Ecological speciation in sympatric palms: 1. Gene expression, selection and pleiotropy

Ecological speciation requires divergent selection, reproductive isolation and a genetic mechanism to link the two. We examined the role of gene expression and coding sequence evolution in this process using two species of Howea palms that have diverged sympatrically on Lord Howe Island, Australia. These palms are associated with distinct soil types and have displaced flowering times, representing an ideal candidate for ecological speciation. We generated large amounts of RNA-Seq data from multiple individuals and tissue types collected on the island from each of the two species. We found that differentially expressed loci as well as those with divergent coding sequences between Howea species were associated with known ecological and phenotypic differences, including response to salinity, drought, pH and flowering time. From these loci, we identified potential 'ecological speciation genes' and further validate their effect on flowering time by knocking out orthologous loci in a model plant species. Finally, we put forward six plausible ecological speciation loci, providing support for the hypothesis that pleiotropy could help to overcome the antagonism between selection and recombination during speciation with gene flow

Micrografting techniques for testing long-distance signalling in Arabidopsis

Grafting in species other than Arabidopsis has generated persuasive evidence for long-distance signals involved in many plant processes, including regulation of flowering time and shoot branching. Hitherto, such approaches in Arabidopsis have been hampered by the lack of suitable grafting techniques. Here, a range of micrografting methods for young Arabidopsis seedlings are described. The simplest configuration was a single-hypocotyl graft, constructed with or without a supporting collar, allowing tests of root–shoot communication. More complex two-shoot grafts were also constructed, enabling tests of shoot–shoot communication. Integrity of grafts and absence of adventitious roots on scions were assessed using plants constitutively expressing a GUS gene as one graft partner. Using the max1 (more axillary growth) and max3 increased branching mutants, it was shown that a wild-type (WT) rootstock was able to inhibit rosette branching of mutant shoots. In two-shoot grafts with max1 and WT shoots on a max1 rootstock, the mutant shoot branched profusely, but the WT one did not. In two-shoot grafts with max1 and WT shoots on a WT rootstock, neither shoot exhibited increased branching. The results mirror those previously demonstrated in equivalent grafting experiments with the ramosus mutants in pea, and are consistent with the concept that a branching signal is capable of moving from root to shoot, but not from shoot to shoot. These grafting procedures will be valuable for revealing genes associated with many other long-distance signalling pathways, including flowering, systemic resistance and abiotic stress responses

Quantification of cyanogenic glycosides in seedlings of three Macadamia (Proteaceae) species

Members of the genus Macadamia contain cyanogenic compounds, which release hydrogen cyanide (HCN) on hydrolysis. Concentrations of releasable cyanide were measured in tissues of mature nuts and seedlings of Macadamia integrifolia Maiden & Betche, M. tetraphylla L.A.S. Johnson and M. ternifolia F. Muell. Root, cotyledon and leaf samples were assayed at several developmental stages from germination to maturation of the first leaves. All samples contained detectable levels of cyanide. Concentrations were low (0.15 µmol g fresh weight) in cotyledons of mature M. integrifolia and M. tetraphylla seeds, corresponding to the edibility of the seeds of these commercial species, and much higher (9.6 µmol g) in the inedible M. ternifolia seeds. Levels in cotyledons of all three species rose dramatically during germination. Root cyanide concentrations of 6-23 µmol g were measured. The immature first leaf of the commercial species contained the highest concentrations (38-77 µmol g). Levels decreased with leaf maturity, correlating with toughening of the leaf and possibly a consequent diminished requirement for cyanide as a herbivory deterrent. The significance of the results with respect to plant-insect interactions is discussed

Ethephon promotion of crop abscission for unshaken and mechanically shaken macadamia

Promotion of fruit abscission in macadamia, Macadamia integrifolia (Proteaceae), has potential to reduce costs associated with prolonged harvesting of late-abscising cultivars. Effects of ethephon [(2-chloroethyl)phosphonic acid] on fruit removal force and crop abscission were monitored at 3 stages of the harvest season on both unshaken and mechanically shaken trees of the late-abscising macadamia cultivar A16. Ethephon application, tree shaking, or a combination of the 2 methods, accelerated crop removal from the tree at all stages during harvest. Early harvest before natural abscission resulted in little or no difference in nut-in-shell and kernel weight, kernel recovery and kernel oil content. Delaying ethephon application or tree shaking until commencement of natural abscission resulted in greater crop removal. Fruit removal force declined naturally towards 1 kgf at this stage, and was further reduced by ethephon application. The most effective approach for harvest acceleration was to reduce fruit removal force, before tree shaking, by spraying trees with ethephon

Routes of ethephon uptake in pineapple (Ananas comosus) and reasons for failure of flower induction

Ethylene-releasing agents such as ethephon (2-chloroethylphosphonic acid) are used widely to induce flowering in pineapples (Ananas comosus (L.) Merrill). However, ethephon treatment is less reliable in summer, particularly if plants are treated on abnormally hot days. [14C]ethephon was used to follow uptake and translocation in leaf tissues. Up to 30% of the ethephon entered the leaf within 4 h, and up to 60% by 24 h. Uptake was dramatically modified by temperature, relative humidity, solution pH, and the surface on which solution droplets were placed. Entry occurred across the leaf cuticle and probably also by way of stomatal pores, and label was recovered at all depths within the leaf. 14C label entered more rapidly through the abaxial epidermis than through the adaxial epidermis. Low-volume spray applications to whole plants resulted in rapidly drying droplets mainly on the adaxial, distal epidermis and were rather ineffective at inducing flowering, possibly because little ethephon or ethylene reaches the shoot apex. High-volume sprays may facilitate ethephon entry because solution accumulates in leaf axils and hence remains in prolonged contact with abaxial epidermis of leaf bases close to the shoot apex. When poured into the center of the plant, 20% of a normal commercial ethephon dose induced full flowering even under adverse temperatures. It is suggested that high-volume evening spraying and avoidance of hot days may reduce the incidence of flowering failure

Effect of 6-BA on nodal explant bud sproutings of Coffea arabica cv. Mundo Novo Efeito de 6-BA na brotação de gemas de explantes nodais de Coffea arabica cv. Mundo Novo

Coffee plants can be micropropagated by nodal bud sprouting using the 6-benzylaminopurine (6-BA) hormone. However, literature reports the use of a wide range of 6-BA, from 0.5 to 88.8 µM L-1. So, this study was performed to narrow that range. Nodal explants of Coffea arabica cv Mundo Novo obtained from in vitro plantlets were inoculated on gelled-MS medium supplemented with different concentrations of 6-BA. Two assays were carried out: in the first one, 6-BA was used at concentrations of 0, 5, 25, 50, and 100 µM L-1, being evaluated at 43 and 123 days. In the second experiment, dosis of 10, 20 and 30 µM L-1, have evaluated at 65 and 100 days. Treatments with 6-BA induced multiple sprouting from the nodal explants, which were best characterized around 100 days after inoculation. The nodal explants grew taller and showed multiple shoots, whereas the effect of 6-BA at 5 to 25 µM L-1 was similar to that with higher concentrations (50 and 100 µM L-1). Nodal explants yielded from 2.9 to 6.0 buds per node, achieving height of 1.3 to 1.5 cm at 5 to 25 µM L-1 of 6-BA, whereas they yielded from 4.3 to 4.9 buds per node but the sprouting grew about 0.8 cm at 50 and 100 µM L-1 of 6-BA. This study indicated that multiple sprouting of lateral buds can be induced by lower concentrations of 6-BA, for example, from 10 to 30 µM L-1, diminishing possible risks of somaclonal variation due to high levels of hormone concentration.<br>O cafeeiro pode ser micropropagado via brotação de gemas laterais, aplicando o regulador de crescimento 6-benzilaminopurina (6-BA). Entretanto, a literatura apresenta ampla variação da dose empregada, desde 0.5 a 88.8 µM L-1. Assim, este estudo visou otimizar doses para explantes nodais do cafeeiro C. arabica cv Mundo Novo. Explantes nodais, obtidos de plântulas cultivadas in vitro, foram inoculados em meio MS geleificado, com adição de diferentes concentrações de 6-BA. Foram feitos dois experimentos: no primeiro, 6-BA foi usado nas doses de 0, 5, 10, 25, 50 e 100 µM L-1 e avaliado aos 43 e 123 dias; no segundo, 10, 20 e 30 µM L-1, avaliado aos 65 e 100 dias após a inoculação dos explantes. Os tratamentos com 6-BA induziram a multibrotação dos explantes nodais, e os resultados foram mais bem caracterizados aos cem dias. Os explantes nodais tratados formaram multibrotações que também atingiram maior altura; todavia, o efeito de 6-BA nas concentrações entre 5 a 25 µM L-1 foi semelhante ao das doses mais elevadas, 50 e 100 µM L-1. As doses de 5 a 25 µM L-1 de 6-BA induziram a brotação de 2,9 a 6,0 gemas por nó, atingindo de 1,3 a 1,5 cm, enquanto os tratamentos de 50 a 100 µM L-1 formaram 3,0 a 4,9 gemas por nó e as suas brotações atingiram cerca de 0,8 cm de altura. Observou-se neste estudo que a multibrotação de explantes nodais de C. arabica cv Mundo Novo pode ser induzida por concentrações menores de 6-BA, entre 10 a 30 µM L-1, diminuindo os riscos de variação somaclonal devido às altas concentrações de hormônio