Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied as a filtration across simplicial complexes (or more simply, a method to measure topological features of spaces across different spatial resolutions), to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. The approach predicts plant family above chance. The application of a persistent homology method, using topological features, to measure leaf shape allows for a unified morphometric framework to measure plant form, including shapes, textures, patterns, and branching architectures.

Plants produce diverse metabolites to cope with the challenges presented by complex and ever-changing environments. These challenges drive the diversification of specialized metabolites within and between plant species. However, we are just beginning to understand how frequently new alleles arise controlling specialized metabolite diversity and how the geographic distribution of these alleles may be structured by ecological and demographic pressures. Here, we measure the variation in specialized metabolites across a population of 797 natural Arabidopsis thaliana accessions. We show that a combination of geography, environmental parameters, demography and different genetic processes all combine to influence the specific chemotypes and their distribution. This showed that causal loci in specialized metabolism contain frequent independently generated alleles with patterns suggesting potential within-species convergence. This provides a new perspective about the complexity of the selective forces and mechanisms that shape the generation and distribution of allelic variation that may influence local adaptation.

Plants face a variety of challenges within their ever-changing environment. Diverse metabolites are central to the plants ability to overcome these challenges. Understanding the environmental and genetic factors influencing the variation in specialized metabolites is the key to understand how plants survive and develop under changing environments. Here we measure the variation in specialized metabolites across a population of 797 natural Arabidopsis thaliana accessions. We show a combination of geography, environmental parameters, demography, and different genetic processes that creates a specific pattern in their accumulation and distribution. By identifying and tracking causal polymorphisms at multiple loci controlling metabolites variation we show that each locus displays extensive allelic heterogeneity with signatures of both parallel and convergent evolutionary processes. These loci combine epistatically and show differing relationships to environmental parameters leading to different distributions. This provides a detailed perspective about the complexity of the forces and mechanisms that shape the accumulation and distribution of a family of specialized metabolites critical for plant fitness.

MS performed the experiments, wrote the manuscript, and processed and analyzed data, AY wrote the manuscript and carried out metabolic analysis, CB carried out genotyping experiments, YC analyzed data, VS analyzed data, SH carried out genotyping and metabolic analysis, EK performed GLS measurements, CK peformed initial gtr1/2 experiment, AL verified analytical methods and assisted with statistical aid, H N-E provided gtr1/2 mutants and initial analysis, DK provided all the GLS mutants and GLS related measurements from the population, RA conceived the experimental design, supervised the work, provided funding, and wrote the manuscript. All authors have reviewed the final version of the manuscript and approved it and therefore are equally responsible for the integrity and accuracy of its content.

Free amino acids (FAAs) and protein-bound amino acids (PBAAs) in seeds play an important role in seed desiccation, longevity, and germination. However, the effect that water stress has on these two functional pools, especially when imposed during the crucial seed setting stage is unclear. To better understand these effects, we exposed Arabidopsis plants at the seed setting stage to a range of water limitation and water deprivation conditions and then evaluated physiological, metabolic, and proteomic parameters, with special focus on FAAs and PBAAs. We found that in response to severe water limitation, seed yield decreased, while seed weight, FAA, and PBAA content per seed increased. Nevertheless, the composition of FAAs and PBAAs remained unaltered. In response to severe water deprivation, however, both seed yield and weight were reduced. In addition, major alterations were observed in both FAA and proteome compositions, which indicated that both osmotic adjustment and proteomic reprogramming occurred in these naturally desiccation-tolerant organs. However, despite the major proteomic alteration, the PBAA composition did not change, suggesting that the proteomic reprogramming was followed by a proteomic rebalancing. Proteomic rebalancing has not been observed previously in response to stress, but its occurrence under stress strongly suggests its natural function. Together, our data show that the dry seed PBAA composition plays a key role in seed fitness and therefore is rigorously maintained even under severe water stress, while the FAA composition is more plastic and adaptable to changing environments, and that both functional pools are distinctly regulated.