Plant Physiology I

Section II: Embryo and Seed Development:

Lecture IX: Seed germination: Orthodox seeds undergo maturation desiccation and some remain
on the mother plant for some time in a fruit or cone. Eventually, however, all seeds are released
from the mother plant and dispersed by the elements or by other living things. It is in the guise of
the seed that the plant is its most resistant to external stress. For example, it is possible to take
many desiccated orthodox seeds and immerse them in liquid nitrogen (-196C), take them out
after one minute or one decade, warm them, water them and they complete germination normally!
Their resistant nature permits seeds to withstand periods of unfavorable environmental
perturbation and sustains life until conditions more favorable to germination and establishment
arise. What is germination? Strictly speaking germination commences when a seed imbibes
water and terminates as soon as the radicle emerges from the seed covers (e.g. testa, nucellus,
perisperm, endosperm). The inappropriate use of the term to also describe radicle protrusion and
seedling establishment has lead to considerable confusion and necessitates covering
GERMINATION and POST-GERMINATION events (which will be covered under the lecture on
dormancy) separately for emphasis.
Anhydrobiosis: Mature, desiccated seeds can have a moisture content of less than 3% by weight
and yet they are alive, they respire, although it is difficult to measure since it occurs at such a
reduced rate. If seeds are capable of completing germination upon the provision of light, heat,
water, and favorable atmospheric conditions, when desiccated they are said to be quiescent. If
they are incapable of completing germination upon entering favorable conditions due to some
internal impediment to radicle protrusion they are said to be dormant. We will discuss seed
dormancy and its implications in the next lecture.
Germination events:
Water uptake: Seed water uptake can be divided into three stages (Fig. 1). Imbibition, the first
stage, is rapid, commencing with the seed being placed on water where it quickly hydrates the
cells and their constituents. This is followed by the lag phase of water uptake, where there is very
little net gain of water. This is not to say that water is not taken up by the seed. In reality there is a
steady state reached between the amount of water lost by the seed due to evaporation and that
taken up by the seed from its surroundings. In natural conditions, the amount of water lost by a
seed that is placed on the surface of the ground can actually exceed the amount it takes up from
the soil and limits germination. Finally, the third phase of water uptake is one of rapid hydration to
support the expansion of the embryo as it completes germination and emerges from the seed
(Fig. 1). Due to the impermeability of the testa or other covers, some seeds cannot take up water
freely when placed in contact with it but must wait for some physical abrasion or partial chemical
breakdown to occur prior to imbibing.
The antagonistic role of ABA and GA in seed germination: Martin Koornneef and co-workers have
isolated many mutants involved in seed development and germination. Two such mutants a
series of abscisic acid deficient or insensitive mutants and several mutants affected in their ability
to synthesize or perceive gibberellic acid have been instrumental in elucidating the fundamental
role the interplay of these two potent plant hormones has on seed development and germination.
ABA is present in the seed during development, peaking in concentration during the
period of maximum stored reserve accumulation. It is currently thought that ABA regulates when
and to what extent storage proteins are synthesized in maize. This theory arises from the
behavior of the ABA deficient (vp5) and ABA insensitive (vp1) mutants of maize which do not
accumulate a specific globulin. Neither the protein nor its mRNA is present in either mutant during
development. Upon application of exogenous ABA, globulin production can be stimulated in the
vp5 mutant but not the vp1. Strangely enough and probably partially based on differences in
storage protein gene promoter characteristics, neither the ABA deficient arabidopsis (aba) or

Leakage
Translation from stored mRNA
DNA repair
Mitochondrial repair
Translation from newly transcribed mRNA
Mobilization of soluble sugars (sucrose and the
raffinose family oligosaccharides)
Mitochondrial synthesis
Mitosis and cytokinesis
Radicle
elongates
Insoluble stored reserves mobilized
Phase 2

Moisture Content

Phase I

Phase 3

Seeds commence
radicle protrusion

Time on water
Seeds placed
on water

Figure 1: Time course of water uptake by an orthodox seed depicting the three stages of
imbibition (phase 1), lag (phase 2), and completion of germination (phase 3) as well as the
cellular events thought to occur in each phase (Redrawn and altered from Bewley 1997, The
Plant Cell, 1055-1066).
tomato (sitw) mutants have deficiencies in storage protein accumulation. However, the
arabidopsis mutants, including those insensitive to ABA (abi1, 2, 3) are all somewhat leaky and
the abi mutants all have higher than normal concentrations of ABA so a lack of abnormal storage
protein accumulation may be due to the presence of some ABA and/or the partial sensitivity of the
embryo to it. Recently, a new ABA insensitive mutant, abi3-3 has been isolated which is totally

insensitive to ABA and this mutant does not accumulate either the 12S or 2S (S = Svedberg Unit,
the rate of sedimentation of a macromolecule in a centrifugal field) storage proteins. ABA is also
known to prevent the precocious germination of embryos in developing seeds. When removed
from the seed, many developing embryos that have attained maximal dry weight or close to it can
complete germination on media if they have been desiccated slightly (5-10% loss of fresh weight).
The ABA in the seed is thought to prevent this from occurring in vivo until the seed commences
maturation desiccation, at which point there is insufficient water available to the seed to permit
the completion of germination.
GA is a potent stimulatory hormone for seed germination. The fact that sever GA deficient
mutants in arabidopsis and tomato will not complete germination without an exogenous supply of
GA indicates how crucial this hormone is to seed germination. The current wisdom is that there is
still sufficient ABA present in the mature desiccated seed to inhibit germination. The newly
synthesized GA made during the lag phase of water uptake overrides this ABA inhibition or
hastens ABA metabolism to the biologically inactive metabolite phaseic acid permitting radicle
protrusion. However, treatments that prevent ABA metabolism such as illumination with far red
light (730 nm) inhibits the completion of germination. Addition of exogenous ABA to seeds also
prevents the completion of germination, seemingly overwhelming the stimulatory effect elicited by
GA biosynthesis or preventing the synthesis of GA altogether.
Cellular events during seed germinationTHE original (and persistent) black box: After phase I
of water uptake, the cells within the seed must reconstitute their lipid membranes, repair damage
to DNA and protein incurred during the quiescent phase, replace extensively damaged
membranes, resume transcription, translation, and respiration, and commence instituting the
sensory machinery to enable it to monitor its external environment to determine if conditions are
favorable for establishment or not. And yet there is not a single visible exterior change
discernable between the live and the dead seed, the desiccated and the imbibed! Even at the
ultrastructural level, very few changes are detectable during seed germination and those only a
few hours prior to the completion of germination. There are, however, three changes that can be
discerned in many seeds that are about to complete germination but have not yet done so. They
are the differentiation of the provascular cylinder of the embryo into xylem and phloem, some very
limited mobilization of stored reserves in the radicle tip and, in endospermic seeds, the micropylar
endosperm, and, again in endospermic seeds, partial cell wall disassembly of the cells of the
micropylar endosperm.
The differentiation of the provasculature into xylem and phloem is necessary in order to
conduct metabolites from stored reserves located in the cotyledons or endosperm into the
growing radicle and hypocotyl. Additionally, this permits the embryo to conduct water taken up
from the soil immediately upon the completion of germination to support continued cell expansion
throughout the embryo. Do not forget that, upon successful completion of germination, the
cotyledons are raised from the surface of the soil, losing any chance to absorb water by diffusion
and therefore are dependent on functioning vasculature for water supply. The ABA insensitive
mutant (abi 3-4) and embryogenesis mutation leafy cotyledon (lec 1-2) both prematurely
differentiate their vasculature while the embryo is developing.
The limited hydrolysis of stored reserves in the radicle tip and micropylar endosperm
adds energy and carbon and nitrogen building blocks to the actively growing radicle during the
commencement of growth. Typically, this replaces the reserves of sucrose and raffinose family
oligosaccharides that have been exhausted during the first stages of germination (Fig. 1). It must
be emphasized that this mobilization of protein and carbohydrate/lipid is in a very limited part of
the seed and the vast majority of stored reserves of protein and carbohydrate/lipid remain
unmobilized until after the radicle has protruded from the seed (i.e. is a post-germinative event,
see below)!
The partial disassembly of the cell wall of the region of the endosperm or other covers
opposite the radicle has been studied extensively in endospermic seeds. Candidate enzymes that

hydrolyse the hemicellulosic or pectin components of the cell wall that have been studied in this
regard include endo--mannanase which randomly cleaves polymers of mannose anywhere
along the backbone into shorter fragments, xylo-glucanases that randomly cleave xyloglucan
polymers, expansins that simply disassociate hemicellulose polymers from para-crystalline
cellulose microfibrils without cleavage, xylo-glucan endotransglycosylases that cleave and then
re-polymerize xyloglucan polymers, and polygalacturonases that cleave single galacturonic acid
moieties from the non-reducing ends of polymers of galacturonic acid (pectin). All of these
hydrolases, transglycosylases, expansins probably play an interactive role in weakening the
resistance of the endosperm or other covers to puncture by the radicle. Hence, it becomes
increasingly more easy for the embryo to push through or between the cells of the covers and
emerge. This decrease in resistance to puncture has been demonstrated for many angiosperm
and one conifer seed. Indeed, the requirement of endosperm weakening to permit radicle
protrusion, in endospermic seeds, is one of the few facets of seed germination that is absolutely
known through direct measurements of resistance to puncture using an instron (instrument
capable of measuring extremely small forces). Additionally, the pleiotropic mutant deficient in
gibberellic acid in both arabidopsis (ga) and tomato (gib-1) does not complete germination unless
the seeds are provided with exogenous gibberellic acid or the testa and endosperm cap are
removed surgically. The endosperm cap of the tomato gib-1 mutant does not weaken as the wild
type does unless exogenous GA is applied. The isolation of gib-1 by Koorneef and co-workers
and the absolute requirement for seed germination in establishing every crop grown the world
over has lead to several attempts to identify other seed germination specific proteins through
mutagenic approaches with some limited success. Mutagenizing seeds in the quest of nongerminating or alter germination phenotypes is an approach embraced by increasingly more labs
as we realize that germination is an extremely complex phenomenon unlikely to be elucidated by
more traditional experimental approaches. However, this same complexity has been used by
other seed scientists to caution that radicle protrusion is so fundamentally important that it is likely
to be comprised of many redundant systems and that a mutagenic approach will ultimately fail
since a backup for a lesion in a pathway will always be available. Only time and experimentation
will determine if those conducting mutagenic studies are destined to be thwarted or not in their
search for a "germination gene". However, recently, some success in this endeavor has been
achieved.
The banyuls (ban) mutant (isolated by Albert et al. 1997 Plant Journal 11: 289-299 in the
Devic lab in France) accumulates flavonoids or their derivatives in the testa resulting in a reddish
tinge to the testa. This also decreases seed germination to about half that of the wild type and
this inhibition is not due to a difference in the spectral quality of the light impacting on the embryo
through the mutant testa. Currently, it is thought that the high amounts of flavonoids in the testa
may in themselves be toxic or at least inhibit normal germination of the embryo, although it would
seem as though, if toxic the embryo should have died prior to attaining maturity. To date there
has been no test of whether excised embryos are all capable of completing germination on
germination media or even if most are, in fact, dead.
The availability of a large assemblage of independent T-DNA tagged lines of arabidopsis
has permitted the screening of arabidopsis seeds for non-germinants by the Caboche lab also in
France (Dubreucq et al. 1996 Mol. Gen. Genet. 252: 42-50). Their intensive screening has led to
only a few non-germinating mutants, one of which is a new loss-of-function mutation at the GA1
locus, as yet unnamed.
There are three mutants that all permit precocious germination in seeds and which all
share the ability to stimulate germinative and/or post-germinative morphological changes in the
developing embryo. The ABA insensitive (abi3-4), fusca (fus3-3), and leafy cotyledon (lec1-2)
mutants of arabidopsis all encode putative transcription factors that, when abnormal, result in the
loss of desiccation tolerance and a propensity for vivipory.
There has been considerably more success in recovering mutants that complete
germination under, what are usually considered, unfavorable conditions. Three mutants of

arabidopsis that produce seed that are capable of completing germination under saline
conditions, resistant to salt (rs), have been isolated (Saleki et al 1993. Plant Physiol. 101: 839845). Strangely enough, tolerance to high salinity was not present in the vegetative stage in any
of the mutants, strictly during germination and early emergence.
A dominant mutant that is resistant to sulfonylurea herbicides (Chlororsulfuron
resistant, Csr-1) was found to contain a lesion in the acetolactate synthase gene (ALS), the
limiting enzyme in branched chain amino acid biosynthesis. This point mutation was found to
prevent sulfonylurea herbicide action on ALS, its usual site of action. Serendipitously, it also
removed ALS from feedback inhibition by its products. This, for an as yet unknown reason, lead
to the ability of seeds to complete germination at cold temperatures. Two schools of thought exist
concerning this ability of the mutant ALS gene to permit completion of germination at lower
temperatures than usual. One is that the accumulation of branched chain amino acids that
occurs with this mutation depletes precursors inhibitory to entry into the cell cycle allowing the
cells of the embryo to commence elongation at colder temperatures where they usually would
not. Conversely, the very accumulation of branched chain amino acids themselves may serve to
stimulate DNA synthesis and permit early entry into the cell cycle with the same result. Needless
to say, both the depletion of inhibitory precursors and accumulation of branched chain amino
acids could work synergistically to permit cell division, embryo elongation, and the completion of
germination at prohibitively cold temperatures.
A gene that was transformed into wild type arabidopsis has been shown to confer to the
seeds the ability to complete germination at colder temperatures than usual. Hayashi et al. 1998
transformed arabidopsis with the choline oxidase gene from a soil bacterium driven by the
cauliflower mosaic virus promoter. Choline oxidase catalyses the conversion of choline to
betaine, a known and potent protectant molecule in bacteria, plants and animals permitting
enhanced stress tolerance. Transgenic arabidopsis plants were capable of imbibing and
completing germination at temperatures that killed wild type plants. Additionally, transformed
plants were capable of accelerated growth at temperatures that severely limited wild type growth
rates. Incidentally, the betaine overproducing plants were also capable of completing germination
and growing under elevated salt.