Plant hormones play important roles as signaling molecules in the regulation of almost all phases of plant development, from seed germination to senescence. For instance, cytokinins (CKs) are involved in the regulation of leaf senescence, apical dominance, shoot development, and root-shoot balance; auxins in apical dominance, phyllotaxis, and root development; abscisic acid (ABA) in seed dormancy and stress response; and gibberellins (GAs) in seed germination and leaf development. Thus, the identification and use of the genes regulating these plant hormone activities are important to the improvement of plant productivity. The contents of these hormones are regulated not only directly by their metabolic and signaling systems, but also indirectly by other apparently unrelated genes, the regulatory interactions of which are not well understood. To find novel genes regulating the contents of the hormones in rice FOX Arabidopsis lines, we established a method for high-throughput analysis of major plant hormones, namely, CKs (tZ, tZR, tZRMP, iP, iPR, iPRMP, cZ, cZR, cZRMP, DZ, DZR, DZRMP, tZ7G, tZ9G, tZOG, tZROG, cZOG, cZROG, iP7G, iP9G, DZ9G), auxins (IAA, IA-Ala, IA-Asp, IA-Phe, IA-Leu, IA-Trp, IA-Ile), ABA, and GAs (GA1, GA3, GA4, GA7, GA9, GA19, GA20, GA24, GA53), from about 100 mg fresh weight of plant tissues. We analyzed lines with morphological abnormalities, namely bushy, hyper apical dominance, lateral shoot, fast growth, large cauline leaf, and large rosette leaf. We also analyzed some "silent phenotype" lines.
Seeds of rice FOX Arabidopsis lines were sterilized with PPM (Plant Cell Technology Inc.) and germinated on MS-agar containing 1% sucrose and 50 µg/mL hygromycin. Plants were grown for 14 days at 22 °C under fluorescent illumination at about 70 µE m-2s-1 with a photoperiod of 16h-light - 8h-darkness. As a control, a transgenic line harboring the pBigS T-DNA region was grown. All material (20-100 mg fresh weight) was harvested, frozen in liquid N, and stored at -80 °C until use.
Hormones were extracted and fractionated by solid-phase extraction as described by Dobrev and Kaminek (2002). Auxins, ABA, and GAs were further purified in a DEAE-cellulose spin column (Vivapure Mini, VivaScience). Almost all extractions were done in triplicate. The hormones were measured by LC-MS (UPLC/Quattro Ultima Pt; Waters) with an ODS column (Aquity UPLC BEH C18, 1.7 µm, 2.1 mm x 50 mm, Waters). Details of measurement conditions will be published elsewhere. To calculate recovery, we added stable-isotope-labeled compounds (OlChemim, Czech Republic) to the extraction solvent. Data are shown as average values (pmol/g fresh weight) with standard deviation.
For convenience of comparison, hormone contents are expressed as the sum of measured species. "Increased" and °»Decreased°… indicate that a transgenic line contains more (>=1.35x) or less (<=0.65x) hormone than the corresponding control. Since the GA content was very small, some data is lacking.