Mechanical and phytochemical protection mechanisms of Calligonum comosum in arid deserts

Abstract

Unlike animals, plants are sessile organisms, lacking circulating antibodies and specialized immune cells and are exposed to various harsh environmental conditions that make them at risk of being attacked by different pathogens and herbivores. Plants produce chemo-signals to respond to the surroundings and be able to distinguish between harmless and harmful signals. In this study, the production of phytochemicals as plant signaling mechanisms and their defensive roles in disease resistance and repelling herbivores are examined in Calligonum comosum. C. comosum is a leafless standalone perennial shrub widespread in sand dunes. The plant has the ability to survive the drastic environmental conditions of the arid/ hyperarid deserts of the Arabia. Structural anatomy and phytochemicals analyses were used to identify both mechanical and chemical defensive mechanisms in C. comosum. Microscopy-based investigations indicated that stems of this species developed hard structures in its outer layers including sclerenchyma and cluster crystals of calcium oxalate (CaOx). Sclerenchyma and CaOx are difficult to be eaten by herbivores and insects and can harm their mouthparts. On the other hand, the plant developed both short-distance (local) and long-distance (systematic over limited sphere) phytochemicals-producing cells located at its outer regions that is surrounding the inner nutrient-rich vascular system (VS). Local chemical was represented by phenolic idioblasts that were released in response to plant cutting. Systematic chemical was represented by toxic volatile oil containing ~50% benzaldehyde derivative (cuminaldehyde). The oil caused strong killing effect on both mammalian cells and microbial pathogens via either direct addition or indirect exposure to its vapor. The plants lost the oil content and allowed fungal growth once cut and dried. The localization of both defensive mechanisms to the outer region of the plant seemed to protect the inner nutrient-rich VS and hence maintained the plant survival. Surprisingly, in relation to traditional folklore use as medicine, local people use only green parts of the plant and only during the winter, where the plant found devoid of volatile oil and phenolic idioblasts. Moreover, it turns into recommendations for local people to avoid any health problems caused by the plant supply.

Fig 1

Calligonum comosum plant grows in the arid desert of UAE at (A and B) winter time and (C and D) fall time [33].

Fig 2. Transverse sections in C. comosum plant.

(A) Diagrammatic over view of the plant section. (B) Plant section under light microscope showed all plant layers. (C and D) Close up view of the plant section under light microscope stained with neutral red and iodine solution, respectively. Cs; Cluster crystals of CaOx, MR; Medullary parenchyma rays, Pi; Phenolic idioblasts, Sc; Sclerenchyma and VO; Volatile oil.

Fig 3. Anatomical structures of C. comosum supporting the plant hardening characteristics.

(A) Transverse section of the plant indicated the location of sclerenchyma cells. (B) Close up view of the plant section indicating the sclerenchyma cells. (C) and (D) Close up view of plant sections at the vascular system showing the existence of (C) unstained and (D) iodine solution-stained starch granules. (E) and (F) Close up view of plant sections showing the presence of clusters crystals of calcium oxalate (CaOx) in (E) the outer region or in (F) the plant powder. Cs; Cluster crystals of CaOx, and Sc; Sclerenchyma.

Fig 4

Transverse sections of C. comosum showing the presence of phenolic idioblasts either (A-C) unstained (red) or (D) ferric chloride solution-stained (black). Pi; Phenolic idioblasts.

Fig 5. Transverse section of C. comosum plant stem showing the location of volatile oil-containing cells group at the outer surface.

(A) Location of volatile oil cells. (C-D) close up view of volatile oil-containing cells. VO; Volatile oil.

Fig 6. Mechanism of release of phenolics and volatile oils from old stem pieces.

(A) C. comosum stem sections showed browning of outer layer when cut and left for 5 hrs. (B) Dried plant pieces left in water showing the release of phenolics outside the plant pieces and the growth of fungus. (C) Plant sections showed the release of oil once heated at 50°C. VO; Volatile oil.

Fig 7. In vitro direct killing activity of plant volatile oil compared to different plant extracts.

(A) MTT mammalian cell damage assay showing the effect of different plant extracts and plant volatile oil on fibroblasts cells compared to doxorubicin as positive control. As shown water extract showed slight damage to mammalian cells (due to phenolics), however oil showed significant damaging effect. (B) Antimicrobial activity of plant extracts in comparison to volatile oil and antibiotics on Gram positive E. coli, Gram negative Staphylococcus aureus, Candida yeasts and spore-forming Asperigillus fungus using micro-dilution assay. Colistin, vancomicin, ketoconazole and amphotricin B were used as positive controls against E. coli, Staph. aureus, Candida and Aspergillus at 3, 3, 1 and 5 μg/ mL, respectively. Each experiment was repeated six times.

Fig 8. In vitro indirect activity of plant volatile oil against both mammalian cells and microbes by exposure to oil vapor.

(A and B) Mammalian fibroblasts and U87 brain cells exposed to oil vapor for 24 hrs. (A) Zone of inhibition of mammalian cells when exposed to oil vapor. (B) Close up view of both cells under light microscope compared to control cells received only solvent buffer. Scale bar = 100 μm. (C) E. coli and Staph. aureus Exposed to oil vapor for 24 hrs.

Fig 9. GC chromatogram of C. comosum oil showing major peaks representing ~50% cuminaldehyde at 11.9 min, ~11% carene-10-al at 12.9 min and ~10% curcumene at 17.7 min.

Fig 10

Transverse section of C. comosum (A) green branches collected in March and its (B) close up view under light microscope.