The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance

Abstract

Nuclear factor Y (NF-Y) is a ubiquitous transcription factor composed of three distinct subunits (NF-YA, NF-YB, and NF-YC). We found that the Arabidopsis thaliana NFYA5 transcript is strongly induced by drought stress in an abscisic acid (ABA)-dependent manner. Promoter:beta-glucuronidase analyses showed that NFYA5 was highly expressed in vascular tissues and guard cells and that part of the induction by drought was transcriptional. NFYA5 contains a target site for miR169, which targets mRNAs for cleavage or translational repression. We found that miR169 was downregulated by drought stress through an ABA-dependent pathway. Analysis of the expression of miR169 precursors showed that miR169a and miR169c were substantially downregulated by drought stress. Coexpression of miR169 and NFYA5 suggested that miR169a was more efficient than miR169c at repressing the NFYA5 mRNA level. nfya5 knockout plants and plants overexpressing miR169a showed enhanced leaf water loss and were more sensitive to drought stress than wild-type plants. By contrast, transgenic Arabidopsis plants overexpressing NFYA5 displayed reduced leaf water loss and were more resistant to drought stress than the wild type. Microarray analysis indicated that NFYA5 is crucial for the expression of a number of drought stress-responsive genes. Thus, NFYA5 is important for drought resistance, and its induction by drought stress occurs at both the transcriptional and posttranscriptional levels.

Figure 1.

Regulation of NFYA5 Expression by Drought Stress and ABA. (A) Real-time PCR assay of the accumulation of NFYA5 gene transcript in Arabidopsis plants in response to drought stress (withholding water from 3-week-old soil-grown plants for the indicated durations) and to ABA (2-week-old seedlings on agar medium). The expression levels were normalized to that of Tub8, and the level of NFYA5 transcript in the controls was set at 1.0. Error bars represent se for three independent experiments. (B) Detection of NFYA5 mRNA in ABA-deficient (aba2-1) or signaling (abi1-1) mutants. The wild type and mutants were grown with sufficient water for 3 weeks, and then water was withheld for 10 d. Twenty micrograms of total RNA from each sample was loaded and hybridized with a ³²P-labeled full-length NFYA5 cDNA probe. Tub8 was used as a loading control, and numbers below each lane indicate the expression level of NFYA5 relative to Tub8.

Figure 2.

NFYA5 Expression Pattern and Transcriptional Regulation. (A) GUS activity in 2-week-old transgenic seedlings on MS-agar medium that were exposed to ABA treatment for 8 h or immersed in water for 8 h (control). (B) NFYA5p:GUS expression pattern in various tissues. The staining was prominent in the vascular tissues (I) and guard cells (II) of leaves. The staining was also visible in floral tissues of the inflorescence (III) and root vascular system (IV). (C) Tissue pattern of NFYA5 transcript accumulation. Total RNA was isolated from various tissues of 4-week-old wild-type plants grown under long-day growth conditions. Real-time RT-PCR quantifications were normalized to the expression of 18S rRNA. Error bars represent se for three independent experiments. (D) Subcellular localization of NFYA5. The NFYA5-YFP fusion construct was expressed in transgenic Arabidopsis under the control of the cauliflower mosaic virus 35S promoter, and the plant roots were observed under a confocal microscope. The photographs were taken in the dark field for yellow fluorescence (I), in the bright field for the morphology of the cells (II), and in combination (III).

Figure 3.

Drought Stress Downregulates a 21-Nucleotide Small RNA That Is Complementary to NFYA5 mRNA. (A) Diagram of the cis-antisense gene pair of NFYA5 and At1g54150. Exons are boxed, and lines between boxes represent introns. Arrow indicates target position of the small RNA. (B) Regulation of the small RNA by drought stress and ABA treatment. miR171 or U6 RNA was probed as a loading control. Numbers below each lane indicate relative expression. (C) Accumulation of the small RNA in various RNA silencing mutants. Forty micrograms of small RNA from each sample was loaded per lane and hybridized with a ³²P-labeled oligonucleotide probe corresponding to the sequence of ASRP1815. miR172, siR255, and U6 were probed as loading controls. (D) NFYA5 mRNA levels in dcl1-7, hen1, and hyl1 and their corresponding wild types. The expression levels were normalized to that of Tub8. Error bars represent se for three independent experiments.

Figure 4.

NFYA5 Is Mainly Regulated by miR169a. (A) Detection of precursor transcripts of the MIR169 family in response to drought stress by real-time RT-PCR. Quantifications were normalized to the expression of Tub8. Error bars represent se for three independent experiments. (B) Diagram of NFYA5 expression constructs. The introduced mutations in the target site of NFYA5 are shown in lowercase letters. Black box in the 3′ UTR indicates the miRNA target site. (C) Coexpression of various combinations of miR169 and NFYA5 expression constructs in N. benthamiana. As a control, NFYA5 was also coexpressed with an unrelated YFP construct. Real-time RT-PCR quantifications were normalized to the expression of 18S rRNA of tobacco. Error bars represent se for three independent experiments. (D) Overexpression of miR169a and miR169c in transgenic Arabidopsis. RNA gel blot analysis of miR169a and miR169c levels in the wild type and two representative transgenic lines. miR171 is shown as a loading control. Numbers below each lane indicate relative expression. (E) Detection of corresponding NFYA5 gene transcripts in 35S:MIR169 transgenic plant lines by real-time RT-PCR. Quantifications were normalized to the expression of Tub8. Error bars represent se for three independent experiments.

Figure 5.

35S:MIR169a Plants Are More Sensitive to Drought Stress. (A) 35S:MIR169a-overexpressing Arabidopsis plants are more sensitive to drought stress. Wild type (Col) and 35S:MIR169a plants were grown in soil with sufficient water for 3 weeks, and then the water was withheld for 8 d. A representative picture is shown. Control, without water withholding. (B) Measurement of stomatal aperture in wild-type and 35S:MIR169a plants. Data are mean ratios of width to length ± se of three independent experiments (n = 40 to 50). (C) Water loss from detached leaves of wild-type and 35S:MIR169a plants. Water loss is expressed as the percentage of initial fresh weight. Values are means from 10 leaves for each of four independent experiments. (D) Anthocyanin content in leaves of Arabidopsis with or without drought treatment for 8 d. Error bars represent se for four independent experiments. FW, fresh weight.

Figure 6.

nfya5 Mutant Plants Are More Sensitive to Drought Stress. (A) Schematic diagram of the T-DNA insertion site in the NFYA5 locus and detection of NFYA5 mRNA by RNA gel blot analysis. Exons are boxed, and lines between boxes represent introns. Twenty micrograms of total RNA from each sample was loaded and hybridized with ³²P-labeled full-length NFYA5 probe. The corresponding ethidium bromide–stained rRNA is shown as a loading control. (B) nfya5 mutant plants are more sensitive to drought stress. Wild-type (Col) and nfya5 plants were grown in soil with sufficient water for 3 weeks, and then the water was withheld for 8 d. A representative picture is shown. (C) Measurement of stomatal aperture in wild-type and nfya5 mutant plants. Data are mean ratios of width to length ± se of three independent experiments (n = 40 to 50). (D) Water loss from detached leaves of wild-type and nfya5 mutant plants. Water loss was expressed as the percentage of initial fresh weight. Values are means from 10 leaves for each of four independent experiments. (E) Anthocyanin content in leaves of Arabidopsis with or without drought treatment for 8 d. Error bars represent se for four independent experiments.

Figure 7.

Improved Drought Resistance in 35S:NFYA5 Plants. (A) Detection of NFYA5 mRNA in 35S:NFYA5 transgenic Arabidopsis. Real-time RT-PCR quantifications were normalized to the expression of Tub8. Error bars represent se (n = 3). (B) Drought resistance of 35S:NFYA5 plants (lines 2, 3, and 5). Wild-type and 35S:NFYA5 Arabidopsis plants were grown in soil with sufficient water for 3 weeks, and then the water was withheld for 14 d. A representative picture is shown. (C) Measurement of stomatal aperture in wild-type and 35S:NFYA5-3 transgenic plants. Data are mean ratios of width to length ± se of three independent experiments (n = 40 to 50). (D) Water loss from detached leaves of wild-type and 35S:NFYA5-3 plants. Water loss was expressed as the percentage of initial fresh weight. Values are means from 10 leaves for each of four independent experiments. (F) Anthocyanin content in leaves of Arabidopsis with or without drought treatment for 14 d. Error bars represent se for four independent experiments.