The Cellular Mechanism Of Sucrose Phloem Unloading In Tuberous Root Of Cassava(Manihot Esculenta Crantz)

Cassava (Manihot esculenta Crantz) is a perennial woody shrub of the family Euphorbiaceae, due to its efficient starch accumulation capacity of the storage root, which currently is not only cultivated as food crop but also as fuel crop in tropical and sub-tropical areas. Efficient accumulation of assimilates not only depends on high photosynthesis rate (source) and translocation capacity (flow), high carbohydrate utilize capacity (sink) of the storage tissues is also necessary. The phloem unloading pathway modulates the development of the sink organs and determines the sink capacity. A growing body of evidence suggests that the dominant phloem unloading pathway is symplasmic, but it is not static, the forms of it can vary considerably, not only between different sink organs of different developmental stages, but even between different tissues, several different pathways may coexist in the plant. Now, it is a mystery as to what factors determine the number of storage roots, storage root formation and starch accumulation capacity. The aim of this study was to reveal the pathway of cassava phloem unloading, and try to analyse the factors and mechanisms underlying tuber yield and efficient starch accumulation. The main results are as follows:1. The main free sugar in cassava phloem exudation is sucrose.In order to determine the main carbohydrate components of cassava phloem sap, firstly we cut off the plant and collected the exudation based on the pressure difference in the phloem, HPLC-ELSD technology was used to analyse the main sugar components. It was found no raffinose, lupeose, sorbitol and mannitol exsit in cassava phloem sap, the main free sugars are sucrose, glucose and fructose, wherein sucrose account for more than a half of the total.2. Based on the anatomical structure under light microscope, five developmental stages of cassava root and the phloem location were determined.Cassava plants grown from stem cuttings the roots are adventitious and they arise from the basal cut surface of the stake, and later these roots develop to make a fibrous root system, called primary fibrous root. A part of primary fibrous roots have the potential to bulk and become storage root, a part of them also have the ability of secondary growth, but stop to bulk due to the genetic and environmental reasons, only function in water and nutrient absorption, which were called secondary fibrous root. Paraffin sections were made to distinct the specific characteristics and developmental stages in cultivar Arg7. Primary fibrous root has a evident primary xylem, cortex tissue account for the most, almost have no starch granules, but have evident proteins in the cortex tissues. Secondary fibrous root has starch granules accumulated in the few parenchyma cells, xylem tissue account for the most, and which was consist of evident vessel cells. Cambium cells were evident in tuberous root, which was closely to the phloem tissue that was difficult distinguish with the inner cortex tissue, as the bulk of the tuber, the number of the xylem parenchyma was increased quickly, the starch granules also increased evidently, almost entire of the tuber tissues were filled with it on the maturation stage.3. More plasmademata were found between kinds of cells of tuberous root than fibrous root, and many symplasmic unloading characteristic were also found in tuberous root instead of fibrous root.Based on the ultrastructure observation, large intercellular space between SE/CC complexes or parenchymal cells was found in primary fibrous root, the number of the companion cells was less than the sevie elements, plasmalemma invagination and incipient plasmolysis were common appeared in companion cells, few palsrnodemata were found, espically the least between SE/CC complexes and phloem parenchymal cells. But the number and density of palsmodemata in tuberous root cells were more than primary fibrous root evidently, the area of various kinds of cells were increased distinctly too (over20times), the intercellular space became smaller, one sieve element was always companied by two companion cells. Starch granules accumulated in the parenchymal cells, until the maturation stage, most of the storage cells were found filled with it.4. The abundant5(6)-carboxyfluorescein (CF) was unloaded in tuberous root of cassava, which proved its main unloading pathway is symplasmic, on the contrary,5(6)-carboxyfluorescein was restricted under the pericycle, which proved its apoplasmic unloading pathway.CF is one of the perfect symplasmic unloading tracers, which were unloaded into the roots of cassava by microjection, based on the same application concentrations and times, CF unloaded in primary fibrous root was confined in the pericycle and phloem strictly, without leak to the inner xylem or the outer cortex. The CF imaging of tuberous root showed that from the stage of elongation, fluorescence was firstly appeared beyond the cambium, more stronger was found in phloem and cortex than xylem, during the swelling stage, more CF was unloaded into the phloem and surrounding tissues, xylem also appeared more. Almost all the tissues of the tuberous root were detected CF fluorescence on maturation stage, from the inner xylem parenchymal cells to the cortex parenchymal cells.5. The relatively high levels of activity of cell wall acid invertase (CWI) and sucrose synthase (SS) in cassava tuberous and fibrous root, represent their main symplasmic and apoplasmic unloading pathways respectively.The activity of the sucrose catabolizing enzymes were analysed, cell wall and soluble acid invertase were showed high activity in primary fibrous root, which were higher remarkable than the invertase activity of three developmental stages of tuberous root. Secondary fibrous root also possesses high CWI activity, but the SAI of it has no differences with SAI in tuberous roots. Sucrose synthase was showed high activity in tuberous root, the activity trend of it in five stages of cassava root was complemented with invertase, especially the CWI. The SS activity of the three stages of tuberous root almost has no changes, but all of them were higher than fibrous root, especially than the primary fibrous root.6. The relatively high levels of mRNA of cell wall acid invertase (CWI) and sucrose synthase (SS) in cassava tuberous and fibrous root, represent their main symplasmic and apoplasmic unloading pathways respectively.Part of sequences of two highly expressed CWI genes were obtained by sequences alignment, reverse transcription PCR and sequencing, the Q-PCR primers of CWI, SAI and SS were designed and were used to amplify. The relative expression was calculated by A ACt method accordingly, CWI and SAI all showed higher expression in fibrous root, especially the CWI, which showed more evident higher expression than the three stages of tuberous root. Even the SAI expression was higher too, but it was not remarkable. SS expression in the tuberous roots showed higher than fibrous roots evidently all, especially the swelling stage of the tuberous root, which showed the peak of it.7. The relatively high levels of protein content of cell wall acid invertase (CWI) and sucrose synthase (SS) in cassava tuberous and fibrous root, represent their main symplasmic and apoplasmic unloading pathways respectively again.The polypeptides of CWI that were selected by above experiment, SAI and SS was speculated and used to prepare the monoclonal antibody of them respectively. According to the western results, the content of CWI in primary and secondary fibrous roots were more than three stages of tuberous root, the molecular was about55K and72k respectively. The SAI molecular content was measured too, which was about95K, but there almost has no differences between the fibrous and tuberous root. The content of SS was showed more in tuberous roots than fibrous root evidently, which was similar with the enzyme activity result, that SS content in five stages of cassava roots was complemented with CWI content in these roots.8. Results of immunogold labeling of acid invertase showed that the amounts of acid invertase was more in fibrous roots than in tuberous root remarkably, which further confirmed the apoplasmic unloading pathway of fibrous root.Results of immunogold labeling showed that the amount of gold particles was dropped dramatically in the tuberous root than the primary fibrous root, there were only a little gold particles combined with CWI on the cell wall of tuberous root. The amount of gold particles combined with CWI in secondary fibrous root was showed little less than primary fibrous root. No gold particles were substantially found in any of the controls without the antiserum or with the preimmune serum controls.