The endodermal tissue layer is found in the roots of vascular plants and functions as a semipermeable barrier, regulating the transport of solutes from the soil into the vascular stream. As a gateway for solutes, the endodermis may also serve as an important site for sensing and responding to useful or toxic substances in the environment. Here, we show that high salinity, an environmental stress widely impacting agricultural land, regulates growth of the seedling root system through a signaling network operating primarily in the endodermis. We report that salt stress induces an extended quiescent phase in postemergence lateral roots (LRs) whereby the rate of growth is suppressed for several days before recovery begins. Quiescence is correlated with sustained abscisic acid (ABA) response in LRs and is dependent upon genes necessary for ABA biosynthesis, signaling, and transcriptional regulation. We use a tissue-specific strategy to identify the key cell layers where ABA signaling acts to regulate growth. In the endodermis, misexpression of the ABA insensitive1-1 mutant protein, which dominantly inhibits ABA signaling, leads to a substantial recovery in LR growth under salt stress conditions. Gibberellic acid signaling, which antagonizes the ABA pathway, also acts primarily in the endodermis, and we define the crosstalk between these two hormones. Our results identify the endodermis as a gateway with an ABA-dependent guard, which prevents root growth into saline environments.

The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCK␣-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an ␣-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCK␣-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1␦ (PP1␦), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1␦, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the ␣-helical leucine repeats. The former was able to interact with PP1␦ only in the phosphorylated state and result in inactivation of PP1␦ activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.

Rho-binding kinase ␣ (ROK␣) is a serine/threonine kinase with multiple functional domains involved in actomyosin assembly. It has previously been documented that the C terminus part of ROK␣ interacts with the N-terminal kinase domain and thereby regulates its catalytic activity. Here we used antibodies against different domains of ROK␣ and were able to reveal some structural aspects that are essential for the specific functions of ROK␣. Antibodies against the kinase domain revealed that this part of the protein is highly complex and inaccessible. Further experiments confirmed that this domain could undergo inter-and intramolecular interactions in a complex manner, which regulates the kinase catalytic activity. Other antibodies that raised against the coiled-coil domain, Rho binding domain, and the pleckstrin homology (PH) domain were all effective in recognizing the native proteins in an immunoprecipitation assay. Only the anti-Rho binding domain antibodies could activate the kinase independent of RhoA. The PH antibodies had no apparent effects on the catalytic activity but were effective in blocking actomyosin assembly and cell contractility. Likewise, mutations of the PH domains can abrogate its dominant negative effects on actin morphology. The subsequent disruption of endogenous ROK localization to the actomyosin network by overexpressing the PH domain is supportive of a role of the PH domain of ROK in targeting the kinase to these structures.Actin cytoskeleton undergoes rapid dynamic changes in response to extracellular signaling cues, and Rho-family GTPases are key mediators in these responses (1-3). In particular, RhoA is responsible for promoting the formation of actin-based stress fibers and focal adhesions, resulting in contractile phenotype in cultured cells treated with lysophosphatidic acid or sphingosine 1-phosphate (4, 5). Two major effectors of this cytoskeletal event have been identified as ROK 1 (ROK/ROCK/ Rho-kinase; Refs. 6 -8) and diaphanous (9, 10), whose cooperative effects upon activation are essential for Rho activities.ROK␣ belongs to a member of a kinase family that includes myotonic dystrophy kinase, myotonic dystrophy kinase-related Cdc42-binding kinase, and citron kinase. In general, they consist of an N-terminal serine/threonine kinase domain that is followed immediately by an extended coiled-coil region and other functional motifs such as GTPase binding, pleckstrin homology (PH), and cysteine-rich domains. In ROK, the C terminus contains an unconventional PH with an internal cysteine-rich motif (6). These multidomain kinases have been reported to be involved in the regulation of some aspects of actin cytoskeleton rearrangement during different cell stages and cytokinesis through their conserved catalytic activities (11)(12)(13)(14).The regulation of the catalytic activity of serine/threonine protein kinases often involves kinase phosphorylation in the activation loop and the hydrophobic motif C-terminal to the kinase domain by autophosphorylation and/or phosphorylation by a hetero...

We have previously described a partial cDNA sequence encoding a RhoGAP protein, GAP25 that is homologous to the recently reported ArhGAP9 and ArhGAP12. We now describe a related new member ArhGAP15 that shares a number of domain similarities, including a pleckstrin homology (PH) domain, a RhoGAP domain and a novel motif N-terminal to the GAP domain. This novel motif was found to be responsible for nucleotide-independent Rac1 binding. Using swop mutants of Rac/Cdc42, we have established that the binding is through the C-terminal half of Rac1. The GAP domain of ArhGAP15 showed speci¢city towards Rac1 in vitro. The PH domain is required for ArhGAP15 to localize to cell periphery and overexpression of the full-length ArhGAP15, but not the mutant with a partial deletion of the PH domain, resulted in an increase in actin stress ¢bers and cell contraction. These morphological e¡ects can be attenuated by the co-expression of dominant negative Rac1 N17 . HeLa cells expressing ArhGAP15 were also resistant to phorbol myristatate acetate treatment, suggesting that ArhGAP15 is a potential regulator of Rac1. ß