Robust increase of leaf size by Arabidopsis thaliana GRF3-like transcription factors under different growth conditions

Abstract

An increase in crop yield is essential to reassure food security to meet the accelerating global demand. Several genetic modifications can increase organ size, which in turn might boost crop yield. Still, only in a few cases their performance has been evaluated under stress conditions. MicroRNA miR396 repress the expression of GROWTH-REGULATING FACTOR (GRF) genes that codes for transcription factors that promote organ growth. Here, we show that both Arabidopsis thaliana At-GRF2 and At-GRF3 genes resistant to miR396 activity (rGRF2 and rGRF3) increased organ size, but only rGRF3 can produce this effect without causing morphological defects. Furthermore, introduction of At-rGRF3 in Brassica oleracea can increase organ size, and when At-rGRF3 homologs from soybean and rice are introduced in Arabidopsis, leaf size is also increased. This suggests that regulation of GRF3 activity by miR396 is important for organ growth in a broad range of species. Plants harboring rGRF3 have larger leaves also under drought stress, a condition that stimulates miR396 accumulation. These plants also showed an increase in the resistance to virulent bacteria, suggesting that the size increment promoted by rGRF3 occurs without an obvious cost on plant defenses. Our findings indicate that rGRF3 can increase plant organ size under both normal and stress conditions and is a valuable tool for biotechnological applications.

Figure 1

Broad control of GRF transcrition factors by miRNA miR396. (a) Phylogenetic tree using the full-length amino acid sequences of the Arabidopsis GRFs constructed by the neighbor joining method. Bootstrap support greater than 50% are indicated on nodes. (b) Scheme representing the exon-intron structure, the localization of the WRC and QLQ protein domains and the miR396-binding site. (c) Scheme representing a typical GRF gene and a detailed view of the interaction of Arabidopsis GRFs with miR396. (d) Distribution of GRFs in representative plants species and the occurrence of miR396 regulation among them.

Figure 2

miR396 limits leaf size in Arabidopsis. (a) Scheme of the multi-miR396 target mimic (9X-MIM396) prepared against miR396. (b,c) Fully expanded leaf 1 size distribution in a population of Arabidopsis primary transgenic plants transformed with the empty vector or with the 9X-MIM396 construct. In panel b, each silhouette of leaf 1 belongs to an independent primary transgenic plant transformed with the indicated vector. In the scatterplots in panel c, each circle indicates the size of a single leaf, while the horizontal solid bar represents the sample median. Bar = 1 cm. (d) Expression levels of miR396 in control plants transformed with the empty vector and 9X-MIM396 #3 plants. The miR396 levels were estimated by small RNA blots and the abundance relative to control plants is indicated by numerals. The data shown are mean ± SEM of three biological replicates. A probe against U6 snRNA was used as a loading and blotting control. The blot shown in the figure is a representative pair of samples from control and 9X-MIM396 #3 plants. (e) Expression levels of GRF2 and GRF3 in control plants transformed with the empty vector and 9X-MIM396 #3 plants. The GRF levels were estimated by RT-qPCR and normalized to control plants. The data shown are mean ± SEM of three biological replicates. Asterisks indicate significant differences from the control plants as determined by Student’s t-test (P < 0.05). (f) Leaf 1 size in control (empty vector) and three independent T3 homozygous transgenic lines transformed with 9X-MIM396.

Figure 3

Superior capacity of rGRF3 compared to rGRF2 in increasing leaf size. (a,b) Fully expanded leaf 1 size distribution in a population of Arabidopsis primary transgenic plants transformed with the empty vector, rGRF2 or rGRF3. In the scatterplots in panel b, each circle indicates the size of a single leaf from an independent T1 transgenic plant, while the horizontal solid bar represents the sample median. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05). Bars = 1 cm. (c) 30-days old plants transformed with the empty vector, rGRF2 or rGRF3. Note the leaf shape changes induced by rGRF2 only (arrowhead), including long and twisted petioles with downward curled leaves. Bars = 1 cm. (d) Expression levels of At-GRF2 and At-GRF3 in homozygous T3 transgenic plants transformed with the empty vector or rGRF2 (rGRF2 #13) or rGRF3 (rGRF3 #1). The GRF levels were estimated by RT-qPCR and normalized to control plants (empty vector). The data shown are mean ± SEM of three biological replicates. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05). (e–g) Leaf 1 size (e), palisade cell size (f) and estimated palisade cell number (g) in selected rGRF2 #13 and rGRF3 #1plants. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05). (h) Fully expanded leaf 1 size distribution in a population of primary transgenic Arabidopsis plants transformed with rGRF2 or rGRF3 under the GRF3 promoter. In the scatterplots each circle indicates the size of a single leaf from an independent T1 transgenic plant, while the horizontal solid bar represents the sample median. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05). Bars = 1 cm.

Figure 4

rGRF3-like genes increase organ size in heterologous species. (a) Area of fully expanded leaf 1 of transgenic Arabidopsis plants expressing At-rGRF3 or selected orthologues from soybean (Gm-rGRF) or rice (Os-rGRF4). The data shown are mean ± SEM of 20 biological replicates. Asterisks indicate significant differences from plants transformed with the empty vector as determined by Student’s t-test (p < 0.05). (b) Leaf 1 of transgenic plants expressing At-rGRF3 or the corresponding orthologues from soybean (Gm-rGRF) or rice (Os-rGRF4). Bar = 1 cm. (c) Wild-type and transgenic Brassica oleracea plants expressing At-rGRF3. To the left, 4 week-old whole plants, in the middle, fully expanded leaf 3 and to the right, a paredermal view of palisade cells from fully expanded leaf 3. White bars = 5 cm; Black bars = 0.05 mm. (d) Size of leaf 3 of 4 week-old wt and rGRF3 transgenic B. oleracea plants. The data shown are mean ± SEM of 10 biological replicates. Asterisks indicate significant differences from the wt as determined by Student’s t-test (P < 0.05). (e) Expression of rGRF3 in B. oleracea transgenic plants as estimated by RT-qPCR. Data are mean ± SEM of 3 biological replicates. (f,g) Seed size of wt and rGRF3 #10 and #7 B. oleracea plants. The data shown in f are mean ± SEM of 30 biological replicates. Asterisks indicate significant differences from the wt control plants as determined by Student’s t-test (P < 0.05). Bar = 2 mm. (h,i) Root architecture (h) and root growth (i) of B. oleracea plants expressing rGRF3. The data shown are mean ± SEM of 6 biological replicates. Asterisks indicate significant differences from wt control plants as determined by Student’s t-test (P < 0.05). Bars = 1 cm.

Figure 5

Response of the miR396-GRF system to drought stress. (a) Decrease in rosette area after a mild drought stress in 19-day-olds control plants transformed with the empty vector. Bar = 1 cm. (b) Premature induction of the MIR396B gene during a mild drought stress. A MIR396B:GUS reporter was used to monitor miR396b expression under normal and drought conditions. Staining was performed in 11 days old plants. Bars = 0.5 mm. (c) Size of the GUS-stained (blue) and non-stained regions (white) measured along the length of the leaves under normal and drought conditions. GUS staining was measured in stained leaves as those of panel b in a defined area along the leaf length. This area is depicted by a yellow box in panel b. The data shown are mean ± SEM of 20 leaves. (d) MiR396 induction after mild drought treatment in developing leaves. miR396 accumulation in total RNA extracted from leaves as those of panel b was estimated by small RNA blots. The abundance relative to control plants is indicated by numerals. The data shown are mean ± SEM of two biological replicates. A probe against U6 snRNA was used as a loading and blotting control. The blot shown in the figure is a representative pair of samples from plants grown in control or drought conditions. (e) Repression of At-GRF3 expression in plants grown under mild drought stress. The chart indicates the At-GRF3 expression levels in plants grown under control and mild drought conditions. The data shown, normalized to the expression value under control conditions, are mean ± SEM of 3 biological replicates. Asterisks indicate significant differences from control plants as determined by Student’s t- test (P < 0.05). (f) Dynamics of leaf 1 growth in control and drought conditions of control (empty vector) or 35S:miR396 #2 plants. Leaf size was monitored from 7 to 19 days after sowing under control and mild drought in the WIWAM platform. The data shown are mean ± SEM of 10 biological replicates.

Figure 6

rGRF3 ameliorates the effect of drought on leaf size. (a) 19 DAS rosettes of control (empty vector) and rGRF3 plants grown under control and drought conditions. Arrowheads indicate leaf 1. Bars = 1 cm. (b) Dynamics of leaf 1 growth from control and rGRF3 plants grown in control and drought conditions. Leaf size was monitored from 7 to 19 days after sowing under control (b) and mild drought (c) in the WIWAM platform. The data shown are mean ± SEM of 10 biological replicates.