1 Macroarray for studying chloroplast gene expression profiles associated with the initial development of wheat

Chloroplasts, as photosynthetic carbon assimilating organelles, play an essential role in plants throughout their development. We have previously devised a mitochondrial macroarray using a set of rice and wheat mitochondrial genes to study their expression associated with biogenesis and initial development of this energy supplying organelle in wheat (1). We now report a chloroplast macroarray that can provide us with a useful means for studying chloroplast gene expression in wheat and other cereals. The chloroplast macroarray was constructed using 60 wheat chloroplast genes excluding tRNA and other small sized genes. In addition, five nuclear encoded and chloroplast-targeted genes and two control nuclear genes were included. Primer sets for the target genes were designed based on the published wheat chloroplast genome sequence (2). Changes in gene expression levels were monitored using RNA isolated from germinating seeds and seedlings at three different stages of development, i.e. at 1st, 2nd and 3rd leaf stages. Remarkable differences and dynamic changes were revealed in the amount of individual transcripts during the initial stages of wheat development. The amount of psbA transcript increased until the 3rd leaf stage. Those of psaA, psaB and psaC also increased significantly. Heterologous hybridisation was successful with cDNAs constructed from barley and rice seedlings, suggesting the versatility of this macroarray system for studies of chloroplast biogenesis and developmental changes in chloroplast gene expression in Gramineae. This system, after refining detailed protocol, can be utilized to depict chloroplast/plastid gene expression dynamics at different developmental stages and under various environmental conditions in cereal