Starch Analysis of HLB-affected and Control Healthy Citrus

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

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

*Corresponding author; phone: (863) 956-8787; email: [email protected]

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 me-tabolism 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 gelati-nization 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 amounts of starch compared to leaves from healthy trees. It has been established that the levels of plant starches and their physical, chemical, and morphological characteristics are highly influenced by conditions ranging from environmental factors, genetic alterations, and pathogen infection. Therefore, differences in starch properties induced by HLB could be used to determine HLB infection. Leaf starch was isolated from HLB-affected trees and from control healthy girdled branches. Starch samples were reacted with 2% iodine and absorption spectra determined before and after separation into amylose and amylopectin. In addition, starch granules were examined morphologically by scanning electron microscopy, polarized light microscopy and their ring structure observed after partial digestion with amylase. Starches from HLB-affected leaves and control leaves did not show con-siderable variations in any of the properties investigated except for their amylase:amylopectin ratio, which was considerably higher in starches from HLB-affected leaves.

One of the main characteristics of HLB-affected citrus trees is the hyperaccumulation of starch in all aerial parts, especially leaves, and the disappearance of such reserves in roots (Etxeberria et al., 2009). Otherwise, detectable amounts of starch in citrus leaves are only sporadically observed as a result of zinc deficiency (Smith, 1974) or girdled branches caused by mechanical injury. According to Schneider (1968), starch accumulates in leaves as the result of the backlog of photoassimilates created by necrotic phloem pockets scattered throughout the foliar vascular system. In

fact, the yellowing leaf mottle symptoms of HLB-affected leaves are believed to result from the disintegration of the chloroplast thylakoid system caused by the bulky starch build-up (Etxeber-ria et al., 2009). Leaf yellowing as a result of thylakoid damage from starch accumulation can be artificially induced by branch girdling of citrus trees (Schaffer et al., 1986).

Recently, Etxeberria et al. (2009) and Folimonova and Achor (2010) reported that starch grains, besides accumulating in virtu-ally every photosynthetic and parenchyma cell of all aerial parts (Etxeberria et al., 2009), are also found in phloem sieve elements (Folimonova and Achor, 2010), a rare condition in higher plants. In support of the observed starch increases, Kim et al. (2009) reported up-regulation of starch biosynthetic enzymes in HLB-affected leaves.

Starch is a natural product of photosynthetic CO2 fixation in green tissues and from conversion of sucrose in storage cells. Formed by α-1,4 glucose linkages, starch chains exists in two forms, the soluble, small linear chain amylose and the highly branched insoluble amylopectin (Wang et al., 1998). In general, plant starch is composed mainly of 30% amylose and 70% amylopectin (Smith et al., 1997), with specific variations among species. It is also well established that the levels of plant starches (Ishak and El-Deeb, 2004; Lebsky and Poghosyan, 2007) and their physical, biochemical, and morphological characteristics are highly influenced by many conditions ranging from environmental factors (Smith and Struckmeyer, 1974), genetic alterations (Craig et al., 1998; Geigenberger et al., 2001), and pathogen infection (Handford and Carr, 2007; Roberts and Wood, 1982; Savile, 1942; Tainter and Lewis, 1982; Técsi et al., 1992).

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

Based on the unusual tissue distribution (Etxeberria et al., 2009), appearance of starch granules in phloem elements (Folimonova and Achor, 2010), and previous reports of pathogen effect on plant starch properties (Handford and Carr, 2007; Roberts and Wood, 1982; Savile, 1942; Tainter and Lewis, 1982; Técsi et al., 1992), we hypothesize that starch from HLB-affected citrus trees will differ in morphology, physical, and/or chemical properties from starch accumulated under the absence of pathological conditions. If confirmed, these differences will further our understanding of the physiological and metabolic effects of HLB on citrus trees, as well as providing a diagnostic tool.

Materials and Methods

PlAnt mAteriAl. Leaf samples from HLB-affected trees were collected from ‘Valencia’ (Citrus sinensis L. Osbeck) trees showing classical symptoms and previously diagnosed HLB-positive by PCR. Samples were taken from six different ‘Valencia’ orange trees grown at the Citrus Research and Education Center in Lake Alfred, FL. Since starch does not accumulate in appreciable amounts under natural conditions, control starch from HLB-negative ‘Valencia’ orange trees were obtained by girdling a 1.5-cm ring of bark tis-sue using a laboratory scalpel. Girdling was necessary to induce starch accumulation for various lengths of time. Leaves of girdled branches were periodically tested for starch content until levels reached those of HLB-affected leaves, approximately 3 months after girdling. Leaf yellowing and sometimes blotchy mottle became evident after 2 months of girdling, with leaf abscission becoming prominent after 6 months.

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.

GrAin morPholoGy. After re-suspension in water, starch samples were filtered under vacuum using cellulose nitrate membrane filter papers (0.2 µm Whatman GmbH) and dried overnight in a vacuum desiccator. The dried starch was mounted on stubs, coated with gold/palladium using a Ladd sputter coater (Ladd Research Industries, Burlington, VT) and viewed using a 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 gran-ules 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 a microscope hot stage Linkam LTS350 adaptor with LNP and TMS93 temperature controllers (Linkam Scientific Instruments, Surrey, UK). The stage was heated from 20 to 100 °C at 1 °C·min–1.

Amylose And AmyloPectin sePArAtion. For the separation of amylose and amylopectin, we followed the procedure described by Santacruz and Åman (2005). Starch samples were solubilized in 1 mL 0.5 M NaOH and heated in boiling water for 30 s. The solubilized samples were fractionated by gel permeation chroma-tography (GPC) in a Sepharose CL-2B column (Sigma CL2B300). The column was (27 cm h × 2.6 cm d) using 0.01 M NaOH as eluent. The flow rate was set at 0.4 mL·min–1 and fractions of 2 mL were collected. Collected fractions were mixed with 0.2 mL of I2-KI solution (2 mg/mL I2, 20 mg/mL KI) and absorbance of the polymer-iodine complex determined at 595 nm. (BioRad model 680). Absorbance spectrum was determined between 350 and 800 nm (UV-VIS Spectrophotometer, Shimadzu UV-1700) 15 min after addition of the I2-KI reagent.

Results

Grain morphologysem. Starch grains from HLB-affected leaves were ap-

proximately 1–3 µm (Fig. 1A). Granules appeared to be more a mixture of discoid, granular, and oval shaped, many of which were more characteristic of storage starches than those isolated from photosynthetic tissues (Zeeman et al., 2002). The size of grains remained reasonably consistent between samples despite the likelihood that the length of infection amongst trees must have differed considerably in our random sampling. Under SEM,

Fig. 1. Scanning electron micrographs of starch grains isolated from HLB-affected leaves (A) and from leaves on control girdled branches (B). Some irregularities, as split grains, are caused by the abrasive method of extraction. Starch granules were purified in a 50% sucrose cushion and centrifugation at 10 × gn for 10 min.

Fig. 1. Scanning electron micrographs of starch grains isolated from HLB-‐‑affectedleaves (A) and from leaves on control girdled branches (B). Some irregularities, as splitgrains, are caused by the abrasive method of extraction. Starch granules were purifiedin a 50% sucrose cushion and centrifugation at 10 x g for 10 min.

Fig. 1. Scanning electron micrographs of starch grains isolated from HLB-‐‑affectedleaves (A) and from leaves on control girdled branches (B). Some irregularities, as splitgrains, are caused by the abrasive method of extraction. Starch granules were purifiedin a 50% sucrose cushion and centrifugation at 10 x g for 10 min.

A

B


starch granules from HLB-affected trees were morphologically indistinguishable from those of girdled branches (Fig. 1B). The slight differences in size in Figure 1 were likely a time effect and not a true distinguishing characteristic since starch populations of smaller size have been obtained from HLB-affected trees.

BirefrinGence. Starch grains viewed under polarized light microscopy equipped with λ-plate displayed the characteristic Maltese cross (Wang et al., 1998), as illustrated by control wheat and rice grains (Fig. 2 A, B, respectively). For both HLB-affected (Fig. 2C) and healthy girdled control starch samples (Fig. 2D) birefringence was comparatively similar and hilum position central throughout the many samples examined. In general, our samples from HLB-affected trees were indistinguishable from girdled branches (Fig. 2 C, D).

Iodine binding stArch extrAct. Unfractionated starch samples from both

HLB-affected and girdled branches were heated and mixed with iodine solution for 15 min before absorption spectra was deter-mined. The absorption spectra for both populations of starches appear virtually identical (Fig. 3). The small variation in the wavelength of maximum absorbance (λmax; HLB = 604.1 and 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 Sepharose CL-2B column resulted in two distinctive peaks (Fig. 4A). The first peak (lower numerical fractions, I) corresponds to amylopectin given its larger molecular size. The smaller amylose

chains eluted afterwards (higher fraction numbers, II). Elution pattern for the two fraction peaks for both the HLB-affected and girdled control starch samples appeared similar. The small differ-ences in the amylose/amylopectin peak ratios of 1.4 ± 0.17 SE for

18

Fig. 2. Light micrographs of starch granules in water viewed under polarized light inconjunction with _ plate. Starch granules from: (A) wheat and (B) rice werecommercially obtained, whereas those from HLB-‐‑affected trees (C) and control girdledbranches (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 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. 3. Absorption spectra of starch samples from leaves obtained from HLB-affected and control healthy, girdled branches. Starch samples were mixed with 2% I2 solution and, after 15 min, OD determined between 350 and 800 nm. Values are the average of 6 samples and are not statistically different (P ≤ 0.05).

λ nm400 600 800

OD

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Girdled

HLB

λ nm

19

??nm400 600 800

OD

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Girdled

HLB

Fig. 3. Absorption spectra of starch samples from leaves obtained form HLB-‐‑affectedand control healthy, girdled branches. Starch samples were mixed with 2% I2 solutionand, after 15 min, OD determined between 350 and 800 nm. Values are the average of 6samples and are not statistically different (p ≤ 0.05).


HLB-affected trees and 1.16 ± 0.07 SE for samples from girdled branches, respectively, were not statistically different (P ≤ 0.05). However, when individual fractions were analyzed for their λmax, a significant shift occurred in the HLB-affected starches (Fig.

4B). For example, amylopectin λmax for fraction 13 from girdled branches was 570 nm compared to that of HLB-affected fraction where values reached 584 nm. Amylose samples (fraction 23) exhibited a similar increase from 604 nm for girdled samples to 615 nm for HLB-associated samples. The differences λmax for the amylopectin and amylose peaks were 14 nm and 11 nm higher in the HLB samples than for the girdled samples, respectively (Fig. 5B). These increases in λmax are an indication of a lengthening in the starch glucan polymer (Bailey and Whelan, 1961).

GelAtinizAtion. Gelatinization of starch grains in response to applied heat was examined microscopically under polarized light and λ-plate. As a reference a wheat grain is presented in Fig. 5. Heat was applied to produce a temperature increase of 1 °C·min–1, and changes recorded every 5 °C, although only relevant micrographs are presented. In wheat starch grains, structural integrity was maintained up to approximately 90 °C when gela-tinization began, as seen by the decline in brightness and loss of the Maltese cross. Similar results were described by Bogracheva et al. (1998) for pea, potato, and maize starch. In samples from control girdled branches, foliar starch began gelatinizing at 85 °C, with complete loss of crystalline structure taking place by the time the temperature reached 90 °C (Fig. 6). However, in starch from HLB-affected trees, gelatinization commenced at approximately 10 °C higher (≈95 °C; Fig. 7) than for girdled samples, with complete gelatinization reached at around 100 °C.

Discussion

In leaves, starch accumulates during the day hours and mobi-lized at times of low photosynthetic activity to maintain a steady carbon supply to heterotrophic tissues. Under normal conditions, citrus leaves accumulate little or no starch (Yelenosky and Guy, 1977) and only noticeable amounts are produced as a result of zinc deficiency (Smith and Struckmeyer, 1974) or girdling (Schaffer et al., 1986). Once accumulated, leaf starch in citrus tends not to be degraded (Goldschmidt and Koch, 1996), although some depletion of the limited starch reserves may occur during the 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. 4. Separation of amylose and amylopectin fractions from starch samples: (A) obtained from leaves of control girdled branches ( ⎯ ) and from HLB-affected leaves (- - -). Separation was obtained using a Sepharose CL2B chromatography (Zeeman et al., 2002), and collected fractions mixed with 2% I2 prior to analysis at 595 nm. Values are the means of 6 experiments with no statistical differences between II/I peak ratios (P ≤ 0.05). (B) Each individual fraction was also analyzed for the wavelength of maximum absorbance λmax. The λmax values (B) were statistically different (P ≤ 0.05).

21

Fig. 5. Polarized-‐‑light micrograph of a wheat grain during gelatinization in 50% glycerol

using a microscope viewed in conjunction of a _ 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.

20

0 20 40

560

580

600

620

??m

ax

540

560

580

600

620

Fraction number

OD

595 nm

0

0.2

0.4

0.6

0.8

1

1.2 Girdled

540

560

580

600

620A

B

HLB

Girdled

HLB

?

??

Fig. 4. Separation of amylose and amylopectin fractions from starch samples (Panel A)obtained from leaves of control girdled branches ( _ ) and from HLB-‐‑affected leaves (-‐‑ -‐‑ -‐‑). Separation was obtained using a Sepharose CL2B chromatography (Zeeman et al.,2002), and collected fractions mixed with 2% I2 prior to analysis at 595 nm. Values arethe means of 6 experiments with no statistical differences between II/I peak ratios (p ≤0.05). (Panel B) Each individual fraction was also analyzed for their wavelength ofmaximum absorbance _max. The _max values (B) were statistically different (p ≤ 0.05)

OD

595

nm

λ max

I

II


Citrus trees affected by HLB accumulate considerable amounts of starch in practically every live cell of aerial organs (Achor et al., 2010; Etxeberria et al., 2009). Not only photosynthetic cells and vascular parenchyma become replete with starch, but phloem elements also develop starch granules (Folimonova and Achor, 2010). This unusual distribution and biosynthetic behavior (Kim et al., 200923), in addition to the several reports of alterations to starch properties caused by pathogen infection (Handford and Carr, 2007; Roberts and Wood, 1982; Savile, 1942; Tainter and Lewis,

1982; Técsi et al., 1992) prompted us to investigate the possibil-ity that HLB-induced starch may possess unique properties that will allow its distinction from starch formed in healthy tissues. Any morphological or physiological abnormality can potentially serve as a diagnostic tool and will provide further understanding of the effects of HLB in citrus trees.

We first concluded that the morphological characteristics of HLB-induced starch are not different in size, shape, and overall appearance from those of healthy branches. We based

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. 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 DFC295 3.0 MP digital camera. The experiment was run in triplicate.

22

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.

23

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 _ 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.


this conclusion on our observations of over 20 micrographs at different magnifications taken from six random trees, of which Fig. 1 is just an example. Starches from both sources appeared to be of various irregular shapes with no distinctive identifiable morphological features. Although some of the morphological irregularities in our samples may have been due to the abrasive nature of the extraction method, a great deal of unevenness was also evident in electron micrographs from whole tissues (Etxeber-ria et al., 2009). In contrast, genetically transformed pea grains, for example, exhibited clear morphological changes (Wang et al., 1997) as were those from potato tubers infected with an unidenti-fied virus (Savile, 1942).

Other possible biochemical differences between starches of HLB-affected trees and girdled branches were based on the ability of starch to react with iodine to produce a range of colors depending on the size of the starch chain. When mixed with iodine, starch chains of about 12 units begin to form a brownish color (with a λmax of 490 nm). As the chain length increases, the complex continues to change in color through brown, red, purple, and blue when the length is roughly 45 DP. Although visibly the color blue does not change any further, the λmax continues to increase up to 645 nm, when DP reaches 350 to 400 (Bailey and Whelan, 1961; Santacruz and Åman, 2005). In our samples, whole starch extracts gave a deep blue color with average maxi-mum absorbance peaks at 616 and 614 nm for HLB and girdled samples, respectively (Fig. 3). Therefore, the combined whole starch fraction does not appear to provide any identifiable variations in the starch molecule. However, clear differences emerged after starch fractions were separated into amylose and amylopectin. When individual fractions were analyzed for λmax, fractions for HLB-affected starch were consistently and significantly higher than those from girdled branches (Fig. 4B), indicating a higher degree of polymerization. The average difference in λmax for the amylopectin (fractions 13) and amylose (fractions 24) was 14 and 11 nm, respectively. Based on detailed studies of the starch molecule length and corresponding absorption spectra (Bailey and Whelan, 1961), we estimate the degree of polymerization for amylopectin and amylose from HLB-affected trees increased by 12 and 45 glucose units, respectively.

The increase in degree of polymerization of the amylopectin chain length, if real, should result in higher crystallinity and consequently higher gelatinization temperatures. Gelatinization experiments (Figs. 5–7) revealed an increase in gelatinization temperature for the HLB-affected starch (Fig. 7) in agreement with previous reports for waxy, high-amylose and low-amylose wheat starch (Da Graca and Korsten, 2004). Although gelatini-zation temperature is affected by many factors including water content, pH, rate of heating, shear stress and the presence of other compounds (Walstra, 2003); all these being constant, the increase in gelatinization temperature can be attributed to a higher crystallinity that is typically associated with higher content of amylopectin. Noda et al. (1998) also noted that gelatinization properties were independent from total amylose content whereas amylopectin molar distribution had a more profound effect on gelatinization. Therefore, it is likely that the higher gelatiniza-tion temperatures noted for HLB-affected starch were the result of the increase in the amylopectin chain length (Fig. 4B) given the negligible effect on in amylose content (Noda et al., 1998).

Starch sporadically accumulates in amounts large enough to produce symptoms analogous to those characteristic of HLB-affected tissues. Starch also accumulates under zinc deficiency, however, it accumulates at low concentrations and the characteristic

foliar symptoms are easily distinguishable from HLB-affected leaves. The results presented here indicate that, although morpho-logically similar, starch grains from leaves affected by HLB are biochemically different from those formed by healthy trees under phloem blockage, a condition frequently caused by mechanical injury. The major biochemical changes brought about HLB are an across-the-board increase in glucan chain lengths and the consequential rise in gelatinization temperature, which combined, reflect the strong effect of HLB in photoassimilate partitioning.

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Starch Analysis of HLB-affected and Control Healthy Citrus