Dynamics of non-structural carbohydrates in developing
leaves, bracts and floral buds of cotton
D. Zhao *, D.M. Oosterhuis
Department of Crop,Soil,and En6ironmental Sciences,Altheimer Laboratory,276Altheimer Dri6e,Uni6ersity of Arkansas,
Fayette6ille,AR72704,USA
Received 29 July 1999; received in revised form 22 October 1999; accepted 26 October 1999
Abstract
Development of cotton (Gossypium hirsutum L.) squares (i.e. floral buds with bracts) is fundamental for yield formation. A 2-year field study was conducted to determine dry weight (DW) accumulations of cotton leaves, floral bracts and floral buds, and the changes in concentrations of non-structural carbohydrates (hexoses, sucrose and starch) in these tissues during square ontogeny as affected by fruiting positions within the plant canopy. During square development, DW accumulation of a subtending sympodial leaf and floral bracts followed a sigmoid growth curve with increasing square age, whereas the DW increase of a floral bud followed an exponential curve. Main-stem node (Node 8, 10 or 12) and branch position (proximal vs. distal) within a plant canopy significantly affected DW accumulations of the leaf, bracts and floral bud. Starch was the dominant non-structural carbohydrate in the three tissues, accounting for more than 65% of total non-structural carbohydrates (TNC). Subtending leaf TNC increased as square age increased. The bracts exhibited a smaller change in TNC than leaves. Non-structural carbohydrate concentration was the lowest in 10-day-old floral buds, and had little change during the first 15 days of square development. Within 5 days prior to anthesis, the floral-bud TNC increased dramatically, tripling at the time of floral anthesis compared with 15-day-old floral buds. Square age and fruiting position significantly affected non-structural carbohydrate concentrations of subtending leaves, bracts, and floral buds. The correlation did not exist between final boll retention and non-structural carbohydrate concentrations of floral buds at different fruiting positions under normal growth conditions. The pattern of floral-bud non-structural carbohydrates during square ontogeny suggests that major events in carbohydrate metabolism occur just prior to anthesis. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Gossypium hirsutum; Floral bud development; Dry matter accumulation; Hexoses; Sucrose; Starch concentration www.elsevier.com/locate/envexpbot
1. Introduction
Yield of field-grown cotton is closely associated with the number of mature bolls (Wells and Meredith, 1984; Heitholt, 1993). The number of bolls per unit land area is partially dependent on * Corresponding author. Tel.: +1-501-575-3955; fax: +
1-501-575-3975.
E-mail address:[email protected] (D. Zhao)
the number of squares (floral buds with bracts) retained by the plants. Therefore, a better under-standing of the initiation, differentiation and devel-opment of cotton squares, and the characteristics of their physiology and biochemistry during devel-opment are important for improving cotton man-agement.
The development of cotton squares is a funda-mental step of fruit development. However, most studies on squares have emphasized the relation between morphology and abscission (McMichael and Guinn, 1980; Ungar et al., 1989). We know much less about the physiology of square forma-tion and growth than any other part of the cotton fruiting cycle (Stewart, 1986). Since only limited studies have been carried out on the physiology of square development, there are many phenomena and questions about cotton fruit development that are not completely understood. For instance, why do cotton plants shed young squares and young bolls, but not white flowers? Why is the abscission of small squares greater than that of large squares? How do the carbohydrate and mineral nutrient profiles of squares change during development? Are there differences in carbohydrate concentrations in floral buds and bracts located at different fruiting positions?
The endogenous hormone balance, photosyn-thetic assimilate supply, and mineral nutrient status of plants are major physiological factors con-trolling fruit abscission (Benedict, 1984). Environ-mental factors inducing fruit abscission include adverse photosynthetic photon flux density, tem-perature, moisture, and nutrition (Guinn, 1974, 1982). McMichael and Guinn (1980) suggested that the sensitivity of cotton square abscission to water stress was greatest during the first week after the squares become visible. Later studies showed that squares smaller than 1 cm in size were more sensitive to stress environments inducing abscission than the squares larger than 1 cm in size (Guinn, 1982; Ungar et al., 1989).
Development of young squares into flowers is an important process of yield development. However, squares often fail to form flowers. If this failure occurs with too many squares abscised, crop yield can be reduced and maturity can be delayed. The hypothesis of this study is that the non-structural
carbohydrate contents of floral buds may be used to explain the differences in the boll retention at different main-stem nodes and fruiting positions. Since the physiological reasons for that failure are not well understood and because there is little information on this topic, the objective of this research was to characterize leaf and square carbo-hydrate concentrations as a function of square age, sympodial branch position (i.e. proximal vs. distal) and vertical main-stem node (i.e. Node 8, 10 or 12) within the canopy.
2. Materials and methods
2.1. Plant culture
The cotton cultivar Deltapine 20 was machine-planted on a moderately well-drained Captina (Typic Fragiudult) silt loam soil at the Arkansas Agricultural Research and Extension Center, Uni-versity of Arkansas in Fayetteville, Arkansas, USA on 4 June 1993 and 17 May 1994. Preplant fertilizer was applied at a rate of 45-30-75 kg N-P-K ha−1,
and an additional side-dressing of 56 kg N ha−1
as ammonium nitrate was applied on 13 July 1993 and 28 June 1994 (early square stage). Control of insects and weeds and furrow irrigation were performed as needed during the growing seasons to minimize plant stress and optimize yield. The field was divided into three blocks (replications), each with two plots for sampling and for determining final boll retention and yield parameters at different fruiting positions. Each plot consisted of ten rows spaced 1 m apart (15 m in length) and oriented in a north – south direction. Plots were hand-thinned to a density of nine plants m−1 row when the
seedlings had three true leaves.
2.2. Sample collection
When the squares at the selected positions first became visible (about 3 mm in diameter), they were individually labeled by tagging main-stem leaves at the respective nodes with dated jewelers tags. Approximately 900 squares at each position from three replications were labeled on the same day. Squares at tagging were considered day 0 in age. During development of the squares, 30 tagged squares were randomly collected at 900 h central daylight saving time (CDST) on each sam-pling date at 5, 10, 15, 20, or 25 days of age until the squares developed into white flowers. Addi-tionally, in 1994 the sympodial leaves subtending Positions 1, 2 and 3 along the sympodium at Node 10 were collected simultaneously with square sampling. The dates of tagging and sam-pling squares at each node and sympodial branch position are shown in Table 1.
The fresh-tissue samples were placed into plas-tic bags and taken to the laboratory on ice. Squares were separated into bracts and floral buds. The floral buds, bracts, and leaves were dried at 90°C for 30 min, followed by 72 h at 70°C in a forced-draft oven before weighing. The entire process from sample collection until initial drying was completed within 2 h to minimize
error in measured sugars. The dried tissues were ground and passed through a 0.5-mm sieve for analysis of non-structural carbohydrates (hexoses, sucrose and starch). In this study, hexose concen-tration represents the sum of glucose and fructose concentrations.
2.3. Non-structural carbohydrate measurements
A modification of the method of Hendrix (1993) was used to extract and quantify the con-centrations of non-structural carbohydrates in the leaves, bracts, and floral buds. Details of entire processes of non-structural carbohydrate extrac-tion and measurement are the same as previously described (Zhao and Oosterhuis, 1998). The sum of hexoses, sucrose and starch was defined as total non-structural carbohydrates (TNC).
2.4. Data analysis
The dry weight (DW) accumulations of leaves, bracts and floral buds, as well as patterns of changes in non-structural carbohydrate concen-trations of these tissues, were plotted as a function of square age. All data were analyzed by analysis of variance with the ANOVA procedures of the statistical analysis system (SAS Inst., Cary, NC).
Table 1
The dates of tagging squares at different main-stem nodes and fruiting positions, sampling times, and heat units accumulated during the period of square development in 1993 and 1994
Sampling (month/day)
Tagging (month/day) Heat unitsb(°C)
Node-positiona
5 Days 10 Days 15 Days 20 Days 25 Days Flower
1993
aThe two numbers represent main-stem node and sympodial branch position, respectively. bDaily heat units (°C)=[(T
max+Tmin)/2]−15°C, whereTmax andTminare the maximum and minimum daily temperatures in degrees Celsius, respectively.
Data of Branch Positions 1 and 2 at Node 10 for each year and non-structural carbohydrates of the three tissues of leaves, bracts, and floral buds in 1994 were also performed with the ANOVA to determine differences between years and among tissues. Correlation coefficients between final boll retention and floral-bud non-structural carbohy-drate content at different positions were also cal-culated. The analyses of variance combined over the 2 years of this study indicated significant differences between years for non-structural car-bohydrates of the bracts and floral buds. There-fore, data were interpreted individually for each year. When the F-value was significant at P5 0.05, the LSD test (P=0.05) was performed.
3. Results
3.1. Dry matter accumulations of lea6es, bracts,
and floral buds
When a square at a given branch position was 5 – 7 days old, the subtending sympodial leaf had just unfolded. Thereafter, leaf DW increased rapidly with increasing square age (Fig. 2(A)) and
reached the greatest DW when the square at the corresponding position became a white flower.
During square development, the increase in bract DW of individual squares with square age was similar to that of the subtending leaf (Fig. 2(B)). The increase in floral bud DW with pro-gressing square age exhibited a typical exponential growth pattern (Fig. 2(C)). Floral bud DW in-creased slowly during the first 15 days of square development. Thereafter, the bud DW increased rapidly and tripled within the last 5 – 8 days of square development (a 20-day-old square to a white flower) compared to a 20-day-old floral bud.
3.2. Non-structural carbohydrates in lea6es
In 1994, hexose, sucrose, starch and TNC con-tents in subtending leaves varied significantly with square age and branch position (PB0.001 – 0.0001), except for sucrose which did not differ among branch positions (Fig. 3). Age and posi-tion also had a significant (PB0.001) interaction
Fig. 3. Changes in non-structural carbohydrate concentrations in the subtending fruiting branch leaves (FBL) of the first three fruiting positions at main-stem Node 10 during square development in 1994. The LSD values are for all combinations of positions and square ages. Each data point is the mean of nine samples from three replications and three subsamples in each replication.
Fig. 2. Changes in dry matter weight of: (A) subtending leaves; (B) bracts; and (C) floral buds of cotton squares at different main-stem nodes and sympodial branch positions during the development in 1993 and 1994. Arrows indicate flowering. The LSD values are for all combinations of nodes, positions, and square ages. Each data point is the mean of 15 leaves or 30 squares from three replications.
not significant during hexose peak (15 – 20 days). Except for a drop at day 20, leaf sucrose con-centration also showed a marked increase with increasing square age. Sucrose concentration in the sympodial leaves was highest just prior to flowering (25-day-old squares) (Fig. 3). Leaf starch concentration also rapidly increased with increased square age and peaked at 5 days before flowering (for the sympodial leaf at Branch Posi-tion 2) or at flowering (for the sympodial leaves at Branch Positions 1 and 3). Of the three sympodial leaves, the leaf at Branch Position 2 exhibited the highest concentrations of starch and TNC, whereas the leaf at Branch Position 1 had the lowest carbohydrate concentrations at the same square age. Starch and TNC were significantly different (PB0.05) between branch positions within the plant canopy (Fig. 3) except for Posi-tions 2 and 3 at 10-day square age and at flowering.
3.3. Non-structural carbohydrates in bracts
Square age, node and branch position signifi-cantly (PB0.01 – 0.0001) affected contents of bract hexoses, sucrose, starch, and TNC. Interac-tive effects on non-structural carbohydrates were also significant (PB0.001 – 0.0001) among age, node, and position.
In 1993, the bract hexose concentration ranged between 4 and 8 g kg−1 DW, accounting for
11 – 15% of the TNC, and was relatively consistent among square ages (Fig. 4). The change over time in bract sucrose concentration showed a greater range (2 – 15 g kg−1 DW) and variation among
sampling dates, nodes or branch positions, com-pared to the bract hexoses. Overall, bract starch concentration accounted for 65 – 76% of the TNC and slowly decreased with increasing square age except for Position 1 of Node 12. There were no consistent differences in concentrations of bract
Fig. 5. Changes in non-structural carbohydrate concentrations in the floral buds during the development of squares at different branch positions of main-stem Nodes 8, 10 and 12 in 1993, and sympodial Branch Positions 1, 2 and 3 of main-stem Node 10 in 1994. White flower occurred at 25 days in 1993 and 28 days in 1994 after first visible appearance of the square. The LSD values are for all combinations of nodes, positions, and square ages. Each data point is the mean of nine samples from three replications and three subsamples in each replication.
hexoses and sucrose between nodes and between branch positions during square development, but averaged the first two branch positions of each node, bract starch and TNC concentrations of squares at Node 8 were significantly higher than those on Nodes 10 and 12 at most sampling dates in 1993.
In 1994, concentrations of hexoses, sucrose and starch in bracts were similar to those in 1993, but their patterns changed over time differently be-tween the years (Fig. 4). Similar to the leaf su-crose, the bract sucrose concentration was more variable than hexoses and starch levels, and the bracts of squares at Branch Position 1 showed the lowest concentrations of starch and TNC (PB 0.05) among the three branch positions (Positions 1, 2 and 3). This might be associated with the shade of main-stem leaves in the upper canopy.
3.4. Non-structural carbohydrates in floral buds
In general, floral-bud non-structural carbohy-drate concentrations (averaged across all square ages) were influenced significantly by node (PB 0.01), branch position (PB0.001) and square age (PB0.0001), except for the starch and TNC in 1994 (Fig. 5). There were significant (PB0.001 – 0.0001) node×branch position, node×age, posi-tion×age, and node×position×age interaction effects on floral-bud hexose, sucrose, starch, and TNC concentrations. Compared with subtending leaves and bracts, floral buds had the highest hexoses and the lowest sucrose concentrations (PB0.05), but starch and TNC concentrations of floral buds were higher than those of bracts and lower than those of subtending leaves (Table 2).
of non-structural carbohydrates in floral buds during square development are shown in Fig. 5. The hexose and sucrose concentrations in floral buds remained relatively constant between 5 and 25 days of square development. During this pe-riod, hexose concentration was 5 – 12 g kg−1DW,
and sucrose 2 – 6 g kg−1 DW. At anthesis, the
floral-bud hexose concentration tripled in 1993 and more than quadrupled in 1994, compared to the floral-bud hexoses of 20-day-old squares. The sucrose in floral buds was less than 10 g kg−1
DW before anthesis, and rose above 60 g kg−1
DW in 1993 and above 35 g kg−1
DW in 1994 (means of squares at all positions). Starch concen-tration was lowest in the buds of 10 – 15-day-old young squares (30 – 50 g kg−1 DW). Thereafter,
floral-bud starch concentration rapidly increased as square age increased, and reached the greatest values at anthesis. During square development, there were no consistent differences in hexose and sucrose concentrations among the squares at the different nodes and branch positions within a year. The squares at Branch Position 2 of Node 12 had the lowest floral-bud starch concentration during the first 15 days of square development in 1993. However, the buds at Branch Position 3 of Node 10 had the highest starch concentration during the early period (5 and 10 days) of square growth in 1994.
Averaged across all square ages and Branch Positions 1 and 2 of Node 10 by the years (data not shown) found significant difference between years in floral-bud non-structural carbohydrate concentrations. Floral buds in 1994 had higher hexoses, lower sucrose, higher starch and higher TNC concentrations (PB0.05) than those in 1993. The differences in bud non-structural
carbo-hydrate concentrations between years were proba-bly associated with temperature during square ontogeny. The heat units during square develop-ment in 1993 were greater than those in 1994 (Table 1). Further study is needed for temperature effects on non-structural carbohydrate contents of cotton tissues.
4. Discussion
Dry matter accumulations of the leaves, bracts, and floral buds decreased significantly as the main-stem node number increased up the plant and branch position increased away from the main stem. The patterns in dry matter accumula-tion in leaves, bracts and floral buds with respect to fruiting position may reflect the formation of sources (leaves) and sinks (floral buds) of differing strength because of increasing competition for assimilate resources as more bolls are set. Such a property of sources and sinks could affect further boll development, and similar correlation among bolls and yield parameters with main-stem node and branch position have been reported (Dan-forth et al., 1990; Jenkins et al., 1990a,b).
During the square development, starch was a dominant non-structural carbohydrate in leaves and accounted for more than 70% of the TNC. Our results are similar to the report by Mauney et al. (1979), who found that leaf starch content accounted for 63% of total leaf carbohydrates for cotton plants grown at normal ambient CO2
con-ditions. The subtending leaves of 10-day-old squares had the lowest non-structural carbohy-drate concentrations. This was probably because the sympodial leaves had just unfolded and were
Table 2
Comparison of hexose, sucrose, starch, and TNC concentrations among the three plant tissues of the subtending leaves, bracts, and floral buds in 1994a
Hexoses (g kg−1DW) Sucrose (g kg−1DW) Starch (g kg−1DW) TNC (g kg−1DW)
not yet fully active photosynthetically (Wullschleger and Oosterhuis, 1990a). Leaf TNC concentration (Fig. 3), as well as leaf size and photosynthetic activity (Wullschleger and Ooster-huis, 1990a; Zhao, 1997), rapidly increased as square age progressed. Therefore, the leaf carbohy-drate content is closely related to leaf photosyn-thetic capacity.
Compared with leaf hexoses and starch, leaf sucrose concentration varied more between branch positions and between sampling dates within a position. Because sucrose is the major form of translocated carbohydrates (Kruger, 1990), the high leaf sucrose prior to flowering may be related to rapid assimilate translocation out of leaves to the rapidly growing subtended squares (Fig. 2(C)). Additionally, increased leaf sucrose concentration at this stage may be associated with an increase in activity of leaf sucrose-phosphate synthetase with leaf age, since there is a close positive correlation between leaf sucrose concentration and activity of that enzyme (Huber, 1983; Hendrix and Huber, 1986).
In cotton, the pattern of starch concentration in leaves over time was similar to that of leaf photo-synthetic rates (Wullschleger and Oosterhuis, 1990a; Zhao, 1997). Stitt (1984) reported that starch accumulation in chloroplasts was primarily a mechanism for storing reduced carbon when the rate of photosynthesis exceeded the capacity of the leaf to export sucrose. Additionally, leaf sucrose export was closely related to both source and sink strengths (Ho et al., 1989). Therefore, starch buildup in cotton leaves in this study was also associated with weak sink activity of squares at this stage (i.e. the supply of assimilates in the leaf exceeded the demand of the square).
The change in non-structural carbohydrate con-centrations of bracts during square development has not been previously reported, although Con-stable and Rawson (1980) investigated the bract photosynthesis of developing squares. Our study showed that the non-structural carbohydrate con-centrations in bracts were similar to those in sympodial leaves from the time of first appearance of the squares (3 mm in size) to 5 days old (Figs. 3 and 4). Thereafter, bracts had a significantly lower TNC concentration than did the leaves. At
20 – 25-day square age, bract TNC concentration was only about one third of the leaf TNC concen-tration. Averaged cross the entire square develop-ment period and the three branch positions in 1994, floral bracts had significantly lower (PB0.001) non-structural carbohydrate concentrations than did the subtending leaves (Table 2). These results suggest that bracts might be a minor source of carbohydrates for squares, compared to sympodial leaves. However, bracts exhibited a much smaller change over time in non-structural carbohydrate concentrations than the leaves did during square ontogeny.
In this study, patterns of non-structural carbohy-drate concentrations of the leaves and bracts during square aging were consistent with the reports of leaf and bract photosynthesis during boll development (Wullschleger and Oosterhuis, 1990b). Those au-thors found that although bracts showed much lower photosynthetic rates per unit area than the leaf did during fruit development, leaf photosyn-thesis declined sharply with age while bract photo-synthesis remained fairly constant. In the current study, bract sucrose represented a higher portion (18%) of bract TNC than that of the leaves (9%; averaged over all sampling dates and fruiting positions in the 2 years). Our results support an earlier speculation that cotton bracts are associated with adjusting assimilate transport (Bhatt, 1988), because a high fraction of sucrose in bracts may be beneficial to carbohydrate translocation from bracts to fruits (Benedict and Kohel, 1975).
dramati-cally 3 days prior to flowering. Large squares rarely shed at this stage, suggesting that as floral bud development approached flowering, rapid synthesis of tissues occurred, requiring large amounts of non-structural carbohydrates. Thus, complex phys-iological and biochemical changes may be occur-ring in the floral bud. Therefore, a close examination of floral-bud non-structural carbohy-drates and other metabolic characteristics in the short time prior to flowering may provide more meaningful insight to the physiological events re-lated to flowering.
Two early physiological theories explaining cot-ton fruit abscission were: (i) the nutritional theory (including mineral nutrients and carbohydrates); and (ii) the hormone balance theory (Benedict, 1984). Furthermore, the growing environments and physiological age of cotton plants considerably influence plant nutrient status and phytohormone levels (Guinn, 1982). The nutritional theory sug-gests that an insufficient supply of assimilates or mineral nutrients is a major factor causing cotton fruit abscission. Under normal growing conditions, fruit abscission increases as the number of fruiting positions increase within the plant canopy (Hei-tholt, 1993). Sympodial Branch Position 1 has significantly higher numbers of bolls than Positions 2 and 3 (Jenkins et al., 1990a,b). Our study showed that although Branch Positions 2 and 3 had an average 30 and 63% decrease in final boll retention, respectively, compared with Branch Position 1 (data not shown), the non-structural carbohydrate concentrations of floral buds showed no consistent differences among fruiting positions. The correla-tion analysis indicated that a significant correlacorrela-tion did not exist between final boll retention and non-structural carbohydrate concentrations of floral buds at different fruiting positions (data not shown). These results are similar to the report by Heitholt and Schmidt (1994) in which the concen-tration of soluble sugars in excised receptacles and ovaries were similar among the first three fruiting positions of a sympodial branch, even though boll retention was higher at sympodial Branch Position 1 than at Position 3. Eaton and Ergle (1953) reported that carbohydrate levels in the stem and young boll did not differ between cotton plants that had different levels of fruit abscission. Therefore,
the non-structural carbohydrate concentration of floral buds cannot explain the variation in boll retention of different fruiting positions under opti-mum environments. Although there were no con-sistent differences in non-structural carbohydrate concentration among fruiting positions in the plant canopy under normal growth conditions, in an-other study Zhao and Oosterhuis (1998) found that 10-day-old squares at Branch Position 3 showed the greatest decrease in TNC concentration among Branch Positions 1, 2 and 3 under shade conditions. Therefore, low non-structural carbohydrate con-tent in floral buds under conditions of stress may be one of the major factors causing square abscis-sion.
In conclusion, starch was the dominant non-structural carbohydrate in cotton leaves, bracts and floral buds. Sucrose concentrations of the leaves and bracts varied more than hexose and starch concentrations of these tissues did. During square development, fruiting position within the plant canopy significantly affected non-structural carbo-hydrate concentrations of leaves and bracts, but had less effect on those of floral buds. Non-struc-tural carbohydrate concentrations of floral buds did not consistently differ among nodes and branch positions during square ontogeny, but the patterns of changes in these carbohydrates with increasing square age were similar among all positions and between years. Floral buds of 10 – 15-day-old young squares had the lowest starch concentration, com-pared to the floral buds of other square ages. The higher abscission of young squares is possibly associated with low starch content of floral buds and poor competition for carbohydrates with large squares and bolls. Floral-bud DW and concentra-tions of all three non-structural carbohydrate com-ponents prior to flowering increased dramatically, suggesting the important metabolic events occur at that time. The results of this study provide a better understanding of cotton square developmental physiology.
Acknowledgements
Sabbe, and Dr J.M. Stewart for their helpful comments and suggestions during this study and manuscript preparation. Published with approval from the Director of the Arkansas Agric. Exp. Stn. No. 98017.
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