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Epidermal anthocyanin production as an indicator of bacterial blight resistance in cotton
Physiological and Molecular Plant Pathology (2002) 61, 189±195 doi:10.1006/pmpp.2002.0434, available online at http://www.idealibrary.com on
Epidermal anthocyanin production as an indicator of bacterial blight resistance in cotton N . K A N G AT H A R A L I N G A M , M A R G A R E T L . P I E R C E , M E L A N I E B. B AY L E S and MARGARET ESSENBERG* Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, U.S.A. (Accepted for publication 12 August 2002) Among near-isogenic upland cotton (Gossypium hirsutum L.) lines, remarkable variation exists in the ability of the leaves to produce a red accessory pigment anthocyanin when challenged by selected isogenic races of the bacterial pathogen, Xanthomonas campestris pv. malvacearum (Smith) Dye. Inoculated areas that developed visible water-soaking that progressed beyond pinpoint-sized dots, a sign of susceptibility, very rarely produced anthocyanin, and on the couple of occasions in which anthocyanin was seen, it was borderline. Inoculated areas that exhibited resistance by developing little or no water-soaking produced various, but obvious, amounts of anthocyanin, with the exception of the cotton line whose only resistance gene is BIn. These results indicate that anthocyanin production by cotton leaves in response to an unsuccessful challenge by the bacterium is a bacterial blight resistance response, but is not essential for resistance. The epidermis of the adaxial surface of the leaves that received direct illumination was the tissue involved in anthocyanin production. The subsidiary cells of the stomatal complex were the initial participants in anthocyanin accumulation. A protective role of anthocyanin-containing cells against damage by infection-related reactive oxygen species and light-activated phytoalexins to the healthy tissues c 2002 Elsevier Science Ltd. All rights reserved. * surrounding infection sites is suggested. Keywords: Gossypium hirsutum L.; cotton; Xanthomonas campestris pv. malvacearum (Smith) Dye; anthocyanin; cyanidin-3-glucoside; water-soaking; bacterial blight; subsidiary stomatal cells; epidermis; host resistance; disease resistance; plant pigment; ¯avonoid; accessory pigment.
INTRODUCTION The bright red, purple, and blue pigments found in higher plants are mostly anthocyanins, a subgroup of the secondary metabolites, the ¯avonoids. In general, production of anthocyanins by plant species is potentiated by light [11, 13]. Anthocyanins are synthesized in the cytosol and transported for storage as an aqueous solution in the vacuoles of mature epidermal cells [3, 21]. Even though foliar anthocyanins are generally found in mature epidermal cells, they may also be localized in mesophyll cells in some species [15]. Until now, no information has been available regarding the kind of epidermal cells that are involved in the initiation of anthocyanin biosynthesis and accumulation in cotton (Gossypium spp.). In higher plants, anthocyanins primarily function in pigmentation, pollination and seed dispersal, protection against u.v.-radiation and other abiotic stresses, antioxidant activity against active oxygen species, protection * To whom all correspondence should be addressed. E-mail: [email protected] Abbreviations used in the text: cfu, colony-forming units; ROS, reactive oxygen species.
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against insect attack, and disease resistance. They can form complexes with other molecules (copigmentation) such as phenols [3] and DNA [28], thereby protecting themselves and sometimes the other molecule against oxidative and other degradative reactions. Anthocyanins can link with many other molecules covalently and otherwise, forming more complex and diverse structures. At present about 200 anthocyanins have been identi®ed as belonging to the natural anthocyanin series [3, 16]. Nevertheless, only a few, well known anthocyanins such as the glycosides of pelargonidin, peonidin, malvidin, cyanidin, delphinidin, and petunidin are widely distributed among higher plants [3, 14]. The signi®cance of anthocyanins as indicators of pathogen and pest resistance has drawn some, but sparse, attention [10, 13, 17±19, 29]. Hedin et al. reported that in cotton leaves the anthocyanin cyanidin-3-glucoside is an important factor in resistance to tobacco budworm, Heliothis virescens F. [17]. Jalali et al. [19] reported that leucoanthocyanin content of bacterial blight-infected resistant cotton leaves was three times that of infected susceptible leaves from an unrelated line. We have observed throughout our studies of cotton's interactions c 2002 Elsevier Science Ltd. All rights reserved. *
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with Xanthomonas campestris pv. malvacearum (Smith) Dye that red pigmentation often accompanies resistance responses. Edwards, in our laboratory, identi®ed the anthocyanin as cyanidin 3-b-D-glucoside [7]. In the present study, seven cotton lines and four strains of X. campestris pv. malvacearum were used under controlled environmental conditions to investigate the relationship between X. campestris pv. malvacearum-induced foliar watersoaking and anthocyanin production. The cotton lines included four near-isogenic lines possessing dierent complements of genes for bacterial blight resistance and dierent levels of resistance. The bacterial strains were four isogenic races developed by De Feyter et al. [5, 6, unpublished work], three of which carry individual genes for avirulence. The cell types in which anthocyanin biosynthesis occurs were also investigated.
MATERIALS AND METHODS
Plants and growth conditions Seven lines of upland cotton (G. hirsutum L.) were used. Ac44E is a plant selection from Ac44 [8]. It possesses no known resistance genes against X. campestris pv. malvacearum and is highly susceptible to this bacterium. Im216 is a broadly and highly resistant line in which several major resistance genes were pyramided [2]. AcIm was derived from a cross between Im216 and Ac44 [1]. After at least 12 generations of selection for immunity to New World races of X. campestris pv. malvacearum, individual plants were backcrossed to Ac44E and subjected to additional screening and selection for immunity. The AcIm plants used in this study were the progeny of a single resistant plant. AcBIn and Acb7 are each homozygous for a single resistance gene, BIn and b7 respectively, in the Ac44E genetic background and are resistant to races of X. campestris pv. malvacearum carrying the corresponding avr genes [8]. AcB4BIn and AcB4b7BIn are lines being developed in our laboratory and possess, respectively, two (B4 and BIn) and three (B4, b7, and BIn) major genes for resistance to bacterial blight. Both lines were derived from crosses among our single-gene, near-isogenic lines AcB4, Acb7, and AcBIn (Bayles, unpublished work) using individual plant and progeny row screening and selection through several generations to verify that the desired genes were present and homozygous. The presence or absence of each gene was con®rmed by screening each progeny plant with the appropriate strain of X. campestris pv. malvacearum carrying the corresponding avr gene. The seven cotton lines were each grown in two replicate pots, each pot containing two plants. They were grown in a Conviron E15 controlled environment growth chamber, providing 14 h light with a maximum
temperature of 308C and 10 h dark with a minimum temperature of 198C as described previously [25]. The youngest fully expanded leaves of four-week-old plants (one leaf per plant) were each inoculated with four dierent races of X. campestris pv. malvacearum.
Bacteria and inoculation method The races employed in this study were X. campestris pv. malvacearum 1003 strains given to us by D.W. Gabriel, University of Florida, Gainesville. Strain 1003 is derived from an African strain that has a wide host range [5]. Plasmid pUFR042 is a vector, carrying no avr genes; strain 1003/pUFR042 is virulent on all of the cotton lines used in this study. The other strains carry individual avr genes cloned from a widely avirulent American strain of X. campestris pv. malvacearum by De Feyter et al. [5, 6, unpublished work]. Strain 1003/pUFR163 carrying avrb7 [6], is virulent on Ac44E, AcBIn, and AcB4BIn, and is avirulent on the other four lines. Strain 1003/pUFR156 carrying avrBIn [6], is virulent on Ac44E and Acb7, and is avirulent on the other ®ve lines. Strain 1003/pUFR123 carrying avrB4 (DeFeyter and Gabriel, unpublished work), is virulent on Ac44E, AcBIn, and Acb7, and is avirulent on the other four lines. The strains were maintained at 708C in 14 % (v/v) glycerol in nutrient broth (Difco Laboratories, Detroit, MI, U.S.A.). The strains were cultured in nutrient broth with rotary aeration at 308C. Actively growing cultures were diluted with sterile distilled water saturated with CaCO3 to 2.5±3.0 106 cfu ml 1 except where otherwise stated. Leaves to be inoculated were covered with plastic bags containing a drop of sterile water for 30±45 min prior to inoculation so that the stomata were open for easy in®ltration with minimum pressure. Inoculation of the fully expanded leaves was carried out using needleless 1-ml plastic syringes loaded with the respective inocula. The inoculation was done on the abaxial surfaces of the leaves at four widely separated locations, with two points of application per location with adequate caution to prevent inoculum spills. Any excess inoculum on the surface was blotted o using sterile paper towels, so that the possibility of cross-contamination was minimized. The inoculated plants were maintained in the growth chamber.
Photography, microscopy, and assessment of red pigmentation and water-soaking Daily observations were made to assess red pigmentation (anthocyanin production) and water-soaking (susceptible bacterial blight symptom). These symptoms were recorded 6, 7, and 10 days post-inoculation using visual grading scales of 0±4. For anthocyanin production, the grading scale was: 0 absence of visible red pigment,
Bacterial blight resistance in cotton 1 mild, 2 moderate, 3 intense, and 4 very intense red pigmentation. For water-soaking, the grading scale was: 0 absence of water-soaking; 1 pinpoint-sized dots; 2 small, round speckles; 3 merged, angular patches; and 4 con¯uent areas of water-soaking. Visual symptoms were photographed using a Pentax ZX-M SLR camera ®tted with a Pentax 50 mm, 1 : 1.7 lens using Kodak Gold 200 ASA ®lm. For microscopy, disks of fresh leaf tissue were mounted in water and examined using a Nikon Optiphot microscope ®tted with a camera. Photographs were taken with the Nikon automatic camera using Kodak Gold 200 ASA ®lm.
RESULTS
Anthocyanin production in epidermal cells Under these test conditions, initial signs of water-soaking in response to X. campestris pv. malvacearum inoculation were observed in cotton leaves during compatible interactions 5±6 days after inoculation, whereas anthocyanin was noted as early as 40 h after inoculation during incompatible interactions. The intensity of anthocyanin production increased with inoculum density over the range 3 103 ±3 106 cfu ml 1. All observations reported here were made following inoculations with 2.5±3.0 106 cfu ml 1. Photographs of the adaxial surfaces of cotton leaves showing inoculated areas and associated anthocyanin distributions are presented in Fig. 1. Microscopic studies consistently revealed that the red anthocyanin was localized in the epidermal cells. A few scattered glandular cells, and an occasional palisade cell produced small amounts of anthocyanin. Mock inoculation with sterile CaCO3 solution elicited no visible watersoaking or anthocyanin production in any of the cotton lines. In the initial stages of anthocyanin production during incompatible cotton/X. campestris pv. malvacearum interactions, the only foliar epidermal cells involved were the three subsidiary or accessory cells of the stomatal apparatus [9]. Their exclusive ability to initiate anthocyanin production was observed within 40 h after inoculation of Im216 (Fig. 2) and AcIm. After 3 days,
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the intervening epidermal cells also actively participated in anthocyanin production. However, the guard cells were rarely found to contain anthocyanin. Under normal growth conditions, epidermal cells with anthocyanin were found only on the adaxial surface of inoculated leaves that received direct illumination.
X. campestris pv. malvacearum-induced water-soaking and anthocyanin levels among cotton lines A semiquantitative grading scale was used to record the levels of anthocyanin in the adaxial surfaces of inoculated leaves, when viewed with the unaided eye (Fig. 1). Microscopic views of those grades are shown in Fig. 3. Macroscopic, abaxial surface views of the water-soaking scale are shown in Fig. 4. Adaxial leaf surfaces of AcBIn and AcIm, showing lesions with all four strains of X. campestris pv. malvacearum with the associated anthocyanin distributions, are presented in Fig. 5. The grades for X. campestris pv. malvacearum-induced water-soaking on the abaxial surfaces and the simultaneous anthocyanin levels on the adaxial surfaces of the leaves of the seven cotton lines 6, 7, and 10 days after inoculation were recorded. A striking pattern of the data emerged when anthocyanin levels were plotted vs. watersoaking levels: all but three points lay on either the X- or the Y-axis. Of the 336 observations made, only three had non-zero values for both water-soaking and anthocyanin. In other words, the two responses were almost mutually exclusive. The means of the four values for each host/ pathogen interaction are presented in Table 1. In the table, the data for incompatible interactions are in italics. Resistance was almost always accompanied by red pigmentation. The only exceptions were some of the inoculated areas in which the only incompatible interaction was between BIn and avrBIn. Line AcBIn, which is resistant to the X. campestris pv. malvacearum strain carrying avrBIn, exhibited no red pigmentation in response to that strain (Fig. 5). Lines AcB4BIn and AcB4b7BIn likewise exhibited resistance to this strain, yet they also accumulated much less anthocyanin in doing so than during their other incompatible interactions.
F I G . 1. Adaxial surface views of X. campestris pv. malvacearum-inoculated cotton leaves, showing the visual anthocyanin scale used in this study. The grades were: 0 absence of visible red pigment; 1 mild; 2 moderate; 3 intense; 4 very intense red pigmentation. Scale: 1.
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F I G . 2. Subsidiary cells of the stomatal apparatus on the adaxial epidermis of a cotton leaf (line Im216) showing initiation of anthocyanin production 40 h post-inoculation with X. campestris pv. malvacearum. Scale bar 25 mm.
DISCUSSION Production of anthocyanins by the adaxial epidermal layer of leaves has not (to our knowledge) been reported to be initiated speci®cally in the subsidiary cells of the stomatal complex. This may be a speci®c feature in cotton, or may be a speci®c response during X. campestris pv. malvacearum/cotton interaction. It is evident that the cotton plant requires illumination for anthocyanin biosynthesis because under low light intensity little anthocyanin was produced, and because only the surface of the leaves exposed to light developed anthocyanin. Usually, this is the adaxial surface. If, however, the abaxial surface is exposed to direct light during development of the resistance response, it develops red pigmentation ( J. A. Hall et al., unpublished work).
Water-soaking in cotton leaves infected with X. campestris pv. malvacearum is a clear indication of host susceptibility and has been used for many years as the basis of a semiquantitative grading scale for bacterial blight resistance in the ®eld [2, 8]. In the present study, anthocyanin grades greater than one always indicated resistance (as de®ned by a growth chamber water-soaking grade less than or equal to one [8]). The near-isogenic relationships of six of the seven cotton lines and the isogenic relationships of the four bacterial strains add strength to the conclusion that it was the incompatible B gene/avr gene interactions that led to anthocyanin production. The three cotton lines carrying resistance gene BIn were resistant to the pathogen strain carrying avrBIn, but produced little or no anthocyanin, indicating that resistance of cotton leaves to X. campestris pv. malvacearum does not always involve anthocyanin production. Line AcBIn consistently showed tissue collapse around and between infection centers, which was not noted in the other lines tested. This resistance without anthocyanin was con®ned to the BIn/avrBIn interaction. The strain carrying avrBIn elicited strong anthocyanin production during the resistance responses in the pyramided, broadly resistant lines AcIm and Im216, whose bacterial blight resistance is determined by a number of genes, but not by BIn [2]. Accumulation of anthocyanins in resistant host plants in response to fungal pathogens has been observed [12, 13, 18]. Although only a little evidence for direct antifungal or antibacterial activity of anthocyanins has been reported [29], in vitro studies have indicated that ¯avonoids such as anthocyanins can function as antioxidants [20, 26, 33]. The speci®c anthocyanin that is of interest in this present study, cyanidin 3-b-D-glucoside, has been clearly shown
F I G . 3. Microscopic views of the distribution of anthocyanin-containing epidermal cells on the adaxial leaf surface (corresponding to the visual grades in Fig. 1). Anthocyanin-containing cells increased in density with grade. Scale bar 25 mm.
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F I G . 4. Abaxial surface views of X. campestris pv. malvacearum-inoculated leaves, showing the visual water-soaking scale used in this study. The grades were: 0 none; 1 pinpoint-sized dots; 2 small, round speckles; 3 merged, angular patches; 4 con¯uent areas. Scale: 1.
to be an active antioxidative agent, comparable to a-tocopherol, a well-known antioxidant [33]. Plant cells are known to generate reactive oxygen species (ROS) under varied environmental stresses including pathogen invasion as reported for the cotton/X. campestris pv. malvacearum system [22, 23]. The rapid generation of ROS during plant±pathogen interactions is often described as an oxidative burst and is believed to be activated at the onset of the hypersensitive response. Hammerschmidt and Nicholson [13] demonstrated that accumulation of anthocyanin in tissues surrounding anthracnose lesions in resistant corn lines was consistently associated with decreased lesion size, and they described this as an indication of host resistance to anthracnose. Hipskind et al. [18] suggested that the anthocyanin protects the healthy cells by forming co-pigmentation complexes with toxic ¯avonoids, sequestering them. Anthocyanin's ability to scavenge ROS from healthy tissues may also contribute to the protecting eect. Another mode of protection that anthocyanins may provide for healthy tissue is ®ltering of harmful u.v.
radiation [30]. The toxicity of the cotton leaf's sesquiterpenoid phytoalexins to the bacterial pathogen [31, 32] and to the foliar tissue itself [27] is activated by the u.v. component of sunlight. It has been shown by Rowlan et al. [27] that anthocyanin-containing epidermal cells of cotton leaves can protect underlying healthy palisade cells from the sunlight-activated toxicity. Even though the mode of action at the infection site is not certain at this time, the present study strongly suggests that anthocyanin production by resistant cotton against challenge by X. campestris pv. malvacearum is a host resistance response. Anthocyanins are not normally produced in epidermal or mesophyll cells of healthy cotton foliage, even when adequate light intensity is available. Mock-inoculated leaves of resistant cotton lines in our preliminary experiments showed little anthocyanin production. Since anthocyanin is produced under illumination, predominantly on the exposed adaxial surface of cotton leaves, its role in protecting the plant against any X. campestris pv. malvacearum-induced tissue damage may be limited, like any other single disease resistance
F I G . 5. Adaxial surface of cotton leaves showing lesions with all four races of X. campestris pv. malvacearum 7 days after inoculation: Lines AcBIn and AcIm are shown. Labels indicate the avr genes carried by each of the races.
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T A B L E 1. A summary of X. campestris pv. malvacearum (Xcm) race-speci®c water-soaking (Ws) and anthocyanin production (Anth) in selected cotton lines Avr gene carried by Xcm strain none avrBIn avrB4 avrb7
Cotton lines Ac44E
AcBIn
Acb7
AcB4BIn
AcB4b7BIn
AcIm3
Im2163
day
WS
Anth
WS
Anth
WS
Anth
WS
Anth
WS
Anth
WS
Anth
WS
Anth
6 7 10 6 7 10 6 7 10 6 7 10
3.00 2.50 4.00 2.50 3.00 4.00 2.25 3.25 4.00 4.00 4.00 4.00
0.252 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.25 3.00 4.00 0.751 1.001 1.001 3.00 3.75 3.75 3.25 3.50 4.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.00 3.25 3.75 1.75 2.50 3.00 1.50 3.25 4.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.50 3.75 3.50
1.00 2.75 3.00 0.00 0.00 0.00 0.00 0.00 0.00 3.50 3.75 4.00
0.00 0.00 0.00 0.75 0.75 0.25 3.00 2.75 3.50 0.252 0.00 0.00
2.75 3.25 3.75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.50 0.50 0.75 1.75 2.00 3.00 2.75 3.25 3.50
2.00 3.75 4.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 2.50 2.75 2.75 3.25 3.75 3.75 2.00 2.50 2.75
1.75 3.00 2.75 0.00 0.00 0.252 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 3.25 2.75 3.00 4.00 3.75 4.00 3.50 3.25 3.75
Values are the means of four observations for each interaction. Data in italics are from incompatible interactions; the others are from compatible interactions. 1 One of the four plants appeared susceptible, showing a water-soaking grade of 3 or 4; the other three plants were resistant (as expected) and showed no water-soaking. 2 One plant of four showed a grade of 1. 3Showed clear anthocyanin production with X. campestris pv. malvacearum races containing avrB4 or avrb7 or avrBIn at 48 h post-inoculation.
mechanism in the broad arsenal of defense mechanisms plants have developed. Studies of resistant responses of cotton cotyledons and leaves to bacterial blight, most of which were performed with plants carrying multiple resistance genes, have identi®ed the following sequence of chemical responses: an oxidative burst that generates both superoxide and hydrogen peroxide [23] and leads to generation of salicylic acid [22], ¯avonoid accumulation in living mesophyll cells at the margin of the inoculated area [4], lipid peroxidation occurring at the same time as hypersensitive cell death (A. Jalloul et al., pers. comm.), ¯avonoid [4] and sesquiterpenoid phytoalexin [24] accumulation in the hypersensitively responding mesophyll cells, and anthocyanin biosynthesis in adaxial epidermal cells (this study). Although the cotton anthocyanin, cyanidin-3-glucoside, is a ¯avonoid, its predominantly epidermal localization indicates that its biosynthesis is regulated dierently than that of the ¯avonoids that were detected principally in mesophyll cells [4]. We are currently pursuing a study to determine the eects of resistance gene pyramiding in a near-isogenic Ac44E background on both the level of resistance to bacterial blight and on the quickness of the various host responses to X. campestris pv. malvacearum listed earlier. We gratefully acknowledge the gift of bacterial strains from Dean W. Gabriel, statistical advice provided by
P. Larry Claypool, and reviews of the manuscript by Claypool, Larry J. Little®eld, and Laval M. Verhalen. Approved for publication by the Director, Oklahoma Agricultural Experimental Station. This research was supported under project OKL01504 and by the Cooperative State Research, Education, and Extension Service, under Agreement No. 97-35303-4625.
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Epidermal anthocyanin production as an indicator of bacterial blight resistance in cotton N . K A N G AT H A R A L I N G A M , M A R G A R E T L . P I E R C E , M E L A N I E B. B AY L E S and MARGARET ESSENBERG* Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, U.S.A. (Accepted for publication 12 August 2002) Among near-isogenic upland cotton (Gossypium hirsutum L.) lines, remarkable variation exists in the ability of the leaves to produce a red accessory pigment anthocyanin when challenged by selected isogenic races of the bacterial pathogen, Xanthomonas campestris pv. malvacearum (Smith) Dye. Inoculated areas that developed visible water-soaking that progressed beyond pinpoint-sized dots, a sign of susceptibility, very rarely produced anthocyanin, and on the couple of occasions in which anthocyanin was seen, it was borderline. Inoculated areas that exhibited resistance by developing little or no water-soaking produced various, but obvious, amounts of anthocyanin, with the exception of the cotton line whose only resistance gene is BIn. These results indicate that anthocyanin production by cotton leaves in response to an unsuccessful challenge by the bacterium is a bacterial blight resistance response, but is not essential for resistance. The epidermis of the adaxial surface of the leaves that received direct illumination was the tissue involved in anthocyanin production. The subsidiary cells of the stomatal complex were the initial participants in anthocyanin accumulation. A protective role of anthocyanin-containing cells against damage by infection-related reactive oxygen species and light-activated phytoalexins to the healthy tissues c 2002 Elsevier Science Ltd. All rights reserved. * surrounding infection sites is suggested. Keywords: Gossypium hirsutum L.; cotton; Xanthomonas campestris pv. malvacearum (Smith) Dye; anthocyanin; cyanidin-3-glucoside; water-soaking; bacterial blight; subsidiary stomatal cells; epidermis; host resistance; disease resistance; plant pigment; ¯avonoid; accessory pigment.
INTRODUCTION The bright red, purple, and blue pigments found in higher plants are mostly anthocyanins, a subgroup of the secondary metabolites, the ¯avonoids. In general, production of anthocyanins by plant species is potentiated by light [11, 13]. Anthocyanins are synthesized in the cytosol and transported for storage as an aqueous solution in the vacuoles of mature epidermal cells [3, 21]. Even though foliar anthocyanins are generally found in mature epidermal cells, they may also be localized in mesophyll cells in some species [15]. Until now, no information has been available regarding the kind of epidermal cells that are involved in the initiation of anthocyanin biosynthesis and accumulation in cotton (Gossypium spp.). In higher plants, anthocyanins primarily function in pigmentation, pollination and seed dispersal, protection against u.v.-radiation and other abiotic stresses, antioxidant activity against active oxygen species, protection * To whom all correspondence should be addressed. E-mail: [email protected] Abbreviations used in the text: cfu, colony-forming units; ROS, reactive oxygen species.
0885-5765/02/$ - see front matter
against insect attack, and disease resistance. They can form complexes with other molecules (copigmentation) such as phenols [3] and DNA [28], thereby protecting themselves and sometimes the other molecule against oxidative and other degradative reactions. Anthocyanins can link with many other molecules covalently and otherwise, forming more complex and diverse structures. At present about 200 anthocyanins have been identi®ed as belonging to the natural anthocyanin series [3, 16]. Nevertheless, only a few, well known anthocyanins such as the glycosides of pelargonidin, peonidin, malvidin, cyanidin, delphinidin, and petunidin are widely distributed among higher plants [3, 14]. The signi®cance of anthocyanins as indicators of pathogen and pest resistance has drawn some, but sparse, attention [10, 13, 17±19, 29]. Hedin et al. reported that in cotton leaves the anthocyanin cyanidin-3-glucoside is an important factor in resistance to tobacco budworm, Heliothis virescens F. [17]. Jalali et al. [19] reported that leucoanthocyanin content of bacterial blight-infected resistant cotton leaves was three times that of infected susceptible leaves from an unrelated line. We have observed throughout our studies of cotton's interactions c 2002 Elsevier Science Ltd. All rights reserved. *
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with Xanthomonas campestris pv. malvacearum (Smith) Dye that red pigmentation often accompanies resistance responses. Edwards, in our laboratory, identi®ed the anthocyanin as cyanidin 3-b-D-glucoside [7]. In the present study, seven cotton lines and four strains of X. campestris pv. malvacearum were used under controlled environmental conditions to investigate the relationship between X. campestris pv. malvacearum-induced foliar watersoaking and anthocyanin production. The cotton lines included four near-isogenic lines possessing dierent complements of genes for bacterial blight resistance and dierent levels of resistance. The bacterial strains were four isogenic races developed by De Feyter et al. [5, 6, unpublished work], three of which carry individual genes for avirulence. The cell types in which anthocyanin biosynthesis occurs were also investigated.
MATERIALS AND METHODS
Plants and growth conditions Seven lines of upland cotton (G. hirsutum L.) were used. Ac44E is a plant selection from Ac44 [8]. It possesses no known resistance genes against X. campestris pv. malvacearum and is highly susceptible to this bacterium. Im216 is a broadly and highly resistant line in which several major resistance genes were pyramided [2]. AcIm was derived from a cross between Im216 and Ac44 [1]. After at least 12 generations of selection for immunity to New World races of X. campestris pv. malvacearum, individual plants were backcrossed to Ac44E and subjected to additional screening and selection for immunity. The AcIm plants used in this study were the progeny of a single resistant plant. AcBIn and Acb7 are each homozygous for a single resistance gene, BIn and b7 respectively, in the Ac44E genetic background and are resistant to races of X. campestris pv. malvacearum carrying the corresponding avr genes [8]. AcB4BIn and AcB4b7BIn are lines being developed in our laboratory and possess, respectively, two (B4 and BIn) and three (B4, b7, and BIn) major genes for resistance to bacterial blight. Both lines were derived from crosses among our single-gene, near-isogenic lines AcB4, Acb7, and AcBIn (Bayles, unpublished work) using individual plant and progeny row screening and selection through several generations to verify that the desired genes were present and homozygous. The presence or absence of each gene was con®rmed by screening each progeny plant with the appropriate strain of X. campestris pv. malvacearum carrying the corresponding avr gene. The seven cotton lines were each grown in two replicate pots, each pot containing two plants. They were grown in a Conviron E15 controlled environment growth chamber, providing 14 h light with a maximum
temperature of 308C and 10 h dark with a minimum temperature of 198C as described previously [25]. The youngest fully expanded leaves of four-week-old plants (one leaf per plant) were each inoculated with four dierent races of X. campestris pv. malvacearum.
Bacteria and inoculation method The races employed in this study were X. campestris pv. malvacearum 1003 strains given to us by D.W. Gabriel, University of Florida, Gainesville. Strain 1003 is derived from an African strain that has a wide host range [5]. Plasmid pUFR042 is a vector, carrying no avr genes; strain 1003/pUFR042 is virulent on all of the cotton lines used in this study. The other strains carry individual avr genes cloned from a widely avirulent American strain of X. campestris pv. malvacearum by De Feyter et al. [5, 6, unpublished work]. Strain 1003/pUFR163 carrying avrb7 [6], is virulent on Ac44E, AcBIn, and AcB4BIn, and is avirulent on the other four lines. Strain 1003/pUFR156 carrying avrBIn [6], is virulent on Ac44E and Acb7, and is avirulent on the other ®ve lines. Strain 1003/pUFR123 carrying avrB4 (DeFeyter and Gabriel, unpublished work), is virulent on Ac44E, AcBIn, and Acb7, and is avirulent on the other four lines. The strains were maintained at 708C in 14 % (v/v) glycerol in nutrient broth (Difco Laboratories, Detroit, MI, U.S.A.). The strains were cultured in nutrient broth with rotary aeration at 308C. Actively growing cultures were diluted with sterile distilled water saturated with CaCO3 to 2.5±3.0 106 cfu ml 1 except where otherwise stated. Leaves to be inoculated were covered with plastic bags containing a drop of sterile water for 30±45 min prior to inoculation so that the stomata were open for easy in®ltration with minimum pressure. Inoculation of the fully expanded leaves was carried out using needleless 1-ml plastic syringes loaded with the respective inocula. The inoculation was done on the abaxial surfaces of the leaves at four widely separated locations, with two points of application per location with adequate caution to prevent inoculum spills. Any excess inoculum on the surface was blotted o using sterile paper towels, so that the possibility of cross-contamination was minimized. The inoculated plants were maintained in the growth chamber.
Photography, microscopy, and assessment of red pigmentation and water-soaking Daily observations were made to assess red pigmentation (anthocyanin production) and water-soaking (susceptible bacterial blight symptom). These symptoms were recorded 6, 7, and 10 days post-inoculation using visual grading scales of 0±4. For anthocyanin production, the grading scale was: 0 absence of visible red pigment,
Bacterial blight resistance in cotton 1 mild, 2 moderate, 3 intense, and 4 very intense red pigmentation. For water-soaking, the grading scale was: 0 absence of water-soaking; 1 pinpoint-sized dots; 2 small, round speckles; 3 merged, angular patches; and 4 con¯uent areas of water-soaking. Visual symptoms were photographed using a Pentax ZX-M SLR camera ®tted with a Pentax 50 mm, 1 : 1.7 lens using Kodak Gold 200 ASA ®lm. For microscopy, disks of fresh leaf tissue were mounted in water and examined using a Nikon Optiphot microscope ®tted with a camera. Photographs were taken with the Nikon automatic camera using Kodak Gold 200 ASA ®lm.
RESULTS
Anthocyanin production in epidermal cells Under these test conditions, initial signs of water-soaking in response to X. campestris pv. malvacearum inoculation were observed in cotton leaves during compatible interactions 5±6 days after inoculation, whereas anthocyanin was noted as early as 40 h after inoculation during incompatible interactions. The intensity of anthocyanin production increased with inoculum density over the range 3 103 ±3 106 cfu ml 1. All observations reported here were made following inoculations with 2.5±3.0 106 cfu ml 1. Photographs of the adaxial surfaces of cotton leaves showing inoculated areas and associated anthocyanin distributions are presented in Fig. 1. Microscopic studies consistently revealed that the red anthocyanin was localized in the epidermal cells. A few scattered glandular cells, and an occasional palisade cell produced small amounts of anthocyanin. Mock inoculation with sterile CaCO3 solution elicited no visible watersoaking or anthocyanin production in any of the cotton lines. In the initial stages of anthocyanin production during incompatible cotton/X. campestris pv. malvacearum interactions, the only foliar epidermal cells involved were the three subsidiary or accessory cells of the stomatal apparatus [9]. Their exclusive ability to initiate anthocyanin production was observed within 40 h after inoculation of Im216 (Fig. 2) and AcIm. After 3 days,
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the intervening epidermal cells also actively participated in anthocyanin production. However, the guard cells were rarely found to contain anthocyanin. Under normal growth conditions, epidermal cells with anthocyanin were found only on the adaxial surface of inoculated leaves that received direct illumination.
X. campestris pv. malvacearum-induced water-soaking and anthocyanin levels among cotton lines A semiquantitative grading scale was used to record the levels of anthocyanin in the adaxial surfaces of inoculated leaves, when viewed with the unaided eye (Fig. 1). Microscopic views of those grades are shown in Fig. 3. Macroscopic, abaxial surface views of the water-soaking scale are shown in Fig. 4. Adaxial leaf surfaces of AcBIn and AcIm, showing lesions with all four strains of X. campestris pv. malvacearum with the associated anthocyanin distributions, are presented in Fig. 5. The grades for X. campestris pv. malvacearum-induced water-soaking on the abaxial surfaces and the simultaneous anthocyanin levels on the adaxial surfaces of the leaves of the seven cotton lines 6, 7, and 10 days after inoculation were recorded. A striking pattern of the data emerged when anthocyanin levels were plotted vs. watersoaking levels: all but three points lay on either the X- or the Y-axis. Of the 336 observations made, only three had non-zero values for both water-soaking and anthocyanin. In other words, the two responses were almost mutually exclusive. The means of the four values for each host/ pathogen interaction are presented in Table 1. In the table, the data for incompatible interactions are in italics. Resistance was almost always accompanied by red pigmentation. The only exceptions were some of the inoculated areas in which the only incompatible interaction was between BIn and avrBIn. Line AcBIn, which is resistant to the X. campestris pv. malvacearum strain carrying avrBIn, exhibited no red pigmentation in response to that strain (Fig. 5). Lines AcB4BIn and AcB4b7BIn likewise exhibited resistance to this strain, yet they also accumulated much less anthocyanin in doing so than during their other incompatible interactions.
F I G . 1. Adaxial surface views of X. campestris pv. malvacearum-inoculated cotton leaves, showing the visual anthocyanin scale used in this study. The grades were: 0 absence of visible red pigment; 1 mild; 2 moderate; 3 intense; 4 very intense red pigmentation. Scale: 1.
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F I G . 2. Subsidiary cells of the stomatal apparatus on the adaxial epidermis of a cotton leaf (line Im216) showing initiation of anthocyanin production 40 h post-inoculation with X. campestris pv. malvacearum. Scale bar 25 mm.
DISCUSSION Production of anthocyanins by the adaxial epidermal layer of leaves has not (to our knowledge) been reported to be initiated speci®cally in the subsidiary cells of the stomatal complex. This may be a speci®c feature in cotton, or may be a speci®c response during X. campestris pv. malvacearum/cotton interaction. It is evident that the cotton plant requires illumination for anthocyanin biosynthesis because under low light intensity little anthocyanin was produced, and because only the surface of the leaves exposed to light developed anthocyanin. Usually, this is the adaxial surface. If, however, the abaxial surface is exposed to direct light during development of the resistance response, it develops red pigmentation ( J. A. Hall et al., unpublished work).
Water-soaking in cotton leaves infected with X. campestris pv. malvacearum is a clear indication of host susceptibility and has been used for many years as the basis of a semiquantitative grading scale for bacterial blight resistance in the ®eld [2, 8]. In the present study, anthocyanin grades greater than one always indicated resistance (as de®ned by a growth chamber water-soaking grade less than or equal to one [8]). The near-isogenic relationships of six of the seven cotton lines and the isogenic relationships of the four bacterial strains add strength to the conclusion that it was the incompatible B gene/avr gene interactions that led to anthocyanin production. The three cotton lines carrying resistance gene BIn were resistant to the pathogen strain carrying avrBIn, but produced little or no anthocyanin, indicating that resistance of cotton leaves to X. campestris pv. malvacearum does not always involve anthocyanin production. Line AcBIn consistently showed tissue collapse around and between infection centers, which was not noted in the other lines tested. This resistance without anthocyanin was con®ned to the BIn/avrBIn interaction. The strain carrying avrBIn elicited strong anthocyanin production during the resistance responses in the pyramided, broadly resistant lines AcIm and Im216, whose bacterial blight resistance is determined by a number of genes, but not by BIn [2]. Accumulation of anthocyanins in resistant host plants in response to fungal pathogens has been observed [12, 13, 18]. Although only a little evidence for direct antifungal or antibacterial activity of anthocyanins has been reported [29], in vitro studies have indicated that ¯avonoids such as anthocyanins can function as antioxidants [20, 26, 33]. The speci®c anthocyanin that is of interest in this present study, cyanidin 3-b-D-glucoside, has been clearly shown
F I G . 3. Microscopic views of the distribution of anthocyanin-containing epidermal cells on the adaxial leaf surface (corresponding to the visual grades in Fig. 1). Anthocyanin-containing cells increased in density with grade. Scale bar 25 mm.
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F I G . 4. Abaxial surface views of X. campestris pv. malvacearum-inoculated leaves, showing the visual water-soaking scale used in this study. The grades were: 0 none; 1 pinpoint-sized dots; 2 small, round speckles; 3 merged, angular patches; 4 con¯uent areas. Scale: 1.
to be an active antioxidative agent, comparable to a-tocopherol, a well-known antioxidant [33]. Plant cells are known to generate reactive oxygen species (ROS) under varied environmental stresses including pathogen invasion as reported for the cotton/X. campestris pv. malvacearum system [22, 23]. The rapid generation of ROS during plant±pathogen interactions is often described as an oxidative burst and is believed to be activated at the onset of the hypersensitive response. Hammerschmidt and Nicholson [13] demonstrated that accumulation of anthocyanin in tissues surrounding anthracnose lesions in resistant corn lines was consistently associated with decreased lesion size, and they described this as an indication of host resistance to anthracnose. Hipskind et al. [18] suggested that the anthocyanin protects the healthy cells by forming co-pigmentation complexes with toxic ¯avonoids, sequestering them. Anthocyanin's ability to scavenge ROS from healthy tissues may also contribute to the protecting eect. Another mode of protection that anthocyanins may provide for healthy tissue is ®ltering of harmful u.v.
radiation [30]. The toxicity of the cotton leaf's sesquiterpenoid phytoalexins to the bacterial pathogen [31, 32] and to the foliar tissue itself [27] is activated by the u.v. component of sunlight. It has been shown by Rowlan et al. [27] that anthocyanin-containing epidermal cells of cotton leaves can protect underlying healthy palisade cells from the sunlight-activated toxicity. Even though the mode of action at the infection site is not certain at this time, the present study strongly suggests that anthocyanin production by resistant cotton against challenge by X. campestris pv. malvacearum is a host resistance response. Anthocyanins are not normally produced in epidermal or mesophyll cells of healthy cotton foliage, even when adequate light intensity is available. Mock-inoculated leaves of resistant cotton lines in our preliminary experiments showed little anthocyanin production. Since anthocyanin is produced under illumination, predominantly on the exposed adaxial surface of cotton leaves, its role in protecting the plant against any X. campestris pv. malvacearum-induced tissue damage may be limited, like any other single disease resistance
F I G . 5. Adaxial surface of cotton leaves showing lesions with all four races of X. campestris pv. malvacearum 7 days after inoculation: Lines AcBIn and AcIm are shown. Labels indicate the avr genes carried by each of the races.
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T A B L E 1. A summary of X. campestris pv. malvacearum (Xcm) race-speci®c water-soaking (Ws) and anthocyanin production (Anth) in selected cotton lines Avr gene carried by Xcm strain none avrBIn avrB4 avrb7
Cotton lines Ac44E
AcBIn
Acb7
AcB4BIn
AcB4b7BIn
AcIm3
Im2163
day
WS
Anth
WS
Anth
WS
Anth
WS
Anth
WS
Anth
WS
Anth
WS
Anth
6 7 10 6 7 10 6 7 10 6 7 10
3.00 2.50 4.00 2.50 3.00 4.00 2.25 3.25 4.00 4.00 4.00 4.00
0.252 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.25 3.00 4.00 0.751 1.001 1.001 3.00 3.75 3.75 3.25 3.50 4.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.00 3.25 3.75 1.75 2.50 3.00 1.50 3.25 4.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.50 3.75 3.50
1.00 2.75 3.00 0.00 0.00 0.00 0.00 0.00 0.00 3.50 3.75 4.00
0.00 0.00 0.00 0.75 0.75 0.25 3.00 2.75 3.50 0.252 0.00 0.00
2.75 3.25 3.75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.50 0.50 0.75 1.75 2.00 3.00 2.75 3.25 3.50
2.00 3.75 4.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 2.50 2.75 2.75 3.25 3.75 3.75 2.00 2.50 2.75
1.75 3.00 2.75 0.00 0.00 0.252 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 3.25 2.75 3.00 4.00 3.75 4.00 3.50 3.25 3.75
Values are the means of four observations for each interaction. Data in italics are from incompatible interactions; the others are from compatible interactions. 1 One of the four plants appeared susceptible, showing a water-soaking grade of 3 or 4; the other three plants were resistant (as expected) and showed no water-soaking. 2 One plant of four showed a grade of 1. 3Showed clear anthocyanin production with X. campestris pv. malvacearum races containing avrB4 or avrb7 or avrBIn at 48 h post-inoculation.
mechanism in the broad arsenal of defense mechanisms plants have developed. Studies of resistant responses of cotton cotyledons and leaves to bacterial blight, most of which were performed with plants carrying multiple resistance genes, have identi®ed the following sequence of chemical responses: an oxidative burst that generates both superoxide and hydrogen peroxide [23] and leads to generation of salicylic acid [22], ¯avonoid accumulation in living mesophyll cells at the margin of the inoculated area [4], lipid peroxidation occurring at the same time as hypersensitive cell death (A. Jalloul et al., pers. comm.), ¯avonoid [4] and sesquiterpenoid phytoalexin [24] accumulation in the hypersensitively responding mesophyll cells, and anthocyanin biosynthesis in adaxial epidermal cells (this study). Although the cotton anthocyanin, cyanidin-3-glucoside, is a ¯avonoid, its predominantly epidermal localization indicates that its biosynthesis is regulated dierently than that of the ¯avonoids that were detected principally in mesophyll cells [4]. We are currently pursuing a study to determine the eects of resistance gene pyramiding in a near-isogenic Ac44E background on both the level of resistance to bacterial blight and on the quickness of the various host responses to X. campestris pv. malvacearum listed earlier. We gratefully acknowledge the gift of bacterial strains from Dean W. Gabriel, statistical advice provided by
P. Larry Claypool, and reviews of the manuscript by Claypool, Larry J. Little®eld, and Laval M. Verhalen. Approved for publication by the Director, Oklahoma Agricultural Experimental Station. This research was supported under project OKL01504 and by the Cooperative State Research, Education, and Extension Service, under Agreement No. 97-35303-4625.
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