Term Paper

on

Genetics of pollination: understanding the mechanism behind plant

reproduction

Subject: Genetics

Paper code: BOT.508

Submitted by: Ishita Sukhija

Reg. no: 23mscbot31

M.Sc. Botany

(2023 – 25)

Submitted to: Dr Pankaj Bhardwaj

Department of Botany

Central University of Punjab, Bathinda

Abstract
For decades we have known that Pollination is the transfer of pollen grains from the stamens
to the stigma but this indeed beautiful process is far beyond just a pollen transfer. From a
geneticist perspective, it’s not only an essential requirement of sexual reproduction in
flowering plants but a lot of genetics concerned with evolution and environment is also a
crucial part of this process. We need to compare the effectiveness of wind and animal
pollination in plants. It needs to be deciphered out if self-pollination is better than
cross-pollination. Flowering plants are observed to evolve a diversity of floral traits that
influence pollination success, where some are responsible for the attraction of animal
pollinators and some make it suitable for wind pollination. It’s fascinating to observe how a
plant rejects its self pollen to promote the genetic diversity of its family - indeed not only
humans but plants are selfish too. Researchers have observed the significant influence of the
environment on plant genetics. Slow and gradual changes in floral traits result in
better-evolved and adaptable plant species. While exploring the potential of pollination
genetics in revolutionizing modern agriculture, geneticists faced some challenges concerned
with the amount and irrelevancy of data. In the Future, all these challenges can be worked out
and researchers can also work on blooming potential opportunities in this field. More studies
in this field can solve the crises of food shortage and can prove to be the solution to many
human-induced problems.

Keywords: Flower, pollination, Floral rewards, floral traits, adaptation, evolution,

self-incompatibility, S-locus, S-RNase, genetic diversity.
1. Introduction
The immensely beautiful and surprising reproductive mechanism of flowering plants has
always attracted the attention of the human race. Flowers have always played a significant
part in our lives, they have always proved that they are more than just an artistic piece of
beauty. It has long been known that through normal breeding and artificial selection, plants
provide food to humans - that is one of the basic necessities of our existence. Technically,
Pollination is the transfer of pollen grains from the stamens to the stigma, which is an
essential requirement of sexual reproduction in flowering plants. With advancements in our
research technologies, it is now possible to explore the molecular developmental basis of
many flower traits, such as the relative heights of stigma and stamens, patterns of
pigmentation within petals, or the production of floral volatiles. All of these traits influence
the ways that different pollen vectors interact with the flower. Yuan et al. commented that the
links observed between genotype to phenotype and resultant pollinator response cited in the
literature are based on “different standards of evidence” and that this can provide us with an
extensive understanding of the genes that govern the interactions between flowers and
pollinators.QTL analyses identified three primary loci, one accounting for most of the
variation in stamen, stigma, and corolla tube length. It is expected that a regulator of cell
division is also present at this locus that is responsible for the change in pollen placement
which is further associated with the pollinator shift.(Hermann K et al. ; 2015).It is believed
that the Genetic analysis of pollen sampled from different locations of a pollinator will aid us
in improving our models of pollen dispersal (Harder and Barrett, 1996). Pollination can
primarily be of two major types,Cross-pollination and Self-pollination. Flowering plants have
evolved a plethora of traits that influence pollination success, including those that are
involved in the correct utilization of self-pollination.(Fattorini, R., & Glover, B. J. ;
2020).It’s observed that floral rewards can increase a plant's reproductive success by
increasing the collection and export of pollen. Floral rewards include a diversity of gifts for
pollinators - Nectar and pollen are some of the prominent nutritive rewards. Some additional
benefits are also observed to be provided to pollinators by plants(Balamurali G. et al.;
2015). Self-incompatibility (SI) is an adaptive plant strategy that promotes genetic diversity
by increasing the chances of cross-pollination and thus reducing the chances of inbreeding
depression. Basically, after pollination, the pistil can recognize genetically related (self) or
different (non-self) pollen grains that fall on it’s stigma surface(Chakraborty, S., Dutta, S.
& Das, M.;2023). Floral isolation can surely be considered a fascinating subject for genetic
speciation studies. Floral traits such as flower color, shape, and reward are comparatively
easy to measure(Kathleen M. Kay et al.; 2009). These selections favoring specialization of
particular pollinator species which is concerned with a specific plant species would turn out
to be fruitful for the evolution of ‘pollination syndromes’, and may also justify why some
plants have floral traits that restrict the entry of only some visitors and pollinators. (Randall
J. Mitchell et al.; 2009). All of these understanding concerned with the genetics of
pollination has immense practical significance in conserving biodiversity, controlling
invasive species, and strengthening the stability of interaction networks. Genetics of
pollination also presents many enthralling challenges and opportunities for the study of
evolution and adaptation.

Fig. 1.1: Diagrammatic visual of different types of pollination.(Self-created)

1.1 Genetic Perspective of Pollination

Flowering plants have evolved to be accompanied by a diversity of traits that interestingly
affect pollination success, these traits are the deciding factors - if plants undergo
self-pollination or cross-pollination. These traits also govern the attraction of animal
pollinators, and the effective use of wind pollination (Fattorini, R., & Glover, B. J.;2020).
According to research studies, For dioecy(unisexual) to evolve from
hermaphroditism(bisexual), the spread of sterility mutations is necessary which are mostly
deleterious to fitness for the organism. It can be deciphered that short pollinator flights might
limit the extent of pollen-mediated gene dispersal that will ultimately alter the genetic
structure of populations (Wright, 1931; Turner et al., 1982). It has been analyzed that in
self-compatible species, the ability of pollinators to visit many flowers of the same kind in a
row increases the opportunity for geitonogamous (among-flower) self-pollination and it
further results in an increase in the selfing rate (Harder and Barrett, 1995, 1996; Snow et
al., 1996; Karron et al., 2009). Wind pollination is considered less productive to plants than
animal pollination, which results in a gradual transition to a better survival strategy for plants
(Owens SJ, Rudall P, eds. 1998). Experimentally, Transgenic lines with manipulation of a
single gene can absolutely be a great idea to test the function of that gene to research
regarding pollinator preferences which would help us to concude genetics of different kinds
of pollinations better. (YW Yuan et al.; 2013)

1.1.1 Self-pollination
Technically, Self-pollination is the phenomenon of a flower being pollinated by pollen from a
flower of the same plant or the pollen of a different flower but from the same plant. A very
high rate of Self-pollination has important genetic and evolutionary impacts those are
responsible for influencing the genetic structure of a population, gene diversity, evolutionary
rates, effective population size, and patterns of evolutionary diversity(Barrett Spencer C.
H.;2010). The molecular mechanism concerned with corolla size reduction in the selfing
species of Capsella has been explored, where the selfing species of Capsella rubella has
flowers with an 85% reduction in petal size, it’s outcrossing species i.e. Capsella grandiflora
bears corolla of normal size. Researchers informed that a region in the intron of the STERILE
APETALA (SAP) gene, that encodes an F-box protein ( Byzova MV, Franken J, Aarts
MGM, de Almeida-Engler J, Engler G, et al.; 1999), was responsible for a 25% reduction
in petal area of C. rubella as compared to C. grandiflora. Also, it has been reported that in
Eichhornia, homostylous(having the same size of stylous) forms that undergo self-pollination
arise because of the major gene changes of stamen position along with further polygenic
changes that resulted in a reduction of flower size (Barrett et al. 2009). Theoretical models
prepared on the co-evolution of selfing and inbreeding depression suggested a general pattern
(Lande & Schemske 1985), this further resulted in significant experimental work on the
connection between selfing rates and the life-history dynamics revolving around inbreeding
depression.

1.1.2 Cross-pollination
Cross-pollination is the phenomenon of transfer of pollen from anther of one plant to the
stigma part of the pistil of another flower. Cross-pollination serves various advantages to
plants and one of the prominent advantages of the same is an increase in genetic diversity
thus providing selection during challenging situations - the basic rule of genetics.(Fattorini,
R., & Glover, B. J.;2020).The higher genetic diversity also results in a great increase in
phenotypic variation promoting a greater diversity of visitors (Barbour et al. 2020). The
most widely accepted hypothesis given by researchers, for the usefulness of dioecy(unisexual
flowers), is that it provides a brilliant escape from the inbreeding depression by preventing
self-fertilization. Theoretical models suggested by researchers identify three major sets of
factors that are important while considering the evolution of dioecy(unisexuality): selfing and
inbreeding depression, the optimal availability of resources to female and male plants, and
the genetic control of sex determination (Charlesworth 1999). Self-incompatibility (SI) is an
adaptive strategy in plants that increases genetic variability by promoting cross-pollination.

1.1.3 Wind pollination V/S Animal pollination

The form of pollination of plants when pollens are carried by the wind is called wind
pollination but when pollens are carried by animals( mainly, insects, birds, and bats) it's
called animal pollination. Animal pollination is technically termed as Zoophily and wind
pollination is termed as Animophily. The strong connection between the environment and
flowering response may work to ensure that plant populations of the same species flower
together, which further increases the chances of successful wind pollination. Wind pollination
is concluded to be less efficient than animal pollination(Owens SJ, Rudall P, et al.; 1998).
Friedman & Barrett ( Friedman J, Barrett SCH., et al.; 2008) informed that the shift from
animal pollination to wind pollination occurs mostly in plant families with unisexual flowers.
These researchers therefore stated that the transition to wind pollination takes place as an
adaptive response to the limited availability of pollinators in the surrounding environment.
According to studies, Wind pollination has evolved at least 65 times from animal-pollinated
ancestral plants (Linder 1998). Researchers have tried to solve the mystery behind the
transition of plants to wind-pollination when it’s so inefficient. They have tried to figure out
why it has evolved repeatedly from animal pollination. After extensive research and
brainstorming, they have hypothesized that wind pollination is likely to evolve when
ecological conditions make animals less available and beneficial as vectors for pollen
transfer. This concludes that in populations having unsatisfactory pollinator service, there is
no proper transfer of pollens and production of seeds, therefore wind pollination evolves as it
provides reproductive assurance and continuation of generation in much the same way as
self-pollination decreases the challenge of pollen limitation( Lord, E. M., & Russell, S. D.;
2002). There are still schools of thought regarding better options for pollination - Some
consider wind pollination to be more reliable but there are others who support animal
pollination over this. Some studies by researchers generally support the hypothesis that
animal pollination increases diversity and variability rates.(Kay, K. M., & Sargent, R. D.;
2009)

1.2 Genetic mechanism behind pollinators' attraction

Nature observes a beautiful give-and-take relationship between flowers and pollinators.
Floral rewards are expected to increase plant reproductive success by selective transfer of
pollen. Like humans, pollinator opts for the flower which serves them more advantage. Many
interesting rewards attract pollinators to a plant. Nectar and pollen are the nutritive rewards
for pollinators, and other additional benefits include heat sources, nesting materials, brooding
sites, sleeping, and finding mates for reproduction(Balamurali G, Krishna S, Somanathan
H.; 2015). Many plants provide Nector to pollinators, and it's composed of sugars, volatile
organic compounds (VOCs), and amino acids(Borghi M, Fernie AR, Schiestl FP,
Bouwmeester HJ.; 2017). Unique and attractive floral scents also play an important role in
attracting pollinators. Floral scents are mainly composed of VOCs. Researchers suggested
that these Floral VOCs are involved in many signaling functions. When emitted from flowers
these VOCs attract pollinators from long distances and influence landing and entrance
patterns. Byers et al. found that allelic variation can be responsible for the interspecific
differences in the OCIMENE SYNTHASE (OS) gene, which further affects the production of
E-β-ocimene - a floral scent, and LIMONENE-MYRCENE SYNTHASE (LMS) gene, which
is responsible for the production of d-limonene and β-myrcene - both of these are floral
scents. Stopping E-β-ocimene production in transgenic M. lewisii plants decreased
bumblebee visitation slowly but significantly, but inhibiting the LMS gene did not affect
bumblebee visits. Within the Mimulus family, M. lewisii is the only plant species that
produces E-β-ocimene. Only mutations in the OS gene were observed to prevent
E-β-ocimene production in other plant species of the same family, which points towards
parallel evolution of loss-of-function alleles. In Mimulus, Bradshaw et al. (1998) did QTL
mapping for specific descriptors traits and found more than 20% of the difference between
different species for flower shape, nectar volume, and flower color patterns. Pollinators are
very choosy when it comes to selecting flowers for pollination. Whitney et al. showed that
bumblebees can differentiate flowers with or without conical cells by ideating the roughness
of the petal surface as a tactile sensing hint.
A field experiment using F2 hybrids informed that anthocyanin and carotenoid content along
with nectar volume, play imperative roles in pollinator preferential selection between the two
plant species, their experiment showed that increased anthocyanin concentration is concerned
with higher hummingbird visitation rate and simultaneously lower bumblebee visitation rate
(Yuan, Y. W., Byers, K. J., & Bradshaw Jr, H. D.;2013). It has been stated since 1900 that
the production of betalains is related to the R and Y loci that influence red V/S yellow (R)
and the presence V/S absence (Y) of color. Also, Loci that are expected to be contributing to
reproductive isolation by pollination preferences based on visual and olfactory rewards, at
last leading to speciation - are currently being identified and studied.
A research study suggested that SWEET9, a member of the SUGARS WILL EVENTUALLY
BE EXPORTED gene family which is located in the plasma membrane, synthesizes a
polypeptide with sucrose transporter activity that is necessary for nectar production.SWEET9
is expected to be concerned with sugar efflux from the nectaries, and a strong role has been
informed for sucrose phosphate synthase in the translocation of nectary starch to aid in the
secretory process. Flowers are dependent on flower visitors i.e. pollinators for their
reproductive success, and these pollinators in turn are dependent on pollen and nectar to
sustain themselves and raise their offspring. Floral rewards play an interesting role in this
reciprocally beneficial interaction.(Borghi, M., Fernie, A. R., Schiestl, F. P., &
Bouwmeester, H. J.;2017).

1.3 Self-incompatibility
Self-incompatibility(SI) is a genetic mechanism where a flower rejects genetically
related(self) pollen considering it incompatible and this is how we derived this word. This
increases genetic diversity in plants by promoting cross-pollination instead of
self-pollination, ultimately reducing inbreeding depression. In self-incompatible plants,
pollination is expected to reduce seed production if self-pollen comes to stigma, it also causes
damage to outcrossed pollen tube growth, increases chances of fruit abortion, and also
reduces chances of pollination success(reviewed in Snow et al., 1996). Gender dimorphism
commonly evolves from self-compatible rather than self-incompatible ancestors, a pattern
consistent with the anti-selfing hypothesis.

Fig. 1.2: Diagrammatic representation of different self-incompatibility systems in plants.

1)Different types of interactions observed in Sporophytic self-incompatibility,2)Different
compatibility types observed in Gametophytic self-incompatibility (GSI) (Self-created with
reference to Chakraborty, S et. al; 2023)

The SI response of pollen is concerned either with the genotype of the diploid parent plants
that produce pollen (sporophytic self-incompatibility, SSI) or the genotype of the haploid
microspores (gametophytic self-incompatibility, GSI). In either of these cases, the
incompatibility mechanism is under the influence of genes located in the multi-allelic S
loci(Chakraborty, S., Dutta, S. & Das, M.; 2023).In the case of GSI, self, and
non-self-pollens are initially allowed to germinate. After that only non-self pollen is allowed
to enter the ovary for fertilization, and the growth of self-pollen is stopped in style. In SSI, a
receptor kinase-based interaction between S LOCUS CYSTEINE RICH (SCR) and S
LOCUS RECEPTOR KINASE (SRK) genes influences the incompatibility response.
According to research studies, at the molecular level, SI is controlled by two tightly linked
genes present in the S locus (Muñoz-Sanz et al. 2020).SSI occurs when even one of the S
proteins present in pollen matches with at least one of the two S proteins present in the pistil.
In the case of GSI, the SI mechanism is concerned with the genotype of the haploid
microspores (Silva and Goring 2001).GSI occurs when the S haplotypes of pollen match
with at least one of the S haplotypes in a heterozygous pistil.
The female factor of the S locus gene is the S LOCUS RECEPTOR KINASE (SRK). Similar
to the SRK component of the pistil, the male factor is the S LOCUS CYSTEINE RICH /S
LOCUS PROTEIN 11 (SCR/ SP11).SCR and SRK proteins ultimately influence and produce
SI responses. In the last 25 years, the molecular characterization of the S-locus in species of
the various economical families including Solanaceae, Plantaginaceae, and Rosaceae has
identified the pistil S determinant of these families as a stylar-expressed RNase, called
S-RNase (Anderson et al., 1986; Sassa et al., 1992; Ushijima et al., 1998). Sometime back
researchers did site-directed mutagenesis of His93 to Asn93, which turned the Petunia plant
incapable of rejecting incompatible self-pollen due to damaged RNase activity.
During these times when the population is increasing uncontrollably, for feeding people, SI
crops are being preferred as seed setting is not desired. Also, since long breeders have
performed a targeted genetic modification of S genes to successfully generate SI lines of SC
plants. Generation of transgenic SI A. thaliana line by transferring both SCR and SRK genes
from sister plant A. lyrata was done many times back (Nasrallah 2002). Because of its
fruitful aspects in both academia and agriculture, SI has been deeply studied in a diversity of
plant species including some prominent crop plants.

1.4 Environmental effect and pollination genetics

Many researchers suggested that the environment influences the genetics of flowers and thus
incredibly affects their pollination. The strong relation between the environment and
flowering response is believed to ensure that local populations of the common plant species
flower together, increasing the chances of successful wind pollination. In tropical habitats,
where seasonal variation is limited and changes in day length are comparatively smaller than
normal, studies have shown that environmental signals, such as the accurate time of sunset or
sunrise, might be enough to result in synchronous flowering(Fattorini, R., & Glover, B.
J.;2020). Researchers suggested that considering genetic diversity is imperative for a better
understanding of the connection between land use and interaction networks between flowers
and pollinators. Also, this understanding has significant value in conserving biodiversity and
improving the stability of interaction networks when the is world facing great challenges of
habitat and diversity loss(Landaverde-González, P., Enríquez, E. & Núñez-Farfán, J.;
2021). Some studies also informed the direct and indirect genetic effects of species
interactions on the composition of plant communities. These genetic effects interact either
through the formation of sub-populations for certain genotypes or by reducing the
environmental effects on the genetic diversity of key species (Franks et al. 2007; Evans et
al. 2008; O'Neill 2008; Keith et al. 2010). These researchers also found that around 85% of
ecological patterns were reduced due to genetic diversity, while only 13% increased its effect
and 8% created new patterns. The higher amount of genetic diversity is equal to abundant
phenotypic variation that further allows a greater diversity of visitors (Barbour et al. 2020).

Fig. 1.3: The three major evolutionary transitions in the Plant reproductive system.(
Self-created with reference to Spencer C. H. Barrett et al.; 2010)

1.5 Evolution in Pollination from a Genetic Perspective

It has been suggested that evolutionary transitions to non-landing pollinators from landing
pollinators involve the loss of the conical cell form (Ojeda DI; et al. 2016). Ojeda et al.
stated that all of the Canary Island species of Lotus had lost conical cells on petals along with
a shift to bird pollination. An in-depth study of bird-pollinated species across the
Macaronesian Islands indicated that plant species that had shifted to a complete dependence
on birds as pollinators had lost conical petal cells as a part of gradual evolution. This also
suggested the evolutionary lability of conical cells.
It is suggested that plants have to face selective pressures including limited availability of
animal pollinators and escape from the floral herbivory. It is believed that selective pressure
for rapid growth and reproduction in plants growing in marginal habitats may also promote
the evolution of selfing, which will allow faster reproduction(Fattorini, R., & Glover, B.
J.;2020). Researchers found some plants that showed coevolution between plants and
pollinators at the species level, where small changes in floral traits or pollinator preferences
are believed to lead to speciation. These kinds of interactions, where both the plant and
animal species are highly specialized, are uncommon and involve pollinating seed consumers
eg. insects, and involves sexual deception. The most beautiful example include pollinating
seed consumer mutualisms of the figs, yuccas, senita cacti, and Phyllantheae, and the
sexually deceptive orchids (Cozzolino & Widmer 2005, Fleming & Holland 1998,
Kawakita & Kato 2009, Pellmyr et al. 1996, Ronsted et al. 2005)

2. Challenges and constraints

Lord, E. M. et al.(2002) faced many challenges and gaps in their understanding of several
areas related to the evolution of plant reproduction strategy. It is suggested that we must
combine candidate gene approaches with genetic mapping preceded by field studies testing
which can provide us with information regarding various floral traits. Studies concerned with
the complete impact of phosphorylation and Ca2+on grass SI response are scarce - we do not
have an in-depth understanding of its complete logic. Even after recent advancements, we are
still unclear about the fact as to how land use and climate change affect plant-pollinator
interactions, especially in the Neotropics region(Dalsgaard 2020). Researchers believe that
with the improvement in technologies, such challenges can be combated.

3. Conclusion
Exploring the imperative properties of the floral phenotype requires more collection of
genetic data based on morphological, developmental, and evolutionary research. Researchers
have tried to integrate genetics with agriculture giving birth to “modern agriculture”. They
have also been successful in this aspect because of recent technical advancements in genetics
and genomic studies, aiding in the identification of genes and mechanisms important for
producing genetic diversity. The relation between genotype and environment is proved to be
evolutionary, as phenotypic evolution is the result of ecological change through the process of
natural selection. Various influencing genes promoting pollinator-associated floral trait
variation have been identified in several plants. Studies and identification of more candidate
plant S genes will help in targeted modification and development of hybrid crops having
better production and survival traits.
4. Future Perspectives
A huge potential lies in comparative molecular analyses of plants like Thalictrum which can
undergo both wind and animal pollination. Identification of more genes and their
corresponding mutations resulting in floral trait variation concerned with pollinator
specificity is important to understand how pollinator shifts occur between closely related
plant species. Only a few studies have been done with plants of agronomical importance. The
yields of crops with high value such as fruits, nuts, and vegetables depend majorly on
pollination success. Therefore, it the important to take into consideration their floral
metabolic traits while doing plant breeding. GLR(GLUTAMATE RECEPTOR-LIKE
channel) gene is an important candidate gene to reveal the role of Ca2+ signaling in SI
response, further studies are important to understand the exact biochemical mechanism and if
its function is haplotype-dependent. Also, future studies are required to understand the
complete influence of phosphorylation and Ca2+on grass SI response. The mechanism behind
the detoxification of S-RNases in compatible pollen also needs to be further studied. Future
studies should work on the hypothesis regarding the collaborative recognition system and
increase in differentiation of new S specificities in plants. Also, more in-depth studies are
required for a better understanding of the dynamics between landscape and the genetic
diversity of key plant species.
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