Functional and phylogenetic analyses of a conserved regulatory program in the phloem of minor veins

Abstract

The minor-vein phloem of mature leaves is developmentally and physiologically distinct from the phloem in the rest of the vascular system. Phloem loading of transport sugars occurs in the minor veins, and consistent with this, galactinol synthase is expressed in the minor veins of melon (Cucumis melo) as part of the symplastic-loading mechanism that operates in this species. A galactinol synthase promoter from melon drives gene expression in the minor-vein companion cells of both transgenic tobacco (Nicotiana tabacum) and Arabidopsis. Neither of these plants use galactinol in the phloem-loading process, implying that the promoter responds to a minor-vein-specific regulatory cascade that is highly conserved across a broad range of eudicotyledons. Detailed analysis of this promoter by truncation and mutagenesis identified three closely coupled sequences that unambiguously modulate tissue specificity. These sequences cooperate in a combinatorial fashion: two promote expression throughout the vascular system of the plant, whereas the third functions to repress expression in the larger bundles. In a complementary approach, phylogenetic footprinting was used to obtain single-nucleotide resolution of conserved sites in orthologous promoters from diverse members of the Cucurbitaceae. This comparative analysis confirmed the importance of the closely coupled sites but also revealed other highly conserved sequences that may modulate promoter strength or contribute to expression patterns outside of the phloem. The conservation of this regulatory design among species that phloem load by different mechanisms supports a model for organismal development in which tissues and cell types are controlled by relatively ancient and conserved paradigms but expression of genes influencing final form and function are relatively plastic.

Figure 1.

CmGAS1 promoter truncations, and resulting GUS activity. A, Promoter truncations (5′) created with convenient restriction endonuclease recognition sites. B, Promoter truncations (5′ and 3′) created by PCR or site-directed mutagenesis and possessing a synthetic, basal promoter consisting of the first 60 bp of the 35s promoter fused to a translational enhancer from TEV (represented by hatched boxes; TT, transcription initiation/translational enhancer). Promoter fragments that confer minor-vein uidA expression, determined in histochemical assays with X-GlcA, are indicated as gray bars, and white bars indicate those that do not. Sequence coordinates are indicated along the top, relative to the CmGAS1 start codon (+1). The uidA reporter gene is indicated in black and is not drawn to scale. Plasmids are labeled to the right of each schematic (pGPTV designation omitted). Relative GUS activities measured in fluorescent assays with 4-methylumbelliferyl β-d-glucuronide hydrate (MUG) as substrate, in arbitrary units, are indicated on the right by black circles; ×15 indicates 15-fold GUS activity relative to a single black circle; ND, not determined; hollow circles, no activity; n > 12 pooled seedlings. The black circle(s) representing GUS activity in plants harboring pGPTVBam101 and pGPTV-TT(–1,333–1025) result from GUS activity in hydathodes, because minor-vein X-GlcA staining was not observed.

Figure 2.

High-resolution mutagenesis of the CmGAS1 promoter and analysis of resulting expression patterns. A, CmGAS1 mutations and resulting expression patterns. CmGAS1 promoter sequences –1,333 to –984 are indicated by the black text. The corresponding mutant sequences, indicated 1 to 35 and separated by spaces, are indicated above in red, italicized text. Blocks I, II, and III, which resulted in altered expression patterns, are enclosed in gray boxes. Within each block, underscored mutant sequences (17, 22, and 32) abolished expression in the minor vein, but retained expression in hydathodes and root apices, overscored mutations (21 and 31) abolished gene expression in all tissues, and the mutation underscored with asterisks (18) resulted in gene expression in all vascular tissue. Mutations surrounding block II resulting in a approximately 50% reduction in staining intensity are enclosed in a white box; n > 20 seedlings. B, Representative plant with uidA expression controlled by the –1,333 to –984 promoter. The observed expression pattern is characteristic of all mutations excluding the blocks enclosed by gray boxes in A. C, uidA expression in hydathodes without minor-vein expression. The imaged plant harbors mutation 17, but is characteristic of those with mutations 22 and 32. D, uidA expression in all veins resulting from mutation 18. E, Absence of uidA expression in all tissues. A plant harboring mutation 21 is shown and is also representative of plants harboring mutation 31.

Figure 3.

Alignment of conserved GAS1 promoter sequences among eight genera in the Cucurbitaceae. A, Graphic representation of alignment constructed with MACAW. Horizontal bars represent sequences from each ortholog, with the location of the ATG start codon indicated by the dashed line with an arrow on the right. Total analyzed sequence lengths upstream of the start codons are indicated on the left. Sequences demonstrating roughly 90% similarity over at least 8 bp among the genera are indicated by vertically aligned black boxes. Gaps introduced to bring sequences into alignment are indicated by the lack of horizontal bars. Aligned sequences enclosed by the box are presented in detail in B. B, Alignment of sequences enclosed by the box in A. Corresponding mutations are indicated above the alignments, using the same pattern of symbols as in Figure 2A. Sequential mutants alternate in upper and lowercase solely to emphasize boundaries. Sequences selected as significant blocks of conservation are indicated in uppercase text. Sequences that are identical among all eight genera are indicated by white text on a black background. See also supplemental data.