Botany Publication and Papers Botany

9-2003 Floral Nectary Fine Structure and Development in Glycine max L. (Fabaceae) Harry T. Horner Iowa State University, [email protected]

Rosaria A. Healy Iowa State University

Teresa Cervantes-Martinez Iowa State University

Reid G. Palmer United States Department of Agriculture

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Recommended Citation Horner, Harry T.; Healy, Rosaria A.; Cervantes-Martinez, Teresa; and Palmer, Reid G., "Floral Nectary Fine Structure and Development in Glycine max L. (Fabaceae)" (2003). Botany Publication and Papers. 51. http://lib.dr.iastate.edu/bot_pubs/51

This Article is brought to you for free and open access by the Botany at Iowa State University Digital Repository. It has been accepted for inclusion in Botany Publication and Papers by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Floral Nectary Fine Structure and Development in Glycine max L. (Fabaceae)

Abstract Floral nectaries of annual cultivated Glycine max develop between the bases of the central gynoecium and lateral stamen ring. Each discoid nectary forms immediately before flower opening and degenerates within 24 h. Three stages of nectary development are identified: preactive, active, and postactive. Preactive and active nectaries are composed of a single-layered epidermis that contains many open stomata, with guard cells having thickened walls, starch-engorged plastids, and other organelles. The am jor portion of each nectary consists of thin-walled special parenchyma cells, each having dense cytoplasm with a nucleus, Golgi bodies and vesicles, mitochondria, plastids, endoplasmic reticulum, many ribosomes, and one or more vacuoles. Fingers of phloem consisting of sieve tubes and companion cells, both with very small wall ingrowths and thought to provide sugars, penetrate the nectary at its base. The fingers originate from 10 vascular bundles with xylem, which innervate the stamen ring peripheral to the nectary. At the beginning of the active stage, special parenchyma around the phloem fingers become highly vacuolated first by each vacuole filling with non-water-soluble material and ribosome-like particles. In many of these cells and in the nonstomata epidermal cells, cytoplasmic bridges are associated with the vacuoles, and straight tubes containing single files of ribosome-like particles occur in the cytoplasm, or traverse plasmodesmata. In addition, bundles of tubules are often seen pressed to the outside of the vacuole tonoplast and in the cytoplasm before and during the time the tonoplast fragments and the vacuole contents mix with the cytoplasmic organelles. These cells then collapse, releasing their contents through the pores of the guard cells and onto the nectary surface. This holocrine secretion is different from that reported for other legume taxa and most other nonlegume taxa and suggests apoptosis. Remaining nectary special parenchyma cells follow the same fate, along with the epidermal cells, so that the entire nectary collapses, leaving only some of the guard cells intact. There are two types of elongate nonglandular trichomes and one type of short five- to seven-celled glandular trichome on the gynoecium adjacent to the nectary. These latter trichomes seem to be developed and functional during the active and postactive stages and following nectary collapse, suggesting that the nectar may consist of a variety of compounds originating from both the nectary and the glandular trichomes.

Keywords floral nectary, Glycine, holocrine secretion, programmed cell death, secretory trichomes, soybean, ultrastructure

Disciplines Agronomy and Crop Sciences | Botany | Plant Biology | Plant Breeding and Genetics

Comments This article is from International Journal of Plant Sciences 164 (2003): 675, doi: 10.1086/377060.

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This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/bot_pubs/51

Floral Nectary Fine Structure and Development in Glycine max L. (Fabaceae) Author(s): Harry T. Horner, Rosaria A. Healy, Teresa Cervantes‐Martinez and Reid G. Palmer Source: International Journal of Plant Sciences, Vol. 164, No. 5 (September 2003), pp. 675- 690 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/10.1086/377060 Accessed: 14-06-2016 14:39 UTC

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This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms Int. J. Plant Sci. 164(5):675–690. 2003. ᭧ 2003 by The University of Chicago. All rights reserved. 1058-5893/2003/16405-0002$15.00

FLORAL NECTARY FINE STRUCTURE AND DEVELOPMENT IN GLYCINE MAX L. (FABACEAE)

Harry T. Horner,1,* Rosaria A. Healy,* Teresa Cervantes-Martinez,* and Reid G. Palmer†

*Department of Botany and Bessey Microscopy Facility, Iowa State University, Ames, Iowa 50011-1020, U.S.A.; and †USDA-ARS CICGR and Departments of Agronomy and Zoology/Genetics, Iowa State University, Ames, Iowa 50011, U.S.A.

Floral nectaries of annual cultivated Glycine max develop between the bases of the central gynoecium and lateral stamen ring. Each discoid nectary forms immediately before flower opening and degenerates within 24 h. Three stages of nectary development are identified: preactive, active, and postactive. Preactive and active nectaries are composed of a single-layered epidermis that contains many open stomata, with guard cells having thickened walls, starch-engorged plastids, and other organelles. The major portion of each nectary consists of thin-walled special parenchyma cells, each having dense cytoplasm with a nucleus, Golgi bodies and vesicles, mitochondria, plastids, endoplasmic reticulum, many ribosomes, and one or more vacuoles. Fingers of phloem consisting of sieve tubes and companion cells, both with very small wall ingrowths and thought to provide sugars, penetrate the nectary at its base. The fingers originate from 10 vascular bundles with xylem, which innervate the stamen ring peripheral to the nectary. At the beginning of the active stage, special parenchyma around the phloem fingers become highly vacuolated first by each vacuole filling with non-water-soluble material and ribosome-like particles. In many of these cells and in the nonstomata epidermal cells, cytoplasmic bridges are associated with the vacuoles, and straight tubes containing single files of ribosome-like particles occur in the cytoplasm, or traverse plasmodesmata. In addition, bundles of tubules are often seen pressed to the outside of the vacuole tonoplast and in the cytoplasm before and during the time the tonoplast fragments and the vacuole contents mix with the cytoplasmic organelles. These cells then collapse, releasing their contents through the pores of the guard cells and onto the nectary surface. This holocrine secretion is different from that reported for other legume taxa and most other nonlegume taxa and suggests apoptosis. Remaining nectary special parenchyma cells follow the same fate, along with the epidermal cells, so that the entire nectary collapses, leaving only some of the guard cells intact. There are two types of elongate nonglandular trichomes and one type of short five- to seven-celled glandular trichome on the gynoecium adjacent to the nectary. These latter trichomes seem to be developed and functional during the active and postactive stages and following nectary collapse, suggesting that the nectar may consist of a variety of compounds originating from both the nectary and the glandular trichomes.

Keywords: floral nectary, Glycine, holocrine secretion, programmed cell death, secretory trichomes, soybean, ultrastructure.

Introduction deal specifically with the simple sugars, such as glucose, fruc- tose, and sucrose, that are a significant and important part of There has been increased interest for the past several decades the nectar (Butler et al. 1972; Baker and Baker 1981; Rosh- in both the development and the functional features of nec- china and Roshchina 1993). Variation in the ratio of xylem taries (Durkee et al. 1981; Durkee 1983; Dafni et al. 1988; and phloem in vasculature of the nectary seems to affect car- Fahn 1988; Beardsell et al. 1989; Figueiredo and Pais 1992; bohydrate composition (Frei 1955; Frey-Wyssling 1955; Esau Zer and Fahn 1992; Rabinowitch et al. 1993; Belmonte et al. 1977). Several studies describe the nectar as containing ad- 1994; Stpiczyn´ ska 1995; Nepi et al. 1996; O’Brien et al. 1996; ditional compounds (Griebel and Hess 1940; Vogel 1969; Gaffal et al. 1998). Some studies focus on the mature stage Baker and Baker 1973, 1975, 1983; Deinzer et al. 1977; when nectar is present or on the composition of the nectar Rodriguez-Arce and Diaz 1992; Roshchina and Roshchina (Percival 1961; Baker and Baker 1981; Rabinowitch et al. 1993; Ecroyd et al. 1995; Ferreres et al. 1996; Cabras et al. 1993; Ecroyd et al. 1995; Davis 1997; Carter et al. 1999; 1999; Petanidou et al. 2000), some of which may serve to Carter and Thornburg 2000; Thornburg et al. 2003; R. G. Palmer, personal communication, 2003). Some of the studies defend the nectar against microbial attack or against insects eating the developing fruit and its included seeds (Carter et al. 1999; Carter and Thornburg 2000; Thornburg et al. 2003). 1 Author for correspondence; telephone 515-294-8635; fax 515- In addition, some taxa have glandular floral and vegetative 294-1337; e-mail [email protected]. trichomes that may be involved in the production of “anti- Manuscript received March 2003; revised manuscript received May 2003. microbial” compounds (Broersma et al. 1972; Levin 1973;

675

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Dimock and Kennedy 1983; Franceschi and Giaquinta 1983; fertile and male-sterile field-grown and greenhouse-grown Rivera 1996). When present together, the nectary and glan- plants of Glycine max L. Merr. (Papilionoideae, Fabaceae) dular trichome secretions may be spatially and functionally were sampled and preserved in several ways to study their timed and, therefore, may jointly contribute to the final nectar anatomy and ultrastructure during nectary development. From composition. these flowers, 100 nectaries were sectioned for light micros- Fabaceae constitute a very important family and include copy, 26 nectaries were sectioned for transmission electron taxa of great worldwide economic importance, such as the microscopy, and 36 nectaries were processed for scanning elec- cultivated soybean. Perusal of the literature indicates that there tron microscopy. Three stages of nectary development (pre- have been relatively few recent studies dealing with the de- active, closed flowers just before opening; active, just-opened velopment, anatomy, and ultrastructure of legume nectaries flowers; and postactive, opened flowers) were defined since (Ancibor 1969; Waddle and Lersten 1973; Gulya´s and Kincsek nectary development and nectar secretion in soybeans are 1982). The more studied cultivated taxa are Lotus corniculatus known to occur for only a brief period of ca. 24 h (Chen et (Murrell et al. 1982), Medicago sativa (Teuber et al. 1980), al. 1987). Phaseolus vulgaris (Webster et al. 1982), Pisum sativum (Ra- zem and Davis 1999), Trifolium pratense (Picklum 1954; Er- Light Microscopy (LM) iksson 1977), Vicia faba (Waddle and Lersten 1973; Davis et al. 1988; Davis and Gunning 1991, 1992, 1993; Stpiczyn´ ska Fresh flowers were processed whole or were dissected by 1995), and Glycine max (Purseglove 1968; Carlson 1973; removing the outer appendages, and sometimes the gynoecium, Waddle and Lersten 1973; McGregor 1976; Erickson and Gar- to expose the nectary and receptacle. The receptacles were ment 1979; Carlson and Lersten 1987; Crozier and Thomas placed in 95% ethanol for several hours, washed in water, and 1993). then transferred to Clorox for several days to remove cyto- Interest in attempting to generate hybrid soybean seeds by plasm (Lersten and Horner 2000). After the specimens were insect cross-pollination (Erickson 1975a, 1975b, 1975c) has clear of pigmented cytoplasm, they were washed thoroughly taken several approaches in our laboratory (Palmer et al. with water, dehydrated through an ethanol series, stained with 2001). We have been working on male sterility systems (Hor- safranin O, and cleared in xylol. The specimens were mounted ner and Palmer 1995; Jin et al. 1997, 1998, 1999; Smith et on slides in Permount, cover slipped, and allowed to dry on al. 2001, 2002), characterizing nectar composition and output a warming tray. The mounted specimens then were observed (R. G. Palmer, personal communication, 2003) and, more re- between crossed polarizers using Olympus BH2 and Zeiss Ax- cently, studying how the floral nectary of the annual cultivated ioplan light microscopes. Images were captured with Zeiss me- soybean develops and functions. dium and high resolution Axiocam digital cameras and were Since the ultrastructure and development of soybean nec- electronically processed and displayed using Adobe PhotoShop taries have not been looked at in any detail ultrastructurally 7.0. (Purseglove 1968; Carlson 1973; Waddle and Lersten 1973; Flowers also were preserved using two different chemical McGregor 1976; Erickson and Garment 1979; Carlson and fixation procedures. (1) Whole flowers were immersed in Lersten 1987; Crozier and Thomas 1993), this study includes formalin–acetic acid–alcohol (FAA; Ruzin 1999) for at least comprehensive information about the changes that occur in 24 h at room temperature (RT). Initially, they were placed the cells and tissues making up the nectary, the subtending under a low vacuum (1 # 10Ϫ1 Torr) to aid in penetration of floral receptacle, the vasculature that innervates the nectary, the FAA. The flowers were stored in FAA until further pro- and the adjacent gynoecium glandular trichomes. Our study cessing. (2) Whole flowers were immersed in RT 2% paraform- shows that soybean nectaries have a different ultrastructure aldehyde and 2% glutaraldehyde in a 0.1 M cacodylate buffer (newly identified structures) and method of secretion (holo- at pH 7.2. These flowers also were placed under a similar crine) than has been reported for any other legume. The soy- vacuum to aid in penetration of the combination fixative. The bean nectary undergoes programmed cell death and ceases to flowers generally were stored overnight (ca. 19 h) in this buf- exist much beyond the pollination stage. These data serve as fered fixative at 4ЊC. After three 20-min buffer washes, the a foundation for observing nectaries in the wild annual soy- flowers were postfixed in 1% osmium tetroxide (OsO4)inthe bean Glycine soja and the wild perennial Glycine species where same buffer and pH for 1–1.5 h at RT. The flowers were rinsed cross-pollination (Brown et al. 1986; Schoen and Brown 1991; with only buffer (to remove unbound OsO4), placed in 5% Fujita et al. 1997) and nectar output (R. G. Palmer, personal aqueous uranyl acetate (UAc) for 1 h (en bloc staining), and communication, 2003) are greater. then rinsed three times with only buffer to remove the unbound UAc. Flowers from fixation procedures 1 and 2 were then Material and Methods dehydrated through a graded ethanol series, 30 min per step, followed by two changes of ultrapure 100% ethanol, 30 min Fresh flowers before, during, and just after flower opening per step. Flowers fixed via 1 were transferred to xylol using (fig. 2) were collected from four different lines: AO2g-2 p several changes, infiltrated with 54Њ–56ЊC melting paraffin # T219H(Y11y11, yellow-green plant, cv. Richland cv. Linman over 2 d, and then pure paraffin for three changes (1 d) before 533);AO2g-3 p CD-5 , yellow-green plant, germinal revertant solidification. Flowers fixed via 2 were infiltrated with either p of T322 (w4-m);AO1-F92 F2 green plant of cv. LR White resin (Bozzola and Russell 1999) or Spurr’s resin # p Minsoy T218H(Y18y18) from cv. Illini; and RGP-MS6 a (Spurr 1969), using an infiltration regime of 3 : 1, 1 : 1, and male-sterile, female-fertile mutant that is nonallelic to ms1 1 : 3 (pure ethanol : resin) for LR White or transferred to pure through ms9 (T-number and gene symbol not assigned). Both acetone first and then acetone : resin for varying extended

This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms HORNER ET AL.—SOYBEAN FLORAL NECTARY DEVELOPMENT 677 times (h/d) for Spurr’s. After complete infiltration with the pure resin, the resin was polymerized for a minimum of 24 h at either 60ЊC (LR White) or 70ЊC (Spurr’s). Paraffin sections were cut 10 mm thick with a steel knife on an AO Spencer rotary microtome. Resin sections were cut 1 mm thick with a glass knife on a Reichert ultramicrotome. Deparaffinized sections were stained with safranin O and fast green, chlorozol black E, or periodic acid–Schiff’s (PAS; Ruzin 1999) technique, the latter for total water-insoluble polysac- charides. Resin sections were stained with toluidine blue O or the PAS technique. Images were digitally captured as previ- ously mentioned.

Scanning Electron Microscopy (SEM) After fixation with procedure 2 and dehydration through 100% ethanol, the flowers were dried in a Denton critical- point dryer (CPD) using liquid carbon dioxide. The sepals, petals, stamens, and gynoecia were removed from each flower (after CPD) to expose the nectary. Each receptacle, with nec- tary and separated gynoecium, was attached to an aluminum stub with an adhesive tab. Silver paint was spread at the bases of the receptacle and extended over the tab to the aluminum stub. The mounted, remaining dissected flower parts with nec- tary were then sputter-coated in a Denton sputter-coater with ca. 12 nm 20 : 80 gold/palladium layer. The specimens were viewed and imaged with a digital JEOL 5800 SEM at either 10 or 15 kV. Saved digital images were processed as described previously. Fig. 1 Drawing of a longitudinal section through an active-stage Transmission Electron Microscopy (TEM) soybean floral nectary and a lower portion of adjacent gynoecium with a thin-walled, elongate, nonglandular trichome and a smaller five- to Flowers fixed with procedure 2 and embedded in LR White seven-celled glandular trichome. Arrows around glandular trichome or Spurr’s resin were first sectioned 1 mm thick in either cross and at the stomatal pores indicate sites of possible secretion. ASP p or longitudinal planes through the nectary and stained, as al- active special parenchyma;C p cuticle ;CT p connective tissue; ready mentioned, and digitally imaged. LM images showing DSP p degenerating and/or degenerated special parenchyma; G p p p p the different stages of nectary development were printed so gynoecium;GC guard cells/stomata;PF phloem fingers; SR p that a lined grid system could be placed on each nectary image staminal ring;ST short five- to seven-celled glandular trichome; STE p sieve tube element;TT p elongate , unicellular, thin-walled to create reference squares for cell/tissue location identification nonglandular trichome;X p xylem . at both the LM and TEM levels. Thin sections (ca. 60 to 80 nm) were cut with a diamond knife and placed on 0.3% results presented here were similar for the nectaries observed Formvar-coated slotted grids. Nectaries were small enough from the four lines. that their entire structure fitted within the grid slot. The sec- The flower receptacle, just below the base of the nectary, tions were stained with 5% aqueous uranyl acetate for four gynoecium, stamen ring, and more peripheral petals and se- 45-s waves in a Pelco microwave, followed by one 60-s wave pals, displays the main vascular bundle and its branches that with a modified Sato’s lead citrate solution (Hanaichi et al. innervate all of these flower organs (fig. 2B). Surrounding these 1986). The sections were then observed with a JEOL 1200EX basal vascular bundles is connective tissue that contains many TEM at 80 kV. Images were captured on Kodak SO-163 film. calcium oxalate crystals, generally one per cell (fig. 2C,2D). The films were digitized with a Umax PowerLook 3000 scan- During the active and postactive stages, additional crystals of ner at 800 dpi, and both types of images, including the LM various types (prismatic, needle-like clusters, and cubical) are and SEM images, were processed in Adobe PhotoShop 7.0 and found within some of the nectary cells, in intercellular spaces, made into plates using Adobe Illustrator 10. and on the outside surface near the base of the collapsed nec- tary. The composition of these crystals is not known. The re- Results ceptacle calcium oxalate crystals do not disappear during nec- tary development and degeneration. The annual soybean nectary develops and is active during a very short period of time, within 24 h before and shortly Preactive Nectary Stage after flower opening (figs. 1, 2A), and then collapses. The nec- tary forms between the central gynoecium and the stamen ring The nectary forms between the bases of the gynoecium and as a basal circular mound (fig. 2B). We identified three stages the stamen ring (fig. 2D,2E). A circular mound of special of nectary development: preactive, active, and postactive. All parenchyma cells is covered by a single layer of epidermis with

This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms HORNER ET AL.—SOYBEAN FLORAL NECTARY DEVELOPMENT 679 distinct stomata, consisting of two guard cells and a pore (fig. ing acropetally further into the nectary. The fingers of sieve 2F). These stomata typically occur all over the surface with elements and companion cells remain intact during this time the slope toward the gynoecium displaying more stomata. A (fig. 3D) and until the entire special parenchyma is in a state distinct, continuous electron-dense cuticle covers all epidermal of collapse. Plasmodesmata occur in the sieve element walls, and guard cells. Nectary parenchyma cells are thin walled with but there are more plasmodesmata in the walls between the one or more large vacuoles, as are the nonstomatal epidermal companion cells and the adjacent special parenchyma cells. cells (fig. 2G). Plasmodesmata occur between adjacent special The plasmodesmata occur either singly or in small groups. The parenchyma cells and between special parenchyma cells and cytoplasmic side of the walls of the sieve elements and com- epidermal cells. The thick-walled guard cells contain promi- panion cells, and to a lesser extent the adjacent special paren- nent plastids filled with starch and other organelles but typi- chyma, display small wall ingrowths (not shown). These in- cally lack plasmodesmata. growths appear to completely cover the inside of the walls but During the earliest part of the preactive nectary stage of are not as pronounced as wall ingrowths of transfer walls development, there are no trichomes on the young gynoecium. described for other taxa. Thin-walled, single-cell, elongated nonglandular trichomes de- The special parenchyma cells undergo several changes before velop soon after, followed by two- to three-celled, elongate collapse and release of their contents. Special parenchyma cells trichomes with very thick walls, and then short five- to seven- farther away from the phloem fingers contain dense peripheral celled glandular trichomes. In addition, early in nectary de- cytoplasm consisting of a nucleus, mitochondria, plastids, velopment, small globular bodies appear in the space between Golgi bodies with vesicles, arrays of rough endoplasmic retic- the young nectary and the gynoecium (fig. 2H). These bodies ulum (RER), smooth endoplasmic reticulum, unassociated ri- vary somewhat in diameter, and their origin is unknown. They bosomes, rather large vesicles or small vacuoles with fibrillar disappear as the preactive nectary enlarges. In sections through material in them, and a larger central vacuole with several the nectary, crystals are observed within the basal region of smaller vacuoles (fig. 3E). No recognizable microbodies/per- the nectary, but most often they occur within a layer two to oxisomes were identified at any stage of development. Special three cells thick below the base of the nectary (fig. 2H). This parenchyma cells near the phloem fingers display plastids con- layer extends from the shared base between the nectary and taining phytoferritin (fig. 3F). At this stage, the lumens of the the stamen ring and the shared base between the nectary and RER are extended and some portions of them are greatly en- gynoecium. larged and display visible contents (fig. 3G). The Golgi bodies have peripheral vesicles (fig. 3G). The central vacuole, in an Active Nectary Stage increasing number of parenchyma cells, is enlarged (fig. 3H) While the flower is still closed, the nectary has enlarged to and contains small ribosome-like particles. These particles oc- its maximum size (fig. 2I). In cross section near the base of cur in circular arrays as seen in sections, as if the particles are the nectary, 10 vascular bundles are evident, from which em- outlining small, unstained spheres (fig. 3I). anate vascular fingers (fig. 2J) that penetrate into the nectary Bundles of tightly packed tubules occur immediately sur- (fig. 2K). The bundles innervating the stamen ring contain rounding the tonoplast, as well as farther away in the cyto- xylem elements (fig. 2G), but the fingers extending into the plasm, of both parenchyma (fig. 3J) and epidermal cells (fig. nectary do not have xylem. The nectary fingers consist of thick- 3K). The bundles in the peripheral cytoplasm contain varying walled sieve elements containing P-protein and sieve plates and numbers of tubules (figs. 3L,4A) as well as the ones pressed companion-like cells, both of which are surrounded by the to the tonoplast (fig. 4B,4C). The bundles of tubules are visible special parenchyma (fig. 3A,3B). The special parenchyma cells only during the active stage. Up to six bundles have been ob- around the fingers are the first to show degeneration (fig. 1, served associated with a single tonoplast in one TEM section, DSP), becoming highly vacuolated (fig. 3C) and then collaps- suggesting that they occur all around the vacuole. The tubules

Fig. 2 Soybean flower buds at time of nectary development and gynoecium and adjacent nectary during early preactive and active stages of development. Stains used in color images are safranin O, C; toluidine blue O, B, D, G, H, J; chlorozol black E, K. A, Stereomicroscopy of soybean unopened and just-opened flower buds depicting timing of preactive, active, and postactive stages of nectary development. Bar p 5.5 mm. B, Light microscopy (LM) of longitudinal section through flower bud showing central gynoecium with young developing trichomes and surrounding young discoid nectary. Staminal ring is next outermost structure. Bar p 100 mm. C, LM of dissected and cleared receptacle of unopened flower bud observed between crossed polarizers, showing exposed discoid nectary (arrows) and large mass of calcium oxalate crystals in parenchyma beneath it. Red coloration is due to safranin O staining. Bar p 100 mm. D, LM of longitudinal section through young nectary. Epidermis and special parenchyma primordia are present. No phloem fingers are visible. Bar p 30 mm. E, Scanning electron microscopy (SEM) of fractured exposed young nectary, between base of central gynoecium with young trichomes, and stamen ring. Bar p 150 mm. F, SEM of portion of young nectary ridge covered with epidermis containing guard cells with some open pores (arrows).Bar p 20 mm. G, LM of longitudinal section of older preactive nectary displaying epidermis with guard cells (arrow), an enlarged central mass of special parenchyma and phloem fingers, and bundle innervating stamen ring that contains xylem (arrowhead). Bar p 50 mm. H, LM of young nectary with several cell layers of parenchyma containing calcium oxalate crystals below its base. Particles are present on epidermis of nectary and adjacent gynoecium (arrows). Bar p 50mm. I, SEM of fully formed active-stage nectary. Bar p 200 mm. J, LM of cross section at base of nectary showing 10 vascular bundles that innervate staminal ring and nectary. Bar p 50 mm. K, LM of longitudinal section through nectary showing phloem fingers (arrows) extending from one vascular bundle that innervates staminal ring (asterisk). Bar p 50 mm.E p epidermis ;G p gynoecium ;N p nectary ; PF p phloem fingers;SP p special parenchyma;SR p staminal ring;T p trichomes ;XL p calcium oxalate crystals.

This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms HORNER ET AL.—SOYBEAN FLORAL NECTARY DEVELOPMENT 681 have an outer diameter of ca. 10.7 nm, but their length is chains of spheres and, later, on the surface of the nectary (fig. unknown. 5E). These spheres seem to coalesce to form a layer covering Associated with the vacuoles and bundles of tubules of both the nectary surface. On the basis of the fixation and processing the special parenchyma cells and the epidermal cells are narrow procedures used, these spheres withstood both aqueous and bridges of cytoplasm that extend from one portion of the cy- ethanol extraction. toplasm into the vacuole and then back to the cytoplasm, thus As an increasing number of special parenchyma cells go encompassing some of the vacuole contents (fig. 4D,4E). through degradation, collapsed strands of cell walls become These cytoplasmic bridges are envisioned as cuplike extensions more prevalent in the main portion of the nectary (fig. 5F). of the cytoplasm. The bridges are sometimes contiguous with The substomatal chambers and pores are filled with material the tubule bundles (fig. 4B). A fine strand of beadlike dense (fig. 5G), possibly one of the non-water-soluble components material (fig. 4E; beads are ca. 10.1 nm in diameter) occurs of the nectar. When the collapse of the nectary nears comple- in each narrow bridge. tion, all of the parenchyma cells and the phloem cells are col- The special parenchyma cells and epidermal cells also con- lapsed and are indistinguishable from each other, except for tain straight tubes, unlike the tubules in the bundles, each with the epidermal cells and the guard cells (fig. 5F,5G). The non- a single strand of ribosome-sized particles inside (fig. 4F). stomata epidermal cells go through the same stages of degen- These tubes either traverse plasmodesmata (fig. 4F) or occur eration as the special parenchyma, and they eventually col- in the cytoplasm (fig. 4G) and sometimes are near and almost lapse. As mentioned earlier, before collapse, the vacuoles perpendicular to the tonoplast (fig. 4F). These tubes are of display characteristic narrow bridges of cytoplasm, cytoplas- unknown length but average 17.8 nm in outer diameter. The mic straight tubes with ribosome-like particles, and tubular ribosome-like particles in the tubes average 17.7 nm in di- bundles. In addition, arrays of small needle-like crystals of ameter, as compared to the cytoplasmic ribosomes, which av- unknown composition occur in the vacuoles of some of the erage 17.6 nm in diameter. degenerating epidermal cells and some of the special paren- As each parenchyma and epidermal cell vacuole enlarges, chyma cells before collapse (fig. 5H, arrows). Crystalline ma- the narrowing peripheral cytoplasm is pressed to the cell wall terial of unknown composition is also observed in the inter- (fig. 4H). Gaps appear in the tonoplast in various places (fig. cellular spaces beneath the guard cells (fig. 5I, arrows). 4H), forming entrances for the cytoplasm to mix with the vacuole contents (fig. 3J). Later, the vacuole disappears as an Postactive Nectary Stage entity, and the mixed contents are bounded by just the plas- malemma and the cell wall (fig. 4I). Many special parenchyma In just-opened flowers, the nectaries are collapsed (fig. 5J), cells at this stage of development appear partially or completely except for the guard cells that remain intact beyond the last collapsed (fig. 4J,4K), some with mixed cytoplasm and vac- stage sampled (fig. 5K,5L). The nectary is now a degenerated uolar contents. However, the thin cell walls do not seem to and collapsed mound that is partially covered with a residue break down or split open. Electron-dense material appears of electron-dense material in which sometimes occur small within and around the collapsed cells (fig. 4L) and in inter- cubical-like crystals of unknown composition, possibly from cellular spaces near the guard cells (fig. 5A). Some of this condensed nectar and other secretions (not shown). material (fig. 4L) looks like compacted ribosomes (ca. 15.6 nm in diameter) that form paracrystalline arrays in some places Presence of Starch in Nectaries during Three (fig. 5B; each ribosome-like particle in the paracrystalline ar- Developmental Stages rays averages 14.8 nm in diameter). These arrays are observed both among completely collapsed and partially collapsed cells. The PAS technique used for localizing non-water-soluble car- About this time, spherical bodies appear (fig. 5C, inset) in bohydrates during the stages of nectary development and de- and around the pores of some of the guard cells (fig. 5D)as generation (fig. 6A–6C) did not indicate any major buildup or

Fig. 3 Portions of soybean nectary during active stage. A, Transmission electron microscopy (TEM) of portion of nectary phloem finger that consists of thicker-walled sieve tube elements and companion cells. Special parenchyma surrounds finger. Bar p 6 mm. B, TEM of sieve plate between two sieve elements. P-protein is visible in elements (arrows).Bar p 600 nm. C, Light microscopy of longitudinal section through active- stage nectary with prominent guard cells in epidermis, mostly on gynoecium side; special parenchyma consisting of many cytoplasmically dense cells with vacuoles; and phloem fingers surrounded by active and degenerating special parenchyma cells (asterisks). Bar p 50 mm. D, TEM of section through a nectary phloem finger surrounded by degenerating special parenchyma cells (asterisks).Bar p 6 mm. E, TEM of cytoplasmically dense thin-walled special parenchyma cells with vacuoles. Bar p 3 mm. F, TEM of plastid in a partially degenerated special parenchyma cell displaying expanded thylakoids, starch grain, and phytoferritin (arrow).Bar p 200 nm. G, TEM of portion of special parenchyma cell displaying nucleus, plastid, mitochondria, Golgi body, expanded RER segments (arrow) with visible contents in lumen, and thin wall.Bar p 600 nm. H, TEM of partially and fully engorged special parenchyma cells displaying vacuoles with unstained material surrounded by ribosome-like particles. Bar p 2 mm. I, TEM of enlarged portion of an engorged special parenchyma cell vacuole with unstained material surrounded by ribosome-like particles that sometimes appear in circular arrays (e.g., three broken circles).Bar p 500 nm. J, TEM of portions of partially engorged special parenchyma cells with large vacuoles. Note packets of tubular bundles adjacent to tonoplasts (arrows).Bar p 2 mm. K, TEM of partially engorged epidermal cell with large vacuole containing ribosome-like particles and surrounded by tubular bundles (arrows). Bar p 2 mm. L, TEM of cross sections of tubular bundles in cytoplasm of a special parenchyma cell away from vacuole tonoplast.Bar p 200 nm.CC p companion cell; PF p phloem fingers;STE p sieve tube element.

This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms HORNER ET AL.—SOYBEAN FLORAL NECTARY DEVELOPMENT 683 depletion of starch. The central gynoecium and tissues below the nonglandular trichomes and appears to be intact. The cu- the nectary had relatively more starch during all of these stages. ticle reaches the base of the glandular trichomes where it ends, and an electron-transparent layer continues over the external Gynoecium Nonsecretory and Secretory Trichomes glandular trichome. These latter three types of trichomes per- Associated with Nectary Development sist throughout the three stages of nectary development and presumably continue to persist (and function) after the nectary During the latter part of preactive nectary development, degenerates. three types of trichomes are initiated from the epidermis of the gynoecium. The first is an elongated, thin-walled, non- glandular, unicellular trichome that contains a large vacuole Discussion with peripheral cytoplasm and a very large nucleus (fig. 6D). The outer surface of the cell wall displays papillae. A distinct The soybean nectary, compared to nectaries described in cuticle covers the entire trichome that is continuous, with the published studies of other cultivated legume taxa, is very un- cuticle covering the gynoecium epidermis, except for the se- usual in its development because it undergoes holocrine se- cretory trichome. The unicellular trichomes occur from near cretion via apoptosis, resulting in the demise of the nectary, the base of the gynoecium to the base of the style (not shown). and it displays several unique ultrastructural features. Our data The second type of elongated, nonglandular trichome con- show that the soybean nectary develops immediately before sists of one or two basal cells with thickened walls and a long flower opening and degenerates within a period of ca. 24 h terminal cell with a much thicker wall than the unicellular (Chen et al. 1987). trichome (fig. 6E). The outer trichome wall also displays pa- During development, the nectary is composed of three tis- pillae. These trichomes contain relatively large nuclei with sues: the epidermis with prominent pairs of guard cells, a cen- large nucleoli and occur in the uppermost part of the gynoe- tral mass of special parenchyma, and fingers of phloem. Studies cium toward the stigma (not shown). of other legume nectaries generally identify these same tissues The third type of trichome develops later than the two elon- and cell types but with a different fate (Picklum 1954; Ancibor gated, nonglandular trichomes and is much smaller and 1969; Waddle and Lersten 1973; Eriksson 1977; Erickson and shorter; it consists of five to seven cells in linear array (fig. 6F). Garment 1979; Teuber et al. 1980; Gulya´s and Kincsek 1982; These latter trichomes are dispersed among the longer trich- Murrell et al. 1982; Webster et al. 1982; Davis et al. 1988; omes from the base (fig. 6G) to the upper part of the gynoe- Davis and Gunning 1991, 1992, 1993; Crozier and Thomas cium. The cells composing this trichome contain dense cyto- 1993; Stpiczyn´ ska 1995; Razem and Davis 1999). plasm with many ribosomes, ER, Golgi bodies, plastids, and The young nectaries of soybean develop directly above re- mitochondria (fig. 6H). The nucleus of each cell is much ceptacle parenchyma that surrounds the main vasculature that smaller than the nucleus in the two types of nonglandular, comes into the flower. This parenchyma contains large num- elongated trichomes, and it is centrally located in each cell. bers of Rosanoffian calcium oxalate crystals similar to those The periclinal walls separating each of the linearly arrayed already described in soybean and other papilionoid taxa (Hor- cells contain many distinct plasmodesmata. The periclinal wall ner and Zindler-Frank 1982a, 1982b; Ilarslan et al. 1997, separating the basal, epidermal initial cell and the next cell has 2001), as do the other flower organs, except for the nectary, fewer plasmodesmata than the other periclinal walls. The com- which rarely contains calcium oxalate crystals. The receptacle position of these latter walls and the external wall of each crystals increase greatly in number as the nectary develops and trichome cell is the same but very different from all other cell persist well beyond the active nectary stage. Several crystal walls of the gynoecium and nectary (fig. 6H). All of these walls layers form early at the base of the young developing nectary, are PAS positive (fig. 6A–6C). No electron-dense cuticle was and the presence of these calcium oxalate crystals in the dif- observed on any of the external walls of these glandular tri- ferent flower parts of a variety of taxa, including soybean, has chomes. The cuticle on the epidermis of the nectary extends been shown numerous times. Crystals associated with the tis- over the nontrichome epidermal cells of the gynoecium and sues in and underlying the nectaries also have been observed

Fig. 4 Portions of soybean nectary during active stage. A, Transmission electron microscopy (TEM) of oblique to almost longitudinal section of tubular bundle in special parenchyma cell away from vacuole tonoplast.Bar p 100 nm. B, TEM of tubular bundle in cross section next to vacuole tonoplast and extending into a cytoplasmic bridge (arrow).Bar p 100 nm. C, TEM of tubular bundle in oblique section next to vacuole tonoplast.Bar p 200 nm. D, TEM of engorged nectary epidermal cell with cytoplasmic bridge extending into vacuole (arrow). Bar p 2 mm. E, TEM of enlarged view of D showing cytoplasmic bridge and strand of beadlike material in it. Note cell wall with plasmodesm with included straight tube containing ribosome-like particles (arrow).Bar p 400 nm. F, TEM of portion of two special parenchyma cells connected by their common walls and plasmodesmata; straight tubes containing ribosome-like particles traverse plasmodesmata. One tube almost touches tonoplast. Bar p 200 nm. G, TEM of special parenchyma cell cytoplasm with straight tubes containing ribosome-like particles.Bar p 300 nm. H, TEM of portion of partially engorged special parenchyma cell with enlarged vacuole and a tonoplast with several separations in it (arrows). Bar p 400 nm. I, TEM of portion of special parenchyma cell whose vacuolar and cytoplasmic contents are mixed and in a state of degeneration. Only plasmalemma remains as boundary membrane. Bar p 3 mm. J, Light microscopy of longitudinal section through active nectary with completely collapsed cells in its central region (arrows). Bar p 20 mm. K, TEM of central portion of nectary with special parenchyma cells in various stages of degeneration and collapse. Bar p 3 mm. L, TEM of central portion of nectary displaying special parenchyma cells partially or completed degenerated and collapsed. Bar p 1.5 mm.

This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms HORNER ET AL.—SOYBEAN FLORAL NECTARY DEVELOPMENT 685 by Ziegler (1956), Davis et al. (1988), Davis and Gunning as well as most nonlegume nectaries (Durkee 1983). We believe (1992, 1993), and Sawidis (1998). One suggestion is that high the phloem fingers innervating the nectary supply the simple levels of calcium ions within the nectary may be inhibitory to sugars that are probably transported to the stomatal pores via the secretory mechanism. This possibility is noteworthy since intercellular spaces. There is relatively little starch that forms the nectary proper generally does not contain any crystals until within the nectary during development, in contrast to many late in its development, when several new types of crystals of other nectaries studied where it is hydrolyzed at anthesis to unknown composition appear, both within the degenerating produce nectar sugars. The soybean nectary starch remains cells and on the surface of the nectary. Even though the earlier- throughout nectary development until the nectary degenerates. formed layer of crystals may be a way of reducing the move- Therefore, we suggest that most of the simple sugars are ment of calcium ions into the nectary, we believe that the formed in other parts of the plant, probably the leaves, and unidentified, later-formed crystals are the result of an accu- transported to the nectary via the vasculature. This process mulation of products, not necessarily calcium oxalate, in the has yet to be confirmed in soybean. degenerating nectary. One additional thought is that the nec- Before the nectary special parenchyma and epidermal cells tary special parenchyma cells have relatively thin walls because undergo programmed cell death, they produce compounds that calcium is sequestered during crystal formation in the recep- engorge their central vacuoles. In addition to the other unique tacle below the nectary. The thin walls would insure rapid and structures already described at this time of development, the easy collapse of the cells and aid in the release of their vacuolar enlarged cytoplasmic rough endoplasmic reticulum (RER) lu- contents. mens in our study (fig. 3G) are similar to those described by In some nectaries analyzed in other studies, there is a high Durkee (1983; fig. 6) as containing protein. We have no cy- magnesium/calcium ratio. In addition, Lu¨ ttge (1977) identified tochemical evidence to show what the lumens in our study cation concentrations in nectar and indicated the presence of contain. Eventually, each central vacuole shows tonoplast deg- antioxidants such as ascorbate in reasonably large amounts. radation that leads to mixing of the cytoplasmic contents with On the basis of these observations, the formation of the crystals the vacuolar contents. This event occurs before each cell col- may be a way of reducing the presence of calcium in the nectary lapses and releases its contents. This method of secretion is tissue via the conversion of ascorbate to oxalate (Loewus 1999; termed holocrine secretion, as opposed to the typical eccrine Horner et al. 2000) and its sequestration by calcium. and granuloeccrine methods of secretion in floral and extra- Soybean nectaries of the annual soybean have an outward floral nectaries described by Durkee (1983) and Fahn (1987). appearance similar to nectaries of other legume taxa. It is a Fahn (1987) studied the stalklike extrafloral nectaries associ- circular mound around the base of the gynoecium, and it is ated with the base of the leaves of Sambucus nigra, where the covered by an epidermis that contains many opened stomata nectariferous cells degenerate after nectar secretion. He termed consisting of two guard cells, primarily on the ridge of the this type of secretion as merocrine and not holocrine. In our nectary and on the slope facing the gynoecium. The guard cells, study, we believe the process of secretion, encompassing sugar once formed and open, seem like autonomous structures since secretion and secretion of the spherical material that appears they retain their turgidity and ultrastructure even after the on the surface of the nectaries, is a direct result of programmed remainder of the nectary special parenchyma, vasculature, and cell death and as such should be considered holocrine. nonstomata epidermal cells have collapsed. No statistical anal- Inside the enlarging vacuoles, circular arrays of ribosome- yses were carried out on the number of stomata, such as by like particles occur, suggesting that they surround circular (e.g., Davis and Gunning (1991, 1992, 1993), but it is evident that spherical in three dimensions) substances similar to the non- there are many stomata on the surface and that they are ca- staining spheres that are exuded from the pores of the guard pable of exuding substances (nectar) from the nectary proper cells. On the basis of our fixation process, these surface spheres as shown in this study. and the vacuole substances do not seem to be water or alcohol Internally, the soybean nectary consists of a special paren- soluble and thus are retained. The chemical composition of chyma that undergoes development and degeneration that is these substances is being investigated. completely different from the other legume nectaries studied The subcellular structures that have been described in the

Fig. 5 Portions of soybean nectary at active and postactive stages. A, Transmission electron microscopy (TEM) of nectary guard cells with residue in pore region (asterisk); interior cells are partially or completely degenerated and collapsed. Bar p 2 mm.GC p guard cells/stomata. B, TEM of portion of central nectary showing almost-collapsed special parenchyma containing individual ribosomes and paracrystalline arrays of ribosomes.Bar p 400 nm. C, Scanning electron microscopy (SEM) of portion of ridge of active nectary showing open guard cells with spherical material on guard cells. Bar p 20mm. Inset is a close-up of guard cells with spherical material. Bar p 10 mm. D, SEM of portion of ridge of nectary showing open guard cell with spherical material emanating from pore as beads. More spherical material covers epidermis. Bar p 5 mm. E, SEM of two guard cells covered with single spheres, spherical beads, and coalesced beads forming a layer. Bar p 4 mm. F, Light microscopy (LM) of longitudinal section of nectary whose special parenchyma is almost collapsed. Epidermal and guard cells are still intact. Bar p 20 mm. G, LM of portion of collapsed nectary ridge showing degenerated special parenchyma, intact epidermal cells, and guard cells with a pore region filled with material. Bar p 30 mm. H, TEM of special parenchyma cell vacuole containing unidentified crystals associated with membranes (arrows).Bar p 400 nm. I, TEM of intact guard cells with normal complement of organelles; partially collapsed nectary. Debris above pore region is partly spherical. Crystals of unknown composition occur in intercellular space (arrows). Bar p 2 mm. J, SEM of mostly collapsed nectary at late active stage. Bar p 200 mm. K, LM of completely collapsed nectary at postactive stage. Guard cells are still intact. Bar p 20 mm. L, TEM of guard cells comparable to those in K showing intact cytoplasm with distinct organelles. Bar p 3 mm.

This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms HORNER ET AL.—SOYBEAN FLORAL NECTARY DEVELOPMENT 687 special parenchyma and epidermal cells during the latter por- or bridges lend themselves to any plausible explanation with tion of the active nectary stage just before the degeneration respect to other cellular events going on during the time they and collapse of the cells are unique. The bundles of tubules are observed, other than the onset of cell degeneration. Ad- surrounding the enlarging special parenchyma and epidermal ditional images and techniques will have to be employed to cell vacuoles could be involved in maintaining the integrity of obtain a better understanding of their significance. these rapidly filling vacuolar sacs until the vacuoles have The fused beads forming layers on the nectary surface most reached a maximum size before the demise of each cell. Durkee certainly are products of the nectary. At least two reports have (1983) showed fibrillar to tubular material within the vacuoles shown substances on nectary surfaces that have been suggested of central cells of petiolar nectaries of Passiflora. We do not to be nectar (Nepi et al. 1996; Gaffal et al. 1998). Nepi et al. know whether this latter material is similar to the tubular (1996) showed both a layer and spheres on the nectary of bundles we observed in the cytoplasm and surrounding the Cucurbita. However, Gaffal et al. (1998) observed both spongy outside of the vacuoles of engorging special parenchyma and and chainlike substances coming from the nectary pores of epidermal cells in our study. Our interpretation at this time is Digitalis. They interpreted the latter to be microorganisms such they are different because of their location and overall as yeast and bacteria growing in the nectar. TEM sections of appearance. the nectary of Digitalis indicated that they were in fact mi- Before and during this period of filling, the ribosome-like croorganisms. We do not believe this is the case with the soy- particles occur within the nonstaining vacuolar space. How- bean nectary beads and layers because our TEM images do ever, breaks seen in the tonoplast could allow for some of the not show any microorganisms. Instead, they show an amor- cytoplasmic ribosomes to enter, and the bundles of tubules phous electron-translucent material. Because this material can would then prevent premature disruption of the vacuole and be seen with the heavy metal coating in the SEM but not readily the cell. These breaks in the tonoplasts were not observed with the TEM, this suggests that it is of low molecular mass earlier in these cells or in other cell types and are not fixation and not stainable with osmium tetroxide or uranyl or lead artifact. In fact, all of the cellular contents (plastids, mito- salts. Until we can cytochemically identify it, we will not know chondria, nuclei, etc.) eventually mix with the vacuolar con- whether this surface substance is really associated with the tents before these cells collapse, suggesting a loss of tonoplast nonstaining substance observed in the engorged special paren- integrity. chyma and epidermal vacuoles as outlined by the ribosome- The presence of the straight, single tubes containing beads like material. This verification is a challenging problem for of ribosome-like particles, along with the vacuolar bridges, is both metabolic and developmental reasons. beyond interpretation at this point. The single tubes, measur- The nectar of soybean is a complex milieu of many com- ing 17.8 nm in outer diameter, could be conduits for the trans- pounds, consisting of sugars, amino acids, proteins, lipids, and fer of ribosomes from cell to cell and as such could be a sig- other compounds that serve both nutritional and protective naling mechanism for synchronized cell activity leading to functions (Griebel and Hess 1940; Vogel 1969; Baker and apoptosis. Franceschi and Giaquinta (1983) observed “mac- Baker 1973, 1975; Deinzer et al. 1977; Rodriguez-Arce and rotubules” measuring 60–70 nm without any visible internal Diaz 1992; Roshchina and Roshchina 1993; Ecroyd et al. contents in the terminal cells of soybean leaf glandular tri- 1995; Ferreres et al. 1996; Cabras et al. 1999; Thornburg et chomes just before their degeneration. We do not know if there al. 2003). In soybean, the origin of these compounds may be is any similarity between the tubes reported in this study and solely from the nectary, or they also may arise from the five- the “macrotubules,” but their different diameters and differ- to seven-celled glandular trichomes associated with the ences in internal contents are difficult to assess even though gynoecium. they were found in glandular cells at or near the time the cells In recent studies, a number of unique proteins have been degenerate. identified in the floral nectar of tobacco (Carter et al. 1999; The cytoplasmic bridges appear to be associated with the Carter and Thornburg 2000; Thornburg et al. 2003). One of bundles of tubules. Neither the single tubes, bundles of tubules, them, Nectarin I, a superoxide dismutase, produces high

Fig. 6 Portions of nectaries stained for non-water-soluble polysaccharides (A–C) and three types of trichomes associated with gynoecium epidermis. Toluidine blue O used in D, E. A–C, Light microscopy (LM) of PAS-stained nectaries to demonstrate presence of starch. There is relatively little starch in the nectary tissues during the three stages, except for the guard cells. A, Preactive nectary. Bar p 30 mm. B, Active nectary. Bar p 30mm. C, Postactive nectary. Bar p 20 mm. D, LM of portion of lower gynoecium epidermis with unicellular, elongate, thin- walled nonglandular trichomes, each with a large nucleus and nucleolus, and short five- to seven-celled glandular trichomes, with each cell displaying a smaller nucleus and nucleolus. Bar p 30 mm. E, LM of portion of upper gynoecium and style with thick-walled, elongate, three- celled, nonglandular trichomes displaying distinct papillae on outer wall surface. Bar p 5 mm. Inset shows basal two cells of ThT trichome with different wall thicknesses and stain affinity, suggesting different wall compositions. Bar p 15 mm. F, Scanning electron microscopy (SEM) of six-celled glandular trichome. Bar p 5 mm. G, SEM of lower portion of gynoecium showing both young unicellular, elongate, thin-walled, nonglandular trichomes interspersed with five- and seven-celled glandular trichomes. As the former trichomes elongate further, glandular trichomes can no longer be seen from surface view. Bar p 30 mm. H, Transmission electron microscopy of longitudinal section of terminal three cells of a five- and seven-celled glandular trichome. Both outer walls and periclinal walls are thin and homogeneous. No cuticle is visible on outer walls, and periclinal walls contain distinct plasmodesmata (arrows). Cytoplasm of each cell contains Golgi bodies with vesicles, RER, small vacuoles, plastids, mitochondria, and central nuclei with distinct nucleolus. Bar p 3 mm.G p gynoecium ;ST p short five- to seven-celled glandular trichome;ThT p elongate , three-celled, thick-walled nonglandular trichome;TT p elongate , unicellular, thin-walled nonglandular trichome.

This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms 688 INTERNATIONAL JOURNAL OF PLANT SCIENCES enough levels of hydrogen peroxide to be considered antimi- dular trichomes on the vegetative leaves have a different de- crobial (Carter and Thornburg 2000). This finding was fol- velopmental fate and secretory products than those on the lowed by using two different methods, nectary-expressed gynoecium. We do support the interpretation of Franceschi cDNAs and hybridization to them, to examine the expression and Giaquinta (1983) that these glandular trichomes may be of defense-related genes in the nectaries. Their results showed producing compounds, possibly volatile in nature, that could that 21% of these cDNAs were defense-related clones, and be involved with microbial and insect deterrence against nectar they were more strongly expressed in floral nectaries than in degradation and/or protection of the young gynoecium. These the rest of the plant (Thornburg et al. 2003). The implication glandular trichomes both in the flower and on the leaves could of these results is that the floral nectar has built-in plant defense also be producing volatiles that attract pollinating insects. compounds against microbial (bacterial and fungal) activity. However, the source of floral volatiles in soybean has not been In soybean nectaries, no recognizable microbodies/peroxi- identified yet. somes were observed that would handle high levels of hydro- The occurrence of the three types of trichomes on the central gen peroxide or other oxidizing compounds through catalase gynoecium next to and above the nectary is possibly of im- activity. This observation, if correct, would allow for the portance both to the gynoecium and the nectary. First, the buildup of oxidizing compounds in the tissues and secreted glandular trichome is the last to develop of the three types but nectar. These compounds, if present, could serve to inhibit seems to be active before the active nectary stage and remains microbial activity as suggested by Thornburg et al. (2003). so beyond the postactive nectary stage. We do not know what Two types of trichomes have been described on the surfaces the chemical nature of the products is, but it must certainly of some of the soybean floral appendages (Carlson and Lersten contribute to the nectar because of its close proximity to the 1987) and on the vegetative stems and leaves (Flores and Es- nectary and its time of development. As some authors suggest pinoza 1977; Franceschi and Giaquinta 1983; Lersten and (Davis 1985; Carter et al. 1999), these trichomes could be Carlson 1987). A third type, a unicellular, thin-walled, non- producing antimicrobial compounds (Carter and Thornburg glandular trichome, also was shown to be present on the gy- 2000; Thornburg et al. 2003) to protect both the nectar and noecium in this study and raises the question of whether this the gynoecium. Second, the two types of long, nonglandular type of trichome and the other three-celled, thick-walled, non- trichomes could act as a mechanical deterrent against some glandular trichome have different functions. invading organisms. They could also serve in a capillary way Franceschi and Giaquinta (1983) conducted a developmen- to hold and move the nectar from the base of the gynoecium tal study in soybean of the five-celled glandular trichomes on where the nectary occurs to near the top of the flower where the young vegetative leaves. They acknowledged a second type pollinating insects probe. of nonglandular trichome, but a detailed description was not The nectary of the annual cultivated soybean is a small but included in their study. Their results show an ultrastructure active gland during the time of anthesis. Even though the vast similar to what we have described with the gynoecium five- majority of field-grown plants are autogamous, the fact that to seven-celled glandular trichomes. They appear to be secre- they are capable of insect cross-pollination indicates that they tory based on the cytoplasmic organelles present, to have spe- have the necessary floral characteristics. As such, there is the cialized outer cell walls with a modified cuticle or a cuticle distinct possibility of increasing cross-pollination by various that was partly separated from the primary walls, and to con- methods and moving toward economically producing hybrid tain many distinct plasmodesmata in the periclinal walls sep- soybean seeds (Palmer et al. 2001). Understanding more about arating the cells from each other. Franceschi and Giaquinta the genes that control the soybean floral nectaries, nectary (1983) identified a cuticle on the outer surface of the primary structure and development, nectar composition and volume, walls of this trichome. Our preliminary observations indicate and floral volatiles (Vainstein et al. 2001) should aid in en- that an electron-dense waxy cuticle does cover the epidermis hancing the potential for increased pollinator attraction. The of all other epidermal cells of the gynoecium and nectary. How- benefits of hybridization, and thus heterosis, could have a ma- ever, we do not observe this cuticle beyond the base of the jor impact on soybean production and use worldwide. glandular trichome. It is replaced by what we interpret as an Several approaches being explored by our research group expanded, possibly porous, somewhat electron-translucent are to assess nectar output and composition, nectary structure layer (with the electron-dense waxy substrate missing) that and development, and increased seed set in wild Glycine per- could allow the passage of secretory products from the tri- ennials and wild annual Glycine soja genotypes to further chome cells. This trichome and the other two types of gynoe- study their characteristics as a means of introducing them into cium trichomes are the focus of another study. the cultivated soybean. The possibility certainly exists that Franceschi and Giaquinta (1983) identified two unique cy- some of these characteristics have been highly reduced by con- toplasmic organelles in the terminal two cells of the leaf glan- tinued autogamy in the cultivated soybean. Learning more dular trichomes in their study, plastids that lack grana and about these characteristics and comparing them to nectaries thylakoids but include distinct crystals of unknown compo- of Glycine taxa that are successful in outcrossing will help in sition and “macrotubules” without contents. In the soybean this approach. glandular trichomes associated with the gynoecium, we did not find plastids with crystals, nor did we find “macrotubules” during the life of the nectary. These contrasting results could Acknowledgments be because the trichomes that were associated with the gy- noecium were not yet at the stage of degeneration (they exist This is a joint contribution of the USDA-ARS CICGR Unit beyond the life of the nectary), or the similar appearing glan- (R. G. Palmer), USDA SGA CRIS Project 3625-21000-029-

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01S (H. T. Horner), and the Iowa Agriculture and Home Eco- preparation of this manuscript. The mention of a tradename nomics Experiment Station, Ames, Iowa 50011, Project 3769 or proprietary product does not constitute a guarantee or war- (R. G. Palmer). The microscopic work was carried out in the ranty of the product by the USDA or Iowa State University Bessey Microscopy Facility. We thank Drs. Nels R. Lersten and and does not imply its approval to the exclusion of other prod- Robert W. Thornburg for their helpful comments during the ucts that may be suitable.

Literature Cited

Ancibor E 1969 Los nectarios florales en Luguminosas–Mimoso´ ideas. Davis AR 1985 The secretion of systemic insecticides in nectar and Darwiniana 15:128–142. some effects on honey bee development. MS thesis. University of Baker HG, I Baker 1973 Amino acids in nectar and their evolutionary Guelph. significance. Nature 241:543–545. ——— 1997 Influence of floral visitation on nectar-sugar composition ——— 1975 Studies of nectar-constitution and pollinator-plant co- and nectary surface changes in Eucalyptus. Apidologie 28:27–42. evolution. Pages 100–140 in LE Gilbert, PH Raven, eds. Coevolution Davis AR, BES Gunning 1991 The modified stomata of the floral of animals and plants. University of Texas Press, Austin. nectary of Vicia faba L. 2. Stomatal number and distribution as ———, eds 1981 Chemical constituents of nectar in relation to pol- selection criteria for breeding for high nectar sugar production. Acta lination mechanisms and phylogeny: biochemical aspects of evolu- Hortic 288:329–334. tionary biology. University of Chicago Press, Chicago. ——— 1992 The modified stomata of the floral nectary of Vicia faba ——— 1983 A brief historical review of the chemistry of floral nectar. L.: development, anatomy and ultrastructure. Protoplasma 164: Pages 126–152 in B Bentley, T Elias, eds. The biology of nectaries. 134–152. Columbia University Press, New York. ——— 1993 The modified stomata of the floral nectary of Vicia faba Beardsell DV, EG Williams, RB Knox 1989 The structure and his- L. 3. Physiological aspects, including comparisons with foliar sto- tochemistry of the nectary and anther secretory tissue of the flowers mata. Bot Acta 106:241–253. of Thryptomene calycina (Lindl.) Atapf (Myrtaceae). Aust J Bot 37: Davis AR, RL Peterson, RW Shuel 1988 Vasculature and ultrastruc- 63–80. ture of the floral and stipular nectaries of Vicia faba (Leguminosae). Belmonte E, L Cardemil, MT Kalin Arroyo 1994 Floral nectary struc- Can J Bot 66:1435–1448. ture and nectar composition in Eccremocarpus scaber (Bignonia- Deinzer ML, PA Thompson, DM Burgett, DL Isaacson 1977 ceae), a hummingbird-pollinated plant of central Chile. Am J Bot Pyrrolizidine alkaloids: their occurrence in honey from tansy rag- 81:493–503. wort (Senecio jacobaea L.). Science 195:497–499. Bozzola JJ, LD Russell 1999 Electron microscopy: principles and Dimock MB, GG Kennedy 1983 The role of glandular trichomes in techniques for biologists. Jones & Bartlett, Boston. the resistance of Lycopersicon hirsutum F. Glabratum to Heliothis Broersma DB, RL Bernard, WH Luckmann 1972 Some effects of soy- zea. Entomol Exp Appl 33:263–268. bean pubescence on populations of the potato leafhopper. J Econ Durkee LT 1983 The ultrastructure of floral and extrafloral nectaries. Entomol 65:78–82. Pages 1–29 in B Bentley, T Elias, eds. The biology of nectaries. Brown AHD, JE Grant, R Pullen 1986 Outcrossing and paternity in Columbia University Press, New York. Glycine argyrea by paired fruit analysis. Biol J Linn Soc 29:283–294. Durkee LT, DJ Gaal, WH Reisner 1981 The floral and extrafloral Butler GD, GM Loper, SE McGregor, JL Webster, H Margolis nectaries of Passiflora. 1. The floral nectary. Am J Bot 68:453–462. 1972 Amounts and kinds of sugars in nectars of cotton (Gossypium Ecroyd CE, RA Franich, HW Kroese, D Steward 1995 Volatile con- spp.) and the time of their nectar secretion. Agron J 64:364–368. stituents of Dactylanthus taylorii flower nectar in relation to flower Cabras PA, C Angioni, C Tuberoso, I Floris, F Reniero, C Guillou, S pollination and browsing by animals. Phytochemistry 40:1387– Ghelli 1999 Homogentisic acid: a phenolic acid as a marker of 1389. strawberry-tree (Arbutus unedo) honey. J Agric Food Chem 47: Erickson EH 1975a Effect of honey bees on yield of three soybean 4064–4067. cultivars. Crop Sci 15:84–86. Carlson JB 1973 Morphology. Pages 17–95 in BE Caldwell, ed. Soy- ——— 1975b Honey bees and soybeans. Am Bee J 115:351–353, 372. beans: improvement, production, and uses. American Society of ——— 1975c Variability of floral characteristics influences honey bee Agronomy, Madison, Wis. visitation to soybean blossoms. Crop Sci 15:767–771. Carlson JB, NR Lersten 1987 Reproductive morphology. Pages 95– Erickson EH, MB Garment 1979 Soya-bean flowers: nectary ultra- 134 in JR Wilcox, ed. Soybeans: improvement, production, and uses. structure, nectar guides, and orientation on the flower by foraging 2d ed. American Society of Agronomy, Madison, Wis. honeybees. J Apic Res 18:3–11. Carter C, R Graham, RW Thornburg 1999 Nectarin I is a novel, Eriksson M 1977 The ultrastructure of the nectary of red clover (Tri- soluble germin-like protein expressed in the nectar of Nicotiana sp. folium pratense). J Apic Res 16:184–193. Plant Mol Biol 41:207–216. Esau K 1977 Anatomy of seed plants. Wiley, New York. Carter C, RW Thornburg 2000 Tobacco Nectarin I: purification and Fahn A 1987 The extrafloral nectaries of Sambucus nigra. Ann Bot characterization of a germin-like, manganese superoxide dismutase (London) 60:299–308. implicated in the defense of floral reproductive tissues. J Biol Chem ——— 1988 Secretory tissues in vascular plants. New Phytol 108: 275:36726–36733. 229–257. Chen LF, MC Albertsen, RG Palmer 1987 Pollen and coenocytic mi- Ferreres F, P Andrade, MI Gil, FA Tomas Barberan 1996 Floral nectar crospore germination in male-fertile and male-sterile soybean. Eu- phenolics as biochemical markers for the botanical origin of heather phytica 36:333–343. honey. Z Lebensm Unters Forsch 202:40–44. Crozier TS, JF Thomas 1993 Normal floral ontogeny and cool Figueiredo AC, M Salome´ Pais 1992 Ultrastructural aspects of the temperature-induced aberrant floral development in Glycine max nectary spur of Limodorum abortivum (L.) Sw (Orchidaceae). Ann (Fabaceae). Am J Bot 80:429–448. Bot 70:325–331. Dafni H, Y Lensky, A Fahn 1988 Flower and nectar characteristics Flores EM, AM Espinoza 1977 Epidermis foliar de Glycine soja Sieb. of nine species of Labiatae and their influence on honeybee visits. y Zucc. Rev Biol Trop 25:263–273. J Apic Res 27:103–114. Franceschi VR, RT Giaquinta 1983 Glandular trichomes of soybean

This content downloaded from 129.186.176.217 on Tue, 14 Jun 2016 14:39:04 UTC All use subject to http://about.jstor.org/terms 690 INTERNATIONAL JOURNAL OF PLANT SCIENCES

leaves: cytological differentiation from initiation to senescence. Bot Palmer RG, J Gai, H Sun, JW Burton 2001 Production and evaluation Gaz 144:175–184. of hybrid soybean. Pages 263–307 in J Janick, ed. Plant breeding Frei E 1955 Die Innervierung der floralen Nektarien dikotyler Pflan- reviews. Vol 21. Wiley, New York. zenfamilien. Ber Schweiz Bot Ges 65:60–114. Percival M 1961 Types of nectar in angiosperms. New Phytol 60:235– Frey-Wyssling A 1955 The phloem supply to nectaries. Acta Bot 281. Neerl 4:358–369. Petanidou T, V Goethals, E Smets 2000 Nectary structure of Labiatae Fujita R, M Ohara, K Okazaki, Y Shimamoto 1997 The extent of nat- in relation to their secretion and characteristics in a Mediterranean ural-pollination in wild soybean (Glycine soja). J Hered 88:124–128. shrub community: does flowering time matter? Plant Syst Evol 225: Gaffal KP,W Heimler, S El-Gammal 1998 The floral nectary of Digitalis 103–118. purpurea L., structure and nectar secretion. Ann Bot 81:251–262. Picklum WE 1954 Developmental morphology of the inflorescence Griebel C, G Hess 1940 The vitamin C content of flower nectar of and flower of Trifolium pratense L. Iowa State J Sci 28:477–495. certain Labiatae. Z Lebensm Unters Forsch 79:168–171. Purseglove JW 1968 Tropical crops: dicotyledons. Vol 1. Longman, Gulya´s S, I Kincsek 1982 Floral nectaries of species of Papilionaceae. London. Acta Biol Szeged 28:53–63. Rabinowitch HD, A Fahn, T Meir, Y Lensky 1993 Flower and nectar Hanaichi T, T Sato, T Iwamoto, J Malavasi-Yamashiro, M Hoshino, attributes of pepper (Capsicum annuum L.) plants in relation to N Mizuno 1986 A stable lead stain by modification of Sato’s their attractiveness to honeybees (Apis mellifera L.). Ann Appl Biol method. J Electron Microsc 35:304–306. 123:221–232. Horner HT, AP Kausch, BL Wagner 2000 Ascorbic acid: a precursor Razem FA, AR Davis 1999 Anatomical and ultrastructural changes of oxalate in crystal idioblasts of Yucca torreyi in liquid root culture. of the floral nectary of Pisum sativum L. during flower development. Int J Plant Sci 161:861–868. Protoplasma 206:57–72. Horner HT, RG Palmer 1995 Mechanisms of male sterility. Crop Sci Rivera GL 1996 Floral trichomes and nectaries in four species of Te- 35:1527–1535. comeae (Bignoniaceae). Darwiniana 34:19–26. Horner HT, E Zindler-Frank 1982a Calcium oxalate crystals and crystal cells in the leaves of Rhynchosia caribaea (Leguminosae: Rodriguez-Arce AL, N Diaz 1992 The stability of beta-carotene in Papilionoideae). Protoplasma 111:10–18. mango nectar. J Agric Univ P R 76:101–102. ——— 1982b Histochemical, spectroscopic, and x-ray diffraction Roshchina VV, VD Roshchina 1993 The excretory function of higher identifications of the two hydration forms of calcium oxalate crystals plants. Springer, Berlin. in three legumes and Begonia. Can J Bot 60:1021–1027. Ruzin SE 1999 Plant microtechnique and microscopy. Oxford Uni- Ilarslan H, RG Palmer, HT Horner 2001 Calcium oxalate crystal id- versity Press, New York. ioblasts in developing seeds of soybean. Ann Bot 88:243–257. Sawidis T 1998 The subglandular tissue of Hibiscus rosa-sinensis nec- Ilarslan H, RG Palmer, J Imsande, HT Horner 1997 Quantitative de- taries. Flora 193:327–335. termination of calcium oxalate in developing seeds of soybean. Am Schoen DJ, AHD Brown 1991 Whole- and part-flower self-pollination J Bot 8:1042–1046. in Glycine clandestina and G. argyrea and the evolution of autogamy. Jin W, HT Horner, RG Palmer, RC Shoemaker 1999 Analysis and Evolution 45:1651–1664. mapping of gene families encoding b-1,3-glucanases of soybean. Smith MA, HT Horner, RG Palmer 2001 Temperature and photo- Genetics 153:445–452. period effects on sterility in a cytoplasmic male-sterile soybean. Crop Jin W, RG Palmer, HT Horner 1997 The genetics and cytology of a Sci 41:702–704. new genic male-sterile soybean, Glycine max (L.) Merr. Sex Plant Smith MA, RG Palmer, HT Horner 2002 Microscopy of a cytoplas- Reprod 10:13–21. mic male-sterile soybean from an interspecific cross between Glycine Jin W, RG Palmer, HT Horner, RC Shoemaker 1998 Molecular map- max and G. soja. Am J Bot 89:417–426. ping of a male-sterile gene in soybean. Crop Sci 38:1681–1685. Spurr AR 1969 A low-viscosity epoxy resin embedding medium for Lersten NR, JB Carlson 1987 Vegetative morphology. Pages 49–94 electron microscopy. J Ultrastruct Res 26:31–43. in JR Wilcox, ed. Soybeans: improvement, production, and uses. 2d Stpiczyn´ ska M 1995 The structure of floral nectaries of some species ed. American Society of Agronomy, Madison, Wis. of Vicia L. (Papilionoideae). Acta Soc Bot Pol 64:327–334. Lersten NR, HT Horner 2000 Types of calcium oxalate crystals and Teuber LR, MC Albertsen, DK Barnes, GH Heichel 1980 Structure macro patterns in leaves of Prunus (Rosaceae: Prunoideae). Plant of floral nectaries of alfalfa (Medicago sativa L.) in relation to nectar Syst Evol 224:83–96. production. Am J Bot 67:433–439. Levin DA 1973 The role of trichomes in plant defence. Q Rev Biol Thornburg RW, C Carter, A Powell, R Mittler, L Rizhsky, HT Hor- 48:3–15. ner 2003 A major function of the tobacco floral nectary is defense Loewus FA 1999 Biosynthesis and metabolism of ascorbic acid in against microbial attack. Plant Syst Evol 238:211–218. plants and of analogs of ascorbic acid in fungi. Phytochemistry 52: Vainstein A, E Lewinsohn, E Pichersky, D Weiss 2001 Floral fra- 193–210. grance: new inroads into an old commodity. Plant Physiol 127:1383– Lu¨ ttge U 1977 Nectar composition and membrane transport of sugar 1389. and amino acids: a review on the present state of nectar research. Vogel S 1969 Flowers offering fatty oil instead of nectar. Paper pre- Apidologie 8:305–319. sented at the eleventh International Botanical Congress, Seattle. McGregor SE 1976 Insect pollination of cultivated crop plants. USDA Agriculture Handbook 496. Washington, D.C. 411 pp. Waddle R, NR Lersten 1973 Morphology of discoid floral nectaries Murrell DC, RW Shuel, DT Tomes 1982 Nectar production and floral in Leguminosae, especially tribe Phaseoleae (Papilionoideae). Phy- characteristics in birdsfoot trefoil (Lotus corniculatus L.). Can J tomorphology 23:152–161. Plant Sci 62:361–371. Webster BD, RM Ross, T Evans 1982 Nectar and the nectary of Pha- Nepi M, F Ciampolini, E Pacini 1996 Development and ultrastructure seolus vulgaris L. Am Soc Hort Sci 107:497–503. of Cucurbita pepo nectaries of male flowers. Ann Bot 78:95–104. Zer H, A Fahn 1992 Floral nectaries of Rosmarius officinalis L.: O’Brien SP, BR Loveys, WJR Grant 1996 Ultrastructure and function structure, ultrastructure and nectar secretion. Ann Bot 70:391–397. of floral nectaries of Chamelaucium uncinatum (Myrtaceae). Ann Ziegler H 1956 Untersuchungen u¨ ber die Leitung und Sekretion der Bot 78:189–196. Assimilate. Planta 47:447–500.

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