Selection and Validation of Candidate Reference Genes for Gene Expression Analysis by RT-qPCR in Rubus

Due to the lack of effective and stable reference genes, studies on functional genes in Rubus, a genus of economically important small berry crops, have been greatly limited. To select the best internal reference genes of different types, we selected four representative cultivars of blackberry and raspberry (red raspberry, yellow raspberry, and black raspberry) as the research material and used RT-qPCR technology combined with three internal stability analysis software programs (geNorm, NormFinder, and BestKeeper) to analyze 12 candidate reference genes for the stability of their expression. The number of most suitable internal reference genes for different cultivars, tissues, and fruit developmental stages of Rubus was calculated by geNorm software to be two. Based on the results obtained with the three software programs, the most stable genes in the different cultivars were RuEEF1A and Ru18S. Finally, to validate the reliability of selected reference genes, the expression pattern of the RuCYP73A gene was analyzed, and the results highlighted the importance of appropriate reference gene selection. RuEEF1A and Ru18S were screened as reference genes for their relatively stable expression, providing a reference for the further study of key functional genes in blackberry and raspberry and an effective tool for the analysis of differential gene expression

Overexpression of RuFLS2 Enhances Flavonol-Related Substance Contents and Gene Expression Levels

As an emerging third-generation fruit, blackberry has high nutritional value and is rich in polyphenols, flavonoids and anthocyanins. Flavonoid biosynthesis and metabolism is a popular research topic, but no related details have been reported for blackberry. Based on previous transcriptome data from this research group, two blackberry flavonol synthase genes were identified in this study, and the encoded proteins were subjected to bioinformatics analysis. RuFLS1 and RuFLS2 are both hydrophobic acidic proteins belonging to the 2OG-Fe(II) dioxygenase superfamily. RuFLS2 was expressed at 27.93-fold higher levels than RuFLS1 in red–purple fruit by RNA-seq analysis. Therefore, RuFLS2-overexpressing tobacco was selected for functional exploration. The identification of metabolites from transgenic tobacco showed significantly increased contents of flavonoids, such as apigenin 7-glucoside, kaempferol 3-O-rutinoside, astragalin, and quercitrin. The high expression of RuFLS2 also upregulated the expression levels of NtF3H and NtFLS in transgenic tobacco. The results indicate that RuFLS2 is an important functional gene regulating flavonoid biosynthesis and provides an important reference for revealing the molecular mechanism of flavonoid accumulation in blackberry fruit

Composition and Antioxidant Activity of Anthocyanins and Non-Anthocyanin Flavonoids in Blackberry from Different Growth Stages

The high nutritional value and unique flavor of blackberries make them a popular food choice among consumers. Anthocyanin content (AC) and non-anthocyanin flavonoid content (NAFC) are important functional components in blackberry. We tested the AC, NAFC, and antioxidant activities of two blackberry—Ningzhi 1 and Hull—during the following ripening stages: green-fruit stage (GFS), color-turning stage (CTS), reddening stage (RDS), and ripening stage (RPS). The results showed that NAFC decreased and AC increased gradually during the ripening stages. The NAFC of Hull blackberry was the highest during GFS (889.74 μg/g), while the AC of Ningzhi 1 blackberry was the highest during RPS (1027.08 μg/g). NAFC was the highest at the initial stage and gradually decreased with ripening. Anthocyanin accumulation mainly occurred during the later ripening stages. These results provide a reference for comparing the NAFC, AC, and antioxidant activity of Ningzhi 1 and Hull and their changes during different ripening stages

Variation in Bioactive Compounds and Antioxidant Activity of <i>Rubus</i> Fruits at Different Developmental Stages

Blackberry and raspberry have high nutritional, health value, and are popular with consumers for their unique flavors. To explore the relationships between nutrient accumulation, antioxidant substance contents in blackberry and raspberry fruits, and fruit growth and development, seven Rubus cultivars were selected, and contents of the main active substance were determined. “Clode Summit” had the highest soluble sugar and fructose contents, “Chester”—the highest total phenol content, and “Bristol’—the highest anthocyanin content. Generally, the contents of flavonoids and total phenols showed a downward trend with the development of fruit in seven Rubus cultivars, and the content of anthocyanins increased rapidly in the later stage of development. Pearson correlation analysis showed extremely significant correlation between antioxidant activity and the contents of vitamin E, total phenols, and flavonoids. Flavonoids were extremely significantly positively correlated with the content of total phenols, and the contents of flavonoids and anthocyanins in various cultivars were highly negatively correlated. Considering the different nutritional ingredients and active antioxidant substance contents, “Clode Summit”, “Bristol”, and “Chester” are recommended for raw consumption, processing, and medicinal purposes, respectively. These results provide a reference for comparing the main active substance contents in different Rubus cultivars and their changes across fruit development stages

Metabolite Profiling and Classification of Highbush Blueberry Leaves under Different Shade Treatments

Blueberry belongs to the genus Vaccinium L. in the Ericaceae and is an economically important shrub that produces small berries that are rich in nutrients. There were differences in the appearance of blueberry leaves under different shade treatments. To explore the differences in metabolites in blueberry leaves under different shading treatments, nontargeted liquid chromatography&ndash;mass spectrometry (LC&ndash;MS) metabonomic analysis was performed. Different shade intensities resulted in significant differences in the contents of metabolites. A total of 6879 known metabolites were detected, including 750 significantly differentially expressed metabolites, including mainly lipids and lipid-like molecules and phenylpropanoid and polyketide superclass members. Based on a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the flavone and flavonol biosynthesis pathways were the most significantly enriched. The results of this study provide a reference and scientific basis for the establishment of a high-quality and high-yield shaded blueberry cultivation system

Physiological and Morphological Responses of Blackberry Seedlings to Different Nitrogen Forms

Blackberries are an emerging third-generation fruit that are popular in Europe, and specific nitrogen (N) supply is an important factor affecting their growth and development. To study the optimal N fertilizer for blackberry seedlings, no N (CK), nitrate (NO3−)–N, ammonium (NH4+)–N and urea were applied to one-year-old ‘Ningzhi 4’ blackberry plants at a key growth period (from May to August) to explore the effects of different N forms on the physiological characteristics. Correlation and principal component analysis were used to determine the relationships between various indexes. Ammonium (NH4+) or urea-fed plants had a better growth state, showed a greater plant height, biomass, SPAD values and enhanced antioxidant enzyme activities and photosynthesis. In addition, NH4+ was beneficial to the accumulation of sugars and amino acids in leaves and roots, and promoted the transport of auxin and cytokinin to leaves. NO3− significantly inhibited root growth and increased the contents of active oxygen, malondialdehyde and antioxidants in roots. Correlation and principal component analysis showed that growth and dry matter accumulation were closely related to the antioxidant system, photosynthetic characteristics, amino acids and hormone content. Our study provides a new idea for N regulation mechanism of blackberry and proposes a scientific fertilization strategy

Effects of Different Light Wavelengths on Fruit Quality and Gene Expression of Anthocyanin Biosynthesis in Blueberry (<i>Vaccinium corymbosm</i>)

Different light wavelengths display diverse effects on fruit quality formation and anthocyanin biosynthesis. Blueberry is a kind of fruit rich in anthocyanin with important economic and nutritional values. This study explored the effects of different light wavelengths (white (W), red (R), blue (B) and yellow (Y)) on fruit quality and gene expression of anthocyanin biosynthesis in blueberry. We found that the B and W treatments attained the maximum values of fruit width, fruit height and fruit weight in blueberry fruits. The R treatment attained the maximum activities of superoxide dismutase (SOD) and peroxidase (POD), and the Y treatment displayed the maximum contents of ascorbic acid (AsA), glutathione (GSH) and total phenol in fruits, thus improving blueberry-fruit antioxidant capacity. Interestingly, there were differences in the solidity–acid ratio of fruit under different light-wavelength treatments. Moreover, blue light could significantly improve the expression levels of anthocyanin biosynthesis genes and anthocyanin content in fruits. Correlation and principal component analysis showed that total acid content and antioxidant enzymes were significantly negatively correlated with anthocyanin content in blueberry fruits. These results provide new insights for the application of light wavelength to improve blueberry fruit quality and anthocyanin content

Fruit Quality and Metabolomic Analyses of Fresh Food Accessions Provide Insights into the Key Carbohydrate Metabolism in Blueberry

Blueberry is a nutrient-rich berry, and its taste and flavor directly determine the consumer preference. Until now, few studies have focused on the comparison of fresh food quality and the key metabolites in superior fresh-eating blueberry cultivars. Herein, fruit quality indicators of 10 highbush blueberry cultivars were evaluated using ‘Bluerain’ as the control. Appearance quality analysis of fruits showed that ‘Brigitta’ had a larger fruit size and ‘Anna’ was the smallest. ‘Anna’ fruits, followed by ‘O′Neal’, had the highest ratio of soluble solids to acidity because of their lowest titratable acidity content. Despite the high soluble sugar content, the antioxidants in ‘Anna’ fruits such as total flavonoids, anthocyanins and vitamin C were lowest among all cultivars, while ‘Duke’ seemed to have opposite patterns. Furthermore, a total of 553 and 557 metabolites were identified by non-targeted metabolomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative ion mode, respectively. Particularly, the numbers of differentially accumulated metabolites (DAMs) were the most between the ‘O′Neal’ vs. ‘Bluerain’ group. The DAMs involved in the metabolic pathways, sesquiterpenoid and triterpenoid biosynthesis, monoterpenoid biosynthesis, galactose metabolism, starch and sucrose metabolism, may be mainly related to the synthesis of flavor and carbohydrate substances. Moreover, the expression patterns of genes involved in sugar metabolism were verified by quantitative real-time PCR (qRT-PCR) analysis in different cultivars. Therefore, the systematical comparison of the quality characteristics, metabolites and expression profiles of related genes in highbush blueberries with good flavor could provide some basis for further research on fresh fruit breeding of blueberries

Effects of Different Nitrogen Forms on Blackberry Fruit Quality

To study the optimal form of nitrogen (N) application and to determine the best harvest date for blackberries, different N fertilizers were applied during the critical growth period of blackberry plants. The results showed that NH4+–N significantly improved the appearance of blackberry fruits, including their size, firmness, and color, and promoted the accumulation of soluble solids, sugars, anthocyanin, ellagic acid, and vitamin C (VC), while fruit treated with NO3−–N accumulated more flavonoids and organic acids and had improved antioxidant capacity. In addition, the fruit size, firmness, and color brightness decreased with the harvest period. While the contents of sugars, anthocyanin, ellagic acid, flavonoids, and VC were higher in the early harvests and then decreased as the season progressed, the total antioxidant capacity and DPPH radical scavenging capacity increased. In all, application of NH4+–N is recommended, as it is more beneficial to fruit appearance, taste, and nutritional quality. Harvests in the early stage help to obtain a good fruit appearance, while harvests in the middle and later stages are more beneficial to fruit taste and quality. This study may help growers to determine the best fertilization scheme for blackberries and choose the appropriate harvest time according to their needs

Cultivation of microalgae–bacteria consortium by waste gas–waste water to achieve CO2 fixation, wastewater purification and bioproducts production

Abstract The cultivation of microalgae and microalgae–bacteria consortia provide a potential efficient strategy to fix CO2 from waste gas, treat wastewater and produce value-added products subsequently. This paper reviews recent developments in CO2 fixation and wastewater treatment by single microalgae, mixed microalgae and microalgae–bacteria consortia, as well as compares and summarizes the differences in utilizing different microorganisms from different aspects. Compared to monoculture of microalgae, a mixed microalgae and microalgae–bacteria consortium may mitigate environmental risk, obtain high biomass, and improve the efficiency of nutrient removal. The applied microalgae include Chlorella sp., Scenedesmus sp., Pediastrum sp., and Phormidium sp. among others, and most strains belong to Chlorophyta and Cyanophyta. The bacteria in microalgae–bacteria consortia are mainly from activated sludge and specific sewage sources. Bioengineer in CBB cycle in microalgae cells provide effective strategy to achieve improvement of CO2 fixation or a high yield of high-value products. The mechanisms of CO2 fixation and nutrient removal by different microbial systems are also explored and concluded, the importance of microalgae in the technology is proven. After cultivation, microalgae biomass can be harvested through physical, chemical, biological and magnetic separation methods and used to produce high-value by-products, such as biofuel, feed, food, biochar, fertilizer, and pharmaceutical bio-compounds. Although this technology has brought many benefits, some challenging obstacles and limitation remain for industrialization and commercializing. Graphical Abstrac