Grown for 11 days in growth chamber (16h/8h light/darkness, 50% humidity, 28°C, approx 40 µmol/s-1/m-2) in soil. M cells collected from second leaves of 3rd leaf emerging stage seedlings.
Extracted molecule
total RNA
Extraction protocol
M protoplasts were isolated as previously described with slight modification; only second leaf blades were used. For full protocol see Markelz, N.H., Costich, D.E., and Brutnell, T.P. (2003). Photomorphogenic responses in maize seedling development. Plant Physiol 133, 1578-1591. RNA was isolated as in Sheehan, M.J., Farmer, P.R., and Brutnell, T.P. (2004). Structure and expression of maize phytochrome family homeologs. Genetics 167, 1395-1405.
Label
Cy3
Label protocol
Followed Genisphere Array 900MPX instructions using buffer 6. cDNA hybridization overnight at 55°C. Washed slides in 2X SSC, 0.5%SDS; 0.5X SSC; 0.05X SSC for 5 min each. First wash buffer was used at 55°C.
Grown for 11 days in growth chamber (16h/8h light/darkness, 50% humidity, 28°C, approx 40 µmol/s-1/m-2) in soil. M cells collected from second leaves of 3rd leaf emerging stage seedlings.
Extracted molecule
total RNA
Extraction protocol
M protoplasts were isolated as previously described with slight modification; only second leaf blades were used. For full protocol see Markelz, N.H., Costich, D.E., and Brutnell, T.P. (2003). Photomorphogenic responses in maize seedling development. Plant Physiol 133, 1578-1591. RNA was isolated as in Sheehan, M.J., Farmer, P.R., and Brutnell, T.P. (2004). Structure and expression of maize phytochrome family homeologs. Genetics 167, 1395-1405.
Label
Cy5
Label protocol
Followed Genisphere Array 900MPX instructions using buffer 6. cDNA hybridization overnight at 55°C. Washed slides in 2X SSC, 0.5%SDS; 0.5X SSC; 0.05X SSC for 5 min each. First wash buffer was used at 55°C.
Hybridization protocol
Followed Genisphere Array 900MPX instructions using buffer 6. cDNA hybridization overnight at 55°C. Washed slides in 2X SSC, 0.5%SDS; 0.5X SSC; 0.05X SSC for 5 min each. First wash buffer was used at 55°C.
Scan protocol
See: Sawers, R.J., Liu, P., Anufrikova, K., Hwang, J.T., and Brutnell, T.P. (2007). A multi-treatment experimental system to examine photosynthetic differentiation in the maize leaf. BMC Genomics 8, 12.
Description
The mutant bundle sheath defective2 (bsd2) lacks the accumulation of Rubisco small and large subunits (Roth et al 1996; Brutnell et al 1999) and cannot perform the Calvin Cycle (Smith et al 1998). The loss of Rubisco may lead to changes in the spatial regulation of typically BS- and M-enriched genes. To detect such changes, gene expression profiles of bsd2 M cells and wild-type M cells from sibling plants were compared using the Maize Oligonucleotide Array (University of Arizona). Tissue and RNA isolations for this experiment were performed as previously described (Covshoff et al 2008). Six biological replicates were conducted in a dye swap arrangement using the Array 900MPX Expression Array Detection Kit and company protocol (Genisphere). References: Brutnell TP, Sawers RJ, Mant A, and Langdale JA (1999). BUNDLE SHEATH DEFECTIVE2, a novel protein required for post-translational regulation of the rbcL gene of maize. Plant Cell 11, 849-864. Covshoff S, Majeran W, Liu P, Kolkman JM, van Wijk KJ, Brutnell TP (2008) Deregulation of maize C4 photosynthetic development in a mesophyll cell-defective mutant. Plant Physiol 146: 1469-1481. Roth R, Hall LN, Brutnell TP, and Langdale JA (1996). bundle sheath defective2, a mutation that disrupts the coordinated development of bundle sheath and mesophyll cells in the maize leaf. Plant Cell 8, 915-927. Smith LH, Langdale JA, and Chollet R (1998). A functional Calvin cycle is not indispensable for the light activation of C4 phosphoenolpyruvate carboxylase kinase and its target enzyme in the maize mutant bundle sheath defective2-mutable1. Plant Physiol 118, 191-197.
Data processing
Feature intensity values were log-transformed, corrected for local background, and filtered to include only those that were at least 2x background. The data was then LOWESS normalized (Sawers et al 2007) and corrected for dye-effect (Smyth 2004). Differentially expressed genes were identified by moderated-t test and the false discovery rate (q) was calculated as previously described (Storey and Tibshirani 2003).
References:
Sawers, R.J., Liu, P., Anufrikova, K., Hwang, J.T., and Brutnell, T.P. (2007). A multi-treatment experimental system to examine photosynthetic differentiation in the maize leaf. BMC
Genomics 8, 12.
Smyth, G.K. (2004). Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genetic Mol Biol 3, Article3.
Storey, J.D., and Tibshirani, R. (2003). Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 100, 9440-9445.
