Starch Analysis of HLB-Affected and Control Healthy Citrus Leaves Reveal Variations in the Amylose/ Amylopectin Ratio

Proc. Fla. State Hort. Soc. 124:69–75. 2011.

Starch Analysis of HLB-affected and Control Healthy Citrus Leaves Reveal Variations in the Amylose/ Amylopectin Ratio

Pedro Gonzalez, Jose Reyes, and Ed Etxeberria* University of Florida, IFAS, Department of Plant Pathology, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850

Additional index words. Citrus sp., plant disease, phloem disease, starch properties, starch accumulation Leaves of HLB (Huanglongbing or citrus greening)-affected branches contain considerably higher levels of starch than those of HLB-unaffected or “healthy trees.” Over-accumulation of starch takes place in photosynthetic cells, vascular parenchyma, ray parenchyma, and even sieve elements. These observations imply strong disturbances in starch metabolism and photoassimilate partitioning brought about by HLB. The elevated starch content, appearance of starch granules in phloem elements, and previous reports of the pathogen effect on starch properties lead us to hypothesize that starch from HLB-affected citrus differs morphologically, physically, and/or chemically from starch accumulated in otherwise healthy leaves. We investigated starch morphology using brightfield, polarized light and SEM and found no morphological differences between HLB-induced starch and that from healthy girdled trees. When reacted with

2% I2, whole starch fractions also showed no significant difference in the absorption spectra (λmax for HLB = 604.1 and girdled 606.2 nm, respectively; n = 6; P ≤ 0.05) nor in their amylose/amylopectin ratio (HLB = 1.4 ± 0.17 and girdled

= 1.16 ± 0.07, P ≤ 0.05) after chromatographic separation. Nevertheless, λmax for individual fractions of HLB-affected leaves increased between 11 to 14 nm indicating a significant increase in the degree of polymerization of chain lengths estimated between 12 to 45 glucose units. The increase in amylopectin chain length was verified by the rise in gelatinization temperature of approximately 10 °C observed by polarized light microscopy. Our results indicate that starch grains from leaves affected by HLB, although morphologically similar, differed biochemically from those formed by healthy trees after phloem blockage caused by mechanical injury.

Leaves from HLB-affected citrus trees accumulate massive fact, the yellowing leaf mottle symptoms of HLB-affected leaves amounts of starch compared to leaves from healthy trees. It are believed to result from the disintegration of the chloroplast has been established that the levels of plant starches and their thylakoid system caused by the bulky starch build-up (Etxeber- physical, chemical, and morphological characteristics are highly ria et al., 2009). Leaf yellowing as a result of thylakoid damage influenced by conditions ranging from environmental factors, from starch accumulation can be artificially induced by branch genetic alterations, and pathogen infection. Therefore, differences girdling of citrus trees (Schaffer et al., 1986). in starch properties induced by HLB could be used to determine Recently, Etxeberria et al. (2009) and Folimonova and Achor HLB infection. Leaf starch was isolated from HLB-affected trees (2010) reported that starch grains, besides accumulating in virtu- and from control healthy girdled branches. Starch samples were ally every photosynthetic and parenchyma cell of all aerial parts reacted with 2% iodine and absorption spectra determined before (Etxeberria et al., 2009), are also found in phloem sieve elements and after separation into amylose and amylopectin. In addition, (Folimonova and Achor, 2010), a rare condition in higher plants. starch granules were examined morphologically by scanning In support of the observed starch increases, Kim et al. (2009) electron microscopy, polarized light microscopy and their ring reported up-regulation of starch biosynthetic enzymes in HLB- structure observed after partial digestion with amylase. Starches affected leaves. from HLB-affected leaves and control leaves did not show con- Starch is a natural product of photosynthetic CO2 fixation in siderable variations in any of the properties investigated except green tissues and from conversion of sucrose in storage cells. for their amylase:amylopectin ratio, which was considerably Formed by α-1,4 glucose linkages, starch chains exists in two higher in starches from HLB-affected leaves. forms, the soluble, small linear chain amylose and the highly One of the main characteristics of HLB-affected citrus trees branched insoluble amylopectin (Wang et al., 1998). In general, is the hyperaccumulation of starch in all aerial parts, especially plant starch is composed mainly of 30% amylose and 70% leaves, and the disappearance of such reserves in roots (Etxeberria amylopectin (Smith et al., 1997), with specific variations among et al., 2009). Otherwise, detectable amounts of starch in citrus species. It is also well established that the levels of plant starches leaves are only sporadically observed as a result of zinc deficiency (Ishak and El-Deeb, 2004; Lebsky and Poghosyan, 2007) and (Smith, 1974) or girdled branches caused by mechanical injury. their physical, biochemical, and morphological characteristics are According to Schneider (1968), starch accumulates in leaves as highly influenced by many conditions ranging from environmental the result of the backlog of photoassimilates created by necrotic factors (Smith and Struckmeyer, 1974), genetic alterations (Craig phloem pockets scattered throughout the foliar vascular system. In et al., 1998; Geigenberger et al., 2001), and pathogen infection (Handford and Carr, 2007; Roberts and Wood, 1982; Savile, 1942; *Corresponding author; phone: (863) 956-8787; email: [email protected] Tainter and Lewis, 1982; Técsi et al., 1992).

Proc. Fla. State Hort. Soc. 124: 2011. 69 Based on the unusual tissue distribution (Etxeberria et al., 2009), Amylose and amylopectin separation. For the separation of appearance of starch granules in phloem elements (Folimonova amylose and amylopectin, we followed the procedure described and Achor, 2010), and previous reports of pathogen effect on plant by Santacruz and Åman (2005). Starch samples were solubilized starch properties (Handford and Carr, 2007; Roberts and Wood, in 1 mL 0.5 M NaOH and heated in boiling water for 30 s. The 1982; Savile, 1942; Tainter and Lewis, 1982; Técsi et al., 1992), solubilized samples were fractionated by gel permeation chroma- we hypothesize that starch from HLB-affected citrus trees will tography (GPC) in a Sepharose CL-2B column (Sigma CL2B300). differ in morphology, physical, and/or chemical properties from The column was (27 cm h × 2.6 cm d) using 0.01 M NaOH as starch accumulated under the absence of pathological conditions. eluent. The flow rate was set at 0.4 mL·min–1 and fractions of 2 If confirmed, these differences will further our understanding of mL were collected. Collected fractions were mixed with 0.2 mL the physiological and metabolic effects of HLB on citrus trees, of I2-KI solution (2 mg/mL I2, 20 mg/mL KI) and absorbance as well as providing a diagnostic tool. of the polymer-iodine complex determined at 595 nm. (BioRad model 680). Absorbance spectrum was determined between 350 Materials and Methods and 800 nm (UV-VIS Spectrophotometer, Shimadzu UV-1700) 15 min after addition of the I2-KI reagent. Plant material. Leaf samples from HLB-affected trees were collected from ‘Valencia’ (Citrus sinensis L. Osbeck) trees showing Results classical symptoms and previously diagnosed HLB-positive by PCR. Samples were taken from six different ‘Valencia’ orange trees Grain morphology grown at the Citrus Research and Education Center in Lake Alfred, SEM. Starch grains from HLB-affected leaves were ap- FL. Since starch does not accumulate in appreciable amounts under proximately 1–3 µm (Fig. 1A). Granules appeared to be more natural conditions, control starch from HLB-negative ‘Valencia’ a mixture of discoid, granular, and oval shaped, many of which orange trees were obtained by girdling a 1.5-cm ring of bark tis- were more characteristic of storage starches than those isolated sue using a laboratory scalpel. Girdling was necessary to induce from photosynthetic tissues (Zeeman et al., 2002). The size of starch accumulation for various lengths of time. Leaves of girdled grains remained reasonably consistent between samples despite branches were periodically tested for starch content until levels the likelihood that the length of infection amongst trees must reached those of HLB-affected leaves, approximately 3 months have differed considerably in our random sampling. Under SEM, after girdling. Leaf yellowing and sometimes blotchy mottle became evident after 2 months of girdling, with leaf abscission becoming prominent after 6 months. A For comparative purposes, commercial starch samples from rice, wheat, and potato were used (rice, S-7260; wheat, S-5127; Sigma, St. Louis, MO). Starch extraction and isolation. Starch granules were extracted from three leaf discs made with a paper hole puncher (27.3 mm2). Leaf tissue was homogenized in 2-mL capped tubes containing 500 µL of water and four metal beads (2.36-mm di- ameter) (Mobio Laboratories, Carlsbad, CA). Homogenization was carried out in two 40-s cycles using a Precellys 24 Tissue Homogenizer (Bertin Technologies, France). The homogenate was placed on top of a 50% sucrose cushion and starch granules precipitated at 1 g. The pellet containing starch granules was washed three times with water in conical microfuge tubes and centrifuged at 10 × gn. The final pellet was dried overnight under vacuum in a desiccator containing calcium sulfate. Starch samples were stored at room temperature in a separate desiccator until use. B Grain morphology. After re-suspension in water, starch samples were filtered under vacuum using cellulose nitrate Fig. 1. Scanning electron micrographs of starch grains isolated from HLB-‐‑affected membrane filter papers (0.2 µm Whatman GmbH) and dried leaves (A) and from leaves on control girdled branches (B). Some irregularities, as split overnight in a vacuum desiccator. The dried starch was mounted on stubs, coated with gold/palladium using a Ladd sputter coater grains, are caused by the abrasive method of extraction. Starch granules were purified (Ladd Research Industries, Burlington, VT) and viewed using a in a 50% sucrose cushion and centrifugation at 10 x g for 10 min. Hitachi S530 SEM (Tokyo, Japan). Polarized light microscopy was performed using a Leica DMLP polarizing microscope with λ-plate at 40× magnification. Gelatinization. Starch gelatinization was characterized by controlled temperature polarized light microscopy. Starch granules were re-suspended in 50% glycerol due to a high mobility of starch granules in water alone. Gelatinization properties of individual starch granules during heating were investigated using Fig. 1. Scanning electron micrographs of starch grains isolated from HLB-affected a microscope hot stage Linkam LTS350 adaptor with LNP and leaves (A) and from leaves on control girdled branches (B). Some irregularities, TMS93 temperature controllers (Linkam Scientific Instruments, as split grains, are caused by the abrasive method of extraction. Starch granules –1 Surrey, UK). The stage wasFig. heated 1. from Scanning 20 to 100 electron °C at 1 °C·min micrographs. were purified of starch in a 50% grains sucrose isolated cushion from and centrifugation HLB-‐‑affected at 10 ×g n for 10 min. leaves (A) and from leaves on control girdled branches (B). Some irregularities, as split 70 grains, are caused by the abrasive method of extraction. Starch granulesProc. Fla. State were Hort. purifiedSoc. 124: 2011. in a 50% sucrose cushion and centrifugation at 10 x g for 10 min. Fig. 2. Light micrographs of starch granules in water viewed under polarized light in conjunction with λ plate. Starch granules from: (A) wheat and (B) rice were commercially obtained, whereas those from HLB-affected trees (C) and control girdled branches (D) were obtained as indicated in Materials and Methods. Fig. 2. Light micrographs of starch granules in water viewed under polarized light in conjunction with _ plate. Starch granules from: (A) wheat and (B) rice were starch granules from HLB-affected trees were morphologically 22 indistinguishable commerciallyfrom those of girdled obtained, branches whereas (Fig. 1B). those The from HLB-‐‑affected trees (C) and control girdled GirdledGirdled slight differences inbranches size in Figure (D) 1 were were obtained likely a time as effect indicated and in Materials1.81.8 and Methods. not a true distinguishing characteristic since starch populations HLBHLB of smaller size have been obtained from HLB-affected trees. 1.61.6 Birefringence. Starch grains viewed under polarized light 1.41.4 microscopy equipped with λ-plate displayed the characteristic

Maltese cross (Wang et al., 1998), as illustrated by control wheat OD OD 1.21.2 and rice grains (Fig. 2 A, B, respectively). For both HLB-affected (Fig. 2C) and healthy girdled control starch samples (Fig. 2D) 11 birefringence was comparatively similar and hilum position central throughout the many samples examined. In general, our 0.80.8 samples from HLB-affected trees were indistinguishable from girdled branches (Fig. 2 C, D). 0.60.6 18

Iodine binding 400400 600600 800800

Starch extract. Unfractionated starch samples from both λ?λ ?nm nmnm HLB-affected and girdled branches were heated and mixed with iodine solution for 15 min before absorption spectra was deter- Fig. 3. Absorption spectra of starch samples from leaves obtained form HLB-‐‑affected mined. The absorption spectra for both populations of starches Fig. 3.and Absorption control healthy,spectra girdledof starch branches. samples from Starch leaves samples obtained were mixed withfrom 2%HLB-2 I solution affected and control healthy, girdled branches. Starch samples were mixed with appear virtually identical (Fig. 3). The small variation in the and, after 15 min, OD determined between 350 and 800 nm. Values are the average of 6 2% Isamples2 solution and and, are after not 15 statistically min, OD different determined (p ≤ 0.05). between 350 and 800 nm. wavelength of maximum absorbance (λmax; HLB = 604.1 and Values are the average of 6 samples and are not statistically different (P ≤ 0.05). girdled 606.2 nm) was statistically insignificant based on the number of replicates (n = 6; P ≤ 0.05). Amylose and amylopectin. Elution of starch fractions in a chains eluted afterwards (higher fraction numbers, II). Elution Sepharose CL-2B column resulted in two distinctive peaks (Fig. pattern for the two fraction peaks for both the HLB-affected and 4A). The first peak (lower numerical fractions, I) corresponds to girdled control starch samples appeared similar. The small differ- amylopectin given its larger molecular size. The smaller amylose ences in the amylose/amylopectin peak ratios of 1.4 ± 0.17 SE for

Proc. Fla. State Hort. Soc. 124: 2011. 71 19 4B). For example, amylopectin λmax for fraction 13 from girdled 1.2 A Girdled 620 branches was 570 nm compared to that of HLB-affected fraction II?? 1 HLB where values reached 584 nm. Amylose samples (fraction 23) exhibited a similar increase from 604 nm for girdled samples to I? 600 0.8 615 nm for HLB-associated samples. The differences λmax for the amylopectin and amylose peaks were 14 nm and 11 nm higher in 0.6 580 the HLB samples than for the girdled samples, respectively (Fig. OD 595 nm

OD 595 nm 0.4 5B). These increases in λmax are an indication of a lengthening in 560 the starch glucan polymer (Bailey and Whelan, 1961). 0.2 Gelatinization. Gelatinization of starch grains in response to applied heat was examined microscopically under polarized 0 540 B light and λ-plate. As a reference a wheat grain is presented in 620 Girdled 620 Fig. 5. Heat was applied to produce a temperature increase of 1 HLB °C·min–1, and changes recorded every 5 °C, although only relevant 600 micrographs are presented. In wheat starch grains, structural 600

max integrity was maintained up to approximately 90 °C when gela- λ

max tinization began, as seen by the decline in brightness and loss of 580 ?

? 580 the Maltese cross. Similar results were described by Bogracheva et al. (1998) for pea, potato, and maize starch. In samples from 560 control girdled branches, foliar starch began gelatinizing at 85 560 °C, with complete loss of crystalline structure taking place by 540 the time the temperature reached 90 °C (Fig. 6). However, in 0 20 40 starch from HLB-affected trees, gelatinization commenced at approximately 10 °C higher (≈95 °C; Fig. 7) than for girdled Fraction number samples, with complete gelatinization reached at around 100 °C.

Fig. 4. SeparationFig. 4. Separation of amylose of and amylose amylopectin and fractions amylopectin from fractions starch samples: from starch samples (Panel A) (A) obtainedobtained from from leaves leaves of control of control girdled girdled branches branches ( ⎯_ ( ) ) andand fromfrom HLB- HLB-‐‑affected leaves (-‐‑ -‐‑ Discussion affected). leaves Separation (- - -). was Separation obtained was using obtained a Sepharose using a Sepharose CL2B chromatograp CL2Bhy (Zeeman et al.,

chromatography2002), and (Zeeman collected et fractionsal., 2002), and mixed collected with2 2% I fractions prior to mixed analysis with at 2% 595 nm.In leaves, Values are starch accumulates during the day hours and mobi- I prior to analysis at 595 nm. Values are the means of 6 experiments with no 2 the means of 6 experiments with no statistical differences between II/I peaklized ratios at (p ≤times of low photosynthetic activity to maintain a steady statistical differences between II/I peak ratios (P ≤ 0.05). (B) Each individual carbon supply to heterotrophic tissues. Under normal conditions, fraction0.05). was (Panel also analyzed B) Each for individualthe wavelength fraction of maximum was also absorbance analyzed for their wavelength. of λmax citrus leaves accumulate little or no starch (Yelenosky and Guy, The λ maximum values ( B absorbance) were statistically max _ . The different max _ values (P ≤ (B) 0.05). were statistically different (p ≤ 0.05) max 1977) and only noticeable amounts are produced as a result of zinc deficiency (Smith and Struckmeyer, 1974) or girdling (Schaffer HLB-affected trees and 1.16 ± 0.07 SE for samples from girdled et al., 1986). Once accumulated, leaf starch in citrus tends not branches, respectively, were not statistically different (P ≤ 0.05). to be degraded20 (Goldschmidt and Koch, 1996), although some However, when individual fractions were analyzed for their λmax, depletion of the limited starch reserves may occur during the a significant shift occurred in the HLB-affected starches (Fig. winter months (Monerri et al., 2011).

Fig. 5. Polarized-light micrograph of a wheat grain during gelatinization in 50% glycerol using a microscope viewed in conjunction of a λ plate. The heating stage with temperature controllers, was heated from 20 to 100 °C at a heating rate of 1 °C per minute. Micrographs were taken using a Leica DFC295 3.0 MP digital camera. Fig. 5. Polarized-‐‑light micrograph of a wheat grain during gelatinization in 50% glycerol

72 using a microscope viewed in conjunction of a _ plate. The heating stageProc. with Fla. State Hort. Soc. 124: 2011.

temperature controllers, was heated from 20 to 100°C at a heating rate of 1°C per

minute. Micrographs were taken using a Leica DFC295 3.0 MP digital camera.

21 Fig. 6. Polarized-light micrograph of a leaf starch grain from a girdled branch during gelatinization in 50% glycerol using a microscope hot stage and viewed in conjunction of a λ plate. The heating stage with temperature controllers was heated from 20 to 100 °C at a rate of 1 °C per minute. Micrographs were taken using a Leica DFC295 3.0 MP digital camera. The experiment was run in triplicate. Fig. 6. Polarized-‐‑light micrograph of a leaf starch grain from a girdled branch during

gelatinization in 50% glycerol using a microscope hot stage and viewed in conjunction

of a _ plate. The heating stage with temperature controllers was heated from 20 to

100°C at a rate of 1°C per minute. Micrographs were taken using a Leica DFC295 3.0

MP digital camera. The experiment was run in triplicate.

Fig. 7. Polarized-light micrograph of a leaf starch grain from a HLB-affected tree during gelatinization in 50% glycerol using a microscope hot stage and viewed in conjunction of a λ plate. The heating stage with temperature controllers was heated from 20 to 100 °C at a rate of 1 °C per minute. Micrographs were taken using a Leica DFC295Fig. 3.0 7. MP Polarized-‐‑light digital camera. The experiment micrograph was run ofin triplicate. a leaf starch grain from a HLB-‐‑affected tree during

gelatinization in 50% glycerol using a microscope hot stage and viewed in conjunction Citrus trees affected by HLB accumulate considerable amounts 1982; Técsi et al., 1992) prompted us to investigate22 the possibil- of starch in practicallyof a _ plate. every Thelive cell heating of aerial stage organs with (Achor temperature et ity that HLB-induced controllers starch was mayheated possess from unique 20 toproperties that al., 2010; Etxeberria et al., 2009). Not only photosynthetic cells will allow its distinction from starch formed in healthy tissues. and vascular100°C parenchyma at a become rate of replete 1°C per with minute. starch, but Micrographsphloem Any morphological were taken or using physiological a Leica abnormality DFC295 3.0 can potentially elements also develop starch granules (Folimonova and Achor, serve as a diagnostic tool and will provide further understanding 2010). This unusualMP digital distribution camera. and biosynthetic The experiment behavior (Kim was runof the in effects triplicate. of HLB in citrus trees. et al., 200923), in addition to the several reports of alterations to We first concluded that the morphological characteristics starch properties caused by pathogen infection (Handford and Carr, of HLB-induced starch are not different in size, shape, and 2007; Roberts and Wood, 1982; Savile, 1942; Tainter and Lewis, overall appearance from those of healthy branches. We based

Proc. Fla. State Hort. Soc. 124: 2011. 73

23 this conclusion on our observations of over 20 micrographs at foliar symptoms are easily distinguishable from HLB-affected different magnifications taken from six random trees, of which leaves. The results presented here indicate that, although morpho- Fig. 1 is just an example. Starches from both sources appeared logically similar, starch grains from leaves affected by HLB are to be of various irregular shapes with no distinctive identifiable biochemically different from those formed by healthy trees under morphological features. Although some of the morphological phloem blockage, a condition frequently caused by mechanical irregularities in our samples may have been due to the abrasive injury. The major biochemical changes brought about HLB are nature of the extraction method, a great deal of unevenness was an across-the-board increase in glucan chain lengths and the also evident in electron micrographs from whole tissues (Etxeber- consequential rise in gelatinization temperature, which combined, ria et al., 2009). In contrast, genetically transformed pea grains, reflect the strong effect of HLB in photoassimilate partitioning. for example, exhibited clear morphological changes (Wang et al., 1997) as were those from potato tubers infected with an unidenti- Literature Cited fied virus (Savile, 1942). Other possible biochemical differences between starches Achor, D.S., E. Etxeberria, N. Wang, S.Y. Folimonova, K.-R. Chung, and of HLB-affected trees and girdled branches were based on the L.G. Albrigo. 2010. Sequence of anatomical symptom observations in ability of starch to react with iodine to produce a range of colors citrus affected with huanglongbing disease. Plant Pathol. J. 9:56–64. depending on the size of the starch chain. When mixed with Bailey, J.M. and W.J. Whelan. 1961. Physical properties of starch. I. iodine, starch chains of about 12 units begin to form a brownish Relationship between iodine stain and chain length. J. Biol. Chem. 236:969–973. color (with a λmax of 490 nm). As the chain length increases, the Bogracheva, T. Y., V.J. Morris, S.G. Ring, and C.L. Hedley. 1998. The complex continues to change in color through brown, red, purple, granular structure of C-type pea starch and its role in gelatinization. and blue when the length is roughly 45 DP. Although visibly Biopolymers 45:323–332. the color blue does not change any further, the λmax continues Craig, J., J.R. Lloyd, K. Tomlinson, L. Barber, A. Edwards, T.L. Wang, to increase up to 645 nm, when DP reaches 350 to 400 (Bailey C. Martin, C.L. Hedley, and A.M. Smith. 1998. Mutations in the gene and Whelan, 1961; Santacruz and Åman, 2005). 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