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

The LAZY1 family expressed in statocytes is likely to regulate the polar auxin transport in response to gravistimulation in gravitropism of roots and shoots and in GSA control of lateral roots.

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.

INTRODUCTION

Plants are able to sense the direction of gravity and alter the orientation of their growth accordingly. In general, primary shoots and roots of vascular plants exhibit negative and positive gravitropism, respectively (Knight, 1806). As described by the starch-statolith hypothesis (Haberlandt, 1900; Sack, 1991), the direction of gravity is perceived mainly by gravity-sensing cells, called statocytes, which detect the sedimentation of starch-accumulating high-density amyloplasts. The resulting gravitropic signal is converted to a biochemical signal within the statocytes (gravity signaling) and then transmitted to responding tissues (auxin transport), where the signal induces the differential growth of the lower and upper surfaces of gravity-responsive organs through the asymmetrical distribution of auxin, as described by the Cholodny-Went theory (Went, 1974; Hart, 1990). Recent molecular genetic studies have provided evidence that strongly supports these hypotheses. Starch accumulation in amyloplasts has been shown to be important for mobility in response to gravistimulation, and amyloplast sedimentation is influenced by large central vacuoles and actin cytoskeletons (Fitzelle and Kiss, 2001; Hashiguchi et al., 2013; Blancaflor, 2013). Moreover, many aspects of the molecular mechanisms for auxin transport, auxin signaling, and auxin response, which are crucial not only for tropisms but also for plant development and growth, have recently been characterized (Sato et al., 2015; Rakusová et al., 2015; Žádníková et al., 2015).

However, the molecular mechanism underlying the change in polar auxin transport following amyloplast sedimentation in statocytes, which bridges the gap between the two long-standing hypotheses, remains unknown (Morita, 2010). In Arabidopsis thaliana, endodermal cells sense gravity in the shoots, whereas columella cells sense gravity in the roots (Fukaki et al., 1998; Blancaflor et al., 1998), and the auxin efflux facilitator PIN3 and the membrane-associated DnaJ domain proteins ALTERED RESPONSE TO GRAVITY1 (ARG1) and ARG1-LIKE2 (ARL2) are reportedly involved in the gravity signaling in statocytes. More specifically, PIN3, which is expressed in both endodermal and columella cells, is thought to relocate to the lower side of statocytes upon reorientation, resulting in a subsequent redistribution of auxin in the responding organs (Harrison and Masson, 2008; Kleine-Vehn et al., 2010; Rakusová et al., 2011). This polar localization of PIN3 has been observed in the endodermal cells of hypocotyls and in the columella cells of roots but has not been reported in the endodermal cells of inflorescence stems. Meanwhile, ARG1 and ARL2, which are both expressed in statocytes, are involved in the gravitropism of hypocotyls and roots (Sedbrook et al., 1999; Boonsirichai et al., 2003), although ARG1 is not involved in gravitropism in inflorescence stems (Fukaki et al., 1997). ARG1 contributes to the polarization of PIN3 distribution in the columella cells of roots (Harrison and Masson, 2008), indicating that ARG1 is involved in gravity signaling by root statocytes.

In this study, we aimed to identify genes involved in the gravity signaling process of inflorescence stems, by performing transcriptome analyses of the endodermis-defective mutants shoot gravitropism1 (sgr1; also known as scarecrow [scr]) (Fukaki et al., 1996) and endodermal amyloplast less1 (eal1) (Fujihira et al., 2000). The sgr1/scr mutant possesses inflorescence stems that lack an endodermis and exhibit agravitropism (Fukaki et al., 1998), whereas eal1, a hypomorphic allele of SGR7/SHORT-ROOT (SHR), retains the ability to form an endodermis-like cell layer but is also agravitropic, indicating that the endodermis-like cells of eal1/shr were not functional statocytes (Morita et al., 2007). Since both SGR1/SCR and SGR7/SHR are transcription factors (Helariutta et al., 2000), we hypothesized that any genes downregulated in both sgr1/scr and eal1/shr would include genes that are expressed in the endodermis and involved in the gravitropism of stems. Among the differentially expressed genes, we focused on two members of the LAZY1 family in Arabidopsis (Yoshihara et al., 2013), namely, AtLAZY1/LAZY1-LIKE1 (LZY1) and AtDRO3/AtNGR1/LZY2. The LAZY1 family contains plant-specific genes with unknown molecular functions that are involved in gravitropism in rice (Oryza sativa), Arabidopsis, maize (Zea mays), and Medicago truncatula (Table 1; Yoshihara and Iino, 2007; Li et al., 2007; Yoshihara et al., 2013; Dong et al., 2013; Uga et al., 2013; Ge and Chen, 2016). Here, we show that LZY1, LZY2, and their paralog AtDRO1/AtNGR2/LZY3 are redundantly involved in both shoot and root gravitropism and that their corresponding proteins play a key role in controlling lateral auxin flow after the reorientation of statocytes. Our findings also demonstrate that the regulation of auxin flow by the three LZY proteins in statocytes influences plant architecture by controlling the growth angle of lateral shoots and lateral roots.

Table 1. Nomenclature of LAZY1 Family Genes.

Name	Gene ID	Previous Name	Reference
LAZY1	Os11g0490600	LAZY1 (LA1)	Yoshihara and Iino (2007); Li et al. (2007)
LZY1	At5g14090	AtLAZY1	Yoshihara et al. (2013)
LZY2	At1g17400	AtNGR1	Ge and Chen (2016)
		AtDRO3	Guseman et al. (2017)
LZY3	At1g72490	AtNGR2	Ge and Chen (2016)
		AtDRO1	Guseman et al. (2017)
LZY4	At1g19115	AtNGR3	Ge and Chen (2016)
		AtDRO2	Guseman et al. (2017)
LZY5	At3g24750	–	Yoshihara et al. (2013)
LZY6	At3g27025	–	Yoshihara et al. (2013)

RESULTS

Role of LZY1, LZY2, and LZY3 in Shoot Gravitropism

To identify genes involved in regulating gravity signaling in shoot statocytes in Arabidopsis, we performed DNA microarray analyses on inflorescence stems of wild-type, sgr1/scr, and eal1/shr mutant plants. Gene expression profiles were then compared among the wild type and mutants. The genes downregulated by more than 5- and 3-fold in the eal1/shr and sgr1/scr mutants were identified (Table 2). Among these genes were two members of the Arabidopsis LAZY1 family: AtLAZY1, which is known to be involved in shoot gravitropism, and At1g17400 (Yoshihara and Iino, 2007; Li et al., 2007; Yoshihara et al., 2013). Here, we refer to AtLAZY1 as LAZY1-LIKE1 (LZY1) and to At1g17400 as LZY2 (Table 1) and focused our subsequent analyses on them and on a paralog LZY3/At1g72490 that shares 66% amino acid sequence identity with LZY2. LZY2 and LZY3 have recently been reported as AtDRO3/AtNGR1 and AtDRO1/AtNGR2, respectively, and investigated for their roles in root gravitropism (Table 1; Ge and Chen, 2016; Guseman et al., 2017). We confirmed that the expression levels of LZY1, LZY2, and LZY3 were significantly reduced in both sgr1/scr and eal1/shr stems by qRT-PCR analyses (Figure 1A). We also evaluated the promoter activities of these LZY genes using GUS activity assays in wild-type plants harboring LZY1p:GUS, LZY2p:GUS, or LZY3p:GUS constructs (Figures 1B; Supplemental Figure 1). We found that LZY1, LZY2, and LZY3 exhibited different expression patterns that partially overlapped and that all three genes were active mainly in the endodermis of both inflorescence stems and hypocotyls.

Table 2. List of Genes Downregulated in Both eal1 and sgr1.

AGI	Annotation	eal1 vs. WT Fold Change (WT/eal1)	sgr1-1 vs. WT Fold Change (WT/sgr1-1)
At2g46640	Tiller angle control 1 (TAC1)	116.82	42.93
At5g14090	LZY1/AtLAZY1	12.93	5.61
At1g17400	LZY2/AtNGR1	11.10	8.05
At5g36925	Unknown protein	10.13	14.48
At4g14980	Cysteine/histidine-rich C1 domain family protein	9.86	6.18
At5g65080	MADS affecting flowering 5 (MAF5)	9.84	4.85
At4g34970	Actin depolymerizing factor 9 (ADF9)	9.30	9.76
At2g26400	Acireductone dioxygenese 3 (ARD3)	7.39	12.24
At5g24900	CYP714A2	7.27	7.85
At5g24910	CYP714A1	6.64	13.13
At1g17700	Prenylated rab acceptor 1.F1 (PRA1.F1)	6.51	5.61
At2g38380	Peroxidase superfamily protein	6.34	3.82
At5g36920	Unknown protein	6.18	9.66
At1g43800	Floral transition at the meristem 1 (FTM1)	5.15	7.02

AGI, gene identification number by the Arabidopsis Genome Initiative. The gene annotation was retrieved from TAIR (http://arabidopsis.org/index.jsp). WT, wild type.

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).

To ascertain the role of the LZY genes in shoot gravitropism of inflorescence stems and hypocotyls, we obtained T-DNA insertion lines (lzy1, GABI_591A12; lzy2, FLAG_199G07; lzy3, SAIL_723_H12) and crossed these to generate multiple mutant lines (Supplemental Figure 2). Although the inflorescence stems of lzy2, lzy3, and lzy2 lzy3 mutant plants did not show significant phenotypic changes in gravitropic responses, lzy1 mutant plants displayed a reduced gravitropic response, and their lateral branches tended to grow in a horizontal direction (Figures 1C and 1D; Yoshihara et al., 2013). The lzy2 mutation enhanced the lzy1 phenotype, but lzy3 did not (Figures 1C and 1D; Supplemental Figure 3A). In addition, lzy1 lzy2 lzy3 triple mutant plants displayed more severe defects in gravitropism than did lzy1 lzy2. Remarkably, the primary shoots of lzy1 lzy2 lzy3 triple mutant plants grew along the ground and showed almost complete loss of the gravitropic capacity for reorientation. In dark-grown hypocotyls, single and double mutants showed slight phenotypic changes in growth directions, indicating retention of gravitropic capability. However, triple mutants exhibited significantly reduced gravitropism (Figure 1E; Supplemental Figure 3B). The phenotypes of the single and multiple lzy mutants demonstrated that the LZY genes have redundant functions and different levels of contribution to shoot gravitropism (LZY1 > LZY2 > LZY3 in the stem; LZY1 ≈ LZY2 ≈ LZY3 in dark-grown hypocotyls). The gravitropic phenotype of the lzy1 lzy2 lzy3 triple mutant was rescued by introducing genomic fragments of LZY2 or LZY3, thus confirming their role in gravitropism (Supplemental Figure 4).

Since organ elongation is essential for tropic responses, we also measured the elongation of inflorescence stems, finding that even the lzy1 lzy2 lzy3 triple mutants elongated normally (Supplemental Figures 5A and 5B). In addition, lzy1 lzy2 lzy3 stems and hypocotyls exhibited positive phototropic responses to unilateral blue light, indicating that lzy1 lzy2 lzy3 shoots retained the capacity for asymmetric organ growth in response to a directional light stimulus (Supplemental Figures 5C and 5D). These observations suggest that these LZY genes are involved in processes that occur before organ elongation during tropic responses.

LZY1, LZY2, and LZY3 Genes Function in the Endodermis in Shoot Gravitropism

Our above analysis using the GUS reporter system to investigate the locations of LZY1, LZY2, and LZY3 expression suggested that the genes function in the shoot endodermis (Figures 1B; Supplemental Figure 1). To investigate whether endodermis-specific LZY expression could rescue the lzy1 lzy2 lzy3 phenotype, the LZY genes were expressed in lzy1 lzy2 lzy3 plants under the control of the SGR1/SCR promoter, which was previously reported to drive expression in the endodermis of both shoots and roots (Wysocka-Diller et al., 2000). We found that the gravitropic phenotype of lzy1 lzy2 lzy3 stems was fully rescued by SCRp:LZY2 and SCRp:LZY3 constructs (Figures 2A and 2B; Supplemental Figure 6A) but was only partially rescued by the SCRp:LZY1 construct (Supplemental Figure 6B), possibly owing to nonoptimal expression of LZY1 when under control of the SCR promoter. Each LZY driven by the SGR1/SCR promoter was also able to rescue the gravitropic phenotype of lzy1 lzy2 lzy3 hypocotyls (Figure 2C). These results indicate that LZY1, LZY2, and LZY3 all function in the statocytes during shoot gravitropism, which suggests that the three LZY genes share redundant molecular functions.

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).

To investigate the role of the LZY genes in statocytes, we first determined whether they influence statocyte development. This analysis showed that the endodermis on the inflorescence stems of lzy1 lzy2 lzy3 plants formed normally and that accumulation of starch in the hypocotyl endodermis was similar to that in the wild type (Supplemental Figure 7). In addition, we observed the relocation of amyloplasts upon reorientation in endodermal cells of the stems of lzy1 lzy2 lzy3 (Nakamura et al., 2015) and found that amyloplasts were sedimented in the direction of gravity at 5 min after reorientation in endodermal cells of both mutant and wild-type plants (Figure 2D). Quantitative comparison of amyloplast sedimentation during the first 5 min after reorientation showed that wild-type and lzy1 lzy2 lzy3 plants behaved similarly (Supplemental Figure 8A). This finding suggests that the LZY genes are likely to be involved in downstream processes of amyloplast-mediated gravity perception in shoot statocytes.

Accordingly, we investigated whether an asymmetric auxin signal could be generated in the inflorescence stems of lzy1 lzy2 lzy3 (Figure 2E), by monitoring IAA5 transcript levels (Taniguchi et al., 2014). A slight difference in IAA5 transcript levels was detected between the upper and lower sides of lzy1 lzy2 lzy3 inflorescence stems at 60 min after reorientation, whereas a >4-fold difference was observed in wild-type stems. Thus, LZY genes play a key role in the statocytes to generate asymmetric auxin distribution in inflorescence stems.

LZY1, LZY2, and LZY3 Genes Have the Same Molecular Function in Root Gravitropism

By analyzing LZY promoter activity, we found that LZY2 and LZY3 were expressed in columella cells, i.e., root statocytes, of both primary and lateral roots, whereas LZY1 was not (Figure 3A; Supplemental Figures 1G to 1L). Plants carrying the double mutation lzy2 lzy3 showed defective root gravitropism, whereas plants with a single or other double combination of lzy mutations showed normal gravitropism (Figure 3B; Supplemental Figure 9A). In addition, there was no significant difference in root gravitropism between lzy2 lzy3 and lzy1 lzy2 lzy3. Thus, LZY1 appears to have little or no role in root gravitropism; this conclusion is consistent with its lack of expression in root columella cells (Figure 3A). The gravitropic phenotype of lzy1 lzy2 lzy3 was rescued by introducing genomic fragments of LZY2 or LZY3, thus confirming their role in root gravitropism (Supplemental Figures 9B and 9C). These results suggest that LZY2 and LZY3 redundantly contribute to root gravitropism.

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.

The LZY2 promoter was active mainly in the columella cells, while LZY3 was active in the stele above the elongation zone as well as in the columella cells in roots. To investigate whether the LZY genes function in root statocytes, the genes were expressed in lzy1 lzy2 lzy3 plants under the control of ACTIN DEPOLYMERIZING FACTOR9 (ADF9), SCR, or SHR promoters. In roots, the promoter activity of ADF9, whose expression was severely decreased in eal1/shr and sgr1/scr (Table 2), was found in statocytes, shoot endodermis, and columella cells of both primary and lateral roots, without detectable expression in other tissues (Supplemental Figure 10). The SCR promoter has previously been shown to promote expression in the endodermis and the quiescent center, while the SHR promoter has been reported to drive expression in the stele of roots; neither promoter is active in the columella cells (Wysocka-Diller et al., 2000; Nakajima et al., 2001). LZY genes driven by the ADF9 promoter were able to rescue the gravitropic phenotype of lzy1 lzy2 lzy3 in roots (Figure 3C; Supplemental Figures 11A to 11C). By contrast, LZY2 and LZY3 driven by the SCR or the SHR promoter failed to rescue the gravitropic phenotype of lzy1 lzy2 lzy3 roots (Supplemental Figures 11D to 11G). These results demonstrate that LZY2 and LZY3 function in the statocytes of roots during gravitropism and that LZY1 has the same molecular function as LZY2 and LZY3 in root statocytes, although LZY1 is not expressed in root statocytes under natural conditions.

We investigated whether the LZY genes influenced the development of statocytes and found that the morphology and starch accumulation behavior of the root caps of both primary and lateral roots were indistinguishable between wild-type and lzy1 lzy2 lzy3 plants (Supplemental Figures 12A to 12H). In addition, amyloplast sedimentation in lzy1 lzy2 lzy3 plants was normal in both root cap columella cells and shoot endodermal cells (Supplemental Figure 12I), suggesting that the LZY genes act downstream of amyloplast-mediated gravity perception in root statocytes as in the shoots. Moreover, asymmetric expression of the auxin responsive marker DR5rev:GFP (Ottenschläger et al., 2003) did not occur in lzy1 lzy2 lzy3 roots at 6 h after reorientation, whereas GFP fluorescence was observed in the lower flank of wild-type roots (Figure 3D). Taken together, these results demonstrate that LZY1, LZY2, and LZY3 share redundant and ubiquitous molecular functions in statocytes of both roots and shoots despite their relatively low sequence similarity; the genes also play a key role in the production of asymmetric auxin distribution in roots and shoots.

We examined whether lzy mutations affected the behavior of PIN3 in the columella cells. In wild-type root columella cells, PIN3 is uniformly distributed but becomes polarized upon reorientation (Harrison and Masson, 2008; Kleine-Vehn et al., 2010). We observed a low level of polarization of PIN3-GFP after reorientation in wild-type columella cells, and we did not detect any significant difference in PIN3-GFP polarization between wild-type and lzy1 lzy2 lzy3 plants (Supplemental Figure 13).

Role of the LZY C-Terminal Region in Gravity Signaling

The lack of recognized functional domains or motifs in LZY family proteins makes it difficult to assign potential molecular functions to these proteins. Here, we investigated their potential functions by analysis of the well-conserved domain between LAZY1 family proteins including OsLA1, ZmLA1, and Arabidopsis LZY proteins. Because the C-terminal 14-amino acid sequence is well conserved among all LAZY1 family members despite relatively low sequence similarity of the overall proteins, we designated the domain as conserved C terminus in LAZY1 family proteins (CCL) (Figure 4A; Supplemental Figure 14). In addition, three Arabidopsis proteins (AtNGR3/At1g19115, At3g24750, and At3g27025) that have CCL domains at the C terminus were designated as LZY4, LZY5, and LZY6, respectively (Table 1). To investigate the role of the CCL in LZY2 and LZY3, genomic fragments of LZY2 and LZY3 lacking the CCL domain were individually expressed in the lzy1 lzy2 lzy3 triple mutant background. We found that the truncated LZY3 protein, for which transcript was detected, did not rescue the gravitropic phenotype of lzy1 lzy2 lzy3 roots, and nor did the truncated LZY2 (Figure 4B; Supplemental Figures 15 and 16A). This observation indicates that the CCL domain is important for the molecular function of both LZY2 and LZY3 in roots.

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).

Next, we investigated the subcellular localization of the LZY proteins. We first established transgenic lines of lzy1 lzy2 lzy3 harboring LZY2p:LZY2-mCherry or LZY3p:LZY3-mCherry, which partially and fully rescued the lzy1 lzy2 lzy3 phenotype, respectively (Supplemental Figures 16B and 16C) as expected of the respective levels of contribution of LZY2 or LZY3 to root gravitropism (Figure 3B). Our observations indicate that LZY2-mCherry and LZY3-mCherry fused proteins are functional in planta. However, mCherry fluorescence was not detectable in root columella cells, possibly because of low abundance or high turnover rate of proteins. Upon transient overexpression of LZY tagged with GFP in Arabidopsis protoplasts, we detected fluorescence mainly at the cell periphery (Supplemental Figure 17). The LZY1-GFP signal was also observed in the nucleus, as reported previously (Yoshihara et al., 2013). LZY3-mCherry signals colocalized with that of a plasma membrane protein, PIN1-GFP, at the cell periphery, indicating that LZY proteins are localized at the plasma membrane, and that only LZY1 is present in the nucleus (Figure 4C). To examine whether the CCL domain, which has relatively high hydrophobicity, plays a role in the subcellular localization of LZY3, we transformed protoplasts with LZY3ΔCCL-mCherry and examined expression of the transgene (Figure 4C). We found that LZY3ΔCCL-mCherry was localized at the plasma membrane, in a similar manner as LZY3-mCherry. By contrast, when CCL-mCherry was expressed in protoplasts and in planta (LZY2p:CCL-mCherry/lzy1 lzy2 lzy3), fluorescence was observed in the cytoplasm of protoplasts and columella cells, indicating that the CCL domain did not contribute to plasma membrane localization (Figure 4C; Supplemental Figure 18A). Overall, our observations indicate that the CCL domain is important for LZY function but not for LZY localization.

When CCL-mCherry was expressed in the lzy1 lzy2 lzy3 background, both primary and lateral roots of the transgenic seedlings grew upward (Figure 4D; Supplemental Figures 18B and 18C). Since the amino acid sequence of LZY2-CCL is identical to that of LZY3-CCL, the effect of CCL-mCherry can be considered to be equivalent between LZY2 and LZY3. When the seedlings were gravistimulated via horizontal orientation, their primary roots grew upward (Figure 4E; Supplemental Figure 18D). The perturbation effect of CCL-mCherry on root gravitropism was also observed in the wild-type background, although it was milder in effect than in the lzy1 lzy2 lzy3 background, suggesting that the effect of CCL-mCherry was influenced by the level of endogenous LZY gene expression (Supplemental Figure 18D). We also observed that amyloplasts were relocated in lzy1 lzy2 lzy3 columella cells expressing LZY3p:CCL-mCherry and found that amyloplasts sedimented normally in the direction of gravity (Figure 4F; Supplemental Figure 8B). These results confirm that mutations of the LZY genes did not affect amyloplast sedimentation and suggest that lzy1 lzy2 lzy3 roots expressing LZY3p:CCL-mCherry recognized the directional signal of gravity, but that the signal was perturbed and resulted in negative gravitropism. These findings also support our conclusion that LZY genes are involved in downstream processes in amyloplast-mediated gravity perception in root statocytes.

We tested whether the perturbed signal causes asymmetric auxin signaling derived from DR5rev:GFP in primary root tips of LZY2p:CCL-mCherry expressing lzy1 lzy2 lzy3 before and after reorientation. Since roots of LZY2p:CCL-mCherry/lzy1 lzy2 lzy3 grew upward, we investigated GFP fluorescence distribution before reorientation. The patterns of GFP signals were indistinguishable between wild-type and transgenic plants (Figure 4G). After reorientation, GFP fluorescence was observed asymmetrically at the lower flank in wild-type roots, whereas additional GFP fluorescence was observed at the upper flank of lzy1 lzy2 lzy3 roots expressing LZY2p:CCL-mCherry (Figure 4G). Given that LZY2p seems to impart columella-specific expression in roots (Figure 3A), it is likely that CCL-mCherry perturbed the gravity signaling in columella cells by interfering with the function of the LZY proteins, thus leading to the disorientation of auxin flow at the root tip.

Effect of lzy Mutations on the Growth Angles of Lateral Shoots and Roots

In addition to abnormal gravitropism in primary shoots and primary roots, we also observed that plants carrying lzy mutations exhibited abnormal growth angles in both lateral shoots and roots. The lateral shoots of lzy1 plants showed a larger growth angle (in almost a horizontal direction) as previously reported (Yoshihara et al., 2013), whereas those of the lzy2, lzy3, and lzy2 lzy3 mutants were similar to wild-type plants (Figure 1C); both lzy2 and lzy3 appeared to enhance the lzy1 phenotype, as found above for gravitropic response of inflorescence stems. We also found that the lateral roots of lzy3 plants exhibited larger growth angles than those of wild-type plants (Figure 5A). Quantitative analyses confirmed these observations and demonstrated that lzy2 enhanced the lzy3 phenotype leading to upward growth of lateral roots, although lzy2 plants were indistinguishable from the wild type (Figure 5B). The growth angle was not affected in lzy1 plants; this observation was consistent with our earlier observation that LZY1 was not expressed in roots (Figure 3A; Supplemental Figures 1G and 1H). More importantly, we also found that the growth angle phenotype of lzy1 lzy2 lzy3 lateral shoots and roots could be rescued by LZY genes expressed in the statocytes. Lateral branches of lzy1 lzy2 lzy3 plants grew downward (Figure 1C); SCRp:LZY2 almost completely rescued the growth angle phenotype in these plants (Figure 2B). With regard to lateral roots, the growth angle phenotype of lzy1 lzy2 lzy3 plants was rescued by columella-specific expression of LZY3 under the control of the ADF9 promoter (Figures 5B and 5C). These results suggest that LZY genes expressed in statocytes play an important role in controlling the growth angle of lateral shoots and roots.

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.

Decrease of LZY Activity Reverses Auxin Flow in Lateral Root Statocytes

Interestingly, the lateral roots of lzy2 lzy3 and lzy1 lzy2 lzy3 plants grew slightly upward (Figure 5B). To investigate this phenotype in detail, we measured the growth angles of lateral roots over 2 mm in length at various growth stages (Supplemental Figure 19A). The growth angles of wild-type lateral roots gradually decreased as the roots grew, and finally they grew almost vertically (Mullen and Hangarter, 2003). By contrast, lzy1 lzy2 lzy3 lateral roots scarcely grew below the horizontal level at any growth stage. To test whether the lateral roots of the triple mutant were capable of responding to reorientation, we measured the growth angles of lateral roots that emerged after turning young seedlings upside down (Figures 5D and 5E). Lateral roots from inverted lzy1 lzy2 lzy3 and wild-type seedlings grew upward and downward, respectively, indicating that the lateral roots of the triple mutant recognized the direction of gravity and then grew in the opposite direction.

We subsequently analyzed whether asymmetric auxin signaling was generated in the lateral roots of lzy1 lzy2 lzy3 growing the opposite direction. It has been reported that asymmetric distribution of auxin signaling by DR5rev:GFP is mostly observed in stage II lateral roots of wild-type plants (Rosquete et al., 2013). We examined whether the growth angle phenotype was present in young lateral roots of submillimeter lengths. Most of the lateral roots of lzy1 lzy2 lzy3 and wild-type plants grew above and below the horizontal level, respectively (Supplemental Figure 19B). Prior to the enlargement of the central columella cells (stage 1), the majority of wild-type and lzy1 lzy2 lzy3 lateral roots displayed symmetric DR5rev:GFP expression (Figures 6A and 6D). During and after the enlargement of the central S2 columella cells (stages 2 and 3), expression of DR5rev:GFP was observed in the lower side of wild-type lateral root caps as reported previously, and the GFP signal was decreased in the upper side of the columella cells (Figures 6B and 6C; Supplemental Figure 20; Rosquete et al., 2013). Interestingly, most lzy1 lzy2 lzy3 lateral roots exhibited significant DR5rev:GFP expression in the upper side of the lateral root cap cells and less GFP expression in the lower side of the columella cells (Figures 6E and 6F). It has been reported that PIN3, which is expressed in columella cells before PIN4 and PIN7 (Rosquete et al., 2013), is involved in the growth angle control. We analyzed the effects of lzy1 lzy2 lzy3 mutations on PIN3 localization in columella cells of lateral roots at early developmental stages. At stage 1, both wild-type and lzy1 lzy2 lzy3 plants showed symmetric PIN3-GFP distribution in columella cells of lateral roots (Figures 6G and 6J). At later stages (stages 2 and 3), PIN3-GFP signal intensity in the lower lateral columella cells in wild-type lateral roots was much higher than in the upper columella cells, although we were unable to observe polarized localization of PIN3-GFP within the columella cells (Figures 6H and 6I; Supplemental Figure 21). By contrast, the signal intensity in the upper lateral columella cells was higher than that of the lower cells in lzy1 lzy2 lzy3 lateral roots (Figures 6K and 6L; Supplemental Figure 21). These asymmetric distributions of PIN3-GFP were in agreement with the asymmetric patterns of DR5rev:GFP and the growth directions of lateral roots. These results demonstrate that LZY genes are required for the flow of auxin toward the direction of gravity in lateral root columella cells. Furthermore, our data also indicate that the decrease in LZY activity causes reversal of the auxin flow in the lateral root columella with recognition of the direction of gravity and that LZY activity affects PIN3 expression pattern in columella cells.

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.

DISCUSSION

Previous studies have reported that LAZY1 and its orthologs are involved in shoot gravitropism of rice (Yoshihara and Iino, 2007; Li et al., 2007), Arabidopsis (Yoshihara et al., 2013), and maize (Dong et al., 2013) and that other LAZY1 gene family members are involved in root gravitropism of rice (DRO1; Uga et al., 2013), M. truncatula (MtNGR), and Arabidopsis (AtLAZY1/LZY1, AtNGR1/LZY2, AtDRO1/AtNGR2/LZY3, and AtNGR3/LZY4; Ge and Chen, 2016; Table 1). In spite of their apparently well-conserved physiological function in gravitropism in various species, the similarities among the entire sequences of LAZY1 family proteins are low (e.g., sequence similarity between LZY1 and LZY2 or LZY3 was around 30%); the proteins share five short conserved regions though have no domain with an identified function (Yoshihara et al., 2013). In this study, we demonstrated that three LZY genes are redundant and share the same molecular function with regard to gravitropism in both shoots and roots (Figures 1 to 3).

In rice coleoptiles, LAZY1 is required for the formation of auxin gradients after reorientation, although it has been unclear in which cells LAZY1 is necessary (Yoshihara and Iino, 2007). We demonstrated in this study that three LZY genes function in the gravity signaling process inside the statocytes of both shoots and roots in Arabidopsis and that they function to positively regulate lateral auxin flow to the direction of gravity upon reorientation (Figures 2 and 3). The statocytes of roots, hypocotyls, and inflorescence stems have distinct developmental origins, and their cellular functions and morphologies are almost completely different, except for their shared role in gravity sensing. However, since amyloplast displacement in the statocytes is used in the gravity perception systems of all organs (Kiss et al., 1989; Weise and Kiss, 1999), it is reasonable that the statocytes of all gravity-responding organs share a common molecular mechanism for gravity signaling. Genes involved in the initial process of gravitropism across all organs have not been reported, except for PHOSPHOGLUCOMUTASE (PGM), which is required for starch accumulation in amyloplasts (Kiss, 2000). In contrast to PGM, LZY genes did not affect amyloplast sedimentation (Supplemental Figure 8). Our results demonstrate that LZY1, LZY2, and LZY3 genes are involved in early gravity signaling processes following amyloplast sedimentation in the statocytes of all organs and, more specifically, that the LZY genes induce auxin flow toward the direction of gravity. Thus, they likely function in processes bridging the gap between those described by the starch-statolith hypothesis and those in the Cholodny-Went theory.

The suggested function of the LZY genes in mediating auxin flow in statocytes in response to reorientation implies the involvement of these genes in the regulation of auxin transporters such as PIN3, which is expressed in statocytes of all organs and is involved in gravitropism in roots and hypocotyls (Friml et al., 2002). In columella cells of primary roots, uniformly distributed PIN3 becomes polarized upon reorientation (Harrison and Masson, 2008; Kleine-Vehn et al., 2010). In addition, polarized localization of PIN3 has been observed in columella cells of young lateral roots (Rosquete et al., 2013). In this study, we did not detect any significant difference in PIN3 polarization between wild-type and lzy1 lzy2 lzy3 primary roots (Supplemental Figure 13). This finding might explain the mild root gravitropism phenotype of lzy1 lzy2 lzy3 plants. As AtNGR3/LZY4 activity is thought to remain in lzy1 lzy2 lzy3 primary roots (Ge and Chen, 2016; Table 1), additional analyses using lzy1 lzy2 lzy3 lzy4 quadruple mutants will be necessary. On the other hand, polarized localization of PIN3-GFP was not detected in columella cells of wild-type and mutant lateral roots in this study. Rather, asymmetry of PIN3-GFP expression in the columella of lateral root was observed (Figure 6). The opposite asymmetric pattern of PIN3 distribution as a consequence of a decrease in LZY gene activity might be an underlying mechanism of the antigravitropic capability (see below). Further investigations are required to elucidate how LZY genes regulate auxin flow in statocytes in response to reorientation and the regulatory mechanisms for asymmetric distribution of PIN3 in the lateral root columella.

The CCL-mCherry construct was shown here to perturb root gravitropism (Figure 4). This effect might have resulted from an excess of the CCL moiety, compared with a low level of endogenous LZY family proteins; this explanation is supported by the observation that the effect of CCL-mCherry was consistently reduced in the presence of endogenous LZY genes (Supplemental Figure 16D). It is also possible that LZY proteins interact with other proteins in gravity signaling via the CCL domain and that CCL-mCherry may interfere with these protein-protein interactions in a competitive manner. Therefore, we speculate that a complete loss of function of the LZY gene family could cause root phenotypes similar to that of CCL-mCherry in the lzy1 lzy2 lzy3 background. A recent study demonstrated that primary roots of the triple mutant atngr1 atngr2 atngr3 (lzy2 lzy3 lzy4 in our nomenclature) grow upward in a similar fashion to that of lzy1 lzy2 lzy3 plants expressing CCL-mCherry (Ge and Chen, 2016), although it is unclear whether AtDRO2/AtNGR2/LZY4 is expressed in columella cells. Further investigations will be necessary to understand the perturbation mechanism of gravitropism by CCL-mCherry in both roots and shoots and will provide valuable information regarding the gravity signaling process in statocytes.

The findings of this study also demonstrate that LZY-mediated gravity signaling in statocytes regulates the growth angles of both lateral roots and shoots, which influence the architecture of the whole plant (Figures 1C, 2B, and 5). The growth angle of organs is maintained at specific angles with respect to gravity (gravitropic set-point angle [GSA]) according to developmental control and environmental factors, a concept that provides a unifying explanation for ortho-, plagio-, and diagravitropism (Digby and Firn, 1995). Recent investigations of growth angle control in the lateral roots and shoots of Arabidopsis suggested that an antagonistic interaction between two balancing auxin-dependent growth components, gravitropism and antigravitropic offset, underlies the mechanism of GSA control (Roychoudhry et al., 2013; Roychoudhry and Kepinski, 2015). Based on the concept of GSA control, the growth angle phenotype of lzy1 lzy2 lzy3 lateral roots and shoots might be the result of an imbalance between gravitropism and antigravitropic offset. This study demonstrated that LZY genes are positive regulators of auxin flow toward the direction of gravity according to amyloplast relocation. Simply thinking, the phenotype caused by the loss of function of such LZY genes would be expected to include loss of responsiveness to reorientation, as in pgm, pin2, or aux1 roots (Kiss et al., 1989; Müller et al., 1998; Chen et al., 1998; Luschnig et al., 1998; Bennett et al., 1996). However, decreased LZY gene activity caused a reversal of the auxin flow in the lateral root, with recognition of the direction of gravity (Figure 6). Downward growth of lateral shoots of lzy1 lzy2 lzy3 plants (Figure 1C) and upward growth of lateral roots of CCL-mCherry expressing lzy1 lzy2 lzy3 plants (Figures 4D and 4E) could be similarly caused by a decrease in LZY activity. Our observations suggest that lateral roots, and possibly lateral shoots, of wild-type plants contain an antigravitropic capability as well as LZY-dependent gravitropism. The antigravitropic capability became obvious following the loss of function of the LZY genes. This conclusion is partly consistent with a previous proposal (Roychoudhry et al., 2013). Primary roots of atngr1 atngr2 atngr3 (lzy2 lzy3 lzy4) and of CCL-mCherry expressing lzy1 lzy2 lzy3 plants also exhibited reverse gravitropism (Ge and Chen, 2016; Figure 4E, Table 1). It is possible that both lateral roots and primary roots have a balance between LZY family-dependent gravitropism and LZY family-independent antigravitropic capability. Thus, the LZY family proteins could play a key role in elucidating a unified mechanism for ortho-gravitropism of primary organs and GSA control of lateral branches via regulation of auxin flow according to amyloplast relocation in statocytes.

METHODS

Plant Materials and Growth Conditions

In this study, we used Arabidopsis thaliana Columbia-0 as the wild-type line, and the T-DNA insertion line lzy1 (GABI 591A12, previously reported as atlazy1) (Yoshihara et al., 2013) was obtained from GABI-Kat (Kleinboelting et al., 2012), whereas the lzy2 (FLAG_199G07, recently reported as atngr1) (Ge and Chen, 2016)) and lzy3 (SAIL_723_H11, recently reported as atngr2) (Ge and Chen, 2016) lines were obtained from the Arabidopsis Biological Resource Center (Samson et al., 2002; Alonso et al., 2003). All T-DNA lines were backcrossed with Col-0 at least three times, and for the Wassilewskija-derived lzy2 line, PCR markers (Toyota et al., 2011) were used to generate progeny with all Col-0-derived chromosomes, except for the bottom of chromosome 1.

Surface-sterilized seeds were sown on MS plates (1× Murashige Skoog salts, 1% [w/v] sucrose, 0.01% [w/v] myoinositol, 0.05% [w/v] MES, and 0.5% [w/v] gellan gum, pH 5.8), incubated in the dark at 4°C for 2 to 3 d, grown at 23°C in a growth chamber with 3350 lm fluorescent light tubes (Panasonic) under continuous light (∼3200 Lux) for 10 to 14 d, transplanted to soil, and grown under continuous light.

Microarray Analysis

Stem samples 1.5 cm long were excised 0.3 to 1.8 cm from apices of primary inflorescence stems grown to 5 to 8 cm, from which lateral organs were removed. At least 10 individuals were used for RNA sample preparations. Total RNA was extracted using the RNeasy Plant Mini kit (Qiagen). Total RNA (0.5 μg) was used for cDNA synthesis and the cRNA labeling reaction with an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Each cRNA was labeled cyanine-3 and cyanine-5 in separate reactions. These labeled RNAs were hybridized to Agilent Arabidopsis 3 Oligo Microarray (Agilent) and washed. Signals were detected with Agilent Technologies Microarray Scanner. Microarray analyses were performed with two or one independent samples for eal1 or sgr1, respectively. Comparison between the wild type and a mutant (eal1 or sgr1) was performed using a dye swap protocol for each sample.

Real-Time qRT-PCR

Total RNA samples from inflorescence stems were prepared for microarray analyses. cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO) from 0.5 μg total RNA. Real-time qPCR was performed using the LightCycler 480 real-time qPCR system (Roche Applied Science), and the KAPA SYBER FAST qPCR kit (Kapa Biosystems) was used for the preparation of real-time qPCR reaction mix. The results of real-time qPCR were analyzed with LightCycler 480 software (Roche Applied Science). ACT8 was used as an internal control. The primers used for real-time qRT-PCR were designed for a specific region of each gene (Universal ProbeLibrary Assay Design Center, Roche) and are summarized in Supplemental Data Set 1A.

Gravitropism Analyses

For the analysis of inflorescence stems, intact plants with primary stems of 4 to 8 cm were grown horizontally under nondirectional dim light at 23°C. For the analysis of hypocotyls, seeds were incubated on MS plates at 4°C for 3 d in the dark, under red fluorescent tubes (FL20S/R-F; National) at room temperature for 1 h to induce germination, and the seedlings were grown vertically at 23°C for 72 h in the dark. For the analysis of roots, the seedlings were vertically grown on MS plates for 5 d, transferred to new MS plates, incubated vertically at 23°C for 1 h under continuous light, and then rotated 90°, and incubated for an additional 12 to 24 h. Photographs were taken at indicated times, and the curvature of the stems and roots and the growth angle of the hypocotyls were measured from the digital images using Image J software (https://imagej.nih.gov/ij/).

GUS Staining

Tissues were fixed in 90% ice-cold acetone for 15 min and then incubated in GUS staining solution (100 mM sodium phosphate, pH 7.0, 10 mM EDTA, 10 mM ferricyanide, 10 mM ferrocyanide, 0.1% Triton X-100, and 2 mM 5-bromo4-chloro-3-indolyl-β-d-glucuronic acid) at 37°C. For whole-mount observation, samples were rinsed with 70% ethanol and cleared in chloral hydrate solution (8 g chloral hydrate, 1 mL glycerol, and 2 mL water). For thin sections, samples were dehydrated in an ethanol series, embedded in Technovit 7100 (Heraeus Kulzer), and sectioned with a microtome. Samples were observed under a light microscope (BX52; Olympus) equipped with a cooled-CCD camera (VB6010; Keyence).

Determination of IAA5 Expression Level by Real-Time qRT-PCR

We evaluated IAA5 expression levels in the upper and lower flanks of gravistimulated inflorescence stems (Taniguchi et al., 2014). Inflorescence stems 4 to 6 cm long were gravistimulated, followed by sampling of stem parts (0.5−3.5 cm from the top) for total RNA extraction with an RNeasy plant mini kit. cDNA was synthesized from 0.5 mg of total RNA treated by ReverTra Ace qPCR RT Master Mix with gDNA Remover according to the manufacturer’s instructions. The KAPA SYBER FAST qPCR kit was used for the preparation of real-time qPCR mix, and then real-time qPCR was performed using the LightCycler 96 real-time PCR system. Based on the results of three technical repeats for three biological replicates, mRNA relative expression levels (in arbitrary units) were determined using standard curves for IAA5 and ACT8 generated by serial dilutions of cDNA. The primers used for the analyses are listed in Supplemental Data Set 1D.

Analysis of Amyloplast Sedimentation

Amyloplast sedimentation after 90° reorientation was analyzed using a vertical stage microscope equipped with a rotatable stage as described by Nakamura et al. (2011). Stem samples were prepared as described previously (Nakamura et al., 2015). With regard to roots, 3- to 5-d-old seedlings grown vertically on an MS plate were placed on a glass slide with surrounding gel and covered with a cover slip. Growing roots were mounted on the vertical stage and preincubated in the vertical position for at least 10 min prior to reorientation. Imaging was performed with a silicon-immersion objective (UPLSAPO60XS, NA 1.30; Olympus). The stage was rotated 90° at time 0 and images were collected every 1 s for 5 min using a CCD camera (CoolSNAP; Nippon Roper). Sequential images were combined into a stack using Image J. Movement of plastids in central S2 columella cells was analyzed with G-Track spot-tracking software (G-Angstrom). Y values were measured to indicate vertical displacement of plastids. Y was the distance in micrometers of the position of the center of brightness of a plastid from its original position at any given time point (Toyota et al., 2013). Measurements were performed using one or two S2 cells from at least three individuals of each genotype.

Expression Analysis of DR5rev:GFP

Surface-sterilized seeds were sown and vernalized on a thin MS gel layer on a cover slip and incubated at 23°C under continuous light- and high-moisture conditions for 2 d. After the primary roots reached to the cover slip, the cover slips were kept vertical for 3 d to induce the roots to grow between the cover slip and MS gel in the direction of gravity. For reorientation, the cover slips were rotated 90° and incubated for 6 h. Confocal images of GFP fluorescence were obtained with FV1000 (Olympus). Seedlings used for observation before reorientation were distinct from those used after reorientation.

Transient Assay with Protoplasts

Plasmids and carrier DNA were introduced into protoplasts generated from Arabidopsis suspension culture (Takeuchi et al., 2000). Confocal images of GFP and mCherry fluorescence were obtained with FV1000.

Analysis of LR Growth Angle

For the growth angle analysis of young lateral roots, seedlings were grown vertically on MS plates for 4.5 d, transferred to new MS plates, and incubated vertically at 23°C for an additional 4.5 d under continuous light. Photographs were taken using an SMZ-U microscope (Nikon), and the angle between the direction of gravity and lateral root tip growth was measured using Image J software. For the analysis of mature lateral roots, the seedlings were grown vertically on MS plates for 12 d, and the angle between the direction of gravity and lateral root tip growth was measured using Image J. For the gravitropic response analysis of lateral roots, the seedlings were grown vertically on MS plates for 4 d, transferred to new MS plates, incubated vertically at 23°C for 0.5 d under continuous light, and then rotated 180° and incubated for additional 4.5 d. Photographs were taken, and the angle between the direction of gravity and lateral root tip growth was measured using Image J software.

Plasmid Construction

We used the Gateway cloning system (Invitrogen) to construct LZY1g, LZY2g, LZY3g, LZY1p:GUS, LZY2p:GUS, LZY3p:GUS, SCRp:LZY1, SCRp:LZY2, SCRp:LZY3, SHRp:LZY1, SHRp:LZY2, SHRp:LZY3, ADF9p:LZY1, ADF9p:LZY2, ADF9p:LZY3, LZY2gΔCCL, LZY3gΔCCL, LZY2p:LZY2-mCherry, LZY3p:LZY3-mCherry, LZY2p:mCherry, LZY3p:mCherry, LZY2p:CCL^LZY2-mCherry, and LZY3p:CCL^LZY3-mCherry. Primers used for cloning are listed in Supplemental Data Set 1.

Genomic fragments covering 1973, 6381, and 4603 bp upstream from the start codon and 485, 1268, and 1095 bp downstream from the stop codon of LZY1, LZY2, and LZY3, respectively, were introduced into pFAST-R01 (Shimada et al., 2010; LZY1g, LZY2g, and LZY3g, Supplemental Data Set 1E). The 1973-, 6381-, and 4603-bp upstream regions from the start codon were used as the promoter regions and fused with DNA fragments containing GUS gene and NOS terminator in the pENTR vector. Subsequently, they were introduced into pFAST-R01 (LZY1p:GUS, LZY2p:GUS, and LZY3p:GUS; Supplemental Data Set 1B). A 3122-bp fragment upstream from the start codon of ADF9 was used as ADF9 promoter and cloned into pBI101.3 (ADF9p:GUS; Supplemental Data Set 1B). The promoter region of SCR has been reported previously (Morita et al., 2002). A 2492-bp fragment ranging from 8 to 2499 bp upstream of start codon of SHR was used as a SHR promoter. The promoter regions of SCR, SHR, and ADF9 were combined with cloning sites and NOS terminator in the pENTR vector. Full-length cDNAs of LZY1, LZY2, and LZY3 were cloned between the promoter and NOS terminator, followed by introducing into pFAST-R01 (SCRp:LZY1, SCRp:LZY2, SCRp:LZY3, SHRp:LZY1, SHRp:LZY2, SHRp:LZY3, ADF9p:LZY1, ADF9p:LZY2, and ADF9p:LZY3; Supplemental Data Set 1C). To construct LZY2gΔCCL and LZY3gΔCCL, plasmid sequences containing genomic region of LZY2 or LZY3 other than the CCL region were amplified with the primers listed in Supplemental Data Set 1F, and amplified fragments were self-ligated to delete the CCL region. The resulting genomic fragments were introduced into pFAST-R01 (LZY2gΔCCL and LZY3gΔCCL). To construct LZY2p:LZY2-mCherry, LZY3p:LZY3-mCherry, LZY2p:mCherry, and LZY3p:mCherry, the GUS region in the pENTR plasmids used for construction of the promoter:GUS series was replaced with CCL-mCherry or mCherry. CCL-mCherry plasmids were constructed using the primers listed in Supplemental Data Set 1G. The resulting pENTR plasmids for the mCherry series were used for Gateway recombination with pFAST-R01 (LZY2p:LZY2-mCherry, LZY3p:LZY3-mCherry, LZY2p:mCherry, and LZY3p:mCherry). To construct LZY2p:CCL-mCherry and LZY3p:CCL-mCherry, pENTR plasmids containing LZY2p:LZY2-mCherry or LZY3p:LZY3-mCherry were amplified using At1g17400c-EcoI-Not-F and mCherry-SacI-R primers listed in Supplemental Data Set 1G to obtain the respective CCL-mCherry fragments. Then, the region of mCherry in the pENTR plasmid containing LZY2p:mCherry or LZY3p:mCherry was replaced with the respective CCL-mCherry fragments. The resulting pENTR plasmids were used for Gateway recombination with pGWB501 (Nakagawa et al., 2007) or pFAST-R01 (LZY2p:CCL-mCherry and LZY3p:CCL-mCherry). Plants were stably transformed with binary vectors carrying these constructs using standard protocols for Agrobacterium tumefaciens (strain GV3101)-mediated transformation (Clough and Bent, 1998).

GFP Imaging in Columella Cells of Lateral Roots

Primary roots containing lateral roots <2.5 mm in length were excised from 10-d-old seedlings expressing DR5rev:GFP or PIN3-GFP. The roots were fixed in 4% paraformaldehyde in phosphate buffer for 30 min in a vertical position. After two washes of 1 min each in phosphate buffer, fixed roots were cleared with ClearSee solution at room temperature for 4 d (Kurihara et al., 2015). Cleared tissues were embedded in ClearSee solution between a glass slide and cover slip. Confocal images of GFP fluorescence were obtained using an FV1000 microscope (Olympus). A previous study performed expression analyses of DR5rev:GFP and PIN3-GFP in wild-type lateral roots at tripartite stages according to asymmetric LR elongation and differentiation (Rosquete et al., 2013); however, this stage classification system was not applicable for analyses of mutants exhibiting defective asymmetric elongation such as lzy mutants. Therefore, we classified lateral root development into tripartite stages according to development of columella cells (Kiss et al., 2002). Stage 1 lateral roots correspond to the type 2 roots producing two rows of columella cells of Kiss et al. (2002), the stage 2 roots correspond to types 3 and 4, in which columella cells are elongating, and stage 3 corresponds to type 5, in which columella cells are fully elongated.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers (AGI codes of Arabidopsis LZY genes are indicated in Table 1): LAZY1, ABI34464; ZmLAZY1, NP_001132334. Germplasm included lzy1, GABI_591A12; lzy2, FLAG_199G07; and lzy3, SAIL_723_H12. The Gene Expression Omnibus accession number for microarray data is GSE101298.

Supplemental Data

• Supplemental Figure 1. Expression pattern of LZY genes.

• Supplemental Figure 2. T-DNA insertion in lzy2 and lzy3 mutants.

• Supplemental Figure 3. Shoot gravitropism in lzy mutants.

• Supplemental Figure 4. Transformation-rescue analyses of lzy mutants.

• Supplemental Figure 5. Growth and phototropic response of lzy mutants.

• Supplemental Figure 6. Transformation-rescue analyses of lzy mutants by endodermis-specific LZY.

• Supplemental Figure 7. Endodermis formation and amyloplast development in lzy triple mutants.

• Supplemental Figure 8. Amyloplast sedimentation in statocytes.

• Supplemental Figure 9. Transformation-rescue analyses in root gravitropism.

• Supplemental Figure 10. ADF9 promoter activity in plants.

• Supplemental Figure 11. Transformation-rescue analyses with statocyte-specific LZY genes.

• Supplemental Figure 12. Root structure and amyloplast development in columella in lzy triple mutants.

• Supplemental Figure 13. PIN3-GFP localization in columella cells in primary roots of lzy triple mutants.

• Supplemental Figure 14. Alignment of LAZY1 family proteins.

• Supplemental Figure 15. Expression analysis of LZY3.

• Supplemental Figure 16. The function of LZY2 lacking CCL and LZY-mCherry fusion gene in root gravitropism.

• Supplemental Figure 17. LZY-GFP localization in protoplast cells.

• Supplemental Figure 18. Expression of CCL-mCherry affects root gravitropism.

• Supplemental Figure 19. The growth angle of lateral roots of lzy1 lzy2 lzy3 triple mutants.

• Supplemental Figure 20. Asymmetric DR5rev:GFP expression in the tips of lateral root.

• Supplemental Figure 21. Localization analysis of PIN3-GFP in the lateral root tips without clearing.

• Supplemental Data Set 1. List of primers used in this study.

Glossary

GSA

gravitropic set-point angle