Gene Expression
Nebulas
Gene Expression NebulasSummary: Basal branching in grasses, or tillering, is an important trait determining both form and function of crops. While similarities exist between eudicot and grass branching programs, one notable difference is that the tiller buds of grasses are covered by the subtending leaf, whereas eudicot buds are typically unconstrained. The current study shows that contact with the leaf sheath represses sorghum bud growth by providing a mechanical signal that cues the bud to refrain from rapid growth. Leaf removal resulted in massive reprogramming of the bud transcriptome that included signatures of epigenetic modifications, and also implicated several hormones in the response. Bud abscisic acid (ABA) transiently increased, then decreased following leaf removal relative to controls, and ABA was necessary to repress bud growth in the presence of the leaf. Jasmonic acid (JA) levels and signaling increased in buds following leaf removal. Remarkably, application of JA to buds in situ promoted growth. The repression of bud growth by leaf contact shares characteristics of thigmomorphogenic responses in other systems, including the involvement of JA, though the JA effect is opposite. The repression of bud growth by leaf contact may represent a mechanism to time tillering to an appropriate developmental stage of the plant.
Overall Design: Analysis of bud transcriptomes at 1, 3 and 6 h with and without removal of the overlying leaf.
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| Growth Protocol: | Seeds were sown singly in 50 mL conical centrifuge tubes modified with a drain hole and filled with LC1 soilless medium supplemented with 7.5g/L 20-20-20 Osmocote fertilizer. Plants were grown in a growth chamber providing 575 mM m-2s-1 of PAR at a R:FR of 13.30 from t5 fluorescent lamps with a photoperiod of 16/8 light dark and 32/22oC day night temperatures. |
| Treatment Protocol: | Treatments began 7 days after sowing. Plants were left intact, or leaf removal was conducted by peeling the entire coleoptile and first leaf from the culm to expose the axillary bud. The dissection was easily accomplished with minimal handling and without touching the bud. Axillary buds from the first leaf axil were harvested at 1, 3 and 6 h after leaf removal. Four replicates of 15 buds were harvested. The three highest quality replicates of each sample (by Bioanalyzer) were used for transcriptome analysis. |
| Extract Protocol: | RNA was extracted using Trizol, the extracted RNA was digested with DNAseI and purified using an RNAeasy kit. |
| Library Construction Protocol: | Libraries were constructed with the Illumina TruSeq kit and 125 bp reads were obtained from an Illumina HiSeq 2500 sequencer. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Specific |