Autotomy in plants: organ sacrifice in Oxalis leaves

Abstract

Autotomy is a self-defence strategy of sacrificing a body part for survival. This phenomenon is widespread in the animal kingdom (e.g. gecko's tail) but was never reported in plants. In this study, we characterize the autotomy mechanism in the leaves of an invasive plant of South African origin, Oxalis pes-caprae. When the leaves and flowers of this plant are pulled, they break easily at their base, leaving the rest of the plant intact. Microscopic observations of the leaves reveal an area of small cells and a marked notch at this designated breaking point. Mechanical analysis showed that the strength statistics of the petioles follow Weibull's function. A comparison of the function parameters confirmed that strength of the tissue at that point is significantly smaller than at other points along the petiole, while the toughness of the tissue at the notch and at mid-petiole are approximately the same. We conclude that leaf fracture in Oxalis is facilitated by an amplification of the far-field stress in the vicinity of local, but abrupt, geometrical modification in the form of a notch. This presents an autotomy-like defence mechanism which involves the sacrifice of vital organs in order to prevent the uprooting of the whole plant.

1. Introduction

Autotomy or self-amputation is the animal behaviour of sacrificing of a body part, typically as a self-defence mechanism to avoid predation. Probably, the best known example of self-amputation is that of the gecko's tail, which was also empirically shown to increase gecko survival under predation [1]. The detachment of a sacrificed body part either occurs after application of external force or is self-inflicted at a moment of imminent danger, while the breaking point occurs at a pre-defined area of weakness [2,3]. As autotomy occurs at a pre-formed breakage point in the tissue, the fracture is clean and heals easily. Often the sacrificed body part regenerates later. This phenomenon is widespread throughout the animal kingdom and has independently evolved multiple times in diverse taxonomic groups (e.g. tails in reptiles [4–6], skin in mice [7], legs in spiders [8], various organs in shell-less molluscs [9], arms in brittle stars [10] and many more). Until now, autotomy has never been reported to occur in plants.

Plants are constantly subjected to herbivory and thus permanently lose and regenerate body parts. In order to optimize the costs, plants employ a combined array of general, permanently active defences and particular, pest-specific defences. The main purposes of plant defences are both repelling and damaging the pest or herbivore and sealing the damaged area and thus preventing subsequent infection [11]. Numerous defence mechanisms were reported in plant aerial parts, including various chemicals [11], hard and unpalatable structures [12,13], defensive coloration [14], etc.

Fracture mechanics is a prime feature of plant–herbivore interactions, as the herbivore has to tear the leaf (or part of it) away from the plant [15]. The easiest way to detach a plant part is by cracking, while the cracks appear according to the applied force and to the material structure of the leaf. Imperfections in the overall shape of the plant structure and in the plant tissue (scratches, notches) result in stress concentration (force per area) at that certain point that generates local fracture. The strength of the leaf is identified as the maximal force divided by the fractured area. A complementary critical aspect is the tissue toughness (work-of-fracture per twice fractured area) which characterizes the resistance of the plant material to crack propagation and ultimate fracture. Various studies deal with the fracture mechanics of plants, the defining parameters and the material structure [15–19].

The current work reports for the first time an autotomy defence mechanism in plants. The phenomenon is studied in Oxalis pes-caprae (African wood-sorrel, Oxalidaceae), a small herbaceous invasive weed, which includes deeply buried underground bulbs with aboveground leaves [20–22]. When a leaf is pulled, the leaf stalk (petiole) is always easily torn at a designated region close to its base (pulvinus), leaving the bulbs and the meristem safely in the ground. This organ sacrifice mechanism is one of the prime reasons why O. pes-caprae is so difficult to eradicate. In the following study, integration of plant science and mechanics of materials is employed to understand the biomechanical mechanism of autotomy in Oxalis.

2. Material and methods

2.1. Plant material

Oxalis pes-caprae L. (syn. Oxalis cernua Thunb.) plants were collected outside, as this is an invasive plant and its collection in the wild in Israel is legal. Oxalis megalorrhiza Jacq. and Oxalis articulata Savigny were obtained at Tel Aviv University botanical garden. Oxalis ‘Otale’ (a patented registered cultivar, number 2439) was obtained from commercial nursery (Yoash–Breeding & Propagation of Ornamental Plants Ltd, Yodfat, Israel). All the plants were grown outdoors in Rishon LeZion, Israel (31° 57′ N, 34° 48′ E) during autumn–winter (their common growth season) in pots filled with gardening mixture. Oxalis bowiei Aiton e×G. Don and Oxalis versicolor L. leaves were collected at a local nursery. Only fully expanded leaves were used in the measurements and microscopic observations.

2.2. Anatomy

For histology, 0.5 mm petiole sections were excised and fixed in FAA (5 : 5 : 90, formalin: acetic acid: 70% ethanol), dehydrated in a graded ethanol series and embedded in wax [23]. Cross sections were cut at 10 µm thickness using a rotary microtome (Leica, Germany) and stained for general observation with either toluidine blue O [24] or alcian green-safranin [25] or ruthenium red for pectins [26]. In addition, hand sections were made using disposable razor blades. The sections were viewed and photographed under a stereo microscope (Olympus SZ2-ILST) equipped with a camera (Olympus LC20).

2.3. Mechanical measurements

Prior to testing, potted plants were transferred to the laboratory and leaves were excised before measurement. To prevent loss of turgor, leaves were tested within 3 min of excision. To identify the failure characteristics of Oxalis leaves (force-to-failure, strength and toughness), tensile tests were performed on two types of samples: intact petioles (with the leaf base) and truncated petioles (with the leaf base removed). The tensile testing was performed using a universal testing machine (Instron 5965) [27] with a 10 N load cell. The petiole ends were strengthened with adhesive gaffer tape in order to increase friction and prevent slipping. Prior to testing, sample length and localized thickness (both at the mid-petiole and in the expected location of abscission at the pulvinus) were measured. For each experiment, a gradual displacement was applied to the sample edges and the reaction force was recorded; the experiment ended when the sample was completely fractured—indicated by a drop of the reaction force to zero.

2.4. Scanning electron microscopy

In order to observe the fractured surface after a tensile experiment was completed, the region of fracture was excised from both intact petioles and truncated petioles and fixed using the methanol method as described by Talbot & White [28]. Afterwards, the samples were critical point-dried in a Critical Point Dryer (CPD-030, Bal-Tec/Leica) and gold coated in a Gold Sputter Coating Unit (Quorum Technologies/Polaron, UK). The samples were observed by low-vacuum scanning electron microscopy (SEM; JSM 5410 LV, Jeol Ltd., Japan).

2.5. Statistical analysis

The statistical analysis of the strength parameter was performed by a customary transformation into Weibull coordinates and performing a linear regression (Matlab R2016A) with R² = 0.9327 for the intact petioles and R² = 0.9825 for the truncated petioles.

As the data for the force-to-fracture were not linearly distributed, Wilcoxon/Kruskal–Wallis nonparametric test (JMP Pro 11 Statistical Software; SAS Institute Inc., Cary, NC, USA) was used to determine differences between treatments.

3. Results

3.1. Structural and material characterization of Oxalis pes-caprae petioles

Oxalis pes-caprae is a bulbous perennial plant. The subterranean stolons carry small bulbs that serve for vegetative reproduction and dispersal. The leaves and flowers grow at the apices of the stolons that poke out of the ground to form dense clumps. The leaves grow in rosettes. Each leaf of the Oxalis pes-caprae is constructed of a leaf base (pulvinus), a long tapering petiole (leaf stalk) and lamina (the broad upper part) (figure 1a–c). Flowers are constructed the same way, but the flower parts are carried at the top of the long stalk instead of the leaf lamina. When pulled by hand, O. pes-caprae leaves and flowers always fracture easily at the pulvinus. Anatomical examination of the pulvinus reveals an abscission zone with small cells (figure 1d). The abscission zone is not active at this stage, and it already appears even in the petioles of very young leaves (Y. Efrat 2009, personal communication). In the inner part of the ‘knee’ of the pulvinus, there is a clearly visible notch (figure 1c,d). When a leaf is pulled, the ‘knee’ straightens and the stress is concentrated at the notch. The notch itself appears suberized (figure 1e). The cell walls at the abscission zone and the vascular bundles are pectin rich (figure 1f). Suberization of the notch area was confirmed by Sudan IV stain (not shown). Interestingly, Charles Darwin mentions pulvini of Oxalis species in the context of leaf movement [29] in his camera lucida image of Oxalis sensitiva pulvinus, where a knee-like form with a notch can be clearly seen.

Figure 1.

Oxalis pes-caprae leaf structure. (a) The whole plant, (b) a leaf, (c) a pulvinus, (d) longitudinal hand section of the pulvinus, toluidine blue O stain, (e) longitudinal section, alcian green safranin stain, (f) longitudinal section, ruthenium red stain for pectins. Arrows indicate the notch in the pulvinus. Note the asymmetric pulvinus structure and the triangular leaf base. P: pulvinus; MP: mid-petiole; AZ: abscission zone; N: notch. (Online version in colour.)

The fractured surfaces at the pulvinus or at the mid-petiole were examined by SEM (figure 2). As can be seen, the pulvinus fracture surface is very smooth, with a clean break of the xylem vessels and the parenchyma cells (figure 2e,f). On the other hand, the mid-petiole surface of a fracture is not smooth and includes distorted xylem vessels and stretched parenchyma (figure 2b,c). Note that in the pulvinus, the vascular bundles are united into one central bundle (figure 2g), while in the mid-petiole they are separated into four separate bundles (figure 2d).

Figure 2.

The fractured surfaces of Oxalis pes-caprae leaves. (a) A whole leaf, (b,c,e,f) SEM images of fractured surfaces, (d,g) anatomical structure of unfractured of the petiole. (b,c,d) Mid-petiole region, (e,f,g) pulvinus (leaf base). Note the disrupted epidermis and the distorted vascular bundles in mid-petiole region fracture (b,c) versus the clean fracture surface of the pulvinus (e,f). P: pulvinus (leaf base); MP: mid-petiole; V: vascular bundle. (Online version in colour.)

3.2. Mechanical characteristics of tensile failure in Oxalis pes-caprae petioles

Tensile experiments were conducted to identify the mechanical characteristics of intact petioles (with the leaf base) as compared to truncated petioles (with the leaf base removed). For both sample types, the force–displacement curves showed a monotonic increase until a maximal force is reached, after which both sample types were suddenly, and completely, fractured (figure 3; electronic supplementary material, figure S1). Both the instantaneous mechanical failure of the Oxalis and the formation of relatively smooth fracture surfaces (figure 2) correspond to failure characteristics of brittle engineering fibre elements [30]. For such elements, local fibre narrowing, surface imperfections (e.g. cracks) and reduction in the material fracture resistance are the customary parameters that reduce the fibre load-bearing capabilities.

Figure 3.

Selected force–displacement curves from tensile experiments on Oxalis pes-caprae leaf petioles. Intact petioles (red) and truncated petioles, with leaf base (pulvinus) removed (blue). (Online version in colour.)

The force-to-fracture (F_max) is clearly the prime functional parameter that promotes the Oxalis autotomy. Following the mechanical testing results, the force-to-fracture for the intact petioles (F_max = 3.17 ± 0.96 N) is significantly lower than the maximal force for truncated petioles (F_max = 4.66 ± 1.3 N) (p ≤ 0.0016; figure 4). Moreover, in the intact petioles, fracture occurred repeatedly in the pulvinus, while in the truncated petioles the fracture appeared at random locations along the mid-petiole region. These specialized failure characteristics of the Oxalis leaves, i.e. low F_max and persistent-localized fracture, are apparently related to biomechanical adaptations of the pulvinus region compared to the rest of the petiole. To isolate the key mechanism that promotes the Oxalis autotomy, the following failure-driving modifications of the pulvinus were analysed: (i) area narrowing at the pulvinus compared to the mid-petiole, which gives rise to the localized force per area and thus reduces the overall force-to-fracture, (ii) sharp-notch geometry that promotes local stress intensifications and (iii) tissue morphology adaptations at the pulvinus that reduce the fracture resistance capabilities.

Figure 4.

The force-to-fracture of Oxalis pes-caprea leaves at the pulvinus and the mid-petiole region. Each column represents the mean (N = 13) + s.e. Significant difference according to Wilcoxon/Kruskal–Wallis test p < 0.0056.

Initially, to investigate whether the area narrowing at the pulvinus leads to its reduced force-to-fracture, the strength of the intact and truncated petioles was calculated by dividing the maximal force by the corresponding fractured area (σ_max = F_max/A_frac). The strength parameter indicates the net force-to-fracture, applied per unit area, and thus eliminates area difference effects between the pulvinus and the remaining petiole. Figure 5 shows the cumulative distribution function, fitted to the set of experimental strength parameters of the intact and truncated petioles. As can be seen, the statistical strength characteristics of the intact petioles follow Weibull's function, $F({\sigma }_{\max })=1-\exp [-{({\sigma }_{\max }/A)}^B]$, with a scale parameter A = 2.73 MPa and a shape parameter B = 2.71. Such a statistical behaviour is tightly related to the weakest-link concept, which is commonly employed to model the failure of engineering fibre elements [30]. Briefly, the fibre is viewed as a chain of links and its strength is dictated by the weakest link in the chain. The strength of an individual link is described via a statistical function which refers to the inherent emergence of stochastic defects in a material increment. Then, the Weibull's function for the fibre strength is obtained analytically by compiling all the failure possibilities of the links in the chain. For the truncated petioles, without the pulvini, the strength statistics follow Weibull's function—but with a scale parameter A = 3.88 MPa and a shape parameter B = 3.30. The difference in scale parameter between the intact and truncated petioles, i.e. with and without the pulvini, corresponds to the difference in their mean strength characteristics. Evidently, the ratio between the mean strength of the intact and truncated petioles is closely related to their corresponding force-to-fracture ratio (less than 5% difference). This indicates that pulvinus narrowing is not the dominating parameter for its reduced force-to-fracture, and thus it plays only a minor role in the Oxalis autotomy functionality.

Figure 5.

Statistical analysis of the strength in Oxalis pes-caprae petioles. Cumulative distribution function of the experimental results for intact petioles (red circles) and truncated petioles, with pulvinus removed (blue circles). Dashed lines represent the corresponding Weibull's function for each set of 13 independent measurements. (Online version in colour.)

Next, we identified whether the pulvinus reduced force-to-fracture is attributed to the sharp-notch geometry (stress intensification effects), or to its locally modified tissue morphology (fracture resistance reduction), or both. For this analysis, the toughness of the intact and truncated petioles was extracted from the mechanical testing results—calculated as the area under the force–displacement curve (figure 3) divided by twice the fractured area [31] ($R=\int F\cdot \mathrm{d}x/(2A_{\mathrm{frac}})$). To avoid diving into the fracture mechanics theory, it is only noted that the strength parameter—discussed above—integrates both stress intensification effects induced by the geometrical changes in the fracture region (e.g. the pulvinus notch shape) and the fracture resistance capabilities of the tissue itself. The toughness parameter, on the other hand, characterizes only the fracture resistance capabilities of the tissue, and it is analogous to its surface energy. Interestingly, the toughness of the intact petioles (R = 3.9 ± 2.4 mJ mm⁻²) was very similar to that of truncated petioles (R = 4.0 ± 1.7 mJ mm⁻²)—which indicates that the abscission zone of the pulvinus possesses equivalent fracture resistance capabilities to those of the mid-petiole region. By combining this outcome with the substantial differences in strength parameter introduced above, it is concluded that the reduced force-to-fracture of the pulvinus originates from local stress intensification effects induced by its notched shape. Therefore, the pulvinus geometry is the key characteristic that provides the Oxalis leaves their autotomy functionality.

3.3. Comparison of several Oxalis species

The leaf fracture in additional Oxalis species that were available to us was also studied (table 1 and figure 6; electronic supplementary material, figure S2). In O. megalorrhiza and O. bowiei, the leaves detach at the leaf base (pulvinus), as in O. pes-caprae. Moreover, O. megalorrhiza leaves were extremely sensitive to shaking and often detached on the way to the laboratory. In O. articulata, O. versicolor and Oxalis ‘Otale’ the leaves do not detach at the base, but rather arbitrarily in the mid-petiole. In Oxalis ‘Otale’, a gardening cultivar, the size of the leaf and the ease of obtaining numerous samples allowed it to be used as a comparative species.

Table 1.

Leaf fracture type in Oxalis species.

autotomy (detaches at pulvinus)	not autotomy (detaches at an arbitrary point along the petiole)
Oxalis pes-caprae	Oxalis corniculata
Oxalis megalorrhiza	Oxalis ‘Otale’
Oxalis bowiei	Oxalis versicolor

Figure 6.

Leaf fracture in four Oxalis sp. (a) Tearing distance from the abscission zone in the pulvinus (leaf base), (b) leaf length.

Examination of the leaf base structure of O. pes-caprae, O. megalorrhiza, O. bowiei and Oxalis ‘Otale’ (figure 7) shows that the detaching leaves have a pulvinus with a notch. In O. pes-caprae, the pulvinus has a bent shape, and there is a single notch in the pulvinus. In O. megalorrhiza and O. bowiei, the pulvinus does not have a clearly bent structure. In O. bowiei, there is a double notch at each side of the pulvinus, and in O. megalorrhiza, there are two notches at each side of the pulvinus. In comparison, the non-detaching leaves of Oxalis ‘Otale’ have a straight structure without a notch.

Figure 7.

Leaf base in four Oxalis species with varying morphologies. Oxalis pes-caprae (a,e), Oxalis bowiei (b,f), Oxalis megalorrhiza (c,g) and Oxalis ‘Otale’ (d,h). (a–d) General appearance, (e–h) Longitudinal hand sections, toluidine blue O stain. Arrows indicate a notch. Note the bent leaf base in Oxalis pes-caprae. Scale bars, 1 mm. AZ: abscission zone; V: vascular bundle. (Online version in colour.)

4. Discussion

Autotomy in animals has been reported in dozens of research papers, for numerous and unrelated species for over more than a hundred years. The cases reported have several common characteristics: (i) the fracture occurs at a pre-defined breakage plane, (ii) the break is clean and heals easily, (iii) usually, the lost part is regenerated later, (iv) often autotomy requires the application of external force, (v) it usually happens quickly [2,3,10,32–35]. In all these cases, autotomy is considered a defensive behaviour of sacrificing an organ in order to save the whole organism. Indeed, it appears that the phenomenon of leaf detachment in O. pes-caprae fits the above description. This is the first time autotomy is reported in plants.

In Oxalis, the autotomy occurs quickly and upon tensile stress. Large herbivores (cows and sheep) harvest the leaves by tension [15]. Sheep are known to ingest O. pes-caprae in large quantities, sufficient to get poisoning [36,37]. Thus, autotomy-like mechanism could serve a protective function minimizing the effect of herbivory by preventing the uprooting of the eaten plant. It is important to mention that leaves do not regenerate from the broken area, but rather grow anew from the meristem. The advantage of having these break-points is that the branch is not pulled or broken so the main apical meristem remains intact and continues producing new leaves [38]. Moreover, the clean cuts at the breaking points minimize water loss and seal the wounded tissue as only one layer of cells is damaged.

Shedding body parts is not rare in the plant world. Abscission layers develop precisely for this purpose [38]. However, the parts shed by abscission are either no longer viable (dry leaves) or shed on purpose, in order to disperse the progeny (fruits). In the case of Oxalis, the detached organs are viable and the autotomy preserves the meristem that will grow other leaves in their stead later. Another common type of losing body parts in plants occurs upon breaking of branches at the branch–stem junction [39–41]. However, branch breaking occurs upon bending loading (rather than tensile alone) which involves effects of both tension at the upper side and compression at the lower side of the junction. In such a case, crack propagation which is often gradual occurs only at the tensile-loaded upper side of the junction [39,42], as opposed to the sudden leaf detachment in Oxalis. From a bio-mechanical aspect, leaf fracture in Oxalis is facilitated by an amplification of the far-field stress in the vicinity of local, but abrupt, geometrical modification in the form of a notch. On the other hand, in the absence of plasticity (as in the present case), such a fracture process is resisted by the material toughness. The comparative mechanical analysis of the intact and truncated petioles indicates that while the toughness of the pulvinus and of the mid-petiole are approximately the same, both the force-to-fracture and the strength that intact petioles can bear are substantially lower compared to the truncated ones. These findings indicate that the notched-like geometrical modification of the pulvinus is the prime characteristic responsible for the autotomy phenomenon in Oxalis.

Morphological shape of the pulvinus (curved and notched) leads to localized amplifications in the stress field which does not happen in the rest of the petiole. Thus, the notched structure plays a central role in leaf detachment. Indeed, while other two Oxalis species exhibiting autotomy possess different pulvinus morphologies, both species have a notched pulvinus; species without such autotomy does not have a notched pulvinus (figure 7).

This plant autotomy differs from that which occurs in a number of animals by not including any active breaking of the sacrificed organ. Among animals, for which the term autotomy was developed, a clear characteristic is that there is an active role the animal usually plays to decide whether to autotomize, and then to subsequently initiate autotomy [6]. Plants, of course, lack such capabilities, as they do not possess a central nervous system and employ different decision mechanisms. In all other aspects, the structure–function phenomena described here serve the plant the same way that ‘classical’ autotomy serves the animals.

In all probability, Oxalis species presented here are not the sole representatives of the plant world that exhibit autotomy. Flower petals of several plant species detach at their base upon tensile force. Sacrifice of petals to protect the reproductive organs could possibly be another example of autotomy. One such remarkable example is that of Verbascum sinuatum petals that detach a few seconds after wounding. It would be extremely interesting to pursue this subject further.

Data accessibility

All the data are available in the paper.

Authors' contributions

I.S.—planning and performing the experiments, data analysis, writing, A.K.—performed biomechanical experiments, A.E.—concept, planning the experiments, data analysis, B.B.O.—data analysis, writing.

Competing Interests

We declare we have no competing interests.

Funding

This research was partially supported by the Israel Science Foundation (grant no. 1429/16).