Roles of amyloplasts and water deficit in root tropisms

Directed growth of roots in relation to a moisture gradient is called hydrotropism. The no hydrotropic response (nhr1) mutant of Arabidopsis lacks a hydrotropic response, and shows a stronger gravitropic response than that of wild type (wt) in a medium with an osmotic gradient. Local application of abscisic acid (ABA) to seeds or root tips of nhr1 increased root downward growth, indicating the critical role of ABA in tropisms. Wt roots germinated and treated with ABA in this system were strongly gravitropic, even though they had almost no starch amyloplasts in the root-cap columella cells. Hydrotropically stimulated nhr1 roots, with or without ABA, maintained starch in the amyloplasts, as opposed to those of wt. Hence, the near-absence (wt) or abundant presence (nhr1) of starch granules does not influence the extent of downward gravitropism of the roots in an osmotic gradient medium. Starch degradation in the wt might help the root sustain osmotic stress and carry out hydrotropism, instead of reducing gravity responsiveness. nhr1 roots might be hydrotropically inactive because they maintain this starch reserve in the columella cells, sustaining both their turgor and growth, and in effect minimizing the need for hydrotropism and at least partially disabling its mechanism. We conclude that ABA and water stress are critical regulators of root tropic responses.     

A possible involvement of autophagy in amyloplast degradation in columella cells during hydrotropic response of Arabidopsis roots

Seedling roots display not only gravitropism but also hydrotropism, and the two tropisms interfere with one another. In Arabidopsis (Arabidopsis thaliana) roots, amyloplasts in columella cells are rapidly degraded during the hydrotropic response. Degradation of amyloplasts involved in gravisensing enhances the hydrotropic response by reducing the gravitropic response. However, the mechanism by which amyloplasts are degraded in hydrotropically responding roots remains unknown. In this study, the mechanistic aspects of the degradation of amyloplasts in columella cells during hydrotropic response were investigated by analyzing organellar morphology, cell polarity and changes in gene expression. The results showed that hydrotropic stimulation or systemic water stress caused dramatic changes in organellar form and positioning in columella cells. Specifically, the columella cells of hydrotropically responding or water-stressed roots lost polarity in the distribution of the endoplasmic reticulum (ER), and showed accelerated vacuolization and nuclear movement. Analysis of ER-localized GFP showed that ER redistributed around the developed vacuoles. Cells often showed decomposing amyloplasts in autophagosome-like structures. Both hydrotropic stimulation and water stress upregulated the expression of AtATG18a, which is required for autophagosome formation. Furthermore, analysis with GFP-AtATG8a revealed that both hydrotropic stimulation and water stress induced the formation of autophagosomes in the columella cells. In addition, expression of plastid marker, pt-GFP, in the columella cells dramatically decreased in response to both hydrotropic stimulation and water stress, but its decrease was much less in the autophagy mutant atg5. These results suggest that hydrotropic stimulation confers water stress in the roots, which triggers an autophagic response responsible for the degradation of amyloplasts in columella cells of Arabidopsis roots.     

Hydrotropism in abscisic acid,wavy, and gravitropic mutants of Arabidopsis thaliana

We have developed experimental systems to study hydrotropism in seedling roots of Arabidopsis thaliana (L.) Heynh. Arabidopsis roots showed a strong curvature in response to a moisture gradient, established by applying 1% agar and a saturated solution of KCl or K(2)CO(3) in a closed chamber. In this system, the hydrotropic response overcame the gravitropic response. Hydrotropic curvature commenced within 30 min and reached 80-100 degrees within 24 h of hydrostimulation. When 1% agar and agar containing 1 MPa sorbitol were placed side-by-side in humid air, a water potential gradient formed at the border between the two media. Although the gradient changed with time, it still elicited a hydrotropic response in Arabidopsis roots. The roots curved away from 0.5-1.5 MPa of sorbitol agar. Various Arabidopsis mutants were tested for their hydrotropic response. Roots of aba1-1 and abi2-1 mutants were less sensitive to hydrotropic stimulation. Addition of abscisic acid restored the normal hydrotropic response in aba1-1 roots. In comparison, mutants that exhibit a reduced response to gravity and auxin, axr1-3 and axr2-1, showed a hydrotropic response greater than that of the wild type. Wavy mutants, wav2-1 and wav3-1, showed increased sensitivity to the induction of hydrotropism by the moisture gradient. These results suggest that auxin plays divergent roles in hydrotropism and gravitropism, and that abscisic acid plays a positive role in hydrotropism. Furthermore, hydrotropism and the wavy response may share part of a common molecular pathway controlling the directional growth of roots.     

Novel hydrotropism mutants of Arabidopsis thaliana and their altered waving response and phototropism.

Roots display positive hydrotropism in response to a moisture gradient, which is important for plants to escape from water stress and regulate the directional growth by interacting with other growth movements such as gravitropism, phototropism and waving response. On Earth, hydrotropism is interfered by gravitropism in particular, so that microgravity conditions or agravitropic mutants have been used for the study of hydrotropism. However, we have recently established an experimental system for the study of hydrotropism in Arabidopsis roots that easily develop hydrotropism in response to moisture gradient by overcoming gravitropism. Using the Arabidopsis system, we isolated hydrotropism mutants named root hydrotropism (rhy). In the present study, we examined the hydrotropism, gravitropism, phototropism, waving response and elongation growth of rhy4 and rhy5 roots that were defective in positive hydrotropism. Interestingly, rhy4 roots curved away from the water source and showed a reduced waving response. Both rhy4 and rhy5 showed normal gravitropism and a slight reduction in phototropism. These results suggest that there is a mutual molecular mechanism underlying hydrotropism, waving response and/or phototropism. Thus, we have obtained novel hydrotropic mutants that will be used for revealing molecular mechanism of root hydrotropism and its interaction with waving response and/or phototropism.     

Salt modulates gravity signaling pathway to regulate growth direction of primary roots in Arabidopsis

Plant root architecture is highly plastic during development and can adapt to many environmental stresses. The proper distribution of roots within the soil under various conditions such as salinity, water deficit, and nutrient deficiency greatly affects plant survival. Salinity profoundly affects the root system architecture of Arabidopsis (Arabidopsis thaliana). However, despite the inhibitory effects of salinity on root length and the number of roots, very little is known concerning influence of salinity on root growth direction and the underlying mechanisms. Here we show that salt modulates root growth direction by reducing the gravity response. Exposure to salt stress causes rapid degradation of amyloplasts in root columella cells of Arabidopsis. The altered root growth direction in response to salt was found to be correlated with PIN-FORMED2 (PIN2) messenger RNA abundance and expression and localization of the protein. Furthermore, responsiveness to gravity of salt overly sensitive (sos) mutants is substantially reduced, indicating that salt-induced altered gravitropism of root growth is mediated by ion disequilibrium. Mutation of SOS genes also leads to reduced amyloplast degradation in root tip columella cells and the defects in PIN2 gene expression in response to salt stress. These results indicate that the SOS pathway may mediate the decrease of PIN2 messenger RNA in salinity-induced modification of gravitropic response in Arabidopsis roots. Our findings provide new insights into the development of a root system necessary for plant adaptation to high salinity and implicate an important role of the SOS signaling pathway in this process.     

Influence of microgravity on root-cap regeneration and the structure of columella cells in Zea mays

We launched imbibed seeds and seedlings of Zea mays into outer space aboard the space shuttle Columbia to determine the influence of microgravity on 1) root-cap regeneration, and 2) the distribution of amyloplasts and endoplasmic reticulum (ER) in the putative statocytes (i.e., columella cells) of roots. Decapped roots grown on Earth completely regenerated their caps within 4.8 days after decapping, while those grown in microgravity did not regenerate caps. In Earth-grown seedlings, the ER was localized primarily along the periphery of columella cells, and amyloplasts sedimented in response to gravity to the lower sides of the cells. Seeds germinated on Earth and subsequently launched into outer space had a distribution of ER in columella cells similar to that of Earth-grown controls, but amyloplasts were distributed throughout the cells. Seeds germinated in outer space were characterized by the presence of spherical and ellipsoidal masses of ER and randomly distributed amyloplasts in their columella cells. These results indicate that 1) gravity is necessary for regeneration of the root cap, 2) columella cells can maintain their characteristic distribution of ER in microgravity only if they are exposed previously to gravity, and 3) gravity is necessary to distribute the ER in columella cells of this cultivar of Z. mays.     

Interactions between Hydrotropism and Gravitropism in the Primary Seminal Roots of Triticum eastivum L.

It has been proposed that hydrotropism interacts with gravitropismin seedling roots; that is, roots which are highly gravitropicshow less hydrotropism (Takahashi and Suge, 1991 PhysiologiaPlantarum 82: 24-31; Takahashi and Scott, 1993 Plant, Cell andEnvironment 16: 99-103). Here, we examine varietal differencesin the hydrotropic response and its interaction with gravitropismin wheat roots. Primary seminal roots of wheat (Triticum aestivumL.) were hydrotropically stimulated by different moisture gradientsestablished by placing wet cheesecloth and saturated solutionsof different salts in closed chambers. From equations obtainedby relative humidity (RH) at different distances from the wetcheesecloth, moisture gradients at the root-tip level were estimatedto be 0·03 to 1·84% RH mm^-1, depending upon thesalt introduced into the chamber. The roots showed positivehydrotropism in response to 0·67% RH mm^-1, and the responseapparently increased as the gradient was strengthened. Whenthe primary seminal roots of 12 cultivars were exposed to amoisture gradient of 1·84% RH mm-1, hydrotropic responsesignificantly differed depending upon the cultivar tested. Amongthe cultivars, the roots of Norin 11, Norin 15, Norin 117, andNorin 125 responded hydrotropically more strongly than the others.These roots, with the exception of Norin 11, showed a less vigorousresponse to gravity compared to the remaining cultivars. However,the roots of Norin 20, Norin 38, and Norin 107 were relativelyunresponsive to both a moisture gradient and to gravity. Thus,the primary seminal roots of wheat respond hydrotropically,and the responsiveness differs among cultivars. However, thevarietal difference in hydrotropic response cannot be explainedsolely by converse differences in responsiveness to gravity.Copyright1995, 1999 Academic Press Cultivar, gravitropism, hydrotropism, primary seminal roots, Triticum aestivum L., wheat     

Complex physiological and molecular processes underlying root gravitropism

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.     

Geoperception in primary and lateral roots of Phaseolus vulgaris (Fabaceae). I. Structure of columella cells

Primary roots of Phaseolus vulgaris (Fabaceae) are positively geotropic, while lateral roots are not responsive to gravity In order to elucidate the structural basis for this differential georesponse, we have performed a qualitative and quantitative analysis of the ultrastructure of columella cells of primary and lateral roots of P. vulgaris. Root systems were fixed in situ so as not to disturb the ultrastructure of the columella cells. The columellas of primary roots are more extensive than those of lateral roots. The volumes of columella cells of primary roots are approximately twice those of columella cells of lateral roots. However, columella cells of primary roots contain greater absolute volumes and numbers of all cellular components examined than do columella cells of lateral roots. Also, the relative volumes of cellular components in columella cells of primary and lateral roots are statistically indistinguishable. The endoplasmic reticulum is sparse and distributed randomly in both types of columella cells. Both types of columella cells contain numerous sedimented amyloplasts, none of which contact the cell wall or form complexes with other cellular organelles. Therefore, positive geotropism by roots must be due to a factor(s) other than the presence of sedimented amyloplasts alone. Furthermore, it is unlikely that amyloplasts and plasmodesmata form a multi-valve system that controls the movement of growth regulating substances through the root cap.     

The Influence of Gravity on the Formation of Amyloplasts in Columella Cells of Zea mays L

Columella (i.e., putative graviperceptive) cells of Zea mays seedlings grown in the microgravity of outer space allocate significantly less volume to putative statoliths (amyloplasts) than do columella cells of Earth-grown seedlings. Amyloplasts of flight-grown seedlings are significantly smaller than those of ground controls, as is the average volume of individual starch grains. Similarly, the relative volume of starch in amyloplasts in columella cells of flight-grown seedlings is significantly less than that of Earth-grown seedlings. Microgravity does not significantly alter the volume of columella cells, the average number of amyloplasts per columella cell, or the number of starch grains per amyloplast. These results are discussed relative to the influence of gravity on cellular and organellar structure.     

Physiological and genetic characterization of hydrotropic mutants of Arabidopsis thaliana.

Roots display positive hydrotropism in response to moisture gradient. Hydrotropism regulates the directional growth by interaction with other growth movements. Using the seedlings of pea, cucumber, maize and wheat, we have revealed that the root cap perceives the moisture gradient and that auxin and calcium are involved in hydrotropism. However, molecular mechanisms for stimulus perception or signal transduction in hydrotropism are still remained unrevealed. To dissect the molecular mechanism underlying hydrotropism in seedling roots, we established a method for screening Arabidopsis mutants defective in root hydrotropism. Among about 20,000 M2 seedlings of Arabidopsis plants treated with EMS, we successfully obtained 12 mutants of which root hydrotropism was reduced to various extents. We named them root hydrotropism (rhy) and examined their gravitropism, phototropism, waving response and elongation growth as well as hydrotropism in roots. Roots of rhy1 mutant showed ahydrotropic response although the other responses and elongation growth of rhy1 mutant were normal. Roots of rhy2 and rhy3 mutants showed a reduced hydrotropism and abnormal responses in gravitropism, phototropism or waving pattern. Genetic analysis of the progeny produced by the backcross of rhy1 mutant to wild type suggested that rhy1 was a recessive mutation. We also examined the map position of the rhy1 locus.     

The promotion of gravitropism in Arabidopsis roots upon actin disruption is coupled with the extended alkalinization of the columella cytoplasm and a persistent lateral auxin gradient

The actin cytoskeleton has been implicated in regulating plant gravitropism. However, its precise role in this process remains uncertain. We have shown previously that disruption of the actin cytoskeleton with Latrunculin B (Lat B) strongly promoted gravitropism in maize roots. These effects were most evident on a clinostat as curvature that would exceed 90 degrees despite short periods of horizontal stimulation. To probe further the cellular mechanisms underlying these enhanced gravity responses, we extended our studies to roots of Arabidopsis. Similar to our observations in other plant species, Lat B enhanced the response of Arabidopsis roots to gravity. Lat B (100 nm) and a stimulation time of 5-10 min were sufficient to induce enhanced bending responses during clinorotation. Lat B (100 nm) disrupted the fine actin filament network in different regions of the root and altered the dynamics of amyloplasts in the columella but did not inhibit the gravity-induced alkalinization of the columella cytoplasm. However, the duration of the alkalinization response during continuous gravistimulation was extended in Lat B-treated roots. Indirect visualization of auxin redistribution using the DR5:beta-glucuronidase (DR5:GUS) auxin-responsive reporter showed that the enhanced curvature of Lat B-treated roots during clinorotation was accompanied by a persistent lateral auxin gradient. Blocking the gravity-induced alkalinization of the columella cytoplasm with caged protons reduced Lat B-induced curvature and the development of the lateral auxin gradient. Our data indicate that the actin cytoskeleton is unnecessary for the initial perception of gravity but likely acts to downregulate gravitropism by continuously resetting the gravitropic-signaling system.     

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.     

Hydrotropism: root growth responses to water

The survival of terrestrial plants depends upon the capacity of roots to obtain water and nutrients from the soil. Directed growth of roots in relation to a gradient in moisture is called hydrotropism and begins in the root cap with the sensing of the moisture gradient. Even though the lack of sufficient water is the single-most important factor affecting world agriculture, there are surprisingly few studies on hydrotropism. Recent genetic analysis of hydrotropism in Arabidopsis has provided new insights about the mechanisms that the root cap uses to perceive and respond simultaneously to moisture and gravity signals. This knowledge might enable us to understand how the root cap processes environmental signals that are capable of regulating whole plant growth.     

How amyloplasts,water deficit and root tropisms interact?

Hydrotropism, the differential growth of plant roots directed by a moisture gradient, is a long recognized, but not well-understood plant behavior. Hydrotropism has been characterized in the model plant Arabidopsis. Previously, it was postulated that roots subjected to water stress are capable of undergo water-directed tropic growth independent of the gravity vector because of the loss of the starch granules in root cap columella cells and hence the loss of the early steps in gravitropic signaling. We have recently proposed that starch degradation in these cells during hydrostimulation sustain osmotic stress and root growth for carrying out hydrotropism instead of reducing gravity responsiveness. In addition, we also proposed that abscisic acid (ABA) and water deficit are critical regulators of root gravitropism and hydrotropism, and thus mediate the interacting mechanism between these two tropisms. Our conclusions are based upon experiments performed with the no hydrotropic response (nhr1) mutant of Arabidopsis, which lacks a hydrotropic response and shows a stronger gravitropic response than that of wild type (WT) in a medium with an osmotic gradient.Key words: starch, water deficit, auxin, abscisic acid, gravitropism, hydrotropismRoots of land plants sense and respond to different stimuli, some of which are fixed in direction and intensity (i.e., gravity) while other vary in time, space, direction and intensity (i.e., obstacles and moisture gradients). Directed growth of roots in relation to a gradient in moisture is called hydrotropism and begins in the root cap with the sensing of the moisture gradient. However, since gravity is an omnipresent accompaniment of Earthly life and many living process have evolved with it as a background constant, it is not surprising that root hydrotropism interacts with gravitropism.¹ The hydrotropic response in Arabidopsis, compare with other plants such as pea and cucumber^2,3 is readily observed even in the presence of gravity.^4,5 When Arabidopsis roots are subjected to a water gradient, such that the source of water is placed 180° opposed to the gravity vector, the roots will grow upwards, displaying positive hydrotropism. Therefore, it has been feasible to isolate so far two Arabidopsis mutants affected in their hydrotropic response.^5,6 Analysis of these mutants reveals new insights of the mechanism of hydrotropism. For one hand, the no hydrotropic response (nhr1) mutant lacks a hydrotropic response, and shows a stronger gravitropic response than that of wt and a modified wavy growth response in a medium with an osmotic gradient.^5,7 On the other hand, the mizu-kussei1 (miz1) mutant did not exhibit hydrotropism and showed regular gravitropism.⁶ Hence, the root hydrotropic response is both linked and unlinked from the gravitropic one. Nonetheless, miz1 roots also showed a reduced phototropism and a modified wavy growth response. This indicates that both MIZ1 and NHR1 are not exclusive components of the mechanism for hydrotropism and supports the notion that the root cap has assessment mechanisms that integrate many different environmental influences to produce a final integrated response.⁸ Thus, the physiological phenomena distinctively displayed by roots in order to forage resources from the environment are the result of integrated responses that resulted from many environmental influences sensed in the root cap.In the course of studying how gravity and water availability affected the perception and assessment of each other in root cap cells that generated the final root tropic response, we found that ABA is a critical regulator of the signal transduction mechanism that integrated these two-root tropisms.⁷ For this, we analyzed the long-term hydrotropic response of Arabidopsis roots in an osmotic gradient system. ABA, locally applied to seeds or root tips of nhr1, significantly increased root downward growth in a medium with an osmotic gradient (root length of nhr1 seedlings grown in this medium were on average 12.5 mm and plus 10 µM ABA were 25.1 mm). On the other hand, WT roots germinated and treated locally with ABA in this system were strongly gravitropic, albeit they had almost no starch in amyloplasts of root cap columella cells. Hydrotropically stimulated nhr1 roots, with or without ABA, maintained starch in amyloplastas, as opposed to those of WT. Therefore, the near-absence (WT) or abundant presence (nhr1) of starch granules does not affect the extent of downward gravitropism of roots in an osmotic gradient medium. Starch degradation in the wt might participate in osmoregulation by which root cells maintain turgor and consequently carry out hydrotropism, instead of reducing gravity responsiveness. In fact, it was just recently published that salt-induced rapid degradation of starch in amyloplasts is not likely the main reason for a negative gravitropic response seen under salt stress, because sos mutant roots of Arabidopsis showed negative gravitropic growth without any apparent rapid digestion of starch granules.⁹ Additionally, the stems of overwintering tubers of Potamogeton pectinatus are capable of elongating much faster in the absence than in the presence of oxygen for up to 14 days and its stems has an enhanced capacity for gravitropic movements in completely anoxic conditions.¹⁰ These authors hypothesized that ABA and starch degradation in the starchy tuber sustained stem cell elongation and cell division as well as differential growth required for the gravitropic response in these aquatic plants. These data taken together suggest that in conditions of anoxia, or water stress, ABA and degradation of starch play a critical role in the ability to survive relatively prolonged periods of unfavorable growth conditions. These players are critical when water or minerals are scarce since they regulate the enhancement of root downward growth. However, since roots can trail humidity gradients in soil, they can modulate their branching patterns (architecture) and thus respond to hydrotropism once a water-rich patch is found. Then the response of plants to gravity is principally one of nutrition (shoots to light, roots to mineral and water) and consequently must be regulated according to the long- and short-term environmental variables that occur during the development of the plant.Differential growth that occurs during the gravitropic and phototropic response has been explained according to the Cholodny-Went hypothesis, which states that the lateral transport of auxin across stimulated plant tissues is responsible for the curvature response.¹¹ Analysis of hydrotropism in some Arabidopsis agravitropic auxin transport mutants has demonstrated that these mutations do not influence their hydrotropic response.⁴ Furthermore, current pharmacological studies using inhibitors also indicated that both auxin influx and efflux are not required for hydrotropic response whereas auxin response is necessary for it.¹² These authors suggested a novel mechanism for auxin in root hydrotropism. Here, we analyzed whether asymmetric auxin distribution takes place across hydrotropically-stimulated roots using transgenic plants carrying a responsive auxin promoter (DR5) driving the expression of β-glucuronidase (GUS) or green fluorescent protein (GFP)^13,14 in wt and nhr1 backgrounds. Wt and nhr1 roots hydrotropically stimulated in a system with air moisture gradient⁵ showed no asymmetric expression of the DR5:: GUS or DR5::GFP (Fig. 1A and B). Nonetheless, nhr1 roots showed a substantial decrease in the signal driven by the DR5::GUS and GFP reporters in humidity saturated conditions (Fig. 1A, part b and B, part b), which might indicate that auxin-induced gene expression in the root cap was inhibited. It remains to be determined the significance of this inhibition in the no hydrotropic response phenotype displayed by nhr1 roots. Determination of the DR5::GUS expression in wt and nhr1 roots growing in an osmotic gradient medium for testing long-term hydrotropism revealed that the GUS signal was to some extent diminished in both wt or in nhr1 roots (Fig. 2C and D) compared to those roots growing in normal medium (Fig. 2A and B). An inhibitor of auxin response reduced hydrotropism,¹² and also inhibited auxin-dependent DR5::GUS expression.¹⁵ However, a decrease of DR5::GUS in wt root tips was not an impediment for developing an hydrotropic response. On the other hand, nhr1 roots also showed a decrease of DR5::GUS expression (Fig. 2B and D) and a complete absence of DR5::GFP (data not shown), which did not influence the extent of downward root gravitropism in water deficit conditions. Therefore, it is difficult to assign a role of auxin-induce gene expression in hydrotropism and further studies are required in order to unravel this issue. Furthermore, it needs to be resolved whether these expression studies oppose the idea that gradients in auxin precede differential growth in response to humidity gradients.Open in a separate windowFigure 1DR5:: GUS (A) and DR5::GFP (B) activity in the wild type NHR1 and nhr1 backgrounds. (A) Root tips hydrostimulated in a system with air moisture gradient (C and D) or grown in a saturated water conditions (A and B) stained with 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic (X-Gluc) acid buffer under the same conditions for 80 min. (B) Root tips hydrostimulated as in (A) (C and D) or grown in a saturated water conditions (A and B) whose green fluorescent signal was visualized by confocal microscopy. Shown are images selected from at least 45 representative root tips. Bar = 29 µm.Open in a separate windowFigure 2Expression of DR5::GUS in wild type NHR1 and nhr1 backgrounds. Roots were hydrotropically stimulated for 8 days in a medium with an osmotic gradient (C and D) or grown in normal medium (A and B) and stained with X-Gluc acid buffer under the same conditions for 80 min. Shown are images selected from at least 50 representative root tips. Bar = 25 µm.Our studies⁷ revealed that ABA is a critical regulator of both root gravitropism and hydrotropism in water deficit conditions, and that the role of auxin under these conditions seems to differ from those observed in several studies thus far published on gravitropism made under well-water conditions. The molecular characterization of NHR1 and from other nhr-like mutants already isolated in our lab will clarify the mechanisms involved in this fascinating tropism.¹⁶     

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type     

Gravitropism in the starch excess mutant of Arabidopsis thaliana

Amyloplasts are hypothesized to play a key role in the cellular mechanisms of gravity perception in plants. While previous studies have examined the effects of starch deficiency on gravitropic sensitivity, in this paper, we report on gravitropism in plants with a greater amount of starch relative to the normal wild type. Thus, we have studied the sex1 (starch excess) mutant of Arabidopsis thaliana, which accumulates extra starch because it is defective in a protein involved in the regulation of starch mobilization. Compared to the wild type (WT), sex1 seedlings contained excess starch in cotyledons, hypocotyls, the root-hypocotyl transition zone, the body of the root, root hairs, and in peripheral rootcap cells. Sedimented amyloplasts were found in both the WT and in sex1 in the rootcap columella and in the endodermis of stems, hypocotyls, and petioles. In roots, the starch content and amyloplast sedimentation in central columella cells and the gravitropic sensitivity were comparable in sex1 and the WT. However, in hypocotyls, the sex1 mutant was much more sensitive to gravity during light-grown conditions compared to the WT. This difference was correlated to a major difference in size of plastids in gravity-perceiving endodermal cells between the two genotypes (i.e., sex1 amyloplasts were twice as big). These results are consistent with the hypothesis that only very large changes in starch content relative to the WT affect gravitropic sensitivity, thus indicating that wild-type sensing is not saturated.     

Hydrotropism: The current state of our knowledge

The response of roots to a moisture gradient has been reexamined, and positive hydrotropism has been demonstrated in recent years. Agravitropic roots of a pea mutant have contributed to the studies on hydrotropism. The kinetics of hydrotropic curvature, interactions between hydrotropism and gravitropism, moisture gradients required for the induction of hydrotropism, the sensing site for moisture gradients, characteristics of hydrotropic signal and differential growth, and calcium involvement in signal transduction have been subjects of these studies. This review summarizes the current state of our knowledge on hydrotropism in roots.     

Hydrotropism: the current state of our knowledge

The response of roots to a moisture gradient has been reexamined, and positive hydrotropism has been demonstrated in recent years. Agravitropic roots of a pea mutant have contributed to the studies on hydrotropism. The kinetics of hydrotropic curvature, interactions between hydrotropism and gravitropism, moisture gradients required for the induction of hydrotropism, the sensing site for moisture gradients, characteristics of hydrotropic signal and differential growth, and calcium involvement in signal transduction have been subjects of these studies. This review summarizes the current state of our knowledge on hydrotropism in roots.     

Root Graviresponsiveness and Cellular Differentiation in Wild-type and a Starchless Mutant of Arabidopsis thaliana

Primary roots of a starchless mutant of Arabidopsis thalianaL. are strongly graviresponsive despite lacking amyloplastsin their columella cells. The ultrastructures of calyptrogenand peripheral cells in wild-type as compared to mutant seedlingsare not significantly different. The largest difference in cellulardifferentiation in caps of mutant and wild-type roots is therelative volume of plastids in columella cells. Plastids occupy12.3% of the volume of columella cells in wild-type seedlings,but only 3.69% of columella cells in mutant seedlings. Theseresults indicate that: (1) amyloplasts and starch are not necessaryfor root graviresponsiveness; (2) the increase in relative volumeof plastids that usually accompanies differentiation of columellacells is not necessary for root graviresponsiveness; and (3)the absence of starch and amyloplasts does not affect the structureof calyptrogen (i.e. meristematic) and secretory (i.e. peripheral)cells in root caps. These results are discussed relative toproposed models for root gravitropism. Arabidopsis thaliana, gravitropism (root), plastids, root cap, stereology, ultrastructure