Euphorbia tirucalli during drought. C₃ leaves, CAM stems and cellular protection

Two organs with different rhythms

A green branch of Euphorbia tirucalli remains a photosynthetic organ even after its small leaves are lost. The succulent stems of this spurge use CAM metabolism, whereas its nonsucculent leaves are described as C₃ photosynthetic organs. This division makes it possible to investigate two arrangements of carbon metabolism within a single plant. The comparison is particularly informative during drought, when water availability, growth and the operating conditions of the photosynthetic apparatus change together. A leaf can consequently respond differently from the adjacent stem [Hastilestari et al. 2013] [Abuelsoud and Papenbrock 2019].

In C₃ photosynthesis, absorbed carbon dioxide enters a cycle of reactions that produces organic compounds. In a typical CAM plant, initial fixation of atmospheric CO₂ occurs mainly at night, while malate, a form of malic acid, accumulates in the tissues. During the day, CO₂ released from malate supplies photosynthesis while stomatal opening is restricted. These microscopic pores in the epidermis connect gas exchange with the loss of water vapour. Separating the stages in time can therefore reduce the water cost of carbon acquisition [Hastilestari et al. 2013].

Water storage and the timing of photosynthesis operate alongside other plant traits. The consequences of dehydration also depend on how growth is allocated between roots and shoots and how living cells maintain their functions. The general role of stored water is discussed in the article on water-storage tissues in succulents. E. tirucalli illustrates an additional dimension particularly clearly. Material belonging to the same species can respond differently to the same water shortage, and different indicators of tolerance need not favour the same genotype [Hastilestari et al. 2013] [Abuelsoud and Papenbrock 2019].

Eight weeks under controlled water availability

In 2013, Hastilestari and colleagues compared material labelled Morocco and Senegal. These names identify two genotypes maintained in an experimental collection. Plants were propagated from cuttings, allowing responses to be compared among individuals of the same material. The experiment began with six-month-old plants and continued for eight weeks. Four levels of volumetric water content in the substrate, abbreviated VWC, were maintained at 25%, 15%, 10% and 5%. Growth results represented five plants in each combination of genotype and water treatment [Hastilestari et al. 2013].

Substrate moisture was monitored, and the water needed to maintain each target level was replenished. Plants were kept in a chamber at 24°C during the day and 20°C at night, with a fourteen-hour light period. The driest treatment therefore represented a defined water availability in a particular substrate and container. It did not represent eight weeks without any watering. VWC expresses water volume as a proportion of substrate volume, and its significance for roots also depends on the properties of that substrate [Hastilestari et al. 2013].

Both genotypes continued to increase in height even at 5% VWC, although their growth was smaller than in the control. After eight weeks, Morocco plants in that treatment reached about 40 cm and Senegal plants about 33 cm. At 25% VWC, both groups reached approximately 54–55 cm. Senegal nevertheless produced more shoot dry mass under both adequate watering and the lowest VWC. Drought was accompanied by a longer taproot and a higher root-to-shoot mass ratio. The response therefore involved both the rate of growth and its allocation within the plant [Hastilestari et al. 2013].

What malate and fluorescence reveal

The researchers measured malate at the end of the dark period and at the end of the day. Stems of both genotypes already showed day–night variation before drought, consistent with CAM. This rhythm was not apparent in the leaves of well-watered plants. After water was restricted to 10% VWC, leaves of both genotypes contained more malate after the night than after the day. At 5% VWC, a significant difference between these times was reported only for Senegal. The response therefore depended on genotype and the severity of water limitation [Hastilestari et al. 2013].

The authors interpreted the leaf response as evidence consistent with possible CAM induction. Malate accumulation alone, however, leaves the source of the carbon being fixed unresolved. The experiment did not measure the daily course of atmospheric CO₂ uptake by leaves. The discussion also considered possible transport of malate from stems, and linked confirmation of the mechanism to further enzyme and gas-exchange studies. The most firmly supported conclusion therefore concerns a change in organic-acid metabolism. A complete leaf switch from C₃ to CAM remains a hypothesis requiring additional measurements [Hastilestari et al. 2013].

Chlorophyll fluorescence was measured separately to calculate Fv/Fm, an indicator of the maximum photochemical efficiency of photosystem II. This photosystem participates in the early stages of using light energy. During prolonged water limitation, Fv/Fm declined in leaves and stems, and the two genotypes responded differently. Such a measurement describes the condition of the photochemical apparatus. It does not directly quantify CO₂ uptake, the overall rate of photosynthesis or the abundance of reactive oxygen species. It connects water stress with photosystem function, while other methods are needed to investigate the causes of the changes [Hastilestari et al. 2013].

From water shortage to oxidative processes

Reactive oxygen species arise during metabolism and can participate in both signalling and damage to cellular components. Hydrogen peroxide, H₂O₂, belongs to this group. Its consequences depend on the cellular context and the balance between production and removal. In 2019, Abuelsoud and Papenbrock investigated how components of the antioxidant system responded to water shortage in leaves and stems of Morocco and Senegal. This system includes enzymes and small compounds involved in limiting oxidative reactions [Abuelsoud and Papenbrock 2019].

The experiment again lasted eight weeks, but compared only a control at 25% VWC with drought at 10% VWC. Six-month-old plants grew at 22°C in a cycle of twelve hours of light and twelve hours of darkness. Each genotype–treatment group contained 30 cuttings, while individual biochemical assays used at least three biological replicates. Leaves and stems were collected and analysed separately. This prevented a whole-plant result from obscuring different responses in the two photosynthetic organs [Abuelsoud and Papenbrock 2019].

H₂O₂ was measured chemically in tissue extracts. A separate assay estimated malondialdehyde, MDA, through its reaction with thiobarbituric acid. MDA served as an indicator of lipid peroxidation, the oxidation of lipids associated with membrane damage. This result must be distinguished from the activity of protective enzymes. High enzyme activity describes a component of the response, whereas the MDA signal approximates one consequence of oxidative processes. Combining these measurements gives a fuller picture than attributing all tolerance to a single compound [Abuelsoud and Papenbrock 2019].

Enzyme responses depend on the organ

Drought increased the activities of ascorbate peroxidase, APX, and the assayed peroxidase, POX, in Morocco stems. These enzymes contribute to hydrogen peroxide removal through reactions involving appropriate electron donors. In Senegal stems, catalase activity, CAT, increased clearly, providing another route for H₂O₂ breakdown. A similar biochemical function was therefore associated with a different pattern of enzyme responses. The results concerned activity measured in extracts and expressed relative to protein content. The study did not directly measure expression of the genes encoding these enzymes [Abuelsoud and Papenbrock 2019].

Activities of glutathione reductase and dehydroascorbate reductase also increased in the stems of both genotypes. These enzymes help regenerate components of the ascorbate–glutathione system, which connects hydrogen peroxide removal with antioxidant turnover. The amount of reduced glutathione, GSH, increased as well. Leaves followed a different pattern. Several enzyme activities declined, while GSH increased in Morocco and decreased in Senegal. The picture of antioxidant defence therefore depends on which organ, compound and regeneration step are measured [Abuelsoud and Papenbrock 2019].

Proline, an amino acid associated with plant stress responses, also accumulated in stems. Its increase was more pronounced in Morocco. The authors nevertheless emphasized that the measured amount of proline could not explain the greater water retention of this material in their experiment. Other potentially relevant osmotic compounds remained unmeasured. This is an important interpretive limit. The simultaneous occurrence of more of a metabolite and a particular plant trait identifies a relationship worth investigating, but does not by itself quantify that metabolite’s contribution to water balance [Abuelsoud and Papenbrock 2019].

Why units and assay type matter

Dehydration changes the proportions of water and dry matter in a tissue. The amount of a compound per gram of dry mass therefore answers a different question from its amount relative to the water present in that tissue. In the 2019 study, stem H₂O₂ expressed per dry mass increased in Morocco and decreased in Senegal. After accounting for water loss, the authors described an increase in both genotypes. This adjustment helps identify the effect of concentration during dehydration, but still concerns the sampled tissue as a whole. It does not determine the concentration in an individual cellular compartment [Abuelsoud and Papenbrock 2019].

The scope of the ORAC assay is equally important. It assessed the ability of an extract to limit radical reactions by monitoring fluorescence from an added probe in a reaction mixture. This differs from chlorophyll fluorescence measured in a living plant. In results expressed per dry mass, ORAC increased in leaves but decreased in stems of both genotypes. After recalculation relative to tissue water, the authors described an increase in stems as well. An ORAC result cannot be equated with either the amount of H₂O₂ or evidence that cellular damage is absent [Abuelsoud and Papenbrock 2019].

MDA results likewise do not support a simple picture of equal damage across all organs. In the dry-mass-normalized figure, a small increase occurred in Morocco leaves, while Senegal leaves showed no such change. For stems, the overall two-factor analysis detected no significant treatment effect on MDA. An increase in one stress indicator can therefore coexist with little change in another. Biological interpretation should retain the organ studied, the unit of measurement and the statistical comparison applied [Abuelsoud and Papenbrock 2019].

Limits of the explanation and relevance to cultivation

The antioxidant study proposed that H₂O₂ accumulation in leaves might contribute to processes leading to their death and shedding during drought. Leaf loss reduces the transpiring surface, while green stems can continue to participate in photosynthesis. The experiment did not, however, test directly whether changing H₂O₂ initiates programmed cell death. It measured biochemical responses at the end of treatment, while the causal mechanism remained a proposed interpretation. Similarly, the labels Morocco and Senegal are insufficient to explain differences as adaptations of entire populations to the climates of particular countries [Abuelsoud and Papenbrock 2019].

For cultivation, the documented distinction between survival, growth and photosynthetic condition is relevant. In the 2013 experiment, plants survived eight weeks at the lowest VWC but grew less and showed lower Fv/Fm. Survival therefore does not define conditions that maintain good plant performance. Applying these moisture percentages to a container in Poland would require accounting for substrate, light, temperature and plant size. The studies support attention to the response of the individual plant, but establish neither a universal watering calendar nor a recommendation to maintain severe drought deliberately [Hastilestari et al. 2013] [Abuelsoud and Papenbrock 2019].

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