Alternative Splicing Confers a Dual Role in Polar Auxin Transport and Drought Stress Tolerance to the Major Facilitator Superfamily Transporter ZIFL1

Major facilitator superfamily (MFS) transporters constitute a major class of transporter in all organisms. They are prevalent in plants; the Arabidopsis thaliana genome contains over 120 genes predicted to encode MFS transporters. However, relatively few have been characterized, such as nitrate transporters related to NRT1 (Forde, 2000) and ZINC-INDUCED FACILITATOR1 (ZIF1; Haydon and Cobbett, 2007). Remy et al. (pages 901–926) present a functional analysis of an Arabidopsis ZIF1 paralog, ZIF-LIKE1 (ZIFL1), and show that two isoforms produced through alternative splicing have distinct roles in polar auxin transport and drought stress tolerance. Using reporter genes to assess expression patterns, the authors found that the ZIFL1 promoter is mainly active in root tissue and stomatal guard cells. ZIFL1 generates three transcripts: ZIFL1.1, predicted to encode the full-size transporter with 12 membrane-spanning segments; and ZIFL1.2 and ZIFL1.3, encoding truncated proteins that lack two N-terminal or two C-terminal membrane-spanning segments, respectively. RT-PCR analysis revealed that roots exclusively express ZIFL1.1/ZIFL1.2, whereas leaves express ZIFL1.3 in addition to the other isoforms. Subcellular localization using yellow fluorescent protein (YFP) fusions showed that ZIFL1.1-YFP localizes to the tonoplast, whereas ZIFL1.3-YFP is targeted to the plasma membrane. These results were further confirmed in colocalization experiments using specific tonoplast and plasma membrane markers and in zifl1 mutant lines carrying ZIFL1.1 fused with green fluorescent protein under control of the native ZIFL1 promoter. Thus, ZIF1.1 appears to function in the tonoplast of root cells, whereas ZIF1.3 is found in the plasma membrane of stomatal guard cells. Phenotypic analysis of zifl1 loss-of-function mutants suggested a role for ZIFL1 in both drought tolerance and polar auxin transport. The mutant plants were hypersensitive to drought stress and showed a range of auxin-related defects, such as in lateral root emergence and root gravitropism. The contribution of the distinct ZIFL1 isoforms to these phenotypes was assessed by overexpressing each ZIFL1 transcript in the wild-type background and by assessing the ability of each isoform to suppress zifl1 mutant defects. These experiments supported the conclusion that ZIFL1.1 modulates root auxin-related processes, whereas ZIFL1.3 plays a role in regulating drought stress tolerance. The authors examined ZILF1 function more specifically in Arabidopsis and yeast cells and found that ZIFL1.1 influences cellular auxin efflux and modulates polar auxin transport in roots, whereas ZIFL1.3 plays no role in auxin-related processes. They hypothesized that ZIFL1.1 might affect polar auxin transport by influencing the activity of the major auxin efflux carrier PIN2. Immunofluoresence detection of PIN2 at the root tip plasma membrane indeed showed that PIN2 stability was significantly decreased by ZIFL1.1 loss of function and enhanced by its overexpression (see Figure). Further experiments in yeast cells showed that both ZIFL1 isoforms catalyze K+- and proton-coupled transport, leading the authors to propose that ZIFL1 isoforms regulate stomatal movements (ZIFL1.3) and polar auxin transport (ZIFL1.1) by modulating K+ and proton fluxes. PIN2 localization in root tips. Treatment with indole-3-acetic acid destabilizes PIN2 in the wild type (Col-0); this effect was exacerbated in zifl1-1 mutants and retarded by ZIFL1.1 overexpression. The eir1-4 mutant lacking PIN2 is shown as a control. c, cortex; e, epidermis; l, lateral root cap. Color coding blue (low) to yellow (high) corresponds to increasing signal intensities. Arrowheads indicate the polarity of PIN2 localization. Bars = 10 μm. (Reprinted from Remy et al. [2013], Figure 8A.) This interesting study provides an example of alternative splicing generating distinct biological functions for a transporter by altering tissue and subcellular localization: one isoform being targeted to the tonoplast of root cells and another to the plasma membrane of stomatal guard cells. Future work will seek to uncover the mechanisms underlying tissue specificity of the ZIFL1 transcripts.