Facile Labeling of Sieve Element Phloem-Protein Bodies Using the Reciprocal Oligosaccharide Probe OGA 488.

Sieve elements of many angiosperms contain structural phloem proteins (P-proteins) that can interact to create large P-protein bodies. P-protein bodies can occlude sieve plates upon injury but the range of functional and physiological roles of P-proteins remains uncertain, in part because of challenges in labeling and visualization methods. Here, we show that a reciprocal oligosaccharide probe, OGA488, can be used in rapid and sensitive labeling of P-protein bodies in Arabidopsis, poplar, snap bean and cucumber in histological sections. OGA488 labeling of knockouts of the two Arabidopsis P-protein-encoding genes, AtSEOR1 and AtSEOR2, indicated that labeling is specific to AtSEOR2. That protein bodies were labeled and visible in Atseor1 knockouts indicates that heterodimerization of AtSEOR1 and AtSEOR2 may not be necessary for P-protein body formation. Double labeling with a previously characterized stain for P-proteins, sulphorhodamine 101, confirmed P-protein labeling and also higher specificity of OGA488 for P-proteins. OGA488 is thus robust and easily used to label P-proteins in histological sections of multiple angiosperm species.

Geometric morphometrics reveals shifts in flower shape symmetry and size following gene knockdown of CYCLOIDEA and ANTHOCYANIDIN SYNTHASE.

BACKGROUND: While floral symmetry has traditionally been assessed qualitatively, recent advances in geometric morphometrics have opened up new avenues to specifically quantify flower shape and size using robust multivariate statistical methods. In this study, we examine, for the first time, the ability of geometric morphometrics to detect morphological differences in floral dorsoventral asymmetry following virus-induced gene silencing (VIGS). Using Fedia graciliflora Fisch. & Meyer (Valerianaceae) as a model, corolla shape of untreated flowers was compared using canonical variate analysis to knockdown phenotypes of CYCLOIDEA2A (FgCYC2A), ANTHOCYANIDIN SYNTHASE (FgANS), and empty vector controls. RESULTS: Untreated flowers and all VIGS treatments were morphologically distinct from each other, suggesting that VIGS may cause subtle shifts in floral shape. Knockdowns of FgCYC2A were the most dramatic, affecting the position of dorsal petals in relation to lateral petals, thereby resulting in more actinomorphic-like flowers. Additionally, FgANS knockdowns developed larger flowers with wider corolla tube openings. CONCLUSIONS: These results provide a method to quantify the role that specific genes play in the developmental pathway affecting the dorsoventral axis of symmetry in zygomorphic flowers. Additionally, they suggest that ANS may have an unintended effect on floral size and shape.

Elaboration of bilateral symmetry across Knautia macedonica capitula related to changes in ventral petal expression of CYCLOIDEA-like genes.

BACKGROUND: Shifts in floral form across angiosperms, particularly from radially symmetrical to bilaterally symmetrical flowers, are often associated with shifts in speciation rates and changes in pollination syndrome. Growing evidence across both rosids and asterids indicates that CYCLOIDEA (CYC)-like transcription factors from the TCP gene family play a role in establishing the dorsoventral pattern of flower symmetry, which affects the development of both the corolla and androecium. Previous studies of CYC-like genes, especially of the CYC2 clade, indicate that these genes are dorsally restricted in bilaterally symmetrical flowers. Also, gene duplication of CYC-like genes often correlates with shifts in floral form in both individual flowers and head-like inflorescences (capitula). RESULTS: Here, we compared the expression patterns of six CYC-like genes from dorsal, lateral, and ventral petals of internal and external florets across capitula of Knautia macedonica (Dipsacaceae). We demonstrate that multiple copies of CYC-like genes are differentially expressed among petal types and between internal and external florets. Across paralogs, there was a general trend toward a reduction in dorsal expression and an increase in ventral expression in internal florets compared to external florets. However, it was in the ventral petals where a statistically significant increase in expression correlates with a less zygomorphic flower. We also show for the first time lateral-specific expression of a CYC-like gene. Additionally, dorsoventral asymmetric expression of a CYC3 paralog indicates that this understudied gene clade is likely also involved in floral symmetry. CONCLUSIONS: These data indicate that the elaboration of bilateral symmetry may be regulated by the dorsoventral gradient of expression, with statistically significant changes in ventral expression correlating with changes in dorsoventral morphological specialization.