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 vectortransformed 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.Plants and cells adapt to changes in the ionic environment as a result of salinity and drought through temporal or sustained regulation of a large number of genes (for review, see Bohnert et al., 1995; Ingram and Bartels, 1996; Bray, 1997), but the molecular mechanisms responsible for this regulation have remained elusive. We have documented coordinated gene regulation in long-term acquired NaCl tolerance in alfalfa (Medicago sativa L.) and rice (Winicov et al., 1989; Winicov, 1991 Winicov, , 1996 and have been interested in defining a functional role for a putative transcription factor, Alfin1, in the altered gene expression in NaCltolerant alfalfa (Winicov, 1993; Bastola et al., 1998).A relatively small number of transcription factors have been identified to date that bind to promoter elements in genes regulated by NaCl/drought stress (for review, see Ingram and Bartels, 1996; Shinozaki and YamaguchiShinozaki, 1997; Winicov and Bastola, 1997), and much of the information has been gene specific. A more complex view of transcriptional regulation is implied by the requirement of a coupling element for stress regulation of the barley HVA22 gene containing the ABA response element (Shen et al., 1996) and the combined role of myc and myb transcriptional activators in ABA-and dehydrationinducible expression of a promoter region of the rd22 gene (Abe et al., 1997). The potential interactions of various factors is compounded further in that transcription factors such as myc and myb belong to extensive multigene families with tissue-specific expression patterns. Nevertheless, recent reports have shown that ecto...

Plant root development is an essential determinant of plant growth and crop yield that could be enhanced by induced changes in the expression of root-specific regulatory factors. We reported previously that Alfin1 binds DNA in a sequence-specific manner and that Alfin1 overexpression in transgenic alfalfa (Medicago sativa L.) enhances expression of the salt-inducible MsPRP2 gene in roots, suggesting that Alfin1 functions to regulate gene expression in roots. Here we show that Alfin1 is an essential gene for root growth and that its overexpression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions. Alfin1-binding sites occur in promoters of genes expressed in roots of a wide variety of plant species and we propose that it is a general root growth regulator. Even though Alfin1 overexpression was under the control of the CaMV 35S promoter, plant shoot growth was not adversely affected. We show further that introduction of the Alfin1 transgene in plants confers a dominant characteristic that significantly increases plant growth and salt tolerance.

A cDNA previously shown to identify a salt-inducible root-specific transcript in Medicago sativa was used to screen an alfalfa library for the corresponding genomic sequence. One positive clone was recovered. The nucleotide sequence of a subclone contained a 329 bp 5' region upstream of the first ATG codon, a 1143 bp coding segment, and a 447 bp 3'-untranslated region interrupted by a single 475 bp intron. Translation of the coding segment, which was designated MsPRP2, suggested it encodes a chimeric 40,569 Da cell wall protein with an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxyl-terminal sequence homologous to nonspecific lipid transfer proteins. The 3'-untranslated region of MsPRP2 contained a sequence similar to one found to destabilize mRNAs transcribed from the elicitor-regulated proline-rich protein gene PvPRP1. Transcription run-on experiments using nuclei from salt-sensitive and salt-tolerant alfalfa callus suggested that the accumulation of MsPRP2 transcripts in salt-tolerant alfalfa cells grown in the presence of salt is due primarily to increased mRNA stability. The MsPRP2 gene thus may be a useful model for studying post-transcriptional salt-regulated expression of cell wall proteins.

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Depending on the number or the length of exposure, application of serotonin can produce either shortterm or long-term presynaptic facilitation of Aplysia sensoryto-motor synapses. The cAMP-dependent protein kinase, a heterodimer of two regulatory and two catalytic subunits, has been shown to become stably activated only during long-term facilitation. Both acquisition of long-term facilitation and persistent activation of the kinase is blocked by anisomycin, an effective, reversible, and specific inhibitor of protein synthesis inAplysia. We report here that 2-hr exposure of pleural sensory cells to serotonin lowers the concentration of regulatory subunits but does not change the concentration of catalytic subunits, as assayed 24 hr later; 5-min exposure to serotonin has no effect on either type of subunit. Increasing intracellular cAMP with a permeable analog of cAMP together with the phosphodiesterase inhibitor isobutyl methylxanthine also decreased regulatory subunits, suggesting that cAMP is the second messenger mediating serotonin action. Anisomycin blocked the loss of regulatory subunits only when applied with serotonin; application after the 2-hr treatment with serotonin had no effect. In the Aplysia accessory radula contractor muscle, prolonged exposure to serotonin or to the peptide transmitter small cardioactive peptide B, both of which produce large increases in intracellular cAMP, does not decrease regulatory subunits. This mechanism of regulating the cAMPdependent protein kinase therefore may be specific to the nervous system. We conclude that during long-term facilitation, new protein is synthesized in response to the facilitatory stimulus, which changes the ratio of subunits of the cAMPdependent protein kinase. This alteration in ratio could persistently activate the kinase and produce the persistent phosphorylation seen in long-term facilitated sensory cells. Sensitization of defensive reflexes in the marine molluskAplysia and the underlying presynaptic facilitation of sensory-to-motor neuron synapses can be short (lasting minutes) or long (lasting days to weeks) (1-3). During acquisition of the long-term process, serotonin, a facilitating transmitter, induces a persistent increase in cAMP-dependent protein phosphorylation in sensory neurons (4). The Aplysia cAMPdependent protein kinase (A-kinase), a heterodimer of two regulatory subunits that inhibit the two catalytic subunits, is activated in both short-term (1, 4) and long-term facilitation (4) of both the gill-and siphon-withdrawal reflex and the tail-withdrawal reflex. The amount of regulatory subunits has been found to be decreased as compared with catalytic subunits in abdominal sensory cells of animals trained for long-term sensitization of the gill-and siphon-withdrawal reflex (5). These changes in regulatory subunits could account for the persistent protein phosphorylation seen in long-term facilitation (4). Evidence has been presented that long-term facilitation (6, 7), behavioral sensitization (8), and persistent phosphorylation...

Alfin1 cDNA, obtained by differential screening of a poly(A)+ library from salt-tolerant alfalfa cells, encodes a novel protein with a Cys4 and His/Cys3 putative zinc-binding domain that suggests a possible role for this protein in transcriptional regulation. We have expressed the cDNA in Escherichia coli and show that the recombinant Alfin1 protein binds DNA in a sequence-specific manner. The DNA recognition sequence was determined from individual clones isolated after four rounds of random oligonucleotide selection in gel retardation assays, coupled with PCR amplification of the selected sequences. The consensus binding site for Alfin1 is shown to contain two to five G-rich triplets with the conserved core of GNGGTG or GTGGNG in clones showing high-efficiency binding. DNA binding of the recombinant Alfin1 was inhibited by EDTA. Alfin1 mRNA was found predominantly in alfalfa roots. Growth of salt-sensitive Medicago sativa L on 171 mM NaCl led to a slight decrease in Alfin1 mRNA, while the salt-tolerant plants showed no decrease in Alfin1 mRNA levels. Interestingly, recombinant Alfin1 binds efficiently to three fragments of the MsPRP2 promoter, each containing consensus sequences identified by the random oligonucleotide selection. Since MsPRP2 transcripts were shown to be root-specific and accumulated in alfalfa roots in a salt-inducible manner, Alfin1 may play a role in the regulated expression of MsPRP2 in alfalfa roots and contribute to salt tolerance in these plants.