Conserved genomic context provides critical information for comparative evolutionary analysis. With the increase in numbers of sequenced plant genomes, synteny analysis can provide new insights into ...gene family evolution. Here, we exploit a network analysis approach to organize and interpret massive pairwise syntenic relationships. Specifically, we analyzed synteny networks of the MADS-box transcription factor gene family using 51 completed plant genomes. In combination with phylogenetic profiling, several novel evolutionary patterns were inferred and visualized from synteny network clusters. We found lineage-specific clusters that derive from transposition events for the regulators of floral development (APETALA3 and PI) and flowering time (FLC) in the Brassicales and for the regulators of root development (AGL17) in Poales. We also identified two large gene clusters that jointly encompass many key phenotypic regulatory Type II MADS-box gene clades (SEP1, SQUA, TM8, SEP3, FLC, AGL6, and TM3). Gene clustering and gene trees support the idea that these genes are derived from an ancient tandem gene duplication that likely predates the radiation of the seed plants and then expanded by subsequent polyploidy events. We also identified angiosperm-wide conservation of synteny of several other less studied clades. Combined, these findings provide new hypotheses for the genomic origins, biological conservation, and divergence of MADS-box gene family members.
Nitrogen fixation by rhizobia is a highly energy-demanding process. Therefore, nodule initiation in legumes is tightly regulated. Environmental nitrate is a potent inhibitor of nodulation. However, ...the precise mechanism by which this agent (co)regulates the inhibition of nodulation is not fully understood. Here, we demonstrate that in
the lipo-chitooligosaccharide-induced accumulation of cytokinins is reduced in response to the application of exogenous nitrate. Under permissive nitrate conditions, perception of rhizobia-secreted signalling molecules leads to an increase in the level of four cytokinins (i.e., iP, iPR, tZ, and tZR). However, under high-nitrate conditions, this increase in cytokinins is reduced. The ethylene-insensitive mutant
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, as well as wild-type plants grown in the presence of the ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine (AVG), is resistant to the inhibition of nodulation by nitrate. This demonstrates that ethylene biosynthesis and perception are required to inhibit nodule organogenesis under high-nitrate conditions.
The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells ...analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.
Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is ...generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION. Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.
Summary
Arbuscular mycorrhizal fungi form the most wide‐spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying ...symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host‐dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host‐ and stage‐dependent manner to contribute to their extremely wide host range. We investigated the expression of SP‐encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA‐seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host‐dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.
Given that nodulating plants do not require exogenous nitrogen fertilizer, engineering nodulation in non-nodulating crops has been a longstanding ambition. From an evolutionary perspective, ...identifying the genetic changes that led to nodulation can provide key engineering targets. The occurrence of nitrogen-fixing root nodule symbiosis with rhizobium or Frankia bacteria is limited to ten plant lineages in four orders: Fagales, Fabales, Rosales, and Cucurbitales. These four orders together form a clade, referred to as the nitrogen-fixing clade. The scattered phylogenetic distribution of nodulating lineages previously led to the hypothesis that nodulation evolved independently multiple times, possibly preceded by a predisposition event at the root of the nitrogen-fixing clade. This thesis presents comparative genomic and transcriptomic analyses to identify genetic changes leading to the evolution of nodulation, as well as innovations in the computational tools required for such analyses. Chapter 2 consists of a review of known molecular mechanisms in two plant-bacteria symbioses (with rhizobia and Frankia) and in two plantfungus symbioses (arbuscular mycorrhizae and ectomycorrhizae). Specifically, I explore to what extent molecular mechanisms are shared between these four symbioses. The two main commonalities in symbiotic signalling are (1) rhizobium and Frankia symbioses are known to co-opt various elements of arbuscular mycorrhizal symbiotic signalling, and (2) plant-secreted flavonoids and strigolactones act as attractants to the symbiont in all four symbioses. Placing the known symbiotic molecular mechanisms in a comparative context will provide a targeted approach at studying the molecular evolution of nodulation.In chapter 3 I studied the molecular evolution of nodulation in the only lineage outside of the legumes that engages in rhizobium symbiosis – Parasponia – and its non-nodulating sister lineage Trema, both from the Cannabaceae family in the order Rosales. I started with assembling and annotating reference genomes for Parasponia andersonii and Trema orientalis using newly generated data, taking care to avoid lineage specific annotation errors. Using targeted and untargeted approaches, I performed a comparative genomic analysis to identify genetic changes that correlate with the nodulation trait. Following the multiple gain hypothesis, I expected to find evidence for a gain of nodulation in Parasponia. However, such evidence could not be found. Instead, I found pseudogenes Trema genomes of genes known to be essential for nodulation, which indicated a loss of the nodulation trait in Trema species. In an extended evolutionary perspective, I analyzed public data for several non-nodulating lineages in the Rosales, revealing consistent gene loss of NOD FACTOR PERCEPTION 2 (NFP2), NODULE INCEPTION (NIN), and RHIZOBIUMDIRECTED POLAR GROWTH (RPG). Combined with the identification of 290 conserved genes that are transcriptionally upregulated in nodules of Parasponia andersonii and the legume Medicago truncatula (order Fabales), I conclude that the evolutionary origin of nodulation lies at least at the root of the Rosales, and that the trait was subsequently lost multiple times in non-nodulating lineages within the nitrogen-fixing clade. As genes do not function in isolation, Chapter 4 extends the perspective of chapter 3 to the evolution of transcriptional networks in nodulation. In model legumes, multiple transcriptional regulators are known to be crucial for nodulation, including NIN
Herbarium genomics is proving promising as next‐generation sequencing approaches are well suited to deal with the usually fragmented nature of archival DNA. We show that routine assembly of partial ...plastome sequences from herbarium specimens is feasible, from total DNA extracts and with specimens up to 146 years old. We use genome skimming and an automated assembly pipeline, Iterative Organelle Genome Assembly, that assembles paired‐end reads into a series of candidate assemblies, the best one of which is selected based on likelihood estimation. We used 93 specimens from 12 different Angiosperm families, 73 of which were from herbarium material with ages up to 146 years old. For 84 specimens, a sufficient number of paired‐end reads were generated (in total 9.4 × 10¹² nucleotides), yielding successful plastome assemblies for 74 specimens. Those derived from herbarium specimens have lower fractions of plastome‐derived reads compared with those from fresh and silica‐gel‐dried specimens, but total herbarium assembly lengths are only slightly shorter. Specimens from wet‐tropical conditions appear to have a higher number of contigs per assembly and lower N50 values. We find no significant correlation between plastome coverage and nuclear genome size (C value) in our samples, but the range of C values included is limited. Finally, we conclude that routine plastome sequencing from herbarium specimens is feasible and cost‐effective (compared with Sanger sequencing or plastome‐enrichment approaches), and can be performed with limited sample destruction.
Summary
Tree stems undergo a massive secondary growth in which secondary xylem and phloem tissues arise from the vascular cambium. Vascular cambium activity is driven by endogenous developmental ...signalling cues and environmental stimuli. Current knowledge regarding the genetic regulation of cambium activity and secondary growth is still far from complete. The tropical Cannabaceae tree Parasponia andersonii is a non‐legume research model of nitrogen‐fixing root nodulation. Parasponia andersonii can be transformed efficiently, making it amenable for CRISPR‐Cas9‐mediated reverse genetics. We considered whether P. andersonii also could be used as a complementary research system to investigate tree‐related traits, including secondary growth. We established a developmental map of stem secondary growth in P. andersonii plantlets. Subsequently, we showed that the expression of the co‐transcriptional regulator PanNODULE ROOT1 (PanNOOT1) is essential for controlling this process. PanNOOT1 is orthologous to Arabidopsis thaliana BLADE‐ON‐PETIOLE1 (AtBOP1) and AtBOP2, which are involved in the meristem‐to‐organ‐boundary maintenance. Moreover, in species forming nitrogen‐fixing root nodules, NOOT1 is known to function as a key nodule identity gene. Parasponia andersonii CRISPR‐Cas9 loss‐of‐function Pannoot1 mutants are altered in the development of the xylem and phloem tissues without apparent disturbance of the cambium organization and size. Transcriptomic analysis showed that the expression of key secondary growth‐related genes is significantly down‐regulated in Pannoot1 mutants. This allows us to conclude that PanNOOT1 positively contributes to the regulation of stem secondary growth. Our work also demonstrates that P. andersonii can serve as a tree research system.
Significance Statement
Parasponia andersonii Pannoot1 CRISPR‐Cas9 knockout mutants are altered for secondary growth development. We show a new role for NBCL genes in tree stem secondary growth and provide a proof of concept that P. andersonii is suited for the investigation of tree‐related traits.
SUMMARY
Photosynthesis is a key process in sustaining plant and human life. Improving the photosynthetic capacity of agricultural crops is an attractive means to increase their yields. While the core ...mechanisms of photosynthesis are highly conserved in C3 plants, these mechanisms are very flexible, allowing considerable diversity in photosynthetic properties. Among this diversity is the maintenance of high photosynthetic light‐use efficiency at high irradiance as identified in a small number of exceptional C3 species. Hirschfeldia incana, a member of the Brassicaceae family, is such an exceptional species, and because it is easy to grow, it is an excellent model for studying the genetic and physiological basis of this trait. Here, we present a reference genome of H. incana and confirm its high photosynthetic light‐use efficiency. While H. incana has the highest photosynthetic rates found so far in the Brassicaceae, the light‐saturated assimilation rates of closely related Brassica rapa and Brassica nigra are also high. The H. incana genome has extensively diversified from that of B. rapa and B. nigra through large chromosomal rearrangements, species‐specific transposon activity, and differential retention of duplicated genes. Duplicated genes in H. incana, B. rapa, and B. nigra that are involved in photosynthesis and/or photoprotection show a positive correlation between copy number and gene expression, providing leads into the mechanisms underlying the high photosynthetic efficiency of these species. Our work demonstrates that the H. incana genome serves as a valuable resource for studying the evolution of high photosynthetic light‐use efficiency and enhancing photosynthetic rates in crop species.
Significance Statement
Hirschfeldia incana is a diploid, wild species, closely related to Brassica crops, which displays remarkably high photosynthesis rates at high irradiance, a desirable trait to improve in crops to increase yield. We propose H. incana as an attractive high‐photosynthesis model species and present the reference genome sequence as a valuable resource for studying the evolution, genetics, and physiology of high photosynthetic light‐use efficiency at high irradiances.
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