Ferritin transgenic Arabidopsis plants
Ten independent transformed T1 Arabidopsis FerEx plants that expressing soybean ferritin protein targeted extracellularly were generated. Total RNA was extracted from these ten transgenic lines and was reverse transcribed to cDNA. The prepared cDNA and the primers (listed in the “Methods” section) were used for the real-time RT PCR analysis, which detected the soybean ferritin transcripts in all ten transgenic lines.
Shoot iron content and biomass yield of transgenic plants
Since iron accumulation is the main plant trait that is essential to the goal of this study, the initial measurement of iron content was conducted using the stems of these ten transformants at their T2 generation. Of these ten transformants, two transformants (FerEX-8a and -10g) showed the highest iron content, and were selected to further process to their T3 generation, for which their homozygosity was confirmed by segregation analysis.
To examine the effects of extracellularly expressed ferritin on plant growth, the FerEX transgenic (FerEX-8a and -10g) and empty vector (EV) control plants were grown in parallel under both H2O-watering and iron-fertilizing conditions. Under distilled H2O-watering conditions, transgenic FerEX plants that overexpressed ferritin extracellularly grew normally as did the EV control plants. Under iron-fertilizing conditions the FerEX transgenic plants showed increased growth compared with the EV control plants, for which the phenotypes of representative plants are shown in Fig. 1a. The average height of FerEX transgenic plants at the senescent stage was 46.2 cm, which was a 12 % (p < 0.05) increase over that of the EV control plants. Meanwhile, the average dry weight of FerEX transgenic plant tissues was 145 mg, which was 18 % (p < 0.05) increase over that of EV control transgenic plants (Fig. 1b). Other studies have reported increases in biomass yields in transgenic plants of Arabidopsis [20], lettuce [25], and tobacco [19] that expressed heterologous ferritin proteins intracellularly. This study, to the best of our knowledge, demonstrated for the first time that similar enhancement phenomenon also exists in Arabidopsis expressing heterologous ferritin extracellularly.
In addition, ICP-OES analysis showed that iron contents in the shoot tissues of FerEX transgenic plants (105–108 ppm in dry matter) was 2.1–2.2 times greater than that of the transgenic EV control plants under normal growth conditions with distilled H2O-watering, which suggests that the FerEX plants can be planted without Fe fertilizing but still hyperaccumulate iron from the soil (Fig. 2a). Similarly, under the iron-fertilizing condition, the iron content in the shoot tissues of the FerEX transgenic plants (527–539 ppm in dry matter), was also approximately 2.1 times that of the EV control plants (Fig. 2b). Note that the above iron content levels in FerEX were slightly higher but not statistically significant different from the previously obtained FerIN transgenic Arabidopsis lines that expressed ferritin intracellularly [26]. The latter (FerIN) accumulated iron to a level of 95–100 ppm under normal H2O-watering condition, and 514–520 ppm under iron-fertilizing condition [26].
PCR and Western blot analyses
As described above, initial real-time RT PCR analysis was conducted on the total RNA and converted cDNA for all the independent transformed T1 Arabidopsis FerEx plants, which detected the soybean ferritin transcripts in all these plants. In addition, PCR analysis of the genomic DNA extract from FerEX transgenic lines (FerEX-8a and -10g) confirmed the integration of the heterologous ferritin transgene into the genome of these lines (Fig. 1c).
The expression of the heterologous ferritin protein in FerEX plants was examined by Western blot analysis, using the cell-wall proteins extracted from the shoot tissues collected at the mid-pod stage and the chicken IgY polyclonal antibody against a synthesized soybean ferritin peptide. The Western blot analyses confirm that expression of soybean ferritin in FerEx transgenic plants resulted in a peptide of the expected molecular mass of 26-kDa in denaturing SDS-PAGE (Fig. 1d), as well as an expected molecular mass of about 600-kDa for the ferritin complex in native PAGE (Fig. 1e).
Iron localization in shoot tissues
Optical and X-ray fluorescence microscopy (XFM) were used to investigate iron accumulation at the cellular scale of tissues. First, fresh cross sections of stems from FerEX transgenic and EV control plants were examined by optical microscopy after Perls’ Prussian blue staining [16]. In the EV control plants, some slight blue staining can be detected within the stem sections (Fig. 3a, b). In contrast, significant blue staining signals were detected within the FerEX transgenic plant stem sections, compared to EV control plants, which indicates iron accumulation at higher levels in FerEX transgenic plants (Fig. 3c, d). Under higher magnification, the iron in the EV control plants is deposited at the interfascicular fiber (IFs) between the cortex and the pith parenchyma cells (Fig. 3b), while in the FerEX transgenic plant sections, the deposition of iron is found around the plant cell walls across the whole section (Fig. 3d).
In addition, the images of senesced stems reveal the existence of some faint blue staining in the interior surface of the cell lumen (which is the lumen side of cell walls) in the EV control (Additional file 1: Figure S1A), which is not surprising as endogenous Arabidopsis ferritin proteins also exist in the host plant. In contrast, iron deposition was observed in the compound corner middle lamella of FerEX transgenic plants (Additional file 1: Figure S1B, white arrow); such extracellular distribution of iron is consistent with the targeted extracellular region for heterologous ferritin expression in the FerEx transgenic plants.
Second, XFM was also used to detect and map iron in 2-micron-thick cross sections cut from EV and FerEX senesced stems (Fig. 4). Results from this highly sensitive elemental mapping technique were in good agreement with those obtained from the Perls’ Prussian blue staining results. XFM maps of iron in the EV stems showed several small, isolated regions of high iron content; however, the iron signal within the cell walls was largely similar to the background over the majority of the image. In contrast, the iron in the FerEX stems (Fig. 4c, d; Additional file 1: Figure S2E–H) could be detected at a level significantly higher than background throughout the cell walls in most regions, but was also observed in elevated concentrations in highly localized areas. The submicron spatial resolution of XFM and the thinness of the sections facilitate the mapping of ions in different cell-wall layers, such as secondary and middle lamella layers [14, 29]. The observation of iron throughout the FerEX cell walls indicates the iron is distributed in both the secondary and compound middle lamella cell-wall layers. This finding suggests that the objective of this study to incorporate iron throughout the entirety wall was generally successful. in addition, similar to the Perls’ Prussian blue staining results, increased iron concentrations could also be detected in some of the corner compound middle lamella regions (Additional file 1: Figure S3, white arrows).
FerEX transgenic Arabidopsis: hot-water pretreatment and co-saccharification
Previously, dilute acid pretreatment was used to assess the pretreatability of FerIN transgenic Arabidopsis [26]. In contrast, this study used hot-water pretreatment to evaluate the pretreatability of the biomass, because it is a greener technology that not only benefits the environment, but also avoids the erosion effect of dilute acid to the reactor and eliminates the downstream step of neutralizing the pretreated biomass residue prior to saccharification. In addition, for comparison purposes, the two previously generated intracellular ferritin-overexpressing (FerIN-2a and -4b) transgenic Arabidopsis plants [26] were also grown, harvested, and pretreated side by side with the FerEX plants.
The dried, ground biomass from iron-fertilized FerEX, FerIN transgenic plants, and the EV control plants was subjected to high-throughput (HTP) hot-water pretreatments at 180 °C, 17.5 min, and after enzymatic saccharification the sugar release was measured. A HTP method that uses 5 mg biomass per well was used because of the small quantity of biomass available from each transgenic plant. The data shows that glucose released after enzymatic hydrolysis from hot-water-pretreated FerEX transgenic plants was similar to FerIN transgenic plants: enhanced 18–21 % more than that found after the hot-water pretreatment and enzymatic hydrolysis of the iron fertilized EV control transgenic plants, which is slightly higher (but not statistically significant) than the extent of enhancement (15–17 %) on glucose release in the FerIN transgenic plants over the EV plants (Fig. 5a).
In contrast, the xylose released from the FerEX transgenic plant biomass was enhanced to a larger extent, i.e., 29–34 % compared eith the EV control plants after hot-water pretreatment and enzyme saccharification, which is significantly higher than the extent of enhancement (14–16 %) on xylose release in FerIN transgenic plants over the EV plant (Fig. 5b). This is a very significant enhancement for the FerEX transgenic plants at these low severity pretreatment conditions. Such observation can be attributed to the facts that FerEX plants accumulate iron in the cell-wall region with a close proximity to cell-wall biopolymers including xylans.
This study showed that FerEX plants enhance more glucose (up to 21 %) and xylose (up to 34 %) release from biomass, which is higher than EV control (Fig. 4); as well as the FerIN plants [26]. Based on the increased shoot dry weight of FerEX plants (18 % more biomass; Fig. 1b) and the sugar release (21 % more glucose and 34 % more xylose; Fig. 5), the sugar yields on a per plant basis is 43 and 58 % more glucose and xylose, respectively, greater than that expected from EV control plants. Theoretically, 100 g of glucose or 100 g of xylose can produce 51.4 g of ethanol [30–32]. Bioethanol production from the improved of sugar release observed in FerEX plants can be calculated following Krishnan et al. [33], in which it was estimated that ethanol yields from glucose and xylose fermentation by recombinant Saccharomyces were typically with 95 and 80 % of the theoretical yield, respectively [33]. A more thorough technoeconomic analysis (TEA) can be conducted in the future after transgenic bioenergy plants expressing ferritin extracellularly are grown at large scale.
Biomass compositional analysis and the implication
It is noteworthy that after hot-water pretreatment and enzymatic digestion of plant biomass, the FerEX plants released 29–34 % more xylose over the EV plant, compared to releasing 18–21 % more glucose over the EV plant (Fig. 5). To address the observed higher extent of xylose release in the FerEX transgenic plants (versus control), we measured the chemical compositions of the harvested EV and FerEX(2) biomasses, using the method described in the "Methods" section. The data revealed that EV biomass contained 33.3 ± 0.6 % cellulose and 12.2 ± 0.4 %, while FerEX(2) biomass contained 33.6 ± 0.3 % cellulose and 12.4 ± 0.3 % (n = 3), with no significant differences in their cellulose and xylan compositions between the two lines. It thus appears that the incorporation of heterologous ferritin into the growing wall does not alter wall composition.
Like most other plant proteins, plant ferritins likely have a frequent turnover rate throughout the plant growth phase. The values of the half-lives of ferritin proteins in various species and cells were reported to be in the range of 3.5–72 h [34–36], and it is safe to assume that the value of the half-life of plant ferritin proteins falls within a similar range. We propose that as the expressed extracellular proteins degrade, the released iron ions are initially deposited onto nascent cellulose, hemicelluloses, and lignins, and then quickly diffuse through the continuous polymer matrix of plant cells. Furthermore, because hemicelluloses lack the crystalline structure of cellulose, their availability, and indeed susceptibility to hydrolysis, is greater than cellulose. Eventually, a softened and continuous, interconnecting network that facilitates chemical transport is established in the walls [14, 37].
In planta iron accumulation versus post-harvest supplementation of iron
Although techno-economic analysis for the presented approach of in planta iron accumulation in delivering iron catalyst to the plant cell wall has not been conducted, the following two factors support the presented approach of in planta iron accumulation may likely be cost efficient and application feasible. First, the localization of iron observed from imaging data supports a new bioprocessing benefit of using a plant-produced ferritin (versus an externally post-harvest exogenously added Fe catalyst), presumably because the accumulated iron ions are in close proximity to cell-wall substrates such as cellulose and hemicellulose in the FerEX transgenic plants. Second, FerEX plants were found to have more shoot biomass than the EV control plants under iron-watering condition (increased by 18 % as indicated in Fig. 1b). Thus, the cost of applying iron fertilization is likely to be offset by the increased plant biomass, as well as the increased pretreatability, digestibility, and the total amount of sugar released.
Comparison of the intracellularly versus extracellularly approaches
The differences between expressing ferritin in plants intracellularly and extracellularly have two considerations. First, considering that most of the metabolic activities of plant cells take place intracellularly, overexpression of ferritin intracellularly has been demonstrated to provide the plants better protection from oxidative stress, iron toxicity, photoinhibition, and pathogens [21, 23, 24, 38]. The consequences of expressing ferritin extracellularly on plant defenses and other stresses are less clear, and remain to be studied further.
Second, these two approaches definitely showed clear contrast in the pattern for iron deposition in live plants. Our previous study clearly showed that the iron was predominantly deposited on the interior surfaces of cell lumen in FerIN plants [26]; and accordingly, we proposed that at the senescent stage, the intracellularly accumulated iron will be released into the internal (lumenal) surface of cell walls from the broken cells at later growth stages. This release would facilitate the interaction between iron co-catalyst and plant biomass during pretreatment and lead to increased biomass pretreatability.
In contrast, expressing ferritin extracellularly in plants allows the delivery of iron into the plant cell wall, presumably embedded in a sandwich pattern as new cell walls are formed layer by layer. Moreover, this deposition may also create a continuous iron ion diffusion pathway along the hemicellulose network throughout the cell-wall matrix. In this regard, the FerEX approach delivers the iron closer to the plant cell-wall matrix which benefits downstream processing.
Our understanding of the chemical mechanism for the iron enhancement of sugar release during dilute acid and hot-water pretreatments of biomass remains limited. However, from in vitro Fourier transform infrared spectroscopy (FTIR) analysis of treated biomass, we have demonstrated that the in muro iron ions during dilute acid pretreatment targets the more recalcitrant chemical bonds in the cell wall, especially the C–O–C and C–H bonds, whereas dilute acid alone targets primarily the glycosyl bonds of polysaccharides [13]. Future studies are warranted to gain deeper understanding of the atomic level interactions between metal ions and cell-wall polymers.
Efficiency of ferritin secretion using signal peptide and future studies
The expression vector pCAMBIA1305.2 that we used in this study was developed by pCAMBIA (http://www.cambia.org) and contains a rice glycine-rich protein (GRP) signal peptide for extracellular targeting. GRP is one of the major secreted proteins that form the plant cell-wall structure [39]. This vector has been widely used and well demonstrated in literature to express target proteins extracellularly in various plant cells and tissues [40, 41].
Particularly, a recently published study used the above GRP signal peptide in pCAMBIA1305.2 vector and GFP to form pCAMBIA1305.2-SP-GFP, which was named as pSBI-MF11 to transform sugarcane callus cells. The GFP fluorescent microscopic images of the callus cells transformed with pSBI-MF11 showed a clear localization of GFP to the apoplastic space [42]. Such observation effectively demonstrated the efficiency of using GRP signal peptide to direct the secretion of GFP protein to extracellular space of plant cells.
So far, to the best of our knowledge, no heterologous plant ferritins had been reported to be fused to GFP and expressed in transgenic ferritin plants [16–21, 23–25, 38, 43, 44]. The mature ferritin is a 24-mer protein assembly, and it was reported that the C termini of many members of the ferritin family tend to be buried during folding [45]. It is unclear if the fusion of a plant ferritin (with a molecular size of approximately 26 kDa for its monomer) to GFP (with a molecular size of approximately 27 kDa) would cause a disruption to the normal self-assembly of ferritin subunits. In addition, the autofluorescence of plant cell wall may also cause interference to microscopic observation of GFP fluorescence. As this complex issue is out of the scope of this study, future studies are warranted to examine the suitability of expressing GFP-ferritin fusion protein in plants for visualizing the localization of target protein.
Future studies for characterizing ferritin expression and function using model microorganisms
This study has demonstrated a correlation between the iron capture (as shown by Perls’ blue staining and XFM data) and ferritin expression (as shown by the Western blotting analysis of extracted cell-wall proteins) on the plant tissue level. Expressing the soybean ferritin gene, SferH-1, in model microorganisms may be attempted in future work to enable a more precise correlation at the protein level. The expression of bullfrog H-subunit ferritin [46], pea-seed ferritin [47] and soybean seed ferritin [48] have been reported in E. coli; in those works, the wild-type ferritins all aggregated as inclusion bodies and were not soluble when heterologously expressed in E. coli at 37 °C. Various approaches, including the engineering of the heterologous wild-type ferritins, the lowering of the induction temperature for cell growth, and/or the coexpression of the chaperone molecules had been used to increase the solubility and function of the expressed ferritins in microorganisms [46–48]. Although similar studies are beyond the scope of our current work presented here, future studies to express engineered soybean ferritin in E. coli or yeast for confirming the iron capture at the protein level should be conducted.
Future applications of co-catalyst-accumulating plants for the bioenergy sector
Our previous data demonstrated the effectiveness of expressing ferritin intracellularly in Arabidopsis to increase the iron accumulation during plant growth; which enhanced the pretreatability and digestibility of the harvested biomass [26]. Importantly, the extracellularly expressed ferritin Arabidopsis plants generated in this study showed a further enhancement the pretreatability and digestibility of the biomass compared with the FerIN transgenic plants (Fig. 5). Together, these studies generating the FerEX plants effectively replace the previous approach of soaking iron containing acid solutions into milled biomass prior to pretreatment, which was time consuming and subject to diffusion limitations. Moreover, this metal co-catalyst accumulation strategy should be “stackable” with other cell-wall engineering approaches, which can lead to a more environment-friendly and economical production of feedstocks for the production of biofuels and biochemicals.
Given the general similarity in cell-wall structure and hemicellulose chemistry, it is feasible that the results shown here for expression of ferritin in Arabidopsis plants will extend to poplar (also dicot) and switchgrass (monocot), the representative bioenergy plants.