Zea mays ZmMybst1 cDNA, encodes a single Myb‐repeat protein with the VASHAQKYF motif

Journal of Experimental Botany, Mar 2003

A cDNA clone from a 4 DAP dissected maize embryo sac encoding a novel Zea mays single‐repeat Myb protein is reported here. This full‐length cDNA contains an ORF of 948 bp. The gene ZmMybst1 contains two introns (1166 and 706 bp) and is a single copy gene. The ZmMybst1 protein shares high sequence identity with the potato Mybst1 protein (58%). Northern blot, RT‐PCR and electronic northern analysis shows that ZmMybst1 is expressed in endosperm between 4 and 30 DAP, coinciding with the period of aleurone cell differentiation and development.

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Zea mays ZmMybst1 cDNA, encodes a single Myb‐repeat protein with the VASHAQKYF motif

Inderjit Singh Mercy 1 Robert B. Meeley 0 Scott E. Nichols 0 Odd-Arne Olsen 1 0 Pioneer Hi-Bred International , PO Box 1004, Johnston , 1A 50131-1004, USA 1 PO Box 5051, Agricultural University of Norway , 1432 Aas, Norway A cDNA clone from a 4 DAP dissected maize embryo sac encoding a novel Zea mays single-repeat Myb protein is reported here. This full-length cDNA contains an ORF of 948 bp. The gene ZmMybst1 contains two introns (1166 and 706 bp) and is a single copy gene. The ZmMybst1 protein shares high sequence identity with the potato Mybst1 protein (58%). Northern blot, RT-PCR and electronic northern analysis shows that ZmMybst1 is expressed in endosperm between 4 and 30 DAP, coinciding with the period of aleurone cell differentiation and development. Introduction Proteins and genes related to the viral Myb protein have been identified in most eukaryotes and are known to be transcriptional regulators (Klempnauer et al., 1982). Vertebrate Myb proteins have been classified into two groups and both contain three characteristic domains. One of these domains; the DNA-binding domain, is comprised of three imperfect repeats of 5052 amino acids (R1, R2 and R3) (Frampton et al., 1989), with three regularly spaced tryptophan residues 1820 amino acids apart, creating a hydrophobic core. Each repeat folds into a helix-turn-helix (HTH) motif (Frampton et al., 1989). The DBD in most plant Mybs contains only two Myb repeats, homologous to the vertebrate R2 and R3 (Martin and Paz-Ares, 1997). Recently, proteins containing three Myb repeats have also been found in plants (Braun and Grotewold, 1999; Kranz et al., 2000). The Arabidopsis genome appears to contain 100 different Myb-like genes, and approximately 80 Myb genes are found in the maize genome (Martin and Paz-Ares, 1997; Rabinowicz et al., 1999). Myb proteins are involved in the regulation of various genes in plants, and roles for Myb proteins in the synthesis of secondary metabolites is well-established (Martin and Paz-Ares, 1997). In maize five Myb genes, namely C1, Pl, P, Zm1, and Zm38, encoding R2R3-type Myb proteins in addition to C1-I, IBP1/IBP2 and RS2 have been identified. In addition, Gomez et al. (2002) identified the single repeat Myb, ZmMRP-1. Random sequencing of cDNA clones from a 4 DAP dissected embryo sac maize cDNA library followed by Genbank BLAST searches identified the maize cDNA clone, ZmMybst1. An electronic northern blot was created using BLAST search, which identified 27 additional ESTs from different developmental stages and tissues. Further sequencing of these clones demonstrated that these clones represent the same transcript. This result strongly suggests that only one ZmMybst1 is expressed in maize. The longest open reading frame (ORF) of the ZmMybst1 cDNA clone is 948 bp, encoding a putative protein of 315 amino acids. A putative translation start site, GGATGACG, and a putative polyadenylation signal, AATAAG, correspond well with known sequences for translation start sites and polyadenylation signals. 5 and 3 untranslated regions of 192 bp and 404 bp, respectively, are found in the clone in addition to a poly(A) tail. Sequencing of Mu-tagged DNA revealed two introns in the ZmMybst1 gene. When DNA blots were hybridized with a full-length ZmMybst1probe, a single hybridizing band of 3.1 kb was detected in EcoRIdigested DNA and Spe1 digestion yielded bands at 5.1 and 11 kb, as expected. Computerized structural predictions suggest that amino acids 87 147 adopt the HTH structure characteristic of a Myb DNA binding domain (DBD). A region spanning amino acids 155 to 200 is rich in both proline and acidic amino acids (Asp and Glu), and may act as a trans-activating domain (TAD). The putative DBD was compared with SHAQKY-motif containing single-repeat Mybs from plants including the maize ZmMRP-1 (Fig. 1A). The derived amino acid sequence of ZmMybst1 is highly similar to Mybst1 from potato (Baranowskij et al., 1994), AtMybst1 from Arabidopsis (Roundsley et al., 1997) and the ZmMRP-1 from maize. The similarity in the DBD domain between Mybst1, ZmMybst1, ZmMRP-1, and AtMybst1 is almost 90%. For three of these the similarity also spans regions outside the DBD (Fig. 1B); 40 of the first 45 amino acids are identical between Mybst1 and ZmMybst1 and they also share a high degree of similarity in the C-terminus. The identity between full-length Mybst1 and ZmMybst1 is 58%, and 44% between AtMybst1 and Mybst1, respectively, but ZmMybst1 is only 23% identical with ZmMRP-1 (Fig. 1B). This clearly shows that ZmMybst1 and ZMRP-1 represents two different proteins and that ZmMybst1 is closer related with Mybst1 and AtMybst1 than with ZmMRP-1. Among the SHAQKY motif 18 Myb proteins, such as CCA1 and LHY proteins share little sequence similarity with ZmMybst1 1 The gene sequence has been submitted by Bankit: the identification number is 513311. 4 To whom correspondence should be addressed. E-mail: . outside the SHAQKY motif. Nevertheless, eight of these; Mybst1, ZmMybst1, ZmMRP-1, MCB1/MCB2, AtMybst1, LeMyb1, AtLeMyb1A, and AtLeMyb1B, share a common DBD (Fig. 1A) and all contain a VASHAQKYF-motif, an extended version of the SHAQKY-motif. Mybst1, AtMybst1 and ZmMybst1 have alanine at positions 114, 161 and 134, respectively. The substitution of tryptophan with alanine in this position seems to be conserved since many Mybs contain alanine at this position (Fig. 1A, B). In other plant Mybs the first trp (W) in R3 is often substituted with other hydrophobic amino acids. According to Ogata et al. (1992), two histidines are important for correct folding of the third repeat of c-Myb; one histidine precedes the last tryptophan while the other is located between the two first tryptophans. In analogy with this, the last alanine in the VASHAQKYF should be the third hydrophobic amino acid. The ZmMybst1 transcript was detected in dissected maize endosperm from 430 DAP and in root, stem, leaf, nucellus, and pericarp (Fig. 2A (upper panel), B). Rehybridization to a maize atubulin probe was used as a loading control (Fig. 2A, lower panel). RT-PCR was also performed and confirmed the presence of a Fig. 2. (A) Upper panel; northern blot probed with a ZmMybst1 c-DNA probe. 500 ng of wt mRNA was used in each lane; whole seed (WS) 0 DAP, endosperm (Endo) 4, 10, 12, 15, 20, and 30 DAP, nucellus and pericarp 4 and 10 DAP. Lower panel; the same northern filter was reprobed with a maize a-tubulin probe to check equal loading of mRNA. (B) RT-PCR with internal primers on mRNA from whole seed (WS): 0 DAP, 4 and 10 DAP endosperm, nucellus and pericarp, leaf (L), root(R) and stem (S), P-positive control (PCR on ZmMybst1 c-DNA), N-negative control (PCR on reaction mix without template). Positions of size-markers are indicated at left. ZmMybst1-specific transcript in all tissues from the northern blot analysis (Fig. 2B). The northern blot results suggest that accumulation of ZmMybst1 transcript is highest in the seed in the early stages of seed development, with a maximum activity around 10 DAP. Northern, RT-PCR and the electronic northern data together demonstrate that the ZmMybst1 transcript is expressed in endosperm from around the time of the completion of endosperm cellularization until past the mid-stage of endosperm maturation. In addition, the transcript is present in most vegetative tissues, indicating no preferential expression in endosperm cell types. Since the ZmMybst1 transcript was isolated in 4 DAP dissected embryo sacs, a role in endosperm development did not seem unlikely for this gene. Many Myb proteins including CPC have been reported to function in epidermal tissue differentiation (Wada et al., 1997). This possibility therefore appeared particularly intriguing, since the aleurone layer is considered by many to be the epidermis of the endosperm. The fact that ZmMybst1 is expressed early in seed development and the proposed role for ZmMRP-1 in the development of transfer cell development strengthens the idea that ZmMybst1 might have an endosperm-related function. References


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Inderjit Singh Mercy, Robert B. Meeley, Scott E. Nichols, Odd‐Arne Olsen. Zea mays ZmMybst1 cDNA, encodes a single Myb‐repeat protein with the VASHAQKYF motif, Journal of Experimental Botany, 2003, 1117-1119, DOI: 10.1093/jxb/erg118