Cloning and Characterization of the Iron-Regulated Transporter (IRT) Genes and Their Transcription Factors in Populus

Iron deficiency causes chlorosis in many plant species, resulting in yield loss and poor quality. Many tree species including poplar are susceptible to iron deficiency. Trees suffering from iron deficiency often show interveinal chlorotic leaves and in severe cases, branches or an entire tree may die. In this study, two trees of Populus tremula L.‘Erecta’ growing near each other but with contrasting leaf color phenotypes were used to study the causes of chlorosis and the mechanisms of tolerance or susceptibility to iron chlorosis in poplar. A leaf analysis revealed that the iron deficiency tolerant tree (PtG) had a higher level of dry matter content, chlorophyll (a+b), Chl a/b ratio, Zn and Fe content than the iron chlorosis susceptible tree (PtY). A hydroponic culture confirmed the differences in aforementioned physiological parameters between PtG and PtY responding to iron deficiency. Two iron-regulated transporter genes (PtIRT1 and PtIRT3), the native promoter of the PtIRT1 gene (PtIRT1-pro), and two basic helix-loop-helix (bHLH) transcription factors (PtFIT and PtIRO) were cloned and characterized for their responses to iron deficiency in PtG and PtY. Deduced amino acid analysis revealed that PtIRT1, PtIRT3, PtFIT, and PtIRO in PtG were identical to those in PtY. Phylogenetic and putative domain analyses showed that PtIRT1, PtFIT, and PtIRO may function in iron homeostasis, while PtIRT3 may play a role in zinc transport in poplar. The expression of PtIRT1 and PtFIT are root-specific and up-regulated by iron deficiency. The expression of a GUS gene derived by PtIRT1-pro in tobacco was also up-regulated by iron deficiency, but was not root-specific. The expression of PtIRT3 is ubiquitous and up-regulated by iron deficiency, but significantly down-regulated by zinc deficiency. A high correlation in the expression between PtFIT and PtIRT1 was observed in PtG, but not in PtY. Transgenic poplars overexpressing PtIRT1 or PtIRT3 did not have enhanced Fe accumulation; however, an enhanced tolerance to iron deficiency was found in transgenic plants overexpressing PtFIT. The results suggested that the transcription factor PtFIT may be involved in iron deficiency response through regulation of PtIRT1 and PtFIT itself may be regulated by other factors in poplar

Cloning and Characterization of the Iron-Regulated Transporter (IRT) Genes and Their Transcription Factors in Populus

Video summarizing Ph.D. dissertation for a non-specialist audience.Consortium for Plant Biotechnology Research (CPBR)Plant SciencesPlant SciencesCollege of Agriculture, Food Systems and Natural Resource

Discovery of Geranylgeranyl Pyrophosphate Synthase (GGPPS) Paralogs from Haematococcus pluvialis Based on Iso-Seq Analysis and Their Function on Astaxanthin Biosynthesis

Haematococcus pluvialis is widely distributed in the world and well known as the richest natural source of astaxanthin that is a strong antioxidant with excellent commercial value. The pathway of astaxanthin biosynthesis in H. pluvialis has been documented as an enzymatic reaction. Several enzymes have been reported, but their isoforms or homologs have not been investigated genome-wide. To better understand the astaxanthin biosynthesis pathway in H. pluvialis, eight candidates of the geranylgeranyl pyrophosphate synthase gene (HpGGPPS) predicted from Iso-seq data were isolated in this study. The length of coding region of these candidates varied from 960 bp to 1272 bp, composing of 7–9 exons. The putative amino acids of all candidates composed the signature domain of GGPPS gene. However, the motifs in the domain region are varied, indicating different bio-functions. Phylogenetic analysis revealed eight candidates can be clustered into three groups. Only two candidates in Group1 encode the synthase participating in the astaxanthin formation. The yield of astaxanthin from these two candidates, 7.1 mg/g (DW) and 6.5 mg/g (DW) respectively, is significant higher than that from CrtE (2.4 mg/g DW), a GGPPS gene from Pantoea ananatis. This study provides a potential productive pathway for astaxanthin synthesis

Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in <i>Chlamydomonas reinhardtii</i>

Anti-lipopolysaccharide factor 3 (ALFPm3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALFPm3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALFPm3 in Chlamydomonas reinhardtii. In this study, signal peptides ARS1 and CAH1 were fused with ALFPm3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALFPm3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALFPm3 could be detected in algal cells and culture medium by immunoblot, meaning that ALFPm3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALFPm3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi, V. alginolyticus, V. anguillarum, and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALFPm3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALFPm3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALFPm3 peptide. Our results provided a new strategy for the secretory production of ALFPm3 with high antibacterial activity in C. reinhardtii, which could improve the application potentiality of ALFPm3 in the aquaculture industry

Improvement of Carotenoids’ Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies

Canthaxanthin is an important antioxidant with wide application prospects, and β-carotene ketolase is the key enzyme involved in the biosynthesis of canthaxanthin. However, the challenge for the soluble expression of β-carotene ketolase is that it hinders the large-scale production of carotenoids such as canthaxanthin and astaxanthin. Hence, this study employed several strategies aiming to improve the soluble expression of β-carotene ketolase and its activity, including selecting optimal expression vectors, screening induction temperatures, adding soluble expression tags, and adding a molecular chaperone. Results showed that all these strategies can improve the soluble expression and activity of β-carotene ketolase in Escherichia coli. In particular, the production of soluble β-carotene ketolase was increased 8 times, with a commercial molecular chaperon of pG-KJE8, leading to a 1.16-fold enhancement in the canthaxanthin production from β-carotene. Interestingly, pG-KJE8 could also enhance the soluble expression of β-carotene ketolase derived from eukaryotic microalgae. Further research showed that the production of canthaxanthin and echinenone was significantly improved by as many as 30.77 times when the pG-KJE8 was added, indicating the molecular chaperone performed differently among different β-carotene ketolase. This study not only laid a foundation for further research on the improvement of β-carotene ketolase activity but also provided new ideas for the improvement of carotenoid production

Enhancement of β-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene β-cyclase

Abstract β-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of β-carotene and α-carotene. Aiming to develop the microalgae with enhanced β-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of β-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant β-carotene), confirming the superior activity of CrtY on β-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased β-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae