P|anta (1993)191:433~439 P l a n t a

�9 Springer-Verlag 1993

Growth and development of sorghum leaves under stress

Spatial and temporal aspects of leaf growth inhibition

Nirit Bernstein*, Wen@ K. Silk, Andr~ L~iuchli Department of Land Air and Water Resources, University of California, Davis, CA 95616, USA

Received: 8 February 1993 / Accepted: 27 March 1993

conditions of NaCI

Abstract. Elevated concentrations of NaC1 in plant growth media cause a reduction in leaf growth. In grass- es, where the leaf growth zone is a small part of the entire leaf, it is important to locate the expanding region and spatially quantify the extent of growth reduction under stress. This will allow comparisons and correlations with possible physiologically important factors. We studied the spatial distribution of growth within the intercalary growth zone of sorghum (Sorghum bicolor [L.] Moench, cv. NK 265) leaves. Salinity (100 mM NaC1) shortened the length of the growth zone from 30 mm in the controls to about 24 mm, and reduced the maximal relative ele- mental growth rate (REG rate). The extent of growth inhibition varied with spatial location along the elonga- tion region. The growth in the basal 3-9 mm of salinized plants was not affected. Young leaves, while still enclosed in the protected whorl of older sheaths and elongating rapidly, were affected more severely by the stress than emerged leaves. The distribution of growth along the leaf growth zone was not steady throughout the elongation period. The length of the elongation region of young non- emerged leaves increased with leaf age and reached a maximum of about 30 mm at leaf emergence. Toward the end of the elongation period, the growth zone shortened for both the control and salinized leaves, and was con- fined to more basal regions. The maximal REG rate in- creased after leaf emergence, the increase being larger in control than salinized leaves. Toward the end of the elon- gation period, when growth was no longer linear with respect to time, the maximum REG rate decreased and its position shifted closer to the leaf base. Growth profiles of leaves 8, 9 and 10 were similar in both control and salinized plants. This suggests that within that time frame of plant development, successive leaves are similarly af- fected by the stress, and that the length of time the plant

Present address: * Institute of Soil and Water, The Volcani Center, P.O. Box 6, Bet-Dagan 50-250, Israel Abbreviations: REG rate = relative elemental growth rate Correspondence to: Dr. N. Bernstein; FAX: 972 (3)9604017

was exposed to a steady level of salinity plays no role in specific leaf inhibition.

Key words: Growth zone - Kinematics - Leaf growth - Salt stress - Sorghum (leaf growth, salt stress)

Introduction

Salinity reduces growth, especially shoot growth and leaf expansion, and delays development of many plants. The mechanism of inhibition of leaf elongation is controver- sial. During long-term exposure, NaC1 inhibition of leaf expansion may be associated with a reduction in leaf turgor caused by low water potentials in the root medi- um; reduction in net CO2 assimilation in the mature part of the leaf; or excessive accumulation of ions, which can induce ion toxicity, ion deficiencies or both (Greenway and Munns 1980; Munns and Termaat 1986; Cramer and Bowman 1991). Specific ions may alter growth directly if they take effect in the growing part of the leaf, or indirect- ly through some influence originating elsewhere in the plant.

To understand better the physiological mechanisms by which leaf expansion is inhibited, it is necessary to study the growing region of the leaf. In grasses, elonga- tion is confined to the basal part of the leaf, which is enclosed within a whorl of encircling older leaf sheaths. Although the growth zone is concealed, grass leaves are well suited for the study of leaf growth processes because of the relatively simple organization of the elongation zone (Volenec and Nelson 1981; Schnyder and Nelson 1988). Expansion is predominantly unidirectional, and a tissue element is displaced away from the leaf base as a result of production of younger tissue and longitudinal growth below it. Elongation is not uniformly distributed throughout the leaf growth zone, but varies as a function of distance from the leaf base (Schnyder and Nelson 1987; Walker 1988; Meiri et al. 1992). The kinematics of leaf growth are known to vary with irradiance (Schnyder

434 N. Bernstein et al.: Sorghum leaf growth under NaC1

and Nelson 1989), genotype (Volenec and Nelson 1981), time of day (Schnyder and Nelson 1988) leaf developmen- tal stage (Schnyder et al. 1990), and leaf number (Meiri et al. 1992).

The observed reduction in final leaf length under salinity can result from a shortened elongation period and/or a reduced leaf growth rate. Furthermore, environ- mental variations could reduce the leaf growth rate by shortening the length of the elongation zone and/or de- creasing local magnitudes of expansion. No information is currently available on salinity effects on the spatial distribution of elongation along leaves.

The objective of the present study was to evaluate quantitatively the inhibitory effects of NaC1 on spatial and temporal distributions of sorghum leaf growth. These detailed assessments of the growth fields will provide the foundation for physiological studies, involv- ing local factors such as solutes, osmotic potential, ions and nutrients, or signals from other plant parts.

Materials and methods

Plant material and growth conditions. Seeds of sorghum (Sorghum bicolor [L.] Moench, cv. NK 265) were germinated on a stainless- steel screen over a 1/10-concentration, aerated, modified Hoagland solution (Epstein 1972). The container holding the screen and seeds was sealed with a plastic wrap to maintain high humidity, and was placed in a growth chamber in the dark, at 27~ After 3 d the seedlings were exposed to a 15-h photoperiod and were kept for an additional 3 d. Seedlings chosen for uniformity were then trans- ferred to 4-L containers, (one seedling per container), and nutrient solution increased to 1/2 concentration, except iron was supplied as 50 laM Fe-EDTA and 20 gM Fe(NH4804)2, and the Na level was elevated to 1 raM. Nutrient solutions were changed every other day. The pH was adjusted daily to 5.7 with 100 mM KOH solution. At any point in time the solution pH did not decrease below pH 5.0. Humidity averaged 65% during the light period and 75% at night. Plants were exposed to a photon fluence rate of 750 gmol �9 m 2. s ' during the 15-h light period. Temperature was kept at 28~ mea- sured at the base of the plant, which is the region of leaf growth.

Salinization. Salinization commenced on day 8 after the beginning of germination, at which time leaf 4 had not yet emerged from the whorl of (enclosing) older leaf sheaths, and leaf 6, if visible as a primordium, had not yet begun rapid elongation. Sodium chloride was increased to 25 mM on the first day of salinization, and later by daily increments of 25 mM - d ' until the desired concentration of 100 mM was reached. The salt was added exactly 1 h after the transition to the dark period, and the solution pH was immediately readjusted.

Measurements of overall leaf elongation and plastochron duration. The growth of a monocotyledonous leaf is predominantly in one direction, from the intercalary meristem at the leaf base. Overall leaf elongation therefore was measured to assess leaf and shoot develop- ment. The length of every visible leaf, from a fixed reference point to the leaf tip, was measured daily using a ruler. The lid of the contain- er which served as the fixed reference point was about 5 mm above the base of the stem. The leaf growth rate was calculated from the daily measurements of leaf length as the slope of the linear regres- sion during the phase of linear leaf growth.

A plastochron, or phyllochron, is the time interval between ini- tiation of two successive leaves. It might be more broadly defined as the interval between corresponding stages of development (Erick- son and Michelini 1957; Klepper et al. 1984). As suggested by Erick- son and Michelini (1957), we defined the reference stage in terms of measurements of leaf length. The plastochron duration was calcu-

lated from the non-destructive leaf-length measurements at the time in which successive leaves reach a reference length of 13 cm.

Leaves were numbered so that later emerging (younger) leaves had higher numbers. Data are averages of eight plants in each treatment. The experiment was repeated three times with similar results. Plant age is counted from the beginning of germination.

Spatial distribution of growth - measurements. Data on the spatial distribution of growth, as a function of distance z, throughout the leaf growth zone were obtained by marking the leaf growth zone with pin pricks and evaluating a short-term displacement of the marks (Schnyder et al. 1987). A series of 13 fine insect-mounting pins (size 00; Hamilton Bell Co., Montvale, N. J., USA) spaced 3 mm apart and mounted on a piece of Plexiglas were used. The pins were longitudinally aligned with the center of the stem and inserted through the sheaths of older outer leaves and the growing region of the young enclosed leaves, and then removed. The plants were pricked at least 3 h after initiation of the light period. Growth was allowed to continue for another 6 h before growing leaves were carefully freed from older enclosing leaves, and the final positions of the pin marks were measured under a dissecting microscope.

Unlike the non-destructive daily leaf measurements which allow calculation of average growth throughout the photoperiod, the marking experiments evaluate day growth. Since rates of elongation are faster at night (Schnyder and Nelson 1988), calculated rates from marking data are expected to be lower than the averaged daily measurements. Each experiment was performed twice, with eight replicates for each treatment. The first prick hole was placed less than 0.6 mm above the base of the leaf, defined as the upper edge of the hair band region in the leaf base (Artschwager 1948).

The effect of the pin pricks on the overall leaf growth was eval- uated by measuring overall leaf elongation over the 6 h immediately following the pricking. Pricking caused the leaf growth rate to be reduced by 34%. Marking affected the elongation rate similarly in both treatments. In other gramineous leaves, a similar inhibition after marking was shown to have a uniform effect, without changing the spatial distribution of relative elemental growth rate (Schnyder et al. 1987, 1990).

Spatial distribution of growth - calculations. Monocotyledonous leaf tissue is made up of cells that are produced in the meristematic region near the leaf base and are continuously pushed away by subsequent cell production and elongation (Boffey et al. 1980). Growth of such a unidirectionally growing organ can be character- ized spatially by the distribution of growth velocity and relative elemental growth rate (Erickson and Sax 1956; Silk and Erickson 1979).

Growth velocity, v(z), the displacement rate of a particle from the leaf base, was calculated as the distance any given hole had shifted from the leaf base per unit time (mm �9 h '). Relative elemental growth rate (REG rate), is the relative growth rate of a leaf segment in one dimension. It is the change that occurs in a length of a leaf segment per unit length, per unit time. The REG rate was calculated with a two-point forward difference formula to estimate the deriva- tive of the displacement velocity curve with respect to position along the leaf.

v(z2) - v(~) REG rate: r(z,) = ~v/Oz

( z 2 - z , )

when v is growth velocity and z is position along the leaf. The REG rate is therefore presented in units of ( m m - m m ' . h ' )= h '. The terms strain rate (Silk 1984) and relative segmental elongation rate (Schnyder and Nelson 1988), have been used synonymously with REG rate.

Leaf elongation rate can be viewed as the integral of REG rate from the basal to the distal limit of the elongation zone.

Results

Overall plant development. E x p o s u r e to 100 m M NaC1 c a u s e d a r e d u c t i o n in leaf g r o w t h ra te a n d final leaf

N. Bernstein et al.: Sorghum leaf growth under NaC1

~ S

~w-. 6

~ 2

30

.~ 1 0

3

0 ~ 2

Contro l A Sal t

I I I

B y

I I I

C

0 r I I

2 4 6 8 10 Leaf no .

Fig. 1A-C. Salinity (100 mM NaC1) effect on sorghum shoot devel- opment as determined by non-destructive daily leaf measurements. A Leaf growth rate. B Final leaf length. C Plastochron. Data are means + SD (n=8)

435

0.2

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I I I

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B o Control Leaf 6

�9 S a l t

Leaf 7

Leaf 8

0 10 20 30

0,1

0.2

r

o.1

r~

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Dis t ance f r o m leaf ba se ( r am)

Fig. 2A,B. Spatial distribution of displacement velocity (A), and REG rate (B), in the elongation zone of sorghum leaves growing at 1 mM NaCl (control) or 100 mM NaC1 (salt). Data were evaluated on day 17 (9 d after treatments began) from displacement of holes in leaves 6, 7 and 8. Leaves 7 and 8 were growing linearly; (leaf 7 had just emerged above the whorl of encircling older leaf sheaths, leaf 8 was not yet visible above the whorl). Leaf 6 had nearly reached its final length and was decreasing in elongation rate. Data are means + SD (n = 5-7)

length (Fig. 1A,B). Plastochrons of leaves formed after leaf 6 were longer in salinized than in control plants (Fig. 1C), which was symptomatic of a general decrease in rate of development, i.e., at a given time, the salinized plants had fewer leaves than the control plants. Control and salinized plants began to differ in their apparent leaf number on day 18 after germination. Thus, day 17 was chosen as the first day of measurement and plants of the two treatments were then considered to be of the same developmental stage.

Spatial distribution of growth along leaves of different de- velopment stages. As in other tissues, growth velocity must increase with distance from the leaf base (Fig. 2), because progressively more tissue is expanding, and the end of the growth zone can be estimated as the point at which the growth velocity no longer increases with posi- tion. Salinity reduced the maximal growth velocity of all growing leaves (i.e.,, 6, 7 and 8) (Fig. 2A). The basal part of the growth zone was less affected by the stress. Salinity appeared to shorten the length of the leaf growth zone of leaves 7 and 8, but not of leaf 6, probably because of its developmental stage. The leaves sampled represent three different developmental stages. Leaf 8 had not yet emerged above the whorl of encircling sheaths, leaf 7 had appeared above the whorl, and leaf 6 was approaching the end of the elongation period, when elongation is no longer linear. Growth velocity was most sensitive to salinity when leaves were elongating linearly at a rapid rate (leaves 7 and 8), and specially sensitive when the leaf was still enclosed in the encircling sheaths (leaf 8). To-

ward the end of the elongation period the sensitivity to salinity was reduced.

Differentiation of the velocity curves with respect to position along the leaf gave the REG rate (Fig. 2B). Con- trol leaves 7 and 8 had similar maximum REG rates of 0.134).15-h -1 with maximum values 14-17 mm above the node. Salinity lowered the maximum by 34-70%, shifted its location closer to the leaf base and shortened the length of the elongation zone by 20-22%. The REG rate of leaf 6 was less affected by salinity, the location of the maximal value and length of the elongation zone were similar in control and salinized leaves.

Effect of plant age on the spatial distribution of growth. We evaluated the leaf growth profiles of plants on day 17 when leaf 7 was just visible above the whorl (Fig. 2), and again on day 20 when the plants were exactly two plas- tochrons older (i.e. leaf 9 was just appearing above the whorl). At the time of the second measurement, the plas- tochron age of the salinized plants was a day behind the control plants (leaf 9 appeared above the whorl a day later than in the control). Growth of leaf 9 of salinized plants was therefore measured for an additional day. This allowed the distribution of growth along control leaf 9 (on day 20), to be compared with salinized leaf 9 on day 20 ("time comparison") and on day 21 ("development comparison") (Fig. 3A). Leaf 10 of the salinized plants was first visible inside the whorl on day 21, a day later than control leaf 10. The growth distribution of control leaf 10 (on day 20) was therefore compared with that of salinized leaf 10 on day 21 (Fig. 3B). Leaf 9 was just

436 N. Bernstein et al.: Sorghum leaf growth under NaCI

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Salt (time comparison) Salt (development comparison)

Leaf 9 ~

Leaf 10

10 2 0 3 0

D i s t a n c e f r o m l e a f b a s e ( m m )

Fig. 3. Spatial distribution of REG rate in the elongation zone of sorghum leaves growing at 1 mM NaC1 (control) or 100 mM NaC1 (salt). Data were evaluated from prick-hole marking experiments of 20- and 2t-d-old plants. Both leaves were growing linearly with respect to time. Leaf 9 of the control emerged above the whorl of encircling older leaf sheaths on day 20; leaf 9 of the salinized plants emerged above the whorl on day 21. Both a 'time comparison' and a 'development comparison' are presented. Leaf 10 was not yet visible above the whorl. It was first identified inside the whorl of the control plants a day earlier than in the salinity treatment. Data are means _+ SD (n=4-7)

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Fig. 4A,B. Demonstration of non-repetitive growth in leaves 7 and 9. Growth velocity and REG rate fields in leaves 7 and 9 were determined when the leaf emerged above the whorl of older leaf sheaths. The plants were grown at 1 mM NaC1 (A; control), or 100 mM NaC1 (B; salt). Data are means _+ SD (n=5 7). The growth pattern of leaves 7 and 9 is not repetitive

appea r ing above the whor l of o lder leaf sheaths and therefore c o r r e s p o n d e d deve lopmen ta l l y to leaf 7 of the first m e a s u r e m e n t (Fig. 2). Leaf 10 was still no t visible above the whorl . Both leaves were g rowing l inearly. Sim- i lar to the day-17 d a t a (Fig. 2), sal ini ty shor tened the length of the e longa t ing region of leaves 9 and 10 and

Table 1. Growth parameters of nonstressed sorghum leaves. The growth was evaluated, from marking data, when each leaf was visi- ble above the whorl of older leaf sheaths. Maximal rate of displace- ment velocity was corrected for growth reduction due to marking, and is therefore the overall rate of leaf elongation. Leaves are num- bered from germination. Values in parentheses indicate standard deviations for a sample of five leaves

Leaf Leaf Length Maximal Maximal No. length of the REG rate rate of

(cm) elongation (mm-h ') displacement zone (mm) velocity

(mm-h 1)

7 10.0 (0.7) 30 0.143 (0.008) 2.60 (0.13) 8 11.3 (0.6) 33 0.090 (0.010) 2.00 (0.09) 9 13.1 (1.0) 33 0.113 (0.016) 1.95 (0.01)

10 14.7 (1.1) 33 0.100 (0.012) 2.15 (0.10)

2 . 5

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~ 0.10

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t3 S a l t �9 Leaf 8 �9 Leaf 10 ( t i m e comparlsonl

Leaf tO (deve lopment comparimon)

A C o n t r o l o Leaf 8

Leaf 10

r , , x b _ ~ , " * ~ , 10 2 0 3 0 0 10 2 0

D i s t a n c e f r o m l e a f b a s e ( m m )

0 . 0 0 I 0 30

Fig. 5A,B. Demonstration of repetitive growth in leaves 8 and 10. Growth velocity and REG rate fields along leaves 8 and 10 were determined when each leaf emerged above the whorl of older leaf sheaths. The plants were grown at 1 mM NaC1 (A; control), or 100 mM NaCI (B; salt). Data are means + SD (n=5-7). Since the curves coincide, growth of leaves 8 and 10 is repetitive

reduced the magn i tude of the R E G rate. Again , sensit ivi- ty to sal ini ty was grea ter in the leaf no t yet visible above the whor l (leaf 10). The length of the e longa t ion zone and the R E G rate of the sal inized leaf increased after the leaf emerged above the whor l of o lder leaf sheaths.

Growth is not repetitive for early-formed leaves. The distr i- bu t i on of g rowth a long leaves 7 and 9 when they are jus t appea r ing above the whor l of enclos ing o lder leaf sheaths is given in Fig. 4. Since the d e v e l o p m e n t of leaf 9 was faster in the con t ro l than the sal inized plant , bo th a " t ime c o m p a r i s o n " and a "deve lopmen t c o m p a r i s o n " were made. The g rowth field of these two leaves was not iden- tical. M a x i m u m R E G rate was lower in leaf 9 than in leaf 7, and the length of the e longa t ing tissue var ied too (Table 1). However , d i s t r ibu t ion of g rowth veloci ty and R E G rates for leaves 8 and 10 were s imilar for bo th the con t ro l and sal inized p lan ts (Fig. 5). Thus we conc lude tha t g rowth is repet i t ive only for leaves 8, 9 and 10.

Changes in growth profile during leaf ontogeny. Informa- t ion in Figs. 2 and 3 indicates tha t the inh ib i to ry effects of

N. Bernstein et al.: Sorghum leaf growth under NaCI 437

salinity are strongest during early stages of leaf develop- ment, before the leaf is visible above the whorl. Thus, we evaluated changes in growth profiles during leaf on- togeny under both control and salinity conditions. Since we found the growth to be repetitive starting with leaf 8, sampling different leaves on a plant as representative of leaf developmental stages will be accurate only if leaf 8 and younger leaves (i.e. 9 and 10) are included. A quanti- tative comparison of different leaf developmental stages is clearest if one follows the salinity effects on leaves of the same insertion number while the leaf develops.

In control and salinized plants, growth of leaf 8 was evaluated - from the first day that the leaf could be iden- tified inside the whorl until it was visible just above it (Fig. 6). Before they emerge, leaves are difficult to mark because they are easily missed by the prick needles (Kemp 1980). This may explain the lack of published data on growth of such young monocotyledonous leaves. The data presented in Fig. 6 were accumulated by careful ver- tical alignment of the prick needles with the center of the leaf whorl. Leaves not fully marked were excluded from the analysis. The length of the elongation zone increased with leaf age during the first stages of leaf development in both the control and salinized leaves. The maximal REG rate value increased when the leaf tip emerged above the whorl of sheaths (between days 2 and 4) in both treat- ments. The increase was much higher in control than in salinized leaves.

Once a leaf was near cessation of elongation, the growth velocity decreased, the length of the growth zone became shortened and the salinity effect became less pro- nounced than in a rapidly growing leaf (Figs. 2, 3). Figure 7 shows the distribution of growth along leaf 6 of control and salinized plants from the time the leaf emerged above the whorl until cessation of elongation. After emergence,

I .=

0

0.20 , , ,

o day1 !

Control ~ ~ day2

0,15 /~'/Ix~w v day4

0.10

0,00 - -

* dayl

Salt - day2

O . 1 5 v day4

0.10

t I

0.00 . ' 10 20 30

Distance f r o m l e a f b a s e (mm)

Fig. 6. Growth field of a young sorghum leaf over time. Spatial distribution of REG rate during the first days of elongation of leaf 8. During days l and 2, the leaf was still enclosed in older leaf sheaths. At the forth day the leaf was just appearing above the whorl. Plants were grown at I mM NaCI (control) or 100 mM NaC1 (salt). Data are means _+ SD (n=4-6) . On day 4 the control maxi- mum REG rate was x 2.5 the rate on days 1 and 2

0.2

0.1

o.o

0 0.2

0.1

i 1

Control Day i o

Day 2 o Day 3 A Day 5 v

S a l t Day I �9

Day 2 -

Day 8 �9

Day 5 �9

0.0 ~ ' 10 20 30

Distance from leaf base ( r a m )

Fig. 7. Growth field of a maturing sorghum leaf over time, Spatial distribution of REG rate along leaf 6 from emergence above the whorl until cessation of elongation. During days 1 and 2 of mea- surements, leaf elongation was linear with time, but was less rapid during days 3 and 5. Plants were grown at 1 mM NaC1 (control) or 100 mM NaCl (salt). Data are means _+ SD (n=5-7)

the control leaf elongated linearly and, as a first approxi- mation, had a steady growth pattern (days 1 and 2, Fig. 7). The growth profile only changed towards the end of the elongation phase, i.e. maximum REG rate was first reduced (on day 3), and then appeared much closer to the leaf base (by day 5). Under saline conditions, the growth profile of rapidly elongating emerged leaves was also steady (days 2 and 3, Fig. 7) and, as in the control plants, toward the end of the elongation phase it became short- ened and confined to the more basal part of the leaf (day 5).

Discussion

Reduction of leaf expansion rate is considered the initial and most sensitive growth response to NaC1 stress (Greenway and Munns 1980). Although much work has been done to study the causes and extent of the growth reduction observed in stressed grass leaves, there are no reports on NaC1 effects on the kinematics of leaf growth.

Growth in a grass leaf is restricted to the small inter- calary meristem at the basal part of the leaf near the point of attachment to the node (Esau 1943). Our results show that NaCI stress imposes a decrease in leaf growth rate by shortening that intercalary leaf growth zone as well as reducing the maximal longitudinal REG rate. Interest- ingly, the REG rate in the basal 6 9 mm of the leaf is unaffected by the stress. This is consistent with the re- sponse of maize root tips to water stress (Sharp et al. 1988).

Sensitivity to salinity changes with leaf ontogeny. The inhi- bition of growth by salinity is largest when leaves are

438 N. Bernstein et al.: Sorghum leaf growth under NaC1

elongating rapidly (leaves 7 and 8 in Fig. 2, leaves 9 and 10 in Fig. 3), and especially before the leaf tip emerges from the whorl of enclosing older leaf sheaths (leaf 8 in Fig. 2, leaf 10 in Fig. 3).

Towards the end of the elongation period, when leaf expansion is no longer linear with time, the salinity effect is less pronounced (leaf 6 in Fig. 2). At that stage of growth the elongation zone is shortened and maximal REG rate is found closer to the leaf base. Leaves then elongate exclusively due to sheath growth and the blade has reached its final length. Schnyder et al. (1990) ob- served that the cell elongation rate throughout the elon- gation zone of ryegrass decreases progressively during these late stages of leaf elongation, and measured a re- duction, although small, in the length of the elongating region.

The direct cause for the highest sensitivity of the non- emerged leaves is not currently understood. Leaf growth in grasses consists of two stages. In the first stage the young leaf is enclosed by sheaths of older leaves and is dependent on the supply of assimilates from the older, more mature leaves. The second stage occurs after the leaf lamina grows out of the protecting outer sheaths. The leaf is then autotrophic as it is exposed to light and is photosynthetically active. It is then no longer totally dependent on the rest of the plant (Friend et al. 1962; Allard and Nelson 1991). Transpiration in the first stage of growth is considerably lower than in the second stage and transport processes vary accordingly. Munns et al. (1988) working with salinized plants followed leaf devel- opment from a primordium into a full leaf. They reported that CI concentrations in the expanding tissue of the stressed leaves were high before leaf emergence above the whorl but subsequently decreased to a level that was sim- ilar to the controls at the time of leaf emergence. Thus, although tissue CI concentration in the various treat- ments did not correlate with the rates of leaf growth throughout the expansion period it may be of special significance during the early stages of leaf elongation, perhaps when cell division is most active. Interestingly, Na + concentrations as well were highest prior to leaf emergence and decreased once the leaf emerged (Munns et al. 1988). No consistent relation was found between Na + concentrations and growth rate throughout the ex- pansion period. Again, Na may have had an effect during the first stage of growth.

The low rate of transpiration in leaves enclosed in the whorl can result in their being deficient in Ca (Wiebe et al. 1977). This problem may become more severe under saline conditions, an issue discussed further in a subse- quent paper.

Munns et al. (1982, 1988) found that NaC1 stress led to increased concentrations of soluble carbohydrates in elongating tissues of barley leaves at the second stage of growth. They concluded that photosynthesis was not a limiting factor in short- or long-term exposures to salini- ty. Volenec and Nelson (1984) reported high concentra- tions of carbohydrates in the growth zone of tall-rescue leaves and suggested that the growth rate of non-stressed leaves is not limited directly by supply of carbohydrates from photosynthesis. We know of no study that com- pared the effect of salinity on the carbohydrate status, or

the water status, of the leaf growth zone during the rapid leaf elongation of the first and second stages of growth.

Repetitive patterns of growth. At a given time, the popula- tion of growing leaves on a plant represents different de- velopmental stages. If growth of these leaves is repetitive, i.e. if the growth-distribution patterns are similar for suc- cessive leaves at the same development stage, studies of physiological processes of leaf growth and development can be simplified. Different leaves on a plant could be sampled at any point in time to infer the ontogenic devel- opment of an individual leaf. Few studies, however, have addressed the question of whether leaf growth is precisely repetitive, although many studies are based on the as- sumption. For example, Meiri et al. (1992) based calcula- tion of deposition rates on the assumption of repetitive growth, and Croxdale and Pappas (1987) measured the levels of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase in a population of Dianthus chinensis leaves to simulate activity at different stages of leaf devel- opment. The plastochron index, which has proved useful in studies of dicotyledonous plant development, implicit- ly assumes repetitive growth. Similarly, in Fig. 2 we demonstrated growth fields of different leaves on a plant as a model for salinity effects on leaves at different stages of development, but we show growth of sorghum leaves 6, 7 and 8 is not repetitive (Fig. 4, 5; Table 1), while growth of leaves 8, 9 and 10 proved repetitive. These results verify the need to demonstrate repetitive growth behavior for a given system.

It is conceivable that the onset of stress conditions will change a repetitive pattern. Interestingly, in sorghum un- der salinity stress, exposure to 100 mM NaC1 inhibited leaves 8-10 similarly, and therefore did not prevent their growth from being repetitive. This implies that longer exposures to steady salinity levels do not change the ex- tent and pattern of leaf growth inhibition.

Spatial variability in growth response to salinity. A recent study evaluated salinity effects on the distribution of growth along cotton roots (Zhong and L/iuchli 1993). The inhibition of the root growth rate was attributed to shortening of the growth zone and reduction of REG rate throughout the entire growing region. In sorghum leaves, under our experimental conditions, salinity shortened the growth zone, but did not affect local rates of elongation in the basal part of the leaf. This is similar to the effect of a dwarfing gene on growth distribution in the wheat leaf (Paolillo and Sorrels 1992). The different responses to the stress may depend on species, stress level, or organ type. However, sensitivity of the entire root growth region to stress is not a general response, since Sharp et al. (1988) demonstrated, that similar to sorghum leaves, the longi- tudinal expansion in the apical part of the growth region of corn roots is insensitive to water stress.

The different spatial response to the stress may also be dependent on duration of exposure to the stress prior to measurement. Our sorghum plants were allowed to grow for 9-13 d in the final salt concentration, before the leaf growth fields were evaluated, while cotton root growth was evaluated 3 8 h after salinization was complete (Zhong and L/iuchli 1993). Since a given cell is displaced

N. Bernstein et al.: Sorghum leaf growth under NaCI 439

away f rom the so rghum leaf base th rough the g rowth zone in 40-50 h, cells tha t made the g rowth zone were p robab ly p roduced while salinization was at a maximum. In co t ton roots, a given cell is displaced away (from a posi t ion 1 m m f rom the apex) t h rough the g rowth zone in abou t 22 h. It is therefore possible that cells that made the roo t g rowth zone were p roduced before salinization had commenced , or while elevating NaC1 concentrat ions. In- terestingly, the length of time a cell has to adjust to a certain salinity level pr ior to the beginning of its e longa- tion, or the level of salinity under which it was produced, may have implications for its g rowth sensitivity to the stress.

Our s tudy shows that the inhibit ion of leaf g rowth by NaCl stress is unequal ly distributed a long the leaf g rowth zone. Such a spatially varying response could be caused by gradients of localized factors within the growing re- gion (i.e., nutr i t ional or water status, t ranspor t and com- pa r tmenta t ion phenomena , or g rowth regulators), or by spatially varying sensitivity of the tissue elements to the cause of damage. N o published informat ion that de- scribes salinity effects on such gradients is currently available; however, Lazof and L/iuchli (1991) detected local variat ions in nutr i t ional status, imposed by salinity, in the shoot meristem and leaf pr imordia of lettuce. If indeed sharp gradients occur, as is the case for element concent ra t ions in non-stressed maize leaves (Meiri et al. 1992) and roots (Sharp et al. 1990), then it is impor tan t to compare local levels of g rowth with the spatial distribu- t ion of factors which are considered to be of impor tance in the g rowth inhibition. "Average" values for large leaf segments will not yield the desired information. In our l abora to ry we have related the spatial pat terns of R E G rate to inorganic solute concent ra t ions and deposi t ion rates. This has al lowed us to evaluate the role of the different mineral elements in inhibiting growth under NaC1 stress (Bernstein 1992).

We thank Dr. M.S. Bret-Harte for critical evaluation of the manuscript and Dr. A Meiri for helpful discussions. This work was supported by Binational Agricultural Research and Development Fund grant No. 1-1485-88.

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