Identification of Pisum sativum L. cytokinin and auxin metabolic and signaling genes, and an analysis of their role in symbiotic nodule development

The present work was designed to study the role of the phytohormones, cytokinin and auxin, in the symbiosis development between pea Pisum sativum L. and Rhizobium leguminosarum bv. viciae. To achieve this aim, the temporal expression patterns of the genes related to the cytokinin and auxin biosynthesis, perception, and response were studied. It was found that most of these genes showed specific transcriptional regulations upon nodulation at various stages following inoculation. Once they were purified using chromatographic methods, the endogenous cytokinins were detected by liquid chromatography-electrospray tandem mass spectrometry (LC[+]ES–MS). Our findings showed that cytokinin and auxin accumulation is tightly regulated during nodulation in order to control nodule organogenesis, infection events, and nodule function.


INTRODUCTION
The interaction between legume plants and bacteria (collectively referred to as rhizobia) results in the formation of a new organ, the root nodule.The key event in establishing legume-rhizobia symbiosis is the binding of bacterial signal molecules, known as Nod factors (NFs), to specific plant receptors, ultimately triggering a signaling pathway.Eventually, this leads to the development of two programs in the root tissues: an infection process in the epidermis and nodule organogenesis in the cortex.Successful implementation of these processes defines the appearance of nitrogenfixing nodules.
The NF signaling pathway is tightly interconnected with hormone regulation (Ferguson and Mathesius, 2014).Previous experimental data indicate that cytokinin and auxin play essential roles in nodulation.Externally applying cytokinin (Libbenga and Harkes, 1973;Cooper and Long, 1994;Mathesius et al., 2000;Heckmann et al., 2011) or auxin transport inhibitors (Hirsch et al., 1989;Mathesius et al., 1998;Rightmyer and Long, 2011;Ng et al., 2015) to plants resulted in the development of nodulelike structures.Moreover, various experiments aimed at reducing the content of cytokinin in legume plants, such as the overexpression of cytokinin oxidase/dehydrogenase genes (CKX), resulted in a reduction in the number of nodules, which also confirmed the positive role played by cytokinin during nodulation (Lohar et al., 2004;Reid et al., 2016).
The key role of cytokinin in the regulation of nodule formation was confirmed following the identification of mutants defective in the cytokinin receptors, LjLHK1/MtCRE1, which are homologs of Arabidopsis AHK4/CRE1 (Suzuki et al., 2001).In loss-of-function hit1 (hyperinfected1) and cre1 (cytokinin response1) mutants, a significant decrease in the number of nodules was found (Gonzalez-Rizzo et al., 2006;Murray et al., 2006Murray et al., , 2007;;Plet et al., 2011).Moreover, the hit1 mutant was characterized by an increased susceptibility to infection (unlike the Medicago cre1 mutant), although infection threads were blocked in the epidermis; this suggests the cytokinin has a negative effect on the infection process (Murray et al., 2007).Conversely, upon constitutive activation of the cytokinin receptor LjLHK1 in the snf2 mutant, the development of nodule-like structures (spontaneous nodules) was observed in the absence of rhizobia (Tirichine et al., 2007).Similarly, in transgenic M. truncatula plants expressing MtCRE1 with replacement in the CHASE domain (L267F), spontaneous nodules appeared (Ovchinnikova et al., 2011).Moreover, partial functional redundancy in nodule development was recently shown for other cytokinin receptors (Held et al., 2014;Boivin et al., 2016).
Following inoculation with rhizobia in model legumes M. truncatula and L. japonicus, it was found that genes enconding the cytokinin receptor MtCRE1/LjLHK1 and other components of the cytokinin phosphorelay pathway, such as the B-type (MtRR1, MtRR2, and MtRR3) and Atype (MtRR4, MtRR5, MtRR8, MtRR9/LjRR6, and MtRR11/LjRR4) response regulators, demonstrated expression activation (Lohar et al., 2006;Gonzalez-Rizzo et al., 2006;Tirichine et al., 2007;Plet et al., 2011;Op den Camp et al., 2011).According to the proposed involvement of cytokinin in the regulation of nodule organogenesis and infection, localization of the LjLHK1 receptor and primary cytokinin response regulators was associated with the dividing cells of the root cortex and epidermis in L. japonicus (Held et al., 2014).In M. truncatula, the pMtCRE1::GUS construct is upregulated in response to Sinorhizobium meliloti inoculation in the pericycle, endodermis, and inner cortical cells (Lohar et al., 2006), as well as the expression of the A-type response regulator MtRR4 (Plet et al., 2011;Vernié et al., 2015).In addition, cytokinin response regulators are activated in the epidermis of M. truncatula in response to inoculation and NF treatment (op den Camp et al., 2011), which is consistent with the transcriptome analysis data (Larrainzar et al., 2015;Jardinaud et al., 2016).
Rhizobial inoculation or NF treatment was shown to affect local auxin accumulation at the site of nodule initiation by altering auxin transport or biosynthesis activation (Mathesius et al., 1998;Boot et al., 1999;Huo et al., 2006;Takanashi et al., 2011;Plet et al., 2011;Suzaki et al., 2012).However, in the M. truncatula cre1 mutant, rhizobia were not able to locally alter auxin transport during nodule initiation, which resulted in the disturbance of auxin accumulation in the roots when compared with the wild-type plants (Plet et al., 2011).Recent studies have shown that cytokinin signaling regulates specific flavonoids, which act as polar auxin transport inhibitors and influence auxin accumulation in cortical cells during the early stages of nodulation (Ng et al., 2015).Similarly, studying the L. japonicus snf2 mutant, with constitutive activation of the cytokinin receptor, suggests that cytokinin signaling positively regulates auxin accumulation; this is correlated with the increased expression of the LjTAR1 gene, which is involved in auxin biosynthesis (Suzaki et al., 2012).Some cytokinin and auxin metabolic genes have been studied in legume plants.The LjIPT1 and LjIPT3 genes encoding isopentenyl transferases in L. japonicus and their homologs MtIPT1 and MtIPT3 in M. truncatula, as well as the cytokinin riboside 5′-monophosphate phosphoribohydrolases (LONELY GUY) (MtLOG1 and MtLOG2) are upregulated during nodulation (Chen et al., 2014;Mortier et al., 2014;Azarakhch et al., 2015).Moreover, decreasing MtLOG1 or LjIPT3 expression using RNA interference resulted in decreased nodulation (Chen et al., 2014;Mortier et al., 2014).Despite these past findings, the role of the various genes involved in cytokinin and auxin metabolism and signaling during symbiotic nodule development in crop legume pea P. sativum L. was not studied comprehensively, with the exception of individual genes (Azarakhch et al., 2015).Therefore, the objective of this work is to study the role of both hormones in the symbiosis development between crop legume pea P. sativum L. and Rhizobium leguminosarum bv.viciae.With this aim, the temporal expression patterns of several genes related to the cytokinin and auxin biosynthesis, perception, and response of were studied.It was shown that most of these genes showed specific transcriptional regulation upon nodulation at various stages following inoculation.

MATERIALS AND METHODS
Plant, bacterial strains, and growth conditions P. sativum L. cv.Frisson seeds were surface sterilized with sulphuric acid for 5 min, washed 3 times with water, transferred on 1% water agar plates and germinated at room temperature in the dark.After germination, 4 days old seedlings were transferred into pots with vermiculite saturated with Jensen medium (van Brussel et al., 1982), grown in a growth chamber at 21°C at 16 h light/8 h dark cycles, 60% humidity.Pea seedlings were inoculated with 1 ml culture of R. leguminosarum bv.viciae CIAM1026 per plant (OD 600 = 0.5), and infected root tissue was harvested at different stages after inoculation.Fragments of main roots corresponding to the socalled susceptible to infection zone or fragments of main roots with nodules were harvested at 1 to 9 days post inoculation (dpi), while the nodules were collected at 14 to 24 dpi.Fragments of noninoculated roots were collected at the same developmental stages.

Quantitative reverse transcription PCR (qRT-PCR) analysis
Total RNA was isolated from roots and nodules using Trizol (Qiagen, Germany) according to the manufacturer's instructions.After a DNase (Thermo Scientific, USA) treatment, the samples were extracted with equal volume of chloroform, and RNA was precipitated from the aqueous phase with 3 M sodium acetate and ethanol and subsequently quantified with a spectrophotometer (Shimadzu UV-1280, Japan).RNA (from 1 to 2.5 μg) was used for cDNA synthesis with the RevertAid Reverse Transcriptase (Thermo Scientific, USA).The quantitative reverse transcription PCR (qRT-PCR) experiments were done on a CFX-96 real-time PCR detection system with C1000 thermal cycler (Bio-Rad Laboratories, USA), and SYBR Green intercalating dye were used for detection (Bio-Rad Laboratories, USA).All reactions were done in triplicate and averaged.Cycle threshold (CT) values were obtained with the accompanying software and data were analyzed with the 2-ΔΔCt method (Livak and Schmittgen, 2001).The relative expression was normalized against the constitutively expressed ubiquitin and actin genes.All primer pairs (Table 1) were designed using Vector NTI program and produced by Evrogen company (www.evrogen.com).PCR amplification specificity was verified using a dissociation curve (55 to 95°C).Each experiment was repeated at least three times with independent biological samples.

Identification and quantification of endogenous cytokinins
The procedure used for the cytokinin analysis reported herein was a little modified from a method described previously (Dobrev and Kamınek, 2002).To start the experiment, 4-day-old pea seedlings were grown in sterile vermiculite (control) and in vermiculite inoculated with R. leguminosarum bv.viciae.Roots and roots with developing nodules (3 days, 7 days, 10 days, and 14 days post inoculation, dpi) were homogenized in liquid nitrogen and extracted overnight (-20°C) in ice-cold methanol/water/formic acid (15:4:1, v/v/v); 5 ml of extraction mixture were used per 1 g of tissue.
Repeat extraction was performed for 2 h in the same conditions.Following centrifugation, the sediments (extracts) were purified on silica Sep-Pak Plus C18 cartridges (Waters, Milford, MA, USA) and evaporated.Before purification, the samples were dissolved in 3 ml of 1 M formic acid.Cytokinin purification was performed on Oasis MCX columns (30 mg Sorbent) (Waters) (Dobrev and Kamınek, 2002).The cytokinin samples were applied to Oasis MCX column pre-conditioned with 5 ml of methanol followed by 5 ml of 1 M formic acid.The column was washed twice with 1 M formic acid and eluted with 0.35 M NH 4 OH, 0.35 M NH 4 OH in 60% methanol and 0.7 M NH 4 OH in 60% methanol solutions.Solvents were evaporated under vacuum.

Mass-spectrometry
The cytokinins in pea root tissues were analyzed using ultraperformance liquid chromatography-electrospray tandem mass spectrometry (LC+-ES/MS).Mass spectra were recorded on a tandem quadrupole mass spectrometer (Waters XEVO TQD flow type) with a positive electrospray ionization sample (ESI+) following prior chromatographic separation of the sample components on an Acquity UPLC BEH C18 column (100, 2.1, and 1.7 mm) using the Waters Ultra Performance Liquid Chromatography (UPLC) Acquity chromatographic system.The mass spectrometer was operated in positive electrospray ionization mode.Gas flow was set to 10 L/min and gas temperature at 300°C.The capillary voltage was set at 3500 V.The fragmentor and collision energy voltage values were optimized for each standard compound.Multiple reaction monitoring (MRM) was used for quantification.Cytokinins were quantified using standards and based on the ratio of the peak areas of the MRM transition for standards.

Statistical methods and computer software
Multiple alignment of nucleotide sequences was performed using Clustal W (Thompson et al., 1994) using Vector NTI Advance 10 (InforMax, http://www.informaxinc.com).MEGA6 was used to generate graphic output of phylogenetic tree.

Identification and analysis of the temporal dynamics of the cytokinin response gene expression in pea during nodulation
An analysis of the model legume M. truncatula genome showed that a family of A-type RR includes 13 members, 5 of which are expressed in the roots upon rhizobial inoculation: MtRR4 (Medtr5g036480), MtRR5 (Medtr7g490310), MtRR8 (Medtr4g106590), MtRR9 (Medtr3g015490), and MtRR11 (Medtr8g038620) (Op de Camp et al., 2011).Subsequent transcriptome analysis revealed increased expression levels of 6 A-type RR genes in response to Nod factor treatment; these genes were MtRR4, MtRR5, MtRR (Medtr3g078613), MtRR8,
A BLAST search in different pea transcriptome databases (http://blast.ncbi.nlm.nih.gov/ and http://bios.dijon.inra.fr/),using sequences of M. truncatula A-type response regulators, was performed to identify the homologous genes in pea.An in silico analysis allowed the identification of full-sized or partial transcripts for 10 A-type RRs (Table 2).Two of these, PsRR4 and PsRR8, corresponded to the previously characterized pea genes (GenBank accession no.KP296699 and KP296703).Since we are interested in searching for those genes that are upregulated during nodulation in pea, we focused on specific pea homologs.The full-length coding sequences of these A-type RRs were verified by polymerase chain reaction (PCR) amplification of cDNA using the primers flanking coding sequences.In addition, a rapid amplification of cDNA ends (RACE) analysis of partial transcripts enabled the identification of their full-length pea cDNAs.As a result, four new full-sized A-type RRs were identified in pea, including PsRR5 (KP296700), PsRR6 (KP296701), PsRR8 (KP296703), PsRR9 (KP296704), and PsRR11.They showed a high level of homology with the corresponding genes from M. truncatula and L. japonicus (Figure 1).
An analysis of the temporal expression dynamics of six A-type RRs, including the PsRR4, PsRR5, PsRR6, PsRR8, PsRR9, and PsRR11 genes, revealed the activation of five of them during nodulation in pea.It was shown that the PsRR5 and PsRR9 regulators are activated upon symbiosis initiation at 1 to 3 days postinoculation (dpi) (Figure 2).The expression of PsRR4 and PsRR8 increased in the roots inoculated with R.   leguminosarum bv.viciae, starting from 5 to 7 dpi and reaching a maximum at 11 to 14 dpi.The expression level of the PsRR4 gene slightly increased in the inoculated roots when compared with the uninoculated control roots (a 1.5 to 3-fold increase, on average), while the expression of PsRR8 showed a higher expression level (a 3 to 5-fold increase, on average).The expression of PsRR11 was initially upregulated at 1 to 3 dpi and continued to increase until it reached a maximum at 14 to 21 dpi (a more than 80-fold increase, on average, when compared with the uninoculated control roots) (Figure 2).Therefore, the expression of the PsRR5, PsRR9, PsRR8, and PsRR11 genes may be useful markers of cytokinin receptor activation in pea at different stages of nodulation (both during the initiation of this process and in nodule development).

Identification of IPT and LOG genes controlling the biosynthesis and activation of cytokinin in pea
To determine the factors that underlie cytokinin receptor stimulation (including cytokinin biosynthesis, activation, or re-localization), we searched for the genes involved in the control of cytokinin metabolism.Of them, we identified the genes that encode for the cytokinin biosynthesis enzymes (isopentenyl transferases, IPTs), and cytokinin-activating enzymes (cytokinin riboside 5′-monophosphate phosphoribohydrolases; LONELY GUYs, LOGs).In plants, these genes are represented by multigene families, so we searched for IPT and LOG gene expression, which had increased during nodulation.
In addition to two known PsIPT1 and PsIPT2 genes (Tanaka et al., 2006), we previously identified four new PsIPT genes in pea, which were designated as PsIPT3-PsIPT6 (GenBank accession no.KP296693, KP296694, KP296695, and KP296696).The nucleotide sequences of the newly identified genes showed a high level of homology with the IPT genes from M. truncatula and L. japonicus.
The expression dynamics of six PsIPT1-PsIPT6 genes in pea roots upon R. leguminosarum bv.viciae inoculation by qRT-PCR was further analyzed.Following inoculation, PsIPT1 and PsIPT2 expression increased in the pea roots at 1 to 3 dpi when compared with the uninoculated control roots.In addition, the expression of PsIPT4 was significantly increased during nodulation, starting from 5 to 7 dai and showing the highest level at 11 to 14 dpi (on average, representing a 10 to 15-fold increase).Moreover, the expression level of PsIPT3 was also upregulated during nodulation, but not as significantly as was observed for PsIPT4 (Figure 3).The expression levels of two other PsIPT5 and PsIPT6 genes did not change significantly upon nodule development (Figure 3).The activation of PsIPT3 and PsIPT4 is consistent with our previously obtained data (Azarakhsh et al., 2015).Therefore, we showed that in pea, two genes (PsIPT1 and PsIPT2) encoding isopentenyl transferases (the rate-limiting enzymes involved in cytokinin biosynthesis) are induced in roots in response to inoculation (1 to 3 dpi), and they may be associated with the early stages of symbiotic development.Similarly, in M. truncatula, the expression of MtIPT4 (Medtr2g022140, the closest homolog of PsIPT2), which encodes isopentenyl transferase, was essentially induced within 3 h of Nod factor treatment (van Zeijl et al., 2015).

GenBank accession number
We also found that the expression of two other PsIPT3 and PsIPT4 genes seems to be connected with the later stages of nodulation.In Lotus, the level of LjIPT3 (the closest homolog of PsIPT3) was significantly induced in response to inoculation with M. loti, and it reached a maximal level at the mature stage (Chen et al., 2014).Similarly, in L. japonicus and M. truncatula, LjIPT1/MtIPT1 (the closest homolog of PsIPT4) increased during nodulation reaching its maximum at later stages (Chen et al., 2014;Azarakhsh et al., 2015).Thus, our results are consistent with those obtained for other legumes, and they suggest that the changes in cytokinin content during symbiosis may be associated with the biosynthesis of these hormones in plants in response to inoculation.These findings strongly suggest the importance of cytokinin at various stages of nodulation in legume plants.
Seven LOG genes (PsLOG1-PsLOG7) were identified by searching all available P. sativum Transcriptome Shotgun Assembly (TSA) databases using the sequences encoding for M. truncatula LOG genes (Table 3).During the next stage of our investigation, we analyzed changes in the expression of LOG genes during nodulation in pea (Figure 4).Significant changes in the level of expression of the PsLOG1, PsLOG2, PsLOG4, and PsLOG5 genes were revealed, starting from 7 dpi, when nodule formation occurred in pea.The maximum expression level of the LOG genes was reached at 11 to 14 dpi (25 to 30 times for PsLOG1, 3 to 5 times for PsLOG2, and 5 to 8 times for PsLOG4 and PsLOG5).As such, based on the analysis of the main genes that control cytokinin metabolism, and which are also involved in the plants' primary response to the action of these hormones, we can infer that cytokinin has a significant impact on both the initial stages of symbiotic interaction and on subsequent nodule development in pea.This is demonstrated by the sequential activation of the cytokine response (RR A-type) genes in pea in response to inoculation with rhizobia.The earliest response of plants to cytokinin was observed at 1 to 3 dpi, and then at 7 to 11 dpi (the period when primordia develop and nodules appear in pea).Cytokinin also played an important role in nodule function, as the expression of the PsRR11 gene reached very high level in the nodules.At the same time, increases in the expression level of the gene that controlled the biosynthesis/activation of cytokinin can serve as the basis for changes in cytokinin content.The early stage may be related to the induction of PsIPT1 and PsIPT2 in pea, when cytokinin receptor activation may be indicated by the increased expression of PsRR5 and PsRR9; furthermore, subsequent stages may be characterized by the activation of PsIPT1, PsIPT3, PsIPT4, PsLOG1, PsLOG2, PsLOG4, and PsLOG5 (that is consistent with the PsRR4, PsRR8, and PsRR11 induction).

Analysis of cytokinins in pea roots upon nodulation
Here, we are interested in determining whether the increase in the expression of the IPT and LOG genes can lead to changes in the content of various cytokinin forms in pea roots upon nodulation.Therefore, the cytokinin content was determined in pea tissues (Dobrev and  Kaminek, 2002;Novak et al., 2008).The results of the analysis of different forms of cytokinins in pea roots during inoculation and in uninoculated roots are presented in Figure 5.The content of transzeatin significantly increased in pea roots in response to inoculation by 3 dai; then, the content of phytohormone trans-zeatin increased again by 10 dpi.At 3 dpi, the content of isopentenyladenine (iPA) also increased in pea roots (Figure 5).The content of other forms of cytokinins (dihydroseatin, cis-zeatin and benzyladenine) did not demonstrate significant changes during nodule development in pea.Thus, we detected a two-stage increase in the content of cytokinin trans-zeatin in the roots of pea during inoculation, which corresponded to a change in the expression of the IPT and LOG metabolic genes.

Identification of pea genes controlling auxin biosynthesis (TAR) and transport (PIN)
The verification of sequences in the National Center for Biotechnology Information (NCBI)  database (Genbank) showed that a few tryptophan aminotransferase-related (TAR1, TAR2, TAR3) genes were previously identified in pea (Tivendale et al., 2012).The expression of these three genes in pea during symbiotic development was analyzed.It was shown that only the TAR2 and TAR3 genes were expressed in the roots and nodules of pea, whereas expression of the TAR1 gene was not evident; there was, also, no significant increase in the expression levels of the TAR2 and TAR3 genes in pea roots upon nodulation (Figure 6).Complete or partial sequences of 10 PsPIN genes were identified by analyzing all available pea TSA databases  4).The names of the pea PIN genes were given according to the known PIN1-PIN11 genes of the model plant, M. truncatula, which showed the highest level of sequence homology.

GenBank accession number
During the next stage of the analysis, the changes in the expression of PIN genes were assessed during pea nodule development (Figure 7).It was found that the level of expression of the PIN2 and PIN4 genes had increased in response to inoculation with rhizobia in the early stages of symbiosis (1 to 3 dpi), while the expression level of the PIN3 gene was significantly suppressed at 1 to 3 dpi followed by increasing at later stages of a nodule formation (9 to 11 dpi) (Figure 7).The expression of other PIN genes did not exhibit noticeable change during symbiosis development (data not shown).Thus, in the early stages of symbiosis (1 to 3 dpi), an increase in cytokinin levels was accompanied by changes in the expression levels of the PIN genes that control auxin transport.
Cytokinin has been reported in literature to influence the distribution and synthesis of auxin in legumes when nodules are formed (Plet et al., 2011;Suzaki et al., 2012).The work of Plet et al. (2011) showed that in the M. truncatula cre1 cytokinin receptor mutant, the polar auxin transport (PAT) was not disrupted in the roots during inoculation (Plet et al., 2011).In the mutant L. japonicus snf2 with a spontaneously activated cytokinin receptor, a positive effect of cytokinin on the accumulation of auxin was also observed.This was associated with an increase in the gene expression of LjTAR1, which is related to the biosynthesis of auxin (Suzaki et al., 2012).
The detected suppression of the PIN3 gene in this study, as well as an increase in the expression level of the PIN2 and PIN4 genes that control auxin transport in pea, are consistent with the observation in legume plants forming non-determined types of nodules, PAT is temporarily disrupted in response to inoculation or NF treatment, and the auxin locally accumulates in the cells of the inner cortex (Mathesius et al., 1998;De Billy et al., 2001;Wasson et al., 2006;Huo et al., 2006).It was believed that PIN3 controls basipetal transport in pea, and that suppression of this is gene related to PAT inhibition, whereas the PIN2 and PIN4 genes in pea can control lateral auxin transport, which determines local auxin accumulation within the cortical cells.Thus, we have shown that changes in cytokinin content, as well as the activation of cytokinin receptor, can occur at several stages of symbiotic development in pea.During the early stages of symbiosis, the increase in cytokinin level occurs in tandem with changes in the expression levels of the PIN genes that control auxin transport.This may be due to the mutual influence of the cytokinin and auxin hormones.In accordance with this concept, in the M. truncatula cre1 mutant impaired in the cytokinin receptor, the rhizobia were not able to locally alter auxin transport and to affect the redistribution of the PIN proteins in response to inoculation when compared with wild-type plants (Plet et al., 2011).Consequently, in the early stages of symbiosis, cytokinin can affect the distribution of PIN proteins, which control auxin transport.A similar effect of cytokinin on the expression and distribution of PIN proteins was observed during the formation of other lateral organs in plants: the lateral roots (Dello Ioio et al., 2007; Laplaze et al., 2007).It is known that upon treating roots with exogenous cytokinin, 6-benzylaminopurine (BAP), or following the overexpression of the IPT genes, changes in lateral root primordia formation were observed, as these structures became flat (Laplaze et al., 2007).In this case, cytokinin affects the expression of PIN genes, which leads to a localized change in auxin distribution (Laplaze et al., 2007).Therefore, during the initial stages of nodule appearance, the mechanisms involved in the control of lateral root organogenesis may be similar with those during nodule organogenesis in legume plants that form an undetermined nodule type.
The activation of cytokinin biosynthesis was also related to later stages of nodule development.These data are consistent with previous results, which were obtained following an analysis of the trans-zeatin content in pea nodules (Syono et al., 1976).Therefore, the cytokinin may also be important in nodule function, but its precise role still remains unclear.

Figure 1 .
Figure 1.Phylogenetic analysis of genes encoding components of the cytokinin signaling pathway (A-type Response Regulators, A-type RR).Phylogenetic tree shows the orthology of the A-type PsRRs with A-type MtRRs.The branches show the bootstrap values.The RRs are named with the first letters of the genus and species: Ps, Pisum sativum; Mt, Medicago truncatula.

Figure 2 .
Figure 2. Relative expression of A-type PsRRs genes in uninoculated plants (NI) and at different days post-inoculation (dpi).The relative expression was normalized against the constitutively expressed ubiquitin and actin genes.Results are means ± SEM of three technical repeats.

Figure 3 .
Figure 3. Quantitative RT-PCR expression analysis of PsIPT1-6 genes in uninoculated plants cv.Frisson (NI) and at different days after inoculation (dai).The relative expression was normalized against the constitutively expressed ubiquitin and actin genes.Results are means ± SEM of three technical repeats.

Figure 7 .
Figure 7. Relative expression of PsPIN genes in uninoculated plants (NI) and at different days post-inoculation (dpi).The relative expression was normalized against the constitutively expressed ubiquitin and actin genes.Results are means ± SEM of three technical repeats.

Table 1 .
List of primers used for PCR.

Table 2 .
BLAST searches in different pea transcriptome databases of the P. sativum A-type Response Regulators (RR) genes.

Table 4 .
BLAST searches in different pea transcriptome databases of the P. sativum PIN genes.