Page 7 - S. Plant biology

Downregulation of PagSAP11 affects abiotic stress responses and
shoot architecture

Su Jin Park, Eun-Kyung Bae , Hyun A Jang, Hyoshin Lee
*
Forest Biotechnology Division, National Institute of Forest Science, Suwon 16631, Korea
BACKGROUND AIM
Plants have evolved defense mechanisms to overcome unfavorable climate conditions In this study, we investigated the molecular and
such as salinity, drought, and extreme temperatures. Such adaptive strategies include fine- physiological characteristics of the novel gene
tuning of stress-associated proteins (SAPs). SAPs contain A20/AN1 zinc-finger domains, PagSAP11 from hybrid poplar (Populus alba × P.
glandulosa).
including an A20 zinc finger at the N-terminus and AN1 zinc finger at the C-terminus. SAPs
contain conserved domains among a variety of plant species, however, their exact
biological functions in plants are not yet fully known.
METHODS

Plant materials
The six-week-old hybrid poplar (P.alba x P.glandulosa) grown in soils and MS media were used.
Generation of transgenic poplars harboring RNAi vectors
To construct the RNAi vector, PagSAP11 was inserted into the pH7GWIWG vector using the Gateway system (Invitrogen, USA). A schematic diagram of vector
construction is shown in Fig. 2. Agrobacterium-mediated plant transformation was performed as previously described by Choi et al. (2005).
Salt treatment
6-week-old poplars were treated with 150 mM NaCl solutions for 20 days.
Drought experiments
Four individual 6-week-old plants were not watered. As the control, four individual plants were normally watered.
RESULTS

Fig. 2. Generation and phenotype of PagSAP11 transgenic poplar
(A) Strategy for construction of expression vector pK7GWIWG2(I), containing the PagSAP11 RNAi. (B) Relative PagSAP11
transcript levels in each transgenic line. The expression level of transgenic plants of lines were confirmed using real-time
Fig. 1. Comparison of the predicted protein sequences of PagSAP11 with other plant qPCR. . (C-E) Phenotype analysis of 6 week old PagSAP11 RNAi transgenic plants. (D) The height analysis in Control and
SAP proteins. PagSAP11 RNAi transgenic plants. (E) The analysis of Branches number in Control and PagSAP11 RNAi transgenic
(A) Multiple sequence alignment of SAP11 amino acids of several plant species. PagSAP11 (Populus plants. All data points are means of three independent replicates, with error bars indicatingSD.
alba × P. glandulosa SAP11), POPTR (Populus trichocarpa), Populus euphratica (XP_011025412.1),
Cassava (MANES_07G004400), Jatropha_curcas (JCGZ_16738), Cucumber(Csa_7G428830).
(B) Phylogenic tree analysis of the amino acid sequences of the SAP11 gene.

Fig. 3. Real time PCR analysis related
genes of the phytohormone auxin
and cytokinin.
(A-B) Auxin-related genes were down-
regulated in PagSAP11 RNAi transgenic
plants. (C-D) The expression level about Fig. 4. Stress resistant characteristics in PagSAP11 RNAi transgenic plants.
cytokinin-related genes was increased in (A) Phenotypic responses of control and PagSAP11 transgenic plants under salt stress. (B) Chlorophyll florescence
PagSAP11 RNAi transgenic plants. (Fv/Fm) of Control and PagSAP11 RNAi transgenic plants under salt stress. (C) Phenotypic responses of control and
PagSAP11 RNAi transgenic plants under drought stress. (D) Chlorophyll florescence (Fv/Fm) of Control and PagSAP11
RNAi transgenic plants under drought stress. All data points are means of three independent replicates, with error
bars indicatingSD.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
Knockdown (RNAi) transgenic poplar plants Choi Y-I, Noh EW, Lee H, Han MS, Lee JS, Choi KS This work was supported in part by the National
exhibited tolerance to salt stress and drought (2005) An efficient and novel plant selectable marker Institute of Forest Science (Project No. FG0702-
stress compared to wild type plants. The RNAi based on organomercurial resistance. J Plant 2018).
Biotechnol 48:351-355.
lines showed increased shoot branching
phenotypes that lead to gain fresh weight. The Yoon S-K, Bae E-K, Lee H, Choi Y-I, Han MH, Choi Contact information
HM, Kang K-S, Park E-J (2018) Downregulation of
RNAi lines exhibited increased expression of stress-associated protein 1 (PagSAP1) increases salt
genes related to auxin signaling, while decreased stress tolerance in poplar (Populus alba × P.
expression of genes involved in cytokinin signaling. glandulosa). Tree 32:823–833. *Corresponding authors: Eun-kyung Bae
Taken together, these results indicate that Lee J, Sun P, Xia Y, Zheng G, Sun J, Jia H (2019) A E-mail: baeek@korea.kr
PagSAP11 represents a promising candidate gene Stress-Associated Protein, PtSAP13, From Populus Address: Forest Bioresources Department, National
for engineering trees with improved stress trichocarpa Provides Tolerance to Salt Stress. Int. J. Institute of Forest Science, 39 Onjeong-ro,
tolerance, which would increase their growth. Mol. Sci. 20:5782. Gwonseon-gu, Suwon, 16631, Korea