Anjun Chen

LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato

To study the function of LeERF1 in ethylene triple response on etiolated seedling, plant development and fruit ripening and softening, LeERF1 gene was introduced into tomato (Lycopersicon esculentum cv. No. 4 Zhongshu) through Agrobacterium-mediated transformation. The sense LeERF1 and anti-sense LeERF1 transgenic tomato were obtained. Overexpression of LeERF1 in tomato caused the typical ethylene triple response on etiolated seedling. In the adult stage, 35S::LeERF1 resulted in morphological changes in the leaves of the LeERF1-sn lines. Anti-sense LeERF1 fruits had longer shelf life compared with wild-type tomato. The results of this manuscript indicated that LeERF1 positively mediated the ethylene signals, while the function of LeERF1 was verified for the first time to be positively related with ethylene triple response on etiolated seedling, plant development and fruit ripening and softening using LeERF1-sn, wt and LeERF1-as tomato.

Computational identification of microRNAs and their targets in wheat (Triticum aestivum L.)

AbstractmicroRNAs (miRNAs) are a class of endogenous, non-coding, short (∼21 nt) RNAs directly involved in regulating gene expression at the post-transcriptional level. Previous reports have noted that plant miRNAs in the plant kingdom are highly conserved, which provides the foundation for identification of conserved miRNAs in other plant species through homology alignment. Conserved miRNAs in wheat are identified using EST (Expressed Sequence Tags) and GSS analysis. All previously known miRNAs in other plant species were blasted against wheat EST and GSS sequences to select novel miRNAs in wheat by a series of filtering criteria. From a total of 37 conserved miRNAs belonging to 18 miRNA families 10 conserved miRNAs comprising 4 families were reported in wheat. MiR395 is found to be a special family, because three members belonging to the same miR395 family are clustered together, similar to animal miRNAs. MiRNA targets are transcription factors involved in wheat growth and development, metabolism,and stress responses.

Lycopene Accumulation Affects the Biosynthesis of Some Carotenoid-related Volatiles Independent of Ethylene in Tomato

For elucidating the regulatory mechanism of ethylene on carotenoid-related volatiles (open chain) compounds and the relationship between lycopene and carotenoid-related volatiles, transgenic tomato fruits in which ACC synthase was suppressed were used. The transgenic tomato fruit showed a significant reduction of lycopene and aroma volatiles with low ethylene production. 6-Methyl-5-hepten-2-one, 6-methyl-5-hepten-2-ol and geranylacetone, which were suspected to be lycopene degradation products, were lower than those in wild type tomato fruits. In order to identify whether lycopene accumulation effects the biosynthesis of some carotenoid-related volatiles independent of ethylene in tomato or not, the capability of both wild type and transgenic tomato fruits discs to convert lycopene into carotenoid-related volatiles was evaluated. The data showed that external lycopene could convert into 6-methyl-5-hepten-2-one and 6-methyl-5-hepten-2-ol in vivo, indicating that the strong inhibition of ethylene production had no effect on enzymes in the biosynthesis pathway of some carotenoid-related volatiles. Therefore, in ACS-suppression transgenic tomato fruits, the low levels of 6-methyl-5-hepten-2-one, 6-methyl-5-hepten-2-ol was due to decreased lycopene accumulation, not ethylene production. Ethylene only affected the accumulation of lycopene, and then indirectly influenced the level of lycopene-related volatiles.

Conserved miRNAs and their targets identified in lettuce (Lactuca) by EST analysis

MicroRNAs (miRNAs) are a newly identified class of endogenous, non-coding, short ( approximately 21nt) RNAs that play important roles in regulating gene expression at post-transcriptional level by targeting mRNA cleavage or translational inhibition in plants and animals. Though there are lots of differences between plant miRNAs and animal miRNAs, most of these tiny RNAs are highly conserved in each kingdom. Here, we show the conserved miRNAs in lettuce (Lactuca) identified using EST (expressed sequence tag) analysis. Namely, all previously known miRNAs in other plant species were blasted against lettuce EST sequences to select novel miRNAs in lettuce by a series of filtering criteria. By this strategy, we found a total of 21 conserved miRNAs belonging to 12 miRNA families. After analyzing the conservation and evolution of lettuce miRNAs and their counterparts in other plant species, we revealed that though miRNAs are highly conserved, some specific sites are more likely to mutate. To confirm the expression of identified miRNAs in lettuce, an RT-PCR approach was employed. Moreover, all identified lettuce miRNAs were used to search their potential target genes by miRU web-server from TIGR database available at http://www.tigr.org and a total of 63 potential targets for 10 identified miRNA families in lettuce were found. Similar to previous works, some miRNA targets are transcription factors involved in lettuce growth and development, metabolism, and stress responses.

Overexpression of SlERF1 tomato gene encoding an ERF-type transcription activator enhances salt tolerance

Ethylene-responsive factors (ERFs) play an important role in plant responses to stresses. For this study, we obtained sense- and antisense-SlERF1 transgenic tomato plants to analyze the function of the SlERF1 gene in tomato plants. Overexpression of SlERF1 in tomato plants enhanced salt tolerance during tomato seedling root development. In addition, tomato seedlings overexpressing SlERF1 showed the higher relative water content and lower MDA content and electrolyte leakage; they accumulated more free proline and soluble sugars, as compared with wild-type and antisense-SlERF1 transgenic tomato plants under salt stress. Moreover, SlERF1 activated the expression of stress-related genes, including LEA, P5CS, DREB3-1, and ltpg2 in tomato plants under salt stress. Thus, SlERF1 played a positive role in the salt tolerance of tomato plants.

Expression and DNA Binding Activity of the Tomato Transcription Factor RIN (Ripening Inhibitor)

Recently, we have found that the accumulation of ripening inhibitor (RIN) protein increased gradually during tomato fruit ripening. Here, the recombinant protein was expressed in Escherichia coli and affinity-purified. The DNA binding activity of renatured RIN protein was tested by electrophoretic mobility shift assay. The results indicated that an optimal expression and purification system was suitable for obtaining active RIN with DNA binding activity.

Expression of a truncated ripening inhibitor (RIN) protein from tomato and production of an anti-RIN antibody

The tomato ripening mutant, ripening inhibitor (rin), whose fruits fails to ripen, has been identified and widely studied. The RIN gene has been cloned. Here we present the expression of a truncated form of the RIN protein from tomato and the preparation of a polyclonal antibody against it. The resulting antibody recognized the RIN of crude protein extracts from different tomato tissues. The protein level of RIN in tomato was detected with this antibody by western blot, which suggested the accumulation of RIN protein increased gradually during tomato fruit ripening.

Analysis of gene functions by a syringe infiltration method of VIGS.

Virus-induced gene silencing (VIGS) is a powerful tool for characterization of functional genes in tomato. In this study, we improved the syringe infiltration method on detached tomato fruits. With PDS (phytoene desaturase gene) used as a reporter gene, harvested mature green tomato fruits were syringe-infiltrated with agrobacterium strain GV3101 containing pTRV1 and pTRV2-LePDS, which resulted in color changing on the fruit surface. The changes in pigments and transcript abundance of PDS were compared between silencing sectors and control. In addition, factors of environmental conditions, such as temperature and humidity, were optimized for gene silencing, and other factors, including volume of agrobacterial culture, were also analyzed. Furthermore, this silencing method was confirmed by silencing LeRIN and LeACS2, which influenced fruit ripening process as positively regulating genes, and LeCTR1, which took part in ethylene signal transduction as a negatively regulating gene. Transcripts of LeRIN, LeCTR1, and LeACS2 were measured in the following analysis of gene silencing. All results indicated that an infiltration-based method is efficient for gene characterization during development and ripening of tomato fruits.