Identification of recognition sequence of ANAC078 protein by the cyclic amplification and selection of targets technique

Abstract

NAC (NAM, ATAF and CUC2) is one of the largest families of transcription factors in the plant genome, but the function and regulation of most NAC genes are still largely unknown. We recently isolated a gene encoding a NAC transcription factor designated ANAC078 from Arabidopsis plants and identified 166 genes upregulated in ANAC078-overexpressing plants compared with the wild-type plants under high-light stress. The cyclic amplification and selection of targets (CASTing) technique showed that the ANAC078 recognition sequence contains T[A/T/C] [A/T/G/C] C[T/G] TG[T/G]G as a DNA-binding site. The recognition sequence identified by this technique was detected in the promoter region of 52 upregulated genes, including the gene for a transcription factor, proteasome subunits, peroxidase and a protein kinase. The findings suggest these genes to be directly targeted by the ANAC078 protein.

Transcription factors regulate the first step of gene expression. The Arabidopsis genome contains genes for more than 1,500 transcription factors,¹ with recent analyses identifying >2,000 transcription factors genes,^2–5 more than that in Drosophila melanogaster and Caenorhabditis elegans. Furthermore, the protein of genes encoding transcription factors in Arabidopsis is 5–10% depending on the database, which is higher than that in D. melanogaster (4.7%) and in C. elegans (3.6%),¹ although it is comparable with that of human (6.0%).⁶ These findings might be because plants, unlike animals, are unable to move and therefore experience numerous forms of environmental stress, such as high-light (HL), drought, high temperature, chilling, salinity and air pollution.

Although transcription factors, such as heat stress transcription factor (Hsf), basic helix-loop-helix (bHLH) and MYB, exist not only in the Saccharomyces cerevisiae, D. melanogaster and C. elegans genomes, but also in the Arabidopsis genome, about half of the Arabidopsis transcription factors, such as AP2-ERF, NAC, Dof, YABBY, WRKY, GARP, TCP, SBP, ABI3-VP1 (B3), EIL and LFY, are plant-specific.^1,7

NAC (NAM, ATAF and CUC2) is one of the largest families of transcription factors in the plant genome, with 106 and 149 members predicted in Arabidopsis and rice, respectively.^8,9 A small number of the Arabidopsis NAC members have been studied,¹⁰ but the function and regulation of most NAC genes are still largely unknown.

We previously isolated a gene encoding a NAC transcription factor named ANAC078 according to the nomenclature established for the Arabidopsis NAC family^11,12 though it has also been designated NTL11.¹³ Furthermore, we reported that the transcript levels of 166 genes in ANAC078-overexpressing Arabidopsis plants (Ox-ANAC078) are increased compared with those in the wild-type plants under HL.¹⁴

To explore in more detail the system by which putative target genes are regulated via ANAC078, we identified the recognition sequence for the protein using the cycle amplification of targets (CASTing) method.^15,16 A His-Trigger factor (TF) tag, as a kind of chaperon, fused with the NAC domain of ANAC078 (TF-NAC domain) was used to select the DNA-binding site from a random pool of double-stranded oligonucleotides. Each round of CASTing consisted of three steps: binding of the double-stranded DNA oligonucleotide pool to the TF-NAC domain protein, removal of unbound DNA, and PCR amplification of DNA that remained bound to the TF-NAC domain protein-Ni sepharose bead complex.

To confirm enrichment of the population with high-affinity binding sites, an electrophoretic mobility shift analysis (EMSA) was performed after each round of CASTing to assess the efficiency of selection. Figure 1 shows the progressive enrichment of the recognition sequence of ANAC078 obtained by this procedure. During the first cycle, the fraction of specific targets within the population was so small that it was not associated with the TF-NAC domain protein. However, a small signal was detected during the two cycles, and thus became a major fraction after four cycles. Figure 2 indicates the sequences obtained after cloning the four-cycle-selected material. Thirty four clones, sequenced independent of each other, contained the T[A/T/C] [A/T/G/C] C[T/G] TG[T/G]G sequence.

Figure 1.

EMSA analysis of CASTing-enriched probes. The material obtained from each cycle of CASTing was radioactively labeled by including [α-³²P]ATP and subjected to EMSA in the presence of 2 µg of bacterially expressed TF-fused ANAC078 NAC domain protein.

Figure 2.

ANAC078-binding sequence. The numbers represent the frequency with which a nucleotide was found at each position (expressed as a percentage of the total).

It has been reported that ANAC019, ANAC055 and ANAC072 bound to the 63-bp promoter region of the ERD1 gene which contained the CATGTG MYC-like motif.¹⁷ The recognition sequence of ANAC019, ANAC055 and ANAC072 was determined as ANN NNN TCN NNN NNN ACA CGC ATGT, containing CATGT and harboring CACG as the core DNA-binding site.¹⁷ Although C[T/G] TG[T/G] similar to CATGT motif was contained in the 3′ region of ANAC078 recognition sequence, no CACG motif was contained in the recognition sequence. Previously, it has been reported that the Arabidopsis NAC1 (ANAC022) protein binds to a 21-bp segment (CTGA CGT AAG GGA TGA CGC AC) within the cauliflower mosaic virus (CaMV) 35S-90 promoter.¹⁸ Duval et al. (2002)¹⁹ reported that purified AtNAM (ANAC018) protein bound to a region of the CaMV 35S promoter between -70 and -76, which was located in the 21-bp segment. No CACG motif was contained in the NAC1 or AtNAM recognition sequence either. Further analysis of the function of the CACG motif in gene regulation via the NAC family is needed.

A search was made for the recognition sequence of ANAC078 in the promoter regions (within 1,000 bp upstream of the coding region) of 166 putative target of ANAC078. The sequence was detected in the putative promoter region of 52 target genes (Suppl. Table 1). These genes encoded transcription factors, proteasome subunits, peroxidase, a protein kinase and so on, suggesting their expression of 52-genes to be directly regulated by ANAC078.

In fact, the transcript levels of transcription factor genes related to flavonoid biosynthesis and the levels of anthocyanins were significantly increased in Ox-ANAC078 plants and reduced in knockout ANAC078 plants (KO-ANAC078) compared with wild-type plants under HL stress.¹⁴ Proteasomes are an essential component of the quality-control system that limits the accumulation of non-functional proteins in the cell.^20–23 Recently, it has been reported that the 20S proteasome plays an important role in responses to several types of stress, such as salinity, and oxidative stress.^24,25 As shown in Supplementary Table 1, there are several genes (AT1G56650, AT1G62085, AT1G73870, AT2G47460, AT3G01600 and AT5G58610) encoding transcription factors. It seems likely that the expression of other ANAC078-target genes which do not have the ANAC078-recognition sequence in their promoter region is regulated by these transcription factor under HL stress.

The present findings and results reported previously suggest that ANAC078 is a key regulator in the induction of not only the defence system, but also the growth and developmental processes of vegetative organs in response to external signals, including environmental stress.

Abbreviations

CASTing

cyclic amplification and selection of targets

EMSA

electrophoretic mobility shift analysis NAC, NAM, ATAF and CUC2

TF

trigger factor

Addendum to: Morishita T, Kojima Y, Maruta T, Nishizawa-Yokoi A, Yabuta Y, Shigeoka S. Arabidopsis NAC transcription factor, ANAC078, regulates flavonoids biosynthesis under highlight. Plant and Cell Physiol. 2009;50:2210–2222. doi: 10.1093/pcp/pcp159.