Davydenko O

Nakamura C

Naydenov N

Siniauskaya M

English

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 1Macroarray for studying chloroplast gene expression profiles associated with the initial development of wheat  Siniauskaya M1, 2, Naydenov N1, 3, Davydenko O2, NakamuraC1 1Laboratory of Plant Genetics, Department of Agroenvironmental Science , Kobe University, Kobe 657-8501, Japan, 2Institute of Genetics and Cytology, Laboratory of Cytoplasmic Inheritance, Akademicheskaya 27, 220072, NAS Belarus, Minsk, Belarus, 3University of Rochester, Box 646  Rochester, NY 14642, USA  ABSTRACT  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 cereals INTRODUCTION  In the last decades molecular biology has made marked advances in understanding many important aspects of biology by providing sequence information of not only nuclear genomes but also chloroplast and mitochondrial genomes in a variety of organisms. Availability of organellar DNA sequences of many land plants has opened the perspectives for the large-scale analysis of organellar gene expression. Organellar microarray and macroarray are two powerful tools for analysing global gene expression profiles of organellar genomes at different developmental stages and under various environmental conditions in plants. In microarray, a set of gene probes corresponding to their genomic sequences, cDNA clones, or synthetic oligonucleotides each corresponding to a single specific coding sequence are spotted at a high density on a chemically coated glass slide and used as the target for different types of RNA or cDNA derived from plants grown under experimentally controlled conditions (3). In macroarray, the same kinds of probes are applied onto nylon membrane. Studies of organellar gene expression therefore have now a tendency to be built upon these two array systems, followed by northern-blot, RT-PCR or real-time PCR analysis for verification of the array data.      Recently plastid microarrays have been reported for Arabidopsis (4), Chlamydomonas (5), Cyanidioschyzon (6), tobacco (7), maize (8) and tomato (9). Using these microarray systems, various biological questions have been addressed such as chloroplast gene expression profiles in mutants deficient in some chloroplast transcription components in Arabidopsis (4). In Chlamydomonas reinhardtii, the specificity of a chloroplast RNA stability mutant was confirmed (5). Chloroplast gene expression profiles under dark-light changes in tobacco (7) and Cyanidioschyzon merolae (6) as well as plastid transcriptomes during fruit development and chloroplast to chromoplast differentiation in tomato (9) were also analysed by this system. In maize, transcript abundance of nuclear, chloroplast and mitochondrial genes was studied at different developmental stages (8).     A macroarray-based approach was also useful for comparative analysis of plastid transcriptomes in wild-type and PEP-deficient tobacco, showing that the plastid genome was completely transcribed in both types of plastids (10).    Previously we have reported the development and successful use of a mitochondrial macroarray bearing a set of rice and wheat mitochondrial genes for studying gene expression profiles associated with mitochondrial biogenesis and initial development of mitochondrial function during germination and early seedling growth in wheat (1). Now we have designed a chloroplast macroarray that can be useful in various studies of chloroplast/plastid gene expression not only in wheat but also in other cereals because it can hybridize successfully with barley and rice cDNA probes. MATERIALS AND METHODS The chloroplast macroarray was designed to cover 60 wheat chloroplast genes, 5 nuclear encoded chloroplast–targeted genes and 2 control nuclear genes. Primer sets  2 for individual genes were developed based on the published wheat chloroplast genome sequence (2) using Gene Runner3 and Olygo3 softwares. The length of amplified gene regions ranged from 200bp to 1,259bp with most probes in the size interval of 250-500bp (the sequences of all primer sets are available upon request to the authors). Amplification was carried out using total DNA and cDNA extracted from common wheat cultivars “Miranovskaya 808” and “Chinese Spring”, respectively. Amplification was performed according to the following procedures: an initial denaturation step for 5 min at 94 oC followed by 25-30 cycles of denaturation for 30-50 sec at 94 oC, then annealing for 30-60 sec at 53-61 oC (primer dependent), elongation for 40-60 sec at 72 oC, with a final elongation for 5 min at 72 oC. The amplified fragments were fractionated by electrophoresis through 1.2 % agarose gel and visualized by staining with ethidium bromide for size verification. Excess primers were removed by a microSpin S-400 MR column (GE Healthcare) according to the manufacturer’s protocol. After estimating concentrations of PCR products using a capillary photometer (NanoDrop Technologies), 50 ng of each dissolved in a total volume of 2.5 µl water and an equal volume of 20 x SSC was added to the nylon membrane (6.5 cm x 5 cm; HybondTM-N+). PCR products were spotted 5 times manually at 1 µl each time onto the nylon membrane. The membranes were treated for denaturation of double stranded DNA according to the standard protocols.    Changes in gene expression profiles were monitored using RNA isolated from seedlings at different stages of development, i.e. embryos just after imbibition (indicated as 1 in all Figures), seedlings at 1st leaf stage on the 4th day  (indicated as 2), 2nd leaf stage on the 8th day (indicated as 3) and 3rd leaf stage on the 11th day of development (shown as 4) after imbibition. Total RNA was extracted with Sepasol-RNAI and cDNA was synthesized using random hexamers and ReverTra Ace (Toyobo, Tokyo, Japan). cDNA labelling and signal detection was performed according to protocols of ECL Direct Nucleic Acid Labelling and Detection Systems (GE Healthcare). After detection step, the membrane was exposed to x-ray film (Fuji) for 3-8 h. Data was quantified by Image J software (http//rsb.info.nih.gov/ij). Mean values were normalized with respect to the actin transcript signal intensity on the same membrane. The experiment was repeated at least twice. RT-PCR was carried out in order to verify the macroarray data.  RESULTS AND DISCUSSION  After testing various experimental conditions, we applied the chloroplast macroarray to monitor changes in chloroplast transcript levels during 11 days of wheat development following imbibition. Germinating embryos and developing seedlings were incubated under 16 h light and 8 h dark conditions. Although considerable fluctuations were noted in transcript levels of individual genes, significant and consistent increases of RNA levels were detected for genes that function in photosystems I (PSI) and II (PSII) after imbibition until the 4th and/or the 8th day of development. Such up-regulated genes included psaA, psaB and psaC in PSI and psbA in PSII. On the other hand, the levels of transcripts of ndh and atp genes initially was high but then showed either no changes or decreased.    Based on the macroarray data, the level of psbA transcripts increased more than 30 % after imbibition till the 1st leaf stage, and then it continued to increase but more slowly to the 2nd and the 3rd leaf stages with a total increase of ca. 70 % (Fig. 1). The result obtained by the macroarray was verified by RT-PCR, although the level of increase estimated by RT-PCR was much greater (ca. 3-fold) (Figs. 2, 3). This is probably due to a lower dynamic range of the array analysis than that of PCR analysis. In Arabidopsis, it was reported that the level of psbA transcript increased two-fold from the youngest (Cot5, 2-days-old) to the oldest cotyledons (Cot30, 27-days-old) (11). Our result showing an increased level of             Fig. 1. Macroarray data for the transcript level of psbA gene during wheat development. For stages, see M & M.             Fig. 2. RT-PCR analysis for estimating the psbA transcript abundance during wheat development. Images of 1. 2 % agarose gels stained with EtBr. For stages, see M & M.  Fig. 3. Graphical view of changes in the transcript level of the psbA gene by RT-PCR evaluated by ImageJ software.  010002000300040005000600070001 2 3 4Developmental stages of wheat 01000020000300004000050000600001 2 3 4 Developmental stages of wheat  1       2      3      4 1     2      3     4 actin (control) Spot signal intensity (raw data) Band intensity (raw data)  3psbA transcript during wheat development thus agreed with the result reported in Arabidopsis.    The transcript levels of genes belonging to PSI at these initial stages were much lower than those of PSII genes. The highest augmentation in the expression levels (≥2-fold) was observed for psaA, psaB and psaC genes from imbibition till the 1st leaf stage. The rate of increase in these transcripts, however, slowed down after the 1st leaf stage until the 2nd leaf stage (Fig. 4).   Fig. 4. Macroarray data for the transcript level of psaA gene during wheat development. For stages, see M & M.    To test versatility of the chloroplast macroarray system, we tried to hybridize wheat chloroplast target genes to cDNAs of barley and rice seedlings and successfully obtained clear hybridization signals with both. Fig. 5 shows an example of the hybridization signal pattern with barley cDNA. Large differences in the abundance of individual gene transcripts was apparent, indicating dynamic changes in the chloroplast gene expression profiles in cereals.               Fig. 5. Wheat plastidial macroarray showing hybridization with cDNA of barley.  CONCLUSION Herein we reported the chloroplast macroarray system that can be applied to analyse chloroplast gene expression during imbibition to early stages of wheat development. After refining the experimental conditions such as probe spotting and signal detection procedures, this macroarray system together with the already established mitochondrial macroarray system (8), should provide useful experimental tools for studying organellar biogenesis in Gramineae.   REFERENCES 1.  Khanam SM, Naydenov NG, Kadowaki K, Nakamura C.  (2007) Mitochondrial biogenesis as revealed by mitochondrial transcript profiles during germination and early seedling growth in wheat. Genes and Genetic Systems 82: 409-420. 2.  Ogihara Y., Isono K., Kojima T., Endo A., Hanaoka M. et al. (2002) Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA. Mol. Genet.  Genomics 266: 740-746. 3.  Kehoe D., Villand P., Somerville S. (1999) DNA microarrays for studies of higher plants and other photosynthetic organisms. Trends in Plant Science 4: 38-41. 4.  Nagashima A., Hanaoka M., Motohashi R., Seki M., Shinozaki K., Kanamaru K., Takahashi H., Tanaka K. 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(2008) Nuclear, chloroplast, and mitochondrial transcript abundance along a maize leaf developmental gradient. Plant Mol. Biol. 66: 33-46. 9.  Kahlau S., Bock R. (2008) Plastid transcriptomics and translatomics of tomato fruit development and chloroplast-to-chromoplast differentiation: chromoplast gene expression largely serves the production of a single protein. The Plant Cell 20: 856-874. 10.  Legen J., Kemp S., Krause K., Profanter B., Herrmann R., Maier R. (2002) Comparative analysis of plastid transcription profiles of entire plastid chromosome from tobacco attributed to wild-type and PEP-deficient transcription machineries. The Plant Journal 31: 171-188. 11.  Zoschke R., Liere K., Borner T. (2007) From seedlings to mature plant: Arabidopsis plastidial genome copy number, RNA accumulation and transcription are differentially regulated during leaf development. The Plant Journal 50: 710-722. 02000 4000 6000 8000 10000120001 2 3Developmental stages of wheat Spot signal intensity (raw data) 

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

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

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