Plant Physiology I

Section II: Embryo and Seed Development:

Lecture IX: Seed Dormancy:
First, dormancy is not a trait that is unique to seeds. Buds too can be dormant in plants that grow
in climates that have seasonal variations in favorable growing conditions. These buds can be on
tubers and bulbs, or be the apical meristems of shoots and roots.
Quiescent vs dormant: Live seeds in which none of the germination events are taking place,
usually due to a low moisture content, are said to be quiescent. They are alive and have
metabolism ongoing at a barely detectable rate but some environmental factor necessary for
germination to commence (usually the addition of water) is necessary for them to commence
germination. Seeds that are in an environment optimal for germination, that is to say they are
provided with ample water, heat, light, and oxygen and yet fail to complete germination are said
to be dormant. The block to the completion of germination is an attribute of the seed itself and
must be removed before the seed will be able to complete germination under favorable
conditions.
Why dormancy?: Since the purpose of the seed is to complete germination and produce the next
generation of plant why some seeds should be shed with an inherent block to germination is not
immediately obvious. It can be argued that the developing embryo of all seeds goes though a
period of dormancy since, upon removal from the seed, slight desiccation, and germination on
nutrient media, many embryos can complete germination whereas they cannot do so in the seed.
This inability to precociously complete germination has been found to be largely due to elevated
ABA amounts in the seed and tissue surrounding it during development on the mother plant. The
advantages of this imposed block to germination during the development of a seed are easily
understandable. But upon dissemination from the mother plant, why would mature seeds be
incapable of completing germination without first undergoing some environmental stimulus and
what evolutionary advantage could this hold? Particularly for plants growing in climates that vary
considerably in their favorability for plant establishment (e.g. seasonal cold, dry, or flooding
variations) immediate germination is not a desirable attribute. There must be some way of
delaying the completion of germination until conditions favorable for the germination process and
seedling establishment are, again, prevalent. Ideally, the environmental variations that occur are
seasonal in nature, with conditions favorable and unfavorable for germination and establishment
cycling yearly (e.g. cold in temperate or boreal climates). In this example, the switch from the
conditions that make it difficult or impossible to complete germination and establish successfully
(winter; freezing temperatures and lack of liquid water) to conditions conducive to completion of
germination and establishment (spring; warmth, rain, and melting ice and snow) can be used as a
cue to alleviate dormancy and permit the seed to complete germination at the start of an optimal
growing period. Seeds from plants of desert annuals do not have the luxury of such a predictable
cycle between a period conducive to establishment and one that is not and must be capable of
reacting quickly to the availability of water at whatever period they get it. In such unpredictable
climates, it is common to have a large variation in the degree of dormancy that individual seeds
from the same plant experience. Hence, some seeds can be released with practically no
dormancy, while others are deeply dormant. This spreads the number of seeds from a particular
plant that are completing germination out over time and maximizes the possibility that some, at
least, will establish successfully.
The role of ABA in inducing/maintaining seed dormancy: There have been innumerable studies of
the types of dormancy exhibited by seeds and the covers or inhibitors responsible for dormancy
imposition. In some cases, the distinction is not clear, as in arabidopsis. Wild type arabidopsis
seeds, at least those of the land race Landsberg erecta (Ler) have coat imposed dormancy. This
dormancy is initiated by an increase in ABA content occurring about halfway through normal seed
development. ABA concentration then declines during the latter phases of development until seed

maturity when very little ABA is present in the seeds. Hence, it is not the presence of the inhibitor
ABA that imposes dormancy on the embryo but the presence of the testa. There have been testa
mutants isolated that show a reduced amount of dormancy. These so-called seed shape
mutants do not need a period of afterripening to be able to complete germination to a high
percentage. It is thought that the mutation, which alters the shape of the testa, also weakens it
physically eliminating it as a barrier to radicle protrusion. However, work with the ABA deficient
mutants of arabidopsis has shown that without ABA, testa imposed dormancy is not initiated and
the embryos complete germination readily! So some aspect of the presence of ABA during
development must trigger changes in the arabidopsis testa and/or embryo that result in dormancy
imposition by this seed part in the mature seed. Elegant work using an ABA deficient mutant was
performed that elucidates where the ABA that is responsible for establishing dormancy arises. Is
the ABA from the mother plant that translocates the hormone to the developing seeds or is it from
the seeds themselves as they develop on the plant? Homozygous mutants (aba) of a recessive
gene imparting ABA deficiency were crossed with wild type (Aba) plants. The resulting progeny
were all heterozygous Aba/aba and were backcrossed to the mutant (aba) (Fig.1). This resulted
in seeds of two genotypes developing on heterozygous plants, plants that could all produce ABA.
The seeds produced by the backcross however were either heterozygotes themselves (Aba/aba)
and capable of producing ABA or homozygous for the recessive mutation (aba/aba) and ABA
deficient. An analysis of seed dormancy showed that dormancy was a trait of only those seeds

aba/aba
(ABA
deficient)

Aba/Aba
(wild type
produces
ABA)

aba/aba G
(ABA
deficient)

Aba/aba E
(Heterozygote
produces
ABA)

ing
dur
er
ent
Lat
pm
elo
dev lant
que e p
sili sam

1:1 segregation
of 2 dormant
Aba/aba heterozygotes to 2
non-dormant
aba/aba homozygote.

G
aba

aba

Aba

Aba/aba

Aba/aba

aba
aba/aba

aba/aba

Punnet
square

Aba/aba E plant
(Heterozygote
produces
ABA)

Figure 1: Seed dormancy in arabidopsis is installed by seed produced ABA during seed
development. Maternal ABA is not responsible for initiating dormancy in seeds produced on the
plant.
that were heterozygous, the homozygotes being without dormancy. If the mother plant was
providing the ABA to the seeds that was responsible for the imposition of dormancy, all seeds
from the back cross would be dormant, regardless of their genotype. Since this was not the case,
the ABA responsible for dormancy imposition in arabidopsis must be seed derived (Fig. 1).
ABA plays a major part in initiating dormancy in seeds, but it is not alone in this process.
Mutants have been isolated that complete germination without a requirement for GA. The seeds
completed germination on media containing inhibitors of GA biosynthesis. Of these mutants,
some were ABA deficient or insensitive but one, spindly, was hypersensitive to GA. This signifies
that the antagonism mentioned in the context of seed germination between these two potent plant
growth regulators is also functional in determining whether a seed is dormant or not. Large
amounts of GA or hypersensitivity to it, lead to a non-dormant phenotype as does the lack of ABA
or insensitivity to it. Based on the phenotype of ga mutants deficient in GA, lack of GA leads to
extreme dormancy that cannot be alleviated without exogenous application of this hormone.
Additionally, the reduced dormancy (rho) mutants of arabidopsis are not deficient, insensitive, or
hypersensitive to either ABA or GA. Their testa color is normal as is testa shape, unlike the
transparent testa (tt) and seed shape mutants, respectively both of which can display reduced
dormancy. The rdo mutations are further divorced from mutations affecting the testa by the
observation that the F1 progeny of a cross between the homozygotic mutant Eand wild-type G
produced a wild type phenotype indicating the mutations affect the embryo and not maternal
tissue (testa).
Metabolism of dormant seeds: There have been many different metabolic pathways hypothesized
to play a role in alleviating dormancy in seeds over the years that this phenomenon has been
studied. The synthesis of nucleic acids or proteins has been implicated as has a switch in
metabolism which alters gas exchange, adenylate charge and/or abundance or uses an
alternative pathway to produce energy and reducing power such as the pentose phosphate
pathway (PPP), reverse glycolysis and cyanide-insensitive pathway. Some of the evidence
accumulated has debunked several of these metabolic changes from having any influence on
dormancy alleviation. Others, such as the PPP, are still under investigation. Support for the PPP
being involved in dormancy alleviation comes from observations that the dormancy of some
species seeds can be alleviated by the application of inhibitors of respiration. Substances that
inhibit terminal oxidation and the tricarboxylic acid pathway are effective in alleviating dormancy
in these seeds as are inhibitors of glycolysis. Additionally, electron acceptors can alleviate
dormancy. These inhibitors are thought to function by diverting cellular oxygen from regular
respiration to the PPP, where it is used to oxidize NADPH to NADP. The electron acceptors
replace oxygen in this capacity and again result in elevated amounts of NADP. However, studies
have shown that the NADP/NADPH ratios are completely unrelated to the dormancy status of the
seeds. Whatever is occurring to alleviate dormancy upon application of inhibitors of respiration or
electron acceptors, it does not appear to be due to elevated NADP amounts.
One of the problems defining metabolic limitations that impose seed dormancy is that
different species seeds behave differently and may alleviate dormancy through a different
metabolic switch than others. Another is accurately determining the dormancy imposing tissue.
For instance, lettuce embryos appear to be constrained by the endosperm and it is this tissue that
imposes dormancy upon them in the absence of light. Illuminate the seed and you alleviate
dormancy. This could occur through either cell wall weakening of the endosperm or increased
embryo thrust permitting them to push through the endosperm opposing their expansion. Excised
embryos complete germination in darkness with no apparent dormancy. However, if they are
placed under water stress, the excised embryos germinated in the dark fail to elongate at much
less sever water deficits than embryos germinated in the light suggesting that light enables the
lettuce embryo to generate more thrust than is possible in the dark. So, the embryos in light have

a lower water potential than embryos germinated in darkness. What is different between the two?
Are the light-germinated-embryos more osmotically active or do they have more extendable cell
walls resulting in lower turgor pressure? Measurements of osmotic potential failed to reveal
differences between light-germinated and dark-germinated embryos. However, a decrease in
turgor pressure has been documented in response to light. This means that the cell walls of the
embryo, when it is illuminated, weaken, allowing the cells to elongate more easily than if the
embryo was held in darkness. The mechanism though which light acts appears to be in a pH
decrease in the apoplast. There is a vacuolar proton ATPase that is upregulated during
germination in tomato, but to date, no report of any of the number of plasmamembrane proton
ATPases known to exist being likewise regulated has been documented. However, isolated
lettuce embryos do seem capable of decreasing the pH of the media they are in if illuminated,
providing strong evidence that such a proton pump does exist.
Phytochrome and Dormancy: One of the more spectacular discoveries in plant physiology
involved the control light quality has on the ability of many species seeds to complete
germination. There are no fewer than 5 phytochromes present in arabidopsis. These
chromophores were first discovered and investigated due to the marked effect phytochrome B
and possibly others, has on lettuce seed germination. When imbibed seeds of lettuce were
illuminated with a period of far-red light, the percentage of seeds that subsequently completed
germination in the dark was very low. If the period of far-red illumination was followed by a period
of red light illumination, the seeds subsequently completed germination to almost 100%. If
nmred light illumination 730
nm by another period of far-red illumination,
however, the 660
second,
was followed
seed germination was again drastically inhibited in the dark. This cycle of germination inhibition
and stimulation can continue ad infinitum until, at some point much advanced in seed
germination, the seeds escape from phytochrome control and complete germination in the dark
regardless of the illumination they perceived last.

Pr

Dark reversion
thermally induced

Lumi-R

Pr

Meta-Fb

Dormancy
alleviation

Pfr

Destruction

Meta-Rb

Meta-Ra
t
dra
y
h
in nly.
g
o
rrin ues
u
c
s
Oc tis

Meta-Fa

ed

Pfr

Lumi-F

The antagonistic nature of red/far red illumination on seed dormancy has led to intensive
Figure 2:A) The photoconversion of phytochrome to an active (Pfr), or inactive form (Pr)
depending on incident light quality, duration in the dark at elevated temperatures, or destruction
over time (Pfr half-life = 1.25 h). B) Detailed schematic of the intermediates through which the two
forms of phytochrome
proceed upon conversion. Thick arrows represent nonphotochemical
A.
steps, thin arrows represent conversions requiring light. Certain reactions proceed only in
hydrated tissue.
investigation of phytochrome states after illumination from light of different spectral qualities.
These investigations led to a model whereby the same chromophore undergoes photoconversion
upon illumination to one of two somewhat stable forms. Pr designates the red light absorbing form
of phytochrome that absorbs light maximally at 660 nm and which is inactive in alleviating
dormancy. Pfr on the other hand is a form of phytochrome that absorbs light maximally at 730 nm
and is active in alleviating dormancy. Photoconversion from Pr to Pfr and vice versa is freely
reversible, requiring only illumination with the appropriate quality of light. Additionally, the Pfr form
is labile, subject to destruction with a half-life of only some 1.25 hours. Pfr is also thermal labile,
reverting in darkness back to Pr at elevated temperatures (Fig. 2). Although their respective
maximas have been indicated, both forms of photochrome absorb over the range from 300-730
nm. Even at 660 nm, where Pr absorbs maximally and is photoconverted to its Pfr form, some
small proportion of Pfr is still capable of absorbing some light and photoreverting back to the Pr
form. This sets up a photoequilibrium between the two forms of phytochrome that will be unique
at eachB.
wavelength and in each mixture of incident light qualities. This photoequilibrium, has
been measured and is designated as Pfr/Ptotal, = . At 660 nm about 80% of the phytochrome
exists in the Pfr form and 20% in the Pr form ( = 0.8). At 730 nm, about 2% of the phytochrome
exists as Pfr ( = 0.02). Since, in nature, light quality is constantly changing due to cloud cover,
vegetation cover, etc. also varies considerably. The seeds of various species have specific
values at which they alleviate dormancy and which have evolved to maximize reproductive
success in their respective climates.
As mentioned previously, there are at least 5 different phytochromes present in
arabidopsis. Of the phytochromes, A and B are by far the best studied, having been cloned and
for which null mutants are available. Studies of the phytochrome A deficient, phytochrome B
deficient, and double mutant, have led to the conclusion that both phytochrome A and B play a
role in seed germination. Phytochrome A, upon absorbing far red light is actually stimulatory for
germination while phytochrome B is the phytochrome that is purportedly responsible for the
photoreversible effect on seed dormancy. Recently, however, the arabidopsis double
phytochrome AB deficient mutant was used to show that seed germination is still under
phytochrome control, signifying that at least a third phytochrome is involved in determining light
regulated seed dormancy in arabidopsis.
Studies with microbeams of intense light at different wavelengths has demonstrated that
phytochrome is present in greatest amounts in the axis of embryos and very little is present in the
cotyledons. This is significant since it is the elongation of the radicle that leads to the completion
of germination and the phytochrome control of dormancy probably should be localized to this
portion of the embryo rather than the cotyledons. No mention was made of whether phytochrome
was also found in the endosperm. This is not crucial to our interpretation below that, in some
seeds at least, phytochrome mediated endosperm wall weakening might control dormancy and
germination. It is fully possible that, even if phytochrome is present only in the axis, its
photoconversion to the active form Pfr elicits a response from the radicle cells that includes the
production and transport of a signal to the cells of the endosperm in the immediate proximity of
the radicle i.e. the micropylar endosperm. This signal would then dictate that the endosperm cells
should produce enzymes that would weaken their cell walls.

Partial cell wall disassembly and dormancy: Recently more interest has been directed to
determining whether or not the cell walls of the endosperm cap region in endospermic seeds
weakens during dormancy alleviation. If the micropylar endosperm does not weaken, as we saw
in the gib-1 mutant of tomato and ga mutant in arabidopsis, the embryo will not complete
germination. There does appear to be some evidence suggesting that endosperm weakening
does have to occur in several species and that this weakening might be prevented in seeds that
are dormant. The enzymes that have been investigated to date all hydrolyze hemicellulose
present in the cell walls of the micropylar endosperm. In Datura ferrox endo--mannanase and a
cellulase, both of whose transcription is under phytochrome control, accumulate prior to radicle
protrusion and endosperm cap weakening. In lettuce, endo--mannanase also appears to be
under phytochrome control and may accumulate prior to radicle protrusion, although this is
contentious. In tomato several forms of endo--mannanase exist, one of which is apparently
specific to the endosperm cap the gene of which is downregulated by ABA and upregulated by
GA prior to the completion of germination.
Secondary (skoto) dormancy: Secondary dormancy is brought about when fully hydrated, mature
seeds experience gravely sub-optimal germination conditions. Some of the conditions that invoke
secondary dormancy include, anoxia, darkness, excessively strong light, higher (lower) than
maximal (minimal) temperatures for the completion of germination, and water stress. These
conditions do not universally elicit secondary dormancy for all species, some species are affected
and other are not. Whatever the mechanism whereby skotodormancy is initiated, ABA does not
appear to be involved, at least not in the response of arabidopsis to higher than maximal
temperatures for germination. Even ABA deficient mutant seeds are capable of skotodormancy.
Secondary dormancy plays a large part in the maintenance of a persistent seed bank of
numerous species. Those seeds that fail to complete germination, for whatever reason, during
the favorable portion of the year for them are prevented from radicle protrusion later due to
unfavorable conditions. Prolonged exposure to these unfavorable conditions elicits secondary
dormancy that persists until the seeds undergo a dormancy alleviating treatment. This treatment
is often overwintering for summer annuals or afterripening during the next summer for winter
annuals. Return to favorable climatic conditions gives the now non-dormant seeds a second
chance to complete germination. Often, secondary dormancy decreases the seeds sensitivity to
chemicals known to promote germination of non-dormant seeds. Such is the case with the small
annual weed, Sisymbrium officinale which decreases its sensitivity to both light and nitrate upon
entering secondary dormancy.
Post-germinative events:
When a seed is planted in soil and successfully completes germination, depending on
where the cotyledons are situated upon the completion of seedling establishment the mode of
germination is described as epigeal, where the cotyledons are raised above the ground on a
hypocotyl, or hypogeal, where the cotyledons remain below the ground. In the case of epigeal
seedling establishment, the hypocotyl expands pushing the shoot apex upward through the soil
enabling it to reach the light. In the case of hypogeal establishment, the hypocotyl does not
elongate or does so only slightly, and the epicotyl does to preponderance of expansion to
penetrate the soil over it.
Upon radicle protrusion, the major stored reserves of the seed are mobilized to support
this rapid growth. In non-endospermic seeds this mobilization keeps pace with the requirements
of the growing seedling. In endospermic seeds however, it behooves the embryo to extract the
nutrients contained within the endosperm as quickly as possible so that, once exhausted it can be
discarded leaving the seedling free to establish. However, if the reserves in the endosperm are
mobilized and drawn into the seedling as sucrose or other osmotically active substances in
amounts that exceed the embryos immediate use, their build up will result in the cells of the
embryo becoming extremely osmotically active. The water potential of the cells will plummet
driving water uptake to the point where turgor pressure would rupture the cells. To avoid this
many endospermic seeds that mobilize their endospermic reserves quickly (e.g. fenugreek)

temporarily store the influx of sucrose as starch in the cotyledons! Starch is an ergastic
substance, not being dissolved in the amyloplast and therefore osmotically inert. Hence, the
embryo re-allocates its reserves from the endosperm which are exterior to the body of the embryo
to the cotyledons which are part of the embryo. Basically, the same reallocation occurs in pea
seeds during development so that, upon dissemination, all the reserves a pea seed requires for
establishment are contained within the embryo.