Global analysis of Two Component System (TCS) member of chickpea and other legume genomes implicates its role in enhanced nodulation

AbstractNodule organogenesis is governed primarily by phytohormone cytokinin. We observed the significant nodulation in chickpea at particular cytokinin concentration (2.5×10−7) which indicated the importance of cytokinin in nodule development. Cytokinin signaling is mediated through the Two Component System (TCS) which comprises of sensor histidine kinases (HKs), histidine phosphotransfer proteins (HPs), and response regulators (RRs). Therefore, we analyzed the interconnection of cytokinin with TCS molecules during root nodule development through global analysis of TCS candidates in legumes with special consideration to cytokinin receptor and Type-B RR member. We have conducted an in depth global analysis of TCS family members in chickpea and other legumes, Medicago and pigeon pea. Higher number of TCS genes were found in Medicago (96), followed by pigeonpea (75) and chickpea (67). A good correlation between TCS members with their corresponding total number of genes were observed in all three-legume species. Collinearity analysis of TCS revealed phylogenetically closer proximity of Cicer to Medicago followed by Glycine than Cajanus. Comprehensive analysis of 3-dimensional structure, genomic organisation and domain arrangement showed a conservation of TCS members within species. In depth investigation showed that HKs were mainly conserved among TCS members in legumes and non-legumes while divergence occurred at level of RRs. Further, Type-B RRs were functionally most diversified in RRs based on phylogeny, syntenic and transcript analysis. Few numbers of segmentally duplicated pair of TCS showed difference in their transcriptional regulation suggesting the functional evolution. For functional characterization the cre1 mutants of (Medicago) were complemented with chickpea cytokinin responsive HKs and nodulation deficient phenotype of mutants were restored. A synchronous cytokinin-induced expression of chickpea cytokinin receptor HKs and CaNIN provides strong relation of cytokinin signaling during nodulation. Furthermore, interesting potential candidate CaRR13 was selected to deduce the underlying molecular mechanism of nodulation, chickpea in specific and legumes in general.

Download Full-text

Silencing of Putative Cytokinin Receptor Histidine Kinase1 Inhibits Both Inception and Differentiation of Root Nodules in Arachis hypogaea

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-17-0144-r ◽

2018 ◽

Vol 31 (2) ◽

pp. 187-199 ◽

Cited By ~ 9

Author(s):

Anindya Kundu ◽

Maitrayee DasGupta

Keyword(s):

Arachis Hypogaea ◽

Root Nodules ◽

Nodule Development ◽

Response Regulators ◽

Cytokinin Signaling ◽

Cortical Cells ◽

Cytokinin Receptor ◽

Undifferentiated State ◽

Mitotic Cyclin ◽

Infected Cells

Rhizobia–legume interaction activates the SYM pathway that recruits cytokinin signaling for induction of nodule primordia in the cortex. In Arachis hypogaea, bradyrhizobia invade through natural cracks developed in the lateral root base and are directly endocytosed in the cortical cells to generate the nodule primordia. To unravel the role of cytokinin signaling in A. hypogaea, RNA-interference (RNAi) of cytokinin receptor histidine-kinase1 (AhHK1) was done. AhHK1-RNAi downregulated the expression of type-A response regulators such as AhRR5 and AhRR3 along with several symbiotic genes, indicating that both cytokinin signaling and the SYM pathway were affected. Accordingly, there was a drastic downregulation of nodulation in AhHK1-RNAi roots and the nodules that developed were ineffective. These nodules were densely packed, with infected cells having a higher nucleo-cytoplasmic ratio and distinctively high mitotic index, where the rod-shaped rhizobia failed to differentiate into bacteroids within spherical symbiosomes. In accordance with the proliferating state, expression of a mitotic-cyclin AhCycB2.1 was higher in AhHK1-RNAi nodules, whereas expression of a retinoblastoma-related (AhRBR) nodule that restrains proliferation was lower. Also, higher expression of the meristem maintenance factor WUSCHEL-RELATED HOMEOBOX5 correlated with the undifferentiated state of AhHK1-RNAi nodules. Our results suggest that AhHK1-mediated cytokinin signaling is important for both inception and differentiation during nodule development in A. hypogaea.

Download Full-text

A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102845 ◽

1988 ◽

Vol 46 ◽

pp. 152-153 ◽

Cited By ~ 1

Author(s):

A. Engel ◽

A. Holzenburg ◽

K. Stauffer ◽

J. Rosenbusch ◽

U. Aebi

Keyword(s):

Membrane Proteins ◽

Flow Rate ◽

Lipid Membranes ◽

Functional Characterization ◽

Dimensional Structure ◽

Electron Crystallography ◽

Peltier Element ◽

Protein Ratio ◽

Precise Control ◽

3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

Download Full-text

The Relationship between AGAMOUS and Cytokinin Signaling in the Establishment of Carpeloid Features

Plants ◽

10.3390/plants10050827 ◽

2021 ◽

Vol 10 (5) ◽

pp. 827

Author(s):

Andrea Gómez-Felipe ◽

Daniel Kierzkowski ◽

Stefan de Folter

Keyword(s):

Regulatory Network ◽

Dependent Manner ◽

Mads Box ◽

Type B ◽

Cytokinin Signaling ◽

Reproductive Structures ◽

Carpel Development ◽

Gene Regulatory ◽

The Relationship ◽

Gynoecium Development

Gynoecium development is dependent on gene regulation and hormonal pathway interactions. The phytohormones auxin and cytokinin are involved in many developmental programs, where cytokinin is normally important for cell division and meristem activity, while auxin induces cell differentiation and organ initiation in the shoot. The MADS-box transcription factor AGAMOUS (AG) is important for the development of the reproductive structures of the flower. Here, we focus on the relationship between AG and cytokinin in Arabidopsis thaliana, and use the weak ag-12 and the strong ag-1 allele. We found that cytokinin induces carpeloid features in an AG-dependent manner and the expression of the transcription factors CRC, SHP2, and SPT that are involved in carpel development. AG is important for gynoecium development, and contributes to regulating, or else directly regulates CRC, SHP2, and SPT. All four genes respond to either reduced or induced cytokinin signaling and have the potential to be regulated by cytokinin via the type-B ARR proteins. We generated a model of a gene regulatory network, where cytokinin signaling is mainly upstream and in parallel with AG activity.

Download Full-text

Down-Regulation of Cytokinin Receptor Gene SlHK2 Improves Plant Tolerance to Drought, Heat, and Combined Stresses in Tomato

Plants ◽

10.3390/plants11020154 ◽

2022 ◽

Vol 11 (2) ◽

pp. 154

Author(s):

Naveed Mushtaq ◽

Yong Wang ◽

Junmiao Fan ◽

Yi Li ◽

Jing Ding

Keyword(s):

Abiotic Stress Tolerance ◽

Receptor Gene ◽

Antioxidant Enzyme Activities ◽

Plant Responses ◽

Plant Tolerance ◽

Combined Stress ◽

Cytokinin Signaling ◽

Down Regulation ◽

Cytokinin Receptor ◽

Combined Stresses

Environmental stresses negatively affect the growth and development of plants. Several previous studies have elucidated the response mechanisms of plants to drought and heat applied separately; however, these two abiotic stresses often coincide in environmental conditions. The global climate change pattern has projected that combined drought and heat stresses will tend to increase in the near future. In this study, we down-regulated the expression of a cytokinin receptor gene SlHK2 using RNAi and investigated the role of this gene in regulating plant responses to individual drought, heat, and combined stresses (drought + heat) in tomato. Compared to the wild-type (WT), SlHK2 RNAi plants exhibited fewer stress symptoms in response to individual and combined stress treatments. The enhanced abiotic stress tolerance of SlHK2 RNAi plants can be associated with increased membrane stability, osmoprotectant accumulation, and antioxidant enzyme activities. Furthermore, photosynthesis machinery was also protected in SlHK2 RNAi plants. Collectively, our results show that down-regulation of the cytokinin receptor gene SlHK2, and consequently cytokinin signaling, can improve plant tolerance to drought, heat, and combined stress.

Download Full-text

Clinical, Immunological, and Functional Characterization of Six Patients with Very High IgM Levels

Journal of Clinical Medicine ◽

10.3390/jcm9030818 ◽

2020 ◽

Vol 9 (3) ◽

pp. 818 ◽

Cited By ~ 1

Author(s):

Vera Gallo ◽

Emilia Cirillo ◽

Rosaria Prencipe ◽

Alessio Lepore ◽

Luigi Del Vecchio ◽

...

Keyword(s):

B Cell ◽

Somatic Hypermutation ◽

Functional Characterization ◽

Class Switch Recombination ◽

Pathogenic Mutation ◽

Potential Candidate ◽

Class Switch ◽

Potential Candidate Gene ◽

Very High

Very high IgM levels represent the hallmark of hyper IgM (HIGM) syndromes, a group of primary immunodeficiencies (PIDs) characterized by susceptibility to infections and malignancies. Other PIDs not fulfilling the diagnostic criteria for HIGM syndromes can also be characterized by high IgM levels and susceptibility to malignancies. The aim of this study is to characterize clinical phenotype, immune impairment, and pathogenic mechanism in six patients with very high IgM levels in whom classical HIGM syndromes were ruled out. The immunological analysis included extended B-cell immunophenotyping, evaluation of class switch recombination and somatic hypermutation, and next generation sequencing (NGS). Recurrent or severe infections and chronic lung changes at the diagnosis were reported in five out of six and two out of six patients, respectively. Five out of six patients showed signs of lymphoproliferation and four patients developed malignancies. Four patients showed impaired B-cell homeostasis. Class switch recombination was functional in vivo in all patients. NGS revealed, in one case, a pathogenic mutation in PIK3R1. In a second case, the ITPKB gene, implicated in B- and T-cell development, survival, and activity was identified as a potential candidate gene. Independent of the genetic basis, very high IgM levels represent a risk factor for the development of recurrent infections leading to chronic lung changes, lymphoproliferation, and high risk of malignancies.

Download Full-text

Pharmacokinetics, Tissue Distribution, and Excretion Study of Cajanonic Acid A in Rats by UPLC-MS/MS

Planta Medica ◽

10.1055/a-1106-6785 ◽

2020 ◽

Vol 86 (05) ◽

pp. 312-318

Author(s):

Li Zhang ◽

Rui Chen ◽

Yujuan Ban ◽

Jin Cai ◽

Jingang Peng ◽

...

Keyword(s):

Tyrosine Phosphatase ◽

Pigeon Pea ◽

Negative Ion ◽

Biological Behavior ◽

Peroxisome Proliferator ◽

Potential Candidate ◽

Peroxisome Proliferator Activated Receptor ◽

Single Reaction Monitoring ◽

Negative Ion Mode

AbstractCajanonic acid A (CAA), a prenylated stilbene derivative extracted from the leaves of pigeon pea (Cajanus cajan), has been reported to possess inhibitory activity on the peroxisome proliferator-activated receptor gamma (PPARγ) and protein tyrosine phosphatase 1B (PTP1B). Its hypoglycemic activity in rats is comparable to that of the approved antidiabetic agent rosiglitazone. Therefore, CAA is a potential candidate for the treatment of type 2 diabetes and a lead compound for the discovery of novel hypoglycemic drugs. To achieve a thorough understanding of the biological behavior of CAA in vivo, our current study was designed to investigate the pharmacokinetics, bioavailability, distribution, and excretion of CAA in rats by UPLC-MS/MS. Chromatographic separation was performed on BEHC18 column (2.1 mm × 50 mm, 1.7 µm). Quantification was performed under the negative ion mode by using single reaction monitoring (SRM) of the transitions of m/z 353.14 → 309.11 for CAA and m/z 269.86 → 224.11 for genistein, respectively. Standard calibration curve showed excellent linearity (r2 > 0.99) within the range of 2 – 800 ng/mL. The accuracies and precisions were within the acceptance limits (all < 20%). CAA was quickly absorbed into bloodstream and distributed rapidly and widely to various tissues. The excretion ratio of CAA in the 3 main pathways via bile, feces, and urine was only 5.17%. These results indicate that CAA was quickly and thoroughly metabolized in vivo and excreted mainly as metabolites.

Download Full-text

Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18000109 ◽

2018 ◽

Vol 74 (2) ◽

pp. 99-104 ◽

Cited By ~ 1

Author(s):

Santhosh Gatreddi ◽

Sayanna Are ◽

Insaf Ahmed Qureshi

Keyword(s):

Functional Characterization ◽

Three Dimensional ◽

Protozoan Parasite ◽

Crystallographic Analysis ◽

Size Exclusion ◽

Dimensional Structure ◽

Diffusion Method ◽

Gene Encoding ◽

Cell Parameters ◽

Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

Download Full-text

Purification, Characterization, and Gene Sequence of Michiganin A, an Actagardine-Like Lantibiotic Produced by the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis

Applied and Environmental Microbiology ◽

10.1128/aem.00639-06 ◽

2006 ◽

Vol 72 (9) ◽

pp. 5814-5821 ◽

Cited By ~ 28

Author(s):

I. Holtsmark ◽

D. Mantzilas ◽

V. G. H. Eijsink ◽

M. B. Brurberg

Keyword(s):

Amino Acids ◽

Three Dimensional ◽

Reversed Phase ◽

Dimensional Structure ◽

Sequence Information ◽

Type B ◽

Clavibacter Michiganensis ◽

Ring Rot ◽

A Genome ◽

Tomato Pathogen

ABSTRACT Members of the actinomycete genus Clavibacter are known to produce antimicrobial compounds, but so far none of these compounds has been purified and characterized. We have isolated an antimicrobial peptide, michiganin A, from the tomato pathogen Clavibacter michiganensis subsp. michiganensis, using ammonium sulfate precipitation followed by cation-exchange and reversed-phase chromatography steps. Upon chemical derivatization of putative dehydrated amino acids and lanthionine bridges by alkaline ethanethiol, Edman degradation yielded sequence information that proved to be sufficient for cloning of the gene by a genome-walking strategy. The mature unmodified peptide consists of 21 amino acids, SSSGWLCTLTIECGTIICACR. All of the threonine residues undergo dehydration, and three of them interact with cysteines via thioether bonds to form methyllanthionine bridges. Michiganin A resembles actagardine, a type B lantibiotic with a known three-dimensional structure, produced by Actinoplanes liguriae, which is a filamentous actinomycete. The DNA sequence of the gene showed that the michiganin A precursor contains an unusual putative signal peptide with no similarity to well-known secretion signals and only very limited similarity to the (only two) available leader peptides of other type B lantibiotics. Michiganin A inhibits the growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of ring rot of potatoes, with MICs in the low nanomolar range. Thus, michiganin A may have some potential in biological control of potato ring rot.

Download Full-text