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expression of a wheat s like rnase ( wrn1 ) cdna during

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expression of a wheat s like rnase ( wrn1 ) cdna during

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									Acta Botanica Sinica 2003, 45 (9): 1071- 1075

http://www.chineseplantscience.com

Expression of a Wheat S-like RNase (WRN1) cDNA During Natural- and Dark-induced Senescence
CHANG Sheng-He1, YING Jia1, ZHANG Ji-Jun1, SU Jun-Ying1, ZENG Ya-Juan1, TONG Yi-Ping2, LI Bin1, LI Zhen-Sheng1*
(1. Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing 100101, China 2. Research Center for Eco-Enviromental Sciences, The Chinese Academy of Sciences, Beijing 100101, China)

Abstract: An S-like RNase cDNA had been isolated from common wheat (Triticum aestivum L.). The transcription of WRN1 mRNA was down-regulated by natural- and dark-induced senescence. But it was not senile-tissue-specific. As the two key histidine residues were replaced, WRN1 may not be active as RNase. Southern blotting analysis showed that WRN1 exists as one of a small gene family in common wheat genome. Key words: wheat S-like RNase (WRN1); leaf senescence; common wheat Senescence is an important and complex phase in the plant life cycle (Delorme et al., 2000). The loss of assimilatory capacity as leaf senescence progresses contributes to limited grain yield, and delayed leaf senescence may increase crop productivity (Miller et al., 1999). Senescence is actively regulated by differential gene expression (Lee et al., 2001). Most of the genes involved in the process of carbon assimilation are down-regulated during leaf senescence (Hensel et al., 1993; Jiang et al., 1993; BuchananWollaston and Ainsworth, 1997), while designated senescence-associated genes (SAGs) are up-regulated during senescence (Lohman et al., 1994; Bleecker and Patterson, 1997; Nam, 1997). During senescence, total RNA declines as RNase activity increases (Blank and McKeon, 1991). The first RNase gene cDNA that has been sequenced is S2-glycoprotein cDNA from tobacco (Anderson et al., 1989). It has close relation to self-incompatibility (Clarke and Newbigin, 1993). In the self-compatible plants, many homologues to the SRNase gene had also been found (Ide et al., 1991; Jost et al., 1991; Taylor and Green, 1991). They were called S-like RNase genes. The S-like RNase genes had been found to be associated with leaf senescence (Bariola et al., 1994) and phosphate starvation (Dodds et al., 1996; Lers et al., 1998). Attempting to identify and characterize genes whose mRNA was related to senescence, we isolated a common wheat S-like RNase (WRN1) cDNA. The identities, patterns of gene expression during dark-induced and natural leaf senescence were discussed.

1 Materials and Methods
1.1 Plant material treatments Seeds of common wheat (Triticum aestivum cv. Xiaoyan 54) were cultured in water for 2 d and planted in Pi-rich media as described previously (Davies et al., 2002). The seedlings were grown in a growth chamber. The photoperiod was 16/8 h light/dark at 22 ¡æ with 85% humidity. After 8 d, seedlings were transferred to complete darkness to induce leaf senescence. Leaves were harvested at different time points after dark treatment. For natural leaf senescence studies, the wheat plants were grown in the field under natural light conditions. During leaf senescence, chlorophyll degrades and yellow area broadens. The flag leaves were harvested at different stages of senescence according to the ratio of the green area to the whole leaf. The first stage was just before the dry tip appeared. 1.2 RNA and DNA extractions Total RNA samples from seeds were extracted as described previously (Gao et al., 2001). Total RNA samples from roots and leaves were isolated using a Trizol Reagent (Gibco, BRL). RNA samples were treated with DNase ¢ñ . Genomic DNA was prepared from wheat seedlings of 14 d old by using a method described previously (Dellaporta et al., 1983). 1.3 Southern blotting analysis DNA samples (30 µg each) were digested with BamH¢ñ , EcoR¢ñand Nde¢ñ Xho¢ñrespectively. The digested , and , DNA samples were fractionated on 0.8% agarose gel and

Received: 2002-11-01 Accepted: 2003-01-09 Supported by the State Key Basic Research and Development Plan of China (G1998010200). *Author for correspondence. E-mail: <zsli@genetics.ac.cn>.

CHANG Sheng-He et al.: Expression of a Wheat S-like RNase (WRN1) cDNA During Natural- and Dark-induced Senescence

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by RT-PCR was performed using two primers (P3: 5¡ä AGAACACTGTTGTAAGGCTCAAC-3¡ä P4: 5¡ä and GAGCTTTACTGCCTCGAACATGG-3¡äspecific for the ) wheat tublin gene. PCR was performed for thirty cycles (94 ¡æ30 s, 55 ¡æ60 s, 72 ¡æ60 s). , , ,

2

Results

2.1 Analysis of WRN1 cDNA and the derived amino acid sequence of WRN1 from common wheat The WRN1 cDNA contained a 738 bp open reading frame, starting at the ATG (nt 34- 26) and terminating at the TGA codons (nt 769- 771) (AF495872). Database searches indicated that the predicated amino acid sequence of WRN1 was 78% identical to that of rice S-like RNase (AF439449). The biggest feature of WRN1 sequence was the replacement of two key histidine residues with glutamine (amino acid site 62), tyrosine (amino acid site 121), respectively (Fig.1). Mutations that resulted in exchange of either histidine in S-RNases leading to the loss of RNase activity had been reported (Lohman et al., 1994). Similar result had also been reported in fungal RNase Rh (Gausing, 2000). Therefore, WRN1 had probably no RNase activity. 2.2 Genomic organization and expression pattern of WRN1 Upon Southern blotting analysis, common wheat genomic DNA samples were digested using BamH¢ñEcoR¢ñ , , Nde¢ñ and Xho¢ñrespectively, which there was no re, striction site in the WRN1 cDNA. The hybridization was performed with the WRN1 cDNA as a probe. Under high stringency conditions, one to three bands were observed. These results indicated that WRN1 belongs to a small gene family (Fig.2). 2.3 Expression of WRN1 cDNA As shown in Fig.3, a fragment (900 bp) was mainly obtained from young leaves, senile leaves (just before dry tip), roots and dry seeds. A tublin-specific fragment (500 bp) was amplified from all organs examined. The intensities were almost the same, indicating that an equal amount of total RNA of each sample was used for RT-PCR amplification. The mRNA level of WRN1 in fully expanded flag leaves was high. The expression of WRN1 was not senescencetissue specific. To investigate the relationship between WRN1 and leaf senescence, its accumulation in senile flag leaves of different stages was compared by semi-quantitative RT-PCR analysis. As shown in Fig.4B, the expression level decreased with the declining of chlorophyll (Chl). Same result had been obtained when the young seedlings were treated in darkness (Fig.4A). WRN1 seemed to be down-regulated by

Fig.2. DNA gel blotting analysis of WRN1 in common wheat. Genomic DNA samples (30 µ each) were digested with the reg striction enzymes BamH¢ñ EcoR¢ñ Nde¢ñand Xho¢ñ , , , respectively. The position and size (kb) of DNA markers are shown on the left side of the graph.

Fig.3. Expression of WRN1 in different organs of wheat with semi-quantitative RT-PCR. Samples of 200 ng of total RNA isolated from roots were cut from 10-day-old seedlings (R); young leaves were cut from 10-day-old seedlings (YL); fully expanded flag leaves were the leaves just before dry tip (FL), dry seeds (DS), immature seeds after flowering 20 d (IS). Reaction products were analyzed by agarose gel electrophoresis.

senescence.

3

Discussion

Leaf senescence is the last stage of development, during which cells undergo a major transition from carbon assimilation and other anabolic reactions to a catabolic

However, the expression level of WRN1 declined with the process of senescence. It seemed that WRN1 had no RNase activity. This can probably be due to the modification of two active-site histidine residues. Similar results had been reported (Jost et al., 1991). References:
Anderson M A, McFadden G I, Bernatzky R, Atkinson A, Orpin T. 1989. Sequence variability of three alleles of the selfincompatibility gene of Nicotiana alata. Plant Cell, 1:483491. Bariola P A, Howard C J, Taylor C B, Verburg M T, Jaglan V D, Green P J. 1994. The Arabidopsis ribonuclease gene RNS1 is Fig.4. Semi-quantitative RT-PCR analysis of WRN1. A. Samples of 200 ng of total RNA isolated from leaves were from seedlings that were treated in darkness for 0 d, 3 d, 5 d, respectively. The bottom line refers to the content of Chl. Reaction products were analyzed by agarose gel electrophoresis. B. Samples of 200 ng of total RNA isolated from senile flag leaves. S1 refers to fully expanded flag leaves (100% green area); S2 refers to senile flag leaves (60%- 100% green area); S3 refers to the senile flag leaves (30%- 60% green area); S4 refers to the senile flag leaves (0%- 30% green area). The bottom line refers to the content of Chl. Reaction products were analyzed by agarose gel electrophoresis. tightly controlled in response to phosphate limitation. Plant J, 6:673- 685. Becker W, Apel K. 1993. Differences in gene expression between natural and artificially induced leaf senescence. Planta, 189:74- 79. Blank A, McKeon T A. 1991. Expression of three RNase activities during natural- and dark-induced senescence of wheat leaves. Plant Physiol, 97:1409- 1413. Bleecker A, Patterson S. 1997. Last exit: senescence, abscission, and meristem arrest in Arabidopsis. Plant Cell, 9:11691179. Buchanan-Wollaston V, Ainsworth C. 1997. Leaf senescence in Brassica napus: cloning of senescence-related genes by subtractive hybridization. Plant Mol Biol, 33:821- 834. Clarke A E, Newbigin E. 1993. Molecular aspects of self-incompatibility in flowering plants. Annu Rev Genet, 27:257- 279. Davies T G E, Ying J, Xu Q, Li Z S, Gordon-Weeks R. 2002. Expression analysis of putative high-affinity phosphate transporters in Chinese winter wheats. Plant Cell Environ, 25: 1325- 1339. Dellaporta S L, Wood J, Hicks J B. 1983. A plant DNA minipreparation. version ¢òPlant Mol Biol Rep, 1:19- 21. . Delorme V G R, McCabe P F, Kim D, Leaver C J. 2000. A matrix metalloproteinase gene is expressed at the boundary of senescence and programmed cell death in cucumber. Plant Physiol, 123:917- 927. Dodds P N, Clarke A E, Newbigin E. 1996. Molecular characterization of an S-like RNase of Nicotiana alata that is induced by phosphate starvation. Plant Mol Biol, 31:227- 238. Gao J W, Liu J Z, Li B, Li Z S. 2001. Isolation and purification of functional total RNA from blue-grained wheat endosperm tissues containing high levels of starches and flavonoids. Plant Mol Biol Rep, 19:185a- 185i. Gausing K. 2000. A barley gene (rsh1) encoding a ribonuclease s-like homologue specifically in young light-grown leaves. Planta, 210:574- 579.

pattern that results in cell death (Hajouj et al., 2000). The catabolic pattern of the senile organ involves chlorophyll (Chl) degradation and chloroplast breakdown (Hajouj et al., 2000). Darkness also hastens leaf yellowing in many plant species, in attached leaves and during artificially induced senescence of detached leaves (Becker and Apel, 1993). However, expression of some senescence-related genes appear to be quite specific to natural senescence, whereas other transcripts are induced in addition to natural senescence (Weaver et al., 1998). WRN1 was down-regulated by both natural senescence and dark-induced senescence in laboratory. Most of the genes involved in the process of carbon assimilation are down-regulated during leaf senescence (Hensel et al., 1993; Jiang et al., 1993; Buchanan-Wollaston and Ainsworth, 1997), while designated senescence-associated genes (SAGs) are up-regulated during senescence (Lohman et al., 1994; Bleecker and Patterson, 1997; Nam, 1997). WRN1 was down-regulated during leaf senescence. It seemed that it was related to carbon assimilation. Total RNA levels decline during senescence as RNase activity increases. Moreover, each RNase has specific function(s) in senescence (Blank and McKeon, 1991).

CHANG Sheng-He et al.: Expression of a Wheat S-like RNase (WRN1) cDNA During Natural- and Dark-induced Senescence Hajouj T, Michelis R, Gepstein S. 2000. Cloning and characterization of a receptor-like protein kinase gene associated with senescence. Plant Physiol, 124:1305- 1314. Hensel L L, Grbic V, Baumgarten D A, Bleecker A B. 1993. Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell, 5:553- 564. Ide H, Kimura M, Arai M, Funatsu G. 1991. The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia). FEBS Lett, 284:161- 164. Jiang C Z, Rodermel S R, Shibles R M. 1993. Photosynthesis, rubisco activity and amount, and their regulation by transcription in senescencing soybean leaves. Plant Physiol, 101: 105- 112. Jost W, Bak H, Glund K, Terpstra P, Beintema J J. 1991. Amino acid sequence of an extracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. Eur J Biochem, 198:1- 6. Lee R, Wang C, Huang L, Chen S G. 2001. Leaf senescence in rice plants: cloning and characterization of senescence upregulated genes. J Exp Bot, 52:1117- 1121.

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Lers A, Khalchitski A, Lomaniec E, Burd S, Green P J. 1998.

S n s e c -n u e RNa e i tmao Plant Mol Biol, e e c n eid c d s s n o t.
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Lohman K N, Gan S, John M C, Amasino R M. 1994. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol Plant, 92:322- 328. Miller J D, Arteca R N, Pell E J. 1999. Senescence-associated gene expression during ozone-induced leaf senescence in Arabidopsis. Plant Physiol, 120:1015- 1023. Nam H G. 1997. The molecular genetic analysis of leaf senescence. Curr Opin Biotechnol, 8:200- 207. Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: a Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press. Talor C B, Green P J. 1991. Genes with homology to fungal and S-gene RNase are expressed in Arabidopsis thaliana. Plant Physiol, 96:980- 984. Weaver L M, Gan S, Quirino B, Amasino R M. 1998. A comparison of the expression patterns of several senescenceassociated genes in response to stress and hormone treatment. Plant Mol Biol, 37:455- 469.

(Managing editor: ZHAO Li-Hui)

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