The seed morphospace, a new contribution towards the multidimensional study of angiosperm sexual reproductive biology.

BACKGROUND: The evolutionary success of flowering plants is associated with the vast diversity of their reproductive structures. Despite recent progress in understanding angiosperm-wide trends of floral structure and evolution, a synthetic view of the diversity in seed form and function across angiosperms is lacking. SCOPE: Here we present a roadmap to synthesise the diversity of seed forms in extant angiosperms, relying on the morphospace concept, i.e. a mathematical representation which relates multiple traits and describes the realised morphologies. We provide recommendations on how to broaden the range of measurable traits beyond mass, by using key morphological traits representative of the embryo, endosperm, and seed coat but also fruit attributes (e.g., dehiscence, fleshiness). These key traits were used to construct and analyse a morphospace to detect evolutionary trends and gain insight into how morphological traits relate to seed functions. Finally, we outline challenges and future research directions, combining the morphospace with macroevolutionary comparative methods to underline the drivers that gave rise to the diversity of observed seed forms. CONCLUSIONS: We conclude that this multidimensional approach has the potential, although still untapped, to improve our understanding of covariation among reproductive traits, and further elucidate angiosperm reproductive biology as a whole.

Seed biology imperative for conservation and restoration of Swertia thomsonii C.B. Clarke-an endemic medicinal plant of Himalaya.

Endemic medicinal plants deserve immediate research priorities as they typically show a limited distribution range, represent few and fragmented populations in the wild and are currently facing anthropogenic threats like overharvesting and habitat degradation. One of the important aspects of ensuring their successful conservation and sustainable utilization lies in comprehending the fundamental seed biology, particularly the dormancy status and seed germination requirements of these plants. Here, we studied the seed eco-physiology and regeneration potential of Swertia thomsonii-an endemic medicinal plant of western Himalaya. We investigated the effect of different pre-sowing treatments, sowing media and sowing depth on seed germination parameters of S. thomsonii. Seeds of S. thomsonii exhibit morphophysiological dormancy (MPD), i.e. when the embryo of the seed is morphologically and/or physiologically immature. Wet stratification at 4 °C for 20 days, pre-sowing treatment with 50 ppm GA3 and pre-sowing treatment with 50 ppm KNO3 were found ideal for overcoming dormancy and enhancing the seed germination of S. thomsonii. Furthermore, seed germination and seedling survival were significantly influenced by pre-sowing treatments, sowing media and sowing depth. The percentage of seed germination and seedling survival got enhanced up to 84-86% and 73-75% respectively when seeds were pre-treated with GA3 or KNO3 and then sown in cocopeat + perlite (1:1) at a depth of 1 cm. The information obtained in the present study outlines an efficient protocol for large-scale cultivation of S. thomsonii thereby limiting the pressure of overexploitation from its natural habitats and may also help in the restoration and conservation of this valuable plant species.

Dormancy cycling: translation-related transcripts are the main difference between dormant and non-dormant seeds in the field.

Primary seed dormancy is a mechanism that orchestrates the timing of seed germination in order to prevent out-of-season germination. Secondary dormancy can be induced in imbibed seeds when they encounter prolonged unfavourable conditions. Secondary dormancy is not induced during dry storage, and therefore the mechanisms underlying this process have remained largely unexplored. Here, a 2-year seed burial experiment in which dormancy cycling was studied at the physiological and transcriptional level is presented. For these analyses six different Arabidopsis thaliana genotypes were used: Landsberg erecta (Ler) and the dormancy associated DELAY OF GERMINATION (DOG) near-isogenic lines 1, 2, 3, 6 and 22 (NILDOG1, 2, 3, 6 and 22). The germination potential of seeds exhumed from the field showed that these seeds go through dormancy cycling and that the dynamics of this cycling is genotype dependent. RNA-seq analysis revealed large transcriptional changes during dormancy cycling, especially at the time points preceding shifts in dormancy status. Dormancy cycling is driven by soil temperature and the endosperm is important in the perception of the environment. Genes that are upregulated in the low- to non-dormant stages are enriched for genes involved in translation, indicating that the non-dormant seeds are prepared for rapid seed germination.

Genetic activity during early plant embryogenesis.

Seeds are essential for human civilization, so understanding the molecular events underpinning seed development and the zygotic embryo it contains is important. In addition, the approach of somatic embryogenesis is a critical propagation and regeneration strategy to increase desirable genotypes, to develop new genetically modified plants to meet agricultural challenges, and at a basic science level, to test gene function. We briefly review some of the transcription factors (TFs) involved in establishing primary and apical meristems during zygotic embryogenesis, as well as TFs necessary and/or sufficient to drive somatic embryo programs. We focus on the model plant Arabidopsis for which many tools are available, and review as well as speculate about comparisons and contrasts between zygotic and somatic embryo processes.

Regulatory-associated protein of TOR (RAPTOR) alters the hormonal and metabolic composition of Arabidopsis seeds, controlling seed morphology, viability and germination potential.

Target of Rapamycin (TOR) is a positive regulator of growth and development in all eukaryotes, which positively regulates anabolic processes like protein synthesis, while repressing catabolic processes, including autophagy. To better understand TOR function we decided to analyze its role in seed development and germination. We therefore performed a detailed phenotypic analysis using mutants of the REGULATORY-ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), a conserved TOR interactor, acting as a scaffold protein, which recruits substrates for the TOR kinase. Our results show that raptor1b plants produced seeds that were delayed in germination and less resistant to stresses, leading to decreased viability. These physiological phenotypes were accompanied by morphological changes including decreased seed-coat pigmentation and reduced production of seed-coat mucilage. A detailed molecular analysis revealed that many of these morphological changes were associated with significant changes of the metabolic content of raptor1b seeds, including elevated levels of free amino acids, as well as reduced levels of protective secondary metabolites and storage proteins. Most of these observed changes were accompanied by significantly altered phytohormone levels in the raptor1b seeds, with increases in abscisic acid, auxin and jasmonic acid, which are known to inhibit germination. Delayed germination and seedling growth, observed in the raptor1b seeds, could be partially restored by the exogenous supply of gibberellic acid, indicating that TOR is at the center of a regulatory hub controlling seed metabolism, maturation and germination.

Measuring metabolic rates of small terrestrial organisms by fluorescence-based closed-system respirometry.

We explore a recent, innovative variation of closed-system respirometry for terrestrial organisms, whereby oxygen partial pressure (PO2 ) is repeatedly measured fluorometrically in a constant-volume chamber over multiple time points. We outline a protocol that aligns this technology with the broader literature on aerial respirometry, including the calculations required to accurately convert O2 depletion to metabolic rate (MR). We identify a series of assumptions, and sources of error associated with this technique, including thresholds where O2 depletion becomes limiting, that impart errors to the calculation and interpretation of MR. Using these adjusted calculations, we found that the resting MR of five species of angiosperm seeds ranged from 0.011 to 0.640 ml g-1 h-1, consistent with published seed MR values. This innovative methodology greatly expands the lower size limit of terrestrial organisms that can be measured, and offers the potential for measuring MR changes over time as a result of physiological processes of the organism.

Genetic dissection of metabolite variation in Arabidopsis seeds: evidence for mQTL hotspots and a master regulatory locus of seed metabolism.

To gain insight into genetic factors controlling seed metabolic composition and its relationship to major seed properties, an Arabidopsis recombinant inbred line (RIL) population, derived from accessions Col-0 and C24, was studied using an MS-based metabolic profiling approach. Relative intensities of 311 polar primary metabolites were used to identify associated genomic loci and to elucidate their interactions by quantitative trait locus (QTL) mapping. A total of 786 metabolic QTLs (mQTLs) were unequally distributed across the genome, forming several hotspots. For the branched-chain amino acid leucine, mQTLs and candidate genes were elucidated in detail. Correlation studies displayed links between metabolite levels, seed protein content, and seed weight. Principal component analysis revealed a clustering of samples, with PC1 mapping to a region on the short arm of chromosome IV. The overlap of this region with mQTL hotspots indicates the presence of a potential master regulatory locus of seed metabolism. As a result of database queries, a series of candidate regulatory genes, including bZIP10, were identified within this region. Depending on the search conditions, metabolic pathway-derived candidate genes for 40-61% of tested mQTLs could be determined, providing an extensive basis for further identification and characterization of hitherto unknown genes causal for natural variation of Arabidopsis seed metabolism.

Seed biology and in vitro seed germination of Cypripedium.

Cypripedium orchids have high horticultural value. The populations of most species are very geographically restricted and they are becoming increasingly rare due to the destruction of native habitats and illegal collection. Reduction of the commercial value through large-scale propagation in vitro is a preferable option to reduce pressure from illegal collection. Cypripedium species are commercially propagated via seed germination in vitro. This review focuses on in vitro seed germination and provides an in-depth analysis of the seed biology of this genus.

Intrapopulation germinability may help the Mediterranean plant species Poterium spinosum L. to cope with climate changes and landscape fragmentation.

Poterium spinosum L. is a key plant species forming typical shrub communities, distributed across the Mediterranean eastern coasts. The conservation of P. spinosum is thus of the utmost importance, especially due to the ever-increasing environmental pressures like climate changes and habitat fragmentation. This study, in particular, investigated for the first time the germination variability of P. spinosum at intrapopulation level, by analysing the germination behavior of five different subpopulations growing along the coasts of Sicily. For a more exhaustive picture of the main drivers of biodiversity loss affecting the distributional area of P. spinosum, the trends of climate and land-cover changes were also studied over the periods 1931-2020 and 1958-2018, respectively. The results found significant intrapopulation variability in P. spinosum, whose germination parameters showed that fruits and seeds from distinct subpopulations respond differently to diverse temperatures. Seeds showed generally higher values of final germination percentage (FGP) compared to fruits, and at higher temperatures: the highest FGP in seeds was 70% at 20 °C, whereas in fruits was 58.2% at 15 °C. The environmental threats showed worrying trends across the study area: during 1931-2020, the average temperature increased by 1.5 °C, whereas the average rainfall declined from 710 to 650 mm. Similarly, in the period 1958-2018, the analysis of the CORINE land-cover changes showed a highly fragmented agricultural landscape, where natural areas were reduced to 2.5-5.0%. Germination variability at intrapopulation level should be considered as a fundamental adaptation strategy, which can increase the reproductive success of P. spinosum under climate and land-cover changes.

Same, same, but different: dissimilarities in the hydrothermal germination performance of range-restricted endemics emerge despite microclimatic similarities.

Seed germination responses for most narrow-range endemic species are poorly understood, imperilling their conservation management in the face of warming and drying terrestrial ecosystems. We quantified the realized microclimatic niches and the hydrothermal germination thresholds in four threatened taxa (Tetratheca erubescens, Tetratheca harperi, Tetratheca paynterae subsp. paynterae and Tetratheca aphylla subsp. aphylla) that are restricted to individual Banded Ironstone Formations in Western Australia. While T. aphylla subsp. aphylla largely failed to germinate in our trials, all other species demonstrated extended hydrothermal time accumulation (186-500°C MPa days), cool minimum temperatures (7.8-8.5°C), but broad base water potential thresholds (-2.46 to -5.41 MPa) under which germination occurred. These slow germination dynamics are suggestive of cool and wet winter months, where soil moisture is retained to a greater capacity in local microsites where these species occur, rather than the warmer and drier conditions in the surrounding arid environment. Hydrothermal time-to-event modelling showed that each species occupied unique hydrothermal germination niches, which correspond with the microclimatic differences the species are exposed to. Our results provide a baseline understanding for environmental and germination thresholds that govern the recruitment, and ultimately the population structure and persistence, of these short-range endemic plants. In addition, our results can aid future conservation, as well as restoration actions such as translocation to bolster population numbers and to mitigate against losses due to anthropogenic disturbance and global environmental change.

Seed dormancy, climate changes, desertification and soil use transformation threaten the Mediterranean endemic monospecific plant Petagnaea gussonei.

This study investigated the germination capacity (endogenous factor) of Petagnaea gussonei (Spreng.) Rauschert, an endemic monospecific plant considered as a relict species of the ancient Mediterranean Tertiary flora. This investigation focused also on the temporal trends of soil-use, climate and desertification (exogenous factors) across the natural range of P. gussonei. The final germination percentage showed low values between 14 and 32%, the latter obtained with GA3 and agar at 10 °C. The rising temperatures in the study area will further increase the dormancy of P. gussonei, whose germination capacity was lower and slower at temperatures higher than 10 °C. A further limiting factor of P. gussonei is its dormancy, which seems to be morpho-physiological. Regarding climate trends, in the period 1931-2020, the average temperature increased by 0.5 °C, from 15.4 to 15.9 °C, in line with the projected climate changes throughout the twenty-first century across the Mediterranean region. The average annual rainfall showed a relatively constant value of c. 900 mm, but extreme events grew considerably in the period 1991-2020. Similarly, the land affected by desertification expanded in an alarming way, by increasing from 21.2% in 2000 to 47.3% in 2020. Soil-use changes created also a complex impacting mosaic where c. 40% are agricultural areas. The effective conservation of P. gussonei should be multilateral by relying on germplasm banks, improving landscape connectivity and vegetation cover, and promoting climate policies.

Effects of Graphene-Based Metal Composite and Urea on Seed Germination and Performance of Berberis chitria Buch.-Ham. ex Lindl.

Being an important source of berberine, Berberis chitria Buch.-Ham. ex Lindl. (Berberidaceae) has high demand in pharmaceutical industries. Its populations are diminishing due to overexploitation, habitat loss, slow-growing nature, and climate change. It is important to develop propagation protocols to sustain its natural populations and ensure its survival in the future. Fertilizers play an essential role in the yield and productivity of different crops. Among others, urea is the most abundantly used fertilizer in crops. Its effects on the yield and survival of medicinal plants are poorly studied. However, it is known that applying urea for a long time affects the soil negatively. Due to these negative effects, alternative fertilizers such as graphene-based metal composite (GMC) are being tested for their efficiency. In the present study, for the first time, we tested the effects of urea and GMC on the germination and performance of B. chitria. GMC showed maximum germination at 30 ppm (75%) and urea at 15 ppm (79%). Findings reveal non-significant effects of GMC and urea on germination and performance of B. chitria, suggesting the use of GMC as an alternative fertilizer.

Reproductive Ecology of the Invasive Alien Shrub Pyracantha angustifolia in the Grassland Biome, South Africa.

Knowledge on reproductive traits of problematic invasive alien plants, such as the woody invasive shrub Pyracantha angustifolia of temperate Chinese origin, can help better manage invasive species. To determine factors contributing to its invasion, we investigated floral visitors and pollen loads, self-compatibility, seed set, seed rain, soil seed banks, and seed longevity in the soil. Generalist insects were recorded visiting flowers and all carried pollen loads of high purity (>70%). Floral visitor exclusion experiments showed that P. angustifolia can set seed (66%) without pollen vectors, although natural pollination resulted in higher fruit set (91%). Fruit count surveys and seed set showed an exponentially increased relationship between seed set and plant size with high natural seed yield (±2 million seeds m-2). Soil core samples revealed a high seed density of 46,400 ± (SE) 8934 m-2 under shrubs, decreasing with distance away from the shrub. Bowl traps stationed under trees and fences confirmed that seeds were efficiently dispersed by animals. Buried seeds survived for less than six months in the soil. Due to high seed production, self-compatibility augmented by generalist pollen vectors, and effective seed dispersal by local frugivores, it is difficult to manage the spread manually. Management of this species should focus on the short life span of seeds.

How do we transport plant species with desiccation-sensitive germplasm in space?

Many useful plant species with potential for plant-based bioregenerative life support systems produce extremophile seeds with tolerance to multiple stressors, including desiccation, which allows for their transport through space in a dried state. However, other valuable species produce desiccation-sensitive seeds or are propagated clonally, and life sciences research in space has not yet addressed the challenge of alternative transport methods in microgravity for such material. Although liquid nitrogen storage is used on Earth for desiccation-sensitive germplasm, it poses atmospheric leakage problems to crewed spacecraft and therefore liquid nitrogen-free cryogenic freezing could be an alternative. Another promising approach is slow growth tissue culture, with subculture intervals extended to months or years through the precise control of the culture environment. Whilst the design of innovative systems for the transport of species with desiccation-sensitive germplasm will be demanding, the prospect still remains for their successful growth beyond Earth.

Recent progress in molecular genetics and omics-driven research in seed biology.

Elucidating the mechanisms that control seed development, metabolism, and physiology is a fundamental issue in biology. Michel Caboche had long been a catalyst for seed biology research in France up until his untimely passing away last year. To honour his memory, we have updated a review written under his coordination in 2010 entitled "Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research". This review encompassed different molecular aspects of seed development, reserve accumulation, dormancy and germination, that are studied in the lab created by M. Caboche. We have extended the scope of this review to highlight original experimental approaches implemented in the field over the past decade such as omics approaches aimed at investigating the control of gene expression, protein modifications, primary and specialized metabolites at the tissue or even cellular level, as well as seed biodiversity and the impact of the environment on seed quality.

L'élucidation des mécanismes qui contrôlent le développement, le métabolisme et la physiologie des graines est une question fondamentale en biologie. Michel Caboche a longtemps été un catalyseur de la recherche en biologie des graines en France jusqu'à son décès prématuré l'année dernière. Pour honorer sa mémoire, nous avons mis à jour une revue écrite sous sa coordination en 2010 intitulée « Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research ¼. Cette revue englobait différents aspects moléculaires du développement des graines, de l'accumulation des réserves, de la dormance et de la germination, qui sont étudiés dans le laboratoire créé par M. Caboche. Nous avons étendu la portée de cette revue pour mettre en évidence des approches expérimentales originales mises en œuvre dans le domaine au cours de la dernière décennie, telles que les approches omiques visant à étudier le contrôle de l'expression des gènes, les modifications des protéines, les métabolites primaires et spécialisés au niveau des tissus ou même des cellules, tout en tenant compte de la biodiversité des graines et de l'impact de l'environnement sur leur qualité.

Assessment of biogeographic variation in traits of Lewis flax (Linum lewisii) for use in restoration and agriculture.

Lewis flax (Linum lewisii) is widely distributed across western North America and is currently used in native ecosystem restoration. There is also growing interest in de novo domestication of Lewis flax as a perennial oilseed crop. To better understand this species and facilitate both restoration and domestication, we used common gardens to assess biogeographical variation in a variety of seed and growth traits from 37 flax accessions, consisting of 35 wild populations from the Intermountain West region, the pre-variety germplasm Maple Grove (L. lewisii) and the cultivar 'Appar' (L. perenne) and related this variation to collection site geography and climate. Results from linear mixed models suggest there is extensive phenotypic variation among populations of Lewis flax within the Intermountain West. Using a multivariate approach, we identify a key suite of traits that are related to latitude and climate and may facilitate adaptation, including flowering indeterminacy, seed mass and stem number. These traits should be taken into account when considering the release of new germplasm for restoration efforts. We also find that Lewis flax seed contains desirably high amounts of alpha-linolenic acid and is otherwise mostly indistinguishable in fatty acid composition from oil-type varieties of domesticated flax (L. usitatissimum), making it a strong candidate for domestication. This study provides fundamental knowledge for future research into the ecology and evolution of Lewis flax, which will inform its use in both restoration and agriculture.

Allelopathic and intraspecific growth competition effects establishment of direct sown Miscanthus.

High yielding perennial crops are being developed as a sustainable feedstock for renewable energy and bioproducts. Miscanthus is a leading biomass crop, but most plantations comprise a sterile hybrid Miscanthus × giganteus that is clonally propagated. To develop new varieties across large areas, rhizome cloning is inefficient, time consuming and expensive. Alternative approaches use seed, and in temperate regions, this has been successfully applied by raising seedlings as plug plants in glasshouses before transfer to the field. Direct sowing has yet to be proven commercially viable because poor germination has resulted in inconsistent stand establishment. Oversowing using seed clusters is a common approach to improve the establishment of crops and it was hypothesized that such an approach will improve uniformity of density in early Miscanthus stands and thereby improve yield. Sowing multiple seeds creates potential for new interactions, and we identified at least two inhibitory mechanisms related to seed numbers. Germinating seed produced allelopathic effects on nearby seed thereby inhibiting plant growth. The inhibitory effect of Miscanthus seed on germination percentages was related to seed number within clusters. An extract from germinating Miscanthus seed inhibited the germination of Miscanthus seed. The extract was analysed by HPLC, which identified a complex mixture including several known allelopathic compounds including proanthocyanidins and vanillic acid. There was also evidence of root competition in soil in a controlled environment experiment. When the experiment on competition was replicated at field scale, the establishment rates were much lower and there was evidence of shoot competition. We conclude that the numbers of seed required to ensure an acceptable level of establishment in the field may be economically impractical until other agronomic techniques are included either to reduce the inhibitory effects of higher seed numbers or to reduce oversowing rates.

High temperature and drought stress cause abscisic acid and reactive oxygen species accumulation and suppress seed germination growth in rice.

Seed germination is one of the most important biological processes in the life cycle of plants, and temperature and water are the two most critical environmental factors that influence seed germination. In the present study, we investigated the roles of the plant hormone abscisic acid (ABA) and reactive oxygen species (ROS) in high temperature (HT) and drought-induced inhibition of rice seed germination. HT and drought stress caused ABA accumulation in seeds and inhibited seed germination and seedling establishment. Quantitative real-time polymerase chain reaction analysis revealed that HT and drought stress induced the expression of OsNCED3, a key gene in ABA synthesis in rice seeds. In addition, ROS (O2•- and H2O2) and malondialdehyde contents were increased in germinating seeds under HT and drought stress. Moreover, we adopted the non-invasive micro-test technique to detect H2O2 and Ca2+ fluxes at the site of coleoptile emergence. HT and drought stress resulted in a H2O2 efflux, but only drought stress significantly induced Ca2+ influx. Antioxidant enzyme assays revealed that superoxide dismutase (SOD), peroxidase, catalase (CAT), and ascorbate peroxidase (APX) activity were reduced by HT and drought stress, consistent with the expression of OsCu/ZnSOD, OsCATc, and OsAPX2 during seed germination. Altogether, these results suggest that ABA and ROS accumulation under HT and drought conditions can inhibit rice seed germination and growth.

Nitrate- and nitric oxide-induced plant growth in pea seedlings is linked to antioxidative metabolism and the ABA/GA balance.

This study looks at the effects of potassium nitrate (KNO3) and sodium nitroprusside (SNP), a nitric oxide (NO)-donor, on the development, antioxidant defences and on the abscisic acid (ABA) and gibberellin (GA) levels in pea seedlings. Results show that 10 mM KNO3 and 50 µM SNP stimulate seedling fresh weight (FW), although this effect is not reverted by the action of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO-scavenger. The KNO3 treatment increased peroxidase (POX) and ascorbate oxidase (AOX) activities. SNP, on the other hand, reduced monodehydroascorbate reductase (MDHAR) activity and produced a significant increase in superoxide dismutase (SOD), POX and AOX activities. The "KNO3 plus cPTIO" treatment increased ascorbate peroxidase (APX), MDHAR, glutathione reductase (GR) and SOD activities, but POX activity decreased in relation to the KNO3 treatment. The "SNP plus cPTIO" treatment increased APX and MDHAR activities, whereas a huge decrease in POX activity occurred. Both the KNO3 and the SNP treatments increased reduced ascorbate (ASC) concentrations, which reached control values in the presence of cPTIO. All treatments increased the dehydroascorbate (DHA) level in pea seedlings, leading to a decrease in the redox state of ascorbate. In the "KNO3 plus cPTIO" treatment, an increase in the redox state of ascorbate was observed. Glutathione contents, however, were higher in the presence of SNP than in the presence of KNO3. In addition, KNO3 produced an accumulation of oxidised glutathione (GSSG), especially in the presence of cPTIO, leading to a decrease in the redox state of glutathione. The effect of SNP on reduced glutathione (GSH) levels was reverted by cPTIO, suggesting that NO has a direct effect on GSH biosynthesis or turnover. Both the KNO3 and SNP treatments produced an increase in GA4 and a decrease in ABA concentrations, and this effect was reverted in the presence of the NO-scavenger. Globally, the results suggest a relationship between antioxidant metabolism and the ABA/GA balance during early seedling growth in pea. The results also suggest a role for KNO3 and NO in the modulation of GA4 and ABA levels and antioxidant metabolism in pea seedlings. Furthermore, this effect correlated with an increase in the biomass of the pea seedlings.