Cytokinins act directly on lateral root founder cells to inhibit root initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.     

The Control of Lateral Root Development in Cultured Pea Seedlings. II. Root Fasciation Induced by Auxin Inhibitors

Lateral root development in cultured seedlings of Pisum sativum (cv. Alaska) was modified by the application of auxin transport inhibitors or antagonists. When applied either to replace the root tip or beneath the cotyledonary node, two auxin transport inhibitors, 2,3,5-triiodobenzoic acid (TIBA) and 3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-α]isoindol-8-one (DPX-1840), increased cell division activity opposite the protoxylem poles. This resulted in the formation of masses of cells, which we are calling root primordial masses (RPMs), 2 to 3 days after treatment. RPMs differed from lateral root primordia in that they lacked apical organization. Some roots however developed both RPMs and lateral roots indicating that both structures were similar in terms of the timing and location of cell division in the pericycle and endodermis leading to their initiation. Removal of the auxin transport inhibitors allowed many of the RPMs to organize later into lateral root primordia and to emerge in clusters. When the auxin, indoleacetic acid (IAA) was added to the growth medium along with DPX-1840, 3 ranks of RPMs now in the form of fasciated lateral roots emerged from the primary root. The auxin antagonist, p-chlorophenoxy-isobutyric acid (PCIB), also induced RPM formation. In contrast to DPX-1840 treatment, the addition of IAA during PCIB treatment caused normal lateral root development.     

Lateral root initiation: one step at a time

Plant growth relies heavily on a root system that is hidden belowground, which develops post-embryonically through the formation of lateral roots. The de novo formation of lateral root organs requires tightly coordinated asymmetric cell division of a limited number of pericycle cells located at the xylem pole. This typically involves the formation of founder cells, followed by a number of cellular changes until the cells divide and give rise to two unequally sized daughter cells. Over the past few years, our knowledge of the regulatory mechanisms behind lateral root initiation has increased dramatically. Here, I will summarize these recent advances, focusing on the prominent role of auxin and cell cycle activity, and elaborating on the three key steps of pericycle cell priming, founder cell establishment and asymmetric cell division. Taken together, recent findings suggest a tentative model in which successive auxin response modules are crucial for lateral root initiation, and additional factors provide more layers of control.     

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT

Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.     

Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana

Plant root systems display considerable plasticity in response to endogenous and environmental signals. Auxin stimulates pericycle cells within elongating primary roots to enter de novo organogenesis, leading to the establishment of new lateral root meristems. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in root branching are not well characterized. We find that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia. Treatment with higher doses of ACC strongly inhibits the ability of pericycle cells to initiate new lateral root primordia, but promotes the emergence of existing lateral root primordia: behaviour that is also seen in the eto1 mutation. These effects are correlated with decreased pericycle cell length and increased lateral root primordia cell width. When auxin is applied simultaneously with ACC, ACC is unable to prevent the auxin stimulation of lateral root formation in the root tissues formed prior to ACC exposure. However, in root tissues formed after transfer to ACC, in which elongation is reduced, auxin does not rescue the ethylene inhibition of primordia initiation, but instead increases it by several fold. Mutations that block auxin responses, slr1 and arf7 arf19, render initiation of lateral root primordia insensitive to the promoting effect of low ethylene levels, and mutations that inhibit ethylene-stimulated auxin biosynthesis, wei2 and wei7 , reduce the inhibitory effect of higher ethylene levels, consistent with ethylene regulating root branching through interactions with auxin.     

Hormonal regulation of lateral root development in Arabidopsis modulated by MIZ1 and requirement of GNOM activity for MIZ1 function

Plant organ development is important for adaptation to a changing environment. Genetic and physiological studies have revealed that plant hormones play key roles in lateral root formation. In this study, we show that MIZU-KUSSEI1 (MIZ1), which was identified originally as a regulator of hydrotropism, functions as a novel regulator of hormonally mediated lateral root development. Overexpression of MIZ1 (MIZ1OE) in roots resulted in a reduced number of lateral roots being formed; however, this defect could be recovered with the application of auxin. Indole-3-acetic acid quantification analyses showed that free indole-3-acetic acid levels decreased in MIZ1OE roots, which indicates that alteration of auxin level is critical for the inhibition of lateral root formation in MIZ1OE plants. In addition, MIZ1 negatively regulates cytokinin sensitivity on root development. Application of cytokinin strongly induced the localization of MIZ1-green fluorescent protein to lateral root primordia, which suggests that the inhibition of lateral root development by MIZ1 occurs downstream of cytokinin signaling. Surprisingly, miz2, a weak allele of gnom, suppressed developmental defects in MIZ1OE plants. Taken together, these results suggest that MIZ1 plays a role in lateral root development by maintaining auxin levels and that its function requires GNOM activity. These data provide a molecular framework for auxin-dependent organ development in Arabidopsis (Arabidopsis thaliana).     

Morphogenetic Effect of the Herbicide Cinch on Arabidopsis thaliana Root Development

Cinch is a morphogenetically active herbicide that inhibits primary root growth and induces abnormal ``nodule-like' lateral roots on Arabidopsis thaliana seedlings. Using 200 nm Cinch, the early stages of lateral root formation occurred along the apical half of the root axis; but once emerged, they were inhibited from further growth. Second-order lateral roots formed at the base of stunted first-order lateral roots after 5 days of Cinch treatment. Results from Cinch experiments suggested that pericycle cells are determined in the meristem to be potential sites of lateral root formation, and the developmental transition point between emerged lateral roots and subsequent growth is inhibited. Results using 2,4-dichlorophenoxyacetic acid and 2,3,5-triiodobenzoic acid suggest that Cinch is not a chemical analog of auxin. Received August 8, 1997; accepted February 23, 1998     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is regulated by auxin in both shoots and roots

The AtNRT1.1 (CHL1) gene of Arabidopsis encodes a dual-affinity nitrate transporter and contributes to both low and high affinity nitrate uptake. Localization studies have shown that CHL1 expression is preferentially targeted to nascent organs and growing regions of roots and shoots in Arabidopsis. In roots, CHL1 expression is concentrated in the tips of primary and lateral roots and is activated during lateral root initiation. In shoots, strong CHL1 expression is found in young leaves and developing flower buds. These findings suggest that CHL1 expression might be regulated by a growth signal such as the phytohormone auxin. To test this, auxin regulation of CHL1 was examined. Using transgenic Arabidopsis plants containing CHL1::GUS/GFP DNA constructs, it was found that treatment with exogenous auxin or introduction of the auxin overproducing mutations (yucca and rooty) resulted in a strong increase in CHL1::GUS/GFP signals in roots and leaves. When mature roots were treated with auxin to induce lateral root formation, CHL1::GFP signals were dramatically enhanced in dividing pericycle cells and throughout primordia development. RNA blot analysis showed that CHL1 mRNA levels in whole seedlings increase within 30 min of auxin treatment. The distribution of CHL1 expression in Arabidopsis roots and shoots was found to be similar to that of DR5::GUS, a synthetic, auxin-responsive gene. These results indicate that auxin acts as an important signal regulating CHL1 expression and contributes to the targeting of CHL1 expression to nascent organs and root tips in Arabidopsis.     

Auxin transport promotes Arabidopsis lateral root initiation

Lateral root development in Arabidopsis provides a model for the study of hormonal signals that regulate postembryonic organogenesis in higher plants. Lateral roots originate from pairs of pericycle cells, in several cell files positioned opposite the xylem pole, that initiate a series of asymmetric, transverse divisions. The auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) arrests lateral root development by blocking the first transverse division(s). We investigated the basis of NPA action by using a cell-specific reporter to demonstrate that xylem pole pericycle cells retain their identity in the presence of the auxin transport inhibitor. However, NPA causes indoleacetic acid (IAA) to accumulate in the root apex while reducing levels in basal tissues critical for lateral root initiation. This pattern of IAA redistribution is consistent with NPA blocking basipetal IAA movement from the root tip. Characterization of lateral root development in the shoot meristemless1 mutant demonstrates that root basipetal and leaf acropetal auxin transport activities are required during the initiation and emergence phases, respectively, of lateral root development.     

Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. CV. Lucknow local

Summary The effect of exogenously fed hormones on hairy root cultures of Cichorium intybus L. ev. Lucknow Local was studied. It was seen that auxin in the presence of low levels of kinetin induces rapid disorganization in hairy root cultures of C. intybus, ultimately to form suspension cultures, and this process was associated with the decrease in coumarin content in the cells. Of various treatments, it was observed that with an increase in the auxin: cytokinin ratio, the biomass decreased with the increase in disorganization index during the culture period of 28 d. The disorganization index was less when the inoculum size was enhanced to 10-fold. The total endogenous indole-3-acetic acid titers and indole-3-acetic acid oxidase activity also decreased with an increase in disorganization index, and was independent of initial inoculum size, with only a magnitude difference. The total coumarin content strictly correlated with growth in all the treatments. In contrast, exogenously supplied gibberellic acid at the 0.5 mg l⁻¹ level enhanced growth, coumarin content, and branching patterns over the control and other treatments on day 28. The exogenously fed growth regulators had an effect on growth, auxin and coumarin biosyntheses, wherein transformed roots treated with increasing concentration of auxin to cytokinin ratios lost their ability for coumarin biosynthesis. The behavior of hairy roots from an Indian cultivar of chicory upon growth regulator treatment is discussed in terms of growth, coumarin and auxin biosyntheses.     

Lateral root initiation by asymmetrical transverse divisions of pericycle cells in adventitious roots ofAllium cepa

Summary In onion adventitious roots cellular events have been identified that indicate that lateral root initiation occurs earlier and nearer the apex than previously documented. Lateral roots are not initiated when a pericycle cell divides periclinally but earlier, when a pair of neighbouring pericycle cells in the same column divide transversely and asymmetrically, with both mitoses close to the end towards the neighbouring pericycle cell. Each cell therefore produces two cells of unequal length. The shorter cells produced by the mother pericycle cells are adjacent, while the longer cells are located above and below the shorter cells. This objective morphological criterion allows clear identification of the site of lateral root initiation. Subsequent to these asymmetric divisions, both the longer pericycle cells again divide transversely and asymmetrically producing more short cells adjacent to the previous ones. The first periclinal division occurs in one of these short pericycle cells.     

Lateral root initiation by asymmetrical transverse divisions of pericycle cells in four plant species:Raphanus sativus,Helianthus annuus,Zea mays,andDaucus carota

Summary In roots ofRaphanus sativus, Helianthus annuus, Zea mays, andDaucus carota, lateral root initiation occurs when a pair of neighbouring elongated and highly vacuolated pericycle cells in the same column almost simultaneously undergo asymmetrical transversal division. This produces a pair of very short pericycle cells lying end-to-end, flanked above and below by two longer cells. This occurs because both mitoses occur close to the ends of the neighbouring pericycle cells. The two longer daughter pericycle cells divide again later. In roots of radish, sunflower, and carrot these cells divide transversely and asymmetrically, producing more short cells adjacent to the previous ones. In corn roots, they undergo oblique divisions. Much later, the first pair of short pericycle cells undergoes periclinal divisions. Although such periclinal divisions of pericycle cells are generally thought to mark lateral root initiation, our results show that the first pair of short neighbouring pericycle cells in the same column offers another morphological criterion which permits identification of the site of lateral root initiation, both earlier and nearer to the apex than previously documented.     

Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle

In angiosperms, root branching requires a continuous re-initiation of new root meristems. Through some unknown mechanism, in most eudicots pericycle cells positioned against the protoxylem change identity and initiate patterned division, leading to formation of lateral root primordia that further develop into lateral roots. This process is auxin-regulated. We have observed that three mutations in the Diageotropica (Dgt) gene in tomato prevent primordium formation. Detailed analysis of one of these mutants, dgt1-1, demonstrated that the mutation does not abolish the proliferative capacity of the xylem-adjacent pericycle in the differentiated root portion. Files of shortened pericycle cells found in dgt1-1 roots were unrelated to primordium formation. Auxin application stimulated this unusual proliferation, leading to formation of a multi-layered xylem-adjacent pericycle, but did not rescue the primordium formation. In contrast to wild type, auxin could not induce any cell divisions in the pericycle of the most distal dgt1-1 root-tip portion. In wild-type roots, the Dgt gene promoter was expressed strongly in lateral root primordia starting from their initiation, and on auxin treatment was induced in the primary root meristem. Auxin level and distribution were altered in dgt1-1 root tissues, as judged by direct auxin measurements, and the tissue-specific expression of an auxin-response reporter was altered in transgenic plants. Together, our data demonstrate that the Dgt gene product, a type-A cyclophilin, is essential for morphogenesis of lateral root primordia, and that the dgt mutations uncouple patterned cell division in lateral root initiation from proliferative cell division in the pericycle.     

Asymmetric cytokinin signaling opposes gravitropism in roots

Plants depend on gravity to provide the constant landmark for downward root growth and upward shoot growth. The phytohormone auxin and its cell‐to‐cell transport machinery are central determinants ensuring gravitropic growth. Statolith sedimentation toward gravity is sensed in specialized cells. This positional cue is translated into the polar distribution of PIN auxin efflux carriers at the plasma membrane, leading to asymmetric auxin distribution and consequently, differential growth and organ bending. While we have started to understand the general principles of how primary organs execute gravitropism, we currently lack basic understanding of how lateral plant organs can defy gravitropic responses. Here we briefly review the establishment of the oblique gravitropic set point angle in lateral roots and particularly discuss the emerging role of asymmetric cytokinin signaling as a central anti‐gravitropic signal. Differential cytokinin signaling is co‐opted in gravitropic lateral and hydrotropic primary roots to counterbalance gravitropic root growth.     

Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis

In plants, the developmental mechanisms that regulate the positioning of lateral organs along the primary root are currently unknown. We present evidence on how lateral root initiation is controlled in a spatiotemporal manner in the model plant Arabidopsis thaliana. First, lateral roots are spaced along the main axis in a regular left-right alternating pattern that correlates with gravity-induced waving and depends on AUX1, an auxin influx carrier essential for gravitropic response. Second, we found evidence that the priming of pericycle cells for lateral root initiation might take place in the basal meristem, correlating with elevated auxin sensitivity in this part of the root. This local auxin responsiveness oscillates with peaks of expression at regular intervals of 15 hours. Each peak in the auxin-reporter maximum correlates with the formation of a consecutive lateral root. Third, auxin signaling in the basal meristem triggers pericycle cells for lateral root initiation prior to the action of INDOLE-3-ACETIC ACID14 (SOLITARY ROOT).     

Improving of oilseed rape lateral root formation by physiological analogues of auxin

The aim of the present work was to study the effect of auxin physiological analogue TA-12 [1-(2-chloroethoksicarbonylmethyl)-4-naphthalenesulfonic acid calcium salt] on the formation of oilseed rape lateral root and on the mitotic activity of apical meristem cells. Spring oilseed rape (Brassica napus L. ssp. oleifera annua Metzg.) cultivar ‘Mascot’ was chosen as a test object. Anatomical, cytological and histological studies on root development suggest that compound TA-12 induces the activity of parent root pericycle cells, stimulates the formation of lateral roots and enhances the division of apical meristem cells. The auxin transport inhibitor 2,3,5-triiodobenzoic acid suppresses the division of apical meristem cells, while this process is restored by the auxin physiological analogue TA-12 and naphthaleneacetic acid. The compound TA-12, by stimulating primary root growth and lateral root induction, optimised the formation of the oilseed rape root system.     

Pericycle cell proliferation and lateral root initiation in Arabidopsis

In contrast with other cells generated by the root apical meristem in Arabidopsis, pericycle cells adjacent to the protoxylem poles of the vascular cylinder continue to cycle without interruption during passage through the elongation and differentiation zones. However, only some of the dividing pericycle cells are committed to the asymmetric, formative divisions that give rise to lateral root primordia (LRPs). This was demonstrated by direct observation and mapping of mitotic figures, cell-length measurements, and the histochemical analysis of a cyclin-GUS fusion protein in pericycle cells. The estimated duration of a pericycle cell cycle in the root apical meristem was similar to the interval between cell displacement from the meristem and the initiation of LRP formation. Developmentally controlled LRP initiation occurs early, 3 to 8 mm from the root tip. Thus the first growth control point in lateral root formation is defined by the initiation of primordia in stochastic patterns by cells passing through the elongation and young differentiation zones, up to where lateral roots begin to emerge from the primary root. Therefore, the first growth control point is not restricted to a narrow developmental window. We propose that late LRP initiation is developmentally unrelated to the root apical meristem and is operated by a second growth control point that can be activated by environmental cues. The observation that pericycle cells divide and lateral root primordia form without intervening mitotic quiescence suggests that lateral organ formation in roots and shoots might not be as fundamentally different as previously thought.     

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.     

Cell-cycle regulation of hydroxyproline-rich glycoprotein HRGPnt3 gene expression during the initiation of lateral root meristems

Analysis of transgenic tobacco plants containing a tobacco hydroxyproline-rich glycoprotein HRGPnt3 gene promoter-β-glucuronidase (GUS) gene fusion (HRGPnt3-uidA) showed that this promoter is active not only in the early stages of initiation of lateral roots as previously described, but also in the initiation of adventitious roots, with similar selective expression in a subset of pericycle cells. HRGPnt3 is also induced during initiation of hairy roots following transformation by Agrobacterium rhizogenes. The auxin indole acetic acid (IAA) induces an increase in the number of characteristic discrete sites of HRGP-nt3 expression. It is shown that these sites are destined to form new root primordia from pericycle cells of both adventitious and main roots. Dose-dependent induction of root meristems by auxin overcomes the limitations of this naturally stochastic process and makes lateral root initiation amenable to biochemical analysis. Quiescent pericycle cells, which are developmentally arrested in the G₂ phase of the cell cycle, rapidly progress into M phase upon mitogenic stimulation. Colchicine and nocodazole, which block completion of mitosis, inhibited the activation of the HRGPnt3 promoter but did not block auxin induction of parA, a marker for de-differentiation in leaf mesophyll cell-derived protoplasts. Hydroxyurea, which inhibits cell-cycle progression at the G₁/S-phase transition and also blocks lateral root initiation, did not inhibit HRGPnt3 induction. Thus, HRGPnt3 induction precedes completion of the first cell division during primordium formation, and is one of the initial steps in a sequential program of gene expression activated upon stimulation of cell division for the development of a new meristem during lateral root initiation.