Identification of fasciclin-like arabinogalactan proteins in textile hemp (Cannabis sativa L.): in silico analyses and gene expression patterns in different tissues.

BACKGROUND: The fasciclin-like arabinogalactan proteins (FLAs) belong to the arabinogalactan protein (AGP) superfamily and are known to play different physiological roles in plants. This class of proteins was shown to participate in plant growth, development, defense against abiotic stresses and, notably, cell wall biosynthesis. Although some studies are available on the characterization of FLA genes from different species, both woody and herbaceous, no detailed information is available on the FLA family of textile hemp (Cannabis sativa L.), an economically important fibre crop. RESULTS: By searching the Cannabis genome and EST databases, 23 CsaFLAs have been here identified which are divided into four phylogenetic groups. A real-time qPCR analysis performed on stem tissues (isolated bast fibres and shivs sampled at three heights), hypocotyls (6-9-12-15-17-20 days-old), whole seedlings, roots, leaves and female/male flowers of the monoecious fibre variety Santhica 27, indicates that the identified FLA genes are differentially expressed. Interestingly, some hemp FLAs are expressed during early phases of fibre growth (elongation), while others are more expressed in the middle and base of the stem and thus potentially involved in secondary cell wall formation (fibre thickening). The bioinformatic analysis of the promoter regions shows that the FLAs upregulated in the younger regions of the stem share a conserved motif related to flowering control and regulation of photoperiod perception. The promoters of the FLA genes expressed at higher levels in the older stem regions, instead, share a motif putatively recognized by MYB3, a transcriptional repressor belonging to the MYB family subgroup S4. CONCLUSIONS: These results point to the existence of a transcriptional network fine-tuning the expression of FLA genes in the older and younger regions of the stem, as well as in the bast fibres/shivs of textile hemp. In summary, our study paves the way for future analyses on the biological functions of FLAs in an industrially relevant fibre crop.

Transcriptomic profiling of hemp bast fibres at different developmental stages.

Bast fibres are long extraxylary cells which mechanically support the phloem and they are divided into xylan- and gelatinous-type, depending on the composition of their secondary cell walls. The former, typical of jute/kenaf bast fibres, are characterized by the presence of xylan and a high degree of lignification, while the latter, found in tension wood, as well as flax, ramie and hemp bast fibres, have a high abundance of crystalline cellulose. During their differentiation, bast fibres undergo specific developmental stages: the cells initially elongate rapidly by intrusive growth, subsequently they cease elongation and start to thicken. The goal of the present study is to provide a transcriptomic close-up of the key events accompanying bast fibre development in textile hemp (Cannabis sativa L.), a fibre crop of great importance. Bast fibres have been sampled from different stem regions. The developmental stages corresponding to active elongation and cell wall thickening have been studied using RNA-Seq. The results show that the fibres sampled at each stem region are characterized by a specific transcriptomic signature and that the major changes in cell wall-related processes take place at the internode containing the snap point. The data generated also identify several interesting candidates for future functional analysis.

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues.

Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.