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2 Centre for Legumes in Mediterranean Agriculture, and 3 Botany Department, University of Western Australia, Nedlands, Australia, 6907
Received for publication June 24, 1999. Accepted for publication March 16, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: cell division • cell separation • ethylene • hydrolytic enzymes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Addicott, 1982
; Sexton and Roberts, 1982
; Osborne, 1989
). Abscission is responsible for the fall of buds, branches, petioles, leaves, flowers, and fruit from plants and can be affected by environmental factors such as temperature, light quality, disease, water stress, and nutrition (Addicott, 1982
). Generally, senescence of the distal organ precedes abscission, but when the process is induced rapidly by exogenous ethylene from artificial sources or from adjacent diseased tissue, senescence may have barely begun before abscission occurs.
Mutants in which the rate or nature of abscission is altered can provide valuable tools for dissecting and understanding the biochemical and structural basis for the process. A number of genotypes in which the rates of organ abscission are altered have been described. The ab gene causes leaf abscission to be delayed in soybean (Probst, 1950
), and the Gr and ih types have a non-yellowing and leaf-retaining phenotype, respectively, in Phaseolus (Honma, Bouwkamp, and Stojianov, 1968
). An induced mutant in Corchorus has enhanced abscission rate (Sen, 1968
), and the Never ripe (Nr) tomato has delayed flower abscission and altered fruit ripening (Tigchelaar, McGlasson, and Buescher, 1978
; Tucker, Schindler, and Roberts, 1984
). The jointless (j) and the lateral suppressor (ls) mutants in tomato fail to shed their flowers or fruit (Roberts, Grierson, and Tucker, 1987
; Szymkowiak and Sussex, 1989
). There is a type of navel orange with altered abscission in leaves and fruit (Zacarias et al., 1993
), and an arrested leaf abscission variety of pubescent birch has been described (Rinne, Tuominen, and Junttila, 1992
).
A new spontaneous mutant was isolated from among plants of Lupinus angustifolius cv. ‘Danja’ (Atkins and Pigeaire, 1993
) that does not abscise any organ despite an apparently normal pattern of growth and senescence. Apart from earlier studies of flower abscission in Lupinus luteus (Van Steveninck, 1957, 1958, 1959
), there has been no detailed study of the process in the genus. Since abscission plays a part in several stages of lupin plant development and defense, new knowledge of abscission should benefit physiological studies that aim to increase plant productivity.
Selection of genotypes with reduced abscission has occurred over centuries in domesticated Gramineae where nearly all wild relatives shed grain by cell separation across living tissues (Osborne, 1989
). Modifications to abscission zone formation or separation may have potential in improving yield or facilitating better harvestability in certain crops. Probst (1950)
reported a delayed abscission genotype in soybean that was considered to offer a desirable trait when included in hay varieties. In certain environments genotypes of Phaseolus with delayed leaf abscission may result in yield advantages (Hardwick, 1979
). Osborne (1989)
suggested that a delay in differentiation of abscission zones in crops such as bean and cotton might provide advantages since early flower-bud abscission is common during transient stress conditions. Since abscission in narrow-leafed lupin occurs primarily among fertilized flowers and small pods (Pigeaire et al., 1992
), alterations to the process may provide benefits with respect to yield, particularly if assimilate supply is sufficient to fill additional pods that otherwise abscise prematurely. Similarly, a delay in abscission of leaves under stress conditions might also provide an advantage if more nutrients were mobilized to reproductive structures instead of being lost from the plant.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), that was identified from a glasshouse population of L. angustifolius cv. ‘Danja’ was crossed with a range of lupin genotypes. Reciprocal crosses between parent and mutant genotypes followed by backcrossing were made in the glasshouse over several growing seasons. Parents, F1, F2, and backcross (BC) populations of each cross were grown together in a shadehouse in the same year and examined for abscission of cotyledons, leaves, petioles, and flowers. Of the several mutants selected from a mutation breeding program, two (Abs2- and Delabs) were used in allelism studies with Abs-. Intercrosses were made between all three abscission mutants: Abs-, Abs2-, and Delabs and segregation observed in F1, F2, and backcrosses.
Mutagenesis
The L. angustifolius genotype cv. ‘Merrit’ was chosen for a mutation breeding study on the basis that it was the recommended, high-yielding narrow-leafed lupin for Western Australia. Approximately 4500 seeds were soaked for 8 h in running tapwater at room temperature and then for 6 h in 0.04 mol/L ethyl methane sulphonate (EMS, 0.43% v/v) in 0.1 mol/L phosphate buffer (pH 7.0) bubbled with air. The seed was washed for 10 h in running tapwater and sown immediately into moist soil in the field. At the end of the growing season (1992) 
15 seeds from each M1 mature plant (main stem and first-order laterals) were harvested. Ten M2 seeds of each M1 plant were sown in 0.5-m rows in 1993. The M2 population consisted of 2460 families ( 24 600 plants). At the end of the season, after M2 selections were made, M3 seed was bulk harvested and a portion sown in the following season in the field. Some further selections were made from among these plants and transplanted to pots in a glasshouse.
Although the effects of EMS on the lupin population was relatively mild (8.0% of M2 families segregating as chlorophyll mutants), the treatment was successful in generating many plants with morphological changes. Three additional mutants with altered abscission (named Delabs, Abs2-, and Mstip) were selected as single plants from separate families in the M2 generation. An additional mutant, DLS, which was dark green, short, and had delayed leaf senescence, was also selected.
Plant material for structural studies
Seeds of L. angustifolius cv. ‘Danja’ and Abs- were sown in 30 cm diameter pots containing 10 kg quartz sand under glasshouse conditions (25° ± 3°C day, 15° ± 3°C night) with natural light. Plants were inoculated with Bradyrhizobium lupini strain WU425 (Nitrogerm® Group G inoculum). Nutrient solution (one-quarter strength Hoaglands without N) was provided twice per week after emergence. Fresh tissues were collected at regular intervals for morphological observation. The course and pattern of organ abscission were recorded for both cv. ‘Danja’ and Abs- in field-grown plants sown in mid-May on sandy soil at Shenton Park, Perth, Western Australia with 200 kg/ha superphosphate applied prior to sowing. Plants were always well nodulated as the site has a history of lupin inoculation.
Definition of stages of natural abscission and collection of material
For anatomical studies of natural abscission, plants were grown through to maturity. The process of abscission at the tissue and cellular level was described by identifying five stages defined on the basis of visual features. Healthy, pre-abscission zone tissues were denoted "Stage 1." "Stage 2" tissues were those where the first signs of abscission, i.e., were faint tan-colored lines traversing the tissue. This was usually accompanied by a discernible yellowing of the distal tissue or of the whole organ distal to the abscission zone. "Stage 3" tissues were those that had clear visual abscission zone formation, with the initial external signs of separation. "Stage 4" was just post separation of the whole organ. "Stage 5" was 2 d after separation and "Stage 6" was 3 wk after separation. In Abs- the equivalent stage relative to the normally abscising genotype was determined visually by the condition and stage of yellowing of the zone and senescence of the distal organ.
For anatomical studies of naturally abscising cotyledons, stage 1 tissue was collected from glasshouse grown plants when seedlings had reached the four-leaf stage. Cotyledons representing subsequent stages were collected from seedlings that had developed to the six-leaf stage. Naturally abscising petioles and leaflets were collected as abscission began to occur in glasshouse-grown plants, 2 wk after flowering (i.e., 75 d after sowing) from lower main stem nodes. Non-abscising (stage 1) petiole/stem and leaflet/petiole pulvinus tissues were collected at the time of flowering on the main stem. Naturally abscising flower pedicels were collected from mid-way along the raceme of the main stem inflorescence.
Ethylene-induced abscission
The same stages of development as those used for natural abscission were defined for ethylene-induced abscission except that stage 2 was defined more by a "water-soaked," dark-green line across the tissue. Stage 5 tissues were collected 8 h post abscission. For ethylene-induced abscission of cotyledons, 14-d-old seedlings (two-leaf stage) were removed from pots with roots intact, placed immediately in water, then trimmed into explants under water to reduce the risk of xylem embolism and dehydration. Explants consisted of intact cotyledons with 15 mm of hypocotyl and 10 mm of stem distal to the cotyledon. They were placed in Elisa immunoassay plate wells containing deionized H2O (DI) and held in air-tight 4-L plastic containers flushed with 10 µL/L ethylene in air at 100 mL/min in growth cabinets at 20°C with continuous light (50 µmol·s-1·m-2). Growth stages for ethylene-treated cotyledons were as follows: stage 1 was healthy pre-abscission zone tissue and stage 2, 3, 4, 5, and 6 had been treated for 36, 44, 56, 72, and 120 h, respectively.
Two types of shoot explant were used for studies of ethylene-induced abscission of petioles. A two-petiole explant was used for initial studies. Subsequently, a six-leaf explant system (D. Osborne, personal communication, Open University, Oxford, UK) was used for comparing petiole abscission only. For two petiole explants, 14-d-old seedlings were collected (as for cotyledons) and explants consisted of the first pair of petioles trimmed each to 20 mm length by removing leaves and some of the petiole. Stem subtending the petioles was trimmed to 20 mm length. The apical meristem was retained but with emerging leaflets removed such that only 5 mm length of new juvenile petiole tissue remained. For six-leaf explants, 30-d-old seedlings (six-leaf stage) were collected with roots, immersed immediately in water, and trimmed to retain 1 cm of hypocotyl. Cotyledons were removed if still attached for Danja and excised at the abscission zone for Abs-. Shoot explants were immersed to a depth of 3 cm in DI and placed in 10 µL/L ethylene atmospheres as for other explants.
Growth stages for ethylene-treated two-petiole explants were as follows: stage 1 was healthy pre-abscission zone tissue, and stages 2, 3, 4, 5, and 6 tissues had been treated for 40, 48, 64, 80, and 120 h, respectively. For six-leaf shoot explants stage 1 was healthy pre-abscission zone tissue, and stages 2, 3, 4, 5, and 6 tissues had been treated for 20, 48, 72, 120, and 144 h, respectively.
Microscopy
Segments of tissues containing abscission zones were fixed in 4% glutaraldehyde, dehydrated in a graded alcohol series, and embedded in glycol methacrylate (Feder and O'Brien, 1968
). Sections (2 µm thick) were stained with 0.5% toluidine blue (pH 4.0) or Periodic-acid Schiff's (PAS) reagent (Jensen, 1962
). Toluidine blue was used to observe general cell organization. Lignified tissues appear blue-green, while other tissues appear deep blue. PAS was used to highlight starch granules that appeared red-black. Fresh sections (10 µm), cut by hand or with the aid of a freezing-stage microtome, were mounted unstained in DI or stained with either toluidine blue or phloroglucinol-HCl. For the latter, sections were mounted in two drops of saturated aqueous phloroglucinol and one drop of 1mol/L HCl was added. Observations and photographs were made rapidly before crystals began to precipitate. Phloroglucinol-HCl was used to highlight lignin (dull red) and cell wall polysaccharide modification (bright red) (Robinson, 1963
). Sudan black B (saturated in 70% ethanol) was used as a counterstain to indicate the presence of suberin. PAS-stained sections were flooded with filtered Sudan black B for 15 min.
For fluorescence microscopy, fresh sections were cut by hand or with a freezing stage microtome, mounted in water, and left unstained for autofluorescence. A Zeiss Axiophot photomicroscope was used with a 100 W HBO 50 Hg lamp light source and UV-G 365 excitation filter.
For electron microscopy, segments of tissue (2 mm long) containing abscission zones were fixed in 4% glutaraldehyde, dehydrated in a graded acetone series, stained in osmium tetroxide (2%), and embedded in Spurr's resin (Spurr, 1969
). Sections (0.1 µm) were cut, stained with uranyl acetate and lead citrate on grids (75/300 copper, slotted, 15 µm thick), and viewed with a JEM (2000FXII) transmission-electron microscope.
Measurement of root cap border cell release
Seeds of Danja and Abs- were germinated aseptically and grown aeroponically in 1-L beakers as described by Hawes and Lin (1990)
. Root tips were agitated in 100-µL DI, and a 10-µL sample was examined using a haemocytometer slide under 40x magnification. Root cap (border) cells, identified as oval to rod-like single cells, were counted.
Measurement of ethylene production from leaflets, explants, and flowers
Tissues to be assayed for ethylene gas evolution were placed in 10-mL serum vials, sealed with rubber septa and incubated for 4 h at 20°C in darkness. Gas samples (1 mL) were withdrawn and analyzed with a Shimadzu gas chromatograph (model GC-8A) fitted with a flame ionization detector and a stainless steel column packed with 80–100 mesh Porapak N (Waters Associates, Sydney, Australia). The column/oven temperature was maintained at 135°C and the injector at 150°C; N2 carrier gas was delivered at 170 mL/min, and H2 and medical air were delivered at 70 and 500 mL/min, respectively. Several replicates of a standard 1 µL/L ethylene were measured with each series of assays.
The effect of ethylene pretreatment of tissues on ethylene production by leaves (Bleecker et al., 1988) was evaluated by preparing whole leaves (lowest leaf pairs from six-leaf stage plants) with 5 mm of petiole immersed in DI water. Leaves were then treated with 10 µL/L ethylene or air in sealed containers for 12 h, flushed in air for 10 min, placed in 10-mL serum vials, and incubated for 4 h at 20°C in darkness. Gas samples (1 mL) were analyzed for ethylene content as above.
Application of chemical stress
Chemical stress was applied to leaf tissue by preparing whole leaves (lowest leaf pairs from six-leaf stage plants) with 5 mm of the petiole immersed in solutions of either 10 mmol/L CuSO4 (Avni et al., 1994
) or 2% (w/v) H2O2 (Ievinsh and Tillberg, 1995
) solutions with DI water as a control. Six replicates of two leaves each were held at 20°C in continuous light, and ethylene production was determined as above 0, 3, 6 and 9 h after sealing the vials.
Application of growth regulators
The sensitivity of cotyledon tissues to 1-aminocyclopropane-1-carboxylic acid (ACC) was assessed by pretreating cotyledons with 1 mmol/L ACC for 8 h and transferring to sealed petri dishes containing DI and storing in darkness for 5 d. Four cotyledons, replicated five times for each genotype, were treated with either ACC solution or DI water for 8 h and then transferred to petri dishes containing 20 mL of DI water. Conductivities of soaking solutions were measured using a conductivity meter by placing 5 mL of solution into the sample chamber. Roots of germinating seeds were treated with ethylene (10 µL/L) or its endogenous precursor, ACC (1-aminocyclopropane-1-carboxylic acid, 20 µmol/L). Germinating seeds (N = 15) of each genotype were placed in moistened growth pouches and allowed to grow for 7 d in air or ethylene-gased plastic containers. Seeds were also germinated in 2% agar containing ACC for 7 d at 20°C.
Plants of Danja and Abs- were grown to the 6–8 leaf stage and treated with foliar ethephon sprays of 0, 100, or 400 mg/L ethephon and also treated with root drenches every second day of 0, 10-6, and 10-5 mol/L ABA. Additional plants of Danja and Abs- were prepared as six-leaf shoots, which were immersed to 3 cm depth in either 0, 10-7, 10-6, 10-5, or 10-4 mol/L ABA solutions. Shoots were then treated with 10 µL/L ethylene in sealed containers under continuous light, and abscission of petioles was recorded periodically. Cotyledon and two-petiole explants placed in DI water in Elisa plates were treated with either NAA (
-naphthalene acetic acid, 0.2%) in lanolin (Van Steveninck, 1958
) or 2 µL NAA (100 µmol/L) solutions and placed in ethylene atmospheres (10 µL/L) under continuous light at 20°C. NAA in lanolin was applied to stem tissue as a ring of 50-µL paste at a point 10 mm below the abscission zone site of the cotyledon or petiole. NAA in solution was applied to the cut stump of the epicotyl above the cotyledons or petioles. Control treatments consisted of either lanolin paste or DI water without NAA added.
Application of drought and waterlogging
Both Danja and Abs- plants were either well watered throughout (control) or droughted for 14 d from the onset of flowering on the main stem. There were four replicate pots containing 10 kg of dry, white quartz sand topped with polythene beads to reduce surface evaporation each with 4 plants/pot, two of which were debranched. Control pots were watered to field capacity (14.0% water content). The droughting treatment was applied by maintaining the soil water content at 25% of field capacity. The water status of each pot was adjusted by weighing daily, and abscission was assessed visually.
Danja and Abs- plants at the 6–8 leaf stage of were subjected to 20 d of waterlogging or no waterlogging. Waterlogging was imposed by placing free-draining pots into trays of water filled to the level of the soil surface in pots. Pots were then returned to non-waterlogged conditions, and abscission was assessed visually.
Grafting experiments
To examine the effect of root genotype on shoot abscission, reciprocal grafts were carried out. Shoots were removed with a V-shaped cut 5 mm above the cotyledons of two-leaf stage seedlings of both Danja and Abs- plants. Controls were Danja shoots grafted onto Danja hypocotyl + roots and similarly for Abs-.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Development of abscission zone tissue under natural conditions
At each site of abscission on cv. ‘Danja’ there was a clearly defined zone of cells at which separation of the organ occurred. Although the detailed structural features of these abscission zones differed between sites, all were characterized by the development of a region of smaller, more densely packed cortical and epidermal cells whose radial walls were relatively more aligned than surrounding distal and proximal tissues. Sections showed that this stage was characterized by anticlinal cell divisions within a layer of 2–4 cells that appeared to develop with more dense cytoplasmic contents. In leaflets this cell layer was only one cell wide. Toluidine blue staining indicated a light-blue band some 3–5 cells wide, distal to the potential fracture plane, which was readily distinguishable from the surrounding purple-stained cells (Fig. 1). This blue color indicates enhanced lignification and was confirmed by characteristic staining with phloroglucinol-HCl (Fig. 2). The bright-yellow autofluorescence of these cells in UV light (Fig. 3) also supports such a view. In addition, the fluorescence micrographs show distinctly the loss of chlorophyll distal to the abscission zone. Cell divisions were followed by amyloplast accumulation in the region of several cell layers comprising the separation layer in cortex and pith parenchyma (Fig. 4). At the cotyledon abscission zone, starch appeared in adjacent tissues even in stage 1 of abscission. Suberin deposition, as evidenced by Sudan black B staining, was seen beginning in stage 3 tissues in cells at and just distal to the region of cell division. Starch accumulation was also visible in bract abscission zones (Fig. 5) but generally not so pronounced at other abscission zone sites and, in leaflets, appeared and then disappeared at stage 4.
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Cell separation began usually with both cortical parenchyma cells just inside the epidermis and the epidermal cells themselves and was initiated more frequently on the adaxial surface. The separation layer was distal to the region of cell division. Electron microscopy indicated dissolution of the middle lamella with primary walls remaining intact. Middle lamella dissolution was regularly followed by dissociation and disorganization of microfibrils of the primary cell walls, especially at the inner region of the abscission zone (Figs. 7–10). Primary walls of parenchyma cells were often still intact just inside the epidermis such that the cells became swollen and rounded. Further toward the inner regions of the zone, there was a greater degree of cell wall distortion and rupture. The distal part of the organ separated with the final shearing of vascular tissue and rapid dehydration and distortion of the proximal cells at the fracture surface. Some cell division activity was seen in stage-4 and-5 tissues in parenchyma that appeared to be a limited form of "protective layer." Sections of abscission zones made 3 wk after the organ was shed showed that several layers of cells below the fracture surface had formed a protective layer and there was no obvious cell division activity after this time.
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At all potential abscission sites the Abs- mutant showed the development of abscission zones that were essentially indistinguishable from those on cv. ‘Danja.’ In Abs- some cell wall distortion with plasmolysis of cells at the separation layer occurred instead of separation (Fig. 9). In this case, some of the distorted cells appeared to swell slightly. Electron microscopy indicated some breakdown of middle lamella that was usually accompanied by distortion of cell walls along the abscission plane. However, disorganization of primary walls was not common but was found to some degree in a few sections (Fig. 10). In the abscission zone of flower bracts in the mutant, cell separation did not occur at all but distortion of new cross walls of recently divided cells was clearly and frequently recorded. Although tissues distal to the "abscission" zones in Abs- did not separate, these organs rapidly lost turgor and shriveled while still attached. Similarly, flowers or small pods, which had aborted, remained attached and dried out. A limited form of protective layer also formed in the Abs- mutant and appeared similar to that in Danja.
Light microscopy of the induced abscission mutants, Abs2-, Delabs, and a stipule and abscission altered mutant (Mstip), confirmed abnormal abscission progression in each. Cotyledon abscission zones in Abs2- was similar to Abs- in that cell divisions without separation of cells occurred with only minor distortion of zone cell walls. In Delabs the abscission zones in cotyledons showed cell divisions with partial cell separation. However, this was not sufficient to cause shedding of cotyledons under natural conditions. Some petiole abscission was observed in glasshouse-grown plants of Delabs. The stipule-altered mutant had incomplete development of abscission zones in both cotyledons and petioles. However, the zone region was quite different from that seen in Danja or even in the other abscission-altered mutants. Cell divisions were not apparent, and the only sign of senescence in the more distal tissues at the abscission zone site was a faint toluidine-blue-staining region similar to, but not as sharply defined, as that in stained sections of Danja or Abs-.
Ethylene-induced abscission
Exposure of two- or six-leaf explants of cv. ‘Danja’ to 10 µL/L ethylene caused rapid and complete abscission of cotyledons, petioles, and leaflets (Fig. 11A–D). However, even 100 µL/L ethylene did not induce abscission of these organs on two-leaf explants of the Abs- mutant. On six-leaf explants only 80% of the petioles abscised in Abs- with 10 µL/L, although not as rapidly as in Danja and without abscission of any leaflets or cotyledons (Fig. 11).
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44 h. Cell expansion appeared to contribute to the separation, since cells in distal tissues did not expand in concert with proximal cells. Separation of cells began with the outer cortex and ended with the xylem being the last to detach, often with the walls of vessels protruding from the proximal face as stretched coils. At stage 4 (immediately post abscission) the fracture surface in Danja showed that many of the cells were intact, highly swollen and spherical, indicating very high turgor. At stage 4, cotyledons on Abs- showed no external signs but could be detached with reduced force. The fracture surface appeared smooth, indicating breakage along a plane of weakness but without the swelling of cells as in Danja. In the mutant after the 96-h ethylene treatment there was evidence of limited cell division and slight swelling of parenchyma cells along a region that would normally form into an abscission layer. No separation or plasmolysis was found even after 120 h (stage 6) ethylene treatment, although some cell division was apparent. Starch granules in parenchyma cells at the abscission layer were observed in ethylene-treated cotyledons of Danja only prior to abscission (early stage 3) and were not seen at other stages. In the two-leaf explant system development of ethylene-induced abscission of petioles resembled closely that seen in cotyledons. Again, the process was dominated by cortical cell swelling, beginning with those closest to the phloem. Some small starch granules were apparent in Danja with a density similar to that seen in cotyledon tissue. However, no cell division was observed at any stage in Danja and, in Abs-, the only changes were as described for cotyledons—limited cell division in a single layer of cells with only minimal distortion of cell walls along the zone at later stages (stage 6 onwards). Starch granules were not seen in Abs- under these conditions.
Abscission of petioles occurred for both Danja and Abs- when six-leaf shoot explants were treated with ethylene. In Abs- abscission was delayed relative to Danja (Fig. 11C, D), but structurally the progression of the process in each was similar. The only other difference was that incomplete abscission of petioles occurred for a greater percentage of explants of Abs- compared with Danja, where complete abscission was almost 100%. Changes in morphology occurred in a similar fashion to those observed in two-leaf explants of Danja treated with ethylene. In both genotypes, abscission sometimes took place distal to the base of stipules rather than the usual proximal site. Secondary abscission zone formation occurred in both genotypes at a position 0.5–1 mm proximal to the normal primary fracture plane (proximal to the base of stipules). While no detailed microscopic examination was made of these secondary abscission zones, macroscopic observations indicated features similar to those of primary zones induced with ethylene except for its lower position. The rate and frequency of secondary zone formation were greater in Danja compared to the Abs- mutant (Fig. 11E).
Ethylene production and response to growth regulators and to treatments causing stress
Endogenous production of ethylene by leaves of the two genotypes and their response to treatment with ethylene, which depressed subsequent evolution and copper sulfate or H2O2, which enhanced ethylene evolution, were not significantly different (Fig. 12A, B). Similarly, roots of seedlings of each genotype exposed to 10 µL/L ethylene or 20 µmol/L ACC were reduced in length, became distorted, and showed thickened hypocotyls (data not shown). Leakage of solutes from ACC-treated cotyledons were also similar for both genotypes (Fig. 12C). Flowers on the main-stem raceme of both genotypes showed similar changes in levels of endogenous ethylene production (Fig. 13A, B) as they proceeded from a bud stage through anthesis to the onset of pod development (stages 5–6). Although the patterns of ethylene evolution were different for flowers that were at the base of the raceme and likely to set pods and those more likely to abort at distal positions, they were similar for the two genotypes.
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Separation of root-cap border cells
The rate of separation of "border cells" from the root tip of both Danja and Abs- was assessed by measuring the number of these cells suspended in water surrounding aeroponically cultured roots. The greatest frequency of border cells was associated with roots 30–35 mm long and a lower frequency with shorter roots (Fig. 15). Both genotypes released a high frequency of separated cells, but the mutant released slightly fewer compared to the roots of Danja.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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in the Abs- plant selection, is designated abs1 (as distinct from the ab gene reported for soybean by Probst [1950]
). One of the new abscission-lacking mutants identified in the present mutation breeding program, Abs2-, was found to be allelic to abs1. Another induced abscission mutant, Delabs, was characterized by a complete lack of cotyledon abscission under normal conditions with abscission of leaflets and flowers reduced. This reduced abscission type was also recessive but not allelic to abs1 and has been designated abs2. The phenotype of Abs2- has yet to be described in detail, but it represents a tool for physiological study and genetic analysis of abscission in lupins. Of potential interest was another mutant with modified stipules (Mstip) which appeared to lack abscission zones at petiole and flower bract bases. The mutant type designated DLS, which was dark green and whose senescence appeared to be delayed and non-yellowing, was also found in preliminary studies with segregating progeny of crosses back to cv. ‘Merrit’ (data not shown) to be conditioned by a single recessive gene. Tissues such as stems, leaves, and pod walls were observed with fluorescence microscopy to have increased chlorophyll levels. Leaves appeared to be retained for longer, but abscission did occur for all organs. This trait may have some similarity with the non-yellowing type in Phaseolus (Honma, Bouwkamp, and Stojianov, 1968
) and with an induced mutant type in Lupinus albus that was similarly dark green and dwarfed and had increased pod dry matter compared to normal types (Huyghe, 1990
). Two enhanced pigment mutants of tomato, both of which showed increased chlorophyll contents have also been reported (Konsler, 1973
; Sanders, Pharr, and Konsler, 1975
; Jarret, Sayama, and Tigchelaar, 1984
; Van Wann, 1995
).
Comparison of natural and ethylene-induced abscission
Abscission zones in L. angustifolius were found to be formed by a region 1–5 cells wide in which there is cell division followed by middle lamella dissolution and cell wall disruption. This allows separation of cortical and epidermal cells with consequent rupture of the vascular tissue. In these regards petiole, pedicel, and leaflet zones of lupin were indistinguishable apart from overall size. The zone regions of cotyledons were slightly different in having much smaller cortical and epidermal cells as a result of division with the cortical cells appearing to be less interlocked (Sexton and Roberts, 1982
) within the zone. No differences were apparent between cell types with respect to lignification in any zone that formed. This general pattern is similar to that described for the natural or induced abscission process in many plant species (Bornman, Spurr, and Addicott, 1967
; Webster, 1970
; Hashim et al., 1980
; Oberholster, Peterson, and Dute, 1991
; Kuang, Peterson, and Dute, 1992
). and, in common with many dicotyledons (Addicott, 1982
; Sexton and Roberts, 1982
), involved the separation of intact cells with minimal rupturing of primary walls.
The ethylene-induced process of rapid dissolution of the middle lamella and disruption of cell walls was similar to that of natural abscission. However, with ethylene treatment cell swelling, particularly of ground parenchyma, always preceded separation for those abscission sites studied. Cell enlargement was not a feature of abscission in lupin cotyledon, petiole, and pedicel zones under natural conditions. Although cells at the separation layer often appeared enlarged, this was only after separation had occurred. Swelling of zone cells has been suggested as a functional component of the abscission process in a number of species (Wright and Osborne, 1974
; Sexton and Redshaw, 1981
; Osborne, 1989
) and in some cases has been attributed to the effect of exogenous ethylene (Wright and Osborne, 1974
; Gaspar et al., 1978
; Jaffe and Goren, 1979
; Polito and Stallmann, 1981
). Osborne (1989)
differentiated between cells that enlarge as a direct result of ethylene (type-2 cells) and adjacent cells (type-1 cells) that enlarge possibly because of an effect of auxin. Different systems of abscission induction can lead to variations in anatomical changes during zone differentiation, and this has been reported by others (Oberholster, Peterson, and Dute, 1991
; Weis et al., 1991
; Zanchin et al., 1993
). It is possible that different turgor conditions are maintained in explants on water agar or DI water under artificial light or in darkness compared to field conditions with intact plants where endogenous ethylene and the hormonal balance as controlled by the whole plant influence the process. A role for cell turgor in abscission has been suggested for many years, and severely water-stressed plants will often not abscise their leaves (Sexton and Roberts, 1982
). This could be due to the requirement for turgor in the process of abscission and/or to the lack of the plants' ability to produce hydrolases under conditions of low turgor or where cells are killed due to severe dehydration.
There was a lack of cell division after separation in lupin as has also been described in several species. No well-defined protective layer could be seen in proximal-zone tissues, even several weeks after separation in whole plants. The abscission scar consisted of a thin region of lignified and possibly suberized, dead cells. In the mutant genotype, this region appeared to be similar but with senescent distal tissues still attached.
Ultrastructurally, lupins show similarities to other species in that there is an increase in starch deposition early in the onset of abscission for both natural and ethylene-induced abscission. However, starch accumulation in L. angustifolius did not appear to be as extensive as reported in other species. Starch is possibly used as an energy source for the production of new proteins (e.g., hydrolases and for formation of new cells prior to and after separation (Oberholster, Peterson, and Dute, 1991
; Kuang, Peterson, and Dute, 1992
; Bornman, Spurr, and Addicott, 1966). There was also an increase in ribosomes and rough endoplasmic reticulum with more prominant dictysomes for cotyledon and petiole abscission zones of lupin, and it might be expected to be similar for the other abscission sites.
Chacterization of the non-abscising lupin genotype
In contrast to all other reported abscission mutants, the Abs- mutant of lupin failed to abscise any organ when whole plants were exposed to exogenous ethylene or ethephon. Similarly, a range of stress treatments, which enhanced endogenous ethylene production, caused organ senescence but not abscission in the mutant. Furthermore, the mutant produced ethylene at rates similar to those of the wild type and showed a range of other responses to ethylene or its precursor ACC, which indicated that, except for abscission, the ethylene physiology of the genotype was no different than its parent. There is good evidence that ACC is translocated to the tops in xylem from the root system (Tudela and Prima-Millo, 1992
), and it is conceivable that endogenous ACC was limiting in the mutant. However, the grafting experiments indicate that neither ACC nor any other root-derived factor associated with abscission of shoot organs was missing in the mutant. Thus, the lesion in Abs- that causes the non-abscission trait is not one related to ethylene production or to a general lack of response to ethylene.
ABA or flooding induced abscission in an arrested leaf abscission mutant of birch (Rinne, Tuominen, and Junttila, 1992
), but in the lupin mutant, neither treatment altered the lack of abscission. Although application of high levels of auxin resulted in senescence of leaves it did not cause abscission of the senesced organs. Thus, it seems unlikely that the mutant phenotype was the result of an alteration in growth regulator balance.
The Abs- mutant of lupin also appears to be different to the jointless (j) mutant of tomato since the latter does not form an abscission zone (Butler, 1936
; Szymkowiak and Sussex, 1989
). Abs- responds to ethylene and to natural senescence signals in that the abscission zone can be seen to develop through a process of cell division and cell wall modification. Senescing tissues are sharply delimited from proximal healthy tissue. The Abs- and normal genotypes show similar staining reactions to phloroglucinol and toluidine blue by cells just distal to the zone, with reduced staining at the separation layer and similar intensity and distribution of autofluorescence. These cytological observations are consistent with enhanced deposition of suberin and lignin and modification of wall polysaccharides (Sexton and Roberts, 1982
) in both.
Many of the ultrastructural changes in cells of the abscission zone prior to the separation stage are similar in normal and Abs- genotypes. However, they differed strikingly in the relative degree to which cell wall disruption occurred at the separation region. In the mutant some dissolution of middle lamella could be seen with distortion of cell walls and a small degree of disorganization of outer fibrillar structure. By comparison in the wild type there was complete loss of the middle lamella and extensive loss of wall organization. It seems reasonable to suppose that limited hydrolysis of wall materials in the mutant precluded cell separation and that its genetic lesion is related to reduced localized expression of hydrolytic enzyme activity.
Prolonged ethylene exposure (5–6 d) did, however, induce abscission of petioles, but never of leaflets or cotyledons, in large (six-leaf) or whole-shoot explants of the mutant. In these explants the abscission zone cells showed features similar to those in the normally abscising genotype consistent with a degree of wall breakdown sufficient for cell separation to proceed. The delay in abscission compared to the parent suggests that the hydrolytic activity was lower or was expressed more slowly in the petiole abscision zone of the mutant. However, if swelling of cells at the separation zone was a feature that contributed to ethylene-induced abscission, the degree to which turgor was maintained in cells proximal to the fracture plane would be a significant factor in determining the frequency of abscission. This might explain why in two-leaf explants the mutant showed no abscission of petioles—the senescing tissues lost turgor so rapidly that the effect of cell swelling at the fracture face was precluded. Additionally, secondary petiole abscission zone formation was seen in both genotypes when whole-shoot or six-leaf explants were held in ethylene for prolonged periods. This phenomenon, reviewed by Osborne (1989)
, is thought to be regulated by both ethylene and auxin. It is possible that a greater auxin gradient is present at the base of petioles in whole shoots (compared to the smaller two-leaf explants) undergoing ethylene-induced abscission and that this alteration changes the course of cell separation.
The Abs- mutant is fully fertile, indicating that cell separation to form viable pollen is not impaired by the mutation. Furthermore, there is apparently no deficiency in the expression of hydrolytic enzymes required for the separation of root border cells. The mutation would therefore appear to involve an enzyme or enzymes specifically associated with abscission zone differentiation. Among such enzymes, a group of endo-ß-1,4-glucanases (cellulases) has been identified as likely to function specifically in the abscission process (Tucker and Milligan, 1991; Kemmerer and Tucker, 1994
; Taylor et al., 1994
). The Abs- lupin mutant thus provides an opportunity to examine more closely the nature and role of these and other hydrolytic enzymes in cell separation associated with abscission.
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FOOTNOTES |
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4 Author for reprint requests (e-mail: clem{at}cyllene.uwa.edu.au
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and Abs- (
). Bars are means ± 1 SE (N = 4)
