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At least 19 records

Nitrogen increases early‐stage and slows late‐stage decomposition across diverse grasslands

1. To evaluate how increased anthropogenic nutrient inputs alter carbon cycling in grasslands, we conducted a litter decomposition study across 20 temperate grasslands on three continents within the Nutrient Network, a globally distributed nutrient enrichment experiment 2. We determined the effects of addition of experimental nitrogen (N), phosphorus (P) and potassium plus micronutrient (K μ ) on decomposition of a common tree leaf litter in a long-term study (maximum of 7 years; exact deployment period varied across sites). The use of higher order decomposition models allowed us to distinguish between the effects of nutrients on early- versus late-stage decomposition. 3. Across continents, the addition of N (but not other nutrients) accelerated early-stage decomposition and slowed late-stage decomposition, increasing the slowly decomposing fraction by 28% and the overall litter mean residence time by 58%. 4. Synthesis. Using a novel, long-term cross-site experiment, we found widespread evidence that N enhances the early stages of above-ground plant litter decomposition across diverse and widespread temperate grassland sites but slows late-stage decomposition. These findings were corroborated by fitting the data to multiple decomposition models and have implications for N effects on soil organic matter formation. For example, following N enrichment, increased microbial processing of litter substrates early in decomposition could promote the production and transfer of low molecular weight compounds to soils and potentially enhance the stabilization of mineral-associated organic matter. By contrast, by slowing late-stage decomposition, N enrichment could promote particulate organic matter (POM) accumulation. Such hypotheses deserve further testing.

Nutrient Network (NutNet)↗

Soil carbon stocks in temperate grasslands differ strongly across sites but are insensitive to decade-long fertilization

We report enhancing soil carbon (C) storage has the potential to offset human-caused increases in atmospheric CO 2 . Rising CO 2 has occurred concurrently with increasing supply rates of biologically limiting nutrients such as nitrogen (N) and phosphorus (P). However, it is unclear how increased supplies of N and P will alter soil C sequestration, particularly in grasslands, which make up nearly a third of non-agricultural land world-wide. Here, we leverage a globally distributed nutrient addition experiment (the Nutrient Network) to examine how a decade of N and P fertilization (alone and in combination) influenced soil C and N stocks at nine grassland sites spanning the continental United States. We measured changes in bulk soil C and N stocks and in three soil C fractions (light and heavy particulate organic matter, and mineral-associated organic matter fractions). Nutrient amendment had variable effects on soil C and N pools that ranged from strongly positive to strongly negative, while soil C and N pool sizes varied by more than an order of magnitude across sites. Piecewise SEM clarified that small increases in plant C inputs with fertilization did not translate to greater soil C storage. Nevertheless, peak season aboveground plant biomass (but not root biomass or production) was strongly positively related to soil C storage at seven of the nine sites, and across all nine sites, soil C covaried with moisture index and soil mineralogy, regardless of fertilization. Overall, we show that site factors such as moisture index, plant productivity, soil texture, and mineralogy were key predictors of cross-site soil C, while nutrient amendment had weaker and site-specific effects on C sequestration. This suggests that prioritizing the protection of highly productive temperate grasslands is critical for reducing future greenhouse gas losses arising from land use change.

59 BASIC BIOLOGICAL SCIENCES↗

Global relationships between crop diversity and nutritional stability

Nutritional stability – a food system’s capacity to provide sufficient nutrients despite disturbance – is an important, yet challenging to measure outcome of diversified agriculture. Using 55 years of data across 184 countries, we assemble 22,000 bipartite crop-nutrient networks to quantify nutritional stability by simulating crop and nutrient loss in a country, and assess its relationship to crop diversity across regions, over time and between imports versus in country production. We find a positive, saturating relationship between crop diversity and nutritional stability across countries, but also show that over time nutritional stability remained stagnant or decreased in all regions except Asia. These results are attributable to diminishing returns on crop diversity, with recent gains in crop diversity among crops with fewer nutrients, or with nutrients already in a country’s food system. Finally, imports are positively associated with crop diversity and nutritional stability, indicating that many countries’ nutritional stability is market exposed.

54 ENVIRONMENTAL SCIENCES↗

Microbial processing of plant remains is co-limited by multiple nutrients in global grasslands

Microbial processing of aggregate-unprotected organic matter inputs is key for soil fertility, long-term ecosystem carbon and nutrient sequestration and sustainable agriculture. In this work, we investigated the effects of adding multiple nutrients (nitrogen, phosphorus and potassium plus nine essential macro- and micro-nutrients) on decomposition and biochemical transformation of standard plant materials buried in 21 grasslands from four continents. Addition of multiple nutrients weakly but consistently increased decomposition and biochemical transformation of plant remains during the peak-season, concurrent with changes in microbial exoenzymatic activity. Higher mean annual precipitation and lower mean annual temperature were the main climatic drivers of higher decomposition rates, while biochemical transformation of plant remains was negatively related to temperature of the wettest quarter. Nutrients enhanced decomposition most at cool, high rainfall sites, indicating that in a warmer and drier future fertilized grassland soils will have an even more limited potential for microbial processing of plant remains.

59 BASIC BIOLOGICAL SCIENCES↗

Stronger fertilization effects on aboveground versus belowground plant properties across nine U.S. grasslands

Abstract Increased nutrient inputs due to anthropogenic activity are expected to increase primary productivity across terrestrial ecosystems, but changes in allocation aboveground versus belowground with nutrient addition have different implications for soil carbon (C) storage. Thus, given that roots are major contributors to soil C storage, understanding belowground net primary productivity (BNPP) and biomass responses to changes in nutrient availability is essential to predicting carbon–climate feedbacks in the context of interacting global environmental changes. To address this knowledge gap, we tested whether a decade of nitrogen (N) and phosphorus (P) fertilization consistently influenced aboveground and belowground biomass and productivity at nine grassland sites spanning a wide range of climatic and edaphic conditions in the continental United States. Fertilization effects were strong aboveground, with both N and P addition stimulating aboveground biomass at nearly all sites (by 30% and 36%, respectively, on average). P addition consistently increased root production (by 15% on average), whereas other belowground responses to fertilization were more variable, ranging from positive to negative across sites. Site‐specific responses to P were not predicted by the measured covariates. Atmospheric N deposition mediated the effect of N fertilization on root biomass and turnover. Specifically, atmospheric N deposition was positively correlated with root turnover rates, and this relationship was amplified with N addition. Nitrogen addition increased root biomass at sites with low N deposition but decreased it at sites with high N deposition. Overall, these results suggest that the effects of nutrient supply on belowground plant properties are context dependent, particularly with regard to background N supply rates, demonstrating that site conditions must be considered when predicting how grassland ecosystems will respond to increased nutrient loading from anthropogenic activity.

59 BASIC BIOLOGICAL SCIENCES↗

Carbon availability, soil pH, and microbial allocation to nitrogen acquisition shape grassland heterotrophic respiration in response to a decade of nitrogen addition

Previous work has found that anthropogenic inputs of nitrogen (N) and phosphorus (P) impact heterotrophic respiration during soil organic matter decomposition in grasslands, a critical pathway through which carbon (C) is lost from soil to the atmosphere. While N addition typically reduces heterotrophic respiration, why the strength and direction of this N effect varies among sites is unclear. Here, to address this, we conducted a 339-day laboratory incubation to measure heterotrophic respiration from nine grasslands across North America that have received 10 years of factorial N and P fertilization. N addition reduced cumulative respiration most at sites with low pH, low microbial allocation towards N acquisition, and high soil C concentration and availability. However, N addition had neutral rather than positive effects on heterotrophic respiration in sites with high pH and decomposer allocation towards N acquisition. Across sites, a decade of N addition reduced heterotrophic respiration by ∼24 %, driven by reductions in microbial biomass. Heterotrophic respiration was less sensitive to P addition, despite its increasing microbial biomass. However, simultaneous N and P addition did ameliorate negative N effects. These results show that previously observed variation in the response of heterotrophic respiration to N addition can be explained by soil C availability and pH status, widely measured factors which can be used to predict how grassland C fluxes may change under continuing nutrient deposition.

N addition↗

Grassland ecosystem type drives AM fungal diversity and functional guild distribution in North American grasslands

Nutrient exchange forms the basis of the ancient symbiotic relationship that occurs between most land plants and arbuscular mycorrhizal (AM) fungi. Plants provide carbon (C) to AM fungi and fungi provide the plant with nutrients such as nitrogen (N) and phosphorous (P). Nutrient addition can alter this symbiotic coupling in key ways, such as reducing AM fungal root colonization and changing the AM fungal community composition. However, environmental parameters that differentiate ecosystems and drive plant distribution patterns (e.g., pH, moisture), are also known to impact AM fungal communities. Identifying the relative contribution of environmental factors impacting AM fungal distribution patterns is important for predicting biogeochemical cycling patterns and plant-microbe relationships across ecosystems. Here, to evaluate the relative impacts of local environmental conditions and long-term nutrient addition on AM fungal abundance and composition across grasslands, we studied experimental plots amended for 10 years with N, P, or N and P fertilizer in different grassland ecosystem types, including tallgrass prairie, montane, shortgrass prairie, and desert grasslands. Contrary to our hypothesis, we found ecosystem type, not nutrient treatment, was the main driver of AM fungal root colonization, diversity, and community composition, even when accounting for site-specific nutrient limitations. We identified several important environmental drivers of grassland ecosystem AM fungal distribution patterns, including aridity, mean annual temperature, root moisture, and soil pH. This work provides empirical evidence for niche partitioning strategies of AM fungal functional guilds and emphasizes the importance of long-term, large scale research projects to provide ecologically relevant context to nutrient addition studies.

59 BASIC BIOLOGICAL SCIENCES↗

Nutrient Stress Detection in Corn Using Neural Networks and AVIRIS Hyperspectral Imagery

AVIRIS image cube data has been processed for the detection of nutrient stress in corn by both known, ratio-type algorithms and by trained neural networks. The USDA Shelton, NE, ARS Variable Rate Nitrogen Application (VRAT) experimental farm was the site used in the study. Upon application of ANOVA and Dunnett multiple comparsion tests on the outcome of both the neural network processing and the ratio-type algorithm results, it was found that the neural network methodology provides a better overall capability to separate nutrient stressed crops from in-field controls.

Estep, Lee↗

Nutrient Stress Detection in Corn, Using Neural Networks and AVIRIS Hyperspectral Imagery

The US Department of Agriculture (USDA) Agricultural Research Service (ARS) Variable Rate (VRAT) Nitrogen Application site in Shelton, Nebraska, represents a well-documented, corn-growing quarter section. The USDA VRAT site is used to systematically study nutrient stress in corn by varying sub-plot application of fertilizer. The field has four replicates of five blocks that vary by nitrogen treatment from 0-kg/ha to 200-kg/ha in 50-kg/ha increments. The treatment blocks are set out in a randomized, complete block design. Typically, the VRAT is planted in a ridge till, monoculture corn and is watered by a central pivot irrigation system on a three-day period. Since water stress can increase spectral reflectance from corn leaves, it is important that the N-application plots be adequately watered so that only nutrient-related stress will predominate. A figure shows imagery of the USDA VRAT site with the fertilizer amounts for each block shown. Low-altitude Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) hyperspectral imagery was acquired over the Shelton, Nebraska, VRAT site on July 22, 1999. The overflight produced 3-meter pixels with 224 spectral bands. Ground personnel supported the mission with measurements at the time of the overflight. The image data was pre-processed at JPL before being sent out to an investigator. The data arrived radiometrically corrected, allowing ready application of an atmospheric correction procedure. The Atmosphere Removal Program (ATREM) was used to perform an atmospheric correction. The AVIRIS imagery after ATREM correction was output as relative reflectance. This relative reflectance file was scaled by an empirical line procedure to provide reflectances that matched closely those measured in the field.

Estep, Lee↗

Physical, resource supply, and biological controls on nutrient processing along the river continuum

Nutrient impairment has led to damages to US surface and groundwater systems in excess of 100 billion dollars per year. Therefore, there is a strong need to develop methods to predict the transport, uptake, and export of nutrients along fluvial networks. We present results that are based on a data-driven mechanistic understanding of three factors that largely control nutrient uptake and export: 1) interactions between transport-related processes (mass transfer to metabolically active zones), 2) resource supply dynamics (nutrient concentration, stoichiometric constraints, etc.), and 3) biological controls (microbial community structure and function). Our results were generated from column experiments conducted along the Jemez River-Rio Grande continuum, which spans four orders of magnitude in mean annual discharge, more than 2000 m in altitude, and more than 500 km of stream longitude. Two resource supply injections were performed on each of the columns, i.e., a nitrate only addition, followed by a stoichiometrically ‘balanced’ 106Carbon:16Nitrogen:1Phosphorus addition. We quantified NO3-N uptake kinetics while constraining three variables: stream order, sediment type and type of injection (N vs stoichiometrically ‘balanced’ C:N:P). Following the laboratory nutrient uptake experiments, the columns were destructively sampled and the contents were homogenized to collect subsamples for DNA sequencing. Amplicon analysis was carried out as described by the Earth Microbiome Protocol for 16s and ITS sequencing.

54 ENVIRONMENTAL SCIENCES↗

Arabidopsis bZIP11 Is a Susceptibility Factor During Pseudomonas syringae Infection

The induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens (e.g., Xanthomonas spp.) use transcription activator-like (TAL) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pv. tomato strain DC3000 lacks TAL effectors yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Because bacteria require other nutrients in addition to sugars for efficient reproduction, we hypothesized that Pseudomonas spp. may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11-regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Biochemistry & Molecular Biology↗

Impoverishing Roots Will Improve Wheat Yield and Profitability Through Increased Water and Nitrogen Use Efficiencies

We report that more than a 60% increase in crop production is required by the 2050's to feed a growing world population. Understanding how plant functional traits and field management affect crop yields has the potential to improve agricultural productivity, minimize economic and environmental losses, and maximize food security. We explored the influence of winter wheat root characteristics and management on winter wheat growth, yield, and profit using a mechanistic and well-tested ecosystem and crop model, ecosys. We applied and further tested ecosys at an agricultural farm growing winter wheat in Ardmore, Oklahoma, United States. The model accurately predicted observed shoot carbon (R 2 = 0.95), soil moisture (R 2 = 0.67), soil temperature (R 2 = 0.91), and yield (percent error = 17%). Numerical optimization experiments were conducted to explore potential improvements of winter wheat yield and profit by modifying root characteristics, including root radius and root:shoot carbon transfer conductance, and fertilizer inputs. Our results show the potential for simultaneously improving winter wheat yields and profits. The optimum conditions were found to be in the range of root radius between 0.1 and 0.3 mm, carbon transfer conductance between 0.004 and 0.01 h -1 , and the currently applied fertilizer rate of 112 kg ha -1 . Under these conditions, improvements in yields and profits of up to approximately 25% and 110%, respectively, were modeled compared to those under baseline root traits. These improvements were achieved by impoverishing root structures, thereby increasing nutrient allocation to grains. Our results also demonstrate and motivate model structures that integrate the complex network of plant physiology, soil nutrient biogeochemistry, hydrology, and management.

54 ENVIRONMENTAL SCIENCES↗

A Single Application of Compost Can Leave Lasting Impacts on Soil Microbial Community Structure and Alter Cross-Domain Interaction Networks

Our current understanding suggests that nutrient management strategies applied to agricultural soils over multiple years are required to cause major and stable shifts in soil microbial communities. However, some studies suggest that agricultural soils can benefit even from sporadic, single additions of organic matter. Here we investigate how single additions of high-quality organic matter can cause significant shifts in microbial soil communities over multiple cropping cycles. We grew radishes in a tropical Oxisol soil for six crop cycles after a single application of a high-nitrogen compost or urea. At planting and before biomass harvest, we sampled soils influenced by the radish rhizosphere and sequenced bacterial and archaeal 16S and fungal ITS rDNA marker genes. We measured microbial richness and diversity, community composition and structure, and constructed correlation networks to predict cross-domain microbial interactions. We found that a single application of compost, compared to urea or control, resulted in a persistent improved plant biomass response and led to sustained changes in the soil microbial community throughout the duration of the 227-day study. Compost altered the structure of both the fungal and prokaryotic microbial communities, introduced new microorganisms that persisted in the resident soil system, and altered soil microbial correlation network structure and hub taxa. In contrast, fertilization with urea did not significantly alter the structure of soil microbial communities compared to the control but reduced network complexity and altered hub taxa. This study highlights the significant impacts that high-quality organic matter fertilization can exert on agricultural soil microbiomes and adds to the growing body of knowledge on using organic fertilizers as a way to steer the soil microbiome toward a healthier soil.

Heisey, Steven↗

Three-dimensional modeling of hyphal fusion, branching, and nutrient transport in filamentous fungi

Fungi exhibit behaviors distinct from other microbes. Filamentous fungi grow by extending complex networks of branched filaments collectively referred to as the mycelium. These networks can expand over large distances and traverse low-nutrient areas by translocating nutrients through the filament network. This spatial characteristic makes filamentous fungi crucial for soil ecosystems, supporting stable microbial communities and promoting plant growth. However, simulating these behaviors is complex. The elongated nature of fungal compartments results in different mechanical interactions compared to the commonly modeled spherical bacteria. These detailed hyphal mechanics require specialized consideration and are often excluded from conventional fungal simulation packages. Additionally, the extensive fungal networks in nature demand computationally intensive simulations, necessitating high-performance algorithms. Therefore, realistic fungi simulations require specialized software. Here, we introduce a fungal modeling expansion to the high-performance biological modelling and interface exchange (bmx) software suite. bmx leverages adaptive mesh refinement in AMReX for chemical diffusion and incorporates a full mechanical model for bacterial cells, accelerated by GPUs. By extending bmx to model filamentous particles, we demonstrate the formation of complex filament networks through interactions like hyphal branching and fusion (anastomosis). We show that the networks produced match real-world fungal structures through various metrics. This work supports computational studies of fungal growth dynamics and can be adapted to investigate the growth of other filamentous structures in biology or materials science. The expanded-BMX package is open-sourced and is available online.

Cell mechanics↗

DNA affinity purification sequencing and transcriptional profiling reveal new aspects of nitrogen regulation in a filamentous fungus

Significance Microorganisms have evolved transcriptional networks to prioritize utilization of available nutrient sources. For filamentous fungi, such as Neurospora crassa , this entails distinguishing between a variety of organic and inorganic nitrogen sources. Here, we transcriptionally profiled the response of N. crassa to a variety of nitrogen sources and used DNA affinity purification sequencing to characterize the role of regulatory genes and their direct downstream targets. We identified a transcription factor responsible for regulating genes involved in amino acid and mannose metabolism. By comparing the genes regulated by transcription factors that regulate specific nitrogen utilization pathways and transcription factors that regulate utilization of all nitrogen sources that require metabolic processing before utilization, we revealed aspects of the nitrogen regulatory network.

59 BASIC BIOLOGICAL SCIENCES↗

Nutrient supply controls the linkage between species abundance and ecological interactions in marine bacterial communities

Nutrient scarcity is pervasive for natural microbial communities, affecting species reproduction and co-existence. However, it remains unclear whether there are general rules of how microbial species abundances are shaped by biotic and abiotic factors. Here we show that the ribosomal RNA gene operon (rrn) copy number, a genomic trait related to bacterial growth rate and nutrient demand, decreases from the abundant to the rare biosphere in the nutrient-rich coastal sediment but exhibits the opposite pattern in the nutrient-scarce pelagic zone of the global ocean. Both patterns are underlain by positive correlations between community-level rrn copy number and nutrients. Furthermore, inter-species co-exclusion inferred by negative network associations is observed more in coastal sediment than in ocean water samples. Nutrient manipulation experiments yield effects of nutrient availability on rrn copy numbers and network associations that are consistent with our field observations. Based on these results, we propose a “hunger games” hypothesis to define microbial species abundance rules using the rrn copy number, ecological interaction, and nutrient availability.

59 BASIC BIOLOGICAL SCIENCES↗

Linking Hydrobiogeochemical Processes and Management Techniques to Close Nutrient Loops in an Arid River

In this study, we explore opportunities to optimize food-energy-water (FEW) resources by closing nutrient loops in arid-land rivers. Specifically, we evaluate source and sink behavior of nitrogen as nitrate (NO 3 -N) in three characteristic channels in the Middle Rio Grande Basin (Delivery and Drain channels associated with agriculture and the main channel of the Rio Grande). All three channels are located downstream of a large wastewater treatment plant that is the main contributor of nutrients to this reach of the Rio Grande. We used a mass balance approach paired with stable isotope analysis to determine sources and processing of NO 3 -N within the channels over time (a year) and through space (along ~15 to 50 km reaches). Results indicated the growing season was an important period of sink behavior for the Delivery channel and the Rio Grande, but the Drain channel was a year-round source. Stable isotope analysis of 15 N and 18 O found a distinct nitrate signature in the Drain associated with biological processing, as well as Rio Grande sites impacted by agricultural outflow, but no equivalent signature was present in the Delivery channel. Based on our findings, we provide recommendations to help close nutrient loops in the study system and analogous aridland irrigation networks by 1) minimizing loss during the transfer of nutrients from wastewater facilities to agricultural areas, and 2) minimizing enrichment to downstream aquatic ecosystems by sequestering nutrients that escape the nutrient loop.

54 ENVIRONMENTAL SCIENCES↗

A novel regulator of the fungal phosphate starvation response revealed by transcriptional profiling and DNA affinity purification sequencing

Cells must accurately sense and respond to nutrients to compete for resources and establish growth. Phosphate is a critical nutrient source necessary for signaling, energy metabolism, and synthesis of nucleic acids, phospholipids, and cellular metabolites. During phosphate limitation, fungi import phosphate from the environment and liberate phosphate from phosphate-containing molecules in the cell. In the model filamentous fungus Neurospora crassa, the phosphate starvation response is regulated by the conserved transcription factor NUC-1. The activity of NUC-1 is repressed by a complex of the cyclin-dependent kinase MDK-1 and the cyclin PREG when phosphate is plentiful. When phosphate is limiting, NUC-1 repression by MDK-1/PREG is relieved by the cyclin-dependent kinase inhibitor NUC-2. We investigated the global response of N. crassa to phosphate starvation. During phosphate starvation, NUC-1 directly activated the expression of genes encoding phosphatases, nucleases, and a phosphate transporter and directly repressed genes associated with the ribosome. Additionally, NUC-1 indirectly activated the expression of an uncharacterized transcription factor, which we named nuc-3. NUC-3 directly repressed the expression of genes involved in phosphate acquisition and liberation after an extended period of phosphate starvation. Additionally, NUC-3 directly repressed the expression of the cyclin-dependent kinase inhibitor nuc-2. Thus, through the combination of NUC-3 direct repression of genes in the phosphate starvation response and nuc-2, an activator of the phosphate starvation response, NUC-3 serves to act as a brake on the phosphate starvation response after an extended period of phosphate starvation. This braking mechanism could reduce transcription, a phosphate-intensive process, under conditions of extended phosphate limitation.IMPORTANCEFungi have evolved regulatory networks to respond to available nutrients. Phosphate is often a limiting nutrient for fungi that is critical for many cellular functions, including nucleic acid and phospholipid biosynthesis, cell signaling, and energy metabolism. The fungal response to phosphate limitation is important in interactions with plants and animals. We investigated the global transcriptional response to phosphate starvation and the role of a major transcriptional regulator, NUC-1, in the model filamentous fungus Neurospora crassa. Our data show that NUC-1 is a bifunctional transcription factor that directly activates phosphate acquisition genes, while directly repressing genes associated with phosphate-intensive processes. NUC-1 indirectly regulates an uncharacterized transcription factor, which we named nuc-3. NUC-3 directly represses phosphate acquisition genes and nuc-2, an activator of the phosphate starvation response, during extended periods of phosphate starvation. Thus, NUC-3 acts as a brake on the phosphate starvation response to reduce phosphate-intensive activities, like transcriptional activation, when phosphate starvation persists.

DNA affinity purification sequencing↗