Constitutive Expression of Rice MicroRNA528 Alters Plant Development and Enhances Tolerance to Salinity Stress and Nitrogen Starvation in Creeping Bentgrass

Abstract

MicroRNA528 (miR528) is a conserved monocot-specific small RNA that has the potential of mediating multiple stress responses. So far, however, experimental functional studies of miR528 are lacking. Here, we report that overexpression of a rice (Oryza sativa) miR528 (Osa-miR528) in transgenic creeping bentgrass (Agrostis stolonifera) alters plant development and improves plant salt stress and nitrogen (N) deficiency tolerance. Morphologically, miR528-overexpressing transgenic plants display shortened internodes, increased tiller number, and upright growth. Improved salt stress resistance is associated with increased water retention, cell membrane integrity, chlorophyll content, capacity for maintaining potassium homeostasis, CATALASE activity, and reduced ASCORBIC ACID OXIDASE (AAO) activity; while enhanced tolerance to N deficiency is associated with increased biomass, total N accumulation and chlorophyll synthesis, nitrite reductase activity, and reduced AAO activity. In addition, AsAAO and COPPER ION BINDING PROTEIN1 are identified as two putative targets of miR528 in creeping bentgrass. Both of them respond to salinity and N starvation and are significantly down-regulated in miR528-overexpressing transgenics. Our data establish a key role that miR528 plays in modulating plant growth and development and in the plant response to salinity and N deficiency and indicate the potential of manipulating miR528 in improving plant abiotic stress resistance.

Figure 1.

Stem-loop RT-qPCR analyses of miR528 expression profiles in response to salt, drought, and N deficiency in wild-type creeping bentgrass leaves. Relative expression levels of the mature miR528 were determined in wild-type plants under salt (A), drought (B), and N deficiency (C) treatment. The relative changes of gene expression were calculated based on the comparative threshold cycle method (Livak and Schmittgen, 2001). UBIQUITIN5 (AsUBQ5) was used as an endogenous control. Data are presented as means of three biological replicates × three technical replicates, and error bars represent se. Asterisks indicate a significant difference of expression levels between untreated and each abiotic-stress treated wild-type plant: **, P < 0.01 and ***, P < 0.001 by Student’s t test.

Figure 2.

Generation and molecular analysis of transgenic creeping bentgrass overexpressing Osa-miR528. A, Schematic diagram of the Osa-miR528 gene overexpression construct, p35S-Osa-miR528/p35S-Hyg. Osa-miR528 is under the control of the Cauliflower mosaic virus (CaMV) 35S promoter and linked to the hygromycin resistance gene, Hyg, driven by the CaMV 35S promoter. LB, Left border; NOS, nopaline synthase terminator; RB, right border. B, PCR analysis to amplify the Hyg gene using genomic DNA of wild-type (WT) and transgenic creeping bentgrass to determine the integration of Osa-miR528 in the host genome. C, Pri-miR528 shows alternative splicing in wild-type rice and transgenic creeping bentgrass. The same splicing pattern of pri-miR528 was observed in both rice and creeping bentgrass. Blue, red, and purple lines represent different splicing patterns. Yellow lines are the locations of pre-miR528. Black arrows indicate the position of mature miR528. D, An additional pair of primers was designed to amplify a common region of the three alternatively spliced pri-miR528 transcripts using semiquantitative RT-PCR analysis to compare the expression levels of primary Osa-miR528 in wild-type and transgenic plants. E, Stem-loop RT-qPCR analysis to detect the expression of mature Osa-miR528 in transgenic and wild-type plants. Relative changes in gene expression were calculated based on the comparative threshold cycle method (Livak and Schmittgen, 2001). ACTIN1 (AsACT1) was used as an endogenous control. Data are presented as means of three technical replicates, and error bars represent se. Asterisks indicate a significant difference of expression levels between the wild type and each transgenic line: **, P < 0.01 and ***, P < 0.001 by Student’s t test.

Figure 3.

Plant tillering and development. A, Ten-week-old wild-type (WT) and transgenic (TG) plants initiated from a single tiller. Bar = 10 cm. B, Two-month-old wild-type and transgenic plants initiated from the same number of tillers were grown in the same 6-inch pot. Bar = 10 cm. C, Closeup of the longest tillers from wild-type and transgenic plants, respectively. Bar = 5 cm. D, All internodes from the representative longest tiller were sliced from top to bottom and arranged from left to right. Bar = 5 cm. E, Top three fully developed leaves from the representative tillers of wild-type and transgenic plants. Bar = 2 cm. F, Cross-section images of wild-type and transgenic leaves. Bar = 200 μm. G, Cross-section images of wild-type and transgenic stems. Bar = 100 μm. H, Statistical analysis of leaf thickness between representative wild-type and transgenic plants (n = 8). I, Statistical analysis of the number of vascular bundles between representative wild-type and transgenic stems (n = 8). J, Tiller number in wild-type and transgenic plants 5 and 10 weeks after initiation from a single tiller (n = 5). K, Total shoot number including both tillers and lateral shoots in wild-type and transgenic plants at 30, 60, and 90 d after initiation from a single tiller (n = 5). L, Average length of the top eight internodes from wild-type and transgenic tillers (n = 6). Data are presented as means, and error bars represent se. Asterisks indicate a significant difference between the wild type and each transgenic line: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t test. Tillers are grass shoots growing out from the crown at the base of the plants. Shoots include both tillers and lateral shoots growing out from the tillers.

Figure 4.

Responses of wild-type controls and transgenics to salinity treatment. A, Wild-type controls (WT) and two transgenic (TG) lines initiated from the same number of tillers were trimmed to the same height before the salt stress test. B, Fully developed wild-type and transgenic plants were subjected to 200 mm NaCl treatment. The performance of wild-type and transgenic plants at 8 d after recovery from a 9-d salt treatment is shown. C, Closeup of representative wild-type and transgenic plants from B. D, Electrolyte leakage values were calculated before and after a 9-d salt treatment. E, Relative water contents were measured before and after a 9-d salt treatment. F, Pro contents of wild-type and transgenic leaves measured before and after a 200 mm NaCl treatment. G to I, Chlorophyll a content (G), chlorophyll b content (H), and total chlorophyll content (I) were measured before and after a 9-d salt treatment. fw, Fresh weight. Data are presented as means (n = 5), and error bars represent se. Asterisks indicate a significant difference between the wild type and each transgenic plant: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t test.

Figure 5.

Na⁺ and K⁺ contents in wild-type and transgenic plants under normal and salt stress conditions. A, Na⁺ relative contents in shoot and root tissues of wild-type (WT) and transgenic (TG) plants before salinity treatment. B, Na⁺ relative contents in shoot and root tissues of wild-type and transgenic plants 9 d after salinity treatment. C, K⁺ relative contents in shoot and root tissues of wild-type and transgenic plants under normal growth conditions. D, K⁺ relative contents in shoot and root tissues of wild-type and transgenic plants 9 d after salinity treatment. E, K⁺:Na⁺ ratio in shoots and roots of wild-type and transgenic plants before a 200 mm NaCl treatment. F, K⁺:Na⁺ ratio in shoots and roots of wild-type and transgenic plants 9 d after salt treatment. G, Shoot K⁺ relative contents in wild-type and transgenic plants before and after salinity stress. H, Root K⁺ relative contents in wild-type and transgenic plants before and after salinity stress. Data are presented as means (n = 3), and error bars represent se. Asterisks indicate significant differences in K⁺ content, Na⁺ content, or K⁺:Na⁺ ratio between the wild type and each transgenic line: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t test.

Figure 6.

Expression levels of AsHAK5 in wild-type and transgenic plants by semiquantitative RT-PCR analysis. A, Expression levels of AsHAK5 in leaf tissues of wild-type (WT) and transgenic (TG) plants under normal growth conditions. B, Expression levels of AsHAK5 in root tissues of wild-type and transgenic plants under normal growth conditions. AsUBQ5 was used as the endogenous control.

Figure 7.

Responses of wild-type and transgenic plants under different concentrations of N solutions. A, Wild-type (WT) and transgenic (TG) plants were trimmed to be uniform before applying N solutions. B, Performance of wild-type controls and three transgenic lines on MS solutions containing 2, 10, or 40 mm N for 4 weeks. C and D, Closeup of wild-type and transgenic shoots under 2 mm (C) and 40 mm (D) N solution treatment for 4 weeks. E and F, Shoot fresh weight (E) and dry weight (F) of wild-type and transgenic plants after 4 weeks of growth with three different N solutions. G, Percentage of shoot total N content of wild-type and transgenic plants measured 4 weeks after applying different N solutions. H, Weight of shoot total N was measured 4 weeks after applying different N solutions. I to K, Plant chlorophyll contents, including chlorophyll a (I), chlorophyll b (J), and total chlorophyll (J), were measured 4 weeks after applying different concentrations of N solutions. fw, Fresh weight. Data are presented as means (n = 4), and error bars represent se. Asterisks indicate significant differences of biomass value, total N weight, or chlorophyll contents between wild-type and transgenic plants: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t test.

Figure 8.

AsNiR gene expression analysis and NiR enzyme assay in wild-type and transgenic plants. A, RT-qPCR analysis of AsNiR transcript levels in wild-type plants (WT) and three transgenic (TG) lines under 10 mm N conditions. AsACT1 was used as an endogenous control. Data are presented as means of three technical replicates and three biological replicates. B, NiR assay in wild-type controls and two transgenic lines before and 2 weeks after N starvation. Data are presented as means of three biological replicates. The error bars represent se. Asterisks indicate significant differences of expression levels or enzyme activities between wild-type and transgenic plants: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t test.

Figure 9.

Putative miR528 target identification in creeping bentgrass. A and B, Expression levels of AsAAO (A) and AsCBP1 (B) in wild-type plants (WT) and three transgenic (TG) lines examined via RT-qPCR. AsUBQ5 was used as an endogenous control. Data are presented as means of three technical replicates, and error bars represent se. Asterisks indicate a significant difference of expression levels between the wild type and each transgenic line: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t test. C, Comparison of miR528 target sites in the putative targets AAO and CBP1 between rice and creeping bentgrass. Asterisks indicate identical RNA sequences. D, Information about the orthologs of the two putative miR528 target genes in rice and Arabidopsis.

Figure 10.

Expression patterns of the two miR528 putative targets under salt and N deficiency conditions through real-time RT-PCR analysis. A, Expression profiles of AsAAO and AsCBP1 in wild-type leaf and root tissues under 200 mm NaCl treatment (0–6 h). B, Expression profiles of AsAAO and AsCBP1 in wild-type leaf and root tissues under N starvation (0 mm N) from 0 to 8 d. AsUBQ5 was used as an endogenous control. Data are presented as means of three technical replicates, and error bars represent se. Asterisks indicate significant differences of gene expression levels between untreated and stress-treated leaf or root tissues: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t test.

Figure 11.

Expression levels of the abiotic stress-related miRNAs and AsNAC60 in wild-type controls and Osa-miR528 transgenic lines. A to C, Expression levels of miR396 (A), miRl56 (B), and miR172 (C) in wild-type (WT) and transgenic (TG) plants revealed through stem-loop RT-qPCR analysis. D, Expression levels of AsNAC60 in the wild type and three transgenic lines through RT-qPCR analysis. Three technical replicates were used for the RT-qPCR analysis. AsUBQ5 was used as an endogenous control. The relative changes of gene expression were calculated based on the 2^−ΔΔCT method (Livak and Schmittgen, 2001). The error bars represent se (n = 3). Asterisks indicate a significant difference of expression levels between the wild-type control and each transgenic line: *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t test.

Figure 12.

Hypothetical model of the molecular mechanisms of miR528-mediated plant abiotic stress response in creeping bentgrass. miR528 is induced during salinity stress but down-regulated under N deficiency. miR528 mediates plant abiotic stress responses through directly repressing the expression of its targets AsAAO and AsCBP1, which regulate the oxidation homeostasis during abiotic stresses. In addition, miR528 positively regulates AsNAC60, AsHAK5, and AsNiR and the gene encoding the antioxidant enzyme CAT, which leads to the enhanced tolerance to salinity stress and N deficiency. Furthermore, expression levels of other stress-related miRNAs are negatively regulated by miR528, suggesting that different miRNAs form a regulatory network to coordinately integrate various signals in response to plant abiotic stress.