Expression of ACC oxidase genes in white clover (Trifolium repens L.) roots in response to phosphate supply : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Molecular Biology at Massey University, Palmerston North, New Zealand
The differential expression of members of the Trifolium repens ACC oxidase (TR-ACO) gene family and accumulation of TR-ACO proteins in white clover roots, and the temporal TR-ACO gene expression and TR-ACO protein accumulation in response to phosphate (Pi) stress has been investigated. Four-node stolon cuttings of wild type and transgenic white clover (designated TR-ACOp::GUS and TR-ACO1p::mGFP5-ER) plants were rooted and acclimatised in Hoagland’s solution, and then subjected to either a Pi sufficiency (1 mM Pi) treatment or a Pi depletion (10 µM Pi) treatment over a designated time course.
Using semi quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqRT-PCR) and gene-specific primers it has been determined that the TR-ACO genes are differentially expressed in the roots of white clover. The TR-ACO1 transcript abundance was greater in the lateral roots when compared to the main roots. By immunodetection analysis using antibodies raised against TR-ACO1, recognition of a protein of expected size (ca. 36 kDa) was also greater in the lateral roots. The tissue-specific localisation of TR-ACO1 promoter activity was investigated first by light microscopy using a single genetic line of white clover transformed with a TR-ACO1p::GUS gene construct, and results then confirmed by confocal microscopy using several genetically independent lines of transgenic plants transformed with a TR-ACO1p::mGFP5 ER gene construct. In these lines, the TR-ACO1 promoter activity was primarily located in the meristem of the main and lateral roots, lateral root primordia as well as in the pericycle of the root with nodes of expression in the emerging lateral roots, suggesting a role for ethylene in the development of young tissues where cells are actively dividing.
In terms of TR-ACO2, greater transcript abundance and protein accumulation of TR-ACO2 were also observed in the lateral roots when compared to the main roots. Histochemical GUS staining of roots of a single genetically-independent line transformed with a TR-ACO2p::GUS construct showed predominant promoter activity in the mature tissues of both the main and lateral roots but not in the meristematic tissues. In contrast, TR-ACO3 showed greater transcript abundance in the main roots relative to the lateral roots, and the promoter activity, as determined using a single genetically- independent line of TR-ACO3p::GUS transformed plants was predominantly in the mature tissues of the main roots
In response to Pi depletion, the members of TR-ACO gene family were temporally expressed in the white clover roots. Using sqRT-PCR, the TR-ACO1 transcript abundance was greater in Pi depleted roots at 12 h and 24 h after Pi depletion in both wild type plants and in the one genetically-independent line of white clover transformed with the TR-ACO1p::mGFP5-ER construct examined. Similarly, by western analysis using both a-TR-ACO1 and commercially available a-GFP antibodies (for the transformed line), a greater accumulation of proteins was consistently observed in Pi depleted roots from the first up to the seventh day after Pi depletion. By confocal microscopy, it was determined for several genetically-independent line of white clover transformed with TR-ACO1p::mGFP5-ER that under Pi depletion more intense GFP fluorescence over a time course of 1 d, 4 d, and 7 d was observed, when compared to plants grown under Pi sufficiency.
For TR-ACO2, there was no significant difference in transcript accumulation and protein accumulation in response to short term Pi depletion of up to seven days. However, at 15 d and 21 d after Pi depletion there was a greater protein accumulation in the roots of Pi depleted plants relative to the Pi sufficient roots. Further, when main and lateral roots were compared, a greater protein accumulation occurred in the lateral roots.
For TR-ACO3, there was no consistent trend of transcript accumulation in response to Pi depletion over a 24 h period. While a marked reduction in transcript accumulation was noted in Pi depleted roots at 1h, 12 h, 24 h, there was an increase in transcript accumulation at 6 h and 18 h after Pi depletion, indicating that factors other than Pi supply may be affecting gene regulation.
Root morphological studies revealed an increase in the main root length and lateral root production in white clover in response to Pi depletion with a greatest growth rate noted between the sixth and ninth day after Pi depletion, and this period overlapped with accumulation of TR-ACO1 protein suggesting a role for ethylene in the Pi stress induced lateral root production in white clover. The differential regulation of the three TR-ACO genes in white clover roots in response to Pi depletion further suggests the divergence in terms of regulation of the ethylene biosynthetic pathway, which may play an important role in fine tuning the responses of plants to particular environmental cues.