Biochemical and Biophysical Research Communications
Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene
Introduction
Serine/arginine-rich (SR) proteins are important splicing factors. These proteins are involved in many biological processes through regulating gene expression [1], [2]. In mammalians, many studies have shown their essential roles in embryonic development since disruption of SR genes could lead to serious defects in development, such as embryonic lethal, largely due to misregulation of target gene expression [2].
Although the functions in splicing and expression patterns of plant SR genes have been extensively studied, their biological roles remain largely unclear [3]. Several studies have suggested that SR proteins are essential for plant development. For example, in Arabidopsis, overexpressing SRp30 or RSZ33 results in morphological and developmental changes [4], [5]. Loss of function in SR-related protein SR45 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens [6], [7]. Recently, SR45 was reported as a negative regulator of glucose and ABA signaling during early seedling development in Arabidopsis [8]. Of note, the biological functions of SR genes are not restricted to development. In some plants, such as Arabidopsis and rice, SR genes are regulated by some abiotic stresses, suggesting their potential roles in plant tolerance to abiotic stresses by regulation of downstream target genes [9], [10].
Among various abiotic stresses, we focused on cadmium (Cd) because it is a non-essential element that negatively affects plant growth and development. In plants, the roots are the main site of Cd absorption and accumulation. Thus, to restrict Cd uptake into the roots is one common mechanism underlying plant Cd tolerance. In this process, Cd transporters located in plasma membrane of root epidermal cells play essential roles and the iron-regulated transporter 1 (IRT1) is believed to be one such Cd transporter [11], [12], [13]. Evidences come from the observation that overexpression of IRT1 in transgenic Arabidopsis and rice (Oryza sativa) results in enhanced sensitivity to Cd due to accumulating high level of Cd [12], [14].
In this study, using a reverse genetic approach, we demonstrate that Cd-upregulated Arabidopsis SR34b gene is a regulator involved in splicing, mRNA stability, and protein accumulation of IRT1 gene, thereby participating in tolerance to Cd in Arabidopsis plants. These observations highlight the roles of SR34b in IRT1 expression and provide clear evidence for SR protein involved in resistance to Cd in plants.
Section snippets
Plant materials and growth conditions
The seeds of Arabidopsis (Col ecotype) were surface-sterilized and sown on 1/2 Murashige and Skoog (MS)-agar plates at 22 °C with 16-h/8-h light/dark cycles.
RNA isolation and cDNA synthesis
Total RNA was extracted from Arabidopsis samples using the RNAprep Pure Plant kit with on-column DNase digestion (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. RNA (2 μg) was used to synthesize the first-strand cDNA with an oligo (dT) primer according to the instruction of PrimeScript™ 1st strand cDNA synthesis
Eight Arabidopsis SR genes respond to Cd stress
We employed RT-PCR analysis to measure the expression of SR genes in Arabidopsis upon Cd exposure. Results showed that the mRNA level of seven SR genes (RS31, RS40, SRZ22, RSZ32, SCL30, SCL30a, and SCL28) was reduced; whereas the SR34b mRNA level was induced (Fig. 1). We selected SR34b for further study because it is the only gene upregulated by Cd among of 17 SR genes.
SR34b T-DNA insertion mutant is sensitive to Cd
To explore the physiological relevance of SR34b in Arabidopsis response to Cd, we established a line homozygous T-DNA insertion
Discussion
In this study, we used a reverse genetic approach to demonstrate that mutation in SR34b causes a moderate decrease in Cd tolerance. sr34b mutant plants have higher Cd2+ uptake rate and accumulated Cd in greater amounts than wild-type. The constitutive accumulation of IRT1 protein in sr34b is responsible for Cd stress-sensitive phenotype of this mutant. Although disruption of SR34b inhibited the IRT1 splicing and then reduced its mRNA level under Cd stress, IRT1 protein accumulation still
Acknowledgments
We are grateful to Prof. Weiwei Zhang (College of life science, Capital Normal University, China) for critical reading and valuable suggestions. This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 31271293 and 30770181) and the Beijing Natural Science Foundation (Grant No. 5112005) to X.Q.
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