Saffron (Crocus sativus L.) corms grown in the field of CSIR-IHBT, Palampur, Himachal Pradesh, India, were used for the study. The corms were planted in October 2020 and harvested in April 2021. After harvesting, the corms were air-dried and sorted according to weight for the experiments. Corms with a weight range of 10–12 g that are considered competent to flower were used for the experimentation. In total 20 corms per treatment were used for each developmental stages. Different tissues of the saffron plant—namely, leaves, roots, corms, and flower tissues (stigma, stamens, and tepals)—were also collected separately for tissue-specific expression of genes.

To understand the role of hormones involved in flowering induction, corms 10–12 g in size were treated with different hormones. Briefly, a hormonal solution of IAA (0.5 mg/l or 2.85 μm), ABA (0.5 mg/l or 1.89 μm), GA (0.5 mg/l or 1.44 μm), and kinetin (0.5 mg/l or 2.32 μm) was prepared in distilled water, and the corms were dipped in solution and then infiltrated for 2 h under vacuum. The control sample was dipped only with distilled water for the same span of time. After treatment, the corms were wiped with tissue, dried, and then stored at 25°C for floral induction in the dark for 90 days. Samples were collected for morphological and gene expression analysis from apical buds after 45 days (stage 1) and 90 days (stage 2) of treatment. Three biological replicates for each treatment were used for the study. The samples were harvested, frozen in liquid nitrogen, and stored at -80°C until further use.

To study the role of hormones in flower formation, flowering competent corms (10–12 g) that were stored at 25°C for 3 months in a growth chamber (June–August) and which already have floral initiation in them were used for the experiment. In early September, after exogenous hormone treatments (IAA, ABA, GA, and kinetin), corms were stored at 15/20°C during the night and day, respectively, with 8/16-h light/dark cycle for flower emergence in the growth chamber. Samples were collected at 45 days’ interval in mid-October (stage III) and at the end of November (stage IV) when flowers emerged and were visible.

Apical bud tissue at 45 days of the developmental stage of large corms were collected and fixed quickly in 4% paraformaldehyde solution. The fixed samples were dehydrated with ethanol series (10%-100%). Samples were stained with 0.05% eosin and replaced by Histo-Clear gradually. Then, the samples were embedded in paraffin and sliced to a thickness of 7–10 µm with Thermo Scientific HM 355S automatic rotary microtome. The sliced samples were photographed under Leica DM2000 optical microscope (Javelle et al., 2011).

The genomic resource (genome sequences) of saffron is not yet available, making it difficult to mine genes involved in the process. In order to study the genes involved in flowering regulation, we selected genes which were previously reported either in saffron or other plants. Briefly, floral integrator genes that comprise FTs, TFL1s, LFY, and floral repressor SVP along with floral homeotic genes (APATELLA, PISTILLATA, SEPATELLA, DROOPING LEAF, etc.) were obtained from in-house and online database and used for the study. The list of genes studied, their source, and the references used in the present work have been listed in Supplementary Table S1 .

Tukey–Kramer multiple-comparison test by GraphPad software (significance level of P < 0.05; GraphPad URL: https://www.graphpad.com) and one-way ANOVA test (significance level of P < 0.05) were used to analyze the real-time PCR and corm morphological data, respectively.

To identify the morphological changes and development of different organs during the flowering process, the apical buds were monitored for 6 months (May–November), and apical bud samples were collected and analyzed after every 45 days from the start of the experiment. The morphological studies suggest that flower induction occurred in the first 45 days (stage 1), whereas the stamen and stigma start to develop between 45 and 90 days (stage 2). Stage 3 is marked with elongation in the stamen and stigma, and tepals development was observed between stage 3 and stage 4. A complete flower with all the organs developed was seen at stage 4 ( Supplementary Figure S1 ).

To study the effect of hormones on flower induction in saffron, dormant corms were treated with different hormones (GA, ABA, IAA, and kinetin) and were analyzed for floral induction process. After 45 days (stage 1), the apical meristem was fixed and used for microscopic analysis after sectioning. The morphological studies suggest that GA and ABA inhibited flower induction as seen in the longitudinal cross-section of the apical bud (small and semi-conical in shape, undifferentiated apical bud meristem tissues) ( Figure 2A ). Corroborating with the results seen after 45 days in ABA and GA treated samples, no floral organ formation can be seen in them after 90 days ( Figure 2A ). However, in the control, IAA- and kinetin-treated apical bud floral induction was prominently visible (differentiated apical bud meristem tissues), which is further confirmed by apical bud images at 90 days (stage 2) after the respective treatment that shows the development of the stamen and stigma ( Figure 2A ). The effect of different hormone treatments was also visible on apical and axillary bud length. IAA and kinetin promoted apical bud growth, which is reflected as increased apical and axillary bud length, whereas a significant reduction in apical and axillary bud length is observed in ABA and GA treated samples ( Figures 2D, E ).

The expressions of floral integrator genes (FTs, TFL1s, LFY, and SVP) were checked during the different stages of the flowering process. The results show that CsatFT1 and CsatFT2 gene expression gradually increased from stage 0 to stage 4 by up to three- and fivefold, respectively ( Figure 3 ). The increase was observed from stage 1 and 2 in CsatFT1 and CsatFT2 genes, respectively, and no significant increase in expression was seen at stage 0 (flowering induction stage). However, CsatFT3 comparatively showed a sixfold increase in stage 1, and then its expression declined in further stages. The CsatFT4 gene transcript levels remained low throughout the stages and rapidly increased by up to ~15 folds in stage 4. The CsatTFL1-1 and CsatTFL1-2 genes showed a similar expression pattern ( Figure 3 ). The transcript of TFLs was higher at stage 0 but reduced rapidly in stage 1 and remained low throughout all the stages except at stage 4, where their expression increased again by more than approximately twofold. CsatLFY gene expression was observed from stage 0, and it gradually increased at further stages. The expression of CsatSVP gene was not found to be significant at the early developmental stages but declined slightly at stage 3 and stage 4 ( Figure 3 ).

The homeotic genes showed a different expression in the developmental stages of saffron, such as the CsatAP3 expression which started to increase from stage 2, had its highest expression by up to approximately fivefold in stage 3, and further showed a small reduction of its transcript levels in stage 4. CsatPISTILLATA-1 and CsatPISTILLATA-2 showed the same expression patterns ( Figure 3 ). The CsatPISTILLATA-1 and CsatPISTILLATA-2 transcripts started to appear from stage 2 and stayed highest by approximately 60- and 30-fold, respectively, in stage 4. The CsatSEP3A and CsatSEP3B genes had different expression patterns. The CsatSEP3A gene expression was highest in stage 2 (approximately 60-fold), and the transcript levels were rapidly decreased by 60%–70% in stage 3 and stage 4. The CsatSEP3B transcripts were also accumulated from stage 2 and went to its highest by ~30-fold in stage 4 ( Figure 3 ). Another gene, DL, showed a gradual increase in expression pattern from stage 1 to stage 3 (approximately sevenfold), while the transcript levels were decreased by 40%–50% in stage 4. The AP1 gene also started to accumulate from stage 1 and declined slightly in stage 3 and stage 4 ( Figure 3 ).

Floral integrator and homeotic genes show tissue-specific expression in plants, suggesting their role in different plant and organ developmental processes. Our previous studies have provided the tissue-specific expression of FT and TFL genes in saffron (Kalia et al., 2022), but it remains unknown for floral homeotic genes and other genes such as SVP and LFY. Thus, we examined the expression of these genes in different tissues. The expression patterns found that CsatDL (dropping leaf) gene was highly and specifically expressed in the stigma tissue ( Supplementary Figure S2 ). The CsatPISTILLATA-2, CsatSEP3A, CsatSEP3B, and CsatAP3 genes were expressed in stamen, stigma, and tepals tissues, whereas CsatPISTILLATA-1 expression was confined to tepals and stigma tissues ( Supplementary Figure S3 ). However, SVP expressions were only observed in leaf and corm tissues of saffron. The CsatLFY gene was expressed in all the tissues studied, with a higher expression in leaf and corm tissues. Similar to CsatLFY, CsatAP1 expression was also found in most of the tissues except in the roots.

Morphological studies identified stage 1 as the flower induction stage in saffron flowering. Hormone treatments also differently affected them. We next examined the expression of previously identified/suggested floral integrator genes to establish a correlation between the effect of hormone on flower induction and organ formation via their transcriptional regulation. FT and LFY genes are positive regulators of floral induction. In our study ABA and GA treatments negatively co-related with flowering induction ( Figure 2A ). ABA and GA significantly reduced the expression of LFY gene in stage 2 ( Figure 4 ). Moreover, ABA reduced the the expression of FT3 gene in stage 1 ( Figure 4 ). No significant effect of ABA and GA was observed on CsatFT1, CsatFT2, and CsatFT4 expression compared with the control. Moreover, ABA significantly induced the expression of CsatTFL1-1 and CsatSVP genes that are considered as floral repressors at floral induction stage 1 ( Figure 4 ). Contrary to this, IAA and kinetin, which promoted flower induction, also promoted the expression of CsatFT3 and CsatLFY genes that are known to be involved in floral induction. At stage 2, most of the positive floral integrator genes were downregulated significantly, while floral repressors were upregulated during this stage in comparison with the control ( Figure 4 ).

Stage 2 was marked as the stamen and stigma differentiation stage ( Figure 2 ). To identify the role of conserved floral homeotic genes, also known as ABCE model genes, and their regulation via different hormones, we studied the expression of these genes during stage 1 and stage 2 of flowering. The expression of the studied genes was not significantly changed during stage 1, and majority of these genes were expressed at low levels except for the CsatAP1 and CsatAP3 genes ( Figure 5 ). The AP3 gene was significantly downregulated in GA treated samples. A significant reduction in the gene expression of CsatPISTILLATA-1 and CsatPISTILLATA-2 (involved in stamen and tepals formation) was observed in ABA and GA treated samples, whereas upregulation was seen in IAA and kinetin treated samples, respectively, at stage 2 ( Figure 5 ). Similarly, members of the CsatSEPTELLA (SEP3A and SEPB) genes were also downregulated in ABA and GA treated samples and upregulated in IAA and kinetin treated samples at stage 2. The dropping leaf (DL)-like gene also showed a significant downregulation in expression compared with the control in ABA and GA treated samples ( Figure 5 ). Overall, the results suggest that hormones that negatively affect flower induction and differentiation suppressed the expression of floral homeotic genes involved in stamen and stigma development at the early stages of flower organ differentiation.

To further investigate the roles of hormones in the flower formation of saffron, hormone treatments were performed in corms that have already induced flowering. After treatment, the corms were observed for flower development mainly flower formation. The results suggest that ABA just like in floral induction negatively affected flower formation. The corms that were treated with ABA showed flower atrophy, and the already initiated flower did not develop further, but the corm produced healthy leaves although with damage to the corms ( Figure 6 ). Similar results were observed in IAA treated corms (flower atrophy with normal leaf development). On the other hand, GA and kinetin treatment accelerated flower formation compared with the control ( Figure 6 ). The flowers in GA and kinetin treated corms showed no major difference in flower formation compared with the control. Interestingly, IAA, which promoted flower induction, suppressed flower formation, whereas GA that inhibited flower induction showed no significant effect on flower formation ( Figure 6 ).

The expression analysis of flower formation genes was carried out at stage 3 and stage 4 of saffron flowering after different hormone treatments. ABA and IAA negatively affected flower formation and also downregulated the expression of ABCE model genes ( Figure 7 ). The expression of CsatAP3, CsatPISTILATTA-1, CsatPISTILATTA-2, CsatSEP3A, CsatSEP3B, and CsatDL-like genes were significantly downregulated in stage 3 and stage 4 in ABA and IAA treated corms ( Figure 7 ). The results correlated with the flower atrophy phenotype observed in corms treated with these hormones. There was no significant upregulation of any of the genes studied in GA and kinetin treated corms in comparison with the control. Moreover, the expression of most of these genes also decreased in GA and kinetin treated corms such as CsatPISTILLATA2, CsatAP3, and CsatDL ( Figure 7 ). The expression of floral homeotic genes suggests that hormones regulate their expression in determining floral organ formation.

In our study, we found that hormones differentially affect the induction and formation of flowers in saffron. GA is the class of hormones which is best documented in the flowering process of Arabidopsis. It enhances the flowering process in Arabidopsis plant (Mutasa-Göttgens and Hedden, 2009), but, on the other hand, restricts flowering in several perennial species (Boss and Thomas, 2002; Garmendia et al., 2019). GA regulates flowering in Arabidopsis by promoting the LFY and FT genes expression (Moon et al., 2003; King et al., 2008). This study has found that GA regulates the flower induction and flower development processes of saffron in distinct ways. GA represses the flower initiation process and promoted the flower development process in saffron. Our results are in accordance with the findings of Renau-Morata et al. (2021), which also predicted by internal GA quantification that GA might inhibit floral induction in saffron. Contrary to this though, the study by Hu et al. (2020) suggested a positive role of GA in promoting floral induction in saffron. The results presented by Hu et al. were based only on transcript analysis, and the contradiction may be due to the different stages of floral induction. It is possible that GA might naturally inhibit floral induction, as in other perennials, and promote flower organ formation. More support to our observation in the role of GA is provided by the transcriptional analysis of floral integrator genes. We observed that GA treatment significantly downregulated the expression of CsatLFY that is a floral integrator gene. In addition to this, GA is known to overcome the chilling requirement that is a must for many plant species to flower (Heggie and Halliday, 2004), but in the case of saffron, ambient high temperatures are required for flowering, and low temperature treatment suppresses flowering (Molina et al., 2005b). This specific temperature requirement for flower induction might be the reason for the divergent role of GA in saffron flowering compared with other flowering plants.

ABA acts antagonistically to GA in various developmental processes, including flowering (Vanstraelen and Benková, 2012; Liu and Hou, 2018). It has a negative effect on flower initiation in Arabidopsis (Wang et al., 2013). However, ABA promotes flowering in Litchi chinensis (Cui et al., 2013). The suppression of flowering by ABA is shown to be mediated by effecting the expression of FT, SVP, and FLC-like genes (Martignago et al., 2020). The effect of ABA on geophytes, including saffron, has not been studied; rather, most of the studies are on leaf senescence. As flowering in saffron is accompanied with sprouting and dormancy release, it is very interesting to see its effects on saffron flowering. Our results also show the inhibitory role of ABA in the flower induction process in saffron and the repression of FT3 and LFY gene expression. In addition to this, ABA treatment also increased the expression of flowering repressor genes CsatTFL1-1 and CsatSVP in stage I of the flower initiation process. TFL and SVP genes have roles in dormancy establishment and release (Singh et al., 2018) other than flowering regulation. All homeotic genes except CsatAP3 also showed downregulation in the initiation process. A negative role of ABA in the flower induction process in saffron is also suggested by Renau-Morata et al. (2021) based on internal hormonal content and the genes involved in ABA signaling. There are not many studies on the hormonal regulation of flowering in saffron, including ABA, although the role of ABA in regulating saffron corm dormancy has been studied (Rubio-Moraga et al., 2014).

Cytokinins have been studied in many plants in connection with flowering with contrasting roles. Cytokinin application in rice reduced the expression of FT1 gene and delayed the flowering time (Cho et al., 2022), whereas in Arabidopsis it promotes flowering via the transcriptional activation of the FT paralog TSF (D'Aloia et al., 2011). Cytokinins are also important for ovule development in Arabidopsis (Higuchi et al., 2004; Bencivenga et al., 2012; Galbiati et al., 2013). However, this study found that cytokinin promotes both flower initiation and the developmental process of saffron by downregulating the flowering repressor gene CsatTFL1-2 and upregulation of flower developmental genes CsatPISTILLATA-1, CsatSEP3A, and CsatSEP3B. Cytokinins have been implicated in dormancy release (Subbaraj et al., 2010; Wu et al., 2019) in several geophytes by the regulation of cell proliferation and division via cell cyclin genes. In calla lilies, a cross-talk between cytokinin and GA regulates dormancy and flowering (Subbaraj et al., 2010). Cytokinin is also essential for in vitro flower development in Panax ginseng (Soon Lee et al., 1991).

Auxins (IAA) are another group of well-known phytohormones which regulate various aspects of plant growth and development (William et al., 2006; Spaepen et al., 2007; Spaepen and Vanderleyden, 2011). It has been found that IAA plays a crucial role in gynoecium development of Arabidopsis (Nemhauser et al., 2000). Our study shows that IAA is able to initiate flowering through activating flowering induction genes FT3 and LFY and suppressing the flower suppressor genes TFL1-2. Auxin also induces the expression of homeotic genes such as SEP3B, PISTILLATA-1, and PISTILLATA-2. In Arabidopsis, auxins regulate LFY expression in promoting flowering (Yamaguchi et al., 2013; Yamaguchi et al., 2016). In tulips, auxin has been identified as the main hormone involved in floral induction (Rietveld et al., 2000). However, unlike kinetin, IAA has a negative impact on the development of flower formation in saffron. Our results are in corroboration with the findings of Renau-Morata et al. (2021) where they also suggested promotion of flowering induction by auxins.