Expression of SULTR2;2 in the Arabidopsis bundle sheath is mediated by a highly conserved positive regulator

The bundle sheath provides a conduit linking veins and mesophyll cells. In C3 Arabidopsis thaliana it also plays important roles in oxidative stress and sulphur metabolism. However, the mechanisms responsible for the patterns of gene expression that underpin these metabolic specialisations are poorly understood. Here we used the A. thaliana SULTR2;2 gene as a model to better understand mechanisms that restrict expression to the bundle sheath. Deletion analysis indicated that the SULTR2;2 promoter contains a short region necessary for expression in the bundle sheath. This sequence acts as a positive regulator and is tolerant to multiple consecutive deletions indicating considerable redundancy in the cis-elements involved. It is highly conserved in SULTR2;2 genes of the Brassicaceae and is functional in the distantly related C4 species Flaveria bidentis that belongs to the Asteraceae. We conclude that expression of SULTR2;2 in the bundle sheath is underpinned by a highly redundant sequence that likely represents an ancient and conserved mechanism found in families as diverse as the Asteraceae and Brassicaceae.


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The evolution of multicellularity is associated with individual cell-types being able to undertake 45 specialised roles within a tissue. In leaves, bundle sheath (BS) cells form a wreath-like structure 46 around the vasculature that appears analogous to the endodermis of roots (Esau, 1965). The role of 47 BS cells is best characterized in C 4 species that partition photosynthesis between the BS and 48 mesophyll cells. In most C 4 plants, after HCO 3 is initially fixed into C 4 acids by phosphoenolpyruvate 49 carboxylase in mesophyll cells, these C 4 acids then diffuse to the BS where CO 2 is released and 50 refixed by Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO). Decarboxylation of C 4 51 acids in the BS generates a high concentration of CO 2 around RuBisCO that suppresses the 52 oxygenase activity of the enzyme and in so doing reduces photorespiration (Hatch, 1987). Thus, in 53 C 4 species, the BS is specialized to allow efficient fixation of CO 2 in the Calvin-Benson-Bassham 54 cycle. In some C 4 plants, the BS is also modified in terms of light capture. For example, in maize and 55 sorghum Photosystem II does not fully assemble in the BS (Kubicki et al., 1994) but components of 56 cyclic electron transport are more abundant in the BS compared with mesophyll cells (Takabayashi 57 et al., 2005). In addition to these changes associated with photosynthesis, the C 4 BS is also modified 58 to preferentially undertake starch synthesis and degradation, as well as the initial steps of sulphur 59 assimilation (Friso et al., 2004).

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In C 3 plants, the role of the BS is less clearly defined. It is thought to help maintain hydraulic 61 integrity of the xylem (Sade et al., 2014), regulate flux of metabolites in and out of the leaf (Leegood, 62 2008) and act as a starch store (Miyake and Maeda, 1976). The C 3 BS is less important for 63 photosynthesis than that of C 4 species. However, although only around 15% of all chloroplasts of 64 the C 3 leaf are found in bundle sheath cells (Kinsman and Pyke, 1998), reducing photosynthesis in 65 these cells compromises growth and seed production (Janacek et al., 2009). Thus, although less 66 photosynthetic than the C 4 BS, this physiological analysis indicates that the BS of C 3 plants is also 67 specialized. This notion is consistent with analysis of gene expression in this cell type. For example 68 quantification of transcripts available for translation indicate that the A. thaliana BS is likely important 69 in sulphur metabolism, glucosinolate biosynthesis, trehalose metabolism and detoxification of active 70 oxygen species (Aubry et al., 2014). In summary, in both C 3 and C 4 plants mechanisms must operate 71 to restrict the expression of some genes to BS cells.

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To date, most studies of the mechanisms responsible for preferential gene expression in the BS

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have occurred in C 4 species (Hibberd and Covshoff, 2010). In the C 4 dicotyledon Flaveria trinervia 74 the glycine decarboxylase P-subunit (GLDPA) gene contains two promoters, one proximal to the 75 coding region, and the other more distal. Activity of the distal promoter is high but not cell-type 76 specific. However, in the presence of the proximal promoter, transcripts derived from the distal 77 promoter are degraded in mesophyll cells by nonsense-mediated RNA decay of incompletely spliced 78 transcripts (Engelmann et al., 2008;Wiludda et al., 2012). Despite the phylogenetic distance 79 between the Brassicaceae and the Asteraceae the GLDPA promoter from F. trinervia is able to 80 generate BS-specific activity in C 3 A. thaliana (Engelmann et al., 2008;Wiludda et al., 2012). In

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This region acts to enhance expression in the BS rather than repressing expression in mesophyll 106 cells. Furthermore, the SULTR2;2 promoter from A. thaliana generates BS specificity in the C 4 107 species F. bidentis that belongs to the Asteraceae. The most parsimonious explanation for this 108 finding is that a common transcription factor is shared by these phylogenetically dispersed species,

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and that it functions in both the C 3 and C 4 BS.

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Nucleotides -2815 to +123 relative to the predicted SULTR2;2 translational start site have 113 previously been reported to generate expression in the BS of A. thaliana (Takahashi et al., 2000).

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We confirmed this finding ( Figure 1A-C). To test if sequence after the predicted start codon is 115 required for expression in the BS a construct that terminated at nucleotide -1 relative to the predicted 116 translational start was generated ( Figure 1D). Staining showed that each construct led to strong 117 accumulation of GUS in the BS of mature rosette leaves ( Figure 1B,C,E,F). Staining was evident in 118 vascular tissue as well as the BS but there was no evidence that GUS accumulated in mesophyll or 119 epidermal cells from either construct ( Figure 1B,C,E,F). Promoter activity was quantified using the 120 MUG fluorimetric assay. GUS activity driven by the construct that contained 603 fewer base pairs at 121 the 5' end but an additional 123 nucleotides of coding sequence was about tenfold higher ( Figure   122 1G). This difference could either be due to a negative regulator located between -3418 and -2185 123 nucleotides, or a positive regulator in sequence downstream of the predicted translational start site.

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A translational fusion between the yellow fluorescent protein (YFP) and the nuclear localized Histone

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2B protein under control of the SULTR2;2 promoter ( Figure 1H) labelled nuclei of BS cells and 126 vascular tissue ( Figure 1I) and indicated that the presence of GUS in the BS was due to gene 127 expression and not diffusion of the dye outwards from to vascular tissue. Consistent with previous 128 reports ( Chytilova et al., 1999) it was noticeable that vascular nuclei were elongated and rod-like 129 compared with the more spherical ones in the BS ( Figure 1I). We conclude that elements between

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The lack GUS accumulation in the BS and loss of promoter activity once region 2 is removed 145 could be because this region contains cis-elements that generate expression specifically in BS cells 146 or because it drives ubiquitous expression but regions 3 to 5 contain elements that restrict activity to

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The cis-regulatory elements necessary for BS-specific expression of AtSULTR2;2 appear to be

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Alignments of sequences 5 kb upstream of each orthologue indicated that with the exception of A.
197 lyrata that contains a 446 nucleotide insertion, region 2 is highly conserved ( Figure 6). However, no 9 short sequences or motifs within this sequence that may restrict expression to the BS could be 199 identified ( Figure 6). Although the results of this alignment therefore do not identify a specific cis- for strength of expression to be reduced. We therefore propose that either multiple independent BS 233 modules contained within this region act additively, or that distinct quantitative elements are co-234 located, and at least partially overlapping with, cis-elements that determine this cell-specificity.

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Redundancy of this sort has previously been reported for the promoter of Phenylalanine Ammonia

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The Mann-Whitney U test was used to determine statistical differences between datasets. Imaging

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Orange arrowheads within the core promoter indicate transcription start sites obtained by 5'-RACE.