Zn2+-Dependent Nuclease Is Involved in Nuclear Degradation during the Programmed Cell Death of Secretory Cavity Formation in Citrus grandis ‘Tomentosa’ Fruits

Zn2+- and Ca2+-dependent nucleases exhibit activity toward dsDNA in the four classes of cation-dependent nucleases in plants. Programmed cell death (PCD) is involved in the degradation of cells during schizolysigenous secretory cavity formation in Citrus fruits. Recently, the Ca2+-dependent DNase CgCAN was proven to play a key role in nuclear DNA degradation during the PCD of secretory cavity formation in Citrus grandis ‘Tomentosa’ fruits. However, whether Zn2+-dependent nuclease plays a role in the PCD of secretory cells remains poorly understood. Here, we identified a Zn2+-dependent nuclease gene, CgENDO1, from Citrus grandis ‘Tomentosa’, the function of which was studied using Zn2+ ions cytochemical localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The full-length cDNA of CgENDO1 contains an open reading frame of 906 bp that encodes a protein 301 amino acids in length with a S1/P1-like functional domain. CgENDO1 degrades linear double-stranded DNA at acidic and neutral pH. CgENDO1 is mainly expressed in the late stage of nuclear degradation of secretory cells. Further spatiotemporal expression patterns of CgENDO1 showed that CgENDO1 is initially located on the endoplasmic reticulum and then moves into intracellular vesicles and nuclei. During the late stage of nuclear degradation, it was concentrated in the area of nuclear degradation involved in nuclear DNA degradation. Our results suggest that the Zn2+-dependent nuclease CgENDO1 plays a direct role in the late degradation stage of the nuclear DNA in the PCD of secretory cavity cells of Citrus grandis ‘Tomentosa’ fruits.

Ontogenesis, histochemistry, and seasonal and luminous environmental characterization of secretory cavities in leaves of Myrcia splendens (Myrtaceae)

Secretory cavities produce compounds that protect plants from herbivory and pathogenic microorganisms. These cavities have been reported in many genera. However, there are few studies on secretory cavity ontogeny in the genus Myrcia (Myrtaceae) as well as the effects of luminosity and seasonality on such secretory cavities. Therefore, the objective of this study was to provide new information regarding the ontogenesis, structure, histochemistry, and effects of seasonality in Myrcia splendens (Sw.) DC. We collected and analyzed leaves from 10 specimens of M. splendens exposed to sun and shade during both the wet and dry seasons. The samples were subjected to standard anatomical techniques for light microscopy. Myrcia splendens has schizo-lysigenous ontogenesis with exudates composed of lipids, essential oils, oil-resins, and alkaloids. The largest secretory cavities were found in leaves exposed to sunlight during the dry season. The presence of lipophilic compounds may be an important strategy for protecting the plant from herbivores. The seasonal variations observed in the leaf’s secretory cavities demonstrate the anatomical plasticity of such species to the availability of light and water. As the total area of the leaf cavities in M. splendens reflects the seasonal variation, this should be taken into account when aiming to grow crops for producing essential oils or for alkaloid extractions.

Ca2+-dependent nuclease is involved in DNA degradation during the formation of the secretory cavity by programmed cell death in fruit of Citrus grandis ‘Tomentosa’

The secretory cavity is a typical structure in Citrus fruit and is formed by schizolysigeny. Previous reports have indicated that programmed cell death (PCD) is involved in the degradation of secretory cavity cells in the fruit, and that the spatio-temporal location of calcium is closely related to nuclear DNA degradation in this process; however, the molecular mechanisms underlying this Ca2+ regulation remain largely unknown. Here, we identified CgCaN that encodes a Ca2+-dependent DNase in the fruit of Citrus grandis ‘Tomentosa’, the function of which was studied using calcium ion localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The results suggested that the full-length cDNA of CgCaN contains an ORF of 1011 bp that encodes a protein 336 amino acids in length with a SNase-like functional domain. CgCaN digests dsDNA at neutral pH in a Ca2+-dependent manner. In situ hybridization signals of CgCaN were particularly distributed in the secretory cavity cells. Ca2+ and Ca2+-dependent DNases were mainly observed in the condensed chromatin and in the nucleolus. In addition, spatio-temporal expression patterns of CgCaN and its protein coincided with the time-points that corresponded to chromatin degradation and nuclear rupture during the PCD in the development of the fruit secretory cavity. Taken together, our results suggest that Ca2+-dependent DNases play direct roles in nuclear DNA degradation during the PCD of secretory cavity cells during Citrus fruit development. Given the consistency of the expression patterns of genes regulated by calmodulin (CaM) and calcium-dependent protein kinases (CDPK) and the dynamics of calcium accumulation, we speculate that CaM and CDPK proteins might be involved in Ca2+ transport from the extracellular walls through the cytoplasm and into the nucleus to activate CgCaN for DNA degradation.

Anatomical structure, flavonoid content, and antioxidant activity of Rhodomyrtus tomentosa leaves and fruits on different age and maturity level

Kuntorini EM, Nugroho LH, Maryani, Nuringtyas TR. 2019. Anatomical structure, flavonoid content, and antioxidant activity of Rhodomyrtus tomentosa leaves and fruits on different age and maturity level. Biodiversitas 20: 3619-3625. Karamunting (Rhodomyrtus tomentosa (Aiton.) Hassk.) is a native plant to southeast Asian countries, and is a well-known medicinal plant used to treat colic diarrhea, wounds, heartburn, abscesses, gynecopathy, and as a pain killer. However, the use of R. tomentosa has not been optimized. This research aimed to observe the anatomical structure, the location, and distribution of flavonoid and to find out the antioxidant activity based on the leaves age and the fruit maturity. Anatomical slides preparation of leaves and fruits were made using the paraffin embedding method with safranin staining. The distribution of flavonoid was analyzed by histochemical test and antioxidant activity was done with DPPH (1,1-diphenyl-2-picrylhydrazyl) method. Leaf anatomical structure shows that the leaf bifacial (dorsiventral) consisted of upper epidermis, mesophyll (palisade and sponge), collateral vascular bundle, parenchyma midrib, abaxial epidermis, globular oil cavity, and non-glandular trichome. Transverse section of green fruit consists of exocarp (thin outer layer), mesocarp (thick inner layer, soft and runny) and endocarp (thin false septa) layers. The histochemical test showed that flavonoid was observed in the leaf epidermis, mesophyll, vascular bundles, secretory cavity, parenchyma and in all fruit types exocarp, mesocarp, endocarp. Antioxidant activity showed that the extract of the young leaves (IC50 = 14.67 ppm) was stronger than the old leaves (IC50 = 19.86 ppm). The antioxidant activity of the purple fruits extract (IC50 = 12.98 ppm) was stronger than the red fruits (IC50 = 28.63 ppm) and the green fruits (IC50 = 48.36 ppm) but it was weaker than quercetin (IC50 = 1.29 ppm). The purple fruit had the highest antioxidant activity compared to other extracts. This information will be useful for developing karamunting as a potential resource of natural antioxidants for functional foods and health products.

Foliar epidermal characters and petiole anatomy of four species of Citrus L. (Rutaceae) in South-western Nigeria

Studies on the leaf epidermal characters and petiole anatomy of four species of Citrus L. viz., C. limon (L.) Burm., C. paradisi Macf., C. reticulata Blanco and C. sinensis (L.) Osbeck were carried out to establish and document their foliar anatomical characters with the aim of separating them from the modified varieties been cultivated globally. Leaf epidermal features that revealed close relationship among these taxa include hypostomatic condition, stomata shape and type, presence of secretory cavity on the adaxial surface, and polygonal shape of epidermal cells. The absence of secretory cavity on the abaxial surface separates C. limon and C. sinensis from others. Petiole anatomy revealed that the outline is flat without ribs in C. sinensis, convex with short ribs in C. reticulata, convex with long ribs in C. paradisi while in C. limon it is circular without ribs. Presence of trichomes and crystals distinguishes C. paradisi from the rest.DOI: http://dx.doi.org/10.3329/bjpt.v19i1.10938Bangladesh J. Plant Taxon. 19(1): 25-31, 2012 (June)