Nectary development in Cleome violacea

Abstract

Nectaries are a promising frontier for plant evo-devo research, and are particularly fascinating given their diversity in form, position, and secretion methods across angiosperms. Emerging model systems permit investigations of the molecular basis for nectary development and nectar secretion across a range of taxa, which addresses fundamental questions about underlying parallelisms and convergence. Herein, we explore nectary development and nectar secretion in the emerging model taxa, Cleome violacea (Cleomaceae), which exhibits a prominent adaxial nectary. First, we characterized nectary anatomy and quantified nectar secretion to establish a foundation for quantitative and functional gene experiments. Next, we leveraged RNA-seq to establish gene expression profiles of nectaries across three key stages of development: pre-anthesis, anthesis, and post-fertilization. We then performed functional studies on five genes that were putatively involved in nectary and nectar formation: CvCRABSCLAW (CvCRC), CvAGAMOUS (CvAG), CvSHATTERPROOF (CvSHP), CvSWEET9, and a highly expressed but uncharacterized transcript. These experiments revealed a high degree of functional convergence to homologues from other core Eudicots, especially Arabidopsis. CvCRC, redundantly with CvAG and CvSHP, are required for nectary initiation. Concordantly, CvSWEET9 is essential for nectar formation and secretion, which indicates that the process is eccrine based in C. violacea. While demonstration of conservation is informative to our understanding of nectary evolution, questions remain. For example, it is unknown which genes are downstream of the developmental initiators CvCRC, CvAG, and CvSHP, or what role the TCP gene family plays in nectary initiation in this family. Further to this, we have initiated a characterization of associations between nectaries, yeast, and bacteria, but more research is required beyond establishing their presence. Cleome violacea is an excellent model for continued research into nectary development because of its conspicuous nectaries, short generation time, and close taxonomic distance to Arabidopsis.

Keywords: Cleomaceae; RNA-seq; VIGS; nectar; nectaries; parallel evolution; transcriptomics.

Figure 1

Cleome violacea flowers at various stages of development. (A) Large undissected floral bud. (B) Large dissected floral bud showing nectary. (C) Newly anthetic flower. (D) Post-anthetic flower with developing fruit. (E) Magnified view of anthetic nectary. Scale bars = 1 mm.

Figure 2

Alcian blue/safranin O-stained sections of Cleome violacea nectaries at pre-anthetic, anthetic and post-anthetic stages. From left to right: (A–C) small, (D–F) medium, and (G–I) large buds in transverse view with proximal-distal indicating relative distance to receptacle. (J–L) Large bud and (M–O) flowers in longitudinal view with lateral-medial indicating relative distance from center. (P, Q) Longitudinal view of 20 μm slices of the same large floral bud with and without vascular tissue, respectively. Scale bars = 250 μm. Sad = adaxial sepal; Sab = abaxial sepal; Pad = adaxial petal; Pab = abaxial petal; s = stamen; g = gynoecium, r = receptacle.

Figure 3

Scanning electron micrographs of whole nectaries from Cleome violacea at (A) pre-anthetic and (B) anthetic stages. (C) Distribution of nectarostomata on pre-anthetic nectary lobe and (D) anthetic nectary lobe. Examples of nectarostomata from (E–G) bud and (F–H) anthetic flowers.

Figure 4

Nectar volume from Cleome violacea flowers taken on first day of anthesis (Day 1) and three days post-anthesis (Days 2, 3, and 4). Averaged value of flowers from 20 plants. Significance measured using paired, one-tailed, student’s t-tests (α < 0.01).

Figure 5

Z-score heatmaps of (A) all differentially expressed transcripts and (B) transcripts with TPM > 100 from Cleome violacea pre-anthetic, anthetic, and post-anthetic nectaries.

Figure 6

A heatmap of phytohormone-related transcripts expressed in pre-anthetic, anthetic, and post-anthetic nectaries of Cleome violacea displayed in log2(TPM). Representative transcripts of genes related to (A) auxin, (B) gibberellic acid (C) jasmonic acid, and (D) ethylene. Significance displayed in brackets. 1 = pre-anthetic; 2 = anthetic; 3 = post-anthetic.

Figure 7

Heatmap of (A) 16s bacterial rRNA and (B) 18s fungal rRNA related transcripts in pre-anthetic, anthetic, and post-anthetic nectaries of Cleome violacea displayed in log2(TPM). Genera and NCBI accessions of respective transcripts for bacteria and fungi outlined in (C, D), respectively.

Figure 8

Cleome violacea flowers from untreated and treatment control groups. (A) Untreated newly anthetic flower and (B) maturing flower. pTRV2-MCS treated flower displaying (C) moderate and (D) mild viral phenotype. (E, F) pTRV2-CvANS treated flowers displaying moderate yellowing petal phenotypes. Scale bars = 1 mm.

Figure 9

Flowers of Cleome violacea treated with pTRV2-CvCRC-CvANS constructs. (A) Flower with strong yellowing phenotype and no nectary. (B) Flower with moderate yellowing phenotype and no nectary. (C) Flower with moderate yellowing phenotype, no nectary, and enlarged gynoecium. (D) Flower with moderate yellowing phenotype and no nectary. (E) Flower with half normal and half yellowing petals with partially absent nectary. (F) Flower with strong yellowing phenotype and reduced lateral nectary lobes. Scale bars = 1 mm.

Figure 10

Flowers of Cleome violacea treated with pTRV2-CvSWEET9-CvANS constructs. (A) Flower with moderate yellowing and nectary with reduced nectar accumulation. (B) Magnified view of nectary in A. (C) Flower with partial yellowing and partial normal phenotype. (D) Flower with near-normal pigmentation and reduced nectar production. (E) Magnified nectary from C displaying decreased nectar accumulation correlating with yellowing phenotype. Scale bars = 1 mm.

Figure 11

Flowers of Cleome violacea treated with pTRV2-CvAG constructs. (A) Flower with repeating perianth whorls. (B) Nectary from flower similar to A with petals removed. (C) Flower with normal adaxial petals, repeating perianth whorls and adaxial nectary. (D) Flower with repeating perianth whorls and distally positioned nectary. (E) Flower with petaloid stamens and adaxial nectary. (F) Flower with repeating perianth whorls and adaxial nectary. White arrowheads indicate nectary position. Scale bars = 1 mm.

Figure 12

Flowers of Cleome violacea treated with pTRV2-CvAG-CvSHP constructs. (A) Flower with partial nectary. (B–E) Flowers with repeating perianth whorls and no nectary. (F) Flower with repeating perianth whorls and partial nectary. Black and white arrowheads represent reduced and absent nectary, respectively. Scale bars = 1 mm.