Abstract
Plant plasma membrane H+-ATPases, such as LHA2, are primary active transporters that play a role in many physiological processes including maintenance of intra- and extracellular pH and cellular expansion. In order to gain a better understanding of plant growth and development this study focused on plant primary and lateral roots specifically looking at two areas of growth that the plasma membrane H+-ATPases effect, the Acid Growth Theory and lateral root initiation. The Acid Growth Theory suggests that the activation of plasma membrane H+-ATPases by auxin causes acidification of the apoplast, which causes loosening of the cell wall allowing turgor driven cell expansion. The other issue dealt with was lateral root initiation; currently there is no definitive early signal that triggers lateral roots. This study supports the hypothesis that activation of the H+-ATPase would cause both cell expansion according to the Acid Growth Theory and would be an early signal in lateral root initiation. To observe adequately the plasma membrane H+-ATPases, the development of two additional constitutively active overexpression lines were studied with two existing overexpression lines in Arabidopsis thaliana. These plant lines insured permanent activation by means of artificial truncation of the autoinhibitory domain and overexpression by coupling the LHA2 gene with the 32S promoter of the cauliflower mosaic virus. Results indicated that there was expression of the LHA2 transgene in Arabidopsis confirmed by RT-qPCR and that expression of the construct produced a fully functional H+ pump protein verified by fluorescent extracellular acidification activity assays using the pH sensitive fluorescent dye, dextran Oregon Green. The extracellular pH assay revealed that all overexpression lines had significantly lower extracellular pH (p<0.05), LHA2-2C at pH 4.73, LHA2-3C at pH 4.61, LHA2-6H at pH 4.44 and LHA2-12K at pH 4.43, compared to the wild-type control at pH 5.01. Phenotypic root results showed significant increases in primary root growth of all single copy homozygous overexpression lines (p<0.01), LHA2-2C, LHA2-3C, LHA2-6H and LHA2-12K with mean growth of 46.6 mm, 49.0 mm, 45.5 mm and 42.3 mm respectively compared to wild-type root growth at 39.2 mm in the 8 day primary root growth experiment. The 8 day lateral root density experiment showed increased densities of lateral roots in 3 of the 4 single copy homozygous overexpression lines (p<0.01). Mean root numbers of LHA2-2C at 1.39 lateral roots/cm (LR/cm), LHA2-3C at 1.49 LR/cm and LHA2-6H with 1.30 LR/cm, when compared to wild-type root at 1.16 LR/cm grown on M/S agar plates in our root growth assays. The wild-type and overexpression lines were also grown with the auxin transport regulators, TIBA and NPA in order to separate the effects of polar auxin flow and endogenous H+ pump activity. These results showed that in the absence of polar auxin flow and endogenous pump activity, effects of the transgenic H+ pumps in the overexpression lines were able to retain growth in primary root length for 3 of the 4 overexpression lines, LHA2-3C, LHA2-6H and LHA2-12K. They also increased retention of lateral root densities in all of the overexpression lines when growing the plant lines on the auxin transport inhibitor, NPA and normalized to the DMSO vehicle control data. Results for plants grown on TIBA and normalized to the DMSO vehicle control, indicated that there were greater retention in lateral root density of the overexpression lines compared to the wild-type but decreased primary root retention which was contrary to our original hypothesis. To date, this study provides some of the most direct evidence that the function of the H+ pump is directly involved with acidification of the apoplast and cell expansion as stated by the Acid Growth Theory. This study also strengthens the hypothesis that the activity of the H+ pump by auxin activation creates transitory cytoplasmic alkalinization events, which may be required as one of the earliest signals to trigger the initiation of lateral roots in plants.