Action spectrum of photoinhibition in leaves of wild type and npq1-2 and npq4-1 mutants of Arabidopsis thaliana

Photoinhibition is light-induced inactivation of PSII. Hypothesesabout the photoreceptor(s) of photoinhibition include the Chlantenna of PSII, manganese of the oxygen-evolving complex (OEC),uncoupled Chl and iron–sulfur centres. We measured theaction spectrum of photoinhibition in vivo from wild-type Arabidopsisthaliana L. and from the npq1-2 and npq4-1 mutants defectivein non-photochemical quenching (NPQ) of excitations of the PSIIantenna. The in vivo action spectrum was found to resemble closelythe in vitro action spectra published for photoinhibition. Wecompared the action spectrum with absorbance spectra of modelcompounds of the OEC complex and other potential photoreceptorsof photoinhibition. The comparison suggests that both manganeseand Chl function as photoreceptors in photoinhibition. In accordancewith the function of two types of photoreceptors in photoinhibition,NPQ was found to offer only partial protection against photoinhibitionat visible wavelengths. The low protective efficiency of NPQsupports the conclusion that the Chl antenna of PSII is notthe only photoreceptor of photoinhibition. Comparison of theaction spectrum of photoinhibition with the emission spectrumof sunlight shows that the UV part of sunlight is responsiblefor the major part of photoinhibition under natural conditions. (Received September 7, 2005; Accepted January 4, 2006)     

A transgene encoding a blue-light receptor, phot1, restores blue-light responses in the Arabidopsis phot1 phot2 double mutant

Phototropins (phot1 and phot2) are suggested to be multifunctional blue-light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses.     

Phospholipase A2beta mediates light-induced stomatal opening in Arabidopsis

Phospholipase A(2) (PLA(2)) catalyses the hydrolysis of phospholipids into lysophospholipids and free fatty acids. Physiological studies have indicated that PLA(2) is involved in stomatal movement. However, genetic evidence of a role of PLA(2) in guard cell signalling has not yet been reported. To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out. Light-induced stomatal opening in PLA(2)alpha knockout plants did not differ from wild-type plants. Plants in which PLA(2)beta was silenced by RNA interference exhibited delayed light-induced stomatal opening, and this phenotype was reversed by exogenous lysophospholipids, which are products of PLA(2). Stomatal opening in transgenic plants that over-expressed PLA(2)beta was faster than wild-type plants. The expression of PLA(2)beta was localized to the endoplasmic reticulum of guard cells, and increased in response to light in the mature leaf. Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta. Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.     

Intraspecific interactions in Arabidopsis thaliana and the stomatal mutants tmm1-1 and sdd1-2

Competition is a major density-dependent factor structuring plant populations and communities in both natural and agricultural systems. Seedlings of the model plant species Arabidopsis thaliana cv. Columbia, and the Columbia-derived stomatal mutants sdd1 and tmm1, were grown under controlled conditions at increasing densities of 1, 10, 20, and 50 plants per pot. We demonstrate significant effects of time (days after planting), density, genotype, density and genotype, and the three-way interaction with time upon several fitness components (plant height, silique number, leaf biomass and flowering stalk biomass) in Columbia and these mutants.     

The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.     

Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2 , display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.
Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis . The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 m M . Stomatal apertures were increased by lowering external Ca²⁺ to 0.05 m M . The apertures of stomates incubated with 10 μ M ABA were not altered by changes in Ca²⁺ from 0.05 to 1.0 m M .
Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.     

Role of vacuolar invertase in regulating Arabidopsis stomatal opening

Guard cells sense and integrate environmental signals to modulate stomatal aperture in response to diverse conditions. In this study, the effect of vacuolar invertase on Arabidopsis stomatal opening was investigated. The technology of enzyme activity detection in situ was used to show that the vacuolar invertase activity was much higher in guard cells than in other epidermal cells. The stomatal aperture of T-DNA insertion mutant in At1g12240 (inv-7) was significantly lower than that in wild-type plants. Increased stomatal aperture was observed in the transgenic Arabidopsis overexpressing cotton vacuolar invertase gene. These results indicated that Arabidopsis stomatal aperture was correlated with vacuolar invertase, and that vacuolar invertase may play an important role in regulating Arabidopsis stomatal opening.     

Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling

In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.     

Light-induced stomatal movement of selected Arabidopsis thaliana mutants

Various Arabidopsis thaliana mutants with defects in phytohormone signal transduction or the reception of light were analysed with regard to their stomatal response in a red, red/blue light irradiation programme. Stomatal response to light was detected with a customized gas exchange measurement device, optimized for the small model plant. Small transpiration-kinetic variations of the two wild-type lines Columbia (Col) and Landsberg erecta (Ler) were observed. A comparison of the mutant lines to the respective wild type revealed significant differences for the phytochrome A (phyA-103), the abscisic acid insensitive (aba3-2) and the auxin resistant (axr1-3) mutant. Furthermore, the zeaxanthin-less mutant line npq1-2 showed no alterations in stomatal response to light.     

Altering arabinans increases Arabidopsis guard cell flexibility and stomatal opening

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The effect of wavelength of light on stomatal opening

The effect of broad band green, blue and red light on stomatal opening of Vicia faba L. (broad bean) leaves was examined. In air, blue light caused greater stomatal opening than red light. In air with green light stomata were only slightly opened. In a nitrogen atmosphere red light caused greater opening than blue light, and green light caused only slight opening. Opening in air or nitrogen atmosphere in red or blue light was inhibited by the uncoupler CCCP, while the photosynthetic inhibitor DCMU inhibited opening in air but not in nitrogen atmosphere. We concluded that more than one light activated metabolic pathway can supply the energy needed to effect stomatal opening and that different pathways are operative under different conditions.     

Reversal of blue light-stimulated stomatal opening by green light

Blue light-stimulated stomatal opening in detached epidermis of Vicia faba is reversed by green light. A 30 s green light pulse eliminated the transient opening stimulated by an immediately preceding blue light pulse. Opening was restored by a subsequent blue light pulse. An initial green light pulse did not alter the response to a subsequent blue light pulse. Reversal also occurred under continuous illumination, with or without a saturating red light background. The magnitude of the green light reversal depended on fluence rate, with full reversal observed at a green light fluence rate twice that of the blue light. Continuous green light given alone stimulated a slight stomatal opening, and had no effect on red light-stimulated opening. An action spectrum for the green light effect showed a maximum at 540 nm and minor peaks at 490 and 580 nm. This spectrum is similar to the action spectrum for blue light-stimulated stomatal opening, red-shifted by about 90 nm. The carotenoid zeaxanthin has been implicated as a photoreceptor for the stomatal blue light response. Blue/green reversibility might be explained by a pair of interconvertible zeaxanthin isomers, one absorbing in the blue and the other in the green, with the green absorbing form being the physiologically active one.     

Stomata from npq1, a zeaxanthin-less Arabidopsis mutant, lack a specific response to blue light

The Arabidopsis mutant npq1, which cannot accumulate zeaxanthin because of a defective violaxanthin deepoxidase, was used to investigate the role of zeaxanthin in the stomatal response to blue light. Neither dark-adapted nor light-treated guard cells or mesophyll cells of the npq1 mutant contained detectable zeaxanthin. In contrast, wild-type guard cells had a significant zeaxanthin content in the dark and accumulated large amounts of zeaxanthin when illuminated. The well-documented red light enhancement of blue light-stimulated stomatal opening, in which increasing fluence rates of background red light result in increased response to blue light, was used to probe the specific blue light response of Arabidopsis stomata. Stomata from the npq1 mutant did not have a specific blue light response under all fluence rates of background red light tested. On the other hand, stomata from leaves of hy4 (cry 1), an Arabidopsis mutant lacking blue light-dependent inhibition of hypocotyl elongation, had a typical enhancement of the blue light response by background red light. The lack of a specific blue light response in the zeaxanthinless npq1 mutant provides genetic evidence for the role of zeaxanthin as a blue light photoreceptor in guard cells.     

RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana

Phototropin 1 (phot1) and phot2, which are blue light receptor kinases, function in blue light-induced hypocotyl phototropism, chloroplast relocation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Previous studies have shown that the proteins RPT2 (for ROOT PHOTOTROPISM2) and NPH3 (for NONPHOTOTROPIC HYPOCOTYL3) transduce signals downstream of phototropins to induce the phototropic response. However, the involvement of RPT2 and NPH3 in stomatal opening and in chloroplast relocation mediated by phot1 and phot2 was unknown. Genetic analysis of the rpt2 mutant and of a series of double mutants indicates that RPT2 is involved in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. Biochemical analyses indicate that RPT2 is purified in the crude microsomal fraction, as well as phot1 and NPH3, and that RPT2 makes a complex with phot1 in vivo. On the other hand, NPH3 is not necessary for stomatal opening or chloroplast relocation. Thus, these results suggest that phot1 and phot2 choose different signal transducers to induce three responses: phototropic response of hypocotyl, stomatal opening, and chloroplast relocation.     

In the light of stomatal opening: new insights into 'the Watergate'

Stomata can be regarded as hydraulically driven valves in the leaf surface, which open to allow CO2 uptake and close to prevent excessive loss of water. Movement of these 'Watergates' is regulated by environmental conditions, such as light, CO2 and humidity. Guard cells can sense environmental conditions and function as motor cells within the stomatal complex. Stomatal movement results from the transport of K+ salts across the guard cell membranes. In this review, we discuss the biophysical principles and mechanisms of stomatal movement and relate these to ion transport at the plasma membrane and vacuolar membrane. Studies with isolated guard cells, combined with recordings on single guard cells in intact plants, revealed that light stimulates stomatal opening via blue light-specific and photosynthetic-active radiation-dependent pathways. In addition, guard cells sense changes in air humidity and the water status of distant tissues via the stress hormone abscisic acid (ABA). Guard cells thus provide an excellent system to study cross-talk, as multiple signaling pathways induce both short- and long-term responses in these sensory cells.     

NDPK2 as a signal transducer in the phytochrome-mediated light signaling

Nucleoside-diphosphate kinase (NDPK) 2 in Arabidopsis has been identified as a phytochrome-interacting protein by using the C-terminal domain of phytochrome A (PhyA) as the bait in yeast two-hybrid screening. The far-red light-absorbing form of phytochrome (Pfr) A stimulates NDPK2 gamma-phosphate exchange activity in vitro. To better understand the multiple functions of NDPK and its role in phytochrome-mediated signaling, we characterized the interaction between phytochrome and NDPK2. Domain studies revealed that PER-ARNT-SIM domain A in the C-terminal domain of phytochrome is the binding site for NDPK2. Additionally, phytochrome recognizes both the NDPK2 C-terminal fragment and the NDPK2 hexameric structure to fulfill its binding. To illustrate the mechanism of how the Pfr form of phytochrome stimulates NDPK2, His-197-surrounding residue mutants were made and tested. Results suggested that the H-bonding with His-197 inside the nucleotide-binding pocket is critical for NDPK2 functioning. The pH dependence profiles of NDPK2 indicated that mutants with different activities from the wild type have different pK(a) values of His-197 and that NDPK2 hyperactive mutants possess lower pK(a) values. Because a lower pK(a) value of His-197 accelerates NDPK2 autophosphorylation and the phospho-transfer between the phosphorylated NDPK2 and its kinase substrate, we concluded that the Pfr form of phytochrome stimulates NDPK2 by lowering the pK(a) value of His-197.     

Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation

In response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2-1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1-1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.