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Genes & Genomics

, Volume 36, Issue 4, pp 427–441 | Cite as

The cloning and characterization of a poplar stomatal density gene

  • Shaneka S. LawsonEmail author
  • Paula M. Pijut
  • Charles H. Michler
Research Article

Abstract

EPIDERMAL PATTERNING FACTOR1 (EPF1) is a well characterized negative regulator of cell division in Arabidopsis thaliana (AtEPF1) where the primary region of localization is the leaf. However, little data have been reported on the role of EPF1 in other plant species. In this study, the EPF1 gene from Arabidopsis and the newly identified poplar ortholog from Populus trichocarpa (PtaEPF1) were overexpressed in a hybrid poplar genotype. We attempted to identify the physiological role of PtaEPF1. Gene overexpression experiments were performed to determine if and how stomatal density (SD) numbers were affected. The poplar 717-1B4 (P. tremula × P. alba) genotype was used in the study. Results presented here suggest that overexpression of PtaEPF1 and AtEPF1 in poplar led to significantly altered SD and also affected transgenic water stress tolerance. Overexpression of AtEPF1 in 717-1B4 led to the most dramatic decrease in SD while overexpression of PtaEPF1 in 717-1B4 significantly increased SD in several transgenic lines, an indication that EPF1 may have additional functions in poplar. Also, abnormalities in leaf morphology were discovered that indicated overexpression of AtEPF1 or PtaEPF1 in poplar triggered aberrant phenotypes not seen in other published Arabidopsis studies, an indication of additional pathway involvement.

Keywords

Stomatal density Tree physiology Poplar Transgenics 

Notes

Acknowledgments

This study was financed by fellowships from the Fred M. van Eck Foundation and the Alliance for Graduate Education and Professoriate (AGEP) at Purdue University. The authors thank Drs. Martin-Michel Gauthier and John Gordon for their comments and critiques of earlier versions of this manuscript. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable.

Conflict of interest

The authors declare no conflict of interest regarding this article.

Supplementary material

13258_2014_177_MOESM1_ESM.tif (311 kb)
Supplemental Fig. 1S Sampling sections on leaves. Visualization of 717-1B4 sampling regions to demonstrate that stomatal densities vary dependent upon sampling location. The region used in this study was region 2 (double circle). Densities given were from a sample leaf from a wild-type 717-1B4 plant
13258_2014_177_MOESM2_ESM.tif (332 kb)
Supplemental Fig. 2S Annotation and comparison of PtaEPF1with AtEPF1. Phytozome website analysis was used to view similarity of the PtaEPF1gene used in this study to AtEPF1. Positioning along the scaffold in either species was also observed
13258_2014_177_MOESM3_ESM.tif (457 kb)
Supplemental Fig. 3S EPF1 family. There were no conserved domains found in any of the sequences presented for EPF1 family members. Despite a low percentage of protein sequence similarity, the poplar homolog sequences have recently been annotated as definitive EPF family protein sequences
13258_2014_177_MOESM4_ESM.tif (189 kb)
Supplemental Fig. 4S RT-PCR of AtEPF1 and PtaEPF1 expression locations. Expression of both the AtEPF1 and PtaEPF1 gene transcripts was highest in the leaves although expression did occur in other locations. The immature leaf primordia nearest the apical meristem (labeled apical meristem in photo) had high expression as did the first mature leaves of both poplar and Arabidopsis (labeled Leaf 1 in photo). Much lower levels of expression were seen in the Arabidopsis rosette, poplar stems (stem base refers to the stem portion directly above the roots and stem tip refers to the uppermost point of the apical meristem), and more mature leaves, such as leaf 15. See illustration on right. PtaEPF1 samples were amplified from poplar cDNA and AtEPF1 samples were amplified from Arabidopsis cDNA. 18S RNA was used as the control
13258_2014_177_MOESM5_ESM.tif (234 kb)
Supplemental Fig. 5S Visualization of leaf primordia and phyllotaxic leaf 1 from 717-1B4. The stomatal density of a 717-1B4 was compared and b graphed. All densities counted were from the abaxial leaf surface. Circles were used to represent individual stomata and were not drawn to scale. Each circle was an accurate depiction of the location where individual stomata were found on the leaf. Each individual square graphic represents 1 mm2. Means with the same letter were not significantly different at p < 0.05. Error bars (±SEM)
13258_2014_177_MOESM6_ESM.tif (309 kb)
Supplemental Fig. 6S Morphological abnormalities. a Ablation of a single guard cell was a phenomenon seen in lines overexpressing PtaEPF1 in 717-1B4 and appears to be the result of PtaEPF1 disruption of asymmetric cell division. b Other transgenic lines demonstrated unusual guard cell positioning where the proximal and distal ends were rotated. Abnormalities such as c malformed and d arrow arrested stomata were also seen. e Trichome densities of transgenics were also observed and photographed for comparison to wild-type. ad Bar = 50 μm e Bar = 300 μm

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Copyright information

© The Genetics Society of Korea 2014

Authors and Affiliations

  • Shaneka S. Lawson
    • 1
    Email author
  • Paula M. Pijut
    • 1
  • Charles H. Michler
    • 1
  1. 1.USDA Forest Service, Northern Research Station, Department of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center (HTIRC)Purdue UniversityWest LafayetteUSA

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