Reverse genetic analysis of gene Pp1s148_40v6 in Physcomitrella patens : an AtMAX2 orthologue?

De Villiers, Ruan Morne (2015-04)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

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.

AFRIKAANSE OPSOMMING: Strigolaktoon is ‘n metaboliet wat deur plante vervaardig word en is redelik onlangs as ’n fitohormoon geklassifiseer. Die klassifikasie as fitohormoon is die gevolg van verskeie studies wat strigolaktoon se rol in die plantstruktuur beklemtoon het. In hierdie studie is daar gebruik gemaak van mutante, soos onderandere die rms lyne, wat gegenereer is in ertjies, wat verskeie kenmerke deel. Sodoende is verskeie komponente van ’n gedeelde molekulêre padweg geïdentifiseer. Daar word tans verstaan dat die sintese van strigolaktoon afhanklik is van die stapsgewyse aksies van ’n isomerase (D27) en twee karotenoïedklewingsdeoksigenases (CCD7 en CCD8). Verder is dit bekend dat strigolaktoon waargeneem word in die plant nukleus deur te assosieer met ’n α/β-vou-hidrolase (D14) wat vervolgens met teikenproteïene bind. Die kompleks word deur ’n F-boks proteïen (MAX2) herken wat daartoe lei dat die teikenproteïen gemerk word vir proteolise; altans, dit is tans die model wat vir hoër plante aanvaar word. Die model briofiet, Physcomitrella patens, word dikwels aangewend in genetiese studies weens dit ’n hoër vatbaarheid vir homoloë rekombinasie het. Om P. patens te benut in navorsing wat die rol van strigolaktoon ondersoek is ook voordelig, aangesien daar reeds ’n Ppccd8Δ mutant beskikbaar is. In vergelyking met die wilde tipe, produseer Ppccd8Δ meer protonemale weefsel en blyk dit dat strigolaktoon die vermoë het om kolonie verspreiding te bekamp. Hier wys ons dat daar ’n enkele kandidaat geen, PpMAX2, in die genoom van die P. patens teenwoordig is wat as ’n homoloog vir die Arabidopsis thaliana MAX2 kan dien. Verder wys ons dat ’n rekombinante GFP:PpMAX2 proteïen wel na die selkern van P. patens selle lokaliseer. ’n Ppmax2:: mutant is gegenereer wat, onverwags, nie die fenotipe van Ppccd8Δ vertoon het nie. Ppmax2:: het ’n onvermoë om protonema te produseer en wy groei eerder aan die produksie van gametofiete. ’n Dubbele mutant, Ppccd8Δ max2Δ, is gegenereer wat ook die fenotipe van Ppmax2:: vertoon het; dus kom ons tot die gevolgtrekking dat die aktiwiteit van PpMAX2 dié van PpCCD8 oorheers. Deur gebruik te maak van ’n GUS verklikkersisteem kon ons aflei dat die aktiwiteit van die PPpMAX2 promotor hoofsaaklik tot die uitdrukking van PpMAX2 in gametofiet weefsel lei. Dit is moontlik dat die aktiwiteit PpMAX2 dus die oorgang van gametofoor weefsel na protonema weefsel te weg bring. Alhoewel strigolaktoon nie die groei van die Ppmax2:: lyn beïnvloed soos vir die PpWT of Ppccd8Δ lyne nie, vermoed ons dat die reaksie slegs in die protonemale weefsel waargeneem sal word. Daar kan tans nie met absolute sekerheid gesê word of PpMAX2 enigsins verbonde met strigolaktoon persepsie in mos is nie, tog vermoed ons dat PpMAX2 ’n primitiewe voorloper vir die gekarakteriseerde MAX2 homoloë van die hoër plante is.

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