Activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses after plants sense an invading pathogen. Here, we show that MPK3 and MPK6, two pathogen-responsive MAPKs, and their upstream MAPK kinases, MKK4 and MKK5, are essential to both stomatal and apoplastic immunity. Loss of function of and , or their upstream and , abolishes pathogen/microbe-associated molecular pattern- and pathogen-induced stomatal closure. Gain-of-function activation of MPK3/MPK6 induces stomatal closure independently of abscisic acid (ABA) biosynthesis and signaling. In contrast, exogenously applied organic acids such as malate or citrate are able to reverse the stomatal closure induced by MPK3/MPK6 activation. Gene expression analysis and in situ enzyme activity staining revealed that malate metabolism increases in guard cells after activation of MPK3/MPK6 or inoculation of pathogen. In addition, pathogen-induced malate metabolism requires functional MKK4/MKK5 and MPK3/MPK6. We propose that the pathogen-responsive MPK3/MPK6 cascade and ABA are two essential signaling pathways that control, respectively, the organic acid metabolism and ion channels, two main branches of osmotic regulation in guard cells that function interdependently to control stomatal opening/closure.

ORCID IDs: 0000-0001-6683-6415 (J.X.); 0000-0002-9910-3119 (J.M.); 0000-0002-4265-7399 (J.L.); 0000-0002-0757-5208 (Q.W.); 0000-0003-2959-6461 (S.Z.)Antimicrobial compounds have critical roles in plant immunity; for example, Arabidopsis thaliana and other crucifers deploy phytoalexins and glucosinolate derivatives in defense against pathogens. The pathogen-responsive MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6 have essential functions in the induction of camalexin, the major phytoalexin in Arabidopsis. In search of cyanide, a coproduct of ethylene and camalexin biosynthesis, we found that MPK3 and MPK6 also affect the accumulation of extracellular thiocyanate ion derived from the indole glucosinolate (IGS) pathway. Botrytis cinerea infection activates MPK3/MPK6, which promote indole-3-yl-methylglucosinolate (I3G) biosynthesis and its conversion to 4-methoxyindole-3-yl-methylglucosinolate (4MI3G). Gain-and loss-of-function analyses demonstrated that MPK3/MPK6 regulate the expression of MYB51 and MYB122, two key regulators of IGS biosynthesis, as well as CYP81F2 and IGMT1/ IGMT2, which encode enzymes in the conversion of I3G to 4MI3G, through ETHYLENE RESPONSE FACTOR6 (ERF6), a substrate of MPK3/MPK6. Under the action of PENETRATION2 (PEN2), an atypical myrosinase, and PEN3, an ATP binding cassette transporter, 4MI3G is converted to extracellular unstable antimicrobial compounds, possibly isothiocyanates that can react with nucleophiles and release the stable thiocyanate ion. Recent studies demonstrated the importance of PEN2/ PEN3-dependent IGS derivatives in plant immunity. Here, we report that MPK3/MPK6 and their substrate ERF6 promote the biosynthesis of IGSs and the conversion of I3G to 4MI3G, a target of PEN2/PEN3-dependent chemical defenses in plant immunity.

Heterotrimeric G proteins have been shown to transmit ultraviolet B (UV-B) signals in mammalian cells, but whether they also transmit UV-B signals in plant cells is not clear. In this paper, we report that 0.5 W m 22 UV-B induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by eliciting a cascade of intracellular signaling events including Ga protein, hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO). UV-B triggered a significant increase in H 2 O 2 or NO levels associated with stomatal closure in the wild type, but these effects were abolished in the single and double mutants of AtrbohD and AtrbohF or in the Nia1 mutants, respectively. Furthermore, we found that UV-B-mediated H 2 O 2 and NO generation are regulated by GPA1, the Ga-subunit of heterotrimeric G proteins. UV-B-dependent H 2 O 2 and NO accumulation were nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGa). In addition, exogenously applied H 2 O 2 or NO rescued the defect in UV-B-mediated stomatal closure in gpa1 mutants, whereas cGa AtrbohD/AtrbohF and cGa nia1 constructs exhibited a similar response to AtrbohD/ AtrbohF and Nia1, respectively. Finally, we demonstrated that Ga activation of NO production depends on H 2 O 2 . The mutants of AtrbohD and AtrbohF had impaired NO generation in response to UV-B, but UV-B-induced H 2 O 2 accumulation was not impaired in Nia1. Moreover, exogenously applied NO rescued the defect in UV-B-mediated stomatal closure in the mutants of AtrbohD and AtrbohF. These findings establish a signaling pathway leading to UV-B-induced stomatal closure that involves GPA1-dependent activation of H 2 O 2 production and subsequent Nia1-dependent NO accumulation.