Starch function in ornamental tobacco floral nectary development

Morphological changes of floral nectary gland and subcellular transition of floral nectary cells of ornamental tobacco LxS8 were exclusively investigated in this study. Enlargement of the floral nectary gland of ornamental tobacco LxS8 that occurs during development is accompanied by a major accumulation of Periodic Acid-Schiff\u27s staining (PAS-staining) starch grains in nectary amyloplasts. Quantification of starch purified from the nectary at various stages of development showed little starch at an early developmental stage, soon thereafter the amount of starch increased dramatically, reaching a peak approximately 24 hours prior to anthesis. After this time, the amount of starch declined dramatically until the flower reached anthesis, suggesting that the accumulated starch was converted to sugars (sucrose, glucose, and fructose) for nectar production that occurs prior to anthesis. A Transmission Electron Microscopy (TEM) study of plastid development further verified that ornamental tobacco nectaries accumulate large amounts of starch during floral development and hydrolyze that starch prior to anthesis. Compositional and structural analyses of nectary starch showed that amylose content and degree of amylopectin branching varied during nectary development. The average chain length in amylopectin was relatively short at early developmental stages, reached maximal length towards the end of nectary development, and was reduced again at anthesis, consistent with the synthesis of an increasingly complex form of starch up to middle stages of nectary development, followed by decreases in starch complexity and amount due to starch degradation in the mature nectary.;Tobacco floral nectaries undergo changes in form and function. As nectaries change from green to orange, a new pigment is expressed. Analysis demonstrated that it is beta-carotene. Plastids undergo dramatic changes. Early in nectary development, they divide and by stage 9 (S9) they are engorged with starch. About S9, nectaries shift from quiescent anabolism to active catabolism resulting in starch breakdown and production of nectar sugars. Starch is replaced by osmiophilic bodies, which contain needle-like carotenoid crystals. Between S9 and S12, amyloplasts are converted to chromoplasts. Changes in carotenoids and ascorbate were assayed and are expressed at low levels early in development; however, following S9 metabolic shift, syntheses of beta-carotene and ascorbate greatly increase in advance of expression of nectar redox cycle. We propose that biosynthesis of these antioxidants is governed by availability of substrate molecules that arise from starch breakdown. These processes and events may be amenable to molecular manipulation to provide a better system for insect attraction, cross pollination, and hybridization.;Thirty-nine different nectary-expressed cDNAs from the nectaries of ornamental tobacco plants that encode 14 different starch metabolic enzymes, 7 different mevalonate metabolic enzymes, 7 different MEP metabolic enzymes, 8 different beta-carotene metabolic enzymes, and 3 different ascorbate metabolic enzymes were identified from ornamental tobacco LxS8. The translated protein sequences were used to identify gene functionality by comparison with well characterized protein sequences from Arabidopsis, maize and solanaceous species. Subsequently RT-PCR or real-time RT-PCR were used to evaluate gene expression throughout nectary development. This analysis revealed that starch metabolism was highly regulated at transcriptional level in ornamental tobacco floral nectary. Three different classes of starch metabolic gene expression pattern appeared. Most starch anabolic genes, including starch synthase 1 (SS1), starch synthase 3 (SS3) and granule-bound starch synthase (GBSS), were expressed early in nectary development (Stage 2 and Stage 6), and the expression was significantly down-regulated after Stage 9. In contrast, the starch catabolic genes, including branching enzyme 1 (BE1), isoamylase 1 (ISA1), alpha-amylase (AMY), beta-amylase (BMY), were not highly expressed at early stages, but were significantly induced by Stage 9 of nectary development. A third class of gene expression that included R1, phosphorylase (PHO), and starch branching enzyme 2 (SBE2) were expressed through nectary development. Immnunodetection shows that ADP-glucose pyrophosphorylase (AGPase) small subunit and sucrose synthase (SuSy) were highly expressed at early stages. Enzyme activity analysis of AGPase also shows that enzyme activity was higher at early stages. Transcript analysis for carotenoid and ascorbate biosynthetic pathways showed that these genes are significantly expressed at S6, prior to the S9 metabolic shift. Thus, formation of antioxidants beta-carotene and ascorbate after the metabolic shift is independent of transcriptional regulation.;We also evaluated the transport of radiolabeled sugars nectar. Consistency in the amounts and ratios of radiolabeled sugars in nectar indicates that the transported photosynthate sugars were subject to metabolic constraints in the nectary tissue prior to their incorporation into nectar. The velocity of radiolabeled sugars accumulated in nectar illustrates that transported photosynthate contributes to the later nectar production. A series of transgenic plants had been generated. The genetic evidence verified that starch regulates the timing and amount of floral nectar