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LAZY Genes Mediate the Effects of Gravity on Auxin Gradients and Plant Architecture

Takeshi Yoshihara, Edgar P. Spalding

Published October 2017. DOI: https://doi.org/10.1104/pp.17.00942

Abstract

A rice (Oryza sativa) mutant led to the discovery of a plant-specific LAZY1 protein that controls the orientation of shoots. Arabidopsis (Arabidopsis thaliana) possesses six LAZY genes having spatially distinct expression patterns. Branch angle phenotypes previously associated with single LAZY genes were here studied in roots and shoots of single and higher-order atlazy mutants. The results identify the major contributors to root and shoot branch angles and gravitropic behavior of seedling hypocotyls and primary roots. AtLAZY1 is the principal determinant of inflorescence branch angle. The weeping inflorescence phenotype of atlazy1,2,4 mutants may be due at least in part to a reversal in the gravitropism mechanism. AtLAZY2 and AtLAZY4 determined lateral root branch angle. Lateral roots of the atlazy2,4 double mutant emerged slightly upward, approximately 10° greater than perpendicular to the primary root axis, and they were agravitropic. Etiolated hypocotyls of the quadruple atlazy1,2,3,4 mutant were essentially agravitropic, but their phototropic response was robust. In light-grown seedlings, the root of the atlazy2,3,4 mutant was also agravitropic but when adapted to dim red light it displayed a reversed gravitropic response. A reversed auxin gradient across the root visualized by a fluorescent signaling reporter explained the reversed, upward bending response. We propose that AtLAZY proteins control plant architecture by coupling gravity sensing to the formation of auxin gradients that override a LAZY-independent mechanism that creates an opposing gravity-induced auxin gradient.

Innate developmental programs and adaptive responses to environmental cues shape plant architecture (Wang and Li, 2008; Dong et al., 2013; Teichmann and Muhr, 2015). Chief among the environmental cues that influence plant architecture is gravity. It orients the primary growth axis as well as the posture of lateral organs, both above and below ground. Typically, the primary growth axes of roots and shoots are vertical, with lateral branches adopting some characteristic angle with respect to the main axis. For example, an Arabidopsis (Arabidopsis thaliana) inflorescence branch typically grows out and up from the main axis at an angle of approximately 40° (Yoshihara et al., 2013). If the plant axis is tipped from the vertical, gravitropism will bend the branch until it achieves its original orientation with respect to the gravity vector. This behavior demonstrates that a plant organ adopts a characteristic gravitational set point angle (Digby and Firn, 1995). Auxin gradients control the differential growth that sets the angle (Rosquete et al., 2013). Limiting the influence of gravity by growing plants in microgravity (Lyon, 1968; Hoson et al., 1999) or on a clinostat (Lyon, 1965; Lyon and Yokoyama, 1966; Yoshihara and Iino, 2006) produces the shapes and architectures that would develop if gravity-response mechanisms were not in operation. Some responses to gravistimulation given after a clinostat treatment are opposite to normal gravitropism, but nonetheless are controlled by auxin gradients (Lyon, 1972; Roychoudhry et al., 2013) established by PIN auxin efflux proteins (Rosquete et al., 2013). This result may indicate greater complexity in the control of growth by gravity than can be presently explained.

The gravity vector is mainly perceived by endodermal cells in shoots (Fukaki et al., 1998), and in roots by the columella cells of the root cap (Sack, 1991; Blancaflor et al., 1998). In both cell types, sedimentation of amyloplasts (Haberlandt, 1900; Němec, 1900) is somehow transduced into the relocalization of PIN proteins, which establishes an auxin gradient across the organ, then differential growth, and a consequential gravitropic bending (Morita, 2010; Strohm et al., 2012).

A protein of unknown biochemical function that participates downstream of a gravity-sensing mechanism was identified by investigating the rice lazy1 mutant, which displays a prostrate growth phenotype due to impaired gravitropism (Abe et al., 1996; Li et al., 2007; Yoshihara and Iino, 2007). The gene affected in a classic maize lazy mutant was recently identified and found to be a homolog of OsLAZY1 (Dong et al., 2013; Howard et al., 2014). Arabidopsis LAZY1 (AtLAZY1) is also required for gravitropism in the inflorescence stem and seedling hypocotyl (Yoshihara et al., 2013). Branch angles are wider in atlazy1 as in oslazy1 (Yoshihara et al., 2013; Sasaki and Yamamoto, 2015) even though branch angle in monocots and dicots are determined by shoot structures that are not anatomically homologous. LAZY1 was shown to be required for auxin redistribution in response to gravitropic stimulation in rice (Oryza sativa; Godbole et al., 1999; Li et al., 2007; Yoshihara and Iino, 2007) and in maize (Zea mays; Dong et al., 2013). OsLAZY1, ZmLAZY1, and AtLAZY1 localize to the nucleus and at the cell periphery, probably including the plasma membrane (Dong et al., 2013; Yoshihara et al., 2013; Howard et al., 2014; Sasaki and Yamamoto, 2015). The rice Deeper Rooting1 (OsDRO1) is also a LAZY family gene (Yoshihara et al., 2013). It controls root gravitropism in both the seminal and crown roots and thus affects general root system architecture (Uga et al., 2013). Likewise, an Arabidopsis LAZY gene named AtDRO1 also affects lateral root orientation (Guseman et al., 2017).

Typical LAZY genes contain regions of conserved sequence I through V. They have been found only in land plant genomes (Yoshihara et al., 2013). They belong to a larger family of genes defined by an IGT [GφL(A/T)GT] motif in region II (Dardick et al., 2013). The rice tiller angle control gene, OsTAC1 (Yu et al., 2007), and its Arabidopsis homolog AtTAC1 (Dardick et al., 2013), are examples of IGT-containing genes that lie outside the LAZY group because they lack a region V, the largest of the conserved domains in LAZY genes (Yoshihara et al., 2013). The predicted protein alignment in Supplemental Figure S1 shows how that criterion distinguishes LAZY from TAC1 genes, which oppositely affect branch or tiller angles in Arabidopsis (Dardick et al., 2013), rice (Yu et al., 2007), maize (Ku et al., 2011), and peach (Prunus persica; Dardick et al., 2013). The Arabidopsis genome contains six LAZY genes, but one contains only the conserved region V (Yoshihara et al., 2013). The original OsLAZY1/Os11g29840 (Li et al., 2007; Yoshihara and Iino, 2007) and ZmLAZY1/GRMZM2G135019 (Dong et al., 2013) possess a region V. Genes recently reported as AtNGR1 (At1g17400), AtNGR2 (At1g72490), and AtNGR3 (At1g19115) by Ge and Chen (2016), and AtDRO1 (At1g72490), AtDRO2 (At1g19115), and AtDRO3 (At1g17400) by Guseman et al. (2017) are identical to genes previously identified as AtLAZY genes using the region V criterion (Yoshihara et al., 2013). Therefore, At1g17400 will be referred to as AtLAZY2, At1g19115 as AtLAZY3, At1g72490 as AtLAZY4, At3g24750 as AtLAZY5, and At3g27025 as AtLAZY6 in this study of AtLAZY gene contributions to tropisms, auxin gradients, and plant architecture controlled by gravity.

RESULTS

The AtLAZY Gene Family Expression Patterns

The native expression pattern of each AtLAZY gene was examined by engineering the β-glucuronidase (GUS) reporter gene (Jefferson et al., 1987) to be controlled by AtLAZY promoters in separate transgenic plants. Multiple transgenic lines were examined for each construct. Figure 1 shows histochemical staining indicative of each gene’s expression pattern. In light grown seedlings, AtLAZY1 promoter activity was apparent throughout the seedling shoot including the vascular system and only very faintly in the root, as shown before (Yoshihara et al., 2013). AtLAZY2/AtNGR1/AtDRO3 was highly expressed in the hypocotyl and root tip. AtLAZY3/AtNGR3/AtDRO2 was expressed in the root tip. AtLAZY4/AtNGR2/AtDRO1 was highly expressed in the lower hypocotyl and root stele. Highest expression of AtLAZY4 was seen in root apex. AtLAZY5 was expressed throughout the root except the apex. AtLAZY6 expression was apparent in the petiole. In dark-grown (etiolated) seedlings, the expression patterns were similar (Fig. 1D; Supplemental Fig. S2). AtLAZY1 was expressed more in the shoot than root. AtLAZY2 was expressed in the cotyledons, hypocotyl, and root tip. AtLAZY3 expression could be seen only in the extreme root tip (Supplemental Fig. S2A). AtLAZY4 was expressed strongest in the cotyledons, hypocotyl stele, root stele, and root tip. AtLAZY5 was expressed in the cotyledon and root except the apex (Supplemental Fig. S2A). AtLAZY6 was expressed in the vicinity of the apical meristem and petiole (Supplemental Fig. S2B). The distinct expression patterns shown in Figure 1 and Supplemental Figure S2 indicated that each gene may function to different extents in roots and shoots.

Figure 1.
Figure 1.

Expression pattern of each AtLAZY gene in light grown and dark grown shoots and roots. A, Histochemical staining of seedlings expressing either pAtLAZY1::GUS, pAtLAZY2::GUS, pAtLAZY3::GUS, pAtLAZY4::GUS, pAtLAZY5::GUS, or pAtLAZY6::GUS grown in white light for 3 d before staining. B, Shoots of the same promoter-reporter lines in (A) grown for 3 d in white light. C, Root apices of the promoter-reporter lines grown in white light for 3 d. D, Etiolated seedlings transformed with the promoter-reporter constructs previously listed and grown in total darkness for 3 d before staining. The images shown are representative of the several transgenic lines created for each construct. Scale bars = 500 μm in A, 200 μm in B, 50 μm in C, and 300 μm in D.

Mutant Analysis

We isolated plants homozygous for T-DNA insertions in five of the AtLAZY genes. The locations of the insertions in each gene are shown in Supplemental Figure S3. A T-DNA insertion line for AtLAZY6 could not be found in publicly available resources. Single and higher-order mutants were grown and their phenotypes were examined. Figure 2A shows that the orientation of seedlings grown in darkness deviated from vertical especially when multiple AtLAZY genes were mutated. The atlazy1,2,3,4 quadruple mutants were frequently horizontal or upside down, indicating a defect in gravitropism. Gravitropism assays based on time-lapse imaging were conducted to investigate the response to reorientation. Seedlings grown in darkness were transferred to a vertical agar plate such that the hypocotyl was vertical, then the plate was rotated 90° to induce gravitropism. Still in darkness, images were collected by computer-controlled cameras using infrared backlight. The angle of the hypocotyl apex was measured from the images. Of the single mutants, atlazy1 showed clearly defective gravitropism and atlazy2 showed a slight defect in the Wassilewskija (Ws) background (Fig. 2B; Supplemental Fig. S4A). The atlazy1 defect was exacerbated by combining it with the atlazy2 and/or atlazy4 mutations (Fig. 2C; Supplemental Fig. S4B). The atlazy1,2,3,4 quadruple mutant responded only very weakly to the gravitropic stimulus (Fig. 2C). Consistent with expression patterns in etiolated hypocotyls (Fig. 1D), AtLAZY1, AtLAZY2, and AtLAZY4 are the most important to gravitropism in the hypocotyl (Fig. 2C).

Figure 2.
Figure 2.

Gravitropism in hypocotyl and root require specific AtLAZY genes. A, Orientation of dark grown seedlings of each genotype on the surface of vertical agar plates. Seedlings were grown in dark for 3 d. Hypocotyls of atlazy1,4, atlazy1,2,4, and atlazy1,2,3,4 often show nonvertical growth. Scale bar, 2 mm. B, Hypocotyl gravitropism in single mutants of AtLAZY genes. Seedlings were grown in dark for 3 d. Gravistimulation was given by rotating plates by 90°. Among single mutants, only atlazy1 displayed a severely impaired gravitropic response. Values shown represent means ± se from 17 to 20 seedlings. Asterisks indicate statistically significant difference from the wild type by Welch’s t test (*P < 0.05; **P < 0.01). C, Hypocotyl gravitropism in higher-order mutants of AtLAZY genes. Gravitropic responses in atlazy1,2,4 and atlazy1,2,3,4 are more severely impaired than in atlazy1. Values shown represent means ± se from 30 to 35 seedlings. Asterisks indicate statistically significant difference from the wild type by Welch’s t test (*P < 0.05; **P < 0.01). D and E, Root gravitropism in single mutants of AtLAZY genes. Seedlings were grown on the surface of vertical agar plates under white light for 3 d. Gravistimulation was given by rotating plates by 90°. Values shown represent means ± se from 38 to 43 seedlings (D) or from 23 to 42 seedlings (E). Asterisks indicate statistically significant difference from the wild type by Welch’s t test (*P < 0.05; **P < 0.01). atlazy4 and atlazy2 showed slight reduction of gravitropic response. F, Root gravitropism in higher-order mutants of AtLAZY genes. atlazy2,3,4 and atlazy1,2,3,4 did not show clear gravitropism. Values shown represent means ± se from 23 to 37 seedlings. Asterisks indicate statistically significant difference from the wild type by Welch’s t test (*P < 0.05; **P < 0.01). WT col, wild-type Columbia; WT Ws, wild-type Wassilewskija.

Etiolated hypocotyls elongate rapidly, making their orientations evident (Fig. 2A) and their gravitropism responses robust (Fig. 2, B and C). In contrast, root growth is slow in etiolated seedlings. Therefore, to examine the impacts of atlazy mutations on root gravitropism, seedlings were grown in white light for 3 d and then placed in front of computer-controlled cameras to collect time series of images from which tip angles could be extracted. The atlazy1, atlazy3, and atlazy5 single mutants displayed a gravitropic response to reorientation that was indistinguishable from the wild type (Fig. 2D). The atlazy4 mutant showed a very slight reduction (Fig. 2D). The atlazy2 mutant displayed a modest reduction in rate of curvature development compared to wild type (Fig. 2E). The severity of gravitropism defects increased as two, three, or four AtLAZY genes were mutated (Fig. 2F). Root tips of atlazy1,2,3,4 mutants did not bend downward. Consistent with expression patterns in root tips (Fig. 1C), AtLAZY2, AtLAZY3, and AtLAZY4 are the most important to gravitropism in the root (Fig. 2F).

Phototropism is a bending growth phenomenon in response to a light gradient that, like gravitropism, is due to the environmental signal causing the hormone auxin to be redistributed across the growing organ (Spalding, 2013). If atlazy mutations impaired the auxin redistribution mechanism or auxin response mechanism shared between gravitropism and phototropism, then defective phototropism would be expected in the agravitropic quadruple mutant. This result was not observed. Instead, phototropism of etiolated quadruple mutant hypocotyls was robust, even faster to develop than the wild type (Fig. 3; Supplemental Movie S1). This indicates that LAZY proteins function in a gravitropism pathway upstream of where light- and gravity-signaling pathways converge on the auxin redistribution mechanism.

Figure 3.
Figure 3.

Phototropism in the atlazy1,2,3,4 mutant is exaggerated compared to wild type. A, Representative images of 3-d-old dark-grown seedlings grown at 0 h or 8 h after the onset of blue light irradiation. The arrows indicate the direction of the continuous unilateral blue light. B, Hypocotyl angle below the hook region was measured from times series of images using HYPOTrace software (Wang et al., 2009). Values shown represent means ± se (n = 10). The time intervals significantly different from the wild type are indicated by brackets and asterisks (Welch’s t test; **P < 0.01).

Reverse Gravitropism and Auxin Gradients

Ge and Chen (2016) recently reported that roots of atlazy2,3,4 (atngr1,2,3) triple mutants display a reverse gravitropism, growing upward. Although they assigned NGR names to the T-DNA mutants in their study, the alleles are identical to those used here. We investigated whether plant culture conditions could be responsible for the difference between the upward root growth behavior reported by Ge and Chen (2016) and the defective gravitropism shown in Figure 2F. In our standard conditions, roots of the triple mutant bent subtly downward rather than upward (Fig. 2F). Roots of triple and quadruple mutants grown in white light then maintained in darkness for 3 h before rotating began to show some evidence of reverse gravitropism toward the end of the 8-h assay in darkness (Supplemental Fig. S5A). This prompted an investigation of light conditions that may affect the root orientation phenotypes of the mutants studied here and by Ge and Chen (2016). Seedlings were grown on vertical agar plates in continuous darkness, in continuous dim red light, or continuous white light for 2 d and then dim red light for 1 d. In each of these conditions, wild-type roots grew mostly straight down as expected but the direction of root tips in atlazy2,3,4 and atlazy1,2,3,4 mutants averaged approximately 130°, where 180° would be pointing vertically upward (Supplemental Fig. S5, B–E). The red-light adapted condition was selected for detailed measurements of gravitropism, to determine if removal of LAZY functions would cause roots to display a reverse form of gravitropism. Roots of wild-type seedlings cultured in white light then shifted to continuous dim red light grew slowly but displayed positive (downward bending) gravitropism upon reorientation. In contrast, roots of atlazy2,3,4 and atlazy1,2,3,4 mutants displayed significant but slower negative (upward bending) gravitropism (Fig. 4A; Supplemental Movie S2). It is well established that redistribution of auxin to the lower flank of a horizontal root is responsible for a growth rate difference between the upper and lower sides, which results in bending (Muday, 2001). Wild-type and atlazy2,3,4 mutant lines expressing the genetically encoded DII-VENUS, which emits a fluorescent signal inversely proportional to auxin signaling activity (Brunoud et al., 2012), were examined by confocal microscopy before or 2 h after 90° rotation. In the wild-type context, the auxin gradient formed as expected from previous results (Band et al., 2012; Brunoud et al., 2012). Figure 4B shows the signal from cells on the upper flank of the root was more than 2-fold higher than from cells on the lower flank. Signal strengths on the left and right sides of vertically maintained roots were not different. In the atlazy2,3,4 background, this auxin reporter indicated a small gradient after reorientation but in the opposite direction (Fig. 4, B and C), consistent with the observed reverse, upward gravitropic bending. A mutated form of the auxin reporter, mDII-VENUS, did not produce an asymmetric signal in vertical or gravistimulated roots (Fig. 4B). The root tips of the mutant were morphologically normal and appeared to contain normal amounts of starch-based statoliths in the gravity-sensing cells (Supplemental Fig. S6). If the reversed gravitropism of the root was due to the modest reversed auxin gradient, pharmacological agents known to suppress the formation of auxin gradients would be expected to suppress the reversed gravitropism as they do normal gravitropism. Supplemental Fig. S7 shows that, as previously reported (Cho et al., 2014), 1-N-naphthylphthalamic acid (NPA), and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) impair gravitropism in the red-light-adapted wild-type seedlings without major effects on growth rate. Treatment of atlazy2,3,4 triple mutant seedlings with either NPA or NPPB impaired the reversed (negative) gravitropism of the root (Fig. 4D) without affecting the growth rate (Fig. 4E). Taken together, these results indicate that AtLAZY proteins function downstream of a gravity-sensing mechanism and upstream of the mechanism that establishes the growth controlling auxin gradient, and upstream of the point where phototropism photoreceptors influence this process (Fig. 5).

Figure 4.
Figure 4.

Reverse auxin gradient produces negative gravitropism in root of atlazy triple mutant. A, Negative root gravitropism in red-light-adapted atlazy2,3,4, and atlazy1,2,3,4 seedlings. Seedlings grown in darkness on the surface of agar plates were rotated by 90° and root tip angles were measured from automatically collected images. Values shown represent means ± se from 11 to 20 seedlings. B, Auxin gradient across the root tip after rotation. Fluorescent intensity of DII-VENUS and mDII-VENUS in wild-type Columbia and atlazy2,3,4 were measured in the apical approximately 160 μm of the root. Fluorescent intensity was measured 2 h after 90° rotation. Values shown represent means ± se of the proportions of the signals detected in the indicated halves of the root tips to those in entire root tips (n = 15 to 22). The fluorescent intensity of DII-VENUS is inversely proportional to the auxin concentration (Brunoud et al., 2012). Note that the atlazy2,3,4 mutant showed higher auxin level in upper side of root tip after rotation (Student’s t test; **P < 0.01). C, Representative images of DII-VENUS fluorescent in root tips. Seedlings were rotated by 90° and images were collected 2 h after rotation. Scale bar, 25 μm. D, Effects of auxin transport inhibitors on negative gravitropism of atlazy1,2,3,4. Red-light-adapted seedlings were transferred on plates containing either 1 μM NPA or 10 μM NPPB. Plates were rotated by 90° at 4 h after transferring. Values shown represent means ± se from 17 to 28 seedlings. Asterisks indicate statistically significant difference from the control by Welch’s t test (*P < 0.05; **P < 0.01). E, Effects of auxin transport inhibitors on root growth of atlazy1,2,3,4. Root lengths were measured in the seedlings used for the gravitropism experiments in Figure 4D. Growth rates were estimated from the differences in root lengths in initial pictures (0 h) and final pictures (8 h). Values shown represent means ± se. Growth rates of samples treated with inhibitors were compared with that of control by Welch’s t test (P > 0.05). Note that neither 1 µM NPA nor 10 µM NPPB inhibited growth. n.s., not significant; WT col, wild-type Columbia.

Figure 5.
Figure 5.

Placement of LAZY protein function in pathways producing bending growth via auxin gradients. LAZY proteins function downstream of a gravity-sensing mechanism but upstream of an auxin redistribution process, and upstream of where unilateral light sensing mechanisms influence auxin redistribution. In parallel, a LAZY-independent pathway transduces gravity signals into an opposing auxin gradient, which can be observed when LAZY proteins are removed by mutation. Thus, LAZY proteins function in a gravity-specific mechanism for creating auxin gradients that bend roots downward and shoots upward. Phototropic signaling does not require LAZY proteins, nor does a gravity-induced reverse gravitropism pathway, but all pathways converge on the mechanism for redistributing auxin across the organ. The question mark indicates that reverse gravitropism of the shoots in plants lacking LAZY functions remains to be demonstrated.

Adult Plant Phenotypes

The AtLAZY mutants were used to investigate the role of LAZY proteins beyond primary growth of seedlings. The specific goal was to assess the extent to which the LAZY mechanism mediated the influence of gravity on lateral organs to affect adult plant morphology. Figure 6 shows that mutation of AtLAZY1 had the previously reported widening effect on inflorescence branch angle (Yoshihara et al., 2013). The atlazy1,2 double mutant showed that disrupting two AtLAZY genes with expression in shoots exacerbated the branch angle phenotype of atlazy1 mutants (Fig. 6). The atlazy1,4 mutant combination also severely impacted shoot architecture (Fig. 6). The inflorescence of the atlazy1,2,4 triple mutant apparently lacked the ability to orient its main axis upright or position its lateral branches at a particular angle.

Figure 6.
Figure 6.

AtLAZY genes control shoot orientation with respect to the gravity vector. Shoot architecture in single and higher-order mutants of AtLAZY genes. Wild-type Columbia, atlazy1, atlazy2, atlazy3, atlazy4, atlazy1,2, atlazy1,3, atlazy1,4, atlazy2,3, atlazy2,4, atlazy3,4, and atlazy1,2,4 are shown. atlazy1,2, atlazy1,4, and atlazy1,2,4 show more exaggerated lazy phenotypes than atlazy1, but atlazy2,4 does not show an obvious phenotype. Scale bars, 3 cm. WT col, wild-type Columbia.

The structure of the root system depended on AtLAZY gene function to a similarly strong degree. Guseman et al. (2017) recently reported a significant contribution of AtLAZY4 (AtDRO1) to the control of lateral root angle. Figure 7, A to C, shows that the lateral roots of atlazy2 and atlazy4 single mutants were significantly less declined than wild type. When these two mutations were combined, the lateral roots were held, on average, above the horizontal (Fig. 7C). The atlazy2,3,4 triple mutant produced lateral roots that appeared to be insensitive to the influence of the gravity vector. Analysis of the lateral root angles of the higher-order mutants was complicated by the fact that the primary root did not grow parallel to the gravity vector (Fig. 7A). But seedlings could be manipulated on agar plates such that the primary root was straight and vertical. In this scenario, atlazy2,4 and atlazy2,3,4 seedlings produced lateral roots that were essentially horizontal (Fig. 7D). When atlazy2,4 seedlings were rotated by 90°, the preexisting lateral roots continued to elongate in their original directions. They did not reorient like the wild type (Fig. 7, E and F).

Figure 7.
Figure 7.

AtLAZY genes control the influence of gravity on branch root angles. A, Representative pictures of 13-d-old seedlings. Seedlings were grown on the surface of an agar-gelled medium containing mineral nutrients (Murashige-Skoog) under constant light. Scale bar, 10 mm. B, Distribution of lateral root angles in different atlazy mutants. Lateral root angles relative to the plumb line were measured at a spot 2 mm away from the base of the lateral root. Polar histogram of lateral root angles in the mutants of AtLAZY genes is shown. Frequencies of lateral root angles in each 22.5° division are shown in the corresponding angular position around a circle (n = 55 to 184). Arrow shows the direction of the plumb line (gravity vector). C, Lateral root angles in the mutants of AtLAZY genes. The absolute deviations from plumb line were recalculated from the data used in Figure 2B. Values shown represent means ± se. Asterisks indicate statistically significant difference by Welch’s t test (P > 0.05; **P < 0.01). D, Angle of lateral roots in atlazy2,3,4 plants formed after the primary root was manually straightened and aligned with the gravity vector by gently pulling it along the agar surface. Values shown represent means ± se of 153 (atlazy2,4) and 97 (atlazy2,3,4) seedlings. E, Lateral roots of atlazy2,4 do not respond to reorientation. Two-week-old seedlings were rotated by 90°. Representative images taken 2 d after the rotation are shown. Black arrows on the lateral roots show the locations and orientations of the root tips at the time of rotation. White arrow shows the direction of gravity vector after rotation. F, Changes in lateral root tip angle after 90° rotation in atlazy2,4. Values shown represent means ± se of 44 (wild-type Columbia) and 42 (atlazy2,4) seedlings. The wild-type roots change growth direction toward the gravity center after rotation but the atlazy2,4 mutant roots continue to grow in the same prerotation direction. n.s., not significant; WT col, wild-type Columbia.

DISCUSSION

These results are attributed to, and in most cases, quantify, the contributions of different AtLAZY genes to the influence of gravity on the shapes of roots and shoots, at seedling and adult stages of development. Some experiments probed the structure of the gravity-signaling pathway. Figure 3 shows that AtLAZY genes act before the point where directional light- and gravity-signaling pathways merge, which is at or before auxin response factors convert the auxin gradient into differential growth (Stowe-Evans et al., 1998; Harper et al., 2000; Correll and Kiss, 2002). Incorporating previous results into the assessment, it now appears that LAZY proteins act downstream of amyloplast sedimentation (Abe et al., 1994), upstream of auxin gradient formation (Godbole et al., 1999; Yoshihara and Iino, 2007; Li et al., 2007), and in a gravity-specific portion of a mechanism for converting environmental signals into differential growth. Although they function upstream of auxin gradient formation, auxin may nonetheless be an important regulator of their function. Uga et al. (2013) found that auxin decreased expression of OsDRO1, a gene that affects root gravity responses and which Yoshihara et al. (2013) had identified as belonging to the LAZY family. It remains to be determined if auxin affects the expression of other LAZY family genes.

Taniguchi et al. (2017) reported function of LAZY genes while our report was being revised. They reported the control of inflorescence stem gravitropism by AtLAZY1, AtLAZY2, and AtLAZY4, supporting the results in Figure 6. They also examined gravitropism in primary and lateral roots and, consistent with our results, found AtLAZY2 and AtLAZY4 to be important. Further, they found that a C-terminal portion of AtLAZY1 reversed primary root gravitropism in atlazy1,2,4. Our current results and the report of Ge and Chen (2016) offer a possible explanation. The C terminus of AtLAZY1, which contains the critical region V, may inhibit the function of AtLAZY3, producing a phenotype like that of the atlazy1,2,3,4 quadruple mutant (Fig. 4). Taniguchi et al. (2017) further showed that AtLAZY genes control the PIN3 expression pattern in the lateral root tip. This can explain the lack of auxin redistribution in lazy mutants (Godbole et al., 1999; Li et al., 2007; Yoshihara and Iino, 2007; Dong et al., 2013) and emergence of reversed auxin redistribution in atlazy2,3,4 (Fig. 4).

The mutants collected and characterized here provide a means to perturb rather specifically a gravity-sensing mechanism controlling plant architecture. When the AtLAZY mechanism(s) was disabled with multiple mutations, a separate mechanism for transducing the gravity vector into an auxin gradient was uncovered. This LAZY-independent mechanism created an oppositely oriented auxin gradient. The reverse gravitropism that ensues, if it also occurs in shoots, could contribute to the enhanced phototropism displayed by atlazy1,2,3,4 mutants (Fig. 3), but enhanced phototropism would be expected in any scenario in which gravitropism is only weakened and not reversed (Vitha et al., 2000; Ruppel et al., 2001; Kiss et al., 2002). No molecular or genetic details of the reverse mechanism demonstrated here and by Ge and Chen (2016) are yet known, but some suggestions can be made. TAC1 appears to act oppositely to LAZY1 by widening tiller angle in rice (Yu et al., 2007), leaf angle in maize (Ku et al., 2011), and branch angles in Arabidopsis and peach (Dardick et al., 2013). Perhaps oppositely functioning genes in the IGT superfamily such as TAC1 and its relatives generate the reverse auxin gradient and gravitropism evident when LAZY genes are removed. A mechanism acting in opposition to the LAZY-dependent mechanism could provide an element of regulation or modulation to gravitropism, which can proceed at different rates with or without a period of overshoot and correction, depending on certain conditions (Brooks et al., 2010).

Because LAZY genes are found only in land plants (Yoshihara et al., 2013), the mechanism they control may have evolved as an adaptation to life removed from the buoyancy of the ancestral aquatic environment. Our results are consistent with the idea that plants accrued multiple gravity-sensing mechanisms over evolutionary time as roots and shoots became specialized for the competitive capture of water, minerals, and light on land (Barlow, 1995). Accordingly, the LAZY-dependent mechanism may have been superimposed on an ancestral LAZY-independent mechanism that now plays a modulatory role by acting in weaker opposition. One caveat to consider is that an Arabidopsis plant lacking all six AtLAZY genes must be obtained to conclude that the mechanism producing reverse gravitropism is formally LAZY-independent. An atlazy6 mutation has not yet been isolated or engineered.

Although AtLAZY6 has not yet been genetically evaluated, the collection of previous and present results enables a fairly comprehensive assessment of function across the family. For example, AtLAZY2 and AtLAZY4 contribute to inflorescence erectness as inferred from the phenotypes of higher-order mutants (Fig. 6). Previously, only LAZY1 was known to affect shoot architecture in Arabidopsis (Yoshihara et al., 2013), rice (Li et al., 2007; Yoshihara and Iino, 2007), and maize (Ku et al., 2011; Dong et al., 2013). The expression of AtLAZY1, AtLAZY2, and AtLAZY4 in etiolated hypocotyls including the endodermis where the cells containing statoliths for gravity sensing are located, is consistent with the genetic evidence that these genes control gravity responses in seedling and adult shoots (Figs. 2 and 6; Supplemental Fig. S4). If expression patterns are reliable indicators of sites of function in this gene family, then AtLAZY6 with expression in petioles, along with AtLAZY1, may control leaf angle. The genetic control of leaf angle is an important topic in crop plant breeding (Ku et al., 2011; Tian et al., 2011). The possibility of a special role for AtLAZY6 in the regulation of leaf angle should be investigated.

A single dominant contributor such as AtLAZY1 in shoots was not evident in roots. Instead, the three genes displaying distinct expression in the gravity-sensing apex of the root (AtLAZY2, AtLAZY3, and AtLAZY4) redundantly contributed to positive gravitropism in the primary root (Fig. 2, D–F). Mutating these genes uncovered the reverse gravitropism phenomenon first reported by Ge and Chen (2016) in a triple mutant apparently identical to the atlazy2,3,4 mutant used in this study. To generate a reverse gravitropism reliable and robust enough to study as in Figure 4, we had to explore different growth conditions, ultimately settling on a light regime that may have been effective by removing the phototropic stimuli, enhancing the auxin transport, and maintaining an adequate root growth rate (Kiss et al., 2003; Boccalandro et al., 2008; Halliday et al., 2009). In lateral roots, apparently only AtLAZY2 and AtLAZY4 are needed to decline their angles relative to the primary root axis from a default orientation that may be slightly greater than horizontal (Fig. 7). Previously, AtLAZY4/AtDRO1 (Guseman et al., 2017) was the only AtLAZY gene family member known to affect lateral root angle similar to OsDRO1. Now that an assessment of expression patterns and functional contributions for most members of a LAZY gene family from a single species has been completed, it may be possible to take a rational approach to engineering different root and shoot architectures by manipulating a small number of LAZY genes in any plant.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The wild types used in all experiments were the Columbia ecotype (Col-0) of Arabidopsis (Arabidopsis thaliana) obtained from the European Arabidopsis Stock Centre and the Wassilewskija ecotype (Ws) from Institut of Jean-Pierre Bourgin. The atlazy1 T-DNA insertion line (GK_591A12) has the Col-0 background and was from NASC. atlazy3 (GK_479C08), and atlazy4 (SAIL_723_H11), and atlazy5 (SAIL_158_H07) also had the Col-0 background and were from the Arabidopsis Biological Resource Center. The atlazy2 (FLAG_199G07) had the Ws background and was from Institut of Jean-Pierre Bourgin. The exact locations of T-DNA insertions for each line were confirmed by sequencing the PCR products amplified with the primer pairs listed on Supplemental Table S1. All single mutants were backcrossed three times with corresponding background wild type. For the creation of multiple mutants with atlazy2 that have a Ws background, atlazy2 was crossed at least five times with the Col-0 background to replace the Ws background with Col-0.

For most of the physiological experiments using seedlings, seeds were surface-sterilized and plated on minimal salt medium (1 mm KCl, 1 mm CaCl2, and 5 mm MES, adjusted to pH 5.7 with BTP, 0.7% [w/v] agar), unless otherwise stated.

For hypocotyl gravitropism, seedlings were grown along the surface of vertical agar plates for 3 d in darkness, and then selected by uniformity and hypocotyl length (6–10 mm). The selected seedlings were transferred and aligned on the surface of new plates so that apical 5 mm to 10 mm of the hypocotyl sticks out from the edge. Gravitropic stimulation was given 2 to 3 h after transferring by rotating the plates by 90°. Dim green light was used for the manipulation of seedlings in darkness.

For hypocotyl phototropism, seedlings were grown along the surface of vertical agar plates for 3 d in darkness. Seedlings were selected by uniformity and hypocotyl length (6–10 mm) and transferred to new plates. Phototropic stimulation was given by irradiating the upper half of the hypocotyl with unilateral continuous blue light. Blue light was supplied from an LED light source (450 ± 70 nm, ELS-450; World Precision Instruments) through fiber optic cable (FO-1000-SMA; World Precision Instruments).

For root gravitropism, seedlings were grown along the surface of vertical agar plates for 3 d under constant light. Gravitropic stimulation was given by rotating the plates by 90°. Dim green light was used for the manipulation of the seedlings in darkness. Red-light-adapted seedlings were obtained by transferring the plates containing 2-d-old seedlings grown under white light to under dim red light. Dim red light was obtained by using a custom-made LED light source (660 ± 20 nm) and the light intensity at plant level was approximately 5 μmol m−2 s−1. The LEDs were part no. UT0373-41 obtained from LEDTech Electronics. The nominal spectral output was confirmed by measurement with a spectroradiometer. For auxin transport inhibitor experiments, red-light-adapted seedlings were transferred to the plates containing 1 μM of NPA (Chem Service) or 10 μm of NPPB (Sigma-Aldrich) 4 h before gravistimulation and incubated in darkness. NPA and NPPB were dissolved in DMSO. Control plates contained the same amount of DMSO (0.1% [v/v]).

To observe lateral root angles, plants were grown on Murashige-Skoog medium supplemented with 1% Suc and B5 vitamins. Two plants were grown on a square petri dish (10 cm × 10 cm). Plates were incubated vertically under constant white light for 13 to 14 d depending on measurements. The angles and lengths were measured on the images by using ImageJ (National Institutes of Health; Schneider et al., 2012). The lateral root angle was measured relative to the plumb line (gravity vector) at 2 mm from the base of the root. The absolute values were used for analysis so that lateral roots emerging from the left and right could be averaged. All statistical tests were performed with the Excel program (Microsoft).

Imaging

The imaging of seedlings was performed according to Wang et al. (2009). In brief, plates were mounted vertically and transverse to the optical axis of a CCD camera (Marlin F-146B; AVT) outfitted with a close-focus zoom lens (model R72; Tokina). An infrared light source (model BL1960-660; Advanced Illumination), having a peak output at 880 nm, was placed behind the petri plate for back illumination. The images for lateral root analysis were obtained by using flat-bed scanner.

Plasmid Construct and Generation of Transgenic Plants

All promoters were obtained by PCR and the resultant fragments were subcloned into pGEM-T-Easy (Promega) for sequence confirmation before proceeding to a further step. All primers used in this study were listed on Supplemental Table S1. The promoter regions of AtLAZY1 homologs were amplified with either HindIII and BamHI (pAtLAZY2; 2.6 kb, pAtLAZY5; 2.5 kb, and pAtLAZY6; 2.8 kb) sites or BamHI and SmaI sites (pAtLAZY3; 0.6 kb, and pAtLAZY4; 2.9 kb) by PCR from genomic Arabidopsis DNA. The resultant fragment was subcloned and recloned into either the HindIII/XbaI site or BamHI/SmaI site in the binary vector, pBI101.1 (Jefferson et al., 1987). The binary vector was transformed into Agrobacterium tumefaciens, GV3101. Arabidopsis plants were transformed using the floral dip methods (Clough and Bent, 1998).

Histochemical Staining

Tissues were immersed in staining solution (100 mm sodium P buffer pH 7.0, 0.1% [v/v] Triton X-100, 0.5 to 2 mm potassium ferrocyanide, 0.5 to 2 mm potassium ferricyanide, and 1 mm X-gluc), vacuum infiltrated for 30 min, and incubated at 37°C for 3 to 24 h. Chlorophyll was removed in 70% (v/v) ethanol at 4°C.

Microscopy

Confocal microscopy was performed on a Elyra LSM 780 laser scanning confocal microscope (Zeiss). Optics employed was a Plan-Apochromat ×20 lens. The sample was excited with the 514-nm laser line for improved YFP, DII-VENUS (Nagai et al., 2002), and channel mode detection was used to record the emission (excitation 514 nm, emission 519 to 620 nm).

Accession Numbers

Genes studied in this article can be found in The Arabidopsis Information Resource and Joint Genome Institute databases under the following accession numbers: At5g14090 (AtLAZY1), At1g17400 (AtLAZY2), At1g19115 (AtLAZY3), At1g72490 (AtLAZY4), At3g24750 (AtLAZY5), At3g27025 (AtLAZY6), LOC_Os11g29840.1 (OsLAZY1), GRMZM2G135019_T01 (ZmLAZY1), At2g46640 (AtTAC1), LOC_Os09g35980.2 (OsTAC1), and GRMZM2G447987_T01 (ZmTAC1).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank the ABRC at Ohio State University for providing pBI101.1 binary vector and the seeds of Arabidopsis T-DNA insertion lines. We also thank the European Arabidopsis Stock Centre (NASC) and the Institut of Jean-Pierre Bourgin (IJPB) in the French National Institute for Agricultural Research for providing the seeds of Arabidopsis T-DNA insertion mutants. The confocal imaging/electron microscopy was performed at the Newcomb Imaging Center, Department of Botany, UW-Madison. We thank Robert Gruener for technical assistance.

Footnotes

  • www.plantphysiol.org/cgi/doi/10.1104/pp.17.00942

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Edgar P. Spalding (spalding{at}wisc.edu).

  • T.Y. and E.P.S. designed experiments; T.Y. performed experiments; T.Y. and E.P.S. interpreted the results; T.Y. and E.P.S. wrote the manuscript.

  • ↵1 This work was supported by the National Science Foundation (grant 1444456 to E.P.S.).

  • ↵[OPEN] Articles can be viewed without a subscription.

  • Received July 12, 2017.
  • Accepted August 14, 2017.
  • Published August 18, 2017.

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LAZY Genes Mediate the Effects of Gravity on Auxin Gradients and Plant Architecture
Takeshi Yoshihara, Edgar P. Spalding
Plant Physiology Oct 2017, 175 (2) 959-969; DOI: 10.1104/pp.17.00942
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