Key steps in a plant's development and adaptation to the environment are the initiation and development of lateral roots (LRs). LR development is regulated by auxin, the major plant hormone promoting LR formation, its counteracting hormones cytokinin, and abscisic acid (ABA). Here, we show that mutating ABSCISIC ACID INSENSITIVE4 (ABI4), which encodes an ABA-regulated AP2 domain transcription factor, results in an increased number of LRs. We show that ABI4 is expressed in roots and that its overexpression impairs LR development. Root expression of ABI4 is enhanced by ABA, and cytokinin and is repressed by auxin. Using hormone response promoters, we show that ABI4 also affects auxin and cytokinin profiles in the root. Furthermore, LR development in abi4 mutants is not altered or inhibited by cytokinin or ABA. Expression of the auxin-efflux carrier protein PIN1 is reduced in ABI4 overexpressors, enhanced in abi4 mutants, and is less sensitive to inhibition by cytokinin and ABA in abi4 mutants than in wild-type plants. Transport levels of exogenously applied auxin were elevated in abi4 mutants and reduced in ABI4 overexpressors. We therefore suggest that ABI4 mediates ABA and cytokinin inhibition of LR formation via reduction of polar auxin transport and that the resulting decrease in root auxin leads to a reduction in LR development.

Summary: A plant's ability to cope with salt stress is highly correlated with their ability to reduce the accumulation of sodium ions in the shoot. Arabidopsis mutants affected in the ABSCISIC ACID INSENSITIVE (ABI) 4 gene display increased salt tolerance, whereas ABI4‐overexpressors are hypersensitive to salinity from seed germination to late vegetative developmental stages. In this study we demonstrate that abi4 mutant plants accumulate lower levels of sodium ions and higher levels of proline than wild‐type plants following salt stress. We show higher HKT1;1 expression in abi4 mutant plants and lower levels of expression in ABI4‐overexpressing plants, resulting in reduced accumulation of sodium ions in the shoot of abi4 mutants. HKT1;1 encodes a sodium transporter which is known to unload sodium ions from the root xylem stream into the xylem parenchyma stele cells. We have shown recently that ABI4 is expressed in the root stele at various developmental stages and that it plays a key role in determining root architecture. Thus ABI4 and HKT1;1 are expressed in the same cells, which suggests the possibility of direct binding of ABI4 to the HKT1;1 promoter. In planta chromatin immunoprecipitation and in vitro electrophoresis mobility shift assays demonstrated that ABI4 binds two highly related sites within the HKT1;1 promoter. These sites, GC(C/G)GCTT(T), termed ABI4‐binding element (ABE), have also been identified in other ABI4‐repressed genes. We therefore suggest that ABI4 is a major modulator of root development and function.

Tomato (Lycopersicon esculantum) ASR1 (abscisic acid stress ripening protein), a small plant-specific protein whose cellular mode of action defies deduction based on its sequence or homology analyses, is one of numerous plant gene products with unknown biological roles that become over-expressed under water- and salt-stress conditions. Steady-state cellular levels of tomato ASR1 mRNA and protein are transiently increased following exposure of plants to poly(ethylene glycol), NaCl or abscisic acid. Western blot and indirect immunofluorescence analysis with anti-ASR1 antibodies demonstrated that ASR1 is present both in the cytoplasmic and nuclear subcellular compartments; approx. one-third of the total ASR1 protein could be detected in the nucleus. Nuclear ASR1 is a chromatin-bound protein, and can be extracted with 1 M NaCl, but not with 0.5% Triton X-100. ASR1, overexpressed in Escherichia coli and purified to homogeneity, possesses zinc-dependent DNA-binding activity. Competitive-binding experiments and SELEX (systematic evolution of ligands by exponential enrichment) analysis suggest that ASR1 binds at a preferred DNA sequence.

Abscisic acid stress ripening 1 (ASR1) is a low molecular weight plant-specific protein encoded by an abiotic stress-regulated gene. Overexpression of ASR1 in transgenic plants increases their salt tolerance. The ASR1 protein possesses a zinc-dependent DNA-binding activity. The DNA-binding site was mapped to the central part of the polypeptide using truncated forms of the protein. Two additional zinc-binding sites were shown to be localized at the amino terminus of the polypeptide. ASR1 protein is presumed to be an intrinsically unstructured protein using a number of prediction algorithms. The degree of order of ASR1 was determined experimentally using nontagged recombinant protein expressed in Escherichia coli and purified to homogeneity. Purified ASR1 was shown to be unfolded using dynamic light scattering, gel filtration, microcalorimetry, circular dichroism, and Fourier transform infrared spectrometry. The protein was shown to be monomeric by analytical ultracentrifugation. Addition of zinc ions resulted in a global change in ASR1 structure from monomer to homodimer. Upon binding of zinc ions, the protein becomes ordered as shown by Fourier transform infrared spectrometry and microcalorimetry, concomitant with dimerization. Tomato (Solanum lycopersicum) leaf soluble ASR1 is unstructured in the absence of added zinc and gains structure upon binding of the metal ion. The effect of zinc binding on ASR1 folding and dimerization is discussed.

ASR1 is a plant‐specific, highly charged, low molecular weight polypeptide. Purified ASR1 was shown to posses sequence specific Zn²⁺‐dependent DNA binding activity (Kalifa et al. Biochemical Journal 381, 373–378, 2004). Steady‐state levels of tomato Asr1 mRNA and protein are transiently increased following exposure of plants to polyethylene glycol, NaCl or abscisic acid. The biological role of ASR1 could not be deduced from sequence analyses or sequence homologies. Tobacco plants over‐expressing tomato ASR1 have a decreased rate of water loss and improved salt tolerance. Upon exposure to salt, ASR1‐over‐expressing plants accumulate less Na⁺ and proline than wild‐type plants, and also results in increased steady‐state levels of other gene products under non‐stressed plant growth conditions. Therefore, ASR1 is probably involved in the regulation of water‐ or salt‐stress‐modulated gene expression.