Variation in phosphorus supply alters nitrogen metabolism in the nodules and roots of Virgilia divaricata, a Cape fynbos indigenous legume from the Cape Floristic Region

Thesis (PhD)--Stellenbosch University, 2016.ENGLISH SUMMARY: This study determined how phosphorus (P) deficiency alters the nitrogen (N) metabolism in the root nodules of Virgilia divaricata (Adamson). The legume is indigenous to nutrient rich forest soils, but is also known to grow across a wide range habitats these including N and P poorer soils of the mature fynbos, a nutrient-poor ecosystem in the Cape Floristic Region of South Africa. Although this implies that the legume has a wide functional tolerance for variable soil N and P levels, it is not known how the plant utilizes inorganic N under variable P supply. This was evaluated in three separate experiments. The first experiment identified the bacterial species that nodulate V. divaricata and their biological N2 fixing (BNF) efficiency during P deficiency. In the experiment, we also integrated the plant C and N metabolism to the N product exported via xylem to the shoots for plant use. Plants were grown at sufficient and low P levels, with four concentrations of inorganic N supply (NH4NO3). At both levels of P, soil N supply reduced the reliance of legumes on BNF. Although the bacterial composition of nodules remained unchanged by P and N supply, the nodule function was greatly altered. In this regard, plants reliant on only N2 at both P levels had higher and more efficient BNF, which resulted in greater plant N. At high P, plants exported more amino acids relative to inorganic N and ureides in their xylem sap, whereas at low P the plants exported more ureides relative to amino acids and NH4. The bacterial tolerance for changes in P and N determined via nodule metabolites and xylem export might be a major factor that underpins the growth of V. divaricata under these variable soil conditions. The second experiment determined whether the P deficiency affects the metabolic status of roots and nodules, and the consequent impact on the routes of N assimilation. The findings show that V. divaricata had a reduced biomass, plant P concentration and BNF during P deficiency. P stressed nodules maintained their P status better than P stressed roots. Furthermore V. divaricata was able to alter C and N metabolism in different ways in roots and nodules, in response to P stress. For both roots and nodules, this was achieved via internal cycling of P, by possible replacement of membrane phospholipids with sulpholipids and galactolipids and increased reliance on the PPi-dependant metabolism of sucrose via UDPG and to Fru-6-P. P stressed roots exported mostly ureides as organic N and recycled amino acids via deamination glutamate dehydrogenase (GDH). In contrast, P stressed nodules largely exported amino acids. Compared to roots, the nodules showed a greater degree of P conservation during low P supply. The third experiment identified the Glutamate dehydrogenase (GDH) transcripts, their relative expressions and activity in P-stressed V. divaricata roots and nodules during N metabolism. GDH might contribute to the functional tolerance of V. divaricata to variable soil N and P levels in the mature fynbos by aminating N via aminating GDH and recycling amino acids via deaminating GDH. The analysis of the GDH cDNA sequences in V. divaricata revealed the presence of GHD 1 and GHD 2 subunits, these corresponding to the GDH1, GDH-B and GDH3 genes of legumes and non-legume plants. The relative expression of GDH1 and GDH2 genes was analysed in the roots and nodules, our results indicate that two subunits were differently regulated depending on the organ type and P supply. Although both transcripts appeared to be ubiquitously expressed in the roots and nodules, the GDH1 transcript evidently predominated over those of GDH2. Furthermore, the higher expression of both GDH transcripts in the nodules than roots in this study may play a role in the ability of nodules to regulate and conserve their internal P better than roots during P deficiency. With regards to GHD activity, both aminating and deaminating GDH activities were induced during P deficiency.AFRIKAANSE OPSOMMING: Hierdie studie bepaal hoe fosfor ( P) tekort die stikstof (N) metabolisme verander in die wortelknoppies van Virgilia divaricata . Die peulplant is inheems aan ryk bosbodems voedingstof, maar is ook bekend om die N en P armer gronde van die volwasse Fynbos, 'n voedingstof -arm ekosisteem in die Kaapse Floristiese Streek van Suid-Afrika binne te val . Hoewel dit impliseer dat die peulplant 'n wye funksionele verdraagsaamheid vir N en P vlakke het, is dit nie bekend hoe die plant anorganiese N onder wisselende P aanbod gebruik nie. Dit is geëvalueer in drie afsonderlike eksperimente. Die eerste eksperiment het die bakteriële spesies wat V. divaricata en hul biologiese N2 vaststelling (BNF) doeltreffendheid tydens P-tekort nodulate, geïdentifiseer. In die eksperiment, het ons ook die C en N metabolisme aan die N produk wat uitgevoer word via xileem na die lote, geintegreerd. Plante is gegroei op voldoende en lae P-vlakke, met vier konsentrasies van anorganiese N. Op beide vlakke van P, grond N toevoer verminder die afhanklikheid van peulplante op BNF. Hoewel die bakteriële samestelling van nodules onveranderd gebly deur P en N toevoer, was die nodule funksie grootliks verander. In hierdie verband, plante afhanklik net N2 by beide P vlakke het hoër en meer doeltreffende BNF, wat gelei het tot 'n groter aanleg N. Met hoë P, het plante meer aminosure relatief tot anorganiese N en ureides in hul xileemsap, terwyl by lae P voer die plante meer ureides relatief tot sure en NH4 amino. Die bakteriële verdraagsaamheid vir veranderinge in P en N via nodule metaboliete en xileem uitvoer kan 'n belangrike faktor onder hierdie veranderlike grondtoestande onderlê. Die tweede eksperiment het bepaal of P-tekort die metaboliese status van die wortels en wortelknoppies affektuur, en die impak op die roetes van N assimilasie. Tydens P- tekort, het V. divaricata plante 'n verminderde biomassa en P konsentrasie. P- tekort wortelknoppies handhaaf hul P status beter as P beklemtoon wortels. V. divaricata kan C en N metabolisme verander in verskillende maniere in reaksie op P stres. Vir beide wortels en wortelknoppies, bereik dit via interne meganisme van P, deur moontlike vervanging van membraanfosfolipiede met sulpholipids en galactolipids en verhoogde afhanklikheid van die PPI-afhanklike metabolisme van sukrose via UDPG en FRU-6-P. Die derde eksperiment het die GDH transkripsies, hul relatiewe uitdrukkings en aktiwiteit in P-beklemtoon wortels en wortelknoppies tydens N metabolisme geïdentifiseer. Glutamaat dehidrogenase (GDH, EG 1.4.2-4) kan bydra tot die funksionele toleransie van V. divaricata veranderlike grond N en P-vlakke in die volwasse fynbos. Die relatiewe uitdrukking van GDH1 en GDH2 gene is ontleed in die wortels en wortelknoppies, wys dat twee subeenhede gereguleer afhangende van die tipe orrel en P aanbod. Alhoewel beide transkripsies verskyn word, is die GDH1 transkipsie oorheers oor GDH2. Verder kan die uitdrukking van beide GDH transkripsies 'n rol speel in die vermoë van wortelknoppies interne P te reguleer. Met betrekking tot GHD aktiwiteit, is albei aminating en deaminating GDH aktiwiteite veroorsaak tydens P-tekort