Auxin enhances grafting success in Carya cathayensis (Chinese hickory)

Abstract

Application of auxin to root stock and scion increases the success rate of grafting in Chinese hickory. The nuts of the Chinese hickory (Carya cathayensis) tree are considered both delicious and healthy. The popularity and high demand result is that the hickory nuts are of very high economical value for horticulture. This is particularly true for the Zhejiang province in eastern China where this tree is widely cultivated. However, there are several difficulties surrounding the hickory cultivation, such as for example long vegetative growth, tall trees, labour-intensive nut picking, and slow variety improvements. These complications form a great bottleneck in the expansion of the hickory industry. The development of an efficient grafting procedure could surpass at least some of these problems. In this study, we demonstrate that application of the auxin indole-3-acetic acid promotes the grafting process in hickory, whereas application of the auxin transport inhibitor 1-N-naphthylphthalamic acid inhibits the grafting process. Furthermore, we have identified hickory genes in the PIN, ABCB, and AUX/LAX-families known to encode influx and efflux carriers in the polar transport of auxin. We show that increased expression of several of these genes, such as CcPIN1b and CcLAX3, is correlating with successful grafting.

Keywords: ABC transporter; LAX; Nut; PIN; Phloem; Polar auxin transport; Xylem.

Fig. 1

Pictorial representation of the leaf primordial development in the grafted plants in control (a), after IAA treatment (b), and NPA treatment (c) at 30 days after grafting (DAG)

Fig. 2

Cladogram including the newly identified hickory CcPIN (a) and CcPILS (b) proteins. PIN and PILS proteins from different plants such as Arabidopsis thaliana (AT), Vitis vinifera (grapevine) (GSVIV), Marchantia polymorpha (Mapoly), Physcomitrella patens (Pp), Selaginella moellendorfii (Smo), Theobroma cacao (Thecc), Prunus persica (Prupe), Glycine max (Glyma), Medicago truncatula (Medtr), Populus trichocarpa (Potri), Sorghum bicolor (Sobic), Ananas comosus (Aco), Oryza sativa (LOC Os), and Aquilegia coerulea (Aqcoe) were retrieved from Phytozome v12 (https://phytozome.jgi.doe.gov). Bootstrap values over 50 are shown. For orientation, the names of the hickory proteins are shown in purple, and the Arabidopsis proteins are in green. Clusters corresponding to different PIN-proteins are shown in different colours. The sequence identifiers are from Phytozome v12 (https://phytozome.jgi.doe.gov)

Fig. 3

Prediction of the transmembrane regions for the selected hickory PIN-proteins through TMHMM2 software (trans~membrane prediction using Hidden Markov Models). The predicted transmembrane is shown in red, the regions predicted to be on the cytoplasmic side are marked in blue, and regions found outside are marked as pink color

Fig. 4

Expression analysis of hickory PIN and PILS genes during different time points of the grafting process (0, 3, 7, and 14 DAG). The transcript abundance was measured under control, and IAA- and NPA-applied conditions. Expression of the CcPIN and CcPILS genes was measured by qRT-PCR with actin as the reference gene. Error bars indicate the standard deviation of at least three different experiments. R rootstock, S scion. The relative significant difference between the samples is marked by small alphabet letters. Different letters indicate significant difference evaluated by Tukey post hoc test with P < 0.05. Sharing of same letter represents no significant difference

Fig. 5

a Cladogram of CcABCB19 with ABCB family members from A. thaliana, O. sativa, and P. trichocarpa. Bootstrap values over 50 are shown. b Analysis of the relative expression level of the putative auxin-transporter CcABCB19 at different time points during grafting (0, 3, 7, and 14 DAG). The samples were analyzed under IAA- and NPA- applied conditions. R rootstock, S scion. Different letters indicate significant difference at P < 0.05, calculated by Turkey’s post hoc test. Same letter represents no significant difference

Fig. 6

a Cladogram of the hickory CcAUX/LAX proteins with AUX/LAX protein family members from A. thaliana (AT), V. vinifera (GSVIV), Solanum lycopersicum (Solyc), Musa acuminate (GSMUA), Brachypodium distachyon (Bradi), M. polymorpha (Mapoly), P. patens (Pp), S. moellendorfii (Smo), Sphagnum fallax (Sphfalx), T. cacao (Thecc), P. persica (Prupe), G. max (Glyma), M. truncatula (Medtr), P. trichocarpa (Potri), S. bicolor (Sobic), A. comosus (Aco), O. sativa (LOC Os), and A. coerulea (Aqcoe). For orientation, the names of the hickory proteins are shown in purple, and the Arabidopsis proteins are in green. Clusters corresponding to different AUX/LAX proteins are shown in different colours. Bootstrap values over 50 are shown. The sequence identifiers are from Phytozome v12 (https://phytozome.jgi.doe.gov). b Relative expression analysis of the CcAUX/LAX genes at different time points during grafting (0, 3, 7, and 14 DAG). The samples were analyzed under IAA- and NPA-applied conditions. R rootstock, S scion. Different lowercase letters represent significant difference with respect to other sample at P < 0.05 analyzed by Tukey’s post hoc test