The impact of stand age and fertilization on the soil microbiome of Miscanthus × giganteus

Yield of the perennial grass Miscanthus × giganteus has shown an inconsistent and unpredictable response to nitrogen (N) fertilizer, yet fertilization underpins the crop’s environmental and economic sustainability. The interactions among soil microbial communities, N availability, and Miscanthus × giganteus and management may explain changes in plant productivity. In this study, soil samples from different stand ages of Miscanthus × giganteus in a replicated chronosequence field trial were used to investigate the effects of stand age and N fertilizer rates on microbial community structure. We hypothesized that there is a definable Miscanthus × giganteus soil microbiome and that this community varies significantly with stand age and fertilization. Our results showed that the main phyla in soil microbial communities, regardless of plant age, are similar but microbial community structures are significantly different. The variation in observed microbial communities generally decreases with older stand ages. The amount of N fertilizer applied also affected the microbial community structure associated with different aged Miscanthus × giganteus. Specifically, the relative abundance of Proteobacteria (Alphaproteobacteria and Gammaproteobacteria) and Acidobacteria (subgroup Gp1) increased shortly after fertilization and was more associated with younger Miscanthus × giganteus. Furthermore, our results show a significant relationship between bacterial α diversity and fertilization rates and that this response is also affected by stand age. Overall, our results emphasize linkages between microbial community structure, plant age, and fertilization in Miscanthus × giganteus

Establishing RNA Interference as a Reverse-Genetic Approach for Gene Functional Analysis in Protoplasts1[C][OA]

Double-stranded (ds)RNA interference (RNAi) is widely used for functional analysis of plant genes and is achieved via generating stable transformants expressing dsRNA in planta. This study demonstrated that RNAi can also be utilized to examine gene functions in protoplasts. Because protoplasts are nongrowing cells, effective RNAi-triggered gene silencing depends not only on a depletion of gene transcripts but also on turnover rates of corresponding polypeptides. Herein, we tested if transient RNAi in protoplasts would result in the depletion of a targeted polypeptide and, because protoplasts have a limited life span, if functional assays of RNAi knockout genes would be feasible in protoplasts. We showed that protoplasts transfection with an in vitro-synthesized dsRNA against Arabidopsis (Arabidopsis thaliana) β-glutamylcysteine synthase (ECS1), a key enzyme in the synthesis of glutathione, resulted in a 95% depletion of ECS1 transcript, a 72% decrease of ECS1 polypeptide, and a 60% drop in glutathione content. These results were comparable with those obtained upon analysis of Arabidopsis seedlings bearing the cad2-1 mutant allele of ECS1. We also improved the procedure for RNAi inactivation of several genes simultaneously. Finally, because we isolated protoplasts from tissues of 14-d-old seedlings instead of 1-month-old mature plants, the described procedure is rapid (as it only takes 20 d from seed planting to functional studies), suitable for analyzing multiple genes in parallel, and independent of cloning dsRNAs into plant expression vectors. Therefore, RNAi in protoplasts complements existing genetic tools, as it allows rapid, cost- and space-efficient initial screening and selection of genes for subsequent in planta studies

Microbiome differences in sugarcane and metabolically engineered oilcane accessions and their implications for bioenergy production

Oilcane is a metabolically engineered sugarcane (Saccharum spp. hybrid) that hyper-accumulates lipids in its vegetable biomass to provide an advanced feedstock for biodiesel production. The potential impact of hyper-accumulation of lipids in vegetable biomass on microbiomes and the consequences of altered microbiomes on plant growth and lipid accumulation have not been explored so far. Here, we explore differences in the microbiome structure of different oilcane accessions and non-modified sugarcane. 16S SSU rRNA and ITS rRNA amplicon sequencing were performed to compare the characteristics of the microbiome structure from different plant compartments (leaf, stem, root, rhizosphere, and bulk soil) of four greenhouse-grown oilcane accessions and non-modified sugarcane. Significant differences were only observed in the bacterial microbiomes. In leaf and stem microbiomes, more than 90% of the entire microbiome of non-modified sugarcane and oilcane was dominated by similar core taxa. Taxa associated with Proteobacteria led to differences in the non-modified sugarcane and oilcane microbiome structure. While differences were observed between multiple accessions, accession 1566 was notable in that it was consistently observed to differ in its microbial membership than other accessions and had the lowest abundance of taxa associated with plant-growth-promoting bacteria. Accession 1566 is also unique among oilcane accessions in that it has the highest constitutive expression of the WRI1 transgene. The WRI1 transcription factor is known to contribute to significant changes in the global gene expression profile, impacting plant fatty acid biosynthesis and photomorphogenesis. This study reveals for the first time that genetically modified oilcanes associate with distinct microbiomes. Our findings suggest potential relationships between core taxa, biomass yield, and TAG in oilcane accessions and support further research on the relationship between plant genotypes and their microbiomes.This article is published as Yang, Jihoon, Thanwalee Sooksa-Nguan, Baskaran Kannan, Sofia Cano-Alfanar, Hui Liu, Angela Kent, John Shanklin, Fredy Altpeter, and Adina Howe. "Microbiome differences in sugarcane and metabolically engineered oilcane accessions and their implications for bioenergy production." Biotechnology for Biofuels and Bioproducts 16, no. 1 (2023): 1-14. DOI: 10.1186/s13068-023-02302-6. Copyright 2023 The Author(s). Attribution 4.0 International (CC BY 4.0). Posted with permission

Microbial linkages to soil biogeochemical processes in a poorly drained agricultural ecosystem

Soil microorganisms mediate biogeochemical processes, but how microbial community composition influences these processes remains contested. We combined monthly sequencing of soil 16S rRNA genes and intensive measurements of nitrogen (N), carbon (C), and iron (Fe) cycling along a topographic gradient in a poorly drained intensive agricultural ecosystem (corn–soybean rotation) in the midwestern United States. Observed microbial composition changed little over time within and among years despite large differences in weather and crop type. Yet, microbial composition varied greatly with topographic location and correlated strongly with moisture, soil organic carbon (SOC), and especially pH. Microbial families, genera, and/or amplicon sequence variants often correlated significantly with measured biogeochemical processes or pools, yet different taxa within the same phylogenetic groups often responded in opposite ways, indicating a lack of ecological coherence among close relatives. Dominant phyla were generally similar across the topographic gradient but specific members showed consistent tradeoffs among locations. Ammonia oxidizing archaea and bacteria sequences varied oppositely with pH across the gradient, but their combined relative abundances remained similar, as did potential nitrification rates. Nitrospira sequences correlated positively with nitrous oxide (N2O) fluxes, suggesting a direct or indirect contribution of nitrification (or possibly comammox) to N2O production. We also found significant linkages between taxonomic groups and redox-sensitive Fe pools, indicating a role for redox variation in structuring microbial communities. Several globally dominant bacteria identified previously correlated significantly with measured biogeochemical variables, providing insights into their possible functional roles. Overall, microbial composition provided a coarse measure of several key biogeochemical functions and implicated taxa that possibly mediate these processes in a widespread agroecosystem of North America.This is a manuscript of an article published as Yu, Wenjuan, Nathaniel C. Lawrence, Thanwalee Sooksa-nguan, Schuyler D. Smith, Carlos Tenesaca, Adina Chuang Howe, and Steven J. Hall. "Microbial linkages to soil biogeochemical processes in a poorly drained agricultural ecosystem." Soil Biology and Biochemistry (2021): 108228. doi:10.1016/j.soilbio.2021.108228. Posted with permission.</p

Microbial linkages to soil biogeochemical processes in a poorly drained agricultural ecosystem

Soil microorganisms mediate biogeochemical processes, but how microbial community composition influences these processes remains contested. We combined monthly sequencing of soil 16S rRNA genes and intensive measurements of nitrogen (N), carbon (C), and iron (Fe) cycling along a topographic gradient in a poorly drained intensive agricultural ecosystem (corn–soybean rotation) in the midwestern United States. Observed microbial composition changed little over time within and among years despite large differences in weather and crop type. Yet, microbial composition varied greatly with topographic location and correlated strongly with moisture, soil organic carbon (SOC), and especially pH. Microbial families, genera, and/or amplicon sequence variants often correlated significantly with measured biogeochemical processes or pools, yet different taxa within the same phylogenetic groups often responded in opposite ways, indicating a lack of ecological coherence among close relatives. Dominant phyla were generally similar across the topographic gradient but specific members showed consistent tradeoffs among locations. Ammonia oxidizing archaea and bacteria sequences varied oppositely with pH across the gradient, but their combined relative abundances remained similar, as did potential nitrification rates. Nitrospira sequences correlated positively with nitrous oxide (N2O) fluxes, suggesting a direct or indirect contribution of nitrification (or possibly comammox) to N2O production. We also found significant linkages between taxonomic groups and redox-sensitive Fe pools, indicating a role for redox variation in structuring microbial communities. Several globally dominant bacteria identified previously correlated significantly with measured biogeochemical variables, providing insights into their possible functional roles. Overall, microbial composition provided a coarse measure of several key biogeochemical functions and implicated taxa that possibly mediate these processes in a widespread agroecosystem of North America

The impact of stand age and fertilization on the soil microbiome of Miscanthus × giganteus

Yield of the perennial grass Miscanthus × giganteus has shown an inconsistent and unpredictable response to nitrogen (N) fertilizer, yet fertilization underpins the crop’s environmental and economic sustainability. The interactions among soil microbial communities, N availability, and Miscanthus × giganteus and management may explain changes in plant productivity. In this study, soil samples from different stand ages of Miscanthus × giganteus in a replicated chronosequence field trial were used to investigate the effects of stand age and N fertilizer rates on microbial community structure. We hypothesized that there is a definable Miscanthus × giganteus soil microbiome and that this community varies significantly with stand age and fertilization. Our results showed that the main phyla in soil microbial communities, regardless of plant age, are similar but microbial community structures are significantly different. The variation in observed microbial communities generally decreases with older stand ages. The amount of N fertilizer applied also affected the microbial community structure associated with different aged Miscanthus × giganteus. Specifically, the relative abundance of Proteobacteria (Alphaproteobacteria and Gammaproteobacteria) and Acidobacteria (subgroup Gp1) increased shortly after fertilization and was more associated with younger Miscanthus × giganteus. Furthermore, our results show a significant relationship between bacterial α diversity and fertilization rates and that this response is also affected by stand age. Overall, our results emphasize linkages between microbial community structure, plant age, and fertilization in Miscanthus × giganteus.This article is published as Ma, Lanying, Fernando Igne Rocha, Jaejin Lee, Jinlyung Choi, Mauricio Tejera, Thanwalee Sooksa-Nguan, Nicholas Neal Boersma, Andrew Vanloocke, Emily Heaton, and Adina Howe. "The impact of stand age and fertilization on the soil microbiome of Miscanthus× giganteus." Phytobiomes Journal (2021). doi: 10.1094/PBIOMES-01-20-0014-FI.</p