Constitutive Overexpression of a Conifer WOX2 Homolog Affects Somatic Embryo Development in Pinus pinaster and Promotes Somatic Embryogenesis and Organogenesis in Arabidopsis Seedlings.

Seyedeh Batool Hassani,Andrea Rupps,Jean-François Trontin,Kurt Zoglauer,Juliane Raschke

doi:10.3389/fpls.2022.838421

Abstract

Although full sequence data of several embryogenesis-related genes are available in conifers, their functions are still poorly understood. In this study, we focused on the transcription factor WUSCHEL-related HOMEOBOX 2 (WOX2), which is involved in determination of the apical domain during early embryogenesis, and is required for initiation of the stem cell program in the embryogenic shoot meristem of Arabidopsis. We studied the effects of constitutive overexpression of Pinus pinaster WOX2 (PpWOX2) by Agrobacterium-mediated transformation of P. pinaster somatic embryos and Arabidopsis seedlings. Overexpression of PpWOX2 during proliferation and maturation of somatic embryos of P. pinaster led to alterations in the quantity and quality of cotyledonary embryos. In addition, transgenic somatic seedlings of P. pinaster showed non-embryogenic callus formation in the region of roots and subsequently inhibited root growth. Overexpression of PpWOX2 in Arabidopsis promoted somatic embryogenesis and organogenesis in a part of the transgenic seedlings of the first and second generations. A concomitant increased expression of endogenous embryogenesis-related genes such as AtLEC1 was detected in transgenic plants of the first generation. Various plant phenotypes observed from single overexpressing transgenic lines of the second generation suggest some significant interactions between PpWOX2 and AtWOX2. As an explanation, functional redundancy in the WOX family is suggested for seed plants. Our results demonstrate that the constitutive high expression of PpWOX2 in Arabidopsis and P. pinaster affected embryogenesis-related traits. These findings further support some evolutionary conserved roles of this gene in embryo development of seed plants and have practical implications toward somatic embryogenesis induction in conifers.

Highlights

With respect to the divergence of angiosperms and gymnosperms, many morphological differences of embryogenesis and embryo development have to be considered (Bowe and Coat, 2000; Cairney et al, 2006; Cairney and Pullman, 2007)
Our results showed that the endogenous expression of P. pinaster homolog to WOX2 (PpWOX2) is enhanced in early proliferating embryos and significantly decreased during somatic embryo (SE) development in P. pinaster
In addition to the conserved homeodomain, many plant WUS/WOX proteins contain a conserved WUS-box (TLPLFP) located downstream of the homeodomain (Figure 1, Haecker et al, 2004; Nardmann and Werr, 2006), including in Pinus pinaster (Alvarez et al, 2018). Such a putative WUS-box was identified in the PpWOX2 protein sequence and shows the same differences in amino acid sequence compared to Arabidopsis (TLPLFPLQP) as other conifers from genera Pinus, Picea and Larix (TLELFPLHP)

Summary

Introduction

With respect to the divergence of angiosperms and gymnosperms, many morphological differences of embryogenesis and embryo development have to be considered (Bowe and Coat, 2000; Cairney et al, 2006; Cairney and Pullman, 2007). Little is known about the function of genes that regulate embryogenesis in conifers (Cairney and Pullman, 2007; De Silva et al, 2008; Trontin et al, 2016a). This is because of particular challenges of conifers. Selected genotypes after field evaluation of somatic seedlings in clonal tests can be propagated from a cryopreserved juvenile stock This is currently the preferred and powerful strategy in conifers to overcome recalcitrance of mature trees (Klimaszewska and Cyr, 2002; Bonga et al, 2010) to achieve clonal propagation of superior genotypes and for gene functional studies by genetic transformation of embryogenic cultures (Klimaszewska et al, 2007, 2009, 2016)

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Discussion

Conclusion