From the Rare
Fruit Club WA
by Barry Madsen
Opening the Door
on the Mysterious Sexual Antics of Pawpaws
Pawpaws
(Carica papaya,
Family Caricaceae, Order Brassicales, commonly called papaya in other
countries) are highly productive fruit trees, native to meso-America
and now grown in tropical and sub-tropical areas worldwide. They are
enjoyed for their pleasant flavours and aromas, and also greatly valued
as a rich source of vitamin A which is a major deficiency problem in
much of the developing world. But for almost all the time they have
been cultivated, growers have had to deal with their strange sexual
behaviour which influences all the parameters of fruit production,
without any real understanding of what's going on and how best to
optimise outcomes.
About 90% of all the flowering plants (angiosperms) have flowers with
both male and female parts in the same flower; these are called
hermaphrodite or bisexual, and are normally self-fertile. Another 5%
have individual male and female flowers on the same plant (monoecious),
and most of the remainder have individual male and female flowers on
separate plants (dioecious). A very small minority such as pawpaw are
trioecious (subdioecious), with separate male, female and hermaphrodite
plants. Seed germination is the principal way in which pawpaws are
propagated and traditionally it has not been possible to determine
whether a given plant will be male, female or hermaphrodite until it
flowers. As only the latter two produce good quality marketable fruit,
this results in much wasted time and resources raising and then
eliminating plants. Only one in 10-20 plants need be male to ensure
sufficient pollination of females, and depending on the climate, one or
other of the bisexual or female plants may be preferred for fruit
production. Bisexual plants are usually preferred in tropical climates
and dioecious in sub-tropical, and this preference may mean other young
plants are sacrificed. Traditional growers were also aware that varying
environmental conditions each season, such as temperature,
precipitation, nitrogen fertilisation, plant age, light intensity,
photoperiod, mite infestation and mechanical injury (eg leaf shredding
after storms or loss of storage tissue from pruning) could cause
strange outcomes, with males sometimes producing fruits, females doing
the equivalent of the virgin birth with fruit production in the absence
of fertilisation (parthenocarpy), females temporarily becoming sterile
or having long peduncles, trees producing normal-shaped fruit one year
and deformed fruit the next, and individual trees changing to have both
female and hermaphrodite flowers (gynomonoecious) or male and
hermaphrodite flowers (andromonoecious).
Although a prominent feature of pawpaw behaviour, sex lability is not
unique to this species and the phenomenon has been observed in more
than 50 species across 25 families including such commonly known plants
as asparagus, cannabis, castor oil, corn, cucumber, holly, orchids,
spinach, summer squash, weeping willow, wheat, white mulberry and wild
grapes. Usually hermaphrodites are more labile than the other sex
forms. Dioecious plants under stressful conditions may convert to
hermaphrodites or the opposite sex, and monoecious plants may change
the ratio of male and female flowers. These changes are usually
consistent with evolutionary adaptations that maximise reproductive
success under difficult conditions, as the stress on females carrying
fruits through to maturity is much greater than males producing pollen
for only a limited time. Generally, stress of one form or another
results in conversion from female forms to male, and optimal conditions
the reverse.
Substantial advances in understanding the pawpaw story only began with
systematic study in the early 1900s. Unravelling the mechanisms
controlling sexual expression illustrates how scientific knowledge
progresses and also how fundamental interpretations of data are always
dependent on techniques available in different eras. While the story is
still incomplete, many key features are now firmly established. Some of
the major steps along the way were:
| • |
In
the 1930-40s breeding experiments of the different sex types showed
that offspring occurred in the following ratios:
male X male – 1 female, 2 males,
female X male – 1 female, 1 male, bisexual X bisexual – 1 female, 2
bisexual,
female X bisexual – 1 female, 1 bisexual, and
bisexual X male – 1 female, 1 male and 1 bisexual.
These non-classical ratios provided support for the existence of a
sex-determining gene that had three forms (alleles, definitions of
common genetic terms are given in the 'More Information' section on the
site) – M from male trees, Mh from hermaphrodite and m from female.
These studies also suggested that male and hermaphrodite trees were
heterozygous, Mm and Mhm resp, and females homozygous, mm. M and Mh
were assumed dominant and m recessive, and dominant combinations MM,
MhMh and MMh were lethal. This interpretation formed the basis of most
theories over the next several decades, but it had to be successively
embellished as new data came to hand. |
| • |
Flowers
determining sex were found to be highly variable (more than 30 types),
particularly amongst hermaphrodites, and in the 1950s it was suggested
they could broadly be classified into 8 basic types, with (i) and (ii)
being male-type, (iii)-(vii) hermaphrodite and (viii) female. The
different types had the following characteristics: |
|
i |
staminate, - unisexual male
flower on long peduncles, 10 stamens present in 2 whorls of 5 each |
|
ii |
teratological
staminate – found on sex-reersing males, with some
degree of carpel initiation and estigial carpels (hair-like processes)
at the base, can fruit in cooler weather |
|
iii |
reduced
elongata - modified elongata flower, aborted pistil and reduced carpel
size, more frequent in warmer conditions. |
|
iv |
elongata
- refers to the shape of the pistil, develops into
pyriform or cylindrical fruit, 5 lateral-fused carpels, and petals
fused two-thirds length |
|
v |
carpelloid
elongata – transformation of the inner 5 stamens into
carpel-like structures. Many sub-types depending on how many stamens
are carpelloid, misshapen fruit |
|
vi |
pentandria
– normal hermaphrodite, stepwise transformation of
stamens to carpels, short corolla tube, 5 stamens in the outer whorl on
long filaments ,globose and furrowed pistil with 5-10 carpels |
|
vii |
carpelloid
pentandria – all 5 stamens in the outer whorl become
carpelloid, producing carpellodic fruit, especially under cool
conditions, original carpels abort and flowers resemble pistillate type |
|
viii |
pistillate
– unisexual flowers, larger than hermaphrodites,
5-carpellate, lack of stamens and the most environmentally stable of
all
types. |
| • |
In
the 1940-60s a gene balance hypothesis proposed that the M and Mh
alleles were on normal chromosomes (autosomes) and the m allele on 'sex
chromosomes', although at that time there was no supporting cytological
evidence of these. The M and Mh regions on the autosomes were
degenerate, missing genes that were necessary for plant development.
This model explained why homozygous dominant MM, MMh and MhMh were
lethal, as heterozygous plants with at least one copy of m were viable. |
| • |
Also
in the 1960s it was suggested that rather than a single gene
controlling sexual expression, a number of these genes resided in a
small region of the 'sex chromosome'. This theory suggested there were
5 genes involved and various combinations of them produced the highly
variable observed behaviour. It was also suggested the existence of
hermaphrodite pawpaw could have been due to human selection ie it was a
relatively recent development in the species. A real problem was that,
like other theories, there was no definitive means of testing them. |
| • |
Another
proposal in the 1960s was that pawpaw sex determination was of the
XX-XY type that we're familiar with in humans and most other mammals;
homozygous XX being female and heterozygous XY being male. It was
further posited that the Y chromosome has a region containing a lethal
factor, and that Mh was a modified form of M while still including the
lethal factor. |
| • |
In
the 1990s a dominant male allele SEX1-M was proposed that promotes
stamen development but suppresses that of the carpels ie it is a
masculinizing factor. The dominant hermaphrodite SEX1-H allele was
suggested to be an intermediate with the ability to induce stamens but
only partially suppress carpels. The recessive female allele sex1-f
promotes carpel development but is a null allele in terms of inducing
stamens. Functional stamens and carpels would develop in heterozygous
SEX1-H/SEX1-f plants, and the lethal factor linked to the SEX1-M and
SEX1-H alleles would explain why homozygous dominant alleles do not
survive and heterozygotes are viable. |
| • |
Finally
in the last 15-20 years, numerous molecular biological studies have
provided unequivocal answers to a number of these perplexing
observations, interpretations and management issues. Up to the present,
none of the vegetative pawpaw features (eg, stem and flower colour)
suggested for their ability to discriminate sex type before flowering
is reliable, and growers still have to put several seedlings in each
planting hole and wait for flowering to know which can be culled.
Modern DNA techniques can discriminate sex type well before this, but
currently are only feasible in a laboratory setting. This could change
in the not too distant future as simple, cheap diagnostic genetic tests
become available. |
Pawpaws have 9 chromosome pairs and a small
genome of 442 million
base pairs with approximately 24,000 genes. The gene count may seem
large but in fact is made up of many repeats, non-functional
pseudogenes and non-expressed DNA. It has been estimated that the
minimum gene count for viability in Angiosperms is about 13,000.
Approximately 96% of the genome has now been sequenced and mapped. The
sex determining region is on an incipient Y chromosome which could not
be differentiated from others using classical techniques. Separation
began about 7 million years ago and the male specific region (MSY) is
only about 10% of Y compared to 95% in humans with more mature
chromosome divergence (240-320 million years ago). For such evolution
to occur, it had to become non-recombinant to allow the MSY to develop
and build up differences from the X. Regions just outside the MSY are
changing about 7 times faster than the genome average, ie they are 'hot
spots' responsible for the rapid changes that have occurred in this
relatively short time. The corresponding MSY region on the X chromosome
is changing much slower. Non-recombinant changes have led to
degeneration and expansion in the MSY, mainly through insertions that
occurred in 2 major evolutionary events, with loss of some key genes
necessary for survival while still being present on the matching X
chromosome; dominant homozygotes are thus lethal and heterozygotes
viable. In breeding experiments the MSY behaves like a single (linked)
genetic unit since it's all on one chromosome and there's no
recombination with X. The hermaphrodite sex chromosome has an
equivalent region (HSY) to MSY that diverged from it only 73,000 years
ago, an incredibly short time in evolutionary history but still
probably too early to be the result of human selection. It consists of
8.1 million bases compared to 3.5Mb on the matching X region, and it
codes for 72 genes compared to 84 on the corresponding X chromosome
region; 50 of these genes are common to both and there are 24 and 14
pseudogenes in the HSY and X regions resp. To date, pawpaw is the only
plant species with fully sequenced and annotated sex chromosomes. The
small pawpaw genome is probably the reason for genetic diversity
between different varieties being relatively minor (correlation
coefficient about 0.9) compared to other flowering plants.
Self-pollinated hermaphrodites (cleistogamy) are just as variable as
open-pollinated dioecious lines, seemingly from rare but still finite
cross-pollination.
Two genes are likely involved as the
initiators of sex determination, one a carpel suppressor or
masculinizing gene for carpel abortion in male flowers, and the other a
stamen suppressor or feminizing gene. These two genes operate in
different time frames - abortion of stamens occurs before initiation of
stamen primordia whereas the male sex determination gene aborts carpels
at later developmental stages. Remnants of the aborted gynoecium are a
feature of the male flower structure, and with good growing conditions,
a few male flowers may not undergo complete carpel abortion and can
form fruit. Arabidopsis thaliana is the closest relative to pawpaw in
the Brassicales that also has sex chromosomes, and this species has
been extensively studied by plant geneticists. Many genome parallels
exist between the two species that indicate the pawpaw female sex
determining gene is likely to be an upstream regulator of 2 genes
called APETALA3 (AP3) and PISTILLATA (PI) that cause early abortion of
stamens. A. thaliana also produces a gene called ATA1 that is expressed
only in male flowers during development and is homologous to similar
genes found in corn and white campion. ATA1 is associated with normal
pollen formation and although it's probably also involved in the
masculinizing pathway in pawpaws it might not be the initial gene that
sets the whole flower development cascade underway. Approximately 180
genes are thought to be involved in producing fully functional flowers.
At
least 2 genes differentiate the M and Mh chromosomes; one controls the
long peduncle on male trees and the other is a masculinizing gene that
controls carpel abortion in male flowers. As embryo abortion occurs
25-50 days after pollination, there is a regulatory gene that's
essential to early embryo development that resides in this region and
has degenerated on both the M and Mh chromosomes but is still
functional in the X region; this is the cause of homozygous dominant
allele lethality. This year (2015) it was reported that one of the key
genes in pawpaw sex differentiation between males and hermaphrodites
and not found on the X is similar to one found in A thaliana called
SHORT VEGETATIVE PHASE (SVP) . SVP has been shown to initiate flower
development, and in pawpaws it is functional in the MSY but not in the
HSY. Thus it would seem that in males, SVP sets in train carpel
abortion whereas in hermaphrodites a varying number of carpels remain
functional and can therefore be fertilised and bear fruit. This whole
field is advancing rapidly and the hope is that it won't be too long
before all the key sex determining genes are identified and
characterised. The evidence so far is that these few genes are the
initial triggers for setting in train male, hermaphrodite or female
development through subsequent genetic, epigenetic and plant hormone
control.
Some examples of other practical developments and possibilities that
have followed this genetic work include:
| • |
Cloning
of a single gene that codes for an enzyme called lycopene beta-cyclase
which determines red fruit flesh colour. In many countries red flesh
pawpaws are preferred over the more usual yellow/orange types and
accordingly fetch higher prices. The allele for red colour, which
produces a non-functional enzyme, is recessive, and this is why
typically with open pollination this feature can be quickly lost and
fruit revert to the less strongly coloured forms. Healthy pro-vitamin A
carotenoids in red flesh fruit consist mainly of lycopene whereas in
the yellow/orange forms with the dominant allele, almost all lycopene
is enzymatically converted to other carotenoid forms. |
| • |
The
accelerated impetus for genetic work on pawpaw stemmed in large part
from the disease Papaya Ring Spot Virus (PRSV). This is a serious
problem in all papaya growing regions worldwide and can devastate whole
orchards. In Hawaii, pawpaw had been one of the top 3 fruit crops and
they were threatened with total collapse of the industry because of the
spread of the virus across all the islands. C. papaya is the only
species in the genus and with a small genome there were limited
opportunities to use classical breeding strategies to develop more
resistance – none was sufficiently successful. Following sequencing of
the PRSV and identification of the viral resistance gene that produced
a coat protein, a major collaborative research effort was undertaken to
develop a transgenic solution by inserting this gene into pawpaw. Sunup
(red flesh) and Rainbow (yellow flesh) became the first such tree crop
fruit to be approved for general release in the US and local growers
quickly moved to incorporate them in production; subsequent approval in
foreign markets such as Japan cemented their place as a valuable
commercial crop and saved the industry. Other major pawpaw producers in
Latin America and South East Asia have found PRSV infections in their
regions are due to different strains, and with assistance and a
technology transfer program are developing their own transgenic
solutions. |
| • |
As
genes are characterised, particularly those in the MSY and HSY, and
their role in plant and fruit development clarified, there is the
prospect of having pure breeding lines that avoid the multi-planting
problem associated with conventional seedling propagation. As a number
of other species in the Brassicales have already been cloned and have
larger genomes with many more genes whose purpose has been identified,
parallel genes can be seen in the C. papaya genome that regulate
features such as precocity, stem height, photoperiod response,
climacteric behaviour, fruit levels of sugar, starch, phytonutrients
and aromatic volatiles, cellulose synthesis for tree strength, fruit
firmness and size, storage properties, disease resistance etc.
Exploitation of this expanding collective knowledge will support
improved economic production of superior fruit with greater control
and/or predictability under varying environmental conditions. |
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