•In legumes, symbiotic leghemoglobins facilitate oxygen diffusion to the bacteroids, but the roles of nonsymbiotic and truncated hemoglobins are largely unknown. Here the five hemoglobin genes of Lotus japonicus have been functionally characterized to gain insight into their regulatory mechanisms.
•Plants were exposed to nitric oxide donors, stressful conditions, and hormones. Gene expression profiling was determined by quantitative PCR, and gene activities were localized using in situ hybridization and promoter–reporter gene fusions.
•The LjGLB1-1, LjGLB2, and LjGLB3-1 mRNA expression levels were very high in nodules relative to other plant organs. The expression of these genes was localized in the vascular bundles, cortex, and infected tissue. LjGLB1-1 was the only gene induced by nitric oxide. Cytokinins caused nearly complete inactivation of LjGLB2 and LjGLB3-1 in nodules and induction of LjGLB1-1 in roots. Abscisic acid induced LjGLB1-1 in nodules and LjGLB1-2 and LjGLB2 in roots, whereas polyamines and jasmonic acid induced LjGLB1-1 only in roots.
•The enhanced expression of the three types of hemoglobins in nodules, the colocalization of gene activities in nodule and root tissues with high metabolic rates, and their distinct regulatory mechanisms point out complementary roles of hemoglobins and strongly support the hypothesis that LjGLB1-1, LjGLB2, and LjGLB3-1 are required for symbiosis.
Hemoglobins (Hbs) are ubiquitous proteins in archaebacteria, eubacteria, and eukaryotes (Vinogradov et al., 2007). In plants, three types of Hbs have been described: symbiotic, nonsymbiotic, and truncated (see reviews by Wittenberg et al., 2002; Hoy & Hargrove, 2008; Dordas, 2009; Jokipii-Lukkari et al., 2009). Symbiotic Hbs include leghemoglobins (Lbs) and some Hbs of actinorhizal plants, and their function is to transport O2 to the microsymbionts of nodules at a low but constant concentration to permit high respiration rates while avoiding nitrogenase inactivation. Recently, the necessity of Lbs for symbiosis has been demonstrated using RNA interference lines of Lotus japonicus (Ott et al., 2005).
Nonsymbiotic Hbs are widespread in plants, from liverworts to dicots, and are expressed in roots, leaves, stems, seeds, and legume nodules. Nonsymbiotic Hbs are hexacoordinate, having a histidine residue that reversibly binds the sixth coordination site of the heme iron, whereas symbiotic Hbs have a typical pentacoordinate heme pocket in both the ferrous and ferric states (Hoy & Hargrove, 2008). Based on phylogenetic analyses, gene expression patterns, and O2-binding properties, nonsymbiotic Hbs are further classified into class 1 Hbs (GLB1s) and class 2 Hbs (GLB2s). Despite hexacoordination, GLB1s have an extreme affinity for O2, which, along with their low concentration in plants, make them unlikely to function as O2 transporters, stores, or sensors (Dordas et al., 2003; Hoy & Hargrove, 2008). Elegant studies involving overexpression of GLB1 from barley (Hordeum vulgare) and Arabidopsis thaliana (AtGLB1) have shown that these proteins are induced by hypoxic conditions and contribute to the maintenance of the energy status of the cells and to enhance the survival and early growth of the plants (Sowa et al., 1998; Hunt et al., 2002). A general function of GLB1s is to modulate nitric oxide (NO) concentration by binding it to the heme group or to cysteine thiol groups. This role is supported by a number of observations, including the accumulation of NO and nitrosyl (NO-heme) complexes in hypoxic cells (Igamberdiev & Hill, 2004) and in plants where GLB1 has been silenced (Hebelstrup et al., 2006), the capacity of GLB1s to scavenge NO by S-nitrosylation of cysteine residues (Perazzolli et al., 2004), and the strong induction of GLB1 genes by nitrate, nitrite, or NO (Sakamoto et al., 2004; Ohwaki et al., 2005; Sasakura et al., 2006).
Much less is known about GLB2s and truncated Hbs (GLB3s). Although GLB2s are more similar to symbiotic Hbs than to GLB1s in terms of amino acid sequences and O2 binding properties, the GLB2 of A. thaliana (AtGLB2) has an O2 dissociation rate that is too slow for efficient O2 transport (Trevaskis et al., 1997). The AtGLB2 gene is expressed in the rosette leaves and is induced by cold and cytokinins (CKs) but not by hypoxia (Trevaskis et al., 1997; Hunt et al., 2001). The GLB3s are phylogenetically similar to a group of bacterial truncated Hbs but are remarkably different from other plant Hbs. They have a tertiary structure based on a 2-on-2 α-helical sandwich in contrast to the classical 3-on-3 globin fold (Wittenberg et al., 2002; Hoy & Hargrove, 2008). The GLB3 of A. thaliana (AtGLB3) exhibits an unusual concentration-independent binding of O2 and CO, and its expression can be detected throughout the plant and is reduced by hypoxia (Watts et al., 2001). As occurs for GLB2s, the function of GLB3s in plants remains obscure, although it has been recently proposed that they could act as NO scavengers during the onset of actinorhizal (Niemann & Tisa, 2008) or arbuscular mycorrhizal (Vieweg et al., 2005) symbioses.
The availability of partial or complete genomic sequences of model and crop legumes allows a molecular study of the three types of Hbs to be undertaken in detail. However, only a few studies have addressed this issue. In soybean (Glycine max), expression of a GLB3 gene is enhanced in mature nodules as compared with other plant organs (Lee et al., 2004), and, in Medicago truncatula, two GLB3 genes have been found which differ in their expression patterns in nodule tissues (Vieweg et al., 2005). In L. japonicus, two nonsymbiotic Hb genes have been previously identified and named LjHb1 and LjHb2 (Shimoda et al., 2005). Because both encode class 1 Hbs, we have redesignated them as LjGLB1-1 and LjGLB1-2, respectively, to avoid confusion with the LjGLB2 gene identified here. The two LjGLB1 genes are functional in all plant tissues and particularly in mature nodules (Shimoda et al., 2005).
The response of the complete set of nonsymbiotic and truncated Hb genes to different stress conditions or signaling compounds such as NO and hormones has not been examined for any legume. This may serve to gain information on the function of these genes, particularly in nodules. In this work, we address these issues using the model legume L. japonicus by characterizing functionally the five LjGLB genes and by determining the spatial expression patterns in nodule tissues.
Materials and Methods
Plant growth and treatments
Seeds of Lotus japonicus (Regel) Larsen cv MG20 were scarified, surface-disinfected, and sown on 0.5% agar plates. After 1 wk, seedlings were inoculated with Mesorhizobium loti strain R7A and transferred after 2 d to aerated hydroponic cultures without combined nitrogen (1 : 4 strength B&D nutrient solution; Broughton & Dilworth, 1971). Plants were grown under controlled-environment conditions (23 : 18°C, 70%, 350 μmol m−2 s−1, 16 h photoperiod). Roots, leaves, nodules, and stems were harvested from plants at 45 d (late vegetative stage), and flowers and fruits from plants at 60 d (pods of c. 3.5 cm; late flowering-fruiting stage). Plant material to be used for expression analysis of LjGLB genes was immersed directly in liquid nitrogen and stored at −85°C, whereas nodules to be used for in situ RNA hybridization were immediately fixed in formaldehyde (see details later).
To investigate the effects of hormones on gene expression, plants were treated for 48 h with 50 μM abscisic acid (ABA), gibberellic acid (GA), jasmonic acid (JA), indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC; the immediate ethylene precursor), CKs (an equimolar mixture of kinetin and 6-benzyl-aminopurine), or polyamines (PAs; an equimolar mixture of spermine, spermidine, and putrescine). Stock solutions of compounds (all from Sigma-Aldrich) were prepared as follows: 100 mM ABA, ACC, or PAs (in 2 ml of ethanol); 100 mM JA or GA (in 2 ml of dimethylsulfoxide); 500 mM kinetin or 6-benzyl-aminopurine (in 200 μl of 1 M NaOH); and 500 mM IAA (in 400 μl of 1 M NaOH). These volumes were then added to 4 l of the hydroponic solution, which was maintained at pH 6.6 for all treatments. Control plants that had grown simultaneously in hydroponics and had been treated with identical concentrations of ethanol, NaOH, or dimethylsulfoxide at the same time-points, were used to correct gene expression values of the hormone treatments.
Several treatments known to up-regulate LjGLB1 genes in roots (Shimoda et al., 2005) were also included in this study to obtain the expression profiles of all the LjGLB genes. To facilitate comparison with this previous study, treatments were applied to roots of nonnodulated seedlings that had been grown for 15 d in 1.5% agar plates (eight seedlings in each square 10 × 10 cm plate) with modified Fahraeus medium containing 0.5 mM NH4NO3 (Boisson-Dernier et al., 2001). Plates were placed vertically under the same controlled-environment conditions described earlier. The nutrient solution was covering only about one-third of the rooting system to avoid anoxia. The plates also contained a filter paper between the agar and the plants to maintain humidity and avoid roots entering the agar.
Sucrose (1%, w/v) was applied for 24 h, whereas the NO-releasing compounds S-nitroso-N-acetyl-DL-penicillamine (SNAP; 500 μM) and sodium nitroprusside (SNP; 250 μM) were applied for 1–24 h. In the latter case, a control treatment with 250 μM of sodium ferricyanide was run in parallel because SNP decomposition produces both NO and ferricyanide during the incubation period (Bethke et al., 2006). Both NO donors were purchased from Sigma-Aldrich. Cold stress was applied by placing the plants at 4°C in a growth chamber. Hypoxic conditions were achieved by spurging plates with N2 for 5 min and then sealing the plates with insulating tape. Both types of stress were applied for 24 h in the dark.
Transformation competent artificial chromosome (TAC) and bacterial artificial chromosome (BAC) genomic libraries of L. japonicus (Table 1) were screened with probes designed using sequence information of TC38673 (LjGLB2), TC39169 (LjGLB3-1), and GO024600 (LjGLB3-2). The nucleotide sequences of the isolated TAC/BAC clones, LjB03I07 (LjGLB2), LjB369G13 (LjGLB3-1), and LjT35N10 (LjGLB3-2), were determined according to the bridging shotgun method (Sato et al., 2008).
Table 1. Exon-intron compositions of Lotus japonicus (LjGLB) and Arabidopsis thaliana (AtGLB) hemoglobin genes
aLengths of exons (E), introns (I), and open reading frames (ORFs) are given in base pairs. Lengths of the three introns are indicated in italic text.
bPredicted lengths and molecular masses of proteins are given in number of amino acid residues and kDa, respectively.
Total RNA was extracted from plant material with the RNAqueous isolation kit (Ambion) and cDNA was synthesized from 1 μg RNA from each sample using DNase-treated RNA with (dT)17 and Moloney murine leukemia virus reverse transcriptase (Promega). Quantitative reverse-transcription (qRT)-PCR analysis was performed with an iCycler iQ instrument using iQ SYBR-Green Supermix reagents (Bio-Rad). Primer sequences (forward and reverse) were as follows: LjGLB1-1 (5′-TCTCACTTCACTTCCATCGCA-3′ and 5′-TCAGTGAAACATGTGCTCCCA-3′), LjGLB1-2 (5′-GGCAGAAAACACAACCACCAT-3′ and 5′-TCACCACCAGAGCTTCTTGCT-3′), LjGLB2 (5′-AAGAAGCCCTGCTGAAAACGT-3′ and 5′-TCCTCGCTCCATTTATCTCCC-3′), LjGLB3-1 (5′-GAGTGGAGTTCTCACATCCAAC-3′ and 5′-GTTTGAAGGCCGAGCTTTTCA-3′), and LjGLB3-2 (5′-GGAACCATTCACGGTACATTCG-3′ and 5′-GCATCTCTCAAACGACACAGCC-3′). The PCR program consisted of an intial denaturation and Taq polymerase activation step of 5 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 60°C. Primer specificity and the absence of contaminating genomic DNA were verified, respectively, by amplicon dissociation curves and by PCR analysis of RNA samples before reverse transcription. The amplification efficiency of primers, calculated by serial dilutions of root, nodule, and leaf cDNAs, was > 80%. Expression levels were normalized using ubiquitin as reference gene and were calculated by the 2−ΔΔCt method (Livak & Schmittgen, 2001). Threshold cycle values were in the range of 15–16 cycles for ubiquitin and 22–29 cycles for the genes of interest. Additional reference genes were included in control experiments to verify that ubiquitin expression was not affected by any of the treatments. These genes were eIF-4A, encoding the eukaryotic initiation factor 4A (Nagata et al., 2008), and PP2A, encoding a subunit of the serine/threonine protein phosphatase 2A (Czechowski et al., 2005). Results were similar with either of the three reference genes.
In situ hybridization
Nodules were fixed in 4% formaldehyde, infiltrated with paraffin (Paraplast X-tra; Sigma-Aldrich), and sectioned (10 μm). Probes were amplified from cDNA of nodules with a PCR program consisting of 95°C for 30 s followed by 35 amplification cycles (95°C for 15 s, 58°C for 30 s, 72°C for 1 min) and an elongation final step of 72°C for 10 min. Primer sequences were as follows (forward and reverse): LjGLB1-1 (5′-TCTTCCAGCTCATGCACTTCC-3′ and 5′-ACTAGCGCTTTGTGAACAGGG-3′), LjGLB1-2 (5′-CAACCCTTTGCTCTCAGTTTCA-3′ and 5′-ACAGCCCCAAAGCAACAATAA-3′), LjGLB2 (5′-AAGCACCTGAGGCAAAGGC-3′ and 5′-CCCCAAGCATTGCTCACTTC-3′), and LjGLB3 (5′-CAAAGCCTGCAGCATAAAGCT-3′ and 5′-GAAATGGTCGATGTCGCCC-3′).
Fragments were cloned into the pGEM-T Easy vectors (Promega), which were linearized by PCR using pUC/M13 primers (Invitrogen) and purified with ‘PCR Clean Up Gel Extraction: NucleoSpin Extract II’ (Macherey-Nagel, Düren, Germany). Linearized fragments were used as template for sense-antisense digoxigenin-labeled riboprobe synthesis using the DIG RNA labeling kit (SP6/T7) (Roche). Paraffin was removed in Histo Clear II (National Diagnostics, Atlanta, GA, USA) and sections were hydrated in a decreasing ethanol series. Sections were digested with 0.13 mg ml−1 Pronase (Roche) at room temperature for 10 min, and the reaction was stopped by adding phosphate-buffered saline. A postfixation treatment was made in 4% formaldehyde in phosphate-buffered saline. Sections were acetylated with acetic anhydride in triethanolamine for 10 min at room temperature to reduce background. Prehybridization was made for 30 min at 50°C with prehybridization buffer (12.5% wash solution (30 mM NaCl, 10 mM Tris-HCl, 100 mM NaH2PO4, 0.5 mM EDTA); 50% formamide; 12.5% of 50% dextran sulfate; 2.5% 50X Denhardt’s in sterile desionized water). Slides were incubated with 250 μl hybridization buffer (12.25% washing solution (30 mM NaCl, 10 mM Tris-HCl, 100 mM Na2HPO4, 0.5 mM EDTA); 49% formamide; 24.5% of 50% dextran sulfate; 2.5% 50X Denhardt’s; 1.25% tRNA 100 mg ml−1; and 2% labeled probe) for 14 h at 50°C. Sections were washed in 50% formamide in saline citrate solution for 1.5 h at 50°C, and digested for 5 min at 37°C with 20 μg ml−1 RNaseA in 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA. Sections were blocked with 0.5% blocking reagent (Roche) in Tris-buffered saline for 15 min at room temperature and further incubated for 2 h with 0.5 μl ml−1 of alkaline phosphatase-conjugated anti-digoxigenin antibody (sheep anti-digoxigenin-AP Fab fragments; Roche). Finally, the signal was visualized after 2.5 h incubation with freshly prepared substrate solution (125 μg ml−1 nitroblue tetrazolium chloride and 188 μg ml−1 5-bromo-4-chloro-3-indolyl phosphate toluidine salt in 78 mM Tris-HCl (pH 9.8), 78 mM NaCl, 38 μM MgCl2). After stopping the reactions, sections were counterstained with Alcian Blue (Fluka, Buchs, Switzerland), mounted with DPX (Fluka), and examined with an inverted compound microscope Leica DMI6000 B (Leica Microsystems, Wetzlar, Germany).
Promoter-reporter gene fusion constructs and plant transformation
To localize the activities of the LjGLB2, LjGLB3-1, and LjGLB3-2 promoters, the 1430 bp, 1761 bp, and 1955 bp upstream of the ATG start codons, respectively, were amplified by PCR using the corresponding genomic clones (Table 1) as templates. Primer sequences were as follows (forward and reverse): LjGLB2 (for 5′-ctgcagTCGAAAATCATATTATTGCGC-3′, PstI site in lowercase, and 5′-ccatggTGTTCCTTTTTTGTTTTCCTT-3′, NcoI site in lowercase); LjGLB3-1 (5′-ctgcAGGTTAAGTCAAGTGGTAAG-3′, PstI site in lowercase, and 5′-ccatggCTTTGAAGTTTGTTTTGC-3′, NcoI site in lowercase); and LjGLB3-2 (5′-ctgcagTCGTGATGCACAAACCTTC-3′, PstI site in lowercase, and 5′-ccatggCTCAAACGACACAGCCTTTG-3′, NcoI site in lowercase). The PCR products were cloned into pGEM-T Easy (Promega) and sequenced to verify that unintended changes had not been introduced. The cloned promoters were excised from pGEM with PstI and NcoI and subcloned into the same sites of the binary vector pGFPGUS+ (Vickers et al., 2007). However, the LjGLB3-2 clone contains an internal PstI site and therefore only 1386 bp upstream of the start codon was subcloned for this promoter. The resulting pGUS-GLB plasmids do not contain the 35S-GFP cassette present in pGFPGUS+, and the 35S-promoter driving GUS is replaced by the corresponding LjGLB promoter.
Hairy root transformation of L. japonicus ecotype ‘Gifu’ with Agrobacterium rhizogenes LBA1334 carrying the pGUS-GLB3-2 vector was performed as described by Díaz et al. (2005). In brief, 5-d-old seedlings were inoculated 2–3 cm below the cotyledons with A. rhizogenes, the hypocotyls were sectioned at the inoculation site, and the roots were discarded. After transformation, seedlings were left to grow for 10 d to allow hairy root emergence, and then plants with transgenic hairy roots were transferred to pots containing vermiculite that had been washed with B&D nutrient solution. Plants were nodulated by adding a 2-d-old culture of M. loti R7A and were grown as described earlier with B&D solution supplemented with 0.25 mM ammonium nitrate. This concentration of combined nitrogen was found to stimulate nodulation in plants grown in pots. At 32 d after plant inoculation with M. loti R7A, nodules from transgenic hairy roots were stained with GUS solution containing 1 mg l−1 X-Gluc (Jefferson et al., 1987). After 20 min of vacuum infiltration, samples were incubated at 37°C in the dark for up to 48 h. After several washes, nodules were embedded in 5% agar, sectioned (80 μm) using a vibrating blade microtome (Leica VT1000 S), and visualized with inverted (Leica DM IL LED) and stereo (Leica M165 FC) microscopes. Nodules from transformed hairy roots generated with A. rhizogenes LBA1334 cells not carrying the pGUS-GLB vector were used as negative controls.
Nonsymbiotic and truncated Hb genes of L. japonicus
The L. japonicus genome contains three genes encoding typical Lbs that are specifically and strongly expressed in nodules (Uchiumi et al., 2002). Like other plants, however, legumes also contain nonsymbiotic and truncated Hbs, about which much less is known in relation to their eventual roles in various organs, notably root nodules. We therefore characterized all the LjGLB genes and determined their expression profiles. A search in genomic libraries and expressed sequence tag databases of L. japonicus allowed us to isolate genomic and cDNA clones containing partial or complete sequences of five LjGLB genes. Phylogenetic analysis of the deduced protein sequences (Supporting Information, Fig. S1), together with gene structures (Table 1), confirmed that we had isolated representatives of the three Hb classes. The dendrogram evidenced a completely separate clade for GLB3s, confirming that these proteins form a distinct family aside from symbiotic and nonsymbiotic Hbs (Wittenberg et al., 2002), as well as closely related clades for the GLB1s and GLB2s. The dendrogram also shows that Lbs are phylogenetically related to nonsymbiotic GLB2s (Fig. S1), in agreement with the similarity in their biochemical properties (Trevaskis et al., 1997). This also supports the hypothesis that Lbs acquired a specialized function in nodules after duplication and divergent evolution of GLB2 (Andersson et al., 1996).
The two LjGLB1 genes are tandemly arranged on chromosome 3, with the same orientation, and their open reading frames share 86% identity. All these data suggest that LjGLB1-1 and LjGLB1-2 originated from a gene duplication event. In addition, we identified one LjGLB2 gene and two LjGLB3 genes. The amino acid sequences of the LjGLB3-1 and LjGLB3-2 proteins are 82% identical.
The five LjGLB genes contain four exons (Table 1), with three introns at homologous positions to those in other Hb genes from vascular and nonvascular plants (Hunt et al., 2001). Exons 2, 3, and 4 of LjGLB1-1, LjGLB1-2, LjGLB2, and their A. thaliana orthologs are of identical or very similar size, which points out a high degree of conservation (Table 1). Likewise, the LjGLB3 and AtGLB3 genes are also of identical size for the first three exons and are only slightly different for exon 4. This exon organization of the LjGLB3 and AtGLB3 genes and the high sequence identities among all plant GLB3s (60–95%) reflect a high degree of conservation among truncated Hbs. This was evident from the dendrogram, in which GLB3s define a completely separate branch (Fig. S1). The deduced LjGLB3 proteins are eight to 17 amino acid residues longer than the GLB1s or GLB2s of L. japonicus and A. thaliana (Table 1). The larger size of GLB3s appears to be a common feature in plants, as opposed to those of bacterial origin, which are typically 20–40 residues shorter than nonsymbiotic Hbs (Vinogradov et al., 2007; Jokipii-Lukkari et al., 2009).
Expression of LjGLB genes in plant organs
Expression profiling of the five genes was carried out by qRT-PCR in plants grown in hydroponic cultures. We found that LjGLB1-1 and LjGLB2 are expressed in roots and more abundantly in nodules, with low expression levels in leaves, stems, flowers, and fruits (Fig. 1). For this gene expression analysis, mature nodules exhibiting pink color, characteristic of Lb expression, and with a minimal size of c. 2 mm, were chosen. The LjGLB1-2 gene is expressed at substantial levels in all plant organs examined, although its transcripts were particularly abundant in leaves and nodules. Expression of LjGLB3-1 was also very high in nodules, with low to undetectable levels in roots and other plant organs, whereas LjGLB3-2 is expressed throughout the plant at similar levels (Fig. 1). In fact, the expression level in nodules of LjGLB3-1 was 20-fold higher than that of LjGLB3-2 (data not shown), consistent with the high expression of the GLB3-1 gene in M. truncatula nodules (Vieweg et al., 2005). These results agree with the observation that nonsymbiotic and truncated Hbs are widely expressed in tissues of A. thaliana (Trevaskis et al., 1997; Watts et al., 2001), soybean (Andersson et al., 1996; Lee et al., 2004), and monocots (Ross et al., 2004).
Notably, one gene representative of each Hb class was highly expressed in nodules. Thus, the mRNA levels of LjGLB1-1, LjGLB2, and LjGLB3-1 were, respectively, 15.5, 2.2, and 6.6 greater in nodules than in roots, with negligible expression in leaves, stems, flowers, and fruits (Fig. 1). These observations strongly suggest that the three types of Hbs are required for nodule functioning and are located in different cellular compartments and/or perform complementary functions. To investigate this further, experiments were conducted to localize the mRNAs of the five genes in nodules and to assess the effects of NO, stress conditions, and hormones on gene expression in roots and nodules.
Localization of Hb gene expression in nodules
The LjGLB transcripts were localized in mature nodules by in situ hybridization using digoxigenin-labeled probes. The probes for LjGLB1-1 and LjGLB1-2 had only 87 and 81 bp, respectively, because of their high sequence homology, whereas those for LjGLB2 and LjGLB3-2 had 301 bp. The LjGLB1-1 and LjGLB1-2 mRNAs were mainly localized to the infected zone and vascular bundles (Fig. 2a,b,d,e). Besides these two localizations, we could detect significant mRNA levels of LjGLB1-1 and LjGLB1-2 in the nodule cortex. In the case of LjGLB1-2, there was no background signal in the cortex (Fig. 2f) and this transcript localization was very precise (Fig. 2d,e). However, in the case of LjGLB1-1, the control probe produced a weak signal in the cortex (Fig. 2c) and hence we cannot rule out some nonspecific signal in this nodule tissue. Although both transcripts share the same localization in nodules, LjGLB1-2 is expressed in all plant organs and its transcript is particularly abundant in leaves, which suggests distinct functions for the LjGLB1-1 and LjGLB1-2 proteins. The LjGLB2 mRNA was preferentially localized in the cortex, although a less intense signal (even undetectable in certain nodule sections) was observed in the vascular bundles and infected zone of nodules (Fig. 2g,h). Essentially the same localization pattern was observed for the LjGLB3-2 mRNA, except that the signal intensity in the vascular bundle tissue was higher than for the LjGLB2 mRNA (Fig. 2j,k). No background signal was seen for the LjGLB2 and LjGLB3-2 mRNAs using control sense probes (Fig. 2i,l).
To complement our results of in situ hybridization for the lesser known genes, LjGLB2, LjGLB3-1, and LjGLB3-2, we generated promoter-reporter gene fusion constructs and visualized promoter (GUS) activities in transgenic roots and nodules at different developmental stages (Figs 3, 4). The LjGLB2 promoter was predominantly active in the tips and vascular bundles of roots (Fig. 3a) and in the cortex, infected zone (especially at the periphery), and vascular bundles of young nodules (Fig. 3b). As the nodules aged, GUS staining was progressively lost in the cortex and infected region (Fig. 3b,c) and could be seen only in the vascular tissue of mature nodules (Fig. 3d). In roots, a similar staining was observed in the tips and vascular bundles for the activities of the LjGLB3-1 (Fig. 4a) and LjGLB3-2 (Fig. 4e) promoters. In nodules, the LjGLB3-1 promoter activity was visualized in the cortex and infected region (Fig. 4b), but as nodules aged the activity was only detectable in the mid- and inner cortex, vascular bundles, and periphery of infected zone (Fig. 4c,d). In young nodules, the LjGLB3-2 promoter activity was also seen in the mid-cortex and vascular bundles, but very faintly in the infected region and intensely at the nodule base (Fig. 4f). As nodules developed, staining was clearly visible only in the mid-cortex and vascular bundles (Fig. 4g,h).
Expression of LjGLB genes in response to NO and stress treatments
Previous work provided compelling evidence that GLB1 proteins are primarily involved in modulating NO concentrations (Igamberdiev & Hill, 2004). Furthermore, some GLB1 genes in L. japonicus and Alnus firma are responsive to NO, and a role for the proteins in NO detoxification during the symbiotic interaction has been proposed (Shimoda et al., 2005; Sasakura et al., 2006). Hence, we determined the expression profiles of the five LjGLB genes in roots treated with two NO donors using different experimental settings (Fig. 5a). The aim was to compare the effects of SNAP and SNP on expression of all the genes, taking LjGLB1-1 as a positive control. Nonnodulated plants that had been grown for 35 d in hydroponics were treated with 250 μM SNP for 1 and 3 h. Because SNP releases not only NO but also ferricyanide, which may be toxic, control plants were treated with the same concentration of ferricyanide. We found that, for incubation times over 6 h, the effect of ferricyanide itself on gene expression was stronger than that seen with SNP. However, there was no detectable effect of ferricyanide after 3 h and hence up-regulation of the LjGLB1-1 gene could be readily observed (Fig. 5a). Interestingly, SNP had no effect on the other four LjGLB genes. The same result was obtained using seedlings grown in Petri dishes for 15 d and treated for 3, 6, or 24 h with SNAP (Fig. 5a).
Plant Hb genes also respond to stress conditions and to sucrose supply. Thus, AtGLB1 is induced by hypoxia and sucrose, and AtGLB2 by cold stress (Trevaskis et al., 1997). The GLB1 gene of A. firma is also induced by cold stress (Sasakura et al., 2006), whereas LjGLB1-1 is induced by hypoxia and cold stress, and LjGLB1-2 by sucrose (Shimoda et al., 2005). We have examined the effects of hypoxia, low temperatures, and sucrose on the expression of the five LjGLB genes in roots of seedlings grown for 15 d in Petri dishes (Fig. 5b). Treatments were applied for 24 h in the dark to avoid possible interference with photosynthetic O2 or sucrose production. The LjGLB1-1 mRNA level was enhanced by c. 3-fold in response to the three treatments. Expression of the other LjGLB genes remained unaffected, with the exceptions of LjGLB1-2, which was slightly up-regulated in response to sucrose, and LjGLB3-1, which was down-regulated under cold stress (Fig. 5b).
Expression of LjGLB genes in response to hormones
Hormones are involved in multiple functions of plants such as organ morphogenesis and stress responses. Because Hbs may act as stress sensors or metabolic modulators in response to developmental or environmental cues, we chose to study the effects of hormones on Hb expression. Information on this issue is still fragmentary, restricted to a few hormones or genes (Hunt et al., 2001; Shimoda et al., 2005). To gain further insight into the regulation of Hb genes, nodulated L. japonicus plants were treated with various hormones and expression levels were determined in roots (Fig. 6) and nodules (Fig. 7). In roots, the LjGLB1-1 mRNA level increased by c. 20-fold in response to ACC, CKs, and PAs, and by c. 5-7 fold after treatment with IAA and JA (Fig. 6). The expression of LjGLB1-2 slightly increased in response to ABA and decreased with ACC and CKs. The LjGLB2 gene was up-regulated 8-12 fold with GA, ABA, and PAs, and down-regulated with ACC and CKs. The effects of hormones on expression of the two LjGLB3 genes were also distinctly different. Expression of LjGLB3-1 was induced in response to ACC (4-fold) and PAs (7.5-fold), whereas it declined with GA application. In contrast, LjGLB3-2 expression remained unchanged after hormonal treatments, with the exception of a significant down-regulation by CKs (Fig. 6). In nodules, there were no significant changes in LjGLB1-2 and LjGLB3-2 for any treatment, whereas LjGLB1-1 expression increased with ABA (6-fold) and ACC (4.8-fold), and, to a lower extent, with PAs (Fig. 7). Most interestingly, supply of CKs to the plants caused nearly complete suppression of LjGLB2 and LjGLB3-1 transcription in nodules. There was also an evident down-regulation of the LjGLB2 gene in nodules upon treatment with PAs (Fig. 7).
A novel finding of this work is that the three classes of Hb genes are highly expressed and colocalize in certain nodule tissues. The most evident case is the colocalization of LjGLB1-1, LjGLB2, LjGLB3-1, and LjGLB3-2 promoter activities and/or transcripts in the vascular bundle cells. These include phloem companion cells and xylem and phloem parenchyma cells, which exhibit high metabolic activity and contain high concentrations of antioxidant enzymes to protect cellular components from reactive oxygen species generated in mitochondria (Rubio et al., 2009). In the vascular bundle cells, high respiratory rates are needed to sustain active transport of metabolites, and therefore the LjGLB proteins may have functions aimed at ensuring adequate ATP synthesis, as reported for the GLB1 of monocots (Igamberdiev & Hill, 2004), and/or to binding specific ligands or signal molecules that are translocated in the vascular bundles.
Phloem and xylem cells are able to generate NO at high rates (Gabaldón et al., 2005; Gaupels et al., 2008), which supports a role for LjGLB1-1 in NO metabolism in vascular bundles. Our localization of LjGLB1-1 promoter activity and expression in nodules agree, in general, with previous results of Shimoda et al. (2009), who reported that the activity of the LjGLB1-1 promoter is localized to the vascular bundles and infected region of mature nodules and is spatially correlated with NO production. Notably, we found that LjGLB1-1 is the only LjGLB gene that responds to NO, suggesting that only specific GLB1s are involved in NO metabolism. In nodules of L. japonicus, a role related to NO scavenging and/or metabolism could be fulfilled by LjGLB1-1 and Lbs because both types of hemoproteins exhibit high affinity for NO in vitro (Seregélyes et al., 2004; Herold & Puppo, 2005) and are able to form nitrosyl complexes in vivo (Mathieu et al., 1998; Dordas et al., 2004; Meakin et al., 2007). The microaerobic conditions, high rates of NO production, and abundance of Lbs in nodules would also be favorable for the operation of a ‘Hb/NO’ cycle, as proposed for hypoxic roots (Igamberdiev & Hill, 2004). The promoters of LjGLB1-1 and, at least in young nodules, of LjGLB2 and LjGLB3-1 were also active in infected cells, which suggests that these genes are involved in the symbiotic interaction. Moreover, high LjGLB3-1 and LjGLB3-2 promoter activities were observed in the mid- and/or inner cortex of nodules. These cell layers show enhanced respiratory activity and antioxidant defenses (Dalton et al., 1998) and are also the site at which the O2 diffusion barrier is located (Minchin et al., 2008). Collectively, these data show that enhanced expression levels of at least one gene of each LjGLB class are associated with tissues having high metabolic activity and strongly suggest a contribution of truncated Hbs to the operation of this barrier. A comparison of these localization results with a previous study of GLB3 gene activity in nodules of M. truncatula (Vieweg et al., 2005) is pertinent here, given the structural and functional differences between indeterminate and determinate nodules. These authors found that the GLB3-1 and GLB3-2 promoters were predominantly active in the infected cells and in the vascular bundles, respectively. Our localization of LjGLB3-2 activity in the vascular tissue and at the base of young nodules is consistent with those observations, albeit in the case of LjGLB3-1 we could only detect weak staining in the infected zone of young nodules and intense staining in the mid- and inner cortex and in the periphery of the infected zone of mature nodules.
The effects of hypoxia, sucrose, and cold stress on the expression of the five LjGLB genes were also examined. We found that LjGLB1-1 was the only gene responsive to the three treatments. This gene was previously reported to be unresponsive to sucrose addition to the rooting medium (Shimoda et al., 2005), a discrepancy that may be the result of the use by these authors of whole plants rather than roots alone, as performed here. On the other hand, the lack of response of LjGLB2 to cold stress is consistent with the observation that the orthologous A. thaliana gene, AtGLB2, is induced by low temperatures in rosette leaves but not in roots (Trevaskis et al., 1997), indicating a tissue-dependent effect.
Our results on the effects of hormones reveal that there exist distinct regulatory mechanisms on Hb expression for each hormone, gene, and plant organ. Several important points follow. First, the LjGLB genes, even those of the same Hb class, display specific expression profiles in response to hormones. Obvious examples are the contrasting effects of ACC and CKs on LjGLB1-1 (induction) and LjGLB1-2 (repression), or the effects of ACC and PAs on LjGLB3-1 (induction) and LjGLB3-2 (no expression change) in roots. Second, hormones may have opposite effects depending on the plant organ. Thus, there was a strong up-regulation of LjGLB1-1 by CKs and of LjGLB2 and LjGLB3-1 by PAs in roots, but down-regulation or no effect in nodules. The increase in LjGLB1-1 expression by CKs was previously observed, albeit less markedly, in L. japonicus seedlings treated with 10 μM benzylaminopurine for 24 h; however, this CK had no effect on LjGLB1-2 expression in whole plants (Shimoda et al., 2005). Third, PAs and JA caused strong up-regulation of LjGLB1-1 in roots. These effects may be mediated by NO because only LjGLB1-1 was responsive to NO and both types of stress-related compounds elicit an NO burst in A. thaliana (Huang et al., 2004; Tun et al., 2006). However, PAs may also operate by NO-independent mechanisms because they also induced the nonresponsive genes LjGLB2 and LjGLB3-1. Fourth, exogenously applied CKs caused strong down-regulation or complete inactivation of LjGLB2 in roots and nodules, LjGLB3-2 in roots, and LjGLB3-1 in nodules. Consequently, cis-acting elements responsive to CKs may be widespread in GLB promoters rather than restricted to GLB2 genes (Hunt et al., 2001), a hypothesis consistent with the identification of such an element in a rice GLB1 promoter (Ross et al., 2004). Apart from the established roles of CKs in A. thaliana and nonlegume plants in the control of meristem differentiation, these hormones are essential in the symbiotic process of legumes (Frugier et al., 2008). The link identified here between nonsymbiotic GBLs and their enhanced expression in nodule tissues may be related to the recruitement of existing root signaling pathways into nodule organogenesis and function.
We thank Loreto Naya and Christine Lelandais for valuable help with the in situ RNA hybridization technique, Javier Abadía for allowing us to use the vibratome blade microtome, Carmen Pérez-Rontomé for assistance in growing the plants and drawing the figures, and three anonymous reviewers for helpful comments on the manuscript. Pilar Bustos-Sanmamed was the recipient of a predoctoral fellowship from the Ministerio de Ciencia e Innovación (MICINN) and Alejandro Tovar-Méndez was the recipient of a postdoctoral contract (JAE-CSIC program). This work was funded by MICINN-FEDER (AGL2008-01298) and Gobierno de Aragón (group A53).