Expression Elements Derived From Plant Sequences Provide Effective Gene Expression Regulation and New Opportunities for Plant Biotechnology Traits.

Plant biotechnology traits provide a means to increase crop yields, manage weeds and pests, and sustainably contribute to addressing the needs of a growing population. One of the key challenges in developing new traits for plant biotechnology is the availability of expression elements for efficacious and predictable transgene regulation. Recent advances in genomics, transcriptomics, and computational tools have enabled the generation of new expression elements in a variety of model organisms. In this study, new expression element sequences were computationally generated for use in crops, starting from native Arabidopsis and maize sequences. These elements include promoters, 5' untranslated regions (5' UTRs), introns, and 3' UTRs. The expression elements were demonstrated to drive effective transgene expression in stably transformed soybean plants across multiple tissues types and developmental stages. The expressed transcripts were characterized to demonstrate the molecular function of these expression elements. The data show that the promoters precisely initiate transcripts, the introns are effectively spliced, and the 3' UTRs enable predictable processing of transcript 3' ends. Overall, our results indicate that these new expression elements can recapitulate key functional properties of natural sequences and provide opportunities for optimizing the expression of genes in future plant biotechnology traits.

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.

Photoactivated phytochromes interact with HEMERA and promote its accumulation to establish photomorphogenesis in Arabidopsis.

Plant development is profoundly regulated by ambient light cues through the red/far-red photoreceptors, the phytochromes. Early phytochrome signaling events include the translocation of phytochromes from the cytoplasm to subnuclear domains called photobodies and the degradation of antagonistically acting phytochrome-interacting factors (PIFs). We recently identified a key phytochrome signaling component, HEMERA (HMR), that is essential for both phytochrome B (phyB) localization to photobodies and PIF degradation. However, the signaling mechanism linking phytochromes and HMR is unknown. Here we show that phytochromes directly interact with HMR to promote HMR protein accumulation in the light. HMR binds more strongly to the active form of phytochromes. This interaction is mediated by the photosensory domains of phytochromes and two phytochrome-interacting regions in HMR. Missense mutations in either HMR or phyB that alter the phytochrome/HMR interaction can also change HMR levels and photomorphogenetic responses. HMR accumulation in a constitutively active phyB mutant (YHB) is required for YHB-dependent PIF3 degradation in the dark. Our genetic and biochemical studies strongly support a novel phytochrome signaling mechanism in which photoactivated phytochromes directly interact with HMR and promote HMR accumulation, which in turn mediates the formation of photobodies and the degradation of PIFs to establish photomorphogenesis.

Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes.

Light plays a profound role in plant development, yet how photoreceptor excitation directs phenotypic plasticity remains elusive. One of the earliest effects of light is the regulated translocation of the red/far-red photoreceptors, phytochromes, from the cytoplasm to subnuclear foci called phytochrome nuclear bodies. The function of these nuclear bodies is unknown. We report the identification of hemera, a seedling lethal mutant of Arabidopsis with altered phytochrome nuclear body patterns. hemera mutants are impaired in all phytochrome responses examined, including proteolysis of phytochrome A and phytochrome-interacting transcription factors. HEMERA was identified previously as pTAC12, a component of a plastid complex associated with transcription. Here, we show that HEMERA has a function in the nucleus, where it acts specifically in phytochrome signaling, is predicted to be structurally similar to the multiubiquitin-binding protein, RAD23, and can partially rescue yeast rad23mutants. Together, these results implicate phytochrome nuclear bodies as sites of proteolysis.

Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains.

At least two of the genes predicted to encode type II PI4K (phosphoinositide 4-kinase) in Arabidopsis thaliana (thale cress), namely AtPI4Kgamma4 and AtPI4Kgamma7, encode enzymes with catalytic properties similar to those of members of the PIKK (phosphoinositide kinase-related kinase) family. AtPI4Kgamma4 and AtPI4Kgamma7 undergo autophosphorylation and phosphorylate serine/threonine residues of protein substrates, but have no detectable lipid kinase activity. AtPI4Kgamma4 and AtPI4Kgamma7 are members of a subset of five putative AtPI4Ks that contain N-terminal UBL (ubiquitin-like) domains. In vitro analysis of AtPI4Kgamma4 indicates that it interacts directly with, and phosphorylates, two proteins involved in the ubiquitin-proteasome system, namely UFD1 (ubiquitin fusion degradation 1) and RPN10 (regulatory particle non-ATPase 10). On the basis of the present results, we propose that AtPI4Kgamma4 and AtPI4Kgamma7 should be designated UbDKgamma4 and UbDKgamma7 (ubiquitin-like domain kinases gamma4 and gamma7). These UBL-domain-containing AtPI4Ks correspond to a new PIKK subfamily of protein kinases. Furthermore, UFD1 and RPN10 phosphorylation represents an additional mechanism by which their function can be regulated.

A naturally occurring recombinant DNA-A of a typical bipartite begomovirus does not require the cognate DNA-B to infect Nicotiana benthamiana systemically.

Species of the genus Begomovirus (family Geminiviridae) found in the western hemisphere typically have a bipartite genome that consists of two 2.6 kb DNA genomic components, DNA-A and DNA-B. We have identified and cloned genomic components of a new tomato-infecting begomovirus from Brazil, for which the name Tomato crinkle leaf yellows virus (TCrLYV) is proposed, and a DNA-A variant of Tomato chlorotic mottle virus (ToCMV-[MG-Bt1]). Sequence analysis revealed that TCrLYV was most closely related to ToCMV, although it was sufficiently divergent to be considered a distinct virus species. Furthermore, these closely related viruses induce distinguishable symptoms in tomato plants. With respect to ToCMV-[MG-Bt1] DNA-A, evidence is presented that suggests a recombinant origin. It possesses a hybrid genome on which the replication compatible module (AC1 and replication origin) was probably donated by ToCMV-[BA-Se1] and the remaining sequences appear to have originated from Tomato rugose mosaic virus (ToRMV). Despite the high degree of sequence conservation with its predecessors, ToCMV-[MG-Bt1] differs significantly in its biological properties. Although ToCMV-[MG-Bt1] DNA-A did not infect tomato plants, it systemically infected Nicotiana benthamiana, induced symptoms of mottling and accumulated viral DNA in the apical leaves in the absence of a cognate DNA-B. The modular rearrangement that resulted in ToCMV-[MG-Bt1] DNA-A may have provided this virus with a more aggressive nature. Our results further support the notion that interspecies recombination may play a significant role in geminivirus diversity and their emergence as agriculturally important pathogens.

Transient gene expression of beta-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment

Studies were carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (Citrus sinensis x Poncirus trifoliata) thin epicotyl sections. The best conditions for transient GUS expression were: M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen and DNA/particle holder and culture of explants in a high osmolarity medium (0.2 M mannitol + 0.2 M sorbitol) 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate) were achieved. This protocol is currently being used for transformation of Carrizo citrange and sweet orange (Citrus sinensis)