Planta (Berl.) 72, 223--231 (1967)

L E A F GI%OWTH O F W H I T E M U S T A R D (SINAPIS IN DIFFERENT ENVIRONMENTS

ALBA)

E. C. HUMPHI~IES Rothamsted Experimental Station, Harpenden, Herts., England Received August 23, 1966

Summary. The numbers of cells and area of fully expanded leaves were determined on successive leaves of Sinapis alba grown either in 8 hr. photoperiod (vegetative plants) or 16 hr. photoperiod (flowering plants) at a constant temperature of 20~ In the 8 hr. photoperiod leaf 9 had the greatest area but leaf 12 had most cells. I n 16 hr. photoperiod leaf 5 had the greatest area but leaf 9 had most cells. The relationship between area and cell number of successive leaves on the main stem fell into 3 distinct phases: in phase (1), cell number increased at a greater rate than leaf area; in phase (2), leaf area decreased while cell number increased; in phase (3), cell number and leaf area decreased proportionally. For an increase in unit area, cell number increased more in 8 ha,. than in 16 hr. photoperiod. Using final area and final cell number of successive leaves, by extrapolation the cell number of unit area of primordium has been deduced. Cell number per unit area increased in successive primordia up to a certain node after which it remained constant at succeeding nodes. It was found that in plants grown under different conditions the cell number per unit area in successive primordia increased at a constant logarithmic rate. That is, cells became progressively smaller. It is concluded that changes in cell size of successive primordia are not influenced by the environment but are under internal control. Introduction B o t h i n t e r n a l a n d e x t e r n a l factors affect leaf growth b u t it is difficult to assess the c o n t r i b u t i o n of each to final leaf size. Also u n k n o w n is exactly how the sequence of events i n a n ageing p r i m o r d i n m affects the size of successive leaves. I n some species e. g. Lycopersicon spp. (WHAL]~Y, 1939) t h e apical m e r i s t e m increases i n size as the p l a n t develops b u t this m a y n o t be generally t r u e (ALLsOPP, 1965). F i n a l size of successive leaves does n o t parallel increasing apical size a n d usually, in a given e n v i r o n m e n t , successive leaves on a stem increase i n size u n t i l a maxim u m is a t t a i n e d after which t h e y become smaller. F o r instance, i n Majestic p o t a t o the 15th leaf on the m a i n stem was u s u a l l y the largest (HvMPm~IES a n d FRenCH, 1961) whereas i n sugar beet m a x i m u m leaf size was b e t w e e n the fifth a n d t e n t h node (ttvMPH~I~s a n d FRENCH, 1965) a n d the largest t o m a t o leaf was a b o u t the f o u r t e e n t h ( C o o P ~ , 1959). Sinapis alba shows a clear sequence of leaf sizes; a n d although the node a t which m a x i m u m size is a t t a i n e d depends on the e n v i r o n m e n t the sequence of leaf size is so regular as to suggest t h a t i n t e r n a l control

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is also concerned. P r e v i o u s l y r e l a t i v e sizes of successive leaves h a v e been s t u d i e d o n l y in c h a n g i n g e n v i r o n m e n t s , so t h a t t h e c o n t r i b u t i o n s of e x t e r n a l a n d i n t e r n a l factors could n o t be s e p a r a t e d . I n t h e w o r k n o w to be d e s c r i b e d cell n u m b e r a n 4 leaf a r e a of successive leaves on t h e m a i n s t e m of Sinapis alba growing u n d e r cont r o l l e d conditions were d e t e r m i n e d a n d t h e results u s e d to d e d u c e cell size in t h e p r i m o r d i a p r o d u c i n g t h e leaves. S. alba requires a t least a 10-hour p h o t o p e r i o d to p r o d u c e flowers (BElcNInlL 1964), a n d b y growing t h e p l a n t s in 8 hr. a n d 16 hr. p h o t o p e r i o d s leaf d e v e l o p m e n t was s t u d i e d on p l a n t s w i t h a n d w i t h o u t flowers.

Methods Plants were grown in cabinets at a light intensity of 1600 f. c. provided by fluorescent tubes at a constant temperature of 20 o and sampled at intervals when particular leaves were fully expanded, determined by frequent measurements of length. In some plants growth was modified by applying CCC to the soil. Only 3 or 4 consecutive leaves were removed from a plant before it was discarded. Cell number and leaf area are the mean of four plants. A fully expanded leaf was removed and its area determined; disks of known area were removed, macerated in a buffered mixture of EDTA and pectinase at 37 o for 2 days, and squirted through a fine pipette which separated the cells without damaging them, and the cells were then counted on a haemocytometer slide (Hvlwem~IES and Wm~EL~R, 1960). Successive leaves were similarly treated as they became fully expanded.

Results Fig. 1 shows areas a n d cell n u m b e r s of successive leaves of p l a n t s g r o w n in 8 hr. p h o t o p e r i o d . Successive leaves b e c a m e p r o g r e s s i v e l y l a r g e r u p t o leaf 9 which h a d t h e g r e a t e s t area, b u t cell n u m b e r increased u p t o leaf 12. I n t h e 16 hr. p l a n t s leaf 5 h a d t h e g r e a t e s t a r e a b u t leaf 9 h a d m o s t cells (Fig. 2). Thus, a l t h o u g h leaf 9 h a d m a n y m o r e cells t h a n leaf 5 its a r e a was less. L e a v e s 6 a n d 13 h a d similar n u m b e r s of cells b u t areas of 70 cm 2 a n d 20 cm ~ respectively. W h e n v a l u e s are p l o t t e d l o g a r i t h m i c a l l y (Fig. 3) t h e r e l a t i o n s h i p b e t w e e n a r e a a n d cell n u m b e r shows 3 d i s t i n c t phases. I n t h e first, cell n u m b e r increases f a s t e r t h a n leaf a r e a (e. g. leaves 1 to 6 in t h e 16 hr. plants). I n t h e second, leaves b e c o m e smaller while cell n u m b e r cont i n u e s t o increase (leaves 7 to 9). I n t h e t h i r d phase, leaf a r e a a n d cell n u m b e r b o t h decrease p r o p o r t i o n a l l y (leaves 10 t o 17). F o r a g i v e n increase in area, in p h a s e 1, cell n u m b e r increases m o r e in a n 8 hr. p h o t o p e r i o d t h a n 16 hr. The processes leading to t h e final size of a leaf were s t u d i e d in d e t a i l in t h e p r i m a r y a n d first t r i f o l i a t e leaves of d w a r f b e a n (I~uMPItRIES a n d W H ~ E L ~ , 1964). I n t h e e a r l y stages of a leaf d e v e l o p i n g f r o m t h e prim o r d i u m u n t i l i t is 1 or 2 e m 2 in area, g r o w t h t a k e s place e n t i r e l y b y cell division a n d a r e a is p r o p o r t i o n a l to cell n u m b e r . B o t h a r e a a n d cell

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n u m b e r increase e x p o n e n t i a l l y a t this stage a n d the a l l o m e t r y formula, y = b x k or log y = l o g b -~ k log x, i n which y = n u m b e r of cells, x = area a n d ]c is a c o n s t a n t , is applicable. The general use of this f o r m u l a 09 10 Z! 12 7.3 Zr 1..5" ZB ZT g81.3

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has been critieised because the value of " k " is sometimes calculated from loa~s of a growth curve t h a t are n o t exponential. This criticism does n o t a p p l y here, a n d as ~he line is inclined a t a n angle of 45 o t h e value of/~ == 1, so t h a t x a n d y have a linear relationship w i t h o u t t r a n s f o r m i n g to logarithms.

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Fig. 4 shows the relation between cell number and area of the primary and the first trifoliate leaf of dwarf bean (HvMPHmv, S and W~v~v,L ~ , 1964). The middle portion of the curve is inclined at 45 o to the ordinates (k = 1). The first part of the curve, which is less steep, applies only to the primary leaf and represents a phase when cells of the embryonic leaf in the seed imbibes water during germination and few cells are dividing. This part of the curve would not apply to a leaf developing from a primordium. The flatter part of the curve at the top is the phase when cells are expanding but few cells are dividing. This phase is common to the development of all leaves whether from a seed or a primordinm. If curves relating number and leaf area for each leaf could be constructed it would be relatively simple to extrapolate from the middle part of the curve to estimate cell number at any particular leaf size and so estimate cell number in unit area of a primordium. This would be very laborious, but using the final area and final cell number for each leaf and extrapolating from each point at an angle of 450 cell number can be deduced for a given area. This is illustrated in the inset of Fig. 4; it is apparent that the true cell number will be underestimated by the amount that cell number and leaf area increase in the third phase. B y doing this for each successive leaf on the plant, cell number per unit area of primordium can be estimated. Cell numbers for unit area (say 1 mm 2) of successive primordia were so calculated for plants grown under the following conditions : 1. 2. 3. 4. 5. 6.

8 hr. 8 hr. 16 hr. 16 hr. in the in the

photoperiod 20 ~ photoperiod 200 plants treated ~dth CCC, photoperiod 20 ~ photoperiod 200 plants treated with CCC, glasshouse in June, glasshouse in September.

Cell number per unit area increased in successive primordia up to a certain node and then cell number became constant for succeeding nodes. Fig. 5 shows the fitted values for the intercept of cell number at constant area (i. e. b in the allometry equation) for the various environments. The value of b increases linearly up to a certain level of insertion (see regression coefficients in Table) and the rate of increase is constant for all the environments investigated (the linear regressions of increase in b are similar). I t is concluded that cell number per unit area of primordium increases in a constant logarithmic proportion up to leaf 12 or 13 and then becomes constant. Increase in cell number is just as regular in plants grown in a fluctuating environment as in plants under a constant environment. The only extensive series of measurements of leaf area and cell number of successive leaves in the literature seems to

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be that of AshBY (1948) for I p o m o e a plants grown in the glasshouse. Fig. 6 shows these results plotted on a logarithmic scale as in Fig. 3. I n AshBY's work cell numbers were estimated by counting epidermal

Leaf Growth of Sinapis alba

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Table. ~egression coe/ficients (rate of increase in log celt number per unit area o/ primordium at successive nodes) appertaining to Fig. 5 Treatment

l%egressioncoefficientof increase in logcell number

16 hour, untreated 16 hour, CCC 8 hour, untreated June, glasshouse September, glasshouse

0.067 -}-0.011 0.069 • 0.002 0.064-~ 0.001 0.068 • 0.004 0.067 -t- 0.016

cells and these numbers have been multiplied by 5, assuming the leaf had 5 layers of cells, to give approximate estimates of cells per leaf. Cell number and leaf area bear a similar relationship to that found in Sinapis ; b increases progressively for the first few nodes and then remains constant. The primordium apparently reaches constant cell number per unit area at a lower node than in Sinapis. Discussion

I t is not possible to say from m y observations whether constant ceil size of the primordium is attained at the same stage that the apical growing point attains a constant size, but anatomical evidence in other species shows that the apical growing point attains a constant size at a certain point in development; moreover, increase in size of apex comes from increase in cell number rather than cell size (LEDr~, 1954). W~ALEY (1939) found a gradual increase in volume of the meristematic region of 3 tomato species accompanied by a diminishing cell size and suggested that the progressive decrease in cell size indicated that cell division proceeded faster than cell enlargement in the meristem. He found that a minimal cell size was attained which then remained constant. This agrees with the deductions made here. WESLEY also found that, as growth proceeded, the nuclear volume diminished but slower than cell size, so that the small cells characteristic of late growth stages had relatively larger nuclei and less cytoplasm than young seedling meristems. He suggested that the change in cell/nucleus ratio may be concerned in stopping growth. Whatever the explanation of the ontogenetie decrease in cell size it must take account of the fact that cells in a single leaf are not constant in size. Cells at the tip of a leaf are usually smaller and grow slower than those at the base (AvErY, 1933). If the above assumptions are justified changes in cell size of the primordium seem to be little affected by changes in the environment: they are similar in 8 hr. or 16 hr. photoperiod and therefore independent of total radiation. Previously it was suggested (see AS]~Bu 1948 for references) that competition for water determined leaf size so that leaves 16 Planta(Berl.), Bd. 72

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E.C. HUMPHRIES:

at the top of a plant were smaller because they are subject to greater water stress. This seems unlikely, because some plants develop large lateral systems at their tops which seem not to be restricted b y lack of water. A good example is the potato which often develops about 20 mainstem leaves and the axi]lary branches about the 17th and 18th nodes are very large. I t seems more likely t h a t cell size becomes restricted with increasing height of insertion of the leaf b y lack of growth factors. Possibly these factors diffuse upwards from the base of the plant. There is some evidence t h a t growth substances originate in the roots (see PATV,, 1966). This idea is supported b y experiments in which removing some leaves on a shoot causes developing leaves to have abnormally large cells. AsK]3Y concluded that, when environmental influences are eliminated, morphological differences between successive leaves remained, and these he attributed to ageing of the apical meristem. This itself is not a reason, but if ageing implies diminishing the supply of essential growth substances, then the explanation m a y be correct. I have att e m p t e d to produce roots on certain internodes of Sinapis to see whether the leaves above the root system were influenced b y these roots but the a t t e m p t failed; b y the time the root system developed the leaves immediately above were too large for their size to be influenced. I t is also possible t h a t the systematic change in cell size happens because cell division proceeds faster t h a n cell enlargement, possibly because celldivision factors are not limiting but cell-enlargement factors are. The results from vegetative (8 hr.) and flowering (16 hr.) plants suggest t h a t the sequence of diminishing cell size in the primordium is not influenced b y induction of the flowering state. Both m y results and ASHBY'S show t h a t two leaves on the plant can have the same cell number but different areas. Could all cells expand to the same extent, the plant would have a much greater leaf area. This has practical implications but applying growth substances to increase leaf area has had only limited success. HUM~H~IES and F~ENCg (1961) increased the area of some potato leaves b y gibberellic acid, which increased both cell size and cell number. There is other evidence t h a t development of the primordium is independent of the current environment. FUL~O~D (1965) found apple leaves were produced at a constant rate over a long period of time. HU~PH~IWS and l ~ w ~ c ~ (1965) found t h a t sugar-beet leaves are also produced at a constant rate for a long time and t h a t the rate the leaves are produced later in life of a sugar-beet plant can be changed b y treating seedlings with growth substances. Similarly, exposing seedlings to different environments for a short period affected the rate leaves were produced during the next 20 weeks (HvMPHRrSS, 1966). The last observations m a y seem to contradict the general theme of this paper t h a t rate of leaf production is independent of environment but as FULFO~D (1OC.

Leaf Growth of Sinapis alba

231

cir.) suggests, the essential factor i n control of m e r i s t e m a t i c a c t i v i t y is the i n h i b i t o r y effect of a d j a c e n t primordia. I f the degree of m u t u a l interference b e t w e e n primordia is altered a t a n early stage of d e v e l o p m e n t w h e n few primordia have developed t h e n the s u b s e q u e n t rate of prod u c t i o n will also be altered. Acknowledgement. I thank Miss LESLEu :F]aEEI~AI~and Mrs. JEA~r GooncttILD for cell counts and leaf area estimations. References ALLSOPP, A. : tteteroblastic development in cormophytes. Handbuch der Pflanzenphysiologie, vol. 15 (1) (ed. R~m_~I)), p. 1172--1221. Berlin-Heidelberg-New York: Springer 1965. Asm3Y, E. : Studies in the morphogenesis of leaves. II. The area, cell size and cell number of leaves of lpomoea in relation to their position on the shoot. New Phytol. 47, 177--195 (1948). AVERY, G. S. : Structure and development of the tobacco leaf. Amer. J. Bot. 20, 565---592 (1933). BE~IER, G.: Etude hlstophysiologique et histochimique de l'6volution du m~rist~me apical de Sinapis alba L., cultiv6 en milieu conditionn~ et en diverses dure6s de jour favorables ou d6favorables &la raise s fleurs. Acad. Roy. Belg., C1. Sci. M~m. in 4 ~ S~r. II 16, 1--149 (1964). CooPv,~, A.J.: Observations on the growth of the leaves of glasshouse tomato plants between March and August. J. Hort. Sci. 84, 104---110 (1959). FULrORD, R. M. : The morphogenesis of apple buds. I. The activity of the apical meristem. Ann. Bot., N.S. 29, 167--180 (1965). ItVMPmXIES,E. C. : Internal control of rate of leaf production in sugar beet. Physiol. Plant. 19, 827---829 (1966). - - , and S.A.W. F~E~c~: Effect of nitrogen supply on the response of Majestic potato to gibberellic acid. Ann. appl. Biol. 49, 331--339 (1961). - - - - A growth study of sugar beet treated with gibberel]ic acid and (2-chloroethyl) trimethylammonium chloride (CCC). Ann. appl. Biol. 55, 159--173 (1965). - - , and A.W. W~]~.LER: The effects of kinetin, gibberellic acid, and light on expansion and cell division on disks of dwarf bean (Phaseolus vulgaris). J. exp. Bot. 11, 81--85 (1960). - - - - Cell division and growth substances in leaves. Reg. Nat. Croissance Vegetale, Gift 505--515 (1964). LEmN, R. B.: The vegetative shoot apex of Zea mays. Amer. J. Bot. 41, 11--17 (1954). PATE, J. S. : Photosynthesising leaves and nodula~ed roots as donors of carbon to protein of the shoot of the field pea (Pisum arvense L.). Ann. Bot., N.S. 30, 93--109 (1966). WH~a~v,r, W. G.: Developmental changes in apical meristems. Proc. nat. Acad. Sci. (Wash.) 25, 445--448 (1939). E. C. Hv~m~iEs Rothamsted Experimental Station Botany Department tIarpendcn, tIerts., Great Britain

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