NO VEIN Mediates Auxin-Dependent Specification and Patterning in the Arabidopsis Embryo,Shoot, and Root

Local efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. Auxin efflux is regulated by dynamic expression and subcellular localization of the PIN auxin-efflux proteins, which appears to be established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means, such as cell fate determination and auxin-independent mechanisms. Here, we show that the Arabidopsis thaliana NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem cell maintenance in the root meristem, as well as for cotyledon outgrowth and separation. nov mutations affect many aspects of auxin-dependent development without directly affecting auxin perception. NOV is required for provascular PIN1 expression and region-specific expression of PIN7 in leaf primordia, cell type–specific expression of PIN3, PIN4, and PIN7 in the root, and PIN2 polarity in the root cortex. NOV is specifically expressed in developing embryos, leaf primordia, and shoot and root apical meristems. Our data suggest that NOV function underlies cell fate decisions associated with auxin gradients and maxima, thus establishing cell type–specific PIN expression and polarity. We propose that NOV mediates the acquisition of competence to undergo auxin-dependent coordinated cell specification and patterning, thereby eliciting context-dependent auxin-mediated developmental responses.     

Endophytic microbes <Emphasis Type="Italic">Bacillus</Emphasis> sp. LZR216-regulated root development is dependent on polar auxin transport in <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Key message

Endophytic microbes Bacillus sp. LZR216 isolated from Arabidopsis root promoted Arabidopsis seedlings growth. It may be achieved by promoting the lateral root growth and inhibiting the primary root elongation.

Abstract

Plant roots are colonized by an immense number of microbes, including epiphytic and endophytic microbes. It was found that they have the ability to promote plant growth and protect roots from biotic and abiotic stresses. But little is known about the mechanism of the endophytic microbes-regulated root development. We isolated and identified a Bacillus sp., named as LZR216, of endophytic bacteria from Arabidopsis root. By employing a sterile experimental system, we found that LZR216 promoted the Arabidopsis seedlings growth, which may be achieved by promoting the lateral root growth and inhibiting the primary root elongation. By testing the cell type-specific developmental markers, we demonstrated that Bacillus sp. LZR216 increases the DR5::GUS and DR5::GFP expression but decreases the CYCB1;1::GUS expression in Arabidopsis root tips. Further studies indicated that LZR216 is able to inhibit the meristematic length and decrease the cell division capability but has little effect on the quiescent center function of the root meristem. Subsequently, it was also shown that LZR216 has no significant effects on the primary root length of the pin2 and aux1-7 mutants. Furthermore, LZR216 down-regulates the levels of PIN1-GFP, PIN2-GFP, PIN3-GFP, and AUX1-YFP. In addition, the wild-type Arabidopsis seedlings in the present of 1 or 5 µM NPA (an auxin transport inhibitor) were insensitive to LZR216-inhibited primary root elongation. Collectively, LZR216 regulates the development of root system architecture depending on polar auxin transport. This study shows a new insight on the ability of beneficial endophytic bacteria in regulating postembryonic root development.

     

Glucose inhibits root meristem growth via ABA INSENSITIVE 5, which represses PIN1 accumulation and auxin activity in Arabidopsis

Glucose functions as a hormone‐like signalling molecule that modulates plant growth and development in Arabidopsis thaliana. However, the role of glucose in root elongation remains elusive. Our study demonstrates that high concentrations of glucose reduce the size of the root meristem zone by repressing PIN1 accumulation and thereby reducing auxin levels. In addition, we verified the involvement of ABA INSENSITIVE 5 (ABI5) in this process by showing that abi5‐1 is less sensitive to glucose than the wild type, whereas glucose induces ABI5 expression and the inducible overexpression of ABI5 reduces the size of the root meristem zone. Furthermore, the inducible overexpression of ABI5 in PIN1::PIN1‐GFP plants reduces the level of PIN1‐GFP, but glucose reduces the level of PIN1‐GFP to a lesser extent in abi5‐1 PIN1::PIN1‐GFP plants than in the PIN1::PIN1‐GFP control, suggesting that ABI5 is involved in glucose‐regulated PIN1 accumulation. Taken together, our data suggest that ABI5 functions in the glucose‐mediated inhibition of the root meristem zone by repressing PIN1 accumulation, thus leading to reduced auxin levels in roots.     

The involvement of auxin in root architecture plasticity in Arabidopsis induced by heterogeneous phosphorus availability

Homogeneous low phosphorus availability was reported to regulate root architecture in Arabidopsis via auxin, but the roles of auxin in root architecture plasticity to heterogeneous P availability remain unclear. In this study, we employed auxin biosynthesis-, transport- and signalling-related mutants. Firstly, we found that in contrast to low P (LP) content in the whole medium, primary root (PR) growth of Arabidopsis was partially rescued in the medium divided into two parts: upper with LP and lower with high P (HP) content or in the reverse arrangement. The down part LP was more effective to arrest PR growth as well as to decrease density of lateral roots (DLR) than the upper LP, and effects were dependent on polar auxin transport. Secondly, we verified that auxin receptor TIR1 was involved in the responses of PR growth and lateral root (LR) development to P supply and loss of function of TIR1 inhibited LR development. Thirdly, effects of heterogeneous P on LRD in the upper part of PR was dependent on PIN2 and PIN4, and in the down part on PIN3 and PIN4, whereas density of total LRs was dependent on auxin transporters PIN2 and PIN7. Finally, heterogeneous P availability altered the accumulation of auxin in PR tip and the expression of auxin biosynthesisrelated genes TAA1, YUC1, YUC2, and YUC4. Taken together, we provided evidences for the involvement of auxin in root architecture plasticity in response to heterogeneous phosphorus availability in Arabidopsis.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> regulates root system architecture by modulating the polar transport and redistribution of auxin

The accumulation and redistribution of the plant hormone auxin plays a crucial role in root development and patterning. Plants can alter their root system architecture (RSA) to adapt to different biotic and abiotic stresses. In addition, reactive oxygen species (ROS), such as H₂O₂, are known to increase in plants undergoing stress. Here, we present evidence that H₂O₂ can regulate auxin accumulation and redistribution through modulating polar auxin transport, leading to changes in RSA. Plants exposed to different concentrations of H₂O₂ formed a highly branched root system with abundant lateral roots and a shorter primary root. Monitoring of the auxin responsive DR5::GUS indicated that auxin accumulation decreased in lateral root primordia (LRP) and emerging lateral root tips. In addition, polar auxin transport, including both basipetal and acropetal transport modulated by AUX1 and PIN protein carriers, was involved in the process. Taken together, our results suggest that H₂O₂ could regulate plastic RSA by perturbing polar auxin transport as a means of modulating the accumulation and distribution of auxin.     

Temporal root responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> L. to chromate reveal structural and regulatory mechanisms involving the SOLITARY ROOT/IAA14 repressor for maintenance of identity meristem genes

The Arabidopsis root system is modified in response to stress generated by high concentrations of nonessential ions such as chromate [Cr(VI)]. In this work, the distribution of auxin and its transporters PIN1 and PIN7, as well as the expression of genes that maintain the identity of the root meristem, were analyzed in Arabidopsis thaliana wild-type (WT) seedlings and in a mutant affected in the SOLITARY ROOT (SLR1/IAA14) locus, which is required for root response to Cr(VI). We show that primary root inhibition, auxin transporter levels, and expression of meristem identity genes were maintained in the slr-1 mutants but not in WT plants in response to Cr(VI) in a time- and concentration-dependent manner. Notably, the outermost single cell layer of the lateral root cap, which normally dies and tends to peel off, remains viable and increases in size following exposure of WT plants, but not slr-1 mutants, to Cr(VI). Our results suggest that (1) the primary root tip senses Cr(VI), (2) the external lateral root cap may play a protective role during Cr(VI) exposure, and (3) Cr(VI) impacts cell division in root meristems via auxin redistribution and SLR1/IAA14 function, influencing the expression of root meristem genes.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.     

Combined in silico/in vivo analysis of mechanisms providing for root apical meristem self-organization and maintenance

Background and Aims

The root apical meristem (RAM) is the plant stem cell niche which provides for the formation and continuous development of the root. Auxin is the main regulator of RAM functioning, and auxin maxima coincide with the sites of RAM initiation and maintenance. Auxin gradients are formed due to local auxin biosynthesis and polar auxin transport. The PIN family of auxin transporters plays a critical role in polar auxin transport, and two mechanisms of auxin maximum formation in the RAM based on PIN-mediated auxin transport have been proposed to date: the reverse fountain and the reflected flow mechanisms.

Methods

The two mechanisms are combined here in in silico studies of auxin distribution in intact roots and roots cut into two pieces in the proximal meristem region. In parallel, corresponding experiments were performed in vivo using DR5::GFP Arabidopsis plants.

Key Results

The reverse fountain and the reflected flow mechanism naturally cooperate for RAM patterning and maintenance in intact root. Regeneration of the RAM in decapitated roots is provided by the reflected flow mechanism. In the excised root tips local auxin biosynthesis either alone or in cooperation with the reverse fountain enables RAM maintenance.

Conclusions

The efficiency of a dual-mechanism model in guiding biological experiments on RAM regeneration and maintenance is demonstrated. The model also allows estimation of the concentrations of auxin and PINs in root cells during development and under various treatments. The dual-mechanism model proposed here can be a powerful tool for the study of several different aspects of auxin function in root.     

Light Quality Regulates Lateral Root Development in Tobacco Seedlings by Shifting Auxin Distributions

Light is an important environmental regulator of diverse growth and developmental processes in plants. However, the mechanisms by which light quality regulates root growth are poorly understood. We analyzed lateral root (LR) growth of tobacco seedlings in response to three kinds of light qualities (red, white, and blue). Primary (1°) LR number and secondary (2°) LR density were elevated under red light (on days 9 and 12 of treatment) in comparison with white and blue lights. Higher IAA concentrations measured in roots and lower in leaves of plants treated with red light suggest that red light accelerated auxin transport from the leaves to roots (in comparison with other light qualities). Corroborative evidence for this suggestion was provided by elevated DR5::GUS expression levels at the shoot/root junction and in the 2° LR region. Applications of N-1-naphthylphthalamic acid (NPA) to red light-treated seedlings reduced both 1° LR number and 2° LR density to levels similar to those measured under white light; DR5::GUS expression levels were also similar between these light qualities after NPA application. Results were similar following exogenous auxin (NAA) application to blue light-treated seedlings. Direct [³H]IAA transport measurement indicated that the polar auxin transport from shoot to root was increased by red light. Red light promoted PIN3 expression levels and blue light reduced PIN1, 3–4 expression levels in the shoot/root junction and in the root, indicating that these genes play key roles in auxin transport regulation by red and blue lights. Overall, our findings suggest that three kinds of light qualities regulate LR formation in tobacco seedlings through modification of auxin polar transport.     

Dichotomization of mycorrhizal and NPA-treated short roots in Pinus sylvestris

Conifers like Scots pine (Pinus sylvestris) have a complicated root system consisting of morphologically and anatomically different root types, of which the short roots have a very limited ability to elongate. Short roots have an important role in nature since they are able to establish ectomycorrhizal symbiosis, in which the growth of fungal mycelium between the epidermal cells and in the intercellular space between cortical cells leads to formation of dichotomous short roots, which may, through further splitting of the meristem, form coralloid root structures. Dichotomous short roots have been suggested to result from changes in either auxin or ethylene concentrations due to the fungal growth inside the root. NPA, the inhibitor of polar auxin transport, enhances the dichotomization of P. sylvestris short root tips similarly to the fungal growth in the root, thus confirming that auxin plays a role in short root morphogenesis. Ethylene is also known to have an important role in the regulation of root morphogenesis. In future the research dealing with the root system and ectomycorrhiza development in P. sylvestris must take into account that both auxin and ethylene are involved and that there is no contradiction in obtaining the same phenotype with both hormones. The expression analysis of PIN proteins, auxin efflux carriers, could give valuable information about the role of auxin transport in regulating the root growth and morphogenesis of coniferous root system and mycorrhiza.Key words: auxin transport, dichotomization, ethylene, mycorrhiza, open meristem, PIN, Pinus sylvestris, short root     

Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of arabidopsis

Background and Aims

Adventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications.

Methods

Expression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type.

Key Results

The accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex.

Conclusions

In ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.     

Shoot-supplied ammonium targets the root auxin influx carrier AUX1 and inhibits lateral root emergence in Arabidopsis

Deposition of ammonium (NH₄⁺) from the atmosphere is a substantial environmental problem. While toxicity resulting from root exposure to NH₄⁺ is well studied, little is known about how shoot‐supplied ammonium (SSA) affects root growth. In this study, we show that SSA significantly affects lateral root (LR) development. We show that SSA inhibits lateral root primordium (LRP) emergence, but not LRP initiation, resulting in significantly impaired LR number. We show that the inhibition is independent of abscisic acid (ABA) signalling and sucrose uptake in shoots but relates to the auxin response in roots. Expression analyses of an auxin‐responsive reporter, DR5:GUS, and direct assays of auxin transport demonstrated that SSA inhibits root acropetal (rootward) auxin transport while not affecting basipetal (shootward) transport or auxin sensitivity of root cells. Mutant analyses indicated that the auxin influx carrier AUX1, but not the auxin efflux carriers PIN‐FORMED (PIN)1 or PIN2, is required for this inhibition of LRP emergence and the observed auxin response. We found that AUX1 expression was modulated by SSA in vascular tissues rather than LR cap cells in roots. Taken together, our results suggest that SSA inhibits LRP emergence in Arabidopsis by interfering with AUX1‐dependent auxin transport from shoot to root.     

SHY2 as a node in the regulation of root meristem development by auxin,brassinosteroids, and cytokinin

In multicellular organisms, the balance between cell division and differentiation determines organ size, and represents a central unknown in developmental biology. In Arabidopsis roots, this balance is mediated between cytokinin and auxin through a regulatory circuit converging on the IAA3/SHORT HYPOCOTYL 2 (SHY2) gene. Here, we show that crosstalk between brassinosteroids (BRs) and auxin occurs in the vascular transition zone to promote root meristem development. We found that BR increases root meristem size by up‐regulating expression of the PINFORMED 7 (PIN7) gene and down‐regulating expression of the SHY2 gene. In addition, BES1 could directly bind to the promoter regions of both PIN7 and SHY2, indicating that PIN7 and SHY2 mediate the BR‐induced growth of the root meristem by serving as direct targets of BES1. Moreover, the PIN7 overexpression and loss‐of‐function SHY2 mutant were sensitive to the effects of BR and could partially suppress the short‐root phenotypes associated with deficient BR signaling. Interestingly, BRs could inhibit the accumulation of SHY2 protein in response to cytokinin. Taken together, these findings suggest that a complex equilibrium model exists in which regulatory interactions among BRs, auxin, and cytokinin regulate optimal root growth.     

Titanium Dioxide Nanoparticles Promote Root Growth by Interfering with Auxin Pathways in <i>Arabidopsis thaliana</i>

TiO₂ nanoparticles (nano-TiO₂) are widely used in the world, and a considerable amount of nano-TiO₂ is released into the environment, with toxic effects on organisms. In the various species of higher plants, growth, including seed germination, root elongation, and biomass accumulation, is affected by nano-TiO₂. However, the underlying molecular mechanisms remain to be elucidated. In this study, we observed that nano-TiO₂ promoted root elongation in a dose-dependent manner. Furthermore, we found that nano-TiO₂ elevated auxin accumulation in the root tips of the auxin marker lines DII-VENUS and DR5:: GUS, and, correspondingly, quantitative real-time PCR analysis revealed that nano-TiO₂ increased the expression levels of auxin biosynthesis- and transportrelated genes. GFP fluorescence observation using transgenic PIN2-GFP indicated that nano-TiO₂ promoted root growth by inducing PIN2 accumulation. Thus, we propose that nano-TiO₂ promote root growth in Arabidopsis thaliana by altering the expression levels of auxin biosynthesis- and transport-related genes.     

Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.     

The short-rooted vitamin B<Subscript>6</Subscript>-deficient mutant <Emphasis Type="Italic">pdx1</Emphasis> has impaired local auxin biosynthesis

The phytohormone auxin regulates many aspects of plant growth and development. Auxin often acts distantly from the site of its biosynthesis and this long-distance-transported auxin is well known to play a critical role in eliciting physiological responses including regulating root development. Auxin can be produced in roots, yet the function of locally synthesized auxin in root growth is unclear. The major auxin in plants, indole 3-acetic acid (IAA), is mainly synthesized through tryptophan (Trp)-dependent pathways that require pyridoxal phosphate (an active form of vitamin B₆)-dependent enzymes. We previously reported that the Arabidopsis vitamin B₆ biosynthesis mutant pdx1 has stunted root growth although the underlying cause is unknown. Here we showed that the pdx1 root is deficient in auxin biosynthesis. By reciprocal grafting of pdx1 and the wild type, we demonstrated that the stunted root growth in pdx1 is caused by a locally generated signal(s) in roots. To test whether auxin might be one such signal, the auxin responsive DR5::GUS reporter was introduced into the mutant. The DR5::GUS activity in pdx1 root tips was greatly reduced compared with that in the wild type although the auxin response was unaltered. pdx1 also suppresses the root hair growth defects in the auxin overproduction mutant yucca. These data indicate that pdx1 is impaired in Trp-dependent auxin biosynthesis, which may contribute to the short-root phenotype of pdx1. We suggest that locally synthesized auxin may play a critical role in postembryonic root growth.