Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Plant Biology
Non Technical Summary
Chilling stress poses a threat to the growth and reproduction of major crop plants including cereals, vegetables and fruits. Recent events of extreme temperature fluctuations have caused yield losses of many agricultural products. Breeding chilling tolerant plants is an effective way to enhance the production under extreme conditions and expand growth area and growth season of crop plants. Earlier studies using natural variations of chilling tolerance in rice has identified CAK1 as a critical gene for chilling tolerance and a CAK1 variant associated with high tolerance. This project aims at using this elite natural variant of CAK1 to improve chilling tolerance in maize and rice. Four major objectives of this research are: 1) determine the function of CAK1 in chilling tolerance in maize, 2) establish the activities of the two CAK1 variants in rice, 3) determine the effectiveness of using CAK1-in4 in chilling tolerance enhancement in maize, 4) identify pathways/processes regulated by CAK1 genes using RNA-Seq. Together, this study will provide genetic materials and gene resources for breeding chilling tolerant crops. It will also forward our understanding of chilling tolerance mechanism and therefore will advance fundamental knowledge of plant adaptation to the environment.
Animal Health Component
50%
Research Effort Categories
Basic
30%
Applied
50%
Developmental
20%
Goals / Objectives
Chilling stress (low but non-freezing temperature) poses a threat to the growth and reproduction of major crop plants including cereals, vegetables and fruits. Recent events of extreme temperature fluctuations have caused yield losses of many agricultural products. Breeding chilling tolerant plants is an effective way to enhance the production under extreme conditions and expand growth area and growth season of crop plants. Compared to conventional breeding, molecular breeding is more effective and faster. However,it is limited by the lack of sufficient knowledge of chilling tolerance and the availability of genes and elite alleles contributing to chilling tolerance in crops.Current knowledge on low temperature responses largely comes from freezing tolerance studies in Arabidopsis thalianawhich is a chilling tolerant plant (Thomashaw, 1999; Hua, 2009). A large number of crop plants includingmaize and ricehave tropical and subtropical origins and arechilling sensitive, unlike Arabidopsis thaliana. My research group has started using rice as a model for chilling tolerance studies. Besides its small genome and ample research resources,rice has rich variations in chilling tolerance resulting froma large number of quantitative trait loci (QTL), making it a good model for chilling tolerance studies. We haveidentified several QTLs usinggenome wide association study for chilling tolerance at seedling stage in rice.One candidate gene for a major QTL for chilling tolerancehas been verified ascasein kinase 1 (CAK1). Using CRISPR/CAS9 method, we generated two knockout mutants of the OsCAK1 gene, and both of them exhibited reduced chilling tolerance compared to the wild type assayed by tolerance index, survival rate and ion leakage. In addition, polymorphisms in the OsCAK1 gene were found to be correlated with chilling tolerance in rice varieties. In particular,a haplotype of OsCAK1 that encodes a protein variant with an extra 4 amino acids is correlated with high chilling tolerance. Furthermore, this CAK1-in4 variant had a higher kinase activity than the other variantwhen assayed in vitro. Therefore, CAK1-in4 is a promising natural variant or elite allele of CAK1 that confers chilling tolerance in rice, and identified one causal gene and a potential allele that is associated with high chilling tolerance. The proposed researchaims at translating this finding from rice into maize to facilitate the breeding of chilling tolerant maize and other crops.Four major objectives of this research are:1. Determine the function of CAK1 in chilling tolerance in maize2. Establish the activities of the two CAK1 variants in rice3. Determine the effectiveness of using CAK1-in4 in chilling tolerance enhancement in maize4. Identify pathways/processes regulated by CAK1 genes using RNA-Seq.
Project Methods
1. Determine the function of CAK1 in chilling tolerance in maize.Maize has one CAK1 gene (we will refer as ZmCAK1) that appears to be orthologous to the rice CAK1 gene based on tree analysis. We will use CRISPR/CAS9 technology (Peng et al 2018) to knockout the activity of ZmCAK1 in maize. Constructs will be made to target the ZmCAK1 gene and transgenic maize plants will be generated via Agrobacterium mediated transformation at the Cornell transformation facility. Transgenic plants will be assayed by PCR and sequencing to identify those with desired mutations at ZmCAK1 that would cause disruption of the gene function. Progenies of the transgenic lines will be selected for CAS9 free and homozygous for the knockout mutations. At least three independent lines will be assayed for chilling tolerance with the wild type plant as control. Survival rate, leaf death, and ion leakage will be measuredduring or after chilling treatment to indicate the level of tolerance.Once a chilling sensitive phenotype is established for these lines, co-segregation analysis among progenies of a heterozygous plant can be used to corroborate the mutant phenotype.2. Establish the activity of the twovariants of OsCAK1 in rice.The OsCAK1-in4 variantwith an extra 4 residues was shown to havea higher protein kinase activity at chilling temperature, and this variation is associated with higher chilling tolerance in the natural population of indica rice. We will use transgenic approachto confirm that this variant of CAK1 indeed could enhancechilling tolerance. We will transformOsCAK1-int4 and OsCAK1 (from the reference accession) into the Oscak1 knockout mutant respectively. Four independent transgenic plants of eachvariant will be assayed for chilling tolerance by both cold tolerance index and survival rate in the second generation. We expect that those with OsCAK1-in4 will have a higher tolerance than those with OsCAK1.3. Determine the effectiveness of using CAK1 variants in chilling tolerance enhancement in maize.We will analyzenatural variants of ZmCAK1 in maize natural populations. Similar adaptation or selection may have occurred in ZmCAK1 as in OsCAK1leading to variationsat the ZmCAK1 gene associated with chilling tolerance. If so, we will transform the high tolerant variant and a reference variant into the ZmCAK1 knockout plants. Transgenic lines will be assayed for chilling tolerance to determine if indeed the variant confers higher chilling tolerance. If no apparent variants could be identified in existing maize varieties, we will generate an equivalent variant of ZmCAK1-in4 by in vitro mutagenesis. The reference ZmCAK1 and ZmCAK1-in4 variants will be transformed into the maize ZmCAK1 knockout plant respectively. Transgenic plants will be assayed for chilling tolerance to determine the effect of ZmCAK1-in4.4. Identify pathways/processes regulated by CAK1 genes using RNA-Seq.To better understand chilling tolerance mechanism, we willanalyze transcriptome changes in theCAK1 mutants to reveal potential pathways and processes associated with chilling tolerance. We will compare the transcription profiles of knockout mutants of OsCAK1 and ZmCAK1 to those of their respective wild types under chilling stress. The cak1 mutants and the wild-type plants will be grown at normal temperature till 3 leaf stage and then be transferred to a low temperature. RNA samples prepared from leaf tissues collected at 0 hour (hr), 3hr and 24hr after chilling will be submitted to Cornell Sequencing Facility where 3'-RNA-Seq will be conducted. Differentially expressed genes (DEGs) between wild type plants and the mutants will be identified in rice and maize respectively. DEGs shared between the Oscak1 and Zmcak1 mutants under chilling will be selected. Specifically enriched GO terms in these DEGs may reveal the pathway(s) that is defective in the cak1 mutants andlikely important for chilling tolerance. This analysis will form the foundation for further mechanistic investigation oftolerance mechanism and provide additional resource for candidate gene selection to improve chilling tolerance.