| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
What's this? |
Physiology and Development |
2Biology Department, Santa Clara University, Santa Clara, California 95053 USA; 3Chemistry Department, University of California, Santa Cruz, California 95064 USA; 4Biology Department, University of California, Santa Cruz, California 95064 USA
Received for publication February 6, 2003. Accepted for publication May 30, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Key Words: Arabidopsis • guard cell • photoreceptor • phototropin • phototropism • stomate • Vicia faba • zeaxanthin
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
; Cashmore et al., 1999
), anthocyanin synthesis (Ahmad et al., 1995
; Wade et al., 2001
), and flowering (Guo et al., 1998
), and PHOT1 and PHOT2, which encode phototropins, the BL photoreceptors involved in phototropism (Christie et al., 1998
; Briggs and Christie, 2002
) and chloroplast movement (Jarillo et al., 2001
; Kagawa et al., 2001
).
The CRY and PHOT genes encode flavoproteins, consistent with the presence of a peak in the UV-A in the action spectra for their responses. Trans-carotenoids, which make up the bulk of the carotenoids in the chloroplast, do not absorb UV-A. Thus, a peak in the UV-A region of the action spectrum has been considered diagnostic for flavoproteins. Recently, we reported the ultraviolet action spectrum for stomatal opening in Vicia faba (Eisinger et al., 2000
). A small peak of activity at around 360 nm was observed, as was a much larger peak at around 280 nm, consistent with a flavoprotein BL photoreceptor in guard cells. This prediction was confirmed by Kinoshita et al. (2001)
, who showed that both PHOT1 and PHOT2 were required for BL-induced stomatal opening.
In contrast, Zeiger and his colleagues have proposed the carotenoid zeaxanthin as the BL photoreceptor of guard cells (Zeiger, 2000
). A key finding in support of the model is that stomata of npq1, a mutant of Arabidopsis blocked at the zeaxanthin de-epoxidase step of zeaxanthin biosynthesis, fail to open in response to blue light, although they show normal red light-induced opening (Frechilla et al., 1999
). More recently, Zeiger and colleagues have determined that green light reverses the effects of blue light on stomatal opening (Frechilla et al., 2000
). By analogy with phytochrome, blue-green photoreversibility of stomatal opening is consistent with a model in which the zeaxanthin chromophore undergoes a trans-to-cis isomerization within the chloroplast, which results in the transmission of a signal to the cytosol, where the response occurs (Zeiger, 2000
). Cis-carotenoids, unlike trans-carotenoids, have an absorption peak in the UV-A region (Molnar and Szabolcs, 1993
). However, according to the model, absorption of light by cis-zeaxanthin should convert the pigment to the inactive trans form, inhibiting stomatal opening, not promoting it. Thus the zeaxanthin model is not in accord with the presence of a small peak of activity in the UV-A region of the action spectrum, but rather supports the participation of phototropins 1 and 2 in UV-A-induced stomatal opening.
The significance of the large peak in the UV-B region of the spectrum is unclear. Treatment with UV-B in the presence of saturating BL failed to cause any additional stomatal opening. Therefore, we initially proposed that the UV-B and BL responses could be mediated by the same protein-pigment molecule, with BL being absorbed by the chromophore and UV-B absorbed by the protein (Eisinger et al., 2000
).
In this report, we follow up our previous observations by investigating the interactions between ultraviolet (UV-B and UV-A) and visible (blue and green) wavelengths in stomatal opening. We also characterize the UV and visible light responses of two xanthophyll-cycle mutants of Arabidopsis, npq1 and aba1. The aba1 mutant is blocked in the synthesis of the enzyme zeaxanthin epoxidase, the enzyme that converts zeaxanthin to violaxanthin, and is characterized by an excess of zeaxanthin (Duckham et al., 1991
; Rock and Zeevaart, 1991
). The responses of the phot1/phot2 double mutant to both UV-B and GL were also determined. Finally, we examined the phototropic bending responses of the phot1/phot2 mutants in response to UV-B. All of the data collected thus far are consistent with a model based on a complex photoreceptor network regulating stomatal opening. The UV-B response in guard cells is mediated by a separate UV-B receptor. In contrast, the UV-B response of phototropism appears to be mediated primarily by phot1 and phot2.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Arabidopsis (mutants and their Col and Ler background) plants were grown in a plant growth chamber at 22°C with a 10-h photoperiod for about a month. Only rosette leaves were used and collected from plants in the vegetative growth stage. Because the leaves were often too small to prepare epidermal peels, stomatal apertures were measured from whole-leaf mounts under glass slides. Whole-leaf mounts gave faster and more reproducible results than epidermal peels.
The UV-light source was a 75-W xenon arc lamp. Specific wavelengths were selected using a Bausch & Lomb (Rochester, New York, USA) high intensity monochromator. For UV experiments, stray visible light was excluded from the monochromater beam using a Corning 7-54 quartz filter (Kopp/Corning, Pittsburgh, Pennsylvania, USA) that transmits only light in the 250–410 nm range. The monochromator was positioned on a shelf above the bench and the beam was directed downwards by means of a front-surface mirror. Fluence rates were determined using a PIN-8 photodiode (United Detector Technologies, Baltimore, Maryland, USA) with extended UV range, which was previously spectrally calibrated against a Kettering 68 (Milton Roy, Florida, USA) thermopile in the 220–700 nm range. The Kettering radiometer calibration is traceable to NBS standard lamps. The calibration was confirmed within 10% using the actinochrome chemical actinometer (Brauer et al., 1983
).
After 1 h of light treatment, the peels were viewed in a Zeiss fluorescence light microscope (Oberkochen, Germany) using a 40x objective and digitally recorded using a Panasonic model WV 850 infrared-sensitive (
800 nm) video camera (New York, New York, USA) and a Packard-Bell PBTV4 video capture card (Sacramento, California, USA). The digitized images were later viewed with Adobe Photoshop (San Jose, California, USA) software, and stomatal apertures were measured using the "Information Palette" feature of that program. A minimum of 50 stomata were measured per light treatment, and each experiment was repeated a minimum of three times.
In experiments involving the combination of two wavelengths, the primary light source was a halogen bulb (Phillips 20 MR 16, Koninklijke Philips Electronics, Somerset, New Jersey, USA) with a Tempax wide band hot mirror filter (Schott Glaswerke, Mainz, Germany). The fluence at tissue level was approximately 500 µmol · m–2 · s–1. Corning glass filters plus hot mirrors or narrow band interference filters were used to provide specific wavelength ranges: 650 nm (630–750 nm); 550 nm (545–555 nm), and 450 nm (400–490 nm). The secondary light source was the xenon arc lamp with monochromator (12 nm bandwidth) described earlier set at 284 nm or 360 nm and providing approximately 0.18 µmol · m–2 · s–1 at tissue level. After an hour of illumination, peels were photographed as described and stomatal apertures were analyzed using Photoshop.
For phototropism experiments, seedlings were grown in potting mixture for 5–6 d in the dark, placed 
2 m from the monochromator on the bench top. The fluence rate at tissue level was approximately 1 µmol · m–2 · s–1 for blue light experiments and 0.1 µmol · m–2 · s–1 for UV-B experiments. A Corning 7-54 filter transmitting only wavelengths between 250 and 410 nm excluded stray visible light in UV-B experiments. Seedlings were irradiated with unilateral blue light (450 nm) or UV-B (280 nm) for 6–24 h and then photographed with a digital camera.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
that GL antagonized the effects of BL in stomatal opening (data not shown). We therefore tested whether GL could block the effects of UV-B on stomatal opening. The results are shown in Fig. 1. Green light alone induced a small amount of opening of Vicia faba stomata. However, when given in combination with UV-B, GL strongly antagonized the stimulatory effects of UV-B on stomatal opening. The ability of GL to antagonize UV-B-induced as well as BL-induced stomatal opening suggests that the fundamental mechanisms of BL-induced and UV-B-induced stomatal opening are the same.
|
60% of the opening induced by blue light, consistent with the previously established action spectrum (Eisinger et al., 2000
). However, when given in combination with BL, UV-A blocked BL-induced opening to the level of the dark control. Thus, UV-A is at least as effective as GL in antagonizing BL-induced opening.
|
|
|
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). Although the effects of BL and UV-B were not found to be additive (Eisinger et al., 2000
), the experiments described in the present study provide strong evidence that the UV-B response is mediated by a separate UV-B receptor. The two main candidates for the BL photoreceptor are the carotenoid zeaxanthin and the flavoproteins, phot1 and phot2.
One of the main arguments used to support the zeaxanthin hypothesis has been the correlation between bulk zeaxanthin levels and the BL response. Pretreating guard cells with increasing fluences of red light enhanced both total zeaxanthin content and the response to a subsequent BL treatment (Frechilla et al., 1999
). Conversely, treatment with 3 mmol/L DTT (dithiothreitol) inhibited zeaxanthin synthesis and also inhibited BL-dependent stomatal opening (Frechilla et al., 1999
). However, our finding that the aba1 mutant, which accumulates zeaxanthin, is less responsive than wild type to BL, suggests that the BL response does not always correlate with bulk zeaxanthin. This finding is consistent with the presence of a small peak in the action spectrum at 360 nm, where the bulk zeaxanthin in the trans form does not absorb (Eisinger et al., 2000
). However, it is possible that a small subset of the total zeaxanthin population is in the cis form and that this represents the physiologically active zeaxanthin BL photoreceptors. In fact, bulk zeaxanthin might actually inhibit the BL response, as occurs in the aba1 mutant, as a result of shielding effects. This overall interpretation is supported by studies with the zeaxanthin-deficient mutant, npq1.
Frechilla et al. (1999)
initially reported that stomata of npq1 failed to open in response to BL, although it showed normal red light-induced opening. Subsequently, both Eckert and Kaldenhoff (2000)
and Kinoshita et al. (2001)
reported that under their conditions, the npq1 mutant had normal BL-induced stomatal opening. In the present study, we were able to confirm the results of Frechilla et al. (1999)
that the stomata of npq1 do not respond to BL. As will be discussed later, the source of the discrepancy in the npq1 results has now been resolved in a way that leaves the zeaxanthin hypothesis intact. In the meantime, however, Kinoshita et al. (2001)
also demonstrated that a phot1/phot2 double mutant lacked a BL response, leading them to conclude that phototropins, rather than zeaxanthin, serve as the BL receptors of guard cells. Thus, there are now two viable candidates for the BL photoreceptor of guard cells: zeaxanthin and the phototropins.
The first major finding of the current study is that stomata of both npq1 and phot1/phot2 open normally in response to UV-B. Because these mutants have normal UV-B-induced stomatal opening, we conclude that the UV-B response is mediated by a separate UV-B receptor. This conclusion is similar to that of a recent report on the regulation of chalcone synthase in Arabidopsis leaf tissue in which there are two regulatory pathways: a BL pathway involving cry1 and a separate UV-B pathway mediated by an unidentified UV-B receptor (Wade et al., 2001
). The authors proposed a photoreceptor network for the regulation of chalcone synthase involving the UV-B receptor, cry1, phyA, and phyB (Wade et al., 2001
). According to the model, phyB specifically antagonizes the UV-B receptor, while both phyA and phyB act synergistically with cry1.
The BL response of stomatal guard cells can be antagonized by GL (Frechilla et al., 2000
; Talbott et al., 2002
). The second major finding of the current report is that GL also antagonizes UV-B-induced opening. We also took advantage of the fact that both the npq1 and phot1/phot2 mutants opened their stomata in response to UV-B to test whether zeaxanthin or the phototropins could also serve as the GL photoreceptor. Although stomata of both mutants opened normally in response to UV-B, neither showed the normal GL antagonism of the UV-B response. Thus, we were able to show that both zeaxanthin and the phototropins are both required for GL inhibition of stomatal opening and that the UV-B receptor is separate from the GL photoreceptor.
As to the identity of the GL photoreceptor, once again the data do not allow us to choose between zeaxanthin and the phototropins because neither of the mutants respond to GL. Zeiger and colleagues (2000)
have proposed a model for zeaxanthin analogous to the phytochrome Pr/Pfr photoreversible reaction. In vitro spectroscopic evidence that BL stimulation and GL inhibition are mediated by a reversible cis-trans isomerization of a pigment in chloroplast membrane has been obtained (Zeiger et al., 2000
). When isolated thylakoids of A. thaliana were irradiated with alternating blue and green pulses, the difference spectrum exhibited a photoreversible bleaching in the blue region and an increase in absorption in the green and UV-A regions. Thylakoids from the zeaxanthin-deficient mutant npq1 lacked the response, and the response could be reconstituted by adding exogenous zeaxanthin to the reaction, but not violaxanthin (Zeiger et al., 2000
).
There is no known blue-green photoreversible reactions in phototropins 1 and 2 comparable to the cis-trans isomerization reaction described for zeaxanthin. Moreover, GL does not antagonize BL during phototropism as it does during stomatal opening (L. Taiz and W. R. Eisinger, unpublished data). Assuming that phot1 and phot2 are the photoreceptors for phototropism, the absence of GL antagonism in phototropism suggests very different photochemical reactions in the two processes. On the other hand, if zeaxanthin is the blue-green photoreversible photoreceptor, how does one explain the lack of GL antagonism in the phot1/phot2 double mutant? If it is assumed that zeaxanthin is restricted to the chloroplast and phototropins are located in the cytosol, physical interaction between the two would seem to be ruled out. Alternatively, a signaling intermediate emanating from the chloroplast might interact either positively or negatively with phototropins, and both partners might be required for the blue-green reversible response. Because GL also antagonizes UV-B, this hypothetical interaction between the chloroplast signal and the phototropins must act downstream of the UV-B receptor, which would also explain why the effects of BL and UV-B are not additive, despite the existence of separate photoreceptors.
As noted earlier, the role of zeaxanthin in blue-light-induced stomatal opening has been challenged by two different laboratories. Both Eckert and Kaldenhoff (2000)
and Kinoshita et al. (2001)
observed normal stomatal opening in npq1 in response to blue light. Their results conflict with the results presented here and with the results of Frechilla et al. (1999)
. More recently, it has been shown that the BL response, measurable under certain conditions, in the npq1 mutant can be reversed by far-red light, suggesting the participation of phytochrome rather than the BL photoreceptor (E. Zeiger, UCLA, personal communication). Phytochrome is also known to be involved in stomatal opening in the orchid Paphiopedilum, whose guard cells lack developed chloroplasts (Talbott et al., 2002
). However, far-red reversal is not observed in wild-type Arabidopsis, suggesting that the phytochrome mechanism of BL-induced opening is normally suppressed by the chloroplast-derived zeaxanthin pathway.
Interestingly, green light causes the opening of Paphiopedilum stomata, and green light-induced opening can be reversed by far-red light (Talbott et al., 2002
). Thus, when the phytochrome pathway is activated (that is, when the zeaxanthin pathway is weak) the green-light response is mediated by phytochrome and is thus far-red reversible.
Could phytochrome be acting as the UV-B receptor? There are currently two arguments against this hypothesis: first, the UV-B stomatal opening pathway is active in the wild type under conditions when the phytochrome pathway appears to be inactive; second, green light acts as an inhibitor of stomatal opening in the UV-B pathway, whereas green light causes stomatal opening in the phytochrome pathway. In other words, green light has opposite effects on stomatal opening in the two pathways. It may be relevant in this regard that in the photoreceptor network proposed for chalcone synthase, PhyB acted as an inhibitor of the UV-B receptor (Wade et al., 2001
).
Figure 11 shows a working model for the photoreceptor network proposed to regulate stomatal opening. A subset of zeaxanthin molecules, perhaps located on the chloroplast inner membrane, can undergo a photoreversible trans-cis isomerization in response to blue and green/UV-A light. In response to blue light, a positive signaling intermediate is transmitted to the cytosol where it interacts with phot1 and phot2. In turn, phot1 and phot2 act as the primary blue/UV-A photoreceptors, which bring about the activation of the plasma membrane H+-ATPase involved in stomatal opening. Green light and UV-A drive the physiologically active zeaxanthin to the inactive trans state, which results in a negative signal in the cytosol, which blocks the action of phot1 and phot2. Because phot1 and phot2 must interact with the positive zeaxanthin signal, any mutation that blocks zeaxanthin synthesis would also block the action of phot1 and phot2. However, the same signal that activates phot1 and phot2 also appears to repress the action of phytochrome. Thus, when the zeaxanthin-signaling intermediate is blocked, as in the case of npq1, blue-light-induced opening can be antagonized by far-red light. Opening in the stomata of Paphiopedalum. which lack chloroplasts, can be stimulated by red light and green light as well, and in both cases the effect can be prevented by far-red light (Talbott et al., 2002
).
|
Finally, it seems likely that such complex networks of photoreceptors will turn out to be the rule rather than the exception in plant photomorphogensis. The control of flowering is another example of photoreceptor networks involving phytochrome and cryptochrome genes. The era of simple linear photoreceptor models would seem to be over.
|
FOOTNOTES |
|---|
5 E-mail: weisinger{at}scu.edu
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Ahmad M. C. Lin A. R. Cashmore 1995 Mutations throughout an Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin accumulation and inhibition of hypocotyl elongation. Plant Journal 8: 653-658[CrossRef][Web of Science][Medline]
Brauer H. D. R. Schmidt G. Gauglitz S. Hubig 1983 Chemical actinometry in the visible (475–610 nm) by meso-diphenylhelinthrene. Photochemistry and Photobiology 37: 595-598[CrossRef]
Briggs W. R. J. M. Christie 2002 Phototropin 1 and phototropin 2: two versatile plant blue-light receptors. Trends in Plant Science 7: 204-210[CrossRef][Web of Science][Medline]
Cashmore A. R. J. A. Jarillo Y.-J. Wu D. Liu 1999 Cryptochromes: blue light receptors for plants and animals. Science 284: 7607-65
Christie J. M. P. Reymond G. K. Powell P. Bernasconi A. A. Raibekas E. Liscum W. R. Briggs 1998 Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism. Science 282: 1698-1701
Duckham S. C. R. S. T. Linforth I. B. Taylor 1991 Abscisic-acid-deficient mutants at the aba gene locus of Arabidopsis thaliana are impaired in the epoxidation of zeaxanthin. Plant, Cell and Environment 14: 601-606[CrossRef]
Eckert M. R. Kaldenhoff 2000 Light-induced stomatal movement of selected Arabidopsis thaliana mutants. Journal of Experimental Botany 51: 1435-1442
Eisinger W. T. Swartz R. Bogomolni L. Taiz 2000 The ultraviolet action spectrum for stomatal opening in broad bean. Plant Physiology 122: 99-105
Frechilla S. L. D. Talbott R. A. Bogomolni E. Zeiger 2000 Reversal of blue light-stimulated stomatal opening by green light. Plant and Cell Physiology 41: 171-176
Frechilla S. J. Zhu L. D. Talbott E. Zeiger 1999 Stomata from npq1, a zeaxanthin-less Arabidopsis mutant, lack a specific response to blue light. Plant and Cell Physiology 40: 949-954
Guo H. H. Yang T. C. Mockler C. Lin 1998 Regulation of flowering time by Arabidopsis photoreceptors. Science 279: 1360-1363
Jarillo J. A. H. Gabrys J. Capel J. M. Alonso J. R. Ecker A. R. Cashmore 2001 Phototropin-related NPL1 controls chloroplast relocation induced by blue light. Nature 410: 9529-9554
Kagawa T. T. Sakai N. Suetsugu K. Oikawa S. Ishiguro T. Kato S. Tabata K. Okada M. Wada 2001 Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response. Science 291: 2138-2141
Kinoshita T. M. Doi N. Suetsugu T. Kagawa M. Wada K. Shimazaki 2001 phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 414: 656-660[CrossRef][Medline]
Molnar P. J. Szabolcs 1993 (Z/E)-photoisomerization of C40-carotenoids by iodine. Journal Chemical Society Perkin Transactions 2: 261-266
Rock C. D. J. A. D. Zeevaart 1991 The aba mutant of Arabidopsis thaliana is impaired in epoxycarotenoid biosynthesis. Proceedings of the National Academy of Sciences 88: 7496-7499
Talbott L. D. J. Zhu S. W. Han E. Zeiger 2002 Phytochrome and blue light-mediated stomatal opening in the orchid, Paphiopedilum. Plant and Cell Physiology 43: 639-646
Wade H. K. T. N. Bibikova W. J. Valentine G. I. Jenkins 2001 Interactions within a network of phytochrome, cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue. Plant Journal 25: 675-685[CrossRef][Web of Science][Medline]
Zeiger E. 2000 Sensory transduction of blue light in guard cells. Trends in Plant Science 5: 183-185[CrossRef][Web of Science][Medline]
Zeiger E. W. Gruszecki S. Frechilla L. Talbott J. T. Zhu T. Swartz R. Bogomolni 2000 Spectroscopic and physiological evidence for a pair of interconverting isomers of zeaxanthin that mediate blue light (BL)-stimulated stomatal opening and its reversal by green light (GL). Plant Physiology Meeting Abstracts 123: 23.
CiteULike Complore Connotea Del.icio.us Digg Facebook Reddit Technorati Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  K. M. Folta and S. A. Maruhnich Green light: a signal to slow down or stop J. Exp. Bot., September 1, 2007; 58(12): 3099 - 3111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zweifel, K. Steppe, and F. J. Sterck Stomatal regulation by microclimate and tree water relations: interpreting ecophysiological field data with a hydraulic plant model J. Exp. Bot., June 1, 2007; 58(8): 2113 - 2131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ban, C. Honda, H. Bessho, X.-M. Pang, and T. Moriguchi Suppression subtractive hybridization identifies genes induced in response to UV-B irradiation in apple skin: isolation of a putative UDP-glucose 4-epimerase J. Exp. Bot., May 1, 2007; 58(7): 1825 - 1834. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Dhingra, D. H. Bies, K. R. Lehner, and K. M. Folta Green Light Adjusts the Plastid Transcriptome during Early Photomorphogenic Development Plant Physiology, November 1, 2006; 142(3): 1256 - 1266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. D. Talbott, J. W. Hammad, L. C. Harn, V. H. Nguyen, J. Patel, and E. Zeiger Reversal by Green Light of Blue Light-stimulated Stomatal Opening in Intact, Attached Leaves of Arabidopsis Operates Only in the Potassium-dependent, Morning Phase of Movement Plant Cell Physiol., March 1, 2006; 47(3): 332 - 339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Folta Green Light Stimulates Early Stem Elongation, Antagonizing Light-Mediated Growth Inhibition Plant Physiology, July 1, 2004; 135(3): 1407 - 1416. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
