The Arabidopsis LAZY1 Family Plays a Key Role in Gravity Signaling within Statocytes and in Branch Angle Control of Roots and Shoots

Abstract

During gravitropism, the directional signal of gravity is perceived by gravity-sensing cells called statocytes, leading to asymmetric distribution of auxin in the responding organs. To identify the genes involved in gravity signaling in statocytes, we performed transcriptome analyses of statocyte-deficient Arabidopsis thaliana mutants and found two candidates from the LAZY1 family, AtLAZY1/LAZY1-LIKE1 (LZY1) and AtDRO3/AtNGR1/LZY2 We showed that LZY1, LZY2, and a paralog AtDRO1/AtNGR2/LZY3 are redundantly involved in gravitropism of the inflorescence stem, hypocotyl, and root. Mutations of LZY genes affected early processes in gravity signal transduction without affecting amyloplast sedimentation. Statocyte-specific expression of LZY genes rescued the mutant phenotype, suggesting that LZY genes mediate gravity signaling in statocytes downstream of amyloplast displacement, leading to the generation of asymmetric auxin distribution in gravity-responding organs. We also found that lzy mutations reversed the growth angle of lateral branches and roots. Moreover, expression of the conserved C-terminal region of LZY proteins also reversed the growth direction of primary roots in the lzy mutant background. In lateral root tips of lzy multiple mutants, asymmetric distribution of PIN3 and auxin response were reversed, suggesting that LZY genes regulate the direction of polar auxin transport in response to gravity through the control of asymmetric PIN3 expression in the root cap columella.

Figure 1.

Arabidopsis LZY Genes Function in Shoot Gravitropism. (A) Transcript levels of LZY1 (left), LZY2 (middle), and LZY3 (right) in inflorescence stems of wild-type Col (black), eal1/shr (gray), and sgr1/scr (blue). Data represent relative values where the mRNA level of each gene in Col was set as 1. Data show mean ± sd of three technical replicates for three independent samples. Asterisks indicate significant differences by Student’s t test compared with the wild type (*P value < 0.01). (B) Expression pattern of the LZY genes. GUS staining of plants expressing LZY1p:GUS (left), LZY2p:GUS (middle), and LZY3p:GUS (right) in inflorescence stems (top) and etiolated hypocotyls (bottom). (C) Aerial parts of 5-week-old plants. (D) Time course of the gravitropic response (mean ± sd) of inflorescence stems after being placed horizontally (n ≥ 12 for each genotype). For statistical analysis, the Tukey-Kramer method was used to compare curvature measurements after 90 (*) and 480 min (**) of horizontal placement. There were significant differences in lzy1, lzy1 lzy2, lzy1 lzy3, and lzy1 lzy2 lzy3 after 90 min compared with Col (P value < 0.05), in lzy1 lzy2 and lzy1 lzy2 lzy3 after 480 min compared with Col (P value < 0.05), between lzy1 and lzy1 lzy2, lzy1 and lzy1 lzy2 lzy3, lzy1 lzy2 and lzy1 lzy3, and lzy1 lzy3 and lzy1 lzy2 lzy3 after 90 min (P value < 0.05), and between lzy1 and lzy1 lzy2, lzy1 and lzy1 lzy2 lzy3, lzy1 lzy2 and lzy1 lzy3, lzy1 lzy2 and lzy1 lzy2 lzy3, and lzy1 lzy3 and lzy1 lzy2 lzy3 after 480 min (P value < 0.05). (E) Growth direction of 3-d-old etiolated hypocotyls, at intervals of 20°. The number of individuals examined for each line is shown within the corresponding circles. Arrow marked with “g” represents the direction of gravity. For statistical analysis, means of the absolute value of the angle between growth direction and horizontal axis were compared. Means not sharing the same symbol (*, †) are significantly different (Tukey-Kramer, P < 0.05). Bars = 100 μm in (B) and 1 cm in (C).

Figure 2.

LZY Genes Regulate Shoot Gravitropism in Statocytes. (A) Time course of the gravitropic response (mean ± sd) of inflorescence stems of the two independent transgenic plants expressing SCRp:LZY2 in lzy1 lzy2 lzy3 background. For statistical analysis, the Tukey method was used to compare curvature measurements after 90 (*) and 480 min (**) of horizontal placement. There were significant differences in SCRp:LZY2 expressing lzy1 lzy2 lzy3_#2 and SCRp:LZY2 expressing lzy1 lzy2 lzy3_#3 compared with lzy1 lzy2 lzy3 (P value < 0.05), but no significant differences compared with Col (P value < 0.05). (B) Aerial parts of 5-week-old plants expressing SCRp:LZY2 in lzy1 lzy2 lzy3 background. (C) Growth direction of 3-d-old etiolated hypocotyls expressing SCRp:LZY1, SCRp:LZY2, and SCRp:LZY3 in the lzy1 lzy2 lzy3 background at intervals of 20°. For statistical analysis, means of the absolute value of the angle between growth direction and horizontal axis were compared. Means not sharing the same symbol (*, †) are significantly different (Tukey-Kramer, P < 0.05). (D) Amyloplasts in the endodermal cells of wild-type and lzy1 lzy2 lzy3 inflorescence stems before (0 min) and after reorientation (5 min). Arrowheads indicate amyloplasts. (E) Relative expression of IAA5 in the upper and lower flanks of wild-type (blue, left panel) and lzy1 lzy2 lzy3 (purple, right panel) inflorescence stems after reorientation. Expression levels were normalized against expression of the actin gene ACT8. Values and bars represent means ± sd of three biological replicates, and differences between the means were assessed for statistical significance using a Student t test (*P < 0.01). Arrows marked with “g” represent the direction of gravity. Bars = 1 cm in (B) and 10 μm in (D).

Figure 3.

LZY Genes Function in Root Gravitropism. (A) Expression patterns of the LZY gene family in roots. GUS staining of roots expressing LZY1p:GUS (left), LZY2p:GUS (middle), and LZY3p:GUS (right). (B) Root gravitropism of 5-d-old seedlings. Root angles were measured at 12 h after a 90° reorientation. The number of individuals examined for each Arabidopsis line is shown within the corresponding circles. Means not sharing the same symbol (*, †) are significantly different (Tukey-Kramer, P < 0.05). (C) Root gravitropism of 5-d-old seedlings expressing ADF9p:LZY1, ADF9p:LZY2, and ADF9p:LZY3 in lzy1 lzy2 lzy3 background. Root angles were measured at 12 h after a 90° reorientation. Means not sharing the same symbol (*, †) are significantly different (Tukey-Kramer, P < 0.05). (D) DR5rev:GFP expression in wild-type and lzy1 lzy2 lzy3 roots before and after 6 h of reorientation. Arrowheads indicate the asymmetric GFP signals. Asymmetric GFP expression was found in the wild type (7 out of 7), whereas symmetric expression was detected in lzy1 lzy2 lzy3 (5 out of 6). Two independent replicates were performed. Arrows marked with g represent the direction of gravity. Bars = 100 μm.

Figure 4.

Important Role of the C-Terminal Region of LZY Proteins in Gravity Signaling. (A) The alignment of C-terminal 14-amino acid sequences of OsLAZY1, ZmLAZY1, and Arabidopsis LAZY1 family. (B) Root gravitropism of 5-d-old seedlings. Root angles were measured at 12 h after a 90° reorientation. Means not sharing the same symbol (*, †) are significantly different (Tukey-Kramer, P < 0.05). (C) Intracellular localization of coexpressed LZY3-mCherry and PIN1-GFP, transiently expressed LZY3ΔCCL-mCherry, and CCL-mCherry in the protoplast cells. (D) Effect of the CCL-mCherry driven by the LZY2 promoter on the direction of root gravitropism in lzy1 lzy2 lzy3. Twelve-day-old seedlings were grown vertically on MS plates. (E) Root gravitropism of 5-d-old seedlings. Root angles were measured at 12 h after a 90° reorientation. Means not sharing the same symbol (*, †, §) are significantly different (Tukey-Kramer, P < 0.05). (F) Amyloplasts in the columella cells of Col (left) and LZY3p:CCL-mCherry in lzy1 lzy2 lzy3 (right) before (0 min) and after reorientation (5 min). Arrowheads indicate amyloplasts. (G) DR5rev:GFP expression in wild-type and LZY2p:CCL-mCherry expressing lzy1 lzy2 lzy3 roots before and after 10 h of reorientation. White and magenta arrowheads indicate GFP signals in the lower side and upper side of lateral root cap, respectively. Wild-type primary roots displayed asymmetric GFP expression (17 out of 19), whereas lzy mutant primary roots showed additional GFP expression in the upper side (10 out of 15). Three independent biological replicates were performed. Each replicate includes data from more than three seedlings. Arrows marked with g represent the direction of gravity. Bars = 5 μm in (C) and (F), 1 cm in (D), and 100 μm in (G).

Figure 5.

LZY Genes Control the Growth Angle of Lateral Roots. (A) Two-week-old seedlings grown vertically on MS plates. (B) Lateral root tip angle of 12-d-old seedlings of each line to the direction of gravity. Statistical analysis was performed compared with Col (*P value < 0.01) and between lzy3 and lzy2 lzy3 (^†P value < 0.01) by Tukey-Kramer method. (C) Twelve-day-old seedlings of Col, lzy1 lzy2 lzy3, and ADF9p:LZY3 expressing lzy1 lzy2 lzy3. (D) Nine-day-old Col and lzy1 lzy2 lzy3 seedlings, rotated 180° at 4 d after germination. Orange arrows indicate the growth direction of lateral roots. (E) Scatterplots of length and growth angle of lateral roots of 9-d-old plants rotated 180° at 4 d after germination. The angle between the direction of gravity and growth direction of lateral root tip was measured. There was a significant difference in the correlation coefficient between Col and lzy1 lzy2 lzy3 (r_Col = −0.420, r_lzy = 0.200, P < 0.01). Black arrows marked with g represent the direction of gravity. Bars = 1 cm.

Figure 6.

LZY Genes Control the Direction of Auxin Transport from Lateral Root Tips. (A) to (F) DR5rev:GFP expression in lateral root tips of Col ([A] to [C]) and lzy1 lzy2 lzy3 ([D] to [F]) at stages 1 ([A] and [D]), 2 ([B] and [E]), and 3 ([C] and [F]) of lateral root development. Stages of lateral root growth were defined as described in Methods. White and magenta filled arrowheads indicate the GFP signals in the lower and upper sides of the lateral root cap, respectively. Arrowheads enclosed by white and magenta dotted lines indicate reduced GFP signals in the upper and lower sides of columella cells, respectively. Asymmetric GFP expression was found in wild-type lateral roots at the stage 2 (14 out of 15) at the stage 3 (15 out of 19) and lzy lateral roots at stage 2 (8 out of 11) and stage 3 (9 out of 11). Three independent biological replicates were performed. Each replicate includes data from more than two seedlings. (G) to (L) PIN3-GFP expression in lateral root tips of wild type ([G] to [I]) and lzy1 lzy2 lzy3 ([J] to [L]) at stages 1 ([G] and [J]), 2 ([H] and [K]), and 3 ([I] and [L]) of lateral root development. White and magenta filled arrowheads indicate asymmetric PIN3 localization in the lower and upper sides of columella cells, respectively. Arrowheads enclosed by white and magenta dotted lines indicate reduced signals in the upper and lower sides of columella cells, respectively. Asymmetric GFP expression was found in wild-type lateral roots at stage 2 (15 out of 22) and stage 3 (10 out of 12) and in lzy lateral roots at stage 2 (11 out of 21) and stage 3 (13 out of 26). Three independent biological replicates were performed. Each replicate includes data from more than three seedlings. Arrows marked with “g” represent the direction of gravity. Red asterisks represent central S2 columella cells. The classification of lateral root development was based on development of columella cells (see Methods). Bars = 20 μm.