Whole-tree and tension wood-associated expression profiles of micrornas in Eucalyptus trees

Abstract

Trees are large, biologically complex multi-cellular organisms that have adapted to terrestrial growth. This places specific demands on their physiology such as the ability to transport water over long distances and the ability to withstand extreme mechanical forces. Wood formation (xylogenesis) is the development of the secondary vascular system within trees, which mainly addresses these two physiological needs. Xylogenesis is a highly ordered developmental process, consisting of a number of overlapping yet distinct developmental phases. These phases are strictly regulated through a combination of biochemical signalling networks and gene expression regulation.<ul> <li>microRNAs (miRNAs) are endogenous non-coding, small (~22 nt) RNAs that function predominantly as negative regulators of gene expression at the post-transcriptional level. They have been implicated in the regulation of plant developmental processes, including determining cell fate in the apical meristem and timing of developmental events such as flowering and leaf morphogenesis. miRNAs have recently been found to have regulatory roles in plants placed under various conditions of abiotic stress such as drought, mechanical stress, cold and high levels of salinity.</li></ul> Trees placed under mechanical stress produce a specialised form of wood called reaction wood. Reaction wood is referred to as tension wood in angiosperms, as it forms on the outside of a bent trunk or branch in order to correct for the non-vertical growth. The formation of tension wood requires extensive reprogramming of wood development processes. This makes tension wood induction an ideal tool to study and refine our understanding of wood development. Recently, miRNA regulation has been implicated in the control of normal and tension wood formation in trees, but the full extent to which miRNAs are involved in tension wood induction is not known. To identify miRNAs potentially involved in the regulation of normal and tension wood development in fast-growing Eucalyptus plantation trees, real-time quantitative polymerase chain reaction (RT-qPCR) and Northern blot analyses were performed for a number of conserved and putatively novel Eucalyptus miRNAs. A total of 12 miRNAs representing 12 distinct miRNA families were profiled, including three novel miRNAs that are putatively specific to Eucalyptus trees. To more fully understand miRNA function in terms of tree development, the abundance profiles of the selected miRNAs were first determined at the whole-tree level. Of the conserved miRNAs profiled, five (miR160, miR166, miR167, miR172, miR408) were found to have abundance profiles consistent with their predicted roles in plant development. At the whole-tree level, miR90, putatively novel to Eucalyptus, was predominately expressed in the mature leaves and flowers. miR90 may target a MADS-box transcription factor which is not required for mature leaf and flower growth. miR408, a regulator of the expression of a plastocyanin gene involved in lignin polymerisation, was expressed at low levels in the immature and mature xylem, where cell lignification is most prominent. In the tension wood, miR166, a known regulator of wood development and miR408 displayed similar increasing abundance over time in tension wood xylem. These profiles support their potential role in wood development. miR160 and miR167, which target auxin response factors responded early to bending stress, with their abundance reaching maximum levels six hrs post-induction before decreasing again. This is consistent with the observed role of auxin response factors as a mechanism to rapidly respond to stimuli, such as bending. The miRNAs abundance profiles generated in this study suggest that some miRNAs do indeed play a role in normal and tension wood development, though not necessarily directly. These results provide further insights into the complex nature of miRNA regulation and their hypothesised roles in wood development. The miRNAs highlighted herein are strong candidates for further functional studies as their abundance profiles and predicted targets are consistent with roles in wood development. Copyright