Proteome Analysis of Vacuoles Isolated from Fig (Ficus carica L.) Flesh during Fruit Development.

Fruit flesh cell vacuoles play a pivotal role in fruit growth and quality formation. In the present study, intact vacuoles were carefully released and collected from protoplasts isolated from flesh cells at five sampling times along fig fruit development. Label-free quantification and vacuole proteomic analysis identified 1,251 proteins, 1,137 of which were recruited as differentially abundant proteins (DAPs) by fold change ≥ 1.5, P < 0.05. DAPs were assigned to 10 functional categories; among them, 238, 186, 109, 93 and 90 were annotated as metabolism, transport proteins, membrane fusion or vesicle trafficking, protein fate and stress response proteins, respectively. Decreased numbers of DAPs were uncovered along fruit development. The overall changing pattern of DAPs revealed two major proteome landscape conversions in fig flesh cell vacuoles: the first occurred when fruit developed from late-stage I to mid-stage II, and the second occurred when the fruit started ripening. Metabolic proteins related to glycosidase, lipid and extracellular proteins contributing to carbohydrate storage and vacuole expansion, and protein-degrading proteins determining vacuolar lytic function were revealed. Key tonoplast proteins contributing to vacuole expansion, cell growth and fruit quality formation were also identified. The revealed comprehensive changes in the vacuole proteome during flesh development were compared with our previously published vacuole proteome of grape berry. The information expands our knowledge of the vacuolar proteome and the protein basis of vacuole functional evolution during fruit development and quality formation.

Extreme sugar accumulation in late fig ripening is accompanied by global changes in sugar metabolism and transporter gene expression.

Female fig (Ficus carica L.) fruit are characterized by a major increase in volume and sugar content during the final week of development. A detailed developmental analysis of water and dry matter accumulation during these final days indicated a temporal separation between the increase in volume due to increasing water content and a subsequent sharp increase in sugar content during a few days. The results present fig as an extreme example of sugar import and accumulation, with calculated import rates that are one order of magnitude higher than those of other sugar-accumulating sweet fruit species. To shed light on the metabolic changes occurring during this period, we followed the expression pattern of 80 genes encoding sugar metabolism enzymes and sugar transporter proteins identified in fig fruit. A parallel comparison with male fig fruits, which do not accumulate sugar during ripening, highlighted the genes specifically related to sugar accumulation. Tissue-specific analysis indicated that the expression of genes involved in sugar metabolism and transport undergoes a global transition.

Ethephon induces coordinated ripening acceleration and divergent coloration responses in fig (Ficus carica L.) flowers and receptacles.

KEY MESSAGE: The regulatory landscape of ethephon-accelerated fig ripening is revealed; flowers and receptacles exhibit opposite responses in anthocyanin accumulation; PG, PL and EXP are suggested key genes in fig softening. Ethephon is used to accelerate fig-fruit ripening for improvement of harvesting efficiency, but the underlying molecular mechanism is still unclear. To elucidate the detailed biological mechanism of ethylene-accelerated fig ripening, fruit in phase II (the lag phase on the double sigmoid growth curve) were treated with ethephon, and reached commercial ripeness 6 days earlier than the nontreated controls. Transcriptomes of flowers and the surrounding receptacles-which together make up the pseudocarp in fig fruit-were analyzed. There were 5189, 5818 and 2563 differentially expressed genes (DEGs) 2, 4 and 6 days after treatment (DAT) in treated compared to control fruit, screened by p-adjust < 0.05 and |log2(fold change) |≥ 2. The DEGs were significantly enriched in plant hormone metabolism and signal transduction, cell-wall modification, sugar accumulation and anthocyanin accumulation pathways. DEGs in the first three pathway categories demonstrated an overall similar expression change in flowers and receptacles, whereas DEGs in anthocyanin pigmentation revealed divergent transcript abundance. Specifically, in both flowers and receptacles, ethephon significantly upregulated 1-aminocyclopropane-1-carboxylate oxidase and downregulated most of the ethylene-response factor genes; polygalacturonase, pectate lyase and expansin were mainly upregulated; two acid beta-fructofuranosidases were upregulated. However, structural genes in the anthocyanin-synthesis pathway were mainly downregulated in female flowers 2 and 4 DAT, whereas they were upregulated in the receptacles. Our study reveals the regulatory landscape of the two tissues of fig fruit in ethylene-induced ripening; the differentially expressed pathways and genes provide valuable resources for the mining of target genes for crucial biological and commercial trait improvement.

Tissue-specific organic acid metabolism in reproductive and non-reproductive parts of the fig fruit is partially induced by pollination.

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part - inflorescence and non-reproductive part - receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids - citrate and malate - were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.

Cytokinin-induced parthenocarpy of San Pedro type fig (Ficus carica L.) main crop: explained by phytohormone assay and transcriptomic network comparison.

KEY MESSAGE: CPPU-induced San Pedro type fig main crop parthenocarpy exhibited constantly increasing IAA content and more significantly enriched KEGG pathways in the receptacle than in female flowers. N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) was applied to San Pedro fig (Ficus carica L.) main crop to induce parthenocarpy; the optimal effect was obtained with 25 mg L-1 application to syconia when female flowers were at anthesis. To elucidate the key expression changes in parthenocarpy conversion, significant changes in phytohormone level and transcriptome of fig female flowers and receptacles were monitored. HPLC-MS revealed increased IAA content in female flowers and receptacle 2, 4 and 10 days after treatment (DAT), decreased zeatin level in the receptacle 2, 4 and 10 DAT, decreased GA3 content 2 and 4 DAT, and increased GA3 content 10 DAT. ABA level increased 2 and 4 DAT, and decreased 10 DAT. CPPU-treated syconia released more ethylene than the control except 2 DAT. RNA-Seq and bioinformatics analysis revealed notably more differentially expressed KEGG pathways in the receptacle than in female flowers. In the phytohormone gene network, GA-biosynthesis genes GA20ox and GA3ox were upregulated, along with GA signal-transduction genes GID1 and GID2, and IAA-signaling genes AUX/IAA and GH3. ABA-biosynthesis gene NCED and signaling genes PP2C and ABF were downregulated 10 DAT. One ACO gene showed consistent upregulation in both female flowers and receptacle after CPPU treatment, and more than a dozen of ERFs demonstrated opposing changes in expression. Our results revealed early-stage spatiotemporal phytohormone and transcriptomic responses in CPPU-induced San Pedro fig main crop parthenocarpy, which could be valuable for further understanding the nature of the parthenocarpy of different fig types.

The distinct ripening processes in the reproductive and non-reproductive parts of the fig syconium are driven by ABA.

The common fig bears a unique closed inflorescence structure, the syconium, composed of small individual drupelets that develop from the ovaries, which are enclosed in a succulent receptacle of vegetative origin. The fig ripening process is traditionally classified as climacteric; however, recent studies have suggested that distinct mechanisms exist in its reproductive and non-reproductive parts. We analysed ABA and ethylene production, and expression of ABA-metabolism, ethylene-biosynthesis, MADS-box, NAC, and ethylene response-factor genes in inflorescences and receptacles of on-tree fruit treated with ABA, ethephon, fluridone, and nordihydroguaiaretic acid (NDGA). Exogenous ABA and ethephon accelerated fruit ripening and softening, whereas fluridone and NDGA had the opposite effect, delaying endogenous ABA and ethylene production compared to controls. Expression of the ABA-biosynthesis genes FcNCED2 and FcABA2, ethylene-biosynthesis genes FcACS4, FcACOL, and FcACO2, FcMADS8, 14, 15, FcNAC1, 2, 5, and FcERF9006 was up-regulated by exogenous ABA and ethephon. NDGA down-regulated FcNCED2 and FcABA2, whereas fluridone down-regulated FcABA2; both down-regulated the ethylene-related genes. These results demonstrate the key role of ABA in regulation of ripening by promoting ethylene production, as in the climacteric model plant tomato, especially in the inflorescence. However, increasing accumulation of endogenous ABA until full ripeness and significantly low expression of ethylene-biosynthesis genes in the receptacle suggests non-climacteric, ABA-dependent ripening in the vegetative-originated succulent receptacle part of the fruit.

Transcriptome analysis and metabolic profiling reveal the key role of α-linolenic acid in dormancy regulation of European pear.

Deciduous trees require sufficient chilling during winter dormancy to grow. To decipher the dormancy-regulating mechanism, we carried out RNA sequencing (RNA-Seq) analysis and metabolic profiling of European pear (Pyrus communis L.) vegetative buds during the dormancy phases. Samples were collected from two cultivars that differed greatly in their chilling requirements: 'Spadona' (SPD), a low chilling requirement cultivar; and Harrow Sweet (HS), a high chilling requirement cultivar. Comparative transcriptome analysis revealed >8500 differentially expressed transcripts; most were related to metabolic pathways. Out of 174 metabolites, 44 displayed differential levels in both cultivars, 38 were significantly changed only in SPD, and 15 only in HS. Phospholipids were mostly accumulated at the beginning of dormancy, sugars between before dormancy and mid-dormancy, and fatty acids, including α-linolenic acid, at dormancy break. Differentially expressed genes underlying previously identified major quantitative trait loci (QTLs) in linkage group 8 included genes related to the α-linolenic acid pathway, 12-oxophytodienoate reductase 2-like, and the DORMANCY-ASSOCIATED MADS-BOX (DAM) genes, PcDAM1 and PcDAM2, putative orthologs of PpDAM1 and PpDAM2, confirming their role for the first time in European pear. Additional new putative dormancy-related uncharacterized genes and genes related to metabolic pathways are suggested. These results suggest the crucial role of α-linolenic acid and DAM genes in pear bud dormancy phase transitions.

Transcriptome analysis unravels spatiotemporal modulation of phytohormone-pathway expression underlying gibberellin-induced parthenocarpic fruit set in San Pedro-type fig (Ficus carica L.).

BACKGROUND: Gibberellin (GA) treatments can induce parthenocarpy in the main crop of San Pedro-type figs, the native non-parthenocarpic fruit, however, the underlying mechanism is still largely unclear. RESULTS: In our study, GA3 was applied to San Pedro-type fig main crop at anthesis. Sharply increased GA3 content was detected in both female flowers and receptacle, along with significantly decreased indole-3-acetic acid (IAA), zeatin and abscisic acid (ABA) levels in female flowers, and increased zeatin peak intensity and earlier ABA peak in receptacles. Transcriptome comparison between control and treatment groups identified more differentially expressed genes (DEGs) in receptacles than in female flowers 2 and 4 days after treatment (DAT); 10 DAT, the number of DEGs became similar in the two tissues. Synchronized changing trends of phytohormone-associated DEGs were observed in female flowers and receptacles with fruit development. Modulation of ethylene and GA signaling and auxin metabolism by exogenous GA3 occurred mainly 2 DAT, whereas changes in auxin, cytokinin and ABA signaling occurred mainly 10 DAT. Auxin-, ethylene- and ABA-metabolism and response pathways were largely regulated in the two tissues, mostly 2 and 10 DAT. The major components altering fig phytohormone metabolic and response patterns included downregulated GA2ox, BAS1, NCED and ACO, and upregulated ABA 8'-h and AUX/IAA. CONCLUSIONS: Thus GA-induced parthenocarpy in fig is co-modulated by the female flowers and receptacle, and repression of ABA and ethylene biosynthesis and GA catabolism might be the main forces deflecting abscission and producing fig parthenocarpy.

High-resolution genetic linkage map of European pear (Pyrus communis) and QTL fine-mapping of vegetative budbreak time.

BACKGROUND: Genomic analysis technologies can promote efficient fruit tree breeding. Genotyping by sequencing (GBS) enables generating efficient data for high-quality genetic map construction and QTL analysis in a relatively accessible way. Furthermore, High-resolution genetic map construction and accurate QTL detection can significantly narrow down the putative candidate genes associated with important plant traits. RESULTS: We genotyped 162 offspring in the F1 'Spadona' x 'Harrow Sweet' pear population using GBS. An additional 21 pear accessions, including the F1 population's parents, from our germplasm collection were subjected to GBS to examine diverse genetic backgrounds that are associated to agriculturally relevant traits and to enhance the power of SNP calling. A standard SNP calling pipeline identified 206,971 SNPs with Asian pear ('Suli') as the reference genome and 148,622 SNPs with the European genome ('Bartlett'). These results enabled constructing a genetic map, after further stringent SNP filtering, consisting of 2036 markers on 17 linkage groups with a length of 1433 cM and an average marker interval of 0.7 cM. We aligned 1030 scaffolds covering a total size of 165.5 Mbp (29%) of the European pear genome to the 17 linkage groups. For high-resolution QTL analysis covering the whole genome, we used phenotyping for vegetative budbreak time in the F1 population. New QTLs associated to vegetative budbreak time were detected on linkage groups 5, 13 and 15. A major QTL on linkage group 8 and an additional QTL on linkage group 9 were confirmed. Due to the significant genotype-by-environment (GxE) effect, we were able to identify novel interaction QTLs on linkage groups 5, 8, 9 and 17. Phenotype-genotype association analysis in the pear accessions for main genotype effect was conducted to support the QTLs detected in the F1 population. Significant markers were detected on every linkage group to which main genotype effect QTLs were mapped. CONCLUSIONS: This is the first vegetative budbreak study of European pear that makes use of high-resolution genetic mapping. These results provide tools for marker-assisted selection and accurate QTL analysis in pear, and specifically at vegetative budbreak, considering the significant GxE and phenotype-plasticity effects.

Chlorophyll metabolism in pollinated vs. parthenocarpic fig fruits throughout development and ripening.

MAIN CONCLUSION: Expression of 13 genes encoding chlorophyll biosynthesis and degradation was evaluated. Chlorophyll degradation was differentially regulated in pollinated and parthenocarpic fig fruits, leading to earlier chlorophyll degradation in parthenocarpic fruits. Varieties of the common fig typically yield a commercial summer crop that requires no pollination, although it can be pollinated. Fig fruit pollination results in larger fruit size, greener skin and darker interior inflorescence color, and slows the ripening process compared to non-pollinated fruits. We evaluated the effect of pollination on chlorophyll content and levels of transcripts encoding enzymes of the chlorophyll metabolism in fruits of the common fig 'Brown Turkey'. We cloned and evaluated the expression of 13 different genes. All 13 genes showed high expression in the fruit skin, inflorescences and leaves, but extremely low expression in roots. Pollination delayed chlorophyll breakdown in the ripening fruit skin and inflorescences. This was correlated with the expression of genes encoding enzymes in the chlorophyll biosynthesis and degradation pathways. Expression of pheophorbide a oxygenase (PAO) was strongly negatively correlated with chlorophyll levels during ripening in pollinated fruits; along with its high expression levels in yellow leaves, this supports a pivotal role for PAO in chlorophyll degradation in figs. Normalizing expression levels of all chlorophyll metabolism genes in the pollinated and parthenocarpic fruit skin and inflorescences showed three synthesis (FcGluTR1, FcGluTR2 and FcCLS1) and three degradation (FcCLH1, FcCLH2 and FcRCCR1) genes with different temporal expression in the pollinated vs. parthenocarpic fruit skin and inflorescences. FcCAO also showed different expressions in the parthenocarpic fruit skin. Thus, chlorophyll degradation is differentially regulated in the pollinated and parthenocarpic fruit skin and inflorescences, leading to earlier and more sustained chlorophyll degradation in the parthenocarpic fruit.

Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees.

MAIN CONCLUSION: Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees-apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.

The ambiguous ripening nature of the fig (Ficus carica L.) fruit: a gene-expression study of potential ripening regulators and ethylene-related genes.

The traditional definition of climacteric and non-climacteric fruits has been put into question. A significant example of this paradox is the climacteric fig fruit. Surprisingly, ripening-related ethylene production increases following pre- or postharvest 1-methylcyclopropene (1-MCP) application in an unexpected auto-inhibitory manner. In this study, ethylene production and the expression of potential ripening-regulator, ethylene-synthesis, and signal-transduction genes are characterized in figs ripening on the tree and following preharvest 1-MCP application. Fig ripening-related gene expression was similar to that in tomato and apple during ripening on the tree, but only in the fig inflorescence-drupelet section. Because the pattern in the receptacle is different for most of the genes, the fig drupelets developed inside the syconium are proposed to function as parthenocarpic true fruit, regulating ripening processes for the whole accessory fruit. Transcription of a potential ripening regulator, FcMADS8, increased during ripening on the tree and was inhibited following 1-MCP treatment. Expression patterns of the ethylene-synthesis genes FcACS2, FcACS4, and FcACO3 could be related to the auto-inhibition reaction of ethylene production in 1-MCP-treated fruit. Along with FcMADS8 suppression, gene expression analysis revealed upregulation of FcEBF1, and downregulation of FcEIL3 and several FcERFs by 1-MCP treatment. This corresponded with the high storability of the treated fruit. One FcERF was overexpressed in the 1-MCP-treated fruit, and did not share the increasing pattern of most FcERFs in the tree-ripened fig. This demonstrates the potential of this downstream ethylene-signal-transduction component as an ethylene-synthesis regulator, responsible for the non-climacteric auto-inhibition of ethylene production in fig.

Genetic and molecular regulation of chilling requirements in pear: breeding for climate change resilience.

Pear (Pyrus spp.) is a deciduous fruit tree that requires exposure to sufficient chilling hours during the winter to establish dormancy, followed by favorable heat conditions during the spring for normal vegetative and floral budbreak. In contrast to most temperate woody species, apples and pears of the Rosaceae family are insensitive to photoperiod, and low temperature is the major factor that induces growth cessation and dormancy. Most European pear (Pyrus Communis L.) cultivars need to be grown in regions with high chilling unit (CU) accumulation to ensure early vegetative budbreak. Adequate vegetative budbreak time will ensure suitable metabolite accumulation, such as sugars, to support fruit set and vegetative development, providing the necessary metabolites for optimal fruit set and development. Many regions that were suitable for pear production suffer from a reduction in CU accumulation. According to climate prediction models, many temperate regions currently suitable for pear cultivation will experience a similar accumulation of CUs as observed in Mediterranean regions. Consequently, the Mediterranean region can serve as a suitable location for conducting pear breeding trials aimed at developing cultivars that will thrive in temperate regions in the decades to come. Due to recent climatic changes, bud dormancy attracts more attention, and several studies have been carried out aiming to discover the genetic and physiological factors associated with dormancy in deciduous fruit trees, including pears, along with their related biosynthetic pathways. In this review, current knowledge of the genetic mechanisms associated with bud dormancy in European pear and other Pyrus species is summarized, along with metabolites and physiological factors affecting dormancy establishment and release and chilling requirement determination. The genetic and physiological insights gained into the factors regulating pear dormancy phase transition and determining chilling requirements can accelerate the development of new pear cultivars better suited to both current and predicted future climatic conditions.

Turned on by heat: differential expression of FT and LFY-like genes in Narcissus tazetta during floral transition.

In Narcissus tazetta, a monocotyledonous bulbous geophyte, floral initiation and differentiation occur within the bulb during the quiescent period in summer, when ambient temperatures are relatively high and the bulb is located underground with no foliage or roots. In many plant species, FLOWERING LOCUS T (FT) and its homologues are considered powerful promoters of flowering. The Narcissus FT gene homologue (NtFT) was isolated, and organ-specific expression patterns of NtFT during the annual cycle and reproductive development under different temperature regimes were analysed using quantitative reverse transcription-PCR (qRT-PCR) and RNA in situ hybridization. During floral induction, NtFT was not expressed in bulb scales, roots, or foliage leaves, but it was detected inside the bulb in the apical meristem and leaf primordia. The expression of another key flowering gene, NLF, the LEAFY homologue in N. tazetta, was also observed only in meristem and leaf primordia within the bulbs; however, its expression did not coincide with that of NtFT during meristem transition to reproductive stage. Under high temperatures (25-30 °C) in the dark, NtFT expression occurred simultaneously with floral induction timing, indicating that floral induction is affected by high temperatures but not by photoperiod or vernalization. Monitoring the apical meristem of Narcissus in February-August of two growing seasons under ambient and controlled storage conditions showed that transition to flowering is temperature dependent and varies between years. Lack of NtFT and NLF expression in foliage leaves suggests that flower initiation control in Narcissus differs from that in common model plants.

Development of a transgenic early flowering pear (Pyrus communis L.) genotype by RNAi silencing of PcTFL1-1 and PcTFL1-2.

Trees require a long maturation period, known as juvenile phase, before they can reproduce, complicating their genetic improvement as compared to annual plants. 'Spadona', one of the most important European pear (Pyrus communis L.) cultivars grown in Israel, has a very long juvenile period, up to 14 years, making breeding programs extremely slow. Progress in understanding the molecular basis of the transition to flowering has revealed genes that accelerate reproductive development when ectopically expressed in transgenic plants. A transgenic line of 'Spadona', named Early Flowering-Spadona (EF-Spa), was produced using a MdTFL1 RNAi cassette targeting the native pear genes PcTFL1-1 and PcTFL1-2. The transgenic line had three T-DNA insertions, one assigned to chromosome 2 and two to chromosome 14 PcTFL1-1 and PcTFL1-2 were completely silenced, and EF-Spa displayed an early flowering phenotype: flowers developed already in tissue culture and on most rooted plants 1-8 months after transfer to the greenhouse. EF-Spa developed solitary flowers from apical or lateral buds, reducing vegetative growth vigor. Pollination of EF-Spa trees generated normal-shaped fruits with viable F1 seeds. The greenhouse-grown transgenic F1 seedlings formed shoots and produced flowers 1-33 months after germination. Sequence analyses, of the non-transgenic F1 seedlings, demonstrated that this approach can be used to recover seedlings that have no trace of the T-DNA. Thus, the early flowering transgenic line EF-Spa obtained by PcTFL1 silencing provides an interesting tool to accelerate pear breeding.

Tobacco Rattle Virus as a Tool for Rapid Reverse-Genetics Screens and Analysis of Gene Function in Cannabis sativa L.

Medical cannabis (Cannabis sativa L.) is quickly becoming a central agricultural crop as its production has continued to increase globally. The recent release of the cannabis reference genomes provides key genetic information for the functional analysis of cannabis genes. Currently, however, the established tools for in vivo gene functional analysis in cannabis are very limited. In this study, we investigated the use of the tobacco rattle virus (TRV) as a possible tool for virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE). Using leaf photobleaching as a visual marker of PHYTOENE DESATURASE (PDS) silencing, we found that VIGS was largely restricted to the agro-infiltrated leaves. However, when agro-infiltration was performed under vacuum, VIGS increased dramatically, which resulted in intense PDS silencing and an increased photobleaching phenotype. The suitability of TRV as a vector for virus-aided gene expression (VAGE) was demonstrated by an analysis of DsRed fluorescence protein. Interestingly, a DsRed signal was also observed in glandular trichomes in TRV2-DsRed-infected plants, which suggests the possibility of trichome-related gene function analysis. These results indicate that TRV, despite its limited spread, is an attractive vector for rapid reverse-genetics screens and for the analysis of gene function in cannabis.

Genome-Wide Analysis of Anthocyanin Biosynthesis Regulatory WD40 Gene FcTTG1 and Related Family in Ficus carica L.

WD40 proteins serve as crucial regulators in a broad spectrum of plant developmental and physiological processes, including anthocyanin biosynthesis. However, in fig (Ficus carica L.), neither the WD40 family nor any member involved in anthocyanin biosynthesis has been elucidated. In the present study, 204 WD40 genes were identified from the fig genome and phylogenetically classified into 5 clusters and 12 subfamilies. Bioinformatics analysis prediction localized 109, 69, and 26 FcWD40 proteins to the cytoplasm, nucleus and other cellular compartments, respectively. RNA-seq data mining revealed 127 FcWD40s expressed at FPKM > 10 in fig fruit. Most of these genes demonstrated higher expression in the early stages of fruit development. FcWD40-97 was recruited according to three criteria: high expression in fig fruit, predicted nuclear localization, and closest clustering with TTG1s identified in other plants. FcWD40-97, encoding 339 amino acids including 5 WD-repeat motifs, showed 88.01 and 87.94% amino acid sequence similarity to apple and peach TTG1, respectively. The gene is located on fig chromosome 4, and is composed of 1 intron and 2 exons. Promoter analysis revealed multiple light-responsive elements, one salicylic acid-responsive element, three methyl jasmonate-responsive elements, and one MYB-binding site involved in flavonoid biosynthesis gene regulation. FcWD40-97 was in the FPKM > 100 expression level group in fig fruit, and higher expression was consistently found in the peel compared to the flesh at the same development stages. Expression level did not change significantly under light deprivation, whereas in leaves and roots, its expression was relatively low. Transient expression verified FcWD40-97's localization to the nucleus. Yeast two-hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays revealed that FcWD40-97 interacts with FcMYB114, FcMYB123, and FcbHLH42 proteins in vitro and in vivo, showing that FcWD40-97 functions as a member of the MYB-bHLH-WD40 (MBW) complex in anthocyanin-biosynthesis regulation in fig. We therefore renamed FcWD40-97 as FcTTG1. Our results provide the first systematic analysis of the FcWD40 family and identification of FcTTG1 in fig pigmentation.

AP2/ERF genes associated with superfast fig (Ficus carica L.) fruit ripening.

Fig fruits have significant health value and are culturally important. Under suitable climatic conditions, fig fruits undergo a superfast ripening process, nearly doubling in size, weight, and sugar content over three days in parallel with a sharp decrease in firmness. In this study, 119 FcAP2/ERF genes were identified in the fig genome, namely 95 ERFs, 20 AP2s, three RAVs, and one soloist. Most of the ERF subfamily members (76) contained no introns, whereas the majority of the AP2 subfamily members had at least two introns each. Three previously published transcriptome datasets were mined to discover expression patterns, encompassing the fruit peel and flesh of the 'Purple Peel' cultivar at six developmental stages; the fruit receptacle and flesh of the 'Brown Turkey' cultivar after ethephon treatment; and the receptacle and flesh of parthenocarpic and pollinated fruits of the 'Brown Turkey' cultivar. Eighty-three FcAP2/ERFs (68 ERFs, 13 AP2s, one RAV, and one soloist) were expressed in the combined transcriptome dataset. Most FcAP2/ERFs were significantly downregulated (|log2(fold change) | ≥ 1 and p-adjust < 0.05) during both normal fruit development and ethephon-induced accelerated ripening, suggesting a repressive role of these genes in fruit ripening. Five significantly downregulated ERFs also had repression domains in the C-terminal. Seven FcAP2/ERFs were identified as differentially expressed during ripening in all three transcriptome datasets. These genes were strong candidates for future functional genetic studies to elucidate the major FcAP2/ERF regulators of the superfast fig fruit ripening process.

Non-photoperiodic transition of female cannabis seedlings from juvenile to adult reproductive stage.

KEY MESSAGE: Vegetative-to-reproductive phase transition in female cannabis seedlings occurs autonomously with the de novo development of single flowers. To ensure successful sexual reproduction, many plant species originating from seedlings undergo juvenile-to-adult transition. This phase transition precedes and enables the vegetative-to-reproductive shift in plants, upon perception of internal and/or external signals such as temperature, photoperiod, metabolite levels, and phytohormones. This study demonstrates that the juvenile seedlings of cannabis gradually shift to the adult vegetative stage, as confirmed by the formation of lobed leaves, and upregulation of the phase-transition genes. In the tested cultivar, the switch to the reproductive stage occurs with the development of a pair of single flowers in the 7th node. Histological analysis indicated that transition to the reproductive stage is accomplished by the de novo establishment of new flower meristems which are not present in a vegetative stage, or as dormant meristems at nodes 4 and 6. Moreover, there were dramatic changes in the transcriptomic profile of flowering-related genes among nodes 4, 6, and 7. Downregulation of flowering repressors and an intense increase in the transcription of phase transition-related genes occur in parallel with an increase in the transcription of flowering integrators and meristem identity genes. These results support and provide molecular evidence for previous findings that cannabis possesses an autonomous flowering mechanism and the transition to reproductive phase is controlled in this plant mainly by internal signals.

Flower development in garlic: the ups and downs of gaLFY expression.

The lack of sexual processes prohibits genetic studies and conventional breeding in commercial cultivars of garlic. Recent restoration of garlic flowering ability by environmental manipulations has opened new avenues for physiological and genetic studies. The LEAFY homologue gaLFY has been shown to be involved in the floral development, while two alternatively spliced gaLFY transcripts are expressed in flowering genotypes. In the present work, quantitative real-time PCR and two techniques of RNA in situ hybridization were employed to analyze spatiotemporal expression patterns of the gaLFY during consequent stages of the garlic reproductive process. Temporal accumulation of gaLFY is strongly associated with reproductive organs, significantly increased during florogenesis and gametogenesis, and is down-regulated in the vegetative meristems and topsets in the inflorescence. The two alternative transcripts of the gene show different expression patterns: a high level of the long gaLFY transcript coincided only with floral transition, while further up-regulation of this gene in the reproductive organs is associated mainly with the short gaLFY transcript. It is concluded that gaLFY is involved at different stages of the sexual reproduction of garlic. These new insights broaden our basic understanding of flower biology of garlic and help to establish conventional and molecular breeding systems for this important crop.