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Effects of Ambient Calcium Concentration on the Deposition of Calcium Oxalate Crystals in Antithamnion (Ceramiales, Rhodophyta)

July 2007
Phycologia 46(4):371-379

DOI:10.2216/06-74.1

Authors:

Curt Pueschel

Binghamton University

John A. West

University of Melbourne

A survey of 18 species of the Ceramiales grown in culture revealed calcium oxalate crystals in Antithamnion antillanum Børgesen, A. callocladum Itono, and A. sparsum Tokida. The needle-shaped crystals were present within the cytoplasm of cells of the indeterminate axes but not in cells of the determinate lateral branches. No such crystals were present in A. nipponicum Yamada & Inagaki. The four species of Antithamnion and three additional members of the Ceramiales that do not normally deposit calcium oxalate were grown in natural seawater culture medium in which calcium concentration was elevated by addition of 5–20 mM calcium chloride. Elevated calcium supply did not induce the deposition of calcium oxalate crystals in species that did not previously exhibit them, and it did not change the crystal size range or the cellular or subcellular localization patterns in species that normally formed crystals. At high calcium concentrations, all species ceased growth whether or not they were able to sequester calcium as an oxalate salt. In a second experiment, the four species of Antithamnion were grown in artificial seawater with reduced calcium concentrations. All appeared healthy and grew well in artificial seawater containing 2.5–10 mM CaCl 2 . In 2.5 mM CaCl 2 , A. antillanum released tetraspores that grew into thalli having abundant calcium oxalate crystals. Even in 1.0 mM CaCl 2 , A. callocladum and A. sparsum continued to grow and deposit calcium oxalate crystals of the size range typical for the species. Thalli of all four species died within 12 days if artificial seawater containing only 0.1 mM CaCl 2 was provided. Calcium oxalate crystals were present in many cells of the dead thalli, indicating that the crystals did not provide a calcium reserve that could be readily mobilized. Culturing the thalli in complete darkness for 14 weeks also did not stimulate mobilization of the crystals. The presence/absence of calcium oxalate crystals over a broad range of calcium concentrations and light intensities suggests that deposition of this mineral is a constitutive feature and thus could serve as a taxonomic character for these species. It also suggests that calcium oxalate deposition in Antithamnion does not have a role in calcium regulation.

Needle-shaped calcium oxalate crystals in the axial cells of the indeterminate branches of three species of Antithamnion. Live specimens, grown in normal culture conditions. The crystals are located in the cytoplasm, which forms a parietal layer and surrounds the nucleus (N), but they are absent from the large central vacuole (V). Polarizing (Fig. 1) and differential interference contrast microscopy (Figs 2–4). Fig. 1. Antithamnion sparsum. Birefringence of the inclusions under polarizing optics demonstrates their crystalline nature and shows their abundance in axial cells. No crystals are present in cells of the determinate lateral branches. Fig. 2. Antithamnion sparsum. Fig. 3. Antithamnion antillanum. Fig. 4. Antithamnion callocadum. Calcium oxalate crystals are smaller than in other species, but their pattern of localization is the same.

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Differential interference contrast microscopy of normal and calcium-enriched cultures. Fig. 5. Antithamnion nipponicum. Calcium oxalate crystals were not found in this species. N 5 nucleus. Fig. 6. Anotrichium tenue. Cellular inclusions (arrow) resemble the needle-like morphology of calcium oxalate crystals, but unlike calcium oxalate they dissolve in sodium hypochlorite. Figs 7, 8. Antithamnion callocadum. Seawater culture medium enriched by addition of 20 mM CaCl 2 . Large axial cells have died, and the basal cells of the determinate lateral branches have penetrated into the lumen, divided, and come into contact (arrows).

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Calcium oxalate crystals persist in Antithamnion thalli cultured in artificial seawater medium (ASW) containing reduced calcium concentrations or in darkness. Differential interference contrast microscopy. Fig. 9. Antithamnion antillanum. Thalli grown in ASW containing 2.5 mM CaCl 2 released spores, and the young sporelings began to form calcium oxalate crystals (arrows) in the axial cells. Treated in sodium hypochlorite to remove organic material. Fig. 10. Antithamnion sparsum. Thalli grown for three weeks in ASW containing only 1 mM CaCl 2 still had calcium oxalate crystals in axial cells. Treated in sodium hypochlorite. Fig. 11. Antithamnion sparsum. Starch grains are absent but calcium oxalate crystals are abundant among the chloroplasts in the parietal cytoplasm of thalli maintained in complete darkness for 14 weeks.

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Effects of ambient calcium concentration on the deposition of calcium oxalate

crystals in Antithamnion (Ceramiales, Rhodophyta)

URT

M. P

UESCHEL

AND

OHN

A. W

EST

Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY, 13902-6000, USA

School of Botany, University of Melbourne, Parkville, Victoria, 3010, Australia

C.M. P

UESCHEL AND

J.A. W

EST

. 2007. Effects of ambient calcium concentration on the deposition of calcium oxalate

crystals in Antithamnion (Ceramiales, Rhodophyta). Phycologia 46: 371–379. DOI: 10.2216/06-74.1

A survey of 18 species of the Ceramiales grown in culture revealed calcium oxalate crystals in Antithamnion antillanum

Børgesen, A. callocladum Itono, and A. sparsum Tokida. The needle-shaped crystals were present within the cytoplasm

of cells of the indeterminate axes but not in cells of the determinate lateral branches. No such crystals were present in A.

nipponicum Yamada & Inagaki. The four species of Antithamnion and three additional members of the Ceramiales that

do not normally deposit calcium oxalate were grown in natural seawater culture medium in which calcium

concentration was elevated by addition of 5–20 mM calcium chloride. Elevated calcium supply did not induce the

deposition of calcium oxalate crystals in species that did not previously exhibit them, and it did not change the crystal

size range or the cellular or subcellular localization patterns in species that normally formed crystals. At high calcium

concentrations, all species ceased growth whether or not they were able to sequester calcium as an oxalate salt. In

a second experiment, the four species of Antithamnion were grown in artificial seawater with reduced calcium

concentrations. All appeared healthy and grew well in artificial seawater containing 2.5–10 mM CaCl

.In2.5mM

CaCl

,A. antillanum released tetraspores that grew into thalli having abundant calcium oxalate crystals. Even in

1.0 mM CaCl

,A. callocladum and A. sparsum continued to grow and deposit calcium oxalate crystals of the size range

typical for the species. Thalli of all four species died within 12 days if artificial seawater containing only 0.1 mM CaCl

was provided. Calcium oxalate crystals were present in many cells of the dead thalli, indicating that the crystals did not

provide a calcium reserve that could be readily mobilized. Culturing the thalli in complete darkness for 14 weeks also did

not stimulate mobilization of the crystals. The presence/absence of calcium oxalate crystals over a broad range of

calcium concentrations and light intensities suggests that deposition of this mineral is a constitutive feature and thus

could serve as a taxonomic character for these species. It also suggests that calcium oxalate deposition in Antithamnion

does not have a role in calcium regulation.

ORDS

:Antithamnion, Calcium, Calcium oxalate, Crystals

INTRODUCTION

Calcium oxalate (CaC

) crystals are widespread

cellular inclusions in embryophytes, occurring in normal

cells and in specialized idioblast cells (Franceschi & Horner

1980; Franceschi & Nakata 2005). One of the proposed

functions of calcium oxalate crystals, especially the bundles

of needle-like raphides, is ecological, that they serve as an

antigrazer defense (Franceschi & Horner 1980). However,

other morphologies of the crystals do not have the same

potential for providing mechanical protection. Physiologi-

cal functions for the calcium oxalate deposits have also

been proposed, and some of these have found increasing

experimental support. Idioblast cells become more abun-

dant if calcium supply to the plant is increased, and

experimental evidence indicates that calcium oxalate

crystals are an inducible sink for calcium and provide the

means by which idioblasts can function in calcium de-

toxification (e.g. Kostman & Franceschi 2000; Pennisi &

McConnell 2001; Volk et al. 2002; Mazen et al. 2003).

However, the physiological function of calcium oxalate

deposits in embryophytes is not entirely settled; calcium

oxalate-deficient mutants of Medicago show no differences

from wild type in their growth and development (Nakata &

McConn 2000).

Reports of calcium oxalate crystals in the algae have been

sporadic and involve mostly green and red seaweeds. The

giant-celled green seaweeds of the Bryopsidales (Friedmann

et al. 1972; Turner & Friedmann 1974; Bo¨hm et al. 1978;

Menzel 1987) and Cladophorales s.l. (Dawes 1969; Leliaert

& Coppejans 2004, 2006) are the subjects of most of these

accounts, but calcium oxalate has also been reported in

a few species of the ubiquitous freshwater genus Spirogyra

(Pueschel 2001). Among the red algae, pyramidal calcium

oxalate crystals were reported for two species of Spyridia

(Klein 1877), and, more recently, Antithamnion kylinii

Gardner and A. defectum Kylin were shown to deposit

needle-like calcium oxalate crystals (Pueschel 1995). Crys-

tals in both species were present within cells of the

indeterminate axes, but they were absent in all cells of the

determinate lateral branches (Pueschel 1995). Cellular

specializations uniquely associated with calcium oxalate

deposition have not been reported for the algae, and

* Corresponding author (curtp@binghamton.edu).

Phycologia (2007) Volume 46 (4), 371–379 Published 5 July 2007

371

certainly no alga is known to have cells comparable to the

specialized calcium oxalate-depositing idioblast cells in

embryophytes. A noteworthy feature shared by Spirogyra

hatillensis Transeau (Pueschel 2001) and A. kylinii and A.

defectum (Pueschel 1995) is that the membrane-bounded

calcium oxalate crystals are present not within the central

vacuole as in embryophytes (Franceschi & Horner 1980;

Franceschi & Nakata 2005) but within the parietal layer of

cytoplasm.

Fundamental questions about the formation and func-

tion of calcium oxalate crystals in the algae remain

unanswered. The concentration of calcium in full-salinity

seawater (35 psu) is about 10 mM (Kennish 2000),

a concentration used experimentally to induce calcium

detoxification mechanisms in vascular plants (e.g. Mazen et

al. 2003). The present study uses a taxonomic survey and

manipulations of calcium concentrations in cultures of

selected red algae to address questions of anatomical

localization, systematic distribution, and possible functions

of calcium oxalate crystals in red algae. The specific goals of

this study were (1) to determine the presence/absence of

calcium oxalate crystals in numerous species of cultured red

algae of the order Ceramiales; (2) to test whether enriching

the calcium concentration of the culture medium can induce

crystal deposition in cultured algae that normally do not

produce calcium oxalate crystals; (3) to determine whether

the anatomical and subcellular patterns of crystal de-

position in species with crystals can be altered by increasing

or decreasing the ambient calcium concentration; (4) to

ascertain the stage of thallus development during which

crystal deposition is first evident; and (5) to determine

whether low ambient calcium concentrations or light

deprivation will induce mobilization of calcium oxalate

crystals.

The proposal that presence or absence of calcium oxalate

crystals could provide a meaningful taxonomic character in

marine algae (Leliaert & Coppejans 2004, 2006) is

considered in the light of our experimental manipulations

of calcium concentrations.

MATERIAL AND METHODS

The cultures examined and their collection data are listed in

Table 1. The cultures are maintained by J. West, University

of Melbourne, Parkville, Victoria, Australia, and are

available upon request. Nomenclature follows AlgaeBase

(http://www.algaebase.org) as of 1 September 2006. All

cultures were grown in Provasoli’s Enriched Seawater

modified according to West (2005) (10 ml enrichment per

litre natural seawater whose salinity was adjusted to 30 psu)

at 19–23uC, 5–20 mmol photons m

cool white

fluorescent lighting, 12:12 photoperiod.

Specimens were examined on a Leica DMRB microscope

with Nomarski or polarizing optics. Images were captured

using a Panasonic F250 colour video camera and recorded

on a Leitz-Pioneer Rewriteable Videodisc Recorder VDR-

V1000P (for details, Pickett-Heaps & West 1998). Bi-

refringent cellular inclusions having the properties of being

resistant to 5% sodium hypochlorite and to 5 N acetic acid

but dissolving rapidly in 1 N hydrochloric acid were

regarded as calcium oxalate crystals (Yasue 1969; Fried-

mann et al. 1972).

The four cultures of Antithamnion, plus Ceramium,

Crouania, and Haloplegma, were selected for testing the

effects of elevated calcium concentrations on calcium

oxalate deposition. They were grown for three weeks in

the same conditions as above and in the same medium

except that CaCl

was added to produce concentrations of

5, 10, and 20 mM above normal, thus approximately 1.5,

2.0, and 3.0 times typical seawater calcium concentrations.

Triplicate cultures were grown in 50–70 ml of medium in

180-ml Corning PyrexHdishes (#3140). The gross appear-

ance of the thalli was monitored, and cellular integrity and

crystal presence were examined microscopically.

To test the effects of reduced calcium concentrations on

calcium oxalate deposition and viability, the four species of

Antithamnion, three of which deposit calcium oxalate, were

grown in reduced calcium concentrations for four weeks.

To obtain reduced calcium levels, CaCl

was added to

artificial seawater (ASW) (cf. McLachlan 1973), which

contained 400 mM NaCl, 10 mM KCl, 20 mM MgSO

20 mM MgCl

, 2 mM NaHCO

, and 10 ml of PES/2

enrichment per litre of medium in Milli-QHpurified water,

to produce final calcium concentrations of 0.1, 1.0, 2.5, 5.0,

7.5, and 10 mM. To minimize culture shock and to reduce

carryover of substantial calcium with the thalli, the two

cultures with the lowest calcium concentrations were started

from specimens that had previously been growing in the

next highest concentration. Artificial seawater with no

calcium additions was also tested. The culture dishes and

environmental conditions were the same as used for the

calcium supplementation experiment, and again three

cultures were used for each set of culture conditions.

Sporelings growing from tetraspores of A. antillanum in

ASW of various calcium concentrations were monitored to

detect the first appearance of calcium oxalate crystals

during thallus development.

A culture of A. sparsum in normal enriched seawater

medium was placed in complete darkness at 19uCfor14

weeks to test the possibility that calcium oxalate would be

mobilized to serve as a carbon source.

RESULTS

A survey of 18 species (Table 1) of ceramialean red algae

grown in enriched natural seawater medium revealed

crystalline inclusions that were birefringent under polariz-

ing optics (Fig. 1) in three of the four species of

Antithamnion examined (Figs 1–4). The crystals were

needle-like, and they were present exclusively in cells of

the indeterminate axes (Fig. 1). Antithamnion sparsum

(Figs 1, 2) had abundant crystals, and most were less than

20 mm long, but some reached 30 mm in length. Crystals in

A. antillanum (Fig. 3) were up to 18 mm long. Crystals were

smallest and least abundant in A. callocladum (Fig. 4); some

were up to 10 mm long, but most were less than 5 mm long.

In all three species, the crystals were located in cytoplasmic

strands across the central vacuole, in the parietal cytoplas-

372 Phycologia, Vol. 46 (4), 2007

mic layer, and in the cytoplasm surrounding the nucleus

(Figs 2–4). These crystals had the same solubilities as those

previously identified as calcium oxalate in Antithamnion

(Pueschel 1995); that is, they were dissolved by dilute

hydrochloric acid but not by concentrated acetic acid or

sodium hypochlorite. Antithamnion nipponicum did not

have crystals (Fig. 5).

Needle-like and polygonal inclusions were found in

Anotrichium tenue (Fig. 6). Based on their morphology,

the needle-like structures were expected to be composed of

calcium oxalate, but they dissolved quickly in sodium

hypochlorite, as did the polygonal inclusions, indicating an

organic rather than a mineral composition.

Augmenting the normal calcium content of seawater

medium with CaCl

at concentrations of as high as 20 mM

did not change patterns of crystal presence in the seven

species tested: the four species of Antithamnion plus

Ceramium,Crouania,andHaloplegma (Table 1). Calcium

oxalate crystals were not induced in any species, including

A. nipponicum, that did not show them under normal

culture conditions. Antithamnion antillanum,A. callocla-

dum, and A. sparsum continued to deposit crystals only in

cells of the indeterminate axis. The size of the crystals in

even the highest calcium concentration was no different

than in the control culture for each species, and crystal

abundance in the higher calcium concentrations was the

same or even less than in the controls without calcium

enrichment.

Most of the seven species exposed to elevated calcium

concentrations grew little under the highest calcium

concentration, and the cells became filled with floridean

starch. Although quantification of growth was not per-

formed, it was plain that there was no correlation of the

ability to deposit calcium oxalate crystals and tolerance of

high calcium conditions. The least affected was A. sparsum;

cultures with high calcium did not accumulate biomass as

quickly as the control, but the thalli appeared normal. At

the other extreme, A. callocladum showed progressive, cell-

specific, calcium-induced distress: cells of the indeterminate

axes became diffusely pink as the phycobilin pigments

leaked from the chloroplasts and then green as the

phycobilins leaked from the cells, leaving only chlorophyll.

By contrast, cells of the determinate lateral branches

maintained a healthy red colour and appeared little

different in the presence of the added calcium, with the

exception of the basal cell of the branch. This cell expanded

into the lumen of the compromised subtending axial cell

(Fig. 7) and formed multicellular, descending processes

(Fig. 8). When the intruding cells of the opposite de-

terminate branches came into contact, they sometimes

fused. Even if the determinate lateral branches appeared

healthy and the thalli were transferred to normal culture

medium, the thallus did not regain its integrity. The cells of

the determinate lateral branches did not develop crystals or

show any other evidence that would suggest conversion

from a determinate axis to an indeterminate axis (Figs 7, 8).

Specimens of the four Antithamnion species were placed

in ASW without calcium, and their loss of colour made the

calcium-deficient cultures distinguishable from control

thalli within 24 hours. Within a week, the calcium-deprived

thalli were nearly colourless. Attempts to revive thalli that

had begun to exhibit pigment loss by transferring them to

normal culture medium were unsuccessful, suggesting that

cell death occurred quickly. Low calcium conditions

affected the cell walls as well as the cell contents, resulting

in the thalli being more delicate and vulnerable to damage

when handled. Consequently, mounting thalli for micro-

scopic examination may have caused premature death of

cells in which mortality was imminent but had not yet

occurred. In the three species of Antithamnion that normally

form calcium oxalate crystals, crystals were present in some

dead axial cells and were absent in others. Antithamnion

nipponicum, which does not deposit calcium oxalate

crystals, did not show symptoms of physiological stress

noticeably more quickly than did the other species.

All four species of Antithamnion appeared healthy and

continued to grow in ASW containing 2.5, 5.0, 7.5, or

10 mM CaCl

. At all these concentrations, tetraspores were

formed and released by A. antillanum. The spore body and

the first few cells of the axis of young sporelings did not

Table 1. Collection information for cultures examined for the presence of calcium oxalate crystals.

Cultures used (J. West culture collection numbers) Collection information

Aglaothamnion tenuissimum Feldmann-Mazoyer 4356 F. Kapraun, Kristineberg Biological Station, Sweden, 15 Aug. 1980

Anotrichium furcellatum (J. Agardh) Baldock 2600 J. West, San Diego Marina, California, 9 Mar. 1982

Anotrichium tenue (C. Agardh) Na¨geli 4360 J. West, Plage de Gatope, New Caledonia, 6 Sep. 2003

Antithamnion antillanum Børgesen 3498 J. West, Cooks Bay, Moorea, French Polynesia, 3 Jul. 1995

A. callocladum Itono 4293 G.H. Kim, Shimoda, Japan, May 1995

A. nipponicum Yamada & Inagaki 4402 G.H. Kim, Kachen, Korea, May 1995

A. sparsum Tokida 4035 G.H. Kim, Kangreung, Korea, Jul. 1986

Antithamnionella ternifolia (Hooker & Harvey) Lyle 3465 J. West, Williamstown, Victoria, Australia, 3 Mar. 1995

Balliella sp. 2656 S. Earle, Aride I. Seychelles, 12 May 1982

Ceramium sp. 3507 J. West, Cooks Bay, Moorea, French Polynesia, 1 Jul. 1995

Claudea batanensis Tanaka 2715 J. West, Siayan I. Batanes, Philippines, 3 Jun. 1986

Crouania attenuata (C. Agardh) J. Agardh 3508 J. West, Cooks Bay, Moorea, French Polynesia, 1 Jul. 1995

Dasya iridescens A.J.K. Millar & Abbott 2400 C. Schlech, Kahala, Oahu, Hawaii, 15 Oct. 1979

Griffithsia monilis Harvey 3445 J. West, Williamstown, Victoria, Australia 13 Feb. 1995

Haloplegma duperreyi Montagne 1521 S. Earle, Chuuk (Truk) I., Micronesia, 9 Nov. 1975

Pterothamnion yezoense (Inagaki) Athanasiadis & Kraft 4401 G.H. Kim, Cheju I., Korea, 14 Aug. 1996

Spermothamnion sp. 4349 J. West, Hienghene River mouth, New Caledonia, 8 Sep. 2003

Zellera tawallina G. Martens 2888 H. Calumpong, Biaras, Catanduanes, Philippines, 14 May 1988

Pueschel & West: Calcium oxalate crystals in Antithamnion 373

Figs. 1–4. Needle-shaped calcium oxalate crystals in the axial cells of the indeterminate branches of three species of Antithamnion. Live

specimens, grown in normal culture conditions. The crystals are located in the cytoplasm, which forms a parietal layer and surrounds the

nucleus (N), but they are absent from the large central vacuole (V). Polarizing (Fig. 1) and differential interference contrast

microscopy (Figs 2–4).

Fig. 1. Antithamnion sparsum. Birefringence of the inclusions under polarizing optics demonstrates their crystalline nature and shows their

abundance in axial cells. No crystals are present in cells of the determinate lateral branches.

Fig. 2. Antithamnion sparsum.

Fig. 3. Antithamnion antillanum.

Fig. 4. Antithamnion callocadum. Calcium oxalate crystals are smaller than in other species, but their pattern of localization is the same.

374 Phycologia, Vol. 46 (4), 2007

Figs. 5–8. Differential interference contrast microscopy of normal and calcium-enriched cultures.

Fig. 5. Antithamnion nipponicum. Calcium oxalate crystals were not found in this species. N 5nucleus.

Fig. 6. Anotrichium tenue. Cellular inclusions (arrow) resemble the needle-like morphology of calcium oxalate crystals, but unlike calcium

oxalate they dissolve in sodium hypochlorite.

Figs 7, 8. Antithamnion callocadum. Seawater culture medium enriched by addition of 20 mM CaCl

. Large axial cells have died, and the

basal cells of the determinate lateral branches have penetrated into the lumen, divided, and come into contact (arrows).

Pueschel & West: Calcium oxalate crystals in Antithamnion 375

deposit calcium oxalate, and, as is typical of more mature

thalli, the apical cell and two or three expanding cells

subtending it did not have crystals. However, by the time

five axial cells were produced, calcium oxalate crystals were

visible in some of the older of these cells. Even in thalli

grown in 2.5 mM CaCl

, the new elongate cells of the

indeterminate axis formed crystals (Fig. 9).

After exposure to ASW containing 1.0 mM CaCl

for

only five days, thalli of A. nipponicum showed loss of colour

in scattered cells of both indeterminate axes and lateral

branches. However, cell death was not pervasive; in

response to loss of their subtending axial cells, the basal

cells of the determinate lateral branches were undergoing

cell division. These same symptoms were apparent in A.

antillanum after one week, but A. callocladum and A.

sparsum retained good colour and morphology, and their

cells had abundant calcium oxalate crystals three weeks

after the cultures were initiated (Fig. 10). For each species,

the size range of the crystals in even the lowest survivable

calcium concentration was no different from that in the

control culture or the high calcium concentration treat-

ments. Unfortunately, spore release did not occur in the A.

antillanum culture medium containing only 1.0 mM CaCl

so the effects of this calcium concentration on sporelings

could not be determined.

Cultures of A. antillanum placed in ASW with only

0.1 mM CaCl

lost colour within three days. After the same

interval, A. callocladum and A. sparsum still appeared to be

in excellent condition and had abundant calcium oxalate

crystals even in the young elongating cells of the in-

determinate axes. However, after one week, the axial cells

of A. antillanum were dead. Apical portions of A. sparsum

thalli died back after a week in 0.1 M CaCl

, but living

axial cells still had calcium oxalate crystals.

Although some loss of thallus colour occurred, Antith-

amnion callocladum,A. sparsum,andA. antillanum still had

calcium oxalate crystals after being cultured at their lowest

survivable calcium concentration (0.1, 1.0, and 2.5 mM,

respectively) for 10 weeks.

After 14 weeks in complete darkness, thalli of A. sparsum

in normal culture medium appeared healthy, but they were

devoid of floridean starch grains. Calcium oxalate crystals

were abundant (Fig. 11).

DISCUSSION

The physiological function of calcium oxalate crystals in

Antithamnion remains unclear. If the ability to precipitate

calcium as an oxalate salt were to serve as a calcium

detoxification mechanism in Antithamnion,asitdoesin

vascular plants (Volk et al. 2002; Mazen et al. 2003), then

the species of Antithamnion with crystals should have been

Figs. 9–11. Calcium oxalate crystals persist in Antithamnion thalli cultured in artificial seawater medium (ASW) containing reduced calcium

concentrations or in darkness. Differential interference contrast microscopy.

Fig. 9. Antithamnion antillanum. Thalli grown in ASW containing 2.5 mM CaCl

released spores, and the young sporelings began to form

calcium oxalate crystals (arrows) in the axial cells. Treated in sodium hypochlorite to remove organic material.

Fig. 10. Antithamnion sparsum. Thalli grown for three weeks in ASW containing only 1 mM CaCl

still had calcium oxalate crystals in

axial cells. Treated in sodium hypochlorite.

Fig. 11. Antithamnion sparsum. Starch grains are absent but calcium oxalate crystals are abundant among the chloroplasts in the parietal

cytoplasm of thalli maintained in complete darkness for 14 weeks.

376 Phycologia, Vol. 46 (4), 2007

more tolerant of high calcium concentrations than the

species that do not deposit calcium oxalate. No such

pattern was apparent. If the calcium oxalate crystals serve

as a calcium reserve, then based on the relative size and

abundance of oxalate crystals in the four species, one would

expect that normal appearance of the thalli and cellular

integrity would be maintained according to the following

ranking: A. sparsum .A. antillanum .A. callocladum .A.

nipponicum. Instead, A. antillanum proved to be the most

sensitive to reduced calcium concentrations. Calcium

oxalate crystals were not mobilized, and the presence of

crystals provided no mitigation of the effects of calcium

deficiency. Disappearance of crystals followed rather than

preceded cell death; crystal dissolution may have been the

result of postmortem bacterial action rather than mobili-

zation by the alga. Specimens of A. sparsum held

in complete darkness for 14 weeks depleted their starch

stores but still had abundant calcium oxalate crystals,

showing that oxalate was not mobilized as a carbon

source.

Calcium is an essential nutrient, as the rapid mortality of

Antithamnion in calcium-free medium demonstrated. Little

is known about minimum calcium requirements for marine

macroalgae, but it has been suggested that the ability of

marine algae to invade habitats of low salinity is

constrained principally by the reduced availability of

calcium in these waters (Yarish et al. 1980; Dawes &

McIntosh 1981). Maintaining sufficient calcium supply

allows the concentration of other ions normally found in

seawater to be substantially reduced (Eppley & Cyrus 1960;

Reed 1984). In our study, the amount of calcium required

for normal growth and reproduction was found to be far

less than the 10 mM calcium found in seawater of full

salinity (Kennish 2000). In A. antillanum, formation and

release of tetraspores and sporeling development took

place in medium containing 2.5 mM CaCl

. Remarkably,

the sporelings deposited calcium oxalate crystals at this

concentration, whereas thalli in 1.0 mM CaCl

showed

extensive cell death within a week. The minimum calcium

concentration required differed among species. Thalli of A.

antillanum and A. nipponicum died quickly after being

placed in ASW having 1.0 mM CaCl

,butA. callocladum

and A. sparsum continued to thrive and had abundant

crystals. Even ASW with only 0.1 M CaCl

did not

prove immediately fatal to A. sparsum. From our experi-

ments, it can be inferred that the minimum calcium

concentrations required by A. nipponicum and A. antillanum

are between 1.0 and 2.5 mM and that those required by

A. callocladum and A. sparsum are between 0.1 and

1.0 mM.

Ascertaining the minimum calcium requirement for

Antithamnion is complicated by the differing responses of

cells within the same thallus. The death of cells of

indeterminate axes of A. callocladum was followed by

intrusion and division of the basal cell of the lateral

filaments, and it was generally observed that cells of the

determinate branches were more tolerant of high and low

calcium concentrations than the axial cells of the in-

determinate filaments. Thus, while some cells died in the

low-calcium environment, other cells of the same thallus

underwent growth and division. All the species of

Antithamnion known to have calcium oxalate crystals

deposit them exclusively in the cytoplasm of cells of the

indeterminate axes; this fact and the differential response of

determinate and indeterminate cells to high and low

concentrations demonstrate that the different cell types

differ in their regulation of calcium.

Kim et al. (1988) studied wound repair in A. nipponicum

and representatives of several other genera of the Cer-

amiales. The death of individual intercalary cells of the

indeterminate axes triggered wound repair by adjacent axial

cells. In A. nipponicum, the determinate lateral filaments

that were subtended by a dead axial cell survived and

became connected to the newly formed replacement axial

cell (Kim et al. 1988). In the present study, calcium stress

killed large numbers of axial cells, so similar wound healing

was not possible. Although the determinate lateral branches

were made physiologically independent by death of their

subtending indeterminate axial cell, the axial cells of the

branches did not develop crystals, nor did they show any

other sign of conversion from determinate to indeterminate

status. As a result, regardless of how well the cells of the

determinate lateral branches endured calcium stress,

extensive death of cells of the indeterminate axes led to

the eventual demise of the thallus.

Calcium detoxification by precipitation of calcium

oxalate appears to be effective in vascular plants (Kost-

man & Franceschi 2000; Pennisi & McConnell 2001; Volk

et al. 2002; Mazen et al. 2003); however, vascular plants

have limited points of entry of calcium and some control

over the path of calcium through the plant. A simple algal

thallus submersed in a calcium-rich marine environment

probably will not be able to control cytosolic calcium

concentration by intracellular precipitation of calcium

oxalate, and we found that crystal depositers as a group

were no more tolerant of high calcium concentrations than

were nondepositers. The possibility that the crystals can act

as a calcium store also can be rejected. We found that

crystals were not mobilized when the thalli were stressed by

low calcium levels and that A. nipponicum, the species

without calcium oxalate crystals, fared better than one

of the three Antithamnion species having crystals. Because

calcium oxalate crystals are constitutive in some species

of Antithamnion, these species must acquire more calcium

than those not depositing crystals. Calcium concentra-

tions in eukaryotic cells are usually in the 30–200-nM

range (e.g. Bush 1993); for example, the steady-state

cytosolic calcium concentration of the brackish water

charophyte Lamprothamnium is 80 nM (Okazaki et al.

2002). Given that seawater contains about 10 mM

calcium, increased calcium acquisition for marine algae

presumably involves less stringent exclusion of calcium ions

by the cells.

Calcium oxalate crystals have been shown to have

systematic value in particular groups of vascular plants

(e.g. Prychid & Rudall 1999). Recent work on members of

the Cladophorales s.l. revealed crystals of diverse morphol-

ogy and chemical nature, including some composed of

calcium oxalate (Leliaert & Coppejans 2004, 2006). Leliaert

and Coppejans (2004) found constancy of features of

calcium oxalate crystals across diverse collections of some

species. They have employed presence/absence of calcium

Pueschel & West: Calcium oxalate crystals in Antithamnion 377

oxalate crystals and morphology of the crystals to aid in

distinguishing species of Cladophoropsis (Leliaert & Cop-

pejans 2006). However, the effects of environmental

conditions on calcium oxalate deposition in these algae

have not been examined experimentally.

Pueschel (1995) examined 12 taxonomically disparate

species of red algae and found oxalate crystals in

Antithamnion defectum and A. kylinii. The present study

more thoroughly sampled the Ceramiales, but among the

18 species surveyed, calcium oxalate crystals were found in

only three of the four species of Antithamnion. Additional

species of Antithamnion also appear to deposit calcium

oxalate crystals. Norris (1987) reported the presence of

acicular crystals in axial cells of A. diminuatum Wollaston,

A. secundum Itono, and A. antillanum [as A. lherminieri

(P.L. Crouan & H.M. Crouan) Bornet ex Nasr] and their

absence in several other species studied. Although the

listing of the crystals’ presence or absence among the

characteristics of each species implies they were considered

to be of systematic value, no discussion was offered as to

the nature of the crystals or their potential value in

systematics (Norris 1987).

Our studies directly tested whether experimental manip-

ulation of ambient calcium concentration had an effect on

the stability of calcium oxalate crystals in seven species of

red algae. Calcium oxalate crystals were not induced by

elevated calcium concentrations in any of the species tested,

nor were the crystals in Antithamnion lost under physio-

logically tolerable low-calcium concentrations; therefore,

crystal presence or absence in this genus (and absence in

several other genera) appears to have the stability required

of a taxonomic character. The size and shape of calcium

oxalate crystals also proved to be stable across the full

range of calcium concentrations, so crystal dimensions may

also have systematic value if the crystal sizes differ

markedly between taxa, as, for example, in A. callocladum

and A. sparsum.

The taxonomy of the genus Antithamnion has been in

flux, with many transfers and synonymies. For example, A.

sparsum and A. defectum were placed in synonymy with A.

densum (Athanasiadis 1996), but Lee et al. (2005) provided

molecular evidence that supports their separation and their

affinities with A. kylinii. All four of these species have

relatively large calcium oxalate crystals (Pueschel 1995;

herein). By contrast, A. nipponicum does not deposit

calcium oxalate, and A. callocladum, with which A.

nipponicum is allied (Lee et al. 2005), has relatively sparse,

small crystals. Antithamnion nipponicum was placed in

synonymy with A. pectinatum (Montagne) Brauner (Atha-

nasiadis 1996), but rbcL sequence data support specific

status (Cho et al. 2005). Antithamnion aglandum Kim & Lee

also has affinities with A. nipponicum (Cho et al. 2005; Lee

et al. 2005). Determination of whether calcium oxalate

crystals are deposited in A. pectinatum and A. aglandum

and, if so, their size and abundance will be of interest. As

molecular data continue to help sort out the species of

Antithamnion, the potential of calcium oxalate crystals as

taxonomic features will become clear.

Our investigations were designed to address cytological,

physiological, and systematic issues related to calcium

oxalate deposition in red algae. We have demonstrated that

calcium oxalate crystals in three species of Antithamnion are

constitutive structures that do not respond in a substantial

way to even drastic variations of ambient calcium levels;

therefore, a role in calcium regulation can be excluded, and

potential use as a systematic character is supported. The

needle-like morphology of the crystals raises the possibility

that they might serve as a defense against grazers, but not

enough is known about grazing of these species to evaluate

this possibility. However, because the crystals occur only in

axial cells, it would appear that the axes would have to be

severed before the mechanical defense would come into

play. Understanding of the function of calcium oxalate

crystals in the biology of the Antithamnion remains elusive.

ACKNOWLEDGEMENTS

J. Pickett-Heaps and T. Spurck generously provided

microscopy facilities and technical expertise at the Univer-

sity of Melbourne, and G.H. Kim provided several algal

cultures. This work was made possible by Australian

Biological Resources Study Grant for 2002–2005 to

J.A.West and G.C. Zuccarello and a University of

Melbourne Visiting Research Scholar Award to C.M.

Pueschel.

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Received 13 September 2006; accepted 1 February 2007

Associate editor: Martha Cook

Pueschel & West: Calcium oxalate crystals in Antithamnion 379

Calcium oxalate mineralisation in the algae

Article

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Living cells of field-collected specimens of the giant-celled marine green alga Chaetomorpha coliformis (Montagne) Kutzing were found to have birefringent cellular inclusions whose composition was determined to be calcium oxalate on the basis of their reactions to diagnostic chemical solubility tests and the Yasue cytochemical staining procedure. The inclusions consisted of individual bipyramidal crystals up to 50 mu m in greatest dimension and variously sized aggregates of much smaller crystals. Some aggregates consisted of rosettes or spheres of radiating elements that resemble embryophyte druses more than any other calcium oxalate deposit yet reported in the algae. Both single crystals and aggregates occurred in the same cells with no discernible pattern to their distributions. The calcium oxalate crystals were anchored on the vacuolar face of the thin cytoplasmic layer rather than being dispersed within the voluminous central vacuole. Light and transmission electron microscopy demonstrated the presence of a nearly continuous layer of small vacuoles between the organelle-rich parietal cytoplasm and the large central vacuole, and the calcium oxalate crystals were associated with this layer of vacuoles. The occurrence of mineralization in the parietal vacuoles, but not the large central vacuole, indicates that differentiation of vacuoles according to ionic contents may be occurring.

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Calcium oxalate is the most widely distributed inorganic crystal in plants, occurring in over 200 plant families. This wide distribution suggests that they constitute an important biomineralization process in plants. The variation in CaOx crystal shape and cell types producing them indicates that crystals may have evolved many times independently in different plant lineages and may probably serve multiple functions. Their distribution among lycophytes and ferns is poorly documented and has led to the assumption that they are rare or absent in these lineages. Only a few reports indicate that CaOx crystals are present in several fern families, but their distribution in this group has never been assessed on a broad scale. Our aim was to determine and compare shape, abundance as well as distribution patterns of calcium oxalate crystals across ferns and lycophytes. Such knowledge is crucial to infer hypotheses about the structural-functional evolution of CaOx-formation in land plants. Our observations were plotted on a phylogenetic tree in order to test whether certain patterns appear on different phylogenetic levels. It was also investigated whether distribution patterns or particular crystal types could be related to fern ecology or potential function(s).

Macroalgae

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Rhodophyta

Chapter

Sep 2017

Rhodophyta, or red algae, comprises a monophyletic lineage within Archaeplastida that includes glaucophyte algae and green algae plus land plants. Rhodophyta has a long fossil history with evidence of Bangia-like species in ca. 1.2 billion-year-old deposits. Red algal morphology varies from unicellular, filamentous, to multicellular thalloid forms, some of which are sources of economically important products such as agar and carrageenan. These species live primarily in marine environments from the intertidal zone to deep waters. Freshwater (e.g., Batrachospermum) and terrestrial lineages also occur. One of the major innovations in the Rhodophyta is a triphasic life cycle that includes one haploid and two diploid phases with the carposporophyte borne on female gametophytes. Red algae are also well known for their contribution to algal evolution with ecologically important chlorophyll-c containing lineages such as diatoms, dinoflagellates, haptophytes, and phaeophytes all containing a red algal-derived plastid of serial endosymbiotic origin. Analysis of red algal nuclear genomes shows that they have relatively small gene inventories of 6,000–10,000 genes when compared to other free-living eukaryotes. This is likely explained by a phase of massive genome reduction that occurred in the red algal ancestor living in a highly specialized environment. Key traits that have been lost in all red algae include flagella and basal body components, light-sensing phytochromes, and the glycosylphosphatidylinositol (GPI)-anchor biosynthesis and macroautophagy pathways. Research into the biology and evolution of red algae is accelerating and will provide exciting insights into the diversification of this unique group of photosynthetic eukaryotes.

Rhodophyta

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Jul 2016

Wound-healing m several filamentous red algae, Ceramiales

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Calcium oxalate crystals in plants

Article

Full-text available

Oct 1980

Calcium (Ca) oxalate crystals occur in many plant species and in most organs and tissues. They generally form within cells although extracellular crystals have been reported. The crystal cells or idioblasts display ultrastructural modifications which are related to crystal precipitation. Crystal formation is usually associated with membranes, chambers, or inclusions found within the cell vacuole(s). Tubules, modified plastids and enlarged nuclei also have been reported in crystal idioblasts. The Ca oxalate crystals consist of either the monohydrate whewellite form, or the dihydrate weddellite form. A number of techniques exist for the identification of calcium oxalate. X-ray diffraction, Raman microprobe analysis and infrared spectroscopy are the most accurate. Many plant crystals assumed to be Ca oxalate have never been positively identified as such. In some instances, crystals have been classified as whewellite or weddellite solely on the basis of their shape. Certain evidence indicates that crystal shape may be independent of hydration form of Ca oxalate and that the vacuole crystal chamber membranes may act to mold crystal shape; however, the actual mechanism controlling shape is unknown. Oxalic acid is formed via several major pathways. In plants, glycolate can be converted to oxalic acid. The oxidation occurs in two steps with glyoxylic acid as an intermediate and glycolic acid oxidase as the enzyme. Glyoxylic acid may be derived from enzymatic cleavage of isocitric acid. Oxaloacetate also can be split to form oxalate and acetate. Another significant precursor of oxalate in plants is L-ascorbic acid. The intermediate steps in the conversion of L-ascorbic acid to oxalate are not well defined. Oxalic acid formation in animals occurs by similar pathways and Ca oxalate crystals may be produced under certain conditions. Various functions have been attributed to plant crystal idioblasts and crystals. There is evidence that oxalate synthesis is related to ionic balance. Plant crystals thus may be a manifestation of an effort to maintain an ionic equilibrium. In many plants oxalate is metabolized very slowly or not at all and is considered to be an end product of metabolism. Plant crystal idioblasts may function as a means of removing the oxalate which may otherwise accumulate in toxic quantities. Idioblast formation is dependent on the availability of both Ca and oxalate. Under Ca stress conditions, however, crystals may be reabsorbed indicating a storage function for the idioblasts for Ca. In addition, it has been suggested that the crystals serve purely as structural supports or as a protective device against foraging animals. The purpose of this review is to present an overview of plant crystal idioblasts and Ca oxalate crystals and to include the most recent literature.

Time-lapse video observations on sexual plasmogamy in the red alga bostrychia

Article

Feb 1998
EUR J PHYCOL

In the red alga Bostrychia moritziana, release of spermatia is triggered by slight osmotic shock; they emerge under pressure apparently generated by swelling of the mucilaginous sheath. Spermatia adhere tenaciously to trichogynes of the carpogonium. Adhesion triggers spermatial mitosis, which is complete in about 30–45 min; there is no cytokinesis and the binucleate spermatium becomes vacuolated. The delicate, dynamic trichogyne cytoplasm contains complex membranous components and vacuoles. At the contact zone, the trichogyne and spermatial wall erode, forming a pore, and cytoplasmic continuity (plasmogamy) is achieved after about 50–70 min. Many trichogynes rupture during these events because of inadequate structural connection with the spermatia. Normally, both spermatial nuclei enter the trichogyne in sequence; rarely, both nuclei enter together. Entrance is rapid, and the nuclei often become thin and greatly elongated as each squeezes through the narrow pore into the trichogyne. Once inside, each nucleus resumes its normal shape as it starts to move steadily along the trichogyne, often with irregular pauses. One nucleus of each pair (not necessarily the first out of the spermatium) migrates along the trichogyne towards the carpogonium base to fertilize the female nucleus; the other moves in the opposite direction, away from the entry site, and it often ends up near the tip of the trichogyne. This same scenario was observed for each of several spermatia contributing nuclei to one trichogyne. Thus, our observations indicate that the two nuclei in each spermatium are differentiated so that only one is capable of fertilization, differentiation being visibly expressed in the direction that the nucleus moves as it enters the trichogyne.

Morphology and classification of the Ceramioideae (Rhodophyta) based on phylogenetic principles

Article

Jan 1996

A. Athanasiadis

Antithamnioid algae have been treated as a natural assemblage for more than a century, being classified in a single genus (Schmitz 1889) or tribe (Hommersand 1963) and more recently divided into two groups, viz. the tribes Antithamnieae and Heterothamnieae (Wollaston 1968). Application of phylogenetic methods based on parsimony and outgroup comparison indicates that the antithamnioids probably represent a paraphyletic assemblage within the subfamily Ceramioideae. They are classified here in the following monophyletic groups: 1) Pterothamnieae trib. nov. (including only Pterothamnion Nageli), 2) Scagelieae trib. nov. (including only Scagelia Wollaston). 3) Dohrnielleae Feldmann-Mazoyer (comprising Acrothamniopsis gen. nov., Antithamnionella Lyle, Trithamnion Wollaston, Callithamniella Feldmann-Mazoyer, and Dohrniella Funk), 4) Antithamnieae Hommersand (comprising Acrothamnion J. Agardh, Antithamnion Nageli, Hollenbergia Wollaston, Macrothamnion J. Agardh, and Perikladosporon gen. nov.), 5) Perithamnieae trib. nov. (comprising Perithamnion J. Agardh and Scageliopsis Wollaston), 6) Ceramieae Schmitz et Hauptfleisch (comprising the genera of the former Ceramieae sensu Kylin 1956 and Hommersand 1963, and also Amoenothamnion Wollaston and Leptoklonion gen. nov.), 7) Heterothamnieae Wollaston (comprising Heterothamnion J. Agardh and Tetrathamnion Wollaston), and 8) Crouanieae Schmitz et Hauptfleisch (comprising the genera of the former Crouanieae sensu Wollaston 1968, 1972c, and 1977b, and also Crouaniella gen. nov.). The subfamily Ceramioideae accommodates taxa with the following characters: a) thallus differentiated into axial filaments of unlimited growth and lateral filaments of limited growth and (whorl-branches) with open or condensed organization, b) thallus regeneration occurring by the production of adventitious axial filaments from whorl-branch cells, c) development of transverse ramification, with whorls of 4 branches of limited growth and progressively either reduced (to 3, 2 or 1) or increased to reach 20 (or more) branches with condensed organization in advanced members, d) development of procarps on basal cells of normal-sized whorl-branches which progressively reduce in length to become 3-, 2-, or 1-celled, c) development of gland cells that touch firmly the mother cell, and f) development of gland cells from branch and branchlet cells. Modification or reduction of some of these features has occurred secondarily in members of the subfamily, which apparently also accommodates the tribes Spyridieae, Griffithsieae, Sphondylothamnieae, and Wrangelieae. This delimitation of the Ceramioideae suggests that even members of the highly specialized families Rhodomelaceae and Dasyaceae belong to this assemblage. The genus Balliella (Delesseriopseae), previously included in the Antithamnieae, lacks the above characters but nevertheless develops gland cells (distally connected to the mother cell), which is one of the reasons that Balliella is here selected as the potential sister-taxon of the Ceramioideae. The study of Balliella cladoderma shows that gland cells originate from axial cells and are later transferred to periaxial cells following the lateral division of the mother (axial) cell. A similar development of procarp evolution is postulated for the Ceramiales, since axial procarps are also demonstrated for the first time in the genus Balliella and in some species of the tribes Pterothamnieae, Scagelieae, and Perithamnieae. Pterothamnieae is the most primitive ceramioid tribe widely distributed in warm and cold temperate regions and is apparently monogeneric, although there is support for placing certain eastern Pacific species in a monophyletic group, which is recognized at subgeneric level, viz., Platythamnion (J. Agardh) stat. nov. Apomorphies of this subgenus, which receives Pterothamnion latiramosum sp. nov. from California, are the tristichous-tetrastichous ramification of major whorl-branches and the heteromorphic transverse ramification; the latter feature appears as a homoplasy in certain North Pacific and North Atlantic species of the subgenus Pterothamnion sect. Pterothamnion, two other species from South Australia and South Africa, in which procarps develop on basal cells of normal-sized whorl-branches and new axes are borne subdichotomously above the whorl-branching. A previously congeneric species from South Australia is transferred to Leptoklonion elongatum (Wollaston) gen. et comb. nov., on the basis of the following features; development of procarps on basal cells of 2-celled whorl-branches, new axes replacing whorl-branches, and sessile tetrasporangia restricted to periaxial cells. Phylogenetically, Amoenothamnion and Leptoklonion share a recent common ancestor with the Ceramieae and are provisionally included in this tribe. Former species of Perithamnion are transferred to Tetrathamnion, which now includes at least 4 species, of which Tetrathamnion myurum (Suhr) comb. nov. from Peru represents the first record of the genus outside Australia. Tetrathamnion is the only genus which is maintained in the former Heterothamnieae, the type genus of which receives Heterothamnion platythaliae sp. nov. from Western Australia. Heterothamnion is clearly distinguished having an endophytic base in species of Fucaceae, but its thallus organization shows considerable variation including species with opposite or transverse development of whorl-branches and transverse or oblique apical divisions. Spermatangial mother cells form branched filaments, borne either along the entire length of whorl-branches or restricted to inner branch cells. Sporangial initials develop by elliptical lateral divisions. Two former species of Antithamnionella are transferred to the new genus Crouaniella, viz. Crouaniella latiaxis (Abbott) comb. nov. from the Caribbean and Crouaniella mcnabbi (Dawson) comb. nov. from the tropics of Pacific America. The genus exhibits 3 whorl-branches per axial cell, independently borne new axes (as a 4th lateral in a whorl of 3 branches), subdichotomously ramified whorl-branches with the divisions starting from the periaxial cell, rhizoidal outgrowths developed from axial cells of the erect frond, and spherical or oblongate tetrasporangia with tetrahedral cleavages borne independently on the bifurcations of whorl-branches. Phylogenetically, Crouaniella belongs to the Crouanieae and its biogeography reflects the geological history of the Panamanian Isthmus. Studies of several species previously referred to Antithamnion support the following transfers: Dasyptilon ptilota (J. D. Hooker et Harvey) comb. nov. from Crozet Islands, Dasyptilon microptilum (Grunow) comb. nov. from St. Paul Island, Grallatoria tanakae (Itono) comb. nov. from Sakurajima (S. Japan), Gymnothamnion nigrescens (J. Agardh) comb. nov. from Port Phillip (Victoria), and Gymnothamnion pteroton (Bornet) comb. nov. from Tangier.

Histochemical identification of calcium oxalate

Article

Jan 1969

TAKAO YASUE

Histochemistry of calcium oxalate has not been definitive. The present author has critically investigated various histochemical methods for calcium salts and finally found a new method advantageous to those hitherto reported. Celloidin models of calcium salts, rat kidneys of oxalosis induced with ethylene glycol and human materials were used for study. New method: Firstly calcium phosphate and carbonate are removed by immersing sections into 5% acetic acid for 30 minutes. Secondly the sections still containing calcium oxalate are made to react with 5% aqueous silver nitrate for 15 minutes, and thirdly the reaction product appears dark brown to black with the use of rubeanic acid (saturated rubeanic acid in 70% alcohol with 2 drops of strong ammonium for 1 minute) that is very sensitive to silver without loosing the definite localization of calcium oxalate. This method has a merit; it does not depend on weather or illumination and takes a shorter time than the Kóssa method. A confident histochemical method for identifying calcium oxalate was established by combining this method with other characteristics of calcium oxalate such as the unstainability with hematoxylin, birefringency, solubility after incineration and gypsum formation. In the present study calcium oxalate was also found to react with lead besides silver and to appear brownish-black when one uses lead nitrate and ammonium polysulphide.

Inducible Calcium Sinks and Preferential Calcium Allocation in Leaf Primordia of Dracaena sanderiana Hort. Sander ex M.T. Mast. (Dracaenaceae)

Article

Dec 2001

The effect of 0, 3, and 7 mM Ca2+ on the allocation and deposition of Ca2+ into intracellular and sub-cuticular periplasmic calcium oxalate (CO) crystals was examined in leaf primordia of rooted cuttings of Dracaena sanderiana Hort. Sander ex M.T. Mast. Crystal development was monitored in two types of cuttings, those rooted in deionized water for 18 months and those rooted in Metro Mix 500 for 6 weeks. Response differed remarkably depending on the type of cutting. Cuttings rooted in deionized water deposited sub-cuticular crystals at the expense of intracellular crystals (raphides). The number of sub-cuticular crystals in leaf primordia of cuttings rooted in deionized water grown in solutions supplemented with either 0, 3, or 7 mM Ca2+ was similar, but crystals were considerably smaller in plants grown in 0 mM Ca2+. Sub-cuticular crystals appeared developmentally earlier in leaf primordia of all cuttings grown in either 3 mM or 7 mM Ca2+ than in cuttings rooted in deionized water grown in 0 mM Ca2+. This finding supports the premise that deposition of sub-cuticular crystals is modulated by Ca2+ levels and could be induced at an earlier ontogenetical stage by raising rhizospheric Ca2+ levels or delayed by lowering rhizospheric Ca2+ levels. The total number of sub-cuticular crystals per epidermal cell did not differ significantly between treatments implying that crystal nucleation sites are predetermined and finite in number. In contrast, the formation of intracellular raphides was highly variable and depended on Ca2+ concentrations. In terms of Ca2+ prioritization, sub-cuticular CO crystals took precedence over intracellular CO raphides.

Temporal Relationship between Cytosolic Free Ca2+ and Membrane Potential during Hypotonic Turgor Regulation in a Brackish Water Charophyte Lamprothamnium succinctum

Article

Sep 2002
PLANT CELL PHYSIOL

Yoshiji Okazaki

Internodal cells of a brackish water charophyte, Lamprothamnium succinctum, regulate turgor pressure in response to changes in external osmotic pressure by modifying vacuolar concentrations of KCl. An increase in cytosolic concentration of free Ca 2 + ([Ca 2 + ] c ) is necessary for the progress of turgor regulation induced by hypotonic treatment. Initial changes in membrane potential and [Ca 2 + ] c upon hypotonic treatment were measured to examine the temporal relationship between the two parameters. Fura-dextran (potassium salt, M r 10,000, anionic) that had been injected into the cytosol was used to measure [Ca 2 + ] c . Membrane potential and membrane conductance under a current-clamp condition were also measured. Decrease in external osmotic pressure by 0.16 Osm induced a simultaneous increase in [Ca 2 + ] c with both depolarization of the membrane and increase in the membrane conductance. Decrease in external osmotic pressure by 0.05 Osm induced a simultaneous increase in [Ca 2 + ] c with membrane depolarization but the increase in membrane conductance started later than the other two processes. There was a close temporal relationship between the increase in [Ca 2 + ] c and membrane depolarization on the initial response of turgor regulation induced by hypotonic treatment.

Fine structure of vacuolar inclusions in the siphonous green alga Chlorodesmis fastigiata (Udoteaceae, Caulerpales) and their contribution to plug formation

Article

Jun 1987

Diedrik Menzel

In Chlorodesmis jastigiata, a remarkable variety of specifically structured material is found in the central vacuole immersed in an ill-defined granular-fibrillar matrix. This material fills the central vacuole throughout the entire length of the dichotomously branched cell of several centimeters' it is most dense in the tip. Most of the vacuolar material is manufactured in the cytoplasm of the apical cell area and then is released gradually into the vacuole. A developmental sequence of the production of osmiophilic granules and their release into the vacuole is proposed. These granules and sphencal fibrous bodies constitute the major components of the vacuolar inclusions. During wound plug formation these components are subject to characteristic redistributions and structural changes, suggesting that they serve specific functions during successive stages of wound plug formation. Paracrystalline chambers around mineral crystals and endophytic bacteria were also observed among the materials in the central vacuole; their function is not known.

A study of the ultrastructure of a green alga, Apjohnia laetevirens Harvey with emphasis on cell wall structure

Article

Jun 1969

Clinton J. Dawes

A light and electron microscopic study on the cell wall and vegetative protoplast of the green alga Apjohnia laetevirens Harvey is presented. Supplementary information on cell wall structure using polarized light and X-ray diffraction and evidence for two types of cross wall formation are also included. The cell wall consists of an amorphous layer, an outer microfibrillar reticulum, numerous inner lamellae of microfibrils oriented in a crossed fibrillar pattern, and a less common layer oriented at 45 degrees to the other fibrillar layers. The parietal cytoplasm contains organelles typical of those found in the Chlorophyta. The filament contains a large vacuole in which are found acicular calcium oxalate crystals. Taxonomic implications of the cytology of Apjohnia are discussed.

Cell and calcium oxalate crystal growth is coordinated to achieve high-capacity calcium regulation in plants

Article

Sep 2000

Crystal idioblasts are cells which are specialized for accumulation of Ca2+ as a physiologically inactive, crystalline salt of oxalic acid. Using microautoradiographic, immunological, and ultrastructural techniques, the process of raphide crystal growth, and how crystal growth is coordinated with cell growth, was studied in idioblasts ofPistia stratiotes. Incorporation of45Ca2+ directly demonstrated that, relative to surrounding mesophyll cells, crystal idioblasts act as high-capacity Ca2+ sinks, accumulating large amounts of Ca2+ within the vacuole as crystals. The pattern of addition of Ca2+ during crystal growth indicates a highly regulated process with bidirectional crystal growth. In very young idioblasts,45Ca2+ is incorporated along the entire length of the needle-shaped raphide crystals, but as they mature incorporation only occurs at crystal tips in a bidirectional mode. At full maturity, the idioblast stops Ca2+ uptake, although the cells are still alive, demonstrating an ability to strictly regulate Ca transport processes at the plasma membrane. In situ hybridization for ribosomal RNA shows young idioblasts are extremely active cells, are more active than older idioblasts, and have higher general activity than surrounding mesophyll cells. Polarizing and scanning electron microscopy demonstrate that the crystal morphology changes as crystals develop and includes morphological polarity and an apparent nucleation point from which crystals grow bidirectionally. These results indicate a carefully regulated process of biomineralization in the vacuole. Finally, we show that the cytoskeleton is important in controlling the idioblast cell shape, but the regulation of crystal growth and morphology is under a different control mechanism.

Effects of Ambient Calcium Concentration on the Deposition of Calcium Oxalate Crystals in Antithamnion (Ceramiales, Rhodophyta)

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