Desiccation of plants is often lethal but is tolerated by the majority of seeds and by vegetative tissues of only a small number of land plants. Desiccation tolerance is an ancient trait, lost from ...vegetative tissues following the appearance of tracheids but reappearing in several lineages when selection pressures favored its evolution. Cells of all desiccation-tolerant plants and seeds must possess a core set of mechanisms to protect them from desiccation- and rehydration-induced damage. This review explores how desiccation generates cell damage and how tolerant cells assuage the complex array of mechanical, structural, metabolic, and chemical stresses and survive.Likewise, the stress of rehydration requires appropriate mitigating cellular responses. We also explore what comparative genomics, both structural and responsive, have added to our understanding of cellular protection mechanisms induced by desiccation, and how vegetative desiccation tolerance circumvents destructive, stress-induced cell senescence.
The seed expressed gene DELAY OF GERMINATION (DOG) 1 is absolutely required for the induction of dormancy. Next to a non‐dormant phenotype, the dog1‐1 mutant is also characterized by a reduced seed ...longevity suggesting that DOG1 may affect additional seed processes as well. This aspect however, has been hardly studied and is poorly understood. To uncover additional roles of DOG1 in seeds we performed a detailed analysis of the dog1 mutant using both transcriptomics and metabolomics to investigate the molecular consequences of a dysfunctional DOG1 gene. Further, we used a genetic approach taking advantage of the weak aba insensitive (abi) 3‐1 allele as a sensitized genetic background in a cross with dog1‐1. DOG1 affects the expression of hundreds of genes including LATE EMBRYOGENESIS ABUNDANT and HEAT SHOCK PROTEIN genes which are affected by DOG1 partly via control of ABI5 expression. Furthermore, the content of a subset of primary metabolites, which normally accumulate during seed maturation, was found to be affected in the dog1‐1 mutant. Surprisingly, the abi3‐1 dog1‐1 double mutant produced green seeds which are highly ABA insensitive, phenocopying severe abi3 mutants, indicating that dog1‐1 acts as an enhancer of the weak abi3‐1 allele and thus revealing a genetic interaction between both genes. Analysis of the dog1 and dog1 abi3 mutants revealed additional seed phenotypes and therefore we hypothesize that DOG1 function is not limited to dormancy but that it is required for multiple aspects of seed maturation, in part by interfering with ABA signalling components.
Over the past few decades seed physiology research has contributed to many important scientific discoveries and has provided valuable tools for the production of high quality seeds. An important ...instrument for this type of research is the accurate quantification of germination; however gathering cumulative germination data is a very laborious task that is often prohibitive to the execution of large experiments. In this paper we present the germinator package: a simple, highly cost-efficient and flexible procedure for high-throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics. The germinator package contains three modules: (i) design of experimental setup with various options to replicate and randomize samples; (ii) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat on a single image; and (iii) curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters. The curve-fitting module enables analysis of general cumulative germination data and can be used for all plant species. We show that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well. In this paper we show the accuracy, reproducibility and flexibility of the germinator package. We have successfully applied it to evaluate natural variation for salt tolerance in a large population of recombinant inbred lines and were able to identify several quantitative trait loci for salt tolerance. germinator is a low-cost package that allows the monitoring of several thousands of germination tests, several times a day by a single person.
Plants are fascinating and complex organisms. A comprehensive understanding of the organization, function and evolution of plant genes is essential to disentangle important biological processes and ...to advance crop engineering and breeding strategies. The ultimate aim in deciphering complex biological processes is the discovery of causal genes and regulatory mechanisms controlling these processes. The recent surge of omics data has opened the door to a system-wide understanding of the flow of biological information underlying complex traits. However, dealing with the corresponding large data sets represents a challenging endeavor that calls for the development of powerful bioinformatics methods. A popular approach is the construction and analysis of gene networks. Such networks are often used for genome-wide representation of the complex functional organization of biological systems. Network based on similarity in gene expression are called (gene) co-expression networks. One of the major application of gene co-expression networks is the functional annotation of unknown genes. Constructing co-expression networks is generally straightforward. In contrast, the resulting network of connected genes can become very complex, which limits its biological interpretation. Several strategies can be employed to enhance the interpretation of the networks. A strategy in coherence with the biological question addressed needs to be established to infer reliable networks. Additional benefits can be gained from network-based strategies using prior knowledge and data integration to further enhance the elucidation of gene regulatory relationships. As a result, biological networks provide many more applications beyond the simple visualization of co-expressed genes. In this study we review the different approaches for co-expression network inference in plants. We analyse integrative genomics strategies used in recent studies that successfully identified candidate genes taking advantage of gene co-expression networks. Additionally, we discuss promising bioinformatics approaches that predict networks for specific purposes.
Quantifying gene expression levels is an important research tool to understand biological systems. Reverse transcription-quantitative real-time PCR (RT-qPCR) is the preferred method for targeted gene ...expression measurements because of its sensitivity and reproducibility. However, normalization, necessary to correct for sample input and reverse transcriptase efficiency, is a crucial step to obtain reliable RT-qPCR results. Stably expressed genes (i.e. genes whose expression is not affected by the treatment or developmental stage under study) are indispensable for accurate normalization of RT-qPCR experiments. Lack of accurate normalization could affect the results and may lead to false conclusions. Since transcriptomes of seeds are different from other plant tissues, we aimed to identify reference genes specifically for RT-qPCR analyses in seeds of two important seed model species, i.e. Arabidopsis and tomato. We mined Arabidopsis seed microarray data to identify stably expressed genes and analyzed these together with putative reference genes from other sources. In total, the expression stability of 24 putative reference genes was validated by RT-qPCR in Arabidopsis seed samples. For tomato, we lacked transcriptome data sets of seeds and therefore we tested the tomato homologs of the reference genes found for Arabidopsis seeds. In conclusion, we identified 14 Arabidopsis and nine tomato reference genes. This provides a valuable resource for accurate normalization of gene expression experiments in seed research for two important seed model species.
The temporal control or timing of the life cycle of annual plants is presumed to provide adaptive strategies to escape harsh environments for survival and reproduction. This is mainly determined by ...the timing of germination, which is controlled by the level of seed dormancy, and of flowering initiation. However, the environmental factors driving the evolution of plant life cycles remain largely unknown. To address this question we have analysed nine quantitative life history traits, in a native regional collection of 300 wild accessions of Arabidopsis thaliana. Seed dormancy and flowering time were negatively correlated, indicating that these traits have coevolved. In addition, environmental–phenotypic analyses detected strong altitudinal and climatic clines for most life history traits. Overall, accessions showing life cycles with early flowering, small seeds, high seed dormancy and slow germination rate were associated with locations exposed to high temperature, low summer precipitation and high radiation. Furthermore, we analysed the expression level of the positive regulator of seed dormancy DELAY OF GERMINATION 1 (DOG1), finding similar but weaker altitudinal and climatic patterns than seed dormancy. Therefore, DOG1 regulatory mutations are likely to provide a quantitative molecular mechanism for the adaptation of A. thaliana life cycle to altitude and climate.
The diversity of annual life cycles is determined by genetic variation for the timing of germination, which is controlled by the level of seed dormancy and by the timing of flowering initiation. Despite the recent progress in understanding the genetic and molecular mechanisms underlying both life history traits in Arabidopsis thaliana, the environmental factors that contribute to maintain life cycle variation remain mostly unknown. In this study we find significant and strong correlations between seed dormancy, flowering time and multiple climatic factors, indicating that both traits have coevolved to adapt annual life cycles to climate.
MAIN CONCLUSION : Besides being an important model to study desiccation tolerance, the induction of desiccation tolerance in germinated seeds may also play an ecological role in seedling ...establishment. Desiccation tolerance (DT) is the ability of certain organisms to survive extreme water losses without accumulation of lethal damage. This was a key feature in the conquering of dry land and is currently found in all taxa including bacteria, fungi, roundworms and plants. Not surprisingly, studies in various fields have been performed to unravel this intriguing phenomenon. In flowering plants, DT is rare in whole plants (vegetative tissues), yet is common in seeds. In this review, we present our current understanding of the evolution of DT in plants. We focus on the acquisition of DT in seeds and the subsequent loss during and after germination by highlighting and comparing research in two model plants Medicago truncatula and Arabidopsis thaliana. Finally, we discuss the ability of seeds to re-establish DT during post-germination, the possible ecological meaning of this phenomenon, and the hypothesis that DT, in combination with dormancy, optimizes seedling establishment.
Main conclusion During re-establishment of desiccation tolerance (DT), early events promote initial protection and growth arrest, while late events promote stress adaptation and contribute to ...survival in the dry state. Mature seeds of Arabidopsis thaliana are desiccation tolerant, but they lose desiccation tolerance (DT) while progressing to germination. Yet, there is a small developmental window during which DT can be rescued by treatment with abscisic acid (ABA). To gain temporal resolution and identify relevant genes in this process, data from a time series of microarrays were used to build a gene co-expression network. The network has two regions, namely early response (ER) and late response (LR). Genes in the ER region are related to biological processes, such as dormancy, acquisition of DT and drought, amplification of signals, growth arrest and induction of protection mechanisms (such as LEA proteins). Genes in the LR region lead to inhibition of photosynthesis and primary metabolism, promote adaptation to stress conditions and contribute to seed longevity. Phenotyping of 12 hubs in relation to re-establishment of DT with T-DNA insertion lines indicated a significant increase in the ability to re-establish DT compared with the wild-type in the lines cbsx4, at3g53040 and at4g25580, suggesting the operation of redundant and compensatory mechanisms. Moreover, we show that re-establishment of DT by polyethylene glycol and ABA occurs through partially overlapping mechanisms. Our data confirm that co-expression network analysis is a valid approach to examine data from time series of transcriptome analysis, as it provides promising insights into biologically relevant relations that help to generate new information about the roles of certain genes for DT.
The depth of seed dormancy can be influenced by a number of different environmental signals, but whether a common mechanism underlies this apparently similar response has yet to be investigated. ...Full-genome microarrays were used for a global transcript analysis of Arabidopsis thaliana Cape Verde Island accession seeds exposed to dry after-ripening (AR), or low temperature, nitrate and light when imbibed. Germination studies showed that the sensitivity of imbibed seeds to low temperature, nitrate and light was dependant upon the length of time spent AR following harvest. Seeds had an absolute requirement for light to complete dormancy release in all conditions, but this effect required an exposure to a prior dormancy relieving environment. Principal component analyses of the expression patterns observed grouped physiological states in a way that related to the depth of seed dormancy, rather than the type of environmental exposure. Furthermore, opposite changes in transcript abundance of genes in sets associated with dormancy, or dormancy relief through AR, were also related to the depth of dormancy and common to different environments. Besides these common quantitative changes, environment-specific gene expression patterns during dormancy relief are also described. For example, higher transcript abundance for genes linked to the process of nitrate accumulation, and nitrate reduction was associated with dormancy relief. The quantity of GA3ox1 transcripts increased during dormancy relief in all conditions, in particular when dormancy relief was completed by exposure to light. This contrasts with transcripts linked to abscisic acid (ABA) synthesis, which declined. The results are consistent with a role for the ABA/gibberellic acid balance in integrating dormancy-relieving environmental signals.
Grasses are among the most resilient plants, and some can survive prolonged desiccation in semiarid regions with seasonal rainfall. However, the genetic elements that distinguish grasses that are ...sensitive versus tolerant to extreme drying are largely unknown. Here, we leveraged comparative genomic approaches with the desiccation-tolerant grass Eragrostis nindensis and the related desiccation-sensitive cereal Eragrostis tef to identify changes underlying desiccation tolerance. These analyses were extended across C4 grasses and cereals to identify broader evolutionary conservation and divergence. Across diverse genomic datasets, we identified changes in chromatin architecture, methylation, gene duplications, and expression dynamics related to desiccation in E. nindensis. It was previously hypothesized that transcriptional rewiring of seed desiccation pathways confers vegetative desiccation tolerance. Here, we demonstrate that the majority of seeddehydration–related genes showed similar expression patterns in leaves of both desiccation-tolerant and -sensitive species. However, we identified a small set of seed-related orthologs with expression specific to desiccation-tolerant species. This supports a broad role for seed-related genes, where many are involved in typical drought responses, with only a small subset of crucial genes specifically induced in desiccation-tolerant plants.
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