Journal of Experimental Botany, Vol. 53, No. 379, pp. 2451-2452,
December 1, 2002
© 2002 Oxford University Press
Isolation and expression of a novel starch-storing cell-specific gene containing the KH RNA binding domain from tobacco-cultured cells BY-2
Received 29 July 2002; Accepted 21 August 2002
1 Plant Functions Laboratory, RIKEN, Hirosawa, Wako-shi, Saitama, 351-0198, Japan
2 Department of Biological Sciences, Faculty of Science, Nara Women’s University, Nara 630-8506, Japan
3 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
4 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan
5 To whom correspondence should be addressed. Fax: +81 48 462 4674. E-mail: yutakam{at}postman.riken.go.jp
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Abstract |
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In cultured Bright Yellow-2 tobacco (Nicotiana tabacum) cells, the depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium induces amyloplast development. This differentiation also includes a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and accelerated by the addition of benzyladenine (BA). Three cDNAs were identified whose transcript levels are specifically increased during differentiation of starch-storing cells using the differential display method, and designated as starch-storing cell induced genes (SCI genes). One of these cDNAs, SCI2 encodes a 285 amino acids long protein with a KH RNA-binding domain. A database search revealed that this protein showed similarity to respective domains of mammalian quaking proteins. 2,4-D addition, which can convert starch-storing cells into dividing cells, to starch-storing BY-2 cells, immediately decreases the SCI2 transcript level, suggesting that SCI2 may have some role in starch-storing cell differentiation in BY-2 cells.
Key words: 2,4-D, benzyladenine, BY-2 cells, differentiation, starch-storing cells, tobacco.
Amyloplasts are mature plastids found in differentiated plant cells, including root caps and storage tissues such as cotyledons, endosperm, and tubers. They play important roles in the synthesis and accumulation of starch as a carbohydrate reserve in storage tissue, and graviperception in the root cap cells. Studies have led to many discoveries about the enzymes involved in starch synthesis (reviewed in Smith et al., 1997), however, the mechanisms that regulate amyloplast development remain unknown.
Generally, starch storage organ development occurs in a series of specific temporal and spatial steps, with a phase of cell division followed by differentiation. These sequential phases cannot be separated, and often occur simultaneously. Moreover, storage organs are highly organized and complex, making it difficult to analyse the mechanisms controlling starch storage in heterotrophic tissues. To overcome this problem, a system was used for inducing amyloplast formation in cultured Bright Yellow-2 (BY-2) tobacco (Nicotiana tabacum) cells (Miyazawa et al., 1999). Conventionally, tobacco BY-2 cells are grown in a liquid culture medium containing auxin (2,4-D), and is characterized by its homogeneity and high growth rate, which are necessary in cell biology studies (Nagata et al., 1992). When BY-2 cells were transferred to an auxin-depleted medium, amyloplast formation was synchronously induced in 2 d (Miyazawa et al., 1999). Isolation and analysis of genes specifically expressed during amyloplast formation will help in the investigation of the mechanisms of differentiation of starch-storing cells from dividing cells.
To isolate genes specifically expressed during amyloplast formation, a differential display method was used (Liang and Pardee, 1992) using random decamer primers (Operon). As nuclear gene expression necessary for amyloplast formation begins 6–12 h after the transfer to 2,4-D-depleted medium (Sakai et al., 1999), four RNA samples were compared at different conditions; stationary-phase cells, 12 h cells cultured in 2,4-D depleted medium (referred to as F-medium, hereafter), 12 h cells cultured in benzyladenine-supplied F-medium (referred to as B-medium, hereafter), and 12 h cells cultured in conventional 2,4-D-supplied medium (referred to as D-medium, hereafter), and isolated specific fragments that are visible only in F-, and B-medium cultured samples, where amyloplast formation occurs.
Three cDNAs were isolated, which were designated as starch-storing cell induced genes (SCI genes). RNA gel-blot analyses confirmed that three transcripts corresponding to three cDNA fragments (SCI1, 2, 3) accumulated to much higher levels in amyloplast-inducing cells than those in non-inducing cells (Fig. 1A). The database was searched for similar nucleotide sequences of these cDNAs using the BLAST algorithm (Altschul et al., 1990). As a result, SCI1 and SCI3 showed a significant similarity with known genes; Arabidopsis ATL6 (Accession No. AF132016), and tobacco cellulose synthase catalytic subunit (Accession No. AF304374) genes, respectively, whereas, SCI2 only showed a significant homology to an unknown predicted protein of Arabidopsis. Following the sequencing of the clones, full-length cDNAs were isolated by 5' and 3' rapid amplification of cDNA ends (SMART RACE cDNA amplification kit. Clontech). SCI2, encodes a 285 amino acid long protein containing KH RNA-binding domain. The KH-domain of SCI2 showed similarity to respective domains of mammalian quaking proteins, which is essential for nervous system myelination and survival of the early embryo in mouse ( Ebersole et al., 1996). Although, significant identities with well-characterized plant genes were not present, a sequence similarity search revealed that there are two orthologues in the Arabidopsis genome whose products are of unknown functions (Accession No. NM_129418, NP_187474).
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The addition of 2,4-D to amyloplast-developing BY-2 cells converts amyloplast into proplastid, and decreases the transcript level of starch-synthesis genes (Miyazawa et al., 1999). To confirm the relationship between starch-storing cell development and the transcript level of SCI2, it was investigated whether 2,4-D application decreases the accumulation of SCI2 mRNA (Fig. 1B). When BY-2 cells were cultured in D-medium, the transcript level of SCI2 was relatively low throughout the culture, whereas, in F-medium, the transcript level increased during the culture. Moreover, when 2,4-D was added to 24 h cells cultured in F-medium, the transcript level immediately decreased. To date, none of the Arabidopsis KH-domain proteins has been characterized (Lorkovi
and Barta, 2002), Along with the fact that mutations in KH-domain proteins very often result in developments defects, these results indicate that the SCI2 product might have a role in starch-storing cell development. |
Acknowledgements |
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The authors thank Yasue Ichikawa and Rie Nakazawa for DNA sequencing (Bioarchitect Research Group, RIKEN). The DDBJ/EMBL/GenBank accession number for the cDNA sequences of SCI2 is AB088637. Yutaka Miyazawa is supported by the Special Postdoctoral Researchers Program of Riken.
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