Characterization of a novel zinc finger transcription factor (TaZnF) from wheat conferring heat stress tolerance in Arabidopsis

Abstract

C3HC4-type zinc finger proteins are known to play important roles in various plant processes including regulation of growth and development, signaling networks, responses to abiotic stresses etc. The current study identifies and explores the involvement of TaZnF in plant stress response, mainly heat stress. TaZnF belongs to C4HC3-type zinc finger transcription factor. Phylogenetic analysis of TaZnF revealed strong sequence similarity to Brachypodium distachyon, a model system for crop species. Gene expression studies have revealed its role under diverse stress conditions including heat and cold conditions. The transcript level of TaZnF was found to be highest in seed and starts at the post anthesis period 3-5DAA, a more sensitive stage resulting in a negative influence on the yield of crop species. TaZnF possesses transcriptional activity. Overexpression of TaZnF in Arabidopsis thaliana conferred improved tolerance to both basal and high-temperature stress as observed from various assays examining their growth and development. The transgenics were recovered and showed early flowering compared to wild-type. They had larger primary roots, more lateral branching, bigger, and more numerous leaves, resulting in heavier fresh weight. Enhanced growth and early recovery resulted in bigger plants with more yield. Additionally, the overexpression Arabidopsis transgenics also showed considerable tolerance to cold and oxidative stress. These observations suggest that TaZnF acts as a positive regulator of thermal stress and thus can be of great significance in understanding and improving temperature stress tolerance in plants.

Keywords: C4HC3-type; Cold stress; High-temperature stress; Oxidative stress; RING zinc finger; Triticum aestivum.

Fig. 1

Domain organization of TaZnF and comparison with its orthologue proteins in other plant species. a Nuclear localization signal prediction of TaZnF by NucPred tool. b Schematic representation of various domains in TaZnF protein by SMART tool. Pink color represent low complexity region, brown color represent RING domain and blue color the trans-membrane region. c Full length protein alignment of TaZnF with its orthologue from monocot and dicot species by Clustalx2

Fig. 2

Phylogenetic analysis of the relationships between TaZnF and its C3HC4-type RING finger genes in other species. The multiple alignment was done using ClustalW2 and the dendrogram was built using MEGA4.0 software with the neighbor-joining and pair-wise deletion options as a consensus of 1000 bootstrap replicates

Fig. 3

Transcription profiling of TaZnF transcription factor under various abiotic stress condition and in various tissues. Change in transcript abundance of TaZnF in a 10-day-old seedlings of PBW343 under heat (37 and 42 °C for 2 h), cold (4 °C for 2 h), NaCl (150 mM for 2 h), and mannitol (2% for 2 h). b Shoot and various tissues from mature plants of PBW343 under heat stress at 37 °C. The transcription level at control, i.e., 0 h of treatment was normalized as 1.0 and the result shown are the means ± SDs of at least three independent experiments

Fig. 4

Transcription profiling of TaZnF transcription factor during high-temperature stress and recovery in various genotypes of Triticum aestivum. Change in transcript abundance of TaZnF in 10-day-old seedlings of various genotypes subjected to high-temperature stress at 37 and 42 °C for 2 h and followed by 2 and 4 h of recovery, a PBW343, b HD2329, c K7903, and d C306. The transcription level of TaZnF at control, i.e., 0 h of treatment was normalized as 1.0 and the results shown are the means ± SDs of at least three independent experiments

Fig. 5

Transcription activation assay of TaZnF protein. a, b Transcriptional activation assay of TaZnF in yeast on SD-W and SD-HW media. Growth of yeast AH109 cells containing TaZnF::pGBKT7 constructs with positive and negative controls on SD-W media and on SD-HW media

Fig. 6

TaZnF overexpressing plants showing increased tolerance to high-temperature stress. One-week-old seedlings of both wild-type and transgenics were given heat stress at 42 °C for 2 h and left for recovery at culture room conditions. a Phenotype observed after 15 days of recovery. b One and half months after transferring the wild-type and transgenic lines to pots. c Transcription profile of TaZnF in wild-type and overexpression transgenic lines of Arabidopsis. The transcription level in wild type (WT) was normalized as 1.0 and the results shown are the means ± SDs of at least three independent experiments

Fig. 7

Arabidopsis transgenics overexpressing TaZnF subjected to high-temperature stress. Effect on various photosynthetic parameters like a photosynthetic efficiency (Fv/Fm). b Effective photosynthetic efficiency (YII); c electron transport rate; d membrane stability index (MSI %); e chlorophyll content (see text for details)

Fig. 8

Effect of heat stress on yield parameters. One-week-old seedlings of both wild-type and transgenics were given heat stress at 42 °C for 2 h and left to recover for almost 2½ month for analysis of various parameters related to yield like a length of siliques; b seed morphology; c number of siliques per plant; d yield per plant

Fig. 9

TaZnF overexpressing Arabidopsis plants showed increased tolerance to oxidative stress. a Oxidative stress response of 14-day-old WT and TaZnF overexpression Arabidopsis seedlings examined by comparing the accumulation of O₂ and H₂O₂ radicals under heat stress, i.e., at 37 °C for 2 h. b Oxidative stress treatment by subjecting leaf of 21-day-old seedlings of both wild and transgenics under by treatment by methyl viologen (50 μM) for 4 h by DAB staining. c Transcription profiling of various oxidative stress-related marker genes in transgenic lines of Arabidopsis plants overexpressing TaZnF. The transcription level in wild-type (WT) was normalized as 1.0 and the result shown are the means ± SDs of at least three independent experiments

Fig. 10

Overexpressing TaZnF plants show increased tolerance to cold stress. a Phenotype observed after cold stress given to 1-week-old seedlings for 24 h and left at recovery for 20 days; b measure of plant height, shoot length and root length; c estimation of fresh weight; d number of flowering plants; e photosynthetic efficiency (Fv/Fm); f membrane stability index (MSI); g proline content. h chlorophyll content measured after stress treatment

Fig. 11

Transcription profiling of marker genes in transgenic lines of Arabidopsis plants overexpressing TaZnF. The transcription level in wild-type (WT) was normalized as 1.0 and the result shown are the means ± SDs of at least three independent experiments