Auxin Involvement in Ceratopteris Gametophyte Meristem Regeneration.

Growth and development of the Ceratopteris hermaphroditic gametophytes are dependent on cell proliferation in the marginal meristem, which when destroyed will regenerate at a new location on the body margin. We established a laser ablation method to destroy a single initial cell in the meristem. Ablation caused the cessation of cell proliferation accompanied by the disappearance of the expression of an auxin synthesis gene (CrTAA2) and a cell proliferation marker gene (CrWOXB). New meristem regeneration occurred within a predictable distance from the original two days post-ablation, signified by cell proliferation and the expression of CrTAA2. Treatment with the naturally occurring auxin indole-3-acetic acid (IAA), synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), or the transport inhibitor naphthylphthalamic acid (NPA) altered positioning of the original marginal meristem toward the apex of the gametophyte. IAA altered positioning of the regenerated meristem after damaging the original meristem. A model of auxin involvement in the positioning of the marginal meristem in Ceratopteris is presented to encompass these results.

Vascular function of the T3/modern clade WUSCHEL-Related HOMEOBOX transcription factor genes predate apical meristem-maintenance function.

BACKGROUND: Plants have the lifelong ability to generate new organs due to the persistent functioning of stem cells. In seed plants, groups of stem cells are housed in the shoot apical meristem (SAM), root apical meristem (RAM), and vascular cambium (VC). In ferns, a single shoot stem cell, the apical cell, is located in the SAM, whereas each root initiates from a single shoot-derived root initial. WUSCHEL-RELATED HOMEOBOX (WOX) family transcription factors play important roles to maintain stem-cell identity. WOX genes are grouped phylogenetically into three clades. The T3WOX/modern clade has expanded greatly in angiosperms, with members functioning in multiple meristems and complex developmental programs. The model fern Ceratopteris richardii has only one well-supported T3WOX/modern WOX gene, CrWUL. Its orthologs in Arabidopsis, AtWUS, AtWOX5, and AtWOX4, function in the SAM, RAM, and VC, respectively. Identifying the function of CrWUL will provide insights on the progenitor function and the diversification of the modern WOX genes in seed plants. RESULTS: To investigate the role of CrWUL in the fern, we examined the expression and function of CrWUL and found it expresses during early root development and in vasculature but not in the SAM. Knockdown of CrWUL by RNAi produced plants with fewer roots and fewer phloem cells. When expressed in Arabidopsis cambium, CrWUL was able to complement AtWOX4 function in an atwox4 mutant, suggesting that the WOX function in VC is conserved between ferns and angiosperms. Additionally, the proposed progenitor of T3WOX genes from Selaginella kraussiana is expressed in the vasculature but not in the shoot apical meristem. In contrast to the sporophyte, the expression of CrWUL in the gametophyte exhibits a more general expression pattern and when knocked down, offered little discernable phenotypes. CONCLUSIONS: The results presented here support the occurrence of co-option of the T3WOX/modern clade gene from the gametophyte to function in vasculature and root development in the sporophyte. The function in vasculature is likely to have existed in the progenitor of lycophyte T3WOX/modern clade genes and this function predates its SAM function found in many seed plants.

A Ceratopteris EXCESS MICROSPOROCYTES1 suppresses reproductive transition in the fern vegetative leaves.

Land plant sexual reproduction involves the transition of cells from somatic to reproductive identity during post-embryonic development. In Arabidopsis, the leucine-rich repeat receptor-like kinase EXCESS MICROSPOROCYTES1 (EXS/EMS1) restricts the number of sporogenous cells during the transition from diploid tissue to haploid spore production by promoting the formation of the tapetum cell layer within the anther. Although all land plants studied contain EMS1 genes, its function is unknown beyond a few angiosperms. In the model fern Ceratopteris (Ceratopteris richardii), we discovered an EMS1 homolog (CrEMS1) that functions to suppress formation of reproductive structures on vegetative leaves of the fern sporophyte, a role not found in angiosperms. Suppression of CrEMS1 by RNAi did not affect sporogenesis on reproductive leaves but did affect antheridium production of the fern gametophyte. Expression patterns of CrEMS1 across developmental stages suggest threshold levels of CrEMS1 control the specification of reproductive organs during both generations of the fern. Additional EMS1 homologs present in the fern genome suggest a dynamic role of EMS1 receptors in the evolution of reproductive development in vascular plants.

Three Genes Define a Bacterial-Like Arsenic Tolerance Mechanism in the Arsenic Hyperaccumulating Fern Pteris vittata.

Arsenic is a carcinogenic contaminant of water and food and a growing threat to human health in many regions of the world. This study focuses on the fern Pteris vittata (Pteridaceae), which is extraordinary in its ability to tolerate and hyperaccumulate very high levels of arsenic that would kill any other plant or animal outside the Pteridaceae. Here, we use RNA-seq to identify three genes (GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (PvGAPC1), ORGANIC CATION TRANSPORTER 4 (PvOCT4), and GLUTATHIONE S-TRANSFERASE (PvGSTF1) that are highly upregulated by arsenic and are necessary for arsenic tolerance, as demonstrated by RNAi. The proteins encoded by these genes have unexpected properties: PvGAPC1 has an unusual active site and a much greater affinity for arsenate than phosphate; PvGSTF1 has arsenate reductase activity; and PvOCT4 localizes as puncta in the cytoplasm. Surprisingly, PvGAPC1, PvGSTF1, and arsenate localize in a similar pattern. These results are consistent with a model that describes the fate of arsenate once it enters the cell. It involves the conversion of arsenate into 1-arseno-3-phosphoglycerate by PvGAPC1. This "chemically trapped" arsenate is pumped into specific arsenic metabolizing vesicles by the PvOCT4 protein. Once inside these vesicles, 1-arseno-3-phosphoglycerate hydrolyses to release arsenate, which is then reduced by PvGSTF1 to arsenite, the form of arsenic stored in the vacuoles of this fern. This mechanism is strikingly similar to one recently described Pseudomonas aeruginosa, whose tolerance to arsenic also involves the biosynthesis and transport of 1-arseno-3-phosphoglycerate, indicating that P. vittata has evolved a simple, bacterial-like mechanism for arsenic tolerance.