Time course of nodule development in the Discaria trinervis (Rhamnaceae) –Frankia symbiosis

The time course of initiation and development of root nodules was investigated in the South American actinorhizal shrub Discaria trinervis (Rhamnaceae). A local strain of Frankia (BCU110501) which was isolated from D. trinervis nodules, was used as inoculum. Inoculated seedlings were periodically studied under the light microscope after clearing with aqueous NaClO. In parallel, semithin and ultrathin sections were analysed by light and electron microscopy. Infection by Frankia BCU110501 involved intercellular penetration among epidermal and cortical root cells. Nodule primordia were detected from 6 d after inoculation, while bacteria were progressing through intercellular spaces of the outer layers of cortical cells. Invasion of host cells by the symbiont occurred 7–9 d after inoculation, and hypertrophy of the primordium cells was associated with Frankia penetration. Root hairs were not deformed during the early events of nodule formation. From 13 to 16 d after inoculation, the proximal cellular zone of the primordia behaved differently from the other tissues after NaClO treatment and remained darkly pigmented. At the same time, differentiation of Frankia vesicles started to occur inside already infected cells. By 16 d after inoculation, spherical vesicles of BCU110501 were homogeneously distributed in the host cells. These vesicles were septate and surrounded by void space. Frankia spores or sporangia were not observed in the nodule tissue. This study has clarified the mode of Frankia penetration in D. trinervis , one of the Rhamnaceae which also includes Ceanothus . The events involved in infection, nodule induction, host-cell infection and vesicle differentiation have been characterized and identified as time-segregated developmental processes in the ontogeny of D. trinervis root nodules.     

Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.     

Isolation of an effective strain of Frankia from nodules of Discaria trinervis (Rhamnaceae)

Frankia strain BCU110501 was isolated from root nodules of the native Patagonian actinorhizal plant Discaria trinervis. The strain was grown on BAP medium with sodium propionate or glucose as carbon sources. Colonies grown in nitrogen-free medium showed branched hyphae bearing polymorphic sporangia and vesicles, which were capable of nitrogen fixation. Old cultures produced a red pigment. The infectivity and effectivity of a Frankia strain isolated from Discaria on its own host, D. trinervis and also in D. chacaye, is reported for the first time. Frankia BCU110501 has physiological properties that are intermediate between categories proposed by Lechevalier et al. (1983) to classify Frankia.     

Regulation of nodulation in the absence of N2 is different in actinorhizal plants with different infection pathways

Root nodulation in actinorhizal plants, like Discaria trinervis and Alnus incana, is subject to feedback regulatory mechanisms that control infection by Frankia and nodule development. Nodule pattern in the root system is controlled by an autoregulatory process that is induced soon after inoculation with Frankia. The final number of nodules, as well as nodule biomass in relation to plant biomass, are both modulated by a second mechanism which seems to be related to the N status of the plant. Mature nodules are, in part, involved in the latter process, since nodule excision from the root system releases the inhibition of infection and nodule development. To study the effect of N(2) fixation in this process, nodulated D. trinervis and A. incana plants were incubated under a N(2)-free atmosphere. Discaria trinervis is an intercellularly infected species while A. incana is infected intracellularly, via root hairs. Both symbioses responded with an increment in nodule biomass, but with different strategies. Discaria trinervis increased the biomass of existing nodules without significant development of new nodules, while in A. incana nodule biomass increased due to the development of nodules from new infections, but also from the release of arrested infections. It appears that in D. trinervis nodules there is an additional source for inhibition of new infections and nodule development that is independent of N(2) fixation and nitrogen assimilation. It is proposed here that the intercellular Frankia filaments commonly present in the D. trinervis nodule apex, is the origin for the autoregulatory signals that sustain the blockage of initiated nodule primordia and prevent new roots from infections. When turning to A. incana plants, it seems likely that this signal is related to the early autoregulation of nodulation in A. incana seedlings and is no longer present in mature nodules. Thus, actinorhizal symbioses belonging to relatively distant phylogenetic groups and displaying different infection pathways, show different feedback regulatory processes that control root nodulation by Frankia.     

Boron requirement in the Discaria trinervis (Rhamnaceae) and Frankia symbiotic relationship. Its essentiality for Frankia BCU110501 growth and nitrogen fixation

The essentiality of boron (B) for nitrogen fixation in heterocystous cyanobacteria and rhizobial symbioses has been widely established. However, nothing is known about the possible involvement of the micronutrient in actinorhizal symbioses. Therefore, the effect of boron (B) deficiency on the establishment of the Discaria trinervis-Frankia BCU110501 symbiosis was investigated. Nodulation was diminished in B-deficient D. trinervis or in plants inoculated with Frankia grown in the absence of B. These poorly nodulated plants showed a reduction of shoot and root weight and small size. Because depletion of the micronutrient during growth of the actinomycete altered the infection capacity of Frankia , we also studied growth, structure and nitrogen fixation of free-living Frankia BCU110501. Growth was delayed in B-deficient BAP media (+N cultures), and completely inhibited in B-deprived N-free BAP media (–N cultures), suggesting that B is required to enhance growth of Frankia and essential for the development of nitrogen fixing activity. Ultrastructural study of B-deficient cells showed an alteration of filament walls both in +N and especially in –N cultures, indicating a possible role of the microelement in the maintenance of these structures. Moreover, the stability of vesicle envelopes was impaired in the absence of B and, hence, nitrogenase occurrence and nitrogen fixation were totally absent. The results show that B is required for both partners to establish an effective symbiosis.     

Local Adaptation of Frankia to Different Discaria (Rhamnaceae) Host Species Growing in Patagonia

Frankia BCU110601 (Da) and Frankia BCU110345 (Dc) were isolated from root nodules of Discaria articulata and Discaria chacaye, respectively; Frankia BCU110501 (Dt) was previously isolated from Discaria trinervis. The strains were identical at the 16S sequence and after analysis of RFLP of 16S and 23S rDNA intergenic region. Diversity was revealed at the molecular level after fingerprint analysis by BOX–polymerase chain reaction. The strains were infective and effective on the original host plants. A cross-inoculation assay intra Discaria genus, including D. trinervis, D. articulata, and D. chacaye, with each of these isolated Frankia strains caused effective symbioses with a similar dry weight in each plant species regardless of the inoculated strain. Nevertheless, a differential degree of recognition was revealed: Homologous symbiotic pairs in the case of D. chacaye–Frankia BCU110345 (Dc), D. articulata–Frankia BCU110601 (Da), and D. trinervis–Frankia BCU110501 (Dt) had faster nodulation rates than heterologous pairs. The differences in nodulation rate would suggest the existence of a subspecific level of recognition within a certain cross-inoculation group, pointing to subspecific adaptation occurring in this actinorhizal symbiosis.     

Infectivity variation of Discaria trinervis-nodulating Frankia in Patagonian soil according to season and storage conditions

Changes in the infectious capacity of soilborne Frankia from the same site may depend on environmental conditions. To test this, we examined the effect of season of sampling, sample storage protocol and storage time. The nodulation capacity of Frankia from rhizospheric soils of Discaria trinervis (Hook et Arn.) Reiche (Rhamnaceae) growing in northwest Patagonia (Argentina) was measured using the most probable number method. Soil samples were collected seasonally and either stored moist at 4°C or air dried at room temperature for few days. Old (air-dried) soil samples were also assayed. All soils nodulated D. trinervis seedlings. Nodulation units (NU) ranged from 44 (spring, moist storage) to about 1 ml⁻¹ of soil (summer moist, and summer and autumn, air-dried storage), with intermediate values in other samples. Soils stored for 12 years, 6 months or 1 week had similar NU. Frankia NU positively correlated with soil water content ( r = 0.6, P < 0.05); therefore, it is likely that soil moisture is a relevant factor regulating soilborne Frankia nodulation ability in Patagonian soils. We suggest that Frankia can remain as spores or grow saprophytically in Patagonian soils.     

Differential sensitivity of nodulation to ethylene in soybean cv. Bragg and a supernodulating mutant

We previously found that the ethylene inhibitor Ag⁺ could overcome the inhibitory effect of nitrate on nodulation of soybean ( Glycine max ) cv. Bragg. The same treatment increased nodulation quantitatively under non-inhibitory conditions, strongly suggesting involvement of ethylene in the control of nodulation in this species. Supernodulation mutants that lack internal autoregulation of nodulation, however, had biosynthesis capacity similar to the wild type. In the present work, the effects of ethylene on nodulation of 'Bragg' and two separate, but allelic, supernodulating mutants ( nts382 and nts1007 ) were compared. The nodulation process appeared much more sensitive than plant growth and development to ethylene, which reduced the number of nodules per plant, but nearly twofold more in the wild type than in the supernodulation mutants. The cause–effect relationship is established by the counteracting effect of Ag⁺ and the fact that the stronger the inhibition by ethylene, the higher the recovery of nodulation ability with the ethylene antagonist. This higher tolerance of or lower sensitivity to ethylene in nts382 persists even under low inoculum dose, where nodule number and mass could be decreased to wild-type levels. Differences between the mutant and the wild type in the triple response test do not appear to support differences in ethylene perception on a whole-plant basis. The results suggest that sensitivity of nodulation to ethylene might have been affected in supernodulation mutants.     

De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard ( Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃. The presence of 20 μ M AgNO₃ or 5 μ M AVG in culture medium containing 5 μ M naphthaleneacetic acid and 10 μ M benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO₃ greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μ M 2-chloroethylphosphonic acid (CEPA), but AgNO_3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO₃ stimulated all 3 parameters of ethylene synthesis. Both AgNO₃ and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO₃. Leaf discs derived from plants grown with AVG or AgNO₃ were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO₃ in the medium was inhibitory to regeneration of those derived from plants grown with AgNO₃.     

Nodule distribution on the roots of actinorhizal Discaria trinervis (Rhamnaceae) growing in pots

Roots of actinorhizal plants can develop nitrogen-fixing nodules with actinomycetic bacteria of the genus Frankia. We aimed to know if unrestricted growth of roots in pots could influence the pattern of nodule development that we had previously observed for Discaria trinervis growing in pouches. Growth pouches, although being a space saving device convenient for the analysis of nodule development, do restrict root growth. Thus, the pattern of root nodule development was analysed in actinorhizal D. trinervis growing in pots with inert substrates. Inoculation of axenic seedlings growing in perlite resulted in clustering of nodules in a defined region of the taproot and upper lateral roots. When surface sterilized seeds were sown in pots containing vermiculite that had been previously inoculated with Frankia cells, nodules were again concentrated in defined portions of the main and lateral roots. Potted plants developed comparable numbers of nodules with respect to plants grown in pouches. However, a significant proportion of nodules appeared in lateral roots. As it was first inferred from field grown plants, these results confirm that D. trinervis plants growing in pots display the same autoregulatory mechanism for nodule formation that was previously observed in growth pouches.     

Water-retentive polymers increase nodulation of actinorhizal plants inoculated with Frankia

Actinorhizal plants form a nodular, nitrogen-fixing root symbiosis with the actinomycete Frankia and are economically and ecologically important due to their ability to improve the nitrogen fertility of disturbed and infertile substrates. In this study, water-retentive polymer inoculum carriers were applied as a root dip. This treatment significantly increased nodulation and in some cases early growth of Alnus glutinosa (L.) Gaertn. and Casuarina equisetifolia var. equisetifolia Forst. & Forst. in a controlled environment and also of A. glutinosa under field conditions. Nodule number and nodule dry weight per plant were at least two to three times greater after 56 to 140 days for plants inoculated with Frankia carried in a water-retentive polymer base compared with plants inoculated with Frankia in water. Nodules on the roots of the plants that were inoculated with Frankia in a polymer slurry were distributed throughout the entire root system, rather than concentrated near the root collar. When amended with water-retentive polymers, actinorhizal plants inoculated with 5- to 10-fold lower titers of Frankia exhibited early growth and nodule numbers equal to or greater than those plants inoculated with standard titers without polymers. The water-retentive, superabsorbent polymers clearly increased the nodulation of two actinorhizal plant species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell biological changes of outer cortical root cells in early determinate nodulation.

In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex. This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant. We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants. Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides [LCOs]) on this phenomenon, and sensitivity of the nodulation process to ethylene. Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L. japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L. japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylene-biosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L. japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L. japonicus.     

Carbon monoxide promotes root hair development in tomato

This study identified the role of CO in regulating the tomato root hair development. Exogenous CO promoted the root hair density and elongation in a concentration-dependent manner. Analysis of cross sections of primary roots also indicated that CO induced the formation of root hairs. Genetic analysis reveals that tomato mutant yg-2 (defective in haem oxygenase-1 activity and intracellular CO generation) displayed a phenotype of delayed root hair development, which however could be reversed by exogenous CO. Further, we analysed LeExt1 :: β -glucuronidase reporter gene for root hair formation and found increasing expression of LeExt1 in the CO-exposed root hairs. Finally, CO was able to act synergistically with auxin, ethylene and NO. It is shown that the effect of CO could be blocked by NPA (auxin transport inhibitor), AVG (ethylene biosynthesis inhibitor), Ag⁺ (ethylene action inhibitor) or cPTIO (NO scavenger). Exposure of tomato roots to CO also enhanced intracellular NO and reactive oxygen species generation in root hairs. Our results suggest that CO would be required for root hair development and may play a critical role in controlling architectural development of plant roots by a putative mechanism of cross-talk with auxin, ethylene and nitric oxide.     

用试管-玻片培养技术研究铵对木麻黄根瘤形成的影响

用植物试管玻片培养技术研究NH_4~ 对细枝木麻黄及Frankia菌株Co01共生体系建立过程的影响。NH_4~ (100,150ppm(NH_4)_2SO_4)通过阻止菌株Cc01与其宿主细枝木麻黄根毛壁的亲和作用来影响结瘤。但NH_4~ 不能阻抑菌株Cc01中结瘤基因pel和cel的表达及纤维素酶和果胶酶活性,且菌丝一旦侵入宿主皮层细胞,并形成根瘤原基及前根瘤,则NH_4~ (250ppm(NH_4)_2SO_4)就不再阻止原基进一步发育为成熟的根瘤。但在这种情况下,NH_4~ 能抑制根瘤的固氮活性。     

Effects of growth regulators on light-dependent anthocyanin production in Zea mays seedlings

Rengel, Z. and Kordan, H. A. 1987. Effects of growth regulators on light-dependent anthocyanin production in Zea mays seedlings.
The effects of ethylene, indolyl- and naphthylacetic acids, zeatin, benzyladenine, gib-berellic acid and triiodobenzoic acid on anthocyanin production in seedlings of Zea mays L. cv. Golden Bantam were investigated. Endogenously produced and exogen-ously supplied ethylene, as well as the other growth regulators tested markedly suppressed anthocyanin formation. Except for triiodobenzoic acid, the other growth regulators stimulated ethylene production, the amounts produced in the light being larger than those in the dark. Absorption of ethylene by permanganate as well as inhibition of ethylene production or action by Co²⁺ or Ag⁺ increased anthocyanin formation in maize seedlings above the level found in the control plants. The inhibiting effect of auxins and cytokinins on anthocyanin production was reversed by Co²⁺ or Ag⁺. In contrast, decreased anthocyanin formation caused by gibberellic acid or triiodobenzoic acid seemed unrelated to ethylene and could not be alleviated by Co²⁺ or Ag+.     

Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [(15)N]NO(3) and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N(2) fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N(2) fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% +/- 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% +/- 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% +/- 6%) than by group IV (81% +/- 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N(2)-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Exogenous Ethylene Inhibits Nodulation of Pisum sativum L. cv Sparkle

Exogenous ethylene inhibited nodulation on the primary and lateral roots of pea, Pisum sativum L. cv Sparkle. Ethylene was more inhibitory to nodule formation than to root growth; nodule number was reduced by half with only 0.07 μL/L ethylene applied continually to the roots for 3 weeks. The inhibition was overcome by treating roots with 1 μm Ag⁺, an inhibitor of ethylene action. Exogenous ethylene also inhibited nodulation on sweet clover (Melilotus alba) and on pea mutants that are hypernodulating or have ineffective nodules. Exogenous ethylene did not decrease the number of infections per centimeter of lateral pea root, but nearly all of the infections were blocked when the infection thread was in the basal epidermal cell or in the outer cortical cells.     

Ethylene: a regulator of root architectural responses to soil phosphorus availability

The involvement of ethylene in root architectural responses to phosphorus availability was investigated in common bean ( Phaseolus vulgaris L.) plants grown with sufficient and deficient phosphorus. Although phosphorus deficiency reduced root mass and lateral root number, main root length was unchanged by phosphorus treatment. This resulted in decreased lateral root density in phosphorus-deficient plants. The possible involvement of ethylene in growth responses to phosphorus deficiency was investigated by inhibiting endogenous ethylene production with amino-ethoxyvinylglycine (AVG) and aerating the root system with various concentrations of ethylene. Phosphorus deficiency doubled the root-to-shoot ratio, an effect which was suppressed by AVG and partially restored by exogenous ethylene. AVG increased lateral root density in phosphorus- deficient plants but reduced it in phosphorus-sufficient plants. These responses could be reversed by exogenous ethylene, suggesting ethylene involvement in the regulation of main root extension and lateral root spacing. Phosphorus-deficient roots produced twice as much ethylene per g dry matter as phosphorus-sufficient roots. Enhanced ethylene production and altered ethylene sensitivity in phosphorus-deficient plants may be responsible for root responses to phosphorus deficiency.     

Characterization of the Lotus japonicus symbiotic mutant lot1 that shows a reduced nodule number and distorted trichomes

We isolated a recessive symbiotic mutant of Lotus japonicus that defines a genetic locus, LOT1 (for low nodulation and trichome distortion). The nodule number per plant of the mutant was about one-fifth of that of the wild type. The lot1 mutant showed a moderate dwarf phenotype and distorted trichomes, but its root hairs showed no apparent differences to those of the wild type. Infection thread formation after inoculation of Mesorhizobium loti was repressed in lot1 compared to that in the wild type. The nodule primordia of lot1 did not result in any aborted nodule-like structure, all nodules becoming mature and exhibiting high nitrogen fixation activity. The mutant was normally colonized by mycorrhizal fungi. lot1 also showed higher sensitivity to nitrate than the wild type. The grown-up seedlings of lot1 were insensitive to any ethylene treatments with regard to nodulation, although the mutant showed normal triple response on germination. It is conceivable that a nodulation-specific ethylene signaling pathway is constitutively activated in the mutant. Grafting experiments with lot1 and wild-type seedlings suggested that the root genotype mainly determines the low nodulation phenotype of the mutant, while the trichome distortion is regulated by the shoot genotype. Grafting of har1-4 shoots to lot1 roots resulted in an intermediate nodule number, i.e. more than that of lot1 and less than that of har1-4. Putative double mutants of lot1 and har1 also showed intermediate nodulation. Thus, it was indicated that LOT1 is involved in a distinct signal transduction pathway independent of HAR1.