Work Ability Index: Psychometric Testing in Aeronautical Industry Workers.

Background: The Work Ability Index (WAI) is an instrument that measures work ability. The wide dispersion of the WAI internationally has led to its adaptation for use in different countries. This study aimed to evaluate the psychometric properties of the Spanish version of the WAI. Methods: A methodological design was used over an opportunistic sample of 233 workers in the aeronautical industry in Spain. Reliability was evaluated through internal consistency. Factorial validity, known groups, and convergent validity were tested. Results: The Cronbach's alpha and item-total correlation indicated an adequate internal consistency. The confirmatory factor analysis, performed to evaluate the factorial validity, found adequate fit indices for a two-factor solution with a high correlation between the factors. Factor 1, "Subjectively estimated work ability and resources", was composed of 3 subscales and factor 2, "Ill-health-related", of 2 subscales. Subscales 4 and 6 had loading in both factors. Workers under 45 years of age obtained higher significant scores than older ones. Convergent validity was also evidenced since WAI was highly correlated with self-assessment of health status. Conclusions: The Spanish version of the WAI has shown evidence of reliability and validity in this study, supporting its use in individual and collective health surveillance by occupational health professionals. The factorial solution that was found has previously been reported in another international context. However, further research is needed to resolve the discrepancies detected in the role of some subscales between other national and international studies.

The Arabidopsis endosperm is a temperature-sensing tissue that implements seed thermoinhibition through phyB.

Seed thermoinhibition, the repression of germination under high temperatures, prevents seedling establishment under potentially fatal conditions. Thermoinhibition is relevant for phenology and agriculture, particularly in a warming globe. The temperature sensing mechanisms and signaling pathways sustaining thermoinhibition are unknown. Here we show that thermoinhibition in Arabidopsis thaliana is not autonomously controlled by the embryo but is rather implemented by the endosperm. High temperature is sensed through endospermic phyB by accelerating its reversion from the active signaling Pfr form into the inactive Pr form, as previously described in seedlings. This leads to thermoinhibition mediated by PIFs, mainly PIF1, PIF3 and PIF5. Endospermic PIF3 represses the expression of the endospermic ABA catabolic gene CYP707A1 and promotes endospermic ABA accumulation and release towards the embryo to block its growth. Furthermore, endospermic ABA represses embryonic PIF3 accumulation that would otherwise promote embryonic growth. Hence, under high temperatures PIF3 exerts opposite growth responses in the endosperm and embryo.

Parental and Environmental Control of Seed Dormancy in Arabidopsis thaliana.

Seed dormancy-the absence of seed germination under favorable germination conditions-is a plant trait that evolved to enhance seedling survival by avoiding germination under unsuitable environmental conditions. In Arabidopsis, dormancy levels are influenced by the seed coat composition, while the endosperm is essential to repress seed germination of dormant seeds upon their imbibition. Recent research has shown that the mother plant modulates its progeny seed dormancy in response to seasonal temperature changes by changing specific aspects of seed coat and endosperm development. This process involves genomic imprinting by means of epigenetic marks deposited in the seed progeny and regulators previously known to regulate flowering time. This review discusses and summarizes these discoveries and provides an update on our present understanding of the role of DOG1 and abscisic acid, two key contributors to dormancy.

The Arabidopsis mature endosperm promotes seedling cuticle formation via release of sulfated peptides.

In Arabidopsis mature seeds, the onset of the embryo-to-seedling transition is nonautonomously controlled, being blocked by endospermic abscisic acid (ABA) release under unfavorable conditions. Whether the mature endosperm governs additional nonautonomous developmental processes during this transition is unknown. Mature embryos have a more permeable cuticle than seedlings, consistent with their endospermic ABA uptake capability. Seedlings acquire their well-sealing cuticles adapted to aerial lifestyle during germination. Endosperm removal prevents seedling cuticle formation, and seed reconstitution by endosperm grafting onto embryos shows that the endosperm promotes seedling cuticle development. Grafting different endosperm and embryo mutant combinations, together with biochemical, microscopy, and mass spectrometry approaches, reveal that the release of tyrosylprotein sulfotransferase (TPST)-sulfated CIF2 and PSY1 peptides from the endosperm promotes seedling cuticle development. Endosperm-deprived embryos produced nonviable seedlings bearing numerous developmental defects, not related to embryo malnutrition, all restored by exogenously provided endosperm. Hence, seedling establishment is nonautonomous, requiring the mature endosperm.

Arabidopsis thaliana Mature Endosperm Dissection and Isolation of Genomic DNA from Mature Seed Tissues.

We describe methods to separate endosperms and embryos from Arabidopsis thaliana mature seeds in large amounts and to isolate high-quality genomic DNA from those tissues. The resulting materials are suitable for analysis of DNA methylation by bisulfite sequencing or histone modifications by chromatin immunoprecipitation (ChIP).

Identification of tannic cell walls at the outer surface of the endosperm upon Arabidopsis seed coat rupture.

The seed coat is specialized dead tissue protecting the plant embryo from mechanical and oxidative damage. Tannins, a type of flavonoids, are antioxidants known to accumulate in the Arabidopsis seed coat and transparent testa mutant seeds, deficient in flavonoid synthesis, exhibit low viability. However, their precise contribution to seed coat architecture and biophysics remains evasive. A seed coat cuticle, covering the endosperm outer surface and arising from the seed coat inner integument 1 cell layer was, intriguingly, previously shown to be more permeable in transparent testa mutants deficient not in cuticular component synthesis, but rather in flavonoid synthesis. Investigating the role of flavonoids in cuticle permeability led us to identify periclinal inner integument 1 tannic cell walls being attached, together with the cuticle, to the endosperm surface upon seed coat rupture. Hence, inner integument 1 tannic cell walls and the cuticle form two fused layers present at the surface of the exposed endosperm upon seed coat rupture, regulating its permeability. Their potential physiological role during seed germination is discussed.

Embryonic Photosynthesis Affects Post-Germination Plant Growth.

Photosynthesis is the fundamental process fueling plant vegetative growth and development. The progeny of plants relies on maternal photosynthesis, via food reserves in the seed, to supply the necessary energy for seed germination and early seedling establishment. Intriguingly, before seed maturation, Arabidopsis (Arabidopsis thaliana) embryos are also photosynthetically active, the biological significance of which remains poorly understood. Investigating this system is genetically challenging because mutations perturbing photosynthesis are expected to affect both embryonic and vegetative tissues. Here, we isolated a temperature-sensitive mutation affecting CPN60α2, which encodes a subunit of the chloroplast chaperonin complex CPN60. When exposed to cold temperatures, cpn60α2 mutants accumulate less chlorophyll in newly produced tissues, thus allowing the specific disturbance of embryonic photosynthesis. Analyses of cpn60α2 mutants were combined with independent genetic and pharmacological approaches to show that embryonic photosynthetic activity is necessary for normal skoto- and photomorphogenesis in juvenile seedlings as well as long-term adult plant development. Our results reveal the importance of embryonic photosynthetic activity for normal adult plant growth, development, and health.

polyamine uptake transporter 2 (put2) and decaying seeds enhance phyA-mediated germination by overcoming PIF1 repression of germination.

Red light promotes germination after activating phytochrome phyB, which destabilizes the germination repressor PIF1. Early upon seed imbibition, canopy light, unfavorable for photosynthesis, represses germination by stabilizing PIF1 after inactivating phyB. Paradoxically, later upon imbibition, canopy light stimulates germination after activating phytochrome phyA. phyA-mediated germination is poorly understood and, intriguingly, is inefficient, compared to phyB-mediated germination, raising the question of its physiological significance. A genetic screen identified polyamine uptake transporter 2 (put2) mutants that overaccumulate polyamines, a class of antioxidant polycations implicated in numerous cellular functions, which we found promote phyA-mediated germination. In WT seeds, our data suggest that canopy light represses polyamines accumulation through PIF1 while red light promotes polyamines accumulation. We show that canopy light also downregulates PIF1 levels, through phyA; however, PIF1 reaccumulates rapidly, which limits phyA-mediated germination. High polyamines levels in decaying seeds bypass PIF1 repression of germination and stimulate phyA-mediated germination, suggesting an adaptive mechanism promoting survival when viability is compromised.

Non-canonical RNA-directed DNA methylation participates in maternal and environmental control of seed dormancy.

Seed dormancy is an adaptive trait preventing premature germination out of season. In a previous report (Piskurewicz et al., 2016) we showed that dormancy levels are maternally inherited through the preferential maternal allele expression in the seed endosperm of ALLANTOINASE (ALN), a negative regulator of dormancy. Here we show that suppression of ALN paternal allele expression is imposed by non-canonical RNA-directed DNA methylation (RdDM) of the paternal ALN allele promoter. Dormancy levels are further enhanced by cold during seed development. We show that DNA methylation of the ALN promoter is stimulated by cold in a tissue-specific manner through non-canonical RdDM, involving RDR6 and AGO6. This leads to suppression of ALN expression and further promotion of seed dormancy. Our results suggest that tissue-specific and cold-induced RdDM is superimposed to parental allele imprints to deposit in the seed progeny a transient memory of environmental conditions experienced by the mother plant.

ICE1 and ZOU determine the depth of primary seed dormancy in Arabidopsis independently of their role in endosperm development.

Seed dormancy is a widespread and key adaptive trait that is essential for the establishment of soil seed banks and prevention of pre-harvest sprouting. Herein we demonstrate that the endosperm-expressed transcription factors ZHOUPI (ZOU) and INDUCER OF CBF EXPRESSION1 (ICE1) play a role in determining the depth of primary dormancy in Arabidopsis. We show that ice1 or zou increases seed dormancy and the double mutant has an additive phenotype. This increased dormancy is associated with increased ABA levels, and can be separated genetically from any role in endosperm maturation because loss of ABA biosynthesis or DELAY OF GERMINATION 1 reverses the dormancy phenotype without affecting the aberrant seed morphology. Consistent with these results, ice1 endosperms had an increased capacity for preventing embryo greening, a phenotype previously associated with an increase in endospermic ABA levels. Although ice1 changes the expression of many genes, including some in ABA biosynthesis, catabolism and/or signalling, only ABA INSENSITIVE 3 is significantly misregulated in ice1 mutants. We also demonstrate that ICE1 binds to and inhibits expression of ABA INSENSITIVE 3. Our data demonstrate that Arabidopsis ICE1 and ZOU determine the depth of primary dormancy during maturation independently of their effect on endosperm development.

Chloroplast Biogenesis Controlled by DELLA-TOC159 Interaction in Early Plant Development.

Chloroplast biogenesis, visible as greening, is the key to photoautotrophic growth in plants. At the organelle level, it requires the development of non-photosynthetic, color-less proplastids to photosynthetically active, green chloroplasts at early stages of plant development, i.e., in germinating seeds. This depends on the import of thousands of different preproteins into the developing organelle by the chloroplast protein import machinery [1]. The preprotein import receptor TOC159 is essential in the process, its mutation blocking chloroplast biogenesis and resulting in albino plants [2]. The molecular mechanisms controlling the onset of chloroplast biogenesis during germination are largely unknown. Germination depends on the plant hormone gibberellic acid (GA) and is repressed by DELLA when GA concentrations are low [3, 4]. Here, we show that DELLA negatively regulates TOC159 protein abundance under low GA. The direct DELLA-TOC159 interaction promotes TOC159 degradation by the ubiquitin/proteasome system (UPS). Moreover, the accumulation of photosynthesis-associated proteins destined for the chloroplast is downregulated posttranscriptionally. Analysis of a model import substrate indicates that it is targeted for removal by the UPS prior to import. Thus, under low GA, the UPS represses chloroplast biogenesis by a dual mechanism comprising the DELLA-dependent destruction of the import receptor TOC159, as well as that of its protein cargo. In conclusion, our data provide a molecular framework for the GA hormonal control of proplastid to chloroplast transition during early plant development.

The plant pathogen Pseudomonas aeruginosa triggers a DELLA-dependent seed germination arrest in Arabidopsis.

To anticipate potential seedling damage, plants block seed germination under unfavorable conditions. Previous studies investigated how seed germination is controlled in response to abiotic stresses through gibberellic and abscisic acid signaling. However, little is known about whether seeds respond to rhizosphere bacterial pathogens. We found that Arabidopsis seed germination is blocked in the vicinity of the plant pathogen Pseudomonas aeruginosa. We identified L-2-amino-4-methoxy-trans-3-butenoic acid (AMB), released by P. aeruginosa, as a biotic compound triggering germination arrest. We provide genetic evidence that in AMB-treated seeds DELLA factors promote the accumulation of the germination repressor ABI5 in a GA-independent manner. AMB production is controlled by the quorum sensing system IQS. In vitro experiments show that the AMB-dependent germination arrest protects seedlings from damage induced by AMB. We discuss the possibility that this could serve as a protective response to avoid severe seedling damage induced by AMB and exposure to a pathogen.

A Maternally Deposited Endosperm Cuticle Contributes to the Physiological Defects of transparent testa Seeds.

Mature dry seeds are highly resilient plant structures where the encapsulated embryo is kept protected and dormant to facilitate its ultimate dispersion. Seed viability is heavily dependent on the seed coat's capacity to shield living tissues from mechanical and oxidative stress. In Arabidopsis (Arabidopsis thaliana), the seed coat, also called the testa, arises after the differentiation of maternal ovular integuments during seed development. We recently described a thick cuticle tightly embedded in the mature seed's endosperm cell wall. We show here that it is produced by the maternal inner integument 1 layer and, remarkably, transferred to the developing endosperm. Arabidopsis transparent testa (tt) mutations cause maternally derived seed coat pigmentation defects. TT gene products encode proteins involved in flavonoid metabolism and regulators of seed coat development. tt mutants have abnormally high seed coat permeability, resulting in lower seed viability and dormancy. However, the biochemical basis of this high permeability is not fully understood. We show that the cuticles of developing tt mutant integuments have profound structural defects, which are associated with enhanced cuticle permeability. Genetic analysis indicates that a functional proanthocyanidin synthesis pathway is required to limit cuticle permeability, and our results suggest that proanthocyanidins could be intrinsic components of the cuticle. Together, these results show that the formation of a maternal cuticle is an intrinsic part of the normal integumental differentiation program leading to testa formation and is essential for the seed's physiological properties.

Primary seed dormancy: a temporally multilayered riddle waiting to be unlocked.

Primary seed dormancy is an important adaptive plant trait whereby seed germination is blocked under conditions that would otherwise be favorable for germination. This trait is found in newly produced mature seeds of many species, but not all. Once produced, dry seeds undergo an aging time period, called dry after-ripening, during which they lose primary dormancy and gradually acquire the capacity to germinate when exposed to favorable germination conditions. Primary seed dormancy has been extensively studied not only for its scientific interest but also for its ecological, phenological, and agricultural importance. Nevertheless, the mechanisms underlying primary seed dormancy and its regulation during after-ripening remain poorly understood. Here we review the principal developmental stages where primary dormancy is established and regulated prior to and during seed after-ripening, where it is progressively lost. We attempt to identify and summarize what is known about the molecular and genetic mechanisms intervening over time in each of these stages.

Dormancy-specific imprinting underlies maternal inheritance of seed dormancy in Arabidopsis thaliana.

Mature seed dormancy is a vital plant trait that prevents germination out of season. In Arabidopsis, the trait can be maternally regulated but the underlying mechanisms sustaining this regulation, its general occurrence and its biological significance among accessions are poorly understood. Upon seed imbibition, the endosperm is essential to repress the germination of dormant seeds. Investigation of genomic imprinting in the mature seed endosperm led us to identify a novel set of imprinted genes that are expressed upon seed imbibition. Remarkably, programs of imprinted gene expression are adapted according to the dormancy status of the seed. We provide direct evidence that imprinted genes play a role in regulating germination processes and that preferential maternal allelic expression can implement maternal inheritance of seed dormancy levels.

Basic Techniques to Assess Seed Germination Responses to Abiotic Stress in Arabidopsis thaliana.

The model organism Arabidopsis thaliana has been extensively used to unmask the molecular genetic signaling pathways controlling seed germination in plants. In Arabidopsis, the normal seed to seedling developmental transition involves testa rupture soon followed by endosperm rupture, radicle elongation, root hair formation, cotyledon expansion, and greening. Here we detail a number of basic procedures to assess Arabidopsis seed germination in response to different light (red and far-red pulses), temperature (seed thermoinhibition), and water potential (osmotic stress) environmental conditions. We also discuss the role of the endosperm and how its germination-repressive activity can be monitored genetically by means of a seed coat bedding assay. Finally we detail how to evaluate germination responses to changes in gibberellin (GA) and abscisic acid (ABA) levels by manipulating pharmacologically the germination medium.

An Endosperm-Associated Cuticle Is Required for Arabidopsis Seed Viability, Dormancy and Early Control of Germination.

Cuticular layers and seeds are prominent plant adaptations to terrestrial life that appeared early and late during plant evolution, respectively. The cuticle is a waterproof film covering plant aerial organs preventing excessive water loss and protecting against biotic and abiotic stresses. Cutin, consisting of crosslinked fatty acid monomers, is the most abundant and studied cuticular component. Seeds are dry, metabolically inert structures promoting plant dispersal by keeping the plant embryo in an arrested protected state. In Arabidopsis thaliana seeds, the embryo is surrounded by a single cell endosperm layer itself surrounded by a seed coat layer, the testa. Whole genome analyses lead us to identify cutin biosynthesis genes as regulatory targets of the phytohormones gibberellins (GA) and abscisic acid (ABA) signaling pathways that control seed germination. Cutin-containing layers are present in seed coats of numerous species, including Arabidopsis, where they regulate permeability to outer compounds. However, the role of cutin in mature seed physiology and germination remains poorly understood. Here we identify in mature seeds a thick cuticular film covering the entire outer surface of the endosperm. This seed cuticle is defective in cutin-deficient bodyguard1 seeds, which is associated with alterations in endospermic permeability. Furthermore, mutants affected in cutin biosynthesis display low seed dormancy and viability levels, which correlates with higher levels of seed lipid oxidative stress. Upon seed imbibition cutin biosynthesis genes are essential to prevent endosperm cellular expansion and testa rupture in response to low GA synthesis. Taken together, our findings suggest that in the course of land plant evolution cuticular structures were co-opted to achieve key physiological seed properties.

Abscisic acid transporters cooperate to control seed germination.

Seed germination is a key developmental process that has to be tightly controlled to avoid germination under unfavourable conditions. Abscisic acid (ABA) is an essential repressor of seed germination. In Arabidopsis, it has been shown that the endosperm, a single cell layer surrounding the embryo, synthesizes and continuously releases ABA towards the embryo. The mechanism of ABA transport from the endosperm to the embryo was hitherto unknown. Here we show that four AtABCG transporters act in concert to deliver ABA from the endosperm to the embryo: AtABCG25 and AtABCG31 export ABA from the endosperm, whereas AtABCG30 and AtABCG40 import ABA into the embryo. Thus, this work establishes that radicle extension and subsequent embryonic growth are suppressed by the coordinated activity of multiple ABA transporters expressed in different tissues.

A seed coat bedding assay to genetically explore in vitro how the endosperm controls seed germination in Arabidopsis thaliana.

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a "seed coat bedding" assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.