Molecular and Functional Characterization of Wheat ARGOS Genes Influencing Plant Growth and Stress Tolerance

Abstract

Auxin Regulated Gene involved in Organ Size (ARGOS) is significantly and positively associated with organ size and is involved in abiotic stress responses in plants. However, no studies on wheat ARGOS genes have been reported to date. In the present study, three TaARGOS homoeologous genes were isolated and located on chromosomes 4A, 4B, and 4D of bread wheat, all of which are highly conserved in wheat and its wild relatives. Comparisons of gene expression in different tissues demonstrated that the TaARGOSs were mainly expressed in the stem. Furthermore, the TaARGOS transcripts were significantly induced by drought, salinity, and various phytohormones. Transient expression of the TaARGOS-D protein in wheat protoplasts showed that TaARGOS-D localized to the endoplasmic reticulum. Moreover, overexpression of TaARGOS-D in Arabidopsis resulted in an enhanced germination rate, larger rosette diameter, increased rosette leaf area, and higher silique number than in wild-type (WT) plants. The roles of TaARGOS-D in the control of plant growth were further studied via RNA-seq, and it was found that 105 genes were differentially expressed; most of these genes were involved in 'developmental processes.' Interestingly, we also found that overexpression of TaARGOS-D in Arabidopsis improved drought and salinity tolerance and insensitivity to ABA relative to that in WT plants. Taken together, these results demonstrate that the TaARGOSs are involved in seed germination, seedling growth, and abiotic stress tolerance.

Keywords: ABA; Arabidopsis; TaARGOS; abiotic stress; transgenic gene; wheat.

FIGURE 1

Cloning regions of the TaARGOSs using genome-specific primers (A) and each primer set was tested on CS nulli-tetrasomic lines (B). Three sets of primers recognized as TaARGOS-4A, -4B, and -4D on chromosomes 4A, 4B, and 4D, respectively. N4AT4B: nullisomic 4A-tetrasomic 4B; N4AT4D: nullisomic 4A-tetrasomic 4D; N4BT4A: nullisomic 4B-tetrasomic 4A; N4BT4D: nullisomic 4B-tetrasomic 4D; N4DT4A: nullisomic 4D-tetrasomic 4A; N4DT4B: nullisomic 4D-tetrasomic 4B; CS: Chinese Spring.

FIGURE 2

Sequence alignment and phylogenetic analysis of the TaARGOSs and other ARGOS proteins. (A) Amino acid sequence alignment of the TaARGOSs and the ARGOS from Arabidopsis AtARGOS (Hu et al., 2003), AtARGOS-like (Hu et al., 2006), AtOSR1 (Feng et al., 2011), and AtOSR2 (Qin et al., 2014), rice OsARGOS (Wang et al., 2009), and maize ZmARGOS1 (Guo et al., 2014) and ZmARGOS8 (Shi et al., 2015). Consensus sequences (100 and 75%) are exhibited in black and gray shading. The black line represents the conserved OSR domain. Red boxes indicate the putative transmembrane helices. (B) Phylogenetic tree based on amino acid sequences indicating the relationships of TaARGOSs with other plant ARGOS proteins. The accession numbers of the amino acid sequences are given in brackets. Twelve wheat protein sequences from the UniProt databases were indicated by dots. ZmARGOS2 is an allele variant of the ZmARGOS1 gene.

FIGURE 3

Alignment of deduced amino acid sequences of three TaARGOS homoeologous genes in wheat and its relatives. The coding sequences of the TaARGOSs in three A genome T. urartu species, three B genome Aegilops speltoides species, three D genome A. tauschii species, three tetraploid T. dicoccoides species, and six common wheat cultivars (T. aestivum L.) were used for sequence analysis. Homology tree was constructed by DNAMAN 7 software.

FIGURE 4

Expression pattern of the TaARGOS genes. (A) Expression pattern analysis by qRT-PCR of the three TaARGOS homoeologous genes in various wheat tissues. Total RNA was isolated from roots, stems, leaves, young spikes (YS), dry seeds, and seedlings at 1–6 days after germination (DAG). (B–I) qRT-PCR assessment of expression patterns of the wheat TaARGOS genes under PEG, NaCl, and exogenous ABA, MeJA, NAA, ACC, or GA₃ treatments. Total RNA was isolated from leaves of wheat seedlings. The β-actin gene was used as an internal reference. Error bars show standard deviation (SD) of three independent biological replicates.

FIGURE 5

Promoter isolation and activity analysis of the TaARGOSs (A) Cis-acting regulatory elements of promoter regions of TaARGOSs. (B) GUS staining of various pTaARGOS-D::GUS transgenic Arabidopsis tissues. GUS staining is mainly localized to leaf veins, hypocotyl, the trichomes of the first true leaves, roots, and flower (arrow). Scale bars = 0.2 mm. (C) 7-day-old Arabidopsis pTaARGOS-D::GUS seedlings were grown on MS plates before transfer to either MS plates (Control), plates with 10% PEG6000, 150mM NaCl, 100 μM ABA, 10 μM MeJA, 50 μM NAA, 10 μM ACC, or 50 μM GA3 and treated for 6 h. Enhanced GUS staining is visible in stresses treated seedlings. Scale bars = 2 mm.

FIGURE 6

Subcellular localization of the TaARGOS protein in wheat protoplasts. Photographs were taken in the light field (A and D), in the dark field for green fluorescence (B and E), and in combination for morphology of the cells (C and F). Bars = 10 μM.

FIGURE 7

Morphological differences between WT and transgenic line at different developmental stages. (A) Phenotype of WT and overexpressing TaARGOS-D seedlings grown for 7, 21, 28, and 56 days, respectively. Plant height (B), fresh weight (C), and silique number (D) of 56-day-old WT and TaARGOS-D transgenic plants (n = 10). (E,F) Number of epidermal cells per area in the adaxial surface of fully expanded fifth rosette leaves of transgenic line and WT plants (n = 5). Bars = 50 μM. Error bars represent the means ± SD. Statistical significance was determined by a Student’s t-test (B–E); significant differences (P ≤ 0.05) are indicated by asterisk.

FIGURE 8

(A) Gene ontology classification for differentially expressed genes (DEGs) in WT and TaARGOS-D transgenic plants. (B) DEGs in TaARGOS-D transgenic and WT Arabidopsis plants by qRT-PCR analysis. Error bars show SD of three replications.

FIGURE 9

Overexpression of TaARGOS-D confers drought and salt tolerance to transgenic Arabidopsis plants. (A–D) Seed germination in WT and TaARGOS-D overexpressing plants. The picture was taken 7 days after sowing. (E) Images of 7-day-old seedlings of WT and three transgenic lines treated with 10% PEG6000 or 150 mM NaCl for 5 days. The fresh weight and root lengths of the seedlings were measured. (F) Phenotypes of WT and three transgenic lines during drought stress. The 4-week-old seedlings (0 days) of WT and transgenic lines were subjected to drought stress treatment by withholding irrigation for 14 days, after which watering was resumed and plants grew for another 2 days. The percentage of survived plants in drought stress was measured. (G) Images of 4-week-old, soil-grown plants (WT and three transgenic lines; 0 days) that were irrigated with water containing 0 or 200 mM NaCl every 3 days for 14 days. The results are means of three replicates ± S.D. Statistical significance was determined by a Student’s t-test; significant differences (P ≤ 0.05) are indicated by asterisk.

FIGURE 10

Proposed model linking overexpression of TaARGOS-D and plant growth and stress tolerance. Red color represents up-regulated genes and blue color represents down-regulated genes. The numbers in brackets indicate the fold change of transcripts.