Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Plants are constantly exposed to adverse environmental conditions including variations in
light intensity and the availability of water resources. These abiotic factors are expected to
worsen as the changing global climate places additional daily and seasonal demands on
plant growth and productivity. As plants are incapable of avoiding stress they have
developed a number of mechanisms to manage and adapt to the unfavourable conditions.
Carotenoids represent one of these mechanisms; with the xanthophylls (oxygenated
carotenes) playing an essential role in photoprotection following exposure to excess light
energy. They are also precursors to the plant hormone abscisic acid (ABA) which plays a
known role in stomatal regulation and thus drought tolerance. Carotenoids have been
identified as potential targets for genetic manipulation to meet the existing nutritional
demands (particularly vitamin A) and to enable plants to survive the climatic variations
predicted. Thorough investigations into the regulation and functioning of each carotenoid
biosynthetic gene in vivo as well as the roles of their encoded proteins are prerequisite.
Within the Grapevine Biotechnology Programme, a number of isoprenoid biosynthetic genes
have been isolated from Vitis vinifera L. cv. Pinotage. From this vast resource two genes
were chosen; namely a lycopene b-cyclase (b-LCY) and 9-cis epoxycarotenoid dioxygenase
(NCED) for detailed in planta analyses to address knowledge gaps in our current
understanding of carotenoid biosynthesis in general, its regulation and the roles of the two
target genes in these processes. Currently, the role of b-LCY within the chloroplasts is not
well known. Although the relationship between NCED overexpression, ABA levels, reduced
stomatal conductance and increased tolerance to water stress has been well-established,
comprehensive physiological analysis of the resulting mutants during conditions of both
water availability and shortage is not well documented. To assess their in planta role,
functional copies of both genes were isolated from Vitis vinifera (cv. Pinotage), characterised
and independently transformed into the genome of the model plant, Arabidopsis thaliana, in
the sense orientation under a constitutive promoter.
In order to investigate these pertinent scientific questions and thus to evaluate the
physiological role of each gene in vivo, a number of technologies were developed and/or
adopted. These included a high-performance liquid chromatography method for profiling the
major plant pigments in leaf tissue, a combination vapour phase extraction and electron
impact-gas chromatography/mass spectrometry method for the phytohormone profiling as
well as various physiological analyses including the use of chlorophyll a fluorescence to
assess the photosynthetic and non-photochemical quenching (NPQ) capacities of the plants.
Overexpression of grapevine b-LCY (Vvb-LCY) decreased lutein levels due to preferential
partitioning of lycopene into the b-branch. This decrease was not met by an increase in
either b-carotene or the xanthophyll cycle pigments implying that Vvb-LCY is not able to
regulate the flow of carbon through the pathway and provides additional evidence to the
fluidity of this pathway whereby pigment levels are continually balanced. The decreased
lutein levels observed under low light (LL) did not compromise the plants’ ability to induce
and maintain NPQ over a wide actinic light range. Vvb-LCY transgenics also had lower neoxanthin levels (and specifically the cis-isomer) under both LL and following exposure to
high light (HL), which could be correlated to an increase in malondialdehyde. Although not
corroborated, a novel and unexpected finding was an essential role for neoxanthin, and
potentially lutein, in preventing or at least reducing lipid peroxidation under HL stress. The
lower neoxanthin amounts may be due to silencing of the Arabidopsis b-LCY by the
Vvb-LCY, as the former may function as a NSY paralog as NSY is not encoded for in the
Arabidopsis genome. Clearly, this study has confirmed that Vvb-LCY partitions the carbon
flux between the a- and b-branches, however, the catalytic action of this enzyme is
dependent on the amount of substrate available and is thus not a regulatory step directing
the flux within the pathway. Enzyme kinetic and detailed transcriptional analyses would
confirm the above findings.
Overexpression of grapevine NCED1 (VvNCED1) increased ABA concentrations, delayed
seed germination and resulted in a slight to severe reduction in the overall plant growth rate.
NCED cleaves the 9-cis xanthophylls regulating ABA synthesis. However, contrary to
expectations, constitutive levels of this regulatory enzyme did not deplete the total and
individual chlorophylls and carotenoids in well-watered plants. Instead the VvNCED1
transgenics simply exhibited a lower chloroplastic pigment complement with no concomitant
effects on their photosynthetic capacity. Of particular interest, well-watered plants
overexpressing the VvNCED1 gene had an increased NPQ capacity of which the thermal
energy dissipation component (qE) was the most significant. It has been speculated that this
NPQ is associated with the phenotype conferred by VvNCED1 overexpression and occurs
independently of the xanthophyll cycle, and specifically zeaxanthin. This study confirmed
that VvNCED1 functions during drought tolerance via ABA regulation of stomatal
conductance. A detailed study was done to understand the plants’ response during water
deficit. Typically, decreases in total and individual carotenoids and the maximum efficiency
of photochemistry (Fv/Fm) as well as the relative water and soil moisture content were
recorded. No changes were recorded in salicylic acid (SA) levels, while indole acetic acid
(IAA) was positively correlated to ABA or vice versa. In contrast, the physiology of VvNCED1
overexpressing lines was largely unaffected, indicating that a reduced stomatal conductance
protects the plants against water stress.
This study has resulted in the isolation and characterisation of a carotenoid biosynthetic gene
(b-LCY) and an abscisic acid synthesising gene (NCED). Significant advancements in our
existing knowledge of the in planta role of both genes have been achieved. We have also
reaffirmed that strict regulatory control and fluidity exists within the carotenoid biosynthetic
pathway whereby individual pigment levels are constantly brought back into balance despite
constitutive expression of one of the pathway gene members. These analyses provide
valuable baseline information about individual genes which can be extended upon with other
omic technologies in order to comprehend the full complexity involved in carotenogenesis.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1081 |
| Date | 03 1900 |
| Creators | Taylor, Kerry Lyn |
| Contributors | Vivier, M. A., Smith, V. R., University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB) |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 9922342 bytes, application/pdf |
| Rights | University of Stellenbosch |
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