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1.  Population genetic structure of two Medicago species shaped by distinct life form, mating system and seed dispersal 
Annals of Botany  2009;103(6):825-834.
Background and Aims
Life form, mating system and seed dispersal are important adaptive traits of plants. In the first effort to characterize in detail the population genetic structure and dynamics of wild Medicago species in China, a population genetic study of two closely related Medicago species, M. lupulina and M. ruthenica, that are distinct in these traits, are reported. These species are valuable germplasm resources for the improvement of Medicago forage crops but are under threat of habitat destruction.
Three hundred and twenty-eight individuals from 16 populations of the annual species, M. lupulina, and 447 individuals from 15 populations of the perennial species, M. ruthenica, were studied using 15 and 17 microsatellite loci, respectively. Conventional and Bayesian-clustering analyses were utilized to estimate population genetic structure, mating system and gene flow.
Key Results
Genetic diversity of M. lupulina (mean HE = 0·246) was lower than that of M. ruthenica (mean HE = 0·677). Populations of M. lupulina were more highly differentiated (FST = 0·535) than those of M. ruthenica (FST = 0·130). For M. lupulina, 55·5 % of the genetic variation was partitioned among populations, whereas 76·6 % of the variation existed within populations of M. ruthenica. Based on the genetic data, the selfing rates of M. lupulina and M. ruthenica were estimated at 95·8 % and 29·5 %, respectively. The genetic differentiation among populations of both species was positively correlated with geographical distance.
The mating system differentiation estimated from the genetic data is consistent with floral morphology and observed pollinator visitation. There was a much higher historical gene flow in M. ruthenica than in M. lupulina, despite more effective seed dispersal mechanisms in M. lupulina. The population genetic structure and geographical distribution of the two Medicago species have been shaped by life form, mating systems and seed dispersal mechanisms.
PMCID: PMC2707894  PMID: 19174379
Medicago lupulina; Medicago ruthenica; microsatellite; genetic diversity; gene flow; forage legume
2.  Magnitude and Timing of Leaf Damage Affect Seed Production in a Natural Population of Arabidopsis thaliana (Brassicaceae) 
PLoS ONE  2012;7(1):e30015.
The effect of herbivory on plant fitness varies widely. Understanding the causes of this variation is of considerable interest because of its implications for plant population dynamics and trait evolution. We experimentally defoliated the annual herb Arabidopsis thaliana in a natural population in Sweden to test the hypotheses that (a) plant fitness decreases with increasing damage, (b) tolerance to defoliation is lower before flowering than during flowering, and (c) defoliation before flowering reduces number of seeds more strongly than defoliation during flowering, but the opposite is true for effects on seed size.
Methodology/Principal Findings
In a first experiment, between 0 and 75% of the leaf area was removed in May from plants that flowered or were about to start flowering. In a second experiment, 0, 25%, or 50% of the leaf area was removed from plants on one of two occasions, in mid April when plants were either in the vegetative rosette or bolting stage, or in mid May when plants were flowering. In the first experiment, seed production was negatively related to leaf area removed, and at the highest damage level, also mean seed size was reduced. In the second experiment, removal of 50% of the leaf area reduced seed production by 60% among plants defoliated early in the season at the vegetative rosettes, and by 22% among plants defoliated early in the season at the bolting stage, but did not reduce seed output of plants defoliated one month later. No seasonal shift in the effect of defoliation on seed size was detected.
The results show that leaf damage may reduce the fitness of A. thaliana, and suggest that in this population leaf herbivores feeding on plants before flowering should exert stronger selection on defence traits than those feeding on plants during flowering, given similar damage levels.
PMCID: PMC3261866  PMID: 22276140
3.  The activity of SnRK1 is increased in Phaseolus vulgaris seeds in response to a reduced nutrient supply 
Phaseolus vulgaris seeds can grow and develop at the expense of the pod reserves after the fruits have been removed from the plant (Fountain etal., 1989). Because this process involves sensing the reduction of nutrients and the remobilisation of pod reserves, we investigated the effect on sucrose non-fermenting related kinase 1 (SnRK1) activity during this process. Bean fruits removed from the plant at 20 days after flowering (DAF) demonstrated active remobilisation of nutrients from the pod to the seeds. After 5 days, the pod dry weight was reduced by 50%. The process was characterized by a rapid degradation of starch, with the greatest decrease observed on day 1 after the fruits were removed. The pod nutrients were insufficient for the needs of all the seeds, and only some seeds continued their development. Those seeds exhibited a transient reduction in sucrose levels on day 1 after the fruits were removed. However, the normal level of sucrose was recovered, and the rate of starch synthesis was identical to that of a seed developed under normal conditions. Removing the fruits from the plant had no effect on the activity of SnRK1 in the pods, whereas in the seeds, the activity was increased by 35%. Simultaneously, a large reduction in seed sucrose levels was observed. The increase in SnRK1 activity was observed in both the cotyledon and embryo axes, but it was higher in the cotyledon. At 20–25 DAF, cotyledons actively accumulate storage materials. It is possible that the increase in SnRK1 activity observed in seeds developed in fruits that have been removed from the plant is part of the mechanism required for nutrient remobilisation under conditions of stress.
PMCID: PMC4030202  PMID: 24860586
nutrient remobilisation; SnRK1; bean seed development
4.  Acorn cotyledons are larger than their seedlings' need: evidence from artificial cutting experiments 
Scientific Reports  2015;5:8112.
Although the consequences of cotyledon removal have been widely studied in oaks producing large acorns, we have little knowledge of at what level cotyledons can be removed without affecting acorn survival and seedling development. In this study, we aimed to test the hypothesis that the amount of energy reserves in cotyledons is more than the demands of seedlings and that large acorns can tolerate seed predation and/or attract seed predators for seed dispersal. Acorn germination rates were not affected even when 60% of cotyledons were cut off at the basal end, suggesting that the energy reserves contained in cotyledons are not essential for acorn survival. Post-cut acorn mass, more than initial acorn mass, appear to be a better predictor of seedling performance, indicating that the energy reserves in cotyledons are sufficient for seedlings. Acorns with large masses sustained cotyledon damage better than small ones with respect to seedling performance. Large acorns were more likely to be dispersed and cached by animals, implying that producing large acorns is more important for oaks to manipulate seed predators and dispersers rather than provide a seedling with cotyledonary reserves.
PMCID: PMC4309963  PMID: 25630843
5.  The Natural Variation of Seed Weight Is Mainly Controlled by Maternal Genotype in Rapeseed (Brassica napus L.) 
PLoS ONE  2015;10(4):e0125360.
Seed weight is a very important and complex trait in rapeseed (Brassica napus L.). The seed weight of rapeseed shows great variation in its natural germplasm resources; however, the morphological, cytological and genetic causes of this variation have remained unclear. In the present study, nine highly pure inbred rapeseed lines with large seed weight variation and different genetic backgrounds were selected for morphological, cytological and genetic studies on seed weight. The results showed the following: (1) Seed weight showed an extremely significant correlation and coordinated variation with seed size (including seed diameter, seed surface area and seed volume), but it showed no significant correlation with bulk density, which suggests that seed weight is determined by size rather than bulk density. (2) Seed weight showed a higher correlation with the cell numbers of seed coats and cotyledons than the cell sizes of seed coats and cotyledons, which suggests that cell number is more tightly correlated with final seed weight. (3) Seed weight was mainly controlled by the maternal genotype, with little or no xenia and cytoplasmic effects. This is the first report on the morphological and cytological causes of seed weight natural variation in rapeseed. We concluded that the natural variation of seed weight is mainly controlled by maternal genotype. This finding lays a foundation for genetic and breeding studies of seed weight in rapeseed and opens a new field of research on the regulation of seed traits in plants.
PMCID: PMC4411071  PMID: 25915862
6.  The Role of the Storage Carbon of Cotyledons in the Establishment of Seedlings of Hymenaea courbaril Under Different Light Conditions 
Annals of Botany  2004;94(6):819-830.
• Background and Aims Hymenaea courbaril (Leguminosae-Caesalpinioideae) is a tree species with wide distribution through all of the Neotropics. It has large seeds (approx. 5 g) with non-photosynthetic storage cotyledons rich (40 %) in a cell wall polysaccharide (xyloglucan) as a carbon reserve. Because it is found in the understorey of tropical forests, it has been considered as a shade-tolerant, late-secondary species. However, the physiological mechanisms involved in seedling establishment, especially regarding the interplay between storage and light intensity, are not understood. In this work, the ecophysiological role of this carbon cotyledon reserve (xyloglucan) is characterized, emphasizing its effects on seedling growth and development during the transition from heterotrophy to autotrophy under different light conditions.
• Methods Seedlings of H. courbaril were grown in environments with different light intensities, and with or without cotyledons detached before xyloglucan mobilization. Development, growth, photosynthesis and carbon partitioning (dry mass and [14C]sucrose) were analysed in each treatment.
• Key Results The detachment of cotyledons was not important for seedling survival, but resulted in a strong restriction (50 % less) of shoot growth, which was the main sink for the cotyledon carbon reserves. Carbon restriction promoted an early maturation of the photosynthetic apparatus without changes in the net CO2 fixation per unit area. The reduced surface area of the first leaves in seedlings without cotyledons was evidence of limited growth and development of seedlings in low light conditions (22 µmol m−2 s−1 photon flux).
• Conclusions There is an increase in the importance of storage xyloglucan in cotyledons for H. courbaril seedling development as light intensity decreases, confirming that this polymer plays a key role in the adaptation of this species to establish successfully in the shadowed understorey of the forest.
PMCID: PMC4242276  PMID: 15514028
Carbon partitioning; forest; light; growth; cell wall; Hymenaea courbaril; photosynthesis; seedling; xyloglucan; storage
7.  Germination Season and Watering Regime, but Not Seed Morph, Affect Life History Traits in a Cold Desert Diaspore-Heteromorphic Annual 
PLoS ONE  2014;9(7):e102018.
Seed morph, abiotic conditions and time of germination can affect plant fitness, but few studies have tested their combined effects on plasticity of plant life history traits. Thus, we tested the hypothesis that seed morph, germination season and watering regime influence phenotypic expression of post-germination life history traits in the diaspore-heteromorphic cold desert winter annual/spring ephemeral Diptychocarpus strictus. The two seed morphs were sown in watered and non-watered plots in late summer, and plants derived from them were watered or not-watered throughout the study. Seed morph did not affect phenology, growth and morphology, survival, dry mass accumulation and allocation or silique and seed production. Seeds in watered plots germinated in autumn (AW) and spring (SW) but only in spring for non-watered plots (SNW). A high percentage of AW, SW and SNW plants survived and reproduced, but flowering date and flowering period of autumn- vs. spring-germinated plants differed. Dry mass also differed with germination season/watering regime (AW > SW > SNW). Number of siliques and seeds increased with plant size (AW > SW > SNW), whereas percent dry mass allocated to reproduction was higher in small plants: SNW > SW > AW. Thus, although seed morph did not affect the expression of life history traits, germination season and watering regime significantly affected phenology, plant size and accumulation and allocation of biomass to reproduction. Flexibility throughout the life cycle of D. strictus is an adaptation to the variation in timing and amount of rainfall in its cold desert habitat.
PMCID: PMC4094427  PMID: 25013967
8.  Inherited variability in multiple traits determines fitness in populations of an annual legume from contrasting latitudinal origins 
Annals of Botany  2009;103(8):1279-1289.
Background and Aims
Variation in fitness depends on corresponding variation in multiple traits which have both genetically controlled and plastic components. These traits are subjected to varying degrees of local adaptation in specific populations and, consequently, are genetically controlled to different extents. In this study it is hypothesized that modulation of different traits would have contrasting relevance for the fitness of populations of diverse origins. Specifically, assuming that environmental pressures vary across a latitudinal gradient, it is suggested that inherited variation in traits differentially determines fitness in annual Lupinus angustifolius populations from contrasting latitudinal origins in western Spain.
Seeds of L. angustifolius from three contrasting origins were grown in a common garden. Traits related to more plastic vegetative growth and more genetically conserved phenology were measured, together with estimates of reproductive success. Fitness was estimated by the number of viable seeds per plant. Structural Equation Models were used to infer causal relationships among multiple traits and fitness, separating the direct and indirect effects of morphological, phenological and reproductive traits.
Key Results
Phenological, vegetative and reproductive traits accounted for most of the fitness variation. Fitness was highest in plants of southernmost origin, mainly due to earlier flowering. Fitness within each seed origin was controlled by variation in different traits. Southern origin plants that grew to a larger size achieved higher fitness. However, plant size in plants of northernmost origin was irrelevant, but early flowering promoted higher fitness. Variation in fruit and seed set had a greater effect on the fitness of plants of central origin than phenological and size variation.
It is concluded that modulation of a functional trait can be relevant to fitness in a given population (i.e. affecting intensity and direction), but irrelevant in other populations. This points to the need to consider integrated phenotypes when trying to unravel local adaptation effects over single traits.
PMCID: PMC2685322  PMID: 19318383
Lupinus; Structural Equation Models; fitness; phenology; functional traits; reproductive success; SLA; seed size
9.  Implications of a long-term, pollinator-mediated selection on floral traits in a generalist herb 
Annals of Botany  2009;104(4):689-701.
Background and Aims
The phenotypic selection of a diverse insect assemblage was studied on a generalist plant species (Paeonia broteroi) in ten flowering seasons, with tests for whether visitor preferences for plants with larger flowers eventually translated into significant differences among plants in visitation rates, seed production, seed mass, seed germination and seedling survival.
Selection gradients were used to assess if selection on flower size contributed to explain differences in visitation rates, seed production and seed mass. First, independent analyses were carried out for each season; then for the ten season as a whole. Seedling emergence and survival were assessed by generalized linear models.
Key Results
Directional selection was found on flower size through visitation rates and seed production, and stabilizing selection through seed mass. Thus, larger flowers were more visited, and produced more, but lighter seeds, than smaller flowers. The results suggest a conflicting selection on flower size through seed number and size. Floral integration found in the study populations was larger than that in populations of a distant region. Finally, seed size did not influence seedling emergence and survival; thus, any advantages of seed size may be constrained under natural conditions before plants become reproductive individuals.
Plants with larger flowers may be benefited by producing more lighter seeds than fewer heavier ones, as they may contribute disproportionately to the seed bank, and have better chances that any descendant could eventually recruit. However, it seems unlikely that differences in flower size and integration found among populations in different regions could have been originated by rapid evolutionary change. First, because of the conflicting selection described; second, because of the remarkably low seedling survival found under natural conditions. Consequently, the influence of pollinator selection alone does not seem to explain differences in flower size and integration.
PMCID: PMC2729625  PMID: 19508980
Paeonia broteroi; long-term selection; conflicting selection; flower size; seed production; generalist pollination
10.  Effects of pollination limitation and seed predation on female reproductive success of a deceptive orchid 
AoB Plants  2014;6:plu031.
This research examines the contribution of plant height, number of flowers, number of stems, as well the joint impacts of mutualists and antagonists on the pollination biology and seed production of the imperiled, deceptive orchid, Cypripedium candidum. We found flowering stem height to be the only morphological feature significant in reproduction, with taller flowering stems simultaneously receiving increased pollination and decreased seed predation. Furthermore we found decreased seed mass in individuals subjected to hand-self pollination treatments. Our results may help explain the factors limiting seed production in other Cypripedium and further emphasize the importance of management in orchid conservation.
For many species of conservation significance, multiple factors limit reproduction. This research examines the contributions of plant height, number of flowers, number of stems, pollen limitation and seed predation to female reproductive success in the deceit-pollinated orchid, Cypripedium candidum. The deceptive pollination strategy employed by many orchids often results in high levels of pollen limitation. While increased floral display size may attract pollinators, C. candidum's multiple, synchronously flowering stems could promote selfing and also increase attack by weevil seed predators. To understand the joint impacts of mutualists and antagonists, we examined pollen limitation, seed predation and the effects of pollen source over two flowering seasons (2009 and 2011) in Ohio. In 2009, 36 pairs of plants size-matched by flower number, receiving either supplemental hand or open pollination, were scored for fruit maturation, mass of seeds and seed predation. Pollen supplementation increased proportion of flowers maturing into fruit, with 87 % fruit set when hand pollinated compared with 46 % for naturally pollinated flowers. Inflorescence height had a strong effect, as taller inflorescences had higher initial fruit set, while shorter stems had higher predation. Seed predation was seen in 73 % of all fruits. A parallel 2011 experiment that included a self-pollination treatment and excluded seed predators found initial and final fruit set were higher in the self and outcross pollination treatments than in the open-pollinated treatment. However, seed mass was higher in both open pollinated and outcross pollination treatments compared with hand self-pollinated. We found greater female reproductive success for taller flowering stems that simultaneously benefited from increased pollination and reduced seed predation. These studies suggest that this species is under strong reinforcing selection to increase allocation to flowering stem height. Our results may help explain the factors limiting seed production in other Cypripedium and further emphasize the importance of management in orchid conservation.
PMCID: PMC4094650  PMID: 24916060
Conservation; orchid; plant reproduction; plant–insect interactions; pollen limitation; pollination ecology; reproductive trade-offs; seed predation; supplemental pollination.
11.  Developmental profiling of gene expression in soybean trifoliate leaves and cotyledons 
BMC Plant Biology  2015;15:169.
Immediately following germination, the developing soybean seedling relies on the nutrient reserves stored in the cotyledons to sustain heterotrophic growth. During the seed filling period, developing seeds rely on the transport of nutrients from the trifoliate leaves. In soybean, both cotyledons and leaves develop the capacity for photosynthesis, and subsequently senesce and abscise once their function has ended. Before this occurs, the nutrients they contain are mobilized and transported to other parts of the plant. These processes are carefully orchestrated by genetic regulation throughout the development of the leaf or cotyledon.
To identify genes involved in the processes of leaf or cotyledon development and senescence in soybean, we used RNA-seq to profile multiple stages of cotyledon and leaf tissues. Differentially expressed genes between stages of leaf or cotyledon development were determined, major patterns of gene expression were defined, and shared genes were identified. Over 38,000 transcripts were expressed during the course of leaf and cotyledon development. Of those transcripts, 5,000 were expressed in a tissue specific pattern. Of the genes that were differentially expressed between both later stage tissues, 90 % had the same direction of change, suggesting that the mechanisms of senescence are conserved between tissues. Analysis of the enrichment of biological functions within genes sharing common expression profiles highlights the main processes occurring within these defined temporal windows of leaf and cotyledon development. Over 1,000 genes were identified with predicted regulatory functions that may have a role in control of leaf or cotyledon senescence.
The process of leaf and cotyledon development can be divided into distinct stages characterized by the expression of specific gene sets. The importance of the WRKY, NAC, and GRAS family transcription factors as major regulators of plant senescence is confirmed for both soybean leaf and cotyledon tissues. These results help validate functional annotation for soybean genes and promoters.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0553-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4492100  PMID: 26149852
Leaf senescence; Cotyledon senescence; Glycine max; Leaf development
12.  Two cytosolic glutamine synthetase isoforms play specific roles for seed germination and seed yield structure in Arabidopsis  
Journal of Experimental Botany  2014;66(1):203-212.
Knockout of glutamine synthetase isogene Gln1;2 reduces nitrogen remobilization and the number and size of siliques and seeds in Arabidopsis. Gln1;1 affects the response of primary root development to exogenous nitrogen.
Nitrogen (N) remobilization from reserves to sinks is essential for seedling establishment and seed production. Cytosolic glutamine synthetase (GS1) is up-regulated during both seed germination and seed filling in plants. However, the specific roles of the individual GS1 isogenes with respect to N remobilization, early seedling vigour, and final seed productivity are not known. In this study, impairment of seed germination and seedling establishment is demonstrated in the single knockout mutant gln1;2, and the double knockout mutant gln1;1:gln1;2. The negative effect of Gln1;2 deficiency was associated with reduced N remobilization from the cotyledons and could be fully alleviated by exogenous N supply. Following reproductive growth, both the single and double Gln1;2-knockout mutants showed decreased seed yield due to fewer siliques, less seeds per silique, and lower dry weight per seed. The gln1;1 single mutant had normal seed yield structure but primary root development during seed germination was reduced in the presence of external N. Gln1;2 promoter–green fluorescent protein constructs showed that Gln1;2 localizes to the vascular cells of roots, petals, and stamens. It is concluded that Gln1;2 plays an important role in N remobilization for both seedling establishment and seed production in Arabidopsis.
PMCID: PMC4265158  PMID: 25316065
Arabidopsis; cytosolic glutamine synthetase; isoform; mutant; nitrogen; seed germination; seed productivity.
13.  Differences in Plastic Responses to Defoliation due to Variation in the Timing of Treatments for Two Species of Sesbania (Fabaceae) 
Annals of Botany  2005;95(6):1049-1058.
• Background and Aims Plastic responses to stress in components of reproduction can have important effects on plant fitness and can vary both within and between species. Responses may also depend on when in the life cycle stress occurs. Here, it is predicted that the timing of initiation of a stress, defoliation, would affect the pattern of plastic responses. These differences should occur because some components of reproduction, such as flower number, are determined earlier in a plant's life than others, such as individual seed mass.
• Methods To test this prediction, 50 % artificial defoliation treatments were initiated at four different times for Sesbania macrocarpa and S. vesicaria. Responses were measured in plant size, number of flowers, number of flowers/plant size, fruit set, number of seeds per fruit, individual seed mass and total seed mass per plant.
• Key Results For S. vesicaria, changes in the timing of stress changed the severity, but not the pattern of response. For S. macrocarpa, plastic responses to defoliation varied strikingly between early and late treatments. Late treatments resulted in over-compensation in this species. Sesbania macrocarpa was generally more plastic than S. vesicaria and the species showed opposite responses for most components of reproduction.
• Conclusions While there were effects of timing of defoliation and differences between species, the nature of these effects did not precisely fit our predictions. Our results suggest that differences in the length and flexibility of the life cycles of the two species allowed for unexpected variation in responses. For example, because flower production continued after the last treatment in S. vesicaria, responses were not constrained to reductions in individual seed mass.
PMCID: PMC4246763  PMID: 15753116
Phenotypic plasticity; components of reproduction; artificial defoliation; reproductive allocation; overcompensation; Sesbania macrocarpa; Sesbania vesicaria
14.  Sexual Dimorphism of Staminate- and Pistillate-Phase Flowers of Saponaria officinalis (Bouncing Bet) Affects Pollinator Behavior and Seed Set 
PLoS ONE  2014;9(4):e93615.
The sequential separation of male and female function in flowers of dichogamous species allows for the evolution of differing morphologies that maximize fitness through seed siring and seed set. We examined staminate- and pistillate-phase flowers of protandrous Saponaria officinalis for dimorphism in floral traits and their effects on pollinator attraction and seed set. Pistillate-phase flowers have larger petals, greater mass, and are pinker in color, but due to a shape change, pistillate-phase flowers have smaller corolla diameters than staminate-phase flowers. There was no difference in nectar volume or sugar content one day after anthesis, and minimal evidence for UV nectar guide patterns in staminate- and pistillate-phase flowers. When presented with choice arrays, pollinators discriminated against pistillate-phase flowers based on their pink color. Finally, in an experimental garden, in 2012 there was a negative correlation between seed set of an open-pollinated, emasculated flower and pinkness (as measured by reflectance spectrometry) of a pistillate-phase flower on the same plant in plots covered with shade cloth. In 2013, clones of genotypes chosen from the 2012 plants that produced pinker flowers had lower seed set than those from genotypes with paler flowers. Lower seed set of pink genotypes was found in open-pollinated and hand-pollinated flowers, indicating the lower seed set might be due to other differences between pink and pale genotypes in addition to pollinator discrimination against pink flowers. In conclusion, staminate- and pistillate-phase flowers of S. officinalis are dimorphic in shape and color. Pollinators discriminate among flowers based on these differences, and individuals whose pistillate-phase flowers are most different in color from their staminate-phase flowers make fewer seeds. We suggest morphological studies of the two sex phases in dichogamous, hermaphroditic species can contribute to understanding the evolution of sexual dimorphism in plants without the confounding effects of genetic differences between separate male and female individuals.
PMCID: PMC3972141  PMID: 24690875
15.  Role of Seed Coat in Imbibing Soybean Seeds Observed by Micro-magnetic Resonance Imaging 
Annals of Botany  2008;102(3):343-352.
Background and Aims
Imbibition of Japanese soybean (Glycine max) cultivars was studied using micro-magnetic resonance imaging (MRI) in order to elucidate the mechanism of soaking injury and the protective role of the seed coat.
Time-lapse images during water uptake were acquired by the single-point imaging (SPI) method at 15-min intervals, for 20 h in the dry seed with seed coat, and for 2 h in seeds with the seed coat removed. The technique visualized water migration within the testa and demonstrated the distortion associated with cotyledon swelling during the very early stages of water uptake.
Key Results
Water soon appeared in the testa and went around the dorsal surface of the seed from near the raphe, then migrated to the hilum region. An obvious protrusion was noted when water reached the hypocotyl and the radicle, followed by swelling of the cotyledons. A convex area was observed around the raphe with the enlargement of the seed. Water was always incorporated into the cotyledons from the abaxial surfaces, leading to swelling and generating a large air space between the adaxial surfaces. Water uptake greatly slowed, and the internal structures, veins and oil-accumulating tissues in the cotyledons developed after the seed stopped expanding. When the testa was removed from the dry seeds before imbibition, the cotyledons were severely damaged within 1·5 h of water uptake.
The activation of the water channel seemed unnecessary for water entry into soybean seeds, and the testa rapidly swelled with steeping in water. However, the testa did not regulate the water incorporation in itself, but rather the rate at which water encountered the hypocotyl, the radicle, and the cotyledons through the inner layer of the seed coat, and thus prevented the destruction of the seed tissues at the beginning of imbibition.
PMCID: PMC2516911  PMID: 18565982
Dry seeds; Glycine max; MRI; seed coat; soaking injury; soybean; testa; role of inner layer of seed coat; water uptake
16.  Retention of cotyledons is crucial for resprouting of clipped oak seedlings 
Scientific Reports  2014;4:5145.
Although resprouting plays an important role in facilitating persistence of damaged seedlings, the functional attributes of cotyledons and taproots during resprouting of 1-year oak seedlings are not well explored. In this study, cotyledons were removed from Quercus mongolica seedlings to explore resprouting in response to simulated disturbance as a function of shoot clipping, and to examine the resprouting ability in relation to timing of clipping and cotyledon removal. Isotope labeling experiments were also performed to evaluate contribution of the cotyledons and taproots to resprouting. Regardless of timing of shoot clipping, seedlings successfully resprouted provided their cotyledons were not detached. Clipped seedlings were less likely to resprout when cotyledons were removed. Seedlings clipped at earlier development stage exhibited higher resprouting capacity than those clipped at later stage. Cotyledon removal, more than timing of clipping, decreased the dry masses of newly-resprouted shoots. However, no significant influences of cotyledon removal and timing of clipping were found on the dry masses of roots, suggesting the importance of cotyledons for resprouting. Roots became functional and accumulated more soil nitrogen after shoot clipping and cotyledon removal, representing a double security-based strategy for the clipped seedlings to resprout despite the importance of cotyledons.
PMCID: PMC4042119  PMID: 24888417
17.  Specific elements of the glyoxylate pathway play a significant role in the functional transition of the soybean cotyledon during seedling development 
BMC Genomics  2007;8:468.
The soybean (Glycine max) cotyledon is a specialized tissue whose main function is to serve as a nutrient reserve that supplies the needs of the young plant throughout seedling development. During this process the cotyledons experience a functional transition to a mainly photosynthetic tissue. To identify at the genetic level the specific active elements that participate in the natural transition of the cotyledon from storage to photosynthetic activity, we studied the transcript abundance profile at different time points using a new soybean oligonucleotide chip containing 19,200 probes (70-mer long).
After normalization and statistical analysis we determined that 3,594 genes presented a statistically significant altered expression in relation to the imbibed seed in at least one of the time points defined for the study. Detailed analysis of this data identified individual, specific elements of the glyoxylate pathway that play a fundamental role during the functional transition of the cotyledon from nutrient storage to photosynthesis. The dynamics between glyoxysomes and peroxisomes is evident during these series of events. We also identified several other genes whose products could participate co-ordinately throughout the functional transition and the associated mechanisms of control and regulation and we described multiple unknown genetic elements that by association have the potential to make a major contribution to this biological process.
We demonstrate that the global transcript profile of the soybean cotyledon during seedling development is extremely active, highly regulated and dynamic. We defined the expression profiles of individual gene family members, enzymatic isoforms and protein subunits and classified them accordingly to their involvement in different functional activities relevant to seedling development and the cotyledonary functional transition in soybean, especially the ones associated with the glyoxylate cycle. Our data suggests that in the soybean cotyledon a very complex and synchronized system of control and regulation of several metabolic pathways is essential to carry out the necessary functions during this developmental process.
PMCID: PMC2234262  PMID: 18093333
18.  Effect of seed position in spikelet on life history of Eremopyrum distans (Poaceae) from the cold desert of north-west China 
Annals of Botany  2010;106(1):95-105.
Background and Aims
Most studies on seed position-dependent effects have focused on germination characteristics. Our aim was to determine the effects of seed position in the spikelet on differences in timing of germination and on the ecological life history of the grass Eremopyrum distans in its cold desert habitat.
For seeds in three spikelet positions, morphology, mass and dormancy/germination characteristics were determined in the laboratory, and seeds planted in field plots with and without watering were followed to reproduction to investigate seedling emergence and survival, plant size and seed production.
Key Results
After maturation, of the seeds within the spikelet, basal ones (group 1) are the largest and have the highest proportion with physiological dormancy, while distal ones (group 3) are the smallest and have the highest proportion of non-dormant seeds. A higher percentage of seeds after-ripened in groups 2 and 3 than in group 1. Seeds sown in the field in early summer and watered at short, regular intervals germinated primarily in autumn, while those under natural soil moisture conditions germinated only in spring. Both cohorts completed their life cycle in early summer. Seeds in group 1 had lower percentages of seedling emergence and higher percentages of seedling survival than those in groups 2 and 3. Also, plants from group 1 seeds were larger and produced more seeds per plant than those from groups 2 and 3.
Seed position-dependent mass was associated with quantitative differences in several life history traits of E. distans. The environmentally enforced (low soil moisture) delay of germination from autumn to spring results in a reduction in fitness via reduction in number of seeds produced per plant.
PMCID: PMC2889797  PMID: 20460387
Eremopyrum distans; physiological dormancy; plant size; seed mass; seed position-dependent effects; seed production; seedling survival
19.  Germination and ultrastructural studies of seeds produced by a fast-growing, drought-resistant tree: implications for its domestication and seed storage 
AoB Plants  2015;7:plv016.
The inner layers of the seed coat that remain attached to the cotyledons probably play a role in seed dormancy of Moringa oleifera. Cotyledons of seeds stored for one year showed no sign of deterioration. In some cells of the three-year-old cotyledons, the membranes of the protein bodies were deteriorated. Cell deterioration was also marked by the collapse of the cell wall adjacent to the intercellular cavity. The decrease in seed viability during storage is associated with the loss in membrane integrity as confirmed by the increase in electrolyte leakage. The longevity of seeds can be extended if they are stored within their fruits under favourable conditions.
Seed ageing during storage is one of the main causes of reduction in seed quality and this results in loss of vigour and failure to thrive. Finding appropriate storage conditions to ameliorate deterioration due to ageing is, therefore, essential. Ultrastructural changes in cellular organelles during storage and seed germination rates are valuable indices of damage that occurs during seed ageing. There is increasing interest in Moringa oleifera Lam. because of its multiple uses as an agroforestry crop. Seeds of this species lose their viability within 6–12 months of harvest but no scientific information is available on the longevity of seed stored in the fruit (capsules). In most undeveloped countries, seeds are still stored inside the fruit by traditional methods in special handmade structures. In this experiment we tried to simulate these traditional storage conditions. Capsules of Moringa were stored at ambient room temperature for 12, 24 and 36 months. The ultrastructure, solute leakage and viability of seed were investigated. The ultrastructure of 1-year-old seed showed no sign of deterioration. It was evident, however, that some cells of the 3-year-old seed had deteriorated. The remnants of the outer and inner two integuments that remain tightly attached to the cotyledons probably play a role in seed dormancy. No significant difference was found between germination percentage of fresh and 1-year-old seed. The germination percentage decreased significantly from 2 years of storage onward. The decrease in seed viability during storage was associated with a loss in membrane integrity which was evidenced by an increase in electrolyte leakage. Our findings indicate that the longevity of M. oleifera seeds can be maintained if they are stored within their capsules.
PMCID: PMC4379585  PMID: 25725083
Deterioration; dormancy; lipid bodies; membrane leakage; protein bodies; seed; storage
20.  The Role of Roots and Cotyledons as Storage Organs in Early Stages of Establishment in Quercus crispula: a Quantitative Analysis of the Nonstructural Carbohydrate in Cotyledons and Roots 
Annals of Botany  2003;92(4):537-545.
Quercus seedlings have hypogeal cotyledons and tap roots, both of which act as storage organs. The importance of the storage function in the two organs may change as the seedling develops. Therefore, changes in carbohydrate reserves in cotyledons and roots of Q. crispula grown under 40 % and 3 % of full light from shoot emergence to the completion of the first leaf flush were monitored. In addition, a shoot‐clipping treatment was performed to examine the relative contribution of the cotyledons and tap roots to resprouting. Cotyledons maintained large amounts of nonstructural carbohydrates during shoot development, and carbohydrates were still present in the cotyledons during the final phase of leaf flush. In addition, a notable increase in the amount of carbohydrates was observed in tap roots before leaf flush at both light levels. Since root development occurred before leaf flush, even in plants grown under 3 % light, the carbohydrate found in them presumably originated from seed reserves and was translocated to roots as storage reserves. When shoots were clipped at the leaf flushing stage, the amount of carbohydrate decreased only in the cotyledons after resprouting, suggesting that cotyledons act as the main storage organs during shoot development stages. However, it could be advantageous as a ‘risk avoidance strategy’ for the seedlings to store reserves in both cotyledons and roots, since cotyledons may be removed by predators during shoot development.
PMCID: PMC4243669  PMID: 12907467
Quercuscrispula Blume; Mizunara oak; seedling; carbohydrate storage; germination stage; hypogeous plant; resprouting; shoot destruction; TNC; seed reserves
21.  Gene expression analysis of flax seed development 
BMC Plant Biology  2011;11:74.
Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.
We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development.
We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.
PMCID: PMC3107784  PMID: 21529361
22.  Flowering time and seed dormancy control use external coincidence to generate life history strategy 
eLife  null;4:e05557.
Climate change is accelerating plant developmental transitions coordinated with the seasons in temperate environments. To understand the importance of these timing advances for a stable life history strategy, we constructed a full life cycle model of Arabidopsis thaliana. Modelling and field data reveal that a cryptic function of flowering time control is to limit seed set of winter annuals to an ambient temperature window which coincides with a temperature-sensitive switch in seed dormancy state. This coincidence is predicted to be conserved independent of climate at the expense of flowering date, suggesting that temperature control of flowering time has evolved to constrain seed set environment and therefore frequency of dormant and non-dormant seed states. We show that late flowering can disrupt this bet-hedging germination strategy. Our analysis shows that life history modelling can reveal hidden fitness constraints and identify non-obvious selection pressures as emergent features.
eLife digest
Plants adjust when they grow, develop flowers and produce, or ‘set’, seeds in response to changes in temperature and day length. It is therefore unsurprising that climate change alters the timing of these important events in plants' lives; for example, many plants are adapting to rising temperatures by flowering earlier and growing for longer.
The environmental signals that control when a plant flowers, and the genes that underlie this process, have been well studied in the model plant Arabidopsis thaliana. This plant's ability to quickly colonize and thrive in disturbed habitats—including agricultural land, construction sites, and waste ground—is partly because some of its seeds lie dormant in the soil, for up to several years, before they start to grow. Whether or not a seed undergoes a period of dormancy is controlled by the temperature that the seeds experienced when they were developing; this in turn is influenced by earlier events, such as when the flowers first developed, and when the plant first started to grow from its seed (a process called germination).
To try to understand these complex interactions, Springthorpe and Penfield developed a computational model of the major events in the life of an Arabidopsis plant. Data collected from Arabidopsis plants that normally germinate in winter and spring were then used to check whether the model could accurately represent what happens in nature.
The analysis confirmed that the timing of seed setting depends mostly on the environmental temperature. Springthorpe and Penfield then showed that plants both flowered and set seed earlier in response to increases in temperature, so that the seeds were shed precisely when the temperature was between 14°C and 15°C.
Springthorpe and Penfield discovered that rise in the average temperature when a plant set seed from 14°C to 15°C had a dramatic effect on the seeds. Almost all of the seeds that developed below 14°C became dormant, while very few of the seeds that developed above 15°C became dormant.
From their findings, Springthorpe and Penfield predict that the temperature control of flowering time has evolved to constrain when seeds are set and ensure that plants produce a mixture of seeds: some that will become dormant, and some that will not. Their findings also show that modelling the whole life history of an organism has the potential to reveal strategies that are not obvious when studying single events in isolation. If the model was extended to include genetic variation across populations of plants, this approach could give new insights into how individual genes help plants adapt to weather and climate.
PMCID: PMC4378508  PMID: 25824056
temperature; phenology; seed dormancy; germination; flowering time; life history; Arabidopsis
23.  Herbivore and Fungal Pathogen Exclusion Affects the Seed Production of Four Common Grassland Species 
PLoS ONE  2010;5(8):e12022.
Insect herbivores and fungal pathogens can independently affect plant fitness, and may have interactive effects. However, few studies have experimentally quantified the joint effects of insects and fungal pathogens on seed production in non-agricultural populations. We examined the factorial effects of insect herbivore exclusion (via insecticide) and fungal pathogen exclusion (via fungicide) on the population-level seed production of four common graminoid species (Andropogon gerardii, Schizachyrium scoparium, Poa pratensis, and Carex siccata) over two growing seasons in Minnesota, USA. We detected no interactive effects of herbivores and pathogens on seed production. However, the seed production of all four species was affected by either insecticide or fungicide in at least one year of the study. Insecticide consistently doubled the seed production of the historically most common species in the North American tallgrass prairie, A. gerardii (big bluestem). This is the first report of insect removal increasing seed production in this species. Insecticide increased A. gerardii number of seeds per seed head in one year, and mass per seed in both years, suggesting that consumption of flowers and seed embryos contributed to the effect on seed production. One of the primary insect species consuming A. gerardii flowers and seed embryos was likely the Cecidomyiid midge, Contarinia wattsi. Effects on all other plant species varied among years. Herbivores and pathogens likely reduce the dispersal and colonization ability of plants when they reduce seed output. Therefore, impacts on seed production of competitive dominant species may help to explain their relatively poor colonization abilities. Reduced seed output by dominant graminoids may thereby promote coexistence with subdominant species through competition-colonization tradeoffs.
PMCID: PMC2920334  PMID: 20711408
24.  Inbreeding Depression under Drought Stress in the Rare Endemic Echium wildpretii (Boraginaceae) on Tenerife, Canary Islands 
PLoS ONE  2012;7(10):e47415.
How climate-change induced environmental stress may alter the effects of inbreeding in patchy populations of rare species is poorly understood. We investigated the fitness of progeny from experimental self- and cross-pollinations in eight populations of different size of Echium wildpretii, a rare endemic plant of the arid subalpine zone of the Canarian island of Tenerife. As control treatments we used open pollination and autonomous selfing. The seed set of open-pollinated flowers was 55% higher than that of autonomously selfed flowers, showing the importance of animal pollination for reproductive success. The seed set, seed mass and germination rate of seedlings of hand-selfed flowers was similar to that of hand-crossed flowers, indicating weak inbreeding depression (seed set –4.4%, seed mass –4.1%, germination –7.3%). Similarly, under normal watering there were no significant effects of inbreeding on seedling survival (–3.0%). However, under low watering of seedlings inbreeding depression was high (survival –50.2%). Seed set of open- and hand-outcrossed-pollinated flowers was higher in large than in small populations, possibly due to more frequent biparental inbreeding in the latter. However, later measures of progeny fitness were not significantly influenced by population size. We predict that increasing drought duration and frequency due to climate change and reductions of population sizes may increase inbreeding depression in this charismatic plant species and thus threaten its future survival in the longer term.
PMCID: PMC3480375  PMID: 23115645
25.  LEA polypeptide profiling of recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance 
Journal of Experimental Botany  2013;64(14):4559-4573.
In contrast to orthodox seeds that acquire desiccation tolerance during maturation, recalcitrant seeds are unable to survive drying. These desiccation-sensitive seeds constitute an interesting model for comparative analysis with phylogenetically close species that are desiccation tolerant. Considering the importance of LEA (late embryogenesis abundant) proteins as protective molecules both in drought and in desiccation tolerance, the heat-stable proteome was characterized in cotyledons of the legume Castanospermum australe and it was compared with that of the orthodox model legume Medicago truncatula. RNA sequencing identified transcripts of 16 homologues out of 17 LEA genes for which polypeptides are detected in M. truncatula seeds. It is shown that for 12 LEA genes, polypeptides were either absent or strongly reduced in C. australe cotyledons compared with M. truncatula seeds. Instead, osmotically responsive, non-seed-specific dehydrins accumulated to high levels in the recalcitrant cotyledons compared with orthodox seeds. Next, M. truncatula mutants of the ABSCISIC ACID INSENSITIVE3 (ABI3) gene were characterized. Mature Mtabi3 seeds were found to be desiccation sensitive when dried below a critical water content of 0.4g H2O g DW–1. Characterization of the LEA proteome of the Mtabi3 seeds revealed a subset of LEA proteins with severely reduced abundance that were also found to be reduced or absent in C. australe cotyledons. Transcripts of these genes were indeed shown to be ABI3 responsive. The results highlight those LEA proteins that are critical to desiccation tolerance and suggest that comparable regulatory pathways responsible for their accumulation are missing in both desiccation-sensitive genotypes, revealing new insights into the mechanistic basis of the recalcitrant trait in seeds.
PMCID: PMC3808335  PMID: 24043848
abi3; Castanospermum australe; desiccation tolerance; late embryogenesis abundant proteins; Medicago truncatula; proteomics; recalcitrant seed; RNAseq.

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