Identification and expression profiles of putative leaf growth related microRNAs in maize (Zea mays L.) hybrid ADA313

Throughout the plant life cycle, growth of new leaves is governed by cell division and cell expansion. During steady-state growth of the maize leaf, these processes are spatially separated between the meristem zone, consisting of dividing cells at the leaf base, the elongation zone, consisting of expanding cells moving upwards from the meristem, and the mature zone containing differentiated mature cells. Increased leaf size can be achieved through increasing cell number or cell size, for example by manipulating the genes controlling the transition between those zones. In this study, microRNA (miRNA) genes, which are a class of endogenous small, non-coding gene regulatory RNAs, were investigated in the growth zones, to gain insight into their role in the transition between cell division and cell expansion. A genome-wide survey was conducted using a miRNA-microarray and 59 miRNA genes were detected to be differentially expressed between the growth zones. miR160, miR166, miR168, miR172, miR319 and miR390 families were significantly up-regulated in the meristem relative to the elongation and mature zones. In contrast, expression of the miR167 and miR396 families was lower in the meristem and higher in the mature zone. Therefore, these were considered to be candidate growth-regulated miRNAs that control cell division processes indirectly by repressing target genes. The miR156, miR166, miR167, miR399, miR408 and miR2275 families were expressed most highly in the elongation zone, and so were classified as elongation-specific, with possible roles in switching from cell division to cell elongation during leaf differentiation. In silico target prediction analysis showed that these miRNAs target several transcription factors and metabolic genes, and a reciprocal relationship between the expression levels of miR319 and miR396 and their targets was confirmed by qRT-PCR. Furthermore, 12 candidate novel miRNAs were identified from the microarray data and computationally verified. Three out of twelve were also validated by qRT-PCR. These findings provide important information regarding the regulatory functions of miRNAs in controlling progression of growth mechanisms.