Synchronization Of Flowering In Cocoa
Introduction
Cocoa production – an industry valued at upwards of $6 billion annually – is an important source of income for 5 million smallholder farmers in developing countries throughout the tropics (World Cocoa Foundation, 2018). The agricultural production systems undergirding this industry, however, face a number of constraints which, if left unaddressed, may leave the sector ill-equipped to face the myriad challenges expected in the coming decades. Persistently low yields and limited opportunities to improve the efficiency of cacao production due to its unusual reproductive physiology are among the important issues to be addressed.
There are many potential avenues by which the these interrelated issues of productivity and efficiency can be pursued. The possibility of synchronizing flowering and fruit set while maintaining or enhancing yield, however, has received little attention from researchers thus far despite several apparent points of entry for pursuing this goal, which we explore below.
What we know about the problem
Environmental (Abiotic) Factors
A suite of environmental factors are employed as signalling mechanisms to regulate the timing, extent and location of developmental (physiological) changes, including floral initiation and guiding patterns of opportunistic growth (Taiz & Zeiger, 2010). Correspondingly, this review considered temperature and altitude; rainfall, soil moisture and relative humidity; soil chemistry and fertility; and light and photoperiod among the abiotic factors which might be manipulatable through management to encourage flowering synchronization.
The available evidence suggests that the management of light intensity may represent the most feasible avenue to achieve maximum flower production within the shortest period of time. Specifically, there is evidence to suggest that sudden increases in light intensity or changes in the spectral composition of light (i. e. changes in wavelength as it filters through the canopy) can stimulate the sudden and more synchronized emergence of a increased number of flowers (Boyer, 1974; Hurd & Cunningham, 1961). Light intensity can then be easily manipulated in a production environment with minimal additional labor or capital investments through the regular pruning of shade trees (with corresponding additional benefits from fertilization and/or mulching)
Physiology/Growth Hormones
For angiosperm reproduction, flowering is the major developmental stage to produce viable offspring. For many plants, this involves a complex network of environmental cues and endogenous signaling that affects phytohormone activation. Though much information has been added to expand the current literature on the topic of hormones such as auxin, cytokinins, gibberellins, abscisic acid and ethylene, direct links of specific hormones, such as cytokinins, to their role in plant flowering remain hazy (Meijon et al. , 2011). The same study found that methylation decreases before flowering, and that cytokinin could be a factor that affects flower initiation via demethylation in Arabidopsis (Meijon et al. , 2011). That does not account for the role of auxin in flowering, as it has an interactive role with cytokinins (Campos-Rivro et al. , 2017). Gibberellins are also known to play a role in floral transition and development (Yamaguchi, 2008). Gibberellins are also linked to the expression of a gene that is responsible for flowering that controls the switch from a vegetative to a reproductive phase during plant development, and its expression is induced by gibberellins (Blazquez et al. 1997).
Abscisic acid is another phytohormone involved in seed physiology and flowering (Shu et al. , 2016; Trivedi et al. , 2016). In some cases, water stress causes early flowering in Arabidopsis, probably by abscisic acid accumulation in response to this stress (Su et al. , 2013, Verslues and Zhu, 2005, Wang et al. , 2013). The loss of water can also trigger a response known as drought escape (DE) (Sherrard and Maherali, 2006), which is a strategy inducing early flowering and seed production before the drought becomes too severe (Su et al. , 2013). Although abscisic acid has been related mostly to water stress, this hormone has also been considered as a floral repressor, such as in the case of Arabidopsis, where it has been shown that applying exogenous ABA delays flowering time (Wang et al. , 2013).
Epigenetics
Phenotypic plasticity is the ability of a genotype to demonstrate different phenotypes under varying conditions, such as when exposed to drought, pathogens and differing soil fertility (Heschel et al. , 2004, Hodge, 2004). It is hypothesised that the heritable variation in these phenotypic traits is due to epigenetic variation (Richards, 2011). Epigenetics can be thought of as the result of communication between genotype and phenotype, whereby the final expression of the locus or chromosome is altered without affecting the DNA of the organism ( Goldberg et. al, 2007). DNA itself is packaged in the nucleus in regions that are loosely packed, known as euchromatin, and regions that are tightly packaged, known as heterochromatin. In regions where the DNA is highly methylated, the region will package tightly into heterochromatin.
These methylation marks can be maintained after each round of replication, as they are a modification made in the DNA template, and are thus heritable, at least in plant species ( Klose, 2014). Previous literature has shown that epigenetic methylation effects are conspicuous in plants, whereby defects in methylation were shown to visibly and heritably alter fertility, flowering time, and leaf and floral morphology in Arabidopsis (Finnegan et al, 1996). By using several methods to study methylation such as methylation-sensitive AFLPs (MS-AFLPs) and chemical in vivo hypomethylation, it was also found that, in dandelions, heritable flowering time variation was caused by DNA methylation events (Wilschut et al, 2016). Cacao itself has shown induced resistance against Black pod disease (Agarwal,1999) suggesting that it is capable of demonstrating other epigenetic traits, such as synchronized flowering.
On the other hand, flowering timing pathways are controlled mostly by floral integrator genes including Flowering Locus T (FT) (Yamaguchi A, 2005). It has been demonstrated in Arabidopsis, and several other flowering plants that FT overexpression causes early flowering and FT-lacking mutants exhibit late flowering ( Kardailsky I, 1999). This FT function is conserved across the plant kingdom and as such can be manipulated to induce flowering on demand. Insect pollinationInsect pollinators play a critical role in increasing cacao production (Forbes and Tobin, 2017 and Toledo et al 2017). A diverse group of insects, such as ceratopogonids, ceciodomyiids, flies, ants, bees, wasps and others, visit the cacao plants. Although ceratopogonid midges are categorized as the main pollinators, diptera (Chloropidae and Phoridae) and Hymenoptera (Eulophidae and Platygastridae) are also considered as potential pollinators (de Schawe et al. 2018). Arnold et al (2018) describes ceratopogonid midge as a specialist pollinator and some species from diptera and hymenoptera as generalist pollinators.
Additionally, ant communities are known to significantly increase the yield of cacao in tropical agroforestry system (Gras et al. 2016 and Clough et al. 2017). Forcipomyia spp. (Diptera: ceratopogonidae) are an important pollinator of cacao plant. F. hardyi, F. quasiingramiare, F. psinolataingrami, F. falcinella and F. ashantii are the species of midges that pollinate the cacao plants. Very little has been done previously to evaluate pollination efficacy of ceratopogonidae midges and no life cycle evaluation of midges has yet been undertaken.
Proposed Experiments
Environmental (Abiotic) Factors
On the basis of past research findings demonstrating the influence of shading and light intensity on floral initiation, we propose a fully multi-factorial, randomized complete block field experiment with treatments related to shade tree management and pruning. To ensure that the effects are consistent despite variability in soil and climate, it would be advisable to include as least four replications and to conduct the experiment over a period of at least three or more years.
B. Physiology/Growth HormonesC EpigeneticsIn studying how epigenetics may be affecting flowering synchronization in caco, it it vital to understand that epigenetic changes vary in most natural systems (Koornneef et al. , 2004). For this reason however, the following experiments will be proposed solely for CCN-51.
One available method is to chemically manipulate the DNA methylations using demethylation agents such as 5-azacytidine (azacitidine) and 5-azadeoxycytidine (decitabine), which work to inhibit the activity of DNA methyltransferases. Differences in flowering patterns of a control population can then be compared to those with varying degrees of demethylation (Bossdorf et al. , 2010).
Another means to study epigenetic variations would be to be to perform Genome‐wide methylation profiling using methylation‐sensitive amplified fragment length polymorphism (MSAP) to study significant DNA methylation polymorphisms within the clonal species that have been exposed to different environments and then to identify methylation alterations associated with response to flowering synchronization (Gao et al. , 2010). A final approach to studying epigenetic effects on flowering would be to create epigenetic recombinant inbred lines (epiRILs). EpiRILs are lines that are created via artificial crossing and maintain the same DNA sequence while differing highly in epigenetics ( Zhang et al, 2013). Previous efforts have succeeded in creating epiRILS which showed significant heritability for flowering time in Arabidopsis (Johannes et al. , 2009). A similar experiment could be done in cacao in order to isolate and identify epigenetic changes that greatly influence cacao flowering synchronization. To manipulate flowering in Arabidopsis via gene editing, a transgenic line with an alcohol-inducible version of FT gene was generated, whereby upon exposure to ethanol vapour FT expression and synchronous flowering was induced. A similar experiment in cacao can be carried out with FT or an orthologue (Yeoh, C. C,2011).
Pollination
The effect of temperature, relative humidity (RH) and light to dark ratio (L: D) on survival and life cycle completion of Forcipomyia spp will be assessed. This will help in understanding the ability of Forcipomyia spp. to survive under different levels of temperature, RH and L: D. Additionally, the pollination efficacy of different species of Forcipomyia on Theobroma cacao (L. ) (Malvaceae) will be assessed. Different species of ceratopogonid midge such as F. hardyi, F. quasiingramiare, F. psinolataingrami, F. falcinella and F. ashantii will be used in this experiment. This will be done in greenhouse settings or with the use of outdoor field cages. Also, Lethality of regularly applied insecticides on different species of Forcipomyia will be assessed. Numerous insecticides are applied in the cacao agro system. It is important to assess the effect of regularly applied insecticides on survival of Forcipomyia spp. of midges. A glass vial contact assay will be done to evaluate the lethality of insecticides on midges.
B. Flowering Physiology and Hormones
Conclusions
A review of the available scientific literature has provided a framework for understanding the environmental, genetic and physiological factors - and their interactions - underlying floral initiation and the cascade of subsequent processes leading ultimately to pod maturation and harvest. With the exception of pollinator population management, the benefits derived from each of the above mentioned lines of inquiry may be partially or wholly redundant and thus substitutable, or they may be additive or even synergistic.
Moreover, each of these approaches carry differing research (fixed) costs, differing implementation (variable) costs, differing time spans for implementation and the realization of benefits, and different likelihoods of success. Despite these limitations a clear opportunity emerges from this research program to identify methods capable of having a profound impact on cocoa productivity and efficiency and supporting the industry as it pursues to secure its environmental, social, and economic sustainability in the coming decades.
