The bundle sheath of rice is conditioned to play an active role in water transport as well as sulfur assimilation and jasmonic acid synthesis

Leaves comprise multiple cell types but our knowledge of the patterns of gene expression that underpin their functional specialization is fragmentary. Our understanding and ability to undertake rational redesign of these cells is therefore limited. We aimed to identify genes associated with the incompletely understood bundle sheath of C3 plants, which represents a key target associated with engineering traits such as C4 photosynthesis into rice. To better understand veins, bundle sheath and mesophyll cells of rice we used laser capture microdissection followed by deep sequencing. Gene expression of the mesophyll is conditioned to allow coenzyme metabolism and redox homeostasis as well as photosynthesis. In contrast, the bundle sheath is specialized in water transport, sulphur assimilation and jasmonic acid biosynthesis. Despite the small chloroplast compartment of bundle sheath cells, substantial photosynthesis gene expression was detected. These patterns of gene expression were not associated with presence/absence of particular transcription factors in each cell type, but rather gradients in expression across the leaf. Comparative analysis with C3 Arabidopsis identified a small gene-set preferentially expressed in bundle sheath cells of both species. This included genes encoding transcription factors from fourteen orthogroups, and proteins allowing water transport, sulphate assimilation and jasmonic acid synthesis. The most parsimonious explanation for our findings is that bundle sheath cells from the last common ancestor of rice and Arabidopsis was specialized in this manner, and since the species diverged these patterns of gene expression have been maintained. Significance statement The role of bundle sheath cells in C4 species have been studied intensively but this is not the case in leaves that use the ancestral C3 pathway. Here, we show that gene expression in the bundle sheath of rice is specialized to allow sulphate and nitrate reduction, water transport and jasmonate synthesis, and comparative analysis with Arabidopsis indicates ancient roles for bundle sheath cells in water transport, sulphur and jasmonate synthesis.


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The rice bundle sheath is specialized for transport but also photosynthesis 153 To gain insight into the genetic basis for functional specialization associated with 154 mesophyll, bundle sheath and veinal cells of rice, laser capture microdissection was used 155 to isolate RNA from these tissues. Paradermal sections allowed the unambiguous 156 identification of mesophyll cells ( Figure 1A), bundle sheath cells containing large vacuoles 157 and fewer chloroplasts ( Figure 1C) and veins ( Figure 1E). To isolate each cell-type with 158 minimal cross contamination, mesophyll cells were first dissected and captured ( Figure 1A Figure 1H) and in veins they were not 164 discernable ( Figure 1I). 165 Three prime mRNA sequencing was performed and from each cell type, 24-36 million 166 reads from four or five biological replicates obtained. After processing to remove low quality 167 reads 13-23 million were quantified against the rice cDNA reference (MSU v7) 168 (Supplemental Table 1). An average of 10,097, 10,083 and 13,648 transcripts were detected 169 in each cell type (Supplemental Table 1). Spearman ranked correlation coefficients for gene 170 expression showed little variation between replicates from each cell type, and that each cell 171 type exhibited distinct patterns of gene expression ( Figure 1J). Principle components 172 analysis also showed close grouping of biological replicates, and that 46.1% of variance was 173 associated with the three cell types whilst that a second component separated the bundle 174 sheath from mesophyll and veins ( Figure 1K). To assess the purity of the tissues sampled, 175 we examined transcript abundance of genes previously reported to be associated with each

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To quantify the extent to which transcript abundance differed between bundle sheath, 189 mesophyll and veinal cells, we performed differential gene expression analysis using  Table 2). Functional enrichment analysis identified three categories over-  Figure 2B). In contrast, transcripts preferential to veins were associated with 213 processes that included RNA biosynthesis, protein homeostasis, lipid metabolism and solute 214 transport (Supplemental Figure 2A). When mesophyll and veins were compared, a greater 215 number of differentially expressed genes were identified with 1,728 and 2,038 transcripts 216 being more abundant in mesophyll and veins respectively (Supplemental Table 2). The 217 expected preferential expression of photosynthesis-related genes in mesophyll cells was 218 detected, and transcripts associated with protein biosynthesis and cellular respiration were 219 more abundant in veins (Supplemental Figure 2C&D). The greater number of differentially 220 expressed genes between mesophyll and veins is consistent with the correlation and PCA 221 analysis ( Figure 1J&K). 222 To further assess patterns of transcript abundance across all three cell types we clustered 223 genes based on expression. 4155 genes defined as being differentially expressed in the 224 pairwise comparisons above were partitioned into six clusters associated with the cell types 225 in which they were preferentially expressed ( Figure 1N). Veins (CV) had the largest (972) 226 whilst bundle sheath cells (CBS) had the fewest (285) number of preferentially expressed 227 genes. Functional enrichment analysis showed that genes in the mesophyll (CM) were over- 228 represented in photosynthesis, coenzyme metabolism and solute transport, whilst CBS were 229 enriched in solute transport, enzyme classification, amino acid metabolism and polyamine 230 metabolism ( Figure 1O). Genes in CV were involved in cellular respiration, polyamine 231 metabolism, carbohydrate metabolism and phytohormone action ( Figure 1O). CBS&M 232 contained genes highly expressed in both mesophyll and bundle sheath, and was over-233 represented in processes including photosynthesis, coenzyme metabolism, carbohydrate 234 metabolism, redox homeostasis, secondary metabolism ( Figure 1O). CBS&V contained genes 235 associated with protein biosynthesis, cellular respiration, and solute transport ( Figure 1O).

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No enriched categories were associated with both the mesophyll and vein cells (CM&V).

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Consistent with their distinct function, vein and mesophyll clusters showed low overlap, but 238 the most abundant transcripts in bundle sheath cells were also either expressed in veins or 239 mesophyll cells. Overall, and associated with their morphology, the data reveal a gradient in 240 photosynthesis-related transcripts from low in veins to high in mesophyll cells. (APC) superfamily were also present (Supplemental Figure 3A).

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The MIP group contains genes encoding water channels (aquaporins) including plasma 252 membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs) (Sakurai et al., 2005).   The data also indicate that gene expression in the bundle sheath is conditioned for 285 synthesis of glutathione, a major sulphur-containing metabolite that plays critical roles in 286 redox homeostasis and heavy metal(loid) detoxification. Biosynthesis of glutathione is 287 catalyzed by γ-glutamylcysteine synthetase (ECS) to generate γ-glutamylcysteine (γ-EC) 288 from glutamate and cysteine, followed by ligation of glycine and γ-EC by glutathione 2C). Rice absorbs both arsenate and arsenite by different transporters, but arsenate needs 296 to be reduced into arsenite before it can be detoxified by phytochelatin. CLT1 has been 297 reported to be critical for rice tolerance to arsenic because it determines phytochelatin 298 biosynthesis and arsenate reduction (Yang et al., 2016). Notably, the arsenate reductase 299 HAC1;1 also showed preferential expression in the bundle sheath, suggesting that this cell 300 type may play an important role in arsenate reduction and detoxification ( Figure 2E).

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As with sulphur assimilation, transcripts encoding some of the pathway allowing nitrate 302 reduction were more highly expressed in the bundle sheath and veinal cells compared with 303 mesophyll cells. Interestingly, this included the nitrate transporters NRT1.4, NRT1.1A, 304 NRT1.2, and NRT2.3, both nitrate reductases (NIA1 and NIA2), nitrite reductase (NIR) and 305 glutamine synthetase (GS1.1). Transcripts encoding NRT2.3, NIA1 and NIA2 were most 306 abundant in bundle sheath cells, while the rest were also highly expressed in veins. In 307 contrast, transcripts encoding glutamine synthetase (GS2) and glutamate synthase (Fd-308 GOGAT) that allow ammonia assimilation in the chloroplast were preferentially expressed in 309 the bundle sheath and mesophyll relative to veinal cells ( Figure 2F, Supplemental Figure 4). 310 These results indicate that gene expression in the rice bundle sheath is also tuned to 311 specialise in nitrate assimilation and amino acid biosynthesis.  Figure 5D). Although transcripts associated the glucose 6-337 phosphate/phosphate translocator (GPT1, GPT2-1) were more abundant in bundle sheath 338 than mesophyll cells, their abundance was low compared with the triose 339 phosphate/phosphate translocator (TPT1) (Supplemental Figure 5D). Together, these data  Figure 6C).

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The bundle sheath specific cluster contained only ten genes that derived from families   Table 5). 480 We compared bundle sheath preferentially expressed genes of rice and Arabidopsis with bundle sheath resembles the mesophyll more than veins indicates that it needs to be re-546 tuned rather than completely re-programmed to achieve this demanding aim. sheath is patterned to facilitate water transport and storage, sulphate and nitrate assimilation.

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It was notable that most of highly expressed aquaporins were preferentially expressed in 590 bundle sheath cells, and included members of the PIP1, PIP2, TIP1 and TIP2 subfamilies.

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It has been reported that water transport activity and plasma membrane localization of PIP1

Plant growth condition and sample preparation 668
The temperate rice (Oryza sativa ssp. japonica) Kitaake was germinated and grown in a 669 mixture of 1:1 topsoil and sand for 2 weeks in a controlled environment growth room.

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Temperature was set to 28°C day, 25°C night, and photoperiod at 12 hr light and 12 hr dark. Competing interests 787 The authors declare that they have no competing interests.          Significantly enriched or depleted mo�fs were iden�fied in each of the cistromes from the 6 gene expression clusters, enrichment was calculated using the regioneR permuta�on tes�ng package (Gel et al., 2016) following mo�f scanning using FIMO to iden�fy mo�fs from the plant Jaspar nonredundant database (Fornes et al., 2020). The Z-scores are shown with a colour scale to show the magnitude of enrichment (dark blue) or deple�on (yellow) for mo�fs that were significant a�er mul�ple tes�ng correc�on. Mo�fs derived from closely related TFs were grouped together for visualisa�on based on their degree of overlap to predicted target sites (e.g. AP2ERFs). The cistrome from cluster C V shows the greatest number of enriched mo�fs, including 13 uniquely enriched, while the C M and C BS cistromes have far fewer.(C) Cluster specific TFs (le� of panel) were mapped to mo�fs (right of panel) they would be most likely to bind based on high protein sequence similarity with the proteins in the Jaspar plant mo�f database. The TFs that mapped to any enriched mo�fs are shown with the mo�f enrichment data. This allows visualisa�on of the intersec�on between TF transcript abundance with poten�al ac�va�on ac�vity. Gene symbols of rice transcrip�on factors were retrieved from funRiceGene database (Yao et al., 2018) but for the symbols not found in the database, symbols of best hit Arabidopsis transcrip�on factors were used and presented in blue. The matching mo�fs show first the best match and then the mo�f group if part of a group as shown in 4B.