer. MiR-211 re-expression suppressed cell growth, cell cycle, migration, and invasion in triple-negative breast cancer (TNBC) cell line MDA-MB231. Luciferase expression from a reporter vector containing the CDC25B -3'UTR was decreased when this construct was transfected with miR-211. The over-expression of miR-211 suppressed the endogenous CDC25B protein level in TNBC cells. For the first time, we demonstrate that miRNA-211 is a direct negative regulator of CDC25B expression in TNBC cells, alters other related target proteins CCNB1 and FOXM1, and then inhibits breast cancer cells growth, migration, and invasion and lead G2/M arrest. The transcriptional loss of miR-211 and the resultant increase in CDC25B expression facilitate increased genomic instability at an early stage of tumor development.

Down-regulated MAC30 expression inhibits proliferation and mobility of human gastric cancer cells.

BACKGROUND: Gastric cancer is one of the most common cancers in the world. MAC30/Transmembrane protein 97 (TMEM97) is aberrantly up-regulated in many human carcinoma cells. However, the function of MAC30 in gastric carcinoma cells is not studied. MATERIAL AND METHODS: To investigate the function of MAC30 in gastric carcinoma, we used RNA silencing technology to knock down the expression of MAC30 in gastric cancer cells BGC-823 and AGS. Real-time quantitative PCR and Western blot were used to analyze the mRNA level and the related protein expression. The localization of MAC30 and lamellipodia was observed by immunofluorescence. The biological phenotypes of gastric cells were examined by cell proliferation assay, cell cycle analysis, apoptosis assay, cell migration and invasion assay. RESULTS: We found that down-regulation of MAC30 expression efficiently inhibited the proliferation of gastric cancer cells. Furthermore, the mobility of gastric cancer cells was also inhibited by down-regulation of MAC30. Moreover, we found that MAC30 knockdown inhibited AKT phosphorylation and reduced the expression of cyclinB1 and WAVE2. CONCLUSION: To our knowledge, this is the first report investigating the effect of MAC30 on growth, cell cycle, migration, and invasion in gastric carcinoma cells via suppressing AKT signaling pathway. MAC30 may be a potential therapeutic target for treatment of gastric carcinoma.

Rootstock-to-scion transfer of transgene-derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry.

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus-resistant transgenic rootstocks developed through siRNA-mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock-to-scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV-hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV-hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long-distance (1.2 m) transfer of PNRSV-hpRNA-derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for 'using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.

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.

Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing.

Prunus necrotic ringspot virus (PNRSV) is a major pollen-disseminated ilarvirus that adversely affects many Prunus species. In this study, an RNA interference (RNAi) vector pART27-PNRSV containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid (triploid) cherry rootstocks, 'Gisela 6' (GI 148-1) and 'Gisela 7'(GI 148-8)', which are tolerant and sensitive, respectively, to PNRSV infection. One year after inoculation with PNRSV plus Prune Dwarf Virus, nontransgenic 'Gisela 6' exhibited no symptoms but a significant PNRSV titre, while the transgenic 'Gisela 6' had no symptoms and minimal PNRSV titre. The nontransgenic 'Gisela 7' trees died, while the transgenic 'Gisela 7' trees survived. These results demonstrate the RNAi strategy is useful for developing viral resistance in fruit rootstocks, and such transgenic rootstocks may have potential to enhance production of standard, nongenetically modified fruit varieties while avoiding concerns about transgene flow and exogenous protein production that are inherent for transformed fruiting genotypes.

Constitutive expression of the K-domain of a Vaccinium corymbosum SOC1-like (VcSOC1-K) MADS-box gene is sufficient to promote flowering in tobacco.

KEY MESSAGE: The K-domain of a blueberry-derived SOC1 -like gene promotes flowering in tobacco without negatively impacting yield, demonstrating potential for manipulation of flowering time in horticultural crops. ABSTRACT: The SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and SOC1-likes, belonging to the MIKC(c) (type II) MADS-box gene subfamily, are major floral activators and integrators of plant flowering. Both MADS-domains and K (Keratin)-domains are highly conserved in MIKC(c)-type MADS proteins. While there are many reports on overexpression of intact MIKC(c)-type MADS-box genes, few studies have been conducted to investigate the effects of the K-domains. In this report, a 474-bp K-domain of Vaccinium SOC1-like (VcSOC1-K) was cloned from the cDNA library of the northern highbush blueberry (Vaccinium corymbosum L.). Functional analysis of the VcSOC1-K was conducted by ectopically expressing of 35S:VcSOC1-K in tobacco. Reverse transcription PCR confirmed expression of the VcSOC1-K in T0 plants. Phenotypically, T1 transgenic plants (10 T1 plants/event) flowered sooner after seeding, and were shorter with fewer leaves at the time of flowering, than nontransgenic plants; but seed pod production of transgenic plants was not significantly affected. These results demonstrate that overexpression of the K-domain of a MIKC(c)-type MADS-box gene alone is sufficient to promote early flowering and more importantly without affecting seed production.

The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry.

KEY MESSAGE: The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering. ABSTRACT: Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6-10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic 'Aurora' and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

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.

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.

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.