Planta (2007) 226:517–527 DOI 10.1007/s00425-007-0501-7
O R I G I N A L A R T I CL E
Mir1-CP, a novel defense cysteine protease accumulates in maize vascular tissues in response to herbivory Lorena Lopez · Alberto Camas · Renuka Shivaji · Arunkanth Ankala · Paul Williams · Dawn Luthe
Received: 14 November 2006 / Accepted: 15 February 2007 / Published online: 10 March 2007 © Springer-Verlag 2007
Abstract When lepidopteran larvae feed on the insectresistant maize genotype Mp708 there is a rapid accumulation of a defensive cysteine protease, Maize insect resistance 1-cysteine protease (Mir1-CP), at the feeding site. Silver-enhanced immunolocalization visualized with both light and transmission electron microscopy was used to determine the location of Mir1-CP in the maize leaf. The results indicated that Mir1-CP is localized predominantly in the phloem of minor and intermediate veins. After 24 h of larval feeding, Mir1-CP increased in abundance in the vascular parenchyma cells and in the thick-walled sieve ele-
ment (TSE); it was also found localized to the bundle sheath and mesophyll cells. In situ hybridization of mRNA encoding Mir1-CP indicated that the primary sites of Mir1CP synthesis in the whorl are the vascular parenchyma and bundle sheath cells. In addition to the phloem, Mir1-CP was also found in the metaxylem of the leaf and root. After 24 h of foliar feeding, the amount of Mir1-CP in the root xylem increased and it appeared to move from xylem parenchyma into the root metaxylem elements. The accumulation of Mir1-CP in maize vascular elements suggests Mir1-CP may move through these tissues to defend against insect herbivores.
Lorena Lopez and Alberto Camas contributed equally to the research and preparation of this manuscript.
Keywords Cysteine protease · Induced plant defense · Spodoptera · Phloem · Thick-walled sieve elements · Xylem · Zea · Plant-herbivore interactions · Mobile signal
L. Lopez · A. Camas · R. Shivaji · A. Ankala Department of Biochemistry and Molecular Biology, Mississippi State University, Box 9650, Mississippi State, MS 39762, USA e-mail:
[email protected] A. Camas e-mail:
[email protected]
Abbreviations Mir1-CP Maize insect resistance 1-cysteine protease FAW Fall armyworm TSE Thick-walled sieve elements
R. Shivaji e-mail:
[email protected] A. Ankala e-mail:
[email protected]
Introduction
P. Williams Corn Host Plant Resistance Laboratory, USDA-ARS, Mississippi State University, Box 9555, Mississippi State, MS 39762, USA e-mail:
[email protected]
The carnivorous plants are a classic example of a plant oVense against arthropods. These plants produce a cocktail of hydrolytic enzymes, including proteases that devour their insect prey (Hooker 1874). Although not as extreme, emerging evidence has demonstrated that other plants use proteases, including the cysteine proteases, in their defense pathways (van der Hoorn and Jones 2004). There are three potential roles for proteases in plant defense, perception of the invader, activation downstream signaling pathways and
D. Luthe (&) Department of Crop and Soil Science, Pennsylvania State University, 116 ASI Building, University Park, PA 16802, USA e-mail:
[email protected]
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execution of a defense response (van der Hoorn and Jones 2004). Although almost every class of protease is involved in some type of defense response, we are primarily interested in the roles of the papain-like cysteine (C1) proteases. There are four papain-like cysteine proteases that have defensive activity in plants. The classic example of a cysteine protease that potentially defends plants against herbivory is papain, which is abundant in papaya (Carica papaya) latex. It has been directly demonstrated that papain or papain-like enzymes in the latex of papaya and Wg (Ficus virgata) inhibited the growth of lepidopteran larvae (Konno et al. 2004). In tomato (Lycopersicum esculentum), the papain-like cysteine protease RCR3, which is found in the apoplast, is required for the resistance gene Cf-2 to recognize the avirulence gene Avr2 of the pathogenic fungus Cladosporium fulvum (Krüger et al. 2002). RD21 is another papain-like cysteine protease that is found in vesicles (ER bodies) in Arabidopsis. The ER bodies Wlled with this protease appeared to accumulate in response to wounding and insect feeding (Matsushima et al. 2002) and hence may be involved in defense. Maize insect resistance 1-cysteine protease (Mir1-CP) is a novel papain-like cysteine protease that rapidly accumulates in the whorls of insect resistant maize genotypes in response to feeding by lepidopteran larvae (Pechan et al. 2000). The growth of fall armyworm (FAW) (Spodoptera frugiperda) larvae that fed on transgenic callus ectopically expressing Mir1-CP was retarded by »80% (Pechan et al. 2000). It has been shown the Mir1CP attacks and damages the insect’s peritrophic matrix and impairs nutrient utilization (Chang et al. 1999; Pechan et al. 2002; Mohan et al. 2005). Mir1-CP is encoded by a single copy gene mir1 on chromosome 6 of the maize genome (Pechan et al. 1999). Mir1-CP has been expressed in an heterologous system and has been shown to have cysteine protease activity (Pechan et al. 2004). In addition, the last 281 bp on the 3⬘ end of mir1 have no matches the databases. Of these 281, 75 bp encode the last 25 amino acids on the C-terminal of Mir1-CP. Bioinformatic analysis predicted that Mir1-CP had some similarity in C-terminal regions with the Citrus tatter virus and apple stem grooving virus movement proteins (MPs) (Fig. 1). In the last 30 amino acids there is a block of 14 amino acids that has »86% similarity with these viral MPs. Potyvirus MPs like HC-Pro, are localized in the phloem and assist in the systemic transport of viruses throughout the plant (Rojas et al. 1997). They often have proteolytic activity (Plisson et al. 2003). In addition to limited similarity to MPs, P-Sort analysis (Nakai and Horton 1999) predicted with 82% certainty that Mir1-CP is a secreted protein. Although many cysteine proteases are localized in the vacuole, these two observations suggested that Mir1-CP is not in that organelle.
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Fig. 1 Alignment of C-terminal amino acids from Mir1-CP (a) viral movement proteins from the citrus tatter leaf virus (b) and apple stemgrooving virus (c). Identical amino acids are shown in black print and conservative amino acids are underlined. Alignment was done using Vector NTI AlignX (Invitrogen). The accession numbers for A, B, and C are AAB70820, BAA03352, and AAP80758
This study was conducted to determine the location of Mir1-CP and mir1 transcripts in the leaves and roots of the insect-resistant genotype Mp708 before and after feeding by FAW larvae. We determined the location of Mir1-CP and mir1 transcripts using immunocytochemistry and in situ hybridization, respectively.
Materials and methods Plant growth and FAW infestation The insect resistant maize (Zea mays) inbred Mp708 (Williams et al. 1990) was used in this study. Plants were grown under greenhouse conditions at the Plant Science Research Center, Mississippi Agricultural and Forest Experiment Station (Mississippi State University) until they were 4–5 weeks old. FAW (S. frugiperda) larvae were reared at the US Department of Agriculture-Agricultural Research Service, Corn Host Plant Research Unit, InsectRearing Laboratory. Approximately third instar larvae were used for infestations. Plant material for microscopy experiments Seven FAW larvae were placed in the whorl of Mp708 plants and allowed to feed. Samples were dissected from the feeding site in the yellow–green whorl sections at 8, 24, and 48 h, and from roots of the same experimental plants. Samples for controls were taken from whorl and roots of uninfested plants. Silver-enhanced light microscopy immunolocalization Tissue segments were prepared for light microscopy by Wxation in 4% paraformaldehyde (v/v) and 1.0% glutaraldehyde (v/v) in 0.1 M phosphate-buVered saline (PBS), pH 7.2 and placed under vacuum for 2 h at room temperature to aid Wxer inWltration. Then samples were placed on a rotating plate overnight at 4°C. After Wxation the leaves were rinsed twice in chilled PBS, 15 min each, dehydrated in a graded ethanol series (30, 50, 75, 95, and 100% ethanol for 15 min each) and embedded in paraYn (Paraplast TX) for
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immunocytochemistry. Sections (10 m) were obtained from paraYn embedded tissue using an American Optical microtome. Then sections were incubated with 5% normal goat serum in blocking buVer [0.01 M PBS buVer containing 0.8% bovine serum albumin (BSA), 0.1% IGSS quality gelatin, pH 7.4] for 1 h at room temperature. After blocking, sections were incubated with anti-Mir1-CP monoclonal serum diluted 1:10 in incubation buVer consisting of 0.8% BSA, 0.1% IGSS quality gelatin, 1% normal goat serum, and 2 mM NaN3 in PBS at pH 7.4 overnight at 4°C. The monoclonal antibody was prepared according to Goding (1980). The sections were then rinsed one time with washing buVer (0.01 M PBS buVer containing 0.8% BSA, 1% IGSS quality gelatin and 2 mM NaN3, pH 7.4) for 5 min and incubated with Auroprobe™ One goat anti-mouse Ig (H + L) (Amersham Biosciences, Piscataway, NJ, USA) diluted 1:50 in incubation buVer for 3 h at room temperature. After several washes, slides were enhanced with silver for 10 min using an IntenSE™ kit (Amersham Biosciences). Then sections were stained with a Harris Hematoxilin solution (Electron Microscope Science) for 5 min. After staining the sections were dehydrated in a graded ethanol series (30, 50, 75, 95, and 100% ethanol for 1 min each), cleared with Citrisolv (Fisher, Atlanta, GA, USA) and mounted using Biomount resin (Electron Microscope Science). Once mounted the sections were examined under bright Weld using an Olympus BX51 microscope equipped with an Olympus DP 70 camera. Digital images were generated using a computerized scanner, and composites of individual prints were assembled using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Silver-enhanced electron microscopy immunolocalization Tissue segments were prepared for transmission electron microscopy by Wxation in 4% paraformaldehyde (v/v) and 0.5% glutaraldehyde (v/v) in 0.1 M PBS (pH 7.2). The tissue was placed under vacuum for 2 h at room temperature and then placed on a rotating plate overnight at 4°C. After Wxation the leaf segments were rinsed twice, 15 min each, in chilled PBS then were dehydrated in a graded ethanol series (30, 50, 75, 95, and 100% ethanol for 15 min each step) and embedded in LR White resin (London Resin Company Ltd., Berkshire, UK) for immunocytochemistry. Ultrathin sections (80–90 nm) were obtained in a Leica Ultracut UCT microtome (Reichert-Jung, Nussloch, Germany), and mounted on Formvar-coated nickel grids (200 m mesh). The sections were Wrst incubated with 5% normal goat serum in blocking buVer (0.01 M PBS buVer containing 0.8% BSA, 0.1% IGSS quality gelatin, pH 7.4) for 1 h at room temperature. After blocking, sections were incubated with anti-Mir1 mouse serum diluted 1:10 in incubation buVer overnight at 4°C. The sections were then
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rinsed one time with washing buVer for 5 min and incubated with Auroprobe One goat anti-mouse serum (Amersham Biosciences) diluted 1:50 in incubation buVer for 4.0 h at room temperature. After several washes, grids were silver treated with enhancer for 5 min using an IntenSE kit (Amersham Biosciences). Then sections were stained with 2% uranyl acetate and lead citrate (Reynolds 1963). The sections were examined and photographed at 60 kV with a JEOL-JEM-100 CX II transmission electron microscope at the Electron Microscope Center (Mississippi State University). Controls were conducted by substituting the antiMir1-CP primary antibody with normal mouse preimmune serum. Electron microscopy immunolocalization for Mir1-CP detection Ultrathin sections (80–90 nm) were mounted on Formvarcoated nickel grids (200 m mesh). Grids were placed in blocking buVer (1% BSA, 0.02% NaN3, and 0.05% Tween20 in Tris-buVer saline pH 7.4) at room temperature for 1 h. After blocking, sections were incubated with anti-Mir1-CP mouse serum diluted 1:10 in blocking buVer at 4°C overnight. Sections were then rinsed through a series of drops of TBS and grids then were incubated with 15 nm protein Agold conjugated secondary antibody (EY Laboratories Inc., San Mateo, CA, USA) in blocking buVer. Excess of gold was washed several times with TBS and then grids were stained with 2% uranyl acetate and lead citrate (Reynolds 1963). The sections were examined and photographed at 60 kV with a JEOL-JEM-100 CX II transmission electron microscope at the Electron Microscope Center (Mississippi State University). In situ hybridization of mir1 RNA In situ hybridization was carried out based on the protocol described by Yang et al. (1998) with minor modiWcations. Mp708 plants infested with FAW larvae for 24 h were used for mir1 in situ hybridization. The controls were uninfested plants. Whorl tissues cut into 5 mm2 segments were immediately Wxed in freshly prepared 4% paraformaldehyde (v/ v) and 0.5% glutaraldehyde (v/v) in 0.1 M PBS (pH 7.2) overnight at 4°C. Then the segments were dehydrated through a graded ethanol series (30, 50, 75, 95, and 100% ethanol for 15 min per step), cleared with Citrisolv (Fisher) and embedded in paraYn. Tissue sections 10 m thick were prepared and spread on microscope slides subbed with Vectabond (Vectorlabs, Burlingame, CA, USA) and baked overnight on a slide warmer set at 42°C. After removing the wax with Citrisolv (Fisher), sections were rehydrated by an ethanol-dilution series (100, 96, 85, 70, 50, and 30% ethanol), then slides were treated in 0.85% NaCl for 2 min and
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digested with Proteinase K for 45 min at 37°C. After a brief wash in 1£ PBS (150 mM NaCl, 10 mM sodium phosphate pH 7.5), sections were dehydrated in an ethanol-dilution series (30, 50, 75, 95, and 100% 1 min per step). In situ hybridization experiments were started after the slides were dried for 1 h. Digoxigenin (DIG)-labeled RNA probes were synthesized using an in vitro transcription kit (Ambion, Huntingdon, UK) according to the manufacturer’s instructions. DNA templates for synthesizing RNA probes were ampliWed by RT-PCR and cloned into a Topo II.1 plasmid (Invitrogen, Carlsbad, CA, USA). The PCR ampliWed products were obtained using the following speciWc primers for mir1: forward primer, 5⬘-CTACTGGATCGTGAAGAA CTCGTG-3⬘ and reverse primer, 5⬘-CACGATGAAA TTTCCCAAGAT-3⬘. To synthesize mir1 ribroprobes the antisense and sense probes were transcribed from SP6 or T7 RNA polymerase promoters, depending on the orientation of the inserted mir1 fragments in the plasmid. Tissue sections Wxed on slides were hybridized in a moist chamber overnight at 43°C in a hybridization solution (50% formamide, 0.3 M Nacl, 0.01 M Tris–HCl pH 6.8, 5 mM EDTA), 1£ Denhardt’s solution, 10% dextran sulfate, 1 mg/ml yeast tRNA, 200 ng/ml probe. The sections were washed twice in 2£ SSC, 50% formamide at room temperature for 30 min. Following stringency washes the sections were incubated with anti-Dig alkaline phosphatase and a Vector Red (Vectorlabs™) work solution was used to stain for the presence of mir1 transcripts. Visualization of the hybridization was done inmmediately according to the manufacturer’s intructions (Vectorlabs). The color reaction was developed in 0.1 M Tris pH 8.5 and examined under bright Weld using an Olympus BX51 microscope equipped with an Olympus DP70 camera. Digital images were generated using a computerized scanner, and composites of individual prints were assembled using Adobe Photoshop (Adobe Systems). Fluorescent images were acquired using a LEICA TCS NT confocal laser scanning microscope (CLSM), inverted model DMIRBE light microscope with a PL Fluotar 20£/0.5 NA and 100£ 1.3 NA oil PL Fluotar objective lens. A FITC/TRITC Wlter set was used with excitation wavelenghts of 488 and 568 nm (Argon and Krypton lasers). A 1,024 £ 1,024 pixel scan-format was used to capture images. Composites of digital images were assembled using Adobe Photoshop (Adobe Systems). Immunoblot analyses Immunoblot analyses of proteins extracted from leaf and root tissues were conducted as previously described (Pechan et al. 2000).
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Fig. 2 Cross-section of a maize leaf showing the large, intermediate, and minor vascular bundles or veins. Each vascular bundle consists of a variable number of vessels, parenchyma cells, and phloem cells and is surrounded by chlorenchymatous bundle sheath cells. Bar indicates 200 m
Results Small and intermediate vascular bundles in the maize leaf The vascular system of the Z. mays L. leaf consists of longitudinal strands interconnected by transverse bundles. When transverse sections of the maize leaf were examined, three types of veins or vascular bundles, small, intermediate, and large, can be seen (Fig. 2). Commonly, one to three intermediate bundles occur between large bundles. Intermediate bundles are separated from one another, and from large bundles by two to seven or more small bundles or minor veins (Russell and Evert 1985). The small and intermediate bundles consist entirely of metaxylem and metaphloem. In maize, two types of sieve elements develop in the phloem of small and intermediate vascular bundles during leaf ontogeny. These are the thin- and thick-walled sieve elements. The thin-walled sieve elements develop earlier than the lateformed metaphloem sieve elements that become the thickwalled sieve elements (TSE). The TSE diVer from the typical sieve elements by having thicker walls and elongate plastids (Walsh 1974). The TSE have abundant cytoplasmic connections with contiguous vascular parenchyma cells. They are not associated with companion cells and commonly are in direct contact with the tracheary elements of the metaxylem (Evert et al. 1978). Moreover, the plastids found in the TSE contain elongate crystalline or quasi-crystalline structures and that appear to be composed of interlaced tubular structures. In addition, the TSE of the longitudinal and transverse veins of the leaf have plasmalemma tubules and are not ligniWed (Evert et al. 1978; Walsh 1974). The small vascular bundles are completely surrounded by chlorenchymatous bundle sheath cells, which in turn, are completely encircled by mesophyll cells. Unlike the large vascular bundles, the intermediate bundles lack large metaxylem vessels and protoxylem, but may have protophloem. Moreover, the metaphloem of intermediate bundles also contains both thinand thick-walled sieve tubes (Russell and Evert 1985).
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Presence of Mir1-CP in the phloem of minor veins in uninfested plants To determine the localization of Mir1-CP prior to insect feeding, we carried out the immunodetection of Mir1-CP in intact yellow–green whorls of insect-resistant maize plants that were not subjected to herbivory. We speculated that Mir1-CP would be present in the control plants because immunoblot analysis conducted in previous research (Pechan et al. 2000) demonstrated that a low amount of Mir1-CP was constitutively present in the yellow–green region in the whorls of resistant plants. Silver-enhancement followed by light microscopy observation of transverse whorl sections indicated that a small amount of Mir1-CP was localized in the phloem of the minor veins prior to insect feeding (Fig. 3a). Mir1-CP was found concentrated in the TSE of some minor veins in the leaves of uninfested plants and occasionally was present in the adjacent vascular parenchyma cells (Fig. 3a). However, Mir1-CP was not detected in the intermediate bundles or large veins of uninfested plants. Accumulation of Mir-CP in vascular parenchyma cells and thick-walled sieve elements (TSE) near the insect wound site in response to larval feeding To examine the distribution of Mir1-CP in response to herbivory, samples were collected from the wound site in the yellow–green region of the whorl after 8, 24, and 48 h of caterpillar feeding. After 8 h of larval feeding, there was a strong signal around the vascular parenchyma cell walls near the TSE in some minor veins (Fig. 3b). The intensity
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of Mir1-CP labeling also was strong in vascular parenchyma cells and labeling was present in the TSE near these cells in the minor vein (Fig. 3c). This labeling pattern suggested that Mir1-CP could be transported from the vascular parenchyma cells adjacent to the xylem vessels to the contiguous TSE. Fig. 3d, shows a region of the whorl adjacent to the larval feeding site. Although the insect consumed a portion of the minor vein, some signal remained in the TSE. After 8 h of larval feeding Mir1-CP was also detected in the TSE of the intermediate veins in the whorl, although it was scarce or absence in the vascular parenchyma cells (data not shown). After 24 h of larval feeding the intensity of Mir1-CP labeling in the whorl increased At 24 h after infestation, additional Mir1-CP accumulated in the intermediate and minor veins. Mir1-CP frequently was localized in the metaxylem vessels close to the TSE (Fig. 3e). The most intense labeling was seen in small veins 24 h after larval feeding, when Mir1-CP accumulation also increased in the TSE and vascular parenchyma cells (Fig. 3e). A variation in labeling intensity was seen in the small veins, where silver staining was strong in some vascular parenchyma cells, TSE, and in the bundle sheath cells of the vein (Fig. 3f). After 48 h of larvae feeding Mir1-CP was predominantly concentrated in the TSE After 48 h of larval feeding, Mir1-CP was most abundant in the TSE of the intermediate and small veins (Fig. 3g, h). At
Fig. 3 Immunolocalization using light microscopy and silver enhancement of Mir1-CP in the yellow–green region of maize whorls. Samples were taken from uninfested plants (a) or the feeding site of plants infested with larvae for 8 h (b–d); 24 h (e, f), and 48 h (g, h). A silverenhanced signal was detected 24 h after insect feeding in the metaxylem near the TSE (tse) and the vascular parenchyma cells (arrowheads) (e). The control (i) was a whorl section treated with preimmune serum. Arrows indicate the presence of Mir1-CP in the TSE (tse), and vascular parenchyma (vp). Other abbreviations are bs bundle sheath, fsw feeding site wound, mx metaxylem, nc nucleus, ph phloem. Bars represent 50 m
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To determine if Mir1-CP was present in the roots, samples from uninfested plants were analyzed on immunoblots (Fig. 4d). The results indicated that Mir1-CP was present in roots of control plants as well as in whorls of plants that were eaten by FAW larvae. To determine the location of
Mir1-CP in the roots, we examined roots from control plants and those that had been infested with larvae for 24 h. In the roots of control plants, a small amount of Mir1-CP was detected the xylem parenchyma cells adjacent to the metaxylem (Fig. 4a, a1). However, after 24 h of larval feeding in the whorl, the abundance of Mir1-CP in the root vascular tissue appeared to increase and it was localized in the lumen of the root metaxylem vessels (Fig. 4b, b1). This change in the labeling pattern suggests that prior to insect feeding, Mir1-CP is synthesized in root xylem parenchyma cells and that after insect feeding in the whorl, it is secreted from these cells to the mature xylem vessels in the roots. When preimmune serum was used as a control, no silver staining or Mir1-CP was detected (Fig. 4c).
Fig. 4 Immunolocalization with silver enhancement of Mir1-CP in maize roots. This shows root vascular tissue taken from the control (a) and treated plants after 24 h of leaf feeding (b). The squares shown in a and b are enlarged in panels a1 and b1. The micrographs (a–c) were from sections immediately above the root tip zone. In the control (a1) label was found close to the cell walls of the xylem parenchyma (xp) adjacent to the metaxylem vessels. After 24 h of leaf feeding (b1) Mir1-CP was detected inside the lumen of metaxylem vessels (mx) of the plant root. Panel c is a root section treated with preimmune serum.
Immunoblot analysis of root and whorl extracts from Mp708 is shown in d. Lane 1, extract from Mp708 callus that is a positive control for Mir1-CP (Pechan et al. 2000); lane 2, a root extract from an uninfested 4-week-old plant; lane 3, whorl extract from a control plant that was not infested with fall armyworm larvae; lane 4, whorl extract from a plant infested with fall armyworm larvae for 24 h. Each lane contained 15 g of protein. Arrowheads show Mir1-CP in xylem cells. Other abbreviations are c cortex, p pith. a and b are in £40 and bars indicate 50 m; a1, b1, and c are in £100 and bars represent 50 m
this time, Mir1-CP accumulation appeared to be greater in the TSE than in the vascular parenchyma cells. These labeling patterns suggest that Mir1-CP could be transported from the vascular parenchyma cells adjacent to the xylem vessels to the TSE. When preimmune serum was used as a control, no silver staining of Mir1-CP was detected (Fig. 3i). After 24 h of leaf feeding, Mir1-CP was detected in the xylem of the root vascular tissue
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Since Mir1-CP was detected in both leaf (Fig. 3e) and root (Fig. 4) metaxylem cells after 24 h of foliar feeding, it is possible that Mir1-CP moves through the xylem sap to the leaf. To test this possibility, both intact maize plants and those with their roots excised were infested with FAW larvae for 24 h. Immunobot analysis of whorl proteins indicated that Mir1-CP was more abundant in infested intact plants than those with detached roots (Fig. 5). The reduction of Mir1-CP in the whorls of plants with detached roots suggests that it might move through xylem sap from the roots to the leaves in response to insect feeding in the whorl. Consequently, the transport of Mir1-CP from the roots might account for a portion of the protease that is found in the whorl vascular tissues. Confocal imaging of the silver-stained whorl section after 24 h of insect feeding Figure 6 shows a confocal laser microscope image taken using conditions to visualize metal particles. It indicates that Mir1-CP is primarily localized in the vascular parenchyma and TSE. Smaller amounts of Mir1-CP appear to be scattered in the bundle sheath and mesophyll cells. However, at this time we do not know if Mir1-CP is transported to these cells from the vascular tissue or vice versa. Mir1-CP was localized in crystalline structures in the plastids of late-formed metaphloem TSE Transmission electron micrographs (TEM) of transverse and longitudinal sections of whorl segments harvested 24 h after larval feeding are shown in Fig. 7. Immunogold labeling was associated with the elongate quasi-crystalline structures that originated from the plastids found in the TSE (Fig. 7a, b). When preimmune serum was used as a control, Mir1-CP was not detected in these structures (data not shown). The long crystalline material appears to be composed of interlaced tubular structures, each with an electron-translucent core and electron-opaque wall (Walsh 1974). The composition of these structures is currently unknown. Because no P-proteins have been reported in maize phloem, they probably are not the P-proteins that
Fig. 5 Immunoblot analysis showing that root removal decreases the abundance of Mir1-CP in the whorls of plants infested with armyworm for 24 h. Lane 1, positive control; lane 2, control plant with roots excised and kept in water (no infestation); lanes 3 and 4, plants with roots excised, kept in water and infested; lane 5, control plant with roots intact, kept in water (no infestation); lane 6, plant with roots intact, kept in water and infested; lane 7, control plant kept in soil with no infestation. Each lane contained 10 g of protein
Fig. 6 Confocal Xuorescence image of Mir1-CP immunogold localization with silver enhancement in a transverse section of the yellow– green portion of the maize whorl 24 h after larval feeding. Most of the green Xuorescence was localized to the metaxylem (mx) vessels, vascular parenchyma cells (vp) and TSE (tse) in the phloem of the small veins. Lower intensity green Xuorescence indicated the presence of Mir1-CP in the bundle sheath (bs) and mesophyll (m) cells. Any label was found in the epidermis (e). This picture was taken with an FV1000 confocal Olympus microscope using a 63£/1.42 objective with £3 zoom. A 488 nm laser excitation with no barrier Wlters was used to achieve reXectance imaging in the Wrst channel for the silver grains. The red color was rhodamine using the 543 nm laser and 577 nm peak emission. The bar indicates 50 m
have been found in the sieve tube elements and phloem of dicotyledonous plants (Singh and Srivastava 1971; Turgeon et al. 1975). Mir1-CP was found in the phloem cell walls in the minor veins Immunogold labeling and TEM was used to further examine the subcellular location of Mir1-CP. After 24 h of larval feeding, it was common to Wnd clusters of immunogoldlabeled Mir1-CP associated with the cell walls between adjacent vascular parenchyma cells and between the vascular parenchyma cells and the TSE (Fig. 7c–e). Mir1-CP was present in the cell walls between vascular parenchyma cells closely associated with the plasmodesmata (Fig. 7c). In the vascular parenchyma cell shown in Fig. 7d, Mir1-CP was detected in plastids with quasi-crystalline structures that were similar to those found in the TSE. These structures were close to the cell wall between the vascular parenchyma and TSE. The cell wall between these two cell types also contained abundant amounts of immunogold-labeled Mir-CP (Fig. 7d). As far as we know, these plastids have not been previously described in the vascular parenchyma cells associated with the TSE in maize. Likewise, Mir-CP
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Fig. 7 Immunolocalization of Mir1-CP in the maize whorl 24 h after larval feeding using transmission electron microscopy. The region surrounding the insect feeding area was used for immunogold localization with silver enhancement. a and b show transverse and longitudinal sections, respectively, and arrows indicate the presence of Mir1-CP associated to the crystalline structures inside the thick walled sieve elements (tse) of the phloem. A transverse section (c) shows Mir1-CP on the walls between two phloem vascular parenchyma cells (vp) and passing through the plasmodesmata (pd) connections between them
(black arrowhead). In d, arrows show Mir1-CP associated with the crystalline proteins in the plastids (p) of a parenchyma cell adjacent to a thick-walled sieve element. Some gold particles are on the cell walls between the vascular parenchyma and the TSE (tse). In e, gold particles in the cell wall of the bundle sheath cells are shown. Other abbreviations are intercellular space (is), thin walled sieve element (tse), and vascular parenchyma (vp). Bars in a and b represent 1 m; in c represents 0.5 m; in d represents 1 m; in e indicates 0.5 m
was detected on the cell wall and in the intercellular spaces between bundle sheath cells in the phloem of the small veins (7e).
Discussion
Mir1-CP transcripts were detected in the phloem vascular parenchyma in the minor and intermediate veins after 24 h of larval feeding To determine the site of Mir1-CP synthesis, we investigated the expression pattern of mir1 transcripts in crosssections of the yellow–green whorl from plants after 24 h of larval feeding. The spatial distribution of mir1 transcripts was analyzed by in situ hybridization and visualized by both bright Weld and confocal Xuorescence microscopy. Antisense-speciWc probes for mir1 mRNA showed strong fuchsia-staining hybridization signals in the bundle sheath cells and vascular parenchyma cells contiguous to the TSE in the intermediate and small veins of the leaf (Fig. 8b). No hybridization signal was detected when sections were treated with the sense probe (Fig. 8a). When examined by Xuorescence microscopy, red Xuorescence was observed in cytoplasm of bundle sheath and phloem vascular parenchyma cells close to TSE of a small vein, no hybridization was detected in the mesophyll cells (Fig. 8c). In agreement with the immunocytochemistry results, in situ hybridization showed that mir1 transcripts are particularly abundant in vascular parenchyma and bundle sheath cells that are close to the vascular parenchyma and TSE where Mir1-CP accumulates after larval feeding.
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In this paper, we report that Mir1-CP, an insect-defense cysteine protease, is localized in the phloem and metaxylem cells of the small and intermediate vascular bundles in the maize leaf. It dramatically accumulated in the TSE during insect feeding, and after 24 and 48 h, the primary location of Mir1-CP was in the TSE. Within the TSE, Mir1-CP accumulated in plastid-like structures containing a quasicrystalline matrix. The association of Mir1-CP with the quasi-crystalline bodies within the TSE has not been studied and their composition in maize is unknown. It is possible that Mir1-CP is stored in these bodies and that they may be transported long distances in the phloem (Thompson and Schulz 1999), but this has not yet been tested. In broad bean, it has been shown that P-protein crystalloids spontaneously assemble and disperse with the sieve elements. This assembly dispersement cycle is dependent on calcium ions (Knoblauch et al. 2001). Dispersement occurs when plasma membrane leakage is induced by mechanical injury (Knoblauch et al. 2001). One could envision that insect chewing might cause similar types of damage and release Mir1-CP from the crystalloid matrix so that it would be free to attack the herbivore. The maize TSE are unusual vascular structures found in phloem and their function has not been fully elucidated. They may be involved in long distance transport within the leaf, or serve as a temporary storage reservoir for excess sucrose that cannot be transported by the other sieve tubes
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Fig. 8 In situ localization of mir1 transcripts in maize leaves after 24 h of larval feeding. Transverse leaf sections were hybridized with sense (a) or antisense (b, c), mir1-speciWc riboprobes. When in situ hybridization was visualized with bright Weld microscopy, areas of hybridization stained fuchsia were observed with VectorRed in the bundle sheath and the vascular parenchyma cells of small veins (b) and intermediate veins (data not shown). Confocal Xuorescence image (c) of the same small vein showed in b following in situ hybridization. In c,
hybridization was detected with a rhodamine Xuorescence Wlter using 568 nm excitation. The red Xuorescence conWrmed that in situ hybridization of mir1 antisense probe is localized in the cytoplasm of the bundle sheath and the vascular parenchyma cell of the vein. No signal was detected in the sieve elements and companion cells of the phloem. Abbreviations are bs bundle sheath, m mesophyll cells, ph phloem, vp vascular parenchyma. The bars in a, b and c represents 10 m
(Evert et al. 1978). The TSE have abundant plasmodesmal connections with one or more contiguous vascular parenchyma cells and are in close contact with the xylem vessels (Evert et al. 1978). They are not associated with companion cells, and lateral connections between thin-walled elements and TSE are absent. Consequently, the only direct cytoplasmic connections of the TSE are with their associated vascular parenchyma cells and between themselves. In situ hybridization indicated that mir1 transcripts were localized in the cytoplasm of the vascular parenchyma and bundle sheath cells near the TSE in the vascular bundles of the small and intermediate veins. This suggests that Mir1-CP is synthesized in these cells and post-translationally moves into the TSE. This is supported by the presence of Mir1-CP in the cell walls between adjacent vascular parenchyma cells, adjoining vascular parenchyma, and TSE cells and adjacent bundle sheath cells. Although we cannot be certain at this time, the appearance of immunogold-labeled Mir1-CP in cell walls near plasmodesmata implies that these structures might be involved in Mir1-CP movement into the TSE. Mir1-CP has a molecular mass of 33 kDa, which is within the size range of proteins that can move through the plasmodesmata (Oparka and Turgeon 1999). There is precedence for this type of movement because a maize pathogenesis-related protein (PRm) was localized to the plasmodesmata between contiguous parenchyma cells in both protoxylem and pith tissue of the vascular cylinder in fungal infected radicles (Murillo et al. 1997). Alternatively, Mir1-CP might interact with the plasmodesmata to increase in the plasmodesmal size exclusion limit (SEL). This type of interaction is required for cell-to-cell transport of viral MPs (Deom et al. 1992; Lucas and Gilbertson 1994). Because the C-terminal of Mir1-CP has similarity to the citrus tatter-leaf virus and apple stem-grooving virus MPs (Yoshikawa et al. 1993;
Ohira et al. 1994), it is possible that this motif could target Mir1-CP to the plasmodesmata and enable it to enlarge the SLE and move into the TSE. In addition to being present in the leaf metaxylem, Mir1CP was also found in the root metaxylem elements. In the roots of control plants, small amounts of Mir1-CP were detected in the xylem parenchyma cells and we initially suspected that it was involved in programmed cell death in the vascular tissue (Beers et al. 2000). However, the abundance of Mir1-CP in the both leaf and root metaxylem elements unexpectedly increased after 24 h of larval leaf feeding. These observations suggest that Mir1-CP is synthesized and transported from the xylem parenchyma to the more developed metaxylem cells after feeding. Its presence in the metaxylem elements suggests that Mir1-CP might be transported in the xylem sap to the leaf. It has been reported that some xylem sap proteins are actively secreted into the xylem stream (Biles and Abeles 1991; Masuda et al. 1999; Sakuta and Satoh 2000; Alvarez et al. 2006) and they may travel to the leaf, where they function in cell wall repair, ligniWcations, and defense (Alvarez et al. 2006) We speculate that some signal triggered by herbivory travels from the leaves to the roots and results in the accumulation of Mir1CP in the root xylem elements. Then, Mir1-CP might travel back to the leaf. In tobacco, jasmonate is transported from the leaves to the roots (Zhang and Baldwin 1997), which induces the expression of putrescine N-methyltransferase (PMT) genes in the root (Shoji et al. 2000). PMT catalyzes the Wrst step in nicotine biosynthesis and nicotine is then transported to the leaf to protect the plant against insect herbivores (Hashimoto and Yamada 1994). It is not uncommon for defensive proteins to be found in vascular Xuids of various plant species (Alvarez et al. 2006; Kehr 2006). Phloem components from cucurbits (Walz et al. 2004) and Ricinus communis L. (Barnes et al. 2004)
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have been examined using proteomic techniques and found to contain a number of proteins that potentially function in herbivore defense. These include lectins, numerous types of protease inhibitors and antioxidant enzymes. It is likely that they and other macromolecules move throughout the plant via the phloem (DannenhoVer et al. 2001; Thompson and Schulz 1999; van Bel et al. 2002; Oparka and Turgeon 1999). In fact, the cucurbit serine protease inhibitor was shown to be graft transmissible (la Cour-Peterson et al. 2005). In tomato, the precursor of defense signaling glycopeptides accumulates in the phloem parenchyma (NarváezVásquez et al. 2005) and presumably the processed peptides move systemically through the phloem. Several of the enzymes in the jasmonic acid biosynthetic pathway also are in the phloem (Hause et al. 2003). Although these enzymes do not directly protect the plant against herbivores, they catalyze the formation of an important defense-signaling molecule. Proteomic analysis of maize xylem exudates indicated that »26% of the proteins present have potential defensive functions (Alvarez et al. 2006). In Arabidopsis both leaf and root tracheary elements contain a papain-like protease, XCP1 that may be involved in programmed cell death or defense (Funk et al. 2002). Maize xylem sap contained a number of hydrolases including a glycosylated cysteine protease with an experimental molecular mass of 33 kDa that is likely a homolog of Mir1-CP (Alvarez et al. 2006). In summary, the results of this study indicated that the insect-defense protease Mir1-CP is present in the leaf and root vascular tissues of maize plants that are resistant to herbivory. Small amounts of Mir1-CP were detected in unwounded plants, but its abundance increased dramatically in the whorls of intact plants, but not those with roots removed, during insect feeding. In the leaves, in situ hybridization indicated that Mir1-CP was synthesized in the vascular parenchyma cells and moves to the TSE. In the roots, Mir1-CP appears to move from the xylem parenchyma into the metaxylem vessels and there is the possibility that it is transported to the leaves in the xylem Xuid. This helps to explain our observation that Mir1-CP accumulation in the whorl in response to larval feeding is greater in plants with intact roots. It also clariWes previous results indicating that Mir1-CP accumulates at the wound site within 1 h of larval feeding (Pechan et al. 2002). This rapid accumulation is faster than that of other plant defense proteins, that appear 8–12 h after herbivory and require transcription and translation for expression (Ryan 2000). Consequently, the presence of Mir1-CP in the vascular tissues most likely facilitates its rapid mobilization to the wound site in response to herbivory. Acknowledgments We thank Bill Monroe and Amanda Lawrence (Mississippi State University Electron Microscope Center) for their
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Planta (2007) 226:517–527 generous help and technical assistance. This research was supported by grant to DSL by the Life Sciences and Biotechnology Institute at Mississippi State University and the National Science Foundation (IBN0236150). This is report J10807 of the Mississippi Agricultural and Forestry Experiment Station.
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