Calcium Distribution and Function in the Glandular Trichomes of Lavandula Pinnata L

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Calcium distribution and function in the glandular trichomes of Lavandula pinnata L

Article in Journal of the Torrey Botanical Society · April 2010 DOI: 10.3159/09-RA-046.1

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Calcium distribution and function in the glandular trichomes of Lavandula pinnata L. Shan-Shan Huang1,2 The Institute of Chinese Medicine of Guangdong Province, Guangzhou, 510520, China Jing-Ping Liao South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China Bruce K. Kirchoff University of North Carolina at Greensboro, Department of Biology, Greensboro, NC 27402

HUANG, S-S. (The Institute of Chinese Medicine of Guangdong province, Guangzhou, 510520, China), J.-P. LIAO (South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China). AND B. K. KIRCHOFF (University of North Carolina at Greensboro, Department of Biology, Greensboro, NC 27402), Calcium distribution and function in the glandular trichomes of Lavandula pinnata L. J. Torrey Bot. Soc. 137: 1–15. 2010.—Calcium distribution during peltate and capitate glandular trichome development in Lavandula pinnata L. was examined with the potassium antimonate precipitation method. In order to establish a role for calcium in the secretory process and elucidate calcium function in the glands, the effects of calcium removal were investigated by treatment with nifedipine (Nif, a calcium channel blocker) and Ethylene glycol-bis (2-aminoethyl ether)-N, N, N9,N9-tetraacetic acid (EGTA, a calcium chelator). Untreated, mature glands accumulate many calcium precipitates in the subcuticular space and adjacent cell wall during secretion. In Nif or EGTA treated plants these precipitates disappear, and the amount of secretory product is drastically reduced. Calcium removal also results in a reduction in gland density, cells with decreased cytoplasmic density, formation of a lax cell wall, abnormal formation of the subcuticular space, thinning of the cuticle, and the presence of multivesicular bodies near the plasma membrane. At the post-secretory stage, calcium precipitates are common on the degenerating organelles. These results support a role for calcium in gland development, secretion, and programmed cell death. Key words: antimonate precipitation, calcium, EGTA, glandular trichome, Lavandula pinnata L., nifedipine, secretion, ultrastructure.

Calcium is an essential element in biological 1996, Taylor and Hepler 1997, Franklin-Tong systems. In its ionic form (Ca2+) it has been 1999, Eckardt 2001, Plieth 2001, Campanoni shown to be crucial in the control of numerous and Blatt 2007). Removal of calcium from the physiological and developmental processes in nutrient supply results in rapid death of cells in plants (Bush 1995, Zhang and Cass 1997, the apical meristem, and a cessation of growth Trewavas and Malho´ 1998, Sanders et al. (Epstein 1972, Gilroy et al. 1993). 1999, Scrase-Field and Knight 2003, Medve- Pollen tubes have been found to respond to dev 2005, Wheeler and Brownlee 2008). For calcium gradients by elongating toward higher instance, the Ca2+ ion is involved in intracel- Ca2+ concentrations in in-vitro assays (Mas- lular events such as protoplasmic streaming, carenhas and Leonarad 1962, Pierson et al. nuclear division, cell division, cell plate for- 1994). When calcium channel blockers are mation, and polarized growth in rhizoids and added to the medium, pollen tube growth is pollen tubes (Takagi and Nagai 1983, Sakai- restrained, suggesting that calcium is acting as Wada and Yagi 1993, Malho´ and Trewavas an intercellular signal (Picton and Steer 1985, Pierson et al. 1994, Herrmann and Felle 1995, Fan et al. 1996, Carol et al. 1997). Consistent 1 We thank the National Natural Science Foun- with this view, elevated cytosolic Ca2+ concen- dation of China (39870087, 30370099, 40332021), trations ([Ca2+] ) have been reported to drive and the Key Scientific Item of Guangdong c (045C013) for financial support. Xing-Lan Xu and docking and fusion of exocytotic vesicles at the Xiao-Ying Hu, instructors in the Electron Micro- tip of pollen tubes (Carol et al. 1997). In a scopy facility of the South China Botanical Garden, related study, Roy et al. (1999) found positive assisted the first author in learning EM. feedback between Ca2+ concentration and 2 Author for correspondence. E-mail: hss@scib. ac.cn exocytosis in lily pollen tubes. According to Received for publication August 6, 2009, and in this work, high concentrations of calcium revised form January 7, 2010. promote exocytosis, thus providing more cell

1 2 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.137 wall substance and causing the circumference As in other Lamiaceae, Lavandula pinnata of the cell membrane to increase, which in turn possesses two kinds of glandular trichomes causes more calcium channels to open (Roy et (Ascensaõ and Pais 1998, Gersbach 2002, al. 1999). Huang et al. 2008). Both capitate and peltate Calcium levels in the anther have also been trichomes are initiated from a single proto- studied and have been found to change with dermal cell, and undergo several cell divisions the stage of development. For instance, when to produce stalk and head cells. However, visualized with potassium antimonate precipi- while there are one to two secretory cells in the tation in photoperiod-sensitive male sterile head of a capitate trichome, there are eight rice, calcium precipitates accumulate in the cells in the head of a peltate trichome (Huang anther walls, on the surface of pollen grains, et al. 2005, 2008). In both cases, gland and in Ubish bodies, but they are less frequent development is very rapid (approximately on the pollen surface at the late microspore 60 h) in comparison to leaf expansion (18– stage (Tian et al. 1998). Later, as starch 25 d, Huang et al. 2008). Three different accumulates in the mature pollen grains, the secretory stages can be recognized: presecre- number of calcium precipitates decreases in tory, secretory and post-secretory (Ascensaõ these cells and increases in the parenchyma- and Pais 1998, Turner et al. 2000, Huang et al. tous cells of the connective tissue (Tian et al. 2005, 2008). The mechanism of secretion is 1998). Changes in calcium distribution during similar to that of other Lamiaceae (Huang et development have also been reported in al. 2008). developing ovules of Nicotiana tabacum (Tian In this study, potassium antimonite precipi- and Russell 1997), pollen grains of Chlorophy- tation is used to investigate loosely bound tum elatum (GO´ rska-Brylass et al. 1997), calcium distribution during both capitate and Torenia fournieri (Chen et al. 2008), and peltate glandular trichome development. The during pollen tube growth of tobacco (Xie et calcium channel blocker nifedipine (Nif) and al. 2005a). the calcium chelator Ethylene glycol-bis (2- Calcium has also been implicated in the aminoethyl ether)-N, N, N9,N9-tetraacetic function of the stylar transmitting tract (Zhao acid (EGTA) are used to elucidate calcium et al. 2004), dividing root cells (Vaughan et al. function during gland development (Williams 1987, Sakai-Wada and Yagi 1993) and poten- 1970, Reiss and Herth 1985, Rosales and tially as a causative factor in Blossom-end rot Brown 1992, Fan et al. 1996, Moysset and of tomato (Suzuki et al. 2003). Despite what is Simoń 1990, Zhang et al. 2000). Using these known from these studies, a role for calcium in calcium antagonists, we investigate the effects the secretion of essential oils has not been of calcium on trichome number, morphology, established. development, and on the secretory process The glandular trichomes of the Lamiaceae itself. are a well-established source of essential oils. Two types of trichomes occur in the family. Material and Methods. MICROPROPAGATION These trichomes are termed peltate or capitate OF APICAL SHOOTS.PlantsofLavandula pinnata depending on the structure of the secretory L. were grown in a greenhouse at the South head (Werker 1993). In both cases, the China Botanical Garden, Guangzhou, China. secretory product accumulates in a subcuticu- Apical shoot explants for the study of calcium lar space outside of the cell wall. The peltate distribution were prepared according to the trichomes produce most of the essential oil, procedure of Huang et al. (2008). Trichome with terpenes comprising the main component development was studied under four growth (Clark et al. 1997, Turner et al. 2000). The conditions: light-grown control, dark-grown capitate trichomes secrete varying amounts of control, 100 mM Nif (maintained in the dark), polysaccharides in addition to some terpenes and 5 mM EGTA (maintained in the light). (Werker 1993). Prior to secretion, the tri- The dark-grown control was necessary be- chomes undergo several cell divisions, and the cause Nif is inactivated by light, so Nif treated cuticle thickens. The subcuticular space forms plants must be maintained in the dark between the cuticle and the cell wall, and (Dentinger 2003). enlarges as the essential oil is secreted (Ascen- saõ et al. 1997, Turner et al. 2000, Gersbach CALCIUM VISUALIZATION. Calcium is present 2002, Huang et al. 2008). in plant tissues in free (ionic), loosely bound, 2010] HUANG ET AL.: CALCIUM IN THE TRICHOMES OF LAVANDULA PINNATA L. 3 and bound states. Visualization of each pool is caused the leaf margins to dry, and some buds strongly technique dependent (Ge et al. 2007). to stop growing. When [Nif] was increased to Although a precise microspectrofluorometric 250 mM, the leaves began to dry out after method is available to measure free cytosolic 10 days and the seedlings died after 15 days. 2+ calcium ([Ca ]c) (Pierson et al. 1994), it is not At 500 mM [Nif] the seedlings died after just possible to use this method for the study of three days. glandular trichomes because of the difficulty in penetrating the cell wall by microinjection. ADMINISTRATION OF EGTA TO SEEDLINGS. In this study we used potassium antimonite to The effects of EGTA were evaluated in label loosely bound calcium, which can be seedlings that were transferred from micro- readily converted to free calcium upon the propagation to potting soil and then to sand. appropriate environmental signal, such as pH Greenhouse grown seedlings approximately (Tian et al. 1998, Ge et al. 2007). Potassium 5 cm in length, and about one month old, antimonate precipitation is a sensitive trans- were transferred to small plastic cups contain- mission electron microscopy (TEM) method ing sand and a complete fertilizer. The long known to label loosely bound calcium transferred seedlings were watered once a (Wick and Hepler 1982, Slocum and Roux day for seven days with 5 mM (pH 6.5) EGTA 1982, Jian et al. 1999). Both calcium localiza- (Sigma, St. Louis, MO, U.S.A.) (Zhang et al. tion and the effects of Nif and EGTA were 2000). The cultures were maintained at 25 6 investigated in capitate and peltate trichomes, 2uC with 16 h day length at a light intensity of but precipitate counts are only reported for 10–20 mmol m22 s21 using Philips TLD 36W/84 peltate trichomes. Detailed counts of calcium bulbs. precipitates were not carried out for the capitate trichomes. Inspection of the TEMs TRANSMISSION ELECTRON MICROSCOPY.After showing calcium localization in these tri- approximately 20 d of growth, 2–3 mm leaves chomes showed the same results as for the were selected from micropropagated or green- peltate trichomes. house-grown buds from of all four treatments (light-grown control, dark-grown control, ADMINISTRATION OF NIF TO SEEDLINGS. The 100 mM Nif, and 5 mM EGTA). The leaves effects of the calcium blocker nifedipine were cut into approximately 1 mm2 pieces (Sigma, St. Louis, MO, U.S.A.) were evalu- and fixed in 2% glutaraldehyde in 0.1 M ated in explants growing on Murashige and potassium phosphate (KH2PO4) buffer (pH Skoog (1962) basal medium supplemented 7.8) containing 1% potassium antimonate with 100 mM Nif dissolved in DMSO at (K2H2Sb2O7?4H2O). Fixation was carried out 1022 M as a preservative. DMSO concentra- for 4 h at 4uC. The fixed pieces were washed in tions , 1021 M were found to have no effect three changes of 1% potassium antimonate in on glandular trichome development. The 0.1 M potassium phosphate buffer (0.5 h cultures were maintained at 25 6 2uC in the each), and post-fixed in 1% osmium tetroxide dark to prevent inactivation of Nif (Dentinger (OsO4)for16hat4uC in the same potassium 2003). Previous work has shown that the lack antimonite/buffer combination (Tian and Rus- of illumination does not effect trichome sell 1997, Tian et al. 2000). The post-fixed development (Huang et al. 2008). tissues were washed in three changes of 0.1 M After evaluating the effects of different Nif potassium phosphate buffer without antimo- concentrations (1, 10, 100, 250, and 500 mM) nite, dehydrated in a graded ethanol series, on seedling growth, 100 mM was selected as and embedded in Spurr’s resin. Ultra-thin the key experimental concentration of Nif. sections (80 nm) were cut using a Leica- Fan et al. (1996) used similar concentrations in Ultracut S ultramicrotome, stained with ur- their investigation of the effects of Nif on anyl acetate and observed with a JEM-1010 pollen germination, pollen tube growth, and TEM at 90 KV (Sajo et al. 2005). the division of the generative nucleus in Precipitates were confirmed as calcium Nicotiana tabacum. When Nif concentration antimonate with a JEOL 2000FX TEM at ([Nif]) was between 1 and 10 mM, L. pinnata 40 kV equipped with an IXRF energy- seedlings grew indefinitely with little effect on dispersive X-ray analysis system. Deconvo- gland development or ultrastructure. Fifteen luted peak ratios reflected a 2:1 ratio of days of treatment with a [Nif] of 100 mM antimony to calcium (Tian and Russell 4 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.137

1997). Two additional controls were also used: Treatment of normally processed sections with 1) potassium antimonate was sometimes the calcium chelator EGTA resulted in the omitted from the solutions during processing removal of the larger precipitates (. 100 nm), to verify that the precipitates were caused by although some smaller precipitates entirely the experimental treatment, and 2) the pre- enclosed in the embedding resin remained. sence of calcium in the precipitates was Osmiophilic bodies were not affected by this confirmed by incubating selected grids, with treatment. treated sections attached, in a solution of 0.1 M EGTA for 1 h at 37uC. Removal of precipi- CHANGES IN CALCIUM DISTRIBUTION DURING tates confirmed the presence of calcium (Tian THE EARLY PRESECRETORY STAGES.Itisnot and Russell 1997). possible to distinguish between capitate and Cuticle thickness was determined from peltate glands at the early presecretory stages. TEM micrographs. Ten images of six post- The number of cells in the trichome increases secretory peltate glandular trichomes from from one to three during this stage. Calcium each of three specimens from the light-grown, abundance is reported as an average of all cells dark-grown, and 100 mM Nif treatments were in the trichome during this period (Table 1). measured. Cuticle thickness was also observed, Calcium precipitates are common at the but not measured, in six EGTA-treated beginning of gland development when the trichomes. gland initial begins to protrude above the The abundance of the calcium precipitates epidermal cells. Deposits are abundant in the in peltate trichomes was determined by cell wall, common in the nucleus, and on the calculating the number of the precipitates per tonoplast and plasma membrane, and uncom- mm2 on each micrograph. Six peltate glandular mon in the mitochondria, cytoplasmic matrix, trichomes were examined at each stage to and plastids (Table 1; Fig. 1A, black dots). At determine calcium abundance (Table 1). the two-celled stage, calcium precipitates decrease in the cell wall, and decrease slightly SCANNING ELECTRON MICROSCOPY. Whole in the plastids, mitochondria and on the leaves (1–5 mm) were selected from all four tonoplast, while remaining relatively constant treatments (light-grown control, dark-grown elsewhere (Fig. 1B). At the three-celled stage, control, 100 mM Nif, and 5 mM EGTA) and calcium precipitates decrease on the plasma fixed in 2% glutaraldehyde in 0.1 M sodium membrane, and decrease slightly on the phosphate buffer (pH 7.2) for 4 h at 4uC. After tonoplast and in the plastids (Fig. 1C). washing in the same buffer, the material was Though the frequency of precipitates is low dehydrated in a graded ethanol series, trans- on the plasma membrane and in the plastids, ferred to amyl acetate and dried in an Emitech mitochondria, and cytoplasm at the tree-cell K850 critical point driver (Machado et al. stage, the precipitates are larger than those 2006). Samples were mounted on stubs, coated observed at earlier stages (Fig. 1C). with gold in a Polaron E5000 sputter coater (Eat-Stgrinftead, UK), and examined with a CALCIUM DISTRIBUTION DURING PELTATE JSM-6360 scanning electron microscope at GLANDULAR TRICHOME DEVELOPMENT. Although 15 kV. we investigated both capitate and peltate Gland density was determined from SEM trichomes, precipitate abundances were not micrographs of abaxial surfaces of whole determined for the capitate trichomes as TEM leaves. Care was taken to measure gland observation suggested that precipitate abun- density at the same stages (1, 2, 3, and 4 mm) dances were similar to those found in peltate in all treatments, as density varies at different trichomes. We report precipitate abundances stages. Three specimens from each treatment, only for peltate trichomes (Table 1). and two leaves per specimen were analyzed. At the presecretory stage, calcium distribu- All of the glands on a leaf were counted. tion in the apical cells (AC) is similar to that at the early presecretory stage, except for on the Results. CONFIRMATION OF PRECIPITATES AS plasma membrane where the number of CALCIUM ANTIMONITE. Omission of potassium precipitates increases significantly, and on the antimonate from the processing solutions tonoplast where there are slightly more resulted in no precipitates, confirming the precipitates (Figs. 2A, B). Calcium precipi- necessity of this chemical for their formation. tates are commonly observed on the cell walls, 2010] UN TA. ACU NTETIHMSOF TRICHOMES THE IN CALCIUM AL.: ET HUANG

Table 1. Calcium distribution during the development of peltate glandula r trichomes in light-grown plants. Mean 6 SE, with assessments of relative abundance: + 5 1–19 precipitates/ mm2 (uncommon); ++ 5 20–39 precipitates/ mm2 (common); +++ 5 40–59 precipitates/ mm2 (abundant); ++++ 5 60 or more precipitates/ mm2 (very abundant). Six glandular trichomes were examined at each stage. Averages of all cells are reported at the early presecretory stage. Averages for the specific cell type are reported at other stages. AC 5 apical cells; BC 5 basal cell; SC 5 stalk cell; SCS 5 subcuticular space.

Early presecretory stage a Presecretory stage Secretory stage Post-secretory stage Iinitial cell Two cells Three cells AC SC BC AC SC BC AC SC BC Cell wall 50 6 4316 2286 4296 4336 496 2526 466 166 1336 4306 3286 3 ++++++++++++ +++++ ++++++ Plasma 30 6 2356 376 1306 4306 476 1286 986 10 306 2306 3246 2 membrane +++++++++++++ ++++++ Cytoplasm 11 6 1116 286 1106 266 166 1000 86 176 046 1 ++++++ + ++ Nucleus 32 6 3336 3326 3286 30 0 000 306 366 166 1 ++ ++ ++ ++ ++ + + Tonoplast 33 6 3266 2226 1276 2116 1106 1000296 496 196 1

++ ++ ++ ++ + + ++ + + PINNATA LAVANDULA Mitochondria 4 6 126 026 026 026 026 0000246 246 046 1 ++++++ ++++ Plastid 6 6 146 026 026 026 00 000 216 146 156 1 +++++ ++++ SCS not not not not not not not 114 6 7 not 000 applicable applicable applicable applicable applicable applicable applicable ++++ applicable

a It is not possible to distinguish between peltate and capitate glandular t richomes a the early presecretory stages. L. 5 6 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.137

FIG. 1. TEM micrographs of light-grown (control) glandular trichomes during the early presecretory stages. It is not possible to distinguish between capitate and peltate glands at these stages. A. The initial cell of a glandular trichome. Calcium precipitates (dark spots, arrows) appear on the cell wall, plasma membrane, vacuole (V) membrane, and in the cytoplasm, nucleus, proplastids (P) and mitochondria (white arrowheads). N: nucleus; Nu: nucleolus; scale 5 1 mm. B. Two-celled stage of a glandular trichome with calcium precipitates (arrows). M: mitochondria; Nu: nucleolus; P: proplastids; V: vacuole; scale 5 2 mm. C. Three-celled stage of a peltate glandular trichome with many large calcium precipitates (arrows) on the cell walls and in the organelles. N: nucleus; P: proplastids; V: vacuole; AC: apical cell; BC: basal cell; SC: stalk cell; scale 5 5 mm. plasma membrane, tonoplast, and in the cell walls of the stalk and basal cells. nucleus, and uncommonly in the cytoplasm, Precipitates remain common on the plasma mitochondria and plastids (Table 1; Fig. 2B). membrane of the apical cells, while they Calcium distribution in the stalk cells (SC) decrease on the plasma membrane of the stalk show this same general pattern (Fig. 2C), cell, and disappear from all other regions of all except that precipitates are less frequently three cells (Fig. 2G). found in the cytoplasm and on the tonoplasts, During the post-secretory stage, as the and are absent from the nucleus. A few organelles begin to degenerate, calcium pre- precipitates are found in, or adjacent to, the cipitates reappear in the cytoplasm and plasmodesmata that link the apical and stalk organelles of all cells, though they are most cells (Fig. 2D). There are fewer precipitates in abundant in the apical cells (Fig. 2H). Cal- the basal cell than in either the apical or stalk cium distributions in the stalk and basal cells cells (Fig. 2C). These differences are especially are similar to each other, with higher concen- apparent in the number of precipitates on the trations in the cell wall and on the plasma cell wall, and plasma membrane, and to a membrane, and lower concentrations on the lesser extent in the plastids. As in the stalk cell, other organelles. Calcium precipitates are no precipitates occur in the nucleus of the absent from the SCS at this stage (Fig. 2H). basal cell. The numbers of precipitates in the cytoplasm, mitochondria and on the tonoplast GLANDULAR TRICHOME DENSITY FOLLOWING are the same in the stalk and basal cells at the NIF AND EGTA TREATMENTS. Nif and EGTA- presecretory stage. treated plants show similar growth and ultra- As secretion begins, the cuticle separates structural defects in their trichomes. The from the cell wall to form the subcuticular effects become worse with increasing concen- space (SCS), and many calcium precipitates tration of these chemicals. Symptoms include appear in the SCS adjacent to the cell walls dry leaf margins, buds with tip burn, the (Fig. 2E). As the SCS fills, more and more cessation of apical growth, and decreased calcium precipitates appear in the SCS glandular trichome density. (Fig. 2F). During the secretory stage the There is little difference in trichome density number of precipitates increases in the cell between light and dark-grown seedlings wall of the apical cells, while decreasing in the (Fig. 3). However, gland density is reduced 2010] HUANG ET AL.: CALCIUM IN THE TRICHOMES OF LAVANDULA PINNATA L. 7 following treatment with Nif and EGTA. normal mean thickness is 200 nm (SE 5 19.91) When [Nif] is between 1–10 mM there appears (Fig. 4F). to be little initial influence on trichome density. However, after the leaves enlarge Discussion. The insoluble precipitates of trichome density of the Nif treated plants is calcium antimonate used in this study facil- less than the controls, suggesting a negative itate visualization of loosely bound calcium effect of Nif on cell division or enlargement of stores that may, under specific conditions, be the trichome cells. With a [Nif] of 100 mM, mobilized and serve as a source of free calcium gland density again decreases, and is less than (Bush 1995, Tian et al. 2000). During the early half that of the controls at leaf maturity stages of gland development, it is likely that (Fig. 3). Treatment with EGTA also results the calcium so visualized is attached to in reduced trichome density. These results are membrane-bound binding sites, and is not free 2+ consistent with the idea that Nif interferes with cytosolic calcium ([Ca ]c). Calcium-binding trichome development, but not with trichome proteins have been shown to be sensitive to initiation. environmental signals and developmental cues and, following the production of specific CHANGES IN ULTRASTRUCTURE AND CALCIUM enzymes, may release free calcium in propor- DISTRIBUTION FOLLOWING TREATMENT WITH tion to the local stores of loosely bound NIF AND EGTA. Experiments with dark- calcium (Weisenseel and Jaffe 1976, Tian et grown control shoots (no Nif or EGTA) show al. 2000). Electrically-gated Ca2+ channels, no effect on calcium distribution in either type which have been described from the tonoplasts of trichome. For example, the precipitate of several species, may further contribute to count for the apical cell of light-grown the release of free calcium (Allen and Sanders secretory stage peltate trichomes is 52 (SE 5 1997, Tian et al. 2000). For these reasons, it is 4, N 5 6), while the same count for dark- likely that the loosely bound calcium visua- grown peltate trichomes is 54 (SE 5 5, N 5 6) lized with potassium antimonate can be read- (Table 2). ily converted into free calcium. The effects of Nif and EGTA treatment are Bush (1995) reviewed the role of Ca2+ pumps similar in both types of trichomes. Following in signaling processes. The pumps act to treatment, the cytoplasmic density of the cells replenish intracellular calcium stores for chan- decreases at all stages (Fig. 4A, B). At the nel-mediated Ca2+ release during signal trans- early presecretory stage almost no calcium duction, supply Ca2+ for biochemical processes precipitates are observed in the gland initials in organelles, maintain low [Ca2+]inthe (Fig. 4A). In a striking difference from un- cytoplasm, furnish Ca2+ for membrane inter- treated glands, parts of the cuticular layer actions such as vesicle transport, membrane separate from the cell wall at the presecretory fusion, and secretion, and can account for stage to form an abnormal SCS (Fig. 4B, spatiotemporal patterns of Ca2+ distribution. arrow). At the secretory stage, abnormal assemblages of vesicles are found adjacent to CALCIUM DISTRIBUTION DURING GLANDULAR the plasma membrane, and may be in the TRICHOME DEVELOPMENT. Glandular trichome process of fusing with it (Fig. 4C). Calcium development in Lavandula pinnata is charac- precipitates are also almost completely absent terized by spatial and temporal changes in from all of the cells at this stage, and from the calcium levels throughout development. In the SCS, which is nearly empty of secretory early stages of gland development many product (Fig. 4D). Large numbers of mito- calcium-induced precipitates are found in the chondria also appear near the plasma mem- initial cells, especially on the cell walls, plasma brane. Finally, at the post-secretory stage, a membrane, tonoplast, and in the nucleus. At few calcium precipitates appear on the mem- the presecretory stage, calcium tends to be branes of the degenerating plastids (Fig. 4E), concentrated in the apical cells, and on the cell the cell walls acquire a lax fibrillar appearance wall and plasma membrane of the stalk cell. (Fig. 4E, arrows), and the cuticular layer is As the trichomes approach maturity and begin thinner than in the controls (Fig. 4F). The to secrete, calcium becomes very abundant in average cuticle thickness is 78 nm (SE 5 8.45) the SCS, abundant in the adjacent cell walls, following Nif treatment, and 97 nm (SE5 and remains common on the plasma mem- 10.36) following EGTA treatment, whereas its brane. It is depleted elsewhere, and almost 8 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.137

FIG. 2. TEM micrographs of light-grown (control) peltate glandular trichomes. A. Presecretory stage, showing calcium distribution (black dots) similar to the early presecretory stage. AC: apical cell; BC: basal cell; N: nucleus; SC: stalk cell; V: vacuole; scale 5 2 mm. B. Enlarged image of the peripheral cytoplasm of two apical cells. Calcium precipitates (arrows) are located on the tonoplast, plasma membrane, plastid (P) membrane, and in the mitochondria (M). CW: cell wall; V: vacuole; scale 5 200 nm. C. Junction of the stalk (SC) and basal cells (BC) at the presecretory stage. Large calcium precipitates (white arrows) are located on the walls of the stalk and basal cells. CW: cell wall; V: vacuole; scale 5 200 nm. D. Calcium precipitates (arrows) in the plasmodesmata (arrowheads) between an apical disk cell (AC) and a stalk cell (SC), at the presecretory stage. CW: cell wall; scale 5 100 nm. E. Secretory stage. Calcium precipitates (arrows) appear in the subcuticular space (SCS) as the gland begins to secrete. C: cuticle; CW: cell wall; scale 5 200 nm. F. Secretory stage. Calcium precipitates (arrows) occur on the plasma membrane, cell wall (CW), and are 2010] HUANG ET AL.: CALCIUM IN THE TRICHOMES OF LAVANDULA PINNATA L. 9

FIG. 3. Mean gland density (mean 6 SE) on the abaxial sides of leaves grown under different treatments (light-grown control, dark-grown control, 10 mMNif,100mM Nif, and EGTA). completely disappears from the stalk and basal lead to a loosening of the fibrillar structure of cells. Finally, at the post-secretory stage, the the cell wall. calcium disappears from the SCS, is irregularly distributed in the cytoplasm of the apical cells, THE ROLE OF CALCIUM IN SECRETION. A good and is common on the periphery of the deal of research has been done on trichome organelles of these cells. This pattern suggests structure in relation to the secretory process in a role for calcium in gland development, order to elucidate the secretory mechanism secretion, and programmed cell death. The (Bosabalidis and Tsekos 1982, Bourett et al. results of the experiments with the calcium 1994, Figueiredo and Pais 1994, Ascensaõ et channel blocker Nif and the calcium chelator al. 1997, Gersbach 2002). This research has EGTA support these conclusions. shown that both the plastids (Wooding and Most of the Ca2+ absorbed by plants is Northcote 1965, Akers et al. 1978), and the found in the cell wall and vacuole (Gilroy et al. endoplasmic reticulum (ER) (Schnepf 1972, 1993, Tian and Russell 1997, Tian et al. 1998). Bourett et al. 1994, Ascensaõ et al. 1997, Although the nature of the interaction be- Turner et al. 2000) participate in the produc- tween Ca2+ and the cell wall is poorly under- tion of the essential oil. stood, a role for Ca2+ in strengthening the wall Exocytosis has been suggested as the by cross-linking the carboxyl groups of the mechanism for transport of the protein-poly- pectic polymers has been proposed (Cleland et saccharide component of the essential oil from al. 1990). In agreement with these findings, we the cell to the subcuticular space in secretory found that both Nif and EGTA treatments trichomes (Ascensaõ and Pais 1998). The r especially abundant in the subcuticular space (SCS) of the apical cells (AC). V: vacuole; scale 5 1 mm. G. Apical cells at the secretory stage. Many calcium precipitates (arrowheads) on the plasma membrane and cell wall (CW), but few in the vacuoles (V) and cytoplasm. P: plastid; scale 5 500 nm. H. Two apical cells at the post-secretory stage. Calcium precipitates (arrowheads) reappear in the cytoplasm and coat the degenerating mitochondria (M) and plastids (P), while they are absent from the subcuticular space (SCS). Scale 5 200 nm. 10 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.137

Table 2. Calcium distribution at the secretory stage of light and dark-grown peltate glandular trichomes. mean 6 SE.

Light-grown Dark-grown AC SC BC AC SC BC Cell wall 52 6 466 166 1546 586 166 1 Plasma Membrane 28 6 486 10 316 396 20 Cytoplasm 0 0 0 0 0 0 Nucleus 0 0 0 0 0 0 Vacuole 0 0 0 0 0 0 Mitochondria 0 0 0 0 0 0 Plastid 0 0 0 0 0 0 SCS 0 114 6 70 0 1106 12 0 terpenoid component may be secreted directly The occurrence of multivesicular bodies from the plastids through the plasma mem- near the plasma membrane of treated cells, brane (Kim and Mahlberg 2003, Turner and suggests that the reduction in secretory prod- Croteau 2004). uct may be due to a disruption of exocytosis. At the secretory stage in untreated plants, Huang et al. (2008) describe the exocytosis of the greatest number of calcium precipitates lipophilic vesicles into the periplasmic space appear in the SCS adjacent to the cell walls. during essential oil secretion of Lavandula Free calcium presumably travels through the pinnata. The presence of the multivesicular stalk and basal cells to the apical cells via the bodies reported here suggests that Nif and plasmodesmata (Robards and Lucas 1990) EGTA may interfere with this secretion until it reaches the SCS and adjacent cell walls. process by interfering with exocytosis. How- This distribution suggests that calcium med- ever there is only one other (rather weak) iates the secretory process. Over two decades report of lipophilic (essential oil?) secretion ago Hepler and Wayne (1985) hypothesized occurring by exocytosis (Gersbach 2002), and that cytosolic free calcium was an obligate multivesicular bodies have also been identified intracellular messenger coordinating cellular as prevacuolar compartments, not secretory responses to numerous developmental cues. vesicles, in tobacco (Tse et al. 2004). These The hypothesis that each developmental cue or facts weaken the supposition that the multi- environmental challenge produces a unique vesicular bodies may be abnormal secretory 2+ [Ca ] c signature, which elicits specific cellular vesicles. Moreover, recycling of membrane responses, was tested and supported by Knight materials through endocytosis has been sug- et al. (1991), and Allen et al. (1999). It seems gested as a stress response that is protective possible that the appearance of calcium pre- against cell death (Levine 2002), and multi- cipitates in the SCS beside the cell wall is vesicular bodies have been shown to be one indicative of such a signature for secretion. way in which this recycling takes place (An et The fact that the amount of secretion was al. 2006). In support of the hypothesis that the reduced by both Nif and EGTA suggests that multivesicular bodies are abnormal secretory calcium plays a role in stimulating secretion in vessels is the fact that those reported here Lavandula pinnata. That we found the same appear to be aggregates of closely appressed result with both calcium antagonists strength- vesicles rather than a system of small vesicles ens this conclusion. Nifedipine regulates enclosed in a single larger one (Levine 2002, plasma membrane Ca2+ channels in excitable Tse et al. 2004, An et al. 2006), and the fact cells, and can inhibit Ca2+ release from that An et al. (2006) could not rule out the intracellular stores (Rosales and Brown possibility that some multivesicular bodies are 1992), while EGTA is a calcium chelator that secretory. binds intercellular calcium (Williams 1970). The role of Ca2+ in carbohydrate exocytosis The reduction of secretion by two compounds has been studied in several systems. A high tip- that act in different ways suggests that the focused [Ca2+] gradient in pollen tubes and effect is due to the interference of these root hairs has been shown to play a role in compounds with calcium metabolism, and is exocytosis (Malho´ and Trewavas 1996, Wy- not a toxic effect of the chemicals. mer et al. 1997, Campanoni and Blatt 2007). 2010] HUANG ET AL.: CALCIUM IN THE TRICHOMES OF LAVANDULA PINNATA L. 11

FIG. 4. TEM micrographs of calcium distribution in peltate glandular trichomes treated with 100 mMNif. A. Early presecretory stage. The initial cell of a glandular trichome with few organelles, decreased cytoplasmic density, and no calcium precipitates. N: nucleus; V: vacuole; scale 5 1 mm. B. Early presecretory, three-celled stage. Abnormal separation of the cuticular layer from the cell wall to form an abnormal subcuticular space (SCS, arrow). Scale 5 2 mm. C. Apical cells at the secretory stage. Anomalous compound vesicles in the vicinity of the plasma membrane. Scale 5 200 nm. D. Apical cell at the secretory stage. Little secretory product occurs in the subcuticular space (SCS, arrowheads), and many mitochondria (M) appear near the plasma membrane. The RER (arrow) has a few ribosomes. CW: cell wall; scale 5 200 nm. E. Post-secretory stage, apical cells. Calcium precipitates (arrows) occur on the outer membrane of the disorganized plastids (P), while the cell wall (CW) has a lax fibrillar appearance. Scale 5 200 nm. F. Comparison of the cuticular layer under three treatments 1) light-grown control, 2) dark-grown control, and 3) 100 mM Nif. C: cuticle; scales 5 100 nm. 12 JOURNAL OF THE TORREY BOTANICAL SOCIETY [VOL.137

Ca2+ is also involved in the regulation of PROGRAMMED CELL DEATH. Calcium is an vesicle fusion in pollen tubes (Hepler et al. important molecule in signaling programmed 1994, Pierson et al. 1996, Carol et al. 1997, cell death (Berridge et al. 1998, Santelia and Monteiro et al. 2005). Lin and Yang (1997) Carafou 1997, Snedden and Fromm 2001, speculated that Rop GTPases might mediate a Medvedev 2005, Ma and Berkowitz 2007), and Ca2+-dependent pathway that leads to exocy- may play a role in the necrosis of the tosis. They suggested that Rop GTPases may trichomes. Its role in programmed cell death carry out this function by regulating the has led some authors to suggest that certain activity of Ca2+-dependent annexins. More patterns of calcium distribution may be recently, it was found that increases in GTP associated with cell death. For instance, in a levels can promote exocytosis (and growth), study of microsporogenesis in male sterile 2+ but do not significantly affect [Ca ]c distribu- Chinese cabbage, Xie et al. (2005b) suggested tion (Camaco and Malho´ 2003). This finding that the high occurrence of calcium precipi- suggests that exocytosis is regulated both by a tates in the tapetum at the binucleate stage 2+ [Ca ]c gradient and the activity of GTPases. signals the beginning of programmed cell Rop GTPases are likely candidates for the death. The appearance of calcium precipitates mediation of Ca2+/GTP crosstalk as shown by on the organelle membranes during the post- knock-out experiments in growing pollen secretory stage may also be associated with tubes (Camaco and Malho´ 2003). programmed cell death. Additional support for a role for calcium in Lavandula pinnata secretion, and possibly in Conclusions. Essential oil secretion in La- exocytosis, comes from TEM observations vandula pinnata is calcium dependent. Changes that reveal that the location of calcium in calcium distribution during development precipitates in the SCS of L. pinnata is similar suggest that calcium also has a role in gland to calcium distribution in actively growing development, and programmed cell death. pollen tubes (Xie et al. 2005a). Removal of calcium with Nif or EGTA results in a reduction in gland density on the abaxial CUTICLE FORMATION. In addition to a surface of leaves, cells with decreased cyto- reduction in secretory product, treatment with plasmic density early in development, forma- either Nif or EGTA leads to a lax cell wall, tion of a lax and fibrillar cell wall, abnormal and a thin cuticle. The thin cuticle indicates formation of the subcuticular space, thinning that the transportation path of the cuticular of the cuticle, reduced secretion, and the material has been disturbed. Since this path- presence of multivesicular bodies near the way likely involves the direct secretion of plasma membrane. The concentration of cuticular material through the plasma membrane to the apoplast (Mahlberg and Kim calcium in the subcuticular space and adjacent 1991, Kim and Mahlberg 1995, Samuels et al. cell wall during the secretory process may be a 2008), we can deduce a relationship between signature for secretion. calcium and these processes (Roy et al. 1999). Literature Cited It is possible that calcium regulates the function of CER5 and/or WBC11, two ATP AKERS, C. P., J. A. WEYBREW, AND R. C. LONG. binding cassette transporters that have been 1978. Ultrastructure of glandular trichomes of leaves of Nicotiana tabacum L. Am. J. Bot. 65: implicated in wax export (Pighin et al. 2004, 282–292. Bird et al. 2007, Samuels et al. 2008). ALLEN,G.J.AND D. SANDERS. 1997. Vacuolar ion It has been suggested that during secretion channels of higher plants. Adv. Bot. Res. 25: cuticle-wall separation occurs at a predeter- 217–252. mined zone of weakness (Bourett et al. 1994, ALLEN, G. J., J. M. KWAK,S.P.CHU,J.LIOPIS,R.Y. TSIEN,J.F.HARPER, AND J. I. SCHROEDER. 1999. Ascensa˜o et al. 1997). In Lavandula pinnata Cameleon calcium indicator reports cytoplasmic both Nif and EGTA treatments lead to an calcium dynamics in Arabidopsis guard cells. abnormal cuticle-wall separation, and early Plant J. 19: 735–747. SCS formation. It is possible that the changes AN, Q., R. HUCKELHOVEN, K.-H. KOGEL, AND A. J. in calcium metabolism caused by these treat- E. VAN BEL. 2006. Multivesicular bodies participate in a cell wall-associated defense response in ments weaken the junction between the cuticle barley leaves attacked by the pathogenic pow- and the wall, which induces early cuticle-wall dery mildew fungus. Cell. Microbio. 8: separation. 1009–1019. 2010] HUANG ET AL.: CALCIUM IN THE TRICHOMES OF LAVANDULA PINNATA L. 13

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