The Arabinogalactan Protein Family of Centaurium erythraea Rafn.

Centaurium erythraea (centaury) is a medicinal plant with exceptional developmental plasticity in vitro and vigorous, often spontaneous, regeneration via shoot organogenesis and somatic embryogenesis, during which arabinogalactan proteins (AGPs) play an important role. AGPs are highly glycosylated proteins belonging to the super family of O-glycosylated plant cell surface hydroxyproline-rich glycoproteins (HRGPs). HRGPs/AGPs are intrinsically disordered and not well conserved, making their homology-based mining ineffective. We have applied a recently developed pipeline for HRGP/AGP mining, ragp, which is based on machine learning prediction of proline hydroxylation, to identify HRGP sequences in centaury transcriptome and to classify them into motif and amino acid bias (MAAB) classes. AGP sequences with low AG glycomotif representation were also identified. Six members of each of the three AGP subclasses, fasciclin-like AGPs, receptor kinase-like AGPs and AG peptides, were selected for phylogenetic and expression analyses. The expression of these 18 genes was recorded over 48 h following leaf mechanical wounding, as well as in 16 tissue samples representing plants from nature, plants cultivated in vitro, and developmental stages during shoot organogenesis and somatic embryogenesis. None of the selected genes were upregulated during both wounding recovery and regeneration. Possible functions of AGPs with the most interesting expression profiles are discussed.

Secondary Somatic Embryogenesis in Centaurium erythraea Rafn.

Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU). A medium containing 0.1 mgL-1 2,4-D and 0.25 mgL-1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL-1 2,4-D and 0.5 mgL-1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury-an emerging model in developmental biology.

Molecular Characterization and Expression of Four Aquaporin Genes in Impatiens walleriana During Drought Stress and Recovery.

Aquaporins comprise a large group of transmembrane proteins responsible for water transport, which is crucial for plant survival under stress conditions. Despite the vital role of aquaporins, nothing is known about this protein family in Impatiens walleriana, a commercially important horticultural plant, which is sensitive to drought stress. In the present study, attention is given to the molecular characterization of aquaporins in I. walleriana and their expression during drought stress and recovery. We identified four I. walleriana aquaporins: IwPIP1;4, IwPIP2;2, IwPIP2;7 and IwTIP4;1. All of them had conserved NPA motifs (Asparagine-Proline-Alanine), transmembrane helices (TMh), pore characteristics, stereochemical properties and tetrameric structure of holoprotein. Drought stress and recovery treatment affected the aquaporins expression in I. walleriana leaves, which was up- or downregulated depending on stress intensity. Expression of IwPIP2;7 was the most affected of all analyzed I. walleriana aquaporins. At 15% and 5% soil moisture and recovery from 15% and 5% soil moisture, IwPIP2;7 expression significantly decreased and increased, respectively. Aquaporins IwPIP1;4 and IwTIP4;1 had lower expression in comparison to IwPIP2;7, with moderate expression changes in response to drought and recovery, while IwPIP2;2 expression was of significance only in recovered plants. Insight into the molecular structure of I. walleriana aquaporins expanded knowledge about plant aquaporins, while its expression during drought and recovery contributed to I. walleriana drought tolerance mechanisms and re-acclimation.

Effects of Exogenous Salicylic Acid on Drought Response and Characterization of Dehydrins in Impatiens walleriana.

Impatiens walleriana is a valued ornamental plant sensitive to drought stress. We investigated whether the foliar application of 2mM salicylic acid (SA) can protect potted I. walleriana plants from drought stress. The plants were divided into: watered plants, drought-stressed plants, watered plants treated with SA and drought-stressed plants treated with SA. The number of flowers and flower buds, relative water content (RWC), contents of malondialdehyde (MDA) and proline (Pro) and the activities of superoxide dismutases, catalases and peroxidases were recorded at different time points. Three dehydrin sequences were identified in de novo assembled leaf transcriptome: IwDhn1, IwDhn2.1 and IwDhn2.2. Drought stress caused wilting, floral abortion, reduction of RWC and increased MDA-an indicator of lipid peroxidation. In response to drought, Impatiens accumulated Pro and induced chloroplastic Cu/ZnSOD and two peroxidase isoforms. The most remarkable drought response was strong induction of IwDhn2.1 and IwDhn2.2. Rehydration restored RWC, Pro level, Cu/ZnSOD activity and dehydrins expression in drought-stressed plants approximately to the values of watered plants.SA had ameliorating effects on plants exposed to drought, including prevention of wilting, preservation of RWC, increased Pro accumulation, modulation of antioxidative activities and remarkable decrease of lipid peroxidation, but without effects on flowers' preservation.

Somatic Embryogenesis in Centaurium erythraea Rafn-Current Status and Perspectives: A Review.

Centaurium erythraea (centaury) is a traditionally used medicinal plant, with a spectrum of secondary metabolites with confirmed healing properties. Centaury is an emerging model in plant developmental biology due to its vigorous regenerative potential and great developmental plasticity when cultured in vitro. Hereby, we review nearly two decades of research on somatic embryogenesis (SE) in centaury. During SE, somatic cells are induced by suitable culture conditions to express their totipotency, acquire embryogenic characteristics, and eventually give rise to somatic embryos. When SE is initiated from centaury root explants, the process occurs spontaneously (on hormone-free medium), directly (without the callusing phase), and the somatic embryos are of unicellular origin. SE from leaf explants has to be induced by plant growth regulators and is indirect (preceded by callusing). Histological observations and culture conditions are compared in these two systems. The changes in antioxidative enzymes were followed during SE from the leaf explants. Special focus is given to the role of arabinogalactan proteins during SE, which were analyzed using a variety of approaches. The newest and preliminary results, including centaury transcriptome, novel potential SE markers, and novel types of arabinogalactan proteins, are discussed as perspectives of centaury research.