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The evolutionary success of higher plants is largely attributed to their tremendous developmental
plasticity, which allows them to cope with adverse conditions. However, because these adaptations
require investments of resources, they must be tightly regulated to avoid unfavourable trade-offs.
Most of the resources required are macronutrients based on carbon and nitrogen. Limitations in the
availability of these nutrients have major effects on gene expression, metabolism, and overall plant
morphology. These changes are largely mediated by the highly conserved master kinase SNF1-RELATED
PROTEIN KINASE1 (SnRK1), which represses growth and induces catabolic processes. Downstream of
SnRK1, a hub of heterodimerising group C and S1 BASIC LEUCINE ZIPPER (bZIP) transcription factors has
been identified. These bZIPs act as regulators of nutrient homeostasis and are highly expressed in
strong sink tissues, such as flowers or the meristems that initiate lateral growth of both shoots and
roots. However, their potential involvement in controlling developmental responses through their
impact on resource allocation and usage has been largely neglected so far. Therefore, the objective of
this work was to elucidate the impact of particularly S1 bZIPs on gene expression, metabolism, and
plant development.
Due to the high homology and suspected partial redundancy of S1 bZIPs, higher order loss-of-function
mutants were generated using CRISPR-Cas9. The triple mutant bzip2/11/44 showed a variety of robust
morphological changes but maintained an overall growth comparable to wildtype plants. In detail
however, seedlings exhibited a strong reduction in primary root length. In addition, floral transition
was delayed, and siliques and seeds were smaller, indicating a reduced supply of resources to the shoot
and root apices. However, lateral root density and axillary shoot branching were increased, suggesting
an increased ratio of lateral to apical growth in the mutant. The full group S1 knockout
bzip1/2/11/44/53 showed similar phenotypes, albeit far more pronounced and accompanied by
growth retardation. Metabolomic approaches revealed that these architectural changes were
accompanied by reduced sugar levels in distal sink tissues such as flowers and roots. Sugar levels were
also diminished in leaf apoplasts, indicating that long distance transport of sugars by apoplastic phloem
loading was impaired in the mutants. In contrast, an increased sugar supply to the proximal axillary
buds and elevated starch levels in the leaves were measured. In addition, free amino acid levels were
increased in bzip2/11/44 and bzip1/2/11/44/53, especially for the important transport forms
asparagine and glutamine. The increased C and N availability in the proximal tissues could be the cause
of the increased axillary branching in the mutants.
To identify bZIP target genes that might cause the observed shifts in metabolic status, RNAseq
experiments were performed. Strikingly, clade III SUGARS WILL EVENTUALLY BE EXPORTED (SWEET)
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genes were abundant among the differentially expressed genes. As SWEETs are crucial for sugar export
to the apoplast and long-distance transport through the phloem, their reduced expression is likely to
be the cause of the observed changes in sugar allocation. Similarly, the reduced expression of
GLUTAMINE AMIDOTRANSFERASE 1_2.1 (GAT1_2.1), which exhibits glutaminase activity, could be an
explanation for the abundance of glutamine in the mutants. Additional experiments (ATAC-seq, DAPseq, PTA, q-RT-PCR) supported the direct induction of SWEETs and GAT1_2.1 by S1 bZIPs. To confirm
the involvement of these target genes in the observed S1 bZIP mutant phenotypes, loss-of-function
mutants were obtained, which showed moderately increased axillary branching. At the same time, the
induced overexpression of bZIP11 in axillary meristems had the opposite effect.
Collectively, a model is proposed for the function of S1 bZIPs in regulating sink tissue development. For
efficient long-distance sugar transport, bZIPs may be required to induce the expression of clade III
SWEETs. Thus, reduced SWEET expression in the S1 bZIP mutants would lead to a decrease in apoplastic
sugar loading and a reduced supply to distal sinks such as shoot or root apices. The reduction in longdistance transport could lead to sugar accumulation in the leaves, which would then increasingly be
transported via symplastic routes towards proximal sinks such as axillary branches and lateral roots or
sequestered as starch. The reduced GAT1_2.1 levels lead to an abundance of glutamine, a major
nitrogen transport form. The combined effect on C and N allocation results in increased nutrient
availability in proximal tissues, promoting the formation of lateral plant organs. Alongside emerging
evidence highlighting the power of bZIPs to steer nutrient allocation in other species, a novel but
evolutionary conserved role for S1 bZIPs as regulators of developmental plasticity is proposed, while
the generation of valuable data sets and novel genetic resources will help to gain a deeper
understanding of the molecular mechanisms involved
Photosynthetic plants have a remarkable ability to modify their metabolism and development according to ever changing environmental conditions. The root system displays continuous growth of the primary root and formation of lateral roots enabling efficient water and nutrient uptake and anchorage of the plant in soil. With regard to lateral roots, development is post-embryonic, originating from the pericycle of the primary root. Coordinated activity of several molecular signalling pathways controlled by the hormone auxin is important throughout all stages of lateral root development.At first, two adjacent Xylem Pole Pericycle (XPP) cells are activated and the nuclei of these cells migrate towards a common cell wall.This is followed by XPP cells acquiring volume thus swelling up.The XPP cells then undergo anticlinal cell division, followed by a series of periclinal and anticlinal divisions,leading to lateral root primordia.These break through the radial cell layers and emerge out the primary root.
Although root system plasticity is well-described in response to environmental cues such as ion nutrition in the soil, little is known on how root development is shaped according to the endogenous energy status of the plant.In this study, we were able to connect limited perturbations in photosynthetic energy supply to lateral root development.We established two experimental systems – treatment with low light and unexpected darkness which led to short-term energy imbalance in the plant.These short perturbations administered, showed an increase in the emerged lateral root density and decrease in root hexose availability and activation of the low energy marker gene ASN1 (ASPARAGINE SYNTHETASE 1).Although not demonstrated, presumably, these disturbances in the plant energy homeo-stasis activates SnRK1 (SNF1 RELATED KINASE 1),an evolutionary conserved kinase mediat-ing metabolic and transcriptional responses towards low energy conditions. In A. thaliana, two catalytic α-subunits of this kinase (SnRK1.α1 and SnRK1.α2) are functionally active and form ternary complexes with the regulatory β- and γ- subunits. Whereas unexpected darkness results in an increase in emerged lateral root density, the snrk1.α1 loss-of-function mutant displayed decrease in emerged lateral root density. As this effect is not that pronounced in the snrk1.α2 loss-of-function mutant, the α1 catalytic subunit is important for the observed lateral root phenotype under short-term energy perturbations. Moreover, root expression patterns of SnRK1.α1:GFP supports a role of this catalytic subunit in lateral root development. Furthermore, the lateral root response during short-term perturbations requires the SnRK1 downstream transcriptional regulator bZIP63 (BASIC LEU-CINE ZIPPER 63), as demonstrated here by a loss-of-function approach. Phenotypic studies showed that in comparison to wild-type, bzip63 mutants displayed decreased lateral root density upon low-light and unexpected darkness conditions. Previous work has demonstrat-ed that SnRK1 directly phosphorylates bZIP63 at three serine residues. Alanine-exchange mutants of the SnRK1 dependent bZIP63 phosphorylation sites behave similarly to bzip63 loss-of-function mutants and do not display increased lateral root density upon short-term unexpected darkness. This data strongly supports an impact of SnRK1-bZIP63 signalling in mediating the observed lateral root density phenotype. Plants expressing a bZIP63:YFP fu-sion protein showed specific localization patterns in primary root and in all developmental stages of the lateral root. bzip63 loss-of-function mutant lines displayed reduced early stage lateral root initiation events under unexpected darkness as demonstrated by Differen-tial Interference Contrast microscopy (DIC) and the use of a GATA23 reporter line. This data supports a role of bZIP63 in early lateral root initiation.
Next, by employing Chromatin Immunoprecitation (ChIP) sequencing, we were able to iden-tify global binding targets of bZIP63, including the auxin-regulated transcription factor (TF) ARF19 (AUXIN RESPONSE FACTOR 19), a well-described central regulator of lateral root development. Additional ChIP experiments confirmed direct binding of bZIP63 to an ARF19 promoter region harboring a G-Box cis-element, a well-established bZIP63 binding site. We also observed that short-term energy perturbation upon unexpected darkness induced tran-scription of ARF19, which was impaired in the bzip63 loss-of-function mutant. These results propose that bZIP63 mediates lateral root development under short-term energy perturba-tion via ARF19.
In conclusion, this study provides a novel mechanistic link between energy homeostasis and plant development. By employing reverse genetics, confocal imaging and high-throughput sequencing strategies, we were able to propose a SnRK1-bZIP63-ARF19 signalling module in integrating energy signalling into lateral root developmental programs.
Host–microbe interactions are the key to understand why and how microbes inhabit specific environments. With the scientific fields of microbial genomics and metagenomics, evolving on an unprecedented scale, one is able to gain insights in these interactions on a molecular and ecological level. The goal of this PhD thesis was to make (meta–)genomic data accessible, integrate it in a comparative manner and to gain comprehensive taxonomic and functional insights into bacterial strains and communities derived from two different environments: the phyllosphere of Arabidopsis thaliana and the mesohyl interior of marine sponges.
This thesis focused first on the de novo assembly of bacterial genomes. A 5–step protocol was developed, each step including a quality control. The examination of different assembly software in a comparative way identified SPAdes as most suitable. The protocol enables the user to chose the best tailored assembly. Contamination issues were solved by an initial filtering of the data and methods normally used for the binning of metagenomic datasets. This step is missed in many published assembly pipelines. The described protocol offers assemblies of high quality ready for downstream analysis.
Subsequently, assemblies generated with the developed protocol were annotated and explored
in terms of their function. In a first study, the genome of a phyllosphere bacterium, Williamsia sp. ARP1, was analyzed, offering many adaptions to the leaf habitat: it can deal with temperature shifts, react to oxygen species, produces mycosporins as protection against UV–light, and is able to uptake photosynthates. Further, its taxonomic position within the Actinomycetales was infered from 16S rRNA and comparative genomics showing the close relation between the genera Williamsia and Gordonia.
In a second study, six sponge–derived actinomycete genomes were investigated for secondary metabolism. By use of state–of–the–art software, these strains exhibited numerous gene clusters, mostly linked to polykethide synthases, non–ribosomal peptide synthesis, terpenes, fatty acids and saccharides. Subsequent predictions on these clusters offered a great variety of possible produced compounds with antibiotic, antifungal or anti–cancer activity. These analysis highlight the potential for the synthesis of natural products and the use of genomic data as screening toolkit.
In a last study, three sponge–derived and one seawater metagenomes were functionally compared. Different signatures regarding the microbial composition and GC–distribution were observed between the two environments. With a focus on bacerial defense systems, the data indicates a pronounced repertoire of sponge associated bacteria for bacterial defense systems, in particular, Clustered Regularly Interspaced Short Palindromic Repeats, restriction modification system, DNA phosphorothioation and phage growth limitation. In addition, characterizing genes for secondary metabolite cluster differed between sponge and seawater microbiomes. Moreover, a variety of Type I polyketide synthases were only found within the sponge microbiomes. With that, metagenomics are shown to be a useful tool for the screening of secondary metabolite genes. Furthermore, enriched defense systems are highlighted as feature of sponge-associated microbes and marks them as a selective trait.