Evaluation of an optimal extraction method for measuring d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) in agricultural soils and its association with soil microbial CO₂ assimilation

Abstract

Assimilating atmospheric carbon (C) into terrestrial ecosystems is recognized as a primary measure to mitigate global warming. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the dominant enzyme by which terrestrial autotrophic bacteria and plants fix CO₂. To investigate the possibility of using RubisCO activity as an indicator of microbial CO₂ fixation potential, a valid and efficient method for extracting soil proteins is needed. We examined three methods commonly used for total soil protein extraction. A simple sonication method for extracting soil protein was more efficient than bead beating or freeze–thaw methods. Total soil protein, RubisCO activity, and microbial fixation of CO₂ in different agricultural soils were quantified in an incubation experiment using ¹⁴C-CO₂ as a tracer. The soil samples showed significant differences in protein content and RubisCO activity, defined as nmol CO₂ fixed g⁻¹ soil min⁻¹. RubisCO activities ranged from 10.68 to 68.07 nmol CO₂ kg⁻¹ soil min⁻¹, which were closely related to the abundance of cbbL genes (r = 0.900, P = 0.0140) and the rates of microbial CO₂ assimilation (r = 0.949, P = 0.0038). This suggests that RubisCO activity can be used as an indicator of soil microbial assimilation of atmospheric CO₂.

Introduction

The anthropogenic increase in atmospheric carbon dioxide (CO₂) is generally believed to significantly contribute to global warming (Lacis et al., 2010). Transferring carbon (C) from the atmosphere to terrestrial ecosystems is a major C sequestration measure (Midgley et al., 2010). Carbon sequestration in terrestrial ecosystems is a result of the assimilation of CO₂ by autotrophic bacteria, algae, and plants (Tabita, 1999) via the Calvin–Benson–Basham (CBB) cycle. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the most abundant protein on Earth (Raven, 2013) and an essential enzyme in the CBB cycle. It fixes CO₂ through the reductive pentose phosphate pathway by combining with CO₂ to form 3-phosphoglycerate (Siegenthaler and Sarmiento, 1993, Sato et al., 2010). RubisCO is a major autotrophic carboxylase in all photosynthetic organisms, and more than 99.5% of the inorganic C assimilated by primary producers (chemolithotrophs as well as photolithotrophs) involves RubisCO (Raven, 2009).

RubisCO enzymes, which exist in a variety of autotrophic organisms including Proteobacteria and Acinobacteria as well as Cyanobacteria, Firmicutes and Chloroflexi (Hügler and Sievert, 2010), have been widely investigated in aquatic ecosystems or laboratory microbial cultivation (Ezaki, 1999, Chakrabarti et al., 2002, Takai et al., 2005, Hügler and Sievert, 2010, Tourova et al., 2010) because of the enzymatic properties of RubisCO that link directly to CO₂ fixation rates. However, relative few studies are concerned with the RubisCO of microbes involved in soil CO₂ assimilation. Carbon fixation genes (cbbL), which encode the RubisCO enzyme, are numerous and widespread in diverse soil ecosystems (Selesi et al., 2007, Videmšek et al., 2009, Yuan et al., 2012, Wu et al., 2013, Xiao et al., 2014).

In comparison to functional gene studies, the analysis of proteins can provide more information about active metabolic pathways (Singleton et al., 2003, Tyson et al., 2004, Ram et al., 2005, Schulze et al., 2005, Wilmes and Bond, 2006, Benndorf et al., 2007, Renella et al., 2014). This protein-based technique can characterize soil enzymes involved in biogeochemical cycling in both natural and polluted soil ecosystems, such as rhizosphere soil, grassland soil, forest soil and contaminated soil (Renella et al., 2002, Singleton et al., 2003, Schulze et al., 2005, Benndorf et al., 2007, Fornasier and Margon, 2007, Wang et al., 2011, Wu et al., 2011). Enzymes are protein molecules with catalytic activities (Ogunseitan, 1997), therefore, the accuracy of the RubisCO activity measurements of field samples depends on obtaining representative extracts of bioactive protein. In the initial efforts to extract protein from sediments and soils, researchers used either cell extraction (recovery of cells from the soil matrix prior to cell lysis) or direct lysis within the soil matrix (Singleton et al., 2003, Barzaghi et al., 2004, Benndorf et al., 2007). Direct extraction of soil protein provides a less biased sample of the soil microbial communities (Singleton et al., 2003, Ogunseitan, 2006).

Bead beating, freeze–thaw and sonication are three commonly used methods for breaking aggregated soil particles and for lysing cells to release protein molecules (Singleton et al., 2003, Ogunseitan et al., 2004, Bastida et al., 2009). However, these soil protein extraction methods are inefficient in the various individual step, with problems of incomplete cell lysis, protein sorption on soil surfaces, and loss or degradation of the protein. Therefore, the primary objective of this work was to establish an optimal method for rapid and efficient soil protein extraction. The extraction method we propose here preserves protein function as much as possible and extracts protein from the entire microbial community, representing the contributions from both prokaryotic and eukaryotic microorganisms. Once the method was established, it was used to examine the activity of soil RubisCO in response to changes in soil microbial CO₂ assimilation rates in different types of agricultural soils using a ¹⁴C-CO₂ tracer experiment. We hypothesized that the measurement of soil RubisCO activity following appropriate calibration can provide a reasonable estimate of the microbial CO₂ fixation potential in the soil.