SGR1, SGR2, SGR3: novel genetic loci involved in shoot gravitropism in Arabidopsis thaliana.

In higher plants shoots show a negative gravitropic response but little is known about its mechanism. To elucidate this phenomenon, we have isolated a number of mutants with abnormal shoot gravitropic responses in Arabidopsis thaliana. Here we describe mainly three mutants: sgr1-1, sgr2-1, and sgr3-1 (shoot gravitropism). Genetic analysis confirmed that these mutations were recessive and occurred at three independent loci, named SGR1, SGR2, and SGR3, respectively. In wild type, both inflorescence stems and hypocotyls show negative gravitropic responses. The sgr1-1 mutants showed no response to gravity either by inflorescence stems or by hypocotyls. The sgr2-1 mutants also showed no gravitropic response in inflorescence stems but showed a reduced gravitropic response in hypocotyls. In contrast, the sgr3-1 mutant was found to have reduced gravitropic responses in inflorescence stems but normal gravitropic responses in hypocotyls. These results suggest that some genetic components of the regulatory mechanisms for gravitropic responses are common between inflorescence stems and hypocotyls, but others are not. In addition, these sgr mutants were normal with respect to root gravitropism, and their inflorescence stems and hypocotyls could carry out phototropism. We conclude that SGR1, SGR2, and SGR3 are novel genetic loci specifically involved in the regulatory mechanisms of shoot gravitropism in A. thaliana.     

SGR2, a phospholipase-like protein, and ZIG/SGR4, a SNARE, are involved in the shoot gravitropism of Arabidopsis

In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively. To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis. The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls. These genes probably are involved in the early step(s) of the gravitropic response. The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion. The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid-preferring phospholipase A1 containing a putative transmembrane domain. This gene family has been reported only in eukaryotes. The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport. Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism.     

Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana

Shoots of higher plants exhibit negative gravitropism. However, little is known about the mechanism or site of gravity perception in shoots. We have identified two loci that are essential for normal shoot gravitropism in Arabidopsis thaliana . Genetic analysis demonstrated that the shoot gravitropism mutants sgr1 and sgr7 are allelic to the radial pattern mutants, scr and shr , respectively. Characterization of the aerial phenotype of these mutants revealed that the primary defect is the absence of a normal endodermis in hypocotyls and inflorescence stems. This indicates that the endodermis is essential for shoot gravitropism and strongly suggests that this cell layer functions as the gravity-sensing cell layer in dicotyledonous plant shoots. These results also demonstrate that, in addition to their previously characterized role in root radial patterning, SCR and SHR regulate the radial organization of the shoot axial organs in Arabidopsis .      

Role of endodermal cell vacuoles in shoot gravitropism

In higher plants, shoots and roots show negative and positive gravitropism, respectively. Data from surgical ablation experiments and analysis of starch deficient mutants have led to the suggestion that columella cells in the root cap function as gravity perception cells. On the other hand, endodermal cells are believed to be the statocytes (that is, gravity perceiving cells) of shoots. Statocytes in shoots and roots commonly contain amyloplasts which sediment under gravity. Through genetic research with Arabidopsis shoot gravitropism mutants, sgr1/scr and sgr7/shr, it was determined that endodermal cells are essential for shoot gravitropism. Moreover, some starch biosynthesis genes and EAL1 are important for the formation and maturation of amyloplasts in shoot endodermis. Thus, amyloplasts in the shoot endodermis would function as statoliths, just as in roots. The study of the sgr2 and zig/sgr4 mutants provides new insights into the early steps of shoot gravitropism, which still remains unclear. SGR2 and ZIG/SGR4 genes encode a phospholipase-like and a v-SNARE protein, respectively. Moreover, these genes are involved in vacuolar formation or function. Thus, the vacuole must play an important role in amyloplast sedimentation because the sgr2 and zig/sgr4 mutants display abnormal amyloplast sedimentation.     

How do plant shoots bend up? — The initial step to elucidate the molecular mechanisms of shoot gravitropism usingArabidopsis thaliana

In higher plants, shoots show a negative gravitropic response. To elucidate the molecular mechanisms of this phenomenon, mutational analyses usingArabidopsis thaliana are in progress. This minireview aims to present recent developments in the genetic analysis of shoot gravitropism in this organism. We focus mainly on our studies on the novelshootgravitropic (sgr) mutants inArabidopsis thaliana that have dramatic defects in shoot gravitropism.     

Involvement of the vacuoles of the endodermis in the early process of shoot gravitropism in Arabidopsis

The endodermal cells of the shoot are thought to be the gravity-sensing cells in Arabidopsis. The amyloplasts in the endodermis that sediment in the direction of gravity may act as statoliths. Endodermis-specific expression of SGR2 and ZIG using the SCR promoter could complement the abnormal shoot gravitropism of the sgr2 and zig mutants, respectively. The abnormalities in amyloplast sedimentation observed in both mutants recovered simultaneously. These results indicate that both genes in the endodermal cell layer are crucial for shoot gravitropism. ZIG encodes AtVTI11, which is a SNARE involved in vesicle transport to the vacuole. The fusion protein of SGR2 and green fluorescent protein localized to the vacuole and small organelles. These observations indicate that ZIG and SGR2 are involved in the formation and function of the vacuole, a notion supported by the results of subcellular analysis of the sgr2 and zig mutants with electron microscopy. These results strongly suggest that the vacuole participates in the early events of gravitropism and that SGR2 and ZIG functions are involved.     

Mg‐dechelatase is involved in the formation of photosystem II but not in chlorophyll degradation in Chlamydomonas reinhardtii

The STAY‐GREEN (SGR) gene encodes Mg‐dechelatase which catalyzes the conversion of chlorophyll (Chl) a to pheophytin (Pheo) a. This reaction is the first and most important regulatory step in the Chl degradation pathway. Conversely, Pheo a is an indispensable molecule in photosystem (PS) II, suggesting the involvement of SGR in the formation of PSII. To investigate the physiological functions of SGR, we isolated Chlamydomonas sgr mutants by screening an insertion‐mutant library. The sgr mutants had reduced maximum quantum efficiency of PSII (F_v/F_m) and reduced Pheo a levels. These phenotypes were complemented by the introduction of the Chlamydomonas SGR gene. Blue Native polyacrylamide gel electrophoresis and immunoblotting analysis showed that although PSII levels were reduced in the sgr mutants, PSI and light‐harvesting Chl a/b complex levels were unaffected. Under nitrogen starvation conditions, Chl degradation proceeded in the sgr mutants as in the wild type, indicating that ChlamydomonasSGR is not required for Chl degradation and primarily contributes to the formation of PSII. In contrast, in the Arabidopsis sgr triple mutant (sgr1 sgr2 sgrL), which completely lacks SGR activity, PSII was synthesized normally. These results suggest that the Arabidopsis SGR participates in Chl degradation while the ChlamydomonasSGR participates in PSII formation despite having the same catalytic property.     

AtLAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence

The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T‐DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch‐angle phenotype and the gene consequently named AtLAZY1. Time‐resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch‐angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch‐angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery.     

The endodermis and shoot gravitropism

Shoots and roots of higher plants exhibit negative and positive gravitropism, respectively. A variety of gravitropic mutants have recently been isolated from Arabidopsis, the characterization of which demonstrates that the molecular mechanisms of the gravitropic responses in roots, hypocotyls and inflorescence stems are different. The cytological and molecular analysis of two mutants, shoot gravitropism 1 (sgrl), which is allelic to scarecrow (scr), and sgr7, which is allelic to short-root(shr), indicate that the endodermis is the site of gravity perception in shoots. These data suggest a new model for shoot gravitropism.     

Spatio-temporal kinematic analysis of shoot gravitropism in Arabidopsis thaliana

Plant shoots can bend upward against gravity, a behavior known as shoot gravitropism. The conventional quantification of shoot bending has been restricted to measurements of shoot tip angle, which cannot fully describe the spatio-temporal bending process. Recently, however, advanced imaging analyses have been developed to quantify in detail the spatio-temporal changes in inclination angle and curvature of the shoot. We used one such method (KymoRod) to analyze the gravitropism of the Arabidopsis thaliana inflorescence stem, and successfully extracted characteristics that capture when and where bending occurs. Furthermore, we implemented an elastic spring theoretical model and successfully determined best fitted parameters that may explain typical bending behaviors of the inflorescence stem. Overall, we propose a data-model combined framework to quantitatively investigate shoot gravitropism in plants.     

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope

The starch‐statolith hypothesis proposes that starch‐filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so‐called ‘static’ or ‘settled’ statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom‐designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild‐type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1  g . sgr2 mutants were able to sense and respond to gravity under 30  g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity‐driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1  g . We found that the correlation between hypergravity‐induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild‐type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the ‘bottom’ of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1  g .     

An Arabidopsis E3 ligase, SHOOT GRAVITROPISM9, modulates the interaction between statoliths and F-actin in gravity sensing

Higher plants use the sedimentation of amyloplasts in statocytes as statolith to sense the direction of gravity during gravitropism. In Arabidopsis thaliana inflorescence stem statocyte, amyloplasts are in complex movement; some show jumping-like saltatory movement and some tend to sediment toward the gravity direction. Here, we report that a RING-type E3 ligase SHOOT GRAVITROPISM9 (SGR9) localized to amyloplasts modulates amyloplast dynamics. In the sgr9 mutant, which exhibits reduced gravitropism, amyloplasts did not sediment but exhibited increased saltatory movement. Amyloplasts sometimes formed a cluster that is abnormally entangled with actin filaments (AFs) in sgr9. By contrast, in the fiz1 mutant, an ACT8 semidominant mutant that induces fragmentation of AFs, amyloplasts, lost saltatory movement and sedimented with nearly statically. Both treatment with Latrunculin B, an inhibitor of AF polymerization, and the fiz1 mutation rescued the gravitropic defect of sgr9. In addition, fiz1 decreased saltatory movement and induced amyloplast sedimentation even in sgr9. Our results suggest that amyloplasts are in equilibrium between sedimentation and saltatory movement in wild-type endodermal cells. Furthermore, this equilibrium is the result of the interaction between amyloplasts and AFs modulated by the SGR9. SGR9 may promote detachment of amyloplasts from AFs, allowing the amyloplasts to sediment in the AFs-dependent equilibrium of amyloplast dynamics.     

Pleiotropic effects of ZmLAZY1 on the auxin-mediated responses to gravity and light in maize shoot and inflorescences

Auxin has been found to control both gravitropism and inflorescence development in plant. Auxin transport has also been demonstrated to be responsible for tropism. Maize, a key agricultural crop, has distinct male (tassel) and female (ear) inflorescence, and this morphogenesis depends on auxin maximum and gradient. The classic maize mutant lazy plant1 (la1) has defective gravitropic response. The mechanism underlining maize gravitropism remains unclear. Recently, we isolated the ZmLA1 gene by map-based cloning, and our findings suggest that ZmLA1 might mediate the crosstalk between shoot gravitropism and inflorescence development by regulating auxin transport, auxin signaling, and auxin-mediated light response in maize. Here, we propose a model describing the ZmLA1-mediated complex interactions among auxin, gravity, light, and inflorescent development.     

Gravitropic response plays an important role in the nutational movements of the shoots of Pharbitis nil and Arabidopsis thaliana

The shoots of a Japanese strain of morning glory ( Pharbitis nil  ) called 'Shidare-asagao' display agravitropic and weeping growth. It has been shown that this shoot agravitropism may be due to the defective differentiation of endodermal cells that contain statoliths. Roots of the weeping morning glory show normal responsiveness to gravity and the shoots are positively phototropic. Shoots of the morning glory cultivar Violet used as a wild type exhibited distinct circumnutation with circular movements that increase as the plants grow. In weeping morning glory, however, nutation was limited to slight back and forth or side to side movements. To determine whether endodermal cells participate in circumnutation through a function that is independent of their role in gravitropism, the nutational movements of various gravitropic mutants of Arabidopsis thaliana were compared. The inflorescences of wild-type Arabidopsis showed relatively large circular movements. Inflorescences of the pgm-1 mutant, which is defective in starch synthesis, showed reduced nutation. Even more seriously affected were the sgr1-1 / scr-3 and sgr7-1 / shr-2 mutants, which are defective in endodermal cell differentiation, and the auxin-resistant axr2-1 mutant showed no significant nutational movements at all. 1- N -naphthylphthalamic acid (NPA) could inhibit Violet circumnutation, supporting the notion that auxin participates in circumnutation. Thus, the gravitropic response is an essential component in plant shoot circumnutation. Endodermal cells are involved in such circumnutation possibly because of their role in inducing the gravitropic response.     

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft‐10 tsf‐1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper‐vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1‐20 mutants. The terminal flower formed in tfl1‐20 mutants converted to a hyper‐vegetative shoot in ft‐10 tsf‐1 mutants. Grafting ft‐10 tsf‐1 or ft‐10 tsf‐1 tfl1‐20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild‐type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft‐10 tsf‐1 mutants the vasculature‐specific misexpression of FT converted the hyper‐vegetative shoot to a normal inflorescence, and in the ft‐10 tsf‐1 tfl1‐20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.     

The Divergent Roles of STAYGREEN (SGR) Homologs in Chlorophyll Degradation

Degradation of chlorophyll (Chl) by Chl catabolic enzymes (CCEs) causes the loss of green color that typically occurs during senescence of leaves. In addition to CCEs, STAYGREEN1 (SGR1) functions as a key regulator of Chl degradation. Although sgr1 mutants in many plant species exhibit a stay-green phenotype, the biochemical function of the SGR1 protein remains elusive. Many recent studies have examined the physiological and molecular roles of SGR1 and its homologs (SGR2 and SGR-LIKE) in Chl metabolism, finding that these proteins have different roles in different species. In this review, we summarize the recent studies on SGR and discuss the most likely functions of SGR homologs.     

Mutations in the gravity persistence signal loci in Arabidopsis disrupt the perception and/or signal transduction of gravitropic stimuli

Gravity plays a fundamental role in plant growth and development, yet little is understood about the early events of gravitropism. To identify genes affected in the signal perception and/or transduction phase of the gravity response, a mutant screen was devised using cold treatment to delay the gravity response of inflorescence stems of Arabidopsis. Inflorescence stems of Arabidopsis show no response to gravistimulation at 4 degrees C for up to 3 h. However, when gravistimulated at 4 degrees C and then returned to vertical at room temperature (RT), stems bend in response to the previous, horizontal gravistimulation (H. Fukaki, H. Fujisawa, M. Tasaka [1996] Plant Physiology 110: 933-943). This indicates that gravity perception, but not the gravitropic response, occurs at 4 degrees C. Recessive mutations were identified at three loci using this cold effect on gravitropism to screen for gravity persistence signal (gps) mutants. All three mutants had an altered response after gravistimulation at 4 degrees C, yet had phenotypically normal responses to stimulations at RT. gps1-1 did not bend in response to the 4 degrees C gravity stimulus upon return to RT. gps2-1 responded to the 4 degrees C stimulus but bent in the opposite direction. gps3-1 over-responded after return to RT, continuing to bend to an angle greater than wild-type plants. At 4 degrees C, starch-containing statoliths sedimented normally in both wild-type and the gps mutants, but auxin transport was abolished at 4 degrees C. These results are consistent with GPS loci affecting an aspect of the gravity signal perception/transduction pathway that occurs after statolith sedimentation, but before auxin transport.