Enantioselective Divergent Syntheses of Cephalotaxus Alkaloids: (-)-Cephalotaxine, (-)-Cephalotine B, and (-)-Fortuneicyclidins A and B.

A new strategy focusing on the last-stage asymmetric assembly of the ring D, which inherently possesses the densest part of stereogenic centers and functional groups in the A/B/C/D ring system of (-)-cephalotaxine, has been developed, in which a novel Rh-catalyzed asymmetric (2 + 3) annulation of tertiary enamides with enoldiazoacetates is designed and explored for enantioselective construction of the crucial cyclopentane ring D bearing a unique spirocyclic aza-quaternary stereocenter. Based on the expeditious access of chiral functionalized building block with the tetracyclic A/B/C/D ring system, a concise enantioselective total synthesis of (-)-cephalotaxine starting from readily available homopiperonyl alcohol has been achieved in nine steps with only two column chromatography purifications. Following the tactical introduction of the Meinwald rearrangement, enantioselective divergent syntheses of (-)-cephalotine B with an additional C3-O-C11 oxo-bridged bond (14 steps), (-)-fortuneicyclidin B with an unprecedented C3-C10 bond (14 steps), and its 2-epimer (-)-fortuneicyclidin A (16 steps) have been also accomplished for the first time.

Iron(III)-catalyzed tandem annulation of indolyl-substituted p-quinone methides with ynamides for the synthesis of cyclopenta[b]indoles.

The unique reactivity of indolyl-substituted p-QMs as a new type of two-carbon synthon has been explored for the first time in a novel iron(III)-catalyzed tandem annulation. This (2+2) annulation/retro-4π electrocyclization/imino-Nazarov cyclization cascade reaction is characterized by an unusual structural reconstruction of indolyl-substituted p-QMs, leading to an expeditious assembly of synthetically important functionalized cyclopenta[b]indoles.

Copper-Catalyzed (4+1) Cascade Annulation of Terminal Alkynes with 2-(Tosylmethyl)anilines: Synthesis of 2,3-Disubstituted Indoles.

A novel strategy based on Cu-catalyzed (4+1) cascade annulation of terminal alkynes as one-carbon synthons with 2-(tosylmethyl)anilines has been developed for the expeditious synthesis of 2,3-disubstituted indoles, in which in situ generations of aza-o-quinone methides and alkynyl-copper(I) species are involved. This annulation provides an effective method for the assembly of synthetically and structurally interesting 2,3-disubstituted indoles.

Tandem (2 + 2) Annulation/Retro-4π Electrocyclization/Imino-Nazarov Cyclization Reaction of p-Quinone Methides with Ynamides: Expeditious Construction of Functionalized Aminoindenes.

A new tandem annulation of p-quinone methides (p-QMs) with ynamides is described. This cascade reaction features a unique combination of (2 + 2) annulation, retro-4π electrocyclization, and imino-Nazarov cyclization, wherein vinyl p-quinone methides (p-VQMs) as one of the key intermediates have been identified chemically. Significantly, an unusual structural reconstruction of p-QMs involving the cleavage of the C5-C6 bond and the late-stage formation of the C4-C6 bond is involved, leading to a methodology development for the construction of functionalized aminoindenes.

Ethylene-induced stomatal closure is mediated via MKK1/3-MPK3/6 cascade to EIN2 and EIN3.

Mitogen-activated protein kinases (MPKs) play essential roles in guard cell signaling, but whether MPK cascades participate in guard cell ethylene signaling and interact with hydrogen peroxide (H2 O2 ), nitric oxide (NO), and ethylene-signaling components remain unclear. Here, we report that ethylene activated MPK3 and MPK6 in the leaves of wild-type Arabidopsis thaliana as well as ethylene insensitive2 (ein2), ein3, nitrate reductase1 (nia1), and nia2 mutants, but this effect was impaired in ethylene response1 (etr1), nicotinamide adenine dinucleotide phosphate oxidase AtrbohF, mpk kinase1 (mkk1), and mkk3 mutants. By contrast, the constitutive triple response1 (ctr1) mutant had constitutively active MPK3 and MPK6. Yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MPK3 and MPK6 physically interacted with MKK1, MKK3, and the C-terminal region of EIN2 (EIN2 CEND). mkk1, mkk3, mpk3, and mpk6 mutants had typical levels of ethylene-induced H2 O2 generation but impaired ethylene-induced EIN2 CEND cleavage and nuclear translocation, EIN3 protein accumulation, NO production in guard cells, and stomatal closure. These results show that the MKK1/3-MPK3/6 cascade mediates ethylene-induced stomatal closure by functioning downstream of ETR1, CTR1, and H2 O2 to interact with EIN2, thereby promoting EIN3 accumulation and EIN3-dependent NO production in guard cells.

UV RESISTANCE LOCUS8 mediates ultraviolet-B-induced stomatal closure in an ethylene-dependent manner.

Although the UV RESISTANCE LOCUS 8 (UVR8)-CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-ELONGATED HYPOCOTYL5 (HY5) signaling pathway, ethylene, hydrogen peroxide (H2O2), and nitric oxide (NO) all participate in ultraviolet-B (UV-B)-triggered stomatal closing, their interrelationship is not clear. Here, we found that UV-B-induced the expression of ethylene biosynthetic genes, production of ethylene, H2O2, and NO, and stomata closing were impaired in uvr8, cop1, and hy5 mutants. UV-B-induced NO production and stomata closing were also defective in mutants for ETHYLENE RESPONSE 1 (ETR1), ETHYLENE INSENSITIVE 2 (EIN2), and EIN3, but UV-B-triggered H2O2 generation was only inhibited in etr1. In either the absence or presence of UV-B, ethylene triggered H2O2 production but not NO generation and stomatal closure in cop1 and hy5, and stomata closing in cop1 and hy5 was induced by NO but not H2O2. Moreover, NO production and stomatal closure were constitutively caused by over-expression of COP1 or HY5 in ein2 and ein3, but not by over-expression of EIN2 or EIN3 in cop1 and hy5. Our data indicate that the UVR8-COP1-HY5 signaling module mediates UV-B-induced ethylene production, ethylene is then perceived by ETR1 to induce H2O2 synthesis. H2O2 induces NO generation and subsequent stomata closing via an EIN2, EIN3, COP1, and HY5-dependent pathway(s).

Heterotrimeric G protein mediates ethylene-induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis.

Heterotrimeric G proteins function as key players in hydrogen peroxide (H2O2) production in plant cells, but whether G proteins mediate ethylene-induced H2O2 production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H2O2 in guard cell ethylene signalling. In wild-type leaves, ethylene-triggered H2O2 synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene-induced H2O2 production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H2O2 production in response to ethylene. Ethylene-triggered H2O2 generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H2O2 rescued the defect of RAN1 and EIN4 mutants or etr1-3 in ethylene-induced H2O2 production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1-1 and etr1-9 in ethylene-induced H2O2 production. Stomata of CTR1 mutants showed constitutive H2O2 production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H2O2, but do generate H2O2 following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene-induced stomatal closure via H2O2 production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H2O2 signalling in guard cells.

The role and interactions of cytosolic alkalization and hydrogen peroxide in ultraviolet B-induced stomatal closure in Arabidopsis.

Cytosolic alkalization has been shown to function as a key player in multiple stimuli-induced stomatal closure, but its role and relationship with hydrogen peroxide (H2O2) in ultraviolet B (UV-B)-induced stomatal closure remains unknown. In this paper, by stomatal bioassay and laser-scanning confocal microscopy, we observed that 0.5 W m(-2) UV-B induced cytosolic alkalization and H2O2 production in guard cells while inducing stomatal closure in Arabidopsis (Arabidopsis thaliana). Butyrate (a weak acid) reduced the cytosolic pH/H2O2 production and prevented stomatal closure by UV-B. Methylamine (a weak base) induced H2O2 production and stomatal closure while enhancing the cytosolic alkalization in guard cells under light alone. The rise in cytosolic pH of wild-type guard cells on exposure to UV-B was evident at 15 min and substantial at 45 min while H2O2 production started to largely increase after 60 min. The failure of UV-B-induced H2O2 production in AtrbohD/F guard cells did not affect the changes of guard cell pH during the first 60 min of UV-B radiation, but largely suppressed cytosolic alkalization after 60 min of UV-B radiation. These results indicate that cytosolic alkalization mediates UV-B-induced stomatal closure via activating H2O2 production and that H2O2 production can feedback-enhance cytosolic alkalization in Arabidopsis guard cells.

Cytosolic alkalisation and nitric oxide production in UVB-induced stomatal closure in Arabidopsis thaliana.

The role and the interrelationship of cytosolic alkalisation and nitric oxide (NO) in UVB-induced stomatal closure were investigated in Arabidopsis thaliana (L.) Heynh. by stomatal bioassay and laser-scanning confocal microscopy. In response to 0.5Wm-2 UVB radiation, the rise of NO levels in guard cells occurred after cytosolic alkalisation but preceded stomatal closure. UVB-induced NO production and stomatal closure were both inhibited by NO scavengers, nitrate reductase (NR) inhibitors and a Nia2-5/Nia1-2 mutation, and also by butyrate. Methylamine induced NO generation and stomatal closure in the wild-type but not in the Nia2-5/Nia1-2 mutant or wild-type plants pretreated with NO scavengers or NR inhibitors while enhancing the cytosolic pH in guard cells under light. NO generation in wild-type guard cells was largely induced after 60min of UVB radiation. The defect in UVB-induced NO generation in Nia2-5/Nia1-2 guard cells did not affect the changes of guard cell pH before 60min of UVB radiation, but prevented the UVB-induced cytosolic alkalisation after 60min of radiation. Meanwhile, exogenous NO caused a marked rise of cytosolic pH in guard cells. Together, our results show that cytosolic alkalisation and NR-dependent NO production coordinately function in UVB signalling in A. thaliana guard cells.