CALIFICACIÓN OBTENIDA E

The negative effect of nitrate is tightly connected to AON (Carroll et al., 1985a,b; Sagan and Gresshoff, 1996; Schnabel et al., 2005; Wopereis et al., 2000; Penmetsa

et al., 2003; Oka-Kira et al., 2005; Magori and Kawaguchi, 2009). We re-analyzed the

effect of nitrate on nodulation in wild type and sunn-4 mutants. A complete inhibition of nodulation on wild type plants was achieved by the addition of 10 mM KNO3and

split-root experiments indicated that this effect involves systemic signals, in accordance to previously published data (Jeudy et al., 2010; Carroll et al., 1985a,b; Sodek and Moura Silva, 1996; Eskew et al., 1989). Nodulation was also more strongly inhibited in the nitrogen-rich roots, than in the nitrogen-deprived roots. Jeudy et al. (2010), explained this effect because a local inhibition acts on top of a systemic inhibition. However, it is equally possible that addition of nitrate activates a concentration gradient of a nodule-inhibiting compound, which is strongest at the place of nitrate addition. Interestingly, nitrate-induced inhibition of nodulation, locally as well as systemically, was reduced in the sunn-4 mutant. Many scientists have explained this partial tolerance by the fact that nitrate-mediated inhibition might involve the AON pathway. Sunn-4 mutants are very strong mutants and in our condition, there was still a clear inhibition of nodulation, locally as well as systemically after nitrate addition. Eskew et al. (1989) described already in 1991 that nodulation of AON mutants is only partially tolerant to nitrate, because very high concentrations of nitrate were able to severely reduce nodule numbers on AON mutants. Hence, it might equally be possible that the lower sensitivity is a consequence of changes in hormone balances in the sunn mutants, whereby auxin levels might be the central actor. If nitrate addition would inhibit nodule formation via reducing the auxin levels in the root, higher nitrate concentrations would be required

Figure 4.17 (facing page): Tree-based alignment of the CLE domain encoded by all MtCLE genes and of the CLE domain of all Arabidopsis CLE genes as well as the nodulation-specific

Figure 4.18: Expression analysis of MtCLE26 (A), MtCLE28 (B), and MtCLE31 (C), by qRT- PCR on cDNA samples of zone-I root tissues of uninoculated plants (NI) and at 4, 6, 8 and 10 dpi. Data and error bars represent means± SD. The experiment was repeated twice with comparable results.

in sunn mutants to reduce the high auxin levels sufficiently to block nodule formation. In Arabidopsis, a link between nitrate and auxin transport has recently been resolved (Krouk et al., 2010; Beeckman and Friml, 2010).

The nitrate effect might act via CLE peptides because in L. japonicus as well as in soybean, nitrate-induced CLE peptide genes were identified, which inhibit nodula- tion in a HAR1/NARK-dependent way (Okamoto et al., 2009; Reid et al., 2011). In the recently released M. truncatula genomic data (Mt3.0 version) 6 more CLE genes (MtCLE26 to MtCLE31) were identified by specialized BLAST searches, besides the 25 (MtCLE1 to MtCLE25) identified before (Mortier et al., 2010). However, none of these MtCLE genes were nitrate-upregulated. As the Mt3.0 version represents about 80 % of the genome, more CLE genes are expected to be found upon completion of the genome sequencing. In the genome of L. japonicus, which is comparable in size to that of M. truncatula (470 Mb) and which has currently been sequenced for 91,3 %, 39

CLE genes were identified (Sato et al., 2008; Okamoto et al., 2009).

In Arabidopsis, gain-of-function analysis has shown that there is a correlation be- tween the function of the CLE peptide and its sequence (Hobe et al., 2003; Ni and Clark, 2006). Based on the sequences of the CLE motif, CLE peptides have been di- vided in three groups (Ito et al., 2006; Mortier et al., 2011). 24 MtCLE peptides were found to belong to group-I, exemplified by CLV3 and which are promoters of cellular differentiation (Ito et al., 2006); 2 MtCLE peptides are related to group-II, exemplified by TDIF and which prevent cellular differentiation and control the rate and orientation

of vascular cell division (Ito et al., 2006; Etchells and Turner, 2010). Finally, 5 MtCLE peptides belong to group-III, to which the M. truncatula, L. japonicus and soybean CLE peptides belong, from which the expression is enhanced during nodulation and which affect nodulation after ectopic overexpression (Mortier et al., 2010, 2011; Okamoto

et al., 2009; Reid et al., 2011). The newly identified group-III CLE peptide genes, MtCLE26 and MtCLE27, might possibly exert a nodulation-related function. Indeed,

for MtCLE26, transcripts were detected by qRT-PCR analysis from 6 dpi on, similar to the group-III MtCLE12 and MtCLE13 genes (Mortier et al., 2010). In contrast, Mt-

CLE27 transcripts were not detected during early nodulation (4 till 10 dpi). Still, an

enhanced transcript level was found in whole root samples carrying 1-month-old nod- ules compared to uninoculated roots. Because this expression pattern was not detected in RNA derived from separate nodules of the same age (data not shown), these results might indicate that MtCLE27 is expressed in the nodulated root tissue rather than in the nodules. This would be a new expression pattern for nodulation-related CLE peptides, but promoter:GUS analysis has to be executed to confirm this result. Also, expression of the group-I type MtCLE28 and MtCLE31 genes was upregulated during nodulation. Group-I CLE peptides are known to cause consumption of the root meristem upon ec- topic addition or overexpression (Okamoto et al., 2009; Mortier et al., 2010, 2011; Reid

et al., 2011). Hence, it seems that a versatile group of CLE genes are activated during

nodulation. Also in soybean nodulation, 6 GmCLE genes, from which 5 had a second copy in the genome, are induced during nodulation (Mortier et al., 2011). We have previously shown that MtCLE13 expression coincides with nodule primordium forma- tion and that, together with MtCLE12, the gene is expressed in the apical meristematic part of the nodule (Mortier et al., 2010). It will be interesting to analyze the expres- sion patterns of the newly identified nodulation-related CLE genes. Moreover, ectopic expression might shed a light on the exact role of MtCLE26, MtCLE27, MtCLE28,

MtCLE29, MtCLE30 and MtCLE31 in nodulated roots.