Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in Arabidopsis

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.     

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.     

ROP3 GTPase Contributes to Polar Auxin Transport and Auxin Responses and Is Important for Embryogenesis and Seedling Growth in Arabidopsis

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.     

MM31/EIR1 promotes lateral root formation in Arabidopsis

Lateral root formation in Arabidopsis provides a model for the study of auxin function. Tryptophan (Trp) is a precursor of the auxin indoleacetic acid (IAA). To study the physiological function of Trp in auxin-related phenotypes, we examined the effect of Trp on lateral root formation. We found that Trp treatment enhanced lateral root formation and, by screening for mutants in which the effect of Trp on lateral root formation was enhanced, we isolated the mm31 mutant. Based on genetic and physiological analyses, we propose that MM31/EIR1 modulates lateral root formation by regulating the IAA polar transport system, and that auxin transport from the shoot to the root regulates lateral root formation.Key words: lateral root formation, Arabidopsis, EIR1, IAA, auxin     

Arabidopsis ASA1 Is Important for Jasmonate-Mediated Regulation of Auxin Biosynthesis and Transport during Lateral Root Formation

Plant roots show an impressive degree of plasticity in adapting their branching patterns to ever-changing growth conditions. An important mechanism underlying this adaptation ability is the interaction between hormonal and developmental signals. Here, we analyze the interaction of jasmonate with auxin to regulate lateral root (LR) formation through characterization of an Arabidopsis thaliana mutant, jasmonate-induced defective lateral root1 (jdl1/asa1-1). We demonstrate that, whereas exogenous jasmonate promotes LR formation in wild-type plants, it represses LR formation in jdl1/asa1-1. JDL1 encodes the auxin biosynthetic gene ANTHRANILATE SYNTHASE α1 (ASA1), which is required for jasmonate-induced auxin biosynthesis. Jasmonate elevates local auxin accumulation in the basal meristem of wild-type roots but reduces local auxin accumulation in the basal meristem of mutant roots, suggesting that, in addition to activating ASA1-dependent auxin biosynthesis, jasmonate also affects auxin transport. Indeed, jasmonate modifies the expression of auxin transport genes in an ASA1-dependent manner. We further provide evidence showing that the action mechanism of jasmonate to regulate LR formation through ASA1 differs from that of ethylene. Our results highlight the importance of ASA1 in jasmonate-induced auxin biosynthesis and reveal a role for jasmonate in the attenuation of auxin transport in the root and the fine-tuning of local auxin distribution in the root basal meristem.     

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.     

Plasticity in Cell Division Patterns and Auxin Transport Dependency during in Vitro Embryogenesis in Brassica napus

In Arabidopsis thaliana, zygotic embryo divisions are highly regular, but it is not clear how embryo patterning is established in species or culture systems with irregular cell divisions. We investigated this using the Brassica napus microspore embryogenesis system, where the male gametophyte is reprogrammed in vitro to form haploid embryos in the absence of exogenous growth regulators. Microspore embryos are formed via two pathways: a zygotic-like pathway, characterized by initial suspensor formation followed by embryo proper formation from the distal cell of the suspensor, and a pathway characterized by initially unorganized embryos lacking a suspensor. Using embryo fate and auxin markers, we show that the zygotic-like pathway requires polar auxin transport for embryo proper specification from the suspensor, while the suspensorless pathway is polar auxin transport independent and marked by an initial auxin maximum, suggesting early embryo proper establishment in the absence of a basal suspensor. Polarity establishment in this suspensorless pathway was triggered and guided by rupture of the pollen exine. Irregular division patterns did not affect cell fate establishment in either pathway. These results confirm the importance of the suspensor and suspensor-driven auxin transport in patterning, but also uncover a mechanism where cell patterning is less regular and independent of auxin transport.     

Mechano-Chemical Aspects of Organ Formation in Arabidopsis thaliana: The Relationship between Auxin and Pectin

How instructive signals are translated into robust and predictable changes in growth is a central question in developmental biology. Recently, much interest has centered on the feedback between chemical instructions and mechanical changes for pattern formation in development. In plants, the patterned arrangement of aerial organs, or phyllotaxis, is instructed by the phytohormone auxin; however, it still remains to be seen how auxin is linked, at the apex, to the biochemical and mechanical changes of the cell wall required for organ outgrowth. Here, using Atomic Force Microscopy, we demonstrate that auxin reduces tissue rigidity prior to organ outgrowth in the shoot apex of Arabidopsis thaliana, and that the de-methyl-esterification of pectin is necessary for this reduction. We further show that development of functional organs produced by pectin-mediated ectopic wall softening requires auxin signaling. Lastly, we demonstrate that coordinated localization of the auxin transport protein, PIN1, is disrupted in a naked-apex produced by increasing cell wall rigidity. Our data indicates that a feedback loop between the instructive chemical auxin and cell wall mechanics may play a crucial role in phyllotactic patterning.     

Glucose Attenuation of Auxin-Mediated Bimodality in Lateral Root Formation Is Partly Coupled by the Heterotrimeric G Protein Complex

Background

Auxin and glucose are both essential elements in normal root development. The heterotrimeric G protein complex in Arabidopsis thaliana, defined as containing alpha (AtGPA1), beta (AGB1), and gamma (AGG) subunits and a GTPase accelerating protein called Regulator of G Signaling 1 protein (AtRGS1), are involved in glucose signaling and regulate auxin transport.

Methodology/Principal Findings

A systems approach was used to show that formation of lateral roots, a process requiring coordinated cell division followed by targeted cell expansion, involves a signaling interaction between glucose and auxin. We dissected the relationship between auxin and glucose action using lateral root formation as the biological context. We found that auxin and glucose act synergistically to yield a complex output involving both stimulatory and antagonist glucose effects on auxin responsiveness. Auxin-induced, lateral-root formation becomes bimodal with regard to auxin dose in the presence of glucose. This bimodality is mediated, in part, by the G protein complex defined above.

Conclusion/Significance

Auxin and glucose are essential signals controlling the rate of cell proliferation and expansion in roots. Auxin promotes the formation of lateral roots and is consequently essential for proper root architecture. Glucose affects the activation state of the heterotrimeric G protein complex which regulates auxin distribution in the root. The bimodality of auxin-induced, lateral-root formation becomes prominent in the presence of glucose and in roots lacking the G protein complex. Bimodality is apparent without added glucose in all loss-of-function mutants for these G protein components, suggesting that the heterotrimeric G protein complex attenuates the bimodality and that glucose inhibits this attenuation through the complex. The bimodality can be further resolved into the processes of lateral root primordia formation and lateral root emergence, from which a model integrating these signals is proposed.     

Low phosphate signaling induces changes in cell cycle gene expression by increasing auxin sensitivity in the Arabidopsis root system

Lateral root development is an important morphogenetic process in plants, which allows the modulation root architecture and substantially determines the plant''s efficiency for water and nutrient uptake. Postembryonic root development is under the control of both endogenous developmental programs and environmental stimuli. Nutrient availability plays a major role among environmental signals that modulate root development. Phosphate (Pi) limitation is a constraint for plant growth in many natural and agricultural ecosystems. Plants posses Pi-sensing mechanisms that enable them to respond and adapt to conditions of limited Pi supply, including increased formation and growth of lateral roots. Root developmental modifications are mainly mediated by the plant hormone auxin. Recently we showed that the alteration of root system architecture under Pi-starvation may be mediated by modifications in auxin sensitivity in root cells via a mechanism involving the TIR1 auxin receptor. In this addendum, we provide additional novel evidence indicating that the low Pi pathway involves changes in cell cycle gene expression. It was found that Pi deprivation increases the expression of CDKA, E2Fa, Dp-E2F and CyCD3. In particular, E2Fa, Dp-E2F and CyCD3 genes were specifically upregulated by auxin in Pi-deprived Arabidopsis seedlings that were treated with the auxin transport inhibitor NPA, indicating that cell cycle modulation by low Pi signaling is independent of auxin transport and dependent on auxin sensitivity in the root.Key words: phosphate signaling, auxin transport, auxin sensitivity, roots     

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.     

N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

Background

N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development.

Methodology/Principal Findings

Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem.

Conclusion/Significance

NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.     

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.     

LjABCB1, an ATP-binding cassette protein specifically induced in uninfected cells of Lotus japonicus nodules

Legume plants develop root nodules through symbiosis with rhizobia, and fix atmospheric nitrogen in this symbiotic organ. Development of root nodules is regulated by many metabolites including phytohormones. Previously, we reported that auxin is strongly involved in the development of the nodule vascular bundle and lenticel formation on the nodules of Lotus japonicus. Here we show that an ATP-binding cassette (ABC) protein, LjABCB1, which is a homologue of Arabidopsis auxin transporter AtABCB4, is specifically expressed during nodulation of L. japonicus. A reporter gene analysis indicated that the expression of LjABCB1 was restricted to uninfected cells adjacent to infected cells in the nodule, while no expression was observed in shoot apical meristems or root tips, in which most auxin transporter genes are expressed. The auxin transport activity of LjABCB1 was confirmed using a heterologous expression system.     

SLOW MOTION Is Required for Within-Plant Auxin Homeostasis and Normal Timing of Lateral Organ Initiation at the Shoot Meristem in Arabidopsis

The regular arrangement of leaves and flowers around a plant''s stem is a fascinating expression of biological pattern formation. Based on current models, the spacing of lateral shoot organs is determined by transient local auxin maxima generated by polar auxin transport, with existing primordia draining auxin from their vicinity to restrict organ formation close by. It is unclear whether this mechanism encodes not only spatial information but also temporal information about the plastochron (i.e., the interval between the formation of successive primordia). Here, we identify the Arabidopsis thaliana F-box protein SLOW MOTION (SLOMO) as being required for a normal plastochron. SLOMO interacts genetically with components of polar auxin transport, and mutant shoot apices contain less free auxin. However, this reduced auxin level at the shoot apex is not due to increased polar auxin transport down the stem, suggesting that it results from reduced synthesis. Independently reducing the free auxin level in plants causes a similar lengthening of the plastochron as seen in slomo mutants, suggesting that the reduced auxin level in slomo mutant shoot apices delays the establishment of the next auxin maximum. SLOMO acts independently of other plastochron regulators, such as ALTERED MERISTEM PROGRAM1 or KLUH/CYP78A5. We propose that SLOMO contributes to auxin homeostasis in the shoot meristem, thus ensuring a normal rate of the formation of auxin maxima and organ initiation.     

Control of root architecture and nodulation by the LATD/NIP transporter

The Medicago truncatula LATD/NIP gene is essential for the development of lateral and primary root and nitrogen-fixing nodule meristems as well as for rhizobial invasion of nodules. LATD/NIP encodes a member of the NRT1(PTR1) nitrate and di-and tri-peptide transporter family, suggesting that its function is to transport one of these or another compound(s). Because latd/nip mutants can have their lateral and primary root defects rescued by ABA, ABA is a potential substrate for transport. LATD/NIP expression in the root meristem was demonstrated to be regulated by auxin, cytokinin and abscisic acid, but not by nitrate. LATD/NIP''s potential function and its role in coordinating root architecture and nodule formation are discussed.Key words: nodule development, lateral root development, root architecture, symbiotic nitrogen fixation, Medicago truncatula, NRT1(PTR) gene familyUnlike most other plants, legumes form two kinds of lateral root organs: lateral roots and nitrogen-fixing root nodules that form in conjunction with compatible symbiotic rhizobium bacteria. Although the morphology and function of these two root organs is distinct, both require the function of the LATD/NIP gene, indicating shared genetic components for these two developmental processes and providing support for a model in which legume nodules evolved from a lateral root blueprint. Both lateral roots and nodules initiate in previously differentiated root cells in response to environmental and developmental cues mediated by hormones. Interestingly, regulation of nodules and lateral roots by hormones is often opposite, allowing formation of one organ or another depending on the conditions.