1. Volume 9, Issue 5, Pages 753-756 (May 2016)
A Role for GIBBERELLIN 2-OXIDASE6 and Gibberellins in Regulating Nectar Production 
Lisa B. Wiesen, Ricci L. Bender, Travis Paradis, Alexie Larson, M. Ann D.N. Perera, Basil J. Nikolau, Neil E. Olszewski, Clay J. Carter 
Molecular Plant 
Volume 9, Issue 5, Pages (May 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
GIBBERELLIN 2-OXIDASE6 and gibberellins play a role in regulating nectary function.
(A1) Normalized mean ATH1 GeneChip probe set signal intensity for Arabidopsis GA2ox6. Original array data for all tissues were described in Kram et al. (2009). ILN, immature lateral nectaries; MLN, mature lateral nectaries; MMN, mature median nectaries.
(A2 and A3) Staining of GUS activity in the nectaries of a stage 14–15 GA2ox6pro::GUS flower; arrowheads point to a lateral nectary (LN).
(A4) RT–PCR analysis of GA2ox6 expression in pre-anthesis (stage 12, pre-nectar secretion) and post-anthesis (stage 14, secretory) flowers. The results shown are representative of three independent biological replicates.
(B) Schematic of the GA2ox6 gene and relative T-DNA insertion sites in ga2ox6-4, ga2ox6-5, and ga2ox6-6 (arrowheads).
(C) RT–PCR expression analysis of GA2ox6 in ga2ox6-4, ga2ox6-5, ga2ox6-6 (30 PCR cycles each), and a representative line expressing GA2ox6 under control of the strong nectary-specific SWEET9 promoter (GA2ox6-OE; 25 PCR cycles). The results shown are representative of three independent biological replicates.
(D) Nectar secretion phenotypes for ga2ox6-4, ga2ox6-5, ga2ox6-6, and GA2ox6-OE; **p < 0.01 relative to wild-type, N = 6, with each biological replicate being individually compared with a unique set of wild-type Arabidopsis plants grown in pots the same tray for each individual genotype.
(E) Nectar production in ga2ox6-4 flowers treated with the GA-synthesis inhibitor paclobutrazol was significantly increased over mock treated flowers (N = 3); **p < 0.01 by two-tailed pairwise t-test.
(F and G) Nectary morphology in Col-0 and ga2ox6-4. Arrowheads indicate the location of lateral nectaries (LN).
(H) Iodine staining of starch in the receptacles and pedicels of stage 12 (just prior to anthesis) Col-0 and ga2ox6-4 flowers; arrowhead points to receptacle and pedicel staining heavily for starch in Col-0.
(I and J) Starch accumulation in the stomata of wild-type (I) and ga2ox6-4 (J) nectaries. Yellow arrowheads point to starch stained stomata on the nectary surface.
(K) qRT–PCR expression analysis of the nectary-enriched genes CWINV4, MYB21, and PIN6 in ga2ox6-4 stage 14 flowers (mean expression level is presented relative to Col-0, N = 3 biological replicates with a minimum of two technical replicates each).
(L) Representative image of the auxin response in lateral nectaries (LN) as observed by DR5::GFP in wild-type (Col-0) and ga2ox6-4.
(M) Quantification of the auxin-dependent GFP signal in wild-type (Col-0), ga2ox6-4, and spy-3, a GA signaling mutant with constitutively elevated GA response. N = 5, **p < 0.01 by two-tailed pairwise t-test.
(N) The spy-3, spy-8, and della pentuple mutants were examined for nectar production because each displays constitutively active GA responses. Conversely, gai has a mutation in the DELLA domain of the DELLA repressor protein GAI, which renders it unable to be degraded in a GA-dependent manner, thus decreasing overall GA signaling. **p < 0.01 relative to wild-type, N = 6, with each biological replicate being individually compared with a unique set of wild-type Arabidopsis plants grown in pots the same tray for each genotype.
(O) Proposed model for the role of GA2ox6 and GA in regulating nectary function. In wild-type flowers (upper panel), GA2ox6 likely limits the accumulation of bioactive gibberellins, thereby allowing full expression of genes involved in nectar production. In particular, the expression level of PIN6 has been positively correlated to both the auxin response and nectar production (Bender et al., 2013). In the case of ga2ox6 flowers (lower panel), decreased expression of GA2ox6 (C) likely leads to an increase in bioactive GA (suggested in E). Elevated GAs, and subsequent downstream signaling, lead to the downregulation of nectary-enriched genes involved in the auxin response and nectar production. For example, ga2ox6-4 flowers had reduced expression of genes known to be involved in nectar production (K), including PIN6. Lower PIN6 levels in turn result in a decreased auxin response in nectaries (L and M), thereby leading to reduced levels of nectar production (D).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions