Expression Differences of Pigment Structural Genes and Transcription Factors Explain Flesh Coloration in Three Contrasting Kiwifruit Cultivars

Abstract

Fruits of kiwifruit cultivars (Actinidia chinensis and A. deliciosa) generally have green or yellow flesh when ripe. A small number of genotypes have red flesh but this coloration is usually restricted to the inner pericarp. Three kiwifruit cultivars having red (‘Hongyang’), or yellow (‘Jinnong-2’), or green (‘Hayward’) flesh were investigated for their color characteristics and pigment contents during development and ripening. The results show the yellow of the ‘Jinnong-2’ fruit is due to the combined effects of chlorophyll degradation and of beta-carotene accumulation. The red inner pericarps of ‘Hongyang’ fruit are due to anthocyanin accumulation. Expression differences of the pathway genes in the inner pericarps of the three different kiwifruits suggest that stay-green (SGR) controls the degradation of chlorophylls, while lycopene beta-cyclase (LCY-β) controls the biosynthesis of beta-carotene. The abundance of anthocyanin in the inner pericarps of the ‘Hongyang’ fruit is the results of high expressions of UDP flavonoid glycosyltransferases (UFGT). At the same time, expressions of anthocyanin transcription factors show that AcMYBF110 expression parallels changes in anthocyanin concentration, so seems to be a key R2R3 MYB, regulating anthocyanin biosynthesis. Further, transient color assays reveal that AcMYBF110 can autonomously induce anthocyanin accumulation in Nicotiana tabacum leaves by activating the transcription of dihydroflavonol 4-reductase (NtDFR), anthocyanidin synthase (NtANS) and NtUFGT. For basic helix-loop-helix proteins (bHLHs) and WD-repeat proteins (WD40s), expression differences show these may depend on AcMYBF110 forming a MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis, instead of it having a direct involvement.

A MYB/bHLH complex regulates tissue‐specific anthocyanin biosynthesis in the inner pericarp of red‐centered kiwifruit Actinidia chinensis cv. Hongyang

“…In addition, similar experiments were conducted to test the possibility of interaction between AcMYB123/AcbHLH42 and the previously identified anthocyanin regulator AcMYBF110 (Liu et al ., ). The BiFC and Co‐IP assays showed that, in contrast to no interaction signal being detected between AcMYB123 and AcMYBF110 (Figure S7a,b), bona fide physical interaction existed between AcMYBF110 and AcbHLH42 (Figure S7a,c).…”

“…To further verify the function of AcMYB123 and AcbHLH42 in anthocyanin biosynthesis, either 35S:: AcMYB123 or 35S : AcbHLH42 , or both, were transiently expressed in N. tabacum leaves via A. tumefaciens ‐mediated transformation. Infiltration of 35S :: AcMYBF110 was used as a positive control (Liu et al ., ). Interestingly, cellular build‐up of anthocyanin was apparently visualized in the microscopic bright‐field in the infiltrated leaf samples either expressing AcMYBF110 alone or co‐expressing AcMYB123 and AcbHLH42 ; by contrast the leaf samples separately transformed with 35S:: AcMYB123 , 35S :: AcbHLH42 or empty vector (pHB) failed to accumulate anthocyanin (Figure a).…”

“…AcMYB44, AcMYB3R‐1 and AcMYB3R‐5 lack the highly conserved motif at the R2R3 domain. To our surprise, the protein sequences from the characterized anthocyanin‐related MYBs, including AcMYB10 (Fraser et al ., ), AcMYB1(Man et al ., ), AcMYBF110 (Liu et al ., ) and AcMYB75 (Li et al ., ), are almost identical (Figures S3b and S4) and mapped to the same annotated gene (Acc00493) of the ‘Red5’ genome (Pilkington et al ., ). Similar analysis showed that all of AcbHLH42, AcGL3 and AcEGL3 are R/B‐like bHLH proteins from the subgroup IIIf possessing N‐terminal MYB interaction region (MIR) with three conserved motifs designated as 11, 18 and 13 box and a C‐terminal basic‐helix1–loop–helix2 (bHLH) domain (Figure S5a) and AcbHLH42 is most closely related to Arabidopsis TT8/AtbHLH042 protein (Figure S5b).…”

“…These results demonstrated that AcMYB123 , AcbHLH42 , AcF3GT1 and AcANS are all necessary for conferring anthocyanin accumulation in the inner pericarp of ‘Hongyang’. To determine the specificity of RNAi silencing, the characterized anthocyanin‐related MYBs, AcMYB1 / AcMYB10 / AcMYB75 / AcMYBF110 (Fraser et al ., ; Man et al ., ; Li et al ., ; Liu et al ., ) and three additional MYB homologs (accession nos Acc16026/Ach00g270991, Acc22332/Ach19g199471 and Acc26115/Ach23g366791) most closely related to AcMYB123 (Figures S3 and S4), as well as three bHLH homologs (accession nos Acc31692/Ach28g321841, Acc20018/Ach18g244201 and Acc09123/Ach08g302211) (Figure S5) most closely related to the AcbHLH42 were included for the qRT‐PCR analyses. Subsequent measurement showed that their expression was nearly unaffected in the RNAi suppressed fruits (Figure S10).…”

“…AcMYBF110 was also shown as an important R2R3‐MYB gene in regulating anthocyanin accumulation in the fruit of red‐fleshed A. chinensis cv. Hongyang, probably by activating the transcription of DFR , ANS and F3GT1 (Liu et al ., ). Upregulation of MYB members ( MYBA1‐1 and MYB5‐1 ) by low temperature could effectively enhance anthocyanin accumulation in kiwifruit during storage through transcriptional activation of ANS1 , ANS2 , DFR1 , DFR2 and UFGT2 (Li et al ., ).…”