Proanthocyanidin Biosynthesis- a Matter of Protection. 3

Proanthocyanidin Biosynthesisa Matter of Protection. 3 4 Richard A. Dixon 1,2 and Sai Sarnala 3 5 1 Hagler Institute for Advanced Studies and Department of Biological Sciences, Texas A and 6 M University, College Station, TX, USA; 2 BioDiscovery Institute and Department of 7 Biological Sciences, University of North Texas, 1155 Union Circle #311428, Denton, TX 8 76203-5017, USA; 3 Texas Academy of Mathematics and Science, TAMS 1155 Union Circle 9 #305309 10 University of North Texas, Denton, TX 76203-5017 11 ORCID ID: 0000-0001-8393-9408 (RAD) 12 13 One-sentence summary: Proanthocyanidins are the second most abundant plant phenolic 14 polymer, but, in spite of intensive investigation, several aspects of their biosynthesis and 15 functions remain unclear 16 17 Author contributions: Both authors contributed to writing this review article. 18 19 BACKGROUND 20 Proanthocyanidins (PAs, also known as condensed tannins) are polymers of flavan-3-ols that 21 bind to proteins and have been ascribed functions as herbivore feeding deterrents and 22 antimicrobial compounds. They provide astringency to fruits and beverages, positively impact 23 human health, and benefit ruminant livestock by improving nitrogen nutrition and providing 24 protection from pasture bloat (McMahon et al., 2000; Dixon et al., 2012; Rauf et al., 2019). 25 Much progress has been made in recent years in understanding the molecular genetic basis of 26 PA biosynthesis. However, there remain difficulties in resolving the chemical labeling pattern 27 of PAs with their proposed biosynthetic pathway, and defining the subcellular sites of 28 biosynthesis. There is also no model that fully explains the cell biological phenotypes of 29 mutations that interrupt the pathway and disturb the accumulation of PAs in the central vacuole. 30 Plant Physiology Preview. Published on August 18, 2020, as DOI:10.1104/pp.20.00973

TT12 (Kitamura et al., 2010). tt19 has higher levels of insoluble PAs than wild-type, and these 156 are reduced in the tt19/tt12 double mutant (Kitamura et al., 2010). To reconcile these findings 157 with the pathways in Figure 1B, Figure 2 presents a model in which reactive PA extension units 158 are stabilized through interaction with TT19, either in soluble complexes or vesicles, and then 159 delivered to pre-vacuole-like vesicles that contain stable starter units, possibly, although 160 perhaps not exclusively, loaded as glycosylated flavan-3-ols by TT12. Sequestration of starter 161 units is required because they themselves can be phytotoxic (Jun et al., 2018). In this model, 162 fusion of the TT19-and TT12-containing structures results in mixing of starter and extension 163 units to initiate oligomerization; the resulting PA dimers and higher oligomers are finally 164 delivered, perhaps by vesicle fusion, to the central vacuole. TT19 is also present in the 165 tonoplast, where it may be associated with transporters for anthocyanins (Sun et al., 2012) or 166 PAs. Consistent with the model in Figure 2, loss of function of LAR results in higher-167 molecular-weight PAs through increasing the ratio of Epi-cys extension units to epicatechin 168 starter units (Liu et al., 2016), and loss of function of TT19 results in higher-molecular-weight 169 PAs formed in the cytosol through interaction of excess "unprotected" extension units with 170 cytosolic epicatechin; this is reversed in the tt19/tt12 double mutant because of the elevated 171 levels of cytosolic starter units (Kitamura et al., 2010). TT13 encodes a tonoplast ATPase 172 necessary for generation of a proton gradient for transport of PA starter units by TT12 173 (Appelhagen et al., 2015). In both tt12 and tt13 mutants, PAs accumulate on the outside of 174 TT12-containing small vacuoles, suggesting that the TT19 complex is targeting the extension 175 units to these vacuoles where the starter units are now backed up; glycosylated epicatechin, 176 presumably cytosolic, accumulates in the tt12 mutant (Appelhagen et al., 2015;Kitamura et al., 177 2010). 178 TT10 encodes a laccase enzyme (AtLAC15 in Arabidopsis) that was originally ascribed 179 a role in PA polymerization, although the non-specific linkage pattern of TT10-catalyzed 180 oligomerization products has led to the hypothesis that the function of the enzyme is more likely 181 the oxidation of pre-formed PAs in the cell wall of the seed coat (Pourcel et al., 2005). TT10 182 can also catalyze lignin polymerization (Liang et al., 2006), and could potentially catalyze the 183 formation of cross-links between PAs and other cell-wall polymers. Whether oxidation by TT10 184 is important for formation of insoluble PAs or whether these simply reflect higher-molecular-185 7 7 weight forms (Liu et al., 2016) remains unclear. In fact, insoluble PAs, although often 186 accounting for more than 50% of the total PA species, remain somewhat poorly characterized. 187 They are easy to quantify by conversion to anthocyanidins by heating in acidic butanol, but this 188 results in the loss of structural information. New methods for analysis of insoluble PAs are 189 clearly required. Although nuclear magnetic resonance approaches are now being applied to PA 190 analysis (e.g. Fryganas et al., 2018), they have yet to provide the structural resolution of similar 191 approaches for the phenylpropanoid polymer lignin (Sette et al., 2011). Increasingly 192 sophisticated mass spectrometry approaches are being applied to PA characterization 193 (Salminen, 2018), but detailed structural features of the internal portions of polymers remain 194

elusive. 195
The role of metabolons in reactions specific for PA biosynthesis, as suggested in Figure  196 2, requires further investigation. It is now well established that the earlier reactions in the 197 flavonoid pathway are organized in metabolons anchored to the endoplasmic reticulum through 198 association with the cytochrome P450 enzymes of the pathway (Nakayama et al., 2019;Waki et 199 al., 2020), but physical interactions between the later enzymes of the pathway have yet to be based on microscopy of tannin-producing cells from across the plant kingdom (Brillouet 2014(Brillouet , 207 2015Brillouet et al 2014a,b). In this model, PA precursors are synthesized in chloroplasts and 208 polymerized in an organelle termed the tannosome, which is derived from thylakoids, and 209 protected during their intracellular journey to the vacuole in "shuttles" bounded by membranes 210 derived from both inner and outer chloroplast envelopes that have budded from the chloroplast 211 (Brillouet et al., 2014a). The shuttles are then incorporated into the vacuole as tannin accretions 212 by invagination of the tonoplast, thus protecting the cell contents from the protein-binding 213 activity of polymerized PAs (Brillouet et al., 2014a). It appears hard at first sight to reconcile 214 the tannosome model with our current understanding of the biochemistry and genetics of PA 215 8 8 different species and/or cell types. It is also hard to reconcile a model in which all reactions of 217 PA biosynthesis occur together in the same subcellular compartment with the asymmetric 218 labeling of PA subunits (Haslam 1977). 219 220 Testing Intracellular Routes of PA Biosynthesis 221 One impediment to a reconciliation of the models derived from cellular, biochemical, and 222 genetic examinations of PA biosynthesis is the lack of an optimal experimental model system. 223 The descriptive studies on the tannosome have used species in which the biochemistry and 224 genetics of PA biosynthesis are less well defined, and which are poorly amenable to genetic 225 manipulation. Furthermore, histological observations provide varying pictures of tannin 226 deposition and vesicles in different cell types (Vio-Michaelis et al., 2020). The species with the 227 best developed genetic tools (Arabidopsis and Medicago) have significant differences in the 228 biochemical pathways to starter units (Arabidopsis possesses neither LAR nor a functional 229 ortholog of Medicago LDOX), and furthermore only accumulate PAs at significant levels 230 during early seed coat development. Poplar (Populus spp.) may be a better model for future 231 studies. In poplar, PAs are produced naturally in leaves, providing large amounts of material for 232 analysis, and the pathway is also inducible by a number of stresses and chemicals (Mellway et 233 al., 2009;Ullah et al., 2019a). Moreover, poplar is genetically transformable and amenable to 234 gene editing (Bewg et al., 2018), and a very large collection of fully sequenced natural variants 235 is available for genome-wide association studies that are already providing new information on 236 multiple traits including the biosynthesis of the phenolic polymer lignin (Chhetri et al., 2019). genes of PA biosynthesis in this species (Sun et al., 2015), but the exact mechanism for 246 formation of the additional A-type linkages remains to be determined. (insect and animal), and pathogen attack. Studies to address such functions benefit from the use 279 of a plant that produces PAs in major organs and tissues, that possesses well-studied ecology 280 and extensive genetic variation, and that is suitable for genetic manipulation to alter PA profiles. 281 Poplar has emerged as a model species that fits these criteria well. Extracts from poplar bark is subject to natural herbivory, for example by the white satin moth Leucoma salicis, or when 310 mechanically wounded (Peters and Constabel, 2002;Tsai et al., 2006), and this induction may 311 protect the trees against unadapted species. However, PAs are often not effective against 312 adapted herbivore species (Barbehenn and Constabel, 2011). In fact, coevolution may cause 313 CTs to become feeding stimulants for some herbivores (Hjältén and Axelsson, 2015). Further 314 research is therefore necessary to elucidate the roles of PAs in herbivore defense, but the use of 315 transgenic models requires careful examination. For example, after a chance outbreak of thrips, 316 transgenic poplar lines expressing high PA levels were found to be more damaged than their 317 wild-type counterparts. This appears to result from reduction in the levels of phenolic Plectosphaerella populi induces transcriptional activation of the PA biosynthetic pathway in 335 poplar leaves (Mellway et al., 2009, Ullah et al., 2019b, Yuan et al., 2012, and PAs and 336 monomeric catechin reduce mycelial growth of P. populi (Ullah et al., 2017(Ullah et al., , 2019b. 337 Overexpression of MYB115 in poplar resulted in a 50% reduction in lesions caused by 338 Dothiorella gregaria, the causative agent of branch canker, whereas lesion numbers were 339 12 12 increased by 137.5% in CRISPR/Cas9-generated myb115 mutant plants (Wang et al., 2017). 340 Both D. gregaria and M. brunnea f. sp. multigermtubi exhibited reduced mycelial growth, 341 shorter hyphae, swollen tips, and fewer hyphal branches on exposure to extracts from MYB115-342 overexpressing plants as compared to control plants (Wang et al., 2017, Yuan et al., 2012, and 343 the improved response to fungal attack was directly attributed to PA accumulation, as MYB115 344 overexpression did not appear to enhance other defense pathways involving genes such as PR5, 345 JAZ10, MYB44, and NPR1 (Wang 2017). 346 . 347 Most PAs in the soil probably originate from leaf litter. Levels in roots tend to be lower, and not 348 correlated with above-ground levels (Dettlaff et al., 2018). Although tannin-rich leaf litter from 349 MYB134-overexpressing poplar did not result in changes in microbial biodiversity, the leaf 350 litter was found to promote the growth of Eocronartium muscicola, a parasite of mosses, which 351 reduced moss proliferation in soil microcosms (Winder et al., 2013). Use of short-term coppiced 352 plants such as poplar for carbon sequestration has been considered (Quinkenstein and Jochheim, 353 2016). Leaf PAs represent a potentially large sink of carbon for greenhouse gas sequestration, 354 but, before attempting to implement increasing PAs as a carbon-reduction strategy, it will be 355 important to consider further their impacts on nutrient recycling, microbial communities, and 356 greenhouse gas emissions from soil. Furthermore, because of the increasingly realized In a classic review entitled "Proanthocyanidins and the lignin connection", Stafford (1998) 394 discussed similarities between lignin and PAs, concluding that, although both polymers often 395 occur together in plants, PAs are unlikely to play major structural roles. This conclusion still 396 appears generally valid, although it was hypothesized that the helicoidal tridimensional 397 structures of PAs in cell walls of some African "resuscitation plants" might provide protection 398 from cell wall cracking under intense desiccation, allowing the plants to recover rapidly 399 following reinstated water availability (Pizzi and Cameron, 1986). Whatever the natural 5-deoxy-flavan-3-ol with a 3´-,4´-,5´-hydroxy-substituted B-ring, Figure 3, compound 1). 408 Epigallocatechin and epigallocatechin gallate (Figure 3, compound 2)

CONCLUDING REMARKS 437
In spite of an apparently near-complete understanding of the biochemical machinery required 438 for PA biosynthesis, how these molecules are assembled is still perplexing (see Outstanding 439 Questions). The complex pathways for metabolic elaboration and sequestration in PA 440 biosynthesis may be necessary for protecting the plant cell against reactive/toxic intermediates, 441 and providing order to a polymerization mechanism that relies on thermodynamic rather than 442 enzymatic control. Furthermore, the spatial and temporal separation of starter and extension unit 443 biosynthesis and accumulation provides an explanation for the difference in labeling of the 444 "upper" and "lower" units in PAs, in spite of their largely shared biochemical pathways. The 445 potential toxicity to the plant of the intermediates and final products of the PA pathway presents 446 challenges to successful metabolic engineering of the pathway. It is well worth attempting to 447 overcome these challenges in view of the potential advances in agriculture, chemical ecology, 448 carbon sequestration, and biomaterials science that this will facilitate.  • PA and anthocyanin precursors are synthesized in metabolons on the cytosolic face of the endoplasmic reticulum.
• Precursors for both PA and anthocyanin pathways are "protected" by the glutathione Stransferase TT19.

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Separation of PA and anthocyanin biosynthesis is likely temporal in cells that make both classes of compounds; TT19 is also involved in anthocyanin biosynthesis.

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PAs polymerize in vesicles during trafficking to the central vacuole.

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The MATE transporter TT12 and associated vacuolar pH ATPase TT13 are required for loading of PA precursors into vacuoles/vesicles.

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Conjugates of PA precursors with cysteine or glucose may represent storage forms of extension and starter units respectively; transport of glucosides to vacuoles requires TT12.
• PA polymerization is likely nonenzymatic, with LAR (when present) regulating chain length through conversion of extension units to starter units.

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PAs ultimately accumulate in tannin accretions as localized regions within the central vacuole B.
THE TANNOSOME MODEL • PAs are synthesized in chloroplasts, on the outer surface of thylakoid membranes

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The thylakoids pearl into 30-nm spheres (tannosomes), which are then converted into tannosome shuttles through envelopment by a new membrane formed from both thylakoid membranes.

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Tannosome shuttles are incorporated into the vacuole by invagination of the tonoplast. The cytosolic localization of ANS, LDOX, ANR, and LAR (there have been some reports that ANS can be found in the chloroplast).

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The localization of the TT19 GST in cytosol and tonoplast.

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The requirement for a tonoplast MATE transporter and proton ATPase for PA accumulation in the vacuole.

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The vesicle accumulation phenotypes of various tt mutants • Impacts on soil microbiome • High PA levels in leaf litter do not grossly affect microbiome biodiversity, but do promote some microbial species with consequences for above-ground species diversity.
• Impacts on carbon sequestration o Poplar appears to have positive attributes (Quinkenstein and Jochheim, 2016), but contribution of PAs requires further investigation. Procyanidins B1-B4 represent the four possible combinations arising from dimerization of 2,3-cis-(-)-epicatechin and 2,3-trans-(+)-catechin, with procyanidins B2 and B3 being the homodimers of epicatechin and catechin, respectively. The % values represent the approximate % of known structures with that particular unit as starter (lower) or extension (upper) unit. The large black arrows signify radiolabel incorporation from trans-cinnamic acid; the upper units incorporate 3-5 times more label than the lower units, and labeled epicatechin labels lower units only in procyanidin B2. B, Scheme for separate origins of starter and extension units in plants with the LDOX/LAR pathway. Green highlighting indicates potential extension units, and the central green box shows the reactive species thus derived (carbocation and quinone methide) and the nucleophiles that can trap them (light blue ovals). Flav-2-en-3,4-diol is proposed as a potential substrate for generation of 2,3-cis-leucocyanidin for epicatechin extension units. In species that possess LAR, expression of this enzyme can determine chain length by converting Epi-cys (extension unit) to epicatchin (starter unit  The model proposes that reactions specific for starter unit formation from LAR occur on freely soluble enzymes, whereas those associated with extension unit formation occur through a hypothetical metabolon associated with a sub-domain of the endoplasmic reticulum (ER) with tethering through the membrane anchor of the F3´H cytochrome P450 enzyme. The products of these reactions (leucoanthocyanidins) are "captured" and protected by the TT19 GST, including through formation of Epi-cys from 2,3,-cis-leucocyanidin. The TT19 complex interacts with vesicles loaded with starter units through the combined activities of a UGT, the MATE transporter TT12, and the proton APTase TT13 (also known as AHA10). Localization of TT19 to the tonoplast may indicates tight association with PA extension units until they are safely loaded into vesicles harboring starter units. Fusion of the structures containing starter and extension units allows non-enzymatic condensation to form PA dimers and higher oligomers during migration of the pre-vacuolar vesicles to ultimately fuse with the central vacuole, where they are finally deposited as tannin accretions. Enzymes, represented by small circles, are as in the legend to Figure 1B, plus C4H, cinnamate 4-hydroxylase. Enzymes and structures circled in green are associated with extension unit formation, in brown with starter unit formation. This model assumes existence of soluble and insoluble forms of DFR, but other spatial or even temporal controls could allow for separation of the pathways