Cyrotogia

45: 113-126,

1980

Thylakoid-Dense

Chloroplasts

of

Callisia

fragrans

Robert Biology Department, Stevens Received June 13, 1978

R.

Wise1

and

Joseph

B.

Harris Point,

University Point, Wisconsin

of Wisconsin-Stevens 54481, U. S. A.

Descriptions grana Porter in diameter or described Valanne light, and (1967) in also thylakoids which In plasts this of is Spinacia. been per 1947, chloroplast Leyon (Kirk

and

micrographs with 1953). 2-100 Although

of thylakoids

chloroplast per plant no work

ultrastructure granum (Spinacia,

report Granick average reported Their it found in the is

40-60 and 4-6m on shape somewhat their has

higher 1967), due to (Hall and per

chloroplasts has g 1974) been forces. although (1973)

and

Tilney-Bassett response

density been variable. continuous of

sedimentation as being (1977) the those found grana of no

different et al.

piano-convex and Descomps size tomato. such increased

Deroche chloroplast Descomps

that protonemata and thylakoids

under

Ceratodon

However, increase large in grana In that

and number due to

Deroche of mineral does found

(1973) per

Bronchart plastid have of and

the

total

Exceptionally (Hall et al.

stacks none which of

deficiencies the in density Callisia

reported per reported

1972).

these has

cases been

chloroplast here. sedimentation fragrans (Lindl). to

approach

study Callisia and commonly

properties, Woods those in the of

shape, (syn. several literature.

and

ultrastructure

of dracaenoides)

the

chlorowere which in the

Tradescantia species Callisia of

inverstigated have been

compared described

higher is a

plants monocot

Commelinaceae.

Materials

and

methods

All for at

plants least six

were months

grown prior

under to

Plant-Gro utilization. Strips of leaf (SFD) one minute and with 50 an mount as and Olympus was

lighting

and

greenhouse

conditions

Fractional Omni-Mixer a was a high speed filtered Sorvall of a in for

centrifugation: a 30 sucrose-ficol-dextran seconds 4-ply centrifuge and microscope at were 250g, randomly 2000, 2500, a or

tissue

were medium

homogenized (Honda on at rotor. were the 100g A

in et al. species. for fresh 5

a Sorvall 1966) The at brei in was

depending centrifuged an HB-4

through RC2-B the pellet 2278

cheesecloth equipped approximately and fresh

minutes mount measured supernatant

made using was

chloroplasts filar made,

randomly The

Zeiss

micrometer. and This again procedure all species

centrifuged

approximately was except repeated Callisia

50

chloroplasts at 500, 1000,

measured 3000,

above. 3500 g

1500,

for

1 U. S. A.

Present

address:

Botany

Department,

Duke

University,

Durham,

North

Carolina

27706,

114

Robert

R.

Wise

and

Joseph

B. Harris

Cytologia

45

where

it After

was each

not

continued fraction was of was

past

1500g. the at represented different the volume next was g higher determined forces. This of g the force. and pellet was The this recorded percentage determined was not

sedimented,

and of the

the the

supernatant total volume of past 1500 molarity (Honda for with sucrose. 5 the et al.

centrifuged pellets it

distribution

chlorplasts g for test: 1966) after pellet Callisia.

among

procedure

continued Sucrose medium at 9150g

Strips with a in 0.20M

of

leaf sucrose,

tissue

were filtered time. of as

homogenized above, This 0.25, then procedure 0.30,

in centrifuged was 0.35,

SFD

minutes same

10-minute sucrose

response concentrations

reand

peated 0.40M

Bovine lisia and ment. organelle in The 1200mg with media tely 0-2 50 for Scanning fixed Specimens PVT-3 in tube were

serum homogenized at

albumin and 9150 g 1966, to and tube 3

(BSA) filtered force for

and as 10

native above. minutes. that of with

leaf

protein The brei Tube

test: was

Leaf placed no was The (Honda

strips in four

of

Caltubes treatof

centrifuged (Honda integrity 2 was

1 received

further protective supernatant et al.

et al. due

considered its content

this native

homogenate leaf SFD replaced protein). medium with 4 was

discarded in

replaced was The

fresh and in pellets was

1966). and treated new

supernatant BSA 1200mg and

discarded supernatant All four

SFD retained

medium and in and were their approximastored

(100mg/ml). BSA (100mg/ml). A were and

tube were of above.

resuspended each pellet Slides

recentrifuged.

fresh

mount

made as were squares

chloroplasts 24 hours electron 4 % were critical osmium

randomly again 50

measured chloroplasts One-cm vapor for acetone with in a

at

randomly from minutes at dried

measured. Callisia room CO2, and scanning leaves temperature. in a Samdri were

microscopy: tetroxide in drier, was a and

30

dehydrated point Tissue

graded coated

series, 75-100A JEOL

with carbon

275-3001 electron

gold-palladium. microscope Transmission vacuum buffer were every buffer by a The at rinsed 10 at several Sorvall pellet infiltrated 0-3 in minutes. 0-3 distilled at

examined

JSM

U-3

15-25

KV. electron with 3% a pH microscopy: glutaraldehyde of 7.2. Following buffer with of rinses. filtered, in chloroplasts cold materials of a and gray or (0-3) and to 1% 7.2 2% was The and agar was ethanol, methods silver DuPont citrate at 50KV. and were diamond examined for One-cm and squares 3% two two of acrolein hours hours tetroxide for were as and into twice (1969). with above again 1 mm in two of with in Callisia in leaf 0.02M tissue cacodylate the of samples buffer were

and 0.02M

fixation, a change 0.02M

cacodylate Post-fixation

osmium continued

cacodylate and followed in water. The dehydrated acetone, and sections MT2-B stained trans-

and

a water

pH

hours then in

leaf

squares

homogenized distilled sedimented. cubes, 100% Ultrathin a Sorvall were HS-8F-1

Omni-Mixer, was agar resuspended pellet series using the index and acetate of of

centrifuged at 60-70 trimmed rinsed of Spurr

solidified in a graded

embedded with refractive

obtained knife. in a

ultramicrotome with mission uranyl electron

glass lead

Sections Hitachi

microscope

1980

Thylakoid-DenseChloroplastsof Calilsiafragrans Results and discussion

115

In order to determine their uniqueness, chloroplasts of Callisia were compared with those from three vascular plants found to be often used in research, Pelargonium, Lycopersicon, and Cyphomandra. Although the study species was a monocot and the reference species were dicots, no distinctive differences have been reported between their plastids and they have been compared before (Laetsch 1971). Previous ultrastructural studies of Cyphomandra (Harris 1979), Pelargonium (Kirk and Tilney-Bassett 1967), and Lycopersicon (Arnott et al. 1969) have been reported.

Fig. The

1. total

Size

distribution (300-400 from

of

Callisia, chloroplasts)

Cyphomandra, for The each

Pelargonium species of was

and divided

Lycopersion into falling 10 classes in

chloroplasts. of 0.6m was

sample ranging

increments

1.8

to

8.4m.

percent

chloroplasts

each

class

computed.

Kirk chloroplasts have dra's sicon Most few

and to

Tilney-Bassett be 4-6m falling in the 4m of in the 5m range. Callisia 9m from in precisely class

(1967) diameter. in while There are range all four quite are this

report Callisia range of

the

average and the 1). from overlap

size three Callisia's Pelargonium between size is

of

higher

plant species

reference and and all 4

chloroplasts bulk are in are the

(Fig. those

CyphomanLycoperspecies. but the

most is

considerable normal somewhat species of were Callisia as far

chloroplasts which When occur

as

concerned

spectacular. subjected chloroplasts to various became sedimenta apparent

chloroplasts an interesting

tion

forces,

characteristic

116

Robert R. Wise and Joseph B. Harris

Cytologia 45

(Fig. 2). A force of 1000g was sufficient to sediment all of the chloroplasts from Callisia, but Lycopersicon had a few small chloroplasts which sedimented with forces of 2500 and 3000g, and both Pelargonium and Cyphomandra had a measurable amount in the 3000g fraction. The cholroplasts of Callisia were sedimented with much less force than those of the reference species.

Fig. 2. Percentof chloroplasts sedimented differentcentrifugal by forces. The three reference specieshad chloroplasts the 2000-3000grange,but all Callisia in chloroplasts were sedimented by 1000g. Because sedimentation rates are a function of size, density, and shape, the latter was investigated for Callisia with scanning electron microscopy (Figs. 3-5). They show the typical piano-convex form (Hall et al. 1974) common to chloroplasts of higher plants. Thus, other explanations for their unusual sedimentation property were sought. To verify that the Callisia chloroplasts were heavier due to their uniqu characteristics, and not to experimental techniques, two tests were undertaken. The sucrose molarity test subjected the chloroplasts of all four species under investigation to five different molar environments: 0.20M, 0.25M, 0.30M, 0.35M, and 0.40M sucrose in the SFD medium (Honda et al. 1966). If any of the chloroplasts were sensitive to osmostic pressure effects then the volume of the centrifuged pellet should have decreased with the increase in sucrose molarity. After an analysis of the data, it was found that this had little or no effect on the volume of the chloroplast pellet of any of the four species. In the bovine serum albumin (BSA) test, the chloroplasts of only Callisia were subjected to four different environments with different combinations of introduced and native proteins to look for non-osmotic pressure effects. The four sets of conditions were: 1) native leaf proteins (those released by homogenizing the leaf) present but BSA

1980

Thylakoid-Dense

Chloroplasts

of Callisia fragrans

117

Figs. 4-6m

3-5. size

Scanning demonstrated.

electron 3

micrographs shows surface relative features

of

Callisia abundance and irregular

chloroplasts. of plastids edges.

Plano-convex per cell. 4

shape and 5

and show

118

Robert

R. Wise and Joseph B. Harris

Cytologia 45

Figs. 6-9. Callisia chloroplasts sedimented with different g forces. 6 is from the 100g fraction, 7 from 250g, 8 from 500g, and 9 from the 1000g fraction. Although a size decrease seemed apparent with increasing g force statistical evaluation showed no difference, and no other differences exist between the fractions. Note grana (G) and starch granules (S).

1980

Thylakoid-Dense

Chloroplasts

of Callisia fragrans

119

Figs. 10-15. Late stages in chloroplast ontogeny in Callisia. 10 is identified as representative of young chloroplasts by prolamellar body (PB), small starch granule (S), and sparse grana (G). 11-14 demonstrate the increase in grana (G), starch granule (S) size, and stromal thylakoids (ST), with a decrease in stroma (SA). 15 is identified as aging by the disassociation of thylakoids and the black, osmiophilic plastoglobuli. Mitochondria (M) appear in 12 and 15.

120

RobertR. Wiseand JosephB. Harris

Cytologia 45

absent, 2) both native leaf proteins and BSA absent, 3) native leaf proteins absent but BSA present, and 4) both native leaf proteins and BSA present. When the data were analyzed, no swelling responses were noted. Small volume changes due to illumination intensities have been reported for some species (Zurzycki and Metzner 1977, Godziemba-Czyz 1975), but this was not investigated in this study. Callisia chloroplasts sediment at relatively low g forces (Fig. 2), and it appears

Fig. 16.

The dense thylakoid system of Callisia chloroplasts. Bifurcating thylakoids starch granule (S), and the peristarch matrix (PM) were observed.

(arrow),

1980

Thylakoid-Dense Chloroplasts Callisia of fragrans

121

that whatever property or properties contribute to this phenomenon are shared by a very high percentage of the plastids. Light microscopy investigations (Figs. 6-9) showed no significant difference in size (see Table 1) or shape between individual plastids at the four sedimentation forces applied. Table 1 shows that the size averages at the same sedimentation forces and although there is decrease in size with increasing g force the differences are not enough to explain the results shown in Fig. 2. Sedimentation of different numbers of chloroplasts with different forces might be explained by variations in internal structure as indicated in Figs. 10-15. Table 1: Relationship between Callisia chloroplast force and requiredfor sedimentation

Several unique ultrastructural features were found in the chloroplasts of Callisia that have not been reported elsewhere. Relatively young ones appear like other developing chloroplasts (Fig. 10), but the increase of both granal and stromal thylakoids continues during development until the plastids are entirely filled with them (Figs. 11-15). This abundance of internal structure might explain why they were sedimented at low g forces: they were more dense because there was so much more to their thylakoid systems than the reference species (Arnott 1969, Harris 1979, Kirk and Tilney-Bassett 1967). The development of the thylakoids seemed to continue to the complete exclusion of the stroma (Fig. 16). At this point, it was somewhat difficult to ascertain if the thylakoids were granal or stromal in nature. This suggests that functional distinctions may then be of little value. Ultrastructure work on Tradescantia spp. chloroplasts (Gyurjan et al. 1977, Keresztes 1971, Keresztes and Faludi-Daniel 1973) does not show this abundance or complexity of the thylakoid system except in relatively rare cases. A peculiar thylakoid/starch grain association seemed to show a distinct peristarch matrix (Figs. 11, 13, 21, and 23). This was not observed in all cases. Figures 14, 16, and 18 show an electrontransparent mass surrounding the starch grains, as is typical for other species. At the perpendicular junctions of two grana, the interrupted thylakoids appeared to end in blind sacs and were not continuous to the other grana (Fig. 17, arrow). Other thylakoids that intersect at lesser angles may come to lie parallel with intersected grana (Fig. 17, left of arrow). The branching of the thylakoids of Callisia deserves special attention. Multiple branches and forks have been found (Fig. 19) between separate grana. Although bifurcations are not uncommon (Hall et al. 1974, Gregory 1971, Wehrmeyer 1964) and have been included in explanations of chloroplast ultrastructure, branching to this degree has not been previously reported. Also present are what may be either a twisting or a multiple branching of thylakoids (Fig. 20). In either case, the activity is another of Callisia chloroplast's peculia-

122

Robert R. Wise and Joseph B. Harris

Cytologia 45

Figs. 17-18. 17, abundant thylakoids and their perpendicular junctions (arrow). At the junction they appeared to end in blind sacs, thus seemed discontinuous. Starch granules (S) and a mitochondrion (M) are indicated. 18, broadening and branching of thylakoids more clearly observed in older chloroplasts, either surrounding a starch granule or free in the stroma (arrows). Also apparent are grana (G) and stromal thylakoids (ST) and the close association between the stromal thylakoids.

1980

Thylakoid-Dense

Chloroplasts

of Callisia fragrans

123

Figs. 19b is

19-21. an

19

and

b, of

high 19a to This

magnification to grana stroma emphasize (arrows). is by seldom the

view the 21, found

(117,000~) system's densely in grana

of

thylakoid 20 grana a (G),

junction and strach it b,

complex. diagonal (S), is and

interpretation of stroma thylakoids (SA).

branching. packed Callisia systems.

arrangement a granular

chloroplasts

where

usually

excluded

extensive

124

Robert R. Wise and Joseph B. Harris

Cytologia

45

Figs. 22-23. 22, numerous thylakoids and grana typical of Callisia chloroplasts. Of particular interest is the plastosome-thylakoid relationship (arrow), where the body appears to be continuous with three thylakoids and is bound by the same membrane that defines them. A second plastosome was sectioned tangentially (below arrow). 23, connections between adjacent grana via numerous stromal thylakoids. This is a mature plastid as evidenced by the plastoglobuli (P) and the abundance of thylakoids. The arrows indicate thylakoid branching around starch granules.

1980

Thylakoid-Dense

Chloroplasts

of

Callisia

fragrans

125

rities. Although been (Figs. in replaced 11-13, protein few by 21). synthesis, how ribosomes of RNA the of the If the thylakoid the and typical would and granularity since Callisia seem DNA to the chloroplasts system), contained when it due is the function investigation. were not undertaken extensive was to found ribosomes, site of with Calvin so stroma it was since (it highly they all having granular function

is mostly stroma chloroplasts deserve content

carbon-fixation little stroma and studies seem

reactions, so and to be few

Carbon and

fixation certainly

studies needed.

Membrane-bound 1978), In with bounds one a and a peculiar (Fig. The

plastosomes plastosome/thylakoid 2.2) three thylakoid The numbers that Spinacia has 15.1 and an by of role of

have

been

reported relationship

in

chloroplasts appeared in be same .

(Harris Callisia.

specimen plastosome. the plastosome. the shows and Callisia 1979) has

parallel membrane this

thylakoids appears

are to is not

seen be

to the

continuous one that

relationship per with in 39.1 chloroplast the Callisia

known in

Counting micrographs Lycopersicon, numerous. (Harris

thylakoids

published of

electron

comparison the thylakoids of

chloroplasts are

Cyphomandra, twice Cyphomandra 1969) 1976) has has 26.9 17.3 as

approximately while et al. and

average

thylakoids/m, (Arnott 1975

thyakoids/m, Spinacia (Sprey

Lycopersicon and Laetsch

thylakoids/m, thylakoids/ m.

Summary

Using scopy, were unique observed.

the

techniques features In size, comparable Callisia in the There a was yet force highly showed of of

of the

fractional chloroplasts and to in those

centrifugation, of their Callisia

light fragrans stages for from of

and

electron (Linda.)

microWoods these

shape,

different

development, Pelargonium, in sedimentation occupied chloroplasts Starch-assoPlastosomes Plastoglobuli by

chloroplasts and properties thylakoid sedimented ciated not were stroma numerous, observed. Cyhomandra. and

were

described differ volume no stroma. were dense very in of

Lycopersicon, others chloroplast

chloroplasts high almost g. There and

relatively was 1000 granular unique

the All

the were

system. by

Callisia fragments. cases. thylakoids.

few most with

were

relationships

Acknowledgements

The for the Pape-r presented Arts, and

authors

wish of Hilkka the

to

express study species,

their Dr.

thanks John

to D.

Dr. Curtis

Robert for and SEM. Academy to

W. critical the This

Freckmann review Institute work of of of was

identification manuscript, Chemistry, in part

Kaustinen

for Wisconsin

technical for of use the

assistance, of their

Appleton, before the

1978

meeting

Wisconsin

Sciences,

Letters.

126

Robert R. Wise and Joseph B. Harris References

Cytologia 45

Arnott, Howard J., Rosso, Samuel W. and Smith, Kenneth M. 1969. Modification of plastid ultrastructure in tomato leaf cells infected with tobacco mosaic virus. J. Ultrastructure Res. 27: 149-167. Bronchart, R. 1967. Effets de la duree des jours sur la structure du chloroplaste d'epinard. In: Le chloroplaste croissance et vieillissement. Edited by C. Sironval. Masson et Cie, Paris. Descomps, S. and Deroche, M. -E. 1973. Action de 1'eclairement continu sur 1'appareil photosynthetique de la Tomate. Physiol. Veg. 11: 615-631. Godziemba-Czyz, J. 1975. Conformational changes in spinach (Spinacia oleracea) leaf chloroplasts in vivo. Acta Soc. Bot. Pol. 44: 277-287. Granick, S. and Porter, K. R. 1947. Stucture of the spinach chloroplast as interpreted with the electron micorscope. Am. J. Bot. 34: 545-550. Gregory, R. P. F. 1971. Biochemistry of Photosynthesis. Wiley Interscience, a division of John Wiley and Sons, Ltd.: London. New York. Gyurjan, I., Nagy, Anna H. and Keresztes. A. 1977. Sturcture and macromolecular composition of defected chloroplasts in variegated leaves of Tradescantia albiflora. Photosynthetica 11: 167-175. Hall, J. D., Barr, R., Al-Abbas, A. H. and Crane, F. L. 1972. The ultrastructure of chloroplasts in mineral-deficient maize leaves. Plant Physiol. 50: 404-409. Hall, J. L., Flowers, T. J. and Roberts, R. M. 1974. Plant Cell Structure and Metabolism. Longman Group Ltd., London. Harris, Joseph B. 1979. Development of a tubular apparatus in chloroplasts of aging Cyphomandra leaves. Cytobios 21: 151-167. Honda, S. I., Hongladarom, Tasani and Laties, G. G. 1966. A new isolation medium for plant organelles. J. Exp. Bot. 17: 460-472. Keresztes, A. 1971. Light microscopic examination of chloroplast mutation in Tradescantia leaves. Acta bot. Acad. Sci. hung. 17: 379-389. - and Faludi-Daniel, Agnes 1973. Ultrastructure, pigment content, and photosynthestic activity of the normal and mutant chloroplasts in developing Tradescantia leaves. Acta biol. Acad. Sci. hung. 24: (3-4): 175-189. Kirk, J. T. O. and Tilney-Bassett, R. A. E. 1967. The Plastids. Their Chemistry, Structure, Growth and Inheritance. W. H. Freeman and Co., London and San Francisco. Laetsch, W. M. 1971. Chloroplast structural relationships in leaves of C4 plants. In: Photosynthesis and Photorespiration. Edited by M. D. Hatch, D. B. Osmond and R. O. Slatyre. John Wiley and Sons, Inc, New York 1971. Leyon, H. 1953. The structure of chloroplasts. An electron microscopical investigation of section. Exp. Cell Res. 4: 371-382. Sprey, B. and Laetsch, W. M. 1975. Chloroplast envelopes of Spinacia oleracea L. I. Polypeptides of chloroplast envelopes and lamellae. Z. Pflanzenphysiol. 75: 38-52. - and -. 1976, Chloroplast envelopes of Spinacia oleracea L. II. Ultrastructure of chloroplast envelopes and lamellae. Z. Pflanzenphysiol. 78: 146-163. Spurr, Arthur R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastructure Res. 26: 31-43. Valanne, Niina. 1977. Effect of continuous light on C02 fixation, chlorophyll content, growth and chloroplast structure in Ceratodon purpureus. Z. Pflanzenphysiol. 81: 347-357. Wehrmaeyer, W. 1964. Zur Klaerung der strukturellen Variabilitat der Chloroplastengrana des Spinats in Profil and Aufsicht. Planta 62: 272-293. Zurzycki, J. and Metzner, H. 1977. Volume changes in chloroplasts in vivo at high densities of blue and red radiation. Photosynthetica 11: 260-267.