Allison R. Kermode

Role of Abscisic Acid in Seed Dormancy

Seed dormancy is an adaptive trait that improves survival of the next generation by optimizing the distribution of germination over time. The agricultural and forest industries rely on seeds that exhibit high rates of germination and vigorous, synchronous growth after germination; hence dormancy is sometimes considered an undesirable trait. The forest industry encounters problems with the pronounced dormancy of some conifer seeds, a feature that can lead to non-uniform germination and poor seedling vigor. In cereal crops, an optimum balance is most sought after; some dormancy at harvest is favored because it prevents germination of the physiologically mature grain in the head prior to harvest (that is, preharvest sprouting), a phenomenon that leads to considerable damage to grain quality and is especially prominent in cool moist environments. The sesquiterpene abscisic acid (ABA) regulates key events during seed formation, such as the deposition of storage reserves, prevention of precocious germination, acquisition of desiccation tolerance, and induction of primary dormancy. Its regulatory role is achieved in part by cross-talk with other hormones and their associated signaling networks, via mechanisms that are largely unknown. Quantitative genetics and functional genomics approaches will contribute to the elucidation of genes and proteins that control seed dormancy and germination, including components of the ABA signal transduction pathway. Dynamic changes in ABA biosynthesis and catabolism elicit hormone-signaling changes that affect downstream gene expression and thereby regulate critical checkpoints at the transitions from dormancy to germination and from germination to growth. Some of the recent developments in these areas are discussed.

Mechanisms of Intracellular Protein Transport and Targeting in Plant Cells

Abstract The specificity of protein targeting processes is the basis of maintaining structural and functional integrity of the cell, enabling the various subcellular compartments to carry out their unique metabolic roles. Studies in plants have progressed markedly in the last 5 years, and many of the specific signals involved in the transport and targeting of proteins to the nucleus, chloroplast, mitochondrion and microbody, and to organelles along the secretory pathway (endoplasmic reticulum [ER], Golgi complex, and vacuole) have been characterized. Exciting prospects include the identification of receptors involved in the recognition of protein targeting signals, mechanisms of vesicle targeting, and the role of mRNA targeting. Although important exceptions exist, a striking feature of the mechanisms and cellular machinery of protein targeting is their universality — among plants, animals, and eukaryotic microorganisms — and even between prokaryotes and eukaryotes. More information is required about the ...

Demethylesterification of Cell Wall Pectins in Arabidopsis Plays a Role in Seed Germination

The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor to investigate PME involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans, but there were no changes in the neutral sugar and uronic acid composition of the cell walls. As compared with wild-type seeds, the PME activities of the overexpressing lines were greatly reduced throughout germination, and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology but to alterations of the micropylar endosperm cells, evident by environmental scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm.

Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of Arabidopsis thaliana (thale cress) and other crucifers, sharing structural similarity with the cupin superfamily of proteins. Cruciferin is synthesized as a precursor in the rough endoplasmic reticulum. Subunit assembly is accompanied by structural rearrangements involving proteolysis and disulfide-bond formation prior to deposition in protein storage vacuoles. The A. thaliana cv. Columbia genome contains four cruciferin loci, two of which, on the basis of cDNA analysis, give rise to three alternatively spliced variants. Using MS, we confirmed the presence of four variants encoded by genes At4g28520.1, At5g44120.3, At1g03880.1 and At1g3890.1 in A. thaliana seeds. Two-dimensional gel electrophoresis, along with immunological detection using anti-cruciferin antiserum and antibodies against phosphorylated amino acid residues, revealed that cruciferin was the major phosphorylated protein in Arabidopsis seeds and that polymorphism far exceeded that predicted on the basis of known isoforms. The latter may be attributed, at least in part, to phosphorylation site heterogeneity. A total of 20 phosphorylation sites, comprising nine serine, eight threonine and three tyrosine residues, were identified by MS. Most of these are located on the IE (interchain disulfide-containing) face of the globulin trimer, which is involved in hexamer formation. The implications of these findings for cruciferin processing, assembly and mobilization are discussed. In addition, the protein phosphatase 2C-impaired mutant, abi1-1, was found to exhibit increased levels of cruciferin phosphorylation, suggesting either that cruciferin may be an in vivo target for this enzyme or that abi1-1 regulates the protein kinase/phosphatase system required for cruciferin phosphorylation.

Water uptake and oil distribution during imbibition of seeds of western white pine (Pinus monticola Dougl. ex D. Don) monitored in vivo using magnetic resonance imaging

Dry or fully imbibed seeds of western white pine (Pinus monticola Dougl. ex D. Don) were studied using high-resolution magnetic resonance imaging (MRI). Analyses of the dry seed revealed many of the gross anatomical features of seed structure. Furthermore, the non-invasive nature of MRI allowed for a study of the dynamics of water and oil distribution during in situ imbibition of a single seed with time-lapse chemical shift selective MRI. During soaking of the dry seed, water penetrated through the seed coat and megagametophyte. The cotyledons of the embryo (located in the chalazal end of the seed) were the first to show hydration followed by the hypocotyl and later the radicle. After penetrating the seed coat, water in the micropylar end of the seed likely also contributed to further hydration of the embryo; however, the micropyle itself did not appear to be a site for water entry into the seed. A model that describes the kinetics of the earlier stages of imbibition is proposed. Non-viable pine seeds captured with MRI displayed atypical imbibition kinetics and were distinguished by their rapid and uncontrolled water uptake. The potential of MR microimaging for detailed studies of water uptake and distribution during the soaking, moist chilling (“stratification”), and germination of conifer seeds is discussed.

Changes in late-embryogenesis-abundant (LEA) messenger RNAs and dehydrins during maturation and premature drying of Ricinus communis L. seeds.

InRicinus communis L. (castor bean) endosperms, two classes of Late Embryogenesis Abundant (Lea) transcripts were first detected during mid-development (at 30–35 days after pollination, DAP) and peaked at 50 DAP, just prior to the onset of desiccation. Most of the Class 1 mRNAs declined substantially during desiccation itself; Class 11 mRNAs remained abundant in the mature dry (60 DAP) seed. Following imbibition, allLea mRNAs abundant in the mature dry seed declined rapidly (within 5–24 h). Premature drying of developing 35-DAP seeds resulted in the loss of storage-protein mRNAs (Leg B Mat I); following rehydration, mRNAs encoding post-germinative proteins (Germ D91, D30 and D38) increased in the endosperm. TheLea mRNAs present in the developing fresh seed at 35 DAP were preserved, but did not increase in response to premature desiccation; upon rehydration theseLea mRNAs declined within 5 h. During seed development, substantial changes occurred in the synthesis of a subset of LEA proteins referred to as ‘dehydrins’; in particular, new dehydrin polypeptides were induced between 40 and 60 DAP. Such proteins were not as evident in prematurely dried endosperms. In contrast to the rapid loss ofLea mRNAs during germination, many of the dehydrin proteins abundant in the dried seed persisted following imbibition or rehydration.

Role of an ABI3 homologue in dormancy maintenance of yellow-cedar seeds and in the activation of storage protein and Em gene promoters

ABI3/VP1 proteins are members of a large group of transcription factors that act as intermediaries in regulating abscisic acid (ABA)-responsive genes during seed development, including those involved in reserve deposition, acquisition of desiccation tolerance and dormancy induction. CnABI3, an ABI3/VP1 gene homologue was recently cloned from yellow cedar, a conifer species that produces seeds that are deeply dormant at maturity. Here, we investigated whether the conifer ABI3/VP1 gene homologue shares characteristics with its angiosperm counterparts. CnABI3 was synthesized exclusively in seeds, with no detectable protein in leaves and roots. Stable expression of the CnABI3 gene in two transgenic tobacco lines previously transformed with chimeric constructs (vicilin and napin 5′ regions linked to a β-glucuronidase (GUS) gene-coding region) showed that the ectopic expression of the CnABI3 protein strongly activated both the vicilin and napin storage protein gene promoters in leaves and other vegetative tissues. GUS activities were up to more than 1000-fold of those in control plants. ABA had a synergistic effect, further enhancing GUS activity levels. When expressed transiently in yellow-cedar embryos, CnABI3 activated the expression of a chimeric Em-GUS gene in the presence of ABA. The role of CnABI3 in dormancy maintenance of yellow-cedar seeds was examined by monitoring the expression of the CnABI3 gene at the mRNA and protein levels before, during and after dormancy termination. CnABI3 protein was present in the megagametophyte and embryo of dormant mature and warm stratified seed, but declined during subsequent moist chilling, a treatment effective in breaking dormancy. In contrast, the protein was preserved (albeit in lower amounts) in seeds subjected to a control treatment (12 weeks in warm, moist conditions) that is ineffective in breaking dormancy. A decline in CnABI3 gene transcripts was also positively correlated with dormancy breakage, but did not occur during moist chilling itself, but rather during subsequent germination, indicating potential control at the post-transcriptional level.

Evolutionarily Conserved Histone Methylation Dynamics during Seed Life-Cycle Transitions

Plants have a remarkable ability to react to seasonal changes by synchronizing life-cycle transitions with environmental conditions. We addressed the question of how transcriptional re-programming occurs in response to an environmental cue that triggers the major life cycle transition from seed dormancy to germination and seedling growth. We elucidated an important mechanistic aspect of this process by following the chromatin dynamics of key regulatory genes with a focus on the two antagonistic marks, H3K4me3 and H3K27me3. Histone methylation patterns of major dormancy regulators changed during the transition to germination and seedling growth. We observed a switch from H3K4me3 and high transcription levels to silencing by the repressive H3K27me3 mark when dormancy was broken through exposure to moist chilling, underscoring that a functional PRC2 complex is necessary for this transition. Moreover, this reciprocal regulation by H3K4me3 and H3K27me3 is evolutionarily conserved from gymnosperms to angiosperms.

Effects of oil sands effluent on cattail and clover: photosynthesis and the level of stress proteins

The oil sands industry located in northeastern Alberta, Canada, generates large volumes of effluent characterized by a high level of dissolved ions and naphthenic acids. The dikes used to store the effluent seep, creating wetlands which are subsequently invaded by obligate wetland flora such as cattail (Typha latifolia L.). The appearance of these wetlands prompted the oil sands industry to consider wetlands as part of their reclamation strategy. However, to ensure long-term viability of such wetlands, the response of the flora to the industrial effluent needed to be determined. To this end, apparent photosynthesis (APS), the level of ribulose-1,5-bisphosphate carboxylase (RuBisCo) large subunit, dehydrin-related polypeptides, and protein disulphide isomerase (PDI) were evaluated in cattail and alsike clover plants (Trifolium hybridum L.) exposed to the oil sands effluent. APS measured in plants impacted by oil sands effluent was significantly higher than that of plants in the non-impacted off-site location. Among the on-site locations, plants growing in the natural wetlands site had higher APS compared to all other sites. The level of RuBisCo was not increased in cattail or clover growing in effluent-contaminated sites indicating that enhanced photosynthesis was not due to greater levels of this enzyme. Dehydrin-related polypeptides were detected only in the roots of cattail and were absent in clover. The polypeptide profile was altered in cattail exposed to oil sands effluent indicating that they were responding to an osmotic stress. The level of PDI was unaffected in the leaves of cattail regardless of the nature of the effluent to which they were exposed. Overall, the data indicate that cattail and clover are adapted to the oil sands effluent, although further studies are needed to assess their long-term ability to survive in the presence of this anthropogenic stress.

Effects of an industrial effluent on plant colonization and on the germination and post-germinative growth of seeds of terrestrial and aquatic plant species.

Major oil sands industrial companies are located in the Athabasca Oil Sands Deposit in northeastern Alberta, Canada. During the process used to extract light crude oil (via hot water digestion and flotation), gypsum is usually added to produce consolidated tails (CT) and CT release water. The vast volumes of process-treated waters (effluent) are held within large dyked tailings ponds. Toward testing viable options for reclamation, various hummock-wetlands systems have been constructed; in addition, natural wetlands (inhabited by obligate wetland plant species) have become established as a result of seeping of the effluents held within the large dyked ponds. Vegetation surveys conducted on and around the industrial site revealed that the constructed wetlands associated with the dyke drainage (effluent treated with phosphorous) and consolidated tails (CT; effluent treated with gypsum) had low biodiversity and were not invaded by many aquatic plants. Although the natural wetland was also not invaded by many aquatic species, it was found to be as diverse as the reference wetlands (i.e. off-site wetlands not exposed to the effluents). Exposure to oil sands effluents had an inhibitory effect on the germination (percent and/or rate) of several plant species (tomato, clover, wheat, rye, pea, reed canary grass, loblolly pine); clover and tomato seed germination were most affected. Two treatments in particular (effluents from the natural on-site wetland and the CT constructed wetland), delayed germination, and also led to reduced fresh weight of seedlings of tomato, wheat, clover and loblolly pine. The osmolarities of the effluents associated with the natural on-site wetland and CT constructed wetland were 712 and 728 mOs/kg, respectively; substituting these effluents with solutions of polyethylene glycol of the same osmotic potentials had a greater inhibitory effect on germination rate. The negative effects of the effluents on seed germination may account for the paucity of aquatic species that invaded the oil sands impacted wetlands. This factor will also be critical in determining the long-term feasibility of hummock-wetland systems.