Vaccination via Chloroplast Genetics: Affordable Protein Drugs for the Prevention and Treatment of Inherited or Infectious Human Diseases.

Plastid-made biopharmaceuticals treat major metabolic or genetic disorders, including Alzheimer's, diabetes, hypertension, hemophilia, and retinopathy. Booster vaccines made in chloroplasts prevent global infectious diseases, such as tuberculosis, malaria, cholera, and polio, and biological threats, such as anthrax and plague. Recent advances in this field include commercial-scale production of human therapeutic proteins in FDA-approved cGMP facilities, development of tags to deliver protein drugs to targeted human cells or tissues, methods to deliver precise doses, and long-term stability of protein drugs at ambient temperature, maintaining their efficacy. Codon optimization utilizing valuable information from sequenced chloroplast genomes enhanced expression of eukaryotic human or viral genes in chloroplasts and offered unique insights into translation in chloroplasts. Support from major biopharmaceutical companies, development of hydroponic production systems, and evaluation by regulatory agencies, including the CDC, FDA, and USDA, augur well for advancing this novel concept to the clinic and revolutionizing affordable healthcare.

HEMERA Couples the Proteolysis and Transcriptional Activity of PHYTOCHROME INTERACTING FACTORs in Arabidopsis Photomorphogenesis.

Phytochromes (phys) are red and far-red photoreceptors that control plant development and growth by promoting the proteolysis of a family of antagonistically acting basic helix-loop-helix transcription factors, the PHYTOCHROME-INTERACTING FACTORs (PIFs). We have previously shown that the degradation of PIF1 and PIF3 requires HEMERA (HMR). However, the biochemical function of HMR and the mechanism by which it mediates PIF degradation remain unclear. Here, we provide genetic evidence that HMR acts upstream of PIFs in regulating hypocotyl growth. Surprisingly, genome-wide analysis of HMR- and PIF-dependent genes reveals that HMR is also required for the transactivation of a subset of PIF direct-target genes. We show that HMR interacts with all PIFs. The HMR-PIF interaction is mediated mainly by HMR's N-terminal half and PIFs' conserved active-phytochrome B binding motif. In addition, HMR possesses an acidic nine-amino-acid transcriptional activation domain (9aaTAD) and a loss-of-function mutation in this 9aaTAD impairs the expression of PIF target genes and the destruction of PIF1 and PIF3. Together, these in vivo results support a regulatory mechanism for PIFs in which HMR is a transcriptional coactivator binding directly to PIFs and the 9aaTAD of HMR couples the degradation of PIF1 and PIF3 with the transactivation of PIF target genes.

Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling.

The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were ~4300-fold higher in C and CN lines than in N, but all accumulated ~150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. The mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering.

RCB initiates Arabidopsis thermomorphogenesis by stabilizing the thermoregulator PIF4 in the daytime.

Daytime warm temperature elicits thermomorphogenesis in Arabidopsis by stabilizing the central thermoregulator PHYTOCHROME INTERACTING transcription FACTOR 4 (PIF4), whose degradation is otherwise promoted by the photoreceptor and thermosensor phytochrome B. PIF4 stabilization in the light requires a transcriptional activator, HEMERA (HMR), and is abrogated when HMR's transactivation activity is impaired in hmr-22. Here, we report the identification of a hmr-22 suppressor mutant, rcb-101, which surprisingly carries an A275V mutation in REGULATOR OF CHLOROPLAST BIOGENESIS (RCB). rcb-101/hmr-22 restores thermoresponsive PIF4 accumulation and reverts the defects of hmr-22 in chloroplast biogenesis and photomorphogenesis. Strikingly, similar to hmr, the null rcb-10 mutant impedes PIF4 accumulation and thereby loses the warm-temperature response. rcb-101 rescues hmr-22 in an allele-specific manner. Consistently, RCB interacts directly with HMR. Together, these results unveil RCB as a novel temperature signaling component that functions collaboratively with HMR to initiate thermomorphogenesis by selectively stabilizing PIF4 in the daytime.

Phytochrome activates the plastid-encoded RNA polymerase for chloroplast biogenesis via nucleus-to-plastid signaling.

Light initiates chloroplast biogenesis by activating photosynthesis-associated genes encoded by not only the nuclear but also the plastidial genome, but how photoreceptors control plastidial gene expression remains enigmatic. Here we show that the photoactivation of phytochromes triggers the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) by stimulating the assembly of the bacterial-type plastidial RNA polymerase (PEP) into a 1000-kDa complex. Using forward genetic approaches, we identified REGULATOR OF CHLOROPLAST BIOGENESIS (RCB) as a dual-targeted nuclear/plastidial phytochrome signaling component required for PEP assembly. Surprisingly, RCB controls PhAPG expression primarily from the nucleus by interacting with phytochromes and promoting their localization to photobodies for the degradation of the transcriptional regulators PIF1 and PIF3. RCB-dependent PIF degradation in the nucleus signals the plastids for PEP assembly and PhAPG expression. Thus, our findings reveal the framework of a nucleus-to-plastid anterograde signaling pathway by which phytochrome signaling in the nucleus controls plastidial transcription.

Mechanism of early light signaling by the carboxy-terminal output module of Arabidopsis phytochrome B.

Plant phytochromes are thought to transduce light signals by mediating the degradation of phytochrome-interacting transcription factors (PIFs) through the N-terminal photosensory module, while the C-terminal module, including a histidine kinase-related domain (HKRD), does not participate in signaling. Here we show that the C-terminal module of Arabidopsis phytochrome B (PHYB) is sufficient to mediate the degradation of PIF3 specifically and to activate photosynthetic genes in the dark. The HKRD is a dimerization domain for PHYB homo and heterodimerization. A D1040V mutation, which disrupts the dimerization of HKRD and the interaction between C-terminal module and PIF3, abrogates PHYB nuclear accumulation, photobody biogenesis, and PIF3 degradation. By contrast, disrupting the interaction between PIF3 and PHYB's N-terminal module has little effect on PIF3 degradation. Together, this study demonstrates that the dimeric form of the C-terminal module plays important signaling roles by targeting PHYB to subnuclear photobodies and interacting with PIF3 to trigger its degradation.