Starch Accumulation in the Megagametophyte of ...

Geonomics, Physiology, and Structure C hap te r 17

Starch Accumulation in the Megagametophyte of Ceratozamia mexicana Brongn. and Zamia furfuracea L.f. María Ydelia Sánchez-Tinoco, José Rubén Ordóñez-Balderas, and E. Mark Engleman Abstract • 251 Resumen • 252 Introduction • 252 Materials and Methods • 254 Results • 255 Discussion • 260 Acknowledgments • 261 Literature Cited • 261

Abstract Megagametophyte maturation in gymnosperms includes nutrient storage and embryo development. In the living Cycadales starch is accumulated before fertilization. Here starch accumulation by the megagametophyte is related to cellularization, archegonia development, fertilization, and embryogeny. Megastrobili, ovules, and seeds were collected from their natural localities and i xed in FAA. Starch was detected using histochemical staining with IKI and PAS. Embryos appeared two years after megastrobilus emergence in Ceratozamia mexicana and one year after this event in Zamia furfuracea. Starch

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grains in both species were structurally similar, but there were diferences in the rate of

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Mem. New York Bot. Gard., Vol. 106 formation. This accumulation of starch was not related to embryo development. During cellularization and diferentiation of archegonia, few starch grains were observed. Later the starch grains were arranged toward the periphery of the cells and increased in size. Before dispersal, but when the archegonia were mature, starch grains were abundant.

Resumen En gimnospermas la maduración del megagametoito incluye el depósito de nutrimentos y desarrollo del embrión. En las Cycadales vivientes se acumula almidón antes de la fecundación. Aquí se analiza la acumulación de almidón en el megagametoito relacionada con celularización, desarrollo de arquegonios, fecundación, y embriogenia. Megastróbilos, óvulos, y semillas fueron recolectados en poblaciones naturales y ijados en FAA. Histoquímicamente se detectó almidón usando IKI y APS. En Ceratozamia mexicana los embriones aparecen dos años después de la emergencia del estróbilo y en Zamia furfuracea, en un año. Estructuralmente en ambas especies la acumulación de almidón fue similar, diiriendo en celeridad. La acumulación de almidón no estuvo relacionada con el desarrollo del embrión. Iniciando la celularización y diferenciación de arquegonios, aparecen pocos granos de almidón. Después, se arreglan hacia la periferia de las células y aumentan de tamaño. Previo a la dispersión, cuando los arquegonios maduran, los granos de almidón abundan.

Introduction The vegetative female gametophyte in cycads consists primarily of reserve tissue that contains abundant starch. The seeds of various species of Cycas (Cycadaceae, Cycadales) are estimated to contain starch reserves that represent approximately 25% of the gametophytic tissue (Pant, 1973) and is a high-value food source. It can sustain the embryo in its early stages and perhaps contributes to the young sporophyte which remains attached to the gametophyte for one to three years in diferent species (Sánchez-Tinoco, 1998). It can also be a food source for other organisms such as insects, fungi, and mammals (including humans) which could be dispersers or predators. Some authors have reported that cycad megagametophytes of Macrozamia communis -1—

L. Johnson and Lepidozamia hopei Regel are consumed by rodents in Australia (Jones,

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1993), and in Mexico for various species of Dioon (Jones, 1993), principally D. edule Lindl.,

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D. spinulosum Dyer & Eichler, Zamia furfuracea L.f., and Z. loddigessi Miq. (GonzálezChristen, 1990, pers. comm.; Vovides, 1990). There are also reports of attacks on the gametophyte of various species of Encephalartos by larvae of a beetle of the genus Antliarhinus. The beetle oviposits on the ovule; when it reaches its irst stages of development, eclusion occurs and the larvae eat the reserve tissue of the gametophyte and destroy it (Jones, 1993). Man also is a consumer of cycad seeds, mainly as lour prepared from gametophytes of various species in diferent parts of the world (Thieret, 1958). The consumption of cycad gametophyte starch requires the removal or denaturalization of toxins (Sánchez-Tinoco, 1998; Brenner et al., 2003). In Mexico, indigenous people and farmers consume the gametophyte of the following species: Ceratozamia robusta Miq., Dioon angustifolium Miq., D. edule Lindl. var. edule, C. microstrobila Vovides & Rees, and D. spinulosum (Vázquez-Torres et al., 1989). In Honduras the seeds of D. mejiae Standl. & L. O. Williams, known as tiusinte, are eaten in tamales, atole, and tortillas (Bonta, 2007). The consumption of the lour of all these species carries a risk of accumulative toxicity, frequently manifested as chronic degenerative disease (Vázquez-Torres et al., 1989). Brenner et al. (2003) mention the existence of neurotoxic compounds in cycads, one of which, beta-methylamineL-alanine (BMAA), could be responsible for the neurodegeneration known as dementia of Guam (Vega & Bell, 1967; Siniscalco Gigliano, 1990; Vovides, 1991; Duncan, 1993; Kurland, 1993; Norstog et al., 1993; Nunn & Ponnusamy, 1993; Brenner et al., 2003). With respect to the timing of starch deposition, Singh and Johri (1972) and Singh (1978), stated that gymnosperm seeds reach maturity when the embryo is structurally complete, the food reserves are adequate, and the seed coat is completely developed. Singh (1978) also highlighted the deposition of nutrients in the gametophyte, stating that during the maturation of the female gametophyte in gymnosperms the storage of reserve nutrients causes a visible microscopic change. Favre-Duchartre (1958) noted that generally the deposition of food reserves in the seed coincides with embryo development, but in Ginkgo biloba L. and the cycads, food reserves are formed in the gametophyte before they are formed in the embryo. Sánchez-Tinoco et al. (2000) reported that ligniication of the seed coat in cycads coincides with cellularization of the gametophyte. This ligniication is completed when the archegonia are formed, starch has accumulated in the gametophyte, and the gametophyte hardens. Thus, nutrients are stored in the gametophyte before dispersal (Sánchez-

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Tinoco et al., 2000).

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Mem. New York Bot. Gard., Vol. 106 The present investigation describes the accumulation of starch in the vegetative megagametophyte of Ceratozamia mexicana and Zamia furfuracea, and relates it to the formation of cell walls, development of archegonia, fertilization, and embryo development.

Materials and Methods Megastrobili, ovules, and seeds were collected in two natural populations of Ceratozamia mexicana. This plant grows in mesophyll mountain forest on “Coacoatzintla” hill, municipality of Coacoatzintla, 13 km due north of Xalapa, Veracruz, México. Zamia furfuracea from coastal sand dune vegetation was located at Ciénega del Sur, municipality of Alvarado, Veracruz, México. For both species specimens were collected at intervals of four to 12 weeks, from the initiation of the ovules until germination (Tables 17-1, 17-2). For both species, female strobili in the process of disintegration and seed dissemination were collected at the time of dispersal. The seeds were sown in a garden in Xalapa, Veracruz. Ceratozamia mexicana was sampled 11 times monthly and Z. furfuracea, three times.

Table 17-1 Collecting time, length, characterization, and development of the ovule and seed of Ceratozamia mexicana. Date and Time Elapsed

Characterization of the Ovule and Seed

Ovule and Seed Development

Sep- 01-94 to Feb-12-95 5 months

Ovules from 0.5 to 12.0 mm. From initiation of the ovules (emergence of the megastrobilus) to closed megastrobilus Ovules from 12.0 to 36.0 mm. From compact megastrobilus, to separated megasporophylls, to disintegration of megastrobilus Seeds planted in a garden

Coenocytic gametophyte

Embryo development

Growth of the suspensor

Embryo development

Development of the body of the embryo Germination

Feb-25-95 to Sep- 01-95 7 months

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Oct- 07-95 1 month Nov-10-95 to Mar- 02-96 4 months Apr-20-96 to Jul-21-96 3 months Aug- 06-96 to Sep- 09-96 1 month

Germination

Diferentiation of archegonia

Mature archegonia

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Table 17-2 Collecting time, length, characterization, and development of the ovule and seed of Zamia furfuracea. Date and Time Elapsed

Characterization of the Ovule and Seed

Ovule and Seed Development

Jun-30- 04 to Aug- 07- 04 2 months

Ovules from 0.5 to 16.0 mm From initiation of the ovules (emergence of the megastrobilus) to closed megastrobilus Ovules from 9.0 to 24.0 mm

Coenocytic gametophyte

Sep- 04- 04 to Sep-29- 04 1 month Oct- 07-98 1 month Nov-13- 04 to Dec-17- 04 1 month Jan-18- 05 1 month Apr-11- 05 to May-11- 05 1 month

Separated megasporophylls Ovules from 16.0 to 25.0 mm Separated megasporophylls Seeds from 18.0 to 28.0 mm Disintegration of megastrobilus Seeds planted in a garden Embryo development

Diferentiation of archegonia Mature archegonia Mature archegonia Growth of the suspensor Mature archegonia Growth of the suspensor Growth of the suspensor

Megastrobili in the earlier stages, ovules, and seeds from diferent plants were dissected and ixed in FAA or CRAF III (Sass, 1958). Free-hand or hand microtome sections were made. Starch was detected with IKI or periodic acid followed by Schif reagent (Locquin & Langeron, 1985). The sections were observed with a light microscope.

Results The time between the initiation of an ovule and the appearance of its embryo was two years for Ceratozamia mexicana and one year for Zamia furfuracea (Figs. 17-1, 17-2). The accumulation of starch grains was similar in both species, but faster in Z. furfuracea. The gametophyte of Ceratozamia mexicana is coenocytic, undergoing free nuclear division from September to February. In February, ive and a half months after megastrobilus emergence, cellularization is active and lasts until May. In May, the gametophyte becomes irm, the hard layer becomes ligniied, and the trichomes of the sarcotesta disappear. The archegonia diferentiate during the following February, and pollen tubes are

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present by June. Seed dispersal occurs in September and October (12 months after the

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Figure 17-1. Starch accumulation in the megagametophyte in relation to the reproductive cycle in Ceratozamia mexicana.

emergence of megastrobili) when the seeds have archegonia or, in some cases, suspensors in their early stages (Fig. 17-1). The suspensor grows for ive months. At the beginning of March, the suspensor occupies a cavity that extends over one-third of the length of the gametophyte. The body of the embryo continues developing during the following four months, and during the months of August and September the seed germinates (24 months after initial megastrobilus emergence) (Fig. 17-1). In Zamia furfuracea the gametophyte is coenocytic from June through September. In September, three and a half months after initial megastrobilus emergence, cellularization is -1—

initiated and lasts for approximately three weeks. In September the gametophyte becomes

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hard and the primordia of archegonia are visible at the beginning of September, four

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Figure 17-2. Starch accumulation in the megagametophyte in relation to the reproductive cycle in Zamia furfuracea.

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Mem. New York Bot. Gard., Vol. 106 months after the emergence of the megastrobilus (Fig. 17-3); in October the archegonia are mature. The hard layer becomes ligniied in November and seed dispersal occurs in December (seven months after the emergence of the megastrobilus), when the seeds develop archegonia and, in some cases, suspensors in their i rst stages of development. Embryo development continues during the following months, and in May, 12 months after megastrobilus emergence, it is assumed that germination occurs (Fig. 17-2). The megagametophytes of these two species go through three stages before reaching archegonium formation. The i rst is the free-nuclear, coenocytic stage that occurs between September and February in Ceratozamia mexicana and, in ZaFigure 17-3. Thick longitudinal section of young seed of Zamia furfuracea in September. The oval object at the micropylar end of the gametophyte may be an archegonium.

mia furfuracea, between June and September. The second stage is cell wall formation that, in C. mexicana, occurs between February and May, and, in Z. furfuracea, lasts

for 25 days in September. The third stage is the accumulation of nutrients (starch grains, proteins, and fats). In C. mexicana this stage begins in May and ends in September; in Z. furfuracea it begins at the end of September and ends in December (Figs. 17-1, 17-2). In Ceratozamia mexicana starch grains are present in all the cells of the female gametophyte. During early May, they are few and small (Fig. 17- 4a) and by June they congregate in the periphery of the cell and increase in size, on average mea sur ing 9 μm in diameter (Fig. 17- 4b). Finally, during the months from July to September they i ll all of the cells (Fig. 17- 4c). The smallest grains are found in the supericial cells. The cells of -1—

the vegetative gametophyte are i lled with starch by September, ive months after starch

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accumulation begins (Fig. 17-1).

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Figure 17- 4. Longitudinal sections of the gametophyte of Ceratozamia mexicana at three stages. Stained with iodine. a. Parenchyma cells with few starch grains (May). b. Peripheral arrangement of starch grains in parenchyma cells ( June). c. Parenchyma cells i lled with starch grains ( July to September). Scale bars = 20 μm.

Figure 17-5. Longitudinal sections of the gametophyte of Zamia furfuracea at four stages. Stained with iodine. a. Beginning of cellularization of the gametophyte (September). b. Peripheral arrangement of starch grains in parenchyma cells (October). c. Parenchyma cells full of starch grains (December). d. Cells 75% full of starch grains in the i rst stages of embryo development (November). Scale bars = 40 μm.

In Zamia furfuracea, the initiation of starch grains occurs at the end of September (Fig. 17-5a); in October they increase in number and accumulate at the periphery of the cell without increasing in size (Fig. 17-5b); by November they occupy almost the whole cell lumen and are larger, mea sur ing 16 μm in diameter (Fig. 17-5d). From December to May the starch grains i ll the whole cell lumen with mostly large grains. The small starch grains (5 μm) are localized in the supericial cells. The megagametophyte cells are full of

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starch in December after two months of accumulation (Fig. 17-5c).

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Mem. New York Bot. Gard., Vol. 106 In both species the accumulation of starch was not related to the development of the embryo since this occurred at the archegonial stage. When cellularization and differentiation of the archegonia occurred, the starch grains appeared and congregated at the periphery of the cells. At the time of dispersal, when the archegonia were fully grown, starch grains occupied the whole cell lumen (Figs. 17-1, 17-2).

Discussion In Ceratozamia mexicana and Zamia furfuracea, it is conirmed that, prior to the presence of the embryo and usually of archegonia, the seed invests in a supply of nutrients and secondary metabolites for protection. The maximum accumulation of nutrients occurs before dispersal. Surely the stage of dispersal is a determining event in the ontogeny of the seeds of the species studied. The relationship between maximum accumulation of nutrients and dispersal (September for C. mexicana and December for Z. furfuracea) could have two possible explanations: (1) causal: the supply of nutrients can only occur at this moment and not before since the plant has been investing its energy and material resources in the construction and development of structures for the support and protection of the reproductive structures (megastrobilus, megasporophylls, seed coat, etc.; (2) consequential: the seed should contain the necessary nutrients for post-dispersal embryo support. In the cellular structure of the mature gametophyte of Ceratozamia mexicana and Zamia furfuracea there are two regions: (1) the peripheral zone with strata of cells rich in lipids, proteins, tannins, and small starch grains; (2) the internal zone with abundant cells perpendicular to the surface which are i lled with large starch grains. This arrangement is similar to the zonation described by Dexheimer (1973, cited by Singh, 1978). He identiied three zones in the mature gametophyte of Ginkgo biloba: the peripheral zone which contains lipids, the central zone which contains lipoproteins, and the internal zone which contains starch in the deeper layers. In Zamia furfuracea but not in Ceratozamia mexicana, there is a pause in starch accumulation before starch grains completely i ll the whole cell. At this stage, which occurred in November, starch grains occupied 75% of the cell lumen, coinciding with the growth of the suspensor (Fig. 17-5d). -1—

Finally, an important i nding of this investigation was that, despite the presence of

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a cuticle, the megagametophyte has no epidermis. The cells of the layer immediately

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interior to the cuticle are called the “supericial cells” (Sánchez-Tinoco, 1998). They differ markedly in size from the other, even more internal cells and they have a pattern of cell division that is diferent from that seen in a typical epidermis.

Acknowledgments The authors acknowledge the assistance of Erika Rosalba Olmedo Vicente, Geraldine Engleman, and Dr. Stephen D. Koch.

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