Identifying the mechanistic basis to nitrogen responsiveness in two contrasting Setaria italica accessions.

Nitrogen (N) is a macronutrient limiting crop productivity with varied requirements across species and genotypes. Understanding the mechanistic basis of N responsiveness by comparing contrasting genotypes could inform the development and selection of varieties with lower N demands, or inform agronomic practices to sustain yields with lower N inputs. Given the established role of millets in ensuring climate-resilient food and nutrition security, we investigated the physiological and genetic basis of nitrogen responsiveness in foxtail millet (Setaria italica L.). We had previously identified genotypic variants linked to N responsiveness, and here, we dissect the mechanistic basis of the trait by examining the physiological and molecular behaviour of N responsive (NRp-SI58) and non-responsive (NNRp-SI114) accessions at high and low N. Under high N, NRp-SI58 allocates significantly more biomass to nodes, internodes and roots, more N to developing grains, and is more effective at remobilising flag leaf N compared to NNRp-SI114. Post anthesis flag leaf gene expression suggests that differences in N induce much higher transcript abundance in NNRp-SI114 than NRp-SI58, a large proportion of which are potentially regulated by APETALA2 (AP2) transcription factors. Overall, the study provides novel insights into the regulation and manipulation of N responsiveness in S. italica.

Protocol to isolate nuclei from Chlamydomonas reinhardtii for ATAC sequencing.

The isolation of sufficient amounts of intact nuclei is essential to obtain high-resolution maps of chromatin accessibility via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Here, we present a protocol for tag-free isolation of nuclei from both cell walled and cell wall-deficient strains of the green model alga Chlamydomonas reinhardtii at a suitable quality for ATAC-seq. We describe steps for nuclei isolation, quantification, and downstream ATAC-seq. This protocol is optimized to shorten the time of isolation and quantification of nuclei.

Epiphytic CAM bromeliads indicate vulnerability of tropical forest communities to climate change.

BACKGROUND AND SCOPE: Vascular epiphytes have a variety of mechanisms to trap and retain water, including crassulacean acid metabolism (CAM). Niche segregation was investigated for epiphytic bromeliads on the tropical Caribbean island of Trinidad, where habitats range from lowland deciduous forests to high-rainfall montane tropical forests, ~1000 m in elevation. METHODS: Four tank-impounding bromeliad epiphytes in the genus Aechmea (Ae. aquilega, Ae. fendleri, Ae. nudicaulis and Ae. dichlamydea) with CAM were mapped across their distinct geographical and elevational zonations in northern Trinidad and Tobago. Species distribution modelling was used to determine environmental limitations for each species. Anatomical and physiological measurements included leaf succulence traits, gas exchange and CAM activity; hydraulic conductance and vulnerability; stomatal sensitivity and quantum yield responses to nocturnal temperature and long-term water deficits. KEY RESULTS: A total of 2876 field observations identified the transitions between the lowland Ae. aquilega and montane Ae. fendleri, occurring >500 m a.s.l. at the drier western end of the Northern Mountain Range and at progressively lower elevations towards the wetter, eastern region. Anatomical and physiological sensitivities of gas exchange, CAM activity and water use, and responses to elevated nocturnal temperatures and drought, were markedly different for Ae. fendleri compared with Ae. aquilega or the ubiquitous Ae. nudicaulis. CONCLUSIONS: The species distribution model highlighted the susceptibility of Ae. fendleri to a changing climate. For each species, physiological and anatomical traits were tailored to environmental tolerances, consistent with specialist or generalist niche preferences. Using Intergovernmental Panel on Climate Change scenarios, we predict that rapid rainfall and temperature changes will lead to the loss of Ae. fendleri and associated lower (and upper) montane forest communities from Trinidad, seriously impacting both biodiversity and critical ecosystem functions here and in other tropical island habitats. Epiphytic bromeliads act as markers for threatened communities, and their physiological tolerances represent key indicators of climate change impacts.

Prospects and perspectives: inferring physiological and regulatory targets for CAM from molecular and modelling approaches.

BACKGROUND AND SCOPE: This review summarizes recent advances in our understanding of Crassulacean Acid Metabolism (CAM) by integrating evolutionary, ecological, physiological, metabolic and molecular perspectives. A number of key control loops which moderate the expression of CAM phases, and their metabolic and molecular control, are explored. These include nocturnal stomatal opening, activation of phosphoenolpyruvate carboxylase by a specific protein kinase, interactions with circadian clock control, as well as daytime decarboxylation and activation of Rubisco. The vacuolar storage and release of malic acid and the interplay between the supply and demand for carbohydrate reserves are also key metabolic control points. FUTURE OPPORTUNITIES: We identify open questions and opportunities, with experimentation informed by top-down molecular modelling approaches allied with bottom-up mechanistic modelling systems. For example, mining transcriptomic datasets using high-speed systems approaches will help to identify targets for future genetic manipulation experiments to define the regulation of CAM (whether circadian or metabolic control). We emphasize that inferences arising from computational approaches or advanced nuclear sequencing techniques can identify potential genes and transcription factors as regulatory targets. However, these outputs then require systematic evaluation, using genetic manipulation in key model organisms over a developmental progression, combining gene silencing and metabolic flux analysis and modelling to define functionality across the CAM day-night cycle. From an evolutionary perspective, the origins and function of CAM succulents and responses to water deficits are set against the mesophyll and hydraulic limitations imposed by cell and tissue succulence in contrasting morphological lineages. We highlight the interplay between traits across shoots (3D vein density, mesophyll conductance and cell shrinkage) and roots (xylem embolism and segmentation). Thus, molecular, biophysical and biochemical processes help to curtail water losses and exploit rapid rehydration during restorative rain events. In the face of a changing climate, we hope such approaches will stimulate opportunities for future research.

GWAS identifies genetic loci underlying nitrogen responsiveness in the climate resilient C4 model Setaria italica (L.).

INTRODUCTION: N responsiveness is the capacity to perceive and induce morpho-physiological adaptation to external and internal Nitrogen (N). Crop productivity is propelled by N fertilizer and requires the breeding/selection of cultivars with intrinsically high N responsiveness. This trait has many advantages in being more meaningful in commercial/environmental context, facilitating in-season N management and not being inversely correlated with N availability over processes regulating NUE. Current lack of its understanding at the physio-genetic basis is an impediment to select for cultivars with a predictably high N response. OBJECTIVES: To dissect physio-genetic basis of N responsiveness in 142 diverse population of foxtail millet, Setaria italica (L.) by employing contrasting N fertilizer nutrition regimes. METHODS: We phenotyped S. italica accessions for major yield related traits under low (N10, N25) and optimal (N100) growth conditions and genotyped them to subsequently perform a genome-wide association study to identify genetic loci associated with nitrogen responsiveness trait. Groups of accessions showing contrasting trait performance and allelic forms of specific linked genetic loci (showing haplotypes) were further accessed for N dependent transcript abundances of their proximal genes. RESULTS: Our study show that N dependent yield rise in S. italica is driven by grain number whose responsiveness to N availability is genetically underlined. We identify 22 unique SNP loci strongly associated with this trait out of which six exhibit haplotypes and consistent allelic variation between lines with contrasting N dependent grain number response and panicle architectures. Furthermore, differential transcript abundances of specific genes proximally linked to these SNPs in same lines is indicative of their N dependence in a genotype specific manner. CONCLUSION: The study demonstrates the value/ potential of N responsiveness as a selection trait and identifies key genetic components underlying the trait in S. italica. This has major implications for improving crop N sustainability and food security.

The Other Mechanisms by Which the Rht Genes Improve the Harvest Index of Wheat.

Uncovering the mechanism that underlies the relationship between crop height and grain yield would potentially inform the strategies for improving wheat with optimal height. The aim of the research reported here was to identify the attributes able to produce wheat yield increases in Rht genotypes without further straw-shortening. Attention was given to examination in a controlled environment the question of the mechanistic foundation that determined the relationship between wheat height and yield in lines (Rht-B1b, Rht-D1b, Rht-B1c, Rht-D1c) compared to wild types in Mercia background. In addition to height reduction, this research revealed three other mechanisms by which the Rht genes may also improve the Harvest Index (HI) of wheat: (i) low Specific Leaf Area (SLA), (ii) increased Mean Residence Time (MRT) of Nitrogen (N), and (iii) increased grain number on spike.

Over-expression of TaDWF4 increases wheat productivity under low and sufficient nitrogen through enhanced carbon assimilation.

There is a strong pressure to reduce nitrogen (N) fertilizer inputs while maintaining or increasing current cereal crop yields. We show that overexpression of TaDWF4-B, the dominant shoot expressed homoeologue of OsDWF4, in wheat can increase plant productivity by up to 105% under a range of N levels on marginal soils, resulting in increased N use efficiency (NUE). We show that a two to four-fold increase in TaDWF4 transcript levels enhances the responsiveness of genes regulated by N. The productivity increases seen were primarily due to the maintenance of photosystem II operating efficiency and carbon assimilation in plants when grown under limiting N conditions and not an overall increase in photosynthesis capacity. The increased biomass production and yield per plant in TaDWF4 OE lines could be linked to modified carbon partitioning and changes in expression pattern of the growth regulator Target Of Rapamycin, offering a route towards breeding for sustained yield and lower N inputs.

Stable isotope signals provide seasonal climatic markers for moss functional groups.

Living moss biomass and archival peat deposits represent key indicators of present and past climatic conditions, but prediction of future climatic impacts requires appropriate marker species to be characterized under a range of contemporary conditions. Stable isotope signals in high latitude moss deposits offer potential climatic proxies. Seasonal changes in δ13C and δ18O of organic material (cellulose) in representative functional groups, and associated photosynthetic activity (as chlorophyll fluorescence) have been compared across East Anglia, UK, as a function of tissue water content. Representative species from contrasting acid bog, heathland, and fen woodland habitats were selected for monthly sampling of recent growth tissues between spring 2017 and autumn 2018, with isotopic signals in purified cellulose compared with tissue water, precipitation, and nearby groundwater signals. Sphagnum and Polytrichum groups, which tend to dominate peat formation, provided contrasting and complementary indicators of seasonal variations in carbon assimilation. Cellulose δ18O signals from Sphagnum spp. demonstrate seasonal variations in source precipitation inputs; carbon isotope signals in Polytrichum spp. indicate evaporative demand and photosynthetic limitation.

Leaf water δ18O reflects water vapour exchange and uptake by C3 and CAM epiphytic bromeliads in Panama.

The distributions of CAM and C3 epiphytic bromeliads across an altitudinal gradient in western Panama were identified from carbon isotope (δ13C) signals, and epiphyte water balance was investigated via oxygen isotopes (δ18O) across wet and dry seasons. There were significant seasonal differences in leaf water (δ18Olw), precipitation, stored 'tank' water and water vapour. Values of δ18Olw were evaporatively enriched at low altitude in the dry season for the C3 epiphytes, associated with low relative humidity (RH) during the day. Crassulacean acid metabolism (CAM) δ18Olw values were relatively depleted, consistent with water vapour uptake during gas exchange under high RH at night. At high altitude, cloudforest locations, C3 δ18Olw also reflected water vapour uptake by day. A mesocosm experiment with Tillandsia fasciculata (CAM) and Werauhia sanguinolenta (C3) was combined with simulations using a non-steady-state oxygen isotope leaf water model. For both C3 and CAM bromeliads, δ18Olw became progressively depleted under saturating water vapour by day and night, although evaporative enrichment was restored in the C3 W. sanguinolenta under low humidity by day. Source water in the overlapping leaf base 'tank' was also modified by evaporative δ18O exchanges. The results demonstrate how stable isotopes in leaf water provide insights for atmospheric water vapour exchanges for both C3 and CAM systems.

Orchestral manoeuvres in the light: crosstalk needed for regulation of the Chlamydomonas carbon concentration mechanism.

The inducible carbon concentration mechanism (CCM) in Chlamydomonas reinhardtii has been well defined from a molecular and ultrastructural perspective. Inorganic carbon transport proteins, and strategically located carbonic anhydrases deliver CO2 within the chloroplast pyrenoid matrix where Rubisco is packaged. However, there is little understanding of the fundamental signalling and sensing processes leading to CCM induction. While external CO2 limitation has been believed to be the primary cue, the coupling between energetic supply and inorganic carbon demand through regulatory feedback from light harvesting and photorespiration signals could provide the original CCM trigger. Key questions regarding the integration of these processes are addressed in this review. We consider how the chloroplast functions as a crucible for photosynthesis, importing and integrating nuclear-encoded components from the cytoplasm, and sending retrograde signals to the nucleus to regulate CCM induction. We hypothesize that induction of the CCM is associated with retrograde signals associated with photorespiration and/or light stress. We have also examined the significance of common evolutionary pressures for origins of two co-regulated processes, namely the CCM and photorespiration, in addition to identifying genes of interest involved in transcription, protein folding, and regulatory processes which are needed to fully understand the processes leading to CCM induction.

Latitudinal Biogeographic Structuring in the Globally Distributed Moss Ceratodon purpureus.

Biogeographic patterns of globally widespread species are expected to reflect regional structure, as well as connectivity caused by occasional long-distance dispersal. We assessed the level and drivers of population structure, connectivity, and timescales of population isolation in one of the most widespread and ruderal plants in the world - the common moss Ceratodon purpureus. We applied phylogenetic, population genetic, and molecular dating analyses to a global (n = 147) sampling data set, using three chloroplast loci and one nuclear locus. The plastid data revealed several distinct and geographically structured lineages, with connectivity patterns associated with worldwide, latitudinal "bands." These imply that connectivity is strongly influenced by global atmospheric circulation patterns, with dispersal and establishment beyond these latitudinal bands less common. Biogeographic patterns were less clear within the nuclear marker, with gene duplication likely hindering the detection of these. Divergence time analyses indicated that the current matrilineal population structure in C. purpureus has developed over the past six million years, with lineages diverging during the late Miocene, Pliocene, and Quaternary. Several colonization events in the Antarctic were apparent, as well as one old and distinct Antarctic clade, possibly isolated on the continent since the Pliocene. As C. purpureus is considered a model organism, the matrilineal biogeographic structure identified here provides a useful framework for future genetic and developmental studies on bryophytes. Our general findings may also be relevant to understanding global environmental influences on the biogeography of other organisms with microscopic propagules (e.g., spores) dispersed by wind.

Identification of Quantitative Trait Loci Relating to Flowering Time, Flag Leaf and Awn Characteristics in a Novel Triticum dicoccum Mapping Population.

Tetraploid landraces of wheat harbour genetic diversity that could be introgressed into modern bread wheat with the aid of marker-assisted selection to address the genetic diversity bottleneck in the breeding genepool. A novel bi-parental Triticum turgidum ssp. dicoccum Schrank mapping population was created from a cross between two landrace accessions differing for multiple physiological traits. The population was phenotyped for traits hypothesised to be proxies for characteristics associated with improved photosynthesis or drought tolerance, including flowering time, awn length, flag leaf length and width, and stomatal and trichome density. The mapping individuals and parents were genotyped with the 35K Wheat Breeders' single nucleotide polymorphism (SNP) array. A genetic linkage map was constructed from 104 F4 individuals, consisting of 2066 SNPs with a total length of 3295 cM and an average spacing of 1.6 cM. Using the population, 10 quantitative trait loci (QTLs) for five traits were identified in two years of trials. Three consistent QTLs were identified over both trials for awn length, flowering time and flag leaf width, on chromosomes 4A, 7B and 5B, respectively. The awn length and flowering time QTLs correspond with the major loci Hd and Vrn-B3, respectively. The identified marker-trait associations could be developed for marker-assisted selection, to aid the introgression of diversity from a tetraploid source into modern wheat for potential physiological trait improvement.

Rubisco and carbon-concentrating mechanism co-evolution across chlorophyte and streptophyte green algae.

Green algae expressing a carbon-concentrating mechanism (CCM) are usually associated with a Rubisco-containing micro-compartment, the pyrenoid. A link between the small subunit (SSU) of Rubisco and pyrenoid formation in Chlamydomonas reinhardtii has previously suggested that specific RbcS residues could explain pyrenoid occurrence in green algae. A phylogeny of RbcS was used to compare the protein sequence and CCM distribution across the green algae and positive selection in RbcS was estimated. For six streptophyte algae, Rubisco catalytic properties, affinity for CO2 uptake (K0.5 ), carbon isotope discrimination (δ13 C) and pyrenoid morphology were compared. The length of the ßA-ßB loop in RbcS provided a phylogenetic marker discriminating chlorophyte from streptophyte green algae. Rubisco kinetic properties in streptophyte algae have responded to the extent of inducible CCM activity, as indicated by changes in inorganic carbon uptake affinity, δ13 C and pyrenoid ultrastructure between high and low CO2 conditions for growth. We conclude that the Rubisco catalytic properties found in streptophyte algae have coevolved and reflect the strength of any CCM or degree of pyrenoid leakiness, and limitations to inorganic carbon in the aquatic habitat, whereas Rubisco in extant land plants reflects more recent selective pressures associated with improved diffusive supply of the terrestrial environment.

Model approaches to advance crassulacean acid metabolism system integration.

This review summarises recent progress in understanding crassulacean acid metabolism (CAM) systems and the integration of internal and external stimuli to maximise water-use efficiency. Complex CAM traits have been reduced to their minimum and captured as computational models, which can now be refined using recently available data from transgenic manipulations and large-scale omics studies. We identify three key areas in which an appropriate choice of modelling tool could help capture relevant comparative molecular data to address the evolutionary drivers and plasticity of CAM. One focus is to identify the environmental and internal signals that drive inverse stomatal opening at night. Secondly, it is important to identify the regulatory processes required to orchestrate the diel pattern of carbon fluxes within mesophyll layers. Finally, the limitations imposed by contrasting succulent systems and associated hydraulic conductance components should be compared in the context of water-use and evolutionary strategies. While network analysis of transcriptomic data can provide insights via co-expression modules and hubs, alternative forms of computational modelling should be used iteratively to define the physiological significance of key components and informing targeted functional gene manipulation studies. We conclude that the resultant improvements of bottom-up, mechanistic modelling systems can enhance progress towards capturing the physiological controls for phylogenetically diverse CAM systems in the face of the recent surge of information in this omics era.

A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii.

A phase-separated, liquid-like organelle called the pyrenoid mediates CO2 fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model alga Chlamydomonas that has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant's phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO2-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

A Roadmap for Lowering Crop Nitrogen Requirement.

Increasing nitrogen fertilizer applications have sustained a growing world population in the 20th century. However, to avoid any further associated environmental damage, new sustainable agronomic practices together with new cultivars must be developed. To date the concept of nitrogen use efficiency (NUE) has been useful in quantifying the processes of nitrogen uptake and utilization, but we propose a shift in focus to consider nitrogen responsiveness as a more appropriate trait to select varieties with lower nitrogen requirements. We provide a roadmap to integrate the regulation of nitrogen uptake and assimilation into varietal selection and crop breeding programs. The overall goal is to reduce nitrogen inputs by farmers growing crops in contrasting cropping systems around the world, while sustaining yields and reducing greenhouse gas (GHG) emissions.

Bryophyte stable isotope composition, diversity and biomass define tropical montane cloud forest extent.

Liverworts and mosses are a major component of the epiphyte flora of tropical montane forest ecosystems. Canopy access was used to analyse the distribution and vertical stratification of bryophyte epiphytes within tree crowns at nine forest sites across a 3400 m elevational gradient in Peru, from the Amazonian basin to the high Andes. The stable isotope compositions of bryophyte organic material (13C/12C and 18O/16O) are associated with surface water diffusive limitations and, along with C/N content, provide a generic index for the extent of cloud immersion. From lowland to cloud forest δ13C increased from -33‰ to -27‰, while δ18O increased from 16.3‰ to 18.0‰. Epiphytic bryophyte and associated canopy soil biomass in the cloud immersion zone was estimated at up to 45 t dry mass ha-1, and overall water holding capacity was equivalent to a 20 mm precipitation event. The study emphasizes the importance of diverse bryophyte communities in sequestering carbon in threatened habitats, with stable isotope analysis allowing future elevational shifts in the cloud base associated with changes in climate to be tracked.

Economic and hydraulic divergences underpin ecological differentiation in the Bromeliaceae.

Leaf economic and hydraulic theories have rarely been applied to the ecological differentiation of speciose herbaceous plant radiations. The role of character trait divergences and network reorganization in the differentiation of the functional types in the megadiverse Neotropical Bromeliaceae was explored by quantifying a range of leaf economic and hydraulic traits in 50 diverse species. Functional types, which are defined by combinations of C3 or Crassulacean acid metabolism (CAM) photosynthesis, terrestrial or epiphytic habits, and non-specialized, tank-forming or atmospheric morphologies, segregated clearly in trait space. Most classical leaf economic relationships were supported, but they were weakened by the presence of succulence. Functional types differed in trait-network architecture, suggesting that rewiring of trait-networks caused by innovations in habit and photosynthetic pathway is an important aspect of ecological differentiation. The hydraulic data supported the coupling of leaf hydraulics and gas exchange, but not the hydraulic safety versus efficiency hypothesis, and hinted at an important role for the extra-xylary compartment in the control of bromeliad leaf hydraulics. Overall, our findings highlight the fundamental importance of structure-function relationships in the generation and maintenance of ecological diversity.

Unexpected Connections between Humidity and Ion Transport Discovered Using a Model to Bridge Guard Cell-to-Leaf Scales.

Stomatal movements depend on the transport and metabolism of osmotic solutes that drive reversible changes in guard cell volume and turgor. These processes are defined by a deep knowledge of the identities of the key transporters and of their biophysical and regulatory properties, and have been modeled successfully with quantitative kinetic detail at the cellular level. Transpiration of the leaf and canopy, by contrast, is described by quasilinear, empirical relations for the inputs of atmospheric humidity, CO2, and light, but without connection to guard cell mechanics. Until now, no framework has been available to bridge this gap and provide an understanding of their connections. Here, we introduce OnGuard2, a quantitative systems platform that utilizes the molecular mechanics of ion transport, metabolism, and signaling of the guard cell to define the water relations and transpiration of the leaf. We show that OnGuard2 faithfully reproduces the kinetics of stomatal conductance in Arabidopsis thaliana and its dependence on vapor pressure difference (VPD) and on water feed to the leaf. OnGuard2 also predicted with VPD unexpected alterations in K+ channel activities and changes in stomatal conductance of the slac1 Cl- channel and ost2 H+-ATPase mutants, which we verified experimentally. OnGuard2 thus bridges the micro-macro divide, offering a powerful tool with which to explore the links between guard cell homeostasis, stomatal dynamics, and foliar transpiration.