Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species

Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal. A crucial component of creating transgenic switchgrass is having the capability of transforming the explants with DNA sequences of interest using vector constructs. However, there are limited options with the monocot plant vectors currently available. With this in mind, a versatile set of Gateway-compatible destination vectors (termed pANIC) was constructed to be used in monocot plants for transgenic crop improvement. The pANIC vectors can be used for transgene overexpression or RNAi-mediated gene suppression. The pANIC vector set includes vectors that can be utilized for particle bombardment or Agrobacterium-mediated transformation. All the vectors contain (i) a Gateway cassette for overexpression or silencing of the target sequence, (ii) a plant selection cassette and (iii) a visual reporter cassette. The pANIC vector set was functionally validated in switchgrass and rice and allows for high-throughput screening of sequences of interest in other monocot species as well.     

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4‐KD, miRNA156‐OE, MYB4‐OE, COMT‐KD and FPGS‐KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second‐ versus the first‐year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second‐year growth of transgenics targeted for wall modification, GAUT4‐KD, MYB4‐OE, COMT‐KD and FPGS‐KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next‐generation bio‐feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.     

Soil microbial community structure and enzymatic activity responses to nitrogen management and landscape positions in switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is usually grown on marginal land for biofuel system, in which nitrogen (N) is an essential management practice, and landscape position is a key topographical factor in impacting the production. However, limited information is available regarding how the N application and landscape positions affect soil microbial communities and enzyme activities under switchgrass. Thus, the specific objective of this study was to evaluate the responses of N rate (high, 112 kg N/ha; medium, 56 kg N/ha; and low, 0 kg N/ha) and landscape positions (shoulder and footslope) on soil biological health under switchgrass field. Data showed that N addition significantly influenced carbon and N fractions. The hot water‐soluble organic carbon (HWC) and nitrogen (HWN) fractions were significantly higher at footslope position than the shoulder position. The amount of total phospholipid fatty acid (PLFA), total bacterial, actinomycetes, gram‐negative and gram‐positive bacteria, total fungi, arbuscular mycorrhizal (AM) fungi, and saprophytes PLFAs were highest with medium and high N rates and footslope position. The N addition increased total PLFAs in N fertilizer treatments, viz. medium (5,946 ng PLFA‐C/g soil) and high N rates (5,871 ng PLFA‐C/g soil). Microbial biomass carbon and nitrogen and enzyme activities (urease, β‐glucosidase, acid phosphatase and arylsulfatase) were significantly enhanced by N fertilization (medium and high N rates) compared to control (low N rates) under footslope position. The urease activity under medium (36.3 µmol N‐NH₄⁺ g^?1 soil hr^?1) and high N rates (31.4 µmol N‐NH₄⁺ g^?1 soil hr^?1) was 42.9% and 23.6% higher than low N rates, respectively. This study suggests that the application of medium N rate in footslope position to switchgrass can enhance the soil biological properties and hence can protect the environment from the excessive use of N fertilizer.     

Predicting soil carbon changes in switchgrass grown on marginal lands under climate change and adaptation strategies

The United States Great Lakes Region (USGLR) is a critical geographic area for future bioenergy production. Switchgrass (Panicum virgatum) is widely considered a carbon (C)‐neutral or C‐negative bioenergy production system, but projected increases in air temperature and precipitation due to climate change might substantially alter soil organic C (SOC) dynamics and storage in soils. This study examined long‐term SOC changes in switchgrass grown on marginal land in the USGLR under current and projected climate, predicted using a process‐based model (Systems Approach to Land‐Use Sustainability) extensively calibrated with a wealth of plant and soil measurements at nine experimental sites. Simulations indicate that these soils are likely a net C sink under switchgrass (average gain 0.87 Mg C ha^?1 year^?1), although substantial variation in the rate of SOC accumulation was predicted (range: 0.2–1.3 Mg C ha^?1 year^?1). Principal component analysis revealed that the predicted intersite variability in SOC sequestration was related in part to differences in climatic characteristics, and to a lesser extent, to heterogeneous soils. Although climate change impacts on switchgrass plant growth were predicted to be small (4%–6% decrease on average), the increased soil respiration was predicted to partially negate SOC accumulations down to 70% below historical rates in the most extreme scenarios. Increasing N fertilizer rate and decreasing harvest intensity both had modest SOC sequestration benefits under projected climate, whereas introducing genotypes better adapted to the longer growing seasons was a much more effective strategy. Best‐performing adaptation scenarios were able to offset >60% of the climate change impacts, leading to SOC sequestration 0.7 Mg C ha^?1 year^?1 under projected climate. On average, this was 0.3 Mg C ha^?1 year^?1 more C sequestered than the no adaptation baseline. These findings provide crucial knowledge needed to guide policy and operational management for maximizing SOC sequestration of future bioenergy production on marginal lands in the USGLR.     

Effects of Switchgrass (Panicum virgatum) Rotations with Peanut (Arachis hypogaea L.) on Nematode Populations and Soil Microflora

A 3-year field rotation study was conducted to assess the potential of switchgrass (Panicum virgatum) to suppress root-knot nematodes (Meloidogyne arenaria), southern blight (Sclerotium rolfsii), and aflatoxigenic fungi (Aspergillus sp.) in peanut (Arachis hypogaea L.) and to assess shifts in microbial populations following crop rotation. Switchgrass did not support populations of root-knot nematodes but supported high populations of nonparasitic nematodes. Peanut with no nematicide applied and following 2 years of switchgrass had the same nematode populations as continuous peanut plus nematicide. Neither previous crop nor nematicide significantly reduced the incidence of pods infected with Aspergillus. However, pod invasion by A. flavus was highest in plots previously planted with peanut and not treated with nematicide. Peanut with nematicide applied at planting following 2 years of switchgrass had significantly less incidence of southern blight than either continuous peanut without nematicide application or peanut without nematicide following 2 years of cotton. Peanut yield did not differ among rotations in either sample year. Effects of crop rotation on the microbial community structure associated with peanut were examined using indices for diversity, richness, and similarity derived from culture-based analyses. Continuous peanut supported a distinctly different rhizosphere bacterial microflora compared to peanut following 1 year of switchgrass, or continuous switchgrass. Richness and diversity indices for continuous peanut rhizosphere and geocarposphere were not consistently different from peanut following switchgrass, but always differed in the specific genera present. These shifts in community structure were associated with changes in parasitic nematode populations.     

Response of soil carbon dioxide fluxes,soil organic carbon and microbial biomass carbon to biochar amendment: a meta‐analysis

Biochar as a carbon‐rich coproduct of pyrolyzing biomass, its amendment has been advocated as a potential strategy to soil carbon (C) sequestration. Updated data derived from 50 papers with 395 paired observations were reviewed using meta‐analysis procedures to examine responses of soil carbon dioxide (CO₂) fluxes, soil organic C (SOC), and soil microbial biomass C (MBC) contents to biochar amendment. When averaged across all studies, biochar amendment had no significant effect on soil CO₂ fluxes, but it significantly enhanced SOC content by 40% and MBC content by 18%. A positive response of soil CO₂ fluxes to biochar amendment was found in rice paddies, laboratory incubation studies, soils without vegetation, and unfertilized soils. Biochar amendment significantly increased soil MBC content in field studies, N‐fertilized soils, and soils with vegetation. Enhancement of SOC content following biochar amendment was the greatest in rice paddies among different land‐use types. Responses of soil CO₂ fluxes and MBC to biochar amendment varied with soil texture and pH. The use of biochar in combination with synthetic N fertilizer and waste compost fertilizer led to the greatest increases in soil CO₂ fluxes and MBC content, respectively. Both soil CO₂ fluxes and MBC responses to biochar amendment decreased with biochar application rate, pyrolysis temperature, or C/N ratio of biochar, while each increased SOC content enhancement. Among different biochar feedstock sources, positive responses of soil CO₂ fluxes and MBC were the highest for manure and crop residue feedstock sources, respectively. Soil CO₂ flux responses to biochar amendment decreased with pH of biochar, while biochars with pH of 8.1–9.0 had the greatest enhancement of SOC and MBC contents. Therefore, soil properties, land‐use type, agricultural practice, and biochar characteristics should be taken into account to assess the practical potential of biochar for mitigating climate change.     

Spatiotemporal assessment of farm‐gate production costs and economic potential of Miscanthus × giganteus,Panicum virgatum L., and Jatropha grown on marginal land in China

Spatially explicit farm‐gate production costs and the economic potential of three types of energy crops grown on available marginal land in China for 2017 and 2040 were investigated using a spatial accounting method and construction of cost–supply curves. The average farm‐gate cost from all available marginal land was calculated as 32.9 CNY/GJ for Miscanthus Mode, 27.5 CNY/GJ for Switchgrass Mode, 32.4 CNY/GJ for Miscanthus & Switchgrass Mode, and 909 CNY/GJ for Jatropha Mode in 2017. The costs of Miscanthus and switchgrass were predicted to decrease by approximately 11%‐15%, whereas the cost of Jatropha was expected to increase by 5% in 2040. The cost of Jatropha varies significantly from 193 to 9,477 CNY/GJ across regions because of the huge differences in yield across regions. The economic potential of the marginal land was calculated as 28.7 EJ/year at a cost of less than 25 CNY/GJ for Miscanthus Mode, 4.0 EJ/year at a cost of less than 30 CNY/GJ for Switchgrass Mode, 29.6 EJ/year at a cost of less than 25 CNY/GJ for Miscanthus & Switchgrass Mode, and 0.1 EJ/year at a cost of less than 500 CNY/GJ for Jatropha Mode in 2017. It is not feasible to develop Jatropha production on marginal land based on existing technologies, given its high production costs. Therefore, the Miscanthus & Switchgrass Mode is the most economical way, because it achieves the highest economic potential compared with other modes. The sensitivity analysis showed that the farm‐gate costs of Miscanthus and switchgrass are most sensitive to uncertainties associated with yield reduction and harvesting costs, while, for Jatropha, the unpredictable yield has the greatest impact on its farm‐gate cost. This study can help policymakers and industrial stakeholders make strategic and tactical bioenergy development plans in China (exchange rate in 2017: 1€ = 7.63￥; all the joules in this paper are higher heat value).     

下层土壤容重对玉米根际土壤微生物数量及微生物量碳、氮的影响

采用微区池栽模拟试验,研究下层(20～40cm 、40～60cm)土壤容重改变后,玉米根际微生物数量、微生物量C(MBC)和微生物量N(MBN)的动态变化规律.结果表明,玉米根际微生物(细菌、放线菌、真菌)数量、MBC和MBN随土层加深和下层土壤容重增加而降低,且相同层次不同容重的处理间差异达显著水平;不同层次土壤根际微生物数量、MBC和MBN既受本土层容重大小影响,也随相邻土层容重增大其降幅增加,且20～40cm土层容重对土壤微生物数量、MBC和MBN的影响远大于40～60cm土层容重.玉米生育期间,三者受下层土壤容重变化和玉米生长发育的双重影响,且玉米的生长发育过程放大了容重对根际微生物数量、MBC和MBN的影响效果.     

Mycorrhizal fungi have a potential role in soil carbon storage under elevated CO2 and nitrogen deposition

In this review, we discuss the potential for mycorrhizal fungi to act as a source or sink for carbon (C) under elevated CO₂ and nitrogen deposition. Mycorrhizal tissue has been estimated to comprise a significant fraction of soil organic matter and below-ground biomass in a range of systems. The current body of literature indicates that in many systems exposed to elevated CO₂, mycorrhizal fungi might sequester increased amounts of C in living, dead and residual hyphal biomass in the soil. Through this process, the fungi might serve as a negative feedback on the rise in atmospheric CO₂ levels caused by fossil fuel burning and deforestation. By contrast, a few preliminary studies suggest that N deposition might increase turnover rates of fungal tissue and negate CO₂ effects on hyphal biomass. If these latter responses are consistent among ecosystems, C storage in hyphae might decline in habitats surrounding agricultural and urban areas. When N additions occur without CO₂ enrichment, effects on mycorrhizal growth are inconsistent. We note that analyses of hyphal decomposition under elevated CO₂ and N additions are extremely sparse but are critical in our understanding of the impact of global change on the cycling of mycorrhizal C. Finally, shifts in the community composition of arbuscular and ectomycorrhizal fungi with increasing CO₂ or N availability are frequently documented. Since mycorrhizal groups vary in growth rate and tissue quality, these changes in species assemblages could produce unforeseeable impacts on the productivity, survivorship, or decomposition of mycorrhizal biomass.     

Soil carbon stocks,deforestation and land‐cover changes in the Western Ghats biodiversity hotspot (India)

Habitat loss and soil organic carbon (SOC) stock variations linked to land‐cover change were estimated over two decades in the most densely populated biodiversity hotspot in the world, in order to assess the possible influence of conservation practices on the protection of SOC. For a study area of 88 484 km², 70% of which lie inside the Western Ghats Biodiversity Hotspot (WGBH), land‐cover maps for two dates (1977, 1999) were built from various data sources including remote sensing images and ecological forest maps. SOC stocks were calculated from climatic parameters, altitude, physiography, rock type, soil type and land‐cover, with a modelling approach used in predictive learning and based on Multiple Additive Regression Tree. The model was trained on 361 soil profiles data, and applied to estimate SOC stocks from predictor variables using a Geographical Information System (GIS). Comparison of 1977 and 1999 land‐cover maps showed 628 km² of dense forests habitat loss (6%), corresponding to an annual deforestation rate of 0.44%. This was found consistent with other studies carried out in other parts of the WGBH, but not with FAO figures showing an increase in forest area. This could be explained by the different forest definitions used, based on ecological classification in the former, and on percentage tree cover in the latter. Unexpectedly, our results showed that despite ongoing deforestation, overall SOC stock was maintained (～0.43 Pg). But a closer examination of spatial differences showed that soil carbon losses in deforested areas were compensated by sequestration elsewhere, mainly in recent plantations and newly irrigated croplands. This suggests that more carbon sequestration in soils could be achieved in the future through appropriate wasteland management. It is also expected that increasing concerns about biodiversity loss will favour more conservation and reinforce the already prevailing protective measures, thus further maintaining C stocks.     

Pathways of mineral‐associated soil organic matter formation: Integrating the role of plant carbon source,chemistry, and point of entry

To predict the behavior of the terrestrial carbon cycle, it is critical to understand the source, formation pathway, and chemical composition of soil organic matter (SOM). There is emerging consensus that slow‐cycling SOM generally consists of relatively low molecular weight organic carbon substrates that enter the mineral soil as dissolved organic matter and associate with mineral surfaces (referred to as “mineral‐associated OM,” or MAOM). However, much debate and contradictory evidence persist around: (a) whether the organic C substrates within the MAOM pool primarily originate from aboveground vs. belowground plant sources and (b) whether C substrates directly sorb to mineral surfaces or undergo microbial transformation prior to their incorporation into MAOM. Here, we attempt to reconcile disparate views on the formation of MAOM by proposing a spatially explicit set of processes that link plant C source with MAOM formation pathway. Specifically, because belowground vs. aboveground sources of plant C enter spatially distinct regions of the mineral soil, we propose that fine‐scale differences in microbial abundance should determine the probability of substrate–microbe vs. substrate–mineral interaction. Thus, formation of MAOM in areas of high microbial density (e.g., the rhizosphere and other microbial hotspots) should primarily occur through an in vivo microbial turnover pathway and favor C substrates that are first biosynthesized with high microbial carbon‐use efficiency prior to incorporation in the MAOM pool. In contrast, in areas of low microbial density (e.g., certain regions of the bulk soil), MAOM formation should primarily occur through the direct sorption of intact or partially oxidized plant compounds to uncolonized mineral surfaces, minimizing the importance of carbon‐use efficiency, and favoring C substrates with strong “sorptive affinity.” Through this framework, we thus describe how the primacy of biotic vs. abiotic controls on MAOM dynamics is not mutually exclusive, but rather spatially dictated. Such an understanding may be integral to more accurately modeling soil organic matter dynamics across different spatial scales.     

Respiratory carbon use and carbon storage in mid‐rotation loblolly pine (Pinus taeda L.) plantations: the effect of site resources on the stand carbon balance

We used estimates of autotrophic respiration (R_A), net primary productivity (NPP) and soil CO₂ evolution (S_ff), to develop component carbon budgets for 12‐year‐old loblolly pine plantations during the fifth year of a fertilization and irrigation experiment. Annual carbon use in R_A was 7.5, 9.0, 15.0, and 15.1 Mg C ha^?1 in control (C), irrigated (I), fertilized (F) and irrigated and fertilized (IF) treatments, respectively. Foliage, fine root and perennial woody tissue (stem, branch, coarse and taproot) respiration accounted for, respectively, 37%, 24%, and 39% of R_A in C and I treatments and 38%, 12% and 50% of R_A in F and IF treatments. Annual gross primary production (GPP=NPP+R_A) ranged from 13.1 to 26.6 Mg C ha^?1. The I, F, and IF treatments resulted in a 21, 94, and 103% increase in GPP, respectively, compared to the C treatment. Despite large treatment differences in NPP, R_A, and carbon allocation, carbon use efficiency (CUE=NPP/GPP) averaged 0.42 and was unaffected by manipulating site resources. Ecosystem respiration (R_E), the sum of S_ff, and above ground R_A, ranged from 12.8 to 20.2 Mg C ha^?1 yr^?1. S_ff contributed the largest proportion of R_E, but the relative importance of S_ff decreased from 0.63 in C treatments to 0.47 in IF treatments because of increased aboveground R_A. Aboveground woody tissue R_A was 15% of R_E in C and I treatments compared to 25% of R_E in F and IF treatments. Net ecosystem productivity (NEP=GPP‐R_E) was roughly 0 in the C and I treatments and 6.4 Mg C ha^?1 yr^?1 in F and IF treatments, indicating that non‐fertilized treatments were neither a source nor a sink for atmospheric carbon while fertilized treatments were carbon sinks. In these young stands, NEP is tightly linked to NPP; increased ecosystem carbon storage results mainly from an increase in foliage and perennial woody biomass.     

Tree seed rain and seed removal,but not the seed bank,impede forest recovery in bracken (Pteridium aquilinum (L.) Kuhn)‐dominated clearings in the African highlands

Considerable areas dominated by bracken Pteridium aquilinum (L.) Kuhn occur worldwide and are associated with arrested forest recovery. How forest recovery is impeded in these areas remains poorly understood, especially in the African highlands. The component processes that can lead to recruitment limitation—including low seed arrival, availability and persistence—are important determinants of plant communities and offer a potential explanation for bracken persistence. We investigated key processes that can contribute to recruitment limitation in bracken‐dominated clearings in the Bwindi Impenetrable National Park, Uganda. We examined if differences in seed rain (dispersal limitation), soil seed bank, or seed removal (seed viability and persistence) can, individually or in combination, explain the differences in tree regeneration found between bracken‐dominated areas and the neighboring forest. These processes were assessed along ten 50‐m transects crossing the forest–bracken boundary. When compared to the neighboring forest, bracken clearings had fewer seedlings (bracken 11,557 ± 5482 vs. forest 34,515 ± 6066 seedlings/ha), lower seed rain (949 ± 582 vs. 1605 ± 335 tree seeds m^?2 year^?1), comparable but sparse soil seed bank (304 ± 236 vs. 264 ± 99 viable tree seeds/m²), higher seed removal (70.1% ± 2.4% vs. 40.6% ± 2.4% over a 3‐day interval), and markedly higher rodent densities (25.7 ± 5.4 vs. 5.0 ± 1.6 rodents per 100 trapping sessions). Camera traps revealed that rodents were the dominant animals visiting the seeds in our seed removal study. Synthesis: Recruitment limitation contributes to both the slow recovery of forest in bracken‐dominated areas, and to the composition of the tree species that occur. Low seed arrival and low persistence of unburied seeds can both explain the reduced density of seedlings found in bracken versus neighboring forest. Seed removal, likely due to rodents, in particular appears sufficient to constrain forest recovery and impacts some species more severely than others.