Transgenic overexpression of the transcription factor alfin1 enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants

Abstract

Alfin1 cDNA encodes a putative transcription factor associated with NaCl tolerance in alfalfa (Medicago sativa L.). The recombinant protein binds DNA in a sequence-specific manner, including promoter fragments of the NaCl-inducible gene MsPRP2. Alfin1 function was tested in transgenic alfalfa under the control of the 35S promoter in the sense and antisense orientations with the endogenous MsPRP2 as a reporter gene. Calli overexpressing Alfin1 were more resistant to growth inhibition by 171 mM NaCl than vector-transformed controls, whereas calli expressing Alfin1 in the antisense orientation were more sensitive to NaCl inhibition. Transgenic plants overexpressing Alfin1 in the sense orientation grew well. In contrast, the antisense transgenic plants grew poorly in soil, demonstrating that Alfin1 expression is essential for normal plant development. Transgenic calli and plant roots overexpressing Alfin1 showed enhanced levels of endogenous MsPRP2 mRNA accumulation. However, MsPRP2 mRNA accumulation was also regulated in a tissue-specific manner, as shown in leaves of transgenic plants overexpressing Alfin1. These results suggest that Alfin1 acts as a transcriptional regulator in plants and regulates MsPRP2 expression in alfalfa. Alfin1 overexpressing transgenic plants showed salinity tolerance comparable to one of our NaCl-tolerant plants, indicating that Alfin1 also functions in gene regulation in NaCl tolerance.

Figure 1

Schematic representation of Alfin1 sense and antisense constructs used in transformation of alfalfa. Restriction sites are as follows: E, EcoRI; H, HindIII; B, BglII; and S, SalI. BR and BL are T-DNA right and left borders, respectively (An et al., 1988).

Figure 2

Northern analysis of Alfin1 and MsPRP2 expression in control and transgenic calli from Alfin1 sense transformants. Lanes 1 and 2, RNA isolated from untransformed NaCl-tolerant callus grown with or without 171 mm NaCl for 4 weeks; lane 3, RNA isolated from untransformed NaCl-sensitive callus; lane 4, RNA isolated from NaCl-sensitive callus transformed with pGA vector (1V); lanes 5 to 9, RNA isolated from NaCl-sensitive callus transformed with Alfin1 sense construct (S1, S2, S4, and S6 are independently transformed lines); and lane 9, RNA isolated from S2-transformed callus grown in 171 mm NaCl. Each lane contained 10 μg of total RNA. Each blot was hybridized sequentially with the following probes: Alfin1, the large EcoRI fragment (Fig. 1); MsPRP2, the carboxy-terminal and 3′-untranslated region fragment (Winicov and Deutch, 1994); and Msc27, the fragment of a constitutively expressed alfalfa gene.

Figure 3

Northern analysis of Alfin1 expression in control and transgenic plants from Alfin1 sense transformants. RNA was isolated from leaves of control and transgenic plants. Lane Con, No. 1 control NaCl-sensitive parent plant for all transformations; lanes S1, S2, and S3, plants transformed with the Alfin1 sense construct and regenerated from transformed callus; and lane V, vector-transformed plant. Each blot was hybridized sequentially with the following probes: Alfin1, large EcoRI fragment (Fig. 1); pGA-vector, EcoRI/BglII fragment from pGA643 to show readthrough of the Alfin1 transgene; and Msc27, fragment of a constitutively expressed alfalfa gene. Each lane contained 10 μg of total RNA.

Figure 4

Northern analysis of Alfin1 and MsPRP2 expression in control and transgenic plants from Alfin1 sense transformants grown in one-quarter-strength Hoagland solution with or without 128 mm NaCl. RNA was isolated from roots and leaves of control plants and plants tested for NaCl tolerance described in Table II legend. Lanes #1, Parent wild-type control; lanes 1V, control transformed with empty vector; lanes #9, NaCl-tolerant plant regenerated from NaCl-tolerant callus; and lanes S1, S2, and S3, parent no. 1 transformed with pGA-sense. The blot was hybridized sequentially with probes as described for Figure 2. Each lane contained 10 μg of total RNA.