Anna Amtmann

Trehalose 6‐phoshpate (Tre6P) is a key signalling molecule that reflects carbon status and integrates it with developmental decision making. Tre6P has been demonstrated to regulate various developmental processes, including vegetative growth, shoot branching, flowering, and root branching. Here, we investigate how vasculature‐derived Tre6P influences root system architecture by expressing heterologous Tre6P synthase or Tre6P phosphatase (TPP) specifically in the plant vasculature. Plants with elevated vascular‐derived Tre6P levels had smaller root systems, reduced sucrose levels, and lower root metabolite levels, whereas vascular TPP overexpression had the opposite effect. Using reciprocal grafting experiments, we identified shoot‐derived vascular Tre6P to be the main driver of these systemic responses. In lines with increased Tre6P in the shoot vasculature, the shoot‐to‐root metabolite ratios were consistently increased, indicating that Tre6P modulates metabolite allocation and utilisation to balance carbon partitioning between shoot and root growth. Our data further suggest that vascular Tre6P contributed to optimising the carbon‐to‐nitrogen ratio to support growth and fitness. Besides this systemic function, this study shows that root‐derived Tre6P also plays a critical local role in modulating root development. Collectively, our results demonstrate that Tre6P in the vasculature functions both systemically and locally to coordinate metabolic status with root growth and architecture.

Photosynthetic cyanobacteria can be utilised in biotechnology as environmentally sustainable cell factories to convert CO₂ into a diverse range of biochemicals. However, a lack of molecular tools available for precise and dynamic control of gene expression hinders metabolic engineering and contributes to low product titres. Optogenetic tools enable light-regulated control of gene expression with high tunability and reversibility. To date, their application in cyanobacteria is limited and transferability between species remains unclear. In this study, we expressed the blue light-repressible YF1/FixJ and the green/red light-responsive CcaS/CcaR systems in Synechococcus sp. PCC 7002 and characterised their performance using GFP fluorescence assays and qRT-PCR. The YF1/FixJ system of non-cyanobacterial origin showed poor performance with a maximum dynamic range of 1.5-fold despite several steps to improve this. By contrast, the CcaS/CcaR system originating from the cyanobacterium Synechocystis sp. PCC 6803 responded well to light wavelengths and intensities, with a 6-fold increased protein fluorescence output observed after 30 min of green light. Monitoring GFP transcript levels allowed us to quantify the kinetics of transcriptional activation and deactivation and to test the effect of both multiple green/red and light/dark cycles on system performance. Finally, we increased CcaS/CcaR system activity under green light through targeted genetic modifications to the pCpcG2 output promoter. This study provides a detailed characterisation of the behaviour of the CcaS/CcaR system in Synechococcus sp. PCC 7002, as well as underlining the complexity of transferring optogenetic tools across species.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step‐by‐step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data‐modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas‐exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

Summary: Early responses of plants to environmental stress factors prevent damage but can delay growth and development in fluctuating conditions. Optimising these trade‐offs requires tunability of plant responsiveness to environmental signals. We have previously reported that Histone Deacetylase Complex 1 (HDC1), which interacts with multiple proteins in histone deacetylation complexes, regulates the stress responsiveness of Arabidopsis seedlings, but the underlying mechanism remained elusive. Here, we show that HDC1 attenuates transcriptome re‐programming in salt‐treated seedlings, and we identify two genes ( LEA and MAF5 ) that inhibit seedling establishment under salt stress downstream of HDC1. HDC1 attenuates their transcriptional induction by salt via a dual mechanism involving H3K9/14 deacetylation and H3K27 trimethylation. The latter, but not the former, was also abolished in a triple knockout mutant of the linker histone H1, which partially mimics the hypersensitivity of the hdc1‐1 mutant to salt stress. Although stress‐induced H3K27me3 accumulation required both H1 and HDC1, it was not fully recovered by complementing hdc1‐1 with a truncated, H1‐binding competent HDC1 suggesting other players or independent inputs. The combined findings reveal a dual brake function of HDC1 via regulating both active and repressive epigenetic marks on stress‐inducible genes. This natural ‘anti‐panic’ device offers a molecular leaver to tune stress responsiveness in plants.

Microorganisms produce extracellular polymeric substances (EPS, also known as exopolysaccharides) of diverse composition and structure. The biochemical and biophysical properties of these biopolymers enable a wide range of industrial applications. EPS from cyanobacteria are particularly versatile as they incorporate a larger number and variety of building blocks and adopt more complex structures than EPS from other organisms. However, the genetic makeup and regulation of EPS biosynthetic pathways in cyanobacteria are poorly understood. Here, we measured the effect of changing culture media on titre and composition of EPS released by Synechocystis sp. PCC 6803, and we integrated this information with transcriptomic data. Across all conditions, daily EPS productivity of individual cells was highest in the early growth phase, but the total amount of EPS obtained from the cultures was highest in the later growth phases due to accumulation. Lowering the magnesium concentration in the media enhanced per-cell productivity but the produced EPS had a lower total sugar content. Levels of individual monosaccharides correlated with specific culture media components, e.g. xylose with sulfur, glucose and N-acetyl-galactosamine with NaCl. Comparison with RNA sequencing data suggests a Wzy-dependent biosynthetic pathway and a protective role for xylose-rich EPS. This multi-level analysis offers a handle to link individual genes to the dynamic modulation of a complex biopolymer. Key points: • Synechocystis exopolysaccharide amount and composition depends on culture condition • Production rate and sugar content can be modulated by Mg and S respectively • Wzy-dependent biosynthetic pathway and protective role proposed for xylose-rich EPS Graphical

The cyanobacteriochrome GAF domains represent a trove of spectral diversity. These proteins are endemic to cyanobacteria and sense the color and power of light. Multiple mechanisms are used to tune the natural absorbance spectrum of the bound bilin chromophore. In practice, these are difficult to identify from the predicted amino acid sequence. Their individual presence rarely yields a consistent and predictable outcome. The absorbance characteristics of the GAF domain are a complex function of many such tuning mechanisms. This implies that a more combinatoric approach to characterizing the diversity of GAF domains would better to predict spectral tunes. We reviewed the literature and constructed a dataset of predicted/confirmed cyanobacteriochrome GAF domains. This dataset was subjected to multiple sequence alignments and 18 GAF domain families were defined. The amino acid sequence similarity correlated well with known spectral characteristics but there were exceptions. A second approach to predict chromotype involved using Principal Component Analysis to characterize the whole domain architectures of cyanobacteriochrome. This approach identified 7 conserved domain architectures, with some variations. These also offered a correlation to the spectral tune of the GAF domains therein, in addition to the 18 GAF families. The three-dimensional structures of 98 spectrally characterized GAF domains were predicted using Phyre2. Subsequent grouping based on distance maps offered an insight into how the general spectral position of the domain is set. Finer tuning is likely to be achieved by means of six key residues within the binding pocket. Taken together, these insights allowed us to carry out a Multiple Correlation Analysis serving as a mathematical summary of the diversity of cyanobacteriochrome GAF domains. This summary or “cyanobacteriochrome atlas” can be used to make spectral predictions on uncharacterized GAF domains.

Summary: Plant adaptation to a desert environment and its endemic heat stress is poorly understood at the molecular level. The naturally heat‐tolerant Brassicaceae species Anastatica hierochuntica is an ideal extremophyte model to identify genetic adaptations that have evolved to allow plants to tolerate heat stress and thrive in deserts. We generated an A. hierochuntica reference transcriptome and identified extremophyte adaptations by comparing Arabidopsis thaliana and A. hierochuntica transcriptome responses to heat, and detecting positively selected genes in A. hierochuntica. The two species exhibit similar transcriptome adjustment in response to heat and the A. hierochuntica transcriptome does not exist in a constitutive heat ‘stress‐ready’ state. Furthermore, the A. hierochuntica global transcriptome as well as heat‐responsive orthologs, display a lower basal and higher heat‐induced expression than in A. thaliana. Genes positively selected in multiple extremophytes are associated with stomatal opening, nutrient acquisition, and UV‐B induced DNA repair while those unique to A. hierochuntica are consistent with its photoperiod‐insensitive, early‐flowering phenotype. We suggest that evolution of a flexible transcriptome confers the ability to quickly react to extreme diurnal temperature fluctuations characteristic of a desert environment while positive selection of genes involved in stress tolerance and early flowering could facilitate an opportunistic desert lifestyle.

Transport of water, ions and signals from roots to leaves via the xylem vessels is essential for plant life and needs to be tightly regulated. The final composition of the transpiration stream before passage into the shoots is controlled by the xylem-adjacent cell layers, namely xylem parenchyma and pericycle, in the upper part of the root. To unravel regulatory networks in this strategically important location, we generated Arabidopsis lines expressing a nuclear tag under the control of the HKT1 promoter. HKT1 retrieves sodium from the xylem to prevent toxic levels in the shoot, and this function depends on its specific expression in upper root xylem-adjacent tissues. Based on FACS RNA-sequencing and INTACT ChIP-sequencing, we identified the gene repertoire that is preferentially expressed in the tagged cell types and discovered transcription factors experiencing cell-type specific loss of H3K27me3 demethylation. For one of these, ZAT6, we show that H3K27me3-demethylase REF6 is required for de-repression. Analysis of zat6 mutants revealed that ZAT6 activates a suite of cell-type specific downstream genes and restricts Na ⁺ accumulation in the shoots. The combined Files open novel opportunities for ‘bottom-up’ causal dissection of cell-type specific regulatory networks that control root-to-shoot communication under environmental challenge.