Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006. / Plants have evolved photosynthetic systems to efficiently harvest sunlight energy for
the production of carbohydrates, but these systems also are extremely susceptible to
an excess of light. To combat the potential damaging effects of light, plants have
developed various mechanisms to control and cope with light stress. These
mechanisms include the movement of either leaves, cells (negative phototaxis) or
chloroplasts to adjust the light-capturing potential, the adjustment of the
light-harvesting antenna size through gene expression or protein degradation, the
removal of excess excitation energy either through an alternative electron transport
pathway or as heat. However, the latter mechanism based on thermal dissipation,
remains the most effective to rid the plant of damaging excess light energy. This
process involves several carotenoid pathway pigments, specifically the de-epoxidised
xanthophyll cycle pigments. The process and extent of thermal dissipation in plants
can be measured and quantified as non-photochemical quenching (NPQ) of
chlorophyll fluorescence by using well-established methodologies. Several
Arabidopsis and Chlamydomonas mutants affected in the xanthophyll cycle have
been isolated. These mutants have provided evidence for the correlation between
the de-epoxidised xanthophyll cycle pigments and NPQ as well as better
understanding of the operation of the xanthophyll cycle and the related carotenoid
biosynthetic enzymes. This key photoprotective role of the xanthophyll cycle is
therefore a promising target for genetic engineering to enhance environmental stress
tolerance in plants. Several genes from the carotenoid biosynthetic pathway of
grapevine (Vitis vinifera L.) were isolated previously in our laboratory. The main aim
of this study was to over-express two xanthophyll cycle genes from grapevine in
Arabidopsis and to analyse the transgenic population with regards to pigment content
and levels as well as certain photosynthetic parameters. The transgenic lines were
compared with wild type Arabidopsis (untransformed) plants and two xanthophyll
cycle mutants under non-limiting conditions as well as a stress condition, specifically
a high light treatment to induce possible photodamage and photoinhibition.
Transgenic Arabidopsis lines over-expressing the two V. vinifera xanthophyll
cycle genes, β-carotene hydroxylase (VvBCH) and zeaxanthin epoxidase (VvZEP),
were established following Agrobacterium transformation. In addition to the
untransformed wild type, two NPQ mutants, npq1 (lacking violaxanthin de-epoxidase)
and npq2 (lacking zeaxanthin epoxidase), were used as controls throughout this
study. The transgenic lines were propagated to a homozygous T3-generation, where
stable integration and expression of the transgenes were confirmed in only 16% and
12% for VvBCH and VvZEP lines, respectively. No phenotypical differences could be
observed for the transgenic lines compared to the wild type, but the npq2 mutant
showed a stunted and ‘wilty’ phenotype, as was previously described. To evaluate the pigment composition of the transgenic lines a reliable and
reproducible method was needed to analyse carotenoids from leafy material. To this
end a new high-performance liquid chromatography (HPLC) method was developed
for the quantitative profiling of eight major carotenoids and chlorophyll a and b.
Emphasis was placed on baseline separation of the xanthophyll pigments, lutein and
zeaxanthin as well as the cis- and trans-forms of violaxanthin and neoxanthin. The
method effectively distinguished Arabidopsis wild type plantlets from the two NPQ
mutant lines (npq1 and 2) and could possibly find application for green leafy tissue
samples in general.
The carotenoid content of the NPQ mutants were in accordance with previous
reports. The lack of zeaxanthin epoxidase activity in the npq2 mutant resulted in the
accumulation of zeaxanthin under both low and high light conditions. This high level
zeaxanthin was found to cause an initial rapid induction of NPQ at low to moderate
light intensities, but this difference disappeared at high light, where zeaxanthin
formation induced considerable NPQ in the wild type. Similarly, the npq1 mutant was
unable to de-epoxidise violaxanthin to zeaxanthin under high light conditions, which
resulted in severe inhibition of NPQ induction. Furthermore, these mutant plantlets
were shown to be more susceptible to photoinhibition compared to that of the wild
type.
The over-expression of VvBCH resulted in a marked increase in the
xanthophyll cycle pool pigments (violaxanthin, antheraxanthin and zeaxanthin) and
reduced β-carotene levels under both low and high light conditions compared to that
the wild type, indicating elevated β-carotene hydroxylase activity possibly due to
over-expression of the VvBCH gene. Similar to the induction of NPQ in the npq2
mutant, the increased levels of zeaxanthin in the VvBCH lines did not offer any
additional photoprotection. This would suggest that the heightened zeaxanthin levels
observed for the VvBCH lines do not necessarily enhance photoprotection, however
may protect the thylakoid membrane against lipid peroxidation as has been shown
previously. The VvZEP lines however, showed reduce levels of zeaxanthin in high
light conditions to that of the wild type, probably due to the competing epoxidation
and de-epoxidation reactions of the xanthophyll cycle. This reduction in zeaxanthin
synthesis in the VvZEP lines resulted in significant reduced NPQ induction compared
that of the wild type, a phenomenon also observed for the npq1 mutant. Similar to
the npq1 mutant, these lines displayed significantly increased photoinhibition, which
may be due to photodamage of the reaction centers if one considers the lowered
photosystem II photochemistry efficiency and reaction center openness of these lines
compared to the wild type. This may suggest that even small reductions in
zeaxanthin amounts can result in an increase in photoinhibition, under high light
conditions.
This study and its results provide fundamental information regarding two
grapevine-derived carotenoid pathway genes and their possible physiological roles.
Moreover, studies like these provide information that is essential when possible biotechnological approaches are planned with this central plant metabolic pathway in
mind. The results highlighted the complex regulation of this pathway, necessitating
attention to flux control, simultaneous manipulation of several pathway genes, and
the measurement of other compounds derived from this pathway when evaluating the
possible applications of the carotenoid pathway of plants.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1987 |
| Date | 03 1900 |
| Creators | Brackenridge, Anika Elma |
| Contributors | Vivier, M. A., Smith, V. R., University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 3409752 bytes, application/pdf |
| Rights | University of Stellenbosch |
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