West Lafayette, Indiana
September 3, 2002

Researchers have discovered how a recently identified family of plant proteins assists in stopping gene function, a finding that may help produce plants resistant to environmental stresses such as saline
soil, drought and cold.

The proteins, AtCPLs, apparently play a crucial role in triggering a gene that controls plants' reactions to stressful conditions, said Purdue University researchers. They, along with collaborators at the University of Arizona, published their findings in two papers appearing in a recent issue of Proceedings of the National Academy of Sciences.

AtCPLs are enzymes of a protein family that in humans controls initiation of gene activation. The family is called the C-terminal domain phosphates family.

Specifically, this enzyme family controls RNA required to produce messenger RNA, the initial product of the gene expression process. RNA, a molecule closely related to DNA, serves as a blueprint that tells cells to manufacture specific proteins.

"This family of proteins, AtCPLs, is undefined in plants," said Mike Hasegawa, co-senior author of a study describing two of the proteins. "The members we examined have both overlapping and unique functions, and this is novel."

Hasegawa, co-senior author Ray Bressan, and their team uncovered the proteins' function by studying mutated Arabidopsis thaliana, a common research plant, to determine its response to the stress of growing in salty soil. The same mutations, called cpl1 and cpl3, also seem to alter response to cold and drought, and alter growth and flowering time.

"It's become the prevailing feeling that when a plant senses its environment and signals to provide defense, the process turns on and off a number of different signal pathways that ultimately control the expression of specific genes that are required for adaptation," said Hasegawa, a horticulture professor.

"This research identifies a new temporal component of gene regulation that occurs after the initiation of transcription of the gene and seems to regulate important stress response processes of plants."

Transcription is when RNA copies and transfers the gene¹s instructions to the cell onto a template of DNA.

Hasegawa, Bressan and their colleagues have mainly focused on plant adaptability to soil salinity. However, by working with a number of different mutations, they have identified genes that are relevant for plant adaptation to other environmental stresses such as cold and drought.

Now the scientists are investigating other proteins that may be involved in plant reaction to environmental stress. They hope to determine the overlapping and unique functions of AtCPL family members so they can use bioengineering to improve plant tolerance for adverse growing conditions.

The other researchers involved in the study in which Hasegawa and Bressan are principal investigators are: research biologist Hisashi Koiwa, Adam Barb, biomedical engineering senior research assistant Fang Li, Michael McCully, post doctoral fellow Irina Sokolchik, Zhizhong Gong, graduate research assistant Altanbadralt Sharkhuu and Yuzuki Manabe, and Shuji Yokoi all of the Purdue Department of Horticulture Center for Plant Environmental Stress Physiology. From the University of Arizona Department of Plant Sciences senior investigator Jianhau Zhu and researchers Liming Xiong, Jian-Kang Zhu, and Byeong-ha Lee. Muppala Reddy of Central Salt and Marine Chemicals Researcher Institute in India also participated in the study.

A National Science Foundation Plant Genome Award and a U.S. Department of Agriculture National Research Initiative Grant provided funding for this project.

Writer: Susan A. Steeves, (765) 496-7481, ssteeves@purdue.edu
Sources:
Paul M. (Mike) Hasegawa, (765) 494-1315, paul.m.hasewaga.1@purdue.edu
Ray Bressan, (765) 494-1336, bressan@hort.purdue.edu

ABSTRACT

Plant Environmental Adaptability

C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development

Hisashi Koiwa*, Adam W. Barb*, Liming XiongÝ, Fang Li*, Michael G. McCully*, Byeong-ha LeeÝ, Irina Sokolchik*, Jianhua Zhu*, Zhizhong Gong*, Muppala Reddyý, Altanbadralt Sharkhuu*, Yuzuki Manabe*, Shuji Yokoi*, Jian-Kang ZhuÝ, Ray A. Bressan*, and Paul M. Hasegawa*§ *Center for Plant
Environmental Stress Physiology, 1165 Horticulture Building, Purdue University, West Lafayette, IN 47907-1165; ÝDepartment of Plant Sciences, University of Arizona, Tucson, AZ 85721; and ýCentral Salt and Marine Chemicals Research Institute, Waghawadi Road, Bhavanagar-364 002, India; Edited by Brian A. Larkins, University of Arizona, Tucson, AZ, and approved April 12, 2002 (received for review June 1, 2001)

Cold, hyperosmolarity, and abscisic acid (ABA) signaling induce RD29A expression, which is an indicator of the plant stress adaptation response. Two nonallelic Arabidopsis thaliana (ecotype C24) T-DNA insertional mutations, cpl1 and cpl3, were identified based on hyperinduction of RD29A
expression that was monitored by using the luciferase (LUC) reporter gene (RD29A::LUC) imaging system. Genetic linkage analysis and complementation data established that the recessive cpl1 and cpl3 mutations are caused by T-DNA insertions in AtCPL1 (Arabidopsis C-terminal domain phosphatase- like) and AtCPL3, respectively. Gel assays using recombinant AtCPL1 and AtCPL3
detected innate phosphatase activity like other members of the phylogenetically conserved family that dephosphorylate the C-terminal domain of RNA polymerase II (RNAP II). cpl1 mutation causes RD29A::LUC hyperexpression and transcript accumulation in response to cold, ABA, and NaCl treatments, whereas the cpl3 mutation mediates hyperresponsiveness only to ABA. Northern analysis confirmed that LUC transcript accumulation also occurs in response to these stimuli. cpl1 plants accumulate biomass more rapidly and exhibit delayed flowering relative to wild type whereas cpl3
plants grow more slowly and flower earlier than wild-type plants. Hence AtCPL1 and AtCPL3 are negative regulators of stress responsive gene transcription and modulators of growth and development. These results suggest that C-terminal domain phosphatase regulation of RNAP II phosphorylation status is a focal control point of complex processes like plant stress responses and development. AtCPL family members apparently have both unique and overlapping transcriptional regulatory functions that differentiate the signal output that determines the plant response.

ABSTRACT

Plant Stress Regulator

Repression of stress-responsive genes by FIERY2, a novel transcriptional regulator in Arabidopsis

Liming Xiong*, Hojoung Lee*, Manabu Ishitani*, Yuko Tanaka*, Becky Stevenson*, Hisashi KoiwaÝ, Ray A. BressanÝ, Paul M. HasegawaÝ, and Jian-Kang Zhu*ý *Department of Plant Sciences, University of Arizona, Tucson, AZ 85721; and ÝCenter for Plant Environmental Stress Physiology,
1165 Horticulture Building, Purdue University, West Lafayette, IN 47907-1165; Edited by William James Peacock, Commonwealth Scientific and Industrial Research Organization, Canberra, Australia, and approved June 24, 2002 (Received for review February 26, 2002)

Low temperature, drought, and high salinity induce the expression of many plant genes. To understand the mechanisms for the transcriptional activation of these genes, we conducted a reporter gene-aided genetic screen in Arabidopsis. Seven allelic mutations in the FIERY2 (FRY2) locus result in significant increases in the expression of stress-responsive genes with the DREyCRT (droughtresponsiveyC-repeat) cis element but non-DREyCRT type stressresponsive genes were less affected. The specific regulation of DREyCRT class of genes by FRY2 appears to be caused by repression of stress induction of the upstream CBFyDREB transcription factor genes. fry2 mutants show increased tolerance to salt stress and to abscisic acid during seed germination but are more sensitive to freezing damage at the seedling stage. FRY2yCPL1 encodes a novel transcriptional repressor harboring two
double-stranded RNAbinding domains and a region homologous to the catalytic domain of RNA polymerase II C-terminal domain phosphatases found in yeast and in animals that regulate gene transcription. These data indicate that FRY2 is an important negative regulator of stress gene transcription and suggest that structured RNA may regulate hormone and stress responses in plants as it does in animals.

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