Main conclusion The DNA-binding protein WHIRLY1, sharing structural similarities with ferritin, plays a role in the formation of iron cofactor proteins within chloroplasts. Abstract Previous studies indicated that barley plants with a knockdown of HvWHIRLY1 containing a minimal amount of the protein are compromised in chloroplast development and photosynthesis, and get chlorotic leaves when grown at high irradiance. Thereby, the leaves display signs of iron deficiency. Metal determination revealed, however, that leaves of WHIRLY1-deficient plants had a regular iron content. Nevertheless, WHIRLY1-deficiency affected the functionality of photosystem II less than that of photosystem I, which has a higher demand for iron. Immunological analyses revealed that components of both photosystems had reduced levels. Additionally, the levels of other chloroplast proteins containing different classes of iron cofactors were lower in the WHIRLY1-deficient plants compared to the wild type. In contrast, the level of the iron sequestering protein ferritin increased in WHIRLY1-deficient lines, whereby high irradiance intensified this effect. RNA analyses showed that the upregulation of ferritin coincided with an enhanced expression of the corresponding gene, reflecting an apparent overload of chloroplasts with free iron. Ferritin and WHIRLY proteins are known to share the same oligomeric structure. Therefore, the high abundance of ferritin in WHIRLY1-deficient plants might be a compensation for the reduced abundance of WHIRLY1. Enhanced expression levels of genes encoding photosynthesis proteins and iron cofactor proteins indicate a demand for protein formation or assembly of protein complexes. The results support a general role of WHIRLY1 in assembly and/or stabilization of chloroplast proteins and, moreover, suggest a specific function in sequestering and supply of iron in chloroplasts.

The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a “classical” bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

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A crucial feature of plant performance is its strong dependence on the availability of essential mineral nutrients, affecting multiple vital functions. Indeed, mineral-nutrient deficiency is one of the major stress factors affecting plant growth and development. Thereby, nitrogen and potassium represent the most abundant mineral contributors, critical for plant survival. While studying plant responses to nutrient deficiency, one should keep in mind that mineral nutrients, along with their specific metabolic roles, are directly involved in maintaining cell ion homeostasis, which relies on a finely tuned equilibrium between cytosolic and vacuolar ion pools. Therefore, in this chapter we briefly summarize the role of the ion homeostasis system in cell responses to environmental deficiency of nitrate and potassium ions. Special attention is paid to the implementation of plant responses via NO3− and K+ root transport and regulation of ion distribution in cell compartments. These responses are strongly dependent on plant species, as well as severity and duration of nutrient deficiency.

The chapter “Mass Spectrometry Data Processing” focuses on the mass spectrometry data processing workflow. The first step consists of processing the raw MS data using conversion of vendor formats to open standards, followed by feature detection, optionally retention time correction and grouping of features across samples leading to a feature matrix amenable for statistical analysis. The metabolomics community has developed several open source software packages capable of processing large-scale data commonly occurring in metabolomics studies. In the second stage, features of interest are identified, i.e., annotated with names of metabolites, or compound classes. Tandem MS or LC-MS/MS fragmentation data provides structural hints. The MS/MS spectra can be used to search in open and commercial spectral libraries. If no reference spectra are available, in-silico annotation tools or more recently machine learning approaches can be used.

The structure of the microtubule cytoskeleton provides valuable information related to morphogenesis of cells. The cytoskeleton organizes into diverse patterns that vary in cells of different types and tissues, but also within a single tissue. To assess differences in cytoskeleton organization methods are needed that quantify cytoskeleton patterns within a complete cell and which are suitable for large data sets. A major bottleneck in most approaches, however, is a lack of techniques for automatic extraction of cell contours. Here, we present a semi-automatic pipeline for cell segmentation and quantification of microtubule organization. Automatic methods are applied to extract major parts of the contours and a handy image editor is provided to manually add missing information efficiently. Experimental results prove that our approach yields high-quality contour data with minimal user intervention and serves a suitable basis for subsequent quantitative studies.

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.

Mass spectrometry coupled with LC (liquid chromatography) separation has developed into a technique routinely applied for targeted as well as for nontargeted analysis of complex biological samples, not only in plant biochemistry. Earlier on, LC‐MS (liquid chromatography–mass spectrometry) was mostly part of the efforts for identification of one or few unknown metabolites of interest as part of a phytochemical study. As a major strategy, unknown compounds had to be purified in sufficient quantities. The purified fractions were then subjected to LC‐MS/MS as part of the structural elucidation, mostly complemented by NMR (nuclear magnetic resonance) analysis. With the advance of mass spectrometry instrumentation, LC‐MS is now widely applied for analysis of crude plant extracts and large numbers (100s to 1000s) of samples. It has become an essential part of metabolomic studies (see Metabolomics), aiming at the comprehensive coverage of the metabolite profiles of cells, tissues, or organs. Owing to the huge chemical diversity of small molecules, conditions for the extraction will restrict the subfraction of the metabolome, which can be actually analyzed. The conditions for LC have to be adjusted to allow good separation of the particular metabolites from the respective extract. Major consideration will be the selection of an appropriate column and suitable eluents, the establishment of gradient profiles, temperature conditions, and so on.

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.