How a developing leaf organises its tissues Leaf development makes sure that the cells for photosynthesis are on the upper side and gas exchange tissue is below. European researchers have investigated how this happens at the molecular level. Food and Natural Resources Fundamental Research © MRS.Siwaporn, Shutterstock Recent research has shown that gradients of small mobile RNAs contribute to developmental patterning. The upper (adaxial) and lower (abaxial) sides of a leaf are sharply divided domains in the leaf resulting in a stable adaxial-abaxial boundary. This polar expression pattern is controlled by two small RNAs and the defined expression in each domain suggests that small RNAs may regulate gene expression via a dose-dependent read-out of the small RNA gradient. The SRNAGRAD (Robust developmental patterns generated by opposing gradients of mobile small RNAs) project has investigated the importance of these small RNA gradients. Specifically, researchers looked at how small RNA gradients are formed and how they create target gene expression patterns. Using transgenic lines expressing artificial microRNA (miRNA) that targeted the abaxial specific ARF3 transcription factor (miRARF), the project team determined how the position and direction of the gradient affected the domain, level and boundary of target gene expression. They also directly visualised the developmental effects using the β-glucuronidase (GUS) reporter system. Results showed that gradients of mobile miRNAs create on-off target gene expression boundaries at various positions in the leaf. Moreover, they suggest that levels of the small RNA affect the position of the expression boundary, consistent with the dose-dependent readout theory. In lines where the gradient is perturbed, the adaxial epidermis-specific (AS2) promoter showed lack of expression robustness and leaf parameters were very variable as compared with the wild type. Also, the definition of the adaxial-abaxial boundary was less straight and not sharply defined. Again, this showed that properly formed RNA gradients are essential for correct development. Further testing involved artificial small RNA targeting green fluorescent protein (GFP), miRGFP, as compared with endogenous small RNA. Studies showed that a gradient was formed and that on-off target expression boundaries were created irrespective of the direction of the gradient. Furthermore, increased miR-GFP levels moved GFP expression towards the abaxial side supporting the premise that expression control is dose-dependent. SRNAGRAD research results could have widespread significance in the plant development research arena. Knowledge of the molecular machinery is the first step in breeding programmes for more efficient production of agricultural crops. Keywords Leaf development, adaxial-abaxial boundary, gene expression, small RNA gradient, SRNAGRAD
