Overexpression of the class I homeodomain transcription factor TaHDZipI‐5 increases drought and frost tolerance in transgenic wheat

Summary Characterization of the function of stress‐related genes helps to understand the mechanisms of plant responses to environmental conditions. The findings of this work defined the role of the wheat TaHDZipI‐5 gene, encoding a stress‐responsive homeodomain–leucine zipper class I (HD‐Zip I) transcription factor, during the development of plant tolerance to frost and drought. Strong induction of TaHDZipI‐5 expression by low temperatures, and the elevated TaHDZipI‐5 levels of expression in flowers and early developing grains in the absence of stress, suggests that TaHDZipI‐5 is involved in the regulation of frost tolerance at flowering. The TaHDZipI‐5 protein behaved as an activator in a yeast transactivation assay, and the TaHDZipI‐5 activation domain was localized to its C‐terminus. The TaHDZipI‐5 protein homo‐ and hetero‐dimerizes with related TaHDZipI‐3, and differences between DNA interactions in both dimers were specified at 3D molecular levels. The constitutive overexpression of TaHDZipI‐5 in bread wheat significantly enhanced frost and drought tolerance of transgenic wheat lines with the appearance of undesired phenotypic features, which included a reduced plant size and biomass, delayed flowering and a grain yield decrease. An attempt to improve the phenotype of transgenic wheat by the application of stress‐inducible promoters with contrasting properties did not lead to the elimination of undesired phenotype, apparently due to strict spatial requirements for TaHDZipI‐5 overexpression.

A transactivation assay in yeast was used to identify the activation domain of TaHDZipI-5. The full-length open reading frame (ORF) or various truncated fragments of TaHDZipI-5 were individually fused in frame with the yeast GAL4 DNA-binding domain in the pGBKT7 vector (Invitrogen, Victoria, Australia). Constructs were transformed into yeast (Saccharomyces cerevisiae strain AH109). Transformed yeast cells were examined on synthetic defined (SD) (-Trp) medium and replica-plated to SD (-Trp / -His) medium. Yeast growth on the SD medium reflected the growth of yeast containing the native activation domain in truncated TaHDZipI-5 sequences.

Analysis of transgenic plants
Three independent T1 lines of transgenic wheat with a single copy of the transgene were selected for primary phenotypic characterisation and seed multiplication. Twelve seeds of control plants (WT) and twelve T1 transgenic seeds from each line were sown into 12-cm square pots filled with coco-peat soil, with one plant per pot, and grown under well-watered conditions in a greenhouse with day/night temperatures of 23 o C (16 hours) and 19 o C (8 hours).
Leaves of three-week old control and transgenic seedlings of each line were sampled for genomic DNA isolation. Plant height, tiller and spike number, seed weight, total dry biomass, seed number, flowering time and single grain weight were recorded for each plant. The transgene copy number was determined by Q-PCR (Fig. S1).
Comparative evaluations of growth and yield components of transgenic T3 lines and control plants grown under well-watered and mild-drought conditions were performed in two large containers filled with a mixture of coco-peat, sand and clay soil (1:1:1) (Shavrukov et al., 2016). Three independent T3 lines of all four transgenics were used for the evaluation of growth and yield components. Untransformed WT plants were used as control. Transgenic plants were grown in two identical containers, one with well-watered conditions and one with slowly increasing drought. In each container, 16 plants of each transgenic line and the same number of WT plants were randomly grown in rows, with eight plants per row. Leaf samples of each plant were collected for DNA/RNA isolation at the three-leaf stage of seedling development.
In the well-watered container, plants were regularly watered until maturity. In the drought-subjected container, plants were regularly watered until mid-tillering and watering stopped thereafter. Plants showed signs of mild wilting at the beginning of flowering. The soil water potential of each container was automatically monitored and recorded by Magpie-3 (Measuring Engineering Australia) using sensors in two depths (10 cm and 30 cm) below the soil surface ( Fig. S1). Growth and yield characteristics of transgenic lines and control plants were monitored in both containers. The data for each measured parameter for each line were statistically analysed using Student t-tests (unpaired, two-tails), and null-segregants were excluded from the analyses in cases where lines were heterozygous.

Drought tolerance test or the survival rate of seedlings under terminal drought
Two independent homozygous lines with minimal differences in a seedling size to those of control plants were used in a drought survival test, conducted in a PC2 glasshouse. WT plants were used as control. Seeds were sown in five 6-inch round pots filled with the same amount of coco-peat soil. Before sowing seeds, the soil in each pot was water-saturated by soaking the pot in water overnight in plastic trays. The following day, pots were removed and drained for 24 hours, and each pot was weighed after drainage. The soil moisture weight was calculated as the difference between the soil weight after drainage and the dry soil weight (measured after incubation for a week at 65 o C). Two plants of each line and WT plants were grown in each pot in the growth room under 24 o C during 16 hours day light and 19 o C during 8 hours darkness.
Plants in each pot were well-watered for three weeks, after which watering was stopped. During the well-watered stage, each pot was weighted daily and water was added if the soil water content was below 80% of soil moisture weight. After 25 to 28 days of drought, plants were rewatered and survival rates were assessed after a three-week recovery.
Frost tolerance test (survival rate of seedlings subjected to frost) Three T3 independent homozygous lines of transgenic plants were used in a frost survival test.
Untransformed WT plants were used as control. Seeds were sown in twelve 6-inch round pots filled with coco-peat soil. One plant of each line and WT plants were grown in a pot (Fig. S2A).
Plants in each pot were well-watered and kept in a PC2 room (24/16 o C of day/night temperature, 16 hours day length) for three weeks and later placed into a cold cabinet (BINDER, Tuttlingen, Germany). Plants in the cold cabinet were exposed to temperatures decreasing gradually from 18 o C to a minimum temperature of -8 o C with 6.5 hours under the lowest temperature, and then slowly returned back to 18 o C (Fig. S2B). Pots were insulated to protect plant roots from frost-damage. Leaf tissues for RNA isolation were collected before the stress application (Fig. S2B). In addition, leaves of plants with stress-inducible promoters and control plants were collected at 4 o C. The ice nucleating agent SNOMAX (Sno-Quip Pty Ltd, Mittagong, NSW, Australia) (2 g/L) was used to spray plants to prevent water crystallisation below 0 o C. After the frost treatment, pots were transferred back to the PC2 growth room for recovery. Survival rates were estimated after two weeks of recovery. TaHDZipI-5 The analysis of the downstream gene expression was performed by Q-PCR, as described by Fletcher et al. (2014). Gene-specific primers from 3'UTRs (Table S1) were used to analyse the expression levels of TaWZY2 (GenBank: EU395844), TaCOR14B (GenBank: AF207546;Tsvetanov et al., 2000), TaRAB15 (GenBank: X59133;King et al., 1992) and TaDREB3 (GenBank: DQ353853;Lopato et al., 2006) genes in three independent control WT plants and three T3 sublines of each of three independent transgenic lines with the constitutive overexpression of TaHDZipI-5. Three technical replicates were used in this experiment.

Legends to supplementary figures
Fig. S1. Soil water tension monitored at 10 cm and 30 cm depths in large containers used for plant growth under well-watered conditions or increasing drought. An arrow (no watering) indicates the point at which watering was withdrawn.       Table S1. List of PCR primers and DNA probes used in this study.   Table S3. Hydrogen bonds of homo-dimeric TaHDZipI-3 and TaHDZipI-5, and hetero-dimeric TaHDZipI-3/TaHDZipI-5 with HDZ1 (5'-CAATCATTGC-3'/5'-GCAATGATTG-3'). Table S4. Characteristics of the T2/T3 progenies of TaHDZipI-5 transgenic lines analysed in large containers under well-watered or mild drought condition.