Progress 04/01/22 to 03/31/25
Outputs
Target Audience:Through professional conferences and publications, we presented research results and interacted with peers in the field of plant growth and development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students and four undergraduate students have been trained in the PI's lab during the report period time. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Determine the role of SlNAC1 in fruit development and ripening. We have generated two SlNAC1-KO transgenic tomato lines using the CRISPR/Cas9-based gene knockout technique. No significant difference was observed through fruit development and ripening at all critical stages, including anthesis, 1cm (8day post anthesis (DPA), immature green, mature green, breaker, pink, light red and red ripe. This result suggests a functional redundancy of SlNAC1 and other NAC members in tomato since there are more than one hundred NAC members in tomato and many of them have been shown to play a role in fruit ripening. In addition, to determine genes differentially regulated by SlNAC1 involved in fruit development and ripening, RNA-seq-based gene expression profiling analyses on various SlNAC1-overexpression fruits have been conducted. More than 120 differentially expressed genes (DEGs) have been identified and analyzed based on patterns of up- and down-regulation in transgenic fruits relative to WT fruits, Particularly, comparison of DEGs regulated by AD1 and AD2 revealed each activation domain differentially modulates fruit ripening. However, it is notable that some of these genes may be indirectly regulated by AD1 and AD2 and are not the direct targets of SlNAC1 as an intact transcription factor. Objective 2. Manipulate SlNAC1 for early fruit ripening without growth detriments. We have generated an SlNAC1 construct (TFM7::SlNAC1Δ191-270) under the control of a fruit-specific promoter to develop tomatoes with normal growth habits and yield, but early ripening fruits. We cloned a 2-kb promoter from the fruit-specific gene TFM7. By qRT-PCR analysis, the TFM7 promoter was verified during fruit development from the 1cm to mature green stage. The TFM7 promoter was fused with the SlNAC1Δ191-270 mutant, resulting in a novel TFM7::SlNAC1Δ191-270 construct that would be specifically expressed in tomato fruits. We used this construct for tissue culture-based transformation and introduced it into tomato cultivar Ailsa Craig (AC). 12 individual TFM7::SlNAC1Δ191-270 transgenic lines have been generated and the presence of transgene has been verified by PCR-based analysis. Three representative TFM7::SlNAC1Δ191-270 transgenic lines have been advanced to T2 population for further analyses. Fruits of TFM7::SlNAC1Δ191-270 transgenic lines reached the red ripe stage 8 - 12 days earlier than WT fruits, and, significantly, transgenic lines were morphologically indistinguishable with the wild-type non-transgenic AC plants.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
Han Lu, Youhong Fan, Yulin Yuan, Xiangli Niu, Bingyu Zhao, Yongsheng Liu, Fangming Xiao (2023). Tomato SlSTK is involved in glucose response and regulated by the ubiquitin ligase SlSINA4. Plant Science. 2023 Mar 13;331:111672. doi: 10.1016/j.plantsci.2023.111672.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Yuan Y, Fan Y, Huang L, Lu H, Tan B, Ramirez C, Xia C, Niu X, Chen S, Gao M, Zhang C, Liu Y, Xiao F (2024). The SINA1-BSD1 Module Regulates Vegetative Growth Involving Gibberellin Biosynthesis in Tomato. Advanced Science. 2024 Aug 27:e2400995. doi: 10.1002/advs.202400995. Online ahead of print. PMID: 39190572
|
Progress 04/01/23 to 03/31/24
Outputs
Target Audience:Through professional conferences and publications, we presented research results and interacted with peers in the field of plant growth and development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student and two undergraduate students have been trained in the PI's lab during the report period time. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We willcontinue to advance TFM7::SlNAC1Δ191-270 transgenic tomato lines to T2 homozygous populations. Once obtained, homozygous TFM7::SlNAC1Δ191-270 transgenic plants will be grown in the greenhouse alongside previously-generated SlNAC1Δ191-270-OX transgenic plants and WT plants for comprehensive phenotypicassays.
Impacts
What was accomplished under these goals?
Objective 1: Determine the role of SlNAC1 in fruit development and ripening. We have generated two SlNAC1-KO transgenic tomato lines using the CRISPR/Cas9-based gene knockout technique. No significant difference was observed through fruit development and ripening at all critical stages, including anthesis, 1cm (8day post anthesis (DPA), immature green, mature green, breaker, pink, light red and red ripe. This result suggests a functional redundancy of SlNAC1 and other NAC members in tomato since there are more than one hundred NAC members in tomato and many of them have been shown to play a role in fruit ripening. In addition, to determine genes differentially regulated by SlNAC1 involved in fruit development and ripening, RNA-seq-based gene expression profiling analyses on various SlNAC1-overexpression fruits have been conducted. More than 120 differentially expressed genes (DEGs) have been identified and analyzed based on patterns of up- and down-regulation in transgenic fruits relative to WT fruits, Particularly, comparison of DEGs regulated by AD1 and AD2 revealed each activation domain differentially modulates fruit ripening. However, it is notable that some of these genes may be indirectly regulated by AD1 and AD2 and are not the direct targets of SlNAC1 as an intact transcription factor. Objective 2. Manipulate SlNAC1 for early fruit ripening without growth detriments. We have generated an SlNAC1 construct (TFM7::SlNAC1Δ191-270) under the control of a fruit-specific promoter to develop tomatoes with normal growth habits and yield, but early ripening fruits. We cloned a 2-kb promoter from the fruit-specific gene TFM7. By qRT-PCR analysis, the TFM7 promoter was verified during fruit development from the 1cm to mature green stage. The TFM7 promoter was fused with the SlNAC1Δ191-270 mutant, resulting in a novel TFM7::SlNAC1Δ191-270 construct that would be specifically expressed in tomato fruits. We used this construct for tissue culture-based transformation and introduced it into tomato cultivar Ailsa Craig (AC). Up to date, more than 10 individual TFM7::SlNAC1Δ191-270 transgenic lines have been generated and the presence of transgene has been verified by PCR-based analysis. Generation of homozygous T2 lines for further characterization is in progress.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Han Lu, Youhong Fan, Yulin Yuan, Xiangli Niu, Bingyu Zhao, Yongsheng Liu, Fangming Xiao (2023). Tomato SlSTK is
involved in glucose response and regulated by the ubiquitin ligase SlSINA4. Plant Science. 2023 Mar 13;331:111672. doi:
10.1016/j.plantsci.2023.111672.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Lu H, Niu X, Fan Y, Yuan Y, Huang L, Zhao B, Liu Y, Xiao F(2023). The extensin protein SAE1 plays a role in leaf senescence and is targeted by the ubiquitin ligase SINA4 in tomato. J Exp Bot. 2023 Sep 29;74(18):5635-5652. doi: 10.1093/jxb/erad242. PMID: 37368909
|
Progress 04/01/22 to 03/31/23
Outputs
Target Audience:Through professional conferences and publications, we presented research results and interacted with peers in the field of plant growth and development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student and two undergraduate students have been trained in the PI's lab during the report period time. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?To determine genes differentially regulated by SlNAC1 and individual ADs involved in fruit development and ripening, we will conduct time-course gene expression profiling of tomato fruits to determine genes regulated by SlNAC1, particularly investigating the precise gene regulation by individual activation domains alone and together in the context of full-length SlNAC1 (Objective 1B). We will also continue to generate TFM7::SlNAC1Δ191-270 transgenic tomato plants. Multiple lines will be verified by PCR and advanced to T2 homozygous populations that will be evaluated to confirm that phenotypes correspond only to the expression of the transgene (objective 2A). Once obtained, homozygous TFM7::SlNAC1Δ191-270 transgenic plants will be grown in the greenhouse alongside previously-generated SlNAC1Δ191-270-OX transgenic plants and WT plants for an extensive phenotype assay (objective 2B).
Impacts
What was accomplished under these goals?
Objective 1: Determine the role of SlNAC1 in fruit development and ripening. Gene expression profiling analyses have shown that SlNAC1 is specifically highly expressed in immature green fruits but otherwise at basal level throughout the fruit development and ripening processes. We have generated two SlNAC1-KO transgenic tomato lines using the CRISPR/Cas9-based gene knockout technique. We used double-guide RNAs (dgRNAs) targeting the second exon of SlNAC1. The SlNAC1-KO-1 and SlNAC1-KO-2 contained a 4 bp deletion at different positions of the second exon of SlNAC1, resulting in a frame shift that generated a premature stop codon. The transgenic lines were advanced to T1 populations to identify individuals that contained homozygous deletion but the Cas9 cassette segregated out. T2 null SlNAC1-KO mutant populations were further generated and used for phenotypic analyses. SlNAC1-KO-1 and SlNAC1-KO-2 lines were grown side-by-side with WT plants in green house conditions. No significant difference was observed through fruit development and ripening at all critical stages, including anthesis, 1cm (8day post anthesis (DPA), immature green, mature green, breaker, pink, light red and red ripe. This result suggests a functional redundancy of SlNAC1 and other NAC members in tomato since there are more than one hundred NAC members in tomato and many of them have been shown to play a role in fruit ripening. In addition, to determine genes differentially regulated by SlNAC1 involved in fruit development and ripening, RNA-seq-based gene expression profiling analyses on various SlNAC1-overexpression fruits has been initiated. Objective 2. Manipulate SlNAC1 for early fruit ripening without growth detriments. In this objective, we aim to engineer an SlNAC1 construct under the control of a fruit-specific promoter to develop tomatoes with normal growth habits and yield, but early ripening fruits. We have cloned a 2-kb promoter from the fruit-specific gene TFM7. The TFM7 promoter has been verified with strong expression during fruit development from the 1cm to mature green stage. The TFM7 promoter has been fused with the SlNAC1Δ191-270 mutant, resulting in a novel TFM7::SlNAC1Δ191-270 construct that will be specifically expressed in tomato fruits. TFM7::SlNAC1Δ191-270 construct has been introduced into Agrobacterium GV3001 strain. Agrobacterium-mediated transformation is in progress. ?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Han Lu, Youhong Fan, Yulin Yuan, Xiangli Niu, Bingyu Zhao, Yongsheng Liu, Fangming Xiao (2023). Tomato SlSTK is involved in glucose response and regulated by the ubiquitin ligase SlSINA4. Plant Science. 2023 Mar 13;331:111672. doi: 10.1016/j.plantsci.2023.111672.
|
|