The Flavonoid Pathway Regulates the Petal

Colors of Cotton Flower

Assignment #2 for Finals

Outline:

A. Introduction
1. Cover page
2. Image of plant with classification(At the back page)

B. Outline Proper:
1. Description of Plant
2. Principles/ Mechanisms involved
3. Conclusion

C. Bibliography
 I. PLANT DESCRIPTION

Cotton (Gossypium spp.) is a staple fiber crop. Although, its place of origin is not known,
West-central and Southern Mexico, North-east Africa and Arabia, and Australia are the primary
centers of diversityDNA sequence data from the existing Gossypium spp. suggests that the
genus arose about 10- 20 million years ago (Wendel & Albert, 1992; Seelanan et al., 1997). Its
antiquity, however, can be traced back to the 4th millennium in India in the ancient village of
Mohenjodaro as was mentioned in the Veda hymn. It is now classified as follows:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Malvales

Family: Malvaceae

Genus: Gossypium

Gossypium comprises of 50 species, but four (G.hirsutum L., G.barbadense L., G.arboreum
L., and G.herbaceum L.) are grown are for commercial purposes (Fryxell, 1992). G.hirsutum
(Upland cotton, 90% of world plantings) and G.barbadense (Pima, or Long-staple cotton), the
new world cottons, are where majority of commercial cotton are extracted. The two other
species, G.arboreum and G.herbaceum, are indigenous to Asia and Africa and are popularly
referred as desi cottons in India. The latter two are also referred to as the old-world cottons as
they were the antiquity cottons being cultivated at the Mohenjodaro. They were popularly used
before the 1990’s.

Cotton plants can grow 6ft in height and develop a semi-wooden stem and branches. It has a
taproot system which grow rapidly to a depth of 20-25cm before the seedling has even
emerged above ground. After emergence and unfolding of cotyledons, lateral roots then
develop; sideways and then downwards. Final depth of root system depends on soil moisture,
aeration, temperature and variety but is usually about 180-200 cm. Under dry growing
conditions, cotton roots have been known to reach a depth of 3-4m. The growth of tap root as
well as lateral root is affected by excessive moisture, hard dry soil layer and degree of soil
alkalinity.
The stem has a growing point at its apex, with an apical bud. As long as this bud remains
active, lateral buds situated below it remain dormant. The main stem carries branches and
leaves but no flowers. Length and number of internodes determine the final height of the plant.
As a rule plant with short internodes is
early maturing. The lateral bud normally remains dormant; but if the axillary bud aborts, it may
develop into a branch. Vegetative branches (monopodia) are morphologically similar to the
main stem. They do not bear flowers or fruits directly, but carry secondary branches (fruiting
branches), that are characterized by their sympodial growth habit.
Leaves are generally hairy and some varieties have glabrous leaves. Hairy leaves cause fewer
difficulties in mechanical harvesting and are more tolerant to jassids, but bear larger
proportions of white fly which apparently finds more sheltered conditions among the leaf hairs.
The leaves have variable lobes. Size, texture, shape and hairiness of leaves vary a great deal.
Nectaries are present on leaf calyx and bracts. Each leaf has two buds at its axis. On all aerial
parts of cotton plants are found internal glands which in different species vary in size, number,
distribution and pigmentation. These glands secrete a volatile oil i.e. gossypol and related
compounds. Gossypol is a polyphenolic yellow pigment and is toxic to non-ruminants. The
presence of gossypol makes cotton seed cake toxic and hence, glandless varieties have been
bred in recent years. However, it has been found that glandless varieties are susceptible to a
wide range of pests over the glanded varieties.

The full-grown seed of cotton is irregularly pear-shaped, varying in size depending on the
variety and conditions of growing. It may be naked or bear short hairs called “fuzz”. All
cultivated cotton seeds bear long fibres named “lint” and a majority of them also have fuzz on
the same seed. The lint is removed by gins while the fuzz remains attached. The colour of fibres
is generally white, but may also be brown or green and that of the seed is usually grey,
brownish or black. The mature seed has two cotyledons folded up that occupy the entire
portion of its cavity. The cotyledons are broad and kidney shaped. Delayed germination in some
of the species and varieties may be due to hard seed coat, closed micropyle and partially filled
cotyledonary-cum-embryonic contents. The germination increases when the seed coat thickness
is reduced by various methods of delinting. The seeds account for about 65 to 70 per cent of
the total yield by weight. The seeds are rich in protein (10-20%) and oil (up to 25%).
The oil content in G.barbadense is higher than G.hirsutum.
As mentioned above, lint and fuzz represent the outgrowths of epidermal cells on seeds. Some
cells continue to lengthen while others stop growing after a time. The former is known as
the lint and the latter is the fuzz. The lint hair is unicellular and its development is phased in
two stages, the first phase is a period of elongation and the second phase is increase in
thickness. A lint cell bulges first, the protoplasm inside turns granular, and the nucleus moves
towards the bulge. The swelling enlarges until it is twice the diameter of the original cell and
the nucleus moves to or near the tip. The elongation of cell may take upto 40 days. There is no
change in thickness. The growth is not regular; slow at first but fast from about the 15th day.
The rate slackens during days and quickens during nights. The cell wall thickens in the second
half of boll maturation. Deposits of cellulose are formed on the inside of primary wall. They are
laid in layers as seen from some fibres showing as many as 25 concentric layers. As soon as the
boll dehisces, the hairs dry, collapse and flatten the cylindrical form, assuming ribbon like shape
and go into spirals. The mature hair is uniform in diameter up to 3/4th length and then
gradually tapers to a point. The length of lint is a varietal character and varies from 15-50mm in
different varieties. Fibre quality traits such as length, fineness and strength are important as
spinning are dependent on these characteristics.

In G.hirsutum bolls are large, generally ranging from 4-5 grams. The general variation in boll
weight is 3-5 g, however in some varieties it can weigh upto 8g. The bolls are pale green,
smoothskinned and with few oil glands. In contrast, bolls ofG.arboreum are smaller (1.5 - 3g),
dark green and covered with numerous glands. Cotton plants by its remarkable auto-regulatory
mechanism shedthe bolls that are in excess of the load capacity of the plant under given
environmental conditions. As a result, the ratio of bolls to total vegetative growth is fairly
constant. In general, varieties or strains with large bolls do not adjust so well to change in
environment and to stress as do types with smaller bolls. Hence, shedding occurs more readily
and to a large extent in the former than in the latter case. Bolls developing under falling
temperature need more days to mature than those growing under rising temperature. The big
bolled American types in India take about 40-50 days while the Asiatic cottons require 35 days.
In G.hirsutum the boll consists of four to five locules each of which contains about 7 mature
seeds. A fair percentage of the ovules remain undeveloped due to nonfertilization, heredity and
environment. These are called “motes”. The size and shape of the bolls differ in diploid and
tetraploid cotton.
Lateral branches arise from the axils of the leaves of main stem and consist of two types viz.,
vegetative and fruiting. Vegetative branches are more vertical and ascending. Fruiting branches
are nearly horizontal. The internodes on the fruiting branches, referred to as sympodial
branches are not straight as in main stem but have a zig zag appearance with the leaves
alternately placed. The flowering and fruiting are dependent on the initiation of sympodial
branches.The timing of the crop for harvest is also determined by the early or late production of
such sympodial branches on the plant body. Very early varieties have their fruiting branches
even at first or second node to the total exclusion of vegetative branching from leaf axils.
Similarly, very late varieties go on producing a very large number of monopodial before
sympodial divergence appear. Relative proportion of vegetative and fruiting branches is
dependent on temperature, day-length, plant density and the rate of boll shedding.

Cotton flowers are extra-axillary, terminal and solitary and are borne on the sympodial
branches. The flower is subtended by an involucre of usually three unequal leaf like bracts.
Bracteoles, alternating with the bracts on the inside of the involucre or standing on either side
of the small bract, may be present. The calyx, consisting of five undiverged sepals, is persistent
and shaped as a shallow cup. The calyx adheres tightly to the base of the boll as it develops.
The corolla is tubular, consisting of five obcordate petals alternating with calyx lobes and
overlapping the next one in the series in a convolute manner. In some species, a spot of
purple, sometimes called ‘petal spot’, is found on the claw (base) of the petals. On the first day
after anthesis (the period when the flower is in full bloom and functional), the corolla changes
into pinkish blue and then into red during succeeding days. It withers and falls off on the third
day, together with the staminal column and stigma leaving the ovary, calyx and involucre intact.
The stamens are numerous and united to form a tubular sheath which surrounds the pistils
except for the exposed portion of style and stigma at the tip. The pistil consists of 3-5
undiverged carpels corresponding to the locular composition of a fully mature dehisced boll. The
ovules are attached to parietal placenta of each locule. The style varies in length and splits near
the apex into three, four or five parts depending on the number of carpels.

Pollination is usually done by water, insects or other animals. Wind is not usually a factor in
pollination since relatively large, heavy, sticky, and watery, so it cannot be easily carried by the
wind. Cross pollination occurs within close vicinity and among insects bess (Apis mellifera) are
the common pollinators of the plant. Pollination usually takes place in the morning when the
cotton flower is in full bloom. In 24-30 hours after pollination, fertilization takes place and the
corolla along with the anthers and the filament fall off the fertilized ovary (Govila, 1969). Boll
development is initially slow and then grows rapidly and steady in the later stage. It takes about
40-50 days from fertilization for the boll bursting, maturation of fibers, and seed formation. The
fruit is a capsule about 1.5 in (3.75 cm) long containing numerous seeds surrounded by a
whitish tomenta (lint and fuzz). The capsule opens at maturity exposing the seeds with their
tomenta which is the cotton boll.

Cotton requires a daily minimum temperature of 16°C for germination and 21°C to 27°C for
proper crop growth. During the fruiting phase, the day temperature ranging from 27°C to 32°C
and cool nights are needed. The sowing season of cotton varies considerably from tract to tract
and is generally early (April-May) in northern India where it is mostly irrigated. It is delayed on
proceeding to down south. It is cultivated largely under rainfed or dryland conditions. An annual
rainfall of atleast 50 centimetre distributed through-out the growing season is required for good
yield. It is mainly raised during tropical monsoon season, although in southern India it is
cultivated during late-monsoon season in winter. The cotton picking period from mid-September
to November must have bright sunny days to ensure a good quality. Cotton can be successfully
grown on all soils except sandy, saline or water logged types since it has developed moderate
tolerance to salinity, and sensitive to chilling or frost, and water-logging.

II. PRINCIPLES/ MECHANISMS INVOLVED

There are important concepts that are involved in the color of the petals of cotton plant.
Generally, cotton contains a variety of colors which can be divided into three color schemes: (1)
cream, which is the most common, (2) Yellow, (3) Red. There are also white flowers, although
it is rare. First, although there are many color varieties, the color of the flower changes
dramatically after blooming. On the first day of anthesis, the color was cream at 11 am, at 12
the color appeared slightly red which grew darker until 6pm and increased more in color at 6am
on the second day of anthesis. This proves that anthocyanin biosynthesis was enhanced during
flower development giving the petals an intense red hue. Along with this, the expression level
of genes of the anthocyanin pathway are consistent with the color change. The related genes,
PAL, CHS, F3H, DFR, FLS, ANR, ANS and, UFGT were abundant in the flower and are affected
by the light. Only the FLS 1, which was related to Flavonol biosynthesis, was high when there
was low anthocyanin, and decreased when anthocyanin accumulated until flower development.

Secondly, it must be understood that flavonoid is the main pigment responsible for
cotton flower color. A loss of flavonoid results in loss of color pigment in the petals. Therefore
the petals tend to appear white while cream colored petals appear in cotton plants rich with
flavonoid. Although, anthocyanin is proven to be the cause of the dramatic color change in
cotton flower, flavonoid is responsible for the initial color of the petals before anthocyanin
accumulation. This indicates that flavonoid is the main pigment responsible for cotton flower
colors including red, cream, and white.

Lastly, genetic and environmental both influence flavonoid metabolism in cotton flower.
It must be noted that flavonoid biosynthesis and anthocyanin biosynthesis go hand in hand.
Since flavonoid and anthocyanin are light dependent, gene expression for the two pigments
would be low without presence of light. Both have low expression in low lighted or shaded
areas.

Anthocyanin is a water soluble pigment which has a diverse role in biochemical and
physiological processes. It has a great impact in the different plant systems, as they can be
found in all plant parts. Anthocyanins are a group of plant pigments responsible for colors
ranging from red to violet and blue (van Tunen & Mol 1991). These pigments accumulate in the
vacuoles of epidermal cell, and both their chroma and hue are dependent on external
conditions, as well as on the pH in the vacuoles (Harborne & Grayer, 1988). Anthocyanins are
able to accumulate in epidermal vacuoles and blend with the plastid pigments to give various
hues that vary with light exposure and night and day temperatures (Sachray et al., 2002). In
most flowers, anthocyanin synthesis occurs with petals growth and is under developmental
control. Anthocyanin biosynthesis, depending on the geography,can be induced by many
factors. The anthocyanin biosynthesis leads to the accumulation of anthocyanin in the plant
body which in the case of cotton flower, in the petal which leads to it changing its color hue
during the anthesis. With the accumulation of anthocyanin, it also leads to the plant’s flower
being short lived since anthocyanin is responsible for plant aging. By this is meant that as
anthocyanin accumulates in the petals, it also speeds up the aging of the aging thus making the
flower relatively short-lived.
 Flavonoid, where anthocyanin belongs to, is found to be the one responsible for cotton
flower color, although the pigment could not be anthocyanin since it would tint the flower red.
In addition to this, anthocyanin is only highly active during anthesis and not before. This would
mean that another flavonoid is responsible for giving the cotton flower a cream hue. In the
research, they made use of F3H, a silencing gene. It silences the expression of flavonoids in
plant growth. Plants inoculated with the gene rendered a white pigment, or a lack of color
compared to plant without the gene which exhibited a cream hue. Although, during anthesis
both group of plants were affected by the accumulation of anthocyanin. This implies that the
flavonoid silenced was not anthocyanin as it was able to manifest it red tint to both group of
plants. The pigment is likely to be related to flavonol, which exhibits a cream hue. Flavonoids
act as attractants to pollinators and symbionts, as sunscreens to protect against UV irradiation,
as allelochemicals, and as antimicrobial and antiherbivory factors. Their importance in plant
biology goes beyond their specific functions within the plant. For example, the early advances in
floral genetics were primarily the result of the ease of screening for mutations impacting
flavonoid-derived flower colors, and the first demonstration of epigenetic gene silencing in
plants was likewise associated with flavonoid biosynthesis (Jorgensen, 1995). Flavonoids have
been ascribed positive effects on human and animal health and are central to the current
interest in “botanicals” for disease therapy and chemoprevention. This was also proven due to
the F3H silencing gene which rendered petals white instead of the normal cream hue. Implying
that the gene which was silenced was the flavonol.

Lastly, the environmental factors affect the gene expression of flavonoids. Flavonoids
are light-dependent. For effective expression, they must be activated by sunlight. When the
plants were shaded, there was an observed less flavonoid, specifically anthocyanin,
accumulation in the flowers thus reducing the red tint. Flavonoids are derived from
phenylalanine, which is catalyzed by the phenylalanine ammonia-lyase (PAL). Then mediated by
chalcone synthase (CHS), Flavanon-3-hydroxylase (F3H), and fluxed into anthocyanin
biosynthesis by dihydroflavano-4-reductase (DFR), anthocyanin synthase (ANS), and UDP-
glucose; flavonoid-3-Oglucosyltransferase (UFGT); fluxed into proanthocyanin by flavanol
biosynthesis by flavanol synthase (FLS) following F3H, or flux via anthocyanin reductase (ANR)
following ANS. The whole process is light-dependent.
III. CONCLUSION

The place of origin of cotton is not known but can be traced to the 4th millennium city of
Mohenjodaro. Cotton have many commercial purposes which lead to many of its variations
today. To better understand how the cotton plant performs, the observed changes of its short-
lived flowers were studied.
It was found out that during anthesis, anthocyanin accumulates in the flower. The period of
anthesis dictates the amount of anthocyanin that accumulates, since this is also monitored by
the presence of light available throughout the day. It becomes more intense in color with the
higher amount of light, since as a flavonoid, anthocyanin is also light-dependent. With this in
mind, they also found out that another flavonoid, flavonol, is responsible for the color of the
flower (before anthocyanin accumulation. Flavonol exhibits a cream hue observed in the cotton
plant. This was seen when F3H silenced Flavonol genes, but not anthocyanin, thus allowing
anthocyanin to continue to accumulate and change the color of the petals during the two-day
anthesis. Lastly, the environmental factors, especially light availability, affect gene expression of
the flavonoid (which are light dependent).
It was shown that flavonoids regulate the expression of color in the cotton flower. It is
responsible for the cream color it exhibits when it blooms, then anthocyanin accumulation
changes the hue to a red color to darker and deeper red until the end of the anthesis when the
plant then start to wither.

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