The development of phosphate stress and its functional consequences in the model legume plant Medicago truncatula

Thesis (MSc)--Stellenbosch University, 2016.ENGLISH ABSTRACT: Phosphate is an abundant nutrient in the soil; however it is mostly bound to other elements that make phosphate unavailable for plant uptake. This bound state makes phosphate the second most limiting nutrient for plant growth. Phosphate is also a nonrenewable mined resource that forms a major constituent of fertiliser given to crops grown in nutrient poor soils. The second most important crop family in agriculture is Leguminosae. In an attempt to to reduse possible nitrogen stress, legumes can form a symbiosis with nitrogen-fixing soil bacteria. This symbiosis, found in the nodules, exchanges fixed nitrogen with host photosynthate and phosphate. The nodules are thus a phosphate sink that place stress on the rest of the plant. Legumes have adapted different ways to optimise the limited available phosphate to continue their own growth while maintaining the adenosine-triphosphate expensive nitrogen-fixing reaction. In this study, we looked at how the genetic model legume, Medicago truncatula Gaertner, has adapted to phosphate stressed conditions as it relied solely on biological nitrogen fixation as a source of nitrogen. In the first treatment, Medicago truncatula seedlings were infected with Sinorhizobium meliloti and received a low concentration of phosphate throughout the growth period. This was done to simulate Medicago truncatula growing in already phosphate deprived soils. The comparisons of biomass and growth, internal free phosphate concentrations, and organic acid and acid phosphatases enzyme activities were done on the above versus below ground tissues. Photosynthesis parameters were also recorded. Above ground tissues responded to phosphate stress with increased activity of bypass enzymes at the steps that required adenosine-triphosphate. While the below ground tissues focused on using acid phosphatases to recycle phosphate. Although the rate of photosynthesis had decreased in the phosphate stressed plants, the efficiency of photosynthesis with the phosphate that was available in the leaves had increased. The second treatment involved the growth of nodulated Medicago truncatula with an optimal phosphate concentration, followed by an induced phosphate stress period. In this manner, soil that had been depleted of phosphate during plant growth was simulated. With the addition of determining differences in activities of nitrogen assimilating enzymes, the above-mentioned comparisons were made on the nodules and roots of the sample plants. Under the induced stress condition, available phosphate was concentrated to the nodules. A possible cause for this was the increase in activity of the organic acid synthesising enzymes present in the nodule. The nitrogen assimilating enzyme activities indicated that stressed nodules may export glutamine rather than asparagine to the roots. Root nitrogen assimilating enzyme activities remained relatively constant during phosphate stress. Reduced nitrogen and carbon content of stressed plants indicated that phosphate had a direct impact on nitrogen fixation. From this study, we deduced that above ground tissues adapted metabolically for improved photosynthesis phosphate use efficiency; while below ground tissues recycle the available phosphate to be used for nitrogen-fixation. After the induction of phosphate stress it was found that the nodules relied on saving available phosphate for nitrogenfixation, while the roots recyled assimilated glutamine to maintain function.AFRIKAANS OPSOMMING: Fosfaat is volop in die grond, maar dit is gebind aan ander elemente soos metale wat die plante hinder om dit op te neem. Hierdie voedingstof is die tweede mees beperkende voedingstof vir die groei van plante. Fosfaat is ‘n gemynde nie-hernubare hulpbron wat ‘n groot deel beslaan van kunsmis. Hiedie hulpbron word veral gebruik in kunsmis wat toegedien word aan grond met lae konsentrasies van voedingstowwe. Peulplante is die tweede mees belangrikste landbou gewas ter wêreld. Om moontlike sitkstofstres te bekamp, vorm peulplante ‘n simbiose met grond bakterieë om wortelknoppies te vorm. Binne die wortelknoppies ruil die bakterieë stikstof, wat hul fikseer vanaf die atmosfeer, vir fosfaat en produkte van fotosintese vanaf die gasheer plant. Die aanwensel van wortelknoppies het ‘n groot aanvaag na fosfaat wat stres plaas op die res van die gasheer plant. Peulplante het in verskeie maniere aangepas tot beperkte fosfaat kondisies om sodoende te kan oorleef en die onkoste van stikstoffiskering te kan handhaaf. Vir hierdie studie het ons gekyk hoe die genetiese model peulplant, Medicago truncatula Gaertner, aangepas is vir fosfaatstres wanneer dit uitsluitlik op biologiese stikstoffiksering moet staatmaak as stikstofbron. Tydens die eerste behandeling was Medicago truncatula saailinge geïnokuleer met Sinorhizobium meliloti en het regdeur hul groeiperiode slegs ‘n lae konsentrasie fosfaat ontvang. In hierdie manier was Medicago truncatula wat in fosfaat arme grond groei gesimuleer. Vergelykings tuseen biomassa en groei; interne fosfaat, koolstof en stikstof konsentrasies; organiese suur produserende en suurfosfatases ensiemaktiwiteit was bepaal op bo- en ondergrondse weefsel. Fotosintese lesings was ook vergelyk tussen plante wat onder fosfaatstres gegroei is teenoor die wat met optimale omstandighede volwassenheid bereik het. Hieruit het ons gevind dat die bogrondse weefsels reageer op fosfaatstres deur aktiwiteit van ensieme, wat reakies wat adenosine trifosfaat vereis vebysteek, te verhoog. Terwyl die ondergrondese weefsels fokus op die herwinning van fosfaat deur die gebruik van suur fofatases. Alhoewel die tempo van fotosintese tydens die fosfaatstres afgeneem het, het die doeltreffendheid van fotosintese tenopsigte van die beskikbare fosfaat aansienlik toegeneem. Tydens the tweede behandeling was geïnokuleerde Medicago truncatula tot volwassenheid gegroei met optimale kondisies en daarna van fosfaat onttrek. Op hierdie wyse is grond wat ‘n verlaging van fosfaat vlakke tydens groei gesimuleer. Die bogenoemde vergelykings was gemaak met die wortelkonoopies en wortels van hierdie plante. Die analiese van die verskille in aktiwiteit van stikstofverwerkendsensieme was bygevoeg tot die analieses van die eerste behandelingseksperiment. Tydens die geïnduseerde stres toestand, word vrye fosfaat gekonsentreer vanaf die wortels na die wortelknoppies. Verhoogde aktiwiteit van ensieme wat die energie benodigende stappe verbysteek in die wortelknoppies help met die besparing van fosfaat vir die optimale werking van die stikstoffikserings- en verwerkingsensieme. Die verskuiwing in aktiwiteit van die stikstofverwerkende ensieme dui daarop dat die wortelknoppies tydens fosfaatstres eerder glutamien as asparagien uitvoer na die wortels. Die wortel stikstofverwerkingsensieme het relatief constant gebly tydens die fosfaatstresperiode en mag daarna toe lei dat die wortels glutamine herwin. Die verminderde koolstof en stikstof inhoud van die plante tydens stress het dus ‘n direkte invloed op stikstoffiksering. Vanuit hierdie studie kon ons aflei dat die metabolisme van plant weefsel wat bo die grond gevind word aangepas word om fosfaat te bewaar vir verhoogde fosfaat gebruik tydens fotosintese; terwyl ondergrondse weefsel die fosfaat herwin vir stikstof-fiksering. Wanneer daar gekyk was na die ondergrondse weefsels na ‘n geïnduseerde fosfaat stres periode, was daar gevind dat die wortelknoppies fosfaat bewaar; terwyl die wortels die geassimileerde glutamien herwin om sodoende hul funksie te kan onderhou.National Research Foundation (NRF)Master