Overexpression of OsTF1L, a rice HD-Zip transcription factor, promotes lignin biosynthesis and stomatal closure that improves drought tolerance

Abstract

Drought stress seriously impacts on plant development and productivity. Improvement of drought tolerance without yield penalty is a great challenge in crop biotechnology. Here, we report that the rice (Oryza sativa) homeodomain-leucine zipper transcription factor gene, OsTF1L (Oryza sativa transcription factor 1-like), is a key regulator of drought tolerance mechanisms. Overexpression of the OsTF1L in rice significantly increased drought tolerance at the vegetative stages of growth and promoted both effective photosynthesis and a reduction in the water loss rate under drought conditions. Importantly, the OsTF1L overexpressing plants showed a higher drought tolerance at the reproductive stage of growth with a higher grain yield than nontransgenic controls under field-drought conditions. Genomewide analysis of OsTF1L overexpression plants revealed up-regulation of drought-inducible, stomatal movement and lignin biosynthetic genes. Overexpression of OsTF1L promoted accumulation of lignin in shoots, whereas the RNAi lines showed opposite patterns of lignin accumulation. OsTF1L is mainly expressed in outer cell layers including the epidermis, and the vasculature of the shoots, which coincides with areas of lignification. In addition, OsTF1L overexpression enhances stomatal closure under drought conditions resulted in drought tolerance. More importantly, OsTF1L directly bound to the promoters of lignin biosynthesis and drought-related genes involving poxN/PRX38, Nodulin protein, DHHC4, CASPL5B1 and AAA-type ATPase. Collectively, our results provide a new insight into the role of OsTF1L in enhancing drought tolerance through lignin biosynthesis and stomatal closure in rice.

Keywords: Oryza sativa; OsTF1L; HD-Zip transcription factor; drought tolerance; lignin biosynthesis; stomatal closure.

Figure 1

Expression patterns and subcellular localization of OsTF1L. (a) Phylogenetic tree created using the neighbour‐joining method in CLC sequence viewer using full‐length amino acid sequences of the rice HD‐ZIP IV proteins. Bootstrap support (100 repetitions) is shown for each node. (b, c) Quantitative RT‐PCR of OsTF1L in various tissues and at different growth stages. DAG, day after germination; L, leaf; R, root; N, node; S, sheath; F, flower. Ubi1 (rice ubiquitin1) expression was used as an internal control. Data bars represent the mean ± SD of two biological replicates, each of which had three technical replicates. (d, e) In situ hybridization analysis of OsTF1L expression. Cross sections of shoot apex from 6‐day‐old seedling were probed with digoxigenin‐labelled antisense or sense strand of OsTF1L RNA. Scale bars, 100 μm. (f) Confocal images of OsTF1L‐GFP in rice protoplasts with DAPI staining. Scale bars, 5 μm.

Figure 2

qRT‐PCR analysis showing the transcript levels of drought‐inducible genes (a), stomatal movement related genes (b) and lignin biosynthetic genes (c) in 1‐month‐old OsTF1 L ^OX and OsTF1 L ^{RNA i} shoots. Ubi1 was used as the reference gene. Data are shown as the mean ± SD of three biological and two technical replicates. (d) qRT‐PCR analysis of OsTF1L expression in 5‐week‐old OsTF1 L ^OX (#17, 23, 24), OsTF1 L ^{RNA i} (#2, 12, 13) and NT plants. Ubi was used as an internal control. Data bars represent the mean ± SD of two biological replicates, each of which had three technical replicates. Asterisks indicate significant differences compared with NT (P < 0.05, one‐way ANOVA).

Figure 3

ChIP‐qPCR and transient protoplast assays for OsTF1L interacting with promoters of the selected genes in ChIP‐seq and RNA‐seq analysis. (a–j) Two‐week‐old OsTF1 L ^OX and NT shoots were used in the ChIP‐qPCR experiments with an anti‐myc antibody. (a–e) Promoter regions showing three PCR‐amplified regions (P1–P3). (f–j) ChIP‐qPCR data show each PCR amplification region of each gene. The relative enrichment was normalized with total input. Data are shown as the mean ± SD of three independent experiments. (k, l) Transient protoplast expression assay using a dual‐luciferase reporter system. (k) Schematic diagram of the effector, internal control and five reporter constructs. (l) Relative fLUC (fLUC/rLUC) activity in rice protoplasts. Data are shown as the mean ± SD of three independent experiments. Asterisks indicate significant differences compared with NT (P < 0.05, Student's t‐test).

Figure 4

OsTF1L overexpression in rice confers drought tolerance. (a) Phenotypes of OsTF1 L ^OX and OsTF1 L ^{RNA i} transgenic rice plants under drought stress at the vegetative stage. Three independent homozygous OsTF1 L ^OX and OsTF1 L ^{RNA i} T₃ lines and nontransgenic (NT) control plants were grown in soil for 5 weeks and exposed to drought for 3 days, followed by re‐watering. (b) Chlorophyll fluorescence (F _v /F _m) of OsTF1 L ^OX and OsTF1 L ^{RNA i} transgenic rice plants and nontransgenic (NT) plants during a 12‐day drought treatment. F _v /F _m values were measured in the dark to ensure sufficient dark adaptation. Data are shown as the mean ± SD (n = 30). (c) Performance index (PI _total) of OsTF1 L ^OX and OsTF1 L ^{RNA i} transgenic plants and NT plants during drought conditions for 12 days. Data are shown as the mean ± SD (n = 30). (d) Agronomic traits of OsTF1 L ^OX and OsTF1 L ^{RNA i} transgenic rice plants grown in field under both normal and drought conditions. Spider plots of yield parameters with three independent homozygous T₄ lines of OsTF1 L ^OX and two independent homozygous T₃ lines of OsTF1 L ^{RNA i} under normal and drought conditions, respectively. Each data point shows a percentage of the means values (n = 30) listed in Table 2. Mean values from NT controls were set at 100% as a reference. CL, culm length; PL, panicle length; NP, number of panicles per hill; NTS, number of total spikelets; NSP, number of spikelets per panicle; NFG, number of filled grains; FR, filling rate; TGW; total grain weight; 1000 GW, 1000 grain weight.

Figure 5

Overexpression of OsTF1L Increasing Stomatal Closure. (a) Scanning electron microscopy images (600×) of the abaxial and adaxial leaf epidermis, where stomata are marked by black arrowheads. Scale bar represents 100 μm. (b) Average stomata numbers per square millimetre calculated from 10 plants for each line. Values are mean ± SD. (c) Comparison of the stomata length between NT and transgenic plants. Twenty stomata were measured for each plant. Ten plants were used for each line. Values are mean ± SD. (d) Scanning electron microscopy images (2000×) of two levels of stomata opening. Scale bar represents 10 μm. (e) The percentage of two levels of stomatal apertures in the leaves of OsTF1 L ^OX , OsTF1 L ^{RNA i} and NT plants under normal and drought stress conditions. Twenty stomata were measured for each plant. Ten plants were used for each line. Values are mean ± SD. Asterisks indicate significant differences compared with NT (P < 0.05, one‐way ANOVA). (f) Relative water content of detached leaves. For each replicate, fully expanded ten leaves of 3‐month‐old mature plants were used for each line. Data are shown as the mean ± SD of three independent lines and two experimental replicates. Asterisks indicate significant differences compared with NT (P < 0.05, one‐way ANOVA).

Figure 6

Accumulation of lignin in OsTF1 L ^OX , OsTF1 L ^{RNA i} transgenic rice and control plants. (a) Lignin contents in 2‐month‐old OsTF1 L ^OX , OsTF1 L ^{RNA i} and nontransgenic (NT) rice plants. Data bars represent the mean ± SD of three biological replicates (n = 3), each of which had two technical replicates. Asterisks indicate significant differences compared with NT (P < 0.05, one‐way ANOVA). (b–g) Transverse hand sections of phloroglucinol‐HCl stained 3‐month‐old mature shoots of NT (left), OsTF1 L ^OX (middle) and OsTF1 L ^{RNA i} (right) plants. (e–g) are enlarged views of the dotted line boxes in (b), (c) and (d), respectively. ls, leaf sheath; is, inflorescence stem; p, pith; va, vascular bundles; ep, epidermis; sc, sclerenchyma tissues.