Author: Customer
Company: ARO

Abstract
The primary group of pigments in plants, comprised of hundred of components, is the
anthocyanin group. Anthocyanins accumulate in the epidermal cell vacuoles in flower
petals and also in other plant organs, such as the stem, leaves and fruit. They are
responsible for colors ranging from pink and red to blue. The stability of these
pigments depends on microenvironmental conditions in the vacuole, such as metal
and flavonoid concentration and pH conditions.
The biosynthetic course of the anthocyanins as part of the phenylpropanoid pathway
has been thoroughly researched and characterized in several model plants, such as
Arabidopsis, petunia and maize sprouts. Contrary to the detailed molecular
information available of the synthesis, there is very little information on the stability
and catabolism of these pigments in plant tissue. Despite this, it is clear that a
degradation process does occur in plant anthocyanins and it is reasonable to assume
that this breakdown process significantly influences the color of agricultural products.
Research conducted on the litchi fruit has shown that there is a breakdown of
anthocyanins in the husk of the fruit during storage. There has also been research
proving an enzymatic breakdown of anthocyanins in blueberry juice. In another study
conducted recently on vines, they show a breakdown of marked anthocyanins
following exposure to high temperatures.
In our laboratory, we have recently characterized an active degradation process of
anthocyanins and proven that there is a need to create new proteins in order to break
down color. As far as we know, this study is the first to describe active degradation, in
planta, of anthocyanins. The Brunfelsia flowers serve as an excellent subject for study
on anthocyanin degradation for several reasons: (1) The flowers undergo a dramatic
and rapid color change from dark purple on the day of their unfurling and then stark
white two days later. This change is the result of decreased levels of pigments in the
tissue (2) This process of anthocyanin breakdown occurs even when the flowers are
detached from the plant on the day of their unfurling and dipped in a sucrose solution
(3) Belonging to the Solanaceae Family, studying Brunfelsia allows us to characterize the proteins or genes by comparing them to other model plants from this family, such as the tomato and tobacco.
Based on studies conducted on wine and fruit juices, likely candidates for enzymatic
anthocyanin degradation are peroxidases. This group of enzymes is usually active in
most cell organelles, including the cell vacuoles. In a preliminary study in our lab, we
saw a clear correlation between general peroxidase activity and anthocyanin
degradation in Brunfelsia. In this study we examined the involvement of isoenzymes
from the peroxidase family in active anthocyanin degradation in the Brunfelsia
flower, and we developed transformation and regeneration systems that will serve to
examine enzymatic activity of these enzymes in the Brunfelsia plant.
There are several isoenzymes of the peroxidase family active in the Brunfelsia flower
and their level of activity varies with the development of the flower and the fading of
its hue. On seeing enzymatic activity in the various plant organs we observed
differential activity of the peroxidase family isoenzymes. The active isoenzymes in
the flower barely appear in the other plant organs. The activity of two isoenzymes was
observed in the mature leaf and to a much lesser degree in a young leaf.
We observed that the activity of several peroxidase isoenzymes increased in
correlation to the pigment degradation. Isolation of those enzymes was conducted by
running soluble protein extracts from the Brunfelsia flower on an Ion exchange and
gel filtration columns. One protein with peroxidase activity was isolated and sent for
LC-MS-MS analysis. The analysis revealed a partial sequence homologous to
peroxidase enzymes. Along with isolating the peroxidase protein, we also isolated a
partial sequence from a peroxidase gene expressed in the Brunfelsia flower. This
sequence is highly homologous with two tomato genes encoding for peroxidase
involved in a process of creating lignin in tomato roots. In comparison to these
sequences, the gene sequence isolated from the Brunfelsia represents around 40% of
the whole gene sequence. As there is no information about anthocyanin-degrading
enzymes of the whole plant in relevant literature, it is possible that the result of this
gene in the Brunfelsia is activity related to pigment breakdown.
The expression of this isolated gene during the flowers development and the
anthocyanin degradation was examined with real time PCR reactions conducted on
the cDNA that originated from the RNA derived from plants in various developmental
stages. The expression level was found to peak several hours before flower opening
and decreased to very low levels by day 2 after opening. These results strengthen the
possibility that the isolated gene product may be involved in anthocyanin degradation.
Continued research of the gene activity of the Brunfelsia plant requires transient
transformation or permanent transformation of the Brunfelsia plant. For this purpose,
transformation and regeneration systems were developed for the Brunfelsia plant.
Transformation systems for flowers were developed using agrobacterium that contain
the GFP reporter gene. The regeneration system was developed by comparing the
reaction of various plant organs to mediums with similar hormonal components. In
this experiment we determined that the optimal plant organ for regeneration is a
young leaf as it has the optimal medium for regeneration.
In summation, in this study we have proven that the activity of several peroxidase
isoenzymes increases in correlation with color fading, and this increase is in relation
to the increase in expression of the isolated peroxidase gene. Furthermore, we have
developed systems that will serve in future study of enzymatic activity in the
Brunfelsia plant.