Keywords

9.1 Introduction

Sweetpotato [Ipomoea batatas (L.) Lam.] is recognized as the seventh most important food crop in the world (FAOSTAT data 2019). It has a global production of approximately 144 million metric tons and is the third most important root or tuber crop after potato (Solanum tuberosum L.) and cassava (Manihot esculenta Crantz) (FAOSTAT 2019). World production is centered in the Asian-Southeast Asian region, with China being the largest producer, while Sub-Saharan Africa ranks second (FAOSTAT 2019). Although leaves are consumed as vegetables in some regions, the fleshy storage roots are the main economically important organ of the crop that is grown in diverse production environments with yields ranging from 4–10 t/ha in SSA (Ngailo et al. 2019) to 60–90 t/ha in the sweetpotato growing regions of Australia (Stirling 2021). Sweetpotato is used as a source of starch, ethanol, and animal fodder in most of Asia while it is considered a subsistence crop in Africa. The USA, Israel, Japan, Australia, New Zealand, and South Africa are among the few countries that grow sweetpotato as a vegetable to market in developed economies. In many production environments, the storage root is also the basis of propagation. Depending on the number of adventitious roots that will be induced to form storage roots, sweetpotato plants will yield either a high number (4–8/plant) of marketable storage roots or a low number of roots that may even be reduced to one very large storage root per plant or no marketable roots at all. Poor shape is another quality variable that renders many roots unmarketable. Due to the underground nature of the crop, the performance of sweetpotato plants can be evaluated only post-factum and the above-ground growth provides little or no indication of crop yield during development. Substantial information is missing with relation to the formation of sweetpotato storage roots in general. There have been recent reviews (Ravi et al. 2009; Tanaka 2016; Yang et al. 2023) of the progress of physiological and molecular mechanisms of storage root formation in sweetpotato. While available anatomical and molecular evidence has significantly increased our understanding of how storage roots form, significant knowledge gaps exist in terms of explaining the variable number of storage roots produced per plant. Thus, the understanding of how adventitious roots transition to storage roots is important not only from the scientific understanding but can lead to practical applications that improve food security and economic sustainability where the sweetpotato is grown.

Hoang et al. (2020a, b) took a cogent approach in synthesizing available evidence on storage root development in root crops by focusing on the vascular cambium as the main driver of secondary growth. Hoang et al. (2020a, b) concluded that the amount and resolution of currently available data for each root crop create gaps as far as pinpointing the key processes responsible for crop yield. This lack of granularity leads to a lack of resolution as regards tissue-specific gene expression patterns despite hundreds of thousands of candidate genes identified. This review coincided with the publication by Hoang et al. (2020a, b) of their work on identifying conserved gene regulatory networks of secondary growth in radish. In this work, they used laser capture microdissection to collect tissue samples for gene expression analysis from multiple time points representing key development stages of radish storage roots. They then compared to putative Arabidopsis orthologues to gain insights about gene regulation pathways in radish. Blomster and Mähönen (2020) reviewed this work and suggested that this tissue-specific radish dataset may well help to elucidate additional regulators radial growth and that the radish storage root could serve as an informative model for storage organ development. Blomster and Mähönen (2020) highlighted the underlying challenge in elucidating the genetic regulation of storage roots of root crops in general and the sweetpotato in particular: the lack of a model system.

This review will highlight studies that focus on vascular cambium as the main driver of storage root formation in sweetpotato. The overarching goal of this review is to integrate classical anatomical benchmarks, physiological studies, and emerging root architecture evidence into recent genetic studies to provide an updated summary of the genetic regulation of storage root formation. Focusing on the cambium enables the integration of various lines of evidence, including the role of hormone signaling. This review will also address carbon partitioning and differential sink strength to explain why under certain conditions, some adventitious roots fail to become storage roots. Further storage root growth is due to the translocation of photosynthates produced in the leaves to the developing storage roots where the sucrose is converted to other forms of carbohydrates. This review will also address the inconsistencies in terminology, specifically as it applies to the root system, as used in the majority of past and recent work that seek to elucidate the genetic regulation of storage root formation and development in sweetpotato. The use of inconsistent root terminology in root biology hinders understanding and scientific progress (Dubrovsky 2022; Zobel and Waisel 2010). Finally, we will present a model of storage root formation that is based on current available evidence. This model will address current gaps in knowledge, in particular offering proposed mechanisms that lead to the failure of some adventitious roots to become storage roots in response to environmental and management variables.

9.2 Anatomical Benchmarks

Classical and recent morphological and anatomical studies have unambiguously defined the anatomical features associated with the transition from primary to secondary growth associated with storage root formation in sweetpotato. Foremost of this is the work of McCormick (1916) that not only set the tone for future work but clearly documented the secondary features associated with storage root thickening, noting the role of primary and secondary cambium (Fig. 9.1c, d). McCormick also noted that there were as many rows of lateral roots as protoxylem points and explains the presence of definite rows of lateral roots on “mature” storage roots (Fig. 9.1b). Artschwager (1924) corroborated the association of primary and secondary cambium with the early differentiation of the fleshy root. Reference was also made about the presence of lateral roots which sit in a scar-like tissue similar to the “potato eye.” In addition, Artschwager (1924) noted that the “parts of the sweetpotato roots that do not become thickened,” the cells between the protoxylem points and the large central cell become lignified. At this point, anatomical studies were largely descriptive. Togari (1950) built on these prior works and documented the roles of nutrients, temperature, water, and light on anatomical cues of storage root development. More importantly, Togari (1950) established the timing of anatomical benchmarks that were associated with storage root formation (Fig. 9.1). Defining these stages was important as it provided a context for gene expression analysis. Esau (1967) synthesized available evidence and described the secondary growth in sweetpotato as a complex type of anomalous growth in fleshy adventitious roots. Esau (1967) used the terms “normal cambium” and “anomalous cambium.” In this work, we will use “primary cambium” and “vascular cambium” interchangeably, to distinguish from “anomalous cambium.”

Fig. 9.1
figure 1

SR1—primary growth, differentiation of protoxylem (5 DAP). SR2—Onset of secondary growth marked by the appearance of vascular cambium (ca) (15–20 DAP). SR3—appearance of anomalous cambium (20–25 DAP)

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Defining these stages is important as it provides context for unraveling signaling networks. To date, anatomical evidence of anomalous or circular cambium development remains the key indicator of the onset of storage root formation. Gene expression studies by nature are time-sensitive and tissue-specific assays and the outcome may be different if sampled at different time points. When Firon et al. (2013) generated transcriptome data from adventitious roots that were either undergoing storage root formation or lignification, they sectioned adventitious roots at the 2.5 cm section of the proximal tip, verified the anatomical features, and classified root tissue samples accordingly.

9.3 Emerging Root Architecture Terminology: Consensus or Conundrum?

Gregory and Wojciechowski (2020) conducted a comprehensive review of the literature of root systems of root and tuber crops and noted that the inconsistency in terminology applied to root systems of these crops was a notable feature of their effort to synthesize the available literature. In particular, they highlighted the incorrect application of terms used to describe the root systems which, with the exception of a few cassava and yam crops grown from seed, all of the root and tuber crops produce adventitious roots (ARs) (Fig. 9.1a, b). They also agreed with prior work (Adu et al. 2018; Villordon et al. 2014) that the term “fibrous roots” is unhelpful and misleading. AR axes (the main root) emerge from stem nodes, basal stems of cells (wound tissues), stolons, and the junction of stem and mother tuber/corm of the crop and lateral roots emerge from these axes. Esau (1967) defined “lateral root” as any root branching from another root. To assist in describing the relationship of lateral roots, lateral root orders are described as “first-order laterals” and from these arise “second-order laterals” and so on (Zobel and Waisel 2010). This distinction is important because lateral roots are functionally and physiologically different from the main axis (or the main root) of the adventitious root where cambium activity associated with storage root development occurs. Lateral roots have diararch or polyarch steles, in contrast with the main root that are typically either pentarch or hexarch steles.

Why is the term “fibrous root” confusing in the context of sweetpotato root systems and storage root formation? In a review of the physiology of the sweetpotato, Kays (1985) discussed separately the subject of root distribution and architecture from sections devoted to lateral roots, “primary fibrous roots,” and “pencil roots.” Kays (1985) described “primary fibrous roots” as emerging largely from tetrarch “thin” adventitious roots although “under adverse conditions, they maybe from pentarch, hexarch, and even septarch thick roots.” Earlier, Wilson and Lowe (1973) reported on the anatomical features of field-grown sweetpotato, corroborated the timing of anatomical benchmarks proposed by Togari (1950), and introduced some variation in terminology. For example, they referred to “tuberous” and “non-tuberous” roots and introduced the term “fibrous roots,” which they defined as uniformly thickened roots with normal secondary growth leading to complete lignification of the stele. In other words, Wilson and Lowe (1973) used the term “fibrous roots” in reference to non-swollen ARs. Belehu et al. (2004) determined that “fibrous roots” were lateral roots and proposed the term to refer to first, second, and third-order lateral roots.

9.4 Sink Strength: Bridges Gap Between Morpho-Anatomical and Molecular Data?

Scientists have repeatedly recognized that species with large below-ground sinks for carbon and with apoplastic mechanisms of phloem loading are likely to be the best candidates for a large response to rising atmospheric CO2 (Miglietta et al. 2000). There is also an increasing consensus that growth or storage sink limitations are possibly major factors constraining responses of plants to elevated CO2 (Miglietta et al. 2000). To understand the problem of regulation of dry matter partitioning by the sinks, there has been substantial interest in a property of a sink, called sink strength, that determines this regulation. Sink strength can be defined as the competitive ability of an organ to receive or attract assimilates (Wareing and Patrick 1975; Wolswinkel 1985; Farrar 1993a). At present, many discussions focus on the question whether the concept of sink strength is a useful one, or a vague and confusing concept (Farrar 1993b). Much confusion is due to lack of a clear definition of sink strength. The actual rate of assimilated import or growth has often been used as a measure of sink strength (Warren-Wilson 1972). When defined in this way, sink strength in fact represents the net result of assimilate flow which may depend on the competitive ability of all sinks on a plant and the assimilates supply (source strength). This is not a useful measure of sink strength, and it is the prime cause why some authors reject the use of the concept of sink strength. Minchin and Thorpe (1993), dismissed sink strength (as measured by the actual import rate) as a misnomer, and other authors (e.g., Patrick 1993) stated that it should be possible to identify a set of parameters to describe a sink's ability to influence assimilate import which are independent of the rest of the plant. More recent evidence in other species also supports the hypothesis that RSA contributes to the determination of sink strength and is consistent with increased upregulation of enzymes involved in starch and sucrose metabolism (reviewed by Hennion et al. 2019). Current available evidence about the role of lateral root emergence and RSA in determining sink strength is consistent with modeling work (Bidel et al. 2000; Thaler and Pages 1998). Linking sucrose synthase (SuSy) activity with RSA in sweetpotato fills significant gaps in our knowledge of storage root formation, strengthens the concept of sink strength, and can contribute to advances in water and nutrient management and contribute to harnessing high-value root traits for crop improvement. New evidence from RSA studies (Bui et al. 2015; Paszkowski and Gutjahr 2013) supports the hypothesis that root architecture is a key determinant of root carbon sink strength, integrating current available molecular, hormonal, nutritional, and morphological evidence that leads to a more comprehensive understanding of storage root formation.

9.5 Genetic Regulation of Vascular Cambium-Driven Storage Root Formation in Sweetpotato: A Synthesis

The overarching goal of this review is to highlight work that defines storage root formation within the context of vascular cambium development, with clear definition of developmental stages either via anatomical features, and well-described root developmental stages and root orders. For clarity, if the term “fibrous roots” is used other than in reference to “lateral roots,” as defined previously, then it will be noted. This review will also highlight genetic data associated with lateral root development, protoxylem development, carbon allocation, and lignification presented within the context of storage root formation as defined in Fig. 9.1.

Table 9.1 highlights studies that specifically define storage root formation within the context of vascular cambium development and with the objective of identifying genes or genetic networks associated with storage root formation in sweetpotato. The work by You et al. (2003) likely represented the first attempt to identify genes associated with storage root formation in sweetpotato. They classified adventitious roots based on thickness and generated a cDNA library based on their definition of “early stage storage roots (0.3–1 cm in diameter).” They sequenced the clones and identified 39 genes putatively involved in gene regulation, signal transduction, and development. Of these 39 genes, IbMADS3 and IbMADS4 were categorically associated with cambium development. Tanaka et al. (2005) used digoxigenin (DIG) labeling to specifically link SRF6 expression to the vascular cambium and anomalous cambium (Table 9.1). This is likely the first evidence linked gene expression data to anatomical location. They also detected genes associated with sugar metabolism, signal transduction, and carotenoid biosynthesis. Kim et al. (2005) used a candidate gene approach to link IbAGL17, a MADS-box gene to increased sink strength of developing adventitious roots. It was also through DIG labeling that Ku et al. (2008) localized IbMADS1 expression to within the stele and lateral root primordia, effectively linking lateral root emergence with storage root formation. In this work, Ku et al. (2008) used “fibrous roots” apparently to refer to adventitious roots in various stages of storage root formation.

Table 9.1 List of genes that have been associated with specific storage root formation stages and benchmarks in sweetpotato
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However, the anatomical evidence clearly showed that the gene expression data was linked to lateral root emergence sites. Ku et al. (2008) concluded that IbMADS1 is an important integrator at the initiation of storage root formation and possibly regulated by a network involving a MADS-box gene in which hormones such as jasmonic acid and cytokinins are trigger factors. Noh et al. (2010) presented DIG labeling evidence that SRD1 expression was localized in cambial cells but no signal was detected in storage parenchyma and xylem vessels. They also presented evidence that SRD1 was responsive to variation in auxin concentration. The work by Tao et al. (2012) likely represented the first use of next-generation RNA sequencing that made possible the investigation of storage root development without genome sequence information. Even though the experimental methodology precluded the identification of genes associated with storage root initiation and early development, Tao et al. (2012) presented evidence of increased SuSy expression associated with expanding storage roots. Firon et al. (2013) likely represented the first work that coupled anatomical confirmation of anomalous cambium with NGS transcriptome data. More importantly, this work specified the specific section of the main adventitious root where the evidence of anomalous cambium was detected and corresponded to the tissue used for RNA extraction. This work provided evidence of upregulation of genes involved in carbohydrate and starch biosynthesis and the downregulation of genes (4CL, CCoAOMT, CAD) involved in lignin biosynthesis in adventitious roots that failed to show evidence of storage root formation. Noh et al. (2013) presented evidence that an expansin-like gene, IbEXP1, was apparently negatively involved in storage root formation by suppressing the proliferation of metaxylem and cambium cells. Wang et al. (2015) used microarray data to generate evidence that starch biosynthesis is upregulated while lignin biosynthesis is downregulated during storage root development. In addition, this work provided evidence that transcription factors that modulate or control root development and lateral root were also detected during storage root development. However, no specific genes were associated with vascular cambium development. Dong et al. (2019) analyzed transcriptome data from different developmental stages based on adventitious root diameter. They used “fibrous roots” to refer to adventitious roots less than 1 cm in diameter and apparently to describe adventitious roots that do not show evidence of vascular or anomalous cambium development. This work identified LBD4, WOX4, and TMO6 were associated with cambium activity. It also identified starch biosynthesis genes, including ADP-glucose pyrophosphorylase (GLGL), starch synthase (SSY), and starch-branching enzyme (GLGB). Singh et al. (2019) focused on the role of GA on storage root formation and provided evidence that GA suppressed cambium development and was associated with lignin biosynthesis. This work also provided evidence that lateral root development was linked to the capacity of an adventitious root to undergo storage root formation. Singh et al. (2019) presented a model accounting for the role of GA in storage root formation that incorporates root architecture attributes like lateral root number and length. However, this model does not provide any links to external management or environmental variables known to suppress storage root formation or favor lignification. Cai et al. (2022) also used adventitious root thickness to classify storage root formation stages to characterize genes associated with storage root formation using NGS. They identified auxin-responsive genes (AUX/IAA, ARF, SAUR, and CH3) that were associated with anomalous cambium activity. Interestingly, they provided evidence that expansin was associated with storage root development, contrary to the evidence presented by Noh et al. (2013). An examination of the methodology used to generate tissue samples in both studies showed significant differences in terms of timing and specificity of tissue collection. Both studies used the term “fibrous roots” to refer to adventitious roots that are not associated with storage root formation. However, Noh et al. (2013) defined the diameter of “fibrous roots” as less than 0.2 cm while Cai et al. (2022), defined this as less than 0.1 cm. Prior work has indicated that adventitious roots around 1 mm can show evidence of storage root formation depending on cultivar and growth conditions (Wilson and Lowe 1973). Furthermore, Noh et al. (2013) did not define the sampling time while Cai et al. (2022) collected storage root samples at 90 days after planting. Both studies did not specify the specific tissue from which samples were collected for RNA extraction. This lack of standard experimental protocols and specificity of tissue sampling complicates the direct comparison of gene expression data. Cai et al. (2022) also developed a model for storage root formation based on their transcriptomic data, highlighting the role of genes hypothesized to be involved in storage root formation and outlining presumptive regulatory pathways. As with the prior cited work, this hypothetical model does not provide alternate pathways for lignification nor proposes any links to known external variables that affect storage root formation like moisture and temperature. Both models highlight the role of starch and sucrose metabolism. The role of starch metabolism in storage root formation is further highlighted by Song et al. (2022) who performed comparative transcriptomics using NGS. They also identified transcription factors possibly associated with storage root formation. They concluded that additional research is needed in order to validate their roles in storage root formation.

Taken together, recent molecular work underscores the role of starch biosynthesis genes in storage root formation. However, due in part to the lack of hypothetical models in storage root formation that account for variation in sink strength among adventitious roots, these genes are merely enumerated and not properly contextualized. Ravi et al. (2009) stated that sink strength determined storage root growth but did not elaborate on how this varied among adventitious roots. Li and Zhang correlated sink strength with greater SuSy in storage root expressed sequence tags (ESTs) than in non-storage root ESTs and correlated with ADP-glucose pyrophosphorylase (AGPase) expression. AGPAse catalyzes the formation of ADP-glucose, the first step dedicated to starch synthesis (reviewed in Hennion et al. 2019). In sweetpotato, cumulative evidence supports the hypothesis that sink strength determines the capacity of adventitious roots to undergo storage root formation (Keutgen et al. 2002; Li and Zhang 2003). Considering prior evidence, it is therefore surprising that the concept of sink strength has been overlooked in most of the reports cited in this review. Hoang et al. (2020a, b) and Zierer et al. (2021) reviewed the physiological and genetic regulation of storage roots in general and specifically addressed the role of sink strength in storage root formation. Prior work assumed that all adventitious roots were phenotypically uniform, and the subject of RSA variability was never accounted for in the context of storage root formation signaling. However, cumulative data from recent research indicate that adventitious roots within the same plant vary in root architectural attributes (main root length, lateral root number, lateral root length, lateral root density) in response to biotic and abiotic variables. These root architectural modifications in turn are associated with the competency of an adventitious root to undergo storage root formation. Evidence presented by Ku et al. (2008) and Singh et al. (2019) underscores the importance of incorporating lateral root measurements in current and future work that seeks to further characterize the genetic regulation of storage root formation in sweetpotato.

A model depicting the synthesis and integration of available molecular evidence of vascular cambium-driven storage root formation into existing anatomical and physiological benchmarks is depicted in Fig. 9.2. Carbon partitioning and sink strength determination are also incorporated into the model, along with pathways hypothesizing the sensing of environmental cues. In this model, the developing root system integrates internal and external signals that in turn determine sink strength of individual storage roots. Adventitious roots that develop into the soil profile characterized as possessing optimal conditions (temperature, moisture, fertility) will develop optimal lateral root architecture, which in turn increases sink strength. It is hypothesized that sucrose is a shoot-derived signal associated with vascular cambium development. On the other hand, adventitious roots that develop into marginal soil conditions fail to develop optimal root architecture, reducing its sink strength, and unable to compete for carbon allocation.

Fig. 9.2
figure 2

Hypothetical model synthesizing current understanding of genetic regulation of storage root formation that integrates sink strength determination and root architectural responses to external management and environmental stimuli

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9.6 Conclusions

It is evident from the current review that the integration of anatomical, morphological, and physiological cues of storage root formation with molecular and genomic evidence will lead to a more complete understanding of the genetic regulation of the role of vascular cambium in sweetpotato storage root formation. Recent technological advances and the availability of reference genomes have led to significant advances in unraveling the genetic regulation of storage root formation in sweetpotato. At the same time, increased attention to the role of vascular cambium in other root crops such as radish and cassava has underscored the need to improve the resolution and tissue specificity of gene expression studies and consolidation of genomic and molecular data. This consolidation will lead to the identification of shared regulatory programs and promote comprehensive studies related to storage root development. These new insights should provide a new benchmark for future studies that seek to further unravel the genetic regulation of sweetpotato storage root formation. Future work should address the lack of standard experimental protocols and tissue specificity which hinders overall progress in our understanding of the genetic regulation of storage root formation in sweetpotato.