The GmFWL1 (FW2‐2‐like) nodulation gene encodes a plasma membrane microdomain‐associated protein

The soybean gene GmFWL1 (FW2‐2‐like1) belongs to a plant‐specific family that includes the tomato FW2‐2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2‐2‐like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain‐associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process.     

Proteomic mapping for legume nodule organogenesis

While genetic screens have identified mutants of the model legume Lotus japonicus that can nodulate in the absence of rhizobia, the lack of a proteome map is a major hindrance to understanding the functional protein networks associated with this nodulation process. In this issue of Proteomics, Dam et al. (Proteomics 2014, 14, 230–240) developed 2D gel‐based reference maps of nodules and roots of Lotus and a spontaneous nodule formation mutant (snf1). Comparative proteomic analysis of roots and two developmental stages of nodules provide useful insights into tissue‐specific mechanisms underlying nodule organogenesis. Additionally, a comparison of interspecies nodule proteomes displays that overlapping and individual mechanisms are associated with legume nodulation.     

Triarabinosylation is required for nodulation‐suppressive CLE peptides to systemically inhibit nodulation in Pisum sativum

Legumes form root nodules to house beneficial nitrogen‐fixing rhizobia bacteria. However, nodulation is resource demanding; hence, legumes evolved a systemic signalling mechanism called autoregulation of nodulation (AON) to control nodule numbers. AON begins with the production of CLE peptides in the root, which are predicted to be glycosylated, transported to the shoot, and perceived. We synthesized variants of nodulation‐suppressing CLE peptides to test their activity using petiole feeding to introduce CLE peptides into the shoot. Hydroxylated, monoarabinosylated, and triarabinosylated variants of soybean GmRIC1a and GmRIC2a were chemically synthesized and fed into recipient Pisum sativum (pea) plants, which were used due to the availability of key AON pathway mutants unavailable in soybean. Triarabinosylated GmRIC1a and GmRIC2a suppressed nodulation of wild‐type pea, whereas no other peptide variant tested had this ability. Suppression also occurred in the supernodulating hydroxyproline O‐arabinosyltransferase mutant, Psnod3, but not in the supernodulating receptor mutants, Pssym29, and to some extent, Pssym28. During our study, bioinformatic resources for pea became available and our analyses identified 40 CLE peptide‐encoding genes, including orthologues of nodulation‐suppressive CLE peptides. Collectively, we demonstrated that soybean nodulation‐suppressive CLE peptides can function interspecifically in the AON pathway of pea and require arabinosylation for their activity.     

Manipulation of two α‐endo‐β‐1,4‐glucanase genes,AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots

Plant endo‐β‐1,4‐glucanases (EGases) include cell wall‐modifying enzymes that are involved in nematode‐induced growth of syncytia (feeding structures) in nematode‐infected roots. EGases in the α‐ and β‐subfamilies contain signal peptides and are secreted, whereas those in the γ‐subfamily have a membrane‐anchoring domain and are not secreted. The Arabidopsis α‐EGase At1g48930, designated as AtCel6, is known to be down‐regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another α‐EGase, AtCel2, is up‐regulated. Here, we report that the ectopic expression of AtCel6 in soybean roots reduces susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduces the susceptibility to SCN. In contrast, in studies on two γ‐EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α‐EGases are likely to be more useful than membrane‐bound γ‐EGases in the development of an SCN‐resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis–H. schachtii interaction as a model for the soybean–H. glycines pathosystem.     

Long‐chain base phosphates modulate pollen tube growth via channel‐mediated influx of calcium

Long‐chain base phosphates (LCBPs) have been correlated with amounts of crucial biological processes ranging from cell proliferation to apoptosis in animals. However, their functions in plants remain largely unknown. Here, we report that LCBPs, sphingosine‐1‐phosphate (S1P) and phytosphingosine‐1‐phosphate (Phyto‐S1P), modulate pollen tube growth in a concentration‐dependent bi‐phasic manner. The pollen tube growth in the stylar transmitting tissue was promoted by SPHK1 overexpression (SPHK1‐OE) but dampened by SPHK1 knockdown (SPHK1‐KD) compared with wild‐type of Arabidopsis; however, there was no detectable effect on in vitro pollen tube growth caused by misexpression of SPHK1. Interestingly, exogenous S1P or Phyto‐S1P applications could increase the pollen tube growth rate in SPHK1‐OE, SPHK1‐KD and wild‐type of Arabidopsis. Calcium ion (Ca²⁺)‐imaging analysis showed that S1P triggered a remarkable increase in cytosolic Ca²⁺ concentration in pollen. Extracellular S1P induced hyperpolarization‐activated Ca²⁺ currents in the pollen plasma membrane, and the Ca²⁺ current activation was mediated by heterotrimeric G proteins. Moreover, the S1P‐induced increase of cytosolic free Ca²⁺ inhibited the influx of potassium ions in pollen tubes. Our findings suggest that LCBPs functions in a signaling cascade that facilitates Ca²⁺ influx and modulates pollen tube growth.     

An (E,E)‐α‐farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect‐induced volatiles

Plant terpene synthase genes (TPSs) have roles in diverse biological processes. Here, we report the functional characterization of one member of the soybean TPS gene family, which was designated GmAFS. Recombinant GmAFS produced in Escherichia coli catalysed the formation of a sesquiterpene (E,E)‐α‐farnesene. GmAFS is closely related to (E,E)‐α‐farnesene synthase gene from apple, both phylogenetically and structurally. GmAFS was further investigated for its biological role in defence against nematodes and insects. Soybean cyst nematode (SCN) is the most important pathogen of soybean. The expression of GmAFS in a SCN‐resistant soybean was significantly induced by SCN infection compared with the control, whereas its expression in a SCN‐susceptible soybean was not changed by SCN infection. Transgenic hairy roots overexpressing GmAFS under the control of the CaMV 35S promoter were generated in an SCN‐susceptible soybean line. The transgenic lines showed significantly higher resistance to SCN, which indicates that GmAFS contributes to the resistance of soybean to SCN. In soybean leaves, the expression of GmAFS was found to be induced by Tetranychus urticae (two‐spotted spider mites). Exogenous application of methyl jasmonate to soybean plants also induced the expression of GmAFS in leaves. Using headspace collection combined with gas chromatography–mass spectrometry analysis, soybean plants that were infested with T. urticae were shown to emit a mixture of volatiles with (E,E)‐α‐farnesene as one of the most abundant constituents. In summary, this study showed that GmAFS has defence roles in both below‐ground and above‐ground organs of soybean against nematodes and insects, respectively.     

When Does the Self-Regulatory Response Elicited in Soybean Root after Inoculation Occur?

The inoculation of soybean (Glycine max L.) roots with Bradyrhizobium japonicum produces a regulatory response that inhibits nodulation in the younger regions of the roots. By exposing the soybean roots to live homologous bacteria for only a short period of time, the question of whether or not early interactions of rhizobia with root cells, prior to infection, elicit this regulatory response has been explored. B. japonicum cells mixed with infective bacteriophages were applied to the roots and then 6 or 24 hours later roots were again inoculated with phage-resistant rhizobia. Mixing of the rhizobia and bacteriophages caused bacterial lysis in 6 to 8 hours and allowed the bacteria to act as live symbionts on the root for only a few hours. However, the interaction of live homologous bacteria with the soybean roots for a few hours did not cause inhibition of nodulation in the younger regions of the roots. Results of these experiments indicate that the self-regulatory response in soybean is not rapidly produced by the early, pre-infection, interactions between rhizobia and the root cells.     

Are common symbiosis genes required for endophytic rice-rhizobial interactions?

Legume plants are able to establish root nodule symbioses with nitrogen-fixing bacteria, called rhizobia. Recent studies revealed that the root nodule symbiosis has co-opted the signaling pathway that mediates the ancestral mycorrhizal symbiosis that occurs in most land plants. Despite being unable to induce nodulation, rhizobia have been shown to be able to infect and colonize the roots of non-legumes such as rice. One fascinating question is whether establishment of such associations requires the common symbiosis (Sym) genes that are essential for infection of plant cells by mycorrhizal fungi and rhizobia in legumes. Here, we demonstrated that the common Sym genes are not required for endophytic colonization of rice roots by nitrogen-fixing rhizobia.     

Identification and functional analysis of the HvD14 gene involved in strigolactone signaling in Hordeum vulgare

In this study, the barley HvD14 gene encoding α/β hydrolase, which is involved in strigolactone (SL) signaling, was identified. Bioinformatics analysis revealed that the identified gene is an orthologue of the D14, AtD14 and PhDAD2 genes that have been described in rice, Arabidopsis thaliana and petunia, respectively. Using TILLING strategy, an hvd14.d mutant that carried the G725A transition, located in the second exon, was identified. This mutation led to the substitution of a highly conserved glycine‐193 to glutamic acid in the conserved fragment of the α/β hydrolase domain of the HvD14 protein. The plants that carry the hvd14.d allele were semi‐dwarf and produced a higher number of tillers in comparison to the wild‐type (WT) parent cultivar. Additionally, the root architecture of mutant plants was affected: the total length of the seminal roots was significantly reduced, and the density of the lateral roots was higher than in the WT. Plants with the hvd14.d allele were insensitive to treatment with GR24, which is the synthetic analogue of SL. Analysis of the indole‐3‐acetic acid (IAA) concentration in the lateral buds showed no differences between the WT and mutant plants. By contrast, the WT seedlings treated with GR24 developed a lower number of tillers, longer primary roots with a reduced number of lateral roots and had an increased concentration of IAA in lateral buds. This paper describes the first barley SL mutant and shows the potential functions of SLs in barley growth and development.     

Neuronal RNA‐binding protein HuD regulates addiction‐related gene expression and behavior

The neuronal RNA‐binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD‐mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuD_OE). When this set was further limited to genes in the knowledgebase of addiction‐related genes database (KARG) that contains predicted HuD‐binding sites in their 3′ untranslated regions (3′UTRs), we found that HuD regulates networks that have been associated with addiction‐like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction‐related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild‐type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuD_OE and wild‐type mice in CPP and found that HuD_OE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction‐related behaviors such as cocaine conditioning and seeking, through increased plasticity‐related gene expression.     

Functional analysis of chimeric lysin motif domain receptors mediating Nod factor‐induced defense signaling in Arabidopsis thaliana and chitin‐induced nodulation signaling in Lotus japonicus

The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1–AtCERK1 and LjNFR5–AtCERK1, were expressed in cerk1‐2, an A. thaliana CERK1 mutant lacking chitin‐induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1‐2 expressing both chimeras accumulated reactive oxygen species, expressed chitin‐responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor‐binding protein), respectively. The chimeras, named OsCERK1–LjNFR1 and OsCEBiP–LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1‐1; nfr5‐2) carrying a GUS (β‐glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF‐induced defense signaling in non‐legumes and chitin‐induced nodulation signaling in legumes.     

Manipulation of Ethylene Synthesis in Roots Through Bacterial ACC Deaminase for Improving Nodulation in Legumes

Symbiotic association between rhizobia and legumes results in the development of unique structures on roots, called nodules. Nodulation is a very complex process involving a variety of genes that control NOD factors (bacterial signaling molecules), which are essential for the establishment, maintenance and regulation of this process and development of root nodules. Ethylene is an established potent plant hormone that is also known for its negative role in nodulation. Ethylene is produced endogenously in all plant tissues, particularly in response to both biotic and abiotic stresses. Exogenous application of ethylene and ethylene-releasing compounds are known to inhibit the formation and functioning of nodules. While inhibitors of ethylene synthesis or its physiological action enhance nodulation in legumes, some rhizobial strains also nodulate the host plant intensively, most likely by lowering endogenous ethylene levels in roots through their 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Co-inoculation with ACC deaminase containing plant growth promoting rhizobacteria plus rhizobia has been shown to further promote nodulation compared to rhizobia alone. Transgenic rhizobia or legume plants with expression of bacterial ACC deaminase could be another viable option to alleviate the negative effects of ethylene on nodulation. Several studies have well documented the role of ethylene and bacterial ACC deaminase in development of nodules on legume roots and will be the primary focus of this critical review.