Abstract
Abiotic stress represents a serious threat affecting both plant fitness and productivity. One of the promptest responses that plants trigger following abiotic stress is the differential expression of key genes, which enable to face the adverse conditions. It is accepted and shown that the cell wall senses and broadcasts the stress signal to the interior of the cell, by triggering a cascade of reactions leading to resistance. Therefore the study of wall-related genes is particularly relevant to understand the metabolic remodeling triggered by plants in response to exogenous stresses. Despite the agricultural and economical relevance of alfalfa (Medicago sativa L.), no study, to our knowledge, has addressed specifically the wall-related gene expression changes in response to exogenous stresses in this important crop, by monitoring the dynamics of wall biosynthetic gene expression. We here identify and analyze the expression profiles of nine cellulose synthases, together with other wall-related genes, in stems of alfalfa plants subjected to different abiotic stresses (cold, heat, salt stress) at various time points (e.g. 0, 24, 72 and 96 h). We identify 2 main responses for specific groups of genes, i.e. a salt/heat-induced and a cold/heat-repressed group of genes. Prior to this analysis we identified appropriate reference genes for expression analyses in alfalfa, by evaluating the stability of 10 candidates across different tissues (namely leaves, stems, roots), under the different abiotic stresses and time points chosen. The results obtained confirm an active role played by the cell wall in response to exogenous stimuli and constitute a step forward in delineating the complex pathways regulating the response of plants to abiotic stresses.
Highlights
The study of biological phenomena requires several sensitive analytical techniques, which can convey detailed information at different depths of organismal complexity, namely tissular, metabolic, genomic
NormFinder ranks translation initiation factor IIA (TFIIA) among the 4 most stable genes when all the tissues are taken into account (Table 1)
The best gene pairs identified by NormFinder are different in some tissues from those identified by geNormPLUS, a finding which has already been reported in other studies e.g. [20] and might be due to the different ranking methodology used by the two softwares: normalization in the leaves requires TFIIA and eIF4A according to geNormPLUS, while NormFinder suggests ADF1 and PAB4, which in the geNormPLUS ranking are the 4th and 7th least stable gene, respectively (Fig. 1)
Summary
The study of biological phenomena requires several sensitive analytical techniques, which can convey detailed information at different depths of organismal complexity, namely tissular, metabolic, genomic. Accurate gene expression analyses rely on several critical aspects and experimental steps (namely RNA purity and integrity, genomic DNA contamination, reverse transcription) and, in the case of relative quantification, on the identification of suitable reference genes for data normalization [2,3]. Those are genes whose expression is stable and not subject to fluctuations across the different conditions tested. This feature is critical, as the choice of inappropriate reference genes can significantly bias the results obtained and lead to misinterpretations of biological events
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