Josefine Nymark Hegelund

Competition between uptake of ammonium and potassium in barley and Arabidopsis roots: molecular mechanisms and physiological consequences.

Plants can use ammonium (NH4+) as the sole nitrogen source, but at high NH4+ concentrations in the root medium, particularly in combination with a low availability of K+, plants suffer from NH4+ toxicity. To understand the role of K+ transporters and non-selective cation channels in K+/NH4+ interactions better, growth, NH4+ and K+ accumulation and the specific fluxes of NH4+, K+, and H+ were examined in roots of barley (Hordeum vulgare L.) and Arabidopsis seedlings. Net fluxes of K+ and NH4+ were negatively correlated, as were their tissue concentrations, suggesting that there is direct competition during uptake. Pharmacological treatments with the K+ transport inhibitors tetraethyl ammonium (TEA+) and gadolinium (Gd3+) reduced NH4+ influx, and the addition of TEA+ alleviated the NH4+-induced depression of root growth in germinating Arabidopsis plants. Screening of a barley root cDNA library in a yeast mutant lacking all NH4+ and K+ uptake proteins through the deletion of MEP1–3 and TRK1 and TRK2 resulted in the cloning of the barley K+ transporter HvHKT2;1. Further analysis in yeast suggested that HvHKT2;1, AtAKT1, and AtHAK5 transported NH4+, and that K+ supplied at increasing concentrations competed with this NH4+ transport. On the other hand, uptake of K+ by AtHAK5, and to a lesser extent via HvHKT2;1 and AtAKT1, was inhibited by increasing concentrations of NH4+. Together, the results of this study show that plant K+ transporters and channels are able to transport NH4+. Unregulated NH4+ uptake via these transporters may contribute to NH4+ toxicity at low K+ levels, and may explain the alleviation of NH4+ toxicity by K+.

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn2+ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn2+ and Cd2+ chelator with capacity to bind 10 Zn2+ ions per C terminus. When AtHMA4 is expressed in a Zn2+-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn2+ and Cd2+ tolerance and lowered Zn2+ and Cd2+ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn2+ and Cd2+ chelator (sensor) and as a regulator of the efficiency of Zn2+ and Cd2+ export. The identification of a post-translational handle on Zn2+ and Cd2+ transport efficiency opens new perspectives for regulation of Zn2+ nutrition and tolerance in eukaryotes.

Review: The role of atomic spectrometry in plant science

Inductively coupled plasma-mass spectrometry (ICP-MS) is the state-of-the-art technique for multi-elemental analysis of plant tissue. It provides a powerful tool in functional genomics, linking altered elemental profiles of mutants with gene expression and function. In addition, with its unmatched sensitivity, ICP-MS enables characterization of the substrate specificity and regulation of membrane transport proteins. Digestion of plant tissue has traditionally represented a bottleneck due to the low capacity of commercially available equipment. However, recent developments in micro-scaled digestion, combined with semi-quantitative analysis and chemometrics, have enabled high-throughput multi-elemental profiling and multivariate classification of large sample sets, thereby supporting a range of new applications in molecular breeding, quality assessment and authenticity testing of plants. Novel hyphenated techniques based on liquid chromatography and ICP-MS (LC-ICP-MS) have significantly improved the understanding of elemental species and their importance for e.g. the bioactivity of metals in plants. Development of procedures for sample pre-treatment, extraction and multi-dimensional separation now allows characterization of important metallo-biomolecules in plants, such as the coordination complexes of phytochelatins, metallothioneins, nicotianamine and inositol phosphates. These are key ligands involved in ion homeostasis, translocation and long-term storage of elements. Much emphasis has also been given to studies of covalently bound Se and As species, primarily due to their impact on human health. LC-ICP-MS has extensively been complemented by molecular mass spectrometry for structural information of biologically relevant species. This review covers the most recent developments in multi-elemental analysis (Part A) and speciation analysis (Part B) in plant science. A number of relevant cases are presented in order to demonstrate how the analytical developments have unravelled the functional roles of elements in plants science. These cases show that ICP-MS is an essential technology in plant metallomic platforms.

Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins believed to play a role in cytosolic zinc (Zn) and copper (Cu) homeostasis. However, evidence for the functional properties of MTs has been hampered by methodological problems in the isolation and characterization of the proteins. Here, we document that barley (Hordeum vulgare) MT3 and MT4 proteins exist in planta and that they differ in tissue localization as well as in metal coordination chemistry. Combined transcriptional and histological analyses showed temporal and spatial correlations between transcript levels and protein abundance during grain development. MT3 was present in tissues of both maternal and filial origin throughout grain filling. In contrast, MT4 was confined to the embryo and aleurone layer, where it appeared during tissue specialization and remained until maturity. Using state-of-the-art speciation analysis by size-exclusion chromatography inductively coupled plasma mass spectrometry and electrospray ionization time-of-flight mass spectrometry on recombinant MT3 and MT4, their specificity and capacity for metal ion binding were quantified, showing a strong preferential Zn binding relative to Cu and cadmium (Cd) in MT4, which was not the case for MT3. When complementary DNAs from barley MTs were expressed in Cu- or Cd-sensitive yeast mutants, MT3 provided a much stronger complementation than did MT4. We conclude that MT3 may play a housekeeping role in metal homeostasis, while MT4 may function in Zn storage in developing and mature grains. The localization of MT4 and its discrimination against Cd make it an ideal candidate for future biofortification strategies directed toward increasing food and feed Zn concentrations.

Golgi Localized Barley MTP8 Proteins Facilitate Mn Transport

Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species.

Barley metallothioneins differ in ontogenetic pattern and response to metals

The barley genome encodes a family of 10 metallothioneins (MTs) that have not previously been subject to extensive gene expression profiling. We show here that expression of MT1a, MT2b1, MT2b2 and MT3 in barley leaves increased more than 50-fold during the first 10 d after germination. Concurrently, the root-specific gene MT1b1 was 1000-fold up-regulated. Immunolocalizations provided the first evidence for accumulation of MT1a and MT2a proteins in planta, with correlation to transcript levels. In developing grains, MT2a and MT4 expression increased 4- and 300-fold over a 28-day-period after pollination. However, among the MT grain transcripts MT2c was the most abundant, whereas MT4 was the least abundant. Excess Cu up-regulated three out of the six MTs expressed in leaves of young barley plants. In contrast, most MTs were down-regulated by excess Zn or Cd. Zn starvation led to up-regulation of MT1a, whereas Cu starvation up-regulated MT2a, which has two copper-responsive elements in the promoter. Arabidopsis lines constitutively overexpressing barley MT2a showed increased sensitivity to excess Cd and Zn but no Cu-induced response. We suggest that barley MTs are differentially involved in intracellular homeostasis of essential metal ions and that a subset of barley MTs is specifically involved in Cu detoxification.

Ethylene resistance in flowering ornamental plants - improvements and future perspectives.

Various strategies of plant breeding have been attempted in order to improve the ethylene resistance of flowering ornamental plants. These approaches span from conventional techniques such as simple cross-pollination to new breeding techniques which modify the plants genetically such as precise genome-editing. The main strategies target the ethylene pathway directly; others focus on changing the ethylene pathway indirectly via pathways that are known to be antagonistic to the ethylene pathway, e.g. increasing cytokinin levels. Many of the known elements of the ethylene pathway have been addressed experimentally with the aim of modulating the overall response of the plant to ethylene. Elements of the ethylene pathway that appear particularly promising in this respect include ethylene receptors as ETR1, and transcription factors such as EIN3. Both direct and indirect approaches seem to be successful, nevertheless, although genetic transformation using recombinant DNA has the ability to save much time in the breeding process, they are not readily used by breeders yet. This is primarily due to legislative issues, economic issues, difficulties of implementing this technology in some ornamental plants, as well as how these techniques are publically perceived, particularly in Europe. Recently, newer and more precise genome-editing techniques have become available and they are already being implemented in some crops. New breeding techniques may help change the current situation and pave the way toward a legal and public acceptance if products of these technologies are indistinguishable from plants obtained by conventional techniques.

Transmembrane nine proteins in yeast and Arabidopsis affect cellular metal contents without changing vacuolar morphology

Transmembrane nine (TM9) proteins are localized in the secretory pathway of eukaryotic cells and are involved in cell adhesion and phagocytosis. The mechanism by which TM9 proteins operate is, however, not well understood. Here we have utilized elemental profiling by inductively coupled plasma mass spectrometry (ICP-MS) to further investigate the physiological function of TM9 proteins. Cellular copper contents in Saccharomyces cerevisiae varied depending on the presence of TM9 homologues from both yeast and Arabidopsis thaliana. A yeast tmn1-3 triple mutant lacking all three yeast endogenous TMNs showed altered metal homeostasis with a reduction in the cellular Cu contents to 25% of that in the wild-type. Conversely, when TMN1 was overexpressed in yeast, cellular Cu concentrations were more than doubled. Both Tmn1p-GFP and Tmn2p-GFP fusion proteins localized to the tonoplast. Yeast vacuolar biogenesis was not affected by the lack or presence of TM9 proteins neither in the tmn1-3 triple mutant nor in TM9 overexpressing strains. Heterologous expression in yeast of AtTMN7, a TM9 homologue from Arabidopsis, affected Cu homeostasis similar to the overexpression of TMN1. In Arabidopsis, the two TM9 homologues AtTMN1 and AtTMN7 were ubiquitously expressed. AtTMN7 promoter constructs driving the expression of GFP showed elevated expression of AtTMN7 in the root elongation zone. It is concluded that TM9 homologues from S. cerevisiae and A. thaliana have the ability to affect the intracellular Cu balance. Tmn1p and Tmn2p operate from the yeast vacuolar membrane without influencing vacuolar biogenesis. A new physiological function of the TM9 family coupled to vacuolar Cu homeostasis is proposed.

Increasing genetic variability in oilseed rape ( Brassica napus ) – Genotypes and phenotypes of oilseed rape transformed by wild type Agrobacterium rhizogenes

Brassica napus (oilseed rape) is a major oil crop worldwide. Due to the short domestication period of oilseed rape the genetic variability is limited compared to other crops. Transfer of rol and aux genes from Agrobacterium rhizogenes is used in horticulture to increase genetic variability. In the current study, we explore transformation by A. rhizogenes as a biotechnological approach in breeding for more branched and shorter oilseed rape. In the 2nd generation of transformed oilseed rape, branch numbers increased significantly by 49% from 7.7 ± 0.4 to 11.5 ± 1.9 when comparing rol+/aux+ plants with WT. Simultaneously, the apical height of plants was reduced by 25% from 81.3 ± 1.9 cm to 62.4 ± 6.7 cm in rol+/aux+ plants at the onset of flowering. Reproductive parameters affecting yield as seed size and number were negatively affected in rol+/aux+ plants. Interestingly, oil composition was changed in rol+/aux+ seeds. Oleic acid (ω9) contents were reduced by more than 3% whereas α-linolenic acid (ω6) increased by more than 25% in mature seeds. To obtain shorter and more branched breeding material of oilseed rape we suggest crossing plants with the rol+/aux+ genotype back into the parental breeding line. This could reduce the negative impact of rol+/aux+ on yield.

Hairy Root Cultures of Rhodiola rosea to Increase Valuable Bioactive Compounds

Rhodiola rosea, commonly known as roseroot, is an arctic and alpine plant distributed on the northern hemisphere. The plant has for long been used ethnobotanically as a means of increasing endurance and as a general cure against several diseases. Nowadays, the medicinal properties of roseroot have been characterized, and some of its important bioactive compounds are salidroside and the rosavinoids rosavin, rosarin, and rosin. The primary effect of the plant has been described as adaptogenic, i.e., providing a nonspecific broad-range response. Recently, R. rosea has increased in popularity which has led to overexploitation in nature, and new bio-sustainable production methods are needed for future production. Transformation with the soil bacterium Agrobacterium rhizogenes is a promising strategy to increase the natural content of bioactive compounds within plants. The increase of the bioactive compounds is caused by the root oncogenic loci (rol) genes, present on the transfer DNA within the bacterial plasmid. The rol genes are integrated in the plant host genome during transformation, causing formation of hairy roots. Other species in the Rhodiola genus have been successfully transformed by A. rhizogenes. However, several optimizations in terms of selection of superior plant lines, explant for transformation, and tissue culture are needed in order for R. rosea to serve as a platform for the production of bioactive compounds in hairy root cultures. Once established, several further measures could be taken to increase the content of bioactive compounds further, in that respect genome editing via the CRISPR/Cas9 system is emerging as a powerful beacon.