Physiological and Molecular Plant Pathology 79 (2012) 71e78
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Physiological and Molecular Plant Pathology
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Characterization of leaf starch from HLB-affected and unaffected-girdled citrus trees
Pedro Gonzalez a, Jose Reyes-De-Corcuera b, Ed Etxeberria a,* a Department of Horticultural Sciences, University of Florida/IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, Florida 33850, USA b Department of Food Science and Human Nutrition, University of Florida/IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, Florida 33850, USA article info abstract
Article history: Starch content in leaves of HLB (Huanglongbing or citrus greening)-affected branches increases sharply Accepted 21 May 2012 compared to those from non-HLB trees. Starch not only over-accumulates in photosynthetic cells, but starch grains become prominent in vascular parenchyma and sieve elements as well. These observations Keywords: imply strong disturbances in starch metabolism and photoassimilate partitioning in HLB-affected trees. Citrus greening Based on the elevated starch content, appearance of starch granules in phloem elements, and previous Candidatus Liberibacter asiaticus reports of the pathogen effect on starch properties, we hypothesized that starch from HLB-affected citrus Starch properties trees may differ morphologically, physically and/or chemically from starch accumulated in otherwise healthy leaves. To obtain starch granules from healthy trees of comparable size to those of HLB-affected trees, we girdled 2-year-old branches and allowed starch to accumulate for 3 months. Starch morphology was investigated under brightfield, polarized light and SEM. HLB-induced starch grains were not morphologically different in size, shape and overall appearance from those of girdled branches. When reacted with 2% I2, no significant difference was observed in the absorption spectra of whole starch fractions (lmax for HLB ¼ 604.1 and girdled 606.2 nm, respectively; n ¼ 6; p 0.05) nor in their amylose/ amylopectin ratio (HLB ¼ 1.4 0.17 and girdled ¼ 1.16 0.07, p 0.05) after chromatographic sepa- ration. However, lmax for individual fractions of HLB-affected leaves increased between 11 and 14 nm indicating a significant increase in the degree of polymerization of chain lengths of 12e45 glucose units. The increase in amylopectin chain length was confirmed 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 were morphologically similar but differed biochemically from those formed by healthy trees after phloem blockage caused by mechanical injury. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction spread has brought renewed interest on this disease given its devastating potential to the world’s citrus industry. Citrus huanglongbing (HLB or citrus greening) is a highly In citrus trees, specific HLB symptoms do not exist. Some destructive, fast spreading disease of citrus. The disease is associ- symptoms such as yellow shoots, leaf blotchy mottle, and lopsided ated with a fastidious, Gram-negative, obligate parasite, phloem- fruits with color inversion and aborted seeds are characteristic, but limited a-protobacterium (Candidatus Liberibacter) [15,22] not yet they do not always occur together in the same tree, they can be fully cultured, although recent attempts have resulted in limited distorted or masked by symptoms of other diseases, or induced by success [10,32]. Long established in eastern Asia and South Africa conditions other than HLB [6]. Amongst other HLB-induced char- [6,9], the disease is now well established in Brazil [7,38], and the acteristics, Schneider [30] noted massive starch build-up in leaves states of Florida [5,18], Louisiana and in Puerto Rico [11]. Its rapid and petioles which otherwise accumulate little or no starch under normal conditions [43]. Detectable amounts of foliar starch are only sporadically observed as a result of zinc deficiency [33] or girdling (as in severed branches). Schneider [30] concluded that starch l Abbreviations: HLB, huanglongbing or citrus greening; max, wavelength of accumulation resulted from the obstruction of photoassimilate maximum absorbance; MES, 4-Morpholineethanesulfonic acid; PCR, Polymerase transport by necrotic phloem pockets scattered throughout the Chain Reaction; SEM, Scanning Electron Microscope. * Corresponding author. Tel.: þ1 863 956 1151. foliar vascular system prompting accelerated rates of starch E-mail address: eetxeber@ufl.edu (E. Etxeberria). synthesis in leaves. In fact, the yellowing leaf mottle symptoms of
0885-5765/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2012.05.002 72 P. Gonzalez et al. / Physiological and Molecular Plant Pathology 79 (2012) 71e78
HLB-affected leaves are believed to result from the disintegration of months of girdling, with leaf abscission becoming prominent after the chloroplast thylakoid system caused by the bulky starch build- 6 months. This ensured that starch granules had reached their up. Similar leaf yellowing as a result of thylakoid damage from maximum size becoming comparable to those from HLB-affected starch accumulation can be artificially induced by branch girdling leaves, thus eliminating any possible effect due to size. of citrus trees [31]. In most instances, leaves from girdled branches For comparative purposes, commercial starch samples from rice, display characteristics that closely resemble those from HLB- wheat and potato were used. These were all purchased from Sigma, affected trees. St. Louis, MO (rice, S-7260; wheat, S-5127; potato, S-2004). Recently, Etxeberria et al. [12] and Folimonova et al. [13] reported that starch grains, besides accumulating in virtually 2.2. Starch extraction and isolation every photosynthetic and parenchyma cell of all aerial parts [12], are also found in phloem sieve elements [13], a rare condition in For the isolation of starch granules from leaf tissue, three tissue higher plants. The profound changes in starch content were later discs from a single leaf, made with a paper hole puncher verified by studies using Fourier transform infrared-attenuated (27.3 mm2), were homogenized in 2-mL capped tubes containing total reflection spectroscopy [20] where divergences in the 500 mL of water and four metal beads (2.36 mm diameter) (Mobio regions attributable to carbohydrate vibrational bands indicated Laboratories, California). Homogenization was carried out in two chemical alterations (amount, type or structure) to the carbohy- 40-s cycles for a total of 80 s using a Precellys 24 Tissue Homoge- drate fractions. In support of the observed starch increases, Kim nizer (Bertin Technologies, France). The homogenate was subse- et al. [23] reported up-regulation of starch biosynthetic enzymes in quently placed on top of a 50% sucrose cushion and starch granules HLB-affected leaves. precipitated at 1 g. The pellet containing starch granules was Starch is a natural product of photosynthetic CO2 fixation in washed three times with water in conical microfuge tubes and green tissues. Formed by a-1,4 glucose linkages, starch exists in two centrifugation at 10 g. The final pellet was dried overnight under forms, the soluble, small linear chain amylose and the highly vacuum in a desiccator containing calcium sulfate. Starch samples branched insoluble amylopectin [42]. In plants, starch is composed were stored at room temperature in a separate desiccator until use. mainly of 30% amylose and 70% amylopectin [34], with specific ratios varying amongst species. It is also well established that the 2.3. Grain morphology levels of plant starches [21,24] and their physical, biochemical and morphological characteristics are highly influenced by many Starch samples, re-suspended in water, were filtered under conditions ranging from environmental factors [35], genetic alter- vacuum using cellulose nitrate membrane filter papers (0.2 mm ations [8,16] and pathogen infection [19,27,29,36,37]. Whatman GmbH) and dried overnight in a vacuum desiccator. The Leaves from accidentally severed (girdled) branches are often dried starch was mounted on stubs and coated with gold/palladium symptomatically indistinguishable from HLB-affected citrus leaves, using a Ladd sputter coater (Ladd Research Industries, Burlington, including the accumulation of high levels of starch. Based on the VT). Samples were viewed using a Hitachi S530 SEM (Tokyo, Japan) unusual tissue distribution [12], appearance of starch granules in and photographed using a Canon EOS Rebel XT digital camera phloem elements [13], and previous reports of pathogen effect on (Tokyo, Japan). plant starch properties [8,19,29,36,37], it is reasonable to expect that starch from HLB-affected citrus trees will differ in morphology, 2.4. Gelatinization physical and/or chemical properties from starch accumulated under the absence of pathological conditions. Starch gelatinization was characterized by controlled tempera- To further understand the physiological and metabolic effects of ture polarized light microscopy. Starch granules were re-suspended HLB on citrus trees, we initiated the present study aimed at char- in 50% glycerol due to a high mobility of starch granules in water acterizing selected properties of starch grains from HLB-affected alone. Polarized light microscopy was performed using a Leica trees and compared these to starch grains obtained from control DMLP polarizing microscope with l plate at 40 magnification girdled trees. Our results indicate that starch from HLB-affected equipped with a Leica DFC295 3.0 MP digital camera. The gelati- trees contain significantly larger starch polymers than starch nization properties of individual starch granules during heating from healthy trees. The longer amylopectin chains were validated were investigated using a microscope hot stage Linkam LTS350 by gelatinization studies in which the gelatinization temperature adaptor with LNP and TMS93 temperature controllers (Linkam for starch from HLB-affected leaves was approximately 8 C higher Scientific Instruments, Surrey, UK). The stage was heated from 20 to than that of control samples from healthy girdled trees. 100 Cat1 C/min.
2. Materials and methods 2.5. Starch digestion (a-amylase treatment)
2.1. Plant material To visualize the internal structure of the starch granules, starch preparations were ground in a liquid N2-cooled mortar to crack Leaf samples from HLB-affected trees were taken from PCR- open the granules. The resulting solutions were treated with 200 diagnosed HLB-positive ‘Valencia’ (Citrus sinensis L. Osbeck) trees units of a-amylase (100 units from Bacillus subtilis and 100 units showing classical symptoms. Samples were taken from six different from Apergillus oryzae; Sigma catalog numbers 10,070 and 10,065, Valencia orange trees grown at the Citrus Research an Education respectively) in 1 mL reaction medium containing 100 mM MES- Center in Lake Alfred, Florida. For control samples, branches of HLB- NaOH buffer (pH 6.0) for 6 h. Partially hydrolyzed starch granules negative Valencia orange trees of equal number were girdled by were collected by centrifugation, washed three times with water excising a 1.5 cm ring of the bark tissue using a laboratory scalpel. A and prepared for SEM observation as described above. light layer of petroleum jelly was applied to the exposed xylem tissue to avoid desiccation. To standardize starch content and size, 2.6. Amylose and amylopectin separation leaves were periodically tested for starch content until levels reached those of HLB-affected leaves, approximately 3 months after For the separation of amylose and amylopectin we followed the girdling. Leaf yellowing and blotchy mottle became evident after 2 procedure described by Santacruz and Åman [28]. Dried samples P. Gonzalez et al. / Physiological and Molecular Plant Pathology 79 (2012) 71e78 73 were solubilized in 1 mL 0.5 M NaOH and heated in boiling water characteristic since starch populations of smaller size have been for 30 s. The solubilized samples were fractionated by gel perme- obtained from HLB-affected trees. Given that blotchy yellowing ation chromatography (GPC) in a Sepharose CL-2B column (Sigma symptoms started to appear between 2 and 3 months after girdling CL2B300). The column was (27 cm h 2.6 cm d) using 0.01 M NaOH and the similarity in grain size between HLB-affected leaves and as eluent. The flow rate was set at 0.4 mL/min and 2 mL fractions of healthy girdled samples, it is reasonable to assume that yellowing were collected. Collected fractions from Sepharose CL-2B were symptoms start appearing when grains reach approximately 2 mm mixed with 0.2 mL of I2-KI solution (2 mg/mL I2, 20 mg/mL KI) and in size. absorbance of the polymereiodine complex was determined using a microplate reader at 595 nm (BioRad model 680). Absorbance 3.1.2. Gelatinization spectrum was determined between 350 and 800 nm (UV-VIS Starch grains viewed under polarized light microscopy equipped Spectrophotometer, Shimadzu UV-1700) 15 min after addition of with l-plate displayed the characteristic Maltese cross and the I2-KI reagent. a prominent hilum [42], as illustrated by control potato, wheat and rice grains (Fig. 2A, B, C, respectively). For both HLB-affected and 3. Results healthy girdled control starch samples, birefringence was compar- atively similar and hilum position central throughout the many 3.1. Grain morphology samples examined (Fig. 2D, E). In general, our samples from HLB- affected trees were indistinguishable from girdled branches, both 3.1.1. SEM consisting of structures with intermediate characteristics between Isolation of starch grains from HLB-affected leaves in a sucrose storage and chloroplastic starches (Fig. 2D, E). density cushion produced populations of granules of approximately 1e3 mm(Fig. 1A, B). The shape of granules was not entirely discoid, 3.1.3. Starch digestion (a-amylase treatment) but appeared to be more a mixture of discoid, granular and oval Hydrolysis of starch grains with a-amylase, which preferentially shaped granules, many of which were more characteristic of digests the amorphous regions of the granule, renders partially storage starches than those isolated from photosynthetic tissues digested grains devoid of amylose depicting mostly the crystalline [44]. The size of grains remained reasonably consistent between amylopectin structure. Storage granules possess two levels of samples despite the likelihood that the length of infection amongst internal structure created by the organization of the amylopectin trees differed considerably in our random sampling. Although molecules, alternating concentric crystalline and amorphous starch samples contained vascular parenchyma granules, these lamella [44]. Semi-crystalline or amorphous amylose areas of the were indistinguishable and were a negligible percent of the entire granule are easier hydrolyzed than the hard crystalline amylopectin leaf blade extract as determined by light and EM analysis (data not ones [14]. Fig. 3A shows a SEM micrograph of a typical enzymatic shown). Starch grains from leaves on healthy girdled branches digestion of a wheat grain after 6 h incubation in 200 units a- reached a similar size at about 3 months after girdling (Fig. 1C, D). amylase. In this type of grain, the crystalline amylopectin concen- When viewed under SEM, populations of starch granules from tric circles are evident with alternating hollow digested amorphous HLB-affected trees were morphologically indistinguishable from amylase gaps. Similar structures were not seen in starch grains those of girdled branches (Fig. 1). Their slight differences in size in from either HLB-affected (Fig. 3B) or control girdled trees (Fig. 3C), Fig. 1 are likely a time effect and not a true distinguishing although some random digestion was evident in both cases.
Fig. 1. Scanning electron micrographs of starch grains isolated from HLB-affected leaves (A, B) and from leaves on control girdled branches (C, D) at two magnifications. 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 g for 10 min. 74 P. Gonzalez et al. / Physiological and Molecular Plant Pathology 79 (2012) 71e78
Fig. 2. Light micrographs of starch granules in water viewed under polarized light in conjunction with l plate. Starch granules from potato (A), wheat (B) and rice (C) were commercially obtained whereas those from HLB-affected trees (D) and control girdled branches were obtained as indicated in Materials and Methods.
3.2. Iodine binding a significant shift occurred in the lmax for the HLB-affected starches (Fig. 5B). For girdled trees, amylopectin (fraction 13) lmax was 3.2.1. Complete starch extract 570 nm compared to HLB-affected where values reached 584 nm. Unfractionated starch samples from both HLB-affected and Amylose samples (fraction 23) exhibited a similar increase from girdled branches were heated and reacted with iodine solution for 604 nm for girdled samples to 615 nm for HLB-associated samples. 15 min before determination of their absorption spectra. When The differences lmax for the amylopectin and amylose peaks were plotted together, the absorption spectra for both populations of 14 nm and 11 nm higher in the HLB samples than for the girdled starches appear virtually identical (Fig. 4). The small variation in the samples, respectively (Fig. 5B). These increases in lmax are an wavelength of maximum absorbance (lmax; HLB ¼ 604.1 and indication of a lengthening in the starch glucan polymer [2]. girdled 606.2 nm) was statistically insignificant based on the number of replicates (n ¼ 6; p 0.05). 3.3. Gelatinization
3.2.2. Amylose and amylopectin Gelatinization of starch grains in response to applied heat was Separation of amylose from amylopectin in a Sepharose CL-2B examined microscopically under polarized light and l plate. The column resulted in two distinctive peaks (Fig. 5A). The first use of glycerol in the medium was needed to stabilize the starch eluting peak (lower numerical fractions, I) corresponds to amylo- grains and avoid any movement during examination. As a reference pectin given its larger molecular size, whereas the smaller amylose for the observed physical changes, a wheat grain is presented in chains eluted afterwards (higher fraction numbers, II). Elution Fig. 6. Heat was applied to produce a temperature increase of 1 C/ pattern for the two fraction peaks for both the HLB-affected and min, and changes recorded every 5 C, although only relevant girdled control starch samples appeared similar. The apparent micrographs are presented. In wheat starch grains, structural differences in the amylose/amylopectin peak ratios of 1.4 0.17 SE integrity was maintained up to approximately 90 C when gelati- for HLB-affected trees and 1.16 0.07 SE for samples from girdled nization began, as seen by the decline in brightness and loss of the branches, respectively, were not statistically different (p 0.05). Maltese cross. Similar results were described by Bogracheva et al. However, when individual fractions were analyzed for their lmax, [4] for pea, potato and maize starch. In samples from control girdled
Fig. 3. Scanning electron micrographs of partially digested starch granules from wheat (A), leaves from HLB-affected (B) and girdled branches (C). Granules were cracked by grinding in liquid nitrogen and then incubated in 200 units amylase for 6 h. P. Gonzalez et al. / Physiological and Molecular Plant Pathology 79 (2012) 71e78 75
Fig. 4. Absorption spectra of starch samples from leaves obtained form HLB-affected and control healthy, girdled branches. Starch samples were mixed with 2% I2 solu- tion 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). branches, foliar starch began gelatinizing at 85 C, with complete loss of crystalline structure taking place by the time the tempera- ture reached 90 C(Fig. 7). However, in starch from HLB-affected trees, gelatinization commenced at approximately 10 C higher (w95 C; Fig. 8) than for girdled samples, with complete gelatini- zation reached at around 100 C.
Fig. 5. Separation of amylose and amylopectin fractions from starch samples (Panel A) 4. Discussion 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% I prior to analysis at 595 nm. In plant leaves, starch accumulates during light hours and is 2 Values are the means of 6 experiments with no statistical differences between II/I peak mobilized at times of low photosynthetic activity to maintain ratios (p 0.05). (Panel B) Each individual fraction was also analyzed for their a steady carbon supply to heterotrophic tissues. Citrus leaves, wavelength of maximum absorbance lmax. The lmax values (B) were statistically however, accumulate little or no starch under normal conditions different (p 0.05). [43], and only noticeable amounts are accumulated as a result of zinc deficiency [35] or girdling [31]. Once accumulated, leaf starch in citrus tends not to be degraded [17], although some depletion of the an example, we concluded that the morphological characteristics of limited starch reserves may occur during the winter months [25]. HLB-induced starch are not different in size, shape and overall Citrus trees affected by HLB accumulate considerable amounts appearance from those of healthy branches. Starches from both of starch in practically every live cell of the aerial tree parts [1,12].In sources appeared to be of various irregular shapes ranging from HLB-affected tissue not only photosynthetic cells and vascular discoid to tubular, with no distinctive identifiable morphological parenchyma become replete with starch, but phloem elements also features. Although some of the morphological irregularities in our develop starch granules [13], a characteristic of seemingly only few samples may have been due to the abrasive nature of the extraction plant families including Rutaceae [3]. The high levels of starch method, a great deal of unevenness was also evident in electron accumulation are not due to the removal of terminal sinks by micrographs from whole tissues previously published by Etxeberria pathogen-induced necrosis as in the case of red clover mottle virus et al. [12]. In contrast, genetically transformed pea grains, for in pea [37], since in Citrus affected by HLB starch accumulation example, exhibited clear morphological changes [41] as were those takes place in spite the presence of adjacent fruit and in vegetative from potato tubers infected with an unidentified virus [29]. In both growth. latter cases, starch was extracted from storage parenchyma tissue This unusual starch distribution and biosynthetic behavior [23], compared to samples reported here where starch had mostly in addition to the many reports of modifications to starch proper- a chloroplastic origin. ties caused by pathogen infection [19,29,36,37], prompted us to The ability of starch to react with iodine to produce a range of investigate the possibility that HLB-induced starch may possess colors depending on the size of the glucan chain was exploited to unique properties that will allow distinction from starch formed in investigate other possible biochemical differences between healthy tissues. Any morphological or physiological abnormality starches of HLB-affected trees and girdled branches. When mixed would allow for further understanding of the effects of HLB in citrus with iodine, starch forms a stable complex with the a-1,4 linked trees and may serve as basis for potential diagnostic purposes. glucans by insertion into the glucan helices. After reaching the Based on our observations of over 20 micrographs at different length where color forms at about 12 units (with a lmax of magnifications taken from six random trees, of which Fig. 1 is just 490 nm), the complex continues to change in color through 76 P. Gonzalez et al. / Physiological and Molecular Plant Pathology 79 (2012) 71e78
Fig. 6. Polarized-light micrograph of a wheat grain during gelatinization in 50% glycerol using a microscope viewed in conjunction of a l plate. The heating stage with temperature control, 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. brown, red, purple and blue when the length is roughly 45 DP The increase in degree of polymerization of the amylopectin (degree of polymerization). Although visibly the color blue does chain length, if consequential, should result in higher crystallinity not change any further, the lmax continues to increase up to and gelatinization temperatures, although not necessarily trans- 645 nm, when DP reaches 350 to 400 [2,28]. In our samples, whole lating into morphological alterations. Gelatinization experiments starch extracts gave a deep blue color with average maximum (Figs. 7 and 8), revealed that the increase in the amylopectin length absorbance peaks at 616 and 614 nm for HLB and girdled samples, for the HLB-affected starch (Fig. 5B) resulted in higher gelatiniza- respectively (Fig. 4). The combined whole starch fraction, there- tion temperature (Figs. 7 and 8) in agreement with previous reports fore, does not appear to provide any identifiable variations in the for waxy, high-amylose and low-amylose wheat starch [39] and starch molecule. However, after starch fractions were separated structural studies by Borgacheva et al. [4] where higher amylo- into amylose and amylopectin, clear differences emerged. When pectin content is indicative of a more organized crystalline struc- individual fractions were analyzed for lmax, fractions for HLB- ture. Although gelatinization temperature is affected by many affected starch were consistently and significantly higher than factors including water content, pH, rate of heating, shear stress those from girdled branches (Fig. 5B), indicating a higher degree of and the presence of other compounds [40]; all these being polymerization. The average difference in lmax for the amylopectin constant, the increase in gelatinization temperature can be attrib- (fractions 13) and amylose (fractions 24) was 14 and 11 nm, uted to a higher crystallinity that is typically associated with higher respectively. Based on detailed studies of the starch molecule content of amylopectin. In detailed studies of starches from various length and corresponding absorption spectra [2], degree of poly- individuals of the same species, Noda et al. [26] also noted that merization increased by 12 and 45 glucose units for amylopectin gelatinization properties were independent from total amylose and amylose, respectively. content whereas amylopectin molar distribution had a more
Fig. 7. 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 l plate. The heating stage with temperature control 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. P. Gonzalez et al. / Physiological and Molecular Plant Pathology 79 (2012) 71e78 77
Fig. 8. Polarized-light micrograph of a leaf starch grain from an HLB-affected tree during gelatinization in 50% glycerol using a microscope hot stage and viewed in conjunction of a l plate. The heating stage with temperature control 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.
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