Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

University of California

University of California, Davis

The responses of Acala cotton (Gossypium hirsutum L.) in California to a range of applied nitrogen (N) treatments were investigated in a 5-year, multisite experiment. The experiment's goals were to identify crop growth and yield responses to applied N and provide information to better assess the utility of soil residual N estimates in improving fertilizer management. Baseline fertilizer application rates for the lowest applied N treatments were based on residual soil nitrate-N (NO3-N) levels determined on soil samples from the upper 0.6 m of the soil collected prior to spring N fertilization and within 1 week postplanting each year. Results have shown positive cotton lint yield responses to increases in applied N across the 56 to 224 kg N/ha range in only 41% (16 out of 39) of test sites. Soil NO3-N monitoring to a depth of 2.4 m in the spring (after planting) and fall (postharvest) indicate most changes in soil NO3- occur within the upper 1.2 m of soil. However, some sites (those most prone to leaching losses of soluble nutrients) also exhibited net increases in soil NO3-N in the 1.2- to 2.4-m depth zone when comparing planting time vs. postharvest data. The lack of yield responses and soil NO3-N accumulations at some sites indicate that more efforts should be put into identifying the amount of plant N requirements that can be met from residual soil N, rather than solely from fertilizer N applications.

Response of Acala cotton to nitrogen rates in the San Joaquin Valley of California.

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatranscriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes.

Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria.

The ecology of fungi lags behind that of plants and animals because most fungi are microscopic and hidden in their substrates. Here, we address the basic ecological process of fungal succession in nature using the microscopic, arbuscular mycorrhizal fungi (AMF) that form essential mutualisms with 70-90% of plants. We find a signal for temporal change in AMF community similarity that is 40-fold stronger than seen in the most recent studies, likely due to weekly samplings of roots, rhizosphere and soil throughout the 17 weeks from seedling to fruit maturity and the use of the fungal DNA barcode to recognize species in a simple, agricultural environment. We demonstrate the patterns of nestedness and turnover and the microbial equivalents of the processes of immigration and extinction, that is, appearance and disappearance. We also provide the first evidence that AMF species co-exist rather than simply co-occur by demonstrating negative, density-dependent population growth for multiple species. Our study shows the advantages of using fungi to test basic ecological hypotheses (e.g., nestedness v. turnover, immigration v. extinction, and coexistence theory) over periods as short as one season.

Strong succession in arbuscular mycorrhizal fungal communities.

Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop noted for its ability to survive water-limiting conditions. Herein, we present an analytical workflow to explore the changes in histone modifications through plant developmental stages and two drought stresses in two sorghum genotypes that differ in their response to drought. Top-down mass spectrometry (MS) is an ideal method to profile histone modifications and distinguish closely related histone proteoforms. We analyzed leaves of 48 plants and identified 26 unique histone proteins and 677 unique histone proteoforms (124 full-length and 553 truncated proteoforms). We detected trimethylation on nearly all H2B N-termini where acetylation is commonly expected. In addition, an unexpected modification on H2A histones was assigned to N-pyruvic acid 2-iminylation based on its unique neutral loss of CO. Interestingly, some of the truncated histones, in particular H4 and H3.2, showed significant changes that correlated with the growth and water conditions. The histone proteoforms could serve as targets in search of chromatin modifiers and ultimately have important ramifications in future attempts of studying plant epigenetic reprogramming under stress.

Top-down mass spectrometry of histone modifications in sorghum reveals potential epigenetic markers for drought acclimation.

Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [ (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants' roots.

Transcriptomic analysis of field-droughted sorghum from seedling to maturity reveals biotic and metabolic responses.

Community assembly of crop-associated fungi is thought to be strongly influenced by deterministic selection exerted by the plant host, rather than stochastic processes. Here we use a simple, sorghum system with abundant sampling to show that stochastic forces (drift or stochastic dispersal) act on fungal community assembly in leaves and roots early in host development and when sorghum is drought stressed, conditions when mycobiomes are small. Unexpectedly, we find no signal for stochasticity when drought stress is relieved, likely due to renewed selection by the host. In our experimental system, the host compartment exerts the strongest effects on mycobiome assembly, followed by the timing of plant development and lastly by plant genotype. Using a dissimilarity-overlap approach, we find a universality in the forces of community assembly of the mycobiomes of the different sorghum compartments and in functional guilds of fungi.

Fungal community assembly in drought-stressed sorghum shows stochasticity, selection, and universal ecological dynamics.

 f. sp.  (FOV) race 4, is a causal agent of Fusarium wilt of cotton ( spp.). This study aimed to characterize the existing distribution and frequency of current field populations of FOV race 4 genotypes in the San Joaquin Valley (SJV) of California and Lower Valley El Paso, Texas and examine representative isolates for aggressiveness during different stages of seedling development. A survey was conducted from 2017 to 2019 across 13 locations in the SJV and one location in El Paso, Texas during 2018. From the SJV, isolates identified as the FOV race 4 T genotype were dispersed across the SJV, while isolates identified as the FOV race 4 N genotype were most frequently isolated from cotton fields in the northern county of Merced. The FOV race 4 isolates from the Texas location were identified as the MT genotype. A selection of representative isolates was evaluated using three inoculation assays, a rolled towel, FOV infested-oat seed, and a root dip inoculation assay to test the isolates' abilities to produce symptoms during seedling stages of cotton development. All isolates tested were capable of producing symptoms on cotton, however isolate aggressiveness varied within and across inoculation assays. In all assays, higher levels of disease development were observed in the moderately susceptible Pima ( L.) cultivars (DP-340 or PYH-830) when compared to the moderately tolerant Upland ( L.) cultivar (FM-2334). However, no correlation was found among the different response variables for the rolled towel assay when compared with the root dip and infested-oat seed assays. These results suggest that different genes are involved in the resistance response during the early seedling development stage measured in the rolled towel assay compared to the later seedling development stages measured during the root dip inoculation and infested-oat seed assays, revealing the complexity of the Fusarium wilt disease and host-plant resistance mechanisms.

Robert Hutmacher

West Side Research and Extension Center

University of California

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Robert Hutmacher

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University of California

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

West Side Research and Extension Center