Sucrose transport, loading and unloading model in cassava Haiyan Wang Institute of Tropical Bioscience & Biotechnology (ITBB), Chinese Academy of Tropical Agricultural Sciences(CATAS) GCP21-II, Kampala, Uganda June 19, 2012
background Sugar transport is important for starch synthesis in aspect of coordination source and sink. source • Phloem loading: assimilates enter into Sieve element-companion cell complex from mesophyll cell. transport • Phloem unloading: assimilates transfer from se/cc complex to sink tissue. sink
Sugar transport strategy: Symplasmic unloading in sink cell A Symplasmic loading in leaf B Apoplasmic unloading in sink cell C Apoplasmic loading in leaf D • Symplasmic pathway: driven by downhill concentration gradient and depending on high plasmodesmal densities . • Apoplasmic pathway: across plasma membrane driven by sugar transporters .
Sucrose transporter Membrane-localized, energy-dependent, H+-symporting sucrose transporters (SUC or SUT). Type 1, high affinity, low capacity; Type 2, lower affinity, for sucrose sensor; Type 4, lower affinity，high capacity;
Cell wall invertase (CWI) and sucrose synthase (SuSy) involve in sucrose transport coherent with apoplasmic and symplasmic transport respectively. (Arnd Sturm, 1999)
Symplasmic transport Ultrastructure of SE-CC complex Fluorescent tracing autoradiography Apoplasmic transport Enzymatic activity mRNA expression Immunolocalization immunobloting SUT、CW-INV、V-INV、SUSY
Sugar component in phloem sap of Arg7 stem Fructose Glucose Sucrose • Sucrose is the main component, it’s concentration is about 570mg/ml, indicating that sucrose is the form of sugar transport in cassava.
Plasmodesmal density of phloem SE-CC complex and adjacent cells in leaf BS B A PC BS SE cc cc SE SE PC PC BS cc BS SE SE C cc BS PP cc CC PD PD A moderate number of plasmodesmata distributed between SE-CC complex and adjacent cell in leaf minor vein. PC SE sieve element; CC companion cell PD plasmodesmata; PC parenchyma cell BS bundle sheath cell
A primary fibrous root primary fibrous root B obvious increase of storage parenchyma cell occurred in tuberous root. Anatomical character of cassava root D tuberous root Cortex C Primary phloem Secondary phloem Cambium Secondary xylem Primary xylem Boxed areas meaning sampling sites for structure obsevations and immunolabeling.
A. primary fibrous root . B. Early stage of tuberizing (E), C. Middle stage of tuberizing (M), D. Late stage of tuberizing (L), Arrows indicate plasmodesmata between sieve element and parenchyma cell. Plasmodesmal densities between different cells in phloem of the root of cassava during the four stages, showed Large amounts of plasmodesmata distributed in tuber root, indicating the existence of symplasmic unloading of sucrose.
Carboxyfluorescein distribution in cassava root A B In tuber roots, CF was widely distributed, indicating large amounts of plasmodesmata and apparently symplasmic phloem unloading of sucrose in tuberous root of cassava. C D Transverse section of the primary fibrous root and tuberous root with carboxyfluorescein fed the primary fibrous root without(A) or with(B) carboxyfluorescein fed; tuberous root without(C) and with (D) carboxyfluorescein fed.
4 2 1 phylogenetic tree 4 MeSUT cDNAs were obtained. From the phylogenetic tree, they were ascribed into three subfamilies, named as:MeSUT1x, MeSUT1y, MeSUT2, MeSUT4 respectively.
MeSUTs expression analysis in Arg7、SC124 Expression analysis in different growth stage of SC124 roots Expression analysis in different growth stage of Arg7 roots LEAF LEAF d d d d d d d d • All MeSUTs is much higher expression in leaf than in tuber root except of 60d early stage in root; • MeSUT1 and MeSUT2 are more active than MeSUT4 no matter in leaf or root; ROOT ROOT MeSUTs are more important in source leaf than in root, which may attribute to apoplasmic phloem loading.
Higher mRNA expression of SuSy and lower expression of CWI in tuberous root than fibrous root suggested symplasmic unloading may predominate in tuber root. Enzymatic activity of SuSy and CWI showed similar trend.
More amounts of SuSy and less amounts of CWI in Westernbloting indicated symplasmic unloading maybe the main pathway in tuber root.
More amounts of cell wall invertase and vacuole invertase and less amounts of sucrose synthase in fiberous root indicated tuber root prefer symplasmic unloading. A CW C fibrous root D tuberous root E: Early stage of tuberizing; M: Middle stage of tuberizing; L: Late stage of tuberizing; FR: fibrous root.
Summary symplasmic pathway, and apoplasmic loading maybe predominate. loading symplasmic unloading predominates during root tuberization. unloading
Ongoing work Combination of autoradiography and fluorescent markers Immunolocalization of MeSUTs Transgenic validation of MeSUTs in leaf and tuber root.