Identification of potato varieties suitable for cold storage and reconditioning: A safer alternative to anti-sprouting chemicals for potato sprouting control.

Low temperature storage as an alternative to anti-sprouting chemicals in potato storage may induce reducing sugars (RS) accumulation (i.e. glucose and fructose) in potato tubers. This phenomenon is called "cold induced sweetening" (CIS) and occurs in certain varieties. CIS leads to a decrease in the organoleptic qualities and darkening of processed potato and the accumulation of toxic molecules such as acrylamide. To identify potato varieties suitable for storage at low temperatures, we screened six commercial processing varieties: Lady Claire (LC), Verdi, Kiebitz (KB), Pirol, Agria and Markies for their CIS characteristics and sprout-forming potential after storage at 4 °C and 8 °C. Our findings reveal that 4 °C storage allows for efficient sprout reduction in all six tested varieties for up to 4.5 months of storage. Three CIS-resistant varieties, namely Verdi, Lady Claire and Kiebitz, were identified as able to be stored for up to four months at 4 °C with limited increase in glucose content. Conversely, Pirol, Agria and Markies showed an increase in glucose content with a decrease in storage temperature and can be considered as CIS-susceptible varieties. After processing into crisps, the CIS-susceptible varieties displayed poor crisp color quality (brown to black color crisps) after storage for two months at 4 °C compared to the storage at 8 °C, whereas the CIS-resistant varieties had good crisp color quality (pale yellow color crisps) after storage at both 4 and 8 °C. Interestingly, the trends of total RS and/or glucose content in the CIS-resistant and in the CIS-susceptible varieties were correlated with the trends in Vacuolar Invertase (VInv) gene expression for most varieties, as well as with the trends in acrylamide content after processing. In addition, reconditioning of Markies variety after storage at 4 °C by gradually increasing the temperature to 15 °C resulted in a significant decrease of VInv transcript levels (reduction of 80 %), acrylamide content (reduction of 75 %) and glucose content when compared to a storage at 4 °C without reconditioning. Those results demonstrate that the reconditioning technique is a key factor for a sustainable potato storage and for improving the quality of processed potatoes.

Combining hybrid nanoflowers with hybridization chain reaction for highly sensitive detection of SARS-CoV-2 nucleocapsid protein.

BACKGROUND: COVID-19 (coronavirus disease 2019) pandemic has had enormous social and economic impacts so far. The nucleocapsid protein (N protein) is highly conserved and is a key antigenic marker for the diagnosis of early SARS-CoV-2 infection. RESULTS: In this study, the N protein was first captured by an aptamer (Aptamer 58) coupled to magnetic beads (MBs), which in turn were bound to another DNA sequence containing the aptamer (Aptamer 48-Initiator). After adding 5'-biotinylated hairpin DNA Amplifier 1 and Amplifier 2 with cohesive ends for complementary hybridization, the Initiator in the Aptamer 48-Initiator began to trigger the hybridization chain reaction (HCR), generating multiple biotin-labeled DNA concatamers. When incubated with synthetic streptavidin-invertase-Ca3(PO4)2 hybrid nanoflower (SICa), DNA concatamers could specifically bind to SICa through biotin-streptavidin interaction with high affinity. After adding sucrose, invertase in SICa hydrolyzed sucrose to glucose, whose concentration could be directly read with a portable glucometer, and its concentration was positively correlated with the amount of captured N protein. The method is highly sensitive with a detection limit as low as 1 pg/mL. SIGNIFICANCE: We believe this study provided a practical solution for the early detection of SARS-CoV-2 infection, and offered a new method for detecting other viruses through different target proteins.

The potential roles of acid invertase family in Dendrobium huoshanense: Identification, evolution, and expression analyses under abiotic stress.

Dendrobium huoshanense, a traditional Chinese medicine prized for its horticultural and medicinal properties, thrives in an unfavorable climate and is exposed to several adverse environmental conditions. Acid invertase (AINV), a widely distributed enzyme that has been demonstrated to play a significant role in response to environmental stresses. However, the identification of the AINV gene family in D. huoshanense, the collinearity between relative species, and the expression pattern under external stress have yet to be resolved. We systematically retrieved the D. huoshanense genome and screened out four DhAINV genes, which were further classified into two subfamilies by the phylogenetic analysis. The evolutionary history of AINV genes in D. huoshanense was uncovered by comparative genomics investigations. The subcellular localization predicted that the DhVINV genes may be located in the vacuole, while the DhCWINV genes may be located in the cell wall. The exon/intron structures and conserved motifs of DhAINV genes were found to be highly conserved in two subclades. The conserved amino acids and catalytic motifs in DhAINV proteins were determined to be critical to their function. Notably, the cis-acting elements in all DhAINV genes were mainly relevant to abiotic stresses and light response. In addition, the expression profile coupled with qRT-PCR revealed the typical expression patterns of DhAINV in response to diverse abiotic stresses. Our findings could be beneficial to the characterization and further investigation of AINV functions in Dendrobium plants.

Sugar metabolism and 14-3-3 protein genes expression induced by arbuscular mycorrhizal fungi and phosphorus addition to response drought stress in Populus cathayana.

Sugar, as a nutrient exchange substance between arbuscular mycorrhizal (AM) fungi and host plants, plays an important role in the abiotic stress response of mycorrhizal plants. This experiment aimed to study the effects of AM fungi and phosphorus (P) addition on the sugar metabolism and 14-3-3 gene expression of Populus cathayana under drought stress. The results showed that drought affects the process of sugar metabolism by increasing the activities of amylase and invertase, resulting in the decrease of starch content in leaves and roots and the accumulation of soluble sugars (including reducing sugar and sucrose) in roots. Under drought stress, the activity or content of sucrose synthetase, sucrose phosphate synthase, acid invertase, alkaline invertase, reducing sugar, soluble sugar, sucrose, and starch in the root showed the best mycorrhizal effect at the 100 mg P level. The expression levels of the 14-3-3 genes (PcGRF10 and PcGRF11) were significantly increased by mycorrhizal induction under drought stress. These levels were positively correlated with SS, SPS, sucrose, and starch phosphorylase in leaves, as well as with almost all sugar metabolism indicators in roots. However, they were negatively correlated with starch content in both leaves and roots. Sugar metabolism and 14-3-3 protein gene expression were induced by AM fungi and P addition in response to drought stress. The 14-3-3 genes induced by AM fungi may be involved in participating in osmotic regulation during drought stress. This study provides a new idea for the mechanism of sugar metabolism of mycorrhizal plants in arid regions.

Identification and expression analysis of invertase family genes during grape (Vitis vinifera L.) berry development under CPPU and GA treatment.

Sugar is crucial for grape berry, whether used for fresh food or wine. However, berry enlargement treatment with forchlorfenuron (N-(2-chloro4-pyridyl)-N'-phenylurea) (CPPU, a synthetic cytokinin) and gibberellin (GA) always had adverse effects on sugar accumulation in some grape varieties, especially CPPU. Therefore exploring the molecular mechanisms behind these adverse effects could provide a foundation for improving or developing technology to mitigate the effects of CPPU/GA treatments for grape growers. In the present study, invertase (INV) family, the key gene controlling sugar accumulation, was identified and characterized on the latest annotated grape genome. Their express pattern, as well as invertase activity and sugar content, were analyzed during grape berry development under CPPU and GA3 treatment to explore the potential role of INV members under berry enlargement treatment in grapes. Eighteen INV genes were identified and divided into two sub-families: 10 neutral INV genes (Vv-A/N-INV1-10) and 8 acid INV genes containing 5 CWINV (VvCWINV1-5) and 3 VIN (VvVIN1-3). At the early development stage, both CPPU and GA3 treatment decreased the hexose level in berries of 'Pinot Noir' grape, whereas the activity of three types inverstase (soluble acid INV, insoluble acid INV, and neutral INV) increased. Correspondingly, most of INV members were up-regulated by GA3 /CPPU application at least one sampling time point during early berry development, including VvCWINV1, 2, 3, 4, 5, VvVIN1, 2, 3 and Vv-A/N-INV1, 2, 5, 6, 7, 8, 10. At maturity, the sugar content in CPPU-treated berries is still lower than that in the control. Soluble acid INV and neutral INV, rather than insoluble acid INV, presented lower activity in CPPU-treated berries. Meanwhile, several corresponding genes, such as VvVIN2 and Vv-A/N-INV2, 8, 10 in ripening berries were obviously down-regulated by CPPU treatment. These results suggested that most of INV members could be triggered by berry enlargement treatment during early berry development, whereas VvVINs and Vv-A/N-INVs, but not VvCWINVs, could be the limiting factor resulting in decreased sugar accumulation in CPPU-treated berries at maturity. In conclusion, this study identified the INV family on the latest annotated grape genome and selected several potential members involving in the limit of CPPU on final sugar accumulation in grape berry. These results provide candidate genes for further study of the molecular regulation of CPPU and GA on sugar accumulation in grape.

Effects of hemp seed and flaxseed on enzyme activity in the broiler chicken digestive tract.

The activity of enzymes in the digestive tract is an important parameter for appropriate digestive tract function. Feed mixtures can be adjusted to support enzymatic activity in different parts of the digestive tract. Flaxseed and hemp seed are commodities and significant sources of nutrition, and their addition to feed could change enzymatic activity in the digestive tract and improve nutritional intake. The aim of this study was to determine the effects of flaxseed, hemp seed and a combination of both on basic enzymes in the polysaccharidase group, such as amylase, cellulase, pectinase, xylanase and inulinase; basic enzymes in the disaccharidase group, including maltase, invertase and lactase; proteinases and lipases in the digestive tract of broiler chickens. During the experiment, the control group was fed a diet without flaxseed or hemp seed. The diet of the second group contained 80 g/kg flaxseed, the diet of the third group contained 40 g/kg hemp seed, and the diets of the fourth to sixth groups contained 80 and 30 g/kg, 80 and 40 g/kg and 80 and 50 g/kg flaxseed and hemp seed, respectively. Enzyme activity was found to depend on the location in the digestive tract and the composition of the feed mixture (P < 0.05). Most enzymatic conversion occurs in the ileum, where the addition of flaxseed and hemp seed to the diet increased most enzyme activities, namely, amylase, cellulase, pectinase, xylanase, maltase, invertase, proteinase and lipase activities. The highest values of enzyme activity were found in groups IV-VI fed a combination of flaxseed and hempseed, especially in chickens fed diet VI (flaxseed and hemp seed at 80 and 50 g/kg). Growth performance results confirmed the enzyme activity results, as the weights of the chickens increased after the addition of flaxseed and/or hemp seed. The findings have economic implications, suggesting that feeding a diet with a combination of flaxseed and hemp seed is beneficial.

[Genotype-Specific Features of Cold-Induced Sweetening Process Regulation in Potato Varieties Nikulinsky, Symfonia, and Nevsky].

In this study, we performed expression analysis of genes associated with cold-induced sweetening in potato tubers: vacuolar invertase (Pain-1), sucrose synthase (SUS4), and invertase inhibitor (InvInh2). Potato varieties Nikulinsky, Symfonia, and Nevsky were used. All three varieties were found to accumulate sugars at low temperatures; the maximum accumulation of reducing sugars was observed at 4°C. It was found that the expression pattern of genes associated with cold-induced sweetening differs depending on the variety and storage duration. The increased expression of vacuolar invertase and its inhibitor is more pronounced at the beginning of storage period, whereas the increased expression of sucrose synthase is more pronounced after 3 months of storage. At early storage periods, high expression of invertase and low expression of inhibitor is observed in the Dutch variety Symfonia, and vice versa in the Russian varieties Nikulinsky and Nevsky. The involvement of the studied genes in the process of cold-induced sweetening is discussed.

Fertilization can modify the enantioselective persistence of penthiopyrad in relation to the co-influence on soil ecological health.

Penthiopyrad is a widely used chiral fungicide for controlling rust and Rhizoctonia diseases. Development of optically pure monomers is an important strategy to realize amount reduction and increment effects of penthiopyrad, wherein, fertilizers as the co-exiting nutrient supplement may alter the enantioselective residues of penthiopyrad in soil. In our study, influences of urea, phosphate, potash, NPK compound, organic granular, vermicompost and soya bean cake fertilizers on enantioselective persistence of penthiopyrad were fully evaluated. This study demonstrated that R-(-)-penthiopyrad dissipated faster than S-(+)-penthiopyrad during 120 days. High pH, available nitrogen, invertase activities and reduced available phosphorus, dehydrogenase, urease, catalase activities were situated to benefit removing the concentrations of penthiopyrad and weakening enantioselectivity in soil. With respect to the impact of different fertilizers on soil ecological indicators, vermicompost contributed to enhanced pH. Urea and compound fertilizer played an absolute advantage in promoting available nitrogen. All fertilizers didn't go against available phosphorus. Dehydrogenase responded negatively to phosphate, potash and organic fertilizers. Urea increased invertase, besides, it and compound fertilizer both diminished urease activity. The catalase activity was not activated by organic fertilizer. Based on all the findings, soil application of urea and phosphate fertilizers was recommended and considered as a better option to exhibit high efficiency for the dissipation of penthiopyrad. The combined environmental safety estimation can effectively guide the treatment of fertilization soils in line with the nutrition requirements and pollution regulation from penthiopyrad.

An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.

Taking glucose as intermediate bridge-signal-molecule for on-site and convenient detection of ochratoxin A in rice with portable glucose meter.

On-site screening of biotoxins is of great importance for food safety. A new electrochemical-biosensing strategy was constructed for ochratoxin A (OTA) detection by direct using ready-made commercial portable-glucose-meter (PGM). Aptamer against OTA was adopted as the recognition probe and pre-immobilized onto the sensing interface. The complementary biotin-modified probe was further decorated by hybridization. Biotinylated invertase was further introduced onto the sensing system with streptavidin, which also acted as the signal amplification unit. The invertase, which was depended on the amount of OTA, produced the glucose from sucrose in the sensing solution. The glucose could be directly and conveniently measured with PGM. Quantitative analysis of OTA was achieved with a linear range from 0.5 ng/mL to 10 ng/mL and detection limit of 0.45 ng/mL. Of significance, it has been successfully applied for OTA analysis in rice with satisfied recoveries. This unique PGM-based electrochemical platform reveals prospective potential in food safety monitoring.

CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.

MAIN CONCLUSION: VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes. CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90-99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars' analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.

Enzyme Activities in Reduction of Heavy Metal Pollution from Alice Landfill Site in Eastern Cape, South Africa.

Heavy metals are unbreakable, and most of them are poisonous to animals and people. Metals are particularly concerning among environmental contaminants since they are less apparent, have extensive effects on ecosystems, are poisonous, and bioaccumulate in ecosystems, biological tissues, and organs. Therefore, there is a need to use biological agents and phytoremediation processes such as enzymes because they have a high potential for effectively transforming and detoxifying polluting substances. They can convert pollutants at a detectable rate and are potentially suitable for restoring polluted environments. We investigated heavy metal concentrations in different soil samples collected in four sections in Alice and determined the enzyme activity levels present in the soil. The Pearson correlation analysis was conducted to check whether there was any relationship between heavy metal concentrations and enzyme activities in the soil. Samples were randomly collected in three weeks, and the microwave digestion method was used for sample treatment and preparation. Quantitation was achieved by inductively coupled plasma mass spectrometry (ICP-MS). The enzyme assay through incubation method was implemented for discovering the four selected enzymes (urease, invertase, catalase, and phosphatase), and their activity levels were examined colorimetrically by colorimetry spectrophotometer. The ICP-MS results revealed 16 predominating elements, namely: Al, Ba, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Sr, and Zn, and the presence of a non-mental, which is phosphorus (P), and a metalloid in the form of silicon (Si) in all soil samples. Significant differences in metal concentrations were observed among the collection sites. The Al, Fe, K, Mg, and Ca concentrations were above WHO's permissible limits. While Ba, Mn, Na, and P were in moderate concentration, Cu, Cr, Co, Zn, Sr, and Ni were in small amounts recorded mostly below the permissible values from WHO. Four soil enzyme activities were determined successfully (urease, invertase, phosphatase, and catalase). A negative non-significant correlation existed between urease, invertase, phosphatase enzyme activity, and the concentration levels of all selected metals (Al, Ba, Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni, Cr, Sr, and Zn. In contrast, the content of catalase activity was associated non-significantly but positively with the range of selected heavy metals. This study suggests proper monitoring of residences' areas, which can provide detailed information on the impact of high heavy metal content on people's health. They are easily dispersed and can accumulate in large quantities in the soil. The necessary implementation of waste management programs will help the municipality adopt a strategy that will promote recycling programs and protect the residence health from this threat.

Genome-Wide Analysis of the RNase T2 Family and Identification of Interacting Proteins of Four ClS-RNase Genes in 'XiangShui' Lemon.

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the 'XiangShui' lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in 'XiangShui' lemon are the same as those within other species. Expression pattern analysis revealed that S3-RNase and S4-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S3-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S1-RNase, S2-RNase, S3-RNase and S4-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S3-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S3-RNase interacted with TOPP4. Therefore, S3-RNase may play an important role in the SI of 'XiangShui' lemon.

Functional characterization and vacuolar localization of fructan exohydrolase derived from onion (Allium cepa).

Fructans such as inulin and levan accumulate in certain taxonomic groups of plants and are a reserve carbohydrate alternative to starch. Onion (Allium cepa L.) is a typical plant species that accumulates fructans, and it synthesizes inulin-type and inulin neoseries-type fructans in the bulb. Although genes for fructan biosynthesis in onion have been identified so far, no genes for fructan degradation had been found. In this study, phylogenetic analysis predicted that we isolated a putative vacuolar invertase gene (AcpVI1), but our functional analyses demonstrated that it encoded a fructan 1-exohydrolase (1-FEH) instead. Assessments of recombinant proteins and purified native protein showed that the protein had 1-FEH activity, hydrolyzing the ß-(2,1)-fructosyl linkage in inulin-type fructans. Interestingly, AcpVI1 had an amino acid sequence close to those of vacuolar invertases and fructosyltransferases, unlike all other FEHs previously found in plants. We showed that AcpVI1 was localized in the vacuole, as are onion fructosyltransferases Ac1-SST and Ac6G-FFT. These results indicate that fructan-synthesizing and -degrading enzymes are both localized in the vacuole. In contrast to previously reported FEHs, our data suggest that onion 1-FEH evolved from a vacuolar invertase and not from a cell wall invertase. This demonstrates that classic phylogenetic analysis on its own is insufficient to discriminate between invertases and FEHs, highlighting the importance of functional markers in the nearby active site residues.

The amounts and ratio of nitrogen and phosphorus addition drive the rate of litter decomposition in a subtropical forest.

Nitrogen (N) and phosphorus (P) control biogeochemical cycling in terrestrial ecosystems. However, N and P addition effects on litter decomposition, especially biological pathways in subtropical forests, remain unclear. Here, a two-year field litterbag experiment was employed in a subtropical forest in southwestern China to examine N and P addition effects on litter biological decomposition with nine treatments: low and high N- and P-only addition (LN, HN, LP, and HP), NP coaddition (LNLP, LNHP, HNLP, and HNHP), and a control (CK). The results showed that the decomposition coefficient (k) was higher in NP coaddition treatments (P < 0.05), and lower in N- and P-only addition treatments than in CK (P < 0.05). The highest k was observed with LNLP (P < 0.05). The N- and P-only addition treatments decreased the losses of litter mass, lignin, cellulose, and condensed tannins, litter microbial biomass carbon (MBC), litter cellulase, and soil pH (P < 0.05). The NP coaddition treatments increased the losses of litter mass, lignin, and cellulose, MBC concentration, litter invertase, urease, cellulase, and catalase activities, soil arthropod diversity (S) in litterbags, and soil pH (P < 0.05). Litter acid phosphatase activity and N:P ratio were lower in N-only addition treatments but higher in P-only addition and NP coaddition treatments than in CK (P < 0.05). Structural equation model showed that litter MBC, S, cellulase, acid phosphatase, and polyphenol oxidase contributed to the loss of litter mass (P < 0.05). The litter N:P ratio was negatively logarithmically correlated with mass loss (P < 0.01). In conclusion, the negative effect of N addition on litter decomposition was reversed when P was added by increasing decomposed litter soil arthropod diversity, MBC concentration, and invertase and cellulase activities. Finally, the results highlighted the important role of the N:P ratio in litter decomposition.

Assessment of dynamic microbial community structure and rhizosphere interactions during bioaugmented phytoremediation of petroleum contaminated soil by a newly designed rhizobox system.

To understand the plant (Vigna unguiculata) and plant-growth promoting bacteria (PGPB) (Microcococcus luteus WN01) interactions in crude oil contaminated soil, experiments were conducted based on the newly designed rhizobox system. The rhizobox was divided into three main compartments namely the rhizosphere zone, the mid-zone, and the bulk soil zone, in accordance with the distance from the plant. Plants were grown in these three-chambered pots for 30 days under natural conditions. The plant root exudates were determined by analyzing for carbohydrates, amino acids, and phenolic compounds. The degradation of alkane, polycyclic aromatic hydrocarbons (PAHs), and total petroleum hydrocarbons (TPHs) were quantified by GC-FID. Soil catalase, dehydrogenase, and invertase activities were determined. The microbial community structure was assessed using denaturing gradient gel electrophoresis (DGGE). Results showed that the inoculation of M. luteus WN01 significantly enhanced cowpea root biomass and exudates, especially the phenolic compounds. Bioaugmented phytoremediation by cowpea and M. luteus promoted rhizodegradation of TPH. Cowpea stimulated microbial growth, soil dehydrogenase, and invertase activities and enhanced bacterial community diversity in oil contaminated soil. The rhizosphere zone of cowpea inoculated with M. luteus showed the highest removal efficiency, microbial activities, microbial population, and bacterial community diversity indicating the strong synergic interactions between M. luteus and cowpea.

This is the first study to characterize the rhizosphere effect of cowpea on microbial activities, population, and community structure in crude oil contaminated soil in the presence and absence of PGPB, M. luteus WN01. The rhizosphere of cowpea was found to be a degradation hotspot where microbial abundance and metabolic activities were most active. Cowpea-M. luteus association can be a good candidate that can be implemented in real field sites.

Transcriptome Profiling Reveals Role of MicroRNAs and Their Targeted Genes during Adventitious Root Formation in Dark-Pretreated Micro-Shoot Cuttings of Tetraploid Robinia pseudoacacia L.

Tetraploid Robinia pseudoacacia L. is a difficult-to-root species, and is vegetatively propagated through stem cuttings. Limited information is available regarding the adventitious root (AR) formation of dark-pretreated micro-shoot cuttings. Moreover, the role of specific miRNAs and their targeted genes during dark-pretreated AR formation under in vitro conditions has never been revealed. The dark pretreatment has successfully promoted and stimulated adventitious rooting signaling-related genes in tissue-cultured stem cuttings with the application of auxin (0.2 mg L-1 IBA). Histological analysis was performed for AR formation at 0, 12, 36, 48, and 72 h after excision (HAE) of the cuttings. The first histological events were observed at 36 HAE in the dark-pretreated cuttings; however, no cellular activities were observed in the control cuttings. In addition, the present study aimed to uncover the role of differentially expressed (DE) microRNAs (miRNAs) and their targeted genes during adventitious root formation using the lower portion (1-1.5 cm) of tetraploid R. pseudoacacia L. micro-shoot cuttings. The samples were analyzed using Illumina high-throughput sequencing technology for the identification of miRNAs at the mentioned time points. Seven DE miRNA libraries were constructed and sequenced. The DE number of 81, 162, 153, 154, 41, 9, and 77 miRNAs were upregulated, whereas 67, 98, 84, 116, 19, 16, and 93 miRNAs were downregulated in the following comparisons of the libraries: 0-vs-12, 0-vs-36, 0-vs-48, 0-vs-72, 12-vs-36, 36-vs-48, and 48-vs-72, respectively. Furthermore, we depicted an association between ten miRNAs (novel-m0778-3p, miR6135e.2-5p, miR477-3p, miR4416c-5p, miR946d, miR398b, miR389a-3p, novel m0068-5p, novel-m0650-3p, and novel-m0560-3p) and important target genes (auxin response factor-3, gretchen hagen-9, scarecrow-like-1, squamosa promoter-binding protein-like-12, small auxin upregulated RNA-70, binding protein-9, vacuolar invertase-1, starch synthase-3, sucrose synthase-3, probable starch synthase-3, cell wall invertase-4, and trehalose phosphatase synthase-5), all of which play a role in plant hormone signaling and starch and sucrose metabolism pathways. The quantitative polymerase chain reaction (qRT-PCR) was used to validate the relative expression of these miRNAs and their targeted genes. These results provide novel insights and a foundation for further studies to elucidate the molecular factors and processes controlling AR formation in woody plants.

Production, immobilization and application of invertase from new wild strain Cunninghamella echinulata PA3S12MM.

AIMS: The objective of this study was to determine the best conditions to produce invertase by Cunninghamella echinulata PA3S12MM and to immobilize and apply the enzyme. METHODS AND RESULTS: The maximum production was verified in 8 days of cultivation at 28°C supplemented with 10 g L-1 apple peel, reaching 1054.85 U ml-1 . The invertase was purified from the DEAE-Sephadex column. The derivative immobilized in alginate-gelatin-calcium phosphate showed reusability >50% for 19 cycles. The derivative immobilized in glutaraldehyde-chitosan showed greater thermostability and at a different pH. The hydrolysis of 15 ml of sucrose 500 g L-1 in a fixed bed reactor (total volume of 31 ml) produced 24.44 µmol min-1 of glucose and fructose at a residence time of 30 min and a conversion factor of 0.5. CONCLUSIONS: The new wild strain C. echinulata PA3S12MM presents high invertase production in medium supplemented with an agro-industrial residue and the immobilized enzyme showed high thermal stability and resistance at a different pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The fungus C. echinulata PA3S12MM is an excellent producer of invertases in Vogel medium supplemented with apple peel. The enzyme is promising for industrial application since it has good performance in reusability and inverted sugar production.

Predictive modeling and sensitivity analysis to estimate the experimental data of inulinase fermentation by Aspergillus niger grown on sugar beet molasses-based medium optimized using Plackett-Burman Design.

The present work aimed to model Aspergillus niger inulinase fermentation performed in the medium using sigmoidal functions, validate the selected models using an independent set of the experimental values, and perform a sensitivity analysis of the selected models. Based on the results, the selected models were Stannard and Fitzhugh models for substrate consumption (R2  = 0.9976 and 0.9974, respectively), Huang model for inulinase production (R2  = 0.9967), Weibull model for invertase-type production (R2  = 0.9963), and modified logistic model for invertase-type activity/inulinase activity ratio (R2  = 0.9292) with high R2 values (>0.90). Kinetics predicted by particularly selected models mentioned above fit well with the experimental kinetic results. Besides, validation of the selected models with an independent set of the experimental data indicated that they gave satisfying results with high R2 values for consumption and production (R2  > 0.90). Sensitivity analysis of the selected models showed that the yielded R2 values (R2  ≥ 0.9775) were in good agreement with those obtained from the selected models. Consequently, A. niger inulinase fermentation was successfully modeled and the selected models were successfully validated with an independent set of the observed data. Besides, the sensitivity analysis also verified the reliability of the selected models. Those models can serve as universal equations to describe the A. niger inulinase fermentation.

Effects of exogenous sucrose and selenium on plant growth, quality, and sugar metabolism of pea sprouts.

BACKGROUND: Pea sprouts are considered a healthy food. Sucrose is a key nutritional factor affecting taste and flavor. Meanwhile, selenium (Se) is an essential micronutrient that plays multiple roles in wide variety of physiological processes and improves crop quality and nutritional value. Nonetheless, the effects of the combination of sucrose and Se treatment on growth, quality, and sugar metabolism of pea sprouts have not been explored. RESULTS: The results revealed that sucrose at 10 mg L-1 obviously increased fresh weight, vitamin C, soluble protein, soluble sugar, fructose, glucose, and sucrose contents. Se treatments also improved nutritional quality, but higher Se (2.5 mg L-1 ) significantly inhibited the growth of seedlings. Interestingly, the combined application of sucrose (10 mg L-1 ) and Se (1.25 mg L-1 ) could effectively promote vitamin C, sucrose, and fructose contents, especially the Se content, compared with Se application alone. Additionally, there were significant differences in the regulation of sugar metabolism between Se alone and combined application of sucrose and Se. Acid invertase and neutral invertase play a pivotal role in the accumulation of soluble sugar under Se treatments alone, and acid invertase might be the key enzyme to limit sugar accumulation under combined application of sucrose and Se. CONCLUSION: The moderate combined application of sucrose (10 mg L-1 ) and Se (1.25 mg L-1 ) more effectively regulated sugar metabolism and improved nutritional quality than Se application alone did. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.