Browsing by Author "De Villiers, Ruan Morne"

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    Reverse genetic analysis of gene Pp1s148_40v6 in Physcomitrella patens : an AtMAX2 orthologue?
    (Stellenbosch : Stellenbosch University, 2015-04) De Villiers, Ruan Morne; Hills, Paul N.; Kossmann, Jens; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: The plant metabolite, strigolactone, has recently gained the status of phytohormone as the result of several studies that implicated its role in plant architecture. These studies would characteristically rely on the use of mutants, such as the rms lines that were generated in peas, that shared several characteristics. This method allowed for the identification of several genetic component of the shared pathway. It is now known that the biosynthesis of strigolactone is dependent on the sequential action of an isomerase (D27) and two carotenoid cleavage deoxygenases (CCD7 and CCD8). Furthermore, it is known that strigolactone perception is localised to the plant nucleus, where it interacts with an α/β-fold hydrolase (D14) which would concomitantly binds to target proteins. The F box protein (MAX2) is able to recognize this proteïen complex. Through a MAX2 dependent mechanism the target protein becomes tagged for proteolysis. However, this model, though intricate, has only really been shown in higher plants. The model bryophyte, Physcomitrella patens, serves as a useful tool in genetic studies due to its predisposition for homologous recombination. More recently it has also gained interest in studies pertaining to strigolactones, which has led to the generation of a Ppccd8Δ mutant. Compared to the wild type, the Ppccd8Δ line produces more protonemal tissue. Furthermore, exogenous strigolactones have also been shown to inhibit colony expansion. Here we shown that there is only a single candidate gene, PpMAX2, present in the P. patens genome that could serve as a homologue for the Arabidopsis thaliana MAX2. Furthermore, we show that a recombinant GFP:PpMAX2 localises to the nucleus of P. patens cells. A Ppmax2:: mutant was generated which, unexpectedly, did not show the phenotype of Ppccd8Δ. Ppmax2:: has an apparent inability to produce protonema and appears to rather dedicate its growth to the production of gametophores. A double mutant, Ppccd8Δ max2Δ was generated which also displayed the characteristic phenotype of Ppmax2::. It seems therefore that the activity of PpMAX2 is able to override that of PpCCD8. By employing a GUS reporter system, we showed that the promoter, PPpMAX2, is predominantly active within gametophore tissues. Taken together, these results suggest that the activity of PpMAX2 facilitates the transition of gametophore tissue to protonema tissue. Although exogenous strigolactones did not appear to affect the growth of the Ppmax2:: line as it did the PpWT or Ppccd8Δ lines, those responses that have been ascribed to strigolactones to date have mostly been observed in protonemal tissue. We therefore suspect that any strigolactone response that might have been elicited in Ppmax2:: would have been masked by its phenotype of predominantly protonemal tissue. We are therefore hesitant to make any sweeping statements in regards to the role PpMAX2 might have in strigolactone perception in P. patens. However, though we suspect that PpMAX2 might not be a true functional homologue for the characterised MAX2 homologues from higher plants, we suspect that it may well be the ancestral predecessor of MAX2.