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. 2024 Jan 18;30(1):81–91. doi: 10.1007/s12298-024-01410-3

Transcriptome wide identification, characterization and expression analysis of PHD gene family in Crocus sativus

Aubid Hussain Malik 1,2, Nargis Khurshaid 1,2,#, Najwa Shabir 1,2,#, Nasheeman Ashraf 1,2,✉
PMCID: PMC10902251  PMID: 38435850

Abstract

Crocus sativus L., of the Iridaceae family, yields world’s most prized spice, saffron. Saffron is well known for its distinctive aroma, odour and colour, which are imputed to the presence of some specific glycosylated apocarotenoids. Even though the main biosynthetic pathway and most of the enzymes leading to apocarotenoid production have been identified, the regulatory mechanisms that govern the developmental stage and tissue specific production of apocarotenoids in Crocus remain comparatively unravelled. Towards this, we report identification, and characterization of plant homeodomain (PHD) finger transcription factor family in Crocus sativus. We also report cloning and characterisation of CstPHD27 from C. sativus. CstPHD27 recorded highest expression in stigma throughout flower development. CstPHD27 exhibited expression pattern which corresponded to the apocarotenoid accumulation in Crocus stigmas. CstPHD27 is nuclear localized and transcriptionally active in yeast Y187 strain. Over-expression of CstPHD27 in Crocus stigmas enhanced apocarotenoid content by upregulating the biosynthetic pathway genes. This report on PHD finger transcription factor family from C. sativus may offer a basis for elucidating role of this gene family in this traditionally and industrially prized crop.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01410-3.

Keywords: Saffron, Apocarotenoid, Transcriptome, PHD finger, CsPHD27

Introduction

Crocus sativus L., which is generally referred to as saffron, is regarded as the most expensive spice on the planet (Baba et al. 2015). Saffron has always been treasured worldwide in different cuisines and traditional medicines (Jose Bagur et al. 2017). Saffron owes its importance to a wide array of volatile and non-volatile compounds and carotenoid derivatives (Tarantilis et al. 1995; Winterhalter and Straubinger 2000). It is the only crop which produces appreciable quantities of unique apocarotenoids such as crocin, picrocrocin, and safranal (Baba et al. 2015). These apocarotenoids are synthesized from the key carotenoid zeaxanthin, through its oxidative cleavage by carotenoid cleavage dioxygenase 2 enzyme (CCD2) (Frusciante et al. 2014). Apocarotenoids in C. sativus accumulate only in stigmas reaching their maximal levels at the scarlet stage of flower development at anthesis and decline afterwards (Baba et al. 2015). Though the biosynthetic pathway of Crocus apocarotenoids has been elucidated up to a substantial level, the regulatory mechanisms which govern their production are not clear. Therefore, great research efforts are being made towards identification and characterization of genes that have potential role in regulating Crocus apocarotenoid biosynthesis.

Towards understanding the regulatory mechanism of apocarotenoid metabolism in Crocus, many transcription factor families have been studied (Bhat et al. 2021; Malik and Ashraf 2017). Zinc finger family is widely present in all eukaryotes including plants and constitutes one of the most predominant transcription factor families in Crocus (Baba et al. 2015). On the basis of the position of the zinc-binding residues, the zinc finger family has been classified into several groups consisting of RING (Really Interesting New Genes), LIM (Lin11, Isl-1, and Mec-3), and plant homeodomain (PHD) (Aasland et al. 1995; Borden and Freemont 1996). Among these groups, PHD-finger proteins are the most common type of zinc finger proteins, each comprising of one or more than one PHD-finger domains (Kaadige and Ayer 2006). A typical PHD-finger domain consists of around 60 amino acids, consisting of a characteristic amino acid core following a structural arrangement as Cys4-His-Cys3 and the same trend is maintained in the RING (Cys3-His-Cys4) and LIM (Cys2-His-Cys5) classes (Aasland et al. 1995; Borden and Freemont 1996). These core amino acid residues chelate two Zn2+ atoms to form a cross-brace structure (Alam et al. 2019). In plants, HAT3.1 from Arabidopsis was the first PHD-finger protein identified (Schindler et al. 1993) and subsequently several PHD genes have been identified in many other plants including Oryza sativa, Zea mays, Brassica rapa, Pyrus bretschneideri, Solanum tuberosum and Populus trichocarpa (Sun et al. 2017; Wang et al. 2015; Alam et al. 2019; Cao et al. 2018; Qin et al. 2019; Hou et al. 2021).

In plants, the PHD domains are known for their role in transcriptional regulation of stress responses and several developmental as well as physiological processes (Tsuchiya and Eulgem 2014; Wang et al. 2015). In Medicago sativa roots, Alfin, a novel zinc-finger protein, binds to the promoter elements of a salt-inducible gene MsPRP2, regulating its expression and enhances salt tolerance in these plants (Bastola et al. 1998). A study in Arabidopsis thaliana reports that an ARID (AT-Rich Interacting Domain)-containing protein, ARID1, binds to the promoter of DUO1 to regulate its expression, thereby promoting sperm cell formation in plants (Zheng et al. 2014). In Arabidopsis, one more study revealed that a PHD-finger protein VIL1 regulates floral identity gene expression; FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M (FLM) and plays an important role in the vernalisation and photoperiod pathways (Sung et al. 2006). Since PHD-finger genes are quite ample in the Zinc Finger Superfamily and play a variety of roles in plant development and growth, we began by exploring them as the potential candidates for carotenoid/apocarotenoid regulation in C. sativus.

Previously we identified zinc finger family genes from Crocus (Malik and Ashraf 2017). In the current study, we have carried out a detailed investigation of the PHD gene family in Crocus and examined transcript profiles of the identified genes in various tissues like corm, stigma, tepal and anther. Further, we also cloned and characterized CstPHD27 gene, which showed high similarity to HOX (homeodomain) gene sequences from other plants. CstPHD27 exhibited highest expression in stigma and was found to be nuclear localized and transcriptionally active. Over-expression of CstPHD27 in Crocus enhanced crocin content by upregulating expression of carotenoid/apocarotenoid pathway genes. This work presents CstPHD27 as potential candidate involved in regulation of apocarotenoid metabolism in Crocus.

Materials and methods

Plant material

Crocus sativus was farmed as reported earlier (Baba et al. 2017) at Indian Institute of Integrative Medicine’s (IIIM) experimental farm located at Srinagar, India. Raised beds were prepared and proper agronomic practices were followed for cultivation of Crocus corms. The stigmas were harvested at yellow, orange and scarlet stages for developmental stage specific gene expression, freeze dried in liquid nitrogen followed by preservation at -80 °C till further experimental use.

Identification and characterisation of PHD finger gene family of Crocus

With the help of BLAST homology results, PHD gene sequences were retrieved from the C. sativus databases developed in our laboratory (Baba et al. 2015) and from Jain et al. 2016. Further validation of all the identified sequences was done through itaK analysis which helps in identification of transcription factors and protein kinases (http://itak.feilab.net/). Conserved Domain Database (https://www.ncbi.nlm.nih.gov/) and MEME Suite (https://meme-suite.org/) online softwares were used for domain and motif analysis respectively. The significant matches were identified at a cut-off E value of 1e−10. For motif analysis, a maximum of 5 motifs were set. The phylogenetic analysis was performed to discern the sequence conformity and evolutionary correlation among Arabidopsis thaliana, Oryza sativa and Crocus sativus PHD finger genes. The evolutionary tree generation was done by MEGA 6 through the neighbour-joining method. The gene phylogeny evaluation was performed by the bootstrap consensus tree inferred from 1000 replicates. Multiple sequence alignment was done using MUSCLE (Tamura et al. 2013). Differential expression based on transcriptome data was done as mentioned in Baba et al. (2015).

RNA isolation and synthesis of cDNA

Total RNA from various tissue samples was isolated using RNeasy Plant mini kit (Qiagen, Germany) and the manufacturer’s recommended protocol was followed. The extracted RNA was separated on 2% agarose gel and quality and quantity were estimated by Nano Drop® ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). To purify the RNA from genomic DNA contamination, DNase I treatment was given. Synthesis of cDNA was then done by Reverse Transcription kit (Thermo Fisher Scientific, USA) through the recommended protocol.

Real-time (qRT-PCR) analysis

Step One Real time PCR machine (Thermo Fisher Scientific, USA) was employed for qRT-PCR. A 20 µL reaction containing 10 µL of 2X SYBR Green Master Mix (Thermo Fisher Scientific, USA), 100 ng of template cDNA and 0.2 µM gene specific primers was set up. Three technical replicates were used for each sample. The reactions were incubated at 95 °C for 20 s and 40 cycles at 95 °C for 15 s and then at 60 °C for 1 min. For each sample, three biological samples were used and for each such sample, three technical replicates were used. For estimating expression at different stages, yellow stage samples were used as control. The relative quantification (2−ΔΔCT) method was used to evaluate the fold change (Livak and Schmittgen 2001). 18 S gene was used as endogenous control. Information about the primer sequences is given in supplementary Table S2.

Sequence analysis and cloning of CstPHD27  gene

The gene sequence of CstPHD27 was retrieved from transcriptome database developed in house (Baba et al. 2015). The gene amplification was done from cDNA using gene specific primers (CstPHD27-F and CstPHD27-R). The cycling parameters for the amplification were 4 min at 95 °C, 32 cycles (30 s at 95 °C, 35 s at 57 °C and 1 min at 72 °C) and a final extension of 10 min at 72 °C. The amplicon was purified from the agarose gel using gel extraction kit (Qiagen, Germany) and sequenced after cloning in pGEM-T Easy vector (Promega, USA).

Subcellular localization

CstPHD27 subcellular localization was determined in epidermal cells of onion. The amplification of full length ORF of CstPHD27 was done with CstPHD27-GFP-F/CstPHD27-GFP-R primers followed by cloning in pCAMBIA1302 vector at BgIII and SpeI restriction sites in frame with GFP reporter gene. The empty vector was used as control. Both the gene construct as well as control were then transformed into the Agrobacterium tumefaciens GV3101 strain and agroinfiltrated into onion peel cells after wounding them with a syringe tip to improve infiltration. The onion peels were then visualized under the confocal microscope after 24-hour incubation in the dark. The agro-infiltration protocol was followed as per Hussain et al. (2022) and Bhat et al. (2021).

Transactivation assay

Complete Open Reading Frame of CstPHD27 was amplified using CstPHD27PGBKT7-F/CstPHD27PGBKT7-R primers and cloned in pGBKT7 (BD) vector using NdeI and BamHI restriction sites. The CstPHD27-BD construct was transformed into Y187 yeast strain using the Yeast Transformation System 2.0 (Takara, Japan). The screening of positive transformants was done through tryptophan deficient synthetic media. After establishing the positive transformations, streaking of constructs on SD/-Trp plates with D-raffinose, D-galactose and X-gal (5-bromo-4-chloro-3-indolyl-β-D galactopyranoside) was done. The streaked plates were observed for the appearance of blue colouration that is accredited to the β-galactosidase reporter gene activation in the host yeast strain. The Y187 cells possessing only the pGBKT7 vector were treated as negative control, and those with pGBKT7-p53 and pGADT7-T vectors were kept as positive control. The transactivation assay was also confirmed by the β-galactosidase assay as described in Hussain et al. (2022), Bhat et al. (2021).

Over-expression of CstPHD27 in C. sativus stigmas

For over-expression studies, CstPHD27 ORF was cloned into pBI121 vector downstream of 35 S CamV promoter. The pBI121-CstPHD27 construct and pBI121 vector alone (negative control) were independently transformed into Agrobacterium tumefaciens GV3101 cells and further agro-infiltrated into C. sativus stigmas using floral dip method as described in Bhat et al. (2021) and Hussain et al. (2022). The transformed stigmas were harvested 72 h after infiltration, frozen using liquid N2 and stored at − 80 °C till use. For each agro-infiltration experiment, a minimum of 10 flowers were used.

Quantification of crocin using HPLC

For crocin quantification, samples were prepared as previously described by Bhat et al. (2021). Briefly, 500 mg of frozen stigmas were crushed in liquid nitrogen. 10 mL of 50 mM Tris-HCl (pH 7.5) with 1 M NaCl was added followed by the addition of equal volume of chloroform. The samples were incubated on ice for 10 min followed by centrifugation at 4 °C for 10 min at 3500×g for phase separation. The upper aqueous phase was used directly for the quantification of crocin by HPLC. The samples were analyzed on a UHPC (Nexera UHPLC Shimadzu, Japan) equipped with an auto-sampler and a PDA detector. The method is described in detail in Bhat et al. 2021 and Hussain et al. 2022.

Results

Identification and motif investigation of PHD finger genes from transcriptome database of Crocus

A total of 91 non-redundant PHD gene sequences were recovered from the Crocus sativus transcriptome databases (Baba et al. 2015 and from Jain et al. 2016). To confirm presence of PHD domains, these gene sequences were further analysed and validated through itaK which grouped them into 4 groups: PHD transcriptional regulators (61 genes); Alfin-like transcription factors (26 genes), ARID transcriptional regulators (1 gene) and HomeoBox Domain (HB-PHD) transcription factors (3 genes) (Supplementary Table S1). To further understand similarities between them, we performed motif analysis on all the genes (Fig. 1). We observed that members of each group had similar motifs. For example, for the HB-PHD transcription factors (3 genes), all the 5 identified motifs belonged to the C2H2 zinc finger subfamily. For the Alfin-like transcription factors (26 genes), all the identified motifs belonged to the nuclear receptors with C4 zinc fingers subfamily. For the PHD transcriptional regulators, the group to which most of our identified sequences belonged (61 genes), the identified motifs belonged to Homeobox genes which are a class of evolutionarily conserved transcription factors.

Fig. 1.

Fig. 1

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Motif analysis of Crocus PHD finger genes with MEME suite. Sequences with similar motifs have been grouped together

Phylogenetic and domain analysis of Crocus PHD gene

To gain an understanding about the Crocus PHD genes, we created a phylogenetic tree with all the 91 identified genes of the PHD family from C. sativus. These genes clustered into 14 clades (Fig. 2). To elucidate the possible function of each phylogenetic cluster, domain analysis was performed using NCBI’s Conserved Domain Database (CDD). All the sequences were analysed for PHD-domains and other associated domains (Fig. 2). For each PHD gene, we observed the presence of one or more additional domains besides the PHD-finger domain (Fig. 2) (Supplementary Fig. S1). For the 61 PHD transcriptional regulator genes, the most significant domain identified was the PHD_SF which was present in 51 sequences. Besides that, the additional domains identified were Jas, PHD2_CHD_SF, PHD_PRHA, FYRN, BRCT_SF, BAH_SF, Agenet, PHDMMD1, Ring-U box SF and PHD Zinc Finger. For the 26 Alfin like transcriptional factor genes, the most significant domains identified were the PHD_AL_Plant (present in 25 sequences) and Alfin_SF (present in 17 sequences). Besides these two domains the other associated domains identified were: PHD_SF, P34_Arc_SF, PRK07373_SF, and y2fc_SF. For the 3 HomeoBox Domain (HBD) transcriptional factor genes, only two domains PHD_PRHA and Homeodomain were identified in all the three sequences. For the ARID Transcriptional regulator gene, the domains identified were PHD_SF and ARID.

Fig. 2.

Fig. 2

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Phylogenetic tree and domain analysis of Crocus PHD finger genes. The evolutionary tree was generated by MEGA 6 software. Domain analysis was done using conserved domain database in NCBI. Different colour boxes represent different domains (Color figure online)

To shed light on the evolutionary relationship of the PHD genes from C. sativus with those of other plant species, an unrooted phylogenetic tree was constructed with PHD gene sequences of Arabidopsis thaliana and Oryza sativa. The phylogenetic analysis grouped the sequences into 10 different clades (Fig. 3). All of the clades consist of representative sequences from all the three species. Further, clade 6 (SC6) was composed of mostly members from Crocus.

Fig. 3.

Fig. 3

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Evolutionary relationship analysis of Crocus and Arabidopsis PHD finger genes.The evolutionary tree was generated through neighbour joining algorithm and bootstrap analysis of 1000 replicates using the MEGA 6 software. Five major clades were formed. Genes from Crocus are indicated by red and those from Arabidopsis by green colour (Color figure online)

Expression patterns of PHD-finger genes in different Crocus tissues

To acquire a basic knowledge about the possible function of Crocus genes, we studied their expression profile in different tissues based on transcriptome data. The results revealed that out of 91 PHD genes, 13 demonstrated significantly higher expression level in the stigma (Fig. 4). Since stigma is the site where apocarotenoids are synthesized, out of these 13 genes, we selected 5 PHD-genes (CstPHD55, CstPHD6, CstPHD27, CstPHD29 and CstPHD35) which showed highest expression in stigma. We investigated their expression at three different stigma developmental stages i.e., yellow, orange and scarlet red using qPCR.

Fig. 4.

Fig. 4

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Heat map showing expression profile of 91 Crocus PHD finger genes in different Crocus tissues by in silico analysis

We observed that CstPHD27 and CstPHD29 expression increased from yellow through orange stages and had maximum expression at scarlet stage (Fig. 5a). We also quantified major apocarotenoid, crocin, at the above-mentioned stages and observed maximum amount at scarlet stage (Fig. 5b) (Supplementary Fig. S2). Therefore, the expression profile of CstPHD27 and CstPHD29 corroborated with apocarotenoid accumulation. Out of these two, CstPHD27 showed higher expression and hence we carried forward our further experimentation with CstPHD27.

Fig. 5.

Fig. 5

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a Quantitative RT-PCR assessment of relative transcript level of five PHD genes from Crocus at different stigma developmental stages (yellow, orange, and scarlet red). Tubulin was used to normalise the transcript levels. The error bars represent the standard deviation of three replicates b Crocin levels at the stages mentioned above quantified using HPLC (Color figure online)

Cloning and sequence analysis of Cst PHD27 gene

CstPHD27 is 2.5 kbp long and codes for 831 amino acids. Sequence homology of CstPHD27 gene revealed that it is related to the HOX gene sequences from other plants. All such sequences consist of a PHD finger domain and an additional domain named as homeodomain [HOX] (Fig. 6). The genes containing these domains are involved in various developmental processes and stress responses (Bhatacharjee et al. 2017). CstPHD27 also has nuclear localization signal indicating that it might be localized in nucleus.

Fig. 6.

Fig. 6

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Sequence alignment of CstPHD27 with representative gene sequences from other plants. PHD Finger domain and Homeodomain have been highlighted by different colours (Color figure online)

CstPHD27 localizes to the nucleus and is transcriptionally active

In order to examine the subcellular localization of the CstPHD27 protein, its full-length ORF was fused in-frame to the N-terminus of the Green Fluorescent Protein coding sequence of pCAMBIA1302 vector. CstPHD27-GFP fusion construct driven by CaMV35S promoter was transiently expressed in the epidermal cells of onion and observed under flourescence microscope. While control vector was present in whole cell, CstPHD27-GFP protein detection was exclusive to the nucleus (Fig. 7a), verifying its localization to the nucleus.

Fig. 7.

Fig. 7

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Sub-cellular localization and transactivation assay of CstPHD27. a The top pictures show GFP build up all over the cell while the bottom images show that GFP-CstPHD27 localizes only to the nucleus. b Transactivation assay showing yeast cells possessing positive control and pGBKT7-CstPHD27-BD construct develops bluish colour due to β-galactosidase reporter gene activation unlike the negative control. (c) Transactivation quantification by β-galactosidase assay

To investigate the transcriptional activity, CstPHD27 was fused to the GAL4 DNA binding domain pGBKT (BD) vector and transformed into Yeast strain Y187. Only the yeast strain harbouring pGBKT7-CstPHD27 and the strain harbouring positive control (pGBKT7-53 and pGADT7-T vectors co-transformed) developed blue colour that is attributed to β-galactosidase reporter gene activation in the host yeast strain. The cells containing only the empty pGBKT7 vector (negative control) could not grow (Fig. 7b). Further, the transactivation was also confirmed by the β-galactosidase assay using ONPG. The Y187 cells with the BD-CstPHD27 construct measured significantly higher β-galactosidase units compared to the negative control (Fig. 7c). The transactivation assay along with the β-galactosidase assay suggest that the CstPHD27 gene is transcriptionally active.

Over-expression of CstPHD27 enhances apocarotenoid content

To confirm role of CstPHD27 in regulating apocarotenoid biosynthesis, it was overexpressed in Crocus stigmas using floral dip method developed in our laboratory (Bhat et al. 2021).The transformed stigmas were used for estimating crocin, one of the major apocarotenoids in Crocus. There was a significant increase in crocin content in 35 S::CstPHD27 plants as compared to plants expressing vector alone (Fig. 8a). To understand the mechanism by which CstPHD27 regulates crocin biosynthesis, we quantified expression of apocarotenoid pathway genes in 35 S::CstPHD27 stigmas and the stigmas which express vector alone. We observed significant upregulation of key pathway genes like lycopene beta cyclase (LBC), beta carotene hydroxylase (BCH) and carotenoid cleavage dioxygenase (CCD2) in 35 S::CstPHD27 stigmas (Fig. 8b). These genes catalyse important steps in carotenoid/apocarotenoid biosynthetic pathway. For example, LBC converts lycopene into beta carotene which is further acted upon by BCH to form zeaxanthin which is the actual precursor for Crocus apocarotenoids. This zeaxanthin is again acted upon by CCD2 which cleaves it into crocin and picrocrocin. Thus enhanced expression of these pathway genes results in enhanced apcoarotenoid content. This confirmed that CstPHD27 regulates apocarotenoid biosynthesis by modulating pathway gene expression.

Fig. 8.

Fig. 8

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Effect of over-expression of CstPHD27 on a crocin content b apocarotenoid pathway gene expression. Crocin was quantified by HPLC of CstPHD27 over-expressing and vector control expressing stigmas. The expression levels of pathway genes were quantified by qPCR. All the values represent means of three independent biological replicates ± S.D. 18-S gene was used as endogenous control

Discussion

Towards understanding regulatory mechanism of carotenoid/apocarotenoid turnover in C. sativus, many efforts were put to identify roles of different transcription factor families (Bhat et al. 2021; Malik and Ashraf 2017; Hussain et al. 2022). To further compliment these efforts, we worked on PHD finger genes and their probable role in Crocus. From the Crocus transcriptome databases (Baba et al. 2015; Jain et al. 2016), 91 non-redundant PHD gene sequences were identified. From Arabidopsis 70; from Oryza sativa 59 and from Solanum tuberosum, 71 PHD genes are known (Fan et al. 2009; Sun et al. 2017; Qin et al. 2019). Further, itaK analysis classified these gene sequences into 4 groups based on the types of domains present (Supplementary Table S1). The largest group comprised of PHD transcriptional regulators. Genes belonging to this group are structurally closest to the RING Domain family which is involved mostly in chromatin remodelling (Bienz 2006). The genes of this group play a key role in regulating developmental processes like patterning, regional specification, and differentiation (Duverger et al., 2008).This implies that chromatin remodelling may be important in the regulation of many biological processes in Crocus. The second largest group comprised of 26 gene sequences and was classified as Alfin like Transcription Factors. This subfamily is similar in structure to the RING-variant domain and is believed to regulate diverse physiological functions such as nucleic acid binding, control of embryonic development as well as cell differentiation (Laity et al. 2001). This protein group was also shown to aid in stress tolerance (Bastola et al. 1998). Therefore, in Crocus also, they might be involved in various stress signaling pathways.

The phylogenetic analysis which was carried out to decipher the evolutionary trends amongst the identified PHD genes, grouped the PHD gene sequences from Crocus into 14 clusters (Fig. 2). Since PHD proteins often bear additional domains (domains other than PHD domain), we also performed domain analysis using NCBI’s Conserved Domain Analysis to identify the domains associated with each cluster (Fig. 2). It was observed that in most cases, genes belonging to a similar cluster, possessed similar domains suggesting that the domains are highly conserved throughout the PHD gene family and the genes within same cluster may have similar functions. To further analyze the probable functions of Crocus PHD genes, we also performed phylogenetic analysis of Crocus PHD genes with PHD gene family from Arabidopsis thaliana and Oryza sativa (Fig. 3). PHD gene family is not yet known from any of the close relatives of Crocus. Therefore, we used Arabidopsis and Oryza sequences for phylogeny. Arabidopsis was taken as representative of dicots and Oryza for that of monocots. Moreover, many PHD genes from Arabidopsis are characterized (Lai et al. 2020; Franco-Echevarría et al. 2022; Diego-Martin et al. 2022) which could help in assigning probable function to Crocus PHD genes. The sequences were grouped into 10 clusters. Most of the clusters consisted of members from all the three plant species suggesting that these genes might have similar roles in all of them. However, in few cases, like in cluster 6 most of the sequences were from Crocus. Such genes might have Crocus specific roles.

To learn more about the possible function of PHD genes in C. sativus, we explored the expression of these genes in silico. Based on the differential expression profile obtained from our transcriptome database, 13 PHD genes showed upregulation in stigma suggesting their possible role in apocarotenoid biosynthesis as stigma is the main site for apocarotenoid biosynthesis (Fig. 4). Out of the 13 genes, 5 genes which showed the highest expression in stigma were selected for developmental stage specific expression using qPCR (Fig. 5a). We observed that CstPHD27 and CstPHD29 showed maximum expression at scarlet stage. This expression pattern was similar to that of accumulation pattern of apocarotenoids. There are many reports which demonstrate that apocarotenoid accumulation in Crocus stigmas increases from yellow through orange stages and reach maximum at scarlet red stage upto anthesis and post anthesis, their levels start to decline (Moraga et al. 2009). In this study also, we quantified crocin levels which is a major apocarotenoid at yellow, orange and red stages. We observed that crocin showed increasing trend from yellow to orange to red stages (Fig. 5B). This accumulation profile matched with expression profile of CstPHD27 and CstPHD29. This suggests that these genes are probably engaged in regulating apocarotenoid biosynthesis. Since, CstPHD27 showed higher expression, we took this gene for validating its function. Most of the PHD finger proteins are transcriptionally active and localized in nucleus (Bienz 2006). CstPHD27 was also found to be located in the nucleus and transcriptionally active (Fig. 7). Further, over-expression of CstPHD27 in Crocus stigmas was shown to enhance crocin content (Fig. 8a). This confirmed that CstPHD27 acts as a positive regulator of apocarotenoid biosynthesis. PHD finger proteins are known to play important roles in chromatin remodeling and transcriptional regulation. They specifically bind histone modifications as an “epigenome reader”, and in turn mediate expression of underlying genes (Alam et al. 2019). To understand the mechanism of CstPHD27, we measured expression of important apocarotenoid pathway genes and observed their upregulation (Fig. 8b). Thus CstPHD27 enhances apocarotenoids by regulating expression of pathway genes. This knowledge can be used for biotechnological intervention in saffron for enhancing its quality in terms of apocarotrenoid content.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 1290 KB) (1.3MB, pdf)

Author contributions

NA designed the experiments. AHM, NK, NS performed the experiments. NA, AHM, NK, and NS analysed the data and wrote the paper. NA provided the chemicals and other lab facilities.

Funding

We acknowledge the grant from the Council of Scientific & Industrial Research (CSIR), Government of India. AHM acknowledges the fellowship from CSIR-India.

Declarations

Conflict of interest

The authors declare that they do not have any conflict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Nargis Khurshaid and Najwa Shabir have contributed equally.

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