Methods

Six C. revoluta plants growing in a managed garden in Angeles City, Philippines, were sourced for stem tissue. The site was an open field with a loam soil (coarse loamy, isohyperthermic, Typic Untipsamment), and the plants received weed control and irrigation, but no other horticultural care. They were 105–120 cm in stem height, and a 2-cm disc of the entire stem was extracted at the height of 50 cm on 1 Aug 2019. The diameter of the six stem discs was 15 to 18 cm. The basal part of each stem was allowed to produce adventitious buds to resume growth, and the detached apical portions were propagated to produce a new plant with adventitious roots. Each stem section was cut into ∼2-cm cubes, dried at 75 °C for 48 h, then stored for chemical analysis.

Six Z. furfuracea plants growing in a managed garden at the University of Guam were sourced. The garden was in full sun with clay loam soil (clayey, gibbsitic, nonacid, isohyperthermic, Lithic Ustorthents). The horticultural care and extraction of the stem disc were as described for C. revoluta. The stems were 34 to 38 cm in height, intact stem discs were obtained at a height of 12 to 15 cm, and the discs were 15 to 19 cm in diameter. Tissue collection was conducted on 10 Aug 2019. The stem sections were cubed and dried as described for C. revoluta.

The tissue from each replication was milled (20-mesh screen). Soluble sugar extraction was conducted using 80 °C water extraction with acetonitrile. The monosaccharides, disaccharides, and starch were quantified by high-performance liquid chromatography (HPLC) refractive index, as previously described (Marler and Cascasan 2018; Marler and Cruz 2020). The oligosaccharides raffinose and stachyose were quantified by HPLC mass spectrometry (Agilent 1290 HPLC, Santa Clara, CA, USA). The concentration of each of the 16 NSCs was compared for the two cycad species by t test.

Results

All 11 monosaccharides were detected in stems of both cycad species (Table 1). Glucose and fructose accounted for 97% of the monosaccharide sugar content. The monosaccharide content of Z. furfuracea stems was roughly twice that of C. revoluta. Disaccharide content of C. revoluta stems exceeded that of Z. furfuracea stems. Sucrose accounted for 96% to 99% of the disaccharide content. Stachyose accounted for almost all of the oligosaccharide content. Total oligosaccharide content for Z. furfuracea was 74% of that for C. revoluta. The mean starch concentration of these cycad stems was 369 mg·g⁻¹, greatly exceeding the concentration of any single sugar (Table 1). The Z. furfuracea stem starch concentration was 73% of that for C. revoluta stems.

Table 1. Nonstructural carbohydrate concentration (mg·g⁻¹) in stem tissue of two cycad species.

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These results illuminated the nature of the relationships among the sugars of these cycad stems. The differences between the species were greatest for the five most abundant sugars. The sugar contents for Z. furfuracea were sucrose = stachyose > glucose > fructose > galactose > arabinose = fucose = maltose = mannose = raffinose = rhamnose = ribose = ribulose = xylose = xylulose. The sugar contents for C. revoluta were stachyose > sucrose > glucose = fructose > maltose > galactose = arabinose = fucose = mannose = raffinose = rhamnose = ribose = ribulose = xylose = xylulose. The relative proportions of monosaccharides, disaccharides, and oligosaccharides were more evenly distributed for Z. furfuracea than for C. revoluta (Fig. 1).

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The starch:sugar quotient was 0.94 for Z. furfuracea and 1.18 for C. revoluta, indicating a greater proportion of the NSCs were readily available in the Z. furfuracea stems. The sum of measured sugars and starch was 642 mg·g⁻¹ for Z. furfuracea and 787 mg·g⁻¹ for C. revoluta, indicating that roughly 70% of the cycad stem dry weight was composed of NSCs.

Discussion

This pilot study has revealed that every sugar in a 15-sugar assay was detected, and stachyose emerged as a dominant sugar in the stems of two cycad species. Stachyose accounted for 47% of the total sugars in C. revoluta and 38% in Z. furfuracea. These two cycad species are widely separated phylogenetically and belong to discrete families (Calonje et al. 2023). Stachyose may be a widespread storage carbohydrate among the taxa representing the Cycadales, and more complete sugar-richness studies with other species are warranted to more fully understand the NSC relations of cycads.

Stachyose and other members of the raffinose family oligosaccharides serve important functions for the plant, such as storage of NSC, stabilization of membrane function, and signaling components of stress tolerance (Kandler and Hopf 1982; Van den Ende 2013; Hartmann and Trumbore 2016). More cycad research on these topics is warranted.

Quantifying the standing pool of sugars is useful for studying sugar storage components, but is not useful for determining ephemeral sugar relations. For example, stachyose was abundant in these cycad stems and each stachyose required the prior biosynthesis of galactose, but the free galactose pool was negligible. This study shows that an experimental approach that quantified the monosaccharide pool without also quantifying the oligosaccharide pool would underestimate galactose synthesis of these cycad plants.

A greater understanding of cycad NSC relations is needed to inform cycad horticulture and conservation. For example, growth of Cycas micronesica leaves and male cones generated declines in stem NSCs, revealing the deployment of these stem NSCs to support the demanding sink activity (Marler and Cruz 2020). This concept was exploited during historical starch extraction from C. revoluta stems, as harvests were timed to occur immediately before a new flush of leaf growth (Thieret 1958). Invasions of the scale Aulacaspis yasumatsui Takagi have caused irreparable harm to the international cycad horticulture trade (Marler et al. 2021), and declines in C. revoluta NSCs correlated with duration of A. yasumatsui infestations (Marler and Cascasan 2018).

Conclusions

Tissues of C. revoluta and Z. furfuracea stems revealed a rich sugar profile and stachyose was detected in concentrations similar to sucrose. Previous reports of cycad carbohydrates underreported sugar richness, underestimated total sugar content, and overestimated the starch:sugar quotient. The stems of these two species were comprised of 64% to 79% NSCs, confirming that cycad stems serve as a source of NSCs to support the plant’s sink activities when needed.