Mine reclamation using biofuel crops: Insights into the microbial ecology of the switchgrass (Panicum virgatum) microbiome

Bioenergy crop production has steadily increased due to growing political support for renewable energy, thus initiating a demand to find alternative agricultural land. An innovative option is the use of marginal soils, such as reclaimed mine lands, to produce bioenergy crops. Switchgrass (Panicum virgatum) is a promising bioenergy crop that can be grown on marginal lands due to its robust growth in various soil types and climates. However, little is known regarding plant-microbe interactions among switchgrass systems within reclaimed mine lands. A study conducted in 2008 grew switchgrass on high- and low- quality reclaimed mine sites (Hampshire and Hobet, respectively) in West Virginia to examine the resilience of switchgrass as a reclamation-friendly bioenergy crop. Switchgrass yields at Hampshire were nearly an order of magnitude higher than Hobet (8.4 Mg ha−1 vs 1.0 Mg ha−1). Within Hampshire, the Cave-in-Rock cultivar yield was approximately 2-fold greater than that of Shawnee (12.9 Mg ha-1 vs. 7.6 Mg ha-1). Here, I sought to illuminate plant-microbial interactions that may account for this drastic shift in cultivar yield by assessing the soil microbial community’s function and composition. I tested two hypotheses: i) that the microbial community’s ability to acquire C, N, and P will be greatest in Hampshire soils compared to that of Hobet and ii) that there will be a cultivar-specific root-associated microbiome that may drive previously observed greater, but differential yields across switchgrass cultivars at Hampshire. I found that reclamation strategy substantially impacts the switchgrass microbiome’s composition as well as its ability to acquire critical nutrients like carbon, nitrogen, and phosphorus. I also found that a functionally, but not necessarily compositionally, unique microbiome exists in the root-associated soils compared to that of the bulk soil. Additionally, there were indicators that organic amendments to the topsoil may induce cultivar-specific soil microbiomes that mediate or facilitate differential yields within Hampshire. Taken together, I suggest that organic amendments to the topsoil during reclamation selects for a cultivar-specific microbiome more adept to acquiring critical nutrients and thus, increases aboveground productivity

Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis

The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy.Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+.Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu

Sperm penetrate jelly but not the vitelline envelope of <i>X</i>. <i>laevis</i> eggs inseminated in BAPTA.

<p>(A) Inseminated eggs were incubated in 0 or 3 mM BAPTA, and with 0 or 3 mM CaCl₂, were stained with Hoechst to visualize the sperm. 20 minutes following insemination, eggs were dejellied and imaged using fluorescence and bright-field microscopy to assess sperm penetration of the vitelline envelope. Representative images document the presence of Hoechst-stained sperm within the vitelline envelope of eggs inseminated in DVF/3 alone (<i>top</i>) or DVF/3 with 3 mM BAPTA and 3 mM CaCl₂ (<i>bottom</i>) (N = 33–56 eggs in 4 experimental trials). By contrast, no Hoechst-stained sperm were evident within the vitelline envelope of eggs inseminated in DVF/3 with 3 mM BAPTA (<i>middle</i>). Scale bars represent 25 μm. Red, dashed line on overlay indicates location of envelope. (B) Incidence of cleavage furrow development of eggs inseminated in DVF/3 with 0 or 3 mM BAPTA, washed after five minutes, and transferred to a final solution as indicated (N = 75–87 eggs in 3 experimental trials).</p

<i>X</i>. <i>laevis</i> embryos developed normally in the absence of added Ca²⁺.

<p>(A) Averaged percentage of embryos that developed cleavage furrows from eggs inseminated in MR/3 or DVF/3 (N = 153–160 eggs in 5 experimental trials). (B) Representative images of a developed <i>X</i>. <i>laevis</i> embryo at the 4-cell stage (top), an undivided egg (middle), and an embryo with faint cleavage furrows (bottom); scale bar = 250 μm.</p

Extracellular Ca²⁺ is required for early embryonic development in <i>X</i>. <i>laevis</i>.

<p>Plots of averaged percentage of embryos that developed from eggs inseminated in DVF/3 with increasing chelator or CaCl₂ concentrations. Each plot was fit with a sigmoidal function. (A) BAPTA concentrations ranged from 10 μM—5 mM (N = 71–190 eggs in 3–5 experimental trials). (B) Varying concentrations of added CaCl₂ ranging from 10 μM—5 mM, with 1 mM BAPTA (N = 74–102 in 3–5 experimental trials). (C) Various EGTA concentrations ranging from 3 μM—3 mM (N = 80–167 in 4–6 experimental trials).</p

Extracellular Ca²⁺ important for the earliest events of embryonic development in <i>X</i>. <i>laevis</i>.

<p>Incidence of cleavage furrow development from eggs inseminated in DVF/3 either with 0 or 3 mM BAPTA. After 30 minutes, inseminated eggs were washed twice and moved to a new solution of DVF/3 with 0 or 3 mM BAPTA, as indicated. Embryos were assessed for the appearance of cleavage furrows 60–90 minutes after transfer (90–120 minutes after sperm addition) (N = 75–85 eggs in 4 experimental trials).</p

Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis

The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy.Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+.Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu