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Fruiting of cotton IV. Nitrogen, abscisic acid, indole-3-acetic acid, and cutout
Field Crops Research, 22 ( 1989 ) 257-266
257
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Fruiting of Cotton IV. Nitrogen, Abscisic Acid, Indole-3-Acetic Acid, and Cutout
G E N E G U I N N and DONALD L. B R U M M E T T
[,'.S. Department o/Agriculture, Agricultural Research Service, Western Cotton Research Lab¢~ratory, Phoenix, Arizona 85040 (U.S.A.) (Accepted 31 May 1989).
ABSTRACT
Guinn, G. and Brummett, D.L., 1989. Fruiting of cotton. IV. Nitrogen, abscisic acid, indole-3acetic acid, and cutout. Field. Crops Res., 22: 257-266. Decreases in growth, flowering rate, and fruit (boll) retention of cotton (Gossypium hirsutum L. ) characterize cutnut, a hiatus in fruiting. Nitrogen deficiency hastens cutout, and has been reported to cause an increase in the concentration of abscisic acid (ABA), a growth-inhibiting hormone. A field experiment was conducted with 'Deltapine 61' cotton to determine possible effects of N deficit on the concentrations of ABA and the growth-promoting hormone, indole-3acetic acid (IAA), in cotton fruiting branches in relation to their growth, flowering rate, and boll retenti()n. Three harvests were made during the fruiting cycle in 1987 to determine if the ABA concentration of fruiting branches increased or IAA concentration decreased as growth decreased during the season. Three harvests were made the next season (1988) to determine possible changes in c~mcentrations of ABA and IAA in flower buds and flowers. Fruiting branches were always sh[)rter ()n low-N than on high-N plants, and fruiting branches that developed late in the season were much shorter than those that developed early. The ABA concentrations in fruiting branches decreased after 9 July and ABA concentrations were always lower in fruiting branches of low-N than high-N plants. Therefore, the results do not support the hypothesis that ABA accumulates in N-deficient cotton fruiting branches and inhibits their growth. Decreasing concentrations of IAA. however, could have been a cause of decreased growth of fruiting branches. The IAA content ()f fruiting branches decreased during the fruiting cycle, and was lower in low-N than in high-N plants except in the last harvest. ABA increased and IAA decreased in 3-day-old bolls as boll relent ion decreased. The concentrations of ABA in flower buds and flowers did not increase during the season and were not affected by N deficiency. Likewise, the concentrations of IAA in flower buds and flowers were not affected by N. The concentration of IAA in flower buds decreased during the season, however. In general, the results support the hypothesis that the IAA of cotton fruiting branches is affected by N deficit, decreases during the fruiting cycle, and may be a factor in the decreases in growth, flowering, and boll retention commonly referred to as cutout.
0378-4290/89/$03.50
© 1989 Elsevier Science Publishers B.V.
258
G. GUINN AND D.L. BRUMMETT
INTRODUCTION
Most modern cotton cultivars exhibit cycles in growth, flowering, and fruit retention. As the plants become loaded with fruit (bolls), growth, flowering rate, and boll retention decrease (Patterson et al., 1978; Guinn, 1985a; Kerby et al., 1987 ). If the decreases are pronounced, this hiatus in growth and fruiting is called 'cutout' (Patterson et al., 1978). Early cutout can limit yield because the plants do not continue to fruit through the growing season. Conversely, complete and permanent cutout at the end of the season could be beneficial by depriving overwintering insect pests of a food source before diapause. Therefore, an understanding of the causes of cutout is important because it may permit cultural practices to be adjusted for optimum duration of fruiting. Earlier results with ethylene may partially explain the decreases in boll retention that occur with increasing boll load. Guinn (1976) reported that the rate of ethylene production in bolls increased during the season as boll load increased. This, and other experiments reported in the same paper, indicated that a shortage of photosynthate (a carbohydrate stress induced by dim light, long warm nights, or competition for sugars by an increasing boll load) increased ethylene production in young bolls. Because ethylene is a potent abscission hormone, these results could, at least partially, explain the decrease in boll retention as boll load increases. Abscisic acid (ABA) and indole-3-acetic acid (IAA) may also affect boll retention. We reported earlier (Guinn and Brummett, 1987 ) that ABA was negatively correlated and that IAA was positively correlated with boll retention. Hormonal changes might also be a factor in the decreased growth and flowering that are the other characteristics of cutout. ABA inhibits growth (Addicott and Lyon, 1969; Creelman and Sabbe, 1976) whereas IAA stimulates nucleic acid synthesis (Holm et al., 1970) and is considered a growth hormone (Galston and Purves, 1960; dela Fuente and Leopold, 1970; Evans, 1984). Creelman and Sabbe (1976) postulated that ABA accumulates as bolls developed and that it inhibits growth of main-stem and fruiting-branch terminals and, therefore, inhibits flowering. Guinn (1985b), however, was not able to find support for their hypothesis. Nitrogen supply affects the duration of growth and fruiting; N deficiency decreases growth rate and causes earlier cutout (Wadleigh, 1944; Hearn, 1975 ). Jones et al. (1974) developed a computer simulation model for estimating the effects of N deficiency in cotton. Their model and results with plants indicated that N deficiency slows growth and increases abscission of flower buds and bolls. Several workers have reported that a N deficiency increases the ABA concentration in leaves (Goldbach et al., 1975; Daie et al., 1979; Radin et al., 1982 ). Therefore, it is possible that a N deficiency hastens cutout, at least in part, because it causes the concentration of ABA to increase. Conversely, N deft-
NITROGEN, ABA, IAA AND CUTOUT IN COTTON
259
ciency might decrease the concentration of IAA and, thereby, slow growth and decrease boll retention. An experiment was conducted to determine whether the concentrations of ABA and IAA in fruiting branches are affected by N supply and whether they change as growth rate decreases during cutout. The ABA and IAA concentrations in flower buds, flowers, and 3-day-old bolls were also measured to determine whether they are affected by N or whether they change during the fruiting cycle as boll retention decreases. MATERIALS AND METHODS
Plant culture 'Deltapine 61' cotton seeds were planted 1 April 1986, 8 April 1987, and 28 April 1988 in a field at the Western Cotton Research Laboratory in Phoenix, Arizona. The soil is an Avondale clay loam (a member of the fine-loamy, mixed, hyperthermic family of Typic Torrifluevents). Plots, 5 × 30 m, were separated by berms to prevent the movement of N during irrigations. Each plot contained four rows spaced 1 m apart. Urea was applied to the high-N plots in 1986 and 1987 to give 150 kg N h a - 1. No fertilizer was applied to the low-N plots in 1986 or 1987. Ammonium phosphate (18:46:0) was applied preplant to all plots in 1988 to give 27 kg N ha -1. Urea was then applied only to the high-N plots to give an additional 123 kg N h a - 1. The N treatment was replicated four times in a randomized complete block to give a total of eight plots each year. The same plots were used in all three years for the low-N treatment. The weather was typical of summer weather in Phoenix: hot, dry, and sunny. All plots were furrow-irrigated with about 13 cm of water approximately every 2 weeks. Because ABA is affected by moisture stress (Radin et al., 1982), moisture status was estimated by measuring leaf water-potentials. Youngest fully expanded mainstem leaves, at about the 4th node down from the apex, were harvested between 12: 00 and 14: 00 h, placed in plastic bags in a moist chest and transported to the laboratory where leaf water-potentials were determined with a pressure chamber (Waring and Cleary, 1967). Nitrogen status of plants was estimated by measuring nitrate in petioles of the first fully expanded mainstem leaf by the method of Cataldo et al. (1975). Insecticides were applied as needed to control insect pests. Flowers were counted and tagged daily (except Saturdays and Sundays) in 8-m segments of one of the center rows of each plot in 1987. Tagged bolls were recovered in October and used to calculate daily boll-retention rates. When possible, 25 fruiting branches were harvested from the two center rows of each plot at 14-day intervals during the 1987 season. Fruiting branches were not harvested in the 8-m segments used for flowering-rate and boll-retention data. The first nodes of fruiting branches that were to be harvested were
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G. GUINN AND D.L. BRUMMETT
tagged on the day of anthesis on 22 June and on 6 and 20 June and on 6 and 20 July. (It was not possible to find 25 white flowers at the first node of fruiting branches on every plot on 20 July; some had as few as 15. ) The fruiting branches were harvested three days after tagging by cutting them at the first node. This gave fruiting branches at the same stage of development and presumably the same age (but not at the same position on the main stem) on three different dates during the season. The fruiting branches were quickly rinsed in tap water and then in cold deionized water and transported to the laboratory in an ice chest for processing. The 3-day-old bolls at the first node of harvested fruiting branches were removed and analyzed separately. All flower buds were removed after the fruiting branches were harvested, and were not included in the analyses of the fruiting branches. Leaves were not removed and so were included in the analyses. No flowers were present on the harvested fruiting branches. We measured the length of the fruiting branches, cut them into short pieces, combined those harvested from each plot on a given date, and quickly froze them at - 80 ° C. Flower buds and flowers were harvested at 14-day intervals in 1988. Flower buds were harvested from the first node of fruiting branches that were one mainstem node above a flower at anthesis. Because fruiting branches develop at intervals of about three days (up the main stem), the flower bud was approximately three days preanthesis when a flower was at anthesis on the preceding fruiting branch. The flower at anthesis was also harvested. Ten flower buds and ten flowers were harvested in each plot, rinsed in tap water and then in cold deionized water, and quickly frozen at - 80 ° C. All tissues were lyophilized, weighed, ground to pass through a 40-mesh screen, and stored in the dark in sealed vials at - 80 ° C. The vials were flushed with N2 before sealing to minimize oxidation of ABA and IAA during storage.
Analysis of free ABA and IAA Free ABA and IAA were purified and measured by a modification of procedures reported previously (Guinn et al., 1986). Samples of 200-1000 mg each were extracted overnight at 4 °C with gentle stirring in 30 mL of 80% aqueous methanol containing 200 mg of butylated hydroxytoluene (BHT) and 100 mg of Na ascorbate L - 1. Approximately 5000 dpm 1 each of 14C-ABA and 14C-IAA were added as internal standards to each sample at the start of extraction. The samples and four 10-mL rinses (with the same extracting solvent) were filtered. The methanol was then removed by rotary flash evaporation at 40 ° C. Lipids and chlorophyll were removed from the aqueous residue by extracting two or three times (depending upon amount of tissue) with 10-mL portions of hexane. The pH was adjusted to 2.8 with 1N H3PO4. ABA and IAA were then exldpm, disintegrationsper minute.
NITROGEN,ABA,IAAAND CUTOUTIN COTTON
261
tracted from the acidified aqueous samples with three 10-mL portions of CH2CI2 containing 10 mg B H T L - 1. About 0.5 mL of water was added, the CH2C12 was removed by rotary flash evaporation, and the residue was mixed with 5 mL of l m M HC1. The acidic aqueous samples were filtered through a 25-ram nylon 66 filter (Dunlap and Guinn, 1989) and into a C18 cartridge (Waters SepPak) 1 preconditioned with pure methanol followed by l m M HC1. The filter and cartridge were rinsed with 10 mL of pure water and then separated. IAA was eluted from the C18 cartridge with 5 mL of methanol into a 100-mL spherical flask containing 50/tg of BHT. The methanol was concentrated by rotary flash evaporation and the residue transferred to a 1.5-mL conical tube. The volume was reduced to about 25 #L under a stream of N2 at 40 ° C. The samples were further purified by high-performance liquid chromatography (HPLC; Guinn and Hendrix, 1985; Guinn et al., 1986). Three columns were used in sequence. The samples were first fractionated on a strong anion exchange (SAX) column that had been in use long enough for retention times to decrease. It was developed with methanol 0.02N acetic acid. The ABA and IAA fractions were collected separately in 1.5-mL conical tubes and evaporated to the aqueous phase under a stream of N2 at 40 ° C. The IAA was further purified on a newer SAX column developed with 80% methanol/0.05N acetic acid. Both fractions were then subjected to HPLC on a C18 column (Alltech Adsorbosphere C18 HS, 5 #m) developed with 60% methanol/0.02N acetic acid. Samples were injected with a bracketing technique (Guinn and Hendrix, 1985 ) to improve resolution, increase sensitivity, and stabilize peak heights. This technique involves placing about 250/tL of a 'weak' solvent such as water in the injection loop just before and just after the sample so that the sample is bracketed with weak solvent. The solutes in the sample are then strongly retained by the stationary phase and accumulate briefly as a narrow band at the head of the column until the weak solvent is displaced by the stronger solvent (60% methanol). Elution of IAA was monitored by native fluorescence, and ABA was monitored by absorbance at 254 nm. The amount of each was estimated by peak heights after correcting for losses during extraction and purification. Recoveries averaged 50-60% for IAA and about 80% for ABA. R E S U L T S AND D I S C U S S I O N
Flowering rate was decreased by N deficit early in the fruiting cycle but then was the same in high-N and low-N plants on 9 and 23 July despite much lower petiole NO3 concentrations in low-N than in high-N plants (Table 1 ). Kerby et al. (1987) also found that N had little effect on flowering rate during the first flowering cycle of 'Acala' cotton. Our high-N plants, however, resumed 1Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment 'by the United States Department of Agriculture.
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G. GUINN AND D.L. BRUMMETT
TABLE 1 Leaf water potentials, petiole nitrate contents, a n d flowering rates on different dates in high-N a n d low-N cotton plants in 19871 Date
25 June High N Low N 9 July . High N Low N 23 July High N Low N
Water potential 2 (MPa)
Petiol nitrate (t NO3 g - l )
Flowering rate (No. m -2 day -1)
- 1.70 ± .03 - 1.75 ± .02
13.0 _+0.8 2.8 + 0.9
3.68 _+0.14 2.52 ± 0.34
- 1.88 +_.05 - 1.80 ± .04
10.5 _+ 1.1 0.9 + 0.2
4.75 ± 0.22 4.42 +_0.15
- 2.10 ± .08 - 2.04 +_.04
4.4 ± 0.2 0.6 ± 0.1
0.40_+ 0.12 0.41 ± 0.17
1Data are averages of four replicates ± SE. 2Plots were irrigated on 19 J u n e a n d on 2, 17, a n d 31 July.
flowering much sooner and flowered much more after cutout than the low-N plants (data not presented). Results obtained in 1986 are not reported because flower buds were not removed from the fruiting branches before they were analyzed for ABA and IAA. Subsequent results indicated that flower buds contain much higher concentrations of IAA than other parts of the fruiting branches (cf. Tables 2 and 4). Therefore, the inclusion of flower buds with fruiting branches confounded the results as the ratio of bud biomass to fruiting-branch biomass changed. The concentration of IAA in combined fruiting branches plus flower buds initially increased because of a relatively greater decrease in biomass of fruiting branches than of IAA-rich buds, then decreased later in the fruiting cycle as the biomass of both decreased (data not presented). Fruiting branches were shorter in low-N than in high-N plants at every harvest (Table 2). They exhibited marked decreases in growth (as measured by length) during the fruiting cycle despite the fact that all fruiting branches were at the same stage of development when harvested (i.e., 3 days after a white flower appeared at the first node). Some fruiting branches had no measurable growth beyond the first node. The decrease in flowering rate lagged behind the decrease in growth of fruiting branches (cf. Tables I and 2), probably because flower buds were initiated several weeks before they opened as flowers. Most of the late flowers occurred only at the first node of fruiting branches, as reported by Kerby et al. (1987). It is unlikely that ABA caused the reduction in growth of fruiting branches. Although the ABA concentration in fruiting branches was higher on 9 July, it was not higher on 23 July than on 25 June even though fruiting branches were
NITROGEN,ABA,1AAANDCUTOUTIN COTTON
263
TABLE 2 Lengths of fruiting branches and concentrations 1of free ABA and IAA in fruiting branches (FB) on different dates in high-N and low-N cotton plants in 19872 Date
June High N LowN 9 July High N LowN 23 July HighN Low N
Lengtha (mm)
ABA (ng g-1 DW)
IAA (ng g-~ DW)
(ng FB-1)
25
89 + 13 57+ 6
531 + 15 483+15
78 + 7 51_+1
66 + 15 29+ 2
54 + 8 31_+5
914_ 31 763_+40
47_+2 39_+1
37_+3 15_+2
574_+ 5 490_+ 8
31_+1 32 _+1
5_+ 0.3 2_+ 0.2
7_+ 0.4 3 _+ 0.2
~Concentrations are expressed on a dry-weight basis. 2Data are averages of four replications___SE. 3Fruiting branches were severed at and measured beyond the first node. very short by 23 July (Table 2). Furthermore, the concentration of ABA in fruiting branches of low-N plants was lower at every harvest than it was in fruiting branches of high-N plants. Therefore, the shorter length of fruiting branches in N-deficient plants was not caused by a higher concentration of ABA inhibiting growth. The lower concentrations of ABA in fruiting branches of N-deficient plants contrast with results obtained by others in which N deficiency increased ABA concentrations in leaves (Goldbach et al., 1975; Daie et al., 1979; Radin et al., 1982). The differences in concentration of IAA in fruiting branches support the hypothesis that a decrease in IAA is one cause of the measured decreases in growth of fruiting branches. The concentration of IAA in fruiting branches decreased during the season as growth (length) of the fruiting branches decreased (Table 2). Fruiting branches of high-N plants initially contained higher concentrations of IAA than the shorter fruiting branches of the low-N plants, b u t the concentrations eventually decreased to the same low levels in both treatments. Because of the difference in weights of fruiting branches, the amount of IAA per fruiting branch (rather than the concentration) was different at every harvest. T h e IAA content decreased from 66 to 7 ng per fruiting branch in high-N plants and from 29 to 3 ng per fruiting branch in low-N plants (Table 2). Changes in gibberellins and cyotokinins may also have affected growth, b u t these were not measured. As expected, boll retention decreased during the season (Table 3 ). The concentration of ABA in bolls increased during the season, possibly because of changes in water status of the plants. The plants became slightly stressed by 23 July (Table 1) even though all harvests were made within a week after
264
G. GUINNAND D.L. BRUMMETT
TABLE 3 Boll retention rates and concentrations of free ABA and IAA in 3-day-old bolls on different dates in high-N and low-N cotton plants in 19871 Date
Boll retention (%)
ABA n g g -~ DW
IAA ng g - 1 DW
ng boll- '
25 J u n e High N Low N 9 July High N Low N 23 J u l y High N Low N
85+2 89 ± 3
1579+51 1580 + 37
76+7 88 + 5
19+2 23 ± 1
43 _+2 42 ± 4
1950 ± 35 1981 + 62
78 + 5 76 ± 4
21 + 1 18 ± 1
26_+3 6+ 2
2102_+ 188 2434 ± 339
52±4 53 ± 5
12_+ 1 9± 1
~Data are averages of four replications ± SE.
irrigation. N supply did not affect the concentration of ABA or IAA in bolls (Table 3 ). The concentration of IAA in bolls decreased by 23 July as the plants entered cutout, and boll retention decreased to very low levels (Table 3). Because the weight of the bolls also decreased, the amount of IAA per boll decreased more than the concentration, especially in the N-deficient plants where the amount decreased from 23 ng of IAA boll- 1 on 25 June to 9 ng boll- 1 on 23 July (Table 3). ABA and IAA both changed in ways that could have caused low boll retention on 23 July. A reason for the difference between high-N and low-N plants in boll retention on 23 July is not apparent, however. The concentration of ABA was higher and the concentration of IAA was lower than on 25 June when boll retention was high, but neither hormone was affected by N level. Abscission is regulated by the balance of several hormones, including ethylene and possibly cytokinins, neither of which was measured in this experiment. Nitrogen had no consistent effect on the concentrations of ABA and IAA in flower buds and flowers (Table 4). Furthermore, ABA concentrations in flower buds and flowers did not change during the season. In contrast, the concentration of IAA in flower buds decreased greatly during the season (Table 4). Bolls contained greater concentrations of ABA than other tissues, and flower buds contained by far the greatest concentrations of IAA. Flowers contained much less IAA than flower buds, but IAA concentration in flowers did not decrease during the season as it did in flower buds (Table 4). In summary, decreased growth of fruiting branches could not be attributed to an effect of N deficit on ABA or to an increase in the concentration of ABA in fruiting branches, flower buds, or flowers during the season. Conversely,
NITROGEN,ABA,IAA ANDCUTOUTIN COTTON
265
TABLE 4 Concentrations (ng g- ~ DW ) of free ABA and IAA in flower buds and flowers of high-N and lowN cotton on different dates in 1988 ~ Date
29 June High N Low N 18 J u l y High N Low N 3 August HighN Low N
ABA
IAA
Buds 2
Flowers
Buds 2
Flowers
1096_+ 27 1126_+ 45
903 _+33 829_+ 18
2567 _+408 3161_+590
96 -+ 9 90_+ 2
1114 _+ 105 1088 _+ 136
853 _+35 822 _+20
1372 + 159 1136 _+ 104
92 _+25 96 + 29
1030_+ 37 1083 _+31
836_+12 812 _+36
742_+ 89 898 _+ 146
117+ 5 99 -+ 9
IData are averages of four replications -+ SE. eFlower buds were approximately 3 days pre-anthesis when harvested.
decreases in the concentration of IAA support the hypothesis that low levels of IAA limit growth of fruiting branches and decrease boll retention as cotton plants enter cutout. Decreases in the free IAA contents of cotton fruiting branches, flower buds, and bolls are consistent with the hypothesis that IAA is a factor in cutout, but IAA may not be the only hormone involved; whether cytokinins may also decrease is to be determined in future studies. ACKNOWLEDGEMENT
We thank Marie P. Eidenbock for measuring leaf water-potentials and petiole nitrate concentrations.
REFERENCES Addicott, F.T. and Lyon, J.L., 1969. Physiology of abscisic acid and related substances. Annu. Rev. Plant Physiol., 20: 139-164. Cataldo, D.A., Haroon, M., Schrader, L.E. and Youngs, V.L., 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicyclic acid. Commun. Soil Sci. Plant Anal., 6: 71-80. Creelman, R.A. and Sabbem W.E., 1976. The effects of exogenous ABA (abscisic acid) upon cotton plant terminal growth. In: J.M. Brown (Editor), Proc. 28th Cotton Improvement Conf., 5 7 January, Las Vegas. National Cotton Council, Memphis, Tenn., pp. 104-107. Daie, J., Seely, S.D. and Campbell, W.F., 1979. Nitrogen deficiency influence on abscisic acid in tomato. HortScience, 14: 261-262. dela Fuente, R.K. and Leopold, A.C., 1970. Time course of auxin stimulations of growth. Plant Physiol., 46: 186-189.
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Dunlap, J.R. and Guinn, G., 1989. A simple purification of indole-3-acetic acid and abscisic acid for GC-SIM-MS analysis by microfiltration of aqueous samples through nylon. Plant Physiol., 90: 197-201. Evans, M.L., 1984. Functions of hormones at the cellular level of organization. In: T.K. Scott {Editor), Encyclopedia of Plant Physiology, New Ser., Vol. 10. Hormonal Regulation of Development. II. The Functions of Hormones from the Level of the Cell to the Whole Plant. Springer, Berlin, pp. 21-79. Galston, A.W. and Purves, W.K., 1960. The mechanism of action of auxin. Annu. Rev. Plant Physiol., 11: 239-276. Goldbach, E., Goldbach, H., Wagner, H. and Michael, G., 1975. Influence of N-deficiency on the abscisic acid content of sunflower plants. Physiol. Plant., 34: 138-140. Guinn, G., 1976. Nutritional stress and ethylene evolution by young cotton bolls. Crop Sci., 16: 89-91. Guinn, G., 1985a. Fruiting of cotton. III. Nutritional stress and cutout. Crop Sci., 25: 981-985. Guinn, G., 1985b. Abscisic acid and cutout in cotton. Plant Physiol., 77: 16-20. Guinn, G. and Brummett, D.L., 1987. Concentrations of abscisic acid and indoleacetic acid in cotton fruits and their abscission zones in relation to fruit retention. Plant Physiol., 83:199 202. Guinn, G. and Hendrix, D.L., 1985. Bracketing, a simple loading technique that increases sample recovery, improves resolution, and increases sensitivity in high performance liquid chromatography. J. Chromatogr., 348: 123-129. Guinn, G., Brummett, D.L. and Beier, R.C., 1986. Purification and measurement of abscisic acid and indoleacetic acid by high performance liquid chromatography. Plant Physiol., 81:997 1002. Hearn, A.B., 1975. Response of cotton to water and nitrogen in a tropical environment. II. Date of last watering and rate of application of nitrogen fertilizer. J. Agric. Sci., Camb., 84: 419-430. Holm, R.E., O'Brien, T.J., Key, L. and Cherry, J.H., 1970. The influence of auxin and ethylene on chromatin-directed ribonucleic acid synthesis in soybean hypocotyl. Plant Physiol., 45:41 45. Jones, J.W., Hesketh, J.D., Kamprath, E.J. and Bowen, H.D., 1974. Development of a nitrogen balance for cotton growth models: A first approximation. Crop Sci., 14: 541-546. Kerby, T.A., Keeley, M. and Johnson, S., 1987. Growth and development of Acala cotton. Univ. Calif. Agric. Exp. Stn. Bull. No. 1921, 13 pp. Patterson, L.L., Buxton, D.R. and Briggs, R.E., 1978. Fruiting in cotton as affected by controlled boll set. Agron. J., 70: 118-122. Radin, J.W., Parker, L.L. and Guinn, G., 1982. Water relations of cotton plants under nitrogen deficiency. V. Environmental control of abscisic acid accumulation and stomatal sensitivity to abscisic acid. Plant Physiol., 70: 1066-1070. Wadleigh, C.H., 1944. Growth status of the cotton plant as influenced by the supply of nitrogem. Ark. Agric. Exp. Stn. Bull. No. 446, 138 pp. Waring, R.H. and Cleary, B.D., 1967. Plant moisture stress: Evaluation by pressure bomb. Science, 155: 1248-1254.
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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Fruiting of Cotton IV. Nitrogen, Abscisic Acid, Indole-3-Acetic Acid, and Cutout
G E N E G U I N N and DONALD L. B R U M M E T T
[,'.S. Department o/Agriculture, Agricultural Research Service, Western Cotton Research Lab¢~ratory, Phoenix, Arizona 85040 (U.S.A.) (Accepted 31 May 1989).
ABSTRACT
Guinn, G. and Brummett, D.L., 1989. Fruiting of cotton. IV. Nitrogen, abscisic acid, indole-3acetic acid, and cutout. Field. Crops Res., 22: 257-266. Decreases in growth, flowering rate, and fruit (boll) retention of cotton (Gossypium hirsutum L. ) characterize cutnut, a hiatus in fruiting. Nitrogen deficiency hastens cutout, and has been reported to cause an increase in the concentration of abscisic acid (ABA), a growth-inhibiting hormone. A field experiment was conducted with 'Deltapine 61' cotton to determine possible effects of N deficit on the concentrations of ABA and the growth-promoting hormone, indole-3acetic acid (IAA), in cotton fruiting branches in relation to their growth, flowering rate, and boll retenti()n. Three harvests were made during the fruiting cycle in 1987 to determine if the ABA concentration of fruiting branches increased or IAA concentration decreased as growth decreased during the season. Three harvests were made the next season (1988) to determine possible changes in c~mcentrations of ABA and IAA in flower buds and flowers. Fruiting branches were always sh[)rter ()n low-N than on high-N plants, and fruiting branches that developed late in the season were much shorter than those that developed early. The ABA concentrations in fruiting branches decreased after 9 July and ABA concentrations were always lower in fruiting branches of low-N than high-N plants. Therefore, the results do not support the hypothesis that ABA accumulates in N-deficient cotton fruiting branches and inhibits their growth. Decreasing concentrations of IAA. however, could have been a cause of decreased growth of fruiting branches. The IAA content ()f fruiting branches decreased during the fruiting cycle, and was lower in low-N than in high-N plants except in the last harvest. ABA increased and IAA decreased in 3-day-old bolls as boll relent ion decreased. The concentrations of ABA in flower buds and flowers did not increase during the season and were not affected by N deficiency. Likewise, the concentrations of IAA in flower buds and flowers were not affected by N. The concentration of IAA in flower buds decreased during the season, however. In general, the results support the hypothesis that the IAA of cotton fruiting branches is affected by N deficit, decreases during the fruiting cycle, and may be a factor in the decreases in growth, flowering, and boll retention commonly referred to as cutout.
0378-4290/89/$03.50
© 1989 Elsevier Science Publishers B.V.
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G. GUINN AND D.L. BRUMMETT
INTRODUCTION
Most modern cotton cultivars exhibit cycles in growth, flowering, and fruit retention. As the plants become loaded with fruit (bolls), growth, flowering rate, and boll retention decrease (Patterson et al., 1978; Guinn, 1985a; Kerby et al., 1987 ). If the decreases are pronounced, this hiatus in growth and fruiting is called 'cutout' (Patterson et al., 1978). Early cutout can limit yield because the plants do not continue to fruit through the growing season. Conversely, complete and permanent cutout at the end of the season could be beneficial by depriving overwintering insect pests of a food source before diapause. Therefore, an understanding of the causes of cutout is important because it may permit cultural practices to be adjusted for optimum duration of fruiting. Earlier results with ethylene may partially explain the decreases in boll retention that occur with increasing boll load. Guinn (1976) reported that the rate of ethylene production in bolls increased during the season as boll load increased. This, and other experiments reported in the same paper, indicated that a shortage of photosynthate (a carbohydrate stress induced by dim light, long warm nights, or competition for sugars by an increasing boll load) increased ethylene production in young bolls. Because ethylene is a potent abscission hormone, these results could, at least partially, explain the decrease in boll retention as boll load increases. Abscisic acid (ABA) and indole-3-acetic acid (IAA) may also affect boll retention. We reported earlier (Guinn and Brummett, 1987 ) that ABA was negatively correlated and that IAA was positively correlated with boll retention. Hormonal changes might also be a factor in the decreased growth and flowering that are the other characteristics of cutout. ABA inhibits growth (Addicott and Lyon, 1969; Creelman and Sabbe, 1976) whereas IAA stimulates nucleic acid synthesis (Holm et al., 1970) and is considered a growth hormone (Galston and Purves, 1960; dela Fuente and Leopold, 1970; Evans, 1984). Creelman and Sabbe (1976) postulated that ABA accumulates as bolls developed and that it inhibits growth of main-stem and fruiting-branch terminals and, therefore, inhibits flowering. Guinn (1985b), however, was not able to find support for their hypothesis. Nitrogen supply affects the duration of growth and fruiting; N deficiency decreases growth rate and causes earlier cutout (Wadleigh, 1944; Hearn, 1975 ). Jones et al. (1974) developed a computer simulation model for estimating the effects of N deficiency in cotton. Their model and results with plants indicated that N deficiency slows growth and increases abscission of flower buds and bolls. Several workers have reported that a N deficiency increases the ABA concentration in leaves (Goldbach et al., 1975; Daie et al., 1979; Radin et al., 1982 ). Therefore, it is possible that a N deficiency hastens cutout, at least in part, because it causes the concentration of ABA to increase. Conversely, N deft-
NITROGEN, ABA, IAA AND CUTOUT IN COTTON
259
ciency might decrease the concentration of IAA and, thereby, slow growth and decrease boll retention. An experiment was conducted to determine whether the concentrations of ABA and IAA in fruiting branches are affected by N supply and whether they change as growth rate decreases during cutout. The ABA and IAA concentrations in flower buds, flowers, and 3-day-old bolls were also measured to determine whether they are affected by N or whether they change during the fruiting cycle as boll retention decreases. MATERIALS AND METHODS
Plant culture 'Deltapine 61' cotton seeds were planted 1 April 1986, 8 April 1987, and 28 April 1988 in a field at the Western Cotton Research Laboratory in Phoenix, Arizona. The soil is an Avondale clay loam (a member of the fine-loamy, mixed, hyperthermic family of Typic Torrifluevents). Plots, 5 × 30 m, were separated by berms to prevent the movement of N during irrigations. Each plot contained four rows spaced 1 m apart. Urea was applied to the high-N plots in 1986 and 1987 to give 150 kg N h a - 1. No fertilizer was applied to the low-N plots in 1986 or 1987. Ammonium phosphate (18:46:0) was applied preplant to all plots in 1988 to give 27 kg N ha -1. Urea was then applied only to the high-N plots to give an additional 123 kg N h a - 1. The N treatment was replicated four times in a randomized complete block to give a total of eight plots each year. The same plots were used in all three years for the low-N treatment. The weather was typical of summer weather in Phoenix: hot, dry, and sunny. All plots were furrow-irrigated with about 13 cm of water approximately every 2 weeks. Because ABA is affected by moisture stress (Radin et al., 1982), moisture status was estimated by measuring leaf water-potentials. Youngest fully expanded mainstem leaves, at about the 4th node down from the apex, were harvested between 12: 00 and 14: 00 h, placed in plastic bags in a moist chest and transported to the laboratory where leaf water-potentials were determined with a pressure chamber (Waring and Cleary, 1967). Nitrogen status of plants was estimated by measuring nitrate in petioles of the first fully expanded mainstem leaf by the method of Cataldo et al. (1975). Insecticides were applied as needed to control insect pests. Flowers were counted and tagged daily (except Saturdays and Sundays) in 8-m segments of one of the center rows of each plot in 1987. Tagged bolls were recovered in October and used to calculate daily boll-retention rates. When possible, 25 fruiting branches were harvested from the two center rows of each plot at 14-day intervals during the 1987 season. Fruiting branches were not harvested in the 8-m segments used for flowering-rate and boll-retention data. The first nodes of fruiting branches that were to be harvested were
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tagged on the day of anthesis on 22 June and on 6 and 20 June and on 6 and 20 July. (It was not possible to find 25 white flowers at the first node of fruiting branches on every plot on 20 July; some had as few as 15. ) The fruiting branches were harvested three days after tagging by cutting them at the first node. This gave fruiting branches at the same stage of development and presumably the same age (but not at the same position on the main stem) on three different dates during the season. The fruiting branches were quickly rinsed in tap water and then in cold deionized water and transported to the laboratory in an ice chest for processing. The 3-day-old bolls at the first node of harvested fruiting branches were removed and analyzed separately. All flower buds were removed after the fruiting branches were harvested, and were not included in the analyses of the fruiting branches. Leaves were not removed and so were included in the analyses. No flowers were present on the harvested fruiting branches. We measured the length of the fruiting branches, cut them into short pieces, combined those harvested from each plot on a given date, and quickly froze them at - 80 ° C. Flower buds and flowers were harvested at 14-day intervals in 1988. Flower buds were harvested from the first node of fruiting branches that were one mainstem node above a flower at anthesis. Because fruiting branches develop at intervals of about three days (up the main stem), the flower bud was approximately three days preanthesis when a flower was at anthesis on the preceding fruiting branch. The flower at anthesis was also harvested. Ten flower buds and ten flowers were harvested in each plot, rinsed in tap water and then in cold deionized water, and quickly frozen at - 80 ° C. All tissues were lyophilized, weighed, ground to pass through a 40-mesh screen, and stored in the dark in sealed vials at - 80 ° C. The vials were flushed with N2 before sealing to minimize oxidation of ABA and IAA during storage.
Analysis of free ABA and IAA Free ABA and IAA were purified and measured by a modification of procedures reported previously (Guinn et al., 1986). Samples of 200-1000 mg each were extracted overnight at 4 °C with gentle stirring in 30 mL of 80% aqueous methanol containing 200 mg of butylated hydroxytoluene (BHT) and 100 mg of Na ascorbate L - 1. Approximately 5000 dpm 1 each of 14C-ABA and 14C-IAA were added as internal standards to each sample at the start of extraction. The samples and four 10-mL rinses (with the same extracting solvent) were filtered. The methanol was then removed by rotary flash evaporation at 40 ° C. Lipids and chlorophyll were removed from the aqueous residue by extracting two or three times (depending upon amount of tissue) with 10-mL portions of hexane. The pH was adjusted to 2.8 with 1N H3PO4. ABA and IAA were then exldpm, disintegrationsper minute.
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261
tracted from the acidified aqueous samples with three 10-mL portions of CH2CI2 containing 10 mg B H T L - 1. About 0.5 mL of water was added, the CH2C12 was removed by rotary flash evaporation, and the residue was mixed with 5 mL of l m M HC1. The acidic aqueous samples were filtered through a 25-ram nylon 66 filter (Dunlap and Guinn, 1989) and into a C18 cartridge (Waters SepPak) 1 preconditioned with pure methanol followed by l m M HC1. The filter and cartridge were rinsed with 10 mL of pure water and then separated. IAA was eluted from the C18 cartridge with 5 mL of methanol into a 100-mL spherical flask containing 50/tg of BHT. The methanol was concentrated by rotary flash evaporation and the residue transferred to a 1.5-mL conical tube. The volume was reduced to about 25 #L under a stream of N2 at 40 ° C. The samples were further purified by high-performance liquid chromatography (HPLC; Guinn and Hendrix, 1985; Guinn et al., 1986). Three columns were used in sequence. The samples were first fractionated on a strong anion exchange (SAX) column that had been in use long enough for retention times to decrease. It was developed with methanol 0.02N acetic acid. The ABA and IAA fractions were collected separately in 1.5-mL conical tubes and evaporated to the aqueous phase under a stream of N2 at 40 ° C. The IAA was further purified on a newer SAX column developed with 80% methanol/0.05N acetic acid. Both fractions were then subjected to HPLC on a C18 column (Alltech Adsorbosphere C18 HS, 5 #m) developed with 60% methanol/0.02N acetic acid. Samples were injected with a bracketing technique (Guinn and Hendrix, 1985 ) to improve resolution, increase sensitivity, and stabilize peak heights. This technique involves placing about 250/tL of a 'weak' solvent such as water in the injection loop just before and just after the sample so that the sample is bracketed with weak solvent. The solutes in the sample are then strongly retained by the stationary phase and accumulate briefly as a narrow band at the head of the column until the weak solvent is displaced by the stronger solvent (60% methanol). Elution of IAA was monitored by native fluorescence, and ABA was monitored by absorbance at 254 nm. The amount of each was estimated by peak heights after correcting for losses during extraction and purification. Recoveries averaged 50-60% for IAA and about 80% for ABA. R E S U L T S AND D I S C U S S I O N
Flowering rate was decreased by N deficit early in the fruiting cycle but then was the same in high-N and low-N plants on 9 and 23 July despite much lower petiole NO3 concentrations in low-N than in high-N plants (Table 1 ). Kerby et al. (1987) also found that N had little effect on flowering rate during the first flowering cycle of 'Acala' cotton. Our high-N plants, however, resumed 1Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment 'by the United States Department of Agriculture.
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TABLE 1 Leaf water potentials, petiole nitrate contents, a n d flowering rates on different dates in high-N a n d low-N cotton plants in 19871 Date
25 June High N Low N 9 July . High N Low N 23 July High N Low N
Water potential 2 (MPa)
Petiol nitrate (t NO3 g - l )
Flowering rate (No. m -2 day -1)
- 1.70 ± .03 - 1.75 ± .02
13.0 _+0.8 2.8 + 0.9
3.68 _+0.14 2.52 ± 0.34
- 1.88 +_.05 - 1.80 ± .04
10.5 _+ 1.1 0.9 + 0.2
4.75 ± 0.22 4.42 +_0.15
- 2.10 ± .08 - 2.04 +_.04
4.4 ± 0.2 0.6 ± 0.1
0.40_+ 0.12 0.41 ± 0.17
1Data are averages of four replicates ± SE. 2Plots were irrigated on 19 J u n e a n d on 2, 17, a n d 31 July.
flowering much sooner and flowered much more after cutout than the low-N plants (data not presented). Results obtained in 1986 are not reported because flower buds were not removed from the fruiting branches before they were analyzed for ABA and IAA. Subsequent results indicated that flower buds contain much higher concentrations of IAA than other parts of the fruiting branches (cf. Tables 2 and 4). Therefore, the inclusion of flower buds with fruiting branches confounded the results as the ratio of bud biomass to fruiting-branch biomass changed. The concentration of IAA in combined fruiting branches plus flower buds initially increased because of a relatively greater decrease in biomass of fruiting branches than of IAA-rich buds, then decreased later in the fruiting cycle as the biomass of both decreased (data not presented). Fruiting branches were shorter in low-N than in high-N plants at every harvest (Table 2). They exhibited marked decreases in growth (as measured by length) during the fruiting cycle despite the fact that all fruiting branches were at the same stage of development when harvested (i.e., 3 days after a white flower appeared at the first node). Some fruiting branches had no measurable growth beyond the first node. The decrease in flowering rate lagged behind the decrease in growth of fruiting branches (cf. Tables I and 2), probably because flower buds were initiated several weeks before they opened as flowers. Most of the late flowers occurred only at the first node of fruiting branches, as reported by Kerby et al. (1987). It is unlikely that ABA caused the reduction in growth of fruiting branches. Although the ABA concentration in fruiting branches was higher on 9 July, it was not higher on 23 July than on 25 June even though fruiting branches were
NITROGEN,ABA,1AAANDCUTOUTIN COTTON
263
TABLE 2 Lengths of fruiting branches and concentrations 1of free ABA and IAA in fruiting branches (FB) on different dates in high-N and low-N cotton plants in 19872 Date
June High N LowN 9 July High N LowN 23 July HighN Low N
Lengtha (mm)
ABA (ng g-1 DW)
IAA (ng g-~ DW)
(ng FB-1)
25
89 + 13 57+ 6
531 + 15 483+15
78 + 7 51_+1
66 + 15 29+ 2
54 + 8 31_+5
914_ 31 763_+40
47_+2 39_+1
37_+3 15_+2
574_+ 5 490_+ 8
31_+1 32 _+1
5_+ 0.3 2_+ 0.2
7_+ 0.4 3 _+ 0.2
~Concentrations are expressed on a dry-weight basis. 2Data are averages of four replications___SE. 3Fruiting branches were severed at and measured beyond the first node. very short by 23 July (Table 2). Furthermore, the concentration of ABA in fruiting branches of low-N plants was lower at every harvest than it was in fruiting branches of high-N plants. Therefore, the shorter length of fruiting branches in N-deficient plants was not caused by a higher concentration of ABA inhibiting growth. The lower concentrations of ABA in fruiting branches of N-deficient plants contrast with results obtained by others in which N deficiency increased ABA concentrations in leaves (Goldbach et al., 1975; Daie et al., 1979; Radin et al., 1982). The differences in concentration of IAA in fruiting branches support the hypothesis that a decrease in IAA is one cause of the measured decreases in growth of fruiting branches. The concentration of IAA in fruiting branches decreased during the season as growth (length) of the fruiting branches decreased (Table 2). Fruiting branches of high-N plants initially contained higher concentrations of IAA than the shorter fruiting branches of the low-N plants, b u t the concentrations eventually decreased to the same low levels in both treatments. Because of the difference in weights of fruiting branches, the amount of IAA per fruiting branch (rather than the concentration) was different at every harvest. T h e IAA content decreased from 66 to 7 ng per fruiting branch in high-N plants and from 29 to 3 ng per fruiting branch in low-N plants (Table 2). Changes in gibberellins and cyotokinins may also have affected growth, b u t these were not measured. As expected, boll retention decreased during the season (Table 3 ). The concentration of ABA in bolls increased during the season, possibly because of changes in water status of the plants. The plants became slightly stressed by 23 July (Table 1) even though all harvests were made within a week after
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G. GUINNAND D.L. BRUMMETT
TABLE 3 Boll retention rates and concentrations of free ABA and IAA in 3-day-old bolls on different dates in high-N and low-N cotton plants in 19871 Date
Boll retention (%)
ABA n g g -~ DW
IAA ng g - 1 DW
ng boll- '
25 J u n e High N Low N 9 July High N Low N 23 J u l y High N Low N
85+2 89 ± 3
1579+51 1580 + 37
76+7 88 + 5
19+2 23 ± 1
43 _+2 42 ± 4
1950 ± 35 1981 + 62
78 + 5 76 ± 4
21 + 1 18 ± 1
26_+3 6+ 2
2102_+ 188 2434 ± 339
52±4 53 ± 5
12_+ 1 9± 1
~Data are averages of four replications ± SE.
irrigation. N supply did not affect the concentration of ABA or IAA in bolls (Table 3 ). The concentration of IAA in bolls decreased by 23 July as the plants entered cutout, and boll retention decreased to very low levels (Table 3). Because the weight of the bolls also decreased, the amount of IAA per boll decreased more than the concentration, especially in the N-deficient plants where the amount decreased from 23 ng of IAA boll- 1 on 25 June to 9 ng boll- 1 on 23 July (Table 3). ABA and IAA both changed in ways that could have caused low boll retention on 23 July. A reason for the difference between high-N and low-N plants in boll retention on 23 July is not apparent, however. The concentration of ABA was higher and the concentration of IAA was lower than on 25 June when boll retention was high, but neither hormone was affected by N level. Abscission is regulated by the balance of several hormones, including ethylene and possibly cytokinins, neither of which was measured in this experiment. Nitrogen had no consistent effect on the concentrations of ABA and IAA in flower buds and flowers (Table 4). Furthermore, ABA concentrations in flower buds and flowers did not change during the season. In contrast, the concentration of IAA in flower buds decreased greatly during the season (Table 4). Bolls contained greater concentrations of ABA than other tissues, and flower buds contained by far the greatest concentrations of IAA. Flowers contained much less IAA than flower buds, but IAA concentration in flowers did not decrease during the season as it did in flower buds (Table 4). In summary, decreased growth of fruiting branches could not be attributed to an effect of N deficit on ABA or to an increase in the concentration of ABA in fruiting branches, flower buds, or flowers during the season. Conversely,
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265
TABLE 4 Concentrations (ng g- ~ DW ) of free ABA and IAA in flower buds and flowers of high-N and lowN cotton on different dates in 1988 ~ Date
29 June High N Low N 18 J u l y High N Low N 3 August HighN Low N
ABA
IAA
Buds 2
Flowers
Buds 2
Flowers
1096_+ 27 1126_+ 45
903 _+33 829_+ 18
2567 _+408 3161_+590
96 -+ 9 90_+ 2
1114 _+ 105 1088 _+ 136
853 _+35 822 _+20
1372 + 159 1136 _+ 104
92 _+25 96 + 29
1030_+ 37 1083 _+31
836_+12 812 _+36
742_+ 89 898 _+ 146
117+ 5 99 -+ 9
IData are averages of four replications -+ SE. eFlower buds were approximately 3 days pre-anthesis when harvested.
decreases in the concentration of IAA support the hypothesis that low levels of IAA limit growth of fruiting branches and decrease boll retention as cotton plants enter cutout. Decreases in the free IAA contents of cotton fruiting branches, flower buds, and bolls are consistent with the hypothesis that IAA is a factor in cutout, but IAA may not be the only hormone involved; whether cytokinins may also decrease is to be determined in future studies. ACKNOWLEDGEMENT
We thank Marie P. Eidenbock for measuring leaf water-potentials and petiole nitrate concentrations.
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Dunlap, J.R. and Guinn, G., 1989. A simple purification of indole-3-acetic acid and abscisic acid for GC-SIM-MS analysis by microfiltration of aqueous samples through nylon. Plant Physiol., 90: 197-201. Evans, M.L., 1984. Functions of hormones at the cellular level of organization. In: T.K. Scott {Editor), Encyclopedia of Plant Physiology, New Ser., Vol. 10. Hormonal Regulation of Development. II. The Functions of Hormones from the Level of the Cell to the Whole Plant. Springer, Berlin, pp. 21-79. Galston, A.W. and Purves, W.K., 1960. The mechanism of action of auxin. Annu. Rev. Plant Physiol., 11: 239-276. Goldbach, E., Goldbach, H., Wagner, H. and Michael, G., 1975. Influence of N-deficiency on the abscisic acid content of sunflower plants. Physiol. Plant., 34: 138-140. Guinn, G., 1976. Nutritional stress and ethylene evolution by young cotton bolls. Crop Sci., 16: 89-91. Guinn, G., 1985a. Fruiting of cotton. III. Nutritional stress and cutout. Crop Sci., 25: 981-985. Guinn, G., 1985b. Abscisic acid and cutout in cotton. Plant Physiol., 77: 16-20. Guinn, G. and Brummett, D.L., 1987. Concentrations of abscisic acid and indoleacetic acid in cotton fruits and their abscission zones in relation to fruit retention. Plant Physiol., 83:199 202. Guinn, G. and Hendrix, D.L., 1985. Bracketing, a simple loading technique that increases sample recovery, improves resolution, and increases sensitivity in high performance liquid chromatography. J. Chromatogr., 348: 123-129. Guinn, G., Brummett, D.L. and Beier, R.C., 1986. Purification and measurement of abscisic acid and indoleacetic acid by high performance liquid chromatography. Plant Physiol., 81:997 1002. Hearn, A.B., 1975. Response of cotton to water and nitrogen in a tropical environment. II. Date of last watering and rate of application of nitrogen fertilizer. J. Agric. Sci., Camb., 84: 419-430. Holm, R.E., O'Brien, T.J., Key, L. and Cherry, J.H., 1970. The influence of auxin and ethylene on chromatin-directed ribonucleic acid synthesis in soybean hypocotyl. Plant Physiol., 45:41 45. Jones, J.W., Hesketh, J.D., Kamprath, E.J. and Bowen, H.D., 1974. Development of a nitrogen balance for cotton growth models: A first approximation. Crop Sci., 14: 541-546. Kerby, T.A., Keeley, M. and Johnson, S., 1987. Growth and development of Acala cotton. Univ. Calif. Agric. Exp. Stn. Bull. No. 1921, 13 pp. Patterson, L.L., Buxton, D.R. and Briggs, R.E., 1978. Fruiting in cotton as affected by controlled boll set. Agron. J., 70: 118-122. Radin, J.W., Parker, L.L. and Guinn, G., 1982. Water relations of cotton plants under nitrogen deficiency. V. Environmental control of abscisic acid accumulation and stomatal sensitivity to abscisic acid. Plant Physiol., 70: 1066-1070. Wadleigh, C.H., 1944. Growth status of the cotton plant as influenced by the supply of nitrogem. Ark. Agric. Exp. Stn. Bull. No. 446, 138 pp. Waring, R.H. and Cleary, B.D., 1967. Plant moisture stress: Evaluation by pressure bomb. Science, 155: 1248-1254.