Thesis (M.S. in botany)--Washington State University, May 2006. / Includes bibliographical references (p. 23-24).

Aging of ray parenchyma cells from the young sap-wood to recently formed heartwood was studied in single stems of Pinus banksiana Lamb., Picea mariana (Mill.) B.S.P., Abies balsamea (L.) Mill, and Populus tremuloides Michx. Season, radial location of cell within sapwood, and cell location vertically within a ray at a given radius were considered as factors which might influence the aging process. A 12 mm increment core was extracted at breast height, from the north aspect of a tree of each species in May and July for moisture content determination. Another set of cores from the south aspect of the same trees was collected in May, June, July, August, October, and November or December. These cores were used to investigate the physiological and cytological properties of living sapwood ray parenchyma cells. Qualitative and quantitative observations were made of the status of ray cells both with light and transmission electron microscopy in order to draw inferences concerning the sapwood/heartwood transformation from the aging of sapwood ray parenchyma cells. The sapwood moisture content of the three conifers studied was higher than that of heartwood, whereas in Populus tremuloides it was lower than that of heartwood. The sapwood moisture content in May was consistently greater than in July. Vitality of the sapwood ray parenchyma cells expressed by a new nuclear elongation index decreased from the outer sapwood towards the heartwood. The survival rate of the cells decreased curvilinearly from the middle sapwood towards the heartwood. At a given sapwood increment, a greater percentage of dead ray parenchyma cells was found among the marginal cells than among the central cells of a ray. No statistically significant difference was found between the vitality of the marginal and central cells, nor between any two contiguous sampling periods with exceptions in Pinus banksiana and Picea mariana between two contiguous sampling periods from July to December. No typical pattern for the distribution of lipid content was found. The pattern of starch distribution displayed significant species, radial, vertical and seasonal variation and showed two general patterns across the sapwood. Pattern A described a decreasing trend from the outer sapwood towards the inner sapwood. Pattern B was characterized by a relatively low starch content both in the outer as well as the inner sapwood. The starch content in Populus tremuloides and the lipid content in Pinus banksiana and Picea mariana displayed no statistically significant difference between marginal and central ray cells. The majority of ray parenchyma cells showed a statistically significant difference between two contiguous sampling periods in starch and lipid contents. There was no inverse relationship between the starch and lipid content over the growing season studied. Young ray parenchyma cells were rich in chromatin and cytoplasm which contained numerous cell organelles. These cells were characterized by amyloplasts which possessed one or more elongated starch granules with thylakoids and osmiophilic globuli, numerous small lipid droplets and mostly rod-like mitochondria. In contrast, aged ray parenchyma cells featured an aggregated, dense nucleus and cytoplasm which contained few cell organelles. These aged cells possessed enlarged swollen starch granules, large lipid droplets or lumps with two staining densities, round shaped mitochondria with inconspicuous cristae and a rough/broken plasmalemma. Some heartwood substances originated from the lipid lumps which appeared frequently in dying ray cells. Based on microscopic observations and measurements of the loss of vitality of ray parenchyma cells, a declining survival rate, the disintegration of cell organelles and the origin of heartwood substances from lipid lumps, it can be concluded that heartwood formation is largely associated with the death of sapwood ray parenchyma cells. The death of these cells is due to the passage of time. / Forestry, Faculty of / Graduate

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. While to date the maize ubiquitin (Ubi1) promoter has been the most effective transgene promoter for sugarcane, there is a high demand for tissue and stage specific promoters for localised transgene expression in the mature culm. The present study sought to characterise genes preferentially expressed in the core and peripheral tissues of the mature culm, which can further be used as research tools for specific promoter isolation. cDNA expression arrays containing 3840 clones from a late stage cDNA library representative of the core and peripheral tissues of the mature culm were prepared. The cDNA expression arrays were then differentially screened in independent hybridisation experiments with radioactively-labeled cDNA representations of core and peripheral tissues of internode 7, and peripheral tissues of internode 10. Comparison of the expression profiles of the arrayed cDNA targets in the three probes led to the identification of 60 tissue-specific, 17 stage-specific and 50 selectively expressed cDNAs within the mature sugarcane culm. ~ESTs of 33 chosen selectively expressed cDNAs with a relatively stronger pattern of expression in the core than in the peripheral tissues revealed sequence homology to a diverse collection of genes in the mature culm. These included genes associated with general cellular metabolism such as protein synthesis, protein modification and structural protein. Also identified were stress-responsive genes. The putative translational products of some of these clones had homologs that are involved in cell-wall structure in other species. These included the [acalin homolog, a lectin, hydroxyproline rich glycoprotein and structured polyprotein C. Many of the cDNAs thought to be involved in cell wall structure or stress related responses also accumulate in a developmental manner in other plants. These may indicate that specific mature culm mRNAs accumulate in response to stresses such as rapid cell expansion or as part of the late developmental program. An unexpected observation was that only one gene associated with sucrose metabolism was identified, namely sucrose synthase. These results confirmed that culm maturation was not controlled by sucrose metabolism despite its distinct physiological characteristic of storing high levels of sugars. ESTs analysis further revealed that sequence homology was not obtained for all the cDNAs exhibiting stage and tissue specific expression in the core and peripheral tissues of the mature culm. These could represent novel genes not only from sugarcane but all plants. Northern analysis demonstrated that 9 putatively identified selectively expressed genes tested so far accumulated specifically in the core and peripheral tissues of the mature culm. No expression was detected in root, leaf, leafroll and internode 3. However, their selective expression in a single internode as observed on the arrays (i.e hybridisation signal intensity being higher in the core than in the peripheral tissue) was not detected on the northern blots. These showed that cDNA expression arrays were not a highcapacity gene expression assay since they were prone to false expression analysis. The validity of results obtained through array screening should always be verified in an independent manner, preferably by the northern hybridisation analysis. Hence, the present study shows that the combination of differential screening, northern blot and DNA sequence analysis permits the rapid characterisation of differentially expressed genes in the core and peripheral tissues of the mature sugarcane culm. These can further be used as research tools for mature culm - specific promoter isolation in the sugarcane. / AFRIKAANSE OPSOMMING: Die doeltreffende uitdrukking van transgene in plantselle is afhanklik van 'n gepaste promotorvolgorde wat stroomop van die geen ingevoeg word. Die Ubi1-promotor van mielies was tot dusver die doeltreffendste transgeenpromotor in suikerriet, maar daar is 'n groot behoefte aan promotors wat weefsel- en ontwikkelingstadium-spesifieke geenuitdrukking kan beheer. Hierdie studie het op die isolering en karakterisering van gene wat selektief in die kern- of periferale stingelweefsel van suikerriet uitgedruk word, gefokus. Hierdie gene sal verder benut kan word om promotors te isoleer. eDNA uitdrukkingsreekse ("expression arrays") van 'n volwasse stingel eDNA biblioteek is voorberei. Hierdie reekse, wat 3840 klone bevat het, is in onafhanklike hibridiseringseksperimente met radioaktiefgemerkte eDNA van onderskeidelik kern- en periferale stingelweefsel van lit 7 en periferale stingelweefsel van lit 10 afgetas. 'n Vergelyking van die uitdrukkingsprofiele van die eDNA teikens in dié drie peilergroepe het tot die identifisering van 60 weefsel-spesifieke-, 17 ontwikkelingstadium-spesifiekeen 50 selektief uitgedrukte eDNAs in die volwasse suikerrietstingel gelei. Uitdrukkingsvolgordemerkers ("ESTs") van 33 geselekteerde eDNAs wat in hoër vlakke in die kern uitgedruk is, se volgordes toon homologie aan 'n wye verskeidenheid gene in die volwasse stingel. Hierdie groep sluit gene in wat met algemene sellulêre ..metabolisme soos proteïensintese, proteïenmodifisering en strukturele proteïene geassosieer is. Spanningsverwante gene is ook hier geïdentifiseer. Die transleringsprodukte van sommige klone het homoloë wat by selwandstruktuur in ander spesies betrokke is, soos die jaealin-homoloog, 'n lektien, hidroksiprolien-ryke glikoproteïen en gestruktureerde poliproteïen C. 'n Wye verskeidenheid eDNAs wat by selwandstruktuur of spanningsverwante reaksies betrokke is, akkumuleer ook in 'n ontwikkelingsafhanklike wyse in ander plante. Dit mag 'n aanduiding wees dat spesifieke mRNAs in die volwasse stingel in reaksie op spanning wat met vinnige seluitsetting gepaardgaan, versamel. Slegs een geen wat met sukrose metabolisme geassosieer is, nl. sukrosesintase, is in hierdie studie geïdentifiseer. Hierdie onverwagte waarneming het bevestig dat, ondanks suikerriet se kenmerkende vermoë om hoë konsentrasies suiker te berg, stingelveroudering nie net met sukrose metabolisme geassosieer kan word nie. Nie al die eDNA-fragmente wat geïsoleer is, het homologie aan ander gene in die internasionale databasisse getoon nie, wat moontlik kan aandui dat nuwe gene suksesvol geïsoleer is. Nege ontwikkelingstadium-spesifieke gene wat slegs in die volwasse stingelweefsels uitgedruk word, is dmv noordelike oordraganalises geïdentifiseer. Geen transkripte van hierdie gene is in die wortels, blaarrol, blare of jong stingel waargeneem nie. Die weefselspesifisiteit wat met die uitdrukkingsreekse waargeneem is, kon nie mbv noordelike orrdraganalises bevestig word nie. Dit mag 'n aanduiding wees dat die uitdrukkingsreekse vals positiewe resultate kan oplewer en dit is raadsaam om voortaan altyd die verkrygde profiele met ander, meer sensitiewe tegnieke, te bevestig. Die studie het aangetoon dat 'n kombinasie van differensiële aftasting, noordelike oordraganalise en DNA-volgordebepaling gebruik kan word om gene wat differensieel uitgedruk word in die volwasse suikerrietstingel, te identifiseer. Hierdie geenfragmente kan nou vir promotorisoleringsdoeleindes aangewend word.