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In addition to MFA, we used a random forest (RF) algorithm to determine whether glycomic profiles can be used to predict if a species is succulent or non-succulent (see Suppl. Methods). Based on CoMPP data alone, the RF algorithm was able to classify species in their respective categories with 90% accuracy (Suppl. Data). The variable importance plot from the RF algorithm identified several cell wall components driving this classification (Figure S3), namely arabinogalactan proteins (AGPs), xylans, low-DM HGs and RG-I (incl. arabinan and galactan side chains). Regarding HGs and RG-I, these results agree with the differences between succulents and non-succulents mentioned above. We also observed drastically lower levels of xylans in the succulents studied, compared to the non-succulent species (Suppl. Data). However, xylans are often found in lignified support tissues (Zhong et al., 2013), and small-stature succulent species such as the ones we studied generally lack these tissues, relying primarily on turgor for support (Niklas, 1992; Gibson, 1996; Bobich and North, 2009). Thus, such differences may not hold for larger succulents. An interesting observation concerns AGPs, a notoriously complex group of cell wall glycoproteins with many suggested functions (Seifert and Roberts, 2007; Silva et al., 2020). LM14 and MAC207, two mAbs that recognize the same or structurally related AGP epitopes (Marzec et al., 2015; Yan et al., 2015), did not yield any signal among succulents despite being present in most non-succulent species tested (Suppl. Data). In contrast, other AGP-targeting mAbs (e.g. JIM13) showed comparable levels between the two groups, likely reflecting the diversity of AGPs and their numerous alleged functions. For instance, periplasmic AGPs have been postulated as stabilizers of the membrane-cell wall continuum and may also act as cell wall plasticizers when they are released from their membrane anchors (Gens et al., 2000; Knox, 2006; Lamport et al., 2006; Liu et al., 2015). The striking differences in signal intensity of the AGP-targeting mAbs we used warrant further exploration into the specific epitopes that they recognize and their functions.

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