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. 2013 Jun 25;14(6):R65.
doi: 10.1186/gb-2013-14-6-r65.

Thermal Stress Effects on Grain Yield in Brachypodium Distachyon Occur via H2A.Z-nucleosomes

Free PMC article

Thermal Stress Effects on Grain Yield in Brachypodium Distachyon Occur via H2A.Z-nucleosomes

Scott A Boden et al. Genome Biol. .
Free PMC article

Abstract

Background: Crop plants are highly sensitive to ambient temperature, with a 1 ºC difference in temperature sufficient to affect development and yield. Monocot crop plants are particularly vulnerable to higher temperatures during the reproductive and grain-filling phases. The molecular mechanisms by which temperature influences grain development are, however, unknown. In Arabidopsis thaliana, H2A.Z-nucleosomes coordinate transcriptional responses to higher temperature. We therefore investigated whether the effects of high temperature on grain development are mediated by H2A.Z-nucleosomes.

Results: We have analyzed the thermal responses of the Pooid grass, Brachypodium distachyon, a model system for crops. We find that H2A.Z-nucleosome occupancy is more responsive to increases in ambient temperature in the reproductive tissue of developing grains compared withvegetative seedlings. This difference correlates with strong phenotypic responses of developing grain to increased temperature, including early maturity and reduced yield. Conversely, temperature has limited impact on the timing of transition from the vegetative to generative stage, with increased temperature unable to substitute for long photoperiod induction of flowering. RNAi silencing of components necessary for H2A.Z-nucleosome deposition is sufficient to phenocopythe effects of warmer temperature on grain development.

Conclusions: H2A.Z-nucleosomes are important in coordinating the sensitivity of temperate grasses to increased temperature during grain development. Perturbing H2A.Z occupancy, through higher temperature or genetically, strongly reduces yield. Thus, we provide a molecular understanding of the pathways through which high temperature impacts on yield. These findings may be useful for breeding crops resilient to thermal stress.

Figures

Figure 1
Figure 1
Higher ambient temperature is not sufficient to induce flowering in Brachypodium distachyon . (a) Plants were grown in one of three photoperiod conditions: SD (14 h light/10 h dark), LD (20 h/4 h) or in LD after shifting from SD. In each condition, plants were grown at either 22°C (grey) or 27°C (black). DNF (did not flower) indicates the non-flowering phenotype of plants grown in SD for 150 days. Values are the mean ± standard error of ten plants. (***P < 0.001). (b) SD grown plants after 50 days at constant temperature of 22°C and 27°C.
Figure 2
Figure 2
Increased ambient temperature reduces grain yield in Brachypodium. (a,b) Fresh weight (a) and dry weight (b) of developing grain from plants grown at either 22/17°C (black line) or 27/22°C (red line) (day/night temperatures). Data are the replicate of 5 replicate plants, including measurements of at least 20 seed. Values are the mean ± standard error (**P < 0.01). (c) Final yield measurements in units of weight per 10 grain for plants transferred from 22/17°C to 27/22°C throughout grain-filling, then transferred back to 22/17°C at 16 days after pollination, compared to plants grown constantly at 22/17°C (***P < 0.001). Data are from 20 biological repeats. Values are the mean ± standard error.
Figure 3
Figure 3
The Brachypodiumtranscriptome responds to changes in ambient temperature. (a) Transcript profiling experiment shows a robust response to changes in ambient temperature in vegetative seedlings. The heat map depicts all differentially expressed genes (DEGs) with at least two-fold change in any of the temperature treatments as determined from two-way ANOVA (P for temperature effect ≤ 0.05). Expression levels of up-regulated genes are in shades of red and of down-regulated genes in shades of green. (b,c) Venn diagrams of total numbers of up-regulated (b) or down-regulated (c) DEGs in vegetative seedlings after 24 h shift to either 22°C (green), 27°C (red), or in both temperatures (yellow). The two-tailed P-values for the significance of the overlap represented under the Venn diagrams have been calculated using Fisher's exact test. (d)HSF23 (green line), HSP70 (black line) and HSP90 (red line) are induced strongly with increasing temperature, in contrast to other heat-shock genes (grey) that do not respond significantly over the temperature range assessed. (e-g) Quantitative real-time PCR (qRT-PCR) analysis of genes that are up-regulated by increasing temperature (e), down-regulated (f) or show constant expression (g) in vegetative seedlings 24 h after temperature-shift. (h)qRT-PCR analysis of up-regulated genes in plants grown constantly at either 17°C, 22°C or 27°C. (i,j)qRT-PCR analysis of genes in developing grain that are up-regulated by increasing temperature (i), or remain constant within the temperature range (j). (k)qRT-PCR analysis of genes that are up-regulated by temperature with known roles in developing grain. Data are from at least three biological replicates.
Figure 4
Figure 4
Nucleosome positioning by H3 ChIP analysis. (a,b)ChIP of cross-linked H3 at the promoter sites of HSF23 (a) and HSP70 (b) reveal well-positioned -1 and +1 nucleosomes. The x-axis indicates the central position of each amplicon relative to the TSS. In each schematic, the promoter (solid line), 5'UTR (white box), exons (black box) and TSS (arrow) are shown. (c-e)ChIP of cross-linked H3 for genes that were detected to be up-regulated (c), constant (d) or down-regulated (e) in response to temperature reveal sites that show strong enrichment of H3 at probable +1 nucleosome sites according to in silicosequence analysis (see Materials and methods). The x-axis indicates the central position of each amplicon relative to the TSS. Values from H3 and mock reactions are shown in black and grey, respectively. Values are the mean ± standard error of three biological replicates.
Figure 5
Figure 5
Identification and nucleosome positioning of BdHTA9 in Brachypodium. (a) An unrooted maximum-likelihood phylogenetic tree of HTA proteins in Brachypodium, Arabidopsis, humans and yeast, constructed using MEGA5 with 100 bootstrap replicates, summarizes the evolutionary relationship among the HTA proteins and the separation in four phylogenetic subfamilies. Branches are drawn to scale with scale bar representing the number of substitutions per site. (b) Reverse-transcriptase PCR analysis of BdHTA1, BdHTA9 and BdHTA11 from leaf, apex and endosperm tissue of Bd21. Two biological replicates are shown. (c,d)ChIP analysis of HTA9:3XFLAG (H2A.Z) at 17°C shows H2A.Z is enriched at the -1 and +1 nucleosomes of HSF23 (c) and HSP70 (d). The x-axis indicates the central position of each amplicon relative to the TSS. In each schematic, the promoter (solid line), 5'UTR (white box), exons (black box) and TSS (arrow) are shown. Mock reactions (grey) were performed on identical tissue from wild-type plants. Values are the mean ± standard error of three biological replicates.
Figure 6
Figure 6
Occupancy of H2A.Z-nucleosomes is reduced at higher ambient temperatures in developing grain but not in vegetative seedlings. (a-c)ChIP analysis of HTA9:3XFLAG (H2A.Z) at 22°C and 27°C in vegetative seedlings at +1 nucleosomes of genes whose expression was up-regulated (a), remained constant (b) or down-regulated (c) upon an increase in temperature. (d-f)ChIP analysis of HTA9:3XFLAG (H2A.Z) at 22/17°C and 22/27°C in developing grain at +1 nucleosomes of genes whose expression was up-regulated (d) in both seedlings and grain, or remained constant (e) upon an increase in temperature. (f)ChIP analysis of HTA9:3XFLAG (H2A.Z) at 22/17°C and 27/22°C for genes with roles in grain development. Mock reactions (grey) were performed on identical tissue from wild-type plants. ***P < 0.001.
Figure 7
Figure 7
Seeds from plants with reduced expression of ARP6 phenocopy seed from plants grown at higher temperature. (a) Relative expression of ARP6 in wild-type (WT; Bd21) and three independent ARP6 RNAi transgenic lines (T1 generation); ***P < 0.001. (b,c) Seed weight measurements (b) and yield per plant (c) in WT (Bd21) at 22/17°C and 27/22°C, as well as three independent transgenic lines at 22/17°C (T1 generation). Data are the mean ± standard error of at least 15 grains (**P < 0.01; ***P < 0.001). (d) A representative spike from WT Bd21 and ARP6 RNAi.1 displaying the empty florets (white arrowheads) that contained aborted grain. Scale bar, 1 cm.
Figure 8
Figure 8
Genes up-regulated by increased ambient temperature are up-regulated in seeds of ARP6 RNAi transgenic lines at 22°C. Expression of genes that are up-regulated in developing grain by transfer from 22/17°C to 27/22°C (Figure 3) are up-regulated in grain of three ARP6 RNAi transgenic lines (T1 generation) grown at 22/17°C. Values are the mean ± standard error of 4 replicates, each containing 6 grain at 8 DAP.

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