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HTT2 promotes plant thermotolerance in Brassica rapa.

ABSTRACT: Numerous regulatory genes participate in plant thermotolerance. In Arabidopsis, HEAT-INDUCED TAS1 TARGET2 (HTT2) is an important thermotolerance gene that is silenced by ta-siR255, a trans-acting siRNA. ta-siR255 is absent from heading Chinese cabbage (Brassica rapa ssp. pekinensis). Our previous attempt to overexpress the endogenous BrpHTT2 gene of heading Chinese cabbage (B. rapa ssp. pekinensis) failed because of cosuppression. In theory, heading Chinese cabbage can overexpress Arabidopsis HTT2 to improve thermotolerance in the absence of ta-siR255-mediated gene silencing and the weak potential of coexpression.To test the potential application of HTT2 in improving crop thermotolerance, we transferred p35S::HTT2 to heading Chinese cabbage. We tested the leaf electrical conductivity, hypocotyl elongation, and survival percentage of p35S::HTT2 plants subjected to high-temperature (38 °C) and heat-shock (46 °C) treatment. The leaf electrical conductivity of p35S::HTT2 seedlings under high temperature decreased but did negligibly change under heat shock. The hypocotyl length of p35S::HTT2 seedlings increased under high temperature and heat shock. The survival rate of p35S::HTT2 seedlings increased under heat shock. BrpHsfs, a subset of heat-shock factor genes, were upregulated in p35S::HTT2 plants under high-temperature and heat shock conditions. In the field, transgenic plants with HTT2 appeared greener and formed leafy heads earlier than wild-type plants.Exogenous HTT2 increased the survival rates of heat-shocked heading Chinese cabbage by promoting thermotolerance through decreasing electrical conductivity and extending hypocotyl length. Our work provides a new approach to the genetic manipulation of thermotolerance in crops through the introduction of exogenous thermotolerance genes.

SUBMITTER: Jiang J

PROVIDER: S-EPMC6011422 | biostudies-other | 2018 Jun

REPOSITORIES: biostudies-other

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HTT2 promotes plant thermotolerance in Brassica rapa.

Jiang Jianxia J Bai Jinjuan J Li Shuxia S Li Xiaorong X Yang Liyong L He Yuke Y

BMC plant biology 20180620 1

<h4>Background</h4>Numerous regulatory genes participate in plant thermotolerance. In Arabidopsis, HEAT-INDUCED TAS1 TARGET2 (HTT2) is an important thermotolerance gene that is silenced by ta-siR255, a trans-acting siRNA. ta-siR255 is absent from heading Chinese cabbage (Brassica rapa ssp. pekinensis). Our previous attempt to overexpress the endogenous BrpHTT2 gene of heading Chinese cabbage (B. rapa ssp. pekinensis) failed because of cosuppression. In theory, heading Chinese cabbage can overexp ...[more]

PMID: 29925322

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Project description:Chinese cabbage (Brassica rapa L. ssp. pekinensis) is sensitive to high temperature, which will cause the B. rapa to remain in a semi-dormancy state. Foliar spray of GB prior to heat stress was proven to enhance B. rapa thermotolerance. In order to understand the molecular mechanisms of GB-primed resistance or adaptation towards heat stress, we investigated the transcriptomes of GB-primed and non-primed heat-sensitive B. rapa 'Beijing No. 3' variety by RNA-Seq analysis. A total of 582 differentially expressed genes (DEGs) were identified from GB-primed plants exposed to heat stress relative to non-primed plants under heat stress and were assigned to 350 gene ontology (GO) pathways and 69 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. The analysis of the KEGG enrichment pathways revealed that the most abundantly up-regulated pathways were protein processing in endoplasmic reticulum (14 genes), followed by plant hormone signal transduction (12 genes), ribosome (8 genes), MAPK signaling pathway (8 genes), homologous recombination (7 genes), nucleotide excision repair metabolism (5 genes), glutathione metabolism (4 genes), and ascorbate and aldarate metabolism (4 genes). The most abundantly down-regulated pathways were plant-pathogen interaction (14 genes), followed by phenylpropanoid biosynthesis (7 genes); arginine and proline metabolism (6 genes); cutin, suberine, and wax biosynthesis (4 genes); and tryptophan metabolism (4 genes). Several calcium sensing/transducing proteins, as well as transcription factors associated with abscisic acid (ABA), salicylic acid (SA), auxin, and cytokinin hormones were either up- or down-regulated in GB-primed B. rapa plants under heat stress. In particular, expression of the genes for antioxidant defense, heat shock response, and DNA damage repair systems were highly increased by GB priming. On the other hand, many of the genes involved in the calcium sensors and cell surface receptors involved in plant innate immunity and the biosynthesis of secondary metabolites were down-regulated in the absence of pathogen elicitors in GB-primed B. rapa seedlings. Overall GB priming activated ABA and SA signaling pathways but deactivated auxin and cytokinin signaling pathways while suppressing the innate immunity in B. rapa seedlings exposed to heat stress. The present study provides a preliminary understanding of the thermotolerance mechanisms in GB-primed plants and is of great importance in developing thermotolerant B. rapa cultivars by using the identified DEGs through genetic modification.

Project description:Two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines, Chiifu (smaple A) and Kenshin (Smaple B), were grown for approximately 4 weeks in a growth chamber at 22degreesC under a 16 h light/8 h dark photoperiod with a photon flux density of 140 umol m-2 s-1. For microarray analysis, plants were exposed to a temperature of -4degreesC for 4 h, and shoots were sampled. Total RNA was isolated from the samples using an Easy-BLUETM Total RNA Extraction Kit (Invitrogen, NY, U.S.A.) and was then purified using an RNeasy MinEluteTM Cleanup Kit (Qiagen, Germany). Mircroarray experiments were carried out using Brassica 24K Oligo Microarray. RNA were prepared from each plant sample and 10 ug of total RNA were used for cDNA synthesis by using Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, NY, U.S.A.). Following cDNA synthesis, remaining RNA was removed by the addition of RNaseA and the cDNA was precipitated following phenol/chloroform extraction. The cDNA pellet was rehydrated and used for Cy3-labelling. For the synthesis of Cy3-labeled target DNA fragments, 1 ug of double strand cDNA was mixed with 40 ul (1 OD) of Cy3-9 mer primers (Sigma-Aldrich) and the total volume was adjusted to 80 ul with deionized water. After heating at 98degreesC for 10 min, 10 uL of dNTP mix (10 mM each) and 2 ul of Klenow fragment (NEB; 50 units/ul) were added and the reaction was incubated at 37degreesC for 2 h. Finally, the reaction was stopped by the addition of 10 ul of 0.5 M EDTA. Labeled DNA fragments were precipitated with isopropanol and rehydrated with water. The concentration of each sample was determined using spectrophotometer. For hybridization, 13 ug of the labeled DNA fragment was mixed with hybridization buffer (NimbleGen) and then hybridized with the microarray using a MAUI hybridization chamber (Biomicro) at 42degreesC for 16 h. Following hybridization, the microarray was washed with wash solution I, II, and III (NimbleGen), and then dried in a dark desiccator. The microarray slide was scanned using GenePix scanner 4000B (Axon) and the spot intensities were analyzed using a NimbleScan (NimbleGen). The normal distribution of Cy3 intensities was tested by qqline. The data was normalized and processed with cubic spline normalization using quantiles to adjust signal variations between chips and with Rubust Multi-Chip Analysis (RMA) using a median polish algorithm implemented in NimbleScan software (NimbleGen). Two biological replicates were carried out.

Dataset Information

HTT2 promotes plant thermotolerance in Brassica rapa.

Publications

HTT2 promotes plant thermotolerance in Brassica rapa.

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