Plant growth and development are remarkably plastic, which helps them overcome the constraints posed by their sessile existence and enables them to thrive in diverse environmental conditions. Developmental plasticity is achieved partly by preserving the self-renewing stem cells within the meristem niches and by post-embryonic initiation of lateral organs from the flanks of the meristem (Powell and Lenhard, 2012). Organ growth is a resultant outcome of two key cellular processes, proliferation, and post-mitotic expansion, the former preceding the latter. Proliferation produces an adequate cell reserve at an early stage of organ growth, and expansion increases cell volume under the force of turgor pressure (Donnelly et al., 1999; Cosgrove, 2005; Gonzalez et al., 2012). For example, mitotic cycle in Arabidopsis and tobacco leaves ceases when their blades reach ∼10% of their final area, triggering endoreduplication-mediated cell expansion that drives the remainder of the blade growth (Poethig and Sussex, 1985; Donnelly et al., 1999).

Though the physical basis of cell expansion through turgor pressure and cell-wall extensibility has been studied in detail (Ray et al., 1972; Braidwood et al., 2014), its genetic regulation remains less clear. This is primarily because of an overlapping occurrence of cell division and expansion in the lateral organs of post-embryonic origin. For example, within the growing primordium of most angiosperm leaves, cell division and expansion coexist in the proximal and distal domains, respectively (Nath et al., 2003; Das Gupta and Nath, 2015). Cells transiting from an actively dividing state to differentiation characterize a dynamic transition zone, flanked by mitotic cells to its proximal end and differentiated cells to its distal end. Perturbations in rate, duration, or distribution of cell proliferation and expansion often affect the final size and shape of organs (Donnelly et al., 1999; Efroni et al., 2008; Andriankaja et al., 2012). Because proliferation and differentiation are linked by a compensatory mechanism (Gonzalez et al., 2012) in post-embryonic organs, it is difficult to uncouple the contribution of cell growth from that of cell division to the final organ morphology. However, the organs of embryonic origin such as hypocotyl and cotyledon serve as an ideal model for cell growth studies since cell division is absent or insignificant during their growth after germination (Tsukaya et al., 1994; Stoynova-Bakalova et al., 2004; Boron and Vissenberg, 2014). Besides, these embryonic organs possess simpler and uniform epidermal cell morphology, making their analysis more tractable. Utilization of these simplifying advantages of hypocotyl and cotyledon has shed light on the molecular players involved in unidirectional and planar cell expansion, respectively. In this review, we highlight recent advances in our understanding of the genetic regulation of post-mitotic cell growth, with an emphasis on CIN-TCP transcription factors and their new-found role in cell expansion.

Because the rigid wall precludes migration of plant cells, organ morphology is greatly influenced by the direction and duration of cell enlargement (Braidwood et al., 2014). Plant cells predominantly show anisotropic expansion, where the rate and direction of growth vary across the cell surface, allowing cells to acquire a variety of forms to limit the magnitude of mechanical stress and simultaneously enhance tissue strength (Johnson and Lenhard, 2011; Braidwood et al., 2014; Sapala et al., 2018). Anisotropic cell growth can be either diffused type where expansion is dispersed over the cell surface, or tip-growth type where expansion occurs only at localized tips (Martin et al., 2001). For example, cells in hypocotyl, stem, and root show growth along the longitudinal direction at their side walls over the entire surface. By contrast, root hairs and pollen tubes exhibit restricted growth only at a single site at the cell tip (Braidwood et al., 2014). In yet another growth pattern, the pavement cells in leaves and cotyledons show multiple growth polarities on segments of their surface, ultimately producing puzzle piece-shaped cells with lobes and indentations. We refer to this type of cell growth as planar growth since the expansion is primarily in the X–Y plane, with little expansion in the orthogonal direction. Distinct cytological properties such as differential deposition of cell-wall material, orientation of cellulose microfibrils, and polarized accumulation of cytoplasmic content determine the mode of cell growth and organ morphology (Braidwood et al., 2014). However, certain cells such as parenchyma of potato tuber or apple fruit display a diffused isotropic mode of growth, forming isodiametric cells compacted together where the cellulose microfilaments are randomly oriented (Cosgrove, 2014).

In most tissue types, rapid increase in cell volume is directly associated with endoreplication cycle where DNA is replicated without cytokinesis or chromosome segregation, leading to enhanced nuclear ploidy level (Melaragno et al., 1993; Edgar and Orr-Weaver, 2001; Breuer et al., 2010). Polyploidization is known to promote ribosome biogenesis, resulting in an increase in protein synthesis and in total cytoplasmic content, which ultimately leads to cell growth (Larkins et al., 2001; Sugimoto-Shirasu and Roberts, 2003). Thus, modulating the onset of endocycle and the duration of its progression substantially influence organ size with a strong correlation with cellular ploidy level. FASCIATA1 (FAS1), a chromatin assembly subunit protein, and E2F TARGET GENE 1 (ETG1), a replisome factor, interfere in the chromatin assembly that triggers endoreplication in plants. Consequently, fas1 and etg1 mutants perform additional rounds of endocycle, leading to larger cells and organs (Ramirez-Parra and Gutierrez, 2007; Takahashi et al., 2010). Similarly, cell cycle inhibitory KIP-RELATED PROTEIN (KRP) genes controlling endoreplication and chromatin structure regulate expression of genes required for cell expansion (Jegu et al., 2013). The quintuple krp mutant exhibits leaves with smaller epidermal cells and KRP-overexpression lines form larger cells, suggesting a direct correlation of KRP abundance and endoreplication-mediated cell growth (Verkest et al., 2005; Cheng et al., 2013). Additional rounds of endoreplication in dark-grown hypocotyl cells serve as a major determinant of de-etiolated seedling growth in contrast to light-grown seedlings (Gendreau et al., 1997). In floral organs, suppressing the transition to endoreduplication in the frill1 mutant, which has a reduced sterol methyltransferase activity, results in expanded cells with enlarged nuclei during the late stage of petal development (Hase et al., 2000). It is also suggested that the combination of phytohormones, nutrients, and environmental signals trigger endoreplication and cell growth (Kondorosi et al., 2000; Sugimoto-Shirasu and Roberts, 2003). For instance, gibberellic acid (GA) and ethylene coordinate with DNA synthesis and endoreplication-mediated hypocotyl cell elongation (Gendreau et al., 1999). Many reports in multiple plant species strongly indicate the presence of endocycle-independent mechanism of cell growth affecting organ size (Sugimoto-Shirasu and Roberts, 2003).

The rigid wall of plant cells – comprised of complex meshwork of cellulose, hemicellulose, pectin, and glycoproteins – accommodates two seemingly contradictory biological functions - providing a strong structural support to the cell and an adjustable elasticity necessary for its growth (Bashline et al., 2014). The turgor pressure triggered by osmotic activity leads to cell-wall loosening and produces enlarged cells with balanced water potential without losing their integrity (Cosgrove, 2005; Seifert and Blaukopf, 2010; Bashline et al., 2014). The physical properties of cell-wall are altered to promote expansion by the activity of several cell-wall-modifying enzymes such as glycosyltransferases, methylesterases, hydroxylases, and hydrolases. These enzymes target major components of the cell wall (Braidwood et al., 2014). For example, the EXPANSIN (EXP) family proteins promote cell-wall loosening by breaking the non-covalent bonds between cellulose microfibrils in an acidic environment and correspondingly produce bigger leaves with larger cells when overexpressed (Cho and Cosgrove, 2000; Marowa et al., 2016). Similarly, pectin methyl esterases hydrolyze ester bond and enhance negative electrostatic charge within the cell wall to provide flexibility and mobility to homo-galacturonan in the cell wall. Mutant analysis of multiple cellulose synthase (CESA) genes has demonstrated the redundant function of these cell-wall synthesis promoting genes in cell expansion in multiple organs (Burn et al., 2002).

Phytohormones play a critical role in promoting cell growth in association with either cell-wall-remodeling enzymes or by enhancing orientated deposition of cellular microfibrils (Wolters and Jurgens, 2009). In addition, auxin, brassinosteroid (BR), and GA promote cell-wall biosynthesis and expansion by activating several regulatory transcription factors. Auxin induces rapid cell expansion in stem, hypocotyl, leaf, and coleoptile by activating a proton (H⁺) pump ATPase in the plasma membrane (PM), resulting in extracellular acidification, an environment favoring the cell-wall loosening enzymes (Rayle, 1973; Rayle and Cleland, 1977, 1992; Perrot-Rechenmann, 2010). The early auxin-responsive SMALL AUXIN UPREGULATED RNA (SAUR) gene family members participate in modulating PM H⁺-ATPase activity through direct interaction and inhibition of PP2C-D protein phosphatases (Spartz et al., 2014). Recent reports have shed light on tissue-specific expression pattern of SAUR genes and their involvement in distinct morphological processes under the regulation of developmental and environmental factors (Ren and Gray, 2015; Stortenbeker and Bemer, 2019). BR, GA, jasmonic acid (JA), and abscisic acid (ABA) also regulate SAUR-mediated cell growth (Ren and Gray, 2015). Auxin, BR, and GA integrate environmental signals through the DELLA-ARF6-BZR1-PIF4 complex to form a central regulatory network and maintain cell elongation by regulating an overlapping set of SAUR genes (Oh et al., 2014; Van Mourik et al., 2017). Mutants deficient in AUXIN RESPONSE FACTORS (ARFs) form shorter hypocotyl compared to the wild type, further supporting the role of auxin response in cell expansion (Reed et al., 2018). Another homeodomain transcription factor HAT2 is also known to promote cell elongation in hypocotyl in response to auxin signaling (Sawa et al., 2002). A recent report suggests that increased endogenous auxin, triggered by a low-sugar state in the cotyledons following seed germination, promotes compensation-mediated cell enlargement (Tabeta et al., 2021). Another recent study demonstrates that constitutively activated salicylic acid (SA) signaling suppresses compensation-induced cell expansion in leaves, thus assigning a role for SA in organ size regulation (Fujikura et al., 2020).

The regulators of the cell-wall extensibility factors described above are studied in less detail. The ARGOS-LIKE (ARL) gene is preferentially expressed in cotyledon, mature root, and leaves and promotes organ size as a result of increased cell expansion (Hu et al., 2006). Similarly, overexpression of ZINC-FINGER HOMEODOMAIN 5 (ZHD5) produces bigger leaves with enlarged epidermal cells by acting on yet unidentified target genes required for cell expansion (Hong et al., 2011). Few more examples of cell expansion regulators include TARGET OF RAPAMYCIN (TOR), CELL CYCLE SWITCH PROTEIN 52 (CCS27A), CYTOCHROME P450, FAMILY 78, SUBFAMILY A (CYP78A6), and ERBB-3 BINDING PROTEIN 1 (EBP1), overexpression of which results in larger organs due to increased cell expansion (Gonzalez et al., 2012; Vanhaeren et al., 2015). The list of the transcription factors that directly or indirectly regulate cell-wall expansion is still growing, with the recent addition of the TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTOR (TCP) family proteins.

Teosinte branched1, cycloidea, proliferating cell factor proteins participate in sequence-specific DNA-binding, transcriptional activation, and protein–protein interaction to regulate diverse developmental processes including flower symmetry, leaf and petal morphogenesis, trichome development, axillary branching, and pathogen defense (Martin-Trillo and Cubas, 2010; Sugio et al., 2014; Danisman, 2016; Sarvepalli and Nath, 2018; Challa et al., 2019). They are distinguished by their non-canonical basic helix-loop-helix (bHLH) domain known as the TCP domain and are present from chlorophyte algae to higher land plants (Navaud et al., 2007; Dhaka et al., 2017). Based on sequence diversity in amino acid residues within the TCP-domain, the 24 TCP proteins encoded by the Arabidopsis genome are divided into the class I subfamily with thirteen members and the class II subfamily with eleven members (Kosugi and Ohashi, 2002; Li, 2015). Eight class II TCPs are collectively called CINCINNATA-like TCPs (CIN-TCPs) that form a subclade and are expressed in the transition zone in leaf primordia where they redundantly suppress proliferation and promote differentiation in dividing cells (Nath et al., 2003; Das Gupta et al., 2014; Sarvepalli and Nath, 2018; Challa et al., 2019). Transcripts of five CIN-TCP genes – TCP2, 3, 4, 10, and 24 – are degraded by the microRNA miR319 (Palatnik et al., 2003), adding an additional level of complexity in CIN-TCP-mediated regulation of leaf morphogenesis. In addition to leaf maturation, CIN-TCPs also regulate leaflet initiation, biotic and abiotic stress tolerance, photomorphogenic seedling growth, phytohormone signaling, flowering time control, cell-wall thickening, etc (Koyama et al., 2007, 2010a, 2017; Navaud et al., 2007; Efroni et al., 2008; Schommer et al., 2008; Sarvepalli and Nath, 2011a; Aguilar-Martinez and Sinha, 2013; Das Gupta et al., 2014; Wang et al., 2014, 2015; Challa et al., 2016, 2019; Kubota et al., 2017; Lei et al., 2017; Zhou et al., 2018, 2019; Fan et al., 2020; Fang et al., 2021). Most developmental processes regulated by CIN-TCPs are linked to their involvement in suppressing proliferation and promoting differentiation of cells. Effects of CIN-TCPs on the exit from division cycle within leaf primordia have been reviewed elsewhere (Sarvepalli and Nath, 2018).

Accumulating evidence in recent years has revealed a direct participation of CIN-TCPs in post-mitotic cell expansion, which could not be elucidated earlier due to extensive functional redundancy among the members, making the loss-of-function analysis less effective (Efroni et al., 2010; Sarvepalli and Nath, 2011a; Koyama et al., 2017). On the other hand, overexpression of miR319-resistant CIN-TCPs in their endogenous expression domain causes embryonic and seedling lethality, making gain-of-function analysis challenging (Palatnik et al., 2003; Koyama et al., 2007). Moreover, a change in cell proliferation in leaf primordia is often accompanied by an opposite change in cell size by a phenomenon called compensation, making the analysis of gene function in cellular regulation difficult to interpret (Horiguchi and Tsukaya, 2011; Hisanaga et al., 2015). Considering the contribution of CIN-TCPs in repressing cell division potential (Challa et al., 2019), it is rather a daunting task to uncouple TCP-dependent cell expansion from compensatory cellular growth in post-embryonic organs such as leaves.

Several class I TCP proteins have also been implicated in cell division and cell growth. For example, TCP20 is enriched in the chromatin regions that encode cyclin CYCB1;1 and several ribosomal proteins, suggesting its role in coupling division and growth of cells (Li et al., 2005). However, we focus on the class II TCP proteins in this review and set aside the discussion on the class I proteins for future.

Like cotyledons, post-embryonic lateral organs such as leaves also display planar growth driven by cell expansion in both transverse and longitudinal axes. Phytohormones such as ethylene and cytokinin promote cell growth along the transverse axes, whereas auxin, BR, and GA promote expansion along longitudinal axes (Mizukami, 2001). These hormones, individually or in combination, affect the organization of cellular and cortical cytoskeleton elements leading to differential expansion rate along growth axes (Blume et al., 2012). In addition, hormones also impact tubulin and actin transcript abundance (Kandasamy et al., 2001; Blume et al., 2012).

Recent findings implicate a role for the CIN-TCP genes in modulating hypocotyl elongation in response to environmental cues such as light and temperature. Multiple alleles of the jaw-D dominant mutation, where overexpression of miR319 downregulates five cognate CIN-TCP targets (TCP2, 3, 4, 10, and 24), show light-hypersensitive effect, where TCP4 and TCP24 suppress photomorphogenesis and promote hypocotyl elongation (Tsai et al., 2014). By contrast to TCP4 and TCP24, TCP2 inhibits hypocotyl elongation specifically under blue light condition by interacting with the blue-light receptor CRYPTOCHROME 1 and activating the transcription of ELONGATED HYPOCOTYL5 (HY5) and HY5-HOMOLOG, genes that promote photomorphogenesis (He et al., 2016). The non-miR319-targeted CIN-TCP members TCP5, TCP13, and TCP17 also promote hypocotyl elongation in response to canopy-shade by directly activating the auxin biosynthetic YUC genes and PIF4/5 (Zhou et al., 2018). TCP5/13/17 promote seedling growth also in response to high temperature by targeting PIF4 activity at both transcriptional and post-transcriptional level (Han et al., 2019; Zhou et al., 2019). Interestingly, TCP5 is expressed in both cotyledon and hypocotyl where it directly enhances local increase in auxin biosynthesis and BR response upon elevated temperature (Bellstaedt et al., 2019; Han et al., 2019). Heat treatment shifts TCP5 expression from leaf blade to petiole, supporting its role in differential cell expansion of petiole and reduced leaf size (Han et al., 2019). Thus, there appears to be a positive correlation between the level of CIN-TCP expression and the extent of cell elongation in embryonic organs such as hypocotyl (Figure 2A). Environmental challenges such as low light intensity and rise in ambient temperature induce unequal growth rates between adaxial and abaxial regions of the petiole cells, leading to hyponastic growth in association with major phytohormones (Millenaar et al., 2009). Chimeric repression of the CIN-TCP member TCP3 shows irregularly differentiated petiole formation, whereas overexpression of TCP5/13/17 displays longer petiole in response to high temperature, consistent with their petiole-specific expression (Koyama et al., 2007; Han et al., 2019). Further, enhanced and reduced activity of TCP1 leads to elongated and shortened petiole, respectively, which is mediated by auxin and BR response (Guo et al., 2010; Koyama et al., 2010b).

The apparent divergent effects of CIN-TCP transcription factors on the morphogenesis of various primordia including flower, axillary meristem, leaf, and petal converge at their inhibitory effect on cell proliferation. This effect is at least in part mediated by directly activating the transcription of KRP cell-cycle inhibitor genes. By advancing the cessation of cell proliferation and consequently triggering differentiation in various primordia, these proteins regulate the shape and size that are manifested in the mature organs. This rather simplified effect of these proteins has now started to expand with the identification of several target genes, interacting partners, and upstream regulatory proteins, presenting a complex regulatory network involving these transcription factors. One such effect is on cell growth of embryonic organs, which grow solely by cell expansion post-germination. Elongation of hypocotyl cells by CIN-TCPs can be explained by YUCCA-mediated auxin biosynthesis at the shoot apex that is transported to the growing hypocotyl (Challa et al., 2016). Though activation of dominant CIN-TCPs within the cell proliferation phase of leaf primordia reduces cell number, their activation beyond the proliferation phase has no effect on the cell or leaf size (Challa et al., 2019). This is perhaps not surprising, considering that the CIN-TCP genes are primarily expressed in developing leaves. However, the failure of the CIN-TCPs to increase cell expansion in leaves is rather intriguing.

Studies emerging over the past decade have elucidated the function of the CIN-TCP proteins as central integrators of environmental, developmental, and hormonal signals to perform several key developmental processes. By participating at multiple stages of organ growth including the duration and extent of cell proliferation, cell-fate transition from division to differentiation, onset of post-mitotic cell growth and scope of cell expansion, CIN-TCPs program diverse organs to achieve final size and shape over a wide range of plant species. To overcome the intertwined relationship between cell division and differentiation, and to study the exact contribution of CIN-TCP genes in cell expansion per se, the embryonic organs such as hypocotyl and cotyledons are useful model organs. However, the sequence of molecular events leading to cell enlargement and the exact kinetics of cell elongation in association with TCP genes awaits detailed elucidation. Finally, understanding the conservation of this CIN-TCP-dependent modulation in post-mitotic cellular growth across species is a challenge for future studies, considering organ size is a major contributor to crop productivity.

MR wrote the first draft of the manuscript and progressively modified it to the final version. KC and KS contributed to writing and corrections. UN corrected the manuscript and guided the other authors to produce the final version. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.