Root and tuber crops (RTCs) are one of the most important crops globally, following some cereals in scale. These crops accumulate photosynthesis products in their underground organs. The bulking of adventitious roots to form storage roots in certain members such as Cassava and Sweet potato results from the expansion, leading to starch storage tissues accumulation along with highly expressed starch biosynthesis genes and downregulation of lignin biosynthesis genes concurrently (Gregory and Wojciechowski 2020). The development of storage organs in RTCs involves a unique secondary growth process where modified stems, roots, or hypocotyls undergoes girth enlargement through cambium-mediated accumulation of vascular tissues, giving rise to a large proportion of xylem parenchyma cells with minimal lignification of fibers or vessels (Banerjee et al. 2006). This suggests a positive correlation between secondary growth and starch accumulation. Sun et al. (2023) reported significant starch content and low cellulose and lignin levels in cassava storage roots, indicating a dynamic transition between structural and storage roots development to facilitate starch accumulation in the parenchymatous tissue. Unlike the secondary cell walls in woody plants, which consist mainly of lignin, cellulose, and hemicellulose in vascular bundles for water and nutrient transport, storage roots of RTCs have high starch contents, displacing lignin and cellulose to marginal levels (Sun et al. 2023). During root bulking in potato, there is a downregulation of fiber formation and lignification, accompanied by high starch accumulation in the storage organ (Singh et al. 2020; Sun et al. 2023). Higher starch accumulation in cassava roots is attributed to a combination of factors such as powerful stem transport capacity, depicted by a higher stem flow rate, high starch synthesis, and high expression of sugar transporter genes but low starch degradation in roots. (Li et al. 2016). Phytohormones have been implicated in playing significant roles in these processes, interacting with various environmental signals to regulate growth, development, starch biosynthesis and accumulation (Cao et al. 2023; Kim and Kim 2005; Sarkar 2008). However, the molecular mechanism regulating secondary growth have been extensively studied on model plants like Arabidopsis thaliana and Populus trichocarpus (Fischer et al. 2019; Rüscher et al. 2021), with limited information on molecular mechanisms regulating secondary growth and starch biosynthesis in tuberous crops.

Transcription factors (TFs) are known to participate in multiple plant growth and developmental processes (Su et al. 2022) as master switches regulating various cellular, biological functions, and signaling pathways in plant growth and development (Jha et al. 2020). Despite the central role of RTCs in food security and industrial raw material, there is limited research and detailed studies on their secondary growth, starch biosynthesis and accumulation regulated by phytohormones and transcription factors, especially in crops cassava and Taro. Previous studies focused on selective members of the group while neglecting some important members. Additionally, many genes encoding key enzymes during secondary growth and starch biosynthesis pathway remain poorly understood. An integrative approach and comprehensive understanding of the hormonal signal transduction, transcription factors activities on secondary growth, and starch biosynthesis-related gene expression would help unravel the molecular mechanism underlying these crucial processes.

Due to this lack of information, in this review, we summarized the available results from previous studies on the selected members that have received research attention. We question the molecular mechanisms underlying hormone content and dynamics during secondary growth, feedback mechanisms between hormones and the transcription factors in transitioning developmental fate from lignification to cambium-mediated parenchyma formation pathways, and propose studying promising gene expressions regulating secondary growth and starch biosynthesis in woody plants sharing significant phylogenetic evolutionary relationship with this crops. This exploration aims to elucidate their possible roles in improving starch quality and yield in RTCs.

The development of storage organs in RTCs is a complex process involving crosstalk among phytohormones, activities of transcription regulators, and gene expression that regulate cell division and elongation processes, ultimately leading to secondary growth (Carluccio et al. 2022; Li et al. 2015; Utsumi et al. 2022). Phytohormone signal transduction is significant factor in regulating the development of RTCs, as these hormones interact with various environmental cues, influencing growth, development, starch biosynthesis and accumulation in RTCs (Cao et al. 2023; Kim and Kim 2005; Sarkar 2008).

Reports also indicate that phytohormone signaling transduction, activities of various transcription factors (TFs), and sugar sensing play significant roles in driving the developmental processes of storage organ (Li and Zhang 2003; Liu et al. 2008; Sojikul et al. 2015). Interestingly, crop yields are primarily determined by secondary growth driven by vascular cambium (Hoang et al. 2020). Hormonal signal transduction has been well studies and results documented in previous works, such as review by Kolachevskaya et al. 2019; Kondhare et al. 2021; Mathura et al. 2023, research articles by Immanen et al. 2016; Yao et al. 2021. In this review, we focus on highlighting the hormonal functions in regulating the development of RTCs’ storage organs and starch biosynthesis in these crops.

Previous research has unveiled significant insight into the regulatory roles of auxin in cambial cell development in woody plants, a topic we emphasize in this review. For instance, it has been hypothesized that auxins act as central coordinators of growth, development, tuberization, cambial cell proliferation, and expansion (Kolachevskaya et al. 2019; Noh et al. 2013). However, the underlying mechanisms of these processes remain poorly understood. It has also been reported that the initiation and maintenance of meristematic stem cells are primarily regulated by auxin signaling (Agusti et al. 2011).

Auxin transport from the shoot apex, where it is synthesized, to the root via POLAR AXIS TRANSPORT (PAT), leads to the formation of a stem cell organizer through the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIP III) transcription factor, which regulates vascular cambium maintenance and activity (Rüscher et al. 2021; Smetana et al. 2019), indicating the regulatory role of auxin in cambium development. The endogenous indole acetic acid (IAA) content increases steadily in the early developmental stage of storage roots (Noh et al. 2010), and a high IAA level has been shown to promote cell division and storage root development (Ravi et al. 2014). However, there is a lack of reports regarding the molecular mechanisms underlying the content and dynamics of auxin at different developmental stages of storage root development. Additionally, most studies have focused on potato and sweet potato, with limited information available on Cassava, Yam, Taro, and other RTCs with differing storage organ developmental pathways.

Cytokinin (CK) emerges as a significant player among phytohormones in regulating storage root formation, acting as a prerequisite for cambial cell proliferation (Nieminen et al. 2008). Rapid increases in trans-zeatin riboside (t-zeatin) have been documented in the early stages of storage root bulking (Tanaka et al. 2008). A study by Jang et al. 2015 observed that cell proliferation activities in the cambium positively correlated with secondary growth and final yields in radish roots, indicating the substantial impacts of CK on nutrient accumulation in this crops. The upregulation of CK-responsive downstream genes suggests that CK plays a crucial role in storage root development by enhancing cell division and differentiation. However, the molecular interactions of CK with other phytohormones, especially auxin, in regulating underground storage organs, remain poorly understood (Matsuo et al. 1983; Yang et al. 2017). Furthermore, while most CK studies have focused on model plants such as Arabidopsis thaliana and Populus trichocarpa, further investigations are necessary to elucidate the functional roles of CK in RTCs.

Chen et al. (2022) reported that abscisic acid (ABA) signaling participated in the formation and development of various crops. Mathura et al. (2023) reported that several ABA signaling gene families such as IbABF3, IbABF4, IbDPBF3, IbDPBF4, IbPYL4, IbSnRK2.1, and IbSnRK2.2, which are crucial for sweet potato tuber development, as evidenced by their significant expression during storage roots development in these crops. Their expression patterns suggest positive roles during tuberization in sweet potatoes. However, debates among researchers persist regarding ABA’s involvement in tuberization, as it is often termed a stress hormone due to its expression coinciding with stresses. The molecular mechanism triggering its signal transduction to regulate tuberization in RTCs remains elusive.

On the aspect of gibberellic acids (GAs), their expression levels were reported to decrease during storage root development, preceded by the downregulation of GIBBERELLIN 20-OXIDASE and GIBBERELLIN 3-DIOXYGENASE (Sun et al. 2015).

Antagonistic relationships between ABA and GAs in regulating tuberization have been reported. GA3 levels increase initially during rapid thickening but decrease as the process progresses, while ABA content gradually increases along with bulking of the tuberous roots in crops such as I. batatas, S. tuberosum, P. notogiseng, and C. speciosa (Grandellis et al. 2016; Li et al. 2019; Noh et al. 2010; Yao et al. 2021). Some studies suggest that CK and GA are direct targets of KNOX1 transcription factor, which reduces expression of gibberellin biosynthesis genes but increases the transcriptions of cytokinin biosynthesis genes (Jasinski et al. 2005; Yang et al. 2018). However, the molecular mechanism regulating the crosstalk between these two phytohormones in regulating tuberization requires further study and wider application to enhance secondary growth and to promote starch accumulation in these crops.

The regulation of tuberization is increasingly recognized as a complex crosstalk between numerous hormonals and non-hormonal factor (Abeytilakarathna 2021; Dutt et al. 2017; Hannapel et al. 2017; Lomin et al. 2023). Crosstalk among phytohormones was predicted to trigger root tuber development (Chen et al. 2022; Chen et al. 2023).

Previous studies, crosstalk have reported crosstalk among various phytohormones in regulating cambial activities. Actions of multiple phytohormones and signaling factors influence cambial activities in stem (Fischer et al. 2019; Sergeeva et al. 2021). Synergistic roles of phytohormones such as Auxin, CK, ABA, GA, ethylene (ET), and jasmonic acid (JA) contribute to root and tuber storage organ development (Dong et al. 2015; Grandellis et al. 2016; Yao et al. 2021). And according to (Zhu et al. 2022).

Expansion of parenchyma cells for starch accumulations is accompanied by the expression of phytohormones such as indole acetic acid (IAA), zeatin riboside (ZR) and ABA in Corns. In cambial cells, CK is reported as the primary phytohormone regulating cambium initiation, while auxin plays a role in cambium maintenance and activities (Immanen et al. 2016; Nieminen et al. 2008). Although both auxin and CK act on the same vascular tissues to regulate the developmental process during different stages, the feedback mechanism controlling the content and dynamics of these two essential phytohormones remain unclear. Further studies are needed to elucidate these intricate regulatory processes in RTCs (Fig. 1).

Figure 1. Schematic diagram of molecular mechanisms behind secondary growth and starch and sucrose metabolism in selected RTCs. The top segment of Fig. 1 illustrates secondary growth regulation via a complex signaling network containing phytohormones and transcription factors. This network coordinates cambial proliferation and vascular tissue differentiation on either side of the cambium. Cytokinins (CKs) stimulate the expression of the PHLOEM EARLY DOF (PEAR) transcription factor, which triggers the expression of the ALTERED PHLOEM DEVELOPMENT (APL) gene, which regulates phloem differentiation. Phloem intercalated with xylem (PXY) is activated by a TDIF signal induced by phloem, which promotes the transcription of WUSCHEL-RELATED HOMEOBOX 4/14 (WOX4/14). This activation then triggers the release lateral organ boundaries domain (LBDs), transcription factors that promote proliferation of the cambium. Meanwhile, auxin transport from shoot apices initiates the formation of a stem organizer via the homeodomain-leucine zipper III (HD-ZipIII) transcription factor, which facilitates xylem differentiation while counteracting CK effects. The lignification of xylem, mediated by gibberellic acid (GA) signaling, is facilitated by vascular-related NAC domains (VNDs) and secondary wall-associated NAC domains (SNDs). The lower segment of Fig. 1 illustrates starch and sucrose metabolism in selected RTCs, detailing the genes involved in the metabolism of sucrose into starch granules, their transport, and their storage in plant storage tissues. CKs, indole-3-acetic acid, GAs, APL, PEAR, JULGI 1, supressor of MAX 4/5, CLAVATA-3/ESR-related 41/44, PXY, LBDs, WOX4/14, brassinosteroids insensitive-2/GSK-3-like kinase, BRI 1 EMS suppressor, HD-ZIPIIIs, VNDs, SNDs, sugar will eventually be exported transporters, sucrose carriers, invertase, hexose transporters, sucrose synthase, uridine diphosphate-glucose, glucose-6-phosphate, PGM (phosphoflucomutase), glucose-1-phosphate, ADP glucose pyrophosphorylase, adenosine diphosphate-glucose, starch synthase (SS), granule bound SS, starch branching enzyme 2, and starch debranching enzyme.

In addition, Denis et al. (2017) reported that gibberellins regulate xylem cell differentiation and lignification. (Xu et al. 1998) observed in potatoes that GA promotes stolon induction, while ABA, Auxin, ethylene, and sugar inhibit stolon formation and induce tuber formation. Downregulations of GA and auxin biosynthesis, together with upregulation of cytokinin biosynthesis, contributes to storage organ formation and enhances the activities of enzymes involved in starch biosynthesis pathway (Ravi et al. 2014; Sergeeva et al. 2000; Sojikul et al. 2015).

In other members of the such as sweet potato and Cassava, Auxin, CK, GA, and ET have been implicated influencing storage root development (Eguchi and Yoshida 2008; Ku et al. 2008; McGregor 2006; Noh et al. 2010; Sojikul et al. 2015; Wang et al. 2006). During cassava roots development, auxin and cytokinin play central roles, with their functional activities enhanced by brassinosteroids (BR), while the expression of JA and ABA inhibit cassava tuberous roots formation (Utsumi et al. 2022). These findings collectively highlight the antagonistic or synergistic roles among various phytohormones in regulating secondary growth and root development in RTCs.

Dynamic changes in auxin, CK and GA signaling levels during RTCs development are prominent events that promote cell division, transitioning from elongation to expansion via parenchymatous cell production for starch accumulation (Zierer et al. 2021). Similar findings were reported by (Immanen et al. 2016) in Populus trichocarpa, highlighting hormone-induced enhancements in wood formation.

While most studies have focused on tree plants where lignification signifies secondary growth and on potatoes with unique storage organ development pathways, there is a need for more research on RTCs with less attention. Detailed studies on the crosstalk among phytohormones, implicated in inducing secondary growth in both woody plants and some RTCs, are essential characterizing their crosstalks and enhancing starch biosynthesis and accumulation. Investigating hormone signaling impacts in these crops can provide valuable insights into optimizing their growth, development, and yield, especially in the context of starch production.

Previous studies have identified and reported genes that regulate secondary growth, starch biosynthesis, and accumulation in some root and tuber crops (RTCs). The expression of these genes can either activate or inhibit secondary growth, starch biosynthesis, and degradation. Several genes are specifically linked to tuber development, with some positively and others negatively regulating this process (Singh et al. 2023). Changes in the expression levels of genes involved in starch biosynthesis and metabolism have been observed, suggesting their role in regulating starch accumulation level (Cai et al. 2023). Interestingly, only a small fraction of transcription factors has been identified as regulators of secondary growth (Jang et al. 2015). This highlights the need for more detailed studies and functional characterization of these genes and transcription factors. Further elucidation of these genes and transcription factors will enhance our understanding of the molecular mechanisms underlying secondary growth and starch biosynthesis in RTCs. This knowledge can lead to targeted genetic interventions aimed at improving crop yield and starch quality in these important agricultural crops.

Transcription factors play pivotal roles as master regulators in various aspects of plant growth and development, including cellular division, differentiation, biological functions, and signal transductions (Su et al. 2022). For instance (Yang et al. 2024) found that StbHLH93 was significantly expressed in the subapical and peri-medullary region of developing potato tubers and decreased tuber number and size were observed in the mutant lacking this transcription factor, indicating its positive regulatory role in potato tuber development.

In cassava, gene expression patterns encoding putative cassava orthologues of VND6, VND7, NST3 and WOX4 were identified in samples enriched in cambium and developing xylem in both fiber and storage roots (Siebers et al. 2017), suggesting their functional roles in cambium- mediated secondary growth for parenchyma formation.

The period of formation of xylem parenchyma cells and the activation of storage starch biosynthesis and accumulations correlates with the upregulated expression of some transcriptional regulators, such as KNOX/BEL, KNAT1, PENNYWISE, WOX and POUND-FOOLISH (PNF) (Kim et al. 2020; Que et al. 2018). Rüscher et al. (2021) also reported that three important transcription factors (WRKY, NAC and bHLH) regulate potato tuber formation, emphasizing their roles in secondary growth and starch accumulation in potato tuber formation. However, these represent only a fraction of gene involved in these processes, highlighting the need for further elucidation and characterization of additional genes related to secondary growth and starch biosynthesis is crucial for advancing research in RTCs. This enhancement would exert demand pressure on source organs, leading to improved starch accumulation and overall crop productivity.

In sweet potato, highly expressed genes related to hormonal signal transduction, starch and sucrose biosynthesis, mitogen associated protein kinase signal transduction, Trihelix transcriptional regulatory genes were reported during tuberous root bulking (Cai et al. 2023). Investigating the expression levels, inter-signal relationship, and feedback mechanism among these regulatory elements in different RTCs would be crucial for understanding their interactive impacts on secondary growth and starch biosynthesis. These investigations should be extended to other root and tuber crops to comprehensively explore their regulatory networks.

Secondary growth regulatory events are intricately linked to nutrient accumulation in root and tuber crops (RTCs), highlighting their complementary nature. Starch, as the principal polysaccharide in RTC storage organ, significantly influences their quality and yield performance (Cao et al. 2023). The accumulation of starch is regulated by multitude of enzymes, including SUCROSE SYNTHASE (SuSy), ADP-GLUCOSE PYROPHOSPHORYLASE (AGPase-an important enzyme regulating starch biosynthesis), GRANULE BOUND STARCH SYNTHASE (GBSS), STARCH BRANCH ENZYME (SBE), STARCH DE-BRANCH ENZYME (DBE) and host of others (Cao et al. 2023; Ding et al. 2020; Greene and Hannah 1998; Kou et al. 2020; Tanaka et al. 2004). While these enzymes are key regulators of starch synthesis, other enzymes have roles in either repressing or degrading starch grains during starch biosynthesis and accumulation. Further study and comprehensive characterization of these enzymes are warranted. For instance, overexpression of MeAPL3 (subunit of AGPase) in cassava, leads to increased dry matter content and starch components but also correlates with high post-harvest physiological deterioration (PPD) (Beyene et al. 2022). This highlights the need to understand the nuanced roles of such enzymes in balancing starch accumulation and post-harvest traits.

Additionally, STORAGE ROOT DELAY (SRD) in cassava, identified as an orthologue of ALPHA-GLUCAN WATER DIKINASE1 (GWD1), whose expression is regulated under conditions of light/dark cycles in leaves that is associated with storage root development. The cassava srd mutant postpones storage root development but manifests normal foliage growth like wild-type plants (Zhou et al. 2017). Further exploration of genes involved in starch repression or degradation pathways will provide valuable insights into optimizing starch accumulation without compromising post-harvest traits in RTCs (Fig. 2).

Figure 2. Diagram showing the DEGs expressed during root and tuber formation for selected RTCs. INV1, Invertase 1; SuSy, sucrose synthase; APL3, ADP-glucose pyrophosphorylase large subunit 3; SBE, starch branching enzyme; GBSS, granule bound starch synthase; DBE, starch debranching enzyme; APS, adenosine 5’-phosphate sulfotransferase; TFL1, terminal flower 1; ABF2/4, ABF/AREB-like transcription factor 2/4; TUB7/19, STIP1 homology and U-box ubiquitin containing protein 7/19; CDF, cycling Dof factor; POTM1, potato mads box 1; miR172, micro-RNA 172; BEL5, BEL1-like protein; POTH1, potato homeodomain 1; SP6A, potato self-pruning 6A; FRUCT, beta-fructofuranosidase; CAD, cinnamyl alcohol dehydrogenase; PAL, phenylalanine ammonia-lyase; CL, isocitrate lyase; CCoAOMT, caffeoyl CoA 3-O methyltransferase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; VIN2, vacuolar invertase 2; SEX4, starch excess 4; AMY, ALPHA amylase; PHS2, alpha glucan phoshorylase 2; C3H, cysteine 3 histidine; 4CL, 4-coumarate CoA ligase; CO, constans; PHYB, phytochrome B; SUT4, sucrose transporter 4; SP5G, self-pruning 5G; XND1, xylem NAC domain 1; KN2/3, knox gene 2/3; PGM, phosphoglucomutase; AGPase, ADP-glucose pyrophosphorylase; and SP (starch phosphorylase)

Similarly, CELL WALL INVERTASE3 (MeCWINT3) in cassava highlights the delicate balance between sugar transport, sucrose hydrolysis, and overall plant physiology. Transgenic plants overexpressing MeCWINT3 accelerates sucrose hydrolysis in leaves by disrupting sugar transport from source to sink, leading to reduced plant height, induction of leaf senescence-related genes, and downregulation of starch biosynthesis enzymes (Yan et al. 2019). However, the upstream transcription factor responsible for regulating these genes and maintaining optimal expression levels without negatively impacting yield and starch content requires further elucidation.

RTCs offer promising prospects for ensuring food security, especially climate change challenges affecting cereal crops. RTCs hold the advantage of providing optimal yields while mediating starvation due to their tolerance to marginal soil, harsh environment factors. Understanding the intricate hormonal signaling pathways and transcription factors governing secondary growth and nutrient accumulation, particularly starch, in these crops is vital for maintaining global food security. A key focus area should be unraveling the molecular mechanisms behind girth expansion, which enhances sink strength, thereby boosting demand pressure for photo-assimilates on source organs. This understanding can lead to improved photosynthesis efficiency, sucrose metabolism, and enhanced starch biosynthesis and accumulation in root and tuber crops.

The process of secondary growth and starch biosynthesis is a complex, involving intrinsic and extrinsic factors that interact to induce the expression of genes related to these distinct yet interrelated events. The intrinsic factors include hormones signal transduction pathways and their crosstalk, as well as transcription factors that regulate downstream gene expression, thereby affecting secondary growth and production xylem parenchymatous cells for starch biosynthesis and accumulation. This crucial process has been selectively studied in a few crop species, but as storage organ developmental fate might vary among crops, inclusive studies involving the most important species in this category are necessary. We thus emphasize the need for comprehensive investigations. Areas of special emphasis, including but not limited to, are highlighted below.

Detail study and understanding of molecular mechanisms regulating tuberous root/stem development and starch biosynthesis can be achieved through the identification and functional characterization of promising target genes that regulate these events under both field and controlled condition.

The signal transduction activities and hormonal balance among important phytohormones such as Auxin, CK, GA, and ABA are crucial, given their dependency on cellular content and their crosstalk. Investigating potential synergistic or antagonistic interactions in regulating secondary growth and starch biosynthesis pathways via transcription factors that either activate or repress their downstream gene transcription should be deeply explored, employing computational biology and interdisciplinary studies to unravel the detail mechanism and their possible crosstalk.

A holistic approach to identify and characterize the complete sets of genes regulating secondary growth, starch biosynthesis, and degradation pathway is essential. This can be achieved by deploying various techniques such as single-cell RNA sequencing, a harmonized-omics approach, and mass-spectrometer studies across all relevant RTCs, facilitating a robust genome-wide association study (GWAS).

Unexpected outcomes, such as the overexpression of starch biosynthesis gene isoforms like Manihot esculenta ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT 3 (MeAPL3), resulting in high starch accumulation and dry matter content but susceptibility to postharvest physiological deterioration (PPD), underscore the need to understand the regulatory genes and their relationships with starch biosynthesis genes in greater detail. Exploring the activities of genes related to storage root delay (SRD) and MeCWINT3 further can enhance starch biosynthesis and accumulation, ultimately improving the quality of starch in root and tuberous crops.

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-RS-2023-00209134) to H.C.

H.C. conceived the review concept and revised and edited the manuscript. H.A.S. and Y.L. wrote this manuscript and added the models.