ESTs analysis in maize developing kernels exposed to single and combined water and heat stresses

Molecular and metabolic response of plants to a combination of two abiotic stresses is unique and cannot be directly extrapolated from the response of plants to each of the stresses individually. cDNA macroarray has become a useful tool to analyze expression profiles and compare the similarities and differences of various expression patterns. A macroarray of approximately 2,500 maize (Zea mays L.) cDNAs was used for transcriptome profiling in response to single and simultaneous application of water and high temperature stress of maize developing kernels at 15 days after pollination. All stress treatments (water stress-WS, heat stress-HS and their combined application-CS) induced changes in expression of 106 transcripts with 54 up-regulated and 52 down-regulated. There were 11 up-regulated and 15 down-regulated transcripts in common for all three stresses. Although these common transcripts showed existence of a mutual mechanism in stress response, the 23 transcripts induced only in CS indicate that plants responded in a different manner when exposed to simultaneous effects of both stresses. A glimpse of functions regulated under WS, HS and CS is provided, and also the common and different responses between individual and simultaneous stresses.

Application of cDNA Macroarray for Simultaneous Detection of 12 Potato Viruses

A complementary DNA (cDNA) macroarray was developed for simultaneous detection of 12 different potato viruses. A suitable region in the viral genome for each was selected for Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus, Potato mop-top virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, Tomato ringspot virus, and Tomato spotted wilt virus, and their respective cDNAs were cloned into plasmid vectors. Capture probes for each virus ranging from 290 to 577 bp were generated by polymerase chain reaction (PCR) and immobilized on a nylon membrane. Total RNAs were extracted from each of these virus infected-plants, and cDNAs were synthesized from the RNA extracts using a random 9-mer primer. Subsequently, PCR reactions were performed using one primer pair for each of the 12 viruses. During PCR, amplified cDNAs were labeled with biotin and used as a target for hybridization analyses on a macroarray membrane. Hybridization signals between capture probes for the 12 viruses and their respective target cDNAs were observed using chemiluminescent or colorimetric detection. In all viruses, hybridization signals with capture probes were detected only when homologous virus targets were examined, and no hybridization to healthy plant extract was observed, facilitating identification of each virus. The results by colorimetric detection agreed with those obtained using chemiluminescence. The macroarray method developed was 5 × 102 to 4 × 106 times more sensitive than enzyme-linked immunosorbent assay and 5 to 5 × 104 times more sensitive than reverse-transcription PCR, except for Alfalfa mosaic virus. Colorimetric detection and substantial reduction in cross-hybridization signals much improved the method compared with other array-based detection methods for practical use.