Jeff Velten

Desiccation Tolerance in Bryophytes: A Reflection of the Primitive Strategy for Plant Survival in Dehydrating Habitats?

Abstract Bryophytes are a non-monophyletic group of three major lineages (liverworts, hornworts, and mosses) that descend from the earliest branching events in the phylogeny of land plants. We postulate that desiccation tolerance is a primitive trait, thus mechanisms by which the first land plants achieved tolerance may be reflected in how extant desiccation-tolerant bryophytes survive drying. Evidence is consistent with extant bryophytes employing a tolerance strategy of constitutive cellular protection coupled with induction of a recovery/repair mechanism upon rehydration. Cellular structures appear intact in the desiccated state but are disrupted by rapid uptake of water upon rehydration, but cellular integrity is rapidly regained. The photosynthetic machinery appears to be protected such that photosynthetic activity recovers quickly. Gene expression responds following rehydration and not during drying. Gene expression is translationally controlled and results in the synthesis of a number of proteins, collectively called rehydrins. Some prominent rehydrins are similar to Late Embryogenesis Abundant (LEA) proteins, classically ascribed a protection function during desiccation. The role of LEA proteins in a rehydrating system is unknown but data indicates a function in stabilization and reconstitution of membranes. Phylogenetic studies using a Tortula ruralis LEA-like rehydrin led to a re-examination of the evolution of desiccation tolerance. A new phylogenetic analysis suggests that: (i) the basic mechanisms of tolerance seen in modern day bryophytes have changed little from the earliest manifestations of desiccation tolerance in land plants, and (ii) vegetative desiccation tolerance in the early land plants may have evolved from a mechanism present first in spores.

Bryophytes as experimental models for the study of environmental stress tolerance: Tortula ruralis and desiccation-tolerance in mosses

The development of a complete understanding of how plants interact with the environment at the cellular level is a crucial step in advancing our ability to unravel the complexities of plant ecology particularly with regard to the role that many of the less complex plants (i.e., algae, lichens, and bryophytes) play in plant communities and in establishing areas for colonization by their more complex brothers. One of the main barriers to the advancement of this area of plant biology has been the paucity of simple and appropriate experimental models that would enable the researcher to biochemically and genetically dissect the response of less complex plants to environmental stress. A number of bryophytes model systems have been developed and they have been powerful experimental tools for the elucidation of complex biological processes in plants. Recently there has been a resurgent interest in bryophytes as models systems due to the discovery and development of homologous recombination technologies in the moss Physcomitrella patens (Hedw.) Brach & Schimp. In this report we introduce the desiccation-tolerant moss Tortula ruralis (Hedw.) Gaert., Meyer, and Scherb, as a model for stress tolerance mechanisms that offers a great deal of promise for advancing our efforts to understand how plants respond to and survive the severest of stressful environments. T. ruralis, a species native to Northern and Western North America, has been the most intensely studied of all bryophytes with respect to its physiological, biochemical, and cellular responses, to the severest of water stresses, desiccation. It is our hope that the research conducted using this bryophyte will lay the foundationfor not only the ecology of bryophytes and other less complex plants but also for the role of desiccation-tolerance in the evolution of land plants and the determination of mechanisms by which plant cells can withstand environmental insults. We will focus the discussion on the research we and others have conducted in an effort to understand the ability of T. ruralis to withstand the complete loss of free water from the protoplasm of its cells.

An in vivo, luciferase-based, Agrobacterium-infiltration assay system: implications for post-transcriptional gene silencing.

An in vivo assay system for analyzing transient luciferase expression in tobacco leaves infused with Agrobacteriumtumefaciens is described. The system makes use of A. tumefaciens harboring T-DNA vectors containing either an intron-containing firefly (Photinus pyralis) luciferase (EC 1.13.12.7) gene or an intron-containing sea pansy (Renilla reniformis) luciferase (EC 1.13.12.5) gene. Single or mixed Agrobacterium lines were infiltrated into leaf tissues (Nicotiana tabacum or Nicotiana benthamiana) through stomatal openings and leaf disks from infused areas floated on reaction buffers specific to each enzyme. Photons emitted were then measured to determine reporter gene activity. Parameters affecting assay reliability and sensitivity were tested, including: buffer composition; bacterial density; infusion location; reaction kinetics; and environmental factors (light and temperature). The resulting in vivo assay system generates results comparable to those obtained using a commercially available in vitro dual-luciferase® reporter gene assay, and reports relative expression levels, as well as induction characteristics, analogous to those obtained using leaf tissue from stably transformed plants harboring the same promoter::gene constructs. Light and temperature were observed to markedly impact transient reporter activities. Co-expression of viral suppressors of post-transcriptional gene silencing (PTGS), HcPro, p19 and AC2, confirms the occurrence of PTGS within infused zones, and provides a convenient mechanism for PTGS analysis. The in vivo transient assay was used to examine the effect on PTGS of factors such as: promoter strength; incubation temperature and double-stranded RNA production. Results from these assays provide insight into the mechanism(s) used by plants to trigger and maintain PTGS.

Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco

The conserved late element (CLE) was originally identified as an evolutionarily conserved DNA sequence present in geminiviral intergenic regions. CLE has subsequently been observed in promoter sequences of bacterial (T-DNA) and plant origin, suggesting a role in plant and plant viral gene regulation. Synthetic DNA cassettes harboring direct repeats of the CLE motif were placed upstream from a −46 to +1 minimal CaMV 35S promoter-luciferase reporter gene and reporter activity characterized in Nicotiana species during both transient and stable expression. A single direct-repeat cassette of the element (2× CLE) enhances luciferase activity by 2-fold, independent of the element’s orientation, while multiple copies of the cassette (4–12× CLE) increases activity up to 10- to 15-fold in an additive manner. Transgenic tobacco lines containing synthetic CLE promoter constructs enhance luciferase expression in leaf, cotyledon and stem tissues, but to a lesser extent in roots. Single nucleotide substitution at six of eight positions within the CLE consensus (GTGGTCCC) eliminates CLE enhancer-like activity. It has been previously reported that CLE interacts with the AC2 protein from Pepper Huasteco Virus (PHV-AC2). PHV-AC2 (also called AL2 or C2) is a member of the transcriptional activator protein, or TrAP, gene family. In transient and stable expression systems PHV-AC2 expression was found to result in a 2-fold increase in luciferase activity, irrespective of the presence of CLE consensus sequences within the reporter’s promoter. These data suggests that the PHV-AC2 protein, instead of interacting directly with CLE, functions as either a general transcriptional activator and/or a suppressor of post-transcriptional gene silencing.

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.

A spontaneous dominant-negative mutation within a 35S::AtMYB90 transgene inhibits flower pigment production in tobacco

BACKGROUND In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation. FINDINGS A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production. CONCLUSIONS Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors.

Construction and Testing of an Intron-Containing Luciferase Reporter Gene From Renilla reniformis

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.

Analysis of RNA-Mediated Gene Silencing Using a New Vector (pKNOCKOUT) and an In Planta Agrobacterium Transient Expression System *

A hairpin RNA (hpRNA) vector, pKNOCKOUT (pKO) has been constructed to facilitate the analysis of posttranscriptional gene silencing (PTGS) in anAgrobacterium-mediated transient expression system developed for tobacco. The pKO binary vector was tested by cloning a firefly luciferase (Photinus pyralis) gene segment in sense (sFLUC), antisense (aFLUC), and inverted repeat (ihpFLUC) orientations. The inverted repeats of the target gene are separated by the castor bean catalase intron (CbCi) and, when transcribed and spliced, produce a self-complementary hpRNA. hpRNA-mediated gene silencing exploits a cellular mechanism that recognizes double-stranded RNA (dsRNA) and subjects it, and homologous mRNA molecules, to sequence-specific degradation. Agrobacteria harboring compatible plasmids, pE1778-RiLUC (Renilla luciferase normalization construct) and pTCaMV35S-FiLUC (functional firefly luciferase test construct), were co-infused with different variants of pKO-FLUC plasmids intoNicotiana benthamiana andNicotiana tobaccum leaf tissues. Reduced firefly luciferase reporter gene activity (from pTCaMV35S-FiLUC) indicated gene silencing and was observed in leaf tissues co-infused with pKO-sFLUC (50% reduction), pKO-aFLUC (85% reduction), or pKO-ihpFLUC (96% reduction) agrobacteria lines. Gene silencing was observed at different times postinfusion in leaves from bothNicotiana species. The hpRNA-mediated interference with FiLUC reporter gene expression, as measured by reduced firefly luciferase activity, was found to also suppress silencing of the cotransferredRenilla reniformis luciferase normalization reporter gene, resulting in reproducibly elevated RiLUC activity. This suppression effect was reduced by lowering the percentage of infused pKO-ihpFLUC agrobacteria without markedly effecting hpRNA-mediated gene silencing of FiLUC (all pKO-ihpFLUC dilutions produced >90% reduction of FiLUC activity). Viral suppressors of PTGS, such as p19 and HcPro, were found to reduce the RNAi effect of hpRNA-mediated gene silencing. To our knowledge, this is the first demonstration that excess amounts of targeted dsRNA can result in nonspecific suppression of PTGS of an unrelated, co-infused reporter gene in plants.

A selection procedure for identifying transgenic cells and embryos of cotton without the use of antibiotics

Transgenic cells containing inserted antibiotic resistance genes and linked genes of interest are routinely selected by exposure to antibiotics. Concerns over the widespread use of antibiotic resistance genes as selectable markers for genetic transformation have motivated researchers to find alternative selection procedures. This study describes the evaluation of an alternative protocol using temperature as the selection tool. In this method, a population of host cells is transformed with a foreign DNA construct that includes at least one gene of interest and an additional sequence encoding a protein that enhances cellular high temperature tolerance. Following transformation, the population of cells is transiently cultured under temperature conditions wherein growth of non-transformed cells is suppressed or prevented, while growth of cells containing the DNA construct continues. Thus, survival and/or significant additional growth is an indication that a cell has been successfully transformed with the DNA construct and can be subsequently recovered for further growth and development. The present study used a heat shock protein (hsp101) gene from Arabidopsis thaliana under the control of a constitutive promoter as a selectable marker; however, alternative potentially suitable genes include: other heat shock proteins; heat shock transcription factors; cold regulated proteins (COR); or protein transcription factors associated with the induction of cold tolerance.