Involvement of ethylene biosynthesis and perception during germination of dormant Avena fatua L. caryopses induced by KAR1 or GA3.

MAIN CONCLUSION: Germination of primary dormant wild oat caused by KAR1 or GA3 is associated with ACC accumulation and increased ethylene production shortly before radicle protrusion as a result of the non-transcriptional and transcriptional activation of ACS and ACO enzymes, respectively. Response to both compounds involves the modulation of ethylene sensitivity through ethylene receptor genes. Harvested Avena fatua caryopses are primary dormant and, therefore, germinated poorly at 20 °C. Karrikin 1 (KAR1), which action probably requires endogenous gibberellins (GAs), and gibberellin A3 (GA3) was found to induce dormant caryopses to germinate. The stimulatory effects were accompanied by the activation of the ethylene biosynthesis pathway and depended on undisturbed ethylene perception. KAR1 and GA3 promoted 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation during coleorhizae emergence and ethylene production shortly prior to the radicle protrusion, which resulted from the enhanced activity of two ethylene biosynthesis enzymes, ACC synthase (ACS) and ACC oxidase (ACO). The inhibitor of ACS adversely affected beneficial impacts of both KAR1 and GA3 on A. fatua caryopses germination, while the inhibitor of ACO more efficiently impeded the GA3 effect. The inhibitors of ethylene action markedly lowered germination in response to KAR1 and GA3. Gene expression studies preceded by the identification of several genes related to ethylene biosynthesis (AfACS6, AfACO1, and AfACO5) and perception (AfERS1b, AfERS1c, AfERS2, AfETR2, AfETR3, and AfETR4) provided further evidence for the engagement of ethylene in KAR1 and GA3 induced germination of A. fatua caryopses. Both AfACO1 and AfACO5 were upregulated, whereas AfACS6 remained unaffected by the treatment. This suggests the existence of different regulatory mechanisms of enzymatic activity, transcriptional for ACO and non-transcriptional for ACS. During imbibition in water, AfERS1b was stronger expressed than other receptor genes. In the presence of KAR1 or GA3, the expression of AfETR3 was substantially induced. Differential expression of ethylene receptor genes implies the modulation of caryopses sensitivity adjusted to ethylene availability and suggests the functional diversification of individual receptors.

Wound-induced expression of DEFECTIVE IN ANTHER DEHISCENCE1 and DAD1-like lipase genes is mediated by both CORONATINE INSENSITIVE1-dependent and independent pathways in Arabidopsis thaliana.

Endogenous JA production is not necessary for wound-induced expression of JA-biosynthetic lipase genes such as DAD1 in Arabidopsis. However, the JA-Ile receptor COI1 is often required for their JA-independent induction. Wounding is a serious event in plants that may result from insect feeding and increase the risk of pathogen infection. Wounded plants produce high amounts of jasmonic acid (JA), which triggers the expression of insect and pathogen resistance genes. We focused on the transcriptional regulation of DEFECTIVE IN ANTHER DEHISCENCE1 and six of its homologs including DONGLE (DGL) in Arabidopsis, which encode lipases involved in JA biosynthesis. Plants constitutively expressing DAD1 accumulated a higher amount of JA than control plants after wounding, indicating that the expression of these lipase genes contributes to determining JA levels. We found that the expression of DAD1, DGL, and other DAD1-LIKE LIPASE (DALL) genes is induced upon wounding. Some DALLs were also expressed in unwounded leaves. Further experiments using JA-biosynthetic and JA-response mutants revealed that the wound induction of these genes is regulated by several distinct pathways. DAD1 and most of its homologs other than DALL4 were fully induced without relying on endogenous JA-Ile production and were only partly affected by JA deficiency, indicating that positive feedback by JA is not necessary for induction of these genes. However, DAD1 and DGL required CORONATINE INSENSITIVE1 (COI1) for their expression, suggesting that a molecule other than JA might act as a regulator of COI1. Wound induction of DALL1, DALL2, and DALL3 did not require COI1. This differential regulation of DAD1 and its homologs might explain their functions at different time points after wounding.

Reference gene selection for molecular studies of dormancy in wild oat (Avena fatua L.) caryopses by RT-qPCR method.

Molecular studies of primary and secondary dormancy in Avena fatua L., a serious weed of cereal and other crops, are intended to reveal the species-specific details of underlying molecular mechanisms which in turn may be useable in weed management. Among others, quantitative real-time PCR (RT-qPCR) data of comparative gene expression analysis may give some insight into the involvement of particular wild oat genes in dormancy release, maintenance or induction by unfavorable conditions. To assure obtaining biologically significant results using this method, the expression stability of selected candidate reference genes in different data subsets was evaluated using four statistical algorithms i.e. geNorm, NormFinder, Best Keeper and ΔCt method. Although some discrepancies in their ranking outputs were noticed, evidently two ubiquitin-conjugating enzyme homologs, AfUBC1 and AfUBC2, as well as one homolog of glyceraldehyde 3-phosphate dehydrogenase AfGAPDH1 and TATA-binding protein AfTBP2 appeared as more stably expressed than AfEF1a (translation elongation factor 1α), AfGAPDH2 or the least stable α-tubulin homolog AfTUA1 in caryopses and seedlings of A. fatua. Gene expression analysis of a dormancy-related wild oat transcription factor VIVIPAROUS1 (AfVP1) allowed for a validation of candidate reference genes performance. Based on the obtained results it can be recommended that the normalization factor calculated as a geometric mean of Cq values of AfUBC1, AfUBC2 and AfGAPDH1 would be optimal for RT-qPCR results normalization in the experiments comprising A. fatua caryopses of different dormancy status.