Apical dominance in saffron and the involvement of the branching enzymes CCD7 and CCD8 in the control of bud sprouting

Abstract

Background: In saffron (Crocus sativus), new corms develop at the base of every shoot developed from the maternal corm, a globular underground storage stem. Since the degree of bud sprouts influences the number and size of new corms, and strigolactones (SLs) suppress growth of pre-formed axillary bud, it was considered appropriate to investigate SL involvement in physiology and molecular biology in saffron. We focused on two of the genes within the SL pathway, CCD7 and CCD8, encoding carotenoid cleavage enzymes required for the production of SLs.

Results: The CsCCD7 and CsCCD8 genes are the first ones isolated and characterized from a non-grass monocotyledonous plant. CsCCD7 and CsCCD8 expression showed some overlapping, although they were not identical. CsCCD8 was highly expressed in quiescent axillary buds and decapitation dramatically reduced its expression levels, suggesting its involvement in the suppression of axillary bud outgrowth. Furthermore, in vitro experiments showed also the involvement of auxin, cytokinin and jasmonic acid on the sprouting of axillary buds from corms in which the apical bud was removed. In addition, CsCCD8 expression, but not CsCCD7, was higher in the newly developed vascular tissue of axillary buds compared to the vascular tissue of the apical bud.

Conclusions: We showed that production and transport of auxin in saffron corms could act synergistically with SLs to arrest the outgrowth of the axillary buds, similar to the control of above-ground shoot branching. In addition, jasmonic acid seems to play a prominent role in bud dormancy in saffron. While cytokinins from roots promote bud outgrowth. In addition the expression results of CsCCD8 suggest that SLs could positively regulate procambial activity and the development of new vascular tissues connecting leaves with the mother corm.

Figure 1

The corm of saffron and the different parts used in this study. (A) Upper view of the corm showing the apical meristem and the axillary buds located in the different nodes. (B) Transversal section of the corm through the apical meristem showing the vascular tissue and the parenchyma of the corm. (C) Bottom view of the corm showing the basal plate which connected the corm to the mother corm, along with the roots. (D) Transversal section of the corm showing the roots and the parenchyma tissue.

Figure 2

Dominance of the apical meristem in nondormant Crocus sativus corms. (A) removal of the apical bud allowed the sprouting of the axillary buds. a) corms with the apical meristems removed. b) intact corms. c) corms with sprouted secondary axillary bud 13 days after apical meristem removal. d) intact corms, as control, 13 days after the initiation of the decapitation experiment. (B) number of axillary buds sprouted or quiescent in each node after decapitation of the apical meristem. In each treatment, 25 corms were decapitated. Error bars represent SD of 25 replicates. (C) removal of the apical bud with or without other corm parts and results in sprouting of axillary buds. In each treatment, 25 corms were decapitated. Error bars represent SD of 25 replicates. (D) vascularization in the sprouted axillary buds of saffron corms (a-c). Absence of vasculature in quiescent axillary buds (d-f). Buds were sectioned by hand and sections were stained for lignin with phloroglucinol-HCl. Lignin staining is red. Pictures were taken under a dissection microscope.

Figure 3

Hormonal contents in different parts of saffron corms. Each column represents the mean ± of two to four replicates of independently harvested plant material. (A) Auxin content in the different samples. (B) Jasmonic acid (JA) content in the different samples. (C) Salicylic acid (SA) content in the analyzed samples.

Figure 4

Analysis of root-extractable strigolactones quantified by a germination bioassay in different in different corm tissues. Germination of P. ramosa seeds induced by extracts from: P, parenchyma; AB, axillary bud; VT2, vascular tissue from axillary buds and R, roots. GR24 (10–9 and 10–10 M) and demineralized water were used as positive and negative controls, respectively. Bars represent the means of two pools from five independent samples (±SE).

Figure 5

Gene structures of CsCCD7 and CsCCD8 and the phylogenetic relationship with CsCCD7 and CsCCD8 homologues from other plant species. (A) The postulated intron/exon structure for CsCCD7 and positions of the introns in orthologues of CsCCD7 is shown in the left side of the figure. In the right side a representative phylogenetic tree of CCD7 proteins from different plant species is shown. (B) The postulated intron/exon structure for CsCCD8 and positions of the introns in orthologues of CsCCD8 is shown at the left side. In the right side a representative phylogenetic tree of CCD8 proteins from different plant species is shown. Exons and introns are shown as boxes and lines, respectively. The present trees were obtained after alignment of full-length CCDs sequences using ClustalW and clustering with the neighbour-joining method. Accession numbers are as follow: SlCCD7 (ACY39882.1), PhCCD7 (ACY01408.1), AcCCD7 (ADP37985.1), ZmCCD7 (NP_001183928.1), RcCCD7 (XP_002511629.1), VvCCD7 (XP_002274198.1), AaCCD7 (ADB64459.1), PsCCD7 (ABD67496.2), GmCCD7 (ADK26570.1), AtCCD7 (NP_182026.4), CcsCCD7 (ADM18968.1), SmCCD7 (XP_002984696.1), OsCCD7 (EAY95081.1), LjCCD7 (ADM88552.1), MtCCD7 (XP_003622555.1), BdCCD7 (XP_003581501.1), PpCCD7 (ADK36680.1), PtCCD8a (XP002309543), PtCCD8b (XP002324797), AcCCD8 (GU206812.1), AtCCD8 (AT4G32810), BdCCD8 (LOC100831734), MtCCD8 (Medtr3 g127920), OsCCD8 (Os01 g0746400), Dad1 (AY743219), RMS1 (AY557342), SbCCD8 (Sb03 g034400), ZmCCD8 (GRMZM2G446858), RcCCD8, SlCCD8 (NP_001266276.1), StCCD8 (XP_006359761.1), CitCCD8a (KD079823), CitCCD8b (XP_006476130, CaCCD8b (XP_004501157), PpCCD8 (ADK36681.1), SmCCD8 (XP_002972693.1), GmCCD8b (XP_003522713), Gm CCD8a (XP_003522713.2). Branch support is under 5000 Bootstrap replicas.

Figure 6

CsCCD7 and CsCCD8 transcript accumulation (normalised to the saffron household gene RSP18). (A) relative gene expression of CsCCD7 in different plant tissues. (B) relative gene expression of CsCCD8 in different plant tissues. AM, apical bud; AB, axillary bud; AB24, axillary bud 24 hr after decapitation; R, adventitious root; VT, vascular tissue from apical meristem; VT2, vascular tissue from secondary buds; SS, senescent stigma; OS, orange stigma; L, leaf. Values are means of three technical replicates ± SE, normalized to the internal control gene.