Candidate transporters

transport. In developing tracheids, sugar transport is an important process as it regulates turgor pressure and controls the import of the raw materials for SCW synthesis. The transporters detected in the proteomic analysis likely play a role in this process, but as we detected in vitro MG transport in non-lignifying BY-2 cells too, it remains possible that a non-specific transporter for glycosylated compounds is mediating this transport and that the expression of this transporter is constitutive.

On the other hand, the transporter could be targeted specifically for MG transport. In this case, we would expect that gene expression of the transporter would have a high correlation with the expression of genes involved in MG biosynthesis. This is the case in sorghum (Sorghum bicolor) where there is co-expression with biosynthetic genes for the cyanogenic glucoside dhurrin, and SbMATE2 which is able to transport dhurrin (Darbani et al., 2016). Accordingly, a co-expression analysis was done using pre- existing, published datasets of gene expression in Norway spruce to validate these candidate PaMGT transporters. For this, I used ConGenIE (Nystedt et al., 2013), a dataset of gene expression data for Norway spruce, a dataset containing expression data for the Norway spruce cell culture in both lignin-forming and non-lignin-forming conditions (Laitinen et al., 2017), Norwood, which contains gene expression data for the developing xylem of Norway spruce (Jokipii-Lukkari et al., 2017), and a dataset of gene expression from tracheids and ray parenchyma cells isolated by laser-capture microdissection (Blokhina et al., 2019). A Pearson correlation analysis was performed to ascertain which genes are co-expressed with monolignol biosynthesis genes in these four datasets. In Norway spruce, the phenylpropanoid and monolignol biosynthesis pathway enzymes are encoded by small gene families. Jokipii-Lukkari et al. (2018) found a set of 12 monolignol biosynthesis enzymes of Norway spruce which represent all the biochemical reactions needed for pCA and CA biosynthesis, and are co- expressed in the developing xylem throughout a year. The expression of these 12 genes was also correlated in the four datasets described above (II). Further evidence for their role in monolignol biosynthesis in the developing xylem of Norway spruce came from the proteomic data: six of the 12 enzymes were detected in xylem membranes, but only two other isoenzymes were present. This supports the designation by Jokipii-Lukkari et al. (2018) that these 12 enzymes participate in monolignol biosynthesis in the developing xylem of Norway spruce. Thus, we decided to use the 12 genes as baits to study whether there are transporters whose expression is tightly linked to these.

In the gene expression correlation analysis, 15 MATE transporters showed expression following the bait gene expression in the cell culture, but only three (MA_94941g0010, MA_10431371g0010, and MA_110289g0010) had co-expression to baits in the ConGenIE data. One of them, MA_94941g0010, correlated with the core lignin biosynthesis genes in the Norwood dataset as well, making it an excellent MGT candidate (II). Altogether, 95 MFS transporters showed gene co-expression with the monolignol pathway bait genes in at least one of the datasets, with the cell culture MFS transporters being the most co-regulated with the monolignol pathway. Only a few MFS transporters were co-expressed with the baits in more than one dataset.

MA_10429543g0010, which was detected in the xylem proteomics as well, as described above, had gene co-expression with the baits in the ConGenIE data and in the cell culture dataset, suggesting a role during lignification. Co-expression with the baits in three datasets was detected for five MFS transporters. The first one (MA_22713g0010) is a homologue of Arabidopsis phosphate transporter 4;2, which localises at plastid membranes and is thought to play a role in plant growth and starch accumulation (Irigoyen et al., 2011). The second and the third (MA_13801g0010 and MA_304122g0010) belong to a sugar porter (SP) subfamily and are homologous to Early Response to Dehydration 6 (ERD6)-like family members. Members of the ERD6- like family transport monosaccharides at the tonoplast via facilitated diffusion or via H⁺ symport (Yamada et al., 2010; Klemens et al., 2014). The fourth and the fifth (MA_10437216g0010 and MA_119405g0010) are members of a Nitrate Transporter 1/Peptide Transporter (NTR1/PTR) family, and various members of this family are capable of transporting nitrate, peptides, phytohormones, and glucosinolates (Corratge-Faillie and Lacombe, 2017).

Although none of the MATE and MFS candidates discussed here meet all the criteria as a PaMGT, it is possible that some of these transporters accept MGs, as the substrate ranges of individual transporters are not fully understood, even in species that are studied most, not to mention in Norway spruce. It is also important to note that not all the MFS transporters that were identified are predicted to be H⁺ antiporters. The structure of a secondary active transporter can be difficult to predict from the protein sequence (Boudger and Vernon, 2010; Forrest et al., 2011) and, even within subfamilies, the transport mechanisms vary. To further study transport, the selected candidates should be tested biochemically after heterologous production, and/or loss- of-function or gain-of-function spruce plants should be created. Gene modification in

Norway spruce has been challenging, as we have experienced personally (III).

However, the methods are being developed continuously. Silencing suitable candidates in other trees, like poplar which also has MGTs, is another good alternative.

ABC transporter-mediated transport was not detected in the biochemical assays.

However, the fact that these transporters have long been discussed as candidates for monolignol transport, as well as the results already obtained with Arabidopsis (Miao and Liu, 2010; Alejandro et al., 2012) encouraged us to also consider ABC transporters in Norway spruce. The proteomic analysis led to the identification of 21 ABC transporters, the gene co-expression analysis resulted in 47 ABC transporters, and six ABC transporters were identified by both strategies. Out of these transporters, we selected ABCB and ABCG transporters as candidates based on their presence in spruce membranes, based on co-expression with monolignol biosynthesis genes, or based on sequence similarity to pCA transporter AtABCG29. There were 14 ABCG, six ABCB, and one ABCD transporters present in the proteomics data (II). Of those, perhaps the most interesting is the ABCG transporter encoded by MA_18770g0010. This protein was present in the xylem membrane proteome and was co-expressed with the bait biosynthetic genes in three datasets. However, the closest Arabidopsis homologue for this transporter, AtABCG40, is known to import abscisic acid (ABA) at the PM (Kang et al., 2010). The homology between these two sequences is 66.5%, which indicates that there may be substantial differences. ABCG, encoded by MA_17319g0020, was present in all the types of membranes analysed and its gene expression was correlated with the monolignol biosynthetic genes used as baits with the ConGenIE expression dataset.

Furthermore, it is homologous to the putative Arabidopsis monolignol transporter ABCG34 identified by Takeuchi et al. (2018). The gene encoding ABCG34 was co- expression with genes known to be involved in lignification and its regulation. Other homologues to ABCG34 (MA_10184926g0010 and MA_10434601g0010) were also present in xylem membranes but were not co-expressed with the baits and, as such, were not included as candidates. MA_134489g0020 and MA_10260477g0010 had ABCG29 as the closest Arabidopsis homologue and were present in the membrane proteomics. Two more ABCGs (MA_135152g0010 and MA_31011g0010) were selected as candidates. The first one was present in the proteomic analysis and the second one was the only ABCG transporter selected as a candidate solely based on its gene co- expression results. ABCB transporters have received only limited attention in the discussion on lignification. But in this work, four ABCB transporters were present in

the proteomic analyses and were selected as candidates (MA_10434957g0010, MA_62683g0010, MA_9415070g0020, and MA_40328g0010). All were co-expressed with monolignol biosynthesis based on the analysis of the ConGenIE data and the data by Blokhina et al. (2019). Two ABCB transporters (MA_138894g0010 and MA_635039g0010) were selected as candidates based solely on their co-expression with monolignol bait genes in ConGenIE and Norwood datasets.

Without a doubt, many of the ABC transporters that I have mentioned here likely play a role in phytohormone transport, as that is one of the important functions of ABC transporters in plants (Borghi et al., 2019). It has been pointed out that local concentrations of hydrophobic molecules may threaten the integrity of the membranes, and ABC transporters may expel these hydrophobic compounds from membranes (Lefèvre and Boutry, 2018). The concentration of phenolic compounds needed to destabilise the structure of the membrane may, however, not be biologically relevant. More modestly, effects on membrane ion permeabilities (Hossain et al., 2021) or the inhibition of membrane enzymes (Sikkema et al., 1995) could have an effect. In addition, a role for phenylpropanoid transporters in the regulation of the phenylpropanoid pathway has been suggested (Biała et al., 2017; Biała and Jasiński, 2018). Even pathway intermediates could be transported, similarly to the way that ABCG10 transports 4-coumarate and liquiritigenin, which are intermediates of the medicarpin biosynthetic pathway (Biała et al., 2017). However, it is not known if such regulatory transport of monolignol biosynthesis intermediates or monolignols exists.

Interestingly, an amino acid permease family (AAP) protein encoded by MA_18076g0010 was also co-expressed with the monolignol biosynthesis baits (II).

AAPs are proton symporters for amino acids at the PM (Fischer et al., 2002). Lignin biosynthesis requires the transport of various intermediates for monolignol biosynthesis pathway within the cell, and this transporter could be involved in the transport of phenylalanine or shikimate, for example, from the plastid.

3.5. Lignification enzymes associated with the membranes