Arabidopsis NHX5 and NHX6 regulate PIN6-mediated auxin homeostasis and growth.

NHX5 and NHX6, endosomal Na+,K+/H+ antiporters in Arabidopsis thaliana, play a vital role in growth and development. Our previous study has shown that NHX5 and NHX6 function as H+ leak to regulate auxin-mediated growth in Arabidopsis. In this report, we investigated the function of NHX5 and NHX6 in controlling PIN6-mediated auxin homeostasis and growth in Arabidopsis. Phenotypic analyses found that NHX5 and NHX6 were critical for the function of PIN6, an auxin transporter. We further showed that PIN6 depended on NHX5 and NHX6 in regulating auxin homeostasis. NHX5 and NHX6 were colocalized with PIN6, but they did not interact physically. The conserved acidic residues that are vital for the activity of NHX5 and NHX6 were critical for PIN6 function. Together, NHX5 and NHX6 may regulate PIN6 function by their transport activity.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

Coordination of tissue cell polarity by auxin transport and signaling.

Plants coordinate the polarity of hundreds of cells during vein formation, but how they do so is unclear. The prevailing hypothesis proposes that GNOM, a regulator of membrane trafficking, positions PIN-FORMED auxin transporters to the correct side of the plasma membrane; the resulting cell-to-cell, polar transport of auxin would coordinate tissue cell polarity and induce vein formation. Contrary to predictions of the hypothesis, we find that vein formation occurs in the absence of PIN-FORMED or any other intercellular auxin-transporter; that the residual auxin-transport-independent vein-patterning activity relies on auxin signaling; and that a GNOM-dependent signal acts upstream of both auxin transport and signaling to coordinate tissue cell polarity and induce vein formation. Our results reveal synergism between auxin transport and signaling, and their unsuspected control by GNOM in the coordination of tissue cell polarity during vein patterning, one of the most informative expressions of tissue cell polarization in plants.

Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis.

The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here, we used Arabidopsis (Arabidopsis thaliana) reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor long-lived microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.

Connective Auxin Transport in the Shoot Facilitates Communication between Shoot Apices.

The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.

Control of vein network topology by auxin transport.

BACKGROUND: Tissue networks such as the vascular networks of plant and animal organs transport signals and nutrients in most multicellular organisms. The transport function of tissue networks depends on topological features such as the number of networks' components and the components' connectedness; yet what controls tissue network topology is largely unknown, partly because of the difficulties in quantifying the effects of genes on tissue network topology. We address this problem for the vein networks of plant leaves by introducing biologically motivated descriptors of vein network topology; we combine these descriptors with cellular imaging and molecular genetic analysis; and we apply this combination of approaches to leaves of Arabidopsis thaliana that lack function of, overexpress or misexpress combinations of four PIN-FORMED (PIN) genes--PIN1, PIN5, PIN6, and PIN8--which encode transporters of the plant signal auxin and are known to control vein network geometry. RESULTS: We find that PIN1 inhibits vein formation and connection, and that PIN6 acts redundantly to PIN1 in these processes; however, the functions of PIN6 in vein formation are nonhomologous to those of PIN1, while the functions of PIN6 in vein connection are homologous to those of PIN1. We further find that PIN8 provides functions redundant and homologous to those of PIN6 in PIN1-dependent inhibition of vein formation, but that PIN8 has no functions in PIN1/PIN6-dependent inhibition of vein connection. Finally, we find that PIN5 promotes vein formation; that all the vein-formation-promoting functions of PIN5 are redundantly inhibited by PIN6 and PIN8; and that these functions of PIN5, PIN6, and PIN8 are independent of PIN1. CONCLUSIONS: Our results suggest that PIN-mediated auxin transport controls the formation of veins and their connection into networks.

Characterization of an allelic series in the MONOPTEROS gene of Arabidopsis.

Patterning of numerous features of plants depends on transduction of the auxin signal. Auxin signaling is mediated by several pathways, the best understood of which relies on the function of the MONOPTEROS (MP) gene. Seven mp mutant alleles have been described in the widely used Columbia background of Arabidopsis: two extensively characterized and five only partially characterized. One of these five mp alleles appears to be extinct and thus unavailable for analysis. We show that two of the four remaining, partially characterized mp alleles reported to be in the Columbia background are in fact not in this background. We extend characterization of the remaining two Columbia alleles of mp, and we identify and characterize four new alleles of mp in the Columbia background, among which the first low-expression allele of mp and the strongest Columbia allele of mp. These genetic resources provide the research community with new experimental opportunities for insight into the function of MP-dependent auxin signaling in plant development.

Control of vein patterning by intracellular auxin transport.

The vein networks of plant leaves are among the most spectacular expressions of biological pattern, and the principles controlling their formation have continually inspired artists and scientists. Control of vein patterning by the polar, cell-to-cell transport of the plant signaling molecule auxin--mediated in Arabidopsis primarily by the plasma-membrane-localized PIN1--has long been known. By contrast, the existence of intracellular auxin transport and its contribution to vein patterning are recent discoveries. The endoplasmic-reticulum-localized PIN5, PIN6, and PIN8 of Arabidopsis define an intracellular auxin-transport pathway whose functions in vein patterning overlap with those of PIN1-mediated intercellular auxin transport. The genetic interaction between the components of the intracellular auxin-transport pathway is far from having been resolved. The study of vein patterning provides experimental access to gain such a resolution-a resolution that in turn holds the promise to improve our understanding of one of the most fascinating examples of biological pattern formation.

Polarity, continuity, and alignment in plant vascular strands.

Plant vascular cells are joined end to end along uninterrupted lines to connect shoot organs with roots; vascular strands are thus polar, continuous, and internally aligned. What controls the formation of vascular strands with these properties? The "auxin canalization hypothesis"-based on positive feedback between auxin flow through a cell and the cell's capacity for auxin transport-predicts the selection of continuous files of cells that transport auxin polarly, thus accounting for the polarity and continuity of vascular strands. By contrast, polar, continuous auxin transport-though required-is insufficient to promote internal alignment of vascular strands, implicating additional factors. The auxin canalization hypothesis was derived from the response of mature tissue to auxin application but is consistent with molecular and cellular events in embryo axis formation and shoot organ development. Objections to the hypothesis have been raised based on vascular organizations in callus tissue and shoot organs but seem unsupported by available evidence. Other objections call instead for further research; yet the inductive and orienting influence of auxin on continuous vascular differentiation remains unique.

Patterning of leaf vein networks by convergent auxin transport pathways.

The formation of leaf vein patterns has fascinated biologists for centuries. Transport of the plant signal auxin has long been implicated in vein patterning, but molecular details have remained unclear. Varied evidence suggests a central role for the plasma-membrane (PM)-localized PIN-FORMED1 (PIN1) intercellular auxin transporter of Arabidopsis thaliana in auxin-transport-dependent vein patterning. However, in contrast to the severe vein-pattern defects induced by auxin transport inhibitors, pin1 mutant leaves have only mild vein-pattern defects. These defects have been interpreted as evidence of redundancy between PIN1 and the other four PM-localized PIN proteins in vein patterning, redundancy that underlies many developmental processes. By contrast, we show here that vein patterning in the Arabidopsis leaf is controlled by two distinct and convergent auxin-transport pathways: intercellular auxin transport mediated by PM-localized PIN1 and intracellular auxin transport mediated by the evolutionarily older, endoplasmic-reticulum-localized PIN6, PIN8, and PIN5. PIN6 and PIN8 are expressed, as PIN1 and PIN5, at sites of vein formation. pin6 synthetically enhances pin1 vein-pattern defects, and pin8 quantitatively enhances pin1pin6 vein-pattern defects. Function of PIN6 is necessary, redundantly with that of PIN8, and sufficient to control auxin response levels, PIN1 expression, and vein network formation; and the vein pattern defects induced by ectopic PIN6 expression are mimicked by ectopic PIN8 expression. Finally, vein patterning functions of PIN6 and PIN8 are antagonized by PIN5 function. Our data define a new level of control of vein patterning, one with repercussions on other patterning processes in the plant, and suggest a mechanism to select cell files specialized for vascular function that predates evolution of PM-localized PIN proteins.

Unique and overlapping expression patterns among members of photosynthesis-associated nuclear gene families in Arabidopsis.

Light provides crucial positional information in plant development, and the morphogenetic processes that are orchestrated by light signals are triggered by changes of gene expression in response to variations in light parameters. Control of expression of members of the RbcS and Lhc families of photosynthesis-associated nuclear genes by light cues is a paradigm for light-regulated gene transcription, but high-resolution expression profiles for these gene families are lacking. In this study, we have investigated expression patterns of members of the RbcS and Lhc gene families in Arabidopsis (Arabidopsis thaliana) at the cellular level during undisturbed development and upon controlled interference of the light environment. Members of the RbcS and Lhc gene families are expressed in specialized territories, including root tip, leaf adaxial, abaxial, and epidermal domains, and with distinct chronologies, identifying successive stages of leaf mesophyll ontogeny. Defined spatial and temporal overlap of gene expression fields suggest that the light-harvesting and photosynthetic apparatus may have a different polypeptide composition in different cells and that such composition could change over time even within the same cell.

Auxin transport-dependent, stage-specific dynamics of leaf vein formation.

For centuries, the formation of vein patterns in the leaf has intrigued biologists, mathematicians and philosophers. In leaf development, files of vein-forming procambial cells emerge from seemingly homogeneous subepidermal tissue through the selection of anatomically inconspicuous preprocambial cells. Although the molecular details underlying the orderly differentiation of veins in the leaf remain elusive, gradually restricted transport paths of the plant hormone auxin have long been implicated in defining sites of vein formation. Several recent advances now appear to converge on a more precise definition of the role of auxin flow at different stages of vascular development. The picture that emerges is that of vein formation as a self-organizing, reiterative, auxin transport-dependent process.

Time-lapse imaging of Arabidopsis leaf development shows dynamic patterns of procambium formation.

The principles underlying the formation of leaf veins have long intrigued developmental biologists. In leaves, networks of vascular precursor procambial cells emerge from seemingly homogeneous subepidermal tissue through the selection of anatomically inconspicuous preprocambial cells. Understanding dynamics of procambium formation has been hampered by the difficulty of observing the process in vivo. Here we present a live-imaging technique that allows visual access to complex events occurring in developing leaves. We combined this method with stage-specific fluorescent markers in Arabidopsis (Arabidopsis thaliana) to visualize preprocambial strand formation and procambium differentiation during the undisturbed course of development and upon defined perturbations of vein ontogeny. Under all experimental conditions, we observed extension, termination and fusion of preprocambial strands and simultaneous initiation of procambium differentiation along entire individual veins. Our findings strongly suggest that progressiveness of preprocambial strand formation and simultaneity of procambium differentiation represent inherent properties of the mechanism underlying vein formation.