An official website of the United States government
Summary Extant land plants consist of two deeply divergent groups, tracheophytes and bryophytes, which shared a common ancestor some 500 million years ago. While information about vascular plants and the two of the three lineages of bryophytes, the mosses and liverworts, is steadily accumulating, the biology of hornworts remains poorly explored. Yet, as the sister group to liverworts and mosses, hornworts are critical in understanding the evolution of key land plant traits. Until recently, there was no hornwort model species amenable to systematic experimental investigation, which hampered detailed insight into the molecular biology and genetics of this unique group of land plants. The emerging hornwort model species,Anthoceros agrestis, is instrumental in our efforts to better understand not only hornwort biology but also fundamental questions of land plant evolution. To this end, here we provide an overview of hornwort biology and current research on the model plantA. agrestisto highlight its potential in answering key questions of land plant biology and evolution.
more »
« less
An Agrobacterium ‐mediated stable transformation technique for the hornwort model Anthoceros agrestis
Summary Despite their key phylogenetic position and their unique biology, hornworts have been widely overlooked. Until recently there was no hornwort model species amenable to systematic experimental investigation.Anthoceros agrestishas been proposed as the model species to study hornwort biology.We have developed anAgrobacterium‐mediated method for the stable transformation ofA. agrestis, a hornwort model species for which a genetic manipulation technique was not yet available.High transformation efficiency was achieved by using thallus tissue grown under low light conditions. We generated a total of 274 transgenicA. agrestislines expressing the β‐glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed.The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon‐concentrating mechanism, RNA editing and land plant evolution in general.
more »
« less
- PAR ID:
- 10446971
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- New Phytologist
- Volume:
- 232
- Issue:
- 3
- ISSN:
- 0028-646X
- Format(s):
- Medium: X Size: p. 1488-1505
- Size(s):
- p. 1488-1505
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Summary Recent studies have shown that correlations between chromatin modifications and transcription vary among eukaryotes. This is the case for marked differences between the chromatin of the mossPhyscomitrium patensand the liverwortMarchantia polymorpha. Mosses and liverworts diverged from hornworts, altogether forming the lineage of bryophytes that shared a common ancestor with land plants. We aimed to describe chromatin in hornworts to establish synapomorphies across bryophytes and approach a definition of the ancestral chromatin organization of land plants.We used genomic methods to define the 3D organization of chromatin and map the chromatin landscape of the model hornwortAnthoceros agrestis.We report that nearly half of the hornwort transposons were associated with facultative heterochromatin and euchromatin and formed the center of topologically associated domains delimited by protein coding genes. Transposons were scattered across autosomes, which contrasted with the dense compartments of constitutive heterochromatin surrounding the centromeres in flowering plants.Most of the features observed in hornworts are also present in liverworts or in mosses but are distinct from flowering plants. Hence, the ancestral genome of bryophytes was likely a patchwork of units of euchromatin interspersed within facultative and constitutive heterochromatin. We propose this genome organization was ancestral to land plants.more » « less
-
Abstract Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48–72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species.more » « less
-
Summary Cuscuta campestris, a stem parasitic plant, has served as a valuable model plant for the exploration of plant–plant interactions and molecular trafficking. However, a major barrier toC. campestrisresearch is that a method to generate stable transgenic plants has not yet been developed.Here, we describe the development of aCuscutatransformation protocol using various reporter genes (GFP, GUS, or RUBY) and morphogenic genes (CcWUS2andCcGRF/GIF), leading to a robust protocol forAgrobacterium‐mediatedC. campestristransformation.The stably transformed and regenerated RUBYC. campestrisplants produced haustoria, the signature organ of parasitic plants, and these were functional in forming host attachments. The locations of T‐DNA integration in the parasite genome were confirmed through TAIL‐PCR. TransformedC. campestrisalso produced flowers and viable transgenic seeds exhibiting betalain pigment, providing proof of germline transmission of the RUBY transgene. Furthermore, RUBY is not only a useful selectable marker for theAgrobacterium‐mediated transformation, but may also provide insight into the movement of molecules fromC. campestristo the host during parasitism.Thus, the protocol for transformation ofC. campestrisreported here overcomes a major obstacle toCuscutaresearch and opens new possibilities for studying parasitic plants and their interactions with hosts.more » « less
-
Summary Integration ofAgrobacterium tumefacienstransferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that inArabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.We conducted quantitative transformation assays of wild‐type andpolQmutantArabidopsisand rice, analyzed T‐DNA/plant DNA junction sequences, and (forArabidopsis) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency ofpolQmutants was c.20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQmutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQmutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway.more » « less
