Identification and expression of cytokinin signaling and meristem identity genes in sulfur deficient grapevine (Vitis vinifera L.)

Abstract

In plants, cytokinin (CK) perception and signaling pathway is composed by a histidine kinase receptor (HK) and a response regulator (RR), the signal being mediated by a histidine phosphotransfer (HPt), as described in Arabidopsis, maize and rice. From database searches we identified in grapevine three HKs, three HPs, four A-type RRs and six B-type RRs, suggesting a common mechanism for grapevine. The phylogenetic analysis of these Vitis genes showed a variable but high degree of homology with Arabidopsis sequences. When sulfate was withdrawn from the culture medium (-S) of in vitro Vitis shoots, we assessed a significant reduction in shoot branching. To ascertain the crosstalk of S status with CK signaling in grapevine, control and -S grown shoots and control, -S and -CK cell suspensions were used as experimental systems. Real-time PCR was elected to quantify the expression of key genes. The expression of CK receptor genes was downregulated in -S cells while not affected in -CK cells. In differentiated shoots no response to -S was observed on those genes. A-type VvRRa4 was downregulated in -S or -CK cells while Vitis B-type RRs did not respond either to CK or S starvation. The results suggest that Vitis CK signaling pathway is affected by -S, although differently according to the model system. Transcription of Vitis apical meristem-identity genes VvWUS, VvCLV and VvSTM and axillary meristem genes VvBRC1, VvBRC2, VvLAS, VvRAX and VvREV was estimated and VvSTM and VvLAS showed to be downregulated in -S. Then, the expression levels of VvSTM and VvLAS make them strong candidates to be associated with the branching pattern of Vitis shoots in -S.

Keywords: apical meristem genes; axillary meristem genes; cytokinin signaling genes; sulfur; vitis.

Figure 1

Pylogenetic tree of (A) HKs, accession numbers from NCBI: Arabidopsis: At CRE1/AHK4, BAB33310; At AHK2, NP_568532; At AHK3, NP_564276; Lupines: L. albus HK1, ABJ74169; Maize: Z. mays HK2, BAD01584; Z. mays HK3, BAD01586; Z. mays HK1, NP_001104859; Rice: O. sativa HK3, AAP53311; O. sativa CRE1, ABF89563; Vitis: V. vinifera, CA O42401; V. vinifera, CA O41188; V. vinifera, CAO66151, (B) HPts, accession numbers from NCBI: Arabidopsis: At AHP1, NP_188788; At AHP2, NP_850649; At AHP3, NP_198750; At AHP4, NP_566544; At AHP5, NP_563684; Maize: Z. mays, BAC 06592; Z. mays, BAA82874; Populus: P. x canadensis HP3, CAH 56501; Rice: O. sativa, ATT69671; O. sativa, BAD87514; O. sativa, EAY75784; Vitis: V. vinifera, CAO71051; V. vinifera, CAN83997; V. vinifera, CAN69375; V. vinifera, CAO45819; (C) A-type RRs, accession numbers from NCBI: Arabidopsis: At RRa3, NP_176202; At RRa4, NP_172517; At RRa5, NP_190393; At RRa6, NP_201097; At RRa7, NP_17339; At RRa8, NP_181663; At RRa9, NP_191263; At RRa15, NP_177627; At RRa16, NP_181599; At RRa17, NP_567037; Maize: Z. mays RRa2, BAA 85113; Z. mays RRa4, BAB20579; Z. mays RRa6, BAB20581; Z. mays RRa5, BAB20580; Rice: O. sativa RRa4, NP_001045420; Vitis: V. vinifera, CAO41517; V. vinifera, CA O42848; V. vinifera, CA O40969; V. vinifera, CAO63061 and (D) B-type RRs accession numbers from NCBI: Arabidopsis: At RRb1, NP_850600; At RRb2, NP_193346; At RRb10, NP_194920; At RRb11, NP_176938; At RRb12, NP_180090; At RRb13, NP_180275; At RRb14, NP_178285; At RRb18, Q9FGT7; At RRb19, NP_175345; At RRb21, NP_196338; Maize: Z. mays RRb8, BAB41137; Z. mays RRb9, BAB55874; Rice: O. sativa RRb10, AAN52739; O. sativa RRb9, BAD72541; O. Sativa, BAC77080; S. asiatica, ABG35774; Vitis: V. vinifera, CAO22750; V. vinifera, CA O40068; V. vinifera, CAN70187; V. vinifera, CAO46698. The circled assignments correspond to sequences analyzed by real time PCR for Vitis gene expression. The phylogenetic tree based on the amino acid sequence was constructed using the PHYLIP programs, PRODIST and NEIGHBOR.

Figure 2

Expression levels of VvCyt1, VvCyt2, VvCyt3, VvHP2, VvHP3, VvRRa3, VvRRa4, VvRRb5 and VvRRb6 as quantified by real time PCR in Vitis cells at day 7 of growth in −S conditions.

Figure 3

Expression levels of VvCyt1, VvCyt2, VvCyt3, VvHP2, VvHP3, VvRRa3, VvRRa4, VvRRb5 and VvRRb6 genes as quantified by real time PCR in Vitis cells at day 7 of growth in −CK conditions.

Figure 4

Expression levels of VvCyt1, VvCyt2, VvCyt3, VvHP2, VvHP3, VvRRa3, VvRRa4, VvRRb5 and VvRRb6 genes as quantified by real time PCR in Vitis shoots after 2 weeks of −S growth conditions.

Figure 5

Expression levels of Vitis apical (VvWUS, VvCLV and VvSTM) and axillary (VvBRC1, VvBRC2, VvLAS, VvRAX and VvREV) meristem genes as quantified by real time PCR in Vitis shoots after 2 weeks of −S growth conditions.