IRLC Research Articles

The complete chloroplast genome of Plateau herb Chesneya acaulis (Fabaceae)

Chesneya acaulis is a perennial herb, which restricts in Xizang (Tibet) of China, Afghanistan, and Pakistan. The complete chloroplast genome was sequenced using the Illumina Hiseq X-Ten platform. The genome lacks an inverted repeat (IR) region, containing 75 protein-coding genes, 29 tRNAs genes, and 4 rRNAs. The overall GC content is 34.6%. A phylogenetic tree based on the whole chloroplast genomes of 15 species indicated that C. acaulis had a close relationship with the genus Hedysarum, and it nested in the inverted repeat-lacking clade (IRLC), of the subfamily Papilionoideae (Leguminosae).

Caught in the Act: Variation in plastid genome inverted repeat expansion within and between populations of Medicago minima.

The inverted repeat (IR) lacking clade (IRLC) is a monophyletic group within the Papilionoideae subfamily of Fabaceae where plastid genomes (plastomes) do not contain the large IR typical of land plants. Recently, an IRLC legume, Medicago minima, was found to have regrown a ~9 kb IR that contained a number of canonical IR genes, and closely related M. lupulina contained an incomplete IR of ~425 bp. Complete plastomes were generated for seven additional species, putative members of the M. minima clade. Polymerase chain reaction was employed to investigate the presence of the IR across M. minima and M. lupulina including individuals of nine and eight Eurasian and North African accessions and 15 and 14 Texas populations, respectively. While no sequence similar to the ~9 kb IR was detected among the seven newly sequenced plastomes, all Eurasian and North African accessions of M. minima contained the IR. Variation in IR extent was detected within and between the Texas populations. Expansions of 13 bp and 11 bp occurred at the boundaries of both IR/small single‐copy regions, and populations had one or the other expansion, but not both. Expansion of the IR was not detected in the accessions from Eurasia and North Africa suggesting recent mutations yielded at least two additional plastid haplotypes in M. minima.

Unexplored Arsenals of Legume Peptides With Potential for Their Applications in Medicine and Agriculture.

During endosymbiosis, bacteria live intracellularly in the symbiotic organ of their host. The host controls the proliferation of endosymbionts and prevents their spread to other tissues and organs. In Rhizobium-legume symbiosis the major host effectors are secreted nodule-specific cysteine-rich (NCR) peptides, produced exclusively in the symbiotic cells. NCRs have evolved in the Inverted Repeat Lacking Clade (IRLC) of the Leguminosae family. They are secreted peptides that mediate terminal differentiation of the endosymbionts, forming polyploid, non-cultivable cells with increased membrane permeability. NCRs form an extremely large family of peptides, which have four or six conserved cysteines but otherwise highly diverse amino acid sequences, resulting in a wide variety of anionic, neutral and cationic peptides. In vitro, many synthetic NCRs have strong antimicrobial activities against both Gram-negative and Gram-positive bacteria, including the ESKAPE strains and pathogenic fungi. The spectra and minimal bactericidal and anti-fungal concentrations of NCRs differ, indicating that, in addition to their charge, the amino acid composition and sequence also play important roles in their antimicrobial activity. NCRs attack the bacteria and fungi at the cell envelope and membrane as well as intracellularly, forming interactions with multiple essential cellular machineries. NCR-like peptides with similar symbiotic functions as the NCRs also exist in other branches of the Leguminosae family. Thus, legumes provide countless and so far unexplored sources of symbiotic peptides representing an enormous resource of pharmacologically interesting molecules.

The synthesis and investigation of the catalytic properties of symmetric Schiff‐base ligand metal complexes

An ongoing project investigating the synthesis and catalytic properties of symmetric heterometallic Schiff‐base ligand complexes has been instigated. A novel symmetric Schiff‐Base ligand metal complex has been synthesized, characterized through IR spectroscopy, NMR spectroscopy, and LC mass spectrometry. The catalytic activity of this Schiff‐Base ligand has been investigated using cyclic voltammetry, mass spectrometric study, and UV‐vis spectroscopy. With the long‐term goal being a publication of results, this study aims to contribute to the fields of inorganic and biochemistry.Support or Funding InformationBarry Goldwater Scholarship Foundation

Symbiotic Outcome Modified by the Diversification from 7 to over 700 Nodule-Specific Cysteine-Rich Peptides.

Legume-rhizobium symbiosis represents one of the most successfully co-evolved mutualisms. Within nodules, the bacterial cells undergo distinct metabolic and morphological changes and differentiate into nitrogen-fixing bacteroids. Legumes in the inverted repeat lacking clade (IRLC) employ an array of defensin-like small secreted peptides (SSPs), known as nodule-specific cysteine-rich (NCR) peptides, to regulate bacteroid differentiation and activity. While most NCRs exhibit bactericidal effects in vitro, studies confirm that inside nodules they target the bacterial cell cycle and other cellular pathways to control and extend rhizobial differentiation into an irreversible (or terminal) state where the host gains control over bacteroids. While NCRs are well established as positive regulators of effective symbiosis, more recent findings also suggest that NCRs affect partner compatibility. The extent of bacterial differentiation has been linked to species-specific size and complexity of the NCR gene family that varies even among closely related species, suggesting a more recent origin of NCRs followed by rapid expansion in certain species. NCRs have diversified functionally, as well as in their expression patterns and responsiveness, likely driving further functional specialisation. In this review, we evaluate the functions of NCR peptides and their role as a driving force underlying the outcome of rhizobial symbiosis, where the plant is able to determine the outcome of rhizobial interaction in a temporal and spatial manner.

Extensive survey of the ycf4 plastid gene throughout the IRLC legumes: Robust evidence of its locus and lineage specific accelerated rate of evolution, pseudogenization and gene loss in the tribe Fabeae.

The genome organization and gene content of plastome (plastid genome) are highly conserved among most flowering plant species. Plastome variation (in size and gene order) is rare in photosynthetic species but size variation, rearrangements and gene/intron losses is attributed to groups of seed plants. Fabaceae (legume family), in particular the subfamily Papilionoideae and the inverted repeat lacking clade (IRLC), a largest legume lineage, display the most dramatic and structural change which providing an excellent model for understanding of mechanisms of genomic evolution. The IRLC comprises 52 genera and ca 4000 species divided into seven tribes. In present study, we have sampled several representatives from each tribe across the IRLC from various herbaria and field. The ycf4 gene, which plays a role in regulating and assembly of photosystem I, is more variable in the tribe Fabeae than in other tribes. In certain species of Lathyrus, Pisum and Vavilovia, all belonging to Fabeae, the gene is either absent or a pseudogene. Our study suggests that ycf4 gene has undergone positive selection. Furthermore, the rapid evolution of the gene is locus and lineage specific and is not a shared character of the IRLC in legumes.

Symbiotic Efficiency of Spherical and Elongated Bacteroids in the Aeschynomene-Bradyrhizobium Symbiosis.

The legume-rhizobium symbiosis is a major supplier of fixed nitrogen in the biosphere and constitutes a key step of the nitrogen biogeochemical cycle. In some legume species belonging to the Inverted Repeat Lacking Clade (IRLC) and the Dalbergioids, the differentiation of rhizobia into intracellular nitrogen-fixing bacteroids is terminal and involves pronounced cell enlargement and genome endoreduplication, in addition to a strong loss of viability. In the Medicago truncatula-Sinorhizobium spp. system, the extent of bacteroid differentiation correlates with the level of symbiotic efficiency. Here, we used different physiological measurements to compare the symbiotic efficiency of photosynthetic bradyrhizobia in different Aeschynomene spp. (Dalbergioids) hosts inducing different bacteroid morphotypes associated with increasing ploidy levels. The strongly differentiated spherical bacteroids were more efficient than the less strongly differentiated elongated ones, providing a higher mass gain to their hosts. However, symbiotic efficiency is not solely correlated with the extent of bacteroid differentiation especially in spherical bacteroid-inducing plants, suggesting the existence of other factors controlling symbiotic efficiency.

A Toolbox for Nodule Development Studies in Chickpea: A Hairy-Root Transformation Protocol and an Efficient Laboratory Strain of Mesorhizobium sp.

A Mesorhizobium sp. produces root nodules in chickpea. Chickpea and model legume Medicago truncatula are members of the inverted repeat-lacking clade (IRLC). The rhizobia, after internalization into the plant cell, are called bacteroids. Nodule-specific cysteine-rich peptides in IRLC legumes guide bacteroids to a terminally differentiated swollen (TDS) form. Bacteroids in chickpea are less TDS than those in Medicago spp. Nodule development in chickpea indicates recent evolutionary diversification and merits further study. A hairy-root transformation protocol and an efficient laboratory strain are prerequisites for performing any genetic study on nodulation. We have standardized a protocol for composite plant generation in chickpea with a transformation frequency above 50%, as shown by fluorescent markers. This protocol also works well in different ecotypes of chickpea. Localization of subcellular markers in these transformed roots is similar to the localization observed in transformed Medicago roots. When checked inside transformed nodules, peroxisomes were concentrated along the periphery of the nodules, while endoplasmic reticulum and Golgi bodies surrounded the symbiosomes. Different Mesorhizobium strains were evaluated for their ability to initiate nodule development and efficiency of nitrogen fixation. Inoculation with different strains resulted in different shapes of TDS bacteroids with variable nitrogen fixation. Our study provides a toolbox to study nodule development in the crop legume chickpea.

Genetic control of compound leaf development in the mungbean (Vigna radiata L.)

Many studies suggest that there are distinct regulatory processes controlling compound leaf development in different clades of legumes. Loss of function of the LEAFY (LFY) orthologs results in a reduction of leaf complexity to different degrees in inverted repeat-lacking clade (IRLC) and non-IRLC species. To further understand the role of LFY orthologs and the molecular mechanism in compound leaf development in non-IRLC plants, we studied leaf development in unifoliate leaf (un) mutant, a classical mutant of mungbean (Vigna radiata L.), which showed a complete conversion of compound leaves into simple leaves. Our analysis revealed that UN encoded the mungbean LFY ortholog (VrLFY) and played a significant role in leaf development. In situ RNA hybridization results showed that STM-like KNOXI genes were expressed in compound leaf primordia in mungbean. Furthermore, increased leaflet number in heptafoliate leaflets1 (hel1) mutants was demonstrated to depend on the function of VrLFY and KNOXI genes in mungbean. Our results suggested that HEL1 is a key factor coordinating distinct processes in the control of compound leaf development in mungbean and its related non-IRLC legumes.

Complete chloroplast genome of Caragana kozlowii, an alpine shrub endemic to China

Endemic to the southeast boundary of Tibet plateau, the medicinal alpine shrub Caragana kozlowii is a threatened species. The genetic and molecular data about it is insufficient. In this paper, the complete chloroplast (cp) genome sequence of C. kozlowii was sequenced by the Illumina sequencing platform. The result suggests that C. kozlowii cp genome is relatively short because it lacks an Inverted Repeat (IR) region. It was 131,274 bp in length, containing one large and one small single copy region (SSC and LSC) of 85,281 and 18,919 bp separated by an inverted repeat region (IR) of 27,074 bp, respectively. It contains unique 110 genes, including 76 protein-coding genes, 30 tRNAs, and 4 rRNAs. Similar to the other Inverted Repeat-Lacking Clade (IRLC) plastomes, rpl22 and rps16 are absent. The cp genome has 34.5% GC content. Phylogenetic analysis based on ten chloroplast genomes indicates that C. kozlowii is closely related to C. rosea.

Functional Genomics and Genetic Control of Compound Leaf Development in Medicago truncatula: An Overview.

Diverse forms of leaves are present in nature. However, the regulatory mechanisms that underpin the development of diverse leaf forms remain enigmatic. The initiation of leaf primordia from the periphery of shoot apical meristem (SAM) requires downregulation of the class 1 knotted-like homeobox KNOXI proteins. In plants with simple leaves, this downregulation is permanent, consistent with leaves being determinant organs. In most of plants with compound leaves, the KNOXI proteins are reactivated in developing leaf primordia, and this reactivation is required for the development of compound leaves in these plants. Surprisingly, in Medicago truncatula and pea (Pisum sativum) that belong to the so-called inverted repeat-lacking clade (IRLC) of legume plants, the KNOXI proteins are not reactivated in leaf primordia and therefore not likely involved in the development of compound leaves in these plants. Instead, the legume FLORICAULA/LEAFY orthologues, UNIFOLIATA (UNI) and SINGLE LEAFLET1 (SGL1), are required for the initiation and development of lateral leaflet primordia in pea and M. truncatula plants, respectively. On the other hand, PALMATE-LIKE PENTAFOLIATA1 (PALM1) encoding a novel Cys(2)His(2) zinc finger transcription factor is required to suppress a morphogenetic activity at the leaf margin by negatively regulating SGL1 gene expression, and FUSED COMPOUND LEAF1 (FCL1) encoding a class M KNOX protein is required for the development of the leaf proximo-distal axis and organ boundary separation in M. truncatula. Thus, these recent studies have shown that SGL1/UNI, FCL1, and PALM1 provide a genetic framework for our understanding of compound leaf development in the legume plants.

The complete chloroplast genome sequence of Glycyrrhiza lepidota (Nutt.) Pursh - An American wild licorice

The wild species are considered as primary and secondary genepools for the world’s most important food crops. Here, we sequenced the complete chloroplast (cp) genome sequence of an American wild licorice, Glycyrrhiza lepidota for the first time to investigate their phylogenetic relationship among inverted-repeat-lacking clade (IRLC) legumes. The total length of the chloroplast genome is 127,939 bp, with 34.2% overall GC content. The chloroplast genome harbors 110 known genes, including 76 protein-coding genes, four ribosomal RNA genes, and 30 tRNA genes. Similar to other closely related plastomes, rpl22 and rps16 are absent. A total of 464 cp microsatellites (cpSSRs) were analyzed in the G. lepidota. The majority of the SSRs in this cp genome are penta-nucleotides (61.6%). Locally collinear blocks (LCBs) identified between the Glycyrrhiza glabra and G. lepidota cp genomes were showed that they were well conserved with respect to gene organization and order. Moreover, the phylogenetic analysis indicates that G. lepidota is closely related to its confamilial counterparts than to the other taxa of the IRLC legumes.

Genome-Wide Sensitivity Analysis of the Microsymbiont Sinorhizobium meliloti to Symbiotically Important, Defensin-Like Host Peptides

ABSTRACTThe model legume species Medicago truncatula expresses more than 700 nodule-specific cysteine-rich (NCR) signaling peptides that mediate the differentiation of Sinorhizobium meliloti bacteria into nitrogen-fixing bacteroids. NCR peptides are essential for a successful symbiosis in legume plants of the inverted-repeat-lacking clade (IRLC) and show similarity to mammalian defensins. In addition to signaling functions, many NCR peptides exhibit antimicrobial activity in vitro and in vivo. Bacterial resistance to these antimicrobial activities is likely to be important for symbiosis. However, the mechanisms used by S. meliloti to resist antimicrobial activity of plant peptides are poorly understood. To address this, we applied a global genetic approach using transposon mutagenesis followed by high-throughput sequencing (Tn-seq) to identify S. meliloti genes and pathways that increase or decrease bacterial competitiveness during exposure to the well-studied cationic NCR247 peptide and also to the unrelated model antimicrobial peptide polymyxin B. We identified 78 genes and several diverse pathways whose interruption alters S. meliloti resistance to NCR247. These genes encode the following: (i) cell envelope polysaccharide biosynthesis and modification proteins, (ii) inner and outer membrane proteins, (iii) peptidoglycan (PG) effector proteins, and (iv) non-membrane-associated factors such as transcriptional regulators and ribosome-associated factors. We describe a previously uncharacterized yet highly conserved peptidase, which protects S. meliloti from NCR247 and increases competitiveness during symbiosis. Additionally, we highlight a considerable number of uncharacterized genes that provide the basis for future studies to investigate the molecular basis of symbiotic development as well as chronic pathogenic interactions.

Janet I. Sprent.

Janet I. Sprent.

Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides

In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.

Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247

Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide-cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis.

Terminal Bacteroid Differentiation Is Associated With Variable Morphological Changes in Legume Species Belonging to the Inverted Repeat-Lacking Clade.

Medicago and closely related legume species from the inverted repeat-lacking clade (IRLC) impose terminal differentiation onto their bacterial endosymbionts, manifested in genome endoreduplication, cell enlargement, and loss of cell-division capacity. Nodule-specific cysteine-rich (NCR) secreted host peptides are plant effectors of this process. As bacteroids in other IRLC legumes, such as Cicer arietinum and Glycyrrhiza lepidota, were reported not to display features of terminal differentiation, we investigated the fate of bacteroids in species from these genera as well as in four other species representing distinct genera of the phylogenetic tree for this clade. Bacteroids in all tested legumes proved to be larger in size and DNA content than cultured cells; however, the degree of cell elongation was rather variable in the different species. In addition, the reproductive ability of the bacteroids isolated from these legumes was remarkably reduced. In all IRLC species with available sequence data, the existence of NCR genes was found. These results indicate that IRLC legumes provoke terminal differentiation of their endosymbionts with different morphotypes, probably with the help of NCR peptides.

Complete plastid genome of Astragalus membranaceus (Fisch.) Bunge var. membranaceus

The complete nucleotide sequence of the Astragalus membranaceus (Fisch.) Bunge var. membranaceus chloroplast genome was reported and characterized in this study. The chloroplast genome is a circular molecule of 123623 bp that belongs to the inverted repeat-lacking clade (IRLC). It comprises 110 genes, including 76 protein-coding genes, four unique rRNAs and 30 tRNAs. Similar to the plastomes of A. membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P. K. Hsiao and other closely related species, rpl22 and rps16 are absent. The phylogenetic analysis of 67 proteins from 29 chloroplast genomes belonging to IRLC provided strong support for the non-monophyly of Galegeae. This genome has provided a wealth of information for distinguishing varieties of A. membranaceus.

Sinorhizobium fredii HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the Inverted Repeat-Lacking Clade legume Glycyrrhiza uralensis.

In rhizobial species that nodulate inverted repeat-lacking clade (IRLC) legumes, such as the interaction between Sinorhizobium meliloti and Medicago, bacteroid differentiation is driven by an endoreduplication event that is induced by host nodule-specific cysteine rich (NCR) antimicrobial peptides and requires the participation of the bacterial protein BacA. We have studied bacteroid differentiation of Sinorhizobium fredii HH103 in three host plants: Glycine max, Cajanus cajan and the IRLC legume Glycyrrhiza uralensis. Flow cytometry, microscopy analyses and viability studies of bacteroids as well as confocal microscopy studies carried out in nodules showed that S. fredii HH103 bacteroids, regardless of the host plant, had deoxyribonucleic acid (DNA) contents, cellular sizes and survival rates similar to those of free-living bacteria. Contrary to S. meliloti, S. fredii HH103 showed little or no sensitivity to Medicago NCR247 and NCR335 peptides. Inactivation of S. fredii HH103 bacA neither affected symbiosis with Glycyrrhiza nor increased bacterial sensitivity to Medicago NCRs. Finally, HH103 bacteroids isolated from Glycyrrhiza, but not those isolated from Cajanus or Glycine, showed an altered lipopolysaccharide. Our studies indicate that, in contrast to the S. meliloti-Medicago model symbiosis, bacteroids in the S. fredii HH103-Glycyrrhiza symbiosis do not undergo NCR-induced and bacA-dependent terminal differentiation.

Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides.

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.