Stomatal density profiling in Vitis vinifera L. using non-destructive field microscopy

Venter, T. L. (2015-12)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: When plants began colonising land ca. 400 million years ago, they needed to adapt structurally to limit water loss from aerial parts. Thus, a waxy cuticle developed on these parts, particularly the leaves, in order to prevent transpiration from these surfaces. This layer is, however, impermeable to carbon dioxide (CO2) which is required as a substrate in photosynthesis. Therefore, the stomata evolved to allow for gaseous exchange to take place. The main function of stomata is to ensure that the amount of CO2 taken up by the plant is balanced with the amount of water available to it. Stomatal development and function has been studied extensively, but few of these studies have been done on Vitis vinifera L. Since the stomatal development process is complex and carefully guided, a lot of these past studies were conducted on the model plant Arabidopsis thaliana in order to attempt to ascertain which genes may be involved in this process, and how. Environmental stimuli have been found to affect both stomatal development and function. These effects can be short-term, in which case stomata will respond to a momentary change in conditions by opening or closing the stomatal pore (change in stomatal function), or long-term, by which the response is more permanent and affects stomatal density and/or size (change in stomatal development). Such factors which have been investigated include CO2 levels, relative humidity, both light quantity and quality, as well as limited water availability. It has been found that changes in response to water-stress are brought about by the increased production of the plant hormone, abscisic acid, in the roots, which is then transported to the leaves in the transpiration stream. For Vitis vinifera L. the effect of light (quantity and quality), CO2 concentration and water-stress on stomatal development and function have been investigated by other researchers. Various methods are used in stomatal research. The most common methods are light and scanning electron microscopy. These methods are both destructive and make use of intact leaf tissue, or epidermal peels and impressions. In this study an adapted microscopy technique was used in order to test whether it would be suitable for conducting stomatal investigations non-destructively over a period of time. Four Vitis vinifera L. cultivars were selected for this study and in-field stomatal investigations were carried out over the period between bunch closure and post-véraison. A portable digital microscope was used to capture images and these were then digitally analysed. The aim was to investigate whether stomatal density differs between cultivars, leaves of a single plant as well as between different positions on a single leaf. In general there were differences found between cultivars, but not all the differences were significant. Younger leaves displayed a higher stomatal density than more mature leaves and the degree of this also varied between cultivars. Little differences were noted over time and between on-leaf positions. The method was successful in conducting the relevant investigations, but it was not without problems and shortcomings. The resolution of the images produced was not sufficient to allow for the calculation of stomatal index and size, but stomatal density could be determined reliably. With the rate at which new technology becomes available, these issues may be minimised or eliminated in the near future, and the application of this method to stomatal investigations expanded.

AFRIKAANSE OPSOMMING: Sedert plante sowat 400 miljoen jaar gelede die aarde se oppervlak begin bedek het, moes hulle struktureel aanpas om die waterverlies vanaf bo-grondse dele te verminder. Sodoende het die waslaag wat op sulke plantdele, veral die blare, voorkom, ontstaan. Hierdie laag is egter ook nie deurlaatbaar vir koolsuurgas (CO2) nie, wat benodig word vir fotosintese. Huidmondjies het dus ontwikkel om die gas-uitruilingsproses moontlik te maak. Die hoofdoel van die huidmondjies is om die hoeveelheid CO2 wat deur die plant opgeneem word, met die hoeveelheid water beskikbaar tot die plant, te balanseer. Daar is reeds baie navorsing gedoen oor huidmondjie-ontwikkeling en -funksie, maar min studies is spesifiek op Vitis vinifera L. gedoen. Aangesien die ontwikkelingsproses van huidmondjies baie kompleks is en noukeurig gereguleer word, is vele studies op die modelplant, Arabidopsis thaliana, uitgevoer in ‘n poging om te probeer uitvind watter gene moontlik die proses beheer, sowel as die manier waarop hierdie regulering bewerkstellig word. Daar is bevind dat beide ontwikkeling en funksie van huidmondjies deur omgewingsfaktore beïnvloed word. Hierdie veranderings kan óf oor die korttermyn geskied deur die opening, of sluiting van die huidmondjies (‘n aanpassing in huidmondjiefunksie), óf op ‘n langtermyn basis deur ‘n verandering in huidmondjiedigtheid en/of -grootte (‘n aanpassing met betrekking tot huidmondjie-ontwikkeling). Laasgenoemde is ‘n meer permanente aanpassing. Sulke omgewingsfaktore wat al in studies gemonitor is sluit in CO2-vlakke, relatiewe humiditeit, ligkwantiteit en -kwaliteit, sowel as watertekort-toestande. Daar is gevind dat laasgenoemde ‘n verandering in huidmondjies teweegbring deur middel van die verhoogde produksie van absisiensuur in die wortels. Hierdie hormoon word dan in die transpirasiestroom na die blare toe vervoer waar die effek bewerkstellig word. Die effek van ligkwaliteit en -kwantiteit, sowel as CO2-vakke en watertekort-toestande op huidmondjie-ontwikkeling en -funksie is al vir Vitis vinifera L. nagevors. Verskeie metodes word in die navorsing van huidmondjies gebruik, waarvan ligmikroskopie en skandeer-elektronmikroskopie die mees algemeen is. Beide hierdie metodes is destruktief en maak gebruik van blaarweefsel, epidermale afdrukke of afgeskilde lagies. In hierdie studie is ‘n aangepaste mikroskopiese metode gebruik om vas te stel of dit suksesvol toegepas kan word om nie-destruktiewe waarnemings van huidmondjies oor ‘n tydperk te kan uitvoer. Vier Vitis vinifera L. kultivars is vir die studie gebruik en metings is oor die tydperk vanaf trossluiting tot na deurslaan gedoen. ‘n Draagbare digitale mikroskoop is gebruik om beelde te neem wat later digitaal geanaliseer kon word. Die doel was om vas te stel of huidmondjiedigtheid tussen kultivars verskil, so wel as om te bepaal of daar variasies hiervan tussen verkillende blare op ‘n enkele plant en ook oor posisies op ‘n enkele blaar is. In die algemeen het kultivars van mekaar verskil, maar die verskille was nie almal beduidend nie. Jonger blare het ‘n hoër huidmondjiedigtheid getoon as die meer volwasse blare. Daar was nie veel variasie in huidmondjiedigtheid oor tyd, of tussen die verskillende posisies op die blare nie. Die metode kon suksesvol toegepas word om die beoogde waarnemings te maak, maar daar was tog probleme en tekortkominge. Die resolusie van die beelde wat verkry is was nie hoog genoeg om die bepaling van huidmondjie-grootte en -indeks moontlik te maak nie, maar huidmondjiedigtheid kon effektief bepaal word. Gegewe die tempo waarteen nuwe tegnologie ontwikkel, kan dit moontlik wees om hierdie probleem in die nabye toekoms aan te spreek. Die toepassing van hierdie metode vir die navorsing van huidmondjies mag dan sodoende uitgebrei word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98139
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