Roles for Class III HD-Zip and KANADI genes in Arabidopsis root development

Meristems within the plant body differ in their structure and the patterns and identities of organs they produce. Despite these differences, it is becoming apparent that shoot and root apical and vascular meristems share significant gene expression patterns. Class III HD-Zip genes are required for the formation of a functional shoot apical meristem. In addition, Class III HD-Zip and KANADI genes function in patterning lateral organs and vascular bundles produced from the shoot apical and vascular meristems, respectively. We utilize both gain- and loss-of-function mutants and gene expression patterns to analyze the function of Class III HD-Zip and KANADI genes in Arabidopsis roots. Here we show that both Class III HD-Zip and KANADI genes play roles in the ontogeny of lateral roots and suggest that Class III HD-Zip gene activity is required for meristematic activity in the pericycle analogous to its requirement in the shoot apical meristem.     

β-glucuronidase as a marker for clonal analysis of tomato lateral roots

In higher plants, the root-shoot axis established during embryogenesis is extended and modified by the development of primary and lateral apical meristems. While the structure of several shoot apical meristems has been deduced by combining histological studies with clonal analysis, the application of this approach to root apical meristems has been limited by a lack of visible genetic markers. We have tested the feasibility of using a synthetic gene consisting of the maize transposable elementActivator (Ac) inserted between a 35S CaMV promoter and the coding region of a -glucuronidase (GUS) reporter gene as a means of marking cell lineages in roots. The GUS gene was activated in individual cells byAc excision, and the resulting sectors of GUS-expressing cells were detected with the histochemical stain X-Gluc. Sectors in lateral roots originated from bothAc excision in meristematic cells and from parent root sectors that bisect the founder cell population for the lateral root initial. Analysis of root tip sectors confirmed that the root cap, and root proper have separate initials. Large sectors in the body of the lateral root encompassed both cortex and vascular tissues. The number of primary initial cells predicted from the size and arrangement of the sectors observed ranged from two to four and appeared to vary between roots. We conclude that transposon-based clonal analysis using GUS expression as a genetic marker is an effective approach for deducing the functional organization of root apical meristems.     

Proteomic analysis of root meristems and the effects of acetohydroxyacid synthase-inhibiting herbicides in the root of Medicago truncatula

Quantitative proteome analyses of meristematic and nonmeristematic tissues from Medicago truncatula primary and lateral roots and meristem tissues from plants treated with acetohydroxyacid synthase-inhibiting herbicides were made. The accumulation of 81 protein spots changed in meristematic and nonmeristematic tissues and 51 protein spots showed significant changes in accumulation in herbicide-treated meristems. Identified proteins indicate two trends, (i) increased accumulation of cell division and redox-mediating proteins in meristems compared to nonmeristematic tissues and (ii) increased accumulation of pathogenesis-related and decreased accumulation of metabolic proteins in herbicide-treated roots.     

Changes in hormonal balance and meristematic activity in primary root tips on the slowly rotating clinostat and their effect on the development of the rapeseed root system

The morphometry of the root system, the meristematic activity and the level of indole-3-acetic acid (IAA), abscisic acid (ABA) and zeatin in the primary root tips of rapeseed seedlings were analyzed as functions of time on a slowly rotating clinostat (1 rpm) or in the vertical controls (1 rpm). The fresh weight of the root system was 30% higher throughout the growth period (25 days) in clinorotated seedlings. Morphometric analysis showed that the increase in biomass on the clinostat was due to greater primary root growth, earlier initiation and greater elongation of the secondary roots, which could be observed even in 5-day-old seedlings. However, after 15 days, the growth of the primary root slowed on the clinostat, whereas secondary roots still grew faster in clinorotated plants than in the controls. At this time, the secondary roots began to be initiated closer to the root tip on the clinostat than in the control. Analysis of the meristematic activity and determination of the levels in IAA, ABA and zeatin in the primary root tips demonstrated that after 5 days on the clinostat, the increased length of the primary root could be the consequence of higher meristematic activity and coincided with an increase in both IAA and ABA concentrations. After 15 days on the clinostat, a marked increase in IAA, ABA and zeatin, which probably reached supraoptimal levels, seems to cause a progressive disturbance of the meristematic cells, inducing a decrease of primary root growth between 15 and 25 days. These modifications in the hormonal balance and the perturbation of the meristematic activity on the clinostat were followed by a loss of apical dominance, which was responsible for the early initiation of secondary roots, the greater elongation of the root system and the emergence of the lateral roots near the tip of the primary root.     

Changes in Phytohormone Status in Stems and Roots after Decapitation of Pea Seedlings

Experiments were performed on the first and second internodes and 4-cm-long apical segments of main roots of pea (Pisum sativum L.) seedlings, grown in the light and decapitated above the second node on the seventh day after seed germination. Endogenous phytohormones were measured by the enzyme-linked immunosorbent assay during three days after decapitation of seedlings. The IAA level in the internodes decreased 2–3 times on the second day after decapitation of seedlings while the cytokinin level increased 5–6 times for zeatin and zeatin riboside (Z and ZR) and 1.5–2 times for isopentenyl adenine and isopentenyl adenosine (IP and IPA). In contrast to internodes, the IP and IPA contents in the roots of decapitated seedlings did not change, but the levels of Z and ZR increased 1.5–2 times compared to intact plant roots. The IAA level in the apical region of root remained almost unchanged after the removal of shoot apex. It was concluded that the apical meristem of the main root is not the site of the cytokinin response to the auxin signal coming from the stem apex and that a slight accumulation of Z and ZR after decapitation is due to upper zones of the root. There was no difference in the content of gibberellin-like substances between the internodes of intact and decapitated seedlings. However, the content of gibberellins (GA) in the root tip decreased after decapitation of seedling, which suggests an essential role of apical bud in supplying the root with GA and/or intermediates for their biosynthesis.     

The Response of Root Meristems to Colchicine and Indol-3yl-acetic acid in Vicia faba L.

The effects of colchicine and IAA treatments on mitotic activityin various root proliferating tissues have been determined.Lateral root primordia were not affected by IAA, though 24 hfollowing treatment mitotic activity was severely inhibitedin the apical meristems of 1-cm-long attached lateral rootsand primary roots. Primordia were also less sensitive to colchicinetreatment than root apical meristems. Thus telophase figureswere present in the former meristems 3 h following treatment,but not in the latter. Primordia and apical meristems respondedto the same extent, however, to the colchicine-induced increasein number of cells in metaphase, anaphase, and telophase, 3h after treatment began. The apparent difference between largeprimordia and root apical meristems in this respect was dueto the failure of colchicine to penetrate the cells of the formerproliferating tissues as rapidly as the latter. IAA was foundto prevent the increased MI found 24 h following colchicinetreatment only in those meristems where IAA inhibited mitoticactivity at this time. IAA treatments, either alone or withcolchicine, were also found to maintain mitotic activity in1-cm-long lateral roots which were excised from the primaryroots 24 h previously. In such laterals which were not treatedwith IAA, MI was zero at 24 h. It is concluded from the datareported in this paper that, during the development of rootapical meristems, changes take place in the response of cellsto factors affecting mitotic activity.     

Transport of benzyl-8-14C-adenine in pea seedlings in relation to stem apical dominance

In intact, decapitated and decapitated indole-3-acetic acid (IAA) treated pea seedlings the translocation of benzyl-8-^l4C-adenin (¹⁴C-BA) from the roots was studied with regard to the release of lateral buds from apex-induced inhibition. In intact plants (controls) a substantial part of the activity was found in the apical part of the epicotyl. Decapitation resulted in the initiation of growth of lateral buds. As early as 24 h after decapitation and application of¹⁴C-BA a significantly higher activity was found in growing lateral buds (cotylars) of decapitated plants than in inhibited ones of intact or IAA-treated decapitated plants. The accumulation of¹⁴C-activity in stump tops of decapitated plants treated with IAA was associated with the thickening growth.     

Effect of Abscisic Acid and Other Plant Hormones on Growth of Apical and Lateral Buds of Seedlings

The effect of abscisic acid (AbA) on the growth of lateral and apical buds was studied in seedlings of Pisum sativum and some other species. The hormone was applied in three different ways: 1) directly to the lateral bud on the second node of decapitated pea seedlings as 5 μI droplets in an ethanolic solution; 2) to the cut surface of decapitated seedlings: 3) to the apical bud of intact plants. AbA directly applied in amounts of 5 to 0.1 μg to the lateral bud of the second node of decapitated seedlings had a strong inhibitory effect on the bud. Application to the cut surface of seedlings decapitated about 5 mm above the second node resulted in slight inhibition of the lateral bud on the second node and in growth promotion of the bud on the first node. When AbA at 10 to 0.1 μg was applied to the apical bud of intact seedlings, the growth of this bud was inhibited but the lateral buds grew out. It is concluded that the release of the lateral buds from apícal dominance is the result of the inhibitory effect of AbA on growth of the apical bud and of low transport of AbA. This conclusion is supported by application of GA₃ and IAA, individually and each combined with AbA.     

Effect of water stress on root meristems in woody and herbaceous plants during the first stage of development

We investigated the effect of water stress on the root system architecture of pine saplings and pea seedlings during the first stage of development. Attention was focused on meristematic tissue situated at the root tip because of the leading role played by the tissue in the planning of root system architecture. The data showed that both species are extremely sensitive and that plants arrest their growth immediately during water stress treatment. When stress treatment was not intense, both species recovered growth but presented modifications in the root system architecture. In pine saplings, the modification in root system architecture was the consequence of fine root meristems not recovering from water stress. The saplings survived by producing new lateral meristems from the cortical tannin zone above the fine root tip. In the case of pea seedlings, the meristematic tissues in the primary root arrested proliferation during water stress although they recovered when the event occurred during the first hours of germination. The response was different when water stress was enforced on older seedlings. In this case, root meristems never completely recovered their proliferation despite the increase in proline content observed in the cells. The modification of root system architecture in pea seedlings depended on the arrest of primary root elongation and the formation of new root laterals. As regards the primary roots, water stress treatment induced along the axis the formation of irregular ‘swellings’ in the cortical zone above the meristematic zone. Anatomical investigations suggested that such swellings may have derived from the changes in elongation direction of derivatives. The formation of new laterals was observed in hydroponic cultures when water stress treatment was enforced slowly and prolonged for a long time. The production of new lateral meristems may have been a similar response of woody and herbaceous plants to water stress conditions. It is not known whether these new meristems present characteristics of resistance to water stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

Some Cytological Changes accompanying the Regeneration of Meristematic Activity at the Apex of Decapitated Roots of Vicia faba L.

The changes that took place in mitotic index (MI), labellingindex (LI) and the relative proportions of interphase nucleiwith different amounts of DNA have been investigated duringthe regeneration of meristematic activity at the apex of rootsof Vicia faba over the 144 h period following removal of thecap and apical mm of the meristem. Measurements were also madeof the corresponding changes that took place as cells were displacedbasally along the root from the apex over the experimental period.In both parts of the root, MI and the relative proportions ofnuclei with different DNA contents changed from levels similarto those at the apex of the controls at the start and end ofthe experiment to levels resembling those found in more matureparts of the root at 24 and 48 h. In contrast to these results,LI declined over the experimental period. These cytologicalchanges were aresult of the development of lateral root primordiain both the apical 2 mm of the decapitated roots and as cellswere displaced out of the meristem into more basal parts ofthe root. It was concluded that the events leading to the regenerationof meristematic activity at the apex of roots from which thecap and apical mm of the meristem were removed, are no differentfrom those which result in lateral formation as cells are displacedbasally along the primary root from the apex, and they takeplace over the same time interval in both systems.     

Effect of Main Root Decapitation on Root Branching in Flax Seedlings

Root branching patterns in intact and decapitated flax (Linum usitatissimumL.) roots were compared. The number of initiated primordia in the control and decapitated roots was similar, but decapitated roots produced an increased number of lateral roots owing to an increase in the number of primordia developed into the laterals. It is suggested that the apical meristem influences lateral root development only at the stage of root emergence from the parent root.     

Root growth, developmental changes in the apex, and hydraulic conductivity for Opuntia ficus-indica during drought

Developmental changes in the root apex and accompanying changes in lateral root growth and root hydraulic conductivity were examined for Opuntia ficus-indica (L.) Miller during rapid drying, as occurs for roots near the soil surface, and more gradual drying, as occurs in deeper soil layers. During 7 d of rapid drying (in containers with a 3-cm depth of vermiculite), the rate of root growth decreased sharply and most root apices died; such a determinate pattern of root growth was not due to meristem exhaustion but rather to meristem mortality after 3 d of drying. The length of the meristem, the duration of the cell division cycle, and the length of the elongation zone were unchanged during rapid drying. During 14 d of gradual drying (in containers with a 6-cm depth of vermiculite), root mortality was relatively low; the length of the elongation zone decreased by 70%, the number of meristematic cells decreased 30%, and the duration of the cell cycle increased by 36%. Root hydraulic conductivity ( L _P) decreased to one half during both drying treatments; L _P was restored by 2 d of rewetting owing to the emergence of lateral roots following rapid drying and to renewed apical elongation following gradual drying. Thus, in response to drought, the apical meristems of roots of O. ficus-indica near the surface die, whereas deeper in the substrate cell division and elongation in root apices continue. Water uptake in response to rainfall in the field can be enhanced by lateral root proliferation near the soil surface and additionally by resumption of apical growth for deeper roots.     

The indirect role of 2,4-D in the maintenance of apical dominance in decapitated sunflower seedlings (Helianthus annuus L.)

When sufficient 2,4-D to maintain apical dominance for at least 21d was applied to the cut stem interface of sunflower seedlings which had been decapitated in the epicotyl, it could not be detected in the vicinity of the inhibited axillary buds 7d after application. Rather the 2,4-D concentrated at the stump apex where it was associated with formation of meristematic tissue. The results indicate an indirect role for 2,4-D in the maintenance of apical dominance in this system, possibly involving the induced meristematic activity.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - [¹⁴C]2,4-D 2,4-dichlorophenoxy[2-¹⁴C]acetic acid - IAA indol-3-yl-acetic acid     

Translocation of14C-abscisic acid from roots into the aboveground part of pea (pisum sativum L.) seedlings

The translocation of¹⁴C-ABA from roots into other parts of the plant was followed in intact and decapitated pea seedlings. In intact plants ABA from roots was translocated above all into the apical part of epicotyl. In decapitated plants the regulative ability of intact apex can be partly simulated by exogenous IAA. The growth of lateral buds occurring after decapitation was associated with an intensive flow of¹⁴C-ABA from roots into released lateral buds as late as 72 h after decapitation,i.e. in the stage of intensive elongation growth of buds.     

Isolation and identification of lateral bud growth inhibitor,indole-3-aldehyde,involved in apical dominance of pea seedlings

A lateral bud growth inhibitor was isolated from etiolated pea seedlings and identified as indole-3-aldehyde. The indole-3-aldehyde content was significantly higher in the diffusates from explants with apical bud and indole-3-acetic acid treated decapitated explants, in which apical dominance is maintained, than in those from decapitated ones releasing apical dominance. When the indole-3-aldehyde was applied to the cut surface of etiolated decapitated plants or directly to the lateral buds, it inhibited outgrowth of the latter. These results suggest that indole-3-aldehyde plays an important role as a lateral bud growth inhibitor in apical dominance of pea seedlings.     

Growth Substances in the Roots of Vicia faba II. THE EFFECTS OF AGEING AND EXCISION OF THE MAIN TAP-ROOT MERISTEM

A quantitative chromatographic study has been made of the changesin the activity and distribution of the main ether-soluble acidauxins of Vicia faba seedling root systems during developmentand resulting from excision of the main tap-root meristem. AnIAA-like auxin (AP (ii)) is apparently synthesized predominantlyin apical and at a lower rate in lateral meristems. Productionseems to stop when meristematic growth stops. Its concentrationin mature extended cells is much lower and may fall to zeroin old cells, suggesting active degradation by an auxin-oxidase.Excision of the main tap-root tip gradually results in a greatlyaugmented production of AP (ii) in lateral meristems, conceivablythe result of correlative growth promotion. A second auxin and root-growth inhibitor (AP (iii)) is presentat higher activity levels than AP (ii) in tap-root meristemsand at the same level in lateral meristems. In mature cellsits activity is much lower than that of AP (ii). In contrastto AP (ii) it accumulates in both tap-root meristems and maturetissue as the root system ages. It could also be produced duringmeristematic growth but is not subsequently degraded. As withAP (ii), excision of the tap-root tip brings about a great increasein its concentration in lateral tips. A third auxin and root-growth accelerator (AP (i)) (accelerator?) is present in lower concentrations in both meristem and maturetissue. Its concentration tends to decrease with ageing and,in lateral meristems, is not affected by tap-root tip excision. It is suggested that AP (ii) produced by the meristem is normallyat suboptimal levels in the extending cells and may be the principalhormone controlling extension growth. AP (iii) accumulationmay account for growth deceleration on ageing. The role of AP(i) remains obscure. It is unlikely that correlative effectsof the taproot tip on lateral root growth are exercised directlyvia these auxins.     

The involvement of the root apex and cytokinins in the control of lateral root emergence in lettuce seedlings

Removal of the apical 3 mm of the primary root of hydroponically-grown lettuce seedlings 3 or 5 days after sowing, prevented further elongation of the root and increased both the number and total length of lateral roots. The length of the lateral zone, i.e. the distance from the base of the parent root to the lateral nearest the tip, except on one occasion, remained the same as the control in both 3 and 5 day treatments, until the length of the decapitated root (which had ceased elongating) became limiting.Zeatin applied via the roots, at a concentration range from 3 × 10^–10 M to 10^–8 M reduced tap root extension growth at all concentrations. Lateral root emergence was enhanced by low zeatin concentrations and retarded by higher ones. In general, the lateral zone length was the same in cytokinin-treated plants as in untreated controls.     

Die for living better: Plants modify root system architecture through inducing PCD in root meristem under severe water stress

Plant root development is highly plastic in order to cope with various environmental stresses; many questions on the mechanisms underlying developmental plasticity of root system remain unanswered. Recently, we showed that autophagic PCD occurs in the region of root apical meristem in response to severe water deficit. We provided evidence that reactive oxygen species (ROS) accumulation may trigger the cell death process of the meristematic cells in the stressed root tips. Analysis of BAX inhibitor-1 (AtBI1) expression and the phenotypic response of atbi1-1 mutant under the severe water stress revealed that AtBI1 and the endoplasmic reticulum (ER) stress response pathway modulate water stress-induced PCD. As a result, the thick and short lateral roots with increased tolerance to the stress are induced. We propose that under severe drought condition, plants activate PCD program in the root apical root meristem, so that apical root dominance is removed. In this way, they can remodel their root system architecture to adapt the stress environment.Key words: Arabidopsis, adaptation, PCD, root system architecture, water stressPlant shoot apical dominance is well known. The axillary buds are inhibited by the growing shoot apical meristem, and they would not grow until the shoot apical meristems are decapitated.¹ The same phenomenon has been found in the roots of dicot plants. Primary roots exhibit apical dominance over lateral roots and are able to penetrate deeply into the soil. Lateral root primordia were rapidly activated when primary root tips of lettuce (Lactuca sativa) were removed.² It is apparent that apical meristem activity in shoots and roots determines lateral organs and the shapes of above ground and root system architecture under normal conditions. Many plants have active meristematic activity in their shoot and root tips through their whole life resulting in indeterminate development of their shoots and primary roots, whereas others generate branches at certain developmental stages when the meristematic activity and apical dominance become low.It has long been known that plants modify their root morphology, orientation and increase root biomass to maximize water and nutrient absorption.^3,4 However, how the root morphology and architecture are changed in response to water shortage and what the underlying mechanisms are largely unknown. Previously, it has been reported that plants, due to their sessile nature, have developed a very important adaptive mechanism, namely hydrotropism to avoid the damage caused by water shortage. Plant roots can sense the moisture gradient and grow toward to water or moisture when they are grown at conditions with non-uniform water distribution.⁵ Recently, we found another key mechanism through which plants can remove root apical dominance and remodel their root system architecture, thus to minimize the damage caused by a uniform severe water shortage condition.⁶Firstly, we found that growth rates of the Arabidopsis plants germinated on normal conditions were reduced when the concentrations of PEG in the growth media was increased, and primary roots of the stressed plants completely ceased growth when the PEG concentrations reached 40% (w/v) in the agar medium, a severe water stress. The results showed that growth cessation of the stressed plants was caused by PCD of the cells in the region of root apical meristems, and the cells underwent autophagic cell death upon the most severe water deficit. Secondly, we demonstrated that AtBI-1, a marker gene which plays a critical role in protecting the cells from ER stress-induced PCD in plants, mediates water stress-induced PCD of the root meristem. Further observation of ROS accumulation in the root tips upon to the severe water stress suggests that the high level of the ROS may disrupt the ER homeostasis and ROS may act as a signal to trigger the PCD. Importantly, we found that the occurrence of PCD of the meristematic cells of the stressed plants promoted the development of lateral roots. These short and tublized lateral roots grew slowly under severe water stress, but they could immediately become normal lateral roots and resume their elongation and after rehydration. Plant growth is subsequently restored to complete their entire life cycle. However, the lateral roots induced by decapitating primary root tips under normal conditions did not continue elongation like the stress induced lateral roots, and they cannot restore their growth after rehydration.Based on these results, we propose that plants can sense the severity of water stress, initiate autophagic PCD of meristematic cells in Arabidopsis root tips through ER stress signaling pathway and stimulate lateral root development (Fig. 1). Death of meristematic cells results in the loss of mitotic cell division activity in meristem and eventual root meristem function. The outcome of PCD caused-loss of root meristem activity is same as the surgical removal of apical root tips. In both cases, lateral root primordia are activated and lateral root emergence is promoted. However, the main difference between water stress induced-loss of root meristem function and surgical decapitation of root tips is that the former induces lateral roots with enhanced stress tolerance plays key roles in post-stress recovery, whereas the latter promotes development of lateral roots do not alter stress response. This implicates that stress-induced loss of meristem function and subsequent occurrence of specified lateral roots are adaptive mechanisms for plants to cope with the severe water stress. In other words, plants induce cell death of root meristem for living better.Open in a separate windowFigure 1A simplified model depicting the role of PCD in root meristem in plastic development of root system architecture in response to water stress.It is known that auxin distribution and maxima play key roles in lateral root initiation and emergence.^7–10 Alteration in auxin polar transport has been proposed as the main reason of decapitation induced lateral root development.¹¹ It is conceivable that auxin is also involved in stress induced-lateral root formation and development, but it is clear that interplay between stress signaling cascades and developmental signalings occurs after perception of the stress signals by plant cells resulting in root system development remodeling. These findings provide novel insights into mechanisms of plants to adapt to the uniform severe water stress at organ, cellular and molecular levels. However, the research of plastic development of root system in response to water stress is still in its infancy. Combinatorial strategies for the investigation of stress induced-PCD of root meristematic cells and subsequent lateral root development will help to uncover the molecular mechanisms underlying this positive response of plants in response to severe water stress. In particular, further study of auxin redistribution under water stress and interaction between auxin and stress hormone signalings in remodeling root system architecture will further our understanding of how developmental plasticity of plant root system is regulated. The results will facilitate the improvement of drought tolerance in crops.     

Hormonal factors controlling the initiation and development of lateral roots

As the first part of a comprehensive study of the hormonal control of lateral root initiation and development, the effect of surgical treatments such as removal of the root tip, one or more cotyledons, the young epicotyl, or combination of these treatments, on the induction and emergence of lateral roots on the primary root of pea seedlings has been examined. Results show that removal of the root tip leads to a rapid but transitory increase in the number of lateral primordia, the largest number arising in the most apical segment of decapitated roots suggesting the accumulation of acropetally moving promoter substances in this region. The cotyledons appear to be the main source of promoter substances for both the induction and emergence of lateral roots, although one or more promoters also appear to be produced in the epicotyl. The data further indicate that the root tip is possibly the source of a substance which moves basipetally and interacts with acropetally moving promoters to regulate the zone for lateral primordia initiation; the root tip also appears to be the source of a powerful inhibitor of lateral root emergence.