CNR转录因子在番茄果实成熟过程中的功能

SPL转录因子在植物中广泛存在并参与植物生长、发育和成熟过程,CNR是SPL转录因子家族中的一个成员,其作用机制尚不清楚.通过RNA-seq、qRT-PCR和染色质免疫共沉淀技术(ChIP)确定转录因子CNR直接作用的新的靶基因,旨在揭示番茄果实成熟过程中CNR的转录调控网络.通过RNA-seq筛选出野生型AC和突变体...     

Acetaldehyde Is a Causal Agent Responsible for Ethanol-Induced Ripening Inhibition in Tomato Fruit

Inhibition of tomato (Lycopersicon esculentum Mill.) fruit ripening by exogenously applied ethanol was shown to be caused by elevated endogenous levels of acetaldehyde (AA). Exposure of excised pericarp discs of mature-green tomato fruit to ethanol or AA vapors produced elevated levels of both compounds in the tissue, but only the levels of AA were associated with ripening inhibition. Ripening inhibition was dependent on both the applied concentration and the duration of exposure. Discs treated with inhibitory levels of AA had levels of ethanol that were elevated but below that associated with inhibition of ripening. The in vivo activity of alcohol dehydrogenase was inhibited 40 to 60% by 4-methylpyrazole (4-MP), a competitive inhibitor of this enzyme. The inhibitory effect of ethanol on ripening was reduced by the simultaneous application of 4-MP. Tissue treated with 4-MP plus AA vapors had higher endogenous levels of AA and ripening was inhibited longer than in tissue without 4-MP. The tissue AA level resulting from ethanol or AA application appears to be the critical determinant of ripening inhibition.     

Temporal and Spatial Distribution of Auxin Response Factor Genes During Tomato Flower Abscission

Abscission facilitates growth and reproduction and improves plant defenses against pathogens. This tightly regulated process is triggered by environmental cues and hormones such as ethylene and auxin. Because auxin is crucial for abscission, auxin response factors (ARFs) may play important roles in this process. Here, we examined changes in gene expression during abscission in tomato, focusing on regulation of genes encoding ARFs. Specifically, we analyzed the pattern of ARF gene expression in tomato flower pedicel explants treated with ethylene, the ethylene blocker 1-methylcyclopropene (1-MCP), or auxin to determine how auxin and ethylene affect ARF gene expression. In addition, we examined the spatial and temporal distribution of IAA during abscission by examining transgenic tomato plants expressing an IAA-inducible promoter fused to the GUS reporter gene (the P5::GUS ‘Chico III’ line). Flower removal from the explants quickly induced abscission by ethylene, which was inhibited by exogenous auxin or 1-MCP. During early abscission, auxin (or 1-MCP) regulated the expression of various ARFs, including ARF1, 2, 3, 4, 5, 7, 8-1, 9, 11, 12, 13, 13-1, 14, and 17, whereas ethylene had the opposite effect on most of these genes. Further analysis shows that during this stage, auxin may mediate the expression of ARF8-1, 9, 11, 12, 13, 13-1, and 14, whereas ethylene may mediate ARF13-1. During the later stage of abscission, ARF2, 8, 10, 11, and 19 were upregulated, and 8-1, 12, 13, and 13-1 were downregulated, compared with nonabscising parts of plants. Fluorometric GUS analysis indicated that GUS activity in the abscission zone remained stable at 4 h and sharply decreased after 8 h until abscission was complete (32 h).     

Cytokinin-Induced Parthenocarpic Fruit Development in Tomato Is Partly Dependent on Enhanced Gibberellin and Auxin Biosynthesis

Fruit set of plants largely depends on the biosynthesis and crosstalk of phytohormones. To date the role of cytokinins (CKs) in the fruit development is less understood. Here, we showed that parthenocarpic fruit could be induced by 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU, an active CK) in tomato ( Solanum lycopersicum cv. Micro-Tom). The fresh weight of CPPU-induced parthenocarpic fruits was comparable with that induced by GA₃. Importantly, CPPU-induced parthenocarpy was found to be compromised by simultaneous application of paclobutrazol (a GA biosynthesis inhibitor), and this effect could be restored by exogenous GA₃. Like pollination, CPPU-induced fruit showed enhanced accumulation of GA₁₊₃ and indole-3-acetic (IAA), which were accompanied by elevated expression of GA biosynthesis genes like SlGPS, SlGA20ox1, SlGA20ox2 and SlGA3ox1, and IAA biosynthesis gene ToFZY. Elevated GAs level in CPPU-induced fruits was also associated with down-regulation of GA inactivation genes, namely SlGA2ox1,2,3,4,5 in comparison with untreated control. These results suggested that CKs may induce parthenocarpy in tomato partially through modulation of GA and IAA metabolisms.     

AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process.     

Deciphering Ascorbic Acid Regulatory Pathways in Ripening Tomato Fruit Using a Weighted Gene Correlation Network Analysis Approach

Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid （AsA）, which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato （Solanum lycopersicum L.） fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty- four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level.     

番茄果实成熟突变体的研究进展

番茄是一类比较重要的经济作物,具有很高的营养价值,长期以来番茄一直是研究肉质果实生长和成熟的模式生物。近年来,许多番茄果实成熟突变体的发现为研究果实成熟机制提供了重要的生物材料,综述了番茄果实成熟的影响因素、番茄果实成熟相关的突变体及基于突变体对果实成熟的相关研究,为今后突变体及果实成熟机制的研究提供参考。     

Tomato Fruit Development and Ripening Are Altered by the Silencing of LeEIN2 Gene

Loss-of-function ethylene insensitive 2 （EIN2） mutations showed ethylene insensitivity in Arabidopsis, which indicated an essential role of EIN2 in ethylene signaling. However, the function of EIN2 in fruit ripening has not been investigated. To gain a better understanding of EIN2, the temporal regulation of LeEIN2 expres- sion during tomato fruit development was analyzed. The expression of LeEIN2 was constant at different stages of fruit development, and was not regulated by ethylene. Moreover, LeEIN2-silenced tomato fruits were developed using a virus-induced gene silencing fruit system to study the role of LeEIN2 in tomato fruit ripening. Silenced fruits had a delay in fruit development and ripening, related to greatly descended expression of ethylene-related and ripening-related genes in comparison with those of control fruits. These results suggested LeEIN2 positively mediated ethylene signals during tomato development. In addition, there were fewer seeds and Iocules in the silenced fruit than those in the control fruit, like the phenotype of parthenocarpic tomato fruit. The content of auxin and the expression of auxin-regulated gene were declined in silenced fruit, which indicated that EIN2 might be important for crosstalk between ethylene and auxin hormones.     

Klf2 Is an Essential Factor that Sustains Ground State Pluripotency

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The PIM-2 Kinase Is an Essential Component of the Ultraviolet Damage Response That Acts Upstream to E2F-1 and ATM

The oncogenic nature ascribed to the PIM-2 kinase relies mostly on phosphorylation of substrates that act as pro-survival/anti-apoptotic factors. Nevertheless, pro-survival effects can also result from activating DNA repair mechanisms following damage. In this study, we addressed the possibility that PIM-2 plays a role in the cellular response to UV damage, an issue that has never been addressed before. We found that in U2OS cells, PIM-2 expression and activity increased upon exposure to UVC radiation (2–50 mJ/cm²), and Pim-2-silenced cells were significantly more sensitive to UV radiation. Overexpression of PIM-2 accelerated removal of UV-induced DNA lesions over time, reduced γH2AX accumulation in damaged cells, and rendered these cells significantly more viable following UV radiation. The protective effect of PIM-2 was mediated by increased E2F-1 and activated ATM levels. Silencing E2F-1 reduced the protective effect of PIM-2, whereas inhibiting ATM activity abrogated this protective effect, irrespective of E2F-1 levels. The results obtained in this study place PIM-2 upstream to E2F-1 and ATM in the UV-induced DNA damage response.     

生长素反应因子作用机制研究进展

概述ARF的结构和功能特点,其与Aux/IAA阻遏子之间的作用方式,以及它在生长素信号转导过程中的调控机制;对ARF基因在不同植物中的分布构成,表达特点,突变体的表型,以及转录后调控的研究现状进行了归纳分析。     

Endogenous Nmnat2 Is an Essential Survival Factor for Maintenance of Healthy Axons

The molecular triggers for axon degeneration remain unknown. We identify endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnat1 and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured Wld ^S neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that Wld^S does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. We conclude that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and propose that, when present, the more long-lived, functionally related Wld^S protein substitutes for Nmnat2 loss after axon injury. Endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders.     

The Ripening of Tomato Fruit as Affected by the Injection of Certain Chemicals

Some chemical agents known to uncouple oxidation from phosphorylationin biological systems were injected into mature green tomatofruit. Limited amounts of the substances accelerated the ripeningof fruit left on the plants but had no effect on picked fruit.L-Methionine, regarded as having a coupling action on oxidativephosphorylation, appeared to lengthen the ripening period. Aswell as speeding up ripening, DNP and CPA were also shown toincrease the activity of polygalacturonase, but not pectinesterase,in unpicked tomato fruit. It is concluded that even when subjectto the action of uncoupling substances, production of enzymesprobably necessary for the furtherance of the ripening processcontinues. The methods by which this process in tomato fruitcould be maintained are examined, and the possibility is discussedthat ‘loose’ coupling is the mechanism by whichan energy source is provided for the endergonic cell processestaking place during ripening.     

利用TIGR番茄基因索引分析挖掘果实成熟相关基因

目前,互联网上提供许多关于基因及其相关信息的数据库,从这些资料库中提取的资料信息,可用于遗传,生化,分子生物学等各种形式的研究。TIGR Tomato Gene Index数据库中的信息为研究番茄果实成熟开辟了新思路。本研究通过对该数据库的分析,发现了一些在番茄果实成熟过程中差异表达的基因。并进一步通过基因芯片软件和文献挖掘的方法对这些基因进行了分析,结果发现了一些新的与果实成熟相关的基因。这对研究番茄果实成熟提供有价值的信息和思路。     

Carboxypeptidase Activity of Tomato Fruit during the Ripening Process and Some Enzymatic Properties

Carboxypeptidase activity of tomato fruit reached a maximum at an early period of ripening. During storage of the fruit at 25°C, the enzyme activity decreased, accompanied by a fall of the pH value of the sap.

The enzyme was apparently localized in the soluble fraction, as determined by differential centrifugation.

The enzyme was optimally active at pH 5.0 ~ 5.5, was most stable at pH 4.5 ~ 6.5, and was strongly inhibited by DFP and HgCl₂, but not by EDTA and 1,10-phenanthroIine. Z-dipeptides containing arginine, proline and several neutral amino acids were hydrolyzed by the enzyme.

The similarity of the enzymatic properties of the present enzyme to those of other plant carboxypeptidases and pig kidney cathepsin A is also discussed.