- Email: [email protected]
Phenotypic expression of a chlorophyll mutant in cowpea (Vigna unguiculata): Environmental influences and effects on productivity
Field Crops Research, 21 (1989) 19-28
19
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
P h e n o t y p i c E x p r e s s i o n of a Chlorophyll Mutant in Cowpea (Vigna unguiculata): E n v i r o n m e n t a l Influences and Effects on P r o d u c t i v i t y W.R. KIRCHHOFF, A.E. HALL and W.H. ISOM
Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 (U.S.A.) (Accepted 4 November 1988)
ABSTRACT Kirchhoff, W.R., Hall, A.E. and Isom, W.H., 1989. Phenotypic expression of a chlorophyll mutant in cowpea (Vigna unguiculata): Environmental influences and effects on productivity. Field Crops Res., 21: 19-28. Phenotypic expression of a chlorophyll-deficientmutation in cowpea was studied to determine the value of this trait for plant breeding. The mutant and its parent were grown under two irradiances in growth chambers, and under two contrasting temperature regimes in glasshouses. In addition, grain-yield and biomass productivity of the mutant and parent were compared under field conditions. Under optimal temperatures, the leaves of the mutant exhibited significantly lower chlorophyll content per unit leaf area and higher chlorophyll-a: b ratio than the parent when grown in low, moderate, or high photon-flux density. Leaf thickness and leaf chlorophyll content per unit leaf area of both genotypes were less for plants grown under low irradiance, but the chlorophyll-a: b ratio was not influenced by irradiance. In contrast, when grown under hot day/night temperatures of 36/29 ° C and high photon-flux density, the mutant exhibited similar chlorophyll content and chlorophyll composition as the parent. There was an association between higher chlorophyll content and lower, more normal, chlorophyll-a: b ratios. This association was also seen in field conditions, where the mutant produced greener leaves during hot weather in mid-summer. In sunny field conditions the mutant produced equivalent average grain-yields over 6 years, and identical biomass to the parent, even though the leaves of the mutant had substantially less chlorophyll during flowering and pod-filling. Instead of considering the mutant to be chlorophylldeficient, it may be more appropriate to state that the parent, and many other cowpeas, have an excess of chlorophyll for growth under sunny conditions.
INTRODUCTION
Selecting for photosynthetic characters could provide a means for breeding crop plants with increased biomass production per unit of solar radiation. However, little progress has been made to date in developing cultivars with increased photosynthetic efficiency, either by empirical breeding with selec0378-4290/89/$03.50
© 1989 Elsevier Science Publishers B.V.
20 tion mainly based upon grain-yield, or by direct selection for photosynthetic characters (Gifford and Jenkins, 1982; Gifford, 1987). Gifford (1987) proposed that in some species there may be an over-investment in chlorophyll due to the unconscious tendency for breeders to select for dark-green foliage. He suggested that it might be worthwhile to breed plants with lower chlorophyll content, thereby releasing a small amount of carbon and nitrogen for redeployment to other tissues. A chlorophyll-deficient mutant and parent have been described recently in cowpea (Kirchhoff et al., 1989a). The expression of chlorophyll deficiency in the leaves of the mutant has been observed to vary during the growing season. Kirchhoff et al. (1989a) showed that elevated chlorophyll-a: b ratio provided a more definitive characterization of the mutant phenotype than did chlorophyll content. In other chlorophyll-b-deficient mutants, it has been reported that chlorophyll content is influenced by growing conditions, especially temperature and light (Nybom, 1955; Alberte et al., 1974; Adedipe and Ormrod, 1975; Hopkins and Walden, 1977; Markwell et al., 1986). Experiments were conducted to determine the extent to which irradiance and temperature influence the chlorophyll content and chlorophyll-a: b ratio of this mutant of cowpea in comparison with the parent. Studies by Kirchhoffet al. (1989b) indicated that the mutant had 13% higher carbon-dioxide assimilation rates at high irradiances and a trend toward lower rates than the parent at low irradiance. This indicates that the mutant may be more productive than the parent in sunny environments. It is also possible that the mutation will decrease productivity, because even though CO2 assimilation rates were slightly higher, the mutation did dramatically reduce lamellar stacking in the chloroplasts, and other aspects of photosynthesis, such as the reduction of NO~- and SO~-, may be impaired. The influence of the chlorophyll-deficient mutation on productivity was examined by evaluating biomass production and grain-yield of the mutant and parent under field conditions. MATERIALSAND METHODS A chlorophyll-deficient mutant of cowpea (M39) and parent, California Blackeye No. 3 (CB3) were studied. They had been shown to differ by a single nuclear gene influencing chlorophyll composition, but have no obvious differences in other characteristics, such as plant habit, leaf shape, floral characteristics, time of flowering, seed characteristics, and resistance to disease (Kirchhoff et al., 1989a).
Contrasting irradiance experiments The mutant and parent were grown in two replicate experiments in two reachin growth chambers which provided 14.75-h daylengths with daytime temper-
21 atures of 30 ° C and nighttime temperatures of 22 ° C. Relative humidity averaged 50% during the day and 60% at night. Contrasting light environments were created within each chamber by installing shade-cloth covers over onehalf of the interior space. Photon flux densities (Fp) were measured with a quantum sensor (Model LI-190s, LI-COR, Lincoln, Neb. ). In the exposed half of each chamber, the Fp (400-700 nm) 15 cm above the pot surface averaged 330/lmol photon m-2 s-1, increasing to an average value of 1100/lmol photon m -2 s -1 15 cm below the light bank. Under the shade-cloth, Fp was approximately one-third of the exposed half of the chamber, with an average value of 110 ]~mol photon m -2 s -1 at 15 cm above the pot. The light quality of each side of each growth chamber was determined using a spectroradiometer (Model 1800, LI-COR, Lincoln, Neb). There were no significant differences in spectral distribution of the light passing through the screens and the light reaching the pot surfaces in the unshaded half-chamber. Plants were grown in 15-1 pots containing pastuerized U.C. Soil Mix II (Matkin and Chandler, 1957) amended with slow-release fertilizer (18:2.6:10 N:P:K, Osmocote, Sierra Chemicals, Milipitas, CA) at a rate of 5 g per pot in the 1st chamber and 25 g per pot in the 2nd chamber, because we had observed mild symptoms of nitrogen deficiency for the 1st planting in the 1st chamber. Three seeds of each genotype were sown into each of four pots in both light environments in both growth chambers. Plants were thinned to 1 seedling per pot within 10 days of planting. Pots were thoroughly watered at sowing and were subsequently supplied with distilled water as needed until harvest. Leaf chlorophyll ( a + b) content and chlorophyll-a:b ratio of recently expanded trifoliate leaves were measured as described by Kirchhoff et al. (1989a). Individual leaf samples from 4 replicate plants of each genotype from each light environment were taken on the 15th day after sowing and approximately every week thereafter for the next 4 weeks. Biomass production was determined 45 days after sowing for plants in the second chamber. The plants were cut at the soil surface, oven-dried at 60 ° C, and then weighed. The plants in the 1st growth chamber grew more slowly, so were allowed to grow for 54 days before an identical biomass harvest was conducted.
Contrasting temperature experiments The mutant and parent were grown in two glasshouses, situated next to each other, which had contrasting temperature regimes. The daily mean maximum/ minimum temperatures in the designated 'optimal house' were 24 °/17 ° C, and in the 'hot house' were 36 °/29 ° C. Light quantity and quality were measured in both houses by a spectroradiometer, and were found not to be significantly different. Plant were grown in 5-1 pots containing pastuerized U.S. Soil Mix II supplemented with 15 g of 18:2.6:10 N:P:K slow-release fertilizer. There were
22 8 pots of each genotype, M39 and CB3. The pots were widely spaced on the benches to provide ample light penetration into the canopy. All pots were thoroughly watered at sowing and then water was withheld until emergence. After emergence, the pots were irrigated with approximately 1-1 half-strength Hoagland's nutrient solution per pot per day. The plants were thinned to 1 seedling per pot at 1 week after planting. Chlorophyll (a + b) content and chlorophyll-a: b ratio of unifoliate and trifoliate leaves were measured every week.
Field experiments The mutant and parent were included in annual performance trials conducted by the Cooperative Extension of the University of California from 1981 through 1986 at the Moreno Valley (1981-1983), South Coast (1984), and Riverside (1985-1986) Field Stations. The trials at each location were conducted under furrow irrigation with optimal management. The experimental design consisted of four randomized blocks. Individual plots consisted of 4 rows, 76 cm apart and 10 m long, with 10 cm between plants. The center 2 rows of each plot were harvested to determine grain-yield. In 1985, shoot biomass production and grain-yield were evaluated prior to the main grain harvest, from 2-m sections taken from 2 rows of each plot. Leaf chlorophyll (a + b) content per unit leaf area and chlorophyll-a:b ratio were measured for recently expanded trifoliate leaves sampled from the upper part of the canopies just prior to flowering in 1986. RESULTS AND DISCUSSION
Response to irradiance Analysis of total shoot-biomass data for M39 and CB3 grown under the moderate and low irradiances showed significant effects of genotype and light environment, but no chamber effects or interactions. Plants in the first chamber which had lower levels of nutrition simply required more time to produce the same biomass as plants in the second chamber. Consequently, data from the two chambers were pooled. Shoot biomass production of M39 was significantly (P < 0.05 ) less ( - 25% ) than CB3 in the shaded halves of the growth chambers but only slightly and nonsignificantly less ( - 9% ) than CB3 in the exposed halves, indicating that the lower chlorophyll content of the m u t a n t may reduce growth of the m u t a n t in extremely low light environments. These results are consistent with the reduced absorption of light and the trend toward lower CO2 assimilation rates in low light by the m u t a n t reported by Kirchhoff et al. (1989b). Average shoot biomass of both genotypes was 35% less under shaded, compared with exposed, conditions.
23
In both shaded and exposed environments, chlorophyll ( a + b) content per unit leaf area was significantly less for the mutant than for CB3 (Fig. 1 ). Also, chlorophyll content per unit leaf area was significantly greater for both genotypes in the exposed environment compared with shade conditions. Differences became smaller 38 and 45 days from planting. Leaves in the exposed environment had greater fresh weight per unit area and were thicker than leaves in the shaded environment. In contrast, chlorophyll-a:b ratio was not influenced by irradiance during growth but was significantly higher for the mutant compared with CB3 (Fig. 2). Earlier studies of inheritance had shown that high chlorophyll-a: b ratio is a more consistent feature of the phenotype of M39 than reduced chlorophyll content (Kirchhoff et al., 1989a). Since differences in irradiance during growth did not influence chlorophyll-a: b ratio, it is likely that they did not influence the expression of the mutation.
1.0
--,I/
?
P
6
£_
4
---"
~ o
"-J~_.___~K" ~o o~ I 0
I
' //
15
I
'
24 31 38 Days from planting
45
Fig. 1. Chlorophyll (a + b ) content per unit leaf area ofa cowpea cultivar, CB3, grown under shaded ( , ) and exposed ( [] ) conditions, and a mutant, M39, grown under shaded ( • ) and exposed ( O ) conditions.
iI if
71
2 %
6
c_
5
ru
4
3
g
2 L S O o~
I
•
I
i
31
3B
I
z/ /t
o 15
24
45
Days from planting
Fig. 2. Chlorophyll a: b ratios of a cowpea cultivar, CB3, grown under shaded (B) and exposed ( [ ] ) conditions, and a mutant, M39, grown under shaded ( • ) and exposed ( O ) conditions.
24
Response to temperature Differences in temperature during growth resulted in a significant genotype X temperature interaction and a strong genotypic effect with respect to leaf chlorophyll content. Chlorophyll (a+b) content per unit leaf area was higher for the m u t a n t in the hot glasshouse compared with the optimal-temperature glasshouse, whereas CB3 had similar higher levels of chlorophyll in both glasshouses (Fig. 3 ). No differences in leaf fresh weight per unit area were observed between the genotypes or glasshouse treatments (Table 1 ). A substantial genotypeXtemperature interaction also was apparent for chlorophyll-a: b ratio in addition to strong individual effects of genotype and temperature. The chlorophyll-a:b ratio of the m u t a n t was not significantly different from CB3 in the hot glasshouse, but was substantially greater for the m u t a n t grown in the optimal-temperature glasshouse (Fig. 4). CB3 maintained a similar proportion of chlorophyll (a + b) in the form of chlorophyll-b in both temperature regimes (Table 1 ). M39 exhibited a similar proportion of
7
5 4 3
g
LSOo~ [
I
z
I
o 14
21 28 35 Days from planting
Fig. 3. Chlorophyll (a+b) content of unifoliate leaves of a cowpea cultivar, CB3, grown at 24/ 17°C ( I ) and 36/29°C ([Z), and a mutant, M39, grown at 24/17°C (@) and 36/29°C (O).
°
6
~o
4
3 c~
if,
I
o
LSD om
I
i4
I
2! 28 35 Days from planting
Fig. 4. Chlorophyll a:b ratios of unifoliate leaves of a cowpea cultivar, CB3, grown at 24/17 °C (.), and 36/29 °C ([]), and a mutant, M39, grown at 24/17 °C (•), and 36/29 °C (O).
25
chlorophyll-b to total chlorophyll as CB3 in the hot house (23.9%) but much less in the optimal-temperature house (16.7%). Apparently, the mutation was more strongly expressed at optimal temperatures than at hot temperatures. When chlorophyll-a: b ratio was plotted as a function of chlorophyll (a + b) content using the data obtained in the optimal-temperature and hot glasshouses (Fig. 5), the normal chlorophyll-a: b ratios were associated with high chlorophyll (a+b) content, the same relationship was observed in the field (Fig. 6). Apparently, the tendency for the mutant to become greener in the field in mid-summer may be a response to increasing temperature, and there is an association between this greening and more normal chlorophyll a: b ratios. TABLE 1 Leaf chlorophyll (leaf-area basis) of cowpea cultivar CB3 and mutant M39 grown under contrasting temperature regimes Genotype
Environment
Chl a: b ratio
Total Chl a + b
Chl b
Chl b (% of total)
Fresh wt (g dm -2)
16.7 23.9 23.5 25.0
1.72 1.86 1.85 1.96
(mgdm -2) M39
24/17°C 36/29 ° C 24/17°C 36/29°C
CB3
LSD (0.05)
5.01 3.19 3.26 3.00
1.90 2.82 4.31 3.81
0.32 0.67 1.01 0.95
0.08
0.12
0.03
Means are from leaves sampled from the 1st, 2nd, and 3rd trifoliate nodes at 35 days after sowing.
10 o
%
8 6
~ c:) g
4
o
~J]~ ~ ~'4m~i' "
1
2
3
4
5
ChloPophyll {a+b} mg dm-2
Fig. 5. Chlorophyll a: b ratio as a function of chlorophyll (a + b) content per unit leaf area from the 1st measurement of each node for the 1st, 2nd, and 3rd trifoliate nodes for a cowpea cultivar, CB3, grown at 36/29°C ( [] ), and 24/17 ° ( . ) , and a mutant, M39, grown at 36/29°C (O), and 24/17°C (O).
26
o C-
oOD
° Oo
O °
0~ 0oO %
O
•
2
4 6 Chlorophyll
'11J ~ , , ,r ,,
8 l0 (a+b) mg elm-2
12
Fig. 6. Chlorophyll a:b ratio as a function of chlorophyll (a + b) content per unit leaf area from fully expanded upper canopy trifoliate leaves of 54-day-old plants of a cowpea cultivar, CB3 ( . ) , and a mutant, M39 ( O ), grown in the field at Riverside in 1986. TABLE2 Leaf chlorophyll (leaf-area and fresh-weight bases) of 54-day-old plants of a cowpea cultivar CB3 and mutant M39 growing in the Riverside Field Station in 1986 Genotype
Chl a Chl b (mgdm -2)
Chl a 4- b
Chl a 4- b (mgg -1)
Chl a: b ratio
Fresh wt (gdm -2)
M39 CB3
4.59 6.92
5.39 8.94
2.13 3.36
5.85 3.42
2.53 2.66
0.80 2.02
All pairs of means are significantly (P < 0.05) different except for leaf fresh-weight/area.
Field experiments Chlorophyll-a: b ratios of the two genotypes under field conditions just prior to flowering (Table 2 ) were similar to the ratios observed in the optimal-temperature glasshouse (Table 1 ). Chlorophyll ( a 4- b) contents per unit leaf area were much higher in the field (Table 2) than in the glasshouse (Table 1 ), due to higher fresh weight per unit area, and the leaves were thicker under field conditions. Chlorophyll (a 4- b) content of the m u t a n t was lower than the parent on both leaf-area and leaf fresh-weight bases, indicating that the concentration of chlorophyll was less in the mutant. Over 6 years of field-testing, M39 produced the same average grain-yield as the parent CB3 (Table 3). The biomass harvest conducted in 1985 indicated no significant differences between the two genotypes, with M39 producing 10 016 kg dry-matter ha -1, and CB3 producing 10 056 kg ha -1, with an LSDo.o5 of 1798 kg ha-1. The apparent harvest indices of the two genotypes, computed as the ratio of grain-yield to total shoot biomass, were identical at 0.45. The majority of the chlorophyll-deficient mutants reported in the literature
27 TABLE 3 Grain yield of cowpea cultivar CB3 and a mutant M39 in field trials conducted at three locations in California Genotype
M39 CB3 LSD
(0.05)
Grain yield (kg ha -1 ) 1981
1982
1983
1984
1985
1986
Mean
SE
3530 2241
2299 2375
1990 2076
2780 2865
5531 6352
4159 3634
3382 3257
539 660
n.a.
391
343
465
745
563
n.a.=notavailable.
are either lethal recessives or grow very slowly and give relative low yields (Hopkins et al., 1980). However, M39 appears to belong to a small group of mutants, including the chlorophyll-deficient mutants of barley (Highkin and Frenkel, 1962), pea (Highkin et al., 1969), soybean (Keck and Dilley, 1970), and the virescent mutant of tobacco (Schmid, 1971), which exhibit growth rates that are comparable to their wild-type parents.
CONCLUSIONS
The expression of the mutation, in terms of chlorophyll-a: b ratio, was not influenced by levels of irradiance during plant growth under optimal temperatures. In contrast, under very high temperatures and high irradiances, the chlorophyll-a:b ratio of the mutant approached that of the normal parent. This effect of temperature explained the tendency of mutant leaves to become greener under field conditions during hot weather in mid-summer, and there was an association between greening and more normal chlorophyll-a: b ratios. The mutant genotype grown under field conditions had pale-green foliage that contained significantly less chlorophyll and substantially higher chlorophyll-a: b ratio than its parent, CB3, while maintaining equivalent grain-yields over 6 years and similar shoot biomass production. These observations suggest that, instead of considering the mutant genotype to be chlorophyll-deficient, it may be more appropriate to consider that the parent, and many other cowpeas, have an excess of chlorophyll for growth under sunny conditions, as has been concluded for some other cultivated species (Gifford, 1987). The present study also suggests that achieving high productivity under sunny conditions does not require close regulation of chlorophyll-a: b ratio and content of leaves.
28 ACKNOWLEDGEMENTS This research was partially supported by the Bean/Cowpea CRSP, USAID G r a n t No. D A N - 4 0 4 8 - G - 5 5 - 2 0 6 5 - 0 0 . T h e o p i n i o n s a n d r e c o m m e n d a t i o n s are t h o s e of t h e a u t h o r s a n d n o t n e c e s s a r i l y t h o s e of U S A I D .
REFERENCES Adedipe, N.O. and Ormrod, D.P., 1975. Effects of light intensity on growth, and chlorophyll, carbohydrate and phosphorous content of the cowpea (Vigna unguiculata (L.)). Biochem. Physiol. Pflanz., 167: 301-309. Alberte, R.S., Hesketh, J.D., Hofstra, G., Thornber, J.P., Naylor, A.W., Bernard, R.L., Brim, C., Endrizzi, J. and Kohel, R.J., 1974. Composition and activity of the photosynthetic apparatus in temperature-sensitive mutants of higher plants. Proc. Nat. Acad. Sci. U.S.A., 71: 2414-2418. Gifford, R.M., 1987. Barriers to increasing crop productivity by genetic improvement in photosynthesis. In: J. Biggins (Editor), Progress in Photosynthesis Research, Vol. IV. Proceedings of the International Photosynthesis Congress, 10-15 August 1986, at Providence, RI, U.S.A. Martinus Nijhoff, Dordrecht, The Netherlands, pp. 377-384. Gifford, R.M. and Jenkins, C.L.D., 1982. Prospects of applying knowledge of photosynthesis towards improving crop production. In: R. Govindjee (Editor), Photosynthesis: Development, Carbon Metabolism and Plant Productivity, Vol. II. Academic Press, New York, pp. 419-457. Highkin, H.R. and Frenkel, A.W., 1962. Studies of growth and metabolism of a barley mutant lacking chlorophyll b. Plant Physiol., 37: 814-820. Highkin, H.R., Boardman, N.K. and Goodchild, D.J., 1969. Photosynthetic studies on a peamutant deficient in chlorophyll. Plant Physiol., 44: 1310-1320. Hopkins, W.G. and Walden, D.B., 1977. Temperature sensitivity of virescent mutants of maize. J. Hered., 68: 283-286. Hopkins, W.G., Hayden, D.B. and Neuffer, M.G., 1980. A light-sensitive mutant in maize (Zea mays L.). I. Chlorophyll, chlorophyll-protein and ultrastructural studies. Z. Pflanzenphysioi., 99: 417-426. Keck, R.W. and Dilley, R.A., 1970. Chloroplast composition and structure differences in a soybean mutant. Plant Physiol., 46" 692-698. Kirchhoff, W.R., Hall, A.E. and Roose, M.L., 1989a. Inheritance of a mutation influencing chlorophyll content and composition in cowpea. Crop Sci., 29: 105-108. Kirchhoff, W.R., Hall, A.E. and Thomson, W.W., 1989b. Gas exchange, carbon isotope discrimination, and chlorophyll ultrastructure of a chlorophyll-deficient mutant of cowpea. Crop Sci., 29: 109-115. Markwell, J.P., Danko, S.J., Bauwe, H., Osterman, J., Gorz, H.J. and Haskins, F.A., 1986. A temperature-sensitive chlorophyll-b-deficient mutant of sweetclover (Melilotus alba). Plant Physiol., 81: 329-334. Matkin, D.A. and Chandler, P.A., 1957. The U.C.-type soil mixes. In: K.F. Baker (Editor), The U.C. System for Producing Healthy Container-Grown Plants. Univ. Calif. Div. Agric. Sci. Agric. Exp. Stn. Ext. Serv. Man., 23: 68-85. Nybom, N., 1955. The pigment characteristics of chlorophyll mutations in barley. Hereditas, 41: 483-498. Schmid, G.H., 1971. Origins and properties of mutant plants: yellow tobacco. In: A. San Pietro (Editor), Methods in Enzymology. Photosynthesis, Vol. 23. Academic Press, New York, pp. 171-194.
19
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
P h e n o t y p i c E x p r e s s i o n of a Chlorophyll Mutant in Cowpea (Vigna unguiculata): E n v i r o n m e n t a l Influences and Effects on P r o d u c t i v i t y W.R. KIRCHHOFF, A.E. HALL and W.H. ISOM
Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 (U.S.A.) (Accepted 4 November 1988)
ABSTRACT Kirchhoff, W.R., Hall, A.E. and Isom, W.H., 1989. Phenotypic expression of a chlorophyll mutant in cowpea (Vigna unguiculata): Environmental influences and effects on productivity. Field Crops Res., 21: 19-28. Phenotypic expression of a chlorophyll-deficientmutation in cowpea was studied to determine the value of this trait for plant breeding. The mutant and its parent were grown under two irradiances in growth chambers, and under two contrasting temperature regimes in glasshouses. In addition, grain-yield and biomass productivity of the mutant and parent were compared under field conditions. Under optimal temperatures, the leaves of the mutant exhibited significantly lower chlorophyll content per unit leaf area and higher chlorophyll-a: b ratio than the parent when grown in low, moderate, or high photon-flux density. Leaf thickness and leaf chlorophyll content per unit leaf area of both genotypes were less for plants grown under low irradiance, but the chlorophyll-a: b ratio was not influenced by irradiance. In contrast, when grown under hot day/night temperatures of 36/29 ° C and high photon-flux density, the mutant exhibited similar chlorophyll content and chlorophyll composition as the parent. There was an association between higher chlorophyll content and lower, more normal, chlorophyll-a: b ratios. This association was also seen in field conditions, where the mutant produced greener leaves during hot weather in mid-summer. In sunny field conditions the mutant produced equivalent average grain-yields over 6 years, and identical biomass to the parent, even though the leaves of the mutant had substantially less chlorophyll during flowering and pod-filling. Instead of considering the mutant to be chlorophylldeficient, it may be more appropriate to state that the parent, and many other cowpeas, have an excess of chlorophyll for growth under sunny conditions.
INTRODUCTION
Selecting for photosynthetic characters could provide a means for breeding crop plants with increased biomass production per unit of solar radiation. However, little progress has been made to date in developing cultivars with increased photosynthetic efficiency, either by empirical breeding with selec0378-4290/89/$03.50
© 1989 Elsevier Science Publishers B.V.
20 tion mainly based upon grain-yield, or by direct selection for photosynthetic characters (Gifford and Jenkins, 1982; Gifford, 1987). Gifford (1987) proposed that in some species there may be an over-investment in chlorophyll due to the unconscious tendency for breeders to select for dark-green foliage. He suggested that it might be worthwhile to breed plants with lower chlorophyll content, thereby releasing a small amount of carbon and nitrogen for redeployment to other tissues. A chlorophyll-deficient mutant and parent have been described recently in cowpea (Kirchhoff et al., 1989a). The expression of chlorophyll deficiency in the leaves of the mutant has been observed to vary during the growing season. Kirchhoff et al. (1989a) showed that elevated chlorophyll-a: b ratio provided a more definitive characterization of the mutant phenotype than did chlorophyll content. In other chlorophyll-b-deficient mutants, it has been reported that chlorophyll content is influenced by growing conditions, especially temperature and light (Nybom, 1955; Alberte et al., 1974; Adedipe and Ormrod, 1975; Hopkins and Walden, 1977; Markwell et al., 1986). Experiments were conducted to determine the extent to which irradiance and temperature influence the chlorophyll content and chlorophyll-a: b ratio of this mutant of cowpea in comparison with the parent. Studies by Kirchhoffet al. (1989b) indicated that the mutant had 13% higher carbon-dioxide assimilation rates at high irradiances and a trend toward lower rates than the parent at low irradiance. This indicates that the mutant may be more productive than the parent in sunny environments. It is also possible that the mutation will decrease productivity, because even though CO2 assimilation rates were slightly higher, the mutation did dramatically reduce lamellar stacking in the chloroplasts, and other aspects of photosynthesis, such as the reduction of NO~- and SO~-, may be impaired. The influence of the chlorophyll-deficient mutation on productivity was examined by evaluating biomass production and grain-yield of the mutant and parent under field conditions. MATERIALSAND METHODS A chlorophyll-deficient mutant of cowpea (M39) and parent, California Blackeye No. 3 (CB3) were studied. They had been shown to differ by a single nuclear gene influencing chlorophyll composition, but have no obvious differences in other characteristics, such as plant habit, leaf shape, floral characteristics, time of flowering, seed characteristics, and resistance to disease (Kirchhoff et al., 1989a).
Contrasting irradiance experiments The mutant and parent were grown in two replicate experiments in two reachin growth chambers which provided 14.75-h daylengths with daytime temper-
21 atures of 30 ° C and nighttime temperatures of 22 ° C. Relative humidity averaged 50% during the day and 60% at night. Contrasting light environments were created within each chamber by installing shade-cloth covers over onehalf of the interior space. Photon flux densities (Fp) were measured with a quantum sensor (Model LI-190s, LI-COR, Lincoln, Neb. ). In the exposed half of each chamber, the Fp (400-700 nm) 15 cm above the pot surface averaged 330/lmol photon m-2 s-1, increasing to an average value of 1100/lmol photon m -2 s -1 15 cm below the light bank. Under the shade-cloth, Fp was approximately one-third of the exposed half of the chamber, with an average value of 110 ]~mol photon m -2 s -1 at 15 cm above the pot. The light quality of each side of each growth chamber was determined using a spectroradiometer (Model 1800, LI-COR, Lincoln, Neb). There were no significant differences in spectral distribution of the light passing through the screens and the light reaching the pot surfaces in the unshaded half-chamber. Plants were grown in 15-1 pots containing pastuerized U.C. Soil Mix II (Matkin and Chandler, 1957) amended with slow-release fertilizer (18:2.6:10 N:P:K, Osmocote, Sierra Chemicals, Milipitas, CA) at a rate of 5 g per pot in the 1st chamber and 25 g per pot in the 2nd chamber, because we had observed mild symptoms of nitrogen deficiency for the 1st planting in the 1st chamber. Three seeds of each genotype were sown into each of four pots in both light environments in both growth chambers. Plants were thinned to 1 seedling per pot within 10 days of planting. Pots were thoroughly watered at sowing and were subsequently supplied with distilled water as needed until harvest. Leaf chlorophyll ( a + b) content and chlorophyll-a:b ratio of recently expanded trifoliate leaves were measured as described by Kirchhoff et al. (1989a). Individual leaf samples from 4 replicate plants of each genotype from each light environment were taken on the 15th day after sowing and approximately every week thereafter for the next 4 weeks. Biomass production was determined 45 days after sowing for plants in the second chamber. The plants were cut at the soil surface, oven-dried at 60 ° C, and then weighed. The plants in the 1st growth chamber grew more slowly, so were allowed to grow for 54 days before an identical biomass harvest was conducted.
Contrasting temperature experiments The mutant and parent were grown in two glasshouses, situated next to each other, which had contrasting temperature regimes. The daily mean maximum/ minimum temperatures in the designated 'optimal house' were 24 °/17 ° C, and in the 'hot house' were 36 °/29 ° C. Light quantity and quality were measured in both houses by a spectroradiometer, and were found not to be significantly different. Plant were grown in 5-1 pots containing pastuerized U.S. Soil Mix II supplemented with 15 g of 18:2.6:10 N:P:K slow-release fertilizer. There were
22 8 pots of each genotype, M39 and CB3. The pots were widely spaced on the benches to provide ample light penetration into the canopy. All pots were thoroughly watered at sowing and then water was withheld until emergence. After emergence, the pots were irrigated with approximately 1-1 half-strength Hoagland's nutrient solution per pot per day. The plants were thinned to 1 seedling per pot at 1 week after planting. Chlorophyll (a + b) content and chlorophyll-a: b ratio of unifoliate and trifoliate leaves were measured every week.
Field experiments The mutant and parent were included in annual performance trials conducted by the Cooperative Extension of the University of California from 1981 through 1986 at the Moreno Valley (1981-1983), South Coast (1984), and Riverside (1985-1986) Field Stations. The trials at each location were conducted under furrow irrigation with optimal management. The experimental design consisted of four randomized blocks. Individual plots consisted of 4 rows, 76 cm apart and 10 m long, with 10 cm between plants. The center 2 rows of each plot were harvested to determine grain-yield. In 1985, shoot biomass production and grain-yield were evaluated prior to the main grain harvest, from 2-m sections taken from 2 rows of each plot. Leaf chlorophyll (a + b) content per unit leaf area and chlorophyll-a:b ratio were measured for recently expanded trifoliate leaves sampled from the upper part of the canopies just prior to flowering in 1986. RESULTS AND DISCUSSION
Response to irradiance Analysis of total shoot-biomass data for M39 and CB3 grown under the moderate and low irradiances showed significant effects of genotype and light environment, but no chamber effects or interactions. Plants in the first chamber which had lower levels of nutrition simply required more time to produce the same biomass as plants in the second chamber. Consequently, data from the two chambers were pooled. Shoot biomass production of M39 was significantly (P < 0.05 ) less ( - 25% ) than CB3 in the shaded halves of the growth chambers but only slightly and nonsignificantly less ( - 9% ) than CB3 in the exposed halves, indicating that the lower chlorophyll content of the m u t a n t may reduce growth of the m u t a n t in extremely low light environments. These results are consistent with the reduced absorption of light and the trend toward lower CO2 assimilation rates in low light by the m u t a n t reported by Kirchhoff et al. (1989b). Average shoot biomass of both genotypes was 35% less under shaded, compared with exposed, conditions.
23
In both shaded and exposed environments, chlorophyll ( a + b) content per unit leaf area was significantly less for the mutant than for CB3 (Fig. 1 ). Also, chlorophyll content per unit leaf area was significantly greater for both genotypes in the exposed environment compared with shade conditions. Differences became smaller 38 and 45 days from planting. Leaves in the exposed environment had greater fresh weight per unit area and were thicker than leaves in the shaded environment. In contrast, chlorophyll-a:b ratio was not influenced by irradiance during growth but was significantly higher for the mutant compared with CB3 (Fig. 2). Earlier studies of inheritance had shown that high chlorophyll-a: b ratio is a more consistent feature of the phenotype of M39 than reduced chlorophyll content (Kirchhoff et al., 1989a). Since differences in irradiance during growth did not influence chlorophyll-a: b ratio, it is likely that they did not influence the expression of the mutation.
1.0
--,I/
?
P
6
£_
4
---"
~ o
"-J~_.___~K" ~o o~ I 0
I
' //
15
I
'
24 31 38 Days from planting
45
Fig. 1. Chlorophyll (a + b ) content per unit leaf area ofa cowpea cultivar, CB3, grown under shaded ( , ) and exposed ( [] ) conditions, and a mutant, M39, grown under shaded ( • ) and exposed ( O ) conditions.
iI if
71
2 %
6
c_
5
ru
4
3
g
2 L S O o~
I
•
I
i
31
3B
I
z/ /t
o 15
24
45
Days from planting
Fig. 2. Chlorophyll a: b ratios of a cowpea cultivar, CB3, grown under shaded (B) and exposed ( [ ] ) conditions, and a mutant, M39, grown under shaded ( • ) and exposed ( O ) conditions.
24
Response to temperature Differences in temperature during growth resulted in a significant genotype X temperature interaction and a strong genotypic effect with respect to leaf chlorophyll content. Chlorophyll (a+b) content per unit leaf area was higher for the m u t a n t in the hot glasshouse compared with the optimal-temperature glasshouse, whereas CB3 had similar higher levels of chlorophyll in both glasshouses (Fig. 3 ). No differences in leaf fresh weight per unit area were observed between the genotypes or glasshouse treatments (Table 1 ). A substantial genotypeXtemperature interaction also was apparent for chlorophyll-a: b ratio in addition to strong individual effects of genotype and temperature. The chlorophyll-a:b ratio of the m u t a n t was not significantly different from CB3 in the hot glasshouse, but was substantially greater for the m u t a n t grown in the optimal-temperature glasshouse (Fig. 4). CB3 maintained a similar proportion of chlorophyll (a + b) in the form of chlorophyll-b in both temperature regimes (Table 1 ). M39 exhibited a similar proportion of
7
5 4 3
g
LSOo~ [
I
z
I
o 14
21 28 35 Days from planting
Fig. 3. Chlorophyll (a+b) content of unifoliate leaves of a cowpea cultivar, CB3, grown at 24/ 17°C ( I ) and 36/29°C ([Z), and a mutant, M39, grown at 24/17°C (@) and 36/29°C (O).
°
6
~o
4
3 c~
if,
I
o
LSD om
I
i4
I
2! 28 35 Days from planting
Fig. 4. Chlorophyll a:b ratios of unifoliate leaves of a cowpea cultivar, CB3, grown at 24/17 °C (.), and 36/29 °C ([]), and a mutant, M39, grown at 24/17 °C (•), and 36/29 °C (O).
25
chlorophyll-b to total chlorophyll as CB3 in the hot house (23.9%) but much less in the optimal-temperature house (16.7%). Apparently, the mutation was more strongly expressed at optimal temperatures than at hot temperatures. When chlorophyll-a: b ratio was plotted as a function of chlorophyll (a + b) content using the data obtained in the optimal-temperature and hot glasshouses (Fig. 5), the normal chlorophyll-a: b ratios were associated with high chlorophyll (a+b) content, the same relationship was observed in the field (Fig. 6). Apparently, the tendency for the mutant to become greener in the field in mid-summer may be a response to increasing temperature, and there is an association between this greening and more normal chlorophyll a: b ratios. TABLE 1 Leaf chlorophyll (leaf-area basis) of cowpea cultivar CB3 and mutant M39 grown under contrasting temperature regimes Genotype
Environment
Chl a: b ratio
Total Chl a + b
Chl b
Chl b (% of total)
Fresh wt (g dm -2)
16.7 23.9 23.5 25.0
1.72 1.86 1.85 1.96
(mgdm -2) M39
24/17°C 36/29 ° C 24/17°C 36/29°C
CB3
LSD (0.05)
5.01 3.19 3.26 3.00
1.90 2.82 4.31 3.81
0.32 0.67 1.01 0.95
0.08
0.12
0.03
Means are from leaves sampled from the 1st, 2nd, and 3rd trifoliate nodes at 35 days after sowing.
10 o
%
8 6
~ c:) g
4
o
~J]~ ~ ~'4m~i' "
1
2
3
4
5
ChloPophyll {a+b} mg dm-2
Fig. 5. Chlorophyll a: b ratio as a function of chlorophyll (a + b) content per unit leaf area from the 1st measurement of each node for the 1st, 2nd, and 3rd trifoliate nodes for a cowpea cultivar, CB3, grown at 36/29°C ( [] ), and 24/17 ° ( . ) , and a mutant, M39, grown at 36/29°C (O), and 24/17°C (O).
26
o C-
oOD
° Oo
O °
0~ 0oO %
O
•
2
4 6 Chlorophyll
'11J ~ , , ,r ,,
8 l0 (a+b) mg elm-2
12
Fig. 6. Chlorophyll a:b ratio as a function of chlorophyll (a + b) content per unit leaf area from fully expanded upper canopy trifoliate leaves of 54-day-old plants of a cowpea cultivar, CB3 ( . ) , and a mutant, M39 ( O ), grown in the field at Riverside in 1986. TABLE2 Leaf chlorophyll (leaf-area and fresh-weight bases) of 54-day-old plants of a cowpea cultivar CB3 and mutant M39 growing in the Riverside Field Station in 1986 Genotype
Chl a Chl b (mgdm -2)
Chl a 4- b
Chl a 4- b (mgg -1)
Chl a: b ratio
Fresh wt (gdm -2)
M39 CB3
4.59 6.92
5.39 8.94
2.13 3.36
5.85 3.42
2.53 2.66
0.80 2.02
All pairs of means are significantly (P < 0.05) different except for leaf fresh-weight/area.
Field experiments Chlorophyll-a: b ratios of the two genotypes under field conditions just prior to flowering (Table 2 ) were similar to the ratios observed in the optimal-temperature glasshouse (Table 1 ). Chlorophyll ( a 4- b) contents per unit leaf area were much higher in the field (Table 2) than in the glasshouse (Table 1 ), due to higher fresh weight per unit area, and the leaves were thicker under field conditions. Chlorophyll (a 4- b) content of the m u t a n t was lower than the parent on both leaf-area and leaf fresh-weight bases, indicating that the concentration of chlorophyll was less in the mutant. Over 6 years of field-testing, M39 produced the same average grain-yield as the parent CB3 (Table 3). The biomass harvest conducted in 1985 indicated no significant differences between the two genotypes, with M39 producing 10 016 kg dry-matter ha -1, and CB3 producing 10 056 kg ha -1, with an LSDo.o5 of 1798 kg ha-1. The apparent harvest indices of the two genotypes, computed as the ratio of grain-yield to total shoot biomass, were identical at 0.45. The majority of the chlorophyll-deficient mutants reported in the literature
27 TABLE 3 Grain yield of cowpea cultivar CB3 and a mutant M39 in field trials conducted at three locations in California Genotype
M39 CB3 LSD
(0.05)
Grain yield (kg ha -1 ) 1981
1982
1983
1984
1985
1986
Mean
SE
3530 2241
2299 2375
1990 2076
2780 2865
5531 6352
4159 3634
3382 3257
539 660
n.a.
391
343
465
745
563
n.a.=notavailable.
are either lethal recessives or grow very slowly and give relative low yields (Hopkins et al., 1980). However, M39 appears to belong to a small group of mutants, including the chlorophyll-deficient mutants of barley (Highkin and Frenkel, 1962), pea (Highkin et al., 1969), soybean (Keck and Dilley, 1970), and the virescent mutant of tobacco (Schmid, 1971), which exhibit growth rates that are comparable to their wild-type parents.
CONCLUSIONS
The expression of the mutation, in terms of chlorophyll-a: b ratio, was not influenced by levels of irradiance during plant growth under optimal temperatures. In contrast, under very high temperatures and high irradiances, the chlorophyll-a:b ratio of the mutant approached that of the normal parent. This effect of temperature explained the tendency of mutant leaves to become greener under field conditions during hot weather in mid-summer, and there was an association between greening and more normal chlorophyll-a: b ratios. The mutant genotype grown under field conditions had pale-green foliage that contained significantly less chlorophyll and substantially higher chlorophyll-a: b ratio than its parent, CB3, while maintaining equivalent grain-yields over 6 years and similar shoot biomass production. These observations suggest that, instead of considering the mutant genotype to be chlorophyll-deficient, it may be more appropriate to consider that the parent, and many other cowpeas, have an excess of chlorophyll for growth under sunny conditions, as has been concluded for some other cultivated species (Gifford, 1987). The present study also suggests that achieving high productivity under sunny conditions does not require close regulation of chlorophyll-a: b ratio and content of leaves.
28 ACKNOWLEDGEMENTS This research was partially supported by the Bean/Cowpea CRSP, USAID G r a n t No. D A N - 4 0 4 8 - G - 5 5 - 2 0 6 5 - 0 0 . T h e o p i n i o n s a n d r e c o m m e n d a t i o n s are t h o s e of t h e a u t h o r s a n d n o t n e c e s s a r i l y t h o s e of U S A I D .
REFERENCES Adedipe, N.O. and Ormrod, D.P., 1975. Effects of light intensity on growth, and chlorophyll, carbohydrate and phosphorous content of the cowpea (Vigna unguiculata (L.)). Biochem. Physiol. Pflanz., 167: 301-309. Alberte, R.S., Hesketh, J.D., Hofstra, G., Thornber, J.P., Naylor, A.W., Bernard, R.L., Brim, C., Endrizzi, J. and Kohel, R.J., 1974. Composition and activity of the photosynthetic apparatus in temperature-sensitive mutants of higher plants. Proc. Nat. Acad. Sci. U.S.A., 71: 2414-2418. Gifford, R.M., 1987. Barriers to increasing crop productivity by genetic improvement in photosynthesis. In: J. Biggins (Editor), Progress in Photosynthesis Research, Vol. IV. Proceedings of the International Photosynthesis Congress, 10-15 August 1986, at Providence, RI, U.S.A. Martinus Nijhoff, Dordrecht, The Netherlands, pp. 377-384. Gifford, R.M. and Jenkins, C.L.D., 1982. Prospects of applying knowledge of photosynthesis towards improving crop production. In: R. Govindjee (Editor), Photosynthesis: Development, Carbon Metabolism and Plant Productivity, Vol. II. Academic Press, New York, pp. 419-457. Highkin, H.R. and Frenkel, A.W., 1962. Studies of growth and metabolism of a barley mutant lacking chlorophyll b. Plant Physiol., 37: 814-820. Highkin, H.R., Boardman, N.K. and Goodchild, D.J., 1969. Photosynthetic studies on a peamutant deficient in chlorophyll. Plant Physiol., 44: 1310-1320. Hopkins, W.G. and Walden, D.B., 1977. Temperature sensitivity of virescent mutants of maize. J. Hered., 68: 283-286. Hopkins, W.G., Hayden, D.B. and Neuffer, M.G., 1980. A light-sensitive mutant in maize (Zea mays L.). I. Chlorophyll, chlorophyll-protein and ultrastructural studies. Z. Pflanzenphysioi., 99: 417-426. Keck, R.W. and Dilley, R.A., 1970. Chloroplast composition and structure differences in a soybean mutant. Plant Physiol., 46" 692-698. Kirchhoff, W.R., Hall, A.E. and Roose, M.L., 1989a. Inheritance of a mutation influencing chlorophyll content and composition in cowpea. Crop Sci., 29: 105-108. Kirchhoff, W.R., Hall, A.E. and Thomson, W.W., 1989b. Gas exchange, carbon isotope discrimination, and chlorophyll ultrastructure of a chlorophyll-deficient mutant of cowpea. Crop Sci., 29: 109-115. Markwell, J.P., Danko, S.J., Bauwe, H., Osterman, J., Gorz, H.J. and Haskins, F.A., 1986. A temperature-sensitive chlorophyll-b-deficient mutant of sweetclover (Melilotus alba). Plant Physiol., 81: 329-334. Matkin, D.A. and Chandler, P.A., 1957. The U.C.-type soil mixes. In: K.F. Baker (Editor), The U.C. System for Producing Healthy Container-Grown Plants. Univ. Calif. Div. Agric. Sci. Agric. Exp. Stn. Ext. Serv. Man., 23: 68-85. Nybom, N., 1955. The pigment characteristics of chlorophyll mutations in barley. Hereditas, 41: 483-498. Schmid, G.H., 1971. Origins and properties of mutant plants: yellow tobacco. In: A. San Pietro (Editor), Methods in Enzymology. Photosynthesis, Vol. 23. Academic Press, New York, pp. 171-194.