Relationship between gibberellin, ethylene and nodulation in Pisum sativum

Authors


Author for correspondence:
James B. Reid
Tel: +61 (03) 6226 2604
Email: jim.reid@utas.edu.au

Summary

  • Gibberellin (GA) deficiency resulting from the na mutation in pea (Pisum sativum) causes a reduction in nodulation. Nodules that do form are aberrant, having poorly developed meristems and a lack of enlarged cells. Studies using additional GA-biosynthesis double mutants indicate that this results from severe GA deficiency of the roots rather than simply dwarf shoot stature.
  • Double mutants isolated from crosses between na and three supernodulating pea mutants exhibit a supernodulation phenotype, but the nodule structures are aberrant. This suggests that severely reduced GA concentrations are not entirely inhibitory to nodule initiation, but that higher GA concentrations are required for proper nodule development.
  • na mutants evolve more than double the amount of ethylene produced by wild-type plants, indicating that low GA concentrations can promote ethylene production. The excess ethylene may contribute to the reduced nodulation of na plants, as application of an ethylene biosynthesis inhibitor increased na nodule numbers. However, these nodules were still aberrant in structure.
  • Constitutive GA signalling mutants also form significantly fewer nodules than wild-type plants. This suggests that there is an optimum degree of GA signalling required for nodule formation and that the GA signal, and not the concentration of bioactive GA per se, is important for nodulation.

Introduction

Legume nodulation is a symbiotic process in which specific soil bacteria called rhizobia invade the roots of legume plants leading to the formation of new organs called nodules (Ferguson et al., 2010). Within the nodules, the rhizobia differentiate and fix atmospheric nitrogen that is transferred to the plant in exchange for carbohydrates. This complex process is orchestrated by a multitude of bacterial and plant signals (Ferguson & Mathesius, 2003). One such signal is the rhizobia-produced Nod factor, a lipochito-oligosaccharide compound that rapidly induces multiple plant signalling cascades required for nodule development (Dénariéet al., 1996; Radutoie et al., 2007). A number of factors have been identified in these signalling cascades, including those that appear to be nodulation-specific and those that have a more general role in plant development, such as the phytohormones (reviewed in Ferguson & Mathesius, 2003; Den Herder & Parniske, 2009; Ding & Oldroyd, 2009; Ferguson et al., 2010).

To help identify the various genes and signalling elements involved in legume nodulation, a number of techniques have been employed. One highly successful method involves generating and selecting mutant plants having altered nodulation phenotypes. Following the isolation of such mutants, various approaches have been implemented to identify the nature of the mutation and the role of its gene product in nodulation. For example, the pea mutant, sym29, which was isolated because it develops an excessive number of nodules (Sagan & Duc, 1996), was later found to have a mutation in a gene encoding for a receptor-like kinase (Krusell et al., 2002). This receptor is thought to have a role in the autoregulation of nodulation (AON) pathway, which is the in-built mechanism that legumes use to control the number of nodules they form (Ferguson et al., 2010). As a result, the characterization of SYM29 helped to establish the molecular mechanism used by legumes to tightly regulate their nodule numbers, supporting the physiological evidence reported previously (Delves et al., 1986).

A novel approach to investigating the signalling elements involved in legume nodulation was later adopted by Ferguson et al. (2005a). These authors investigated the nodulation phenotypes of already well-characterized phytohormone mutants whose genes and gene products had been identified, but whose nodulation phenotypes had not yet been examined. One of their findings suggested that the bioactive form of gibberellin (GA), GA1, is required for normal nodulation in pea. This was demonstrated using a number of different pea lines deficient in GA1 as a result of mutations in genes acting in the GA biosynthesis pathway. This includes ls-1, lh-2 and na-1, all of which formed significantly fewer nodules than their wild-type (Ferguson et al., 2005a). Of these mutant lines, na-1, which has the most severe GA1 deficiency because of a mutation in an ent-kaurenoic acid oxidase that acts early in the GA biosynthetic pathway (Davidson et al., 2003), was found to form the fewest number of nodules (Ferguson et al., 2005a). Interestingly, the few nodules that did form on na-1 plants were morphologically aberrant, being small, white and probably not functional. Both the number and appearance of na-1 nodules could be completely restored to that of its wild-type following the application of a bioactive GA, GA3 (Ferguson et al., 2005a). In addition, grafting the lh-2 mutant to a wild-type rootstock or scion resulted in nodule numbers similar to that of wild-type self grafts (Ferguson et al., 2005a). This finding indicates that normal GA concentrations in either the rootstock or scion are sufficient for nodule development.

A direct role for GAs acting in nodule development was also reported by Lievens et al. (2005). Using the semiaquatic legume, Sesbania rostrata, these authors demonstrated that GAs were involved in the formation of infection pockets and infection threads, two structures required by the rhizobia to colonize the plant. Using in situ hybridization, the authors elegantly demonstrated that SrGA20ox1, a gene encoding the GA 20-oxidase enzyme that acts towards the later stages of GA biosynthesis, functions downstream of Nod factor signalling in the nodulation pathway. GAs were also found to be involved in the induction of cortical cell divisions and nodule primodium formation in S. rostrata (Lievens et al., 2005). Moreover, nodule formation could be prevented in this species following the addition of GA biosynthesis inhibitors.

More recently, Maekawa et al. (2009) reported on their findings using Lotus japonicus and a predominately GA-application approach to study nodulation. Interestingly, whereas Ferguson et al. (2005a) and Lievens et al. (2005) reported a requirement for GAs in legume nodule development, Maekawa et al. (2009) concluded that GAs are inhibitory to nodule formation at an early stage of development. However, using the GA-overproducing pea mutant, sln, and also using GA application studies, Ferguson et al. (2005a) had also reported that, like low GA concentrations, excessive GA concentrations are inhibitory to nodule development. Ferguson et al. (2005a) suggested that an optimum GA concentration is required for effective nodule development and that concentrations significantly above or below this optimum are inhibitory to the process. It is likely that this optimum varies with both the species being investigated and the growing parameters being employed. It is also highly likely that this optimum varies temporally and spatially throughout the nodulation process, as indicated by the SrGA20ox1 in situ hybridization results of Lievens et al. (2005).

Here, we report on our studies using a variety of pea mutants (Table 1) to further investigate the role of GA in legume nodulation. The GA biosynthetic pathway, including the function of the genes of the various GA mutants investigated here, is illustrated in Fig. 1. The histology of na-1 nodules was examined and the nodulation phenotypes of a number of double mutants were identified, including those of newly generated lines possessing mutations in both the GA biosynthetic pathway and the AON pathway. The role of ethylene in the phenotype of the na-1 mutant was also investigated, as this mutant exhibits many traits associated with elevated concentrations of ethylene, such as short, thick roots and internodes and a reduced capacity for nodulation (Drennan & Norton, 1972; Goodlass & Smith, 1979; Lee & LaRue, 1992; Ferguson et al., 2005b). Findings from these studies revealed a potentially intriguing interaction between GA deficiency and ethylene overproduction.

Table 1.   Overview of the various pea (Pisum sativum) lines investigated
GenotypeLine number or crossMutated gene productEffect of mutationPhenotypeReferences
NA1766 × 1769Wild-type
na-11766 × 1769ent-kaurenoic acid oxidaseSeverely reduced bioactive GAsExtreme dwarf with short, thick rootsYaxley et al. (2001), Davidson et al. (2003), Ferguson et al. (2005a)
NA LA CRY187Wild-type
NA la cry-s197DELLA proteinsConstitutive GA responseElongated internodesReid et al. (1983), Potts et al. (1985), Weston et al. (2008)
na-1 la cry-s188ent-kaurenoic acid oxidase; DELLA proteinsSeverely reduced bioactive GAs and constitutive GA responseElongated internodesReid et al. (1983), Potts et al. (1985), Weston et al. (2008)
LH LE LS107Wild-type
lh-1 LE LS511ent-kaurene oxidaseReduced bioactive GAsDwarfReid (1986), Yaxley et al. (2001), Davidson et al. (2004), Ferguson et al. (2005a)
LH le-3 LS5839GA 3-oxidaseBioactive GAs reduced in shoot, normal in rootDwarfIngram et al. (1984), Yaxley et al. (2001), Ferguson et al. (2005a)
lh-1 LE ls-1181 × 511Copalyl diphosphate synthase; ent-kaurene oxidaseBioactive GAs presumably severely reduced in shoot and rootExtreme dwarf with short, thick rootsReid (1986)
lh-1 le-3 LS511 × 5839ent-kaurene oxidase; GA 3-oxidaseBioactive GAs presumably severely reduced in shootsExtreme dwarfDavidson et al. (2005)
Frisson Wild-type
sym28P64UnknownUnable to autoregulate nodulationSuper-nodulationDuc & Messager (1989), Sagan & Duc (1996)
nod3P79UnknownUnable to autoregulate nodulationSuper-nodulationDuc & Messager (1989), Sagan & Duc (1996)
sym29P88Receptor kinaseUnable to autoregulate nodulationSuper-nodulationSagan & Duc (1996), Krusell et al. (2002)
Figure 1.

 The gibberellin (GA) biosynthetic pathway (arrows) and GA signalling (inhibition of DELLA proteins) in pea (Pisum sativum). Grey-shaded boxes show the sites of action of relevant mutants investigated in this report.

Materials and Methods

Plant growing conditions

An overview of the various plant lines used in this report, including their mutated genes, gene products and phenotypes, is provided in Table 1. In addition, the GA biosynthetic pathway, including the function of the mutated genes of the various GA mutants investigated in this report, is illustrated in Fig. 1. Plants were grown as outlined in Ferguson et al. (2005a) unless otherwise stated. For nodulation studies, seeds were sown in 100 mm (500 ml) ‘Space Saver’ pots (Reko, Monbulk, Australia) and for all other studies, seeds were sown in 200 mm (2 l) ‘Plastamatic’ pots (Garden City Plastics, Melbourne, Australia). At the time of sowing, each pot was provided with either 25 ml (nodulation studies) or 150 ml (chemical-treatment studies) of Rhizobium leguminosarum bv. viciae 128C53K (Nitragin® Inoculants; Liphatech Inc., Milwaukee, WI, USA) grown in yeast-mannitol broth and diluted with water to c. OD600 0.01, which represents 5 × 106 cells ml−1.

Creating double-mutant lines

Pollen of nod3, sym28 and sym29 (lines P79, P64 and P88, respectively) was collected and transferred to individual flowers of na-1 plants (line 1766 × 1769). The resulting F1 seed of these three crosses was sown and the subsequent F2 seed was then also collected and sown. Using this F2 generation of these crosses, nod3 na-1, sym28 na-1 and sym29 na-1 putative double mutants were identified based on the combined identification of extremely dwarf shoots and supernodulating phenotypes at 25 d (Table 4). Confirmation of the double-mutant genotypes and phenotypes was made in the F3 to F5 generations.

Identifying nodulation phenotypes

Plants were harvested at 25 d, allowing the nodules to develop to a stage where they could be accurately assessed. Harvested plants were rinsed in water, severed at the cotyledonary node, and the nodules were subsequently assessed, counted and removed. The cotyledons were discarded and the roots, shoots and nodules of each plant were placed in an oven at 60°C for a minimum of 3 d in order to obtain their DW. Double-mutant lines consisting of na-1 and any of the supernodulating mutations, nod3, sym28 or sym29, yielded very few seeds. Owing to this poor yield, seedlings from these F2 plants could not be sacrificed because their seeds were required to perpetuate the genotypes. Instead, the nodulation phenotypes of these lines were determined by gently pulling back the soil and examining their roots. In the F3 and subsequent generations, the root system of the double mutants was fully excavated to verify these observations.

Assessing the role of ethylene in na

The role of ethylene in the phenotype of the na-1 mutant and its wild-type was investigated by treating plants with either 100 ml of water (control), 0.1 mg ml−1 of the ethylene precursor, ACC (Sigma-Aldrich), or 0.03 mg ml−1 of the ethylene biosynthesis inhibitor AVG (i.e. 0.2 mg ml−1 Retaine© containing 15% AVG, Sigma-Aldrich®). Treatments were initially administered 3 d after planting, following which they were repeated twice per week until harvest at 20 d. Upon harvest, the plants were rinsed of soil and their nodules assessed as already described. The shoot length, in addition to the length of the longest secondary and tertiary lateral roots was measured. The total number of secondary lateral roots was recorded, as was the number of tertiary lateral roots located on each of the upper (i.e. closest to the crown) six secondary lateral roots.

Measuring endogenous ethylene concentrations

For ethylene analysis, NA and na-1 plants were grown four per pot under glasshouse conditions until 21 d old. The plants were then placed in sealed 25 l Perspex chambers. After a further 24 h under glasshouse conditions, three replicate 500 μl samples of headspace gas were withdrawn using a gas-tight syringe. As only four chambers were available, two pots of each genotype were analysed on three different days and the values presented in Fig. 4 are average ethylene concentrations obtained from all six replicates. GC-MS was performed as described by Foo et al. (2006). After analysis the plants were removed from the pots and whole-plant FW was determined.

Assessing gene expression levels

Whole shoots and whole-root systems were collected from three pools of six to eight plants of 21-d-old NA and na-1 and frozen in liquid nitrogen. RNA was extracted from c. 100 mg of tissue with RNeasy Mini Kit (Qiagen). The RNA was quantified by spectrophotometry and cDNA was synthesized from 4.5 μg of RNA with Superscript II (Invitrogen) according to the manufacturer’s instructions. cDNA was diluted and duplicate, real-time PCR reactions were performed using Dynamo SYBR Green Master Mix (Geneworks, Hindmarsh, Australia) in a Rotor Gene 2000 (Corbett, San Francisco, USA). Real-time PCR was performed using 100–200 pmol of each primer as described by Foo et al. (2006), and actin was monitored as a control. Relative gene expression levels were calculated as described by Foo et al. (2005).

Histological analysis

Nodules of na-1 and its wild-type, NA, were excised, serially sliced into 3 μm longitudinal sections, stained with toluidine blue, viewed and photographed as outlined in Ferguson & Reid (2005).

Results

Histology of GA-deficient na-1 nodules

The nodule histology of the wild-type, NA plants (Fig. 2) was similar to that reported for other wild-type lines of pea (Bond, 1948; Newcomb et al., 1979; Ferguson & Reid, 2005). The outer cortex of the nodule enclosed the three nodule histological zones – the meristematic, invasion and infected zones – in addition to the nodule peripheral vasculature (Fig. 2). The cells of the infected zone were enlarged, and appear to have been invaded by the bacteria.

Figure 2.

 Nodule histology of NA (wild-type) (a-b) and na-1 (c–g) pea (Pisum sativum) nodules. (b, c) Magnified view of the nodule region exhibiting the transition between the various histological zones of NA (b) and na-1 (c). C, cortex; V, vasculature; M, meristem; IZ, invasion zone; I, infected zone; U, unenlarged cells of the invasion zone. Bars, 200 μm (a, d–g); 50 μm (b, c). Note the scales of (a) and (d–g) are similar, demonstrating the slightly thicker roots and markedly reduced nodule structures of the na-1 mutant.

By contrast, the nodule histology of the GA1-deficient, na-1, was uncharacteristic of pea nodules. The meristem was reduced in size, often not extending across the entire breadth of the nodule, and the vasculature was generally absent (Fig. 2). Moreover, the infected zone was greatly diminished. The cells of this zone were reduced in number and were not enlarged, when compared with those of NA nodules (Fig. 2). As a result, the overall size of na-1 nodules was significantly reduced compared with NA nodules (Fig. 2), despite the roots of the mutant appearing thicker than those of NA (Fig. 2; Yaxley et al., 2001; Ferguson et al., 2005a).

The role of ethylene in the na-1 phenotype

Effects of treatments with an ethylene precursor or inhibitor  Previously, it was demonstrated that GA application could restore the nodule, root and shoot phenotypes of the na-1 mutant to that of its wild-type, NA (Ferguson et al., 2005a). These findings clearly identify a requirement for GAs in the growth and development of this GA-deficient mutant. However, many traits of untreated na-1 plants, such as short, thick roots and internodes are also characteristic of pea plants exposed to high concentrations of ethylene (Goodlass & Smith, 1979; Lee & LaRue, 1992; Ferguson et al., 2005b). Moreover, at elevated concentrations, ethylene is widely recognized as a potent inhibitor of nodule development in species such as pea (Drennan & Norton, 1972; Goodlass & Smith, 1979; Peters & Chris-Estes, 1989; Lee & LaRue, 1992) and has been reported to have roles in regulating legume nodule numbers, nodule positioning on the root, infection thread formation, Nod factor-induced gene expression and calcium spiking (Heidstra et al., 1997; Penmetsa & Cook, 1997; Oldroyd et al., 2001; Sun et al., 2006; Gresshoff et al., 2009). Thus, the role of ethylene in the phenotype of the na-1 mutant was examined.

Application of the ethylene precursor, ACC, significantly reduced the nodule numbers, the tertiary root numbers and the shoot length of wild-type NA plants (Fig. 3; Table 2). This is consistent with previous reports detailing the effects of ethylene on pea (Goodlass & Smith, 1979; Lee & LaRue, 1992; Ferguson et al., 2005b). By contrast, no significant differences were observed in any of the traits investigated on ACC-treated, na-1 plants (Fig. 3; Table 2). Thus, the extreme phenotype of the GA-deficient na-1 mutant was not exaggerated further by the presence of additional ethylene, probably indicating that the mutant is already responding to its elevated ethylene concentrations (to be described further).

Figure 3.

 Wild-type NA (a) and mutant na-1 (b) pea (Pisum sativum) plants treated with (left to right) water (control), aminoethoxyvinyl glycine (AVG, ethylene biosynthesis inhibitor) or 1-amino-cyclopropane 1-carboxylic acid (ACC, ethylene biosynthesis precursor). Excised secondary roots of NA water-treated control plants (c), na-1 water-treated control plants (d) and AVG-treated na-1 plants exhibiting numerous aberrant nodule structures (e). Bars, 5 cm (a, b); 1 cm (c–e).

Table 2.   Nodule and root numbers, and root and shoot lengths and DWs, of 20-d-old NA and na-1 pea (Pisum sativum) lines treated with the ethylene biosynthesis precursor (1-amino-cyclopropane 1-carboxylic acid, ACC) or an inhibitor of ethylene biosynthesis (aminoethoxyvinyl glycine, AVG)
LineTreatmentNumberLength (cm)DW (mg)
NodulesSecondary rootsTertiary rootsShootTaprootSecondary rootTertiary rootRootShoot
  1. Indicated are the number of nodules and secondary lateral roots per plant, the average number of tertiary lateral roots located on the six uppermost secondary lateral roots, the lengths of the shoot and the longest secondary and tertiary lateral roots per plant, and the root and shoot system DWs of NA and na plants treated with either water (control), ACC or AVG.

  2. Plants were inoculated with Rhizobium leguminosarum at the time of sowing. Results are means ± SE (= 6). Values for each trait followed by an * are significantly different from those of their respective control treatment at the 0.001 level.

NAControl116 ± 11.3100 ± 4.320 ± 1.532.8 ± 0.925.3 ± 1.024.2 ± 1.05.9 ± 0.6246 ± 15366 ± 28
ACC29 ± 4.1*92 ± 5.511 ± 0.7*22.5 ± 0.7*26.2 ± 1.820.8 ± 0.44.2 ± 0.2223 ± 19284 ± 13
AVG124 ± 8.2103 ± 0.615 ± 1.233.9 ± 1.027.3 ± 1.728.0 ± 0.54.2 ± 0.4222 ± 6371 ± 14
na-1Control1 ± 0.757 ± 0.86 ± 0.53.5 ± 0.215.4 ± 0.37.1 ± 0.30.6 ± 0.1140 ± 6143 ± 10
ACC0 ± 0.048 ± 2.25 ± 0.53.2 ± 0.114.4 ± 0.98.7 ± 0.51.6 ± 0.2147 ± 6122 ± 6
AVG36 ± 4.7*66 ± 3.05 ± 0.44.0 ± 0.117.8 ± 0.88.0 ± 0.40.7 ± 0.1137 ± 4194 ± 15

Treatment with the ethylene biosynthesis inhibitor, AVG, caused a highly significant, 36-fold increase in the number of nodules that formed on mutant na-1 plants (Table 2). However, the nodules that formed maintained the aberrant morphology that is characteristic of na-1 plants (Fig. 3; Ferguson et al., 2005a). By contrast, the AVG treatment employed only caused a small, insignificant, increase in the nodule numbers of wild-type NA plants (Table 2). Apart from promoting the nodule numbers of na-1, AVG application did not significantly affect any of the other shoot or root characteristics investigated on NA or na-1 plants (Fig. 3; Table 2). Therefore, AVG appears to directly promote nodule formation in na-1 plants, and the reduced capacity of na-1 to form nodules may be the result, in part, of elevated ethylene production.

Endogenous ethylene concentrations  In order to establish if na-1 plants overproduce ethylene, uninoculated wild-type and na-1 plants with six to eight leaves expanded were transferred in their pots to Perspex chambers, sealed and kept under glasshouse conditions for 24 h. Subsequently, a sample of headspace gas was removed and ethylene evolution of the whole plant was determined by GC-MS as described by Foo et al. (2006). Mutant na-1 plants evolved c. two-fold more ethylene than wild-type plants of a similar developmental stage on a FW basis (< 0.05; Fig. 4). In addition to being statistically significant, an increase of this magnitude may be biologically significant, especially if larger differences occur in localized regions of the root. Further, exogenous application of ethylene at concentrations as low as 0.1 μl l−1 has been shown to affect developmental processes in pea, including nodulation where a threshold level for initiation may exist (Lee & LaRue, 1992; Ferguson et al., 2005b). Headspace gas from chambers containing pots filled only with soil were also collected and did not indicate any significant production of ethylene from nonplant sources.

Figure 4.

 Endogenous ethylene level emitted by 21-d-old NA and na-1 pea (Pisum sativum) plants. Values are means ± SE (= 3).

Expression levels of ethylene biosynthetic genes  To identify the molecular mechanism through which GA deficiency may up-regulate ethylene production, the transcript abundances of two genes involved in the biosynthetic pathway of ethylene were assessed. ACC synthase acts to convert the compound S-adenosyl-methionine into ACC, which is then subsequently converted by ACC oxidase into ethylene. In pea, two genes encoding ACC synthases (PsACS1, PsACS2) and one encoding ACC oxidase (PsACO1) have been characterized (Peck et al., 1998). No difference in PsACS2 transcript abundance was detected between wild-type and na-1 plants (data not shown). However, the level of both PsACS1 and PsACO1 was found to be significantly elevated (five- to 10-fold) in the shoots of na-1 compared with those of NA (Fig. 5). This indicates that the reduced GA concentration of na-1 may promote ethylene production by up-regulating the expression of these ethylene biosynthetic genes in the shoot.

Figure 5.

 Relative transcript abundances of the ethylene biosynthesis genes PsACS1 (ACCsynthase 1) (a) and PsACO1 (ACC oxidase 1) (b) in the shoot and root systems of 21-d-old NA (open bars) and na-1 (closed bars) pea (Pisum sativum) plants. Values are means ± SE, (= 3).

By contrast, the transcript abundance of PsACS1 was decreased in the roots of na-1 compared with those of NA, while no significant change in PsACO1 was detected in the roots of na-1 plants (Fig. 5). Although there was no elevated transcript level of these ethylene biosynthesis genes in na-1 roots, the clear promotion of nodule formation in AVG-treated na-1 plants (Fig. 3) indicates that ethylene may have a role in suppressing na-1 nodulation.

Effect of dwarf shoot stature on nodule number

The na-1 mutant develops an extremely dwarf shoot (Davidson et al., 2003). The reduced shoot stature, and therefore reduced surface area, would result in a diminished photosynthetic capacity, which may be responsible for some aspects of the mutant phenotype. However, the AVG-treatment experiments detailed earlier indicate that a pea plant having a severely dwarf shoot is in fact able to initiate the formation of many nodule structures. To further investigate the influence of dwarf shoot on nodule development, the nodulation phenotypes of two additional extreme dwarfs, the lh-1 ls-1 and lh-1 le-3 double mutants, were examined.

Mutations in lh, which encodes ent-kaurene oxidase, and ls, which encodes ent-copalyl diphosphate synthase, result in reduced GA1 concentrations throughout the entire plant (Reid, 1986; Ait-Ali et al., 1997; Yaxley et al., 2001; Davidson et al., 2004). By contrast, mutations in le, which encodes a GA 3-oxidase, reduce GA1 concentrations in the shoot, but not in the root (Ingram et al., 1984; Yaxley et al., 2001). A further GA 3-oxidase gene preferentially expressed in the roots compared with LE is probably responsible for these root/shoot differences (Weston et al., 2008, 2009). Consequently, lh-1 ls-1 double mutants have an additive phenotype resembling that of na-1 plants, resulting from severe GA1 deficiency causing extreme dwarfism in both shoots and roots (Fig. 6; Davidson et al., 2005). lh-1 le-3 double mutants also exhibit dwarf shoots attributable to severe GA1 deficiency in the shoot (Fig. 6). However, root development is much less affected in this mutant combination, presumably as a result of only one of the mutations, lh-1, affecting GA production in the roots. Therefore, by investigating both of these double-mutant genotypes, we were able to examine if GA deficiency limits nodulation indirectly as a result of shoot dwarfism or more directly by limiting GA concentrations in the roots.

Figure 6.

 Root and shoot phenotype of 25-d-old (left to right) le-3 LS LH, LE LS lh-1, LE LH LS (wild-type), lh-1 le-3 LS and LE lh-1 ls-1 pea (Pisum sativum) plants. Bar, 5 cm.

As previously reported (Ferguson et al., 2005a), single-mutant le-3 plants formed a similar number of nodules to wild-type plants (Table 3). Single-mutant plants possessing the lh-1 mutation also formed a similar number of nodules to wild-type plants (Table 3). This is different from previous studies with another allele of lh (lh-2), which exhibited small but significant reductions in nodule numbers (Ferguson et al., 2005a). This discrepancy may be a result of the lh-1 mutation reducing GA concentrations in the roots to a lesser degree than the lh-2 mutation (Davidson et al., 2004). The size, appearance and location on the root system of the nodules of lh-1 and le-3 single mutants were similar to that found in wild-type plants (Table 3; Fig. 6).

Table 3.   Root, shoot and nodule dry weights and nodule numbers per root and shoot dry weight of 32-d-old wild-type and lh-1, le-3, lh-1 le-3 and lh-1 ls-1 mutants
GenotypeDWNumber of nodules
Shoot (mg)Root (mg)Nodule total (mg)Nodule average (mg)Per plantPer mg shoot DWPer mg root DW
  1. Pea (Pisum sativum) plants were inoculated with Rhizobium leguminosarum at the time of sowing. Results are means ± SE (= 6). Values for each mutant trait followed by an * are significantly different from that of the wild-type at the 0.05 level.

LH LE LS359 ± 14142 ± 623.3 ± 4.80.31 ± 0.0881 ± 40.23 ± 0.020.58 ± 0.04
lh-1 LE LS238 ± 14*130 ± 819.4 ± 2.20.23 ± 0.05102 ± 170.44 ± 0.080.82 ± 0.17
LH le-3 LS252 ± 18*117 ± 9*30.0 ± 4.10.33 ± 0.0491 ± 100.36 ± 0.03*0.78 ± 0.06*
lh-1 le-3 LS118 ± 7*69 ± 6*7.8 ± 1.9*0.17 ± 0.0345 ± 6*0.37 ± 0.04*0.66 ± 0.10
lh-1 LE ls-153 ± 5*57 ± 6*0.0*0*0*0*0*

The lh-1 ls-1 double mutant exhibited extremely underdeveloped root and shoot systems that were significantly reduced in DW compared with those of the wild-type (Fig. 6; Table 3). This is consistent with previous studies using this genotype (Reid, 1986; Davidson et al., 2005). These lh-1 ls-1 mutant plants failed to develop any nodules (Table 3).

The lh-1 le-3 double mutant also exhibited a strong dwarf shoot phenotype. However, in contrast to the lh-1 ls-1 mutant, the number of nodules that formed on the lh-1 le-3 mutant was only reduced by 50% compared with that of wild-type plants (Fig. 6; Table 3; Ferguson et al., 2005a). In fact, on both a shoot and root DW basis, lh-1 le-3 mutant plants produced a similar number of nodules to that of wild-type plants. Moreover, lh-1 le-3 double-mutant plants possessed a much longer and more branched root system than lh-1 ls-1 or na-1 plants, although the root size and DW were still reduced somewhat compared with those of the wild-type and the respective single-mutant parents (Figs 3b, 6; Ferguson et al., 2005a). The individual nodule DW of lh-1 le-3 plants was not significantly different from that of its wild-type, or the le-3 and lh-1 single-mutant lines. In addition, lh-1 le-3 nodules were similar in appearance to those of the wild-type (Table 3). This is in contrast to na-1 plants, which only develop very small, aberrant nodules (Fig. 3d; Table 2; Ferguson et al., 2005a), and lh-1 ls-1 plants, which in an independent experiment also appeared to produce a small number of aberrant nodules similar in morphology to na-1 nodules (data not shown). Taken together, this study indicates that although severely GA-deficient dwarf shoots may have a negative impact on nodulation, a dramatic effect only occurs in roots that are strongly GA-deficient.

Nodulation phenotype of GA-deficient, supernodulating lines

Legumes tightly control the number of nodules they form via the AON pathway (Ferguson et al., 2010). Mutants unable to regulate this process form an excessive number of nodules, termed hyper- or super-nodulation. In pea, three separate loci, NOD3, SYM28 and SYM29, have been identified that are involved in AON, and lesions in these genes result in a supernodulation phenotype (Duc & Messager, 1989; Sagan & Duc, 1996; Krusell et al., 2002).

To determine whether several nodule structures could be induced to form on a severely GA-deficient plant, and to investigate the role of GA in AON, double-mutant plants were created by crossing each of nod3, sym28 and sym29 with na-1. Putative double-mutant plants were subsequently isolated in the resulting F2 populations of each of the three crosses by selecting for an extreme-dwarf shoot (na-1) and supernodulating root phenotype (nod++) (Table 4). Confirmation that these plants were double mutants was achieved by growing progenies from supernodulating F2 or F3 plants that were heterozygous at the NA locus (i.e. they exhibited a wild-type shoot length phenotype). The resulting short na plants must be double mutants and their nodule phenotype was the same as the putative double-mutant F2 plants described (Fig. 7). Additional confirmation of the na sym29 double-mutant genotype was achieved by PCR of the sym29 gene (J. L. Weller, unpublished).

Table 4.   Pea (Pisum sativum) shoot and nodulation phenotypes of F2 segregates from crosses between the supernodulating mutants, sym28, sym29 and nod3 and the severely GA-deficient mutant, na-1
CrossShoot genotypeNodulation phenotype
nod+nod++Total
  1. Shoot phenotypes were scored as wild-type (NA) and extreme dwarf (na-1) shoots. Plants that developed a similar number of nodules to wild-type plants were scored as nod+ (NOD3, SYM28 and SYM29), and plants that developed an excessive number of nodules (supernodulating) were scored as nod++ (nod3, sym28 or sym29). Phenotypes were based on comparisons between the F2 segregates, their single-mutant parents and the wild-type lines of these single-mutant parents at 25 d. Identification of double mutants was confirmed in the F3 to F5 generations.

  2. Plants were inoculated with Rhizobium leguminosarum at the time of sowing. Note that plants possessing the na-1 mutation that are not nod++ are considered as nod+, despite the fact that they produced few to no nodule structures.

nod3 × na-1NA69473
na-112315
Total81788
sym28 × na-1NA441054
na-112416
Total561470
sym29 × na-1NA31536
na-110111
Total41647
Figure 7.

 Root phenotype of sym29 (a), na-1 sym29 (b) and na-1 (c) pea (Pisum sativum) mutants 25 d after inoculation with Rhizobium leguminosarum. (d) Close-up of na-1 sym29 lateral roots exhibiting numerous aberrant nodule structures. Bars, 2 cm (a–c); 1 cm (d).

The root and shoot systems of the nod3 na-1, sym28 na-1 and sym29 na-1 double mutants appeared similar in size, thickness and stature to that of their na-1 parent (Fig. 7; data not shown). Owing to their similar appearance, only one genotype (sym29 na-1) is shown in Fig. 7 as a representative of all three nod++ double-mutant lines. Interestingly, each of the double mutants exhibited numerous nodule structures, which is highly uncharacteristic of na-1, but typical of supernodulating lines (Fig. 7d; Table 4; data not shown). However, the individual nodule structures of these double mutants were similar in appearance to the aberrant nodules observed on na-1 mutants (Fig. 7d; Ferguson et al., 2005a). Thus, although a lack of a functional AON pathway did induce the formation of multiple nodule structures on a severely GA-deficient background, these nodules did not develop properly.

Nodulation phenotype of constitutive GA signalling mutants

Recent reports have indicated that elevated GA concentrations caused by exogenous GA application can inhibit nodule development (Ferguson et al., 2005a; Maekawa et al., 2009). To further investigate the effect of elevated GAs in nodule development, we employed a genetic approach that enabled us to avoid exogenously supplying GAs. This was achieved by examining the nodulation phenotypes of la cry-s mutant plants, which exhibit a constitutive GA response because of a lack of DELLA proteins (Reid et al., 1983; Potts et al., 1985; Weston et al., 2008).

As has been previously reported (Reid et al., 1983; Potts et al., 1985), la cry-s plants exhibited an increased shoot length and shoot DW compared with wild-type plants (Fig. 8, Table 5). However, the root system of la cry-s plants was similar to that of wild-type plants (Fig. 8, Table 5). Interestingly, la cry-s plants formed significantly fewer nodules than corresponding wild-type plants on a per-plant and a per-root or per-shoot DW basis (Table 5). Since previous studies have demonstrated that elevated concentrations of GAs can be inhibitory to nodule development (Ferguson et al., 2005a; Maekawa et al., 2009), the reduced nodule numbers of the la cry-s mutants may be a result of their heightened GA-signalling response caused by their lack of DELLA proteins, which mimics elevated GA concentrations.

Figure 8.

 Root and shoot phenotype of 25-d-old (left to right) NA LA CRY (wild-type), NA la cry-s and na-1 la cry-s pea (Pisum sativum) plants. Bar, 5 cm.

Table 5.   Root, shoot and nodule DWs and nodule numbers per root and shoot DW of 25-d-old la cry-s and na-1 la cry-s mutants and their wild-type
GenotypeDWNumber of nodules
Shoot (mg)Root (mg)Nodule total (mg)Nodule average (mg)Per plantPer mg shoot DWPer mg root DW
  1. Pea (Pisum sativum) plants were inoculated with Rhizobium leguminosarum at the time of sowing. Results are means ± SE (= 8). Values for each mutant trait followed by an * are significantly different from those of their respective wild-type at the 0.05 level.

NA LA CRY192 ± 1076 ± 620.0 ± 0.80.16 ± 0.008127 ± 90.66 ± 0.041.73 ± 0.13
NA la cry-s276 ± 19*93 ± 99.1 ± 2.4*0.12 ± 0.02068 ± 12*0.25 ± 0.04*0.76 ± 0.12*
na-1 la cry-s284 ± 13*106 ± 9*8.3 ± 1.4*0.13 ± 0.009*61 ± 9*0.22 ± 0.04*0.63 ± 0.13*

Although la cry-s mutants formed fewer nodules, the appearance and location of their nodules were similar to those of the wild-type. Moreover, the average individual nodule DW of the la cry-s mutants was also similar to that of the wild-type (Table 5), indicating that the nodules of the mutant were not reduced in size.

Interestingly, the addition of the na-1 mutation to a la cry-s background did not affect any of the traits assessed. Indeed, na-1 la cry-s mutants displayed slender shoots and their nodule number and size were similar to those of NA la cry-s plants (Table 5; Fig. 8). This shoot phenotype is consistent with previous characterization studies involving these lines (Reid et al., 1983; Potts et al., 1985). Collectively, these findings indicate that GA signalling via the DELLA proteins, as opposed to the actual level of the hormone itself, influences nodule development.

Discussion

A novel interaction between GA and ethylene

Phytohormones are well-established regulators of plant development. They can work together, in either a synergistic or antagonistic fashion, and may regulate the concentrations or activities of other phytohormones by what is commonly referred to as hormone cross-talk (Reid & Ross, 2003). For example, many reports have indicated that ethylene can act by modulating GA action and/or concentrations, in processes such as phytochrome-mediated stem elongation, root growth and flood tolerance (Rijnders et al., 1997; Archard et al., 2003; Pierik et al., 2004; Vriezen et al., 2004; Foo et al., 2006). However, there do not appear to be any reports illustrating the reverse interaction, that is, a modulation of ethylene production by GA.

We found the GA1-deficient, na-1 mutant produced nearly twice the amount of ethylene emitted by comparable wild-type plants, indicating that GA1 can suppress ethylene production (Fig. 4). This regulation appears to be modulated by increases in the expression of the ethylene biosynthetic genes, PsACS1 and PsACO1, in the shoot (Fig. 5). The fact that na-1 plants failed to respond to ACC application indicates that the mutant is responding to its elevated endogenous concentration of ethylene. This enhancement in the biosynthesis of ethylene in na-1 plants could be a result of stress associated with GA deficiency or may reflect a more direct relationship between GA concentrations and ethylene production.

Ethylene is a strong inhibitor of nodule formation in pea (Drennan & Norton, 1972; Peters & Chris-Estes, 1989; Lee & LaRue, 1992; Gresshoff et al., 2009). Application of the ethylene biosynthesis inhibitor, AVG, significantly increased the number of nodule structures on na-1 plants (up to 36-fold) compared with that observed on untreated na-1 control plants (Fig. 3; Table 2). This finding indicates that the reduced number of nodule structures typically observed on na-1 plants could be the result, in part, of elevated ethylene concentrations. However, because the nodules of AVG-treated na-1 mutants failed to develop normally, the elevated concentration of ethylene does not appear to be the cause of the abnormal na-1 nodule structures. Moreover, AVG application did not rescue other aspects of the na-1 mutant root phenotype. Together with previous studies showing that application of the bioactive GA3 can restore the number and appearance of na-1 mutant nodules to wild-type (Ferguson et al., 2005a), this study suggests a direct requirement for GAs in nodule growth.

Identifying a direct role for GA in nodule development

Recent reports have indicated that GA is required for nodule development (Ferguson et al., 2005a; Lievens et al., 2005). GA deficiency (e.g. in na-1 plants) results in reduced nodule formation, although in many studies it is not clear whether GA directly promotes nodule formation. Indeed, it is often difficult to separate the role of GA deficiency from that of reduced shoot stature, which is a typical characteristic of a plant with diminished GA concentrations.

In pea, shoot dwarfism resulting from reduced shoot GA concentrations alone does not necessarily impair nodule development. Mutant le-3 plants, which are deficient in GA1 in the shoot but not in the root, develop a dwarf shoot but have roots and nodules that are similar in appearance to those of the wild-type (Table 3; Ingram et al., 1984; Yaxley et al., 2001; Ferguson et al., 2005a). In addition, grafting studies have indicated that GA-deficient dwarf lh-2 scions do not suppress nodule development in wild-type roots (Ferguson et al., 2005a).

The use of double mutants provided further insight into the relative importance of shoot stature and GA concentrations in nodule development. The nod3 na-1, sym28 na-1, and sym29 na-1 double mutants exhibited extremely dwarfed shoots, yet each formed numerous nodule structures. Moreover, AVG treatment significantly increased the number of nodules that formed on na-1 plants without significantly affecting the extremely dwarfed shoot stature of the mutant. However, although the nodule number of these double-mutant and AVG-treated na-1 plants increased, the nodule structures maintained the aberrant morphology that is typical of na-1 mutant plants.

In addition, in lh-1 ls-1 and lh-1 le-3 double mutants, the difference in their nodule numbers is likely to be at least partially related to the GA content of their root systems. Both lh-1 le-3 and lh-1 ls-1 exhibit strongly dwarfed shoots as a result of severe GA deficiency in the shoot (Fig. 6, Table 3). However, there are still quite high nodule numbers observed in lh-1 le-3 double mutants, while the lh-1 ls-1 plants formed few to no nodules. This may be a result of the lh-1 ls-1 mutations leading to a much greater impairment in GA synthesis in the roots than the lh-1 le-3 mutant combination because of the expression of other members of the GA 3-oxidase gene family in the latter combinations (Weston et al., 2008). Collectively, these findings confirm that numerous nodule structures can indeed form on pea plants having extremely dwarf, GA-deficient shoots. These results suggest that the nodule number of the plant is directly influenced by root GA signalling and is not simply a reflection of shoot stature.

GA signalling and nodulation

Constitutive GA signalling because of a lack of DELLA proteins caused by the la cry-s mutant combination (Weston et al., 2008) resulted in a reduced number of nodules, although these nodules were normal in appearance (Fig. 8; Table 5). This is consistent with previous studies, where increasing the GA concentration via GA application (Ferguson et al., 2005a; Lievens et al., 2005; Maekawa et al., 2009) or a mutation in a GA-catabolism gene (sln; Ferguson et al., 2005a) also led to reduced nodule numbers. This may be a case of supraoptimal GA concentrations or signalling inhibiting nodule formation directly. Alternatively, enhanced GA signalling brought about by elevated concentrations or perception of GAs may redistribute nutrients and energy required for nodule growth to other regions of the plant, such as stems.

It is interesting to note that la cry-s na-1 triple-mutant plants, which are GA-deficient as a result of the na-1 mutation, developed similar shoot and root system and nodules to those of la cry-s double-mutant plants, which are not GA-deficient (Fig. 8; Table 5). This indicates that severely GA-deficient na-1 plants can develop numerous nodules if GA signalling is impaired by the la cry-s mutations. The fact that constitutive GA signalling allows for the development of nodules in the absence of normal GA concentrations clearly implies that GA signalling, rather than the GA concentration itself, is a requirement of nodule development.

GAs are required for nodule development

Plants with markedly GA-deficient root systems such as na-1 and lh-1 ls-1 show little or no nodulation. However, such plants are capable of initiating many nodules under appropriate genetic or chemical treatment. For example, sym28 na-1, sym29 na-1, nod3 na-1 double mutants and AVG-treated na-1 plants all developed many nodule structures (Figs 2, 7). This indicates that GAs are not essential for the initial stages of nodulation in pea. However, all of these plants also possessed the aberrant nodule phenotype that is characteristic of na-1 plants (Figs 2, 7). This finding suggests that GAs may be required for the late growth, elongation and expansion stages of nodule development (reviewed in Ferguson et al., 2010).

Investigations into the histology of na-1 nodules revealed major disruptions in nodule development (Fig. 2; Ferguson et al., 2005a). The absence of a developed vascular system and the stunted cells of the infected zone in na-1 nodules suggest that GAs are required for their development. The majority of the affected cells are located in a region of the nodule that forms late in nodule development. In addition, the meristems of na-1 nodules appeared poorly developed. Similar disruptions in meristem development have been reported in the nodules of S. rostrata following the application of the GA biosynthesis inhibitor, chlormequat chloride (Lievens et al., 2005), indicating a role for GAs in the establishment and/or persistence of the nodule meristem.

The studies presented here also show that AON does not operate via suppressing GA concentrations to regulate nodule numbers. Had this been the case, the GA-deficient na-1 sym28, na-1 sym29 and na-1 nod3 double mutants would not have exhibited a supernodulating phenotype (Fig. 7; Table 4; data not shown).

Conclusions

This report demonstrates that the GA1 concentration and GA signalling play an important role in nodulation. Studies with both GA-deficient and constitutive GA signalling mutants indicate that a fine balance of GA is required for proper nodule development, as both reduced and elevated GA1 concentrations and/or signalling can inhibit nodule formation. Our findings show that GA can clearly act in the roots themselves to promote nodulation. GA does not appear to act downstream of AON to regulate nodule numbers, although GA1 may influence nodule initiation, at least in part, by suppressing ethylene concentrations. Future studies will focus on identifying the precise steps in the nodulation signalling pathways that are regulated by GAs and ethylene.

Acknowledgements

We thank Teresa Clark and Hobart Pathology for help with sectioning, Ian Cummings, Tracy Winterbottom and Noel Davies for technical assistance and the Australian Research Council for financial support. Additional support was provided to B.J.F. by the Australian Research Council Centre of Excellence for Integrative Legume Research, the Thomas Crawford Memorial Scholarship, and the Tasmanian International Research Scholarship.

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