IPD3, a master regulator of arbuscular mycorrhizal symbiosis, affects genes for immunity and metabolism of non-host Arabidopsis when restored long after its evolutionary loss.

Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the model Arabidopsis thaliana. To explore if elements of this apparently beneficial trait are still present and could be reactivated we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state, which partially mimics AMF exposure in non-inoculated plants. Our results indicate that molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.

Long- and short-read sequencing methods discover distinct circular RNA pools in Lotus japonicus.

Circular RNAs (circRNAs) are covalently closed single-stranded RNAs, generated through a back-splicing process that links a downstream 5' site to an upstream 3' end. The only distinction in the sequence between circRNA and their linear cognate RNA is the back splice junction. Their low abundance and sequence similarity with their linear origin RNA have made the discovery and identification of circRNA challenging. We have identified almost 6000 novel circRNAs from Lotus japonicus leaf tissue using different enrichment, amplification, and sequencing methods as well as alternative bioinformatics pipelines. The different methodologies identified different pools of circRNA with little overlap. We validated circRNA identified by the different methods using reverse transcription polymerase chain reaction and characterized sequence variations using nanopore sequencing. We compared validated circRNA identified in L. japonicus to other plant species and showed conservation of high-confidence circRNA-expressing genes. This is the first identification of L. japonicus circRNA and provides a resource for further characterization of their function in gene regulation. CircRNAs identified in this study originated from genes involved in all biological functions of eukaryotic cells. The comparison of methodologies and technologies to sequence, identify, analyze, and validate circRNA from plant tissues will enable further research to characterize the function and biogenesis of circRNA in L. japonicus.

Emergent molecular traits of lettuce and tomato grown under wavelength-selective solar cells.

The integration of semi-transparent organic solar cells (ST-OSCs) in greenhouses offers new agrivoltaic opportunities to meet the growing demands for sustainable food production. The tailored absorption/transmission spectra of ST-OSCs impacts the power generated as well as crop growth, development and responses to the biotic and abiotic environments. To characterize crop responses to ST-OSCs, we grew lettuce and tomato, traditional greenhouse crops, under three ST-OSC filters that create different light spectra. Lettuce yield and early tomato development are not negatively affected by the modified light environment. Our genomic analysis reveals that lettuce production exhibits beneficial traits involving nutrient content and nitrogen utilization while select ST-OSCs impact regulation of flowering initiation in tomato. These results suggest that ST-OSCs integrated into greenhouses are not only a promising technology for energy-neutral, sustainable and climate-change protected crop production, but can deliver benefits beyond energy considerations.

Re-engineering a lost trait: IPD3, a master regulator of arbuscular mycorrhizal symbiosis, affects genes for immunity and metabolism of non-host Arabidopsis when restored long after its evolutionary loss.

Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the model Arabidopsis thaliana. To explore why an apparently beneficial trait would be repeatedly lost, we generated Arabidopsis plants expressing a constitutively active form of Interacting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost from Arabidopsis along with the AM host trait. We characterize the transcriptomic effect of expressing IPD3 in Arabidopsis with and without exposure to the AM fungus (AMF) Rhizophagus irregularis, and compare these results to the AM model Lotus japonicus and its ipd3 knockout mutant cyclops-4. Despite its long history as a non-AM species, restoring IPD3 in the form of its constitutively active DNA-binding domain to Arabidopsis altered expression of specific gene networks. Surprisingly, the effect of expressing IPD3 in Arabidopsis and knocking it out in Lotus was strongest in plants not exposed to AMF, which is revealed to be due to changes in IPD3 genotype causing a transcriptional state which partially mimics AMF exposure in non-inoculated plants. Our results indicate that despite the long interval since loss of AM and IPD3 in Arabidopsis, molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture.

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes.

BACKGROUND: Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency. RESULTS: We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T1 generation of any crop transformed via Agrobacterium. We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes. CONCLUSION: This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.

Can the Environment have a Genetic Basis? A Case Study of Seedling Establishment in Arabidopsis thaliana.

The timing of seed germination determines the environment experienced by a plant's most vulnerable life stage-the seedling. Germination is environmentally cued, and genotypes can differ in their sensitivity to environmental cues. When genotypes differ in their response to cues, and when cues accurately predict the postgermination environment, the postgermination environment experienced by seedlings can itself have a genetic basis and potential to evolve. We tested for genetic differences in the postgermination environment using Arabidopsis thaliana genotypes that vary in seed dormancy, a trait known to alter germination time. We dispersed seeds into the field in 5 seasonal cohorts over 1.5 years, observed germination timing for 5297 individuals, and measured the soil temperature and moisture experienced by individuals throughout their life cycle. We found that genotypes differed in the environments they experienced during seedling establishment. This environmental variation occurred because genotypes differed in their environmental sensitivity to germination cues, and pregermination cues were correlated with postgermination environments. Seeds exhibited temporal habitat selection by germinating into a nonrandom subset of environmental conditions available, and seed dormancy increased the consistency of habitat selection. Strikingly, the postgermination environment affected fitness by altering the probability of seedling survival such that genotypes that engaged in stronger habitat selection were less likely to reach reproduction. Our results suggest that environmentally cued development may be a widespread mechanism by which genotypes can differ in the environment they experience, introducing the possibility that the environment itself can be inherited and can evolve.

Canalization of Seasonal Phenology in the Presence of Developmental Variation: Seed Dormancy Cycling in an Annual Weed.

Variation in the developmental timing in one life stage may ramify within and across generations to disrupt optimal phenology of other life stages. By focusing on a common mechanism of developmental arrest in plants-seed dormancy-we investigated how variation in flowering time influenced seed germination behavior and identified potential processes that can lead to canalized germination behavior despite variation in reproductive timing. We quantified effects of reproductive timing on dormancy cycling by experimentally manipulating the temperature during seed maturation and the seasonal timing of seed dispersal/burial, and by assessing temperature-dependent germination of un-earthed seeds over a seasonal cycle. We found that reproductive timing, via both seed-maturation temperature and the timing of dispersal, strongly influenced germination behavior in the weeks immediately following seed burial. However, buried seeds subsequently canalized their germination behavior, after losing primary dormancy and experiencing natural temperature and moisture conditions in the field. After the complete loss of primary dormancy, germination behavior was similar across seed-maturation and dispersal treatments, even when secondary dormancy was induced. Maternal effects themselves may contribute to the canalization of germination: first, by inducing stronger dormancy in autumn-matured seeds, and second by modifying the responses of those seeds to their ambient environment. Genotypes differed in dormancy cycling, with functional alleles of known dormancy genes necessary for the suppression of germination at warm temperatures in autumn through spring across multiple years. Loss of function of dormancy genes abolished almost all dormancy cycling. In summary, effects of reproductive phenology on dormancy cycling of buried seeds were apparent only as long as seeds retained primary dormancy, and a combination of genetically imposed seed dormancy, maternally induced seed dormancy, and secondary dormancy can mitigate variation in germination behavior imposed by variation in reproductive phenology.

Adjusting phenotypes via within- and across-generational plasticity.

Contents 343 I. 343 II. 343 III. 347 IV. 348 348 References 348 SUMMARY: There is renewed interest in how transgenerational environmental effects, including epigenetic inheritance, contribute to adaptive evolution. The contribution of across-generation plasticity to adaptation, however, needs to be evaluated within the context of within-generation plasticity, which is often proposed to contribute more efficiently to adaptation because of the potentially greater accuracy of progeny than parental cues to predict progeny selective environments. We highlight recent empirical studies of transgenerational plasticity, and find that they do not consistently support predictions based on the higher predictive ability of progeny environmental cues. We discuss these findings within the context of the relative predictive ability of maternal and progeny cues, costs and constraints of plasticity in parental and progeny generations, and the dynamic nature of the adaptive value of within- and across-generation plasticity that varies with the process of adaptation itself. Such contingent and dynamically variable selection could account for the diversity of patterns of within- and across-generation plasticity observed in nature, and can influence the adaptive value of the persistence of environmental effects across generations.

Multiple paths to similar germination behavior in Arabidopsis thaliana.

Germination timing influences plant fitness, and its sensitivity to temperature may cause it to change as climate shifts. These changes are likely to be complex because temperatures that occur during seed maturation and temperatures that occur post-dispersal interact to define germination timing. We used the model organism Arabidopsis thaliana to determine how flowering time (which defines seed-maturation temperature) and post-dispersal temperature influence germination and the expression of genetic variation for germination. Germination responses to temperature (germination envelopes) changed as seeds aged, or after-ripened, and these germination trajectories depended on seed-maturation temperature and genotype. Different combinations of genotype, seed-maturation temperature, and after-ripening produced similar germination envelopes. Likewise, different genotypes and seed-maturation temperatures combined to produce similar germination trajectories. Differences between genotypes were most likely to be observed at high and low germination temperatures. The germination behavior of some genotypes responds weakly to maternal temperature but others are highly plastic. We hypothesize that weak dormancy induction could synchronize germination of seeds dispersed at different times. By contrast, we hypothesize that strongly responsive genotypes may spread offspring germination over several possible germination windows. Considering germination responses to temperature is important for predicting phenology expression and evolution in future climates.