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Cells have sophisticated RNA-directed mechanisms to regulate genes, destroy viruses, or silence transposable elements (TEs). In terrestrial plants, a specialized non-coding RNA machinery involving RNA polymerase IV (Pol IV) and small interfering RNAs (siRNAs) targets DNA methylation and silencing to TEs. Here, we present a bioinformatics protocol for annotating and quantifying siRNAs that derive from long terminal repeat (LTR) retrotransposons. The approach was validated using small RNA northern blot analyses, comparing the species Arabidopsis thaliana and Brachypodium distachyon. To assist hybridization probe design, we configured a genome browser to show small RNA-seq mappings in distinct colors and shades according to their nucleotide lengths and abundances, respectively. Samples from wild-type and pol IV mutant plants, cross-species negative controls, and a conserved microRNA control validated the detected siRNA signals, confirming their origin from specific TEs and their Pol IV-dependent biogenesis. Moreover, an optimized labeling method yielded probes that could detect low-abundance siRNAs from B. distachyon TEs. The integration of de novo TE annotation, small RNA-seq profiling, and northern blotting, as outlined here, will facilitate the comparative genomic analysis of RNA silencing in crop plants and non-model species.

The cellular level processes cell division and cell expansion form a crucial level linking regulatory processes at the molecular level to whole plant growth rates and organ size and shape. With the rapid progress in molecular profiling, quantification of cellular activities becomes increasingly important to determine sampling strategies that are most informative to understand the molecular basis for organ and plant level phenotypes. Inversely, to understand phenotypes caused by genetic or environmental perturbations it is crucial to know how the cell division and expansion parameters are affected spatially and temporally. Kinematic analyses provide a powerful and rigorous mathematical framework to quantify cell division and cell expansion rates. In dicotyledonous leaves, these processes are primarily changing over time, resulting in division, expansion, and mature phases of development. Monocotyledonous leaves have a persistent spatial gradient, with an intercalary meristem where division takes place, an expansion zone, and a mature part of the leaf. Here we describe in detail how to perform kinematic analyses in leaves of the model species Arabidopsis thaliana and in the leaves of the monocotyledonous crop species Zea mays. These methods can readily be used and adapted to suit other species using relatively standard equipment present in most laboratories. Importantly, the obtained results can be used to design sampling techniques for proliferating, expanding and mature cells.

Plant growth is readily analysed at the macroscopic level by measuring size and/or mass. Although it is commonly known that the rate of growth is determined by cell division and subsequent cell expansion, relatively few studies describing growth phenotypes include studies of the dynamics of these processes. Kinematic analyses provide a powerful and rigorous framework to perform such studies and have been adapted to the specific characteristics of various plant organs. Here we describe in detail how to perform these analyses in root tips and leaves of the model species Arabidopsis thaliana and in the leaves of the monocotyledonous crop species, Zea mays. These methods can be readily used and adapted to suit other species in most laboratories.