Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype

Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1-1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1-1 mutant root. No significant differences between har1-1 mutant and wild-type plants were detected with respect to root growth responses to 1-aminocyclopropane1-carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1-1 mutant but not the wild-type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non-symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.     

Induction of localized auxin response during spontaneous nodule development in Lotus japonicus

In leguminous plants, rhizobial infection of the epidermis triggers proliferation of cortical cells to form a nodule primordium. Recent studies have demonstrated that two classic phytohormones, cytokinin and auxin, have important functions in nodulation. The identification of these functions in Lotus japonicus was facilitated by use of the spontaneous nodule formation 2 (snf2) mutation of the putative cytokinin receptor LOTUS HISTIDINE KINASE 1 (LHK1). Analyses using snf2 demonstrated that constitutive activation of cytokinin signaling causes formation of spontaneous nodule-like structures in the absence of rhizobia and that auxin responses are induced in proliferating cortical cells during such spontaneous nodule development. Thus, cytokinin signaling positively regulates the auxin response. In the present study, we further investigated the induction of the auxin response using a gain-of-function mutation of Ca²⁺/calmodulin-dependent protein kinase (CCaMK) that causes spontaneous nodule formation. We demonstrate that CCaMK^T265D-mediated spontaneous nodule development is accompanied by a localized auxin response. Thus, a localized auxin response at the site of an incipient nodule primordium is essential for nodule organogenesis.     

Split-root study of autoregulation of nodulation in the model legume Lotus japonicus

We used a split-root system to determine the timing for induction of the autoregulation of nodulation (AUT) in Lotus japonicus (Regel) Larsen after inoculation with Mesorhizobium loti. The signal took at least five days for full induction of AUT and inhibition of infection thread formation. Strain ML108 (able to nodulate but unable to fix nitrogen) induced full AUT, but ML101 (unable to nodulate or to fix nitrogen) did not induce autoregulation. These results indicate that Nod factor-producing strains induce AUT, but that the nitrogen fixed by rhizobia and supplied to the plant as ammonia does not elicit the AUT in L. japonicus.     

Production of (+)-5-deoxystrigol by Lotus japonicus root culture

Lotus japonicus roots, cultured in a modified B5 medium, produced and secreted germination stimulants that induced Striga hermonthica seed germination. The germination-inducing activity was detected both in the roots and the culture filtrate. Following bioassay-guided purification procedures, an active compound was isolated from hexane extracts of the roots and the culture filtrate. Based on chromatographic behaviour on HPLC, and 1H NMR, UV, MS and CD spectroscopic analyses, the germination stimulant was identified as (+)-5-deoxystrigol.     

Additional cause for reduced JA-Ile in the root of a Lotus japonicus phyB mutant

Light is critical for supplying carbon for use in the energetically expensive process of nitrogen-fixing symbiosis between legumes and rhizobia. We recently showed that root nodule formation in phyB mutants [which have a constitutive shade avoidance syndrome (SAS) phenotype] was suppressed in white light, and that nodulation in wild-type is controlled by sensing the R/FR ratio through jasmonic acid (JA) signaling. We concluded that the cause of reduced root nodule formation in phyB mutants was the inhibition of JA-Ile production in root. Here we show that the shoot JA-Ile level of phyB mutants is higher than that of the wild-type strain MG20, suggesting that translocation of JA-Ile from shoot to root is impeded in the mutant. These results indicate that root nodule formation in phyB mutants is suppressed both by decreased JA-Ile production, caused by reduced JAR1 activity in root, and by reduced JA-Ile translocation from shoot to root.     

百脉根嫁接方法的优化及其在结瘤自调控研究中的应用

嫁接试验是研究结瘤自调控的有效方法。本文优化了百脉根的嫁接方法,并应用该方法初步研究了百脉根突变体rel3根瘤数目减少的机理。结果表明,rel3突变体根瘤数目减少的表型是由来自于茎的信号决定。     

Knockdown of LjALD1, AGD2‐like defense response protein 1, influences plant growth and nodulation in Lotus japonicus

The discovery of the enzyme L,L‐diaminopimelate aminotransferase(LL‐DAP‐AT, EC 2.6.1.83) uncovered a unique step in the L‐lysine biosynthesis pathway in plants. In Arabidopsis thaliana, LL‐DAP‐AT has been shown to play a key role in plant‐pathogen interactions by regulation of the salicylic acid(SA) signaling pathway. Here, a full‐length cDNA of LL‐DAP‐AT named as LjALD1 from Lotus japonicus(Regel)Larsen was isolated. The deduced amino acid sequence shares 67% identity with the Arabidopsis aminotransferase AGD2‐LIKE DEFENSE RESPONSE PROTEIN1(AtALD1) and is predicted to contain the same key elements: a conserved aminotransferase domain and a pyridoxal‐5'‐phosphate cofactor binding site.Quantitative real‐time PCR analysis showed that LjALD1 was expressed in all L. japonicus tissues tested, being strongest in nodules. Expression was induced in roots that had been infected with the symbiotic rhizobium Mesorhizobium loti or treated with SA agonist benzo‐(1, 2, 3)‐thiadiazole‐7‐carbothioic Researchacid. LjALD1 Knockdown exhibited a lower SA content, an increased number of infection threads and nodules, and a slight reduction in nodule size. In addition, compared with wild‐type,root growth was increased and shoot growth was suppressed in LjALD1 RNAi plant lines. These results indicate that LjALD1 may play important roles in plant development and nodulation via SA signaling in L. japonicus.     

Invasion of Lotus japonicus root hairless 1 by Mesorhizobium loti involves the nodulation factor-dependent induction of root hairs

In many legumes, including Lotus japonicus and Medicago truncatula, susceptible root hairs are the primary sites for the initial signal perception and physical contact between the host plant and the compatible nitrogen-fixing bacteria that leads to the initiation of root invasion and nodule organogenesis. However, diverse mechanisms of nodulation have been described in a variety of legume species that do not rely on root hairs. To clarify the significance of root hairs during the L. japonicus-Mesorhizobium loti symbiosis, we have isolated and performed a detailed analysis of four independent L. japonicus root hair developmental mutants. We show that although important for the efficient colonization of roots, the presence of wild-type root hairs is not required for the initiation of nodule primordia (NP) organogenesis and the colonization of the nodule structures. In the genetic background of the L. japonicus root hairless 1 mutant, the nodulation factor-dependent formation of NP provides the structural basis for alternative modes of invasion by M. loti. Surprisingly, one mode of root colonization involves nodulation factor-dependent induction of NP-associated cortical root hairs and epidermal root hairs, which, in turn, support bacterial invasion. In addition, entry of M. loti through cracks at the cortical surface of the NP is described. These novel mechanisms of nodule colonization by M. loti explain the fully functional, albeit significantly delayed, nodulation phenotype of the L. japonicus ROOT HAIRLESS mutant.     

Symbiotic Nitrogen Fixation at the Late Stage of Nodule Formation in Lotus japonicus and Other Legume Plants

Lotus japonicus nodules are reviewed, and current research data on Nod-35 (uricase) gene, including that of Lotus japonicus as an example of a late nodulin gene are presented. Received 5 September 2000/ Accepted in revised form 2 October 2000     

Structure and development of stomata on the primary root of Ceratonia siliqua L

Stomata of various sizes are produced on the primary root of Ceratonia siliqua L. Most are generated during embryogenesis, prior to seed desiccation. They can be detected on the dry embryo in a wide zone just above the root tip. Initially, large stomata are formed. These have the ability to induce divisions of their neighbouring cells, creating particular cell patterns around them. Later, small perigenous stomata are generated. As the root grows following seed germination, the stomatal zone overlaps with that of the root hairs. Although root stomata of C. siliqua undergo a structural differentiation that seems almost identical to that of the elliptical stomata formed on leaves, they are unable to move and remain permanently open. Polarizing microscopy of fully differentiated stomata and young stomata at the stage of stomatal pore formation revealed deposition of radial cellulose microfibril systems on their periclinal walls. However, these systems were less developed than those on leaf stomata, a feature that might be responsible for their inactivity. Besides, plastids of the root guard cells (GCs) do not differentiate into chloroplasts but function solely as amyloplasts. Root stomata have a short life span. During rapid and intense root growth, GCs cannot keep pace with the elongation of their neighbouring rhizodermal cells. They therefore split in their mid-region, transversely to the stoma axis. The two parts of the transversely torn stoma are dragged apart and a large opening is formed on the root surface, just above the substomatal cavity. The root stomata, together with these openings, may facilitate increased gaseous exchange during respiration and/or an increased transfer of some nutrients and water in the rapidly growing primary root.     

Arabinogalactan proteins during the development of soybean root nodules

In soybean (Glycine max (L.) Merr.) root nodules the level of hydroxyproline-containing molecules is developmentally regulated. Hydroxyproline accumulates in both nodule cortex and medulla. In the cortex, the hydroxyproline is mainly localized in the cell wall, presumably as extensin, but in the medulla it is mainly in the soluble fraction as an arabinogalactan protein (AGP). Nodule-specific AGPs are present at early nodulation. The highest concentration of AGP is in the nodule medulla, followed by nodule cortex, uninfected roots, leaves, flowers, pods and seeds. Root nodules and all organs of the soybean plant that were tested were found to express a tissue-specific set of arabinogalactan proteins.Abbreviation AGP Arabinogalactan protein     

Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation

Post-translational hydroxylation of peptide-bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co-substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan-proteins, which are involved in the processes of wall stiffening, signalling and cell proliferation. Allium cepa L. roots treated with 3,4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly affected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascorbate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular “secretory” peroxidase activity (−73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed. Received: 14 October 1998 / Accepted: 31 May 1999     

根系发育的营养调控及对其生境的影响

根系发育对土壤中无机营养的供应和分配的变化非常敏感,本文综述了N、P、K等营养影响根系的发育进程如分枝、根毛产生、直径、生长角度、结瘤作用和簇生根的形成等的机制。营养的供应对根系发育的影响既可以是直接的、是外部营养浓度改变的结果、也可以是间接的、是植物内部营养状况变化的结果。直接途径引起暴露在营养供应中的那部分根系的发育反应;间接途径引起系统的反应,并且似乎依赖于来自冠部的长距离的信使。讨论了最新所了解的内外营养感受(sensing)的机制,长距离信号的可能特征,激素在营养形态发生反应中的作用,根系性状的基因型差异和遗传特性,以及植物根系在生态恢复、防止环境污染、全球气候变化和资源可持续发展中的作用。