Alkaloids represent a highly diverse group of compounds that are related only by the occurrence of a nitrogen atom in a heterocyclic ring. Plants are estimated to produce approximately 12,000 different alkaloids, which can be organized into groups according to their carbon skeletal structures. Alkaloid biosynthesis in plants involves many catalytic steps, catalyzed by enzymes that belong to a wide range of protein families. The characterization of novel alkaloid biosynthetic enzymes in terms of structural biochemistry, molecular and cell biology, and biotechnological applications has been the focus of research over the past several years. The application of genomics to the alkaloid field has accelerated the discovery of cDNAs encoding previously elusive biosynthetic enzymes. Other technologies, such as large-scale gene expression analyses and metabolic engineering approaches with transgenic plants, have provided new insights into the regulatory architecture of alkaloid metabolism.

Alkaloid Biosynthesis: Metabolism and Trafficking

Heavy metals are naturally occurring in the earth‘s crust but anthropogenic and industrial activities have led to drastic environmental pollutions in distinct areas. Plants are able to colonize such sites due to several mechanisms of heavy metal tolerance. Understanding of these pathways enables different fruitful approaches like phytoremediation and biofortification. Therefore, this review addresses mechanisms of heavy metal tolerance and toxicity in plants possessing a sophisticated network for maintenance of metal homeostasis. Key elements of this are chelation and sequestration which result either in removal of toxic metal from sensitive sites or conduct essential metal to their specific cellular destination. This implies shared pathways which can result in toxic symptoms especially in an excess of metal. These overlaps go on with signal transduction pathways induced by heavy metals which include common elements of other signal cascades. Nevertheless, there are specific reactions some of them will be discussed with special focus on the cellular level.

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How plants cope with heavy metals

Plant phospholipid signaling: “in a nutshell”

Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy (Papaver somniferum), which produces several benzylisoquinoline alkaloids, and Madagascar periwinkle (Catharanthus roseus), which accumulates an array of monoterpenoid indole alkaloids, have emerged as the premier organisms used to study plant alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and monoterpenoid indole alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and monoterpenoid indole alkaloid biosynthesis in opium poppy and C. roseus is presented.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2008.03438.x

Opium poppy and Madagascar periwinkle: model non‐model systems to investigate alkaloid biosynthesis in plants

Calcium is one of the essential nutrients for growth and development of plants. It is an important component of various structures in cell wall and membranes. Besides some fundamental roles under normal condition, calcium functions as a major secondary-messenger molecule in plants under different developmental cues and various stress conditions including salinity stress. Also changes in cytosolic pH, pH(cyt), either individually, or in coordination with changes in cytosolic Ca(2+) concentration, [Ca(2+)](cyt), evoke a wide range of cellular functions in plants including signal transduction in plant-defense responses against stresses. It is believed that salinity stress, like other stresses, is perceived at cell membrane, either extra cellular or intracellular, which then triggers an intracellular-signaling cascade including the generation of secondary messenger molecules like Ca(2+) and protons. The variety and complexity of Ca(2+) and pH signaling result from the nature of the stresses as well as the tolerance level of the plant species against that specific stress. The nature of changes in [Ca(2+)](cyt) concentration, in terms of amplitude, frequency and duration, is likely very important for decoding the specific downstream responses for salinity stress tolerance in planta. It has been observed that the signatures of [Ca(2+)](cyt) and pH differ in various studies reported so far depending on the techniques used to measure them, and also depending on the plant organs where they are measured, such as root, shoot tissues or cells. This review describes the recent advances about the changes in [Ca(2+)](cyt) and pH(cyt) at both cellular and whole-plant levels under salinity stress condition, and in various salinity-tolerant and -sensitive plant species.

Cytosolic calcium and pH signaling in plants under salinity stress

Intracellular pH signals in the induction of secondary pathways--The case of Eschscholzia californica

The function of a Galpha protein in the elicitation of phytoalexin (benzophenanthridine) biosynthesis was characterized in cultured cells of California poppy (Eschscholzia californica). Both the decrease of Galpha content via antisense transformation and the expression of recombinant anti-Galpha single-chain antibodies strongly impaired the induction of alkaloid biosynthesis by low elicitor concentrations. All transgenic cell types were deficient in two elicitor-triggered early signal events: activation of phospholipase A2 (PLA2) and efflux of vacuolar protons. The lacking H+ efflux could be restored (1) by adding lysophosphatidylcholine (LPC), a product of PLA2 activity, to vacuoles in situ and (2) by exposing intact cells to isotonic, near-neutral HEPES buffers. The latter treatment induced alkaloid biosynthesis in the absence of elicitor and in Galpha-deficient cells. We conclude that Galpha mediates the stimulation of PLA2 by low elicitor concentrations and that the resulting peak of LPC initiates a transient efflux of vacuolar protons. In this way, an acidic peak of the cytoplasmic pH is generated that causes the expression of enzymes of phytoalexin production independent of the hypersensitive response.

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The Gα Protein Controls a pH-Dependent Signal Path to the Induction of Phytoalexin Biosynthesis in Eschscholzia californica

Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).

Regulatory interaction of the Gα protein with phospholipase A2 in the plasma membrane of Eschscholzia californica

In cultured cells of California poppy (Eschscholzia californica), lysophosphatidylcholine (LPC) triggers a signal path that finally induces alkaloid biosynthesis. LPC is transiently generated by elicitor-activated phospholipase A2 of the plasma membrane. Externally added LPC is rapidly acylated by a membrane-bound enzyme that shows the highest specific activity in the purified plasma membrane. The fatty acid incorporated into the sn-2 position of LPC is preferentially linoleic (18:2), which is the most abundant acyl component in the PC species of Eschscholzia cells, but a minor component of the pool of free fatty acids. The fatty acid at the sn-1 position of LPC is less important for substrate specificity. The capacity of LPC acylation by intact cells or isolated plasma membranes by far exceeds the rate of LPC generation by activated phospholipase A2 and is not limited by the availability of acyl donors. Metabolites other than phosphatidylcholine (PC) were not significantly produced from labeled LPC within 20 min, indicating that lysophospholipases are not significantly contributing to the short-time metabolism of LPC. It is concluded that reacylation to PC is the dominating process in the detoxication of LPC and ensures the transient character of its steady state concentrations, even at maximum phospholipase A2 activities.

Wieland Schwartze

Papers

Intracellular pH signals in the induction of secondary pathways--The case of Eschscholzia californica

The Gα Protein Controls a pH-Dependent Signal Path to the Induction of Phytoalexin Biosynthesis in Eschscholzia californica

Regulatory interaction of the Gα protein with phospholipase A2 in the plasma membrane of Eschscholzia californica

The signal molecule lysophosphatidylcholine in Eschscholzia californica is rapidly metabolized by reacylation