Previous studies have showed that UV-B can stimulate closure as well as opening of stomata. However, the mechanism of this complex effect of UV-B is not clear. The purpose of this paper is to investigate the role and the interrelationship of H2O2 and NO in UV-B-induced stomatal closure in broad bean (Vicia faba L.). By epidermal strip bioassay and laser-scanning confocal microscopy, we observed that UV-B-induced stomatal closure could be largely prevented not only by NO scavenger c-PTIO or NO synthase (NOS) inhibitor l-NAME, but also by ascorbic acid (ASC, an important reducing substrate for H2O2 removal) or catalase (CAT, the H2O2 scavenger), and that UV-B-induced NO and H2O2 production in guard cells preceded UV-B-induced stomatal closure. These results indicate that UV-B radiation induces stomatal closure by promoting NO and H2O2 production. In addition, c-PTIO, l-NAME, ASC and CAT treatments could effectively inhibit not only UV-B-induced NO production, but also UV-B-induced H2O2 production. Exogenous H2O2-induced NO production and stomatal closure were partly abolished by c-PTIO and l-NAME. Similarly, exogenous NO donor sodium nitroprusside-induced H2O2 production and stomatal closure were also partly reversed by ASC and CAT. These results show a causal and interdependent relationship between NO and H2O2 during UV-B-regulated stomatal movement. Furthermore, the l-NAME data also indicate that the NO in guard cells of Vicia faba is probably produced by a NOS-like enzyme.

SummaryAccumulating data indicate that strigolactones (SLs) are implicated in the response to environmental stress, implying a potential effect of SLs on stomatal response and thus stress acclimatization. In this study, we investigated the molecular mechanism underlying the effect of SLs on stomatal response and their interrelation with abscisic acid (ABA) signaling.The impact of SLs on the stomatal response was investigated by conducting SL-feeding experiments and by analyzing SL-related mutants. The involvement of endogenous ABA and ABA-signaling components in SL-mediated stomatal closure was physiologically evaluated using genetic mutants. Pharmacological and genetic approaches were employed to examine hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) production.SL-related mutants exhibited larger stomatal apertures, while exogenous SLs were able to induce stomatal closure and rescue the more widely opening stomata of SL-deficient mutants. The SL-biosynthetic genes were induced by abiotic stress in shoot tissues. Disruption of ABA-biosynthetic genes, as well as genes that function in guard cell ABA signaling, resulted in no impairment in SL-mediated stomatal response. However, disruption of MORE AXILLARY GROWTH2 (MAX2), DWARF14 (D14), and the anion channel gene SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) impaired SL-triggered stomatal closure. SLs stimulated a marked increase in H 2 O 2 and NO contents, which is required for stomatal closure.Our results suggest that SLs play a prominent role, together with H 2 O 2 /NO production and SLAC1 activation, in inducing stomatal closure in an ABA-independent mechanism.

SUMMARYBrassinosteroids (BRs) are essential for plant growth and development; however, whether and how they promote stomatal closure is not fully clear. In this study, we report that 24-epibrassinolide (EBR), a bioactive BR, induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering a signal transduction pathway including ethylene synthesis, the activation of Ga protein, and hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H 2 O 2 and NO levels, necessary for stomatal closure in the wild type. These effects were abolished in mutant bri1-301, and EBR failed to close the stomata of gpa1 mutants. Next, we found that both ethylene and Ga mediate the inductive effects of EBR on H 2 O 2 and NO production. EBR-triggered H 2 O 2 and NO accumulation were canceled in the etr1 and gpa1 mutants, but were strengthened in the eto1-1 mutant and the cGa line (constitutively overexpressing the G protein a-subunit AtGPA1). Exogenously applied H 2 O 2 or sodium nitroprusside (SNP) rescued the defects of etr1-3 and gpa1 or etr1 and gpa1 mutants in EBR-induced stomatal closure, whereas the stomata of eto1-1/AtrbohF and cGa/AtrbohF or eto1-1/nia1-2 and cGa/nia1-2 constructs had an analogous response to H 2 O 2 or SNP as those of AtrbohF or Nia1-2 mutants. Moreover, we provided evidence that Ga plays an important role in the responses of guard cells to ethylene. Ga activator CTX largely restored the lesion of the etr1-3 mutant, but ethylene precursor ACC failed to rescue the defects of gpa1 mutants in EBR-induced stomatal closure. Lastly, we demonstrated that Ga-activated H 2 O 2 production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H 2 O 2 production in mutant Nia1-2. Exogenously applied SNP rescued the defect of AtrbohF in EBR-induced stomatal closure, but H 2 O 2 did not reverse the lesion of EBR-induced stomatal closure in Nia1-2. Together, our results strongly suggest a signaling pathway in which EBR induces ethylene synthesis, thereby activating Ga, and then promotes AtrbohF-dependent H 2 O 2 production and subsequent Nia1-catalyzed NO accumulation, and finally closes stomata.

In flowering plants, EMS1 (Excess Microsporocytes 1) perceives TPD1 (Tapetum Determinant 1) to specify tapeta, the last somatic cell layer nurturing pollen development. However, the signaling components downstream of EMS1 are relatively unknown. Here, we use a molecular complementation approach to investigate the downstream components in EMS1 signaling. We show that the EMS1 intracellular domain is functionally interchangeable with that of the brassinosteroid receptor BRI1 (Brassinosteroid Insensitive 1). Furthermore, expressing EMS1 together with TPD1 in the BRI1 expression domain could partially rescue bri1 phenotypes, and led to the dephosphorylation of BES1, a hallmark of active BRI1 signaling. Conversely, expressing BRI1 in the EMS1 expression domain could partially rescue ems1 phenotypes. We further show that PpEMS1 and PpTPD1 from the early land plant Physcomitrella patens could completely rescue ems1 and tpd1 phenotypes, respectively. We propose that EMS1 and BRI1 have evolved distinct extracellular domains to control different biological processes but can act via a common intracellular signaling pathway.

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Previous studies have shown that cytokinins and auxins can induce the opening of stomata. However, the mechanism of stomatal opening caused by cytokinins and auxins remains unclear. The purpose of this paper is to investigate the relationship between hydrogen peroxide (H2O2) levels in guard cells and stomatal opening induced by cytokinins and auxins in Vicia faba. By means of stomatal bioassay and laser-scanning confocal microscopy, we provide evidence that cytokinins and auxins reduced the levels of H2O2 in guard cells and induced stomatal opening in darkness. Additionally, cytokinins not only reduced exogenous H2O2 levels in guard cells caused by exposure to light, but also abolished H2O2 that had been generated during a dark period, and promoted stomatal opening, as did ascorbic acid (ASA, an important reducing substrate for H2O2 removal). However, unlike cytokinins, auxins did not reduce exogenous H2O2, did not abolish H2O2 that had been generated in the dark, and therefore did not promote reopening of stoma induced to close in the dark. The above-mentioned effects of auxins were similar to that of diphenylene iodonium (DPI, an inhibitor of the H2O2-generating enzyme NADPH oxidase). Taken together our results indicate that cytokinins probably reduce the levels of H2O2 in guard cells by scavenging, whereas auxins limit H2O2 levels through restraining H2O2 generation, inducing stomatal opening in darkness.

Recently, in animals, carbon monoxide (CO), like nitric oxide (NO), was implicated as another important physiological messenger or bioactive molecule. Previous researches indicate that heme oxygenase (HO)-1 (EC 1.14.99.3) catalyzes the oxidative conversion of heme to CO and biliverdin IXa (BV) with the concomitant release of iron. However, little is known about the physiological roles of CO in plant, especially in stomatal movement of guard cells. In the present paper, the regulatory role of CO during stomatal movement in Vicia faba was surveyed. Results indicated that, like sodium nitroprusside (SNP), CO donor hematin induced stomatal closure in dose- and time-dependent manners. These responses were also proved by the addition of gaseous CO aqueous solution with different concentrations, showing for the first time that CO and NO exhibit similar regulation role in the stomatal movement. Moreover, our data showed that 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)/N(G)-nitro-L-arginine-methyl ester (L-NAME) not only reversed stomatal closure by CO, but also suppressed the NO fluorescence induced by CO, implying that CO-induced stomatal closure probably involves NO/nitric oxide synthase (NOS) signal system. Additionally, the CO/NO scavenger hemoglobin (Hb) and CO-specific synthetic inhibitor zinc protoporphyrin IX (ZnPPIX), NO scavenger cPTIO and NOS inhibitor L-NAME reversed the darkness-induced stomatal closure and NO fluorescence. These results show that, maybe like NO, the levels of CO in guard cells of V. faba is higher in dark than that in light, HO-1 and NOS are the enzyme systems responsible for generating endogenous CO and NO in darkness, respectively, and that CO being from HO-1 mediates darkness-induced NO synthesis in guard cells' stomatal closure of V. faba.

The role of nitric oxide (NO) in the ultraviolet-B radiation (UV-B)-induced reduction of in vitro pollen germination and tube growth of Paulownia tomentosa Steud. was studied. Results showed that exposure of the pollen to 0.4 and 0.8 W m(-2) UV-B radiation for 2 h resulted in not only the reduction of pollen germination and tube growth but also the enhancement of NO synthase (NOS, EC 1.14.13.39) activity and NO production in pollen grain and tube. Also, exogenous NO donors sodium nitroprusside and S-nitrosoglutathione inhibited both pollen germination and tube growth in a dose-dependence manner. NOS inhibitor N(G)-nitro-l-Arg-methyl eater (l-NAME) and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) not only largely prevented the NO generation but also partly reversed the UV-B-inhibited pollen germination and tube growth. These results indicate that UV-B radiation inhibits pollen germination and tube growth partly via promoting NO production in pollen grain and tube by a NOS-like enzyme. Additionally, a guanylyl cyclase inhibitor 6-anilino-5,8-quinolinequinone (LY-83583) prevented both the UV-B- and NO donors-inhibited pollen germination and tube growth, suggesting that the NO function is mediated by cyclic guanosine 5'-monophosphate. However, the effects of c-PTIO, l-NAME and LY-83583 on the UV-B-inhibited pollen germination and tube growth were only partial, suggesting that there are NO-independent pathways in UV-B signal networks.