Central to understanding fruit development is to elucidate the processes mediating a successful transition from pre-pollination ovaries to newly set fruit, a key step in establishing fruit yield potential. In tomato, ...Central to understanding fruit development is to elucidate the processes mediating a successful transition from pre-pollination ovaries to newly set fruit, a key step in establishing fruit yield potential. In tomato, cell wall invertase （CWIN） LIN5 and its inhibitor INH1 are essential for fruit growth. However, the molecular and cellular basis by which they exert their roles in ovary-to-fruit transition remains unknown. To address this issue, we conducted a study focusing on ovaries and fruitlets at 2 days before and 2 days after anthesis, respectively. In situ hybridization analyses revealed that LIN5 and INH1 exhibited a dispersed expression in ovaries compared with their phloem-specific expression in fruitlets. Remarkably, LIN5 and INH1 proteins were immunologically co-localized to cell walls of sieve elements （SEs） in ovaries immediately prior to anthesis and in young fruitlets, but were undetectable in provascular bundles of younger ovaries. A burst in CWlN activity occurred during ovary-to-fruit transition. Interestingly, the ovaries, but not the fruit- lets, exhibited high expression of a defective invertase, SldeCWIN1, an ortholog of which is known to enhance inhibition of INH on CWlN activity in tobacco. Imaging of a fluorescent symplasmic tracer indicated an apoplasmic phloem unloading pathway operated in ovaries, contrary to the previously observed symplasmic unloading pathway in fruit pericarp. These new data indicate that （1） a phloem-specific patterning of the CWIN and INH mRNAs is induced during ovary-to-fruit transition, and （2） LIN5 protein functions specifically in walls of SEs and increases its activity during ovary-to-fruit transition, probably to facilitate phloem unloading and to generate a glucose signal positively regulating cell division, hence fruit set.展开更多
In this study, we produced tomato plants overexpressing an invertase inhibitor gene （Sly-INH） from tomato using a simple and efficient transient transformation system. Compared with control plants, the expression of...In this study, we produced tomato plants overexpressing an invertase inhibitor gene （Sly-INH） from tomato using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction （PCR）. Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase （CWIN）, which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato.展开更多
利用含有过表达抑制子载体的根癌农杆菌分别在番茄源叶和果实中进行瞬时表达,快速验证转化酶抑制子(inhibitors of invertases,INH)转基因的表达情况及对果实发育过程中转化酶活性的影响。Micro-Tom在源叶注菌处理后,Lin6的表达含量增加...利用含有过表达抑制子载体的根癌农杆菌分别在番茄源叶和果实中进行瞬时表达,快速验证转化酶抑制子(inhibitors of invertases,INH)转基因的表达情况及对果实发育过程中转化酶活性的影响。Micro-Tom在源叶注菌处理后,Lin6的表达含量增加,表明转化酶在植株中响应病菌侵染;但转INH在源叶几乎没有明显表达变化。果实注射后,注射空菌(EHA105)菌液和空载体(pBI121-2A11)菌液后,转化酶(invertases,Inv)基因Lin5、Lin6表达量没有明显改变,但Inv的活性明显升高1倍左右,这表明转录数量并不能反映翻译后的蛋白质量;在注射转基因(p1300-2A11-INH)菌液后3d,INH的表达明显增加,是其它处理的数倍,这是由于在果实特异性启动子2A11的作用下,INH在果实中特异地表达。而且注射p1300-2A11-INH菌液3～5d后,Inv的活性显著下降,尤其胶质胎座中最为明显。这一结果表明INH主要在翻译后水平调控Inv的活性。展开更多
There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of...There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Posttranslational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular invertase in plant defen展开更多
文摘Central to understanding fruit development is to elucidate the processes mediating a successful transition from pre-pollination ovaries to newly set fruit, a key step in establishing fruit yield potential. In tomato, cell wall invertase （CWIN） LIN5 and its inhibitor INH1 are essential for fruit growth. However, the molecular and cellular basis by which they exert their roles in ovary-to-fruit transition remains unknown. To address this issue, we conducted a study focusing on ovaries and fruitlets at 2 days before and 2 days after anthesis, respectively. In situ hybridization analyses revealed that LIN5 and INH1 exhibited a dispersed expression in ovaries compared with their phloem-specific expression in fruitlets. Remarkably, LIN5 and INH1 proteins were immunologically co-localized to cell walls of sieve elements （SEs） in ovaries immediately prior to anthesis and in young fruitlets, but were undetectable in provascular bundles of younger ovaries. A burst in CWlN activity occurred during ovary-to-fruit transition. Interestingly, the ovaries, but not the fruit- lets, exhibited high expression of a defective invertase, SldeCWIN1, an ortholog of which is known to enhance inhibition of INH on CWlN activity in tobacco. Imaging of a fluorescent symplasmic tracer indicated an apoplasmic phloem unloading pathway operated in ovaries, contrary to the previously observed symplasmic unloading pathway in fruit pericarp. These new data indicate that （1） a phloem-specific patterning of the CWIN and INH mRNAs is induced during ovary-to-fruit transition, and （2） LIN5 protein functions specifically in walls of SEs and increases its activity during ovary-to-fruit transition, probably to facilitate phloem unloading and to generate a glucose signal positively regulating cell division, hence fruit set.
基金supported by the National Natural Science Foundation of China(Nos.31372054 and 30971999)the State Key Laboratory of Plant Physiology and Biochemistry Open Project of China(No.SKLPPBKF1404)
文摘In this study, we produced tomato plants overexpressing an invertase inhibitor gene （Sly-INH） from tomato using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction （PCR）. Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase （CWIN）, which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato.
文摘利用含有过表达抑制子载体的根癌农杆菌分别在番茄源叶和果实中进行瞬时表达,快速验证转化酶抑制子(inhibitors of invertases,INH)转基因的表达情况及对果实发育过程中转化酶活性的影响。Micro-Tom在源叶注菌处理后,Lin6的表达含量增加,表明转化酶在植株中响应病菌侵染;但转INH在源叶几乎没有明显表达变化。果实注射后,注射空菌(EHA105)菌液和空载体(pBI121-2A11)菌液后,转化酶(invertases,Inv)基因Lin5、Lin6表达量没有明显改变,但Inv的活性明显升高1倍左右,这表明转录数量并不能反映翻译后的蛋白质量;在注射转基因(p1300-2A11-INH)菌液后3d,INH的表达明显增加,是其它处理的数倍,这是由于在果实特异性启动子2A11的作用下,INH在果实中特异地表达。而且注射p1300-2A11-INH菌液3～5d后,Inv的活性显著下降,尤其胶质胎座中最为明显。这一结果表明INH主要在翻译后水平调控Inv的活性。
文摘There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Posttranslational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular invertase in plant defen
