林业科学  2017, Vol. 53 Issue (3): 40-48   PDF    
DOI: 10.11707/j.1001-7488.20170305
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文章信息

张鹤华, 李艳芳, 聂佩显, 王红阳, 张凌云
Zhang Hehua, Li Yanfang, Nie Peixian, Wang Hongyang, Zhang Lingyun
蓝莓果实同化物韧皮部卸载路径与糖代谢酶活性
Phloem Unloading Pathway of Photosynthates and Sucrose-Metabolizing Enzymes Activities in Vaccinium corymbosum Fruit
林业科学, 2017, 53(3): 40-48.
Scientia Silvae Sinicae, 2017, 53(3): 40-48.
DOI: 10.11707/j.1001-7488.20170305

文章历史

收稿日期:2016-04-15
修回日期:2016-07-28

作者相关文章

张鹤华
李艳芳
聂佩显
王红阳
张凌云

蓝莓果实同化物韧皮部卸载路径与糖代谢酶活性
张鹤华1, 李艳芳1, 聂佩显2, 王红阳1, 张凌云1    
1. 北京林业大学森林培育与保护教育部重点实验室 北京 100083;
2. 山东省农业科学院果树研究所 泰安 271000
摘要:【目的】韧皮部卸载和韧皮部后运输在调节蔗糖在库器官间的分配、维持果实的库强方面起着至关重要的作用,而且很大程度上决定着果实的产量和质量。本文研究目的是明确蓝莓同化物韧皮部卸载的机制与糖代谢机制。【方法】以5年生高丛蓝莓品种‘喜来’(‘Sierra’)为研究对象,对各个发育时期的蓝莓果实韧皮部的超微结构进行观察,并综合运用荧光染料活细胞示踪与激光共聚焦扫描显微镜技术实时观察果实内韧皮部同化物卸载路径,运用高效液相色谱等技术,测定分析蓝莓果实可溶性糖含量及相关代谢酶的活性变化等。【结果】对韧皮部细胞进行的超微结构观察显示,在蓝莓果实整个发育期,果实韧皮部SE/CC(筛管伴胞复合体)与周围薄壁细胞之间均未发现胞间连丝,从而形成了共质体隔离,但在薄壁细胞之间、薄壁细胞与果肉细胞之间存在大量胞间连丝。荧光染料CF[5(6)-羧基荧光素]的活细胞示踪试验表明,果实发育过程中,CF均被严格限制在韧皮部中,没有扩散到周围的薄壁细胞。这些结果证实蓝莓果实同化物以质外体卸载途径为主,韧皮部后运输存在着活跃的共质体途径,大量胞间连丝的存在有利于薄壁细胞之间以及薄壁细胞与果肉细胞之间的物质交换。进一步酶活性测定结果显示,在蓝莓果实的整个发育期,转化酶活性保持在较高水平,为证实蓝莓果实同化物以质外体卸载为主提供了证据。对糖代谢相关酶活性分析显示,在果实发育的各个阶段,蓝莓果实的可溶性糖主要以积累果糖和葡萄糖为主,蔗糖酶的分解活性始终大于合成活性;在果实发育中后期,可溶性酸性转化酶和中性转化酶活性进一步升高,显示果实发育后期果肉细胞内进行着活跃的蔗糖分解、转化及贮藏过程,且糖积累与转化酶、蔗糖合酶和蔗糖磷酸合酶等酶活性成正相关。在蓝莓果实发育过程中,蔗糖代谢相关酶的综合作用是影响蓝莓果实中可溶性糖积累的重要因子。【结论】蓝莓果实同化物以质外体卸载途径为主,韧皮部后运输存在着活跃的共质体途径。蓝莓果实主要以积累果糖和葡萄糖为主,且糖积累与转化酶、蔗糖合酶和蔗糖磷酸合酶等酶活性成正相关。
关键词:蓝莓    果实    同化物    质外体卸载    糖代谢酶    
Phloem Unloading Pathway of Photosynthates and Sucrose-Metabolizing Enzymes Activities in Vaccinium corymbosum Fruit
Zhang Hehua1, Li Yanfang1, Nie Peixian2, Wang Hongyang1, Zhang Lingyun1    
1. Key Laboratory of Forest Silviculture and Conservation of Ministry of Education, Beijing Forestry University Beijing 100083;
2. The Institute of Pomology, Shandong Academy of Agricultural Sciences Tai'an 271000
Abstract: 【Objective】Phloem unloading and postphloem transport play pivotal parts in the regulation of the distribution of sucrose in the storage organs and maintenance the sink of fruit, and largely determine the yield and quality of the crop. The objective of this study was to clarify the mechanism of phloem unloading and glucose metabolism in the phloem of blueberry.【Method】Vaccinium corymbosum 'Sierra'(5-year-old Highbush blueberry) was used for observation of the ultrastructure of the phloem of blueberry in each development period. Fluorescent dye tracer and laser scanning confocal microscopy were used to observe the unloading path of the phloem in the fruit timely. Determination of soluble sugar content and related metabolic enzyme activity in blueberry fruit by HPLC.【Result】An ultrastructural investigation of phloem tissue in blueberry fruit showed that the fruit phloem SE/CC complex is symplasmically isolated from surrounding parenchyma cells over fruit development, whereas a large number of piasmodesma exist between parenchyma cells and pulp cells. Confocal laser scanning images of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands, not spread to the surrounding parenchyma cells, during the whole fruit development.These results provided a clear evidence for the predominance method of the assimilation of blueberry fruit is the unloading pathway over blueberry fruit development and symplasmic pathway present in postphloem transport. A large number of cells are beneficial to the material exchange between parenchyma cells, parenchyma cells and flesh cells. Activity assay showed that, invertase activity remained at a high level, it provides evidence to confirm the apoplasmic phloem unloading pathway at blueberry fruit development. In addition, carbohydrate metabolism related enzyme activity assay shows that a great amount of glucose and fructose primarily accumulated during blueberry ripening and sucrose enzyme decomposition activity was always greater than synthetic activity. During the late stage of fruit development, the activity of soluble acid invertase and neutral invertase was increased, the activity of sucrose decomposition, transformation and storage in the fruit pulp cell was revealed, the accumulation process is positively correlated with the activities of invertase, sucrose synthase and sucrose phosphate synthase enzymes. In the process of fruit development, the comprehensive effect of sucrose metabolism related enzymes is an important factor affecting the accumulation of soluble sugar in blueberry fruit.【Conclusion】These results provided clear evidence for the predominance method of the assimilation of blueberry fruit is the unloading pathway over blueberry fruit development and symplasmic pathway present in postphloem transport. A large amount of glucose and fructose primarily accumulated in blueberry and sugar accumulation was positively correlated with invertase, sucrose synthase and sucrose phosphate synthase.
Key words: Vaccinium corymbosum    fruit    photosynthate    apoplasmic unloading    sucrose-metabolizing enzymes    

光合产物在经济库器官如果实、种子间的分配和运输受到诸多生理过程的调节,如光合效率、同化物在源叶的装载、韧皮部长距离转运、库器官韧皮部的卸载及韧皮部后运输等 (Oparka,1990Patrick,1997Braun et al., 2014),其中,韧皮部卸载及后运输对于同化物在各竞争库之间的分配、维持果实库强起着重要作用,且很大程度上决定着作物的产量和质量 (Fisher et al., 1996Patrick,1997Viola et al., 2001)。因此研究同化物韧皮部卸载的细胞学路径,对于提高碳水化合物向果实中运输、阐明糖积累机制至关重要 (Clearwater et al., 2012)。

在过去20多年中,对同化物卸载路径在多种植物和不同类型库器官中进行了研究,尤其在营养库中研究较多。大多数研究认为,在营养库如茎尖 (Patrick,1997Imlau et al., 1999)、营养叶 (Roberts et al., 1997Haupt et al., 2001)、块茎 (Oparka et al., 2000Viola et al., 2001) 等器官中,同化物的卸载主要以共质体为主。但最近也有相反的报道,Bihmidine等 (2015)发现高粱 (Sorghum bicolor) 茎中存在着质外体途径。在生殖贮藏库如果实中,卸载路径则呈现出多样性和复杂性,随着不同果实类型及果实不同发育时期而改变。例如,苹果 (Malus domestica)、桃 (Amygdalus persica) 在整个果实发育时期以质外体卸载路径为主 (Zhang et al., 2004Zanon et al., 2015),柑桔 (Citrus) 和荔枝 (Litchi chinensis) 韧皮部后运输中存在着质外体途径 (Koch et al., 1990Wang et al.,2015);而更多种类的果实在不同发育时期经历了卸载路径的转变,如番茄 (Solanum lycopersicum) 在发育早期以共质体为主,后期则以质外体为主 (Ruan et al., 1995Patrick et al.,1996),而葡萄 (Vitis vinifera) 在始熟期经历了从共质体到质外体的改变过程 (Zhang et al., 2006)。本实验室在对枣 (Ziziphus jujuba) 的研究中也发现果实在发育过程中同化物卸载经历了由质外体—共质体—质外体转变的过程 (Nie et al.,2010)。另外,同化物卸载在库器官不同组织部位也存在着差异。Wu等 (2004)报道核桃 (Juglans regia) 果皮是以质外体卸载为主,而种子中则以共质体为主。这些结果显示了果实韧皮部卸载路径及机制的多样性和复杂性。此外,果实中糖积累除了受到卸载路径影响之外,与蔗糖代谢相关的酶活性也是决定库强大小的关键因子,尤其对于积累高浓度糖的果实 (Oparka,1990Oparka et al., 2000)。

蓝莓为杜鹃花科 (Ericaceae) 越橘属 (Vaccinium) 植物,是一种营养价值及保健价值极高的经济作物,其花青苷含量为众水果之首 (Prior et al.,2005顾姻等,2001)。虽然目前国内外已经有大量关于蓝莓的研究,但是大多集中于其生物学特性、贮藏加工技术、营养价值、医疗功效等方面 (Gordillo et al., 2009Montalba et al., 2010李亚东等,2014),有关蓝莓果实同化物运输及糖积累机制尚不清楚。为明确蓝莓同化物韧皮部卸载与糖代谢机制,本研究以高丛蓝莓 (Vaccinium corymbosum) 品种‘喜来’(‘Sierra’) 为试验材料,对各个发育时期的蓝莓果实韧皮部进行超微结构观察,并综合运用荧光染料活细胞示踪与激光共聚焦扫描显微镜技术实时观察果实韧皮部同化物卸载路径;运用高效液相色谱等技术,测定分析蓝莓果实可溶性糖含量及相关代谢酶的活性变化等。

1 材料与方法 1.1 试验材料

于2013—2015年生长季在山东省泰安市圣田农林科技开发有限公司蓝莓基地进行处理及取样。以5年生高丛蓝莓品种‘喜来’(‘Sierra’) 为研究对象,选取生长健康、长势较为一致的植株为试材。从盛花后第10天开始取材,每隔7天采样1次,共采样10次,随机自树体的东、西、南、北4个方向采集生长时期一致的健康果实,装入自封袋,立即存于冰盒中带回实验室。随机选取20个,准确称量果实鲜质量,绘制果实发育动态,之后将果实用液氮速冻,-80 ℃保存备用。

1.2 蓝莓果实韧皮部细胞超微结构的观察

参照Nie等 (2010)的方法。

1.3 胞间连丝密度的测定

胞间连丝密度测定参照Nie等 (2010)Kempers等 (1998)的方法。

1.4 CFDA荧光示踪与Texas-Red标记

羧基荧光素酯 (carboxyfluorescein-diacetate,CFDA) 标记参考Zhang等 (2004)的方法,略有改动。选取健康、向阳的果穗,将荧光染料CFDA通过果穗所在的茎引入韧皮部。具体方法为:将棉线穿过Eppendorf管底部,用细针小心穿过茎的韧皮部 (注意不要伤到木质部),用移液枪取适量的EDTA (2.5 mmol·L-1) 溶液滴入伤口处,并涂上凡士林,接着向Eppendorf管中加入200 μL浓度为1 mg·mL-1的CFDA溶液。为防止CFDA见光分解,用锡箔纸将Eppendorf管包住。48 h或72 h后,采摘果穗并立即置于冰盒中,带回实验室进行徒手切片,激光共聚焦扫描显微镜 (CLSM, ZEISS LSM 510 Meta) 下观察。为了区别韧皮部和木质部,将部分被CFDA标记过的果实果柄浸泡于1 mg·mL-1 Texas-Red溶液中,避光标记40 min左右,立即进行徒手切片,置于CLSM下观察,分别在488 nm、543 nm激发光下观察荧光染料5(6)-羧基荧光素 (carboxyfluorescein,CF) 与Texas-Red的标记情况。在本研究中,将CFDA引入蓝莓果实的韧皮部,标记48 h后,激光共聚焦显微镜下观察CF在果实维管束中的运动情况。CFDA为CF的酯类形式,引入后在活细胞内降解为CF,在488 nm下激发产生荧光。

1.5 可溶性糖含量测定

将1 g蓝莓果实放入研钵中加入液氮磨碎,置于10 mL的离心管,加入6 mL 80%的乙醇,80 ℃下温浴30 min,12 000 r·min-1(4 ℃) 下离心15 min,取上清液,用0.22 μm过滤膜过滤2次,进行HPLC (高效液相色谱法) 分析。各个发育时期果实重复测定3次。根据保留时间测定蔗糖、果糖和葡萄糖的含量,重复3次。利用液相色谱仪 (戴安P680测定系统) 进行测定,色谱柱采用氨基酸柱。色谱条件:柱温35 ℃,检测池温度35 ℃,流速0.8mL·min-1;采用Waters 2414示差检测器;检测波长为520 nm;流动相为乙腈:重蒸水=80:20(V:V);每次进样为30 μL。

1.6 糖类代谢相关酶活性测定

将5 g蓝莓果实放入研钵中加入液氮磨碎,加入3倍样品体积的10%的TCA溶液,置于-20 ℃过夜;8 000 r·min-1(4 ℃) 离心30 min,弃掉上清,收集沉淀;将沉淀溶于等体积的预冷丙酮,混匀,4 ℃离心 (8 000 r·min-1,15 min),弃上清液,并真空干燥,保存备用;使用ELISA试剂盒上样前,加入裂解液 (2.7 g尿素,0.2 g CHAPS,溶于去离子水中至终体积5 mL),混匀后室温放置30 min,然后4 ℃离心 (8 000 r·min-1,15 min),取上清液并暂时保存于4 ℃备用。分别利用不同的ELISA检测试剂盒提取相应的酶,分别设空白孔 (不加样品及酶标试剂,其余各步操作相同)、标准孔、待测样品孔,所有的酶待测样品孔先加10 μL样本提取液 (样本研磨液上清),再加40 μL样本稀释液,标准孔加标准样50 μL,经过温浴、配液、洗涤、显色等步骤,37 ℃下反应60 min,加入终止液50 μL,终止反应 (此时蓝色立即转为黄色)。用酶标仪在450 nm下测定吸光度OD值 (加终止液后15 min之内测定)。各个发育时期酶活性的测定均重复4次。

测定不同浓度梯度的标准品溶液的OD值,利用EXCEL软件绘制标准曲线,通过标准溶液的标准曲线得到标准曲线方程,再根据曲线方程与测定溶液的OD值计算得到各样品的浓度,从而得到各个酶的活性。

1.7 数据处理

用SPSS统计分析软件对数据进行分析,采用SIGMAPLOT 10.0软件进行制图。

2 结果与分析 2.1 果实生长发育动态

准确称量不同发育时期蓝莓果实鲜质量,绘制生长发育曲线 (图 1)。蓝莓果实的生长发育曲线为双S型,发育周期约为75天。大致可以分为以下4个时期:缓慢生长期,即花后10~30天左右;迅速生长期,即花后30~50天左右;减缓生长期,为花后50~65天左右;成熟前增长期,为花后65~75天左右。为方便后续试验的进行,试验取材为3个时期,果实发育早期 (即缓慢生长期)、发育中期 (即迅速生长期) 和发育后期 (包括减缓生长期和成熟前增长期)。

图 1 果实生长发育曲线 Fig.1 The development curve of fruits
2.2 蓝莓果实韧皮部的超微结构观察

蓝莓果实为肉质浆果,在结构上属于假果 (李亚东等,2014),其可食用的中果皮部分发育所需碳水化合物主要通过萼片心皮维管束运输。为了研究不同发育时期果实中筛管伴胞复合体同周围细胞如韧皮薄壁细胞及果肉细胞之间胞间连丝情况,取果肉中多个维管组织部位进行包埋、超薄切片,并进行超微结构观察,统计不同类型细胞间胞间连丝密度。如图 2所示,在蓝莓果实不同发育时期,果实中筛管伴胞复合体与周围薄壁细胞间均未发现胞间连丝,从而形成共质体隔离 (图 2 A,B,G)。相反,韧皮薄壁细胞之间则存在丰富的胞间连丝 (图 2 C,D,E)。其中,在果实发育早期,韧皮薄壁细胞之间胞间连丝最为丰富,约为1 μm 2.3个,随着果实发育,胞间连丝密度略有下降,中后期分别为1 μm 1.5个和1.8个;在韧皮薄壁细胞与果肉细胞之间的胞间连丝亦存在同样的变化趋势 (表 1)。在果实发育后期,筛管伴胞复合体之间观察到少量胞间连丝的存在 (图 2H)。在结构上,在果实各个发育时期,相对于筛分子中缺少细胞器、只存在一些不定型的丝状物质,伴胞中则胞质致密、染色较深,其中富含线粒体、高尔基体、叶绿体和内质网等细胞器,液泡化程度不同,有的存在中央大液泡且有的伴胞细胞壁内陷产生多泡体。在韧皮薄壁细胞中则存在丰富的线粒体和囊泡,显示细胞中活跃的合成和代谢活动。

图 2 果实不同发育时期维管束超微结构 Fig.2 The ultrastructure of vascular bundle of fruits at different developmental stages SE-CC:筛管伴胞复合体;PP:韧皮薄壁细胞;PD:胞间连丝;FP:果肉细胞;V:液泡;ER:内质网;M:线粒体;N:细胞核;ICS:细胞间隙;SE:筛分子;CC:伴胞;G:高尔基体;VE:囊泡;C:叶绿体;CW:细胞壁。A:果实发育早期SE-CC复合体及周围韧皮薄壁细胞 (PP) (Bar=5 μm);B:果实发育中期SE-CC复合体及周围韧皮薄壁细胞 (Bar=1 μm);C:果实发育早期韧皮薄壁细胞及胞间连丝纵切图 (Bar=2 μm);D:果实发育中期韧皮薄壁细胞及胞间连丝 (Bar=1 μm);E:果实发育后期韧皮薄壁细胞及胞间连丝 (Bar=1 μm);F:果实发育后期韧皮薄壁细胞与果实细胞之间胞间连丝 (FP)(Bar=1 μm);G:果实发育后期SE-CC复合体与周围韧皮薄壁细胞 (Bar=1 μm);H:果实发育后期SE-CC之间存在胞间连丝 (Bar=1 μm)。 SE-CC: The complex of sieve tubes and companion cells; PP: Phloem parenchyma cell; PD: Plasmodesmata; FP: Flesh parenchyma cell; V: Vacuole; ER: Endoplasmic reticulum; M: Mitochondrion; N: Nucleus; ICS: Intercellular space; SE: Sieve element; CC: Companion cells; G: Golgi apparatus; VE: Vesicles; C: Chloroplast; CW: Cell wall. A: The SE-CC complex and surrounding phloem parenchyma cells (PP) of fruits at the early stage (Bar=5 μm); B:The SE-CC complex and surrounding phloem parenchyma cells of fruits at the middle stage (Bar=1 μm); C: The longitudinal section of phloem parenchyma cells at the early stage, showing the plasmodesmata (Bar=2 μm); D: The phloem parenchyma cells at the middle stage, showing the plasmodesmata (Bar=1 μm); E:The phloem parenchyma cell at the late stage, showing the plasmodesmata (Bar=1 μm); F: Between phloem parenchyma cells and fruit cells in the late stage, showing the plasmodesmata (FP)(Bar=1 μm); G: The SE-CC complex and surrounding phloem parenchyma cells in the late stage (Bar=1 μm); H: The plasmodesmata between the SE-CC complex in the late stage (Bar=1 μm).
表 1 果实不同发育时期胞间连丝密度 Tab.1 The density of plasmodesmata in fruits at different developmental stages
2.3 荧光染料CF在果实内运输及Texas-Red标记情况

图 3为蓝莓果实不同发育时期,荧光染料CF在果实不同部位及不同类型维管束中的分布与卸载情况。在果实不同发育时期,CF均被严格地限制在果实韧皮部中,没有卸出到周围的薄壁细胞中,无论是在果柄中 (图 3bi, bii, biii) 还是在发育早期的果实维管束 (图 3ai, aii, aiii),抑或在果肉主脉维管束 (图 3di, dii) 还是细脉维管束中 (图 3ci, cii, ciii),说明果实韧皮部同周围的薄壁细胞之间存在共质体隔离,在果实整个发育过程中,同化物的卸载采用质外体卸载方式,且没有发生卸载路径的转变,这个结果与超微结构观察到的结果是一致的。

图 3 果实不同发育时期的CF荧光示踪 Fig.3 The CF fluorescent tracing in different developmental stages of blueberry fruits 将羧基荧光素酯 (CFDA) 引入果柄韧皮部,处理48 h后,取果实进行徒手切片后在激光共聚焦扫描显微镜 (CLSM, ZEISS LSM 510 Meta) 下观察。ai, ci, di分别为果实发育早期、中期和后期维管束纵切荧光图; aii, cii为对应的ai, ci在明视野下的透射图;aiii, ciii, dii是对应的荧光图和明视野的叠加图。bi为果柄部位的横切图; bii为明视野图; biii为二者叠加图。e为引入的Texas-red (红色) 和CF荧光在维管束中的分布。木质部为图中Texas-Red标记的红色荧光,韧皮部为CF标记的绿色荧光。 Carboxyfluorescein-diacetate (CFDA) was introduced into the phloem of fruit pedicel. After treatment with 48 h, the sample was sliced freehand and observed under CLSM. ai, ci and di show bundle fluorography at the early, middle and late developmental stages, respectively. aii and cii represent the corresponding bright field in ai and ci. aiii, ciii and dii are the overlaid pictures from fluorescence and bright field. bi is the cross section of fruit pedicel and bii is the bright field picture; biii shows the two superimposed picture from bi and bii. e shows the distribution of Texas-red (red) and CF fluorescence in vascular bundle. Red fluorescence shows the xylem and green fluorescence shows the phloem.

为了进一步验证以上试验结果的可靠性,在将CFDA引入果实韧皮部的同时,用木质部导管示踪剂Texas-Red对CFDA标记过的维管束进行了标记。如图 3e所示,木质部为图中Texas-Red标记的红色荧光,韧皮部为CF标记的绿色荧光,结果显示引入的CF是在韧皮部中运输,从而说明本研究的试验方法及结果是可靠的。

2.4 糖代谢相关酶活性分析

蔗糖合成酶类的净活性是反映代谢酶综合作用的重要指标。蔗糖合成酶类的活性为蔗糖合成酶与蔗糖磷酸化酶活性之和,蔗糖分解酶类的活性为3种转化酶[可溶性酸性转化酶 (SAI)、细胞壁结合酸性转化酶 (CWI) 与中性转化酶 (NI)]活性之和,蔗糖代谢酶的净活性为前者的活性之和减去后者的活性之和。从图 4A可知,在蓝莓整个果实发育期,蔗糖酶的分解活性始终大于其合成活性,酶的净活性为负值;从图 4B可知,果实发育的各个时期,果实内蔗糖酶在盛花后17天时分解活性最高,为11.964 U·g-1 FW;从图 4C可知,合成酶的活性变化的总趋势是上升的,但是一直低于分解酶类的活性。这些结果说明在果实发育各个阶段,蔗糖酶的分解活性大于合成活性,主要以积累果糖和葡萄糖为主。

图 4 果实不同发育时期关键代谢酶活性变化 Fig.4 The changes of metabolic enzymes activities during fruit development SS:蔗糖合成酶;SPS:蔗糖磷酸合成酶;SAI:可溶性酸性转化酶;CWI:细胞壁结合酸性转化酶;NI:中性转化酶 SS: Sucrose synthase; SPS: Sucrose phosphate synthase; SAI: Soluble acid invertase; CWI: Cell wall bound invertase; NI: Neutral invertase.

对果实代谢关键酶进行分析 (图 4D) 表明,蔗糖合成酶 (SS) 和蔗糖磷酸合成酶 (SPS) 活性的变化趋势表现为早期活性较低,花后18天至果实成熟,其活性持续升高,到果实成熟时达到最大值0.258 U·g-1 FW和0.069 U·g-1FW。整个发育过程中,SS的活性始终高于SPS活性。

转化酶,又名蔗糖酶或β-呋喃果糖苷酶,可以催化蔗糖代谢中的蔗糖不可逆地分解为果糖与葡萄糖。转化酶包括可溶性酸性转化酶 (SAI)、细胞壁结合酸性转化酶 (CWI) 与中性转化酶 (NI)。对SAI、CWI与NI的活性测定结果如图 4E显示,在果实发育过程中,3种酶活性变化趋势较为一致。果实发育早期,3种酶活性均略有下降,至花后25天左右,降至最低值,之后大幅升高。其中SAI,CWI,NI的活性在花后30天时达到最大值,分别为6.37,2.21,4.23 U·g-1 FW,之后略有下降后维持在较为稳定的水平,直到果实成熟。

2.5 蓝莓果实发育过程中可溶性糖含量与糖代谢相关酶活性的相关性分析

图 5可知,果实发育早期,果糖和葡萄糖含量均较低,但果糖含量明显高于葡萄糖含量,约为葡萄糖含量的4.3倍。随着果实发育,到花后30天时,2种糖含量急剧增加,到果实成熟时 (花后73天),果糖与葡萄糖的含量均达到最大,分别为43.64,39.99 mg·g-1 FW。相对地,果实发育过程中,蔗糖含量变化较小,在盛花后66天时出现1个含量高峰,为3.92 mg·g-1 FW。由此可见,蓝莓果实中可溶性糖主要以积累果糖和葡萄糖为主。

图 5 果实不同发育时期可溶性糖含量变化 Fig.5 The changes of soluble sugar content during fruit development

蓝莓果实发育的10个不同时期可溶性糖含量与糖代谢相关酶活性的Pearson相关性分析 (表 2) 表明,果糖和葡萄糖含量与蔗糖合成酶、蔗糖磷酸合成酶、转化酶等酶活性呈现正相关关系,其中与蔗糖合成酶、蔗糖磷酸合成酶活性达到显著水平 (P<0.01)。这些结果说明,在蓝莓果实发育过程中,蔗糖代谢相关酶的综合作用是影响蓝莓果实中可溶性糖积累的重要因子。

表 2 蓝莓果实发育过程中可溶性糖含量与糖代谢相关酶活性的相关性分析 Tab.2 The correlation of soluble sugar content and metabolic enzymes activities in blueberry fruit development
3 讨论

在本研究中,超微结构观察提供的细胞学证据显示,在蓝莓果实发育的整个时期,果实韧皮部筛管伴胞 (SE/CC) 复合体与周围薄壁细胞之间均未发现胞间连丝,从而形成了共质体隔离。目前,研究认为韧皮部SE/CC复合体包括中间细胞、转移细胞和普通细胞3种类型的伴胞,其中中间细胞是特化为适于共质体卸出的标志结构,而转移细胞和普通细胞是适于质外体卸出的标志性结构。对蓝莓的观察发现伴胞的胞质浓密、染色较深,在韧皮薄壁细胞与SE/CC复合体相邻的部位,细胞间隙较大且伴胞质膜内陷形成囊泡,从而说明蓝莓果实韧皮部的伴胞属于普通细胞,这种结构与筛管伴胞复合体到薄壁细胞的质外体运输相呼应。CF是一种“膜不透性”的荧光指示剂,被广泛用于韧皮部运转及卸载路径的标记 (Roberts et al., 1997),通过放射性自显影技术,已经证实CF的运输方式与同化物的卸载运输方式相似 (Viola et al., 2001)。本试验中进一步用荧光染料CF的活细胞示踪结果表明,在蓝莓果实发育过程中,CF均被严格地限制在果实韧皮部中,没有扩散到周围的薄壁细胞,无论是在CFDA引入后的48 h或72 h,CF始终被限制在韧皮部中,没有发生卸出,这与超微结构观察结果一致。这些结果表明蓝莓果实发育过程中,筛管伴胞复合体同周围薄壁细胞存在共质体隔离,同化物主要以质外体形式卸载。这与作者在苹果果实中观察到的结果类似,即苹果果实整个发育时期均采取质外体卸载方式 (Zhang et al., 2004)。郑国琦等 (2015)在宁夏枸杞 (Lycium barbarum) 中也发现同样现象,其他报道也显示部分果实中存在着随果实发育卸载路径发生转变,如核桃 (Wu et al., 2004)、冬枣 (Ziziphus jujuba cv. Dongzao)(Nie et al., 2010)、葡萄 (Zhang et al., 2006)、黄瓜 (Cucumis sativus)(Hu et al., 2011)、梨 (Pyrus spp.)(Zhang et al., 2014)、甜高粱 (Sorghum bicolor)(Bihmidine et al., 2015Milne et al., 2015) 等。

另外,值得注意的是,在整个果实发育过程中,虽然筛管伴胞复合体同周围薄壁细胞存在共质体隔离,但韧皮薄壁细胞之间以及韧皮薄壁细胞与果肉细胞之间则存在丰富的胞间连丝,尤其在果实发育早期最为丰富。以往研究发现,韧皮部卸载途径的改变可能会导致胞间连丝的数量和密度的变化,这对于库组织的发育和功能研究是非常重要的 (Patrick,1997Oparka et al., 1999)。胞间连丝在胚胎、营养生长和生殖发育等植物发育的各个阶段是必不可少的 (Burch-Smith et al., 2016),本文研究结果表明蓝莓果实同化物的韧皮部后运输存在着活跃的共质体途径,大量胞间连丝的存在有利于薄壁细胞之间以及薄壁细胞与果肉细胞之间的物质交换。蓝莓果实韧皮部薄壁细胞和果肉薄壁细胞的细胞质中均有线粒体、内质网、高尔基体、叶绿体、液泡的存在,说明这些细胞中具有旺盛的物质代谢和运转过程,与宁夏枸杞一致 (郑国琦等,2015)。蔗糖是同化物在韧皮部中运输的主要物质形式,植物果实蔗糖积累受到蔗糖的合成、运输、分配和在果实中的代谢等过程的共同作用,蔗糖代谢与积累涉及到的关键酶包括转化酶、蔗糖磷酸合成酶和蔗糖合成酶,它们对于果实内糖分的积累具有重要的作用 (张中霞等,2011),蔗糖在果实中的分解强度是增强库强、提高糖卸载能力、保证新合成的蔗糖由源到库不断运输的重要环节 (Rotundo et al., 2009齐红岩等,2012)。其中,细胞壁酸性转化酶被认为与同化物的质外体卸载相关,主要负责质外空间蔗糖的分解 (Zhang et al.,2001),对维持蔗糖浓度梯度、保持库强起到重要作用 (Chandra et al., 2012)。在蓝莓中,在果实整个发育期,转化酶活性保持在较高水平,支持了质外体卸载的证据。蔗糖合成酶在蔗糖代谢过程中,既可以催化蔗糖分解又可以催化蔗糖的合成,蔗糖磷酸合成酶可以催化蔗糖的合成,以促进蔗糖的积累。同葡萄 (闫梅玲等,2010) 类似,本研究发现蓝莓果实可溶性糖主要以积累果糖和葡萄糖为主。相对于蓝莓果实发育过程中转化酶较高的活性,蔗糖合成酶和蔗糖磷酸合成酶活性较低。酶净活性分析显示,蔗糖酶的分解活性始终大于合成活性。在果实发育中后期,可溶性酸性转化酶和中性转化酶活性进一步升高,显示果实发育后期果肉细胞内进行着活跃的蔗糖分解、转化及贮藏过程。与葡萄 (闫梅玲等,2010) 中略有不同的是,蓝莓果糖和葡萄糖的积累与蔗糖合成酶、蔗糖磷酸合成酶及转化酶等酶活性均成正相关。

4 结论

在蓝莓果实发育过程中,蓝莓果实同化物以质外体卸载途径为主,同化物的韧皮部后运输存在着活跃的共质体途径,蔗糖酶的分解活性大于合成活性,主要以积累果糖和葡萄糖为主,且可溶性糖的积累与转化酶、蔗糖合成酶及蔗糖磷酸合成酶等酶活性均成正相关。该结论为蓝莓果实的发育及品质形成的研究提供理论依据。

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