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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 559 records · Page 31
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

An evolutionary algorithm for designing microbial communities via environmental modification

Despite a growing understanding of how environmental composition affects microbial communities, it remains difficult to apply this knowledge to the rational design of synthetic multispecies consortia. This is because natural microbial communities can harbour thousands of different organisms and environmental substrates, making up a vast combinatorial space that precludes exhaustive experimental testing and computational prediction. Here, we present a method based on the combination of machine learning and metabolic modelling that selects optimal environmental compositions to produce target community phenotypes. In this framework, dynamic flux balance analysis is used to model the growth of a community in candidate environments. A genetic algorithm is then used to evaluate the behaviour of the community relative to a target phenotype, and subsequently adjust the environment to allow the organisms to approach this target. We apply this iterative process to thousands of in silico communities of varying sizes, showing how it can rapidly identify environments that yield desired taxonomic compositions and patterns of metabolic exchange. Moreover, this combination of approaches produces testable predictions for the assembly of experimental microbial communities with specific properties and can facilitate rational environmental design processes for complex microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

An ascomycete H4 variant with an unknown function

Histone variants leading to altered nucleosome structure, dynamics and DNA accessibility occur frequently, albeit rarely for H4. We carried out a comprehensive in silico scrutiny of fungal genomes, which revealed the presence of a novel H4 variant (H4E) in the ascomycetes, throughout the Pezizomycotina, in basal species of the Taphrinomycotina and also in the Glomeromycota. The coding cognate genes show a specific intron/exon organization, different from H4 canonical genes. H4Es diverge from canonical H4s mainly in the N- and C-terminal extensions, showing marked differences in the distribution and number of Lys and Arg residues, which may result in novel post-translational modifications. In Aspergillus nidulans (Pezizomycotina, Eurotiomycetes) the H4E variant protein level is low in mycelia. However, the encoding gene is well expressed at 37°C under nitrogen starvation. H4E localizes to the nucleus and interacts with H3, but its absence or overexpression does not result in any detectable phenotype. Deletion of only one of the of the two canonical H4 genes results in a strikingly impaired growth phenotype, which indicates that H4E cannot replace this canonical histone. Thus, an H4 variant is present throughout a whole subphylum of the ascomycetes, but with hitherto no experimentally detectable function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Extending evolutionary forecasts across bacterial species

Improving evolutionary forecasting requires progressing from studying repeated evolution of a single genotype under identical conditions to formulating broad principles. These principles should enable predictions of how similar species will adapt to similar selective pressures. Evolve-and-resequence experiments with multiple species allow testing forecasts on different biological levels and elucidating the causes for failed predictions. Here, we show that forecasts for adaptation to static culture conditions can be extended to multiple species by testing previous predictions for Pseudomonas syringae and Pseudomonas savastanoi. In addition to sequence divergence, these species differ in their repertoire of biofilm regulatory genes and structural components. Consistent with predictions, both species repeatedly produced biofilm mutants with a wrinkly spreader phenotype. Predominantly, mutations occurred in the wsp operon, with less frequent promoter mutations near uncharacterized diguanylate cyclases. However, mutational patterns differed on the gene level, which was explained by a lack of conservation in relative fitness of mutants between more divergent species. The same mutation was the most frequent for both species suggesting that conserved mutation hotspots can increase parallel evolution. This study shows that evolutionary forecasts can be extended across species, but that differences in the genotype–phenotype–fitness map and mutational biases limit predictability on a detailed molecular level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

EndofungalMycetohabitans(formerlyBurkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus,Rhizopus microsporus. The one known secreted effector family fromMycetohabitansconsists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenicXanthomonasandRalstoniaspp. to activate host genes that promote disease. These ‘BurkholderiaTAL-like (Btl)’ proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of twoMycetohabitansstrains revealed that the single Btl in oneMycetohabitans endofungorumstrain enhances fungal membrane stress tolerance, while others in aMycetohabitans rhizoxinicastrain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nineMycetohabitansspp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal–bacterial metagenomes. We show thatbtlgenes are present across diverseMycetohabitansstrains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested thatMycetohabitansmight contain more species than previously recognized. Within our data set, Btl proteins were more conserved acrossM. rhizoxinicastrains than acrossM. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial–fungal symbioses in myriad ways.

Genetics & Heredity↗
DOE OSTI2021

Large-scale Genetic Characterization of a Model Sulfate-Reducing Bacterium

ABSTRACTSulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources, and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

Trotter, Valentine V↗
DOE OSTI2024

Major impacts of widespread structural variation on sorghum

Genetic diversity is critical to crop breeding and improvement, and dissection of the genomic variation underlying agronomic traits can both assist breeding and give insight into basic biological mechanisms. Although recent genome analyses in plants reveal many structural variants (SVs), most current studies of crop genetic variation are dominated by single-nucleotide polymorphisms (SNPs). The extent of the impact of SVs on global trait variation, as well as their utility in genome-wide selection, is not yet understood. In this study, we built an SV data set based on whole-genome resequencing of diverse sorghum lines (n = 363), validated the correlation of photoperiod sensitivity and variety type, and identified SV hotspots underlying the divergent evolution of cellulosic and sweet sorghum. In addition, we showed the complementary contribution of SVs for heritability of traits related to sorghum adaptation. Importantly, inclusion of SV polymorphisms in association studies revealed genotype–phenotype associations not observed with SNPs alone. Three-way genome-wide association studies (GWAS) based on whole-genome SNP, SV, and integrated SNP + SV data sets showed substantial associations between SVs and sorghum traits. The addition of SVs to GWAS substantially increased heritability estimates for some traits, indicating their important contribution to functional allelic variation at the genome level. Our discovery of the widespread impacts of SVs on heritable gene expression variation could render a plausible mechanism for their disproportionate impact on phenotypic variation. This study expands our knowledge of SVs and emphasizes the extensive impacts of SVs on sorghum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes

Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation phenotype may have been prevalent and subsequently lost by laboratory culturing in other Firmicutes currently considered as asporogenic. Cryptosporulation might thus represent an ancestral but unstable and adaptive developmental state in Firmicutes that is under selection under harsh environmental conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Divergence in heritable life history traits suggests potential for local adaptation and trade‐offs associated with a coal ash disposal site

Abstract Globally, human activities have resulted in rapid environmental changes that present unique challenges for wildlife. However, investigations of local adaptation in response to simultaneous exposure to multiple anthropogenic selection pressures are rare and often generate conflicting results. We used an in situ reciprocal transplant design within a quantitative genetic framework to examine how adaptive evolution and phenotypic plasticity contribute to the persistence of an amphibian population inhabiting an environment characterized by high levels of multiple toxic trace elements. We found evidence of phenotypic divergence that is largely consistent with local adaptation to an environment contaminated with multiple chemical stressors, tied to potential trade‐offs in the absence of contaminants. Specifically, the population derived from the contaminated environment had a reduced risk of mortality and greater larval growth and in the contaminated environment, relative to offspring from the naïve population. Further, while survival in the uncontaminated environment was not compromised in offspring from the contaminant‐exposed population, they did show delayed development and reduced growth rates over larval development, relative to the naïve population. We found no evidence of reduced additive genetic variation in the contaminant‐exposed population, suggesting long‐term selection in a novel environment has not reduced the evolutionary potential of that population. We also saw little evidence that past selection in the ASH environment had reduced trait plasticity in the resident population. Maternal effects were prominent in early development, but we did not detect any trends suggesting these effects were associated with the maternal transfer of toxic trace elements. Our results demonstrate the potential for adaptation to multiple contaminants in a wild amphibian population, which may have facilitated long‐term persistence in a heavily impacted environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Contrasting patterns of sequence variation in steelhead populations reflect distinct evolutionary processes

Abstract Multiple evolutionary processes influence genome‐wide allele frequencies and quantifying effects of genetic drift, and multiple forms of selection remain challenging in natural populations. Here, we investigate variation at major effect loci in contrast to patterns of neutral drift across a wide collection of steelhead ( Oncorhynchus mykiss ) populations that have declined in abundance due to anthropogenic impacts. Whole‐genome resequencing of 74 populations of steelhead revealed genome‐wide patterns (~8 million SNPs) consistent with expected neutral population structure. However, allelic variation at major effect loci associated with adult migration timing (chromosome 28: GREB1L / ROCK1 ) and age at maturity (chromosome 25: SIX6 ) reflected how selection has acted on phenotypic variation in contrast with neutral structure. Variation at major effect loci was influenced by evolutionary processes with differing signals between the strongly divergent Coastal and Inland lineages, while allele frequencies within and among populations within the Inland lineage have been driven by local natural selection as well as recent anthropogenic influences. Recent anthropogenic effects appeared to have influenced the frequency of major effect alleles including artificial selection for specific traits in hatchery stocks with subsequent gene flow into natural populations. Selection from environmental factors at various scales has also likely influenced variation for major effect alleles. These results reveal evolutionary mechanisms that influence allele frequencies at major effect loci that are critical for conservation of phenotypic traits and life history variation of this protected species.

Willis, Stuart↗
DOE OSTI2022

Trading water for carbon in the future: Effects of elevated CO 2 and warming on leaf hydraulic traits in a semiarid grassland

Abstract The effects of climate change on plants and ecosystems are mediated by plant hydraulic traits, including interspecific and intraspecific variability of trait phenotypes. Yet, integrative and realistic studies of hydraulic traits and climate change are rare. In a semiarid grassland, we assessed the response of several plant hydraulic traits to elevated CO 2 (+200 ppm) and warming (+1.5 to 3°C; day to night). For leaves of five dominant species (three graminoids and two forbs), and in replicated plots exposed to 7 years of elevated CO 2 , warming, or ambient climate, we measured: stomatal density and size, xylem vessel size, turgor loss point, and water potential (pre‐dawn). Interspecific differences in hydraulic traits were larger than intraspecific shifts induced by elevated CO 2 and/or warming. Effects of elevated CO 2 were greater than effects of warming, and interactions between treatments were weak or not detected. The forbs showed little phenotypic plasticity. The graminoids had leaf water potentials and turgor loss points that were 10% to 50% less negative under elevated CO 2 ; thus, climate change might cause these species to adjust their drought resistance strategy away from tolerance and toward avoidance. The C4 grass also reduced allocation of leaf area to stomata under elevated CO 2 , which helps explain observations of higher soil moisture. The shifts in hydraulic traits under elevated CO 2 were not, however, simply due to higher soil moisture. Integration of our results with others' indicates that common species in this grassland are more likely to adjust stomatal aperture in response to near‐term climate change, rather than anatomical traits; this contrasts with apparent effects of changing CO 2 on plant anatomy over evolutionary time. Future studies should assess how plant responses to drought may be constrained by the apparent shift from tolerance (via low turgor loss point) to avoidance (via stomatal regulation and/or access to deeper soil moisture).

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Genetic Basis of Non–Photochemical Quenching and Photosystem II Efficiency Responses to Chilling in the Biomass Crop Miscanthus

Miscanthus holds a promise as a biocrop due to its high yield, perenniality and ability to grow on infertile soils. However, the current commercial biomass production of Miscanthus is mostly limited to a single sterile triploid clone of M. × giganteus. Nevertheless, parental species of M. × giganteus, Miscanthus sacchariflorus and Miscanthus sinensis contain vast genetic diversity for crop improvement. With M. sacchariflorus having a natural geographic distribution in cold-temperate northeast China and eastern Russia, we hypothesised that it has substantial variation in physiological response to chilling. Using a semi-high-throughput method, we phenotyped 209 M. sacchariflorus genotypes belonging to six genetic groups for non-photochemical quenching (NPQ) and photosystem II efficiency (ΦPSII) kinetics under warm and chilling treatments in three growing seasons. In response to the chilling treatment, all genetic groups exhibited an increase in NPQ induction rate indicating faster activation of NPQ in light. Notably, under chilling, the Korea/NE China/Russia 2x and N China 2x groups stood out for the highest NPQ rate in light and the highest steady-state NPQ in light. This NPQ phenotype may contribute adaptation to chilling during bright, cold mornings of spring and early autumn in temperate climates, when faster NPQ would better protect from oxidative stress. Such enhanced adaptation could expand the growing season and thus productivity at a given location or expand the range of economically viable growing locations to higher latitudes and altitudes. A genome-wide association study identified 126 unique SNPs associated with NPQ and ΦPSII traits. Among the identified candidate genes were enzymes involved in the ascorbate recycle and shikimate pathway, gamma-aminobutyric acid and cation efflux transporters. Identifying natural variation and genes involved in NPQ and ΦPSII kinetics considerably enlarges the toolbox for breeding and/or engineering Miscanthus with optimised photosynthesis under warm and chilling conditions for sustainable feedstock production for bioenergy.

09 BIOMASS FUELS↗
DOE OSTI2026

Miscanthus giganteus Biolistic Transformation Using the Visible RUBY Red Marker Gene to Monitor Transformation Efficiency

Miscanthus × giganteus ( M × g ) is a high-yielding perennial C4 bioenergy crop, but genetic improvement by breeding is constrained by triploid sterility and clonal propagation. Improving genetic transformation methods for M × g would provide opportunities for advantageous trait introgression. Use of an easy to phenotype reporter gene is a promising strategy to improve transformation processes and efficiency. This study presents an efficient novel method for biolistic transformation of inflorescence-derived callus in M × g and demonstrates its efficacy using RUBY, a betalain-based noninvasive reporter that is visible throughout the transformation process. RUBY expression ( Zea mays codon optimized) was visible from callus stage through plantlet development into maturity. RUBY expressing independently transformed plants were confirmed by hygromycin phosphotransferase ELISA and by genomic PCR demonstrating that the RUBY phenotype is sufficient for screening transformants. The Zea mays codon optimized hygromycin selection marker was driven by previously established promoters for Miscanthus, ZmUBI and 2×35S, while the RUBY gene expression was controlled by a known Zea mays C4 promoter, Brachypodium UBI10, newly employed in Miscanthus. The construct containing the 2×35S promoter for hygromycin had a 15.1% transformation efficiency while the ZmUBI promoter had a 20.5% transformation efficiency. This study provides a novel, highly efficient protocol for successful biolistic transformation of M × g for stable expression. This study also demonstrates that RUBY expression can be used as a convenient and powerful monitor of transformation in ongoing and future work to engineer M × g into an improved bioproduct feedstock.

RUBY↗
DOE OSTI2020

Whole genome resequencing reveals genomic regions associated with thermal adaptation in redband trout

Abstract Adaptation to local environments involves evolution of ecologically important traits and underlying physiological processes. Here, we used low coverage whole‐genome resequencing (lcWGR) on individuals to identify genome regions involved in thermal adaptation in wild redband trout Oncorhynchus mykiss gairdneri , a subspecies of rainbow trout that inhabits ecosystems ranging from cold montane forests to high elevation deserts. This study includes allele frequency‐based analyses for selective sweeps among populations, followed by multiple association tests for specific sets of phenotypes measured under thermal stress (acute and chronic survival/mortality; high or low cardiac performance groups). Depending on the groups in each set of analyses, sequencing reads covered 43%–75% of the genome at ≥15× and each analysis included millions of SNPs across the genome. In tests for selective sweeps among populations, a total of six chromosomal regions were significant. The further association tests for specific phenotypes revealed that the region on chromosome 4 was consistently the most significant and contains the cerk gene (ceramide kinase). This study provides insight into a potential genetic mechanism of local thermal adaptation and suggests c erk may be an important candidate gene. However, further validation of this cerk gene is necessary to determine if the association with cardiac performance results in a functional role to influence thermal performance when exposed to high water temperatures and hypoxic conditions.

Chen, Zhongqi↗
DOE OSTI2024

Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus , Pseudomonas aeruginosa , and Francisella tularensis

Abstract Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli , finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate‐dependent resistance or susceptibility . Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa ; FTsmr from Francisella tularensis ; and SAsmr from Staphylococcus aureus . This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate‐specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate‐gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

Biochemistry & Molecular Biology↗
DOE OSTI2020

Xylella fastidiosa causes transcriptional shifts that precede tylose formation and starch depletion in xylem

Abstract Pierce's disease (PD) in grapevine ( Vitis vinifera ) is caused by the bacterial pathogen Xylella fastidiosa . X. fastidiosa is limited to the xylem tissue and following infection induces extensive plant‐derived xylem blockages, primarily in the form of tyloses. Tylose‐mediated vessel occlusions are a hallmark of PD, particularly in susceptible V. vinifera . We temporally monitored tylose development over the course of the disease to link symptom severity to the level of tylose occlusion and the presence/absence of the bacterial pathogen at fine‐scale resolution. The majority of vessels containing tyloses were devoid of bacterial cells, indicating that direct, localized perception of X. fastidiosa was not a primary cause of tylose formation. In addition, we used X‐ray computed microtomography and machine‐learning to determine that X. fastidiosa induces significant starch depletion in xylem ray parenchyma cells. This suggests that a signalling mechanism emanating from the vessels colonized by bacteria enables a systemic response to X. fastidiosa infection. To understand the transcriptional changes underlying these phenotypes, we integrated global transcriptomics into the phenotypes we tracked over the disease spectrum. Differential gene expression analysis revealed that considerable transcriptomic reprogramming occurred during early PD before symptom appearance. Specifically, we determined that many genes associated with tylose formation (ethylene signalling and cell wall biogenesis) and drought stress were up‐regulated during both Phase I and Phase II of PD. On the contrary, several genes related to photosynthesis and carbon fixation were down‐regulated during both phases. These responses correlate with significant starch depletion observed in ray cells and tylose synthesis in vessels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Growth–defense trade‐offs and yield loss in plants with engineered cell walls

Summary As a major component of plant secondary cell walls, lignin provides structural integrity and rigidity, and contributes to primary defense by providing a physical barrier to pathogen ingress. Genetic modification of lignin biosynthesis has been adopted to reduce the recalcitrance of lignified cell walls to improve biofuel production, tree pulping properties and forage digestibility. However, lignin‐modification is often, but unpredictably, associated with dwarf phenotypes. Hypotheses suggested to explain this include: collapsed vessels leading to defects in water and solute transport; accumulation of molecule(s) that are inhibitory to plant growth or deficiency of metabolites that are critical for plant growth; activation of defense pathways linked to cell wall integrity sensing. However, there is still no commonly accepted underlying mechanism for the growth defects. Here, we discuss recent data on transcriptional reprogramming in plants with modified lignin content and their corresponding suppressor mutants, and evaluate growth‐defense trade‐offs as a factor underlying the growth phenotypes. New approaches will be necessary to estimate how gross changes in transcriptional reprogramming may quantitatively affect growth. Better understanding of the basis for yield drag following cell wall engineering is important for the biotechnological exploitation of plants as factories for fuels and chemicals.

Ha, Chan Man↗
DOE OSTI2022

Structural organization of the spongy mesophyll

Summary Many plant leaves have two layers of photosynthetic tissue: the palisade and spongy mesophyll. Whereas palisade mesophyll consists of tightly packed columnar cells, the structure of spongy mesophyll is not well characterized and often treated as a random assemblage of irregularly shaped cells. Using micro‐computed tomography imaging, topological analysis, and a comparative physiological framework, we examined the structure of the spongy mesophyll in 40 species from 30 genera with laminar leaves and reticulate venation. A spectrum of spongy mesophyll diversity encompassed two dominant phenotypes: first, an ordered, honeycomblike tissue structure that emerged from the spatial coordination of multilobed cells, conforming to the physical principles of Euler’s law; and second, a less‐ordered, isotropic network of cells. Phenotypic variation was associated with transitions in cell size, cell packing density, mesophyll surface‐area‐to‐volume ratio, vein density, and maximum photosynthetic rate. These results show that simple principles may govern the organization and scaling of the spongy mesophyll in many plants and demonstrate the presence of structural patterns associated with leaf function. This improved understanding of mesophyll anatomy provides new opportunities for spatially explicit analyses of leaf development, physiology, and biomechanics.

3D↗