Phloem unloading and sugar accumulation in jujube fruits

Abstract:【Objective】The transportation and distribution of photoassimilates is an important factor deter⁃mining fruit yield and quality. Recently, the physiological mechanisms of photoassimilate unloading fromsieve elements and phloem after transportation have been a heat subject in plant physiology and cell biology. Phloem unloading pathways, symplastic or apoplastic, may differ among plant species. Phloem unload⁃ing pathway in different organs of the same plant and in the same organ at different developmental stages,can be apoplasmic or symplasmic pathways or conversable between the two. Jujube is a very importanteconomic horticultural crop, and its yield and quality is determined by sugar accumulation and composi⁃tion in fruit. Phloem unloading is one of the key steps in accumulation of sugars in fruit. The phloem unloading pathways and their relationship to sugar accumulation remain unclear in Chinese jujube cultivarsfor various uses (dry processing, fresh consumption or multiple uses). In this study, we analyzed the un⁃loading pathways in relation to sugar accumulation in fruits of different types of Chinese jujube.【Methods】Phloem unloading pathways and sugar contents were studied in cultivar‘Muzao’for drying processing,‘Goutouzao’for multiple uses, and two wild sour jujubes from Qingjian and Xingtai with electron micros⁃copy and high performance liquid chromatography (HPLC). The fresh jujube fruits were cut into smallpieces of 5 mm × 5 mm × 2 mm (length × width × height), quickly put into a serum bottle containing 2%glutaraldehyde, vacuum pumped for 40 min, fixed at 4 ℃ for 24 h, rinsed with 0.2 mol of phosphate buffer, and post-fixed with 1% osmium acid for 2 h at 4 ℃. After ethanol dehydration processes and propyl⁃ene oxide transition, the samples were embedded with Spurr resin, polymerized at 68 ℃ for 8 h. The em⁃bedded blocks were shaped and sliced with an ultra-thin microtome into sections with thickness of 0.5nm, which were stained with uranium acetate and lead citrate before observed under an HT7700 transmis⁃sion electron microscopy. 1.0 g jujube powder was put into a 50 mL Conical flask with stopper, to which40 mL 80% ethanol solution was added. After ultrasonic extraction for 20 min at 45 ℃, the suspensionwas centrifuged at freezing temperature and 3 500 g for 10 min. The supernatant was transferred to a 150mL beaker, while the residue was re-suspended with 30 mL 80% ethanol solution. The extraction proce⁃dures were repeated twice, and the supernatants were pooled and rotary evaporated to dryness at 45 ℃,and then re-dissolved with 100 mL pure water. After filtration through a 0.22 μm filter membrane, analy⁃sis of sugar content was conducted using a HITACHI-2000 High Performance Liquid Chromatography.【Results】The results showed that fresh weights of jujube and wild jujube fruits followed a“double S”curve, which could be divided into three stages: the early, the middle and the late stages. In the earlystage, the relative growth rate of fruit was 3.50, which was highest in the whole growth period, with the fast⁃est increase in fruit fresh weight and rapid cell division. In the middle stage, the fresh weight growth ratewas 0.44. In the late stage, the fruit fresh weight growth rate was only 0.18. Structural investigationsshowed that plasmodesmata density in SE/CC complex and the surrounding parenchyma cells changed obviously. Plasmodesmata in‘Muzao’were observed between SE/CC complex and its surrounding phloem pa⁃renchyma cells in different developmental stages. In the middle stage, plasmodesmata density (2.48 μm^-1)was clearly higher than in the early and the late stages. There was no significant difference in plasmodes⁃mata density between the early and the late stages. A large number of plasmodesmata in‘Muzao’werefound in the early stage, its density was higher (1.07 μm^-1). With the fruit development, the plasmodesmata number gradually reduced to 0.71 μm^-1 in the middle stage, and plasmodesmata were hardly observablein SE/CC complex in the late stage. There was no difference in plasmodesmata in phloem parenchymacells in different developmental stage, and this pattern was also found in‘Goutouzao’. Some plasmodes⁃mata were observed in the three developmental stages‘Goutouzao’in three tissues. Density of plasmodes⁃mata between SE/CC complex and its surrounding parenchyma cells in different developmental stages wasslightly lower than that in‘Muzao’. Plasmodesmata density was higher (0.68 μm-1) in the late stage, butplasmodesmata between SE/CC complex and its surrounding phloem parenchyma cells were hardly ob⁃served in the early and the middle stages in sour jujube from Qingjian. Within SE/CC complex plasmodesmata were hardly observed in early stage, and their density was 0.78 μm- 1 in middle stage but decreasedgradually later. This trend was also observed in the sour jujube from Xingtai, where plasmodesmata densityin the SE/CC complex reduced. There were more plasmodesmata between SE/CC complex and its sur⁃rounding phloem parenchyma cells the sour jujube from Xingtai in the early and the middle stages, butthey were hardly observable in the late stage. However, in SE/CC complex, plasmodesmata were hardly ob⁃servable in the early stage, but their density in middle stage reached a maximum of 0.98 μm-1 and then gradually decreased to 0.56 μm-1 in the late stage. There were numerous plasmodesmata in phloem parenchymacells in different developmental stages in two wild jujubes. Fruit mainly accumulated glucose and fructose inthe early and middle stages while accumulated sucrose mainly at the late stage. Fructose content in‘Goutou⁃zao’increased first and then decreased to a relatively low level. The change in fructose content in‘Muzao’showed an increasing trend. Glucose content showed a slow increasing trend during the development of‘Goutouzao’, while in‘Muzao’, glucose content fluctuated. Changes in sucrose content during fruit devel⁃opment were in a similar pattern in‘Goutouzao’and‘Muzao’. Previous results indicated that the solublesugar content was lower in the wild jujube fruits.【Conclusion】These results suggested that there is no difference in phloem unloading pathways between jujube cultivars for different uses, whose phloem unloading follows apoplasmic-symplasmic-apoplasmic pathways. Although a small number of plasmodesmatawere observed in different use of wild jujube, apoplasmic pathway might be dominant. In the aspect of sug⁃ar accumulation, in the early stage sucrose is unloaded by the apoplasmic pathway and decomposed intofructose and glucose in the phloem parenchyma. Therefore, glucose and fructose are accumulated in theearly stage. In the middle stage, the sucrose unloading follows a symplasmic pathway through plasmodesmata, and the content of sucrose slowly increase. In the late stage, sucrose accumulates rapidly with thedecrease in fructose and glucose. The three sugars in the wild jujubes are low throughout fruit development. Thus, sugar contents in jujube cultivars are significantly higher than in wild jujubes. High sugar accumulation might be related to symplasmic phloem unloading.