Inter-species functional compatibility of the Theobroma cacao and Arabidopsis FT orthologs: 90 million years of functional conservation of meristem identity genes

Background In angiosperms the transition to flowering is controlled by a complex set of interacting networks integrating a range of developmental, physiological, and environmental factors optimizing transition time for maximal reproductive efficiency. The molecular mechanisms comprising these networks have been partially characterized and include both transcriptional and post-transcriptional regulatory pathways. Florigen, encoded by FLOWERING LOCUS T (FT) orthologs, is a conserved central integrator of several flowering time regulatory pathways. To characterize the molecular mechanisms involved in controlling cacao flowering time, we have characterized a cacao candidate florigen gene, TcFLOWERING LOCUS T (TcFT). Understanding how this conserved flowering time regulator affects cacao plant’s transition to flowering could lead to strategies to accelerate cacao breeding. Results BLAST searches of cacao genome reference assemblies identified seven candidate members of the CENTRORADIALIS/TERMINAL FLOWER1/SELF PRUNING gene family including a single florigen candidate. cDNA encoding the predicted cacao florigen was cloned and functionally tested by transgenic genetic complementation in the Arabidopsis ft-10 mutant. Transgenic expression of the candidate TcFT cDNA in late flowering Arabidopsis ft-10 partially rescues the mutant to wild-type flowering time. Gene expression studies reveal that TcFT is spatially and temporally expressed in a manner similar to that found in Arabidopsis, specifically, TcFT mRNA is shown to be both developmentally and diurnally regulated in leaves and is most abundant in floral tissues. Finally, to test interspecies compatibility of florigens, we transformed cacao tissues with AtFT resulting in the remarkable formation of flowers in tissue culture. The morphology of these in vitro flowers is normal, and they produce pollen that germinates in vitro with high rates. Conclusion We have identified the cacao CETS gene family, central to developmental regulation in angiosperms. The role of the cacao’s single FT-like gene (TcFT) as a general regulator of determinate growth in cacao was demonstrated by functional complementation of Arabidopsis ft-10 late-flowering mutant and through gene expression analysis. In addition, overexpression of AtFT in cacao resulted in precocious flowering in cacao tissue culture demonstrating the highly conserved function of FT and the mechanisms controlling flowering in cacao.

RNAi-mediated repression of dormancy-related genes results in evergrowing apple trees

DORMANCY ASSOCIATED MADS-box (DAM) and SHORT VEGETATIVE PHASE (SVP) genes have been implicated in the regulation of winter dormancy in perennials. Ectopic expression of apple (Malus ×domestica ‘Royal Gala’) DAM and SVP genes delays budbreak and constrains lateral shoot outgrowth. In this study, we used RNAi interference (RNAi) to simultaneously target all apple DAM and SVP genes to study their role and mode of action in the regulation of bud dormancy, budbreak and flowering. A synthetic construct carrying a hairpin fragment assembled from sequences specific to coding regions of three DAM and two SVP genes was used to generate transgenic lines. Reduced expression of DAM/SVP genes resulted in delayed leaf senescence and abscission in autumn, failure to enter bud dormancy in winter, and continual growth of new leaves regardless of the season for over 3 years. Precocious flowering but normal flower morphology, fertility and fruit development were observed. The non-dormant phenotype was associated with modified phytohormone composition. The content of gibberellins (GAs) and jasmonates (JAs) was significantly increased in terminal buds of RNAi lines compared to wildtype plants, accompanied by elevated expression of the key GA biosynthesis pathway gene GIBBERELLIN 20 OXIDASE-2 (MdGA20ox-2) along with the FLOWERING LOCUS T gene MdFT2. The key mediator of plasmodesmatal closure, MdCALLOSE SYNTHASE 1 (MdCALS1) was repressed in RNAi lines. This study provides functional evidence for the role of DAM/SVP genes in vegetative phenology of apple, and paves the way for production of low-chill varieties suitable for growth in warming climates.

Sweet cherry rootstocks.

All commercial sweet cherry trees are either budded or grafted. This chapter deals with sweet cherry rootstocks. Rootstocks are used for several purposes: (i) ease for propagating and producing more trees of a superior cultivar; (ii) better adaptation to particular soil or site characteristics; and (iii) the potential improvement of production due to additional traits like precocious flowering, higher productivity, and greater or reduced scion vigor as appropriate. Unfortunately, no one rootstock can satisfy all the requirements for consistently producing high yields of large, firm fruit of premium quality. Growers are advised to consider carefully the effects of each specific scion-rootstock combination as a function of environmental and cultural practices when replanting an orchard. Selecting the proper rootstock depends not only on the management skills of the grower, but also on the scion cultivar, training system, and site climate and soil selected for the orchard. Dwarfing, semi-dwarfing and even semi-vigorous rootstocks have major economic advantages over full-size rootstocks. The development of these new, precocious rootstocks has been almost as significant to the sweet cherry industry as to the apple industry several decades ago. When compared to Mazzard, Colt and even Mahaleb, size-controlling rootstocks have allowed sweet cherry growers an opportunity to plant high density, pedestrian orchards that become profitable more quickly, are more readily protected with orchard covering systems, and promote greater labor efficiency, easier management, and a safer and more productive work environment.

The vascular targeted citrus FLOWERING LOCUS T3 gene promotes non-inductive early flowering in transgenic Carrizo rootstocks and grafted juvenile scions

Shortening the juvenile stage in citrus and inducing early flowering has been the focus of several citrus genetic improvement programs. FLOWERING LOCUS T (FT) is a small phloem-translocated protein that regulates precocious flowering. In this study, two populations of transgenic Carrizo citrange rootstocks expressing either Citrus clementina FT1 or FT3 genes under the control of the Arabidopsis thaliana phloem specific SUCROSE SYNTHASE 2 (AtSUC2) promoter were developed. The transgenic plants were morphologically similar to the non-transgenic controls (non-transgenic Carrizo citrange), however, only AtSUC2-CcFT3 was capable of inducing precocious flowers. The transgenic lines produced flowers 16 months after transformation and flower buds appeared 30–40 days on juvenile immature scions grafted onto transgenic rootstock. Gene expression analysis revealed that the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and APETALA1 (AP1) were enhanced in the transgenics. Transcriptome profiling of a selected transgenic line showed the induction of genes in different groups including: genes from the flowering induction pathway, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family genes, and jasmonic acid (JA) pathway genes. Altogether, our results suggested that ectopic expression of CcFT3 in phloem tissues of Carrizo citrange triggered the expression of several genes to mediate early flowering.

SHAGGY-like kinase 12 regulates flowering through mediating CONSTANS stability in Arabidopsis

Photoperiod is a major environmental cue that determines the floral transition from vegetative to reproductive development in flowering plants. Arabidopsis thaliana responds to photoperiodic signals mainly through a central regulator CONSTANS (CO). Although it has been suggested that phosphorylation of CO contributes to its role in photoperiodic control of flowering, how this is regulated so far remains unknown. Here, we report that a glycogen synthase kinase-3 member, SHAGGY-like kinase 12 (SK12), plays an important role in preventing precocious flowering through phosphorylating CO. Loss of function of SK12 causes early flowering. SK12 expression in seedlings is decreased during the floral transition, and its expression in vascular tissues is required for repressing flowering. SK12 interacts with and phosphorylates CO at threonine 119, thus facilitating CO degradation. Our findings suggest that site-specific phosphorylation of CO by SK12 is critical for modulating the photoperiodic output for the floral induction in Arabidopsis.

Identification, Characterization and Functional Analysis of C-Class Genes Associated with Double Flower Trait in Carnation (Dianthus caryphyllus L.)

Flowers with more petals are of more ornamental value. It is well known that AGAMOUS (AG) is the core member of the C-class gene which plays an essential role in double flower formation and identification of stamens and carpels in Arabidopsis thaliana. We searched C-class genes in the genome of the carnation, and found two AG orthologs (DcaAGa, DcaAGb). Phylogenetic analysis showed that the two genes were closely related to the euAG subclade. Then we searched the genomes of other Caryophyllales plants (Beta vulgaris, Spinacia oleracea, Chenopodium quinoa) for C-class genes, and found that their C-class genes all belonged to the euAG subclade. Semi-quantitative PCR (sq-PCR) analysis indicated that the expression of DcaAG genes in the single flower phenotype was higher than that in the double flower phenotype. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that the expressions of DcaAG genes in the flower bud were significantly different from those in the root, stem, and leaf between the single and double flower phenotype carnations, and that DcaAG genes were specifically expressed in the stamen and carpel of carnation. Moreover, the expression of other floral organ identity genes (AP1 and AP2, PI and AP3, SEP1 and SEP3 corresponding to the A-, B-, and E-class of genes, respectively) showed no significant difference in all floral organs between the single and double flower phenotype carnations, suggesting that C-class (DcaAG) genes might play an important role in the double flower phenotype in carnation. Petal loss or decrease, precocious flowering, silique shortening, and seed sterility were observed in 35S::DcaAGa and 35S::DcaAGb transgenic Arabidopsis plants. All these results show that DcaAG genes might affect the petal number negatively and have a specific function in stamen and carpel development in carnation.