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.

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.

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.

Anti-Microbial, Thermal, Mechanical, and Gas Barrier Properties of Linear Low-Density Polyethylene Extrusion Blow-Molded Bottles.

Microbial contamination can occur on the surfaces of blow-molded bottles, necessitating the development and application of effective anti-microbial treatments to mitigate the hazards associated with microbial growth. In this study, new methods of incorporating anti-microbial particles into linear low-density polyethylene (LLDPE) extrusion blow-molded bottles were developed. The anti-microbial particles were thermally embossed on the external surface of the bottle through two particle deposition approaches (spray and powder) over the mold cavity. The produced bottles were studied for their thermal, mechanical, gas barrier, and anti-microbial properties. Both deposition approaches indicated a significant enhancement in anti-microbial activity, as well as barrier properties, while maintaining thermal and mechanical performance. Considering both the effect of anti-microbial agents and variations in tensile bar weight and thickness, the statistical analysis of the mechanical properties showed that applying the anti-microbial agents had no significant influence on the tensile properties of the blow-molded bottles. The external fixation of the particles over the surface of the bottles would result in optimum anti-microbial activity, making it a cost-effective solution compared to conventional compounding processing.

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.

Quantitative inheritance of crop timing traits in interspecific hybrid Petunia populations and interactions with crop quality parameters.

The leaf unfolding rate (i.e., development rate) and the number of nodes forming prior to floral initiation are 2 factors determining production times for floriculture crops. Wild relative species of the cultivated petunia (Petunia x hybrida Vilm.) that exhibited faster development rates than modern cultivars and may therefore be useful genetic sources to develop cultivars with decreased production time were identified. Three interspecific F(2) families, Petunia exserta Stehmann x P. axillaris (Lam.) Britton et al., P. x hybrida 'Mitchell' x P. axillaris, and P. axillaris x P. integrifolia (Hook.) Schinz & Thell. all exhibited transgressive segregation for development rate and node number below the first flower. Development rate and time to flower segregated independently in all families. Leaf number below the first flower was positively correlated with leaf unfolding rate in all families except P. axillaris x P. integrifolia. Time to flower was positively correlated with flower bud number in the P. x hybrida 'Mitchell' x P. axillaris and P. axillaris x P. integrifolia families only. Based on these results, wild Petunia germplasm should be useful for developing petunia cultivars with reduced crop production times, but some negative effects on crop quality parameters may need to be overcome.

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.

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.

Transcript Profile of Flowering Regulatory Genes in VcFT-Overexpressing Blueberry Plants.

In order to identify genetic components in flowering pathways of highbush blueberry (Vaccinium corymbosum L.), a transcriptome reference composed of 254,396 transcripts and 179,853 gene contigs was developed by assembly of 72.7 million reads using Trinity. Using this transcriptome reference and a query of flowering pathway genes of herbaceous plants, we identified potential flowering pathway genes/transcripts of blueberry. Transcriptome analysis of flowering pathway genes was then conducted on leaf tissue samples of transgenic blueberry cv. Aurora ('VcFT-Aurora'), which overexpresses a blueberry FLOWERING LOCUS T-like gene (VcFT). Sixty-one blueberry transcripts of 40 genes showed high similarities to 33 known flowering-related genes of herbaceous plants, of which 17 down-regulated and 16 up-regulated genes were identified in 'VcFT-Aurora'. All down-regulated genes encoded transcription factors/enzymes upstream in the signaling pathway containing VcFT. A blueberry CONSTANS-LIKE 5-like (VcCOL5) gene was down-regulated and associated with five other differentially expressed (DE) genes in the photoperiod-mediated flowering pathway. Three down-regulated genes, i.e., a MADS-AFFECTING FLOWERING 2-like gene (VcMAF2), a MADS-AFFECTING FLOWERING 5-like gene (VcMAF5), and a VERNALIZATION1-like gene (VcVRN1), may function as integrators in place of FLOWERING LOCUS C (FLC) in the vernalization pathway. Because no CONSTAN1-like or FLOWERING LOCUS C-like genes were found in blueberry, VcCOL5 and VcMAF2/VcMAF5 or VRN1 might be the major integrator(s) in the photoperiod- and vernalization-mediated flowering pathway, respectively. The major down-stream genes of VcFT, i.e., SUPPRESSOR of Overexpression of Constans 1-like (VcSOC1), LEAFY-like (VcLFY), APETALA1-like (VcAP1), CAULIFLOWER 1-like (VcCAL1), and FRUITFULL-like (VcFUL) genes were present and showed high similarity to their orthologues in herbaceous plants. Moreover, overexpression of VcFT promoted expression of all of these VcFT downstream genes. These results suggest that VcFT's down-stream genes appear conserved in blueberry.

Overexpression of blueberry FLOWERING LOCUS T is associated with changes in the expression of phytohormone-related genes in blueberry plants.

Flowering locus T (FT) is a primary integrator in the regulation of plant flowering. Overexpressing a blueberry (Vaccinium corymbosum L.) FT gene (VcFT) (herein VcFT-OX) resulted in early flowering and dwarfing in 'Aurora' plants (herein 'VcFT-Aurora'). In this study, we found that VcFT-OX reduced shoot regeneration from leaf explants. To investigate the potential roles of the phytohormone pathway genes associated with VcFT-OX, differentially expressed (DE) genes in leaf tissues of 'VcFT-Aurora' plants were annotated and analyzed using non-transgenic 'Aurora' plants as a control. Three DE floral genes, including the blueberry SUPPRESSOR of Overexpression of constans 1 (VcSOC1) (gibberellin related), Abscisic acid responsive elements-binding factor 2 (VcABF2) and protein related to ABI3/VP1 (VcABI3/VP1) (ethylene-related), are present under both the phytohormone-responsive and the dwarfing-related Gene Ontology terms. The gene networks of the DE genes overall showed the molecular basis of the multifunctional aspects of VcFT overexpression beyond flowering promotion and suggested that phytohormone changes could be signaling molecules with important roles in the phenotypic changes driven by VcFT-OX.