A metabolic modeling-based framework for predicting trophic dependencies in native rhizobiomes of crop plants.

The exchange of metabolites (i.e., metabolic interactions) between bacteria in the rhizosphere determines various plant-associated functions. Systematically understanding the metabolic interactions in the rhizosphere, as well as in other types of microbial communities, would open the door to the optimization of specific predefined functions of interest, and therefore to the harnessing of the functionality of various types of microbiomes. However, mechanistic knowledge regarding the gathering and interpretation of these interactions is limited. Here, we present a framework utilizing genomics and constraint-based modeling approaches, aiming to interpret the hierarchical trophic interactions in the soil environment. 243 genome scale metabolic models of bacteria associated with a specific disease-suppressive vs disease-conducive apple rhizospheres were drafted based on genome-resolved metagenomes, comprising an in silico native microbial community. Iteratively simulating microbial community members' growth in a metabolomics-based apple root-like environment produced novel data on potential trophic successions, used to form a network of communal trophic dependencies. Network-based analyses have characterized interactions associated with beneficial vs non-beneficial microbiome functioning, pinpointing specific compounds and microbial species as potential disease supporting and suppressing agents. This framework provides a means for capturing trophic interactions and formulating a range of testable hypotheses regarding the metabolic capabilities of microbial communities within their natural environment. Essentially, it can be applied to different environments and biological landscapes, elucidating the conditions for the targeted manipulation of various microbiomes, and the execution of countless predefined functions.

Cytonuclear interactions modulate the plasticity of photosynthetic rhythmicity and growth in wild barley.

In plants, the contribution of the plasmotype (mitochondria and chloroplast) in controlling the circadian clock plasticity and possible consequences on cytonuclear genetic makeup have yet to be fully elucidated. A genome-wide association study in the wild barley (Hordeum vulgare ssp. spontaneum) B1K collection identified overlap with our previously mapped DRIVERS OF CLOCKS (DOCs) loci in wild-cultivated interspecific population. Moreover, we identified non-random segregation and epistatic interactions between nuclear DOCs loci and the chloroplastic RpoC1 gene, indicating an adaptive value for specific cytonuclear gene combinations. Furthermore, we show that DOC1.1, which harbours the candidate SIGMA FACTOR-B (SIG-B) gene, is linked with the differential expression of SIG-B and CCA1 genes and contributes to the circadian gating response to heat. High-resolution temporal growth and photosynthesis measurements of B1K also link the DOCs loci to differential growth, Chl content and quantum yield. To validate the involvement of the Plastid encoded polymerase (PEP) complex, we over-expressed the two barley chloroplastic RpoC1 alleles in Arabidopsis and identified significant differential plasticity under elevated temperatures. Finally, enhanced clock plasticity of de novo ENU (N-Ethyl-N-nitrosourea) -induced barley rpoB1 mutant further implicates the PEP complex as a key player in regulating the circadian clock output. Overall, this study highlights the contribution of specific cytonuclear interaction between rpoC1 (PEP gene) and SIG-B with distinct circadian timing regulation under heat, and their pleiotropic effects on growth implicate an adaptive value.

Slow release of a synthetic auxin induces formation of adventitious roots in recalcitrant woody plants.

Clonal propagation of plants by induction of adventitious roots (ARs) from stem cuttings is a requisite step in breeding programs. A major barrier exists for propagating valuable plants that naturally have low capacity to form ARs. Due to the central role of auxin in organogenesis, indole-3-butyric acid is often used as part of commercial rooting mixtures, yet many recalcitrant plants do not form ARs in response to this treatment. Here we describe the synthesis and screening of a focused library of synthetic auxin conjugates in Eucalyptus grandis cuttings and identify 4-chlorophenoxyacetic acid-L-tryptophan-OMe as a competent enhancer of adventitious rooting in a number of recalcitrant woody plants, including apple and argan. Comprehensive metabolic and functional analyses reveal that this activity is engendered by prolonged auxin signaling due to initial fast uptake and slow release and clearance of the free auxin 4-chlorophenoxyacetic acid. This work highlights the utility of a slow-release strategy for bioactive compounds for more effective plant growth regulation.

Functional analysis of the apple fruit microbiome based on shotgun metagenomic sequencing of conventional and organic orchard samples.

Fruits harbour abundant and diverse microbial communities that protect them from post-harvest pathogens. Identification of functional traits associated with a given microbiota can provide a better understanding of their potential influence. Here, we focused on the epiphytic microbiome of apple fruit. We suggest that shotgun metagenomic data can indicate specific functions carried out by different groups and provide information on their potential impact. Samples were collected from the surface of 'Golden Delicious' apples from four orchards that differ in their geographic location and management practice. Approximately 1 million metagenes were predicted based on a high-quality assembly. Functional profiling of the microbiome of fruits from orchards differing in their management practice revealed a functional shift in the microbiota. The organic orchard microbiome was enriched in pathways involved in plant defence activities; the conventional orchard microbiome was enriched in pathways related to the synthesis of antibiotics. The functional significance of the variations was explored using microbial network modelling algorithms to reveal the metabolic role of specific phylogenetic groups. The analysis identified several associations supported by other published studies. For example, the analysis revealed the nutritional dependencies of the Capnodiales group, including the Alternaria pathogen, on aromatic compounds.

Deep transcriptomic study reveals the role of cell wall biosynthesis and organization networks in the developing shell of peanut pod.

BACKGROUND: Peanut (Arachis hypogaea L.) belongs to an exceptional group of legume plants, wherein the flowers are produced aerially, but the pods develop under the ground. In such a unique environment, the pod's outer shell plays a vital role as a barrier against mechanical damage and soilborne pathogens. Recent studies have reported the uniqueness and importance of gene expression patterns that accompany peanut pods' biogenesis. These studies focused on biogenesis and pod development during the early stages, but the late developmental stages and disease resistance aspects still have gaps. To extend this information, we analyzed the transcriptome generated from four pod developmental stages of two genotypes, Hanoch (Virginia-type) and IGC53 (Peruvian-type), which differs significantly in their pod shell characteristics and pathogen resistance. RESULTS: The transcriptome study revealed a significant reprogramming of the number and nature of differentially expressed (DE) genes during shell development. Generally, the numbers of DE genes were higher in IGC53 than in Hanoch, and the R5-R6 transition was the most dynamic in terms of transcriptomic changes. Genes related to cell wall biosynthesis, modification and transcription factors (TFs) dominated these changes therefore, we focused on their differential, temporal and spatial expression patterns. Analysis of the cellulose synthase superfamily identified specific Cellulose synthase (CesAs) and Cellulose synthase-like (Csl) genes and their coordinated interplay with other cell wall-related genes during the peanut shell development was demonstrated. TFs were also identified as being involved in the shell development process, and their pattern of expression differed in the two peanut genotypes. The shell component analysis showed that overall crude fiber, cellulose, lignin, hemicelluloses and dry matter increased with shell development, whereas K, N, protein, and ash content decreased. Genotype IGC53 contained a higher level of crude fiber, cellulose, NDF, ADF, K, ash, and dry matter percentage, while Hanoch had higher protein and nitrogen content. CONCLUSIONS: The comparative transcriptome analysis identified differentially expressed genes, enriched processes, and molecular processes like cell wall biosynthesis/modifications, carbohydrate metabolic process, signaling, transcription factors, transport, stress, and lignin biosynthesis during the peanut shell development between two contrasting genotypes. TFs and other genes like chitinases were also enriched in peanut shells known for pathogen resistance against soilborne major pathogens causing pod wart disease and pod damages. This study will shed new light on the biological processes involved with underground pod development in an important legume crop.

Thermal plasticity of the circadian clock is under nuclear and cytoplasmic control in wild barley.

Temperature compensation, expressed as the ability to maintain clock characteristics (mainly period) in face of temperature changes, that is, robustness, is considered a key feature of circadian clock systems. In this study, we explore the genetic basis for lack of robustness, that is, plasticity, of circadian clock as reflected by photosynthesis rhythmicity. The clock rhythmicity of a new wild barley reciprocal doubled haploid population was analysed with a high temporal resolution of pulsed amplitude modulation of chlorophyll fluorescence under optimal (22°C) and high (32°C) temperature. This comparison between two environments pointed to the prevalence of clock acceleration under heat. Genotyping by sequencing of doubled haploid lines indicated a rich recombination landscape with minor fixation (less than 8%) for one of the parental alleles. Quantitative genetic analysis included genotype by environment interactions and binary-threshold models. Variation in the circadian rhythm plasticity phenotypes, expressed as change (delta) of period and amplitude under two temperatures, was associated with maternal organelle genome (the plasmotype), as well as with several nuclear loci. This first reported rhythmicity driven by nuclear loci and plasmotype with few identified variants, paves the way for studying impact of cytonuclear variations on clock robustness and on plant adaptation to changing environments.

Fine-Mapping the Branching Habit Trait in Cultivated Peanut by Combining Bulked Segregant Analysis and High-Throughput Sequencing.

The growth habit of lateral shoots (also termed "branching habit") is an important descriptive and agronomic character of peanut. Yet, both the inheritance of branching habit and the genetic mechanism that controls it in this crop remain unclear. In addition, the low degree of polymorphism among cultivated peanut varieties hinders fine-mapping of this and other traits in non-homozygous genetic structures. Here, we combined high-throughput sequencing with a well-defined genetic system to study these issues in peanut. Initially, segregating F2 populations derived from a reciprocal cross between very closely related Virginia-type peanut cultivars with spreading and bunch growth habits were examined. The spreading/bunch trait was shown to be controlled by a single gene with no cytoplasmic effect. That gene was named Bunch1 and was significantly correlated with pod yield per plant, time to maturation and the ratio of "dead-end" pods. Subsequently, bulked segregant analysis was performed on 52 completely bunch, and 47 completely spreading F3 families. In order to facilitate the process of SNP detection and candidate-gene analysis, the transcriptome was used instead of genomic DNA. Young leaves were sampled and bulked. Reads from Illumina sequencing were aligned against the peanut reference transcriptome and the diploid genomes. Inter-varietal SNPs were detected, scored and quality-filtered. Thirty-four candidate SNPs were found to have a bulk frequency ratio value >10 and 6 of those SNPs were found to be located in the genomic region of linkage group B5. Three best hits from that over-represented region were further analyzed in the segregating population. The trait locus was found to be located in a ~1.1 Mbp segment between markers M875 (B5:145,553,897; 1.9 cM) and M255 (B5:146,649,943; 2.25 cM). The method was validated using a population of recombinant inbreed lines of the same cross and a new DNA SNP-array. This study demonstrates the relatively straight-forward utilization of bulk segregant analysis for trait fine-mapping in the low polymeric and heterozygous germplasm of cultivated peanut and provides a baseline for candidate gene discovery and map-based cloning of Bunch1.

Gene expression profiling during seed-filling process in peanut with emphasis on oil biosynthesis networks.

Pod-filling is an important stage of peanut (Arachis hypogaea) seed development. It is partially controlled by genetic factors, as cultivars considerably vary in pod-filling potential. Here, a study was done to detect changes in mRNA levels that accompany pod-filling processes. Four seed developmental stages were sampled from two peanut genotypes differing in their oil content and pod-filling potential. Transcriptome data were generated by RNA-Seq and explored with respect to genic and subgenomic patterns of expression. Very dynamic transcriptomic changes occurred during seed development in both genotypes. Yet, general higher expression rates of transcripts and an enrichment in processes involved "energy generation" and "primary metabolites" were observed in the genotype with the better pod-filling ("Hanoch"). A dataset of 584 oil-related genes was assembled and analyzed, resulting in several lipid metabolic processes highly expressed in Hanoch, including oil storage and FA synthesis/elongation. Homoeolog-specific gene expression analysis revealed that both subgenomes contribute to the oil genes expression. Yet, biases were observed in particular parts of the pathway with possible biological meaning, presumably explaining the genotypic variation in oil biosynthesis and pod-filling. This study provides baseline information and a resource that may be used to understand development and oil biosynthesis in the peanut seeds.

Evolutionary Conservation and Divergence of Gene Coexpression Networks in Gossypium (Cotton) Seeds.

The cotton genus (Gossypium) provides a superior system for the study of diversification, genome evolution, polyploidization, and human-mediated selection. To gain insight into phenotypic diversification in cotton seeds, we conducted coexpression network analysis of developing seeds from diploid and allopolyploid cotton species and explored network properties. Key network modules and functional associations were identified related to seed oil content and seed weight. We compared species-specific networks to reveal topological changes, including rewired edges and differentially coexpressed genes, associated with speciation, polyploidy, and cotton domestication. Network comparisons among species indicate that topologies are altered in addition to gene expression profiles, indicating that changes in transcriptomic coexpression relationships play a role in the developmental architecture of cotton seed development. The global network topology of allopolyploids, especially for domesticated G. hirsutum, resembles the network of the A-genome diploid more than that of the D-genome parent, despite its D-like phenotype in oil content. Expression modifications associated with allopolyploidy include coexpression level dominance and transgressive expression, suggesting that the transcriptomic architecture in polyploids is to some extent a modular combination of that of its progenitor genomes. Among allopolyploids, intermodular relationships are more preserved between two different wild allopolyploid species than they are between wild and domesticated forms of a cultivated cotton, and regulatory connections of oil synthesis-related pathways are denser and more closely clustered in domesticated vs. wild G. hirsutum. These results demonstrate substantial modification of genic coexpression under domestication. Our work demonstrates how network inference informs our understanding of the transcriptomic architecture of phenotypic variation associated with temporal scales ranging from thousands (domestication) to millions (speciation) of years, and by polyploidy.

A Transcriptome Profile for Developing Seed of Polyploid Cotton.

Cotton ranks among the world's important oilseed crops, yet relative to other oilseeds there are few studies of oil-related biosynthetic and regulatory pathways. We present global transcriptome analyses of cotton seed development using RNA-seq and four developmental time-points. Because Upland cotton (Gossypium hirsutum L.) is an allopolyploid containing two genomes (A/D), we partitioned expression into the individual contributions of each homeologous gene copy. Data were explored with respect to genic and subgenomic patterns of expression, globally and with respect to seed pathways and networks. The most dynamic period of transcriptome change is from 20-30 d postanthesis (DPA), with about 20% of genes showing homeolog expression bias. Co-expression analysis shows largely congruent homeolog networks, but also homeolog-specific divergence. Functional enrichment tests show that flavonoid biosynthesis and lipid related genes were significantly represented early and later in seed development, respectively. An involvement of new features in oil biosynthesis was found, like the contribution of DGAT3 (diacylglycerol acyltransferase) to the total triglyceride expression pool. Also, catechin-based and epicatechin-based proanthocyanidin expression are reciprocally biased with respect to homeolog usage. This study provides the first temporal analysis of duplicated gene expression in cotton seed and a resource for understanding new aspects of oil and flavonoid biosynthetic processes.