Analysis of bZIP Transcription Factor Family and Their Expressions under Salt Stress in Chlamydomonas reinhardtii

Abstract

The basic leucine-region zipper (bZIP) transcription factors (TFs) act as crucial regulators in various biological processes and stress responses in plants. Currently, bZIP family members and their functions remain elusive in the green unicellular algae Chlamydomonas reinhardtii, an important model organism for molecular investigation with genetic engineering aimed at increasing lipid yields for better biodiesel production. In this study, a total of 17 C. reinhardtii bZIP (CrebZIP) TFs containing typical bZIP structure were identified by a genome-wide analysis. Analysis of the CrebZIP protein physicochemical properties, phylogenetic tree, conserved domain, and secondary structure were conducted. CrebZIP gene structures and their chromosomal assignment were also analyzed. Physiological and photosynthetic characteristics of C. reinhardtii under salt stress were exhibited as lower cell growth and weaker photosynthesis, but increased lipid accumulation. Meanwhile, the expression profiles of six CrebZIP genes were induced to change significantly during salt stress, indicating that certain CrebZIPs may play important roles in mediating photosynthesis and lipid accumulation of microalgae in response to stresses. The present work provided a valuable foundation for functional dissection of CrebZIPs, benefiting the development of better strategies to engineer the regulatory network in microalgae for enhancing biofuel and biomass production.

Keywords: Chlamydomonas reinhardtii; bZIP transcription factors; lipid accumulation; photosynthesis; salt stress; transcriptional regulation.

Figure 1

Phylogenetic analysis of C. reinhardtii and Arabidopsis basic leucine-region zipper (bZIP) proteins. ClustalW was employed to align the protein sequences of 17 CrebZIPs and 11 AtbZIPs representing subgroups A to I, S, and U in Arabidopsis. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA7.0 software. The evolutionary distances were computed using the Poisson correction method with the number of amino acid substitutions per site as the unit. All positions containing gaps and missing data were excluded.

Figure 2

Analysis of the conserved domain in CrebZIP proteins. The conserved motifs in the CrebZIP proteins were identified with MEME software. Conserved sites in the key conserved domain (motif 1) are indicated by “←”.

Figure 3

The Exon-intron organization of CrebZIP genes. The exons and introns are represented by yellow boxes and horizontal black lines, respectively. Untranslated regions are shown with blue boxes. The length of CrebZIP genes are indicated by the horizontal axis (kb).

Figure 4

Distribution of CrebZIP gene family members on the C. reinhardtii chromosomes. The chromosome number is indicated at the top of each chromosome. Values next to the bZIP genes indicate the location on the chromosome.

Figure 5

The effects of salt stress on (a) cell growth and (b) total lipid content of C. reinhardtii. Cell samples harvested after 0, 24, and 48 h cultivation from both the salt treatment and the control were used for measurements of cell growth and lipid content. Each value is the mean ± SD of three biological replicates. Asterisks indicate statistical significance (* 0.01 < p < 0.05, ** p < 0.01) between control and salt-treated cells according to the Tukey’s test.

Figure 6

Nile Red staining lipid drops in C. reinhardtii under (a) salt stress and (b) the control treatments. The cell and lipid droplet colors were visualized with red and yellow, respectively, under fluorescent light.

Figure 7

The effect of salt stress on pigment contents of C. reinhardtii. Cell samples harvested after 0, 24, and 48 h cultivation from both the salt treatment and the control were used to measure the contents of chlorophyll a, Chlorophyll b, and carotenoid. Each value is the mean ± SD of three biological replicates. Asterisks indicate statistical significance (* 0.01 < p < 0.05, ** p < 0.01) between control and salt-treated cells according to the Tukey’s test.

Figure 8

The effects of salt stress on (a) the maximum photo-chemical efficiency (F_v/F_m) and (b) non-photochemical quenching co-efficiency (NPQ) of C. reinhardtii. Cell samples harvested after 0, 24, and 48 h cultivation from both the salt treatment and the control, were used for measurements of F_v/F_m and NPQ. Each value is the mean ± SD of three biological replicates. Asterisks indicate statistical significance (* 0.01 < p < 0.05, ** p < 0.01) between control and salt-treated cells according to the Tukey’s test.

Figure 9

Expression profiles of six CrebZIP genes in C. reinhardtii under salt stress. Expression of CrebZIP genes was determined by qRT-PCR using total RNA from microalgae cells sampled at different time points of salt treatment. The α-tubulin gene was used as the internal reference gene. Each value is the mean ± SD of six biological replicates. Asterisks indicate statistical significance (* 0.01 < p < 0.05, ** p < 0.01) between control and salt-treated cells according to the Tukey’s test.