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Plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTORS (TCP) transcription factors have versatile functions in plant growth, development and response to environmental stress. Despite blueberry's value as an important fruit crop, the TCP gene family has not been systematically studied in this plant. The current study identified blueberry TCP genes (VcTCPs) using genomic data from the tetraploid blueberry variety 'Draper'; a total of 62 genes were obtained. Using multiple sequence alignment, conserved motif, and gene structure analyses, family members were divided into two subfamilies, of which class II was further divided into two subclasses, CIN and TB1. Synteny analysis showed that genome-wide or segment-based replication played an important role in the expansion of the blueberry TCP gene family. The expression patterns of VcTCP genes during fruit development, flower bud dormancy release, hormone treatment, and tissue-specific expression were analyzed using RNA-seq and qRT-PCR. The results showed that the TB1 subclass members exhibited a certain level of expression in the shoot, leaf, and bud; these genes were not expressed during fruit development, but transcript levels decreased uniformly during the release of flower bud dormancy by low-temperature accumulation. The further transgenic experiments showed the overexpression of VcTCP18 in Arabidopsis significantly decreased the seed germination rate in contrast to the wild type. The bud dormancy phenomena as late-flowering, fewer rosettes and main branches were also observed in transgenic plants. Overall, this study provides the first insight into the evolution, expression, and function of VcTCP genes, including the discovery that VcTCP18 negatively regulated bud dormancy release in blueberry. The results will deepen our understanding of the function of TCPs in plant growth and development. [ABSTRACT FROM AUTHOR]

• Identification and cloning of metal transport-related Nramp genes in blueberry. • VcNramp4 and VcNramp6 are involved in the absorption and transport of Fe by plants. • VcNramp4, VcNramp6 and VcNramp8.2 were Zn and Cd transporters in blueberry. The natural resistance-associated macrophage protein (Nramp) family comprises crucial membrane transport proteins that play vital roles in the transport and reuse of heavy metal ions in plants. Limited information is currently available on the Nramp family in blueberry (Vaccinium spp.; Vc). Elucidation of the mechanism underlying mineral-element deficiency and heavy metal toxicity in blueberry is essential for the sustainable development of the blueberry industry. In this study, three Nramp genes (VcNramp4, VcNramp6 , and VcNramp8.2) were cloned and identified from blueberry, and their functions were analyzed in transgenic Arabidopsis thaliana and yeast. Sequence analysis showed that the VcNramp genes encoded 321–455 amino acids and contained 6–8 transmembrane structural domains. Expression analysis showed that VcNramp genes were significantly up-regulated under iron (Fe) deficiency. The expression of VcNramp genes was most significantly up-regulated in the roots, by 2.05-fold relative to the control group. VcNramp4 and VcNramp6 expression in yeast and A. thaliana alleviated Fe deficiency stress. Interestingly, yeast expressing VcNramp6 showed a 1.13- to 1.38-fold increase in Fe accumulation at different medium pH levels, which was significantly higher than that of yeast expressing the other VcNramp genes. All VcNramp genes respond to changes in Fe ion concentration. Among them, VcNramp6 can effectively ameliorate Fe deficiency symptoms in plants. The expression of VcNramp genes exhibited distinct tissue specificity. Notably, the expression levels of VcNramp6 and VcNramp8.2 in the stems was only 2 %–10 % of that in the roots. The results illustrated the Fe nutrient inefficiency of blueberry. Furthermore, all identified VcNramp genes responded to mild zinc or cadmium stress, but their expression levels decreased under severe zinc or cadmium stress. [ABSTRACT FROM AUTHOR]

• At pH 8.0, H+ influx and H+ efflux were restricted in blueberry roots. • In blueberry roots and leaves, most VcHAs had lowest transcript levels at pH 8.0. • ATP proton pump activity was increased in Arabidopsis over-expressing (OE) VcHAs. • A. thaliana OE lines tolerated alkaline stress due to increased H+ pumping. Blueberry (Vaccinium spp.) favors acid soils (pH 4.0–5.5), but the reasons for this strong preference are unclear. Root plasma membrane proton pumps (PM H+-ATPases - HAs) promote the efflux of hydrogen ions (H+) and thus promote nutrient absorption, thereby reducing stress associated with alkaline soils. In this study, 'Jewel' blueberry (V. corymbosum) was grown in media with a range of pH values (pH 5.0, pH 7.0, pH 8.0). After 30 days of growth, the physiological responses and the transcript levels of VcHAs (PM H+-ATPases) were analyzed. Eight VcHA genes were genetically transformed into Arabidopsis thaliana for functional verification. The results showed that (1) blueberry plants grew best at pH 5.0. The root activity at pH 5.0 was more than 1.6 times that at pH 8.0. In the pH 8.0 treatment, H+ influx and efflux from blueberry roots were limited. (2) Different VcHA genes showed diverse expression patterns. Among them, VcHA8 was only expressed in the root at pH 5.0. Except for VcHA11.2 in the roots, the transcript levels of VcHAs in roots and leaves were lowest in the pH 8.0 treatment. (3) Transgenic Arabidopsis thaliana plants over-expressing VcHA4.1, VcHA4.2, VcHA4.3, VcHA5, VcHA10.1, VcHA10.2, VcHA11.1, or VcHA11.2 showed stronger root activity than that of wild-type in all pH treatments. The root activity of the line overexpressing VcHA5 was 96 times that of the control. Compared with control plants, the phenotypic analyses of the VcHAs transgenic plants revealed enhanced levels of plant height, leaf size and chlorophyll content. The H+-ATPase activity of Arabidopsis overexpressing VcHA10.2 was 44 times that of the control. VcHAs not only significantly increased the hydrolytic activity and ATP proton pump activity in Arabidopsis but also increased H+ efflux, leading to cytoplasmic alkalinization in root epidermal cells. Over-expression of VcHAs significantly increased H+ pumping in Arabidopsis roots under alkaline rhizosphere stress, confirming that these genes encode functional proteins that are capable of acidifying the rhizosphere. VcHA10.2 and VcHA5 had the strongest ability to pump H+. The expression of VcHAs was inhibited in blueberry plants growing in high-pH media. This may explain why blueberry grows best in acid soils. These results help to explain why blueberry favors acid soils and may be useful for developing strategies to cultivate this crop in a wider range of soil types. [ABSTRACT FROM AUTHOR]

• This study explored the detailed function of members of the ZIP family in blueberry plant. • VcZIP6–VcZIP8 participated in the uptake of Fe and likely contributed to Fe uptake and transportation in plants. • VcZIP1, VcZIP7, VcZIP8 and VcZIP11 were Zn transporters in blueberry. • VcZIP1 and VcZIP7 could mediate the transport of Cd in yeast. Blueberry (Vaccinium spp., Vc) plants are highly susceptible to Fe deficiency stress. An understanding of the mechanisms involved in Fe as well as other mineral ions uptake and transportation is critical for the improvement of mineral ions use efficiency in the field practices. The functional roles of the VcZIPs in mineral ions uptake and transportation were not well understood. To elucidate the functions of ZIP transporter family that is involved in the uptake and transport of metal elements in blueberry, VcZIPs such as VcZIP1 , VcZIP2 , VcZIP6 , VcZIP7 , VcZIP8 and VcZIP11 were cloned and identified from blueberry genome. The results of functional complementarity and heterologous expression of yeast mutant verified that most of VcZIPs had the ability to transport Fe and Zn, and VcZIP1 and VcZIP7 could mediate the transport of Cd in yeast. Furthermore, higher chlorophyll contents and bigger crown diameter sizes were observed in VcZIP6 , VcZIP7 and VcZIP8 over-expressed Arabidopsis thaliana at Fe deficient treatments. In this study, VcZIP1 , VcZIP2 , VcZIP6 – VcZIP8 and VcZIP11 were Fe-deficient responsive genes in blueberry. VcZIP6–VcZIP8 participated in the uptake of Fe and likely contributed to Fe uptake and transportation in plants. VcZIP1, VcZIP7, VcZIP8 and VcZIP11 were involved transportation Zn in blueberry. Comparatively, higher Zn-transport abilities were observed in VcZIP1 and VcZIP7. [ABSTRACT FROM AUTHOR]