1. Introduction

Homeobox transcription factors (TFs) containing homeodomain proteins are separated into 14 families, including the WUSCHEL -related homeobox (WOX) TF family [1,2,3]. The domain features a helix–loop–helix–turn–helix (HTH) structure that contains 60–66 amino acids crucial for specific functions in plants [4,5,6]. Two alpha-helices are intricately associated with DNA by a short turn, defined as the HTH motif [7]. WOX proteins usually contain a strongly conserved homeodomain that is highly specific to DNA binding proteins and may act as repressors or activators [3,8]. Homeodomain proteins are widely identified in monocot and dicot plants [9]. The model plant Arabidopsis contains 15 WOX members, which are divided into three clades based on their evolutionary relationship: modern/WUS, intermediate, and ancient clades [9]. Arabidopsis WOX members have been extensively studied, and their orthologs have exhibited diverse functions in numerous development processes [10,11,12,13], along with rice and maize [8,12]. Besides Arabidopsis, genome-wide studies have also been conducted on the WOX gene families of woody plants such as walnut [10], physic nut [14], grapes [15,16], peach, pear, apricot [17], coffee [18], and poplar [19]. In these plants, WOX genes are involved in vital regulatory networks that link the developmental mechanisms in plants, including shoot apical meristem, lateral organ development, plant stem cell maintenance, and floral determinacy [5,20,21,22].

In Arabidopsis, AtWUS and AtWOX1 determine floral meristem identity and maintenance [23,24,25]. AtWOX2, AtWOX6, AtWOX8, and AtWOX9 regulate ovule development and enable the development of cotyledon boundaries, eggs, and zygotes [26]. AtWOX3 targets pathways that promote flower organ primordia and leaf margin development [27]. AtWOX7 plays an important role in lateral root development and sugar (sucrose and glucose) status in Arabidopsis [28]. AtWOX11/AtWOX12 is involved in cell fate transition and root organogenesis [29]. Some WOX genes in Arabidopsis are involved in hormone signaling transduction pathways. For example, AtWOX4, AtWOX5, and AtWOX11 regulate auxin signaling that determines lateral organ and apical root growth [19,20,30]. WOX1 homologs have been demonstrated to control leaf blade outgrowth in Zea mays, Petunia hybrida, Medicago truncatula, and Nicotiana tabacum [5,31,32]. Leaf blade outgrowth is controlled by the WOX domain [33]. Recently, 12 WOX proteins were identified in walnut; JrWOX3a and JrWOX3b enable leaf development. PpWUS and PpWOX5 regulate embryo development in Pinus pinaster [3,10]. In Norway spruce, PaWOX3 promotes lateral organ outgrowth in conifers [34]. In recent years, several previous studies reported that WOX genes respond to abiotic stresses and hormone treatment, including Oraza sativa, Gossypium, and Brassica napus [8,23,35,36,37]. However, the response of WOX genes during abiotic stress has not been studied adequately in citrus.

Several previous studies indicated that the WUS gene is one of the most important genes in the WOX gene family and involved in numerous important developmental processes including size of shoot meristem, somatic embryo, as well as adventitious shoot and lateral leaf formation [5,38,39]. For example, AtWUS is crucial for shoot apical meristem maintenance to replace the function of WOX1 and PRESSED FLOWER (PRS) [5]. The ectopic overexpression of AtWUS in tobacco is involved in stem cell fate and lateral leaf formation [40]. In Medicago truncatula, the AtWUS homolog HEADLESS (HDL) is also involved in leaf development [31]. WUS gene functions downstream of the CLAVATA3 (CLV3) signaling pathway [41]. The WUS gene is expressed in the organizing center and enhances CLV3 expression in stem cells. Likewise, CLV3 negatively regulates meristem size by suppressing WUS expression [42,43]. WUS interacts with CYCLOIDEA 2 (CYC2) and regulates reproductive organ development (ovary, stigma, and style) in Chrysanthemum morifolium [44]. In the embryonic columella, WOX5 and CYCD3;3/CYCD1;1 facilitate cell proliferation, and CYCD3 plays a major role in normal cell division [45]. The WUS orthologs STERILE AND REDUCE TILLERING (MOC3/SRT1) and TILLERS ABSENT1/MONOCULM 3 are involved in bud formation and female fertility of rice [46]. The WUS gene regulates histone acetylation and interferes with HISTONE DEACETYLASE (HDAC) activity, which stimulates the auxin signaling pathway in stem cells [38]. In addition, WUS expression is indirectly repressed by AGMOUS (AG) and stimulates expression of the zinc finger TF C2H2 type (KNUCKLES), which in turn suppresses WUS expression directly or indirectly involved in the maintenance of floral meristem cells [5]. Thus far, functional characterization of WUS genes has been studied in other plants but is rarely studied in citrus.

The above-mentioned studies report that WOX TFs primarily affect plant development by regulating the expression of downstream genes. Notably, WOX protein cloning and functional analyses were predominantly focused on model plants such as Arabidopsis. However, there have been relatively few studies on the regulatory and genetic development role of the WOX family in citrus. The availability of the citrus genome database gives us a valuable genetic resource to study specific sweet orange genes [47,48]. A thorough screening of the citrus database allowed us to find the evolutionary, regulatory, and developmental role of WOX orthologs in sweet orange. In the current study, we identified 11 putative WOX members in Citrus sinensis. Tissue-specific expression patterns and co-expression profiles of CsWOXs under water deficit and floral inductive conditions were comprehensively studied. The subcellular localization, transactivation activity, and DNA-binding ability confirmed that CsWUS may be a TF. Moreover, CsWUS overexpression and the virus-induced gene silencing (VIGS) assay revealed new insights into floral organ development, stem cell activity, and leaf development in citrus. In addition, yeast two-hybrid assays and DNA-protein interactions confirmed the complex involvement of CsWUS in developmental regulatory networks.

This entry is adapted from the peer-reviewed paper 10.3390/ijms22094919