During the early stages of seed development, Arabidopsis (Arabidopsis thaliana) endosperm is syncytial and proliferates rapidly through repeated rounds of mitosis without cytokinesis. This stage of endosperm development is important in determining final seed size and is a model for studying aspects of cellular and molecular biology, such as the cell cycle and genomic imprinting. However, the small size of the Arabidopsis seed makes high-throughput molecular analysis of the early endosperm technically difficult. Laser capture microdissection enabled high-resolution transcript analysis of the syncytial stage of Arabidopsis endosperm development at 4 d after pollination. Analysis of Gene Ontology representation revealed a developmental program dominated by the expression of genes associated with cell cycle, DNA processing, chromatin assembly, protein synthesis, cytoskeleton-and microtubule-related processes, and cell/organelle biogenesis and organization. Analysis of core cell cycle genes implicates particular gene family members as playing important roles in controlling syncytial cell division. Hormone marker analysis indicates predominance for cytokinin signaling during early endosperm development. Comparisons with publicly available microarray data revealed that approximately 800 putative early seed-specific genes were preferentially expressed in the endosperm. Early seed expression was confirmed for 71 genes using quantitative reverse transcription-polymerase chain reaction, with 27 transcription factors being confirmed as early seed specific. Promoter-reporter lines confirmed endosperm-preferred expression at 4 d after pollination for five transcription factors, which validates the approach and suggests important roles for these genes during early endosperm development. In summary, the data generated provide a useful resource providing novel insight into early seed development and identify new target genes for further characterization.

BackgroundArabidopsis thaliana is a useful model organism for deciphering the genetic determinants of seed size; however the small size of its seeds makes measurements difficult. Bulk seed weights are often used as an indicator of average seed size, but details of individual seed is obscured. Analysis of seed images is possible but issues arise from variations in seed pigmentation and shadowing making analysis laborious. We therefore investigated the use of a consumer level scanner to facilitate seed size measurements in conjunction with open source image-processing software.ResultsBy using the transmitted light from the slide scanning function of a flatbed scanner and particle analysis of the resulting images, we have developed a method for the rapid and high throughput analysis of seed size and seed size distribution. The technical variation due to the approach was negligible enabling us to identify aspects of maternal plant growth that contribute to biological variation in seed size. By controlling for these factors, differences in seed size caused by altered parental genome dosage and mutation were easily detected. The method has high reproducibility and sensitivity, such that a mutant with a 10% reduction in seed size was identified in a screen of endosperm-expressed genes. Our study also generated average seed size data for 91 Arabidopsis accessions and identified a number of quantitative trait loci from two recombinant inbred line populations, generated from Cape Verde Islands and Burren accessions crossed with Columbia.ConclusionsThis study describes a sensitive, high-throughput approach for measuring seed size and seed size distribution. The method provides a low cost and robust solution that can be easily implemented into the workflow of studies relating to various aspects of seed development.

Epigenetic reprogramming occurs during gametogenesis as well as during embryogenesis to reset the genome for early development. In flowering plants, many heterochromatic marks are maintained in sperm, but asymmetric DNA methylation is mostly lost. Asymmetric DNA methylation is dependent on small RNA but the re-establishment of silencing in embryo is not well understood.Here we demonstrate that small RNAs direct the histone H3 lysine 9 dimethylation during Arabidopsis thaliana embryonic development, together with asymmetric DNA methylation. This de novo silencing mechanism depends on the catalytic domain of SUVH9, a Su(Var)3-9 homolog thought to be catalytically inactive.