Ancient Memories of Seeds: ABA-Dependent Growth Arrest and Reserve Accumulation.

Genetic studies of seed maturation regulators, combining transcriptomics and network analysis, suggest the significance of genetic diversification for maturation programs, particularly in seed plants. By contrast, analogs of the maturation programs, such as dormancy and desiccation tolerance, are also found in non-seed plants. It is thus conceivable that seeds recalled or renovated ancient programs of stress-induced growth arrest that were already present in ferns, bryophytes, and possibly in algae. This opinion article discusses the origins and genetic diversification of the abscisic acid (ABA)-dependent seed maturation programs by addressing questions provoked by recent findings about the DELAY OF GERMINATION1 (DOG1) family proteins, which regulate reserve accumulation, dormancy, and/or desiccation tolerance in seeds in a gene-specific or redundant manner.

Ancient and recent gene duplications as evolutionary drivers of the seed maturation regulators DELAY OF GERMINATION1 family genes.

The DELAY OF GERMINATION1 (DOG1) family genes (DFGs) in Arabidopsis thaliana are involved in seed dormancy, reserve accumulation, and desiccation tolerance. Decoding the molecular evolution of DFGs is key to understanding how these seed programs evolved. This article demonstrates that DFGs have diverged in the four lineages DOG1, DOG1-LIKE4 (DOGL4), DOGL5 and DOGL6, whereas DOGL1, DOGL2 and DOGL3 arose separately within the DOG1 lineage. The systematic DFG nomenclature proposed in this article addresses the current issues of inconsistent DFG annotation and highlights DFG genomic synteny in angiosperms. DFG pseudogenes, or collapsed coding sequences, hidden in the genomes of early-diverging angiosperms are documented here. They suggest ancient birth and loss of DFGs over the course of angiosperm evolution. The proposed models suggest that the origin of DFG diversification dates back to the most recent common ancestor of living angiosperms. The presence of a single form of DFG in nonflowering plants is discussed. Phylogenetic analysis of gymnosperm, lycophyte, and liverwort DFGs and similar genes found in mosses and algae suggests that DFGs diverged from the TGACG motif-binding transcription factor genes before the divergence of the bryophyte lineage.

DELAY OF GERMINATION 1-LIKE 4 acts as an inducer of seed reserve accumulation.

More than 70% of global food supply depends on seeds. The major seed reserves, such as proteins, lipids, and polysaccharides, are produced during seed maturation. Here, we report that DELAY OF GERMINATION 1-LIKE 4 (DOGL4) is a major inducer of reserve accumulation during seed maturation. The DOGL family proteins are plant-specific proteins of largely unknown biochemical function. DOGL4 shares only limited homology in amino acid sequence with DOG1, a major regulator of seed dormancy. DOGL4 was identified as one of the outstanding abscisic acid (ABA)-induced genes in our RNA sequencing analysis, whereas DOG1 was not induced by ABA. Induction of DOGL4 caused the expression of 70 seed maturation-specific genes, even in germinating seeds, including the major seed reserves ALBUMIN, CRUCIFERIN and OLEOSIN. Although DOG1 affects the expression of many seed maturation genes, the major seed reserve genes induced by DOGL4 are not altered by the dog1 mutation. Furthermore, the reduced dormancy and longevity phenotypes observed in the dog1 seeds were not observed in the dogl4 mutants, suggesting that these two genes have limited functional overlap. Taken together, these results suggest that DOGL4 is a central factor mediating reserve accumulation in seeds, and that the two DOG1 family proteins have diverged over the course of evolution into independent regulators of seed maturation, but retain some overlapping function.

A repressor complex silencing ABA signaling in seeds?

Seed dormancy is induced primarily by abscisic acid (ABA) and maintained through elevated levels of ABA sensitivity in seeds. The core mechanisms of ABA-imposed seed dormancy are emerging, but it is still unclear how these blockages in seeds are eliminated during after-ripening, or what molecular events in imbibed seeds are responsible for the initial stages of germination induction. Some pieces of evidence suggest that a repressor complex, which potentially triggers seed germination through the suppression of ABA signaling components, might be present in seeds. The usual suspect, protein phosphatase 2C, which inactivates kinases and shuts down ABA signaling in the major dormancy pathway, is possibly associated with this complex. Other members, such as WD40 proteins and histone deacetylase subunits, homologs of which are found in the flowering repressor complex, perhaps constitute this complex in seeds. The repressor activity could counteract the dormancy mechanisms in an overwhelming manner, through well-coordinated inactivation and turnover of germination-suppressing transcription factors, which is probably accompanied by chromatin silencing and transcriptional repression of the transcription factor target genes. This review provides a perspective on a putative seed germination-inducing repressor complex, including its possible modes of action and upstream regulators.

Seed germination and dormancy: The classic story, new puzzles, and evolution.

This review highlights recent progresses in seed germination and dormancy research. Research on the weakening of the endosperm during germination, which is almost a classic theme in seed biology, was resumed by α-xylosidase studies. Strong genetic evidence was presented to suggest that the quality control of xyloglucan biosynthesis in the endosperm (and the embryo) plays a critical role in germination. Further analyses on the endosperm and the adjacent layers have suggested that the cutin coat in the endosperm-testa interphase negatively affects germination while the endosperm-embryo interphase produces a sheath that facilitates germination. These progresses significantly advanced our understanding of seed germination mechanisms. A breakthrough in dormancy research, on the other hand, revealed the unique abscisic acid signaling pathway that is regulated by DELAY OF GERMINATION1 (DOG1). The detailed analysis of DOG1 expression uncovered the intriguing story of reciprocal regulation of the sense-antisense pair, which generated new questions. Recent studies also suggested that the DOG1 function is not limited to dormancy but extended through general seed maturation, which provokes questions about the evolution of DOG1 family proteins. Seed biology is becoming more exciting with the classic stories being revitalized and new puzzles emerging from the frontier.

Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds.

Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA-stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE-binding factor) expression in Arabidopsis Columbia-0 seeds, which caused 9- to 73-fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non-dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre-harvest sprouting during crop production, and therefore contributes to translational biology.

Induction of 9-cis-epoxycarotenoid dioxygenase in Arabidopsis thaliana seeds enhances seed dormancy.

Full understanding of mechanisms that control seed dormancy and germination remains elusive. Whereas it has been proposed that translational control plays a predominant role in germination, other studies suggest the importance of specific gene expression patterns in imbibed seeds. Transgenic plants were developed to permit conditional expression of a gene encoding 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, using the ecdysone receptor-based plant gene switch system and the ligand methoxyfenozide. Induction of NCED6 during imbibition increased ABA levels more than 20-fold and was sufficient to prevent seed germination. Germination suppression was prevented by fluridone, an inhibitor of ABA biosynthesis. In another study, induction of the NCED6 gene in transgenic seeds of nondormant mutants tt3 and tt4 reestablished seed dormancy. Furthermore, inducing expression of NCED6 during seed development suppressed vivipary, precocious germination of developing seeds. These results indicate that expression of a hormone metabolism gene in seeds can be a sole determinant of dormancy. This study opens the possibility of developing a robust technology to suppress or promote seed germination through engineering pathways of hormone metabolism.

Mechanisms of hormonal regulation of endosperm cap-specific gene expression in tomato seeds.

The micropylar region of endosperm in a seed, which is adjacent to the radicle tip, is called the 'endosperm cap', and is specifically activated before radicle emergence. This activation of the endosperm cap is a widespread phenomenon among species and is a prerequisite for the completion of germination. To understand the mechanisms of endosperm cap-specific gene expression in tomato seeds, GeneChip analysis was performed. The major groups of endosperm cap-enriched genes were pathogenesis-, cell wall-, and hormone-associated genes. The promoter regions of endosperm cap-enriched genes contained DNA motifs recognized by ethylene response factors (ERFs). The tomato ERF1 (TERF1) and its experimentally verified targets were enriched in the endosperm cap, suggesting an involvement of the ethylene response cascade in this process. The known endosperm cap enzyme endo-ß-mannanase is induced by gibberellin (GA), which is thought to be the major hormone inducing endosperm cap-specific genes. The mechanism of endo-ß-mannanase induction by GA was also investigated using isolated, embryoless seeds. Results suggested that GA might act indirectly on the endosperm cap. We propose that endosperm cap activation is caused by the ethylene response of this tissue, as a consequence of mechanosensing of the increase in embryonic growth potential by GA action.

Modulation of wheat grain dormancy by introducing the recombinant abscisic acid-stimulated abscisic acid biosynthesis gene.

Pre-harvest sprouting of cereals greatly reduces yield and quality of the grains. Abscisic acid (ABA) is an essential phytohormone for the induction and maintenance of seed dormancy. In this study, the ABA responsive promoter-driven ABA biosynthesis gene system was introduced to common wheat (Triticum aestivum L.) to enhance ABA production in the embryos and pre-harvest sprouting tolerance of the grains. This system consists of a wheat ABA responsive element containing Early-Methionine-labelled (EM) promoter and a sorghum 9-cis-epoxycarotenoid dioxygenase (SbNCED) gene which encodes an ABA biosynthesis rate-limiting enzyme. Twenty-three independent single-insertion lines were obtained, from which five homozygous lines showing various SbNCED expression levels were selected. Correlations were observed between SbNCED expression, ABA accumulation in the embryos and enhanced dormancy levels of the grains. The engineered wheat grains exhibited a few day-delay in germination, which should be effective in reducing pre-harvest sprouting damage. However, the increase in ABA levels in the recombinant grains was moderate, which explains why germination was not completely suppressed. Further analysis indicated a concomitant increase in the expression of the ABA catabolic enzyme gene TaABA8'OH1 and in the levels of isoleucine-conjugated jasmonic acid, implying the presence of possible negative feedback regulation in the innate system, which should be overcome for future technology development. These findings advance an understanding of the regulatory mechanisms of hormone metabolism in seeds and facilitate the development of pre-harvest sprouting tolerance in cereal grains.

Seed traits and genes important for translational biology--highlights from recent discoveries.

Seeds provide food, feed, fiber and fuel. They are also an important delivery system of genetic information, which is essential for the survival of wild species in ecosystems and the production of agricultural crops. In this review, seed traits and genes that are potentially important for agricultural applications are discussed. Over the long period of crop domestication, seed traits have been modified through intentional or unintentional selections. While most selections have led to seed traits favorable for agricultural consumption, such as larger seeds with higher nutritional value than the wild type, other manipulations in modern breeding sometimes led to negative traits, such as vivipary, precocious germination on the maternal plant or reduced seed vigor, as a side effect during the improvement of other characteristics. Greater effort is needed to overcome these problems that have emerged as a consequence of crop improvement. Seed biology researchers have characterized the function of many genes in the last decade, including those associated with seed domestication, which may be useful in addressing critical issues in modern agriculture, such as the prevention of vivipary and seed shattering or the enhancement of yields. Recent discoveries in seed biology research are highlighted in this review, with an emphasis on their potential for translational biology.

MicroRNA gene regulation cascades during early stages of plant development.

MicroRNAs (miRNAs) regulate various developmental programs of plants. This review focuses on miRNA involvement in early events of plant development, such as seed germination, seedling development and the juvenile to adult phase transition. miR159 and miR160 are involved in the regulation of seed germination through their effects on the sensitivity of seeds to ABA. miR156 and miR172 play critical roles in the emergence of vegetative leaves at post-germinative stages, which is important for the transition to autotrophic growth. The phase transition from the juvenile to adult stage in both monocots and dicots is also regulated by miR156 and miR172. In these early developmental processes, there are miRNA gene regulation cascades where the miR156 pathway acts upstream of the miR172 pathway. Moreover, targets of miR156 and miR172 exert positive feedback on the expression of MIR genes that suppress themselves. The early events of plant development appear to be controlled by complex mechanisms involving sequential expression of different miRNA pathways and feedback loops among miRNAs and their target genes.

microRNA, seeds, and Darwin?: diverse function of miRNA in seed biology and plant responses to stress.

microRNAs (miRNAs) are small, single-stranded RNAs that down-regulate target genes at the post-transcriptional level. miRNAs regulate target genes by guiding mRNA cleavage or by repressing translation. miRNAs play crucial roles in a broad range of developmental processes in plants. Multiple miRNAs are present in germinating seeds and seedlings of Arabidopsis, some of which are involved in the regulation of germination and seedling growth by plant hormones such as abscisic acid (ABA) and auxin. The involvement of miRNAs in ABA responses is not limited to the early stages of plant development but seems to be important for general stress responses throughout the plant life cycle. This Darwin review summarizes recent progress in miRNA research focusing on seed and stress biology, two topics which were of interest to Charles Darwin.

The Long-Standing Paradox of Seed Dormancy Unfolded?

There has been a long-standing question in seed research, why cyanide, a respiration inhibitor, breaks seed dormancy. While the alternative respiratory pathway and reactive oxygen species have been suggested to be part of the mechanism, the cell biological and mechanistic significance of this paradox remains unclear. The outcomes of recent research on mitochondrial RNA processing for the subunits of the electron transport chain complexes seem to offer a logical explanation. This opinion article attempts to integrate the accumulating evidence of mitochondrial involvement in ABA signaling with the frontier of seed research on DELAY OF GERMINATION1, a master regulator of dormancy, to present a coherent model for ABA signaling in seeds, which could also address the old paradox in seed research.

Jasmonic acid and ethylene are involved in the accumulation of osmotin in germinating tomato seeds.

The expression of SlNP24 encoding osmotin was studied in germinating tomato seeds Solanum lycopersicum L. cv. Moneymaker. The results show that the accumulation of the transcripts of SlNP24 and its potential upstream regulator TERF1 encoding an ethylene response factor was induced by ethylene and methyl jasmonate in germinating tomato seeds. There was no effect of gibberellins on the expression of the genes studied. The expression of SlNP24 was localized in the micropylar region of the endosperm of tomato seeds. The promoter of tomato osmotin was active in the endosperm cells of transgenic Arabidopsis thaliana seeds, which contain reporter genes under control of SlNP24 promoter. The activity of SlNP24 promoter in A. thaliana reporter line seeds was visible when the expression of its ortholog gene in A. thaliana (AtOMS34) was observed. The mechanism of induction and a possible role of NP24 in germinating tomato seeds are discussed.

Seed Biology Updates - Highlights and New Discoveries in Seed Dormancy and Germination Research.

An understanding of the biology of seeds has been greatly advanced in recent years. The progresses, particularly in the field of seed dormancy and germination research, have been made at a remarkable speed. Some of the possible epigenetic mechanisms, including an involvement of non-coding RNA, which were predicted for DELAY OF GERMINATION1 just a few years ago, have now been demonstrated with strong molecular and genetic evidence. Imprinting, or parent-of-origin-specific gene silencing/expression, which was characterized particularly for developing seeds, was also found in imbibed seeds and suggested for dormancy mechanisms. Hormone biology in seeds, which is the most advanced and almost a traditional area of seed research, also presents a new dimension. Upstream regulators of hormone metabolism and hormone transporters, such as abscisic acid and gibberellin influx/efflux carriers, have been identified. Characterization of the novel posttranslational modification pathways, including the N-end rule and S-nitrosylation pathways, which play a critical role in turnover of the major hormone signal transduction proteins, also expanded our knowledge about the complexity of hormone signaling in seeds. These progresses made at the molecular level are significant steps toward a better understanding of how seeds translate soil and other environmental signals into their internal hormone biology and make an important decision to stay dormant or commence with germination.

Prevention of Preharvest Sprouting through Hormone Engineering and Germination Recovery by Chemical Biology.

Vivipary, germination of seeds on the maternal plant, is observed in nature and provides ecological advantages in certain wild species, such as mangroves. However, precocious seed germination in agricultural species, such as preharvest sprouting (PHS) in cereals, is a serious issue for food security. PHS reduces grain quality and causes economical losses to farmers. PHS can be prevented by translating the basic knowledge of hormone biology in seeds into technologies. Biosynthesis of abscisic acid (ABA), which is an essential hormone for seed dormancy, can be engineered to enhance dormancy and prevent PHS. Enhancing nine-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting enzyme of ABA biosynthesis, through a chemically induced gene expression system, has successfully been used to suppress germination of Arabidopsis seeds. The more advanced system NCED positive-feedback system, which amplifies ABA biosynthesis in a seed-specific manner without chemical induction, has also been developed. The proofs of concept established in the model species are now ready to be applied to crops. A potential problem is recovery of germination from hyperdormant crop grains. Hyperdormancy induced by the NCED systems can be reversed by inducing counteracting genes, such as NCED RNA interference or gibberellin (GA) biosynthesis genes. Alternatively, seed sensitivity to ABA can be modified to rescue germination using the knowledge of chemical biology. ABA antagonists, which were developed recently, have great potential to recover germination from the hyperdormant seeds. Combination of the dormancy-imposing and -releasing approaches will establish a comprehensive technology for PHS prevention and germination recovery.

A gel diffusion assay for visualization and quantification of chitinase activity.

Higher plants, bacteria, fungi, insects, and crustaceans all produce chitinases. Chitinase genes in many organisms are currently under investigation. Chitinase activity is usually assayed with radiolabeled or fluorogenic substrates. We developed a simple, inexpensive, nonradioactive gel-diffusion assay for chitinase that can be used to screen large numbers of samples. In this assay, chitinase diffuses from a small circular well cut in an agarose or agar gel containing the substrate glycol chitin, a soluble, modified form of chitin. Chitinase catalyzes the cleavage of glycol chitin as it diffuses through the gel, leaving a dark, unstained circular zone around the well, because the fluorescent dye calcofluor binds only to undigested chitin. Sample activities can be determined from linear regression of log-standard enzyme concentration versus the zone diameter of internal standards on each Petri dish used for a diffusion assay.

Seed dormancy and germination-emerging mechanisms and new hypotheses.

Seed dormancy has played a significant role in adaptation and evolution of seed plants. While its biological significance is clear, molecular mechanisms underlying seed dormancy induction, maintenance and alleviation still remain elusive. Intensive efforts have been made to investigate gibberellin and abscisic acid metabolism in seeds, which greatly contributed to the current understanding of seed dormancy mechanisms. Other mechanisms, which might be independent of hormones, or specific to the seed dormancy pathway, are also emerging from genetic analysis of "seed dormancy mutants." These studies suggest that chromatin remodeling through histone ubiquitination, methylation and acetylation, which could lead to transcription elongation or gene silencing, may play a significant role in seed dormancy regulation. Small interfering RNA and/or long non-coding RNA might be a trigger of epigenetic changes at the seed dormancy or germination loci, such as DELAY OF GERMINATION1. While new mechanisms are emerging from genetic studies of seed dormancy, novel hypotheses are also generated from seed germination studies with high throughput gene expression analysis. Recent studies on tissue-specific gene expression in tomato and Arabidopsis seeds, which suggested possible "mechanosensing" in the regulatory mechanisms, advanced our understanding of embryo-endosperm interaction and have potential to re-draw the traditional hypotheses or integrate them into a comprehensive scheme. The progress in basic seed science will enable knowledge translation, another frontier of research to be expanded for food and fuel production.