Michael Krogh Jensen

The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signalling

TFs (transcription factors) are modular proteins minimally containing a DBD (DNA-binding domain) and a TRD (transcription regulatory domain). NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] proteins comprise one of the largest plant TF families. They are key regulators of stress perception and developmental programmes, and most share an N-terminal NAC domain. On the basis of analyses of gene expression data and the phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and the divergent C-termini. Nine of the ten NAC domains analysed bind a previously identified conserved DNA target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analysed is dependent on the C-terminal region for transactivational activity. In silico analyses show that the NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder. Furthermore, ANAC019 was identified as a new positive regulator of ABA (abscisic acid) signalling, conferring ABA hypersensitivity when ectopically expressed in plants. Interestingly, ectopic expression of the ANAC019 DBD or TRD alone also resulted in ABA hypersensitivity. Expression of stress-responsive marker genes [COR47 (cold-responsive 47), RD29b (responsive-to-desiccation 29b) and ERD11 (early-responsive-to-dehydration 11)] were also induced by full-length and truncated ANAC019. Domain-swapping experiments were used to analyse the specificity of this function. Chimaeric proteins, where the NAC domain of ANAC019 was replaced with the analogous regions from other NAC TFs, also have the ability to positively regulate ABA signalling. In contrast, replacing the ANAC019 TRD with other TRDs abolished ANAC019-mediated ABA hypersensitivity. In conclusion, our results demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.

Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in bakers yeast Saccharomyces cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains. We applied our genome engineering tool for an exploratory analysis of all possible single, double, triple, quadruple and quintuple gene disruption combinations to search for strains with high mevalonate production, a key intermediate for the industrially important isoprenoid biosynthesis pathway. Even though we did not overexpress any genes in the mevalonate pathway, this analysis identified strains with mevalonate titers greater than 41-fold compared to the wild-type strain. Our findings illustrate the applicability of this highly specific and efficient multiplex genome engineering approach to accelerate functional genomics and metabolic engineering efforts.

In situ mineralization of nitorgen and phosphorus of arctic soils after perturbations simulating climate change

Seasonal net nitrogen (N) and phosphorus (P) mineralization was investigated at Abisko, Swedish Lapland in soils of a subarctic heath and in soils of a colder (by about 4° C), high altitude fellfield by (a) using in situ soil incubation in soils which had been shaded or subjected to two levels of increased temperature, combined with (b) reciprocal transplantation of soils between the two sites. Proportionally large and significant net seasonal mineralization of N, in contrast to non-significant P mineralization, was found in untransplanted and transplanted fellfield soil. In contrast, P was mineralized in proportionally large amounts, in contrast to low N mineralization, in the transplanted and untransplanted heath soil. The differences indicate that P was strongly immobilized in relation to N at the fellfield and that N was more strongly immobilized than P in the heath soil. The immobilization in both soils remained high even after a temperature change of 4–5° C experienced by transplanted soils. Air temperature increases of up to 4–5° C in greenhouses resulted in a soil temperature increase of 1–2° C and did not cause any extra increase of net N and P mineralization. The results suggest that soil temperature increases of up to 2° C, which are likely to occur by the end of the next century as an effect of a predicted 4–5° C rise in air temperature, have only small effects on net mineralization in at least two characteristic tundra soils. These effects are probably smaller than the natural fluctuation of plant available nutrients from site to site, even within the same plant community. A further soil temperature increase of up to 4–5° C may enhance decomposition and gross mineralization, but the rate of net mineralization, and hence the change of nutrient availability to the plants, depends on the extent of microbial immobilization of the extra nutrients released.

Structure, function and networks of transcription factors involved in abiotic stress responses.

Transcription factors (TFs) are master regulators of abiotic stress responses in plants. This review focuses on TFs from seven major TF families, known to play functional roles in response to abiotic stresses, including drought, high salinity, high osmolarity, temperature extremes and the phytohormone ABA. Although ectopic expression of several TFs has improved abiotic stress tolerance in plants, fine-tuning of TF expression and protein levels remains a challenge to avoid crop yield loss. To further our understanding of TFs in abiotic stress responses, emerging gene regulatory networks based on TFs and their direct targets genes are presented. These revealed components shared between ABA-dependent and independent signaling as well as abiotic and biotic stress signaling. Protein structure analysis suggested that TFs hubs of large interactomes have extended regions with protein intrinsic disorder (ID), referring to their lack of fixed tertiary structures. ID is now an emerging topic in plant science. Furthermore, the importance of the ubiquitin-proteasome protein degradation systems and modification by sumoylation is also apparent from the interactomes. Therefore; TF interaction partners such as E3 ubiquitin ligases and TF regions with ID represent future targets for engineering improved abiotic stress tolerance in crops.

Transcriptional regulation by an NAC (NAM–ATAF1,2–CUC2) transcription factor attenuates ABA signalling for efficient basal defence towards Blumeria graminis f. sp. hordei in Arabidopsis

ATAF1 is a member of a largely uncharacterized plant-specific gene family encoding NAC transcription factors, and is induced in response to various abiotic and biotic stimuli in Arabidopsis thaliana. Previously, we showed that a mutant allele of ATAF1 compromises penetration resistance in Arabidopsis with respect to the non-host biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh). In this study, we have used genome-wide transcript profiling to characterize signalling perturbations in ataf1 plants following Bgh inoculation. Comparative transcriptomic analyses identified an over-representation of abscisic acid (ABA)-responsive genes, including the ABA biosynthesis gene AAO3, which is significantly induced in ataf1 plants compared to wild-type plants following inoculation with Bgh. Additionally, we show that Bgh inoculation results in decreased endogenous ABA levels in an ATAF1-dependent manner, and that the ABA biosynthetic mutant aao3 showed increased penetration resistance to Bgh compared to wild-type plants. Furthermore, we show that ataf1 plants show ABA-hyposensitive phenotypes during seedling development and germination. Our data support a negative correlation between ABA levels and penetration resistance, and identify ATAF1 as a new stimuli-dependent attenuator of ABA signalling for the mediation of efficient penetration resistance in Arabidopsis upon Bgh attack.

The HvNAC6 transcription factor: a positive regulator of penetration resistance in barley and Arabidopsis

Pathogens induce the expression of many genes encoding plant transcription factors, though specific knowledge of the biological function of individual transcription factors remains scarce. NAC transcription factors are encoded in plants by a gene family with proposed functions in both abiotic and biotic stress adaptation, as well as in developmental processes. In this paper, we provide convincing evidence that a barley NAC transcription factor has a direct role in regulating basal defence. The gene transcript was isolated by differential display from barley leaves infected with the biotrophic powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh). The full-length cDNA clone was obtained using 5′-RACE and termed HvNAC6, due to its high similarity to the rice homologue, OsNAC6. Gene silencing of HvNAC6 during Bgh inoculation compromises penetration resistance in barley epidermal cells towards virulent Bgh. Complementing the effect of HvNAC6 gene silencing, transient overexpression of HvNAC6 increases the occurrence of penetration resistant cells towards Bgh attack. Quantitative RT-PCR shows the early and transient induction of HvNAC6 in barley epidermis upon Bgh infection. Additionally, our results show that the Arabidopsis HvNAC6 homologue ATAF1 is also induced by Bgh and the ataf1-1 mutant line shows decreased penetration resistance to this non-host pathogen. Collectively, these data suggest a conserved role of HvNAC6 and ATAF1 in the regulation of penetration resistance in monocots and dicots, respectively.

CRISPR/Cas9 advances engineering of microbial cell factories.

One of the key drivers for successful metabolic engineering in microbes is the efficacy by which genomes can be edited. As such there are many methods to choose from when aiming to modify genomes, especially those of model organisms like yeast and bacteria. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing with special emphasis on their application for metabolic engineering of yeast and bacteria. Also, examples of how nuclease-deficient Cas9 has been applied for RNA-guided transcriptional regulation of target genes will be reviewed, as well as tools available for computer-aided design of guide-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering.

ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin‐immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G]CGT as ATAF1 consensus binding sequences. Co‐expression analysis across publicly available microarray experiments identified 25 genes co‐expressed with ATAF1. The promoter regions of ATAF1 co‐expressors were significantly enriched for ATAF1 binding sites, and TTGCGTA was identified in the promoter of the key abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP‐qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis.

Effects of carbohydrate amendments on nutrient partitioning, plant and microbial performance of a grassland-shrub ecosystem

Nutrient partitioning, microbial and plant nutrient assimilation and biomass production were investigated after addition of carbon of low and high decomposability (sawdust and sugar, respectively) to the soil of a non-agricultural mixed grassland-shrub ecosystem at intervals during one growing season. The aim was to investigate how the energy source available for soil microorganisms, and the quality of the source, interacted with ecosystem nutrient circulation and plant performance. By the end of the season, sugar, but not sawdust amendment, had reduced the pools of soil inorganic N and P strongly whereas the pools of N and P in the microbial biomass had increased. Nutrient uptake and biomass production of herbs, but not of shrubs, declined to about one third of that in non-sugar amended soils, and tissue N and P concentrations declined. Total P in circulation between the soil inorganic, the microbial and the plant biomass pools was unchanged, whereas N declined as a result of a comparatively larger decrease in plant biomass N and soil inorganic N than the simultaneous gain in microbial biomass N. The results show, firstly, that microbial nutrient immobilization and competition with plants for inorganic soil nutrients can affect plant nutrient uptake and reduce biomass production strongly if microbial activity is stimulated by an increase of the available carbon in the soil. Secondly, the lack of increase of P or, for N, the decline of the amounts in circulation between the biological pools and the soil inorganic pool indicates that ecosystem gross mineralization was not stimulated when extra labile carbon became available. Hence, the increased microbial demand for nutrients without any stimulation of nutrient mineralization depleted the soil inorganic nutrient pool, led to reduced plant nutrient uptake and biomass production, and to a higher proportion of ecosystem nutrients allocated to the microbial biomass.

CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.