Molecular genetic approaches to understanding and manipulating seed development in Arabidopsis thaliana

Background: Arabidopsis 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. Results: By 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. Conclusions: This 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. Background More food will need to be produced during the next 50 years than in the entire history of humankind. Therefore, increasing crop yields is a major challenge for the 21 century. Since most of the world’s food calories come from seed, one way to meet this challenge is to create plants with more and larger seeds. Arabidopsis thaliana is a useful model organism for studying seed development due to its ease of cultivation and extensive genetic and community resources available. Thus far, only a handful of genes are known to be directly involved in determining Arabidopsis seed size [1-8]. Genes that regulate seed size can be discovered by screening for mutants or by quantitative trait loci (QTL) analysis to identify the genes that underlie the natural variation in seed size between different accessions. Alonso-Blanco et al. [9] performed a QTL analysis on recombinant inbred lines (RILs) from crosses between the small seeded Landsberg erecta (Ler) accession and the large seeded Cape Verde Islands (Cvi) accession. Seed weight and length QTL were mapped as well as those affecting maternal factors that contribute to seed size (such as seed number and leaf size). Six QTL affecting seed size, without significant effects on the maternal plant, were identified [9]. The genes/alleles underlying these QTL have not been determined, which is an important step if they are to be applied in a biotechnological context. Only a few Arabidopsis mutants have been identified that directly affect seed size. These mutants reveal that both endosperm and integument * Correspondence: richard.macknight@otago.ac. nz † Contributed equally Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand Full list of author information is available at the end of the article Herridge et al. Plant Methods 2011, 7:3 http://www.plantmethods. com/content/7/1/3 PLANT METHODS © 2011 Herridge et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. growth are involved in seed size determination in Arabidopsis [10]. HAIKU1, 2 (IKU1 and 2) and MINISEED3 (MINI3) act in the same pathway to control early endosperm proliferation and subsequent seed size at maturity [2,4]. MINI3 and IKU2 are in close proximity to two quantitative trait loci (QTL) discovered by AlonsoBlanco et al. [9] suggesting that they may play an important role in the natural variation observed between accessions [4]. SHORT HYPOCOTYL UNDER BLUE1 (SHB1) binds the promoters of IKU2 and MINI3. However, the shb1 mutant only has a minor effect on seed size [7], suggesting that there may be other regulators of seed size upstream of this pathway. The AUXIN RESPONSE FACTOR2 (ARF2) and TRANSPARENT TESTA GLABRA2 (TTG2) genes affect seed size via integument cell elongation [5,11]. Crossing ttg2 and iku2 mutants reveals that, although the genes operate in independent pathways, cross-talk occurs between the integument and endosperm to determine final seed size [10]. Parental genome dosage can also affect seed size. In interploidy crosses, a seed size effect is observed when the ratio of maternal to paternal genomes is altered in the endosperm, with over-representation of paternal genomes resulting in larger seeds whereas the opposite is true for maternal genomes [12]. A similar phenomenon is found when reciprocal crosses of met1 mutants are performed, suggesting that DNA methylation plays an important role in seed size determination, via the action of imprinted genes in the endosperm and also hypomethylation in the integuments [13,14]. A major aim of our laboratory is to discover the molecular mechanisms that regulate seed size. We have developed a sensitive, high-throughput method of measuring seed size using a scanner and particle analysis software. Furthermore, by identifying and taking into account certain maternal factors that contribute to seed size we were able to reduce variation, enabling routine detection of subtle differences in size. We show that the approach is capable of identifying differences in seed size due to mutation, parental genome dosage, natural variation and detection of a number of novel seed size QTL. Results A document scanner and open source image analysis software provide a low cost and reliable means of rapidly measuring seed size We investigated whether a document scanner could provide a means of rapidly screening a large number of Arabidopsis plants for seed size phenotypes with a high degree of sensitivity. Using a commercially available scanner with a resolution of 1200 dpi combined with image analysis software we were able to quickly obtain accurate measurements of seed size. The slide-holder included with the scanner enabled 24 samples to be scanned simultaneously, reducing the amount of labour required (Figure 1A). A major factor with regard to the ease of processing was the use of transmitted light.