Application of Pseudomonas fluorescens to Blackberry under Field Conditions Improves Fruit Quality by Modifying Flavonoid Metabolism

Application of a plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens N21.4, to roots of blackberries (Rubus sp.) is part of an optimised cultivation practice to improve yields and quality of fruit throughout the year in this important fruit crop. Blackberries are especially rich in flavonoids and therefore offer potential benefits for human health in prevention or amelioration of chronic diseases. However, the phenylpropanoid pathway and its regulation during ripening have not been studied in detail, in this species. PGPR may trigger flavonoid biosynthesis as part of an induced systemic response (ISR) given the important role of this pathway in plant defence, to cause increased levels of flavonoids in the fruit. We have identified structural genes encoding enzymes of the phenylpropanoid and flavonoid biosynthetic pathways catalysing the conversion of phenylalanine to the final products including flavonols, anthocyanins and catechins from blackberry, and regulatory genes likely involved in controlling the activity of pathway branches. We have also measured the major flavonols, anthocyanins and catechins at three stages during ripening. Our results demonstrate the coordinated expression of flavonoid biosynthetic genes with the accumulation of anthocyanins, catechins, and flavonols in developing fruits of blackberry. Elicitation of blackberry plants by treatment of roots with P.fluorescens N21.4, caused increased expression of some flavonoid biosynthetic genes and an accompanying increase in the concentration of selected flavonoids in fruits. Our data demonstrate the physiological mechanisms involved in the improvement of fruit quality by PGPR under field conditions, and highlight some of the genetic targets of elicitation by beneficial bacteria.     

Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.     

Apple 1-Aminocyclopropane-1-Carboxylic Acid Synthase Genes, MdACS1 and MdACS3a, are Expressed in Different Systems of Ethylene Biosynthesis

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is one of the key regulatory enzymes involved in the synthesis of ethylene. Climacteric fruit ripening is accompanied by increased ethylene production, in which ethylene biosynthesis is changed from system 1 to system 2. In apple, at least four members of the ACS gene family have been identified, two of which, MdACS1 and MdACS3a, have been studied extensively due to their specific expression in fruit tissue. However, the regulatory role of MdACS1 and MdACS3a in the ethylene biosynthesis system is unknown. Here we addressed this issue by investigating ACS expression in ripening apple fruits. Expression analysis in ‘Golden Delicious’ and ‘Red Fuji’ fruits, in combination with treatments of 1-MCP (1-methylcyclopropene, an ethylene inhibitor) and Ethephon (an ethylene releaser) has demonstrated that MdACS3a and MdACS1operate in system 1 and system 2 ethylene biosynthesis, respectively.     

Physiological and molecular analyses of early and late Coffea arabica cultivars at different stages of fruit ripening

Coffee quality is strongly influenced by a great number of factors, among which the fruit ripening stage at harvest time has a major influence on this feature. Studies comprising ethylene production and the regulation of ethylene biosynthesis genes during the ripening process indicate that ethylene plays an important role on coffee fruit ripening. Coffee early cultivars usually show a more uniform ripening process although little is known about the genetic factors that promote the earliness of ripening. Thus, in order to better understand the physiological and genetic factors involved in the regulation of ripening time, and consequently ripening uniformity, this study aimed to analyze ethylene and respiration patterns during coffee ripening, as well as to analyze ACC oxidase, an ethylene biosynthesis enzyme, gene expression, in fruits of early (Catucaí 785-15) and late (Acauã) coffee cultivars. Coffee fruits were harvested monthly from 124 days after flowering (end of February), until complete maturation (end of June). Dry matter, moisture content, color, respiratory rate and ethylene production analysis were performed. In silico analysis identified a coffee ACC oxidase gene (CaACO-like) and its expression was analyzed by real-time PCR. Dry matter and relative water content constantly increased and gradually decreased, respectively, during fruit ripening, and the color analysis enabled the observation of the earliness in the ripening process displayed by Catucaí 785-15 and its higher fruit ripening uniformity. The results obtained from the CaACO-like expression analysis and respiration and ethylene analysis suggest that the differences in ripening behavior between the two coffee cultivars analyzed in this study may be related to the differences in their capacity to produce ethylene, with fruits of Catucaí 785-15 and Acauã showing a typical and an attenuated climacteric phase, respectively, which may have lead to differences in their ripening time and uniformity.     

Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits

Xyloglucan endotransglucosylase/hydrolase (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan modifying enzyme, has been proposed to have a role during tomato and apple fruit ripening by loosening the cell wall. Since the ripening of climacteric fruits is controlled by endogenous ethylene biosynthesis, we wanted to study whether XET activity was ethylene-regulated, and if so, which specific genes encoding ripening-regulated XTH genes were indeed ethylene-regulated. XET specific activity in tomato and apple fruits was significantly increased by the ethylene treatment, as compared with the control fruits, suggesting an increase in the XTH gene expression induced by ethylene. The 25 SlXTH protein sequences of tomato and the 11 sequences MdXTH of apple were phylogenetically analyzed and grouped into three major clades. The SlXTHs genes with highest expression during ripening were SlXTH5 and SlXTH8 from Group III-B, and in apple MdXTH2, from Group II, and MdXTH10, and MdXTH11 from Group III-B. Ethylene was involved in the regulation of the expression of different SlXTH and MdXTH genes during ripening. In tomato fruit fifteen different SlXTH genes showed an increase in expression after ethylene treatment, and the SlXTHs that were ripening associated were also ethylene dependent, and belong to Group III-B (SlXTH5 and SlXTH8). In apple fruit, three MdXTH showed an increase in expression after the ethylene treatment and the only MdXTH that was ripening associated and ethylene dependent was MdXTH10 from Group III-B. The results indicate that XTH may play an important role in fruit ripening and a possible relationship between XTHs from Group III-B and fruit ripening, and ethylene regulation is suggested.     

Monochromatic light increases anthocyanin content during fruit development in bilberry

Background

Light is one of the most significant environmental factors affecting to the accumulation of flavonoids in fruits. The composition of the light spectrum has been shown to affect the production of phenolic compounds during fruit ripening. However, specific information on the biosynthesis of flavonoids in fruits in response to different wavelengths of light is still scarce. In the present study bilberry (Vaccinium myrtillus L.) fruits, which are known to be rich with anthocyanin compounds, were illuminated with blue, red, far-red or white light during the berry ripening process. Following the illumination, the composition of anthocyanins and other phenolic compounds was analysed at the mature ripening stage of fruits.

Results

All the three monochromatic light treatments had significant positive effect on the accumulation of total anthocyanins in ripe fruits compared to treatment with white light or plants kept in darkness. The elevated levels of anthocyanins were mainly due to a significant increase in the accumulation of delphinidin glycosides. A total of 33 anthocyanin compounds were detected in ripe bilberry fruits, of which six are novel in bilberry (cyanidin acetyl-3-O-galactose, malvidin acetyl-3-O-galactose, malvidin coumaroyl-3-O-galactose, malvidin coumaroyl-3-O-glucose, delphinidin coumaroyl-3-O-galactose, delphinidin coumaroyl-3-O-glucose).

Conclusions

Our results indicate that the spectral composition of light during berry development has significant effect on the flavonoid composition of ripe bilberry fruits.

     

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.