1.

Corvallis OR

Oregon State University Dept. Botany and Plant Pathology

Carroll, K.A., Kulhanek, D., Fowler, J. and Rivin, C.

Microarray evidence for ABA – GA antagonism during embryo maturation

Complex developmental processes are often controlled by the interplay of positive and antagonistic, or modulating, signaling pathways. The switch between embryogenesis and the maturation phase of maize embryo development involves the interaction of antagonistic signaling pathways governed by abscisic acid (ABA) and Gibberellic acids (GAs). Abscisic acid (ABA) is a highly conserved hormone signal required to induce maturation phase in developing plant embryos. During embryonic development in cereals, bioactive GAs accumulate, peaking prior to the ABA peak that initiates maturation phase. Although ABA’s role in maturation is highly conserved in plants, a preceding GA peak is found only in cereals, and its significance is unclear. We have previously used both genetics and manipulation of hormone levels in culture to support our idea that the pre-maturation GA peak antagonizes ABA in controlling maturation-phase processes in maize . In these studies, we found that ABA-deficient kernels are viviparous (germinating precociously on the ear) and desiccation-sensitive, but that mutants deficient in both ABA and GA exhibit the wild-type phenotypes of quiescence and desiccation tolerance. Thus, the early GA peak may either intercept the ABA signaling pathway to modulate ABA sensitivity, or participate in a negative regulatory mechanism to suppress maturation independently of ABA.

The wildtype behavior of ABA / GA double-deficiency embryos suggests that gene expression in this genotype is more like that in wildtypes than in ABA-deficient mutants. To test this proposition, we collected early maturation (stage 3) embryos from two types of ears: 1) ears segregating for vp5 (ABA-deficient kernels) and 2) d1 homozygotes (bioactive GA deficient) segregating for vp5 kernels. mRNA was isolated from sibling wildtype and vp5 homozygous embryos from each type of ear for comparison by microarray analysis. A loop-design hybridization scheme was used to compare the message profiles of the four genotypes, using the maize oligonucleotide array produced by the University of Arizona. Bioconductor and Limma software packages were used to identify genes with significantly different expression based on a adjusted P value p< 0.05.

In a comparison of wildtype and ABA-deficient sibling embryos at Stage 3 of embryogenesis, 75 moderate – to –highly expressed genes were found to be significantly different in expression between the normal and hormone-deficient condition. Of these genes, 70 were also found to be significantly different in a comparison of sibling embryo mRNAs from the double ABA / GA vs single ABA-deficient ears, a 93% overlap in expression patterns indicating that gene expression in the double hormone mutants is very similar to wildtype on a broad scale. The differentially expressed genes included well-known maturation genes like the storage globulins and LEA proteins previously shown to be regulated by ABA and the Vp1 transcription factor, but a wide variety of other genes, not known to be ABA regulated, also appeared in this gene set.

1. White CN and Rivin CJ: Gibberellins and seed development in maize. II. Gibberellin synthesis inhibition enhances abscisic acid signaling in cultured embryos. Plant Physiol 122: 1089-97 (2000).

2. White CN, Proebsting WM, Hedden P and Rivin CJ: Gibberellins and seed development in maize. I. Evidence that gibberellin/abscisic acid balance governs germination versus maturation pathways. Plant Physiol 122: 1081-8 (2000).

2.

Corvallis OR

Oregon State University Dept. Botany and Plant Pathology

Carroll, KA and Rivin, C.

Practical advice on using the maize oligonucleotide microarray

Microarrays have become a popular method to monitor gene expression levels on a genomic scale. We have been using the array produced by the Maize Oligonucleotide Array Project at University of Arizona. We have generally followed the protocols provided on the project website ((), and we have also tried modifications. Based on our experience with microarray experiments we have the following recommendations for people who may be interested in starting a microarray experiment. Please feel free to contact us if you have any questions or would like more information.

1. Successful modifications to the project protocols. We used the protocols for cRNA targets provided on the website with the following alterations.

a. During RNA purification step we adjusted the total elution volume adjusted to 100 ul (65 1st, 45 2nd) instead of the recommended 60 ul. Our yields ranged from 20-40 ug aRNA.

b. We experienced up to 50% reduction in yield during the Cy Dye coupling step due to cRNA adherence to the column. To help alleviate this we used 50ºC DEPC water for the elution steps and also heated the entire column during the elution for ~ 5 minutes in a 50ºC hybridization oven. This increased the yield of labeled aRNA to about 80%.

c. In fear of washing the oligos off the microarray slides we opted to skip the rehydration steps as recommended in the protocol under DNA Probe Immobilization and simply cross-linked and washed the slides as described.

2. Use of aliens as a control feature for cRNA targets. Aliens are control RNAs that can be added to the total RNA as a standard for data normalization and scanning. Stragene alien sequences 1-10 are printed on the maize array. To take advantage of this control, we used mRNA spikes from the Stratagene SpotReport® Alien® cDNA Array Validation System in our amplification and hybridizations. In our hands, the aliens created problems during scanning as they drastically reduced the signals from other features. We also found that the aliens could not be used to manually adjust the scanner for equal red and green intensities. Our core facility has a Perkin Elmer ScanArray 4000 and Genepix software for microarray scanning and analysis. Using this scanner and software the auto PMT setting were found to be optimal for adjusting the signal intensities for all scans (the saturation levels were adjusted from the default settings of .05% to .005 % when using the auto PMT setting).

3. Use of Dyesaver for fluor preservation. Dyesaver, by Genesphere, is a toluene-based material coating which is applied to the slides after hybridization and washing. It is recommended to help preserve the fluorochromes from degradation, especially the Cy5 which is more easily degraded than the Cy3 dye. Our experience is that Dyesaver is expensive and may not be necessary for repeated scanning. As an experiment, we used the “practice” slides supplied to us by the Maize Array Project for two identical hybridizations, one with Dyesaver and the other without. The data from both slides produced similar results. The slide without the Dyesaver was scanned at least 4 times with only a minor loss in fluor intensity, using the auto PMT setting, with laser power settings between 70 – 90%. Slides that were coated in Dyesaver did maintain their integrity for several months, unlike untreated slides which expire rapidly. The major disadvantages of Dyesaver were the toxic toluene fumes which made it unpleasant to work with, high evaporation rate of the dye during storage drastically reducing the number of slides on which one can actually use the dye, and the overall green hue it gives to the slides.

4. Data analysis using Bioconductor freeware (bioconductor.org), which uses the R computing environment (), requires writing customized Perl scripts. The main advantage to using Bioconductor is that it is one of the most powerful software packages to use for microarray statistical analysis. The main disadvantage, however, is that it uses a command driven user interface and therefore is not user unfriendly for most scientists. We needed to create customized scripts effectively filter and normalize our data. In Bioconductor we used the Limma package (also available at ) which uses the empirical Bayesian method to create a linear model to evaluate genes with significant differential expression. We would be happy to share our scripts for filtering, normalization and linear model analysis.