The effect of different carbon sources on biofouling in membrane fouling simulators: microbial community and implications.

Biofouling is a problem affecting the operation of nanofiltration systems due to the complexity of the carbon matrix affecting bacteria and biofilm growth. This study used membrane fouling simulators to investigate the effects of five different carbon sources on the biofouling of nanofiltration membranes. For all the carbon sources analyzed, the increase in pressure drop was most accelerated for acetate. The use of acetate as the single carbon source produced less adenosine triphosphate but more extracellular polymers than glucose. The microbial community was analyzed using 16 s rRNA. The use of more than a single carbon source produced an increase in bacteria diversity even at similar concentrations. The relative abundance of proteobacteria was the highest at the phylum level (95%) when a single carbon source was added. Additionally, it was found that the use of different carbon sources produced a shift in the microbial community, affecting the biofouling and pressure drop on membranes.

microRNA172 targets APETALA2 to regulate flavonoid biosynthesis in apple (Malus domestica).

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red coloration and anthocyanin accumulation in various tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and qRT-PCR analyses. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.

PpIAA1 and PpERF4 form a positive feedback loop to regulate peach fruit ripening by integrating auxin and ethylene signals.

The signaling pathways of both auxin and ethylene regulate peach fruit ripening via the Aux/IAA and ERF transcription factors, respectively. However, the molecular mechanisms that coordinate both auxin and ethylene signals during peach fruit ripening remain unclear. In this study, we show that PpIAA1 and PpERF4 act as key players in a positive feedback loop, and promote peach fruit ripening by directly binding to and enhancing the activity of target gene promoters. PpIAA1 increased the expression of the ethylene biosynthesis gene PpACS1. Furthermore, PpERF4 enhanced the transcription of PpACO1 and PpIAA1 genes by binding to their promoters. Additionally, PpIAA1 and PpERF4 bound to each other to form a complex, which then enhanced the transcription of abscisic acid biosynthesis genes (PpNCED2 and PpNCED3) and the fruit softening gene (PpPG1) to levels higher than those achieved by each transcription factor individually. Moreover, overexpression of PpIAA1 in tomato accelerated fruit ripening and shortened the fruit shelf-life by increasing the production of ethylene and the expression levels of ripening regulator genes. Collectively, these results advance our understanding of the molecular mechanisms underlying peach fruit ripening and softening via auxin and ethylene signaling pathways.

The NAM/ATAF1/2/CUC2 transcription factor PpNAC.A59 enhances PpERF.A16 expression to promote ethylene biosynthesis during peach fruit ripening.

Peach is a typical climacteric fruit that releases ethylene during fruit ripening. Several studies have been conducted on the transcriptional regulation of ethylene biosynthesis in peach fruit. Herein, an ethylene response factor, PpERF.A16, which was induced by exogenous ethylene, could enhance ethylene biosynthesis by directly inducing the expression of 1-aminocyclopropane-1-carboxylic acid synthase (PpACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (PpACO1) genes. Moreover, the NAM/ATAF1/2/CUC2 (NAC) transcription factor (TF) PpNAC.A59 was coexpressed with PpERF.A16 in all tested peach cultivars. Interestingly, PpNAC.A59 can directly interact with the promoter of PpERF.A16 to induce its expression but not enhance LUC activity driven by any promoter of PpACS1 or PpACO1. Thus, PpNAC.A59 can indirectly mediate ethylene biosynthesis via the NAC-ERF signaling cascade to induce the expression of both PpACS1 and PpACO1. These results enrich the genetic network of fruit ripening in peach and provide new insight into the ripening mechanism of other perennial fruits.

Diverse Functions of IAA-Leucine Resistant PpILR1 Provide a Genic Basis for Auxin-Ethylene Crosstalk During Peach Fruit Ripening.

Auxin and ethylene play critical roles in the ripening of peach (Prunus persica) fruit; however, the interaction between these two phytohormones is complex and not fully understood. Here, we isolated a peach ILR gene, PpILR1, which encodes an indole-3-acetic acid (IAA)-amino hydrolase. Functional analyses revealed that PpILR1 acts as a transcriptional activator of 1-amino cyclopropane-1-carboxylic acid synthase (PpACS1), and hydrolyzes auxin substrates to release free auxin. When Cys137 was changed to Ser137, PpILR1 failed to show hydrolase activity but continued to function as a transcriptional activator of PpACS1 in tobacco and peach transient expression assays. Furthermore, transgenic tomato plants overexpressing PpILR1 exhibited ethylene- and strigolactone-related phenotypes, including premature pedicel abscission, leaf and petiole epinasty, and advanced fruit ripening, which are consistent with increased expression of genes involved in ethylene biosynthesis and fruit ripening, as well as suppression of branching and growth of internodes (related to strigolactone biosynthesis). Collectively, these results provide novel insights into the role of IAA-amino acid hydrolases in plants, and position the PpILR1 protein at the junction of auxin and ethylene pathways during peach fruit ripening. These results could have substantial implications on peach fruit cultivation and storage in the future.

Identification of gene co-expression networks and key genes regulating flavonoid accumulation in apple (Malus × domestica) fruit skin.

Anthocyanin provides a red color for apple and health benefit for human. To better understand the molecular mechanisms of regulating apple color formation, we analyzed 27 transcriptomes of fruit skin from three cultivars 'Huashuo' (red-skinned), 'Hongcuibao' (red-skinned), and 'Golden Delicious' (yellow-skinned) at 0, 2, and 6 days after bag removal. Using pairwise comparisons and weighted gene co-expression network analyses (WGCNA), we constructed 17 co-expression modules. Among them, a specific module was negatively correlated to anthocyanin accumulation. The genes in the module are enriched in flavonoid biosynthesis pathways. These pathway genes were used to construct gene co-expression network of anthocyanin accumulation. Finally, a R2R3-MYB repressor designated MdMYB28 was identified as a key hub gene in the anthocyanin metabolism network. During the anthocyanin accumulation of apple fruit skin reaching a peak, MdMYB28 expression level was negatively correlated with the anthocyanin content. MdMYB28 was shown to directly bind to the promoter of MdMYB10 in yeast one-hybrid analyses. Over-expression of MdMYB28 decreased the anthocyanin biosynthesis in tobacco flower petals, suggesting that MdMYB28 acts as a negatively regulator of anthocyanin biosynthesis.

Citrus mangshanensis Pollen Confers a Xenia Effect on Linalool Oxide Accumulation in Pummelo Fruit by Enhancing the Expression of a Cytochrome P450 78A7 Gene CitLO1.

The aroma quality of citrus fruit is determined by volatiles that are present at extremely low levels in the citrus fruit juice sacs; it can be greatly improved by increasing volatiles. In this study, we showed that the contents of cis- and trans-linalool oxides were significantly increased in the juice sacs of three pummelos artificially pollinated with the Citrus mangshanensis (MS) pollen. A novel cytochrome P450 78A7 gene (CitLO1) was significantly upregulated in the juice sacs of Huanong Red pummelo pollinated with MS pollen in comparison to that with open pollination. Compared to wild-type tobacco Bright-Yellow2 cells, transgenic cells overexpressing CitLO1 promoted a 3- to 4-fold more conversion of (-)-linalool to cis- and trans-linalool oxides. Overall, our results suggest that MS pollen has a xenia effect on pummelo fruit aroma quality, and CitLO1 is a linalool oxide synthase gene that played an important role in the xenia effect.

Peach ethylene response factor PpeERF2 represses the expression of ABA biosynthesis and cell wall degradation genes during fruit ripening.

Ethylene response factors (ERFs) are known to regulate fruit ripening. However, the ERF regulatory networks are not clear. In this study, we have shown that peach (Prunus persica) PpeERF2 regulates fruit ripening through suppressing the expression of two ABA biosynthesis genes (PpeNCED2, PpeNCED3) and a cell wall degradation gene (PpePG1). The transcript levels of PpeERF2 in fruit were opposite to that of PpeNCED2, PpeNCED3 and PpePG1 during ripening and in response to various ripening treatments. PpeERF2 was found to bind to the PpeNCED2, PpeNCED3 and PpePG1 promotors as demonstrated by yeast one-hybrid (Y1H) and EMSA assays; and further found to repress the promoter activities of the three genes in tobacco leaf tissues after Agrobacterium infiltration. Taken together, these results provide new information for a better understanding of the crosstalk network between ethylene signaling, cell wall degradation and ABA biosynthesis during fruit ripening.

Cit1,2RhaT and two novel CitdGlcTs participate in flavor-related flavonoid metabolism during citrus fruit development.

Neohesperidosides are disaccharides that are present in some flavonoids and impart a bitter taste, which can significantly affect the commercial value of citrus fruits. In this study, we identified three flavonoid-7-O-di-glucosyltransferase (dGlcT) genes closely related to 1,2-rhamnosyltransferase (1,2RhaT) in citrus genomes. However, only 1,2RhaT was directly linked to the accumulation of neohesperidoside, as demonstrated by association analysis of 50 accessions and co-segregation analysis of an F1 population derived from Citrus reticulata × Poncirus trifoliata. In transgenic tobacco BY2 cells, over-expression of CitdGlcTs resulted in flavonoid-7-O-glucosides being catalysed into bitterless flavonoid-7-O-di-glucosides, whereas over-expression of Cit1,2RhaT converted the same substrate into bitter-tasting flavonoid-7-O-neohesperidoside. Unlike 1,2RhaT, during citrus fruit development the dGlcTs showed an opposite expression pattern to CHS and CHI, two genes encoding rate-limiting enzymes of flavonoid biosynthesis. An uncoupled availability of dGlcTs and substrates might result in trace accumulation of flavonoid-7-O-di-glucosides in the fruit of C. maxima (pummelo). Past human selection of the deletion and functional mutation of 1,2RhaT has led step-by-step to the evolution of the flavor-related metabolic network in citrus. Our research provides the basis for potentially improving the taste in citrus fruit through manipulation of the network by knocking-out 1,2RhaT or by enhancing the expression of dGlcT using genetic transformation.

PbrmiR397a regulates lignification during stone cell development in pear fruit.

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell-based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl- and guaiacyl-lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild-type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.

KEY MESSAGE: Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 µg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 µg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 µg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway.

Ethylene is the major effector of ripening in many fleshy fruits. In apples (Malus x domestica) the addition of ethylene causes a climacteric burst of respiration, an increase in aroma, and softening of the flesh. We have generated a transgenic line of 'Royal Gala' apple that produces no detectable levels of ethylene using antisense ACC OXIDASE, resulting in apples with no ethylene-induced ripening attributes. In response to external ethylene these antisense fruits undergo a normal climacteric burst and produced increasing concentrations of ester, polypropanoid, and terpene volatile compounds over an 8-d period. A total of 186 candidate genes that might be involved in the production of these compounds were mined from expressed sequence tags databases and full sequence obtained. Expression patterns of 179 of these were assessed using a 15,720 oligonucleotide apple microarray. Based on sequence similarity and gene expression patterns we identified 17 candidate genes that are likely to be ethylene control points for aroma production in apple. While many of the biosynthetic steps in these pathways were represented by gene families containing two or more genes, expression patterns revealed that only a single member is typically regulated by ethylene. Only certain points within the aroma biosynthesis pathways were regulated by ethylene. Often the first step, and in all pathways the last steps, contained enzymes that were ethylene regulated. This analysis suggests that the initial and final enzymatic steps with the biosynthetic pathways are important transcriptional regulation points for aroma production in apple.

Transgenic tobacco and apple plants expressing biotin-binding proteins are resistant to two cosmopolitan insect pests, potato tuber moth and lightbrown apple moth, respectively.

Tobacco (Nicotiana tabacum cv. Samsun) and apple (Malus x domestica cv. Royal Gala) plants expressing avidin or strepavidin were produced using Agrobacterium tumefaciens-mediated transformation. ELISA assays showed that avidin expression ranged from 3.1 to 4.6 microM in tobacco and from 1.9 to 11.2 microM in apple and streptavidin expression ranged from 11.4 to 24.5 microM in tobacco and from 0.4 to 14.6 microM in apple. Expressed at these levels, both biotin-binding proteins conferred a high level of insect resistance on transformed tobacco plants to larval potato tuber moth (PTM), Phthorimaea operculella (Zeller) (fam. Gelechiidae) and on apple plants to larvae of the lightbrown apple moth (LBAM) Epiphyas postvittana (Walker) (fam. Tortricidae). More than 90% of PTM larvae died on tobacco plants expressing either avidin or streptavidin genes within 9 days of inoculation. Mortality of LBAM larvae was significantly higher (P < 0.05) on three avidin-expressing (89.6, 84.9 and 80.1%) and two streptavidin-expressing (90 and 82.5%) apple plant lines than on non-transformed control plants (14.1%) after 21 days. Weight of LBAM larvae was also significantly reduced by feeding on all apple shoots expressing avidin and on apple shoots expressing streptavidin at levels of 3.8 microM and above.