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An autoradiographic study of root cell differentiation in zea mays
Life Sciences Vol . 8, Part II, pp . 683-687, 1989 . Printed in Great Britain.
Pergamon Press
AN AUTORADIOGRAPHIC STUDY OF ROOT CELL DIIrFERENTIATION IN ZEA hIAYS Bruce Pachtar, J. Nitre, and Leonard Amoral# Department of Biology, Oradnate School of Arte and Scienoea, New York University, Netr York 10003, and* Department of Pathology, Methodist Hospital, Brooklyn, N.Y, 1121: (Received 21 February 1989 ; in final form 27 March 1989) The use of root tips for the cytological study of plant cell differentiafion is advantageoua in that the transition of its cell types is represented in any lonPitudinal section, such aec'tions having clear demarcated $ones of meristam and root cap (basal, central and apical :zones). are highly specialized and short lived,
The apical root cap celle
Aa one proceeds from the meriatematic
region to the apical root cap region, two morphological changea in their reanective nuclei are evident : srucleoli decr~ase in volume . cell volume (1).
the chromatin becomes highly condensed, and the Concurrent with these changea is an increase in
Previous atudiea pertaining to nuclear differences during
differentiation in root cello have augeestad that DNA amounts par nucleus increase from merietematic to apical root cap region (2, 3) .
These findings
thus led to the question, "Dose the syntheaia of R~1A and protein undQrgo acco~apanying altsrationa during the process of cell differentiation, and if ao, what would this relationship auggeat?", With these questions in mind, intact Zea maw roots w~sre groin in one half strength Hoagland'a solution containing either of the followings
H3-TdR (S .A,
0.36 c/mM), H3-UdR (S .A, 5,0 c/selS), end A3-Leuoine (3,9 . 2.0 o/Sr~li) at a done of 1,0 ysc/cc for periods of 30 sainutea, 30 minutes, and 1 hour respectively, and aubaequently ohaaed for 2 hours in one half strength Hoagland'a éolntion . Roots ~rare fi.~oad in Camay's fizative, e~ebedded in paraffin and 5 micro longitudinal sections were node . Autoradiograma were perpared by the esaulaiondipping procedures (b), using NTH3 emulsion, lncubeted at 4°C for 10 days and developed and stained with haematoxylin end eoatn through the emulsion . 883
The
684
ROOT CELL DIFFERENTIATION
Vol . 8, No . 14
cross sectional area of nucleoli, nuclei and cytoplasm for each of the above $ones were obtained by aicrometer techniouea (5) .
The mean areas were deter-
mined fran a minimum of 100 cells per region. The number of grains (corrected for backround) over nucleoli, nuclei and cytoplasm for each of the aforemenboned regions divided by their respective areas results in the activity of that cellular constituent . The results presented in Table 1 auPaest that the amount of DVA synthesised in the basal root cap nuclei (nuclei of the cap initials) is higher than that of the meristematic region, and this is in agreement with the findings of others (1, 3) .
This increase in grain co~mts is a consequence of increased
DNA amounts . TABLE 1 a Standard Error The Mean Number of Graina/Cell/Region Incorporating Labeled Thymidine Mean Number of Grains/Cell Meriatematic Region
1l~2=6 .7a
Bsaal Root Cap Region
196_3 .3
The results presented in Figure 1 indicate that 1) nucleolar RA activity increases from meriatematic to the central root cap region and falls to aero in the apical sons, ard, 2) micleolar protein activity parallels that of RNA . In eaameh as these increases are similar, the incr~aaed activities noted may represent increased nucleolar organiser regions afforded bY polyploidy (6) from mariat~natic to central root cap regions . The activity of RNA synthesis in the nucleus, as danonstrated in !~igure 1 decreases from the region of the meristem to that of the apic~l root cap region. This decrease in activity is similar to that observed in systems xhich undergo
Vol . 8, No . 14
ROOT CELL DIFFERENTIATION
685
"end point" differentiation, i .e ., cells xhich differentiate to a final etaRe, and die .
The increase in the amount of protein synthesis observed (also
Figure 1) from the meristematic to the central root cap region may be the result of endoploidT, in that increases in DNA amounts vould be paralelled xith increases in nuclear histone .
7001 auc~Eaus
rlo a
n
< â
â u
wuc~EUs
CYTOPLAS~1
s" 0 r1 N~ _J ~~ IIAERISTEM
r~~A~
r-7 N ~~CÂP~f
r~ CA
Fia . 1 Histogram shoving the numtrr of grains per unit area (activity), over the nucleolus, mucleua and cytoplasw of celle Tram the meristem and root cap (basal,
686
ROOT CELL DIFFERENTIATION
Vol. 8, No . 14
central and apical regions) . The clear beta refer to H3-UdR, whereas the stripped bars refer to H3-d.eucine. The synthesis of cytoplasmic protein (Figure 1) increases fraw the meriatematic to the basal root cap region and decreases aarkedly in the central and apical root cap region . This decrease may be due to the possible eeeaation of mitotic activity of these cells .
Figure 1 also shone the increase of synthe-
sized RNA in the cytoplasm fran the meriatem to the basal root cap region, Since the activity of nucleolar RNA is greatest in the central root cap region, whereas cytoplaamic RIdA is least, it may be that the latter is the result of the inhibited transfer of nucleolar RWA to the cytoplasa of the esme cell .
The
increased cytoplasmic activity of RNA and protein fray the rieriatma to the basal root cap region is in accord with the asaumntion that cytoplasmic RNA and protein is abundant in rapidly dividing cells (7), ouch as the basal root cap cells . In summary, our findings suggest that as differentiation proceeds fray the iaerist~astic to the apical root cap regions, alterations in DHA, RNA and protein synthesis occur .
These observed alterations in our opinion suggest
that : 1) The increased amount of nucleolar protein found in the ce~ral root cap nucleus represents en accaaipaniraent to the increased amount of DNA . 2) The increased nucleolar activity found in the central root cap region may represent an increase in the amour of nucleolar organiser material which would be the result of the process of endoploidy . 3) The decreased amount of cytoplaamic RkA of the central root cap cell may be the result of the inhibition of transfer of nucleolar RNA to the cytoplasm. 4) ?he corked differences observed in the apical root cap region may represent cells which have reached the end point in their differentiation,
Vol . 8, No. 14
ROOT CELL DIFFERENTIATION
687
References 1.
J. Clause, J, p~tl . Hot. 9, 229 (1958) .
2.
H . Sui.ft, Proc, Natl .
3.
W.A . Jensen, L.i, Kavaljian and S, Marsinot, Sxptl. Cell Rea. 20, 361 (1960) .
!< .
L.F . Helanger and C.F, ieblond, Endocrinology 39, 8 (19116),
5.
D.P . Hloch, R.A, N'acquigg~ S .D . Hrock and J. Wu, J . Cell Hiol . 33, b51 (1967) . --
6.
K.~Lin, Chromosome T, 3110 (1955) .
7.
T. Casperason and J. Schahs, Rature 142, 2911 (1938) .
Aced Sci ., Wash . 36, 6lî3 (1950) .
Pergamon Press
AN AUTORADIOGRAPHIC STUDY OF ROOT CELL DIIrFERENTIATION IN ZEA hIAYS Bruce Pachtar, J. Nitre, and Leonard Amoral# Department of Biology, Oradnate School of Arte and Scienoea, New York University, Netr York 10003, and* Department of Pathology, Methodist Hospital, Brooklyn, N.Y, 1121: (Received 21 February 1989 ; in final form 27 March 1989) The use of root tips for the cytological study of plant cell differentiafion is advantageoua in that the transition of its cell types is represented in any lonPitudinal section, such aec'tions having clear demarcated $ones of meristam and root cap (basal, central and apical :zones). are highly specialized and short lived,
The apical root cap celle
Aa one proceeds from the meriatematic
region to the apical root cap region, two morphological changea in their reanective nuclei are evident : srucleoli decr~ase in volume . cell volume (1).
the chromatin becomes highly condensed, and the Concurrent with these changea is an increase in
Previous atudiea pertaining to nuclear differences during
differentiation in root cello have augeestad that DNA amounts par nucleus increase from merietematic to apical root cap region (2, 3) .
These findings
thus led to the question, "Dose the syntheaia of R~1A and protein undQrgo acco~apanying altsrationa during the process of cell differentiation, and if ao, what would this relationship auggeat?", With these questions in mind, intact Zea maw roots w~sre groin in one half strength Hoagland'a solution containing either of the followings
H3-TdR (S .A,
0.36 c/mM), H3-UdR (S .A, 5,0 c/selS), end A3-Leuoine (3,9 . 2.0 o/Sr~li) at a done of 1,0 ysc/cc for periods of 30 sainutea, 30 minutes, and 1 hour respectively, and aubaequently ohaaed for 2 hours in one half strength Hoagland'a éolntion . Roots ~rare fi.~oad in Camay's fizative, e~ebedded in paraffin and 5 micro longitudinal sections were node . Autoradiograma were perpared by the esaulaiondipping procedures (b), using NTH3 emulsion, lncubeted at 4°C for 10 days and developed and stained with haematoxylin end eoatn through the emulsion . 883
The
684
ROOT CELL DIFFERENTIATION
Vol . 8, No . 14
cross sectional area of nucleoli, nuclei and cytoplasm for each of the above $ones were obtained by aicrometer techniouea (5) .
The mean areas were deter-
mined fran a minimum of 100 cells per region. The number of grains (corrected for backround) over nucleoli, nuclei and cytoplasm for each of the aforemenboned regions divided by their respective areas results in the activity of that cellular constituent . The results presented in Table 1 auPaest that the amount of DVA synthesised in the basal root cap nuclei (nuclei of the cap initials) is higher than that of the meristematic region, and this is in agreement with the findings of others (1, 3) .
This increase in grain co~mts is a consequence of increased
DNA amounts . TABLE 1 a Standard Error The Mean Number of Graina/Cell/Region Incorporating Labeled Thymidine Mean Number of Grains/Cell Meriatematic Region
1l~2=6 .7a
Bsaal Root Cap Region
196_3 .3
The results presented in Figure 1 indicate that 1) nucleolar RA activity increases from meriatematic to the central root cap region and falls to aero in the apical sons, ard, 2) micleolar protein activity parallels that of RNA . In eaameh as these increases are similar, the incr~aaed activities noted may represent increased nucleolar organiser regions afforded bY polyploidy (6) from mariat~natic to central root cap regions . The activity of RNA synthesis in the nucleus, as danonstrated in !~igure 1 decreases from the region of the meristem to that of the apic~l root cap region. This decrease in activity is similar to that observed in systems xhich undergo
Vol . 8, No . 14
ROOT CELL DIFFERENTIATION
685
"end point" differentiation, i .e ., cells xhich differentiate to a final etaRe, and die .
The increase in the amount of protein synthesis observed (also
Figure 1) from the meristematic to the central root cap region may be the result of endoploidT, in that increases in DNA amounts vould be paralelled xith increases in nuclear histone .
7001 auc~Eaus
rlo a
n
< â
â u
wuc~EUs
CYTOPLAS~1
s" 0 r1 N~ _J ~~ IIAERISTEM
r~~A~
r-7 N ~~CÂP~f
r~ CA
Fia . 1 Histogram shoving the numtrr of grains per unit area (activity), over the nucleolus, mucleua and cytoplasw of celle Tram the meristem and root cap (basal,
686
ROOT CELL DIFFERENTIATION
Vol. 8, No . 14
central and apical regions) . The clear beta refer to H3-UdR, whereas the stripped bars refer to H3-d.eucine. The synthesis of cytoplasmic protein (Figure 1) increases fraw the meriatematic to the basal root cap region and decreases aarkedly in the central and apical root cap region . This decrease may be due to the possible eeeaation of mitotic activity of these cells .
Figure 1 also shone the increase of synthe-
sized RNA in the cytoplasm fran the meriatem to the basal root cap region, Since the activity of nucleolar RNA is greatest in the central root cap region, whereas cytoplaamic RIdA is least, it may be that the latter is the result of the inhibited transfer of nucleolar RWA to the cytoplasa of the esme cell .
The
increased cytoplasmic activity of RNA and protein fray the rieriatma to the basal root cap region is in accord with the asaumntion that cytoplasmic RNA and protein is abundant in rapidly dividing cells (7), ouch as the basal root cap cells . In summary, our findings suggest that as differentiation proceeds fray the iaerist~astic to the apical root cap regions, alterations in DHA, RNA and protein synthesis occur .
These observed alterations in our opinion suggest
that : 1) The increased amount of nucleolar protein found in the ce~ral root cap nucleus represents en accaaipaniraent to the increased amount of DNA . 2) The increased nucleolar activity found in the central root cap region may represent an increase in the amour of nucleolar organiser material which would be the result of the process of endoploidy . 3) The decreased amount of cytoplaamic RkA of the central root cap cell may be the result of the inhibition of transfer of nucleolar RNA to the cytoplasm. 4) ?he corked differences observed in the apical root cap region may represent cells which have reached the end point in their differentiation,
Vol . 8, No. 14
ROOT CELL DIFFERENTIATION
687
References 1.
J. Clause, J, p~tl . Hot. 9, 229 (1958) .
2.
H . Sui.ft, Proc, Natl .
3.
W.A . Jensen, L.i, Kavaljian and S, Marsinot, Sxptl. Cell Rea. 20, 361 (1960) .
!< .
L.F . Helanger and C.F, ieblond, Endocrinology 39, 8 (19116),
5.
D.P . Hloch, R.A, N'acquigg~ S .D . Hrock and J. Wu, J . Cell Hiol . 33, b51 (1967) . --
6.
K.~Lin, Chromosome T, 3110 (1955) .
7.
T. Casperason and J. Schahs, Rature 142, 2911 (1938) .
Aced Sci ., Wash . 36, 6lî3 (1950) .