HbBIN2 Functions in Plant Cold Stress Resistance through Modulation of HbICE1 Transcriptional Activity and ROS Homeostasis in Hevea brasiliensis.

Cold stress poses significant limitations on the growth, latex yield, and ecological distribution of rubber trees (Hevea brasiliensis). The GSK3-like kinase plays a significant role in helping plants adapt to different biotic and abiotic stresses. However, the functions of GSK3-like kinase BR-INSENSITIVE 2 (BIN2) in Hevea brasiliensis remain elusive. Here, we identified HbBIN2s of Hevea brasiliensis and deciphered their roles in cold stress resistance. The transcript levels of HbBIN2s are upregulated by cold stress. In addition, HbBIN2s are present in both the nucleus and cytoplasm and have the ability to interact with the INDUCER OF CBF EXPRESSION1(HbICE1) transcription factor, a central component in cold signaling. HbBIN2 overexpression in Arabidopsis displays decreased tolerance to chilling stress with a lower survival rate and proline content but a higher level of electrolyte leakage (EL) and malondialdehyde (MDA) than wild type under cold stress. Meanwhile, HbBIN2 transgenic Arabidopsis treated with cold stress exhibits a significant increase in the accumulation of reactive oxygen species (ROS) and a decrease in the activity of antioxidant enzymes. Further investigation reveals that HbBIN2 inhibits the transcriptional activity of HbICE1, thereby attenuating the expression of C-REPEAT BINDING FACTOR (HbCBF1). Consistent with this, overexpression of HbBIN2 represses the expression of CBF pathway cold-regulated genes under cold stress. In conclusion, our findings indicate that HbBIN2 functions as a suppressor of cold stress resistance by modulating HbICE1 transcriptional activity and ROS homeostasis.

Improving the Activity of Tryptophan Synthetase via a Nucleic Acid Scaffold.

Tryptophan synthetase (TSase), which functions as a tetramer, is a typical enzyme with a substrate channel effect, and shows excellent performance in the production of non-standard amino acids, histamine, and other biological derivatives. Based on previous work, we fused a mutant CE protein (colistin of E. coli, a polypeptide with antibacterial activity) sequence with the sequence of TSase to explore whether its catalytic activity could be enhanced, and we also analyzed whether the addition of a DNA scaffold was a feasible strategy. Here, dCE (CE protein without DNase activity) protein tags were constructed and fused to the TrapA and TrapB subunits of TSase, and the whole cell was used for the catalytic reaction. The results showed that after the dCE protein tag was fused to the TrapB subunit, its whole cell catalytic activity increased by 50%. Next, the two subunits were expressed separately, and the proteins were bound in vitro to ensure equimolar combination between the two subunits. After the dCE label was fused to TrapB, the activity of TSase assembled with TrapA also improved. A series of experiments revealed that the enzyme fused with dCE9 showed higher activity than the wild-type protein. In general, the activity of assembly TSase was optimal when the temperature was 50 °C and the pH was about 9.0. After a long temperature treatment, the enzyme maintained good activity. With the addition of exogenous nucleic acid, the activity of the enzyme increased. The maximum yield was 0.58 g/L, which was almost three times that of the wild-type TSase (0.21 g/L). The recombinant TSase constructed in this study with dCE fusion had the advantages of higher heat resistance and higher activity, and confirmed the feasibility of adding a nucleic acid scaffold, providing a new idea for the improvement of structurally similar enzymes.

Thermoplastic moulding of regenerated silk.

Early insights into the unique structure and properties of native silk suggested that ß-sheet nanocrystallites in silk would degrade prior to melting when subjected to thermal processing. Since then, canonical approaches for fabricating silk-based materials typically involve solution-derived processing methods, which have inherent limitations with respect to silk protein solubility and stability in solution, and time and cost efficiency. Here we report a thermal processing method for the direct solid-state moulding of regenerated silk into bulk 'parts' or devices with tunable mechanical properties. At elevated temperature and pressure, regenerated amorphous silk nanomaterials with ultralow ß-sheet content undergo thermal fusion via molecular rearrangement and self-assembly assisted by bound water to form a robust bulk material that retains biocompatibility, degradability and machinability. This technique reverses presumptions about the limitations of direct thermal processing of silk into a wide range of new material formats and composite materials with tailored properties and functionalities.

HbSnRK2.6 Functions in ABA-Regulated Cold Stress Response by Promoting HbICE2 Transcriptional Activity in Hevea brasiliensis.

Low temperature remarkably limits rubber tree (Hevea brasiliensis Muell. Arg.) growth, latex production, and geographical distribution, but the underlying mechanisms of Hevea brasiliensis cold stress response remain elusive. Here, we identified HbSnRK2.6 as a key component in ABA signaling functions in phytohormone abscisic acid (ABA)-regulated cold stress response in Hevea brasiliensis. Exogenous application of ABA enhances Hevea brasiliensis cold tolerance. Cold-regulated (COR) genes in the CBF pathway are upregulated by ABA. Transcript levels of all five HbSnRK2.6 members are significantly induced by cold, while HbSnRK2.6A, HbSnRK2.6B, and HbSnRK2.6C can be further activated by ABA under cold conditions. Additionally, HbSnRK2.6s are localized in the cytoplasm and nucleus, and can physically interact with HbICE2, a crucial positive regulator in the cold signaling pathway. Overexpression of HbSnRK2.6A or HbSnRK2.6B in Arabidopsis extensively enhances plant responses to ABA and expression of COR genes, leading to increased cold stress tolerance. Furthermore, HbSnRK2.6A and HbSnRK2.6B can promote transcriptional activity of HbICE2, thus, increasing the expression of HbCBF1. Taken together, we demonstrate that HbSnRK2.6s are involved in ABA-regulated cold stress response in Hevea brasiliensis by regulating transcriptional activity of HbICE2.

Deciphering metabolic responses of biosurfactant lichenysin on biosynthesis of poly-γ-glutamic acid.

Poly-γ-glutamic acid (γ-PGA) is an extracellularly produced biodegradable polymer, which has been widely used as agricultural fertilizer, mineral fortifier, cosmetic moisturizer, and drug carrier. This study firstly discovered that lichenysin, as a biosurfactant, showed the capability to enhance γ-PGA production in Bacillus licheniformis. The exogenous addition of lichenysin improved the γ-PGA yield up to 17.9% and 21.9%, respectively, in the native strain B. licheniformis WX-02 and the lichenysin-deficient strain B. licheniformis WX02-ΔlchAC. The capability of intracellular biosynthesis of lichenysin was positively correlated with γ-PGA production. The yield of γ-PGA increased by 25.1% in the lichenysin-enhanced strain B. licheniformis WX02-Psrflch and decreased by 12.2% in the lichenysin-deficient strain WX02-ΔlchAC. Analysis of key enzyme activities and gene expression in the TCA cycle, precursor glutamate synthesis, and γ-PGA synthesis pathway revealed that the existence of lichenysin led to increased γ-PGA via shifting the carbon flux in the TCA cycle towards glutamate and γ-PGA biosynthetic pathways, minimizing by-product formation, and facilitating the uptake of extracellular substrates and the polymerization of glutamate to γ-PGA. Insight into the mechanisms of enhanced production of γ-PGA by lichenysin would define the essential parameters involved in γ-PGA biosynthesis and provide the basis for large-scale production of γ-PGA.

Propofol alleviates cisplatin-related cognitive impairment.

Chemotherapy-related cognitive impairment (CRCI) is commonly reported following the administration of chemotherapeutic agents and comprises a wide variety of neurological problems. Many patients after chemotherapy need further surgery under anesthesia. Thus, in this study, we examined whether propofol, one of the most commonly used anesthetics in surgery, could further affect the cognitive abilities in mouse CRCI models. The mice were injected intraperitoneally with cisplatin (2 mg/kg/day) for continuous 10 days and showed significantly reduced body weights. After 10 days reconversion, mice with cisplatin injection showed impaired memory retention in the inhibitory avoidance (IA) task, mimicking the CRCI in patients. Then, we found that a single injection of propofol with the sub-anesthetic dosage (50 mg/kg) but not the anesthetic dosage (250 mg/kg) could significantly alleviate the cisplatin-induced memory impairment. These results imply the possible clinical application of propofol, especially at the sub-anesthetic dosage, in the surgery of patients after chemotherapy.

Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide.

Phosphoproteomics studies have reported phosphorylation at multiple sites within collagen, raising the possibility that these post-translational modifications regulate the physical or biological properties of collagen. In this study, molecular dynamics simulations and experimental studies were carried out on model peptides to establish foundational principles of phosphorylation of Ser residues in collagen. A (Gly-Xaa-Yaa)11 peptide was designed to include a Ser-containing sequence from type I collagen that was reported to be phosphorylated. The physiological kinase involved in collagen phosphorylation is not known. In vitro studies showed that a model kinase ERK1 (extracellular signal-regulated protein kinase 1) would phosphorylate Ser within the consensus sequence if the collagen-like peptide is in the denatured state but not in the triple-helical state. The peptide was not a substrate for FAM20C, a kinase present in the secretory pathway, which has been shown to phosphorylate many extracellular matrix proteins. The unfolded single chain (Gly-Xaa-Yaa)11 peptide containing phosphoSer was able to refold to form a stable triple helix but at a reduced folding rate and with a small decrease in thermal stability relative to the nonphosphorylated peptide at neutral pH. These biophysical studies on model peptides provide a basis for investigations into the physiological consequences of collagen phosphorylation and the application of phosphorylation to regulate the properties of collagen biomaterials.

Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding.

Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa)n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502GFPGER507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI.

Lichenysin production is improved in codY null Bacillus licheniformis by addition of precursor amino acids.

Lichenysin is categorized into the family of lipopeptide biosurfactants and has a variety of applications in the petroleum industry, bioremediation, pharmaceuticals, and the food industry. Currently, large-scale production is limited due to the low yield. This study found that lichenysin production was repressed by supplementation of extracellular amino acids. The global transcriptional factor CodY was hypothesized to prevent lichenysin biosynthesis under an amino acid-rich condition in Bacillus licheniformis. Thus, the codY null strain was constructed, and lichenysin production was increased by 31.0% to 2356 mg/L with the addition of precursor amino acids, and the lichenysin production efficiency was improved by 42.8% to 98.2 mg/L• h. Correspondingly, the transcription levels of the lichenysin synthetase gene lchAA, and its corresponding regulator genes comA, degQ, and degU, were upregulated. Also, the codY deletion enhanced biosynthesis of lichenysin precursor amino acids (Gln, Ile, Leu, and Val) and reduced the formation of byproducts, acetate, acetoin, and 2,3-butanediol. This study firstly reported that lichenysin biosynthesis was negatively regulated by CodY and lichenysin production could be further improved with the precursor amino acid amendment in the codY null strain.

Neuroprotective effects of HTR1A antagonist WAY-100635 on scopolamine-induced delirium in rats and underlying molecular mechanisms.

BACKGROUND: Limited surveys have assessed the performance of 5-hydroxytreptamine receptor 1A and its antagonist WAY-100635 in pharmacological manipulations targeting delirium therapies. The purpose of this paper was to assess the central pharmacological activity of WAY-100635 in a rat model of scopolamine-induced delirium and its underlying mechanism. RESULTS: A delirium rat model was established by intraperitoneal injection of scopolamine and behavioral changes evaluated through open field and elevated plus maze experiments. Concentrations of monoamines in the hippocampus and amygdalae were detected by high performance liquid chromatography. The effect of WAY-100635 on the recovery of rats from delirium was assessed by stereotactic injection of WAY-100635 and its mechanism of action determined by measuring mRNA and protein expression via real time PCR and western blotting methods. The total distance and the number of crossing and rearing in the elevated plus maze test and the time spent in the light compartment in the dark/light test of scopolamine-treated rats were significantly increased while the percentage of time spent in the open arms was decreased, showing the validity of the established delirium rat model. The measurement of the concentrations of noradrenaline, 3,4-dihydroxyphenylacetic acid, the homovanillic acid, 5-hydroxy-3-indoleacetic acid and serotonin concentrations in the cerebrospinal fluid (CSF) of scopolamine-induced delirium rats were significantly increased. The intra-hippocampus and intra-BLA injections of WAY-100635 improved the delirium-like behavior of rats by significantly reducing the expression of NLRP3 inflammasome and the release of IL1-ß and IL8 into CSF. CONCLUSIONS: Taken together, these findings indicate that WAY-100635 may exert a therapeutic effect on post-operative delirium by controlling neurotransmission as well as suppressing neuroinflammation in the central nervous system.

5-HT(1A) receptor antagonist improves behavior performance of delirium rats through inhibiting PI3K/Akt/mTOR activation-induced NLRP3 activity.

Postoperative delirium is a common complication that often results in poor outcomes in surgical and elderly patients. Accumulating evidences suggest that the pathophysiology of delirium results from multiple neurotransmitter system dysfunctions. To further clarify the effects of the selective serotonin (5-HT) (1A) antagonist WAY-100635 on the behaviors in scopolamine induced-delirium rats and to explore the molecular mechanism, in this study, we investigated the change of monoamine levels in the cerebrospinal fluid (CSF) and different brain regions using high-performance liquid chromatography and assessed the behavioral retrieval of delirium rats treated with WAY-100635. It was found that 5-hydroxy-3-indoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid, and homovanillic acid concentrations in the CSF of scopolamine-induced delirium rats were significantly increased, among which 5-HIAA was also increased in hippocampus and basolateral amygdala (BLA), and 5-HT(1A) receptor was significantly higher in the hippocampuses and BLA than other brain regions. Furthermore, intrahippocampus and intra-BLA stereotactic injection of WAY-100635 improved the delirium-like behavior of rats. Mechanistically, after WAY-100635 treatment, significant reduction of IL-1ß release into CSF and NOD-like receptor family, pyrin domain containing 3 (NLRP3) expression, phosphorylated phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), and S6K was observed. Altogether, these results suggest that delirium rats induced by scopolamine may be correlated with an increased cerebral concentration of 5-HT and dopamine neurotransmitters system; the selective 5-HT(1A) antagoniszts can reverse the delirium symptoms at some extent through tendering PI3K/Akt/mammalian target of rapamycin complex 1 (mTOR) activation-induced NLRP3 activity and then reducing IL-1ß release.

[Magnetic resonance guided focused ultrasound surgery for pain palliation of bone metastases: early experience of clinical application in China].

OBJECTIVE: To evaluate the safety and efficacy of magnetic resonance guided focused ultrasound surgery (MRgFUS) in treatment for pain palliation of bone metastases. METHODS: Eighty-one patients of painful bone metastases were volunteered to screen for this study in Shanghai General Hospital from June 2014 to February 2015. Twenty-three patients among them were treated by MRgFUS, who was more than 18-years old, having the ability to fully understand the informed consent of the research, suffering with pain of numeric rating scale (NRS) ≥ 4, non-received radiotherapy or chemotherapy for pain palliation of bone metastases in the past two weeks. The NRS, the standard question of Brief Pain Inventory (BPI-QoL), and the standard question of Europe Organization for Research and Treatment of Cancer Quality of Life Questionnaire- Bone Metastases 22 (EORTC QLQ-BM22) were respectively recorded before and 1-week, 1-month, 3-month after the treatment. The related adverse events of MRgFUS were observed and recorded in 3 months after the treatment as well. RESULTS: (1)Twenty-three metastatic bone tumor lesions of 23 patients were treated by MRgFUS, the treatment data was as follows: the mean treatment time was (88 ± 33) minutes, the mean sonication number was 13 ± 8. (2) Adverse events included: pain in therapy area 3/23, which spontaneous relieving within one week; numbness in lower limb (1/23), which relieved after physiotherapy. (3) The NRS of before treatment and at 1-week, 1-month, and 3-month after treatment respectively was 6.0 ± 1.5, 3.7 ± 1.7,3.1 ± 2.0, and 2.2 ± 1.0,which significantly decreased after the treatment (P<0.01). (4) The BPI-QoL score of before treatment and at 1-week, 1-month, and 3-month after treatment respectively was 39 ± 16, 27 ± 18, 26 ± 18, and 21 ± 18, which significantly decreased after the treatment (P<0.01). (5) The EORTC QLQ-BM22 score of before treatment and at 1-week, 1-month, and 3-month after treatment respectively was 52 ± 13, 44 ± 12, 42 ± 12, and 39 ± 12, which also significantly decreased after the treatment (P<0.01). CONCLUSIONS: MRgFUS can be used as a non-invasive, safe, and effective method for treating painful bone metastases. Its clinical benefits of pain palliation and patient's quality of life improving are sustained after the treatment at least to 3 months.

Improvement of lichenysin production in Bacillus licheniformis by replacement of native promoter of lichenysin biosynthesis operon and medium optimization.

Lichenysin is a biodegradable surfactant with huge potential for recovering crude oil from the oil reservoir. The current production of lichenysin is made through fermentation from wild strain of Bacillus licheniformis, which is limited by low yield. The aim of this work was to improve lichenysin-producing capability of a wide strain B. licheniformis WX-02. Lichenysin produced from WX-02 was first extracted, purified, and identified. Through the substitution of the promoter of lichenysin biosynthesis operon, the mutants B. licheniformis WX02-P43lch, WX02-Pxyllch, and WX02-Psrflch were constructed with the constitutive promoter (P43), the xylose-inducible promoter (P xyl ), and the surfactin operon promoter (P srf ), respectively. A consistent change trend was observed between lichenysin production and lchAA gene transcription, confirming the strength of the promoters as an important factor for lichenysin synthesis. Among the three mutants, WX02-Psrflch produced the highest lichenysin yield. The production by the mutant WX02-Psrflch was further improved with the optimization of the major medium components including glucose, NH4NO3, and Na2HPO4/KH2PO4. Under 30 g/L glucose, 5 g/L NH4NO3, and 80 mM/60 mM Na2HPO4/KH2PO4, the strain WX02-Psrflch produced 2,149 mg/L lichenysin, a 16.8-fold improvement compared to that of wild strain WX-02.

Exploring techniques for vision based human activity recognition: methods, systems, and evaluation.

With the wide applications of vision based intelligent systems, image and video analysis technologies have attracted the attention of researchers in the computer vision field. In image and video analysis, human activity recognition is an important research direction. By interpreting and understanding human activity, we can recognize and predict the occurrence of crimes and help the police or other agencies react immediately. In the past, a large number of papers have been published on human activity recognition in video and image sequences. In this paper, we provide a comprehensive survey of the recent development of the techniques, including methods, systems, and quantitative evaluation towards the performance of human activity recognition.

Current Insights on the Diverse Structures and Functions in Bacterial Collagen-like Proteins.

The dearth of knowledge on the diverse structures and functions in bacterial collagen-like proteins is in stark contrast to the deep grasp of structures and functions in mammalian collagen, the ubiquitous triple-helical scleroprotein that plays a central role in tissue architecture, extracellular matrix organization, and signal transduction. To fill and highlight existing gaps due to the general paucity of data on bacterial CLPs, we comprehensively reviewed the latest insight into their functional and structural diversity from multiple perspectives of biology, computational simulations, and materials engineering. The origins and discovery of bacterial CLPs were explored. Their genetic distribution and molecular architecture were analyzed, and their structural and functional diversity in various bacterial genera was examined. The principal roles of computational techniques in understanding bacterial CLPs' structural stability, mechanical properties, and biological functions were also considered. This review serves to drive further interest and development of bacterial CLPs, not only for addressing fundamental biological problems in collagen but also for engineering novel biomaterials. Hence, both biology and materials communities will greatly benefit from intensified research into the diverse structures and functions in bacterial collagen-like proteins.

Genome sequence of Bacillus licheniformis WX-02.

Bacillus licheniformis is an important bacterium that has been used extensively for large-scale industrial production of exoenzymes and peptide antibiotics. B. licheniformis WX-02 produces poly-gamma-glutamate increasingly when fermented under stress conditions. Here its genome sequence (4,270,104 bp, with G+C content of 46.06%), which comprises a circular chromosome, is announced.

Complete Genome Sequence of Brachybacterium sp. Strain NBEC-018, Isolated from Nematode-Infected Potatoes.

Brachybacterium species are ubiquitous Gram-positive bacteria. Here, we report the complete genome sequence of Brachybacterium sp. strain NBEC-018. The strain was isolated from rotten potatoes that were infected with potato rot nematodes (Ditylenchus destructor). The genome sequence will be beneficial to clarify the ecological role of Brachybacterium species.

Sulfenylation of ENOLASE2 facilitates H2O2-conferred freezing tolerance in Arabidopsis.

H2O2 affects the expression of genes that are involved in plant responses to diverse environmental stresses; however, the underlying mechanisms remain elusive. Here, we demonstrate that H2O2 enhances plant freezing tolerance through its effect on a protein product of low expression of osmotically responsive genes2 (LOS2). LOS2 is translated into a major product, cytosolic enolase2 (ENO2), and sometimes an alternative product, the transcription repressor c-Myc-binding protein (MBP-1). ENO2, but not MBP-1, promotes cold tolerance by binding the promoter of C-repeat/DRE binding factor1 (CBF1), a central transcription factor in plant cold signaling, thus activating its expression. Overexpression of CBF1 restores freezing sensitivity of a LOS2 loss-of-function mutant. Furthermore, cold-induced H2O2 increases nuclear import and transcriptional binding activity of ENO2 by sulfenylating cysteine 408 and thereby promotes its oligomerization. Collectively, our results illustrate how H2O2 activates plant cold responses by sulfenylating ENO2 and promoting its oligomerization, leading to enhanced nuclear translocation and transcriptional activation of CBF1.

Acaricidal Activity and Field Efficacy Analysis of the Potential Biocontrol Agent Bacillus vallismortis NBIF-001 against Spider Mites.

In recent years, spider mites have caused considerable economic losses to global agriculture. However, currently available management strategies are limited because of the rapid development of resistance. In this study, Bacillus vallismortis NBIF-001 was isolated and evaluated for its acaricidal activity. NBIF-001 exhibited a significant lethal effect on spider mites within 48 h. The median lethal concentration (LC50) of the culture powders (3.2 × 1010 CFU/g) was 50.2 µg/mL for Tetranychus urticae (red form), 18.0 µg/mL for T. urticae (green form), and 15.7 µg/mL for Panonychus citri (McGregor). Cultivation optimisation experiments showed that when the number of spores increased, fermentation toxicity also increased. Moreover, field experiments demonstrated that NBIF-001 performed well in the biocontrol of P. citri, which showed a similar corrected field efficacy with the chemical control (67.1 ± 7.9% and 71.1 ± 6.4% after 14 days). Genomics analysis showed that NBIF-001 contains 231 factors and seven gene clusters of metabolites that may be involved in its acaricidal activity. Further bioassays of the fermentation supernatants showed that 50× dilution treatments killed 72.5 ± 5.4% of the mites in 48 h, which was similar with those of the broth. Bioassays of the supernatant proteins confirmed that various proteins exhibited acaricidal activity. Five candidate proteins were expressed and purified successfully. The bioassays showed that the small protein BVP8 exhibited significant acaricidal activity with an LC50 of 12.4 µg/mL (T. urticae). Overall, these findings suggest that B. vallismortis NBIF-001 is a potential biocontrol agent for spider mite management.

Complete Genome Sequence of Bacillus badius NBPM-293, a Plant Growth-Promoting Strain Isolated from Rhizosphere Soil.

Bacillus species have a long history of widespread use in biocontrol and crop growth-promoting fields. Here, we present the genome sequence of the rhizobacterium B. badius NBPM-293. The genome sequence will provide valuable information for a better understanding of the mechanism of plant growth promotion.