Functional Analysis of Salt-Inducible Proline Transporter of Barley Roots

Abstract

We cloned a cDNA encoding Hordeum vulgare Proline Transporter (HvProT) from salt-stressed barley roots by differential display. HvProT was 2,161 bp long and had an open reading frame encoding 450 amino acids. The deduced amino acid sequence of HvProT was similar to those of proline transporter proteins of rice (65.7%), Arabidopsis (57.7%) and tomato (42.0%). Northern blot analysis showed that the transcript level of HvProT was induced in roots at 30 min after 200 mM NaCl treatment and its peak was observed at 3 h. However, the transcript level was very low in leaves and did not increase by salt stress. The expression level of Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), encoding a key enzyme of proline synthesis, was induced later than HvProT by salt stress. A transport assay using a yeast with mutation in proline uptake revealed that HvProT was a transporter with high affinity for L-proline (K(m) = 25 microM). HvProT was found to be a unique transporter with high affinity for L-proline. Since its transport activity was dependent on the pH gradient, HvProT was suggested to be a H(+)/amino acid symporter. In situ hybridization analysis showed that the HvProT mRNA was strongly expressed in root cap cells under salt stress. HvProT might play an important role in the transport of proline to root tip region urgently upon salt stress.

The AtProT Family. Compatible Solute Transporters with Similar Substrate Specificity But Differential Expression Patterns

“…AtProT2 expression, like that of HvProT, was highest in roots where it might be involved in uptake of compatible solutes from the soil or for retrieval of apoplastic amino acids delivered to the roots via the phloem (Schobert and Komor, 1987;Ueda et al, 2001). In maize roots subjected to low water potential, transport was shown to be the main source of Pro in the tip region (Verslues and Sharp, 1999;Raymond and Smirnoff, 2002).…”

“…In barley, both Gly betaine and Pro are accumulated in leaves in response to salt stress (Zuniga et al, 1989). However, the barley homolog HvProT is a specific Pro transporter that does not recognize Gly betaine, GABA, or other amino acids (Ueda et al, 2001).…”

“…The steady-state mRNA levels of the Arabidopsis ProT2 and the three mangrove ProT homologs (AmT1-3) increased under salt stress conditions, as do the concentrations of Pro and Gly betaine, respectively (Waditee et al, 2002). Furthermore, HvProT expression in barley roots was increased by salt stress treatments (Ueda et al, 2001). In tomato, expression of LeProT1 was shown to be confined to pollen and expression correlated with elevated Pro levels.…”

“…In the last few years, genes encoding transporters for the compatible solutes Pro and Gly betaine were isolated from a number of plant species, including Arabidopsis (AtProT1 and AtProT2; Rentsch et al, 1996), tomato (Lycopersicon esculentum; LeProT1-3; Schwacke et al, 1999), rice (Oryza sativa; OsProT; Igarashi et al, 2000), barley (Hordeum vulgare; HvProT; Ueda et al, 2001), orach (Atriplex hortensis; AhProT1; accession no. AAF76897), and mangrove (Avicennia marina; AmT1-3; Waditee et al, 2002).…”

Proline Metabolism and Its Implications for Plant-Environment Interaction

“…Interestingly, a proline-specific transporter induced by salt stress specifically in barley root tip has been identified (Ueda et al, 2001). Tissue-specific overexpression of this transporter in the Arabidopsis root tip led to increased proline content of the root apex (0-5 mm) and increased growth under control conditions (Ueda et al, 2008), although the effects of such targeted overexpression on growth under stress conditions were not tested.…”