Kinetic Characterization of 100 Glycoside Hydrolase Mutants Enables the Discovery of Structural Features Correlated with Kinetic Constants

The use of computational modeling algorithms to guide the design of novel enzyme catalysts is a rapidly growing field. Force-field based methods have now been used to engineer both enzyme specificity and activity. However, the proportion of designed mutants with the intended function is often less than ten percent. One potential reason for this is that current force-field based approaches are trained on indirect measures of function rather than direct correlation to experimentally-determined functional effects of mutations. We hypothesize that this is partially due to the lack of data sets for which a large panel of enzyme variants has been produced, purified, and kinetically characterized. Here we report the k_cat and K_M values of 100 purified mutants of a glycoside hydrolase enzyme. We demonstrate the utility of this data set by using machine learning to train a new algorithm that enables prediction of each kinetic parameter based on readily-modeled structural features. The generated dataset and analyses carried out in this study not only provide insight into how this enzyme functions, they also provide a clear path forward for the improvement of computational enzyme redesign algorithms.     

A novel double T-DNA system for producing stack and marker-free transgenic plants

This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7 containing seven target genes was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of two intact T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least seven target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.     

Metabolic regulation of ammonia emission in different senescence phenotypes of Nicotiana tabacum

In order to reveal the character of ammonia emission in senescent tobacco (Nicotiana tabacum), the content of NH₄⁺, total nitrogen, and soluble protein, and the activities of nitrogen metabolism-related enzymes were measured in leaves of a quick-leaf-senescence phenotype ZY90 and a slow-leaf-senescence phenotype NC89. Compared with NC89, ZY90 had a higher NH₄⁺ accumulation, a lower glutamine synthetase activity, and a significantly higher stomatal ammonia compensation point, and ammonia emission during 40 to 60 d after leaf emergence. During senescence, the quick-leafsenescence phenotype was characterized by nitrogen re-transfer by ammonia emmission, whereas the slow-leafsenescence phenotype by nitrogen re-assimilation. The ammonia emission was primarily regulated by glutamine synthetase activity, apoplastic pH, and NH₄⁺ content.     

Crosstalk of nitric oxide with calcium induced tolerance of tall fescue leaves to high irradiance

Calcium ion (Ca²⁺) is essential secondary messenger in plant signaling networks. In this study, the effect of Ca²⁺ on oxidative damage caused by a high irradiance (HI) was investigated in the leaves of two cultivars of tall fescue (Arid3 and Houndog5). Pretreatment of the tall fescue leaves with a CaCl₂ solution significantly increased Ca²⁺ content and intrinsic HI tolerance due to a decreased ion leakage and content of malondialdehyde, hydrogen peroxide, and superoxide radicals. Moreover, the activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase increased in both the cultivars in the presence of Ca²⁺ under the HI stress. In contrast, treatments with a Ca²⁺ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or a plasma membrane Ca²⁺ channel blocker LaCl₃ reversed these effects. On the other hand, a pronounced increase in nitric oxide synthase-like activity and NO release by exogenous Ca²⁺ treatment was observed in the tolerant Arid3 plants after exposure to the HI, whereas only a small increase was observed in more sensitive Houndog5. Moreover, the inhibition of NO production by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or N^ω-nitro-L-arginine blocked the protective effect of exogenous Ca²⁺, whereas the inhibition of Ca²⁺ by EGTA or LaCl₃ had no influence on the protective effect of NO. The results indicate that NO might be involved in the Ca²⁺-induced activities of antioxidant enzymes further protecting against HI-induced oxidative damage. This protective mechanism was found to be more efficient in Arid3 than in Houndog5.     

Combined effect of ethylene- and salicylic acid-signaling insensitive mutation on Arabidopsis response to low temperature

The roles of ethylene (ET) or salicylic acid (SA) in plant response to low temperature (LT, 5 °C) have been implicated. However, the combined effect of ET- and SA-signaling on plant growth and metabolism under LT remains to be evaluated. In this study, we comparatively analyzed the response of Arabidopsis ethylene insensitive (ein) 2-1 (an ET insensitive mutant), nonexprressor of pathogenesis relative (npr)1-1 (an SA insensitive mutant) and double mutant ein2-1/npr1-1 plants to LT. The results show that a LT of 5 °C induced plant growth retardation to a less degree in ein2-1, an intermediate degree in npr1-1, but a much larger in ein2-1/npr1-1 compared to the wild-type (WT) plants. The LT susceptibility of the ein2-1/npr1-1 plants was correlated to a lower net photosynthetic rate and proline content, and a higher content of H₂O₂ and malondialdehyde and electrolyte leakage relative to the WT plants. Lower activities of superoxide dismutase, peroxidase, and catalase, as well as a lower glutathione content and a ratio of its reduced form to its oxidized form were also observed in the double mutant plants as compared with the WT plants. However, at normal conditions (23 °C), all the tested physiological and biochemical parameters were comparable between the ein2-1/npr1-1 and WT plants, and plant growth was even better in the double mutant than in the WT plants. On the contrary, most of the above-mentioned parameters were advantageous in the ein2-1 and npr1-1 plants over the WT plants under the LT conditions. These data suggest that a parallel function or physiological redundancy of nonexpressor of pathogenesis relative 1 and ethylene insensitive 2 existed in the Arabidopsis plant response to the LT. On the other hand, an interaction between ET- and SA-signaling occurred during this process.     

Nitrogen deprivation induces cross-tolerance of Poa annua callus to salt stress

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (–N+NaCl). Malondialdehyde (MDA) and H₂O₂ content and Na⁺/K⁺ ratio significantly increased under the 600 mM NaCl treatment but less under the–N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H⁺-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the–N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H₂O₂ and Na⁺/K⁺ ratio dramatically increased, whereas PM H⁺-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H₂O₂ scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na⁺ over-uptake.     

Involvement of polar auxin transport in the inhibition of Arabidopsis seedling growth induced by Stenotrophomonas maltophilia

A wide range of microorganisms found in the rhizhosphere are able to regulate plant growth and development, but little is known about the mechanism by which epiphytic microbes inhibit plant growth. Here, an epiphytic bacteria Stenotrophomonas maltophilia, named as LZMBW216, were isolated and identified from the potato (Solanum tuberosum L. cv. Da Xi Yang) leaf surface. They could decrease primary root elongation and lateral root numbers in Arabidopsis seedlings. The inhibitory effects of LZMBW216 on plant growth were not due to a reduced indole-3-acetic acid (IAA) content, as exogenously applied IAA did not recover the inhibition. Furthermore, LZMBW216 did not affect the expression of DR5::GUS and CycB1;1::GUS. However, we found that LZMBW216 exhibited little effect on the primary root elongation in the pin2 mutant and on the lateral root numbers in the aux1-7 mutant. Moreover, LZMBW216 decreased expressions of AUX1 and PIN2 proteins. Together, these results suggest that root system architecture alterations caused by LZMBW216 may involve polar auxin transport.     

Hydrogen peroxide induces oxidative stress and the mitochondrial pathway of apoptosis in RAT intestinal epithelial cells (IEC-6)

In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H₂O₂), IEC-6 cells were subjected to 20 μmol/L H₂O₂ and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes. Results: Significant morphology damage was caused by exposure to H₂O₂, and results showed that ROS generation significantly increased (P < 0.01). The activity of superoxide dismutase decreased significantly (P < 0.05), malondialdehyde content increased (P < 0.05), and expression of both catalase and glutathione peroxidase decreased significantly (P < 0.05) in the H₂O₂ treatment group. Mitochondrion membrane potential was reduced, cytochrome released into the cytoplasm and caspase-9 and caspase-3 were significantly increased (P < 0.01) after treatment with H₂O₂. Moreover, the ratio of Bax/Bcl-2 and apoptosis were significantly increased (P < 0.01) in the H₂O₂ group. In conclusion, the present study indicated that the mitochondrial pathway plays a vital role in H₂O₂ induced IEC-6 cell apoptosis.