Expression of MtCBF4 improved salt tolerance and activated two CBF target genes in M. truncatula. (a) RFP fluorescence in A. rhizogenes-transformed M. truncatula roots was observed with fluorescence microscopy 2 weeks after inoculation. In empty vector as well as d35S:MtCBF4 transgenic composite plants, RFP was observed in corresponding bright-field images (left). Wild-type plants were used as a control. Bars = 200 μm. (b) Representative examples of MtCBF4-overexpressing A. rhizogenes-transformed roots 1 week after transfer to control medium (left) or medium containing 100 mM NaCl (right). Black lines indicate the position of root tips at the moment of transfer. An empty pRedRoot vector was used as a control. Bars = 0.5 cm. (c) Primary root length of transgenic roots was measured from the point of transfer to salt-containing medium (100 mM NaCl) or normal medium after 1 week. A representative example of three replications is shown (n > 30 per construct and condition per experiment). Asterisk indicates that these plants had significantly longer root length under 100 mM NaCl than control plants (Student's t-test, **P < 0.01). (d) and (e) Expression of MtCBF4 and potential targets in transiently transfected M. truncatula leaves. RNA from leaves transformed with an empty vector (V) or d35S:MtCBF4 construct (S) were used for qRT-PCR after 48 h of transfection. For salt treatment, the leaves were treated with 100 mM NaCl for 6 h (Salt), and ddH2O treatment was used as a control (CK). Histograms show relative quantification of the transgene and the putative targets (MtCAS15 and MtCAS31). Data represent means and SE of three replications.