Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence

37Citations
Citations of this article
86Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence. It was significantly up-regulated following ABA treatment, supported by ABA-responsive elements in its promoter, but it was not up-regulated during dark-induced senescence. The C-termini of proteins closely related to HvNAC005 showed overall high divergence but also contained conserved short motifs. A serine- and leucine-containing central motif was essential for transcriptional activity of the HvNAC005 C-terminus in yeast. Over-expression of HvNAC005 in barley resulted in a strong phenotype with delayed development combined with precocious senescence. The over-expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling pathways. Binding of HvNAC005 to promoter sequences of putative target genes containing the T[G/A]CGT core motif was shown by direct protein-DNA interactions of HvNAC005 with promoters for two of the up-regulated genes. In conclusion, HvNAC005 was shown to be a strong positive regulator of senescence and so is an obvious target for the fine-tuning of gene expression in future attempts to improve nutrient remobilization related to the senescence process in barley.

Figures

  • Fig. 1. HvNAC005 expression levels, as determined by qRT-PCR, following ABA treatment and darkness in barley ‘Golden Promise’ plants. The qRT-PCR results were normalized to the expression level of 18S rRNA. Gene expression levels (–∆∆Ct, i.e. log2 scale) was determined relative to untreated control leaves. (A) ABA treatment of 2nd leaves of 3-week-old plants with either 0.1 or 1.0 mM ABA. (B) Three days of dark treatment, i.e. coverage with tin foil, of 2nd leaves of 3-week-old plants. – Rubisco ss, Rubisco small subunit (GenBank: AB020943); PP2C, protein phosphatase 2C (GenBank: BM816007); HvNAC005 (GenBank: AK251058); HvNAC013 (GenBank: AK376297); HvNAC027 (GenBank: FR819765); CYSPEP, papain cysteine peptidase (GenBank: AK358908.1). Error bars represent SE. */**/***, P <0.05/0.025/0.001 (t test, R software version 3.0.1, R Core Team, 2015).
  • Fig. 2. Features of the NAC domain proteins in the subgroups NAC-a5 and -a6 (Shen et al., 2009). (A) Phylogenetic tree of HvNAC005-related proteins belonging to the NAC-a5 and -a6 subgroups (Supplementary Table S2). The tree was based on the N-terminal NAC domains of the proteins and constructed using the ClustalX software. ANAC060 (subgroup NAC-d) was included as an outgroup. D, dicot; M, monocot. (B) C-terminal parts corresponding to the NAC domains in (A), highlighting conserved MEME amino acid motifs. (C) WEBlogos of the six MEME motifs shown in (B). The height of a letter in the WebLogo reflects its relative frequency at the given position in the motif. (This figure is available in colour at JXB online).
  • Fig. 5. Representation of genes in the MapMan top BINs for all genes (dark+light grey) and the 2630 differentially expressed genes (dark grey), respectively, in the Agilent microarray experiment for HvNAC005 overexpressing plants, line HvNAC005-OE-26. Asterisks indicate significant overrepresentation among differentially expressed genes for individual categories determined using a hypergeometric test (R software version 3.0.1, R Core Team, 2015).
  • Fig. 7. Validation by qRT-PCR of gene expression levels in barley HvNAC005 over-expressing lines for selected SAGs and genes selected from the microarray experiment (Supplementary Table S3). Ct values were normalized to 18S rRNA expression and the results (–∆∆Ct, i.e. log2 scale) are shown relative to expression levels in null-transgenic lines as controls. Where available, the fold change in the microarray experiment is shown in the bottom of the figures. (A) Results from leaves of 2-week-old seedlings. (B) Results from 3-d-old roots of single seeds germinated on moist filter paper. SaDH, saccharopine dehydrogenase (GenBank: AJ508229.2); Nuclease I, BnucI (GenBank: AB028448.1); ZOG, putative zeatin O-glucosyltransferase (GenBank: AK364192.1); ZIM domain protein, jasmonate ZIM domain-containing protein (GenBank: AK358513.1); NCED1: 9-cis-epoxycarotenoid dioxygenase (GenBank: AK365103.1); PP2C–1, protein phosphatase 2C (GenBank: AK367469.1); PP2C–2, protein phosphatase 2C (GenBank: AK374059.1); 2OG-Fe(II) oxygenase, oxoglutarate/iron-dependent dioxygenase (GenBank: AK355337.1); SGR, staygreen protein (GenBank: AK361634.1); PaO, pheophorbide a oxygenase (GenBank: AK358479.1); Amino acid permease, amino acid permease (GenBank: AK361903.1); AP/ EREBP, APETALA2 and ethylene responsive element binding protein (GenBank: AK374826.1).( a) The microarray probe for HvNAC005 was in the 3′-UTR and, hence, did not detect the transcript of the HvNAC005 transgene.( b) The SaDH and Nuclease I genes were not represented among the microarray probes.(c) The signal for the PP2C-2 microarray probe was filtered out during data analysis. Error bars represent SE. *, P <0.05 (Limma test; Smyth, 2005).
  • Fig. 8. EMSA assays with the DNA binding NAC domain of HvNAC005 and the promoter sequences of two genes selected from the genes up-regulated in the microarray experiment. Glutathione-S-transferase (GST) was included as a negative control. (A) EMSA results with the 1000 bp promoter of the ZOG gene (EnsemblPlants: MLOC_61801.1; GenBank: AK364192.1). A number of other NAC transcription factors were tested on this promoter fragment but were, for the sake of clarity, excluded from the figure. (B) EMSA results with three promoter fragments of a ZIM domain protein (EnsemblPlants: MLOC_61774.1; GenBank: AK358513.1). EMSA assays were performed at least in triplicate. Between 100 and 200 ng HvNAC005 we observed 'smear'and precipitation in the wells, which obscured the number of possible protein–DNA complexes. Thus, to obtain the best qualitative result, the best two titrations were merged.

References Powered by Scopus

MEME Suite: Tools for motif discovery and searching

7577Citations
N/AReaders
Get full text

Jasmonates: Biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany

1963Citations
N/AReaders
Get full text

A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat

1284Citations
N/AReaders
Get full text

Cited by Powered by Scopus

Major genes determining yield-related traits in wheat and barley

156Citations
N/AReaders
Get full text

The biotechnological importance of the plant-specific NAC transcription factor family in crop improvement

122Citations
N/AReaders
Get full text

Roles of potential plant hormones and transcription factors in controlling leaf senescence and drought tolerance

115Citations
N/AReaders
Get full text

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Cite

CITATION STYLE

APA

Christiansen, M. W., Matthewman, C., Podzimska-Sroka, D., O’Shea, C., Lindemose, S., Møllegaard, N. E., … Gregersen, P. L. (2016). Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence. Journal of Experimental Botany, 67(17), 5259–5273. https://doi.org/10.1093/jxb/erw286

Readers over time

‘16‘17‘18‘19‘20‘21‘22‘23‘240481216

Readers' Seniority

Tooltip

PhD / Post grad / Masters / Doc 38

69%

Researcher 11

20%

Professor / Associate Prof. 3

5%

Lecturer / Post doc 3

5%

Readers' Discipline

Tooltip

Agricultural and Biological Sciences 29

54%

Biochemistry, Genetics and Molecular Bi... 17

31%

Nursing and Health Professions 4

7%

Social Sciences 4

7%

Save time finding and organizing research with Mendeley

Sign up for free
0