Overexpression of Arabidopsis FLOWERING LOCUS T (FT) gene improves floral development in cassava (Manihot esculenta, Crantz)

Cassava is a tropical storage-root crop that serves as a worldwide source of staple food for over 800 million people. Flowering is one of the most important breeding challenges in cassava because in most lines flowering is late and non-synchronized, and flower production is sparse. The FLOWERING LOCUS T (FT) gene is pivotal for floral induction in all examined angiosperms. The objective of the current work was to determine the potential roles of the FT signaling system in cassava. The Arabidopsis thaliana FT gene (atFT) was transformed into the cassava cultivar 60444 through Agrobacterium-mediated transformation and was found to be overexpressed constitutively. FT overexpression hastened flower initiation and associated fork-type branching, indicating that cassava has the necessary signaling factors to interact with and respond to the atFT gene product. In addition, overexpression stimulated lateral branching, increased the prolificacy of flower production and extended the longevity of flower development. While FT homologs in some plant species stimulate development of vegetative storage organs, atFT inhibited storage-root development and decreased root harvest index in cassava. These findings collectively contribute to our understanding of flower development in cassava and have the potential for applications in breeding

Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis

Plant stem-cell pools, the source for all organs, are first established during embryogenesis. It has been known for decades that cytokinin and auxin interact to control organ regeneration in cultured tissue. Auxin has a critical role in root stem-cell specification in zygotic embryogenesis, but the early embryonic function of cytokinin is obscure. Here, we introduce a synthetic reporter to visualize universally cytokinin output in vivo. Notably, the first embryonic signal is detected in the hypophysis, the founder cell of the root stem-cell system. Its apical daughter cell, the precursor of the quiescent centre, maintains phosphorelay activity, whereas the basal daughter cell represses signalling output. Auxin activity levels, however, exhibit the inverse profile. Furthermore, we show that auxin antagonizes cytokinin output in the basal cell lineage by direct transcriptional activation of ARABIDOPSIS RESPONSE REGULATOR genes, ARR7 and ARR15, feedback repressors of cytokinin signalling. Loss of ARR7 and ARR15 function or ectopic cytokinin signalling in the basal cell during early embryogenesis results in a defective root stem-cell system. These results provide a molecular model of transient and antagonistic interaction between auxin and cytokinin critical for specifying the first root stem-cell niche

Subcellular localization of micrornas by microrna in situ hybridization (miR-ISH)

MicroRNAs (miRNAs) are 22-nucleotide RNA sequences that regulate up to 60% of the mammalian transcriptome. Although canonical miRNA-induced silencing complex-mediated messenger RNA degradation occurs in the cytoplasm, miRNAs have been described in other subcellular compartments with potentially novel functions. Currently, there are limited methodologies for visualizing RNA locations within cells to elucidate mechanisms and pathways of miRNA biogenesis, transport, and function. Here, we describe a simple and rapid miRNA in situ hybridization method that can be combined with standard immunofluorescence procedures for subcellular localization of mature and precursor miRNAs