Hypericum perforatum L. commonly known as Saint John’s Wort (SJW) is an economically important medicinal plant known for accumulating its valuable bioactive compounds in a compartmentalized fashion. The dark glands are very rich in hypericin, and translucent glands are filled with hyperforin. The antibiotic properties of the afore mentioned bioactive compounds make it hard to establish tissue regeneration protocols essential to put in place a transformation platform that is required for testing gene function in this challenging species. In this study, we report the establishment of a regeneration and root induction cycle from different types of explants. The regeneration cycle was set up for the continuous supply of roots and leaf explants for downstream transformation experiments. The most effective medium to obtain multiple shoot-buds from node cultures was MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium supplemented with 0.5 mg L−1 6-Benzylaminopurine (BAP) and 0.5 mg L−1 indole-3-butyric acid (IBA). The same combination yielded copious amounts of shoots from root and leaf explants as well. For rooting the elongated shoots, MS medium devoid of plant growth regulators (PGRs) was sufficient. Nevertheless, addition of a low amount of IBA improved the quantity and quality of roots induced. Additionally, the roots obtained on a medium containing IBA readily developed shoot buds.

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Salinity poses a serious threat to global agriculture and human food security. A better understanding of plant adaptation to salt stress is, therefore, mandatory. In the non-photosynthetic cells of the root, salinity perturbs oxidative balance in mitochondria, leading to cell death. In parallel, plastids accumulate the jasmonate precursor cis (+)12-Oxo-Phyto-Dienoic Acid (OPDA) that is then translocated to peroxisomes and has been identified as promoting factor for salt-induced cell death as well. In the current study, we probed for a potential interaction between these three organelles that are primarily dealing with oxidative metabolism. We made use of two tools: (i) Rice OPDA Reductase 7 (OsOPR7), an enzyme localised in peroxisomes converting OPDA into the precursors of the stress hormone JA-Ile. (ii) A Trojan Peptoid, Plant PeptoQ, which can specifically target to mitochondria and scavenge excessive superoxide accumulating in response to salt stress. We show that overexpression of OsOPR7 as GFP fusion in tobacco (Nicotiana tabacum L. cv. Bright Yellow 2, BY-2) cells, as well as a pretreatment with Plant PeptoQ can mitigate salt stress with respect to numerous aspects including proliferation, expansion, ionic balance, redox homeostasis, and mortality. This mitigation correlates with a more robust oxidative balance, evident from a higher activity of superoxide dismutase (SOD), lower levels of superoxide and lipid peroxidation damage, and a conspicuous and specific upregulation of mitochondrial SOD transcripts. Although both, Plant PeptoQ and ectopic OsOPR7, were acting in parallel and mostly additive, there are two specific differences: (i) OsOPR7 is strictly localised to the peroxisomes, while Plant PeptoQ found in mitochondria. (ii) Plant PeptoQ activates transcripts of NAC, a factor involved in retrograde signalling from mitochondria to the nucleus, while these transcripts are suppressed significantly in the cells overexpressing OsOPR7. The fact that overexpression of a peroxisomal enzyme shifting the jasmonate pathway from the cell-death signal OPDA towards JA-Ile, a hormone linked with salt adaptation, is accompanied by more robust redox homeostasis in a different organelle, the mitochondrion, indicates that cross-talk between peroxisome and mitochondrion is a crucial factor for efficient adaptation to salt stress.

Mannan is a class of cell wall polysaccharides widespread in the plant kingdom. Mannan structure and properties vary according to species and organ. The cell walls of cereal grains have been extensively studied due to their role in cereal processing and to their beneficial effect on human health as dietary fiber. Recently, we showed that mannan in wheat (Triticum aestivum) grain endosperm has a linear structure of β-1,4-linked mannose residues. The aim of this work was to study the biosynthesis and function of wheat grain mannan. We showed that mannan is deposited in the endosperm early during grain development, and we identified candidate mannan biosynthetic genes expressed in the endosperm. The functional study in wheat was unsuccessful therefore our best candidate genes were expressed in heterologous systems. The endosperm-specificTaCslA12 gene expressed in Pichia pastoris and in an Arabidopsis thaliana mutant depleted in glucomannan led to the production of wheat-like linear mannan lacking glucose residues and with moderate acetylation. Therefore, this gene encodes a mannan synthase and is likely responsible for the synthesis of wheat endosperm mannan.

The contribution of carbon assimilation and allocation and of invertases to the stimulation of adventitious root formation in response to a dark pre-exposure of petunia cuttings was investigated, considering the rooting zone (stem base) and the shoot apex as competing sinks. Dark exposure had no effect on photosynthesis and dark respiration during the subsequent light period, but promoted dry matter partitioning to the roots. Under darkness, higher activities of cytosolic and vacuolar invertases were maintained in both tissues when compared to cuttings under light. This was partially associated with higher RNA levels of respective genes. However, activity of cell wall invertases and transcript levels of one cell wall invertase isogene increased specifically in the stem base during the first two days after cutting excision under both light and darkness. During five days after excision, RNA accumulation of four invertase genes indicated preferential expression in the stem base compared to the apex. Darkness shifted the balance of expression of one cytosolic and two vacuolar invertase genes towards the stem base. The results indicate that dark exposure before planting enhances the carbon sink competitiveness of the rooting zone and that expression and activity of invertases contribute to the shift in carbon allocation.

Various cleavage products of C40 carotenoid substrates are formed preferentially or exclusively in roots. Such apocarotenoid signaling or regulatory compounds differentially induced in roots during environmental stress responses including root colonization by arbuscular mycorrhizal fungi include ABA, strigolactones and C13 α-ionol/C14 mycorradicin derivatives. The low carotenoid levels in roots raise the question of whether there is a regulated precursor supply channeled into apocarotenoid formation distinct from default carotenoid pathways. This review describes root-specific isogene components of carotenoid pathways toward apocarotenoid formation, highlighting a new PSY3 class of phytoene synthase genes in dicots. It is clearly distinct from the monocot PSY3 class co-regulated with ABA formation. At least two members of the exclusive dicot PSY3s are regulated by nutrient stress and mycorrhization. This newly recognized dicot PSY3 (dPSY3 vs. mPSY3 from monocots) class probably represents an ancestral branch in the evolution of the plant phytoene synthase family. The evolutionary history of PSY genes is compared with the evolution of MEP pathway isogenes encoding 1-deoxy-d-xylulose 5-phosphate synthases (DXS), particularly DXS2, which is co-regulated with dPSY3s in mycorrhizal roots. Such stress-inducible isoforms for rate-limiting steps in root carotenogenesis might be components of multi-enzyme complexes committed to apocarotenoid rather than to carotenoid formation.

Benzophenanthridine alkaloids are strong antimicrobials of Papaveraceae and attractive lead compounds for drug development. The cytotoxicity of these compounds requires the producing plant to limit the pathogen-triggered burst of biosynthesis. Cells of Eschscholzia californica excrete early benzophenanthridines to the cell wall, followed by re-uptake and reduction in the cytoplasm by the detoxifying enzyme sanguinarine reductase. We now discovered that this enzyme is a core component of self-control in alkaloid production. RNAi-based silencing of sanguinarine reductase gave rise to mutants that either show a complete stop of elicitor-triggered alkaloid production or a burst of biosynthesis that severalfold surpasses the wild type level. These unexpected phenotypes reflect impacts of substrate or product of sanguinarine reductase: the substrate, sanguinarine, inhibits phospholipase A2 at the plasma membrane, an initial component of the signal path towards expression of biosynthetic enzymes. The product, dihydrosanguinarine, inhibits enzymes of early biosynthesis, prior to reticuline formation. By tuning these steady states, sanguinarine reductase adjusts the capacity of alkaloid biosynthesis: a minimum activity is sufficient to prevent the blockade of the induction pathway by sanguinarine, while the full activity of the same enzyme causes a limitation of the biosynthetic flow via dihydrosanguinarine.

Apocarotenoids are a class of compounds that play important roles in nature. In recent years, a prominent role for these compounds in arbuscular mycorrhizal (AM) symbiosis has been shown. They are derived from carotenoids by the action of the carotenoid cleavage dioxygenase (CCD) enzyme family. In the present study, using tomato as a model, the spatio-temporal expression pattern of the CCD genes during AM symbiosis establishment and functioning was investigated. In addition, the levels of the apocarotenoids strigolactones (SLs), C13 α-ionol and C14 mycorradicin (C13/C14) derivatives were analyzed. The results suggest an increase in SLs promoted by the presence of the AM fungus at the early stages of the interaction, which correlated with an induction of the SL biosynthesis gene SlCCD7. At later stages, induction of SlCCD7 and SlCCD1 expression in arbusculated cells promoted the production of C13/C14 apocarotenoid derivatives. We show here that the biosynthesis of apocarotenoids during AM symbiosis is finely regulated throughout the entire process at the gene expression level, and that CCD7 constitutes a key player in this regulation. Once the symbiosis is established, apocarotenoid flux would be turned towards the production of C13/C14 derivatives, thus reducing SL biosynthesis and maintaining a functional symbiosis.

Glucosinolates are a diverse class of nitrogen- and sulfur-containing secondary metabolites. They are rapidly hydrolyzed on tissue disruption to a number of biologically active compounds that are increasingly attracting interest as anticarcinogenic phytochemicals and crop protectants. Several glucosinolate-derived isothiocyanates are potent chemopreventive agents that favorably modulate carcinogen metabolism in mammals. Methylsulfinylalkyl isothiocyanates, in particular the 4-methylsulfinylbutyl derivative, are selective and potent inducers of mammalian detoxification enzymes such as quinone reductase (QR). Cruciferous plants including Arabidopsis thaliana (L.) Heyhn, synthesize methylsulfinylalkyl glucosinolates, which are derived from methionine. Using a colorimetric assay for QR activity in murine hepatoma cells and high performance liquid chromatography (HPLC) analysis of desulfoglucosinolates, we have demonstrated a strong positive correlation between leaf QR inducer potency and leaf content of methionine-derived glucosinolates in various A. thaliana ecotypes and available glucosinolate mutants. In a molecular genetic approach to glucosinolate biosynthesis, we screened 3000 chemically mutagenized M2 plants of the Columbia ecotype for altered leaf QR inducer potency. Subsequent HPLC analysis of progeny of putative mutants identified six lines with significant and heritable changes in leaf glucosinolate content and composition.