A molecular mechanism unique to hydrotropism in roots

Plants are sessile in nature and must respond to various environmental cues to regulate their growth orientation. Root hydrotropism, a response to moisture gradients, has been considered to play an important role in drought avoidance. Nonetheless, the processes underlying hydrotropism in roots have remained obscure until recently because of the interfering effect of gravitropism. To shed light on root hydrotropism, we isolated and analyzed two Arabidopsis mutants, mizu-kussei (miz) 1 and 2, that have abnormal hydrotropic responses but normal responses to gravity. MIZ1 encodes a protein of unknown function with a conserved domain at its C-terminus. MIZ2 encodes a guanine-nucleotide exchange factor for ADP-ribosylation factor-type G proteins, which has been identified as GNOM. These findings suggest that roots possess molecular mechanisms essential for hydrotropism but independent of gravitropism. One of such mechanisms involves vesicle transport unique to hydrotropism in roots. Here we summarize recent progress on the molecular mechanism of root hydrotropism and the roles of MIZ1 and MIZ2.     

An altered hydrotropic response (ahr1) mutant of Arabidopsis recovers root hydrotropism with cytokinin

Roots are highly plastic and can acclimate to heterogeneous and stressful conditions. However, there is little knowledge of the effect of moisture gradients on the mechanisms controlling root growth orientation and branching, and how this mechanism may help plants to avoid drought responses. The aim of this study was to isolate mutants of Arabidopsis thaliana with altered hydrotropic responses. Here, altered hydrotropic response 1 (ahr1), a semi-dominant allele segregating as a single gene mutation, was characterized. ahr1 directed the growth of its primary root towards the source of higher water availability and developed an extensive root system over time. This phenotype was intensified in the presence of abscisic acid and was not observed if ahr1 seedlings were grown in a water stress medium without a water potential gradient. In normal growth conditions, primary root growth and root branching of ahr1 were indistinguishable from those of the wild type (wt). The altered hydrotropic growth of ahr1 roots was confirmed when the water-rich source was placed at an angle of 45° from the gravity vector. In this system, roots of ahr1 seedlings grew downward and did not display hydrotropism; however, in the presence of cytokinins, they exhibited hydrotropism like those of the wt, indicating that cytokinins play a critical role in root hydrotropism. The ahr1 mutant represents a valuable genetic resource for the study of the effects of cytokinins in the differential growth of hydrotropism and control of lateral root formation during the hydrotropic response.     

Intensity of hydrostimulation for the induction of root hydrotropism and its sensing by the root cap

Roots of Pisum sativum L. and Zea mays L. were exposed to different moisture gradients established by placing both wet cheesecloth (hydrostimulant) and saturated aqueous solutions of various salts in a closed chamber. Atmospheric conditions with different relative humidity (RH) in a range between 98 and 86% RH were obtained at root level, 2 to 3mm from the water-saturated hydrostimulant. Roots of Silver Queen corn placed vertically with the tips down curved sideways toward the hydrostimulant in response to approximately 94% RH but did not respond positively to RH higher than approximately 95%. The positive hydrotropic response increased linearly as RH was lowered from 95 to 90%. A maximum response was observed at RH between 90 and 86%. However, RH required for the induction of hydrotropism as well as the responsiveness differed among plant species used; gravitropically sensitive roots appeared to require a somewhat greater moisture gradient for the induction of hydrotropism. Decapped roots of corn failed to curve hydrotropically, suggesting the root cap as a major site of hydrosensing.     

Diversity of root hydrotropism among natural variants of Arabidopsis thaliana

Root gravitropism affects root hydrotropism. The interference intensity of root gravitropism with root hydrotropism differs among plant species. However, these differences have not been well compared within a single plant species. In this study, we compared root hydrotropism in various natural variants of Arabidopsis under stationary conditions. As a result, we detected a range of root hydrotropism under stationary conditions among natural Arabidopsis variants. Comparison of root gravitropism and root hydrotropism among several Arabidopsis natural variants classified natural variants that decreased root hydrotropism into two types; namely one type that expresses root gravitropism and root hydrotropism weaker than Col-0, and the other type that expresses weaker root hydrotropism than Col-0 but expresses similar root gravitropism with Col-0. However, root hydrotropism of all examined Arabidopsis natural variants was facilitated by clinorotation. These results suggested that the interference of root gravitropism with root hydrotropism is conserved among Arabidopsis natural variants, although the intensity of root gravitropism interference with root hydrotropism differs.

     

Root hydrotropism of an agravitropic pea mutant, ageotropum

We have partially characterized root hydrotropism of an agravitropic pea mutant, ageotropum (from Pisum sativum L. cv. Weibull's Weitor), without interference of gravitropism. Lowering the atmospheric air humidity inhibited root elongation and caused root curvature toward the moisture-saturated substrate in ageotropum pea. Removal of root tips approximately 1.5 mm in length blocked the hydrotropic response. A computer-assisted image analysis showed that the hydrotropic curvature in the roots of ageotropum pea was chiefly due to a greater inhibition of elongation on the humid side than the dry side of the roots. Similarly, gravitropic curvature of Alaska pea roots resulted from inhibition of elongation on the lower side of the horizontally placed roots, while the upper side of the roots maintained a normal growth rate. Gravitropic bending of Alaska pea roots was apparent 30 min after stimulation, whereas differential growth as well as curvature in positive root hydrotropism of ageotropum pea became visible 4–5 h after the continuous hydrostimulation. Application of 2,3,5-triiodobenzoic acid or ethyleneglycol-bis-( β -aminoethylether)-N,N,N',N'-tetraacetic acid was inhibitory to both root hydrotropism of ageotropum pea and root gravitropism of Alaska pea. Some mutual response mechanism for both hydrotropism and gravitropism may exist in roots, although the stimulusperception mechanisms differ from one another.     

Induction of hydrotropism in clinorotated seedling roots of Alaska pea,Pisum sativum L.

Roots of the agravitropic pea (Pisum sativum L.) mutantageotropum show positive hydrotropism, whereas roots of Alaska peas are hydrotropically almost non-responsive. When the gravitropic response was nullified by rotation on clinostats, however, roots of Alaska peas showed unequivocal positive hydrotropism in response to a water potential gradlent. These results suggest that roots of Alaska peas possess normal ability to respond hydrotropically and their weak hydrotropic response results from a counteracting effect of gravitropism.     

MIZ1-regulated hydrotropism functions in the growth and survival of Arabidopsis thaliana under natural conditions

Background and Aims

Root hydrotropism is a response to water-potential gradients that makes roots bend towards areas of higher water potential. The gene MIZU-KUSSEI1 (MIZ1) that is essential for hydrotropism in Arabidopsis roots has previously been identified. However, the role of root hydrotropism in plant growth and survival under natural conditions has not yet been proven. This study assessed how hydrotropic response contributes to drought avoidance in nature.

Methods

An experimental system was established for the study of Arabidopsis hydrotropism in soil. Characteristics of hydrotropism were analysed by comparing the responses of the miz1 mutant, transgenic plants overexpressing MIZ1 (MIZ1OE) and wild-type plants.

Key Results

Wild-type plants developed root systems in regions with higher water potential, whereas the roots of miz1 mutant plants did not show a similar response. This pattern of root distribution induced by hydrotropism was more pronounced in MIZ1OE plants than in wild-type plants. In addition, shoot biomass and the number of plants that survived under drought conditions were much greater in MIZ1OE plants.

Conclusions

These results show that hydrotropism plays an important role in root system development in soil and contributes to drought avoidance, which results in a greater yield and plant survival under water-limited conditions. The results also show that MIZ1 overexpression can be used for improving plant productivity in arid areas.     

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.     

植物根向水性反应研究进展

根的向水性生长是指植物通过根尖感知土壤中的水分梯度, 向着水势较高区域生长的一种生物学特性, 这一特性对植物从土壤中有效获取水分极为重要。植物向水性研究已成为当前植物学研究的热点领域, 但对于调控这一生理反应的分子机制仍知之甚少。目前的研究表明, MIZ1和GNOM作为植物向水性反应的重要调节因子, 正向调控植物根的向水性生长。此外, 植物激素、光、ROS及钙离子也参与调节植物的向水性反应。该文将从向水性的研究历史、调控基因以及内外因素等方面进行阐述, 便于读者全面了解植物向水性研究进展, 以期为向水性研究提供新思路。     

Hormonal interactions during root tropic growth: hydrotropism versus gravitropism

Terrestrial plants have evolved remarkable morphological plasticity that enables them to adapt to their surroundings. One of the most important traits that plants have acquired is the ability to sense environmental cues and use them as a basis for governing their growth orientation. The directional growth of plant organs relative to the direction of environmental stimuli is a tropism. The Cholodny–Went theory proposes that auxin plays a key role in several tropisms. Recent molecular genetic studies have strongly supported this hypothesis for gravitropism. However, the molecular mechanisms of other tropisms are far less clear. Hydrotropism is the response of roots to a moisture gradient. Since its re-discovery in 1985, root hydrotropism has been shown to be common among higher plant species. Additionally, in some species, gravitropism interferes with hydrotropism, suggesting that both shared and divergent mechanisms mediating the two tropisms exist. This hypothesis has been supported by recent studies, which provide an understanding of how roots sense multiple environmental cues and exhibit different tropic responses. In this review, we focus on the overlapping and unique mechanisms of the hormonal regulation underlying gravitropism and hydrotropism in roots.     

Characterization of hydrotropism: the timing of perception and signal movement from the root cap in the agravitropic pea mutant ageotropum

In this study, ageotropum pea mutant was used to determine the threshold time for perception of an osmotic stimulation in the root cap and the time requirement for transduction and transmission of the hydrotropic signal from the root cap to the elongation region. The threshold time for the perception of an osmotic stimulation was compared to current estimates of threshold times for graviperception in roots. The time required for transduction and transmission in the hydrotropic response of ageotropum was compared to the time requirement in the gravity response of Alaska pea roots. We determined that threshold time for perception of an osmotic stimulation in the root cap is very rapid, occurring in less than 2 min following the application of sorbitol to the root cap. Furthermore, a single 5 min exposure of sorbitol to the root cap fully induced a hydrotropic response. We also found that transduction and transmission of an osmotic stimulus requires 90-120 min for movement from the root cap to more basal tissues involved in differential growth leading to root curvature. The very rapid threshold time for perception of root hydrotropism is similar to those times reported for root gravitropism. However, the time required for the transduction and transmission of an osmotic stimulation from the root cap is significantly longer than the time required in gravitropism. These results suggest that there must exist some differences between root hydrotropism and gravitropism in either the rate or mechanisms of transduction and transmission of the tropistic signal from the root cap.     

玉米初生根向水性诱导优化试验研究

为了研究湿度梯度对根系向水性反应的影响,采用Takahashi and Scott于1993年创建的方法,设置以下3个试验:1)向水性诱导物不同倾斜角试验;2)根系距向水性诱导物不同距离试验;3)根尖距底部饱和K2CO3溶液不同距离试验。同时,还研究了根长和根系延伸速率对根系向水性弯曲的影响。结果表明,用饱和K2CO3溶液控制湿度时根系的向水性弯曲度明显大于纯水。随着诱导物倾斜角的增大,向水性弯曲增强。与距诱导物3 mm和6 mm相比,根系直接接触诱导物时表现出最大的向水性反应。与根尖距底部盐溶液6 cm相比,相距4 cm时向水性弯曲度增大,这些与根尖周围的湿度梯度增大有关。当根长为1.0、1.5、2.0、2.5、3.0 cm时,短根比长根表现出更大的向水性反应,这可能与其较慢的延伸速率为根系对湿度梯度的反应提供了更充足的时间有关。为了验证这个假说,用相同长度的根系、通过控制不同温度进行试验,结果表明根系的向水性弯曲随温度升高而降低。可见,玉米初生根的向水性反应受环境和根系发育阶段两方面影响。当根系相距诱导物较近、根系周围的湿度梯度较大时,根系向水性反应更强。而且,具有较小延伸速率根系的向水性反应更大。考虑到干旱条件下根系伸长慢、且土壤中湿度梯度大,因而可以认为干旱条件下根系的向水性生长在玉米吸收水分中有重要作用。同时,对根系向水性诱导方法的优化有助于其生理机制的进一步研究。     

Induction of hydrotropism in clinorotated seedling roots of Alaska pea, Pisum sativum L

Roots of the agravitropic pea (Pisum sativum L.) mutant ageotropum show positive hydrotopism, whereas roots of Alaska peas are hydrotropically almost non-responsive. When the gravitropic response was nullified by rotation on clinostats, however, roots of Alaska peas showed unequivocal positive hydrotropism in response to a water potential gradient. These results suggest that roots of Alaska peas possess normal ability to respond hydrotropicallly and their weak hydrotropic response results from a counteracting effect of gravitropism.     

GNOM-Mediated Vesicular Trafficking Plays an Essential Role in Hydrotropism of Arabidopsis Roots

Roots respond not only to gravity but also to moisture gradient by displaying gravitropism and hydrotropism, respectively, to control their growth orientation, which helps plants obtain water and become established in the terrestrial environment. As gravitropism often interferes with hydrotropism, however, the mechanisms of how roots display hydrotropism and differentiate it from gravitropism are not understood. We previously reported MIZU-KUSSEI1 (MIZ1) as a gene required for hydrotropism but not for gravitropism, although the function of its protein was not known. Here, we found that a mutation of GNOM encoding guanine-nucleotide exchange factor for ADP-ribosylation factor-type G proteins was responsible for the ahydrotropism of Arabidopsis (Arabidopsis thaliana), miz2. Unlike other gnom alleles, miz2 showed no apparent morphological defects or reduced gravitropism. Instead, brefeldin A (BFA) treatment inhibited both hydrotropism and gravitropism in Arabidopsis roots. In addition, a BFA-resistant GNOM variant, GN^M696L, showed normal hydrotropic response in the presence of BFA. Furthermore, a weak gnom allele, gnom^B/E, showed defect in hydrotropic response. These results indicate that GNOM-mediated vesicular trafficking plays an essential role in hydrotropism of seedling roots.Stationary growth is a distinct feature of plants and distinguishes them from other organisms. Plants have evolved a variety of mechanisms for responding to environmental cues, which enables them to survive in the presence of limited resources or environmental stresses. One of the most important growth adaptations plants have acquired is tropism, growth response that involves bending or curving of plant organs toward or away from a stimulus. For example, roots display tropisms in response to environmental cues such as gravity, light, touch, and moisture (Darwin and Darwin, 1880; Takahashi, 1997; Correll and Kiss, 2002; Monshausen et al., 2008). Gravitropism has been the subject of intense study, while other tropic responses of roots have been less well characterized. There is some evidence of hydrotropism in roots, but this response has proven difficult to differentiate from gravitropism, as the latter always interferes with hydrotropism (Jaffe et al., 1985; Takahashi, 1994; Takahashi, 1997). The demonstration of true hydrotropism in roots has facilitated the identification of some of the physiological aspects of hydrotropism and its existence in a wide range of plant species. However, the underlying mechanisms that regulate hydrotropism remain unknown. The limited supply of water and precipitation in many parts of the world greatly affects agriculture and ecosystems. Elucidating the molecular mechanism of hydrotropism in roots is therefore important not only for understanding how terrestrial plants adapt to changes in moisture, but also for improving crop yields and biomass production.The isolation and analysis of hydrotropism-deficient mutants using the model plant species Arabidopsis (Arabidopsis thaliana) represents a potent tool for dissecting the molecular mechanism of hydrotropism. Previously, we isolated an ahydrotropic mutant of Arabidopsis, mizu-kussei1 (miz1), and showed that MIZ1 encodes a protein of unknown function (Kobayashi et al., 2007). In light of both the physiological features of hydrotropism, as well as what we have learned from genetic studies of other tropisms, it is unlikely that miz1 alone governs the hydrotropic response. In support of this, we have identified a second ahydrotropic mutant, miz2, a unique allele of gnom that confers ahydrotropic but not agravitropic growth, which implies distinct roles of vesicular trafficking between hydrotropism and gravitropism in roots.     

Characterization of hydrotropism: the timing of perception and signal movement from the root cap in the agravitropic pea mutant ageotropum.

In recent years, experiments have demonstrated that the gravity response of roots can be separated from the hydrotropic response by using the agravitropic pea mutant ageotropum. Though this mutant has been a useful tool for demonstrating the existence of the hydrotropic response of roots, little is known about how perception, transduction, transmission, and the growth response is accomplished. In this study, we have used the ageotropum mutant to investigate both the threshold time for perception of an osmotic stimulation and the minimum time required for signal transduction and transmission in roots following an osmotic stimulation at the root cap. In addition, we have compared the threshold times and signal transmission times of hydrotropism in the ageotropum roots to the gravity response of Alaska pea roots.     

Involvement of Arabidopsis thaliana phospholipase Dζ2 in root hydrotropism through the suppression of root gravitropism

Root hydrotropism is the phenomenon of directional root growth toward moisture under water-deficient conditions. Although physiological and genetic studies have revealed the involvement of the root cap in the sensing of moisture gradients, and those of auxin and abscisic acid (ABA) in the signal transduction for asymmetric root elongation, the overall mechanism of root hydrotropism is still unclear. We found that the promoter activity of the Arabidopsis phospholipase Dζ2 gene (PLDζ2) was localized to epidermal cells in the distal root elongation zone and lateral root cap cells adjacent to them, and that exogenous ABA enhanced the activity and extended its area to the entire root cap. Although pldζ2 mutant root caps did not exhibit a morphological phenotype in either the absence or presence of exogenous ABA, the inhibitory effect of ABA on gravitropism, which was significant in wild-type roots, was not observed in pldζ2 mutant roots. In root hydrotropism experiments, pldζ2 mutations significantly retarded or disturbed root hydrotropic responses. A drought condition similar to that used in a hydrotropism experiment enhanced the PLDζ2 promoter activity in the root cap, as did exogenous ABA. These results suggest that PLDζ2 responds to drought through ABA signaling in the root cap and accelerates root hydrotropism through the suppression of root gravitropism.     

Auxin response, but not its polar transport, plays a role in hydrotropism of Arabidopsis roots

Plants are sessile in nature, and need to detect and respond to many environmental cues in order to regulate their growth and orientation. Indeed, plants sense numerous environmental cues and respond via appropriate tropisms, and it is widely accepted that auxin plays an important role in these responses. Recent analyses using Arabidopsis have emphasized the importance of polar auxin transport and differential auxin responses to gravitropism. Even so, the involvement of auxin in hydrotropism remains unclear. To clarify whether or not auxin is involved in the hydrotropic response, Arabidopsis seedlings were treated with inhibitors of auxin influx (3-chloro-4-hydroxyphenylacetic acid), efflux (1-naphthylphthalemic acid and 2,3,5-triiodobenzoic acid), and response (p-chlorophenoxyisobutylacetic acid), and their effects were examined on both hydrotropic and gravitropic responses. In agreement with previous reports, gravitropism was inhibited by all the chemicals tested. By contrast, only an inhibitor of the auxin response (p-chlorophenoxyisobutylacetic acid) reduced hydrotropism, whereas inhibitors for influx or efflux of auxin had no effect. These results suggest that auxin response, apart from its polar transport, plays a definite role in hydrotropic response, and will evoke a new concept for the auxin-mediated regulation of tropisms.     

Water potential, turgor and cell wall properties in elongating tissues of the hydrotropically bending roots of pea (Pisum sativum L.)

The hydrotropic bending of roots of an ageotropic pea mutant, ageotropum, was studied in humid air in a chamber with a steady humidity gradient. We examined the effects of atmospheric humidity around the root on the water status of root tissues, as well as the wall growth and the hydraulic properties of the elongating tissues. Atmospheric humidity at the surface of the root was clearly lower on the side orientated towards the air with lower humidity than on the side orientated towards the air with higher humidity. However, there were no differences in water potential and osmotic potential between the tissues that faced air with higher and lower humidities in the elongating and mature regions. Plastic extensibility was higher in the tissues that faced the air with lower humidity than in the tissues that faced the air with higher humidity. No differences in turgor pressure and yield threshold were observed between the tissues that faced air with higher and lower humidities. Therefore, the extensibility of the cell wall appeared to be responsible for the different growth rates of tissues in root hydrotropism. A further probable cause of the hydrotropical bending of roots is changes in the hydraulic conductance in the elongating tissues. Since the hydrotropic bending of roots occurred only when a root tip was exposed to a humidity gradient, hydrotropism might occur after perception of a difference in humidity by the root tip, with accompanying changes in cell wall extensibility and hydraulic conductance.     

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.