Myogenesis is determined by a set of myogenic differentiation factors that are, in turn, regulated by a number of peptide growth factors. During embryonic mouse tongue formation, transforming growth factor alpha (TGF alpha), epidermal growth factor (EGF), and their cognate receptor (EGFR) are co-expressed spatially and temporally with desmin, a muscle-specific structural protein. This investigation tested the hypothesis that TGF alpha directly regulates the myogenic program in developing tongue myoblasts. Mandibular processes from the first branchial arch of embryonic day 10.5 (E10.5) mouse embryos were microdissected and explanted into an organ culture system using serumless chemically defined medium. Exogenous TGF alpha at 10 and 20 ng/ml specifically increased the amount of desmin expression and the number of desmin-positive cells without affecting the general growth and development of the mandibles. This inductive response was detected as early as 2 days after treatment and sustained up to 9 days in culture. EGFR antisense oligonucleotides (30 microM) as well as tyrphostin (80 microM) were able to negate TGF alpha-induced up-regulation of desmin expression. These data indicate that autocrine and/or paracrine action of TGF alpha promotes tongue myogenesis, and that this action is mediated through functional kinase activity of the EGFR. We speculate that the myogenic program in the developing mouse tongue is dependent upon growth factor mediated cell-cell communication of mesenchymal cells originating from the occipital somites and ectomesenchymal cells originating from the cranial neural crest.
Cellular responses to abiotic stress involve multiple secondary messengers including reactive oxygen species (ROS), Ca2+, phytohormones such as abscisic acid (ABA) and chloroplast-to-nucleus retrograde signals such as 3’-phosphoadenosine 5’-phosphate (PAP). Mechanism(s) by which these messengers, produced in different subcellular compartments, intersect for cell regulation remain enigmatic. Here we demonstrate a mechanistic link enabling ABA and the chloroplast retrograde signal PAP to coordinate both chloroplast and plasma membrane ROS production. In whole leaves, PAP alters various ROS-related processes including plasmodesmal permeability as well as responses to ozone and the bacterial elicitor flg22, but largely quenches ROS during oxidative stress. Conversely, in guard cells, both PAP and ABA induce a ROS burst in both chloroplasts via photosynthetic electron transport, and the apoplast via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). Both subcellular ROS sources were necessary for ABA- and PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD, instead of RBOHF, for apoplastic ROS production. We identify calcium-dependent protein kinases (CPKs) transcriptionally induced by PAP as the post-translational activators of RBOHD-mediated ROS production. CPK13, CPK32, and CPK34 concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two Serine (S) residues, including S120 which is also targeted by the core ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpressing the PAP-induced CPKs rescues stomatal closure inost1.Our data identify chloroplasts, as sources and mediators of ROS and retrograde signals such PAP, to be critical environmental sensors and focal node in the multifaceted cellular stress response network.Significance StatementThe chloroplast is an important node to coordinate multiple plant signaling pathways in response to stresses such as drought. However, how does it function in specialized cells for which carbon fixation is secondary? Here we show that the chloroplast retrograde signal 3’-phosphoadenosine 5’-phosphate (PAP) plays multiple roles in reactive oxygen species (ROS) signaling and homeostasis. While PAP suppresses ROS in photosynthetic tissue, surprisingly PAP induces ROS in chloroplasts and extracellular space of guard cells to induce stomatal closure. We decipher how PAP-induced proteins activate both extracellular ROS production and anion channels for stomatal closure, thus providing a mechanism by which chloroplasts provide a strategic complement to canonical hormonal pathways in regulating plant physiological responses in specialized cells.
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