The tobacco chloroplast YCF4 gene is essential for transcriptional gene regulation and plants photoautotrophic growth

Abstract

A tobacco chloroplast hypothetical open reading frame 4 (YCF4) has been reported as a non-essential assembly factor for photosynthesis based on an incomplete knockout of YCF4, just 93 of 184 amino acids from the N-terminus were knocked out. On the other hand, we removed the complete sequence of YCF4 from tobacco chloroplasts and observed that ΔYCF4 plants were unable to survive photoautotrophically as their growth was hampered in the absence of an external carbon supply, clearly showing that the YCF4 is essential for photosynthesis. Initially, the aadA gene was introduced into the tobacco plastome replacing the complete YCF4 gene through homologous recombination events. The replacement of YCF4 with aadA was confirmed by PCR and Southern blot analysis in ΔYCF4 plants. Homoplasmic ΔYCF4 plants had a light green phenotype, and the leaves became pale yellow as the plants grew older. The structure of chloroplasts of ΔYCF4 mutants of light green phenotype was studied using a transmission electron microscope (TEM), and the micrographs demonstrated structural anomalies in the chloroplasts; including shape, size, and grana stacking compared to the wild-type plants. Further, transcriptome analysis revealed that the expression of PSI, PSII, and ribosomal genes remained unchanged in ∆YCF4 plants. On the other hand, transcriptome levels of rbcL (Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit), LHC (Light-Harvesting Complex), and ATP Synthase (atpB and atpL) decreased, indicating that the YCF4 has the function(s) in addition to assembling the photosynthetic complex. This was confirmed by in-silico protein-protein interactions of full-length YCF4 as well as 93 and 91 of 184 amino acids from N- and C-termini of the full-length protein, which revealed that the C-terminus (91 aa) of YCF4 is important in interacting with other chloroplast proteins. These findings provide genetic support for the plastid YCF4 gene’s critical role in regulating the plastid gene expression and assembling the photosynthetic complex.

Comparative plastome analysis of the sister genera Ceratocephala and Myosurus (Ranunculaceae) reveals signals of adaptive evolution to arid and aquatic environments

“…In particular, the seven genes ( clpP , rps7 , rps12 , rps14 , rps16 , rps19 , and ycf2 ) are only found to have signals of positive selection in the Ceratocephala plastome, implying that these seven genes might have been associated with the adaptation of Ceratocephala to arid environments. Zhong et al [ 36 ] also found that the ycf2 was gene under positive selection in Helianthus tuberosus (Asteraceae), which grows in saline, alkaline and dry conditions as a widely cultivated plant in Northwest China. The two genes ( rpl32 and ycf4 ) are only found to have signals of positive selection in the Myosurus plastome, implying that they might have been associated with the adaptation of Myosurus to aquatic environments.…”

Plastome Data of Red Currant and Gooseberry Reveal Potential Taxonomical Issues within the Ribes Genus (Grossulariaceae)

“…The ycf4 gene has been described as a highly conserved protein in plants, green algae, and cyanobacteria. Recently, a study on Nicotiana tabacum showed that the chloroplast ycf4 gene is essential for transcriptional gene regulation and plant photoautotrophic growth [52]. Positive selection signatures in ycf4 and clpP show that these genes may have played important roles in the speciation of Ribes species and their adaptation to diverse environments.…”

Introductory Chapter: Biology and Biotechnological Applications of Chloroplasts

“…A collection of genes called hypothetical chloroplast open reading frames (ycfs) is still unknown to have all of its specialized roles fully understood. Some of these ycfs have been classified as non-essential, while others have been classified as essential elements [5].…”