Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels.

In plant biotechnology and basic research, chloroplasts have been used as chassis for the expression of various transgenes. However, potential unintended side effects of transgene insertion and high-level transgene expression on the expression of native chloroplast genes are often ignored and have not been studied comprehensively. Here, we examined expression of the chloroplast genome at both the transcriptional and translational levels in five transplastomic tobacco (Nicotiana tabacum) lines carrying the identical aadA resistance marker cassette in diverse genomic positions. Although none of the lines exhibits a pronounced visible phenotype, the analysis of three lines that contain the aadA insertion in different locations within the petL-petG-psaJ-rpl33-rps18 transcription unit demonstrates that transcriptional read-through from the aadA resistance marker is unavoidable, and regularly causes overexpression of downstream sense-oriented chloroplast genes at the transcriptional and translational levels. Investigation of additional lines that harbour the aadA intergenically and outside of chloroplast transcription units revealed that expression of the resistance marker can also cause antisense effects by interference with transcription/transcript accumulation and/or translation of downstream antisense-oriented genes. In addition, we provide evidence for a previously suggested role of genomically encoded tRNAs in chloroplast transcription termination and/or transcript processing. Together, our data uncover principles of neighbouring effects of chloroplast transgenes and suggest general strategies for the choice of transgene insertion sites and expression elements to minimize unintended consequences of transgene expression on the transcription and translation of native chloroplast genes.

Sulfur deficiency-induced genes affect seed protein accumulation and composition under sulfate deprivation.

Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (-S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under -S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency.

Loss of inner-envelope K+/H+ exchangers impairs plastid rRNA maturation and gene expression.

The inner-envelope K+ EFFLUX ANTIPORTERS (KEA) 1 and 2 are critical for chloroplast development, ion homeostasis, and photosynthesis. However, the mechanisms by which changes in ion flux across the envelope affect organelle biogenesis remained elusive. Chloroplast development requires intricate coordination between the nuclear genome and the plastome. Many mutants compromised in plastid gene expression (PGE) display a virescent phenotype, that is delayed greening. The phenotypic appearance of Arabidopsis thaliana kea1 kea2 double mutants fulfills this criterion, yet a link to PGE has not been explored. Here, we show that a simultaneous loss of KEA1 and KEA2 results in maturation defects of the plastid ribosomal RNAs. This may be caused by secondary structure changes of rRNA transcripts and concomitant reduced binding of RNA-processing proteins, which we documented in the presence of skewed ion homeostasis in kea1 kea2. Consequently, protein synthesis and steady-state levels of plastome-encoded proteins remain low in mutants. Disturbance in PGE and other signs of plastid malfunction activate GENOMES UNCOUPLED 1-dependent retrograde signaling in kea1 kea2, resulting in a dramatic downregulation of GOLDEN2-LIKE transcription factors to halt expression of photosynthesis-associated nuclear-encoded genes (PhANGs). PhANG suppression delays the development of fully photosynthesizing kea1 kea2 chloroplasts, probably to avoid progressing photo-oxidative damage. Overall, our results reveal that KEA1/KEA2 function impacts plastid development via effects on RNA-metabolism and PGE.

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas.

Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.

Commonalities and differences of chloroplast translation in a green alga and land plants.

Chloroplast gene expression is a fascinating and highly regulated process, which was mainly studied on specific genes in a few model organisms including the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii) and the embryophyte (land) plants tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). However, a direct plastid genome-wide interspecies comparison of chloroplast gene expression that includes translation was missing. We adapted a targeted chloroplast ribosome profiling approach to quantitatively compare RNA abundance and translation output between Chlamydomonas, tobacco and Arabidopsis. The re-analysis of established chloroplast mutants confirmed the capability of the approach by detecting known as well as previously undetected translation defects (including the potential photosystem II assembly-dependent regulation of PsbH). Systematic comparison of the algal and land plant wild-type gene expression showed that, for most genes, the steady-state translation output is highly conserved among the three species, while the levels of transcript accumulation are more distinct. Whereas in Chlamydomonas transcript accumulation and translation output are closely balanced, this correlation is less obvious in embryophytes, indicating more pronounced translational regulation. Altogether, this suggests that green algae and land plants evolved different strategies to achieve conserved levels of protein synthesis.

Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker.

The unicellular green alga Chlamydomonas reinhardtii has become an invaluable model system in plant biology. There is also considerable interest in developing this microalga into an efficient production platform for biofuels, pharmaceuticals, green chemicals and industrial enzymes. However, the production of foreign proteins in the nucleocytosolic compartment of Chlamydomonas is greatly hampered by the inefficiency of transgene expression from the nuclear genome. We have recently addressed this limitation by isolating mutant algal strains that permit high-level transgene expression and by determining the contributions of GC content and codon usage to gene expression efficiency. Here we have applied these new tools and explored the potential of Chlamydomonas to produce a recombinant biopharmaceutical, the HIV antigen P24. We show that a codon-optimized P24 gene variant introduced into our algal expression strains give rise to recombinant protein accumulation levels of up to 0.25% of the total cellular protein. Moreover, in combination with an expression strain, a resynthesized nptII gene becomes a highly efficient selectable marker gene that facilitates the selection of transgenic algal clones at high frequency. By establishing simple principles of successful transgene expression, our data open up new possibilities for biotechnological research in Chlamydomonas.

Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii.

The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high-level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.