Interaction of nitric oxide with auxin and ethylene signalling in Arabidopsis root gravitropism

Gravitropism is required for the appropriate alignment of the plant root and shoot. According to the Cholodny-Went hypothesis, gravity induces asymmetric accumulation of auxin in the lower side of the root tissue. More recent studies on root gravitropism have also shown asymmetric accumulation of nitric oxide (NO) in response to gravity, and suggested the involvement of ethylene and NO signalling as well as auxin. Hence, this project aimed to investigate how NO, auxin and ethylene signalling interact in root growth, development and gravitropism. Arabidopsis mutants with defects in these hormonal signals were used in gravistimulation experiments. As expected, Col-0 (WT) plants displayed root bending 2 h after gravistimulation, but auxin mutants (aux1, axr2 and axr3) did not exhibit any root bending in response to gravistimulation. Roots of ethylene mutants showed reduced root bending compared to WT. Nitrate reductase mutants nia1 and nia2 also revealed reduced root bending, and nia1 showed slower bending than nia2. Exogenous application of the auxin NAA and the NO donor SNAP increased gravitropic bending. The application of the ethylene precursor ACC reduced root bending in the presence of NO, but increased bending in the absence of NO.The localization of NO in response to gravitropism was investigated using confocal microscopy. Gravitropism induced asymmetric accumulation of NO in WT in the lower side of the bending zone of roots, whereas auxin mutants aux1 and axr2 localised NO ubiquitously in the root. NIA1 (nitrate reductase 1) transcript levels in WT root tips were measured using qPCR. The NIA1 transcript starts to accumulate after gravistimulation, reaching a two fold higher level after 2 h, before gradually subsiding. The findings suggested that functional auxin signalling is a prerequisite for NO signalling, and that NIA1 mediated NO induces the root bending.To ascertain the sub-cellular localization of NIA1 in response to gravity and other hormonal interactions, NIA1 transcriptional and translational mGFP4 reporter constructs were made and transformed into the WT and auxin, ethylene and NR mutant plants. Integration of the reporter construct in the plant genome was confirmed by PCR and sequencing. Initial confocal experiments showed the successful expression of NIA1-driven mGFP4 fluorescence in roots and stomatal guard cells. Further experiments with these transgenic lines will be needed to examine the role of NO in gravitropism and cross-talk with other phytohormones