# Genome-wide identification and characterization of WOX gene family in saffron (Crocus sativus L.) and their roles in stress response, development and callus formation

## TL;DR

This study identifies and characterizes WOX genes in saffron, revealing their roles in stress response, development, and callus formation.

## Contribution

The study provides the first genome-wide analysis of WOX genes in saffron and their functional roles in stress and development.

## Key findings

- Twenty CsWOX genes were identified in saffron, with collinear and syntenic relationships revealed.
- CsWUS and CsWOX13B expression decreased under cold stress but showed recovery trends.
- CsWOX11 and CsWUS were upregulated in callus formation, with distinct expression patterns observed.

## Abstract

The shoot apical meristem (SAM) of saffron (Crocus sativus L.) plays a critical role in floral transition and development. WUSCHEL-related homeobox (WOX) genes are key regulators of stem cell activity and SAM size across diverse species, including Arabidopsis thaliana, Oryza sativa Japonica, Gossypium hirsutum L. and the hybrid poplar clone 84K (Populus alba × P. glandulosa).

WOX genes were identified and analyzed for phylogeny, gene structure, conserved motifs, cis-regulatory elements, chromosomal location, collinearity and duplication. Expression patterns were examined in different tissues, cold stresses, hormonal stress and callus formation, with key genes validated by real-time quantitative PCR.

In this study, we identified 20 CsWOX genes based on the triploid genome of saffron. Intraspecific collinearity analysis revealed two collinear pairs (CsWOX7/CsWOX5 and CsWOX13A/CsWOX13B) in saffron. Furthermore, interspecific synteny was established between AtWUS in A. thaliana and CsWUS in saffron, as well as between saffron and rice (OsWOX9/CsWOX5, OsWOX9/CsWOX7, and OsWOX2/CsWOX3). Transcriptome analysis was conducted to investigate the expression patterns of CsWOX genes in the SAM under temperature and hormonal stresses. Cold treatment (9°C) significantly downregulated CsWUS expression during stages S1–S6, while the expression of CsWOX13B and CsWOX4 exhibited a recovery trend during stages S4–S6. Although Gibberellic Acid (GA) and Abscisic Acid (ABA) promoted the differentiation of apical meristems into floral meristems, most CsWOX genes remained largely unresponsive or showed only transient responses to these hormones. Additionally, CsWOX13A/B expression increased progressively throughout the four stages of stigma development, peaking at stage 4. In corm-derived callus, CsWUS and CsWOX11 were significantly upregulated at the non-shoot-forming (NSF) and shoot-forming (SF) callus stages. Notably, while CsWOX11 exhibited a similar expression pattern across both corm and stigma sources, CsWUS showed a distinct, progressive increase in expression during the NSF and SF stages of stigma-derived callus.

This study analyzed the potential functions of different WOX genes from multiple perspectives, including temperature stress, hormonal regulation, growth and development, and callus tissue culture. These findings provided a foundation for further investigation into the mechanisms by which candidate WOX genes regulate the saffron SAM.