Publications - Cell and Metabolic Biology

Medicago truncatula, owing to its small diploid genome (∼500 Mbp), short life cycle, and high natural diversity makes it a good model plant and has opened the door of opportunities for scientists interested in studying legume biology. But over the years, challenges are also being faced for genetic manipulation of this plant. Many genetic manipulation protocols have been published involving Agrobacterium tumefaciens, a pathogen causing tumor disease in plants. These protocols apart from being difficult to achieve, are also time consuming. Nowadays, an easy, less time consuming and highly reproducible Agrobacterium rhizogenes based method is in use by many research groups. This method generates composite plants having transformed roots on a wild‐type shoot. Here, stable transformed lines that can be propagated over time are not achieved by this method, but for root‐development or root–microbe interaction studies this method has proven to be a useful tool for the community. In addition, transformed roots can be propagated by root organ cultures (ROCs), wherein transformed roots are propagated on sucrose containing media without any shoot part. Occasionally, even stable transgenic plants can be regenerated from transgenic roots. In this chapter, developments and improvements of various transformation protocols are discussed. The suitability of composite plants is highlighted by a study on mycorrhization of transformed and non‐transformed roots, which did not show differences in the mycorrhization rate and developmental stages of the arbuscular mycorrhizal (AM) fungus inside the roots as well as in transcript accumulation and metabolite levels of roots. Finally, applications of the A. rhizogenes based transformation method are discussed.

Plant glandular trichomes are epidermal differentiations that are dedicated to the production of specialized metabolites, which constitute a first line of defense against pathogens and herbivores. The secretions of these metabolic factories are chemically very diverse, including of terpenoid, fatty acid, or phenylpropanoid origins. They find uses in various industrial areas, for example as pharmaceutical, flavor, or fragrance ingredients or as insecticides. Recent progress in the elucidation of biosynthesis pathways for these compounds has opened up novel opportunities for metabolic engineering in microorganisms as well as in plants.

Transcription activator‐like effectors (TALEs) can be programmed to bind specific DNA sequences. This property was used to construct libraries of synthetic TALE‐activated promoters (STAPs), which drive varying levels of gene expression. After a brief description of these promoters, we explore how these STAPs can be used for various applications in plant synthetic biology, in particular for the coordinated expression of multiple genes for metabolic engineering and in the design and implementation of gene regulatory networks.

Environmental stress is one of the major factors reducing crop productivity. Due to the oncoming climate changes, the effects of drought and high light on plants play an increasing role in modern agriculture. These changes are accompanied with a progressing contamination of soils with heavy metals. Independent of their nature, environmental alterations result in development of oxidative stress, i.e. increase of reactive oxygen species (ROS) contents, and metabolic adjustment, i.e. accumulation of soluble primary metabolites (amino acids and sugars). However, a simultaneous increase of ROS and sugar concentrations ultimately results in protein glycation, i.e. non-enzymatic interaction of reducing sugars or their degradation products (α-dicarbonyls) with proteins. The eventually resulting advanced glycation end-products (AGEs) are known to be toxic and pro-inflammatory in mammals. Recently, their presence was unambiguously demonstrated in vivo in stressed Arabidopsis thaliana plants. Currently, information on protein targets, modification sites therein, mediators and mechanisms of plant glycation are being intensively studied. In this chapter, we comprehensively review the methodological approaches for plant glycation research and discuss potential mechanisms of AGE formation under stress conditions. On the basis of these patterns and additional in vitro experiments, the pathways and mechanisms of plant glycation can be proposed.

Plant secondary metabolites, also termed specialized plant metabolites, currently comprise more than 200 000 natural products that are all based on a few biosynthetic pathways and key primary metabolites. Some pathways like flavonoid and terpenoid biosynthesis are universally distributed in the plant kingdom, whereas others like alkaloid or cyanogenic glycoside biosynthesis are restricted to a limited set of taxa. Diversification is achieved by an array of mechanisms at the genetic and enzymatic level including gene duplications, substrate promiscuity of enzymes, cell‐specific regulatory systems, together with modularity and combinatorial aspects. Specialized metabolites reflect adaptations to a specific environment. The observed diversity illustrates the heterogeneity and multitude of ecological habitats and niches that plants have colonized so far and constitutes a reservoir of potential new metabolites that may provide adaptive advantage in the face of environmental changes. The code that connects the observed chemical diversity to this ecological diversity is largely unknown. One way to apprehend this diversity is to realize its tremendous plasticity and evolutionary potential. This chapter presents an overview of the most widespread and popular secondary metabolites, which provide a definite advantage to adapt to or to colonize a particular environment, making the boundary between the “primary” and the “secondary” old fashioned and blurry.

For centuries, progress in biological research has been connected to the development of tools and equipment that allow new insights into the living matter. The invention of and improvements in optical systems were very important because exceeding the limits of the optical resolution of the human eye delivered new insights into tissues, cells, and subcellular compartments on the one hand and cellular processes on the other. Even the very first light microscopes, developed at the beginning of the seventeenth century, enabled the discovery of “Cells as little boxes” by Robert Hooke, and of bacteria by Antoni van Leeuwenhoek. Since then, many aspects of microscopes have been improved and new illumination, staining, and detection methods have been developed in order to increase the optical resolution. In this chapter, we describe the principles and possibilities of the use of microscopes in biology, as well as specific methods of preparing biological materials in order to obtain optimum microscopic images with an appropriate scientific message. Further, emphasis is given on staining techniques used for biological materials including transgenic approaches that use the wide variance of fluorescent proteins.

BackgroundJasmonates are important regulators in plant responses to biotic and abiotic stresses as well as in development. Synthesized from lipid-constituents, the initially formed jasmonic acid is converted to different metabolites including the conjugate with isoleucine. Important new components of jasmonate signalling including its receptor were identified, providing deeper insight into the role of jasmonate signalling pathways in stress responses and development.ScopeThe present review is an update of the review on jasmonates published in this journal in 2007. New data of the last five years are described with emphasis on metabolites of jasmonates, on jasmonate perception and signalling, on cross-talk to other plant hormones and on jasmonate signalling in response to herbivores and pathogens, in symbiotic interactions, in flower development, in root growth and in light perception.ConclusionsThe last few years have seen breakthroughs in the identification of JASMONATE ZIM DOMAIN (JAZ) proteins and their interactors such as transcription factors and co-repressors, and the crystallization of the jasmonate receptor as well as of the enzyme conjugating jasmonate to amino acids. Now, the complex nature of networks of jasmonate signalling in stress responses and development including hormone cross-talk can be addressed.

Cleavage products of carotenoids (apocarotenoids) exert a variety of often poorly characterized functions in roots and in rhizospheric interactions of plants with both symbionts and parasites. They are generated by regiospecific cleavage enzymes (CCDs, NCEDs) that act in a single or sequential way on C40 carotenoids. Among such apocarotenoids are one well‐known phytohormone controlling drought stress response networks (abscisic acid, ABA) and a newly discovered class of growth regulators involved in adaptive reactions to nutrient stress (strigolactones, SL). A third class of apocarotenoids consists of derivatives of a cyclic cyclohexenone (CH) and a linear mycorradicin (MR) type. They accumulate abundantly as part of a so‐called yellow pigment complex in roots colonized by arbuscular mycorrhizal (AM) fungi. Mycorrhizal phenotypes of pathway knockdown and loss‐of‐function mutants are reviewed in order to clarify the role of the three apocarotenoid classes for the AM symbiosis. One case of pathway interconnection between SL and CH/MR biogenesis through CCD7 is discussed along with other implications for interplay between pathways. SLs appear to preferentially affect early steps of root colonization by AM fungi and thus colonization levels. In contrast, accumulation of CH/MR derivatives is associated with arbuscule formation. Arbuscules are transient structures, which undergo constant degradation and reformation (turnover). Colocalization of CH/MR derivatives with degrading arbuscules and other observations suggest a phytoalexin‐like function in a plant‐controlled degradation of degenerating or poorly functional arbuscules. A model is presented, which proposes maintenance of high levels of functional arbuscules delivering phosphate through plant management of their rapid turnover.

Following the description of two separate pathways for isoprenoid precursor biosynthesis in plants, a new level of complexity has been introduced by the discovery of two divergent gene classes encoding the first enzyme of the plastidial methylerythritol phosphate (MEP) pathway. These nonredundant 1-deoxy-d-xylulose 5-phosphate synthase (DXS) isogenes are differentially expressed in such a way that DXS1 appears to serve housekeeping functions, whereas DXS2 is associated with the production of specialized (secondary) isoprenoids involved in ecological functions. Examples of the latter are apocarotenoid formation in roots colonized by arbuscular mycorrhizal fungi and mono- or diterpenoid biosynthesis in trichomes. Knockdown of DXS2 genes can specifically suppress secondary isoprenoid formation without affecting basic plant functions. Analyzing DXS isogenes along the progression of land plant evolution shows separation in structure and complementary expression already at the level of gymnosperms, which is maintained in all angiosperms except Arabidopsis.

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