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Institute
Sonstige beteiligte Institutionen
Maintaining the balance between CO2 uptake and transpiration is important for plants and depends on tightly controlled turgor changes caused by the activity of various anion and cation channels. These channels are part of signaling cascades triggered, for example, by phytohormones such as ABA (abscisic acid) and JA (jasmonate), both of which act during drought stress in guard cells. In addition, JA is known to be involved in the plant's response to pathogen attack or wounding.
GORK (guard cell outward rectifying K+ channel) is the only known outward rectifying K+ channel in guard cells and therefore responsible for K+ efflux during stomatal closure.
In the course of this work it could be demonstrated by stomatal aperture assays, that GORK is an essential part of JA-induced stomatal closure. This is true for both triggers, leaf wounding as well as direct MeJA (methyl jasmonate) application. Patch clamp experiments on guard cell protoplasts backed this finding by revealing GORK K+ outward currents as a target of JA signaling in guard cells. As cytosolic Ca2+ signals are known to be involved in both ABA as well as JA signaling, the interaction of GORK with Ca2+-dependent kinases was examined consequently. An antagonistic regulation of GORK by
CIPK5-CBL1/9 complexes and ABI2 was identified by DEVC (double electrode voltage clamp) and protein-protein interaction experiments and backed up by in vitro kinase assays. Patch-clamp recordings on guard cell protoplasts of cipk5-2 kinase loss-of-function mutant revealed the importance of CIPK5 for JA-triggered stomatal closure via activation of GORK. The interaction of different CDPKs (Ca2+-dependent protein kinases) with GORK was also investigated.
Besides Ca2+ signaling also ROS (reactive oxygen species) production is essential in ABA and MeJA signaling. In DEVC experiments a reversible effect of ROS on GORK channel activity could be demonstrated, which could be one piece in the explanation of those ROS effects in ABA and MeJA signaling.
Genetic foundation of unrivaled survival strategies - Of water bears and carnivorous plants -
(2018)
All living organisms leverage mechanisms and response systems to optimize reproduction, defense, survival, and competitiveness within their natural habitat. Evolutionary theories such as the universal adaptive strategy theory (UAST) developed by John Philip Grime (1979) attempt to describe how these systems are limited by the trade-off between growth, maintenance and regeneration; known as the universal three-way trade-off. Grime introduced three adaptive strategies that enable organisms to coop with either high or low intensities of stress (e.g., nutrient deficiency) and environmental disturbance (e.g., seasons). The competitor is able to outcompete other organisms by efficiently tapping available resources in environments of low intensity stress and disturbance (e.g., rapid growers). A ruderal specism is able to rapidly complete the life cycle especially during high intensity disturbance and low intensity stress (e.g., annual colonizers). The stress tolerator is able to respond to high intensity stress with physiological variability but is limited to low intensity disturbance environments. Carnivorous plants like D. muscipula and tardigrades like M. tardigradum are two extreme examples for such stress tolerators. D. muscipula traps insects in its native habitat (green swamps in North and South Carolina) with specialized leaves and thereby is able to tolerate nutrient deficient soils. M. tardigradum on the other side, is able to escape desiccation of its terrestrial habitat like mosses and lichens which are usually covered by a water film but regularly fall completely dry. The stress tolerance of the two species is the central study object of this thesis. In both cases, high througput sequencing data and methods were used to test for transcriptomic (D. muscipula) or genomic adaptations (M. tardigradum) which underly the stress tolerance. A new hardware resource including computing cluster and high availability storage system was implemented in the first months of the thesis work to effectively analyze the vast amounts of data generated for both projects. Side-by-side, the data management resource TBro [14] was established together with students to intuitively approach complex biological questions and enhance collaboration between researchers of several different disciplines. Thereafter, the unique trapping abilities of D. muscipula were studied using a whole transcriptome approach. Prey-dependent changes of the transcriptional landscape as well as individual tissue-specific aspects of the whole plant were studied. The analysis revealed that non-stimulated traps of D. muscipula exhibit the expected hallmarks of any typical leaf but operates evolutionary conserved stress-related pathways including defense-associated responses when digesting prey. An integrative approach, combining proteome and transcriptome data further enabled the detailed description of the digestive cocktail and the potential nutrient uptake machinery of the plant. The published work [25] as well as a accompanying video material (https://www.eurekalert.org/pub_releases/ 2016-05/cshl-fgr042816.php; Video credit: Sönke Scherzer) gained global press coverage and successfully underlined the advantages of D. muscipula as experimental system to understand the carnivorous syndrome. The analysis of the peculiar stress tolerance of M. tardigradum during cryptobiosis was carried out using a genomic approach. First, the genome size of M. tardigradum was estimated, the genome sequenced, assembled and annotated. The first draft of M. tardigradum and the workflow used to established its genome draft helped scrutinizing the first ever released tardigrade genome (Hypsibius dujardini) and demonstrated how (bacterial) contamination can influence whole genome analysis efforts [27]. Finally, the
M. tardigradum genome was compared to two other tardigrades and all species present in the current release of the Ensembl Metazoa database. The analysis revealed that tardigrade genomes are not that different from those of other Ecdysozoa. The availability of the three genomes allowed the delineation of their phylogenetic position within the Ecdysozoa and placed them as sister taxa to the nematodes. Thereby, the comparative analysis helped to identify evolutionary trends within this metazoan lineage. Surprisingly, the analysis did not reveal general mechanisms (shared by all available tardigrade genomes) behind the arguably most peculiar feature of tardigrades; their enormous stress tolerance. The lack of molecular evidence for individual tardigrade species (e.g., gene expression data for M. tardigradum) and the non-existence of a universal experimental framework which enables hypothesis testing withing the whole phylum Tardigrada, made it nearly impossible to link footprints of genomic adaptations to the unusual physiological capabilities. Nevertheless, the (comparative) genomic framework established during this project will help to understand how evolution tinkered, rewired and modified existing molecular systems to shape the remarkable phenotypic features of tardigrades.
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)
8
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