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. 2007;35(1):203-13.
doi: 10.1093/nar/gkl1068. Epub 2006 Dec 7.

Exploring membrane-associated NAC transcription factors in Arabidopsis: implications for membrane biology in genome regulation

Sun-Young Kim  1 , Sang-Gyu Kim, Youn-Sung Kim, Pil Joon Seo, Mikyoung Bae, Hye-Kyung Yoon, Chung-Mo Park
Affiliations

Exploring membrane-associated NAC transcription factors in Arabidopsis: implications for membrane biology in genome regulation

Sun-Young Kim et al. Nucleic Acids Res. 2007.

Abstract

Controlled proteolytic cleavage of membrane-associated transcription factors (MTFs) is an intriguing activation strategy that ensures rapid transcriptional responses to incoming stimuli. Several MTFs are known to regulate diverse cellular functions in prokaryotes, yeast, and animals. In Arabidopsis, a few NAC MTFs mediate either cytokinin signaling during cell division or endoplasmic reticulum (ER) stress responses. Through genome-wide analysis, it was found that at least 13 members of the NAC family in Arabidopsis contain strong alpha-helical transmembrane motifs (TMs) in their C-terminal regions and are predicted to be membrane-associated. Interestingly, most of the putative NAC MTF genes are up-regulated by stress conditions, suggesting that they may be involved in stress responses. Notably, transgenic studies revealed that membrane release is essential for the function of NAC MTFs. Transgenic plants overexpressing partial-size NAC constructs devoid of the TMs, but not those overexpressing full-size constructs, showed distinct phenotypic changes, including dwarfed growth and delayed flowering. The rice genome also contains more than six NAC MTFs. Furthermore, the presence of numerous MTFs is predicted in the whole transcription factors in plants. We thus propose that proteolytic activation of MTFs is a genome-wide mechanism regulating plant genomes.

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Figures

Figure 1
Figure 1
NTL genes and protein structures of NTLs. (A) Arabidopsis NTL genes. The presence of the TMs in the Arabidopsis NAC transcription factors was examined in the membrane protein database ARAMEMNON. At least thirteen NAC members were predicted to be MTFs. NTL12 and NTL13 are equivalent to NTM1 and NTM2, respectively (7). (B) NTL gene structures. They consist of 3–7 exons. (C) Protein structures of NTLs. The highly conserved NAC domains are present in their N-terminal regions (black boxes). The α-helical TMs (open boxes) are located in their far C-terminal regions.
Figure 2
Figure 2
Phylogenetic analysis of NTLs. (A) Phylogenetic relationships. The phylogenetic relationships were inferred using the PHYLIP program. The NTL proteins are apparently classified into 4 phylogenetic subgroups (I-IV). (B) NTL pairs with close relationships.
Figure 3
Figure 3
Expression patterns of NTLs and their responses to abiotic stresses. (A) Tissue-specific expression patterns. Total RNA samples were separately prepared from cauline leaves (CL), flowers (F), shoot apexes (SA), rosette leaves (RL), roots (R) and stems (ST). A tubulin gene (TUB) was included as a control for constitutive expression. (B) Growth stage-dependent expression patterns. Plants were harvested at the indicated time intervals throughout the life span for total RNA extraction. (C) Effects of abiotic stresses on NTL expression. Plants grown for 2 weeks on MS-agar plates were incubated at 4°C (cold) for 12 h or 37°C (heat) for 1 h before harvesting plant materials. To examine the effects of drought stress, plants were dehydrated on dry 3 MM paper for 30 or 60 min. CBF2 was included as a positive control. (D) NaCl effects on NTL expression.
Figure 4
Figure 4
Regulation of NTL expressions by H2O2, MMS and growth hormones. (A) Effects of H2O2. Plants were subjected to a vacuum/release cycle in hydroponic MS media supplemented with 50 mM H2O2 and subsequently grown under normal growth conditions for the indicated time periods. (B) Effects of MMS. Plants were incubated in hydroponic MS media containing 100 p.p.m. MMS for the indicated time periods. (C) Growth hormone effects. Plants that were grown for 2 weeks on MS-agar plates were incubated in hydroponic MS media supplemented with appropriate growth regulators. They were used at the final concentrations of 100 μM for ABA and indole-3-acetic acid (IAA), 50 μM for 1-aminocyclopropane-1-carboxylic acid (ACC), BA and paclobutrazol (PAC), 20 μM for gibberellic acid (GA) and 1-N-naphthylphthalamic acid (NPA), and 1 μM for brassinolide (BL) and brassinazol (BRZ).
Figure 5
Figure 5
NTL6 transgenic plants. (A) NTL6 constructs used for Arabidopsis transformation. The numbers in parentheses are those of amino acid residues. (B) NTL6 transgenic plants. Ten-day-old plants are displayed. Overexpression of the transgenes was confirmed by RT–PCRs. (C) Statistics of transgenic plants. TL indicates the number of total transgenic lines obtained, and PL indicates the number of transgenic plants showing the phenotype shown in (B). (D) Localized expression of NTL6 in guard cells. (E) NTL6 induction by SA. Ten-day-old wild-type plants were treated with SA at a final concentration of 0.1 mM for 2 h or 6 h. (F) Defense gene induction in the 35S::6ΔC transgenic plants. Two representative transgenic plants (1 and 2) were analyzed.
Figure 6
Figure 6
NTL8 transgenic plants. (A) NTL8 constructs used for Arabidopsis transformation. (B) NTL8 transgenic plants. Two types of the 35S::8ΔC transgenic plants were isolated: one type has a normal morphology but is late flowering (35S::8ΔC-1), and the other type shows reduced growth as well as severe late flowering (35S::8ΔC-2). Inlet shows an enlarged view of the 35S::8ΔC-2 transgenic plant. (C) Flowering time measurements of the 35S::8ΔC-1 transgenic plants. Thirty plants were measured and averaged for each plant group. Bars denote standard error of the mean. Statistical significance was determined using a student t-test (P < 0.01). (D) ΔC transgene expression. The severity of phenotypic alterations in the 35S::8ΔC transgenic plants is proportional to the level of the ΔC transgene expression. (E) Expression of flowering time genes. The transcript levels were analyzed by RT–PCR-based Southern blot hybridization.
Figure 7
Figure 7
Processing of Arabidopsis and rice NTLs. (A) Analysis of transcriptional activities. P, positive control (full-size GAL4); N, negative control (DNA-binding domain); 8ΔC-N and 8ΔC-C, N-terminal and C-terminal regions of 8ΔC, respectively. Three independent measurements were averaged (P < 0.01). (B) NTL processing. The myc-NTL gene fusion constructs were infiltrated into N.benthamiana leaves. The full-size (arrow heads) and the processed (arrows) NTLs were immunologically detected using an anti-myc antibody. (C) Membrane-association of NTL8. Aliquots of each fraction, which have been processed as described previously (7), were subject to western blot analysis (upper panel) using an anti-myc antibody. The Commassie-stained membrane is displayed at the bottom. T, total extract; S, soluble fraction; M, membrane fraction; B, buffer-extracted fraction; SD, SDS-extracted fraction; F, final membrane fraction. (D) ABA effects on NTL6 processing. Plants were treated with 100 μM ABA for 6 h. A part of Commassie-stained gel is shown at the bottom. (E) Two putative mechanisms for releasing MTFs. Controlled cleavage of MTFs either by intramembrane proteases (RIP) or ubiquitin/proteasome-dependent processing (RUP) is schematically shown. This diagram is modified from Hoppe et al. (2).
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