Journal of Experimental Botany, Vol. 51, No. 346, pp. 895-900,
May 2000
© 2000 Oxford University Press
Seed coat cell turgor in chickpea is independent of changes in plant and pod water potential
1 Centre for Legumes in Mediterranean Agriculture, University of Western Australia, Nedlands, WA 6907, Australia
2 CSIRO Plant Industry, Private Bag, PO Wembley WA 6014, Australia
Received 6 January 2000; Accepted 21 January 2000
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Abstract |
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Turgor pressure in cells of the pod wall and the seed coat of chickpea (Cicer arietinum L.) were measured directly with a pressure probe on intact plants under initially dry soil conditions, and after the plants were irrigated. The turgor pressure in cells of the pod wall was initially 0.25 MPa, and began to increase within a few minutes of irrigation. By 2–4 h after irrigation, pod wall cell turgor had increased to 0.97 MPa. This increase in turgor was matched closely by increases in the total water potential of both the pod and the stem, as measured by a pressure chamber. However, turgor pressure in cells of the seed coat was relatively low (0.10 MPa) and was essentially unchanged up to 24 h after irrigation (0.13 MPa). These data demonstrate that water exchange is relatively efficient throughout most of the plant body, but not between the pod and the seed. Since both the pod and the seed coat are vascularized tissues of maternal origin, this indicates that at least for chickpea, isolation of the water relations of the embryo from the maternal plant does not depend on the absence of vascular or symplastic connections between the embryo and the maternal plant.
Key words: Turgor pressure, pod wall, water stress, irrigation.
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Introduction |
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A number of studies have suggested that the turgor pressure (
p) of the seed coat of legumes is maintained nearly constant as part of a physiological mechanism to maintain carbon import to the developing embryo. This turgor–homeostat model (Patrick and Offler, 1995
) proposes that above some minimum level of seed coat cell p, the rate of assimilate unloading (efflux) from the seed coat cells into the embryonic and seed coat apoplast is positively correlated with the p of the seed coat cells. Hence if the assimilate demand by the embryo increases, the concentration of solutes in the apoplast will decrease, leading to an increase in the p of the seed coat cells and a corresponding increase in the rate of assimilate efflux from the seed coat cells. This increased efflux could regulate both the apoplastic s and the seed coat cell p back to approximately the initial level (Patrick and Offler, 1995). Consistent with this hypothesis, it has been reported that the p of seed coat cells which were bathed in a solution initially responded to a step change in the s of the bathing solution, but then returned within about 20 min to the initial level of p (Thorpe et al., 1993; Zhang et al., 1996). In both cases the measurements were made on surgically modified seed coats using the empty seed coat (Thorne and Rainbird, 1983) or a similar technique, and the step changes in p were in the order of 0.07 MPa. However, on intact seed coats of chickpea (Cicer arietinum) and faba bean (Vicia faba), large changes in cell p (0.1–0.4 MPa in chickpea) could be achieved by wetting or drying the seed, and no evidence for a homeostatic response of seed coat p was found (Shackel and Turner, 1998).
Many studies have presented evidence that seed and/or embryo is independent of plant , although after reviewing much of this evidence, Bradford concluded that psychrometric determinations of seed may be unreliable (Bradford, 1994). In soybean, it has been reported that seed remained constant as the water potential of the leaf and pod wall decreased with the development of water deficits (Westgate and Grant, 1989). Yeung and Brown (Yeung and Brown, 1982) found that during seed development, seed coat in P. vulgarisremained constant between -0.6 and -0.9 MPa, however, they also found that embryo decreased to -1.8 MPa. Bradford suggested that an apoplastic barrier to solutes may explain the independence of seed from the of its environment (Bradford, 1994). In wheat, evidence was found that a barrier to apoplastic transport occurred in the chalaza, a small zone of cells between the maternal vascular tissue and the nucellus (Wang and Fisher, 1994). Thus, one unresolved issue is whether or not the independence of seed from plant can be confirmed in situ.
Field studies with chickpea have shown that the water potential of the pod, measured with the pressure chamber technique, was higher than that of the leaf in adequately watered plants, but similar to that of the leaf in water-stressed plants (Leport et al., 1999). However, it is not clear whether the pressure chamber measures the water potential of the pod wall, the developing seed or some weighted mean of the two and, therefore, whether the water potential of the seed remained independent of the pod wall and remainder of the plant as the soil water potential decreased. As the water relations of the developing seed play such a fundamental role in seed filling, and seed filling is sensitive to water shortage (Davies et al., 1999), the present study was initiated to describe the water relations of the chickpea seed in situ, and to determine how it is influenced by the water relations of the parent plant.
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Materials and methods |
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Seeds of chickpea (Cicer arietinum L. cvs Tyson and Kaniva) were inoculated with commercial strains of Bradyrhizobium and planted in 4.0 l plastic pots containing a commercial potting soil. A single plant per pot was grown from July to November 1996, in a naturally lit, evaporatively cooled glasshouse (temperature 10–25 °C) in Perth, Western Australia. Plants were irrigated as needed to maintain wet soil conditions, which, for mature plants, was typically daily. Twice weekly, pods were tagged when visible (about 3 mm long), and most measurements were made on seeds aged from 2–3 weeks after pod set. This corresponded to a developmental stage in which the pod was fully expanded, the embryo had filled the seed coat, and the rate of seed dry weight accumulation was rapid (Davies et al., 1999
). For this stage it is estimated that the seed coat cells were 75% of their final size. The plants used for experiments were irrigated in the evening, the soil was covered with aluminium foil to reduce surface evaporation, and no further irrigation was applied. Preliminary tests showed that about 3 d of no irrigation were required to reach a predawn leaf water potential (leaf) of about -1 MPa. On the evening of the third day, the test plant was moved from the glasshouse into the laboratory and all leaves to be used for water potential measurements were enclosed in foil-covered black plastic envelopes. The envelopes were used to prevent leaf transpiration and allow leaf to equilibrate with the water potential of the stem (stem) at the point of the insertion of the leaf xylem (Begg and Turner, 1970). The stem and the water potential of the pod (pod) were measured using the pressure chamber technique (Turner, 1988). Preliminary tests under laboratory conditions indicated that covering had a small effect on the measured of the leaves, but no detectable effect on the measured of the pods, so only leaves were covered. These tests also showed that both stem and pod were quite uniform throughout a plant, with no detectable differences among stems or for different positions along a stem. Hence, a pod was selected for pressure probe measurements, and 2–4 pods at a similar developmental stage were identified throughout the plant, and these pods and the adjacent leaves were used to follow stem and pod during each experiment. The protocol for each experiment was to determine the stem and pod from these selected organs and the seed coat and pod cell turgor (see below) from the single selected pod. After measurements were made on the plant in dry soil the plant was irrigated by pouring sufficient water over the soil surface to bring the entire soil volume to saturation as evidenced by drainage.
The methods used to determine the turgor (p) in cells of intact, attached seed coats in chickpea have been described (Shackel and Turner, 1998). Briefly, a section of pod wall was removed from one side of the pod, exposing the seed without disturbing the dorsal or ventral pod sutures or the attachment of the seed to the pod. The pod was mounted on a ball-and-socket swivel under a long-distance-objective, vertically-illuminated microscope for p measurements (Fig. 1) and all operations were performed under 100% relative humidity (RH). It was possible to adjust the position of the pod so that the p of cells in both the seed coat and an undisturbed section of pod wall could be measured using the pressure probe technique (Steudle, 1993). In cross-section, the pod walls were 400–500 µm thick, with approximately spheroidal epidermal and sub-epidermal cells of about 50 µm in overall diameter from the epidermis to a depth of about 250 µm. At this point there was a 50 µm thick layer of apparently dense tissue that could not be penetrated by the glass microcapillary of the pressure probe. All measurements of pod wall cell p came from epidermal cells or sub-epidermal cells external to this dense tissue layer. Unlike the p of seed coat cells (Shackel and Turner, 1998), the p of pod wall cells showed no response to changes in the local RH of the air (data not shown).
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In a total of nine experiments, measurements made before irrigation and those made 2–4 h after irrigation were pooled to give one value for each parameter and time interval for each experiment. In four of these experiments, additional values of seed coat
p were measured 24 h after irrigation, using a previously unmeasured seed at a similar developmental stage as the seed used the previous day.
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Results |
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After 3 d of no irrigation, different plants exhibited different levels of
stem in the laboratory, but all plants showed a clear and relatively rapid recovery of stem following irrigation, similar to the that of the plant shown in Fig. 2C. The p of pod wall cells also showed a clear increase which began within a few minutes of irrigation (Fig. 2B). This was in contrast to the p of seed coat cells, which showed no apparent change following irrigation (Fig. 2A). For both seed coat and pod wall cells, the short-term variations in the measurement of p within each individual cell were a consequence of the need to confirm that the probe tip was not plugged, as reported previously (Shackel and Turner, 1998). For the plant shown in Fig. 2, there was substantial agreement in the values of stem and pod measured both before and after irrigation (Fig. 2C), and this agreement was found in all experiments (Fig. 3). There was also general agreement in the magnitudes of the increases in both stem and pod wall p following irrigation (Fig. 2), but as pod wall p reached values on the order of 1 MPa, it became increasingly difficult to obtain stable readings of p from the same cell before a decline was observed, and so the frequency of obtaining reliable measurements of pod wall p from 2–4 h after irrigation was low (Fig. 2B).
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The plant-to-plant variability in
stem before irrigation was greater than that at 2–4 h after irrigation (Table 1), probably reflecting differences among plants in the degree to which soil water had been depleted during drying in the glasshouse. However, the 0.85 MPa average increase which occurred in stem as a result of irrigation was large, and was similar to the 0.72 MPa increase that occurred in the p of the pod wall cells (Table 1). Similar to the data shown in Fig. 1
, there was little change in seed coat cell p, which remained about 0.1 MPa, even 24 h after irrigation (Table 1). In order to account for some of the plant-to-plant variation in water status before irrigation, the change (
) in p from before irrigation to after irrigation was compared to the corresponding change that occurred in stem for each experiment. This comparison showed substantial agreement between stem and pod wall p across all experiments, but not between stem and seed coat p (Fig. 4). The somewhat lower pod wall p than expected in some experiments, may have been related to the increased difficulty of obtaining stable p measurements after irrigation, and may indicate that some cell damage was occurring during penetration. Hence, even though pod wall cell p was stable in the short term (minutes), it may have been an underestimate (Shackel et al., 1987).
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Discussion |
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An important question regarding reproductive development in plants is the extent to which the water status of reproductive organs is isolated from that of the parent plant. Based on 3H2O labelling studies of pods in cowpea (Vigna unguiculata [L.] Walp.), Pate et al. (Pate et al., 1985
) stated that ‘only very slow and ineffective exchanges of water occur between symplastic and apoplastic compartments of an organ, tissue, or transport pathway’, and recognized that their evidence was ‘contrary to the widely held view that recently acquired water equilibrates rapidly with existing pools of water in all compartments of a plant tissue’. The results in this study have shown that water, recently acquired in the roots through irrigation, has an almost immediate effect on the p of the cells comprising the pod wall, indicating that the exchange of water, at least in terms of its thermodynamic potential, is quite rapid and effective in both symplastic and apoplastic compartments throughout the chickpea plant. The very close agreement in the measured by the pressure probe and pressure chamber in this (Fig. 4) and earlier studies (Shackel et al., 1987) is strong evidence that the pressure chamber is thermodynamically sound, contrary to concerns about the pressure chamber raised previously (Zimmerman et al., 1994; Canny, 1995).
The values of stem observed in the present study covered a considerable portion of the range expected in plants subjected to water deficits in the field (Leport et al., 1999). Over this range it is clear that pod and stem were very similar (Fig. 2), and the minimal difference that was exhibited at low was in a direction contrary to that which might be expected for a stem-to-pod gradient associated with pod transpiration and/or pod or seed growth. Covering of the pod to reduce water loss also had no influence on pod, indicating that the rate of pod transpiration is low, consistent with the low stomatal frequencies and no measurable net photosynthesis in pods reported previously (Leport et al., 1999). Short- to medium-term changes in stem, pod, and pod wall cell turgor as a result of irrigation, were also very similar (Fig. 2), clearly indicating a good hydraulic continuity extending from the xylem of the stem to the cells of the pod wall.
In contrast to the straightforward response of pod wall cell p to changes in the water status of the parent plant, p in the cells of the seed coat, which is also a vascularized structure of the parent plant, was remarkably unchanged by irrigation (Fig. 4). It is generally agreed that the developing embryo has no direct symplastic connection to the surrounding parental tissue, and that water, carbon and nutrients must pass through the apoplast to reach the embryo (Thorne, 1985; Bradford, 1994). In addition to this symplastic isolation of the embryo however, results from this study indicate that in chickpea the seed as a whole is isolated from the parent plant by a barrier to water exchange located between the pod wall and the seed coat. Xylem-mobile dyes, labelled water, phosphorus, and sucrose have been used to show that the xylem penetrates minimally into the seed coat of legumes and suggests that water and nutrients enter the seeds via the phloem (Pate et al., 1985; Thorne, 1985; Bradford, 1994). Bradford has postulated that a semipermeable apoplastic membrane exists between the seed and the rest of the plant that prevents solute loss from the seed while allowing water efflux from the seed (Bradford, 1994). Such a barrier is consistent with the finding in this study that seed coat cell p remains unchanged despite changes in the water status of the parent plant. However, if the function of the proposed apoplastic barrier is to retain solutes in the apoplast of the seed coat as a whole, then this model is difficult to reconcile with the rapid responses of seed coat cell turgor to wetting and drying reported previously (Shackel and Turner, 1998), The enigma is that water exchange (i.e. water potential equilibrium) within the seed coat appears to be both rapid and effective, whereas water exchange between the seed coat and the parent plant is not. It may be that the apoplastic solutes retained by the barrier proposed by Bradford (Bradford, 1994) are restricted to a relatively small zone within the seed coat, comparable to the apoplastic barrier in the small chalazal zone of wheat (Wang and Fisher, 1994) and that diffusion out of this zone is counterbalanced by continued solute uptake and recycling. Such a system would probably require co-ordinated regulation of the hydraulic properties of the cells responsible for the recycling, to prevent water potential equilibration along the recycling pathway. In this respect, the hydraulic isolation between the seed coat and the rest of the parent plant may be an interesting target system in which to study the potential importance of aquaporins (Maurel, 1997), or similar membrane active substances, to whole plant function.
The values of turgor pressure measured in cells of the seed coat remained around 0.1 MPa both before and after plant and pod rehydration. These values are comparatively low, but consistent with those in chickpea that were not subjected to water deficits (Shackel and Turner, 1998) and those estimated for the seed coat of Phaseolus vulgaris (Zhang et al., 1996). The turgor homeostat model (Patrick and Offler, 1995) has been proposed as a mechanism for maintaining steady assimilate transport to the seed under varying conditions of supply and demand, but the results of this study suggest that homeostatic conditions may be maintained within the seed coat simply by its isolation as a whole from the rest of the plant. The maintenance of seed coat cell p during water deficits suggests that the influence of water deficits on the reduction in seed size (Davies et al., 1999) may arise at least initially from a reduction in assimilate supply rather than from a reduced seed p and a reduced capacity of the sink to utilize assimilates. It is interesting to note that turgor maintenance is generally regarded as important for the maintenance of growth under water-limiting conditions, and it is often assumed that turgor should be maintained near its maximum, by processes such as osmotic adjustment (Hsiao et al., 1984). If homeostasis of turgor, however, in actively growing zones for instance, is more important than achieving high levels of turgor, then it may also be appropriate to consider down-regulation of turgor under conditions of high water availability. For instance, fruits are known to accumulate very high levels of solute as a part of normal development, and are known to split or crack under conditions of increased water availability (Opara et al., 1997), presumably a result of the forces generated by turgor. Because of the large ratio of symplastic to apoplastic volume in plant tissues, regulating the level of solutes in the apoplastic space may be an efficient mechanism to avoid excessive turgor, particularly in systems where a large amount of solute uptake or production may be typical. Apoplastic solutes have been suggested as playing an important role in reducing turgor in fruits (Shackel et al., 1991), in sugarcane (Welbaum and Meinzer, 1990) and halophytes (Clipson et al., 1985), all of which produce or accumulate large amounts of solute.
The present study has demonstrated a close coupling of pod to plant water status, but a clear isolation of seed from pod water status. This is consistent with observations in other legumes, using indirect methods, that the turgor in the seed coat remains relatively constant despite changes in the water potential of its environment. This homeostasis may be part of a mechanism to ensure continued seed filling and assimilate redistribution even when low water potentials have reduced the current availability of assimilates from the leaves.
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Acknowledgments |
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We thank Drs Tim Colmer, David Turner and Jane Gibbs for their help and loan of equipment that made this study possible and Drs Hossein Behboudian, Kent Bradford, and Jairo Palta for comments on the manuscript. Ken Shackel thanks the Centre for Legumes in Mediterranean Agriculture for financial support and the CSIRO Center for Mediterranean Agricultural Research for the use of facilities.
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Notes |
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3 Present address and to whom correspondence should be sent: Department of Pomology, University of California, Davis, CA 95616–8683, USA. Fax: +1 530 752 8502. E-mail: kashackel{at}ucdavis.edu
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