(http://www.noble.org/medicago/ecotypes.html). Inrecent years,
M. truncatula
has been recognized as anexcellent legume model in view of its small, diploid ge-nome, self fertility, and short life cycle, as well as avail-ability of various genomic and genetic tools (Barkeret al., 1990; Bell et al., 2001; Young et al., 2005). All thesetraits make
M. truncatula
suitable for identifying genesthat could improve agronomic performances such asabiotic stress resistance (Cook, 1999).Stress responses involve alterations in gene expres-sion (Tester and Davenport, 2003), suppressive sub-tractive hybridizations, and array technologies usingcDNAs or oligonucleotides are increasingly beingused to monitor global gene expression changes invarious plants in response to abiotic stresses (Ozturket al., 2002; Seki et al., 2002; Bartels and Sunkar, 2005).However, few reports analyzed differences in geneexpression between salt-sensitive and salt-tolerantgenotypes in plants (Walia et al., 2005). Althoughsuch genes are useful in the dissection of genotype-specific regulatory pathways and mechanisms of salttolerance, they usually represent only a fraction of allsalt-regulated genes, and isolating them is a challeng-ing task. The mechanisms underlying the genotype-dependent difference in expression of such genes arelargely unknown.Two genotypes (108-R and Jemalong A17) wereshown to have a different adaptation to salt stress(Merchan et al., 2003). We have developed, in the
M.truncatula
108-R genotype, a dedicated macroarraycontaining genes linked to salt stress and recoveryresponses in roots (Merchan et al., 2007). Here, weaimed to identify genes involved in genotype-specificregulatory pathways and mechanisms of salt acclima-tion. A comparison of molecular and physiologicalresponses to salt stress in sensitive and tolerant
M.truncatula
genotypes (108-R and Jemalong A17, re-spectively) was performed. Expression analysis usingthe dedicated macroarray revealed several genes po-tentially associated with the contrasting responses tosalt stress in these plants. Among them, we identifieda homolog of the
COLD
-
REGULATEDA1
(
CorA1
;
MtCorA1
;Labergeetal.,1993)gene,whichisaputativetarget of MtZpt2 transcription factors (TFs; Merchanet al., 2007). Accordingly, these TFs showed higher ex-pression levels in the tolerant variety. Overexpressionof
MtZpt2
-
1
or
MtZpt2
-
2
in roots of the salt-sensitivevariety allowed significant increase in root growthspecifically under salt stress conditions, suggesting arole of these pathways in the differential adaptiveresponse to salt stress of these
M. truncatula
genotypes.
RESULTSEvaluation of Salt Stress Growth Responses of Two
M. truncatula
Genotypes
We have examined root growth and dry weight biomass in two genotypes of the model legume
M. truncatula
(108-R and Jemalong A17) in responseto different salt stress conditions. Root length wasmeasured after 5 d of growth on a rich medium(Fahra¨eus; Truchet et al., 1985) containing variousconcentrations of NaCl (Fig. 1). Root length of
M.truncatula
108-R was significantly reduced after NaCltreatments in the range of 90 to 150 m
M
, as comparedwith plants grown without salt (Kruskal and Wallistest,
P
,
0.01;
n
5
20; Fig. 1A). However, root length in Jemalong A17 was negatively affected by salinity onlyat 150 m
M
(Kruskal and Wallis test,
P
,
0.01;
n
5
20;Fig. 1A). Altogether, we could identify a differentialresponse to salt stressbetween the 108-R and JemalongA17 genotypes. As shown in Figure 1B,
M. truncatula
Jemalong A17 grewwellathigh salinitylevels (120 m
M
NaCl), while 108-R plants grew more slowly underthese salt conditions. Similarly, salt treatment re-duced the root dry weight more noticeably for thevariety 108-R already after a 60 m
M
NaCl treatment,whereas it was only significantly affected at 150 m
M
in Jemalong A17 (Kruskal and Wallis test,
P
,
0.01;
n
5
20; Fig. 1C, left). No significant differences were,however, found for leaf dry weight biomass of eachvariety and in all treatments, suggesting greater saltsensitivity of the root than of the aerial part at thisearly postgermination stage (Fig. 1C, middle). Theratio of root dry weight to leaf dry weight (as per-centage of control) reveals the relative effects of increasing NaCl concentration for each genotype(Fig. 1C, right). As expected, ratio values for
M.truncatula
108-R were significantly reduced with in-creased salt treatments (60–150 m
M
NaCl; Kruskaland Wallis test,
P
,
0.01;
n
5
20). In contrast, this ratiowas constant for the tolerant genotype, at least up to120 m
M
NaCl. These results suggest that root dryweight is significantly different between the twogenotypes at medium and high salt concentrations(60–150 m
M
).Root growth performance was also assessed on alow-nitrogen medium (‘‘i’’; Blondon, 1964) to exam-ine the impact of the medium on the range of vari-ability for salinity tolerance between these twogenotypes of
M. truncatula
. At the same salinityconcentration, root length of 108-R was even moresignificantly affected by salt treatment when com-pared with the results obtained in the rich medium.This difference is likely to be a consequence of min-imal nutrient capacity of the ‘‘i’’ medium that maypotentiate salt stress effects on growth. Statisticallysignificant differences between 108-R and JemalongA17 genotypes were found for all assayed salinityconditions (Fig. 1D).
M. truncatula
108-R growth wasalready reduced by more than 50% at 90 m
M
of salt,and an NaCl concentration of 150 m
M
nearly abol-ished root growth. This negative effect of salt on rootgrowthwaslesspronouncedinJemalongA17,inagree-ment with the results obtained on rich (Fahra¨eus)medium (Fig. 1A).These various parameters allowed us to monitor theeffects of salt treatments on plant growth and furtherrevealed differential root growth responses between
de Lorenzo et al.1522 Plant Physiol. Vol. 145, 2007
Leave a Comment