Crassulas under the microscope. Leaf anatomy, water storage and CAM photosynthesis

Four species in one comparison

Lisa Anne Jones compared the aquatic species Crassula helmsii, Crassula multicava from shaded habitats, and two crassulas associated with semi-arid regions of southern Africa, Crassula ovata and Crassula socialis. This selection made it possible to compare organ organization in closely related plants occurring under different water conditions. The familiar cultivated jade plant was examined alongside plants differing in growth form, leaf size and spatial arrangement. Membership of one genus provided common ground, while specific tissue traits formed the basis of comparison. [Jones 2011]

The material comprised 48 cuttings taken from four stock plants. They were maintained for approximately five months in a greenhouse, in a mixture of perlite and compost. The medium supporting Crassula helmsii was kept waterlogged. Leaf and stem sections were then prepared, tissues were stained and surface structures were counted. Leaf water content was measured separately. Replication described variation within the greenhouse material examined. The origin of the cuttings from a small number of stock plants also defines the scale at which the findings describe variation within entire species. [Jones 2011]

Two measures of succulence

The most intuitive question concerns the quantity of water held per unit leaf area. The author weighed fresh leaves, dried them and calculated the mass of water lost. This was related to the measured blade area. On this basis, Crassula ovata ranked first, at approximately 3 g of water per dm². The corresponding values were approximately 1.5 g for Crassula multicava, 1 g for Crassula socialis and 0.1 g per dm² for Crassula helmsii. These are approximate readings from the graph for plants under the experimental conditions. They illustrate the difference between a thick leaf holding substantial reserves and the more delicate organ of an aquatic species. [Jones 2011]

A second question concerns water reserves relative to the quantity of organic matter used to construct the leaf. After drying, the samples were ashed to separate that fraction from the mineral residue. On this basis, Crassula socialis had the highest mean, followed by Crassula ovata, Crassula multicava and Crassula helmsii. The difference between the first two species was statistically inconclusive. The change in ranking follows from the choice of denominator. A large reservoir per unit area can accompany a relatively substantial mass of dry tissue. Another leaf holds less water per unit area but more relative to its organic matter. Both indices describe useful, distinct aspects of succulence. [Jones 2011]

The leaf surface as a protective system

All four species had a single-layered leaf epidermis. Its outer surface carried a cuticle, a layer restricting water movement. The thickness of this covering differed among species and between the two sides of the blade. The thinnest upper-surface cuticle occurred in Crassula helmsii. The thickest belonged to Crassula multicava, at nearly twice the thickness found in the two crassulas from semi-arid habitats. This result illustrates the multiple dimensions of adaptation. Origin in a drier environment and the thickness of a single protective layer can form different combinations, depending on other leaf traits. [Jones 2011]

Jones also considered protection against fungi as a possible function of the thick cuticle in the species from more humid habitats. This is an interpretation of the observed structure based on general knowledge of cuticles. The experiment itself documented its thickness and distribution. In Crassula socialis, hairs along the leaf margins provided an additional surface feature. It was the only species with such hairs in the sampled group. Anatomy therefore reveals different combinations of protective elements that can subsequently be investigated in relation to actual water loss, exposure to light and interactions with microorganisms. [Jones 2011]

Stomata and hydathodes serve different roles

Stomata occurred on both leaf surfaces in all four species. This arrangement is termed amphistomatic. The stomata lay level with the epidermis. The highest density occurred in aquatic Crassula helmsii. The number per unit area describes potential gas-exchange pathways, whereas the flow at a particular moment also depends on pore opening. The review by Males and Griffiths shows how closely stomatal behaviour in CAM plants is linked to time of day, metabolism and water availability. Interpreting a micrograph therefore involves distinguishing a structural trait from the current functional state. [Jones 2011] [Males and Griffiths 2017]

Hydathodes were larger pores associated with endings of the vascular system. In the study they occurred much less frequently than stomata. Their density on the upper surface was highest in Crassula ovata. In Crassula helmsii, they were identified only on the lower surface, whereas the other species had them on both sides. Jones discussed the possible involvement of these structures in taking up water deposited on leaves, drawing on earlier work. Her own results provided a map of hydathode distribution and structure. The actual water flux through these structures requires a separate physiological measurement. [Jones 2011]

The stem supports and protects

Stems of Crassula ovata showed secondary thickening and a periderm, a secondary covering tissue. Its cork layer contains suberin, which helps restrict water movement through cell walls. The developed stem contributes to the shrubby habit and supports heavy leaves. Secondary thickening also occurred in Crassula multicava and Crassula socialis. In aquatic Crassula helmsii, the sampled shoots had a different organization, with air spaces in their tissues. The arrangement of these spaces is relevant to gas distribution within submerged organs. [Jones 2011]

Chloroplasts occurred in stem tissues of three species, including beneath the periderm of Crassula ovata. They were absent from the sampled stems of Crassula socialis. Jones related this difference to its compact habit and the shielding of shoots by leaves. Chloroplast presence identifies cells equipped with photosynthetic machinery. The contribution of the entire stem to the plant’s carbon balance is established through measurements of gas exchange and metabolism. Microscopy helps identify where to investigate these processes and how differently organized organs may participate in plant function. [Jones 2011]

Stained sections also revealed tannin-containing cells and amyloplasts, plastids associated with starch storage. These structures occurred in the leaves and stems of all four species. In the jade plant, tannin-containing cells were particularly concentrated at leaf margins and around hydathodes. Jones considered their potential contribution to protection against intense radiation. The presence of starch also shows that tissues store products of carbon metabolism. A leaf section therefore reveals several kinds of reserves and protective structures together. Their distribution helps explain organ organization, while their quantities and changes through the day require measurements appropriate to each compound. [Jones 2011]

Daily metabolism uses cellular storage capacity

CAM, or crassulacean acid metabolism, describes the temporal separation of stages in carbon fixation. In its classical expression, phosphoenolpyruvate carboxylase participates in initial carbon fixation at night. The resulting organic acids, predominantly malic acid, accumulate in mesophyll-cell vacuoles. During the day, they are decarboxylated and the released CO₂ becomes available to Rubisco. Large cellular storage capacity is therefore relevant to the organization of the carbon-compound reservoir. Water stored in a leaf and its nocturnal acid pool are connected with cell structure, but each reserve requires its own appropriate measurement. [Males and Griffiths 2017]

Males and Griffiths distinguish four classical phases of gas exchange. Nocturnal carbon uptake is followed by a shorter morning period, daytime use of internal reserves and, under favourable conditions, renewed stomatal opening in late afternoon. The expression of each phase changes with the environment. When water becomes restricted, recycling CO₂ produced by respiration can become more important. Under extremely restricted exchange with the surroundings, the plant uses an internal carbon cycle. This describes different modes of CAM presented in the physiological review. Their contribution in a particular crassula is established by measuring daily patterns of gas exchange and acidity. [Males and Griffiths 2017]

From a leaf section to the whole plant

The central result of the four-crassula comparison is the coexistence of different combinations of traits. Crassula ovata combined high water storage per unit leaf area with a developed supporting stem. Crassula multicava stood out for its thick cuticle. Crassula socialis had marginal hairs and a high mean water content relative to organic matter. Crassula helmsii represented the different organization of an aquatic plant. Each result becomes clearer when the organ, measurement method and growing conditions are specified. This approach connects a crassula’s appearance with its functioning while preserving the distinction between anatomical and physiological evidence. [Jones 2011] [Males and Griffiths 2017]