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

Abstract

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.

Transcriptomics, Targeted Metabolomics and Gene Expression of Blackberry Leaves and Fruits Indicate Flavonoid Metabolic Flux from Leaf to Red Fruit

“…The overexpression of the isoform RuPAL1 over the RuPAL2 in fruits (Figure 8) is consistent with the complex role of this enzyme in directing the carbon flux to the flavonol pathway in key points of fruit ripening (Garcia-Seco et al, 2015; Dastmalchi et al, 2017) and further supports the initial activation of the flavonol synthesis in fruits, together with the presence of cholrogenic acid identified therein (Table 4). Interestingly, the almost two-fold expression of RuDFR and RuLAR in fruits suggests that accumulation of catechins (Table 3) is taking place in fruits, as these compounds are usually accumulated in seeds (Zhang et al, 2014) and is supported by the presence of catechins only in fruits (Tables 4, 5), probably synthetized from the quercetins originated in leaves.…”

“…Interestingly, the almost two-fold expression of RuDFR and RuLAR in fruits suggests that accumulation of catechins (Table 3) is taking place in fruits, as these compounds are usually accumulated in seeds (Zhang et al, 2014) and is supported by the presence of catechins only in fruits (Tables 4, 5), probably synthetized from the quercetins originated in leaves. The downregulation of RuANS is consistent with the mid ripe status of blackberries, that still have not started to massively accumulate anthocyanins (Garcia-Seco et al, 2015). …”

“…Most studies on blackberry and other berries (Paredes-López et al, 2010) have been done on fruits since they are the edible part and therefore, hold the health-potential. The biosynthesis of flavonols and anthocyanins in blackberry fruits along maturation has already been described, demonstrating an increased biosynthesis of anthocyanins in the black ripen stage (Garcia-Seco et al, 2015). On the other hand, it may be anticipated that blackberry leaves also have an active secondary metabolism, in which flavonols must be abundant to participate in adaptation to environmental changes.…”

“…In a previous work a ripening study was performed, showing changes in flavonol-anthocyanin pathway core genes expression, and bioactive accumulation in the three stages (green, red, black) of maturation in fruit (Garcia-Seco et al, 2015). In this work, fruit flavonoid, and anthocyanin biosynthesis was demonstrated.…”

“…Transcription factors play an important role in flavonoid biosynthesis (Matsui et al, 2007) regulating some genes co-expression to stimulate or inhibit the accumulation of flavonoids. Based on a previous study on blackberry (Garcia-Seco et al, 2015), homologous regulatory genes ( RuMYB1, RuMYB3, RuMYB4, RuMYB5, RuMYB6, RuTT2 ) were found and their expression was also evaluated (Figure 9). …”