Breaking boundaries: a novel role for CUC genes in sex determination in cucurbits

Abstract

This article comments on:

Segura M, García A, Gamarra G, Benítez A, Iglesias-Moya J, Martínez C, Jamilena M. 2024. An miR164-resistant mutation in the transcription factor gene CpCUC2B enhances carpel arrest and ectopic boundary specification in Cucurbita pepo flower development. Journal of Experimental Botany 75, 1948–1966.

Elucidating the mechanisms underlying sex determination in flowers is not only essential for a better understanding of plant reproductive biology but it also has important agronomic implications as it underpins crop breeding efforts. In this issue, Segura et al. (2024) show that in Cucurbita pepo, the NAC-family transcription factor-coding gene CUP-SHAPED COTYLEDON 2B (CpCUC2B), which is post-transcriptionally regulated via the conserved miR164 miRNA pathway, is a critical determinant for sex.

Plant sexual systems are highly diverse and have long been a fascinating subject of study among biologists (Harkess and Leebens-Mack, 2017). Most angiosperms, or flowering plants, are hermaphrodites wherein both male and female organs are present in a single individual (Bachtrog et al., 2014). In this case, hermaphroditism can take a broad range of forms, from the classic ‘perfect’ flower with functional male and female organs found within single flowers of an individual (bisexual flower), to monoecy with separate male and female flowers occurring on an individual plant (unisexual flowers) (Dellaporta and Calderon-Urrea, 1993; Pannell, 2017). Gynodioecy and androdioecy are also forms of plant hermaphroditism where individuals produce hermaphroditic and/or only female or only male flowers, respectively (Masuda and Akagi, 2023). Dioecy, or the development of individual male and female plants, is considered to have evolved from hermaphroditism. In fact, it is generally considered that all current forms of sexual expression in flowering plants evolved from an ancestral hermaphroditic progenitor and that distinct sexes evolved repeatedly and only relatively recently (Dellaporta and Calderon-Urrea, 1993; Charlesworth, 2002; Bachtrog et al., 2014; Masuda and Akagi, 2023). This could be seen as in direct contrast to well-established sex determination mechanisms among animals, particularly in mammals, wherein sexuality can be attributed solely to X and Y sex chromosomes (XX for females and XY for males) derived from a master-switch gene (Sry) in ancestral autosomes (Foster and Graves, 1994; Bachtrog et al., 2014). Thus, sexual determinacy is more variable and fluid for plants when compared with other organisms (Tanurdzic and Banks, 2004; Diggle et al., 2011; Bachtrog et al., 2014).

Arrested development: monoecious sex determination in C. pepo

Various species have emerged as models for elucidating the control of sex determination in plants, such as white campion (Silene latifolia) and sorrel (Rumex) where plant sex chromosomes were initially discovered from as early as 1923 (Westergaard, 1958; Harkess and Leebens-Mack, 2017; Masuda and Akagi, 2023). More recently, various agronomically valuable species have also made this list, including melon (Cucumis melo), papaya (Carica papaya), strawberry (Fragaria spp.), persimmon (Diospyros lotus), and garden asparagus (Asparagus officinalis), to name a few (Byers et al., 1972; Boualem et al., 2008; Spigler et al., 2008; Yu et al., 2008; Akagi et al., 2014; Harkess et al., 2015; Harkess and Leebens-Mack, 2017; Zhang et al., 2022). Apart from melon, other members of the Cucurbitaceae family are also widely used in sex determination studies, including C. pepo which encompasses the agriculturally important squash, pumpkin, and zucchini morphotypes (Martínez and Jamilena, 2021). Cucurbita pepo is a monoecious member of the Cucurbitaceae that exhibits three distinct stages of sexual development (Fig. 1C). Upon reaching reproductive maturity, it goes through an initial male phase, producing only unisexual male flowers. This is followed by a hermaphroditic mixed phase with male and female flowers alternately produced. The final stage is a female phase comprising a majority of, or exclusively, unisexual female flowers (Schilling et al., 2020; Martínez and Jamilena, 2021; Segura et al., 2024). For monoecious plants such as C. pepo, this type of sex determination mechanism can be considered as ‘developmental’ wherein sexuality is not specifically controlled by sex chromosomes but rather through the activity of various regulatory factors leading to the arrest of either carpel development to promote the male phase, or stamen development to promote the female phase (Pannell, 2017; Martínez and Jamilena, 2021). Among those regulatory factors, the phytohormone ethylene plays a central role as it has been demonstrated that ethylene biosynthesis and signalling determine female development across cucurbits (Pannell, 2017; Schilling et al., 2020; Martínez and Jamilena, 2021).

Fig. 1.

Sexual development in wild-type (WT) Cucurbita pepo. (A) Graphical representation of the unisexual flowers of C. pepo. The calyx is represented by the inverted yellow triangle. Female and male flowers are distinguished by the shape of their peduncles (green). Female flowers have an elongated oval peduncle while male flowers have a straight and slender peduncle. (B) CpCUC2B is upstream of the late ethylene biosynthesis and response pathway. CpCUC2B expression is post-transcriptionally regulated by miR164 and suppresses female flower development. CpCUC2B also probably acts synergistically with the ethylene perception and response genes CpETR1/2 for overall sex determination. (C) Graphical representation of C. pepo sexual developmental phases (read from bottom to top). Sexual development is preceded by an initial male phase (blue bracket and text) where only male flowers are formed. The phase when female flowers first form is considered as the moment when the promotion of female flowering commences (boxed text in pink). The developmental events initiated by the regulatory pathway in (B) probably occur during this phase. From this point, a mixed male and female phase (purple bracket and text) proceeds until the final phase featuring mostly, or exclusively, female flowers, considered as the female phase (pink bracket and text). Figure generated using BioRender.

Masters of sex: transcription factors, miRNA, and hormones as regulators of sex determination

In this issue, the study by Segura et al. (2024) introduces the C. pepo NAC-family transcription factor-coding gene CUP-SHAPED COTYLEDON 2B (CpCUC2B) as a new transcriptional regulator involved in the sex determination pathway in zucchini. CpCUC2B is a homologue of the Arabidopsis thaliana CUC2 gene that is well known for specifying and defining organ boundaries along with CUC1 and CUC3 during embryogenesis, shoot apical meristem formation, flower development, and leaf development (Mallory et al., 2004; Maugarny et al., 2016). The current study by Segura and colleagues is the first description of CUC genes playing a role in sex determination, indicating that CUC gene functions in other plant families can expand and diverge to include control of reproductive development. Using a forward genetics approach, the group initially identified the semi-dominant cuc2b mutant (Fig. 2C). cuc2b plants display mostly male flowers as a result of a prolonged male phase and delayed female flowering. Flower development was also altered in cuc2b mutants, namely during floral meristem specification resulting in increased floral organ numbers and fused floral organ phenotypes (Figs 1A, 2A). The authors showed that the causative mutation in cuc2b is located in exon 3 of CpCUC2B. Notably, they found that this mutation is in the binding site of members of the miR164 family of miRNAs, which are known to post-transcriptionally regulate NAC-domain genes, including Arabidopsis CUC2 (Mallory et al., 2004; Sieber et al., 2007). In Arabidopsis, it has been shown that the miR164-dependent regulation fine-tunes CUC gene expression and is essential for proper organ boundary establishment and formation (Laufs et al., 2004; Nikovics et al., 2006; Adam et al., 2011). In C. pepo, the mutation in the miR164-binding site renders CpCUC2B resistant to miR164 regulation, leading to a gain of CpCUC2B function. The observed nearly androecious C. pepo phenotype in cuc2b therefore suggests that CpCUC2B promotes male flowering by arresting carpel development (Figs 1B, 2B). The results of additional experiments, in which double mutants between cuc2b and ethylene biosynthesis or signalling mutants were analysed, further imply that CpCUC2B is also involved in suppressing the arrest of stamen development and that the interaction of CUC2B with the ethylene response pathway is complex and probably differs in male and female reproductive organs. Overall, the results suggest that the interplay between ethylene, transcription factors, and miRNA-mediated post-transcriptional pathways is a key determinant of monoecious sex determination.

Fig. 2.

Sexual development in the C. pepo cuc2b mutant. (A) Graphical representation of the unisexual flowers in cuc2b. Wild-type flowers develop along with double flowers exhibiting a fused calyx (inverted yellow triangles) and fused peduncles (green oval or elongated bar structure) phenotypes for both the female and male flowers. (B) cuc2b is a gain-of-function mutation that suppresses the late ethylene pathway produced by CpACS27 and CpACO1A, so promoting the development of stamens which are inactive as miR164 is unable to post-transcriptionally repress CpCUC2B. (C) Graphical representation of cuc2b sexual developmental phases (read from bottom to top). In cuc2b, the male phase is extended as a result of a delayed female flowering transition. The mixed female phase is also delayed, and the overall result is a nearly androecious phenotype with a reduced number of female flowers. Both the wild-type and fused flowers are depicted here. Figure generated using BioRender.

Moving beyond limits: understanding plant sex determination for better plant productivity and resilience

Uncovering the genetic and molecular mechanisms underlying sex determination in flowering plants is essential for a comprehensive understanding of how sex evolved throughout living systems. No less important and profound is this knowledge for how we can better adapt plants, especially those that we rely on for sustenance and livelihood (such as many C. pepo morphotypes), in a world with increasingly adverse climatic conditions and food insecurity. The work by Segura and colleagues adds a novel factor in the current sex determination framework for cucurbits that will form the basis for future activities aimed at improving plant productivity and resilience. Strategies in improving crop pollination success, yield, and fruit quality, and implementing cropping and breeding approaches are just some examples in our agricultural systems that would stand to benefit from similar advances in plant sex determination studies (Masuda and Akagi, 2023). To understand how sex determination and overall reproductive success are affected by abiotic and biotic stresses will also be of significant interest in the face of erratic weather events brought on by climate change and in improving crop productivity to keep pace with unprecedented global population growth.

Conflict of interest

The authors declare no conflict of interest regarding this publication.

Funding

Work in our laboratory is supported by a grant (21/FFP-P/10205) from Science Foundation Ireland.