Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 15

Early molecular studies reported the expression of a limited number of genes that were differentially expressed during a particular developmental process or phase. Available genomic tools, i.e. an extensive kiwifruit EST database (Crowhurst et al., 2008) and the first draft of the kiwifruit genome sequence (Huang et al., 2013), have begun to generate more data. An international consortium was formed in 2015 to generate new information for this draft (Crowhurst et al., 2016). Other recent genomic resources include a restriction site-associated DNA (RAD)-based linkage map (Scaglione et al., 2015), a comparative database for kiwifruit genomics (Yue et al., 2015) and the first complete chloroplast genome sequence in this family (Yao et al., 2015), whose evolution was recently analysed (Wang et al., 2016). With some of this information, Avsar and Aliabadi (2015) identified putative microRNAs and Li et al. (2015) established the expression profiles of anthocyanin biosynthesis and accumulation genes.

An important part of functional genomic studies is the use of quantitative polymerase chain reaction (qPCR) analysis to determine the expression at the transcriptional level of those genes identified using genomic tools. For obtaining accurate relative expression data in qPCR, appropriate reference genes for normalization must be used. Petriccione et al. (2015) assessed nine candidate reference genes in A. deliciosa leaves during the first period of interaction (13 days post inoculation) with Psa. Ferradás et al. (2016) conducted a systematic study of eight primer pairs corresponding to four candidate reference genes into five plant sample sets (mature leaves, axillary buds, stigmatic arms, fruit flesh and seeds) commonly used in kiwifruit gene expression analyses.

The most extensive studies of functional genomics were related to fruit development and ripening, e.g. the role of ethylene during ripening. Synthesis and signalling of ethylene in relation to these processes (in particular kiwifruit ripening) have been addressed (Yin et al., 2008, 2009, 2010; Atkinson et al., 2011; Murakami et al., 2014; McAtee et al., 2015; Nieuwenhuizen et al., 2015). From the draft sequence of the kiwifruit genome (Huang et al., 2013), 105 novel ERF genes were identified, belonging to different subfamilies, but all of which displayed a single AP2/ERF domain. Gene expression analysis identified eight of these AdERF genes as highly associated with kiwifruit ripening and softening, including four putative activators and four putative repressors (Zhang et al., 2016).

Yang et al. (2007) reported that α-expansin genes are upregulated by exposure to ethylene or expressed during fruit softening (Richardson et al., 2011). On the other hand, the study of the expression profiles of several lignin-related genes indicated that lignin accumulates during kiwifruit cold storage (Li et al., 2017), thus reducing fruit edibility and quality. Hu et al. (2016) identified and characterized five genes as being potentially involved in starch degradation during kiwifruit postharvest ripening.

Flavour, colour and vitamin content are important traits related to kiwifruit ripening. Günther et al. (2011) investigated changes in steady-state transcript levels of Actinidia acyltransferases (ATs) to select sequences, potentially encoding for alcohol ATs that could be involved in ester biosynthesis. They evaluated their involvement in flavour-related fruit ester formation, identifying increased transcript levels specific in ripe fruit and regulated by ethylene. These authors also found that the production of aroma-related esters decreased in response to cold storage with time, but they were restored by ethylene treatment (Günther et al., 2015). Terpenes and their derivatives are related to kiwifruit aroma. Nieuwenhuizen et al. (2015) correlated terpinolene production in ripe A. arguta fruit with expression of terpene synthase1 (AaTPS1). Comparative promoter analysis allowed them to identify transcription factors as key for TPS expression. C6-aldehydes have been also reported as active flavour notes in kiwifruit; their production is mediated by LOX. Zhang et al. (2009) described how several LOX encoding genes that are related to ripening and sensitive to ethylene are related to the release of fruit ripening-associated aromatic compounds.

High content of vitamin C is an important feature of kiwifruit. Bulley et al. (2009) determined that the high concentration of ascorbate found in the fruit of A. eriantha is probably due to the expression of GDP-mannose-3′,5′-epimerase (GME) and GDP-L-galactose guanyltransferase (GGT) genes. Overexpression of GGT in Arabidopsis or transient expression in tobacco leaves of GME and GGT together confirm that GGT catalyses a major control point of ascorbate biosynthesis. Another enzyme involved in ascorbate synthesis is GDP-galactose phosphorylase (GGP). Li et al. (2014) found some correlation between the expression of GGP and ascorbate concentration during early fruit development.

New kiwifruit cultivars with different flesh colour have been introduced, and red-fleshed cultivars are probably the most interesting. Anthocyanins, carotenoids and chlorophylls are responsible for different flesh colours. Montefiori et al. (2011) identified two glycosyltransferases (F3GT1 and F3GGT1) as responsible for much of the anthocyanin diversity. While F3GGT1 is responsible for the end product of the pathway, F3GT1 is the key enzyme regulating the accumulation of anthocyanin in red-fleshed cultivars. Li et al. (2015) conducted a phylogenetic analysis of the kiwifruit genome followed by examination of the transcript levels of putative anthocyanin genes, finding differential expression levels during kiwifruit development. They also used phylogenetic analysis to identify nine transcription factors potentially involved in anthocyanin metabolism from transcriptome data. It has been shown that storage at low temperature stimulates anthocyanin accumulation in kiwifruit by regulating structural and regulatory genes involved in anthocyanin biosynthesis (Li et al., 2017).

Studies of flowering regulation are extremely important due to the dependence of this species on pollination. Walton et al. (2001) identified homologues of the Arabidopsis floral meristem identity genes LEAFY and APETALA1, and in situ hybridization analysis revealed that flowering occurs through two growing seasons. Later, Varkonyi-Gasic et al. (2011) described floral development in kiwifruit at the molecular level, identifying nine Actinidia MADS-box genes as genetic markers for inflorescence and flower development, potentially having impact on kiwifruit growth, phase change and time of flowering. The role of kiwifruit FT, described in other species as a floral integrator, but also CEN, BFT or FD genes were explored by Varkonyi-Gasic et al. (2013) and Voogd et al. (2017), suggesting their roles in the integration of developmental and environmental cues that affect dormancy, bud break and flowering. However, functional and expression analyses of kiwifruit SOC1-like genes, also described as a floral integrator, suggested that they may not have a role in the transition to flowering but may affect the duration of dormancy (Voogd et al., 2015). Other kiwifruit MADS-box genes with homology to Arabidopsis SVP have been identified by Wu et al. (2012b), suggesting that they may have distinct roles during bud dormancy and flowering. A kiwifruit protein inversely correlated with spring bud break has been identified (Wood et al., 2013).

Wu et al. (2014) demonstrated that SVP3 reduced anthocyanin accumulation in petals of transgenic A. eriantha in a quantitative manner. The underlying mechanism was the reduced expression of the key structural gene F3GT1 (Montefiori et al., 2011) and the transcriptional repression of regulatory MYB transcription factors, i.e. MYB110a, a key gene required for kiwifruit petal pigmentation (Fraser et al., 2013).

The regulation of the timing of flowering is necessary to ensure that sexual reproduction occurs at an appropriate time to enable cross-pollination. In many plant species, the timing of flowering depends on seasonal cues, i.e. photoperiod and temperature. With kiwifruit, exposure to winter temperatures is essential for synchronized bud break and efficient flowering (Walton et al., 2009), but it is less clear if photoperiod plays any role in flowering. Ferradás et al. (2017) identified genes encoding kiwifruit phytochromes and cryptochromes and determined their daily and annual expression patterns, showing that they were significantly highly expressed from late floral development until full bloom and fitting with floral evocation, closely matching the peaks of expression of AcFT- and AcCO-like genes.

Kiwifruit usually has >1000 seeds in each fruit. Studies on pollen–pistil interaction have determined that PCD occurs in female tissues after anthesis and is accelerated by pollination (Ferradás et al., 2014). Several genes related to ethylene biosynthesis and signalling show a correlation with PCD signs in unpollinated and pollinated flowers. The only exception is ACO5 that is only expressed at the end of effective pollination period or after pollen tube passage, indicating its possible implication in regulating senescence processes in other organs, i.e. ovaries or petals (Ferradás et al., in press).

Several traits related to biotic and abiotic stresses have been recently addressed, e.g. bacterial canker of kiwifruit caused by a virulent strain of Psa. Psa affects mainly kiwifruit cultivars with gold flesh, although it can also affect green-fleshed cultivars (‘Hayward’). A sample of genes representing both the basal defence and the resistance gene-mediated defence pathways were characterized by Fraser et al. (2015). Li et al. (2016) identified nucleotide-binding sites (NBS)-encoding resistance genes in the kiwifruit genome. Corsi et al. (2017) detected changes in the transcription profiles of two genes involved in the metabolic pathway of systemic acquired resistance (SAR) (pathogenesis-related proteins 1 and 5) in response to chitosan treatment of kiwifruit plants inoculated with Psa. Wang et al. (2017) identified circular RNAs as a mechanism of the species-specific response to Psa; Yin et al. (2012) showed that AdERF genes differentially respond to abiotic stresses during fruit postharvest storage, and some of them have been identified as acetylsalicylic acid-responsive genes (Yin et al., 2013). Li et al. (2013a) established that GGP expression in two kiwifruit species is regulated by light or abiotic stress via the relative cis-elements in their promoters. Waterlogging tolerance can be important in fruit production; Zhang et al. (2015a) have identified up to 14,843 unigenes differentially expressed during waterlogging, representing an important resource for understanding the molecular mechanisms of the waterlogging response. Zhang et al. (2016) also cloned and established the implication of pyruvate decarboxylase 1 gene (AdPDC1) in waterlogging stress. Liu et al. (2016) cloned two dehydroascorbate reductases, correlating them with increased L-ascorbic acid concentration and tolerance of salinity.