Optical Properties of the Pod Wall of the Pea (Pisum sativum L.). III. The Effects of Senescence and Pod Desiccation

]. Plant PbysioL VoL 143. pp. 218-221 (1994)

Optical Properties of the Pod Wall of the Pea (Pisum sativum L.) Ill. The Effects of Senescence and Pod Desiccation DAVID

N. PRicE, MARIA E. DoNKIN, and ANGELA WATSON

The University of Plymouth, Dept. of Biological Sciences, Drake Circus, Plymouth, Devon, PL4 SAA, U.K. Received August 3, 1993 · Accepted October 13, 1993

Summary The transmission, reflection and absorption spectra of pods of Pisum sativum L. variety JI 141 were measured at various stages of senescence. These optical properties were correlated with the percentage water content and levels of chlorophyll a, b and total chlorophyll of the pod wall at each stage. Photomorphogenic effects of pod senescence on the seed environment were deduced from changes in the ratio of 660/730 nm light transmitted through the pod wall. Structural aspects of the final stages of pod desiccation were also investigated using scanning electron microscopy. The significance of these results are discussed in relation to the use of optical properties to monitor pod moisture content and the photoenvironment of the senescing pod as it affects 1) phytochrome levels before final seed desiccation and 2) near UV light transmission to the drying seed.

Key words: Pisum sativum, optical properties, pod wal~ desiccation, senescence. Introduction Seed development in Pisum sativum consists of three phases, all of which are limited by low moisture content (Le Deunff and Rachidian, 1988). The first phase corresponds to the formation of the embryo and surrounding structure, the second phase to the filling of the cotyledons and the third phase to desiccation on the mother plant. At a moisture content of approximately 55 % the seed has reached physiological maturity. During the third phase the fresh weight of the seed rapidly decreases to a moisture content of around 14% (Le Deunff and Rachidian, 1988). Senescence of the pod and subsequent desiccation are also an essential phase in development of Pisum sativum, desiccation of the pod tissues is necessary for protein to be mobilised and translocated to the seeds (Pate and Flinn, 1977). As the pod desiccates there may also be changes in the optical properties of the pod wall, which would influence the quantity and quality of light reaching the maturing seeds. Kendrick (1976) and Cresswell and Grime {1981) have pointed out the importance of light quality on maturation of wild seeds and the controlling effects of dehydration. © 1994 by Gustav Fischer Verlag, Stuttgart

We have previously developed a technique to measure the optical properties of the pod wall of legumes, and have estimated the photomorphogenic environment within mature pea pods (Donkin and Price, 1990). We have also used this technique to look at varietal and species differences in the optical properties of the pod wall (Donkin, Price and Watson, 1993). In this paper we have used these techniques to follow the change in the optical properties of one variety of Pisum sativum with age and subsequent desiccation of the wall tissues. These changes may have implications for the final stages of development of the seed.

Materials and Methods Plant Material Pisum sativum L. John Innes Accession line 141, a variety with a conventional green pod, was used throughout this investigation. The plants were grown in a glasshouse until flowering {temperature range 14-18°C) then transferred to a growth room with a controlled temperature of 15° C and 13 h light (96 JJ.molesm- 2 s- 1).

Optical properties of the pea pod wall III.

Stages ofDevelopment and Senescence The pods were collected from JI 141 at three stages of development described previously (Donkin, Price, and Watson, 1993) these are referred to as S1, S2 and S3 and four stages of senescence and desiccation. These are referred to in the text as indicated below: Dl: Pods beginning to dehydrate, turning slightly yellow; Seeds at maximum fresh weight; Rough pod texture; Approximately 20 to 25 days post anthesis. D2: Pods moderately/completely yellow; Rough textured and fragile pod; Seeds beginning to dehydrate; 25 to 30 days post anthe-

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wall was observed using a JEOL JSM-5200 scanning electron microscope.

Results

The percentage water contents of JI 141 pods at different stages of development are shown in Figure 1. Percentage wa-

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D3: Pods moderately brown; Rough textures and fragile pod; Seeds wrinkled and shrunken; 30 to 35 days post anthesis. D4: Pods completely brown; Pods thin and fragile; Seeds hard, small and wrinkled; > 35 to 40 days post anthesis.

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Spectrophotometric Measurements Measurements of transmission, reflection and absorption were carried out with a Pye-Unicam SP 8-100 spectrophotometer fitted with a diffuse reflectance accessory and interfaced to a British Broadcasting Corporation Microcomputer, Model B as described previously (Donkin and Price, 1990).

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Five replicate pods were used for each measurement. Each pod was carefully split open and the peas were removed before dry weights were determined by drying in an oven at 120° C for 24 h.

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Fig.1: The line graph shows the percentage water content of JI 141 pods at four stages of senescence (Dl to D4). The points are the means of 5 replicates and the bars show the standard error of the mean. The bar graph shows chlorophyll a [Ill], chlorophyll b (open bars) and total chlorophyll [\\\] levels of JI 141 pods at the same four stages of senescence. The bars show the standard error of the mean (3 to 5 replicates).

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Fig. 2: Percentage transmission, reflection and absorption properties of JI 141 pods from stage 3 to stage D4 of senescence. Solid line, stage 3; Dotted line, D1; Short dashed line, D2; Long dashed line, D3; Dot-dashed line, D4.

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DAVID N. PRicE, MARIA E. DoNKIN, and ANGELA WATSON

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Fig. 3: Transmission ratio at 660/730 nm through pods of Jl 141 peas at stage 53 through to D4 stage of senescence. Data taken from the specta in Figure 2 A.

ter content remains above 80 % until the second stage of desiccation (D2} when it starts to decrease, and then decreases sharply between stages D2 and D3 to reach its lowest point at stage D4 when the pod has reached a stable level of water content. The transmission, reflection and absorption spectra of Jl 141 pods at stage 3 of development compared with the four stages of desiccation are shown in Figure 2. The spectra show that there is an increase in transmission and reflection with pod age with an accompanying decrease in pod wall absorption. It is also obvious from Figure 2 that there is gradual decline in the spectral quality of the light transmitted through the pod wall with age and also in the light reflected and absorbed by the pod wall. Figure 2 C shows that the typical chlorophyll absorption spectrum with a peak in the red and blue has diminished by D3 and D4 to a general absorption increasing towards the UV. The calculated areas under the curves from Figure 2 show that the overall absorption value of the pod wall decreases from 75.6% for a fully mature pod to approximately 50% for a D3 and D4 senescent pod. The overall transmission almost doubles from a value of 12.6% in the mature pod to 22.5% by D2 and then falls again to 15.7% by the final stage of drying out. Reflection, however, increases consistently from 11.9% to 33.4% by D4. When the data on water contents at the four stages of desiccation from Figure 1 were compared with the areas under the percentage reflection curve and a regression analysis was carried out, a correlation coefficient of -0.977 and an R2 value of 95.49 were obtained. A comparison of the chlorophyll a and b levels and the total chlorophyll at the four stages of desiccation are also shown in Figure 1. It is apparent that chlorophyll a decreases consistently with the stages of desiccation, whereas chlorophyll b remains constant over the first three stages and does not decrease until the final stage. Total chlorophyll decreases slowly over the first three stages and then rapidly between stage D3 and D4. The chlorophyll alb ratio decreases from

Plate 1: Scanning electron micrograph of a section across the pod wall of a fully desiccated}! 141 pod at stage D4 of senescence. OE, outer epidermis; IE, inner epidermis; S, sclerenchyma layers; MC, collapsed mesocarp cells; VB, vascular bundle.

1.49 (± 0.13} at D1 to a minimum value of 0.70 (± 0.03} at D3, with a mature pod having a level of 2.0. The transmission ratio at 660 nm/730 nm taken from the spectra in Figure 2 as described by Blanke (1990} is shown in Figure 3. The ratio tends to increase throughout senescence but levels off slightly at the final stages between D2 to D3 and D3 to D4. The ratio starts at S3 quite low at 0.36 and increases by D4 to a value (0.92} approaching that of sunlight (approximately 1.0}. The structure of the tissues in a fully desiccated pod (at D4} is shown in Plate 1 which is a scanning electron micrograph taken of the cut surface of the pod wall. The plate shows the arrangement of tissues from outer to inner epidermis, with the sclerenchyma layers and collapsed mesocarp layer shown in some detail. The sclerenchyma layer in the dried pod (Plate 1} makes up approximately 50% of the pod wall thickness compared with only 7 % in a young pod of Jl 14116days post anthesis (Price, Smith, and Hedley, 1988}. Discussion

Protein reserve accumulation in the final stages of the developing pea seed requires the pod to be senescencing. The

Optical properties of the pea pod wall Ill.

longer the period of dehydration the more protein will be mobilized and translocated to the seed (Pate and Flinn, 1977). Pod desiccation, which is achieved by the restriction of water supply (Le Deunff and Rachidian, 1988), also helps seed drying, which is essential for the deposition of reserves. The data in Figure 1 show a sharp decrease in pod water content between stages D2 and D3 in agreement with results for Solara and Finale varieties of pea (Le Deunff and Rachidian, 1988). The changes in water content of the pod wall are closely mirrored by changes in the pod optical properties at each stage. Transmission tends to increase with senescence up to stage D3 with a general loss in the chlorophyll spectrum but there is a slight decrease at D4 perhaps due to changes in optical properties as the pod wall tissues collapse in the final drying stage (Plate 1). Absorption spectra show a gradual loss in the chlorophyll spectrum with stages of drying. This agrees with the loss of chlorophyll shown in Figure 1, the absorption spectrum showing virtually no peaks at stage D4 corresponding with the very low chlorophyll levels measured in the pod wall. The reflection spectrum shows an increase in overall reflection with pod age and a change from a spectrum typical of chlorophyll containing tissue (Donkin and Price, 1990) to a much higher non-specific reflection spectrum. The reflection of light from the pod surface appears to show the best correlation with the water content of the pod wall at each stage of desiccation (data taken from Fig. 1). Figure 2 A shows that throughout all stages of desiccation up to D4 the pod wall still acts as an effective filter for near UV (350-400nm) radiation despite an overall increase in transmission of the visible spectrum. This is probably due to the persistence of the sclerenchyma layer which could provide continued protection of the embryo from damaging UV radiation even after mesocarp collapse. The spectral evidence of chlorophyll disappearance as the pod wall dries is confirmed by the chlorophyll analyses in Figure 1. However, chlorophyll a appears to be lost more quickly than chlorophyll b, which results in a change in the chlorophyll alb ratio from 1.49 to 0.70 at D3, compared with a level of about 2.0 in a mature stage 3 pod (Donkin and Price, 1990). The chlorophyll changes observed are probably related to the relative rates of degradation of chlorophyll a and b. Chlorophyll a declines more rapidly than chlorophyll b in climacteric fruit due to chlorophyllase activity (Rhodes and W ooltorton, 1967), and chlorophyll alb ratio has been shown to decrease with fruit development in apples (Phan, 1975; Knee, 1972). Comparisons of the transmission ratio at 660/730 nm show an increase from 0.36 in green tissue (53) to a much higher ratio by D 2. This may be explained to some extent by loss of chlorophyll in the pod wall. However other factors such as the loss of air/water interfaces may also be important. The evidence that the ratio rises over the early stages of senescence would mean that the pea seeds are subjected in the

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drying pod to light which is more red rich than in a green pod, which would affect the phytochrome equilibrium causing more Pfr to be formed. The pod is almost completely desiccated by D3. This would be accompanied by a rapid dehydration of the seeds (Le Deunff and Rachidian, 1988) which would then the phytochrome in its final Pfr form. It would appear in Pisum sativum the timing of the increase in the 660/730 nm ratio before complete seed desiccation is advantageous for the final state of the seed and its subsequent germination. Acknowledgements

We would like to acknowledge the data on pod water contents which was supplied by Miss Tracy Tompkins. We are also grateful to Mr. Tony Hull and the staff of Rumleigh Experimental Station for growing the peas, and to Mrs. Jane Green and Dr. Roy Moate at the University of Plymouth Electron Microscopy Unit for help with the scanning electron microscopy.

References ARNoN, D. R.: Copper enzymes in chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BLANKE, M.: Carbon economy of the grape inflorescence 4. Light transmission into grape berries. Vitic. Enol. Sci. 45, 21-23 (1990}. CRESSWELL, E. G. and J.P. GRIME: Induction of light requirement during seed development and its ecological consequences. Nature 291, 583-585 (1981}. DoNKIN, M. E. and D. N. PRICE: Optical properties of the pod wall of the pea (Pisum sativum L.): I. General Aspects J. Plant Physiol. 137, 29-35 (1990). DoNKIN, M. E., D. N. PRicE, and A. WATSON: Optical properties of the pod wall of the pea (Pisum sativum L.): II Varietal differences and species comparisons. J. Plant Physiol. 141, 347-352 (1993). KENDRICK, R. E.: Photocontrol of seed germination. Science Progress (Oxford) 63, 347-367 (1976}. KNEE, M.: Anthocyanin, carotenoid and chlorophyll changes in the peel of Cox's Orange Pippin apples during ripening on and off the tree. J. Exp. Botany 23, 84-96 ( 1972). LE DEUNFF, Y. and Z. RAcHIDIAN: Interruption of water delivery at physiological maturity is essential for seed development, germination and seedling growth in pea, Pisum sativum L. J. Exp. Botany 39, 1221-1230 (1988). PATE, J. S. and A.M. FLINN: Fruit and Seed Development. In: The Physiology of the Garden Pea (Ed. SuTCLIFFE,]. F. andJ. S. PATE. pp. 435-443. Academic Press, London (1977). PHAN, C. T.: Occurrence of active chloroplasts in the internal tissues of apples. Their possible role in fruit maturation. Colloques Internationaux CNRS, 238, 49-55 (1975}. PRicE, D. N., C. M. SMITH, and C. L. HEDLEY: The effect of the gp gene on fruit development in Pisum sativum L. I. Structural and physical aspects. New Phytologist 110, 261-269 (1988). RHODES, M. J. C. and L. S. C. WooLTORTON: The respiration climacteric in apple fruits: The action of hydrolytic enzymes in peel tissue during the climacteric period in fruit detached from the tree. Phytochemistry 6, 1- 12 (1967).