Global rice production had reached 660 million dry metric tons in 2009, along with 800 million metric tons of straw, and continues to grow at a steady rate [32
]. Rice straw contains a high cellulose content (approximately 45%), and is a suitable resource for the large-scale production of biofuels to replace fossil fuels and to reduce environmental pollution caused by agricultural wastes and burning of fossil fuels [33
]. Use of cell wall-degrading enzymes from microorganisms for the conversion of cellulosic material to sugars is a limiting step at present. Thus, large-scale production of effective cellulose hydrolytic enzymes is key to the biofuels industry. The expression of heterologous proteins in transgenic plants is an established technology. Consequently, the expression of foreign proteins has been successfully applied in plant systems at various levels, including industrially useful enzymes, viral proteins, pharmaceutical proteins, polypeptides (including antibodies) and various structural proteins [34
]. The factors influencing the level of accumulation for each of these protein classes vary. Although success depends on the characteristics of the individual protein, protein accumulation has been particularly successful when targeted to the cell wall (apoplast compartment), vacuole or endoplasmic reticulum [11
This study demonstrated that the E1
gene of A. cellulolyticus
integrated into the genome of transgenic rice plants (Figure ) can be properly transcribed (Figures and ) and translated into an active enzyme and accumulated at high levels (Figures , , and ). We noticed that some of the transgenic rice plants with high E1 activities exhibited a short stature and flowered earlier than wild type plants. However, in a previous study, transgenic rice overexpressing A. cellulolyticus
did not show any deleterious effect on plant growth and development [24
]. The differences in E1 effect on rice plant growth and development between the present study and that of Oraby et al
] could in part stem from expression level. The highest expression level achieved in our transgenic lines was 6.1% of the total leaf soluble protein, higher than that reported by Oraby et al
]. The controversy regarding the effects of E1 on plant growth and development has been reported previously [11
]. It also could be related to the difference in the primary structure of cell walls and its composition between monocot (rice) and dicot (tobacco) plants [39
] or the position effects of E1
transgene insertion in the genome of transgenic plants. Further study is needed to address the effect of growth temperature on the growth and development of E1 transgenic rice. Most importantly, the E1 protein produced in rice retains its thermostability and can be purified by a simple heat treatment (Figure ). Also significant is the fact that it remains active in tissues after a long period of storage at room temperature and the presence of E1 in the rice straw increases its hydrolytic efficiency (Figure ).
The A. cellulolyticus
E1 activity could be detected in all tissues of all five transgenic lines. Its activities in the leaves ranged from 60,000 pmol MU/mg protein/min to 79,000 pmol MU/mg protein/min in the homozygous plants derived from the line with the highest expression level (Figure ). The large (ten-fold) variation in expression among the five transgenic lines containing E1
(Figure ) was presumably a position effect, as all transgenic lines had one copy of insertion (Figure ). This highlights the importance of generating as many transgenic plants as possible for selecting high expression lines with a normal phenotype. Transformation of rice with the E1 expression vector under the control of a constitutive Mac promoter [30
] with the tobacco pathogenesis related signal peptide [21
] allowed the heterologous E1 protein to be secreted to and accumulated in the apoplastic compartment at high levels. This is consistent with previous studies reporting that the apoplast can serve as a storage site for large quantities of functional foreign proteins [7
]. Based on the zymogram PAGE (Figure ) and western blot analysis data (Figure ), E1 protein in all tissue extracts of the transgenic plants was partially degraded to its catalytic domain polypeptide of 38 kDa (Figure ), in agreement with the observation of previous studies [7
]. Obviously, the degraded protein still retains its catalytic activity. The partial degradation of E1 protein may be due to the sensitivity of the cellulose-binding domain to proteases in plant extracts. Further study will be necessary to illustrate the E1 protein degradation pattern in plant tissues.
Although the Mac promoter is known to drive constitutive expression of transgenes in plants and the E1
transcript was detected in all tissues, including the mature seed, floret, leaf, stem and root of transgenic rice (Figures ,, and ), much higher expression levels were found in green tissues, such as leaf, floret and stem (Figure ). However, a relative large amount of E1 protein was detected in the root soluble protein sample in the western blot analysis (Figure ), but a relatively low E1 specific enzyme activity was found in the enzyme activity assay (Figure ). This is due to the fact that an equal amount of the total soluble protein for each sample was used in both analyses for comparison but root extract contains a relative low amount of other soluble protein and some potential inhibitors to the activity of E1. The highest amount of E1 protein accumulation, in the leaves of our transgenic rice plants, was up to 6.1% of the total leaf soluble protein (Figure ), higher than the 4.9% in the transgenic rice plants reported by Oraby et al
], in which the cauliflower mosaic virus 35S promoter was used to drive the expression of the bacterial E1
gene in rice. Thus, the Mac promoter may be more effective for E1 protein production. However, the position of the gene insertion into the genome apparently has a strong influence on transgene expression. With a conservative estimate of ten metric tons of rice straw produced per hectare per year and a 5% E1 protein content in the tissues, 30 kg of very pure E1 protein can be produced annually from a hectare of padding field.
The high thermostability and protease-resistance of A. cellulolyticus
E1 protein allows the enzyme to remain active in the tissues for a long period of time. Most importantly, as demonstrated in the digestion experiment with cultured CGF (Figure ), direct expression of A. cellulolyticus
E1 in the transgenic rice straw increases the hydrolytic efficiency of cellulose during saccharification, which can reduce the amount of hydrolytic enzyme needed for the conversion of cellulosic biomass to fermentative sugars. A recent study with transgenic tobacco and maize suggests that the expression of A. cellulolyticus
E1 during cell wall construction may alter the inherent recalcitrance of the cell wall [26
]. All of these features show the great potential of using transgenic plants as a bioreactor for the large scale production of cellulases and reducing the cost for enzymes through tissue autohydrolysis.