Journal of Experimental Botany, Vol. 51, No. 353, pp. 2009-2020,
December 2000
© 2000 Oxford University Press
Original Papers |
Using 1H magnetic resonance imaging and complementary analytical techniques to characterize developmental changes in the Zantedeschia Spreng. tuber
1 Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand
2 The Horticulture and Food Research Institute of New Zealand, Private Bag 11 030, Palmerston North, New Zealand
Received 21 March 2000; Accepted 6 July 2000
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Abstract |
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Nuclear magnetic resonance imaging (MRI) and complementary analytical techniques were used to examine tissue structure and developmental changes occurring during active growth in the root tuber of Zantedeschia Spreng. cv. Chianti, a commercially significant cut flower. Plants were observed during the period of leaf senescence and tuberization at the end of the first growth cycle of micropropagated plantlets and, following cool storage to break endodormancy, during development occurring after the replanting of ecodormant tubers. MRI distinguished two distinct regions within the tuber, and the differences in the binding state of water in the two regions were reflected in differences in tissue morphology and function. An abundance of free water was observed in tissue comprised of large parenchyma cells, at the base of the tuber. This tissue appeared to be involved in maintaining the viability of the plant during the period of dormancy, a function indicated primarily by increased metabolic activity in this tissue during dormancy, and reduced metabolic activity during periods of active growth. In contrast, water was more tightly bound in tissue comprised of small parenchyma cells. This tissue appeared to operate as a region for dynamic carbohydrate storage. The initial increase in the free water content of this tissue during the growth phase was linked to the mobilization of starch during canopy development. The subsequent decrease in free water in the remainder of the growth period was linked to the reaccumulation of starch while the tuber functioned as a sink for photosynthate prior to canopy senescence.
Key words: Dormancy, MRI, tuberization, Zantedeschia, calla.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Species of Zantedeschia Spreng. (family Araceae) are typically winter-dormant, herbaceous, perennial monocotyledons. Each plant has an underground storage organ known as a root tuber. Vegetative buds on the crown of the tuber enter a period of dormancy from the time when leaf production ceases prior to leaf senescence in late summer (Funnell, 1994a
). This is characterized by a short period of endogenously imposed dormancy (endodormancy). In the field, the time when endodormancy ends coincides with winter. The cool temperatures and lack of water experienced during this season induce ecodormancy, and the resumption of growth is suppressed until winter has passed (Funnell, 1994b). Numerous Zantedeschia cultivars make significant contributions to international floriculture industries. However, little is known about the dynamic nature of the changes in metabolism, starch mobilization and water status taking place within the Zantedeschia tuber during dormancy and active growth, and how this relates to deleterious physiological processes such as ‘calcification’ (hardening of tubers) or pathogen invasion. Both these factors have a negative impact on commercial production. To provide a basis for understanding these problems, a magnetic resonance imaging (MRI) study was undertaken of the commercially important hybrid Zantedeschia cv. Chianti, using tubers of a size suitable for imaging.
MRI has been applied to a variety of plant tissues, and employed to monitor processes such as growth, ripening and disorder development in horticultural produce (Sarafis et al., 1992; Clark et al., 1997; Faust et al., 1997). Storage organs examined by MRI include potato and the bulbs of Allium, Lilium and Tulipa (Chen et al., 1989; MacFall and Johnson, 1994; Roh et al., 1996; Okubo et al., 1997; Zemah et al., 1999). Images produced by proton MRI are topological representations of the mobile water fractions in soft-tissue specimens. Image intensities at discrete locations throughout the sample are sensitive to both instrument settings and sample-dependent factors, such as proton spin density (or water content) and underlying relaxation processes. Relaxation, the unique processes associated with the NMR phenomenon, are typically described by the terms T1 (spin-lattice or longitudinal relaxation) and T2 (spin-spin or transverse relaxation). These define the rate at which protons return to their ground-state following perturbation by a radio frequency pulse, and are influenced by a number of factors including solution composition, solution concentration, pH, viscosity, and cell structure (Clark et al., 1998). MRI enables the molecular environment of water molecules to be probed at sub-millimolar resolution. Coupled with quantitative imaging, which facilitates separation of the component contributions of proton spin density and relaxation in image intensity (Kuchenbrod et al., 1995), it is possible to compile a detailed description of how this environment changes with time, and without destroying the sample in the process.
Because the underlying physiological basis of contrast in magnetic resonance images is dependent upon the biological system under observation, interpretation of images is best carried out in conjunction with measurement of other physiological parameters. Therefore, in this study, MRI techniques were complemented with classical analysis of plant growth and development, a histological study of tuber tissue, and a qualitative and quantitative study of carbohydrate distribution. The spatial distribution of metabolic activity in the tubers by physical staining with tetrazolium salts was also investigated. Using this technique it was possible to demonstrate a strong correlation between free water content and metabolic activity when comparing MR images, with tissue at the same location subsequently stained to confirm their viability (Iwaya-Inoue et al., 1996).
Since the transitions between phases of growth and dormancy constitute major events in the ontogeny of the Zantedeschia plant, it was presumed that these changes would be reflected in the tuber tissue. It was hypothesized that the abundance and binding state of water within the tuber would change as the plant went through its annual growth cycle. Further, it was expected that changes in the abundance and binding state of water would relate to changes in other physiological phenomena such as tissue organization, carbohydrate content and general metabolism.
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Materials and methods |
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Plant material and growing conditions
Tuber growth and development in Zantedeschia cv. Chianti (a hybrid of undocumented parentage) was studied for a total of 23 weeks during two periods of active growth. Observations started mid-way through the first growth cycle following ex-flasking of the tissue culture plantlets (177 d after planting), and included tuberization, and leaf and root senescence as tubers entered endodormancy (233 d after planting). Following cool storage, the now ecodormant tubers were replanted, and a second growth cycle was studied for 105 d, during which the plants developed root systems and leaf canopies and bloomed. For the first growth cycle (Cycle 1), plants were supplied by a commercial nursery (Bloomz, NZ), where they were grown in protected cultivation. Irrigation was withheld from the crop 193 d after planting to accelerate plant senescence, and the tubers were lifted from their growing medium 212 d after planting, in preparation for storage. For the second growth cycle (Cycle 2), tubers that had completed their first growth cycle before being stored at 8 °C for 15 weeks were dipped in a water emulsion of N-(phenylmethyl)-1H-purine-6-amine (0.057 g l-1) and gibberellic acid (0.057 g l-1) (Promalin, Nufarm) in keeping with commercial practice to enhance flowering (Funnell et al., 1988
). They were then planted in individual pots on the surface of a peat-based growing medium as described previously (Clemens et al., 1998). Tubers were covered with 40 mm of pumice (expanded volcanic rock fragments) to facilitate lifting for analysis. Plants were watered by capillary irrigation, and grown in a temperature-controlled glasshouse (20/30 °C, min/max).
Destructive harvests and analyses
Tubers were chosen randomly for destructive harvests at 2- and 3-weekly intervals for plants in Cycle 1 and Cycle 2, respectively. These were used for investigation of plant growth parameters, water distribution within tubers using MRI, and tissue structure. Measurements were made of maximum tuber diameter and fresh weights (FW) and dry weights (DW) of roots, shoots and tubers. To measure DW, samples were thinly sliced (
100 mg FW), and freeze-dried to constant weight (48 h). The number of replications per harvest was not less than five plants. In addition, for harvests in Cycle 1 after 177 d, and in Cycle 2 after 63 d, FW and DW measurements were taken and moisture content calculated for small samples (100 mg FW) taken from the top, middle and base of each tuber on the axis of the primary bud. (For the purposes of this paper, the ‘top’ of the tuber refers to that region most closely situated to the growing points (dormant buds and actively expanding shoots), whereas ‘base’ refers to that region of the tuber most distant from the growing points. Medial longitudinal images have been orientated so that these regions are towards the top and bottom of figures, respectively.)
Of the five plants taken for growth analysis at each harvest, tubers of three of these were imaged by 1H-MRI (200 MHz) in a system consisting of a 4.7 Tesla widebore cryomagnet (89 mm bore) and custom-designed hardware and software. Individual tubers were positioned in a 40 mm birdcage coil to image a medial longitudinal plane containing the primary bud, using a Hahn spin-echo pulse sequence. Eleven image slices of this plane were acquired sequentially according to a programmed script. For calculation of T1, the repetition time (TR) varied from 100, 200, 400, 800, 1600, to 3200 ms whilst the echo time (TE) was held constant (25 ms). For calculation of T2, TR was held constant (3200 ms) while TE was varied in 15 ms increments from 25 to 100 ms. Image acquisition (n=2 acquisitions per slice) required 3 h per sample for a complete set of images. Individual images were acquired as 256x256 data arrays with a field-of-view of 36 mm and a slice thickness of 3 mm.
Calculated images of T1, T2 and pseudo-proton spin density were determined by computation as described previously (Clark et al., 1998). Data from the images themselves (areas and region-of-interest measurements) were obtained using standard image processing tools in the public-domain software package, NIH-IMAGE (ver. 1.61, available at zippy.nimh.nih.gov). For investigation of tuber tissue structure, tuber metabolism, qualitative distribution of starch in complete tuber cross-sections, and quantitative analysis of carbohydrates in different regions within the tuber, samples were taken at three significant developmental stages. These were: during tuber endodormancy at the end of the first growth cycle (233 d in Cycle 1); during tuber ecodormancy, immediately before tubers were replanted for the commencement of the second growth cycle (0 d in Cycle 2); and at flowering, late in the second growth cycle (101 or 105 d in Cycle 2).
To investigate tuber tissue structure, samples were taken from a core of tissue in the medial longitudinal plane, from beneath the primary bud to the base of the tuber. Sample tissue was fixed in 30% formalin, ethanol and acetic acid (FAA) under vacuum for 15 min, and then at atmospheric pressure overnight. Following 3x2 h washes in 70% ethanol, samples were taken through an ethanol–TBA series (70, 85, 95, then 100% ethanol) with 2 h intervals. Samples were transferred to 1:1 TBA/paraffin oil for 1 h, then infiltrated with wax at 60 °C, before being sectioned at 10 µm thickness. Mounted sections were taken through a stain schedule that included safranin to stain lignified tissue, and fast green to stain cytosolic components. Specimens were viewed with a light microscope with or without a polarized filter. Tetrazolium trichloride (TTC) staining was used for the cytochemical demonstration of the succinate dehydrogenase enzyme system as an indicator of metabolism in tuber tissue (Rosa and Tsou, 1961). Tubers were bisected through the same median longitudinal plane that had been used for imaging. The face of one half was stained with TTC using a staining protocol developed from those recommended for testing seed viability (Grabe, 1970). Samples were washed in Milli-Q water for 30 min to clean exudate from cut surfaces, and then stained in a 0.7% TTC solution for 3 h at 21 °C. The face of the second half was washed and stained for 1 min in a solution of 0.3% iodine in 0.6% potassium iodide, then destained in running water for 1 min. A Hewlett-Packard flatbed colour scanner and IBM PC was used to record digital images of cut surfaces before and after staining with TTC or iodine.
The concentration of starch, sucrose, glucose, and fructose was determined for tissue (100–400 mg FW) taken from the top, middle and base of the tubers, directly beneath the primary bud. Soluble sugars were extracted using a modified method of Hasslemore and Roughan (Hasslemore and Roughan, 1976). Carbohydrate concentrations (mg g-1 FW) were determined enzymatically with sucrose/D-fructose/D-glucose and starch kits (Boehringer Mannheim Biochemicals), with the methodology altered to use micro-amounts and a plate reader (Gapper, 1998). Known concentrations of D-glucose, D-fructose, sucrose, and starch were taken through the respective assay procedures to produce standard curves (absorption at 340 nm against sample concentration). Extraction efficiency was determined by taking samples of known concentration through the respective sample extraction and assay procedures, then comparing the determined concentration to the actual. Determined sample concentrations were adjusted accordingly.
Statistical analyses
Quantitative data was analysed using the Statistical Analysis System. One-way analysis of variance (ANOVA) was conducted for each variable as a function of time, with the probability that the data at each time interval within a variable was the same as each of the others being calculated to establish the statistical significance of trends observed in average values. Probability less than 0.05 confirmed a significant difference between two means. As this test was only appropriate for the analysis of normally distributed data, a Shampiro-Wilks test was used to confirm the normality of data at different times within each variable. All trends in plant growth parameters, T1 and T2, and carbohydrate concentration of tuber tissue were statistically significant as indicated by the above tests, unless mentioned otherwise in the text.
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Results |
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Plant development
During the latter stages of Cycle 1 (177–233 d after planting), shoots senesced completely, and root DW declined to <0.1 g per plant. At the same time, tuber DW increased significantly (P<0.05) throughout the observation period (Fig. 1A
). Tuber diameter also increased significantly (P<0.01) from 28.6 to 31.5 mm in the 28 d prior to lifting, but no further increase occurred after this (Fig. 1B). In general, whole tuber moisture content decreased over the observation period (from 77% to 72%), the greatest decrease occurring in the first 14 d of observation (Fig. 1C). At the start of the observation period there was 20% difference in moisture content between tissue at the top (65.8%) and the base of the tuber (85.3%). Shoot and root moisture contents were 90% until plants were lifted.
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5) of Zantedeschia cv. Chianti tubers as a function of time during Cycle 1 and Cycle 2. (A, D) Tuber dry weight, (B, E) tuber diameter, and (C, F) tuber moisture content.Immediately following replanting of tubers in Cycle 2, leaf and root DW increased steadily and significantly with each harvest, reaching 40 g and 18 g after 84 d, respectively. A steady moisture content of
95% was maintained in roots, and shoot moisture content increased from 89.6% to 93.4% in the first 21 d, then decreased to 87.1% over the remainder of the observation period.
As in Cycle 1, tubers exhibited trends in biomass accumulation in Cycle 2 contrasting with those of shoots and roots. In the first 63 d, tuber DW approximately halved from 2.7 g to 1.3 g. However, this loss was rapidly reversed in the following 21 d (Fig. 1D
). During the period of decline in tuber DW, tuber diameter did not vary significantly. However, tuber diameter increased from 34.8 mm to 43.1 mm as tuber DW increased in the latter stages of Cycle 2 (Fig. 1E).
The decline in tuber weight followed by its increase was accompanied by opposing trends in tuber moisture content, whole tuber moisture content increasing from 75.0% to 89.7% in the first 63 d, and then decreasing to 84% in the next 21 d (Fig. 1F). Separate measurements in the top, middle and basal regions of the tuber just before the transition between tuber dry weight decline and increase indicated that moisture content was similar in the top and middle regions (82.4% and 81.8%, respectively), and lower than that in the basal region (90.2%).
Preliminary MR measurements
To optimize instrument settings for use during the study, a series of images was obtained about orthogonal planes centred on the middle of a tuber. A network of strands, 0.3–0.7 mm in diameter, distributed randomly throughout the tuber interior was the dominant feature in transverse image sections (Fig. 2A). The strands were superimposed on a featureless background which varied in intensity from near-black (absence of signal) in the centre of the tuber, to grey (some signal) 6–8 mm inside the tuber surface, to white (high signal) in a band up to 1.5 mm thick extending part-way round the tuber perimeter.
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Inspection of longitudinal images (Fig. 2B
) revealed that the strands were in fact an interlaced network of transport vessels, present at high concentration beneath the bud and extended throughout the tuber (confirmed by histological examination, see below). Distinction between the central void, where no signal could be detected, and parenchyma tissue between void and surface where some signal was evident, was also apparent in these images.
Lack of NMR signal can be attributed to factors other than the absence of mobile water molecules. These are frequently localized magnetic effects arising either from the porous nature of the sample (known as susceptibility effects), or the presence of more powerful magnetic moments associated with paramagnetic ions (Gadian, 1995). Further exploratory investigations were carried out to determine whether these were pertinent here. Measurements on imaged tubers that were subsequently dissected revealed no significant difference between porosity (Jensen et al., 1969) in tissue from the centre of the tuber (1.4%) and that in the outer region around the periphery (2.8%). Paramagnetic ions in biological systems likely to affect the proton MR signal include Mn, Fe, Cu, and Ni. The relative trace metal concentrations (mg kg-1 FW) in the centre and peripheral tissues were Mn (20 and 9), Fe (279 and 474), Cu (2.0 and 2.2), and Ni (0.3 and 0.5), respectively. Respective moisture contents were 60.8% and 71.9%. These experiments implied that localized magnetic effects were unlikely to be responsible for the lack of signal in the centre of the tuber. The Zantedeschia tuber is not a hyperaccumulator of trace metal ions, and the differences in trace metal concentrations and porosity between the two regions are not pronounced. However, water was indeed present. This suggested relaxation processes at that stage in the tuber's growth cycle were too fast to detect a signal at our lowest echo time setting of 25 ms.
MRI in Cycle 1
Calculated maps for T1 and T2 relaxation for the period between 177 and 233 d after planting in Cycle 1 are shown in Fig. 3. Tuber images were characterized by a region of comparatively long T1 and T2 in tissue at the base of the tuber, and a region of shorter T1 and T2 in the lower-middle tissue. A region of relaxation time too short for the instrument to measure (below 50 ms for T1 and 15 ms for T2) covered the remainder of the middle tissue, as well as the tissue at the top of the tuber just below the crown. This made up 30% of the total tuber cross-sectional area 177 d after planting, and increased steadily to 41.4% after 233 d. T1 in the vascular tissue was similar to that found in middle tissue, but T2 times were closer to those found in basal tissue.
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During Cycle 1, T1 increased along a similar trend in the basal and middle regions, from 1320 to 1990 ms, and 780 to 1330 ms, respectively (Fig. 4A
). Approximately 90% of the increase in T1 in the basal tissue occurred in the first 14 d of observation. The decrease in T1 in basal tissue after lifting was marginally significant (P=0.046), but was non-significant (P=0.12) in the middle tissue (Fig. 4A). Changes in T2 in the basal tissue during the observation period were not significant (P>0.1), and T2 was therefore constant at 50 ms throughout this period (Fig. 4B). Similarly, T2 in middle tissue was constant at 16 ms (Fig. 4B).
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MRI in Cycle 2
Following the period of cold storage between Cycles 1 and 2, T1 increased by 200 ms in basal tissue, but decreased by 100 ms in middle tissue (Fig. 4C). With this being the case, T1 times in the basal tissue (1950 ms) were approximately twice that in middle tissue (960 ms) at the start of Cycle 2. No significant change was observed for T2 in any region between growth cycles (Fig. 4D). T2 in the basal tissue (42 ms) was approximately three times that in the middle tissue (17 ms). Relaxation times remained too short to measure towards the top of the tuber (Fig. 5A, D).
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T1 in the basal tissue remained at
2000 ms for the first 21 d, then declined significantly (P<0.05) to 1300±50 ms over the next 21 d, and remained constant for the rest of the observation period. In the middle tissue, T1 increased to 1440±60 ms in the first 21 d and remained between 1200 and 1400 ms for the remainder of the observation period (Fig. 4C).
T2 in the base and middle tissue followed a similar trend over the observation period from planting until flowering after 84 d, although the former was consistently 20 ms higher than the latter. In the 63 d following planting, a significant increase of 20 ms was observed in basal and middle tissue giving T2 of 61 and 38 ms, respectively (P<0.02). This was followed by a significant decline in T2 in these tissues, to 45 and 27 ms, respectively (P<0.02) (Fig. 4D).
Averaged over all tubers imaged, the area of the tuber for which relaxation times were too short to measure declined from 21.4% at planting to 0% over the first 63 d of the experiment. This transition was visualized in the T1 and T2 maps as a ‘in-filling’ of the central void in the tuber cross-section (Fig. 5). Although none of the three tubers imaged at planting had measurable relaxation times throughout upper-middle and top tissues, this was not the case for two of the three tubers imaged 21 d after planting. After 63 d, all three of the tubers imaged had measurable relaxation times throughout these tissues. However, by 84 d, relaxation times were measurable throughout only two of the three tubers harvested, and the average area in which relaxation time was too short to measure increased once more.
T1 in tissue towards the top of the tubers was 920±20 ms, and T2, 15±0.4 ms 21 d after planting, both shorter than relaxation times in any other tissue region (Fig. 4C). There was no significant change in T1 in these tissues over the remainder of the observation period. There was, however, a significant increase in T2 between 21 d and 63 d, to 25±2 ms, and this was followed by a significant decline to 17±1 ms over the next 21 d (Fig. 4D).
Tissue structure
Sections taken from tubers in Cycles 1 and 2 showed that tissues were comprised predominantly of thin-walled, isodiametric parenchyma cells. The morphology of these parenchyma cells was not uniform within the tuber, and separation of cells into two regions of distinct morphology was evident in all tubers. The first region was immediately below the apical buds, and radiated downwards to cover the bulk of the medial longitudinal section of the tuber. Tissue immediately below the primary bud was typically comprised of small parenchyma cells that contained a small amount of green-stained material, including occasional starch granules (Fig. 6A). The unstained intracellular space in most of these cells could have been one of two things, depending on the cell in question. It was either a large vacuole, or an artefact of plasmolysis caused by an inadequate tissue fixation technique. Differentiated vascular elements were present within this parenchyma, the lignified spiral xylem thickenings stained red with safranin.
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Further below the crown, cells were slightly larger and progressively more densely filled with stained material (Fig. 6B
). The unstained region around the periphery of most cells was almost certainly an artefact of plasmolysis, indicated by occasional contact points between plasmalemma and cell wall plasmodesmata. Starch granules were abundant in these cells, and were frequently observed in groups within amyloplasts (Fig. 6B). Occasionally, calcium oxalate raphide or druse crystals were observed in this tissue. These were held in the vacuoles of comparatively large parenchyma cells that contained no other stained material. Vascular differentiation was still in evidence in this tissue, although it was notably less common than immediately below the buds.
The second morphologically distinct tissue region was toward the base of the tuber. Between this and the first region, parenchyma cell morphology went through a considerable and rapid transition. Cells were notably larger in basal tissue than elsewhere in the tuber. Stained intracellular material thinned to near absence, with starch granules becoming very rare (Fig. 6C).
As plants progressed through Cycles 1 and 2, changes were observed in the ratio of different tuber tissue morphologies to one another (Fig. 7A). In endodormant tubers at the end of Cycle 1, the region of large parenchyma at the base of the tuber was very narrow. This remained the case after storage and replanting in Cycle 2 (up to 63 d). After this time, the band of large parenchyma broadened to make up a larger proportion of the tuber cross-section. As Cycle 2 progressed, vascular differentiation within the tuber developed further, becoming more pronounced throughout the small parenchyma tissue. This was especially so immediately below the apical bud, where the top of the tuber grew further outwards with the progressive development of shoots and the vasculature leading into them.
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Staining of fresh, complete medial longitudinal tuber sections with I2 gave a qualitative description of the location of starch within the tuber (Fig. 7B
). At no time was I2 staining seen in the large parenchyma tissue at the base of the tuber. In endodormant tubers at the end of Cycle 1, I2 staining was observed in all but the very top of small parenchyma below the primary bud, and right to the boundary between the small and large parenchyma tissues. In the ecodormant tuber before planting in Cycle 2, a larger region of small parenchyma immediately below the bud, and a portion of the small parenchyma tissue towards the base did not stain. After 101 d in Cycle 2, when tubers had gained considerable biomass, I2 stained only the top half of the small parenchyma tissue, including that leading into the growing stem.
TTC staining indicated that the distribution of metabolic activity was not uniform within the tuber, regardless of whether the tubers were endodormant, ecodormant after storage, or flowering. The distribution of metabolic activity was dynamic with respect to the plant's stage of development (Fig. 7C). In endodormant tubers, intense red staining was seen predominantly in basal tissue, but also to an extent in the lower-middle tissue. There was also some staining in the top tissue immediately below the primary bud. By contrast, intense staining in ecodormant tubers was limited to the tissue at the top of the tuber, with basal and lower-middle tissue staining less intensely. Furthermore, in tubers nearing the end of flowering, staining was all but absent from basal and lower-middle tissue, but very intense in upper-middle tissue and tissue leading into the stem. Intense staining of the vascular material was common to tubers at all stages of development.
Quantitative analysis of starch and soluble sugars
Quantitative assays were conducted for starch, sucrose, glucose, and fructose in the top, middle and basal tissue of tubers in states of endodormancy, ecodormancy and at flowering. In the tissue at the top of the tuber, starch concentration was maintained at a similar, high level between endodormancy and ecodormancy (Fig. 8A, B). Between replanting and flowering in Cycle 2, starch concentration in this tissue decreased to a quarter of its previous level during endo- and ecodormancy (Fig. 8C). A similar trend was observed for starch in the middle issue, although the concentration was consistently 10–15 mg g-1 FW higher than in the top tissue. In the basal tissue, starch concentration was much lower than that found in top and middle tissue (Fig. 8).
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) and sucrose (
) concentrations (mean±standard error, n=3) in tissue taken from the top, middle and base of Zantedeschia cv. Chianti tubers during (A) endodormancy (Cycle 1, 233 d), (B) ecodormancy (Cycle 2, 0 d), and (C) at flowering (Cycle 2, 105 d).In top and middle tissue, sucrose concentration remained relatively constant between endo- and ecodormancy (Fig. 8A
, B), then increased by 5 mg g-1 FW between replanting and flowering in Cycle 2 (Fig. 8C). These increases were statistically significant (P<0.05). There was little difference in sucrose concentration between top and middle tissue. Sucrose concentration in the basal tissue was lower than that in the top and middle tissue, and was not affected by the growth cycle (Fig. 8). In contrast, glucose and fructose concentrations were relatively unaffected by either position within the tuber or the developmental stage of the plant. The concentration presence of these soluble carbohydrates within tuber tissue was comparatively low (<1 mg g-1 FW) at all times.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant development trends observed in this study were consistent with those reported for Zantedeschia by other workers (Clark and Boldingh, 1991
; Funnell and Go, 1993; Clemens et al., 1998). The transition from a net loss to a gain in tuber mass observed partway through Cycle 2 has been referred to as the ‘source–sink transition’ (Funnell, 1994a). This represents a change in tuber function from a source of stored resources for shoot development, to a sink for surplus photosynthate from the mature canopy. Changes in other developmental parameters were also focused around the tuber source/sink transition. The change from loss to gain of tuber dry weight was accompanied by a change from gain to loss of tuber moisture content, and tuber diameter only began to increase significantly once the transition had been made. These data suggest a linear change in starch concentration in small parenchyma tissue between planting of ecodormant tubers and flowering. However, because no samples were analysed between these two times, starch concentration at the source/sink transition (the theoretical low-point for stored reserves) was unknown. Funnell, who sampled intensively throughout the growth period, observed a decrease in total tuber starch content until the source–sink transition, then an increase beyond it (Funnell, 1994a). It is reasonable to assume this same trend in starch concentration occurred in tubers of plants in this study.
Relaxation time in tuber tissue was reflective of cell morphology and tissue function. The region of comparatively long relaxation time at the base of the tuber corresponded to the region of large parenchyma cells that contained little visible material and had high moisture content. The region of short or very short T2 times in the middle and top of the tuber corresponded to the tissue comprised of small parenchyma cells that contained more visible material, and had lower moisture content. The same was true of T1 during endo- and ecodormancy, and to a lesser extent shortly after ecodormancy was relieved, at which time T1 became somewhat homogenous throughout the tuber. The difference in relaxation time between the small and large parenchyma tissues is indicative of differences in the binding state of the water present, and is consistent with observations in MRI studies of a wide variety of plant materials (Williamson et al., 1992; Veres et al., 1993; Ishida et al., 1994; Maas and Line, 1995; Glidewell et al., 1997). Both T1 and T2 decrease with increasing solute concentration and viscosity within cellular space. Because this results in greater interaction between water and other molecules, there is a reduction in the amount of free water present (Gadian, 1995).
Overall, the T2 component of relaxation was the better indicator of the distribution of the two tissue morphologies. MRI studies of mature fruits provide useful comparisons for the correlation between free water and tissue morphology observed in Zantedeschia tubers. Abundant free water (as indicated by long T1) was observed in the parenchyma surrounding the seed envelope, pericarp, and locular cavities of mature tomato fruits (Ishida et al., 1994). However, there was little free water in the membranous seed envelope, seeds or columella. As in the present study, a strong correlation between T2 and the size of parenchyma cells within ripe grape berries has been identified (Glidewell et al., 1997). Radially arranged striations within the T2 map corresponded to the distribution and alignment of panels of cells in the inner mesocarp. Similarly, T2 was a better indicator of the difference in parenchyma morphology than T1. Longer T1 was observed in the pith than in the cortex of ripe strawberry ‘fruits’, both of which are comprised of parenchyma cells (Maas and Line, 1995). These authors suggested a linkage between the differences in free water the tissues contained and their functional separation. The pith mediates transport of photosynthate from leaves to the cortex, whereas the cortex is the destination sink. Such functional separation between tissues is evident in the Zantedeschia tuber, given that small parenchyma appear to operate as a storage facility for photosynthate, whereas large parenchyma do not. The fundamental differences in structure and function between these two tissues are a foundation, therefore, upon which changes in the aqueous environment over the course of plant development may be rationalized.
Changes in the binding state of water within small parenchyma towards the top of the tuber correlated with changes in the concentration of starch in this tissue. Both T1 and T2 (and therefore free water) increased as starch concentration decreased following the release of ecodormancy. In a review of the structure of starch granules in plant tissue, reasons were suggested as to why starch affects the moisture and free water content, and hence relaxation parameters, in plant tissue (Buleon et al., 1998). Starch granules are porous structures exhibiting both an external and an internal surface. Water is absorbed into the porous interior, where it is able to interact with the amorphous starch matrix. This induces higher molecular mobility of the starch, and swelling of the starch granule at the expense of the mobility of the water involved. As such, not only does the proportion of starch to cellular volume affect the space available for intracellular water, but it also affects the proportion of this water that has a close association with starch, and as such is in a highly bound state. The trends in relaxation time in small parenchyma tissue of the Zantedeschia tuber in the growth period after the release of ecodormancy are, therefore, adequately explained in terms of the mobilization of stored starch before the source–sink transition, and its reaccumulation after it.
The changes in the binding state of water observed in the Zantedeschia tuber during plant development are consistent with similar observations in a variety of fruits as they mature (Bennett et al., 1987; Williamson et al., 1992; Ishida et al., 1994; Maas and Line, 1995). A clear distinction exists between the development of a fruit structure that culminates in terminal maturation and subsequent senescence, and the cyclic development pattern of the Zantedeschia tuber, which is maintained from one season to the next. However, common ground exists in that both processes represent ontogenic progression. With changes in tissue composition occurring throughout development, the binding state and, therefore, relaxation parameters of water also experience change.
In contrast to the small parenchyma tissue, there was no evidence to suggest that the large parenchyma tissue at the base of the Zantedeschia tuber operated as a sink for photosynthate. A possible functional role for the large parenchyma became apparent only when trends in distribution of free water and starch were observed in conjunction with trends in the distribution of metabolic activity within the tuber. During endo- and ecodormancy, metabolic activity was largely confined to tissue that contained free water, i.e. that in which starch was less abundant and water relaxation was long enough to be measured. However, at the latter stages of flowering (and presumably well before), higher metabolic activity was found only in the area that contained starch. It was notable that metabolic activity was comparatively high in large parenchyma during endodormancy, but less so during ecodormancy, and minimal during the active growth period. Such trends suggest that the large parenchyma at the base of the tuber maintained a high level of metabolic activity during dormancy, using carbohydrates mobilized from the periphery of the small parenchyma tissue. In this way, the viability of the plant was maintained during the dormant phase.
Water within buds of fruit trees is also held in a tightly bound state during endodormancy, and, with transition to ecodormancy, the water becomes comparatively free (Faust et al., 1991; Rowland et al., 1992; Liu et al., 1993; Gardea et al., 1994; Erez et al., 1998). Given this, changes in the binding state of water within Zantedeschia tubers between endo- and ecodormancy were anticipated. However, such changes were not observed, matching a lack of significant change in physiological parameters, such as moisture content and carbohydrate concentration. On the basis of these observations, one might conclude that the regulation of endodormancy in Zantedeschia tubers is the functional domain of the shoot meristems at the crown of the tuber, and not of the tuber per se. Limited detail was contained in the MR images of primary buds at the scan resolutions of 140–160 µm used in this study. The resolution here was dictated by the necessity to image an entire tuber. It would be possible to obtain superior MR images of this site by dissecting the buds and imaging in a smaller coil at higher field strength, if one was prepared to sacrifice physiological function.
This study provides the first spatial description of the binding state of water within the Zantedeschia tuber. It is also one of a few MRI reports to investigate an underground storage organ. The trends in distribution of free water and metabolic activity observed in Zantedeschia tubers are not in accordance with those observed in tulip bulb scales by other authors (Iwaya-Inoue et al., 1996). These researchers observed a transition from base-level metabolic activity related to tight binding of water in tulip bulb scales during dormancy, to high-level metabolic activity related to abundant free water once chilling relieved dormancy. This was clearly not the case for Zantedeschia tubers, in which there appeared to be a negative relationship between the distribution of metabolic activity and starch during dormancy, which was reversed during the active growth phase. This relationship would appear to be sensible given that mobilization and synthesis of starch requires ATP (Pozueta-Romero et al., 1999), a product of respiration, and that modifications to starch reserves occur predominantly during the active growth phase. The clear operational differences between the tulip bulb and Zantedeschia tuber, despite their common function, demonstrate the need for care in the interpretation of contrast changes in magnetic resonance images. The ability to interpret contrasting changes in biological samples during a developmental process depends on a broad knowledge of the physiology of that process, rather than of its discrete elements (Maas and Line, 1995).
In conclusion, the hypothesis that the distribution of free water within Zantedeschia tuber tissue would change during the course of the growth cycle, and that such changes would reflect changes in tissue composition related to its function, was supported. Changes in water relaxation parameters were attributed to an interaction between water and starch molecules, and a negative relationship between starch and moisture content. In addition, the investigation demonstrated morphological and functional separation between the two major tissues found in the Zantedeschia tuber. Small parenchyma cells comprised the bulk of the tuber. This tissue was clearly identified as a region for dynamic carbohydrate storage that supported vegetative development after the release of ecodormancy, before being replenished once the canopy matured. In contrast, tissue at the base of the tuber was comprised of large parenchyma cells, and appeared to be involved in maintaining the viability of the plant system between phases of active vegetative and reproductive development.
While this study focused on one cultivar only, findings are almost certainly applicable to other Zantedeschia. Given the ability of MRI to visualize pathogenic invasion of plant tissue (Goodman et al., 1992; Pearce et al., 1994; Maas and Line, 1995; Glidewell et al., 1997), this technique should prove useful in further studies to characterize the invasion of Zantedeschia by commercially significant pathogens, such as Erwinia sp.
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Thanks are expressed to Dr Dan Cohen, Professor Paula Jameson, Ms Liz Nickless, Professor Rod Thomas, and the staff of Bloomz New Zealand for their contributions to this project, and to the JP Skipworth and Massey University Alumni Scholarships for financial support of AR.
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3 To whom correspondence should be addressed. Fax: +64 6 350 5688. E-mail: J.Clemens{at}massey.ac.nzAbstractNuclear
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