pin2 mutant agravitropic root phenotype is conditional and nutrient-sensitive.

Plants have the capacity to sense and adapt to environmental factors using the phytohormone auxin as a major regulator of tropism and development. Among these responses, gravitropism is essential for plant roots to grow downward in the search for nutrients and water. We discovered a new mutant allele of the auxin efflux transporter PIN2 that revealed that pin2 agravitropic root mutants are conditional and nutrient-sensitive. We describe that nutrient composition of the medium, rather than osmolarity, can revert the agravitropic root phenotype of pin2. Indeed, on phosphorus- and nitrogen-deprived media, the agravitropic root defect was restored independently of primary root growth levels. Slow and fast auxin responses were evaluated using DR5 and R2D2 probes, respectively, and revealed a strong modulation by nutrient composition of the culture medium. We evaluated the role of PIN and AUX auxin transporters and demonstrated that neither PIN3 nor AUX1 are involved in this process. However, we observed the ectopic expression of PIN1 in the epidermis in the pin2 mutant background associated with permissive, but not restrictive, conditions. This ectopic expression was associated with a restoration of the asymmetric accumulation of auxin necessary for the reorientation of the root according to gravity. These observations suggest a strong regulation of auxin distribution by nutrients availability, directly impacting root's ability to drive their gravitropic response.

Auxin triggers pectin modification during rootlet emergence in white lupin.

Emergence of secondary roots through parental tissue is a highly controlled developmental process. Although the model plant Arabidopsis has been useful to uncover the predominant role of auxin in this process, its simple root structure is not representative of how emergence takes place in most plants, which display more complex root anatomy. White lupin is a legume crop producing structures called cluster roots, where closely spaced rootlets emerge synchronously. Rootlet primordia push their way through several cortical cell layers while maintaining the parent root integrity, reflecting more generally the lateral root emergence process in most multilayered species. In this study, we showed that lupin rootlet emergence is associated with an upregulation of cell wall pectin modifying and degrading genes under the active control of auxin. Among them, we identified LaPG3, a polygalacturonase gene typically expressed in cells surrounding the rootlet primordium and we showed that its downregulation delays emergence. Immunolabeling of pectin epitopes and their quantification uncovered a gradual pectin demethylesterification in the emergence zone, which was further enhanced by auxin treatment, revealing a direct hormonal control of cell wall properties. We also report rhamnogalacturonan-I modifications affecting cortical cells that undergo separation as a consequence of primordium outgrowth. In conclusion, we describe a model of how external tissues in front of rootlet primordia display cell wall modifications to allow for the passage of newly formed rootlets.

Dynamic Development of White Lupin Rootlets Along a Cluster Root.

White lupin produces cluster roots in response to phosphorus deficiency. Along the cluster root, numerous short rootlets successively appear, creating a spatial and temporal gradient of developmental stages that constitutes a powerful biological model to study the dynamics of the structural and functional evolution of these organs. The present study proposes a fine histochemical, transcriptomic and functional analysis of the rootlet development from its emergence to its final length. Between these two stages, the tissue structures of the rootlets were observed, the course of transcript expressions for the genes differentially expressed was monitored and some physiological events linked to Pi nutrition were followed. A switch between (i) a growing phase, in which a normal apical meristem is present and (ii) a specialized phase for nutrition, in which the rootlet is completely differentiated, was highlighted. In the final stage of its determinate growth, the rootlet is an organ with a very active metabolism, especially for the solubilization and absorption of several nutrients. This work discusses how the transition between a growing to a determinate state in response to nutritional stresses is found in other species and underlines the fundamental dilemma of roots between soil exploration and soil exploitation.

Pangenome of white lupin provides insights into the diversity of the species.

White lupin is an old crop with renewed interest due to its seed high protein content and high nutritional value. Despite a long domestication history in the Mediterranean basin, modern breeding efforts have been fairly scarce. Recent sequencing of its genome has provided tools for further description of genetic resources but detailed characterization of genomic diversity is still missing. Here, we report the genome sequencing of 39 accessions that were used to establish a white lupin pangenome. We defined 32 068 core genes that are present in all individuals and 14 822 that are absent in some and may represent a gene pool for breeding for improved productivity, grain quality, and stress adaptation. We used this new pangenome resource to identify candidate genes for alkaloid synthesis, a key grain quality trait. The white lupin pangenome provides a novel genetic resource to better understand how domestication has shaped the genomic variability within this crop. Thus, this pangenome resource is an important step towards the effective and efficient genetic improvement of white lupin to help meet the rapidly growing demand for plant protein sources for human and animal consumption.

High-quality genome sequence of white lupin provides insight into soil exploration and seed quality.

White lupin (Lupinus albus L.) is an annual crop cultivated for its protein-rich seeds. It is adapted to poor soils due to the production of cluster roots, which are made of dozens of determinate lateral roots that drastically improve soil exploration and nutrient acquisition (mostly phosphate). Using long-read sequencing technologies, we provide a high-quality genome sequence of a cultivated accession of white lupin (2n = 50, 451 Mb), as well as de novo assemblies of a landrace and a wild relative. We describe a modern accession displaying increased soil exploration capacity through early establishment of lateral and cluster roots. We also show how seed quality may have been impacted by domestication in term of protein profiles and alkaloid content. The availability of a high-quality genome assembly together with companion genomic and transcriptomic resources will enable the development of modern breeding strategies to increase and stabilize white lupin yield.

Tissue-specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology.

Recent advances in the study of plant developmental and physiological responses have benefited from tissue-specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue-specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5-fluorocytosine (5-FC) into 5-fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY-UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell-autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5-FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell-specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type-specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development.

Anatomical and hormonal description of rootlet primordium development along white lupin cluster root.

Cluster root (CR) is one of the most spectacular plant developmental adaptations to hostile environment. It can be found in a few species from a dozen botanical families, including white lupin (Lupinus albus) in the Fabaceae family. These amazing structures are produced in phosphate-deprived conditions and are made of hundreds of short roots also known as rootlets. White lupin is the only crop bearing CRs and is considered as the model species for CR studies. However, little information is available on CRs atypical development, including the molecular events that trigger their formation. To provide insights on CR formation, we performed an anatomical and cellular description of rootlet development in white lupin. Starting with a classic histological approach, we described rootlet primordium development and defined eight developmental stages from rootlet initiation to their emergence. Due to the major role of hormones in the developmental program of root system, we next focussed on auxin-related mechanisms. We observed the establishment of an auxin maximum through rootlet development in transgenic roots expressing the DR5:GUS auxin reporter. Expression analysis of the main auxin-related genes [TIR, Auxin Response Factor (ARF) and AUX/IAA] during a detailed time course revealed specific expression associated with the formation of the rootlet primordium. We showed that L. albus TRANSPORT INHIBITOR RESPONSE 1b is expressed during rootlet primordium formation and that L. albus AUXIN RESPONSE FACTOR 5 is expressed in the vasculature but absent in the primordium itself. Altogether, our results describe the very early cellular events leading to CR formation and reveal some of the auxin-related mechanisms.

A protocol combining multiphoton microscopy and propidium iodide for deep 3D root meristem imaging in rice: application for the screening and identification of tissue-specific enhancer trap lines.

BACKGROUND: The clear visualization of 3D organization at the cellular level in plant tissues is needed to fully understand plant development processes. Imaging tools allow the visualization of the main fluorophores and in vivo growth monitoring. Confocal microscopy coupled with the use of propidium iodide (PI) counter-staining is one of the most popular tools used to characterize the structure of root meristems in A. thaliana. However, such an approach is relatively ineffective in species with more complex and thicker root systems. RESULTS: We adapted a PI counter-staining protocol to visualize the internal 3D architecture of rice root meristems using multiphoton microscopy. This protocol is simple and compatible with the main fluorophores (CFP, GFP and mCherry). The efficiency and applicability of this protocol were demonstrated by screening a population of 57 enhancer trap lines. We successfully characterized GFP expression in all of the lines and identified 5 lines with tissue-specific expression. CONCLUSIONS: All of these resources are now available for the rice community and represent critical tools for future studies of root development.

Immunoprofiling of Rice Root Cortex Reveals Two Cortical Subdomains.

The formation and differentiation of aerenchyma, i.e., air-containing cavities that are critical for flooding tolerance, take place exclusively in the cortex. The understanding of development and differentiation of the cortex is thus an important issue; however, studies on this tissue are limited, partly because of the lack of available molecular tools. We screened a commercially available library of cell wall antibodies to identify markers of cortical tissue in rice roots. Out of the 174 antibodies screened, eight were cortex-specific. Our analysis revealed that two types of cortical tissues are present in rice root seedlings. We named these cell layers "inner" and "outer" based on their location relative to the stele. We then used the antibodies to clarify cell identity in lateral roots. Without these markers, previous studies could not distinguish between the cortex and sclerenchyma in small lateral roots. By immunostaining lateral root sections, we showed that the internal ground tissue in small lateral roots has outer cortical identity.

A plausible mechanism, based upon Short-Root movement, for regulating the number of cortex cell layers in roots.

Formation of specialized cells and tissues at defined times and in specific positions is essential for the development of multicellular organisms. Often this developmental precision is achieved through intercellular signaling networks, which establish patterns of differential gene expression and ultimately the specification of distinct cell fates. Here we address the question of how the Short-root (SHR) proteins from Arabidopsis thaliana (AtSHR), Brachypodium distachyon (BdSHR), and Oryza sativa (OsSHR1 and OsSHR2) function in patterning the root ground tissue. We find that all of the SHR proteins function as mobile signals in A. thaliana and all of the SHR homologs physically interact with the AtSHR binding protein, Scarecow (SCR). Unlike AtSHR, movement of the SHR homologs was not limited to the endodermis. Instead, the SHR proteins moved multiple cell layers and determined the number of cortex, not endodermal, cell layers formed in the root. Our results in A. thaliana are consistent with a mechanism by which the regulated movement of the SHR transcription factor determines the number of cortex cell layers produced in the roots of B. distachyon and O. sativa. These data also provide a new model for ground tissue patterning in A. thaliana in which the ability to form a functional endodermis is spatially limited independently of SHR.

PHIV-RootCell: a supervised image analysis tool for rice root anatomical parameter quantification.

We developed the PHIV-RootCell software to quantify anatomical traits of rice roots transverse section images. Combined with an efficient root sample processing method for image acquisition, this program permits supervised measurements of areas (those of whole root section, stele, cortex, and central metaxylem vessels), number of cell layers and number of cells per cell layer. The PHIV-RootCell toolset runs under ImageJ, an independent operating system that has a license-free status. To demonstrate the usefulness of PHIV-RootCell, we conducted a genetic diversity study and an analysis of salt stress responses of root anatomical parameters in rice (Oryza sativa L.). Using 16 cultivars, we showed that we could discriminate between some of the varieties even at the 6 day-olds stage, and that tropical japonica varieties had larger root sections due to an increase in cell number. We observed, as described previously, that root sections become enlarged under salt stress. However, our results show an increase in cell number in ground tissues (endodermis and cortex) but a decrease in external (peripheral) tissues (sclerenchyma, exodermis, and epidermis). Thus, the PHIV-RootCell program is a user-friendly tool that will be helpful for future genetic and physiological studies that investigate root anatomical trait variations.

New insights into Fe localization in plant tissues.

Deciphering cellular iron (Fe) homeostasis requires having access to both quantitative and qualitative information on the subcellular pools of Fe in tissues and their dynamics within the cells. We have taken advantage of the Perls/DAB Fe staining procedure to perform a systematic analysis of Fe distribution in roots, leaves and reproductive organs of the model plant Arabidopsis thaliana, using wild-type and mutant genotypes affected in iron transport and storage. Roots of soil-grown plants accumulate iron in the apoplast of the central cylinder, a pattern that is strongly intensified when the citrate effluxer FRD3 is not functional, thus stressing the importance of citrate in the apoplastic movement of Fe. In leaves, Fe level is low and only detected in and around vascular tissues. In contrast, Fe staining in leaves of iron-treated plants extends in the surrounding mesophyll cells where Fe deposits, likely corresponding to Fe-ferritin complexes, accumulate in the chloroplasts. The loss of ferritins in the fer1,3,4 triple mutant provoked a massive accumulation of Fe in the apoplastic space, suggesting that in the absence of iron buffering in the chloroplast, cells activate iron efflux and/or repress iron influx to limit the amount of iron in the cell. In flowers, Perls/DAB staining has revealed a major sink for Fe in the anthers. In particular, developing pollen grains accumulate detectable amounts of Fe in small-size intracellular bodies that aggregate around the vegetative nucleus at the binuclear stage and that were identified as amyloplasts. In conclusion, using the Perls/DAB procedure combined to selected mutant genotypes, this study has established a reliable atlas of Fe distribution in the main Arabidopsis organs, proving and refining long-assumed intracellular locations and uncovering new ones. This "iron map" of Arabidopsis will serve as a basis for future studies of possible actors of iron movement in plant tissues and cell compartments.

The Arabidopsis YELLOW STRIPE LIKE4 and 6 transporters control iron release from the chloroplast.

In most plant cell types, the chloroplast represents the largest sink for iron, which is both essential for chloroplast metabolism and prone to cause oxidative damage. Here, we show that to buffer the potentially harmful effects of iron, besides ferritins for storage, the chloroplast is equipped with specific iron transporters that respond to iron toxicity by removing iron from the chloroplast. We describe two transporters of the YELLOW STRIPE1-LIKE family from Arabidopsis thaliana, YSL4 and YSL6, which are likely to fulfill this function. Knocking out both YSL4 and YSL6 greatly reduces the plant's ability to cope with excess iron. Biochemical and immunolocalization analyses showed that YSL6 resides in the chloroplast envelope. Elemental analysis and histochemical staining indicate that iron is trapped in the chloroplasts of the ysl4 ysl6 double mutants, which also accumulate ferritins. Also, vacuolar iron remobilization and NRAMP3/4 expression are inhibited. Furthermore, ubiquitous expression of YSL4 or YSL6 dramatically reduces plant tolerance to iron deficiency and decreases chloroplastic iron content. These data demonstrate a fundamental role for YSL4 and YSL6 in managing chloroplastic iron. YSL4 and YSL6 expression patterns support their physiological role in detoxifying iron during plastid dedifferentiation occurring in embryogenesis and senescence.

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots.

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.

Metal movement within the plant: contribution of nicotianamine and yellow stripe 1-like transporters.

Background Since the identification of the genes controlling the root acquisition of iron (Fe), the control of inter- and intracellular distribution has become an important challenge in understanding metal homeostasis. The identification of the yellow stripe-like (YSL) transporter family has paved the way to decipher the mechanisms of long-distance transport of Fe. Scope Once in the plant, Fe will systematically react with organic ligands whose identity is poorly known so far. Among potential ligands, nicotianamine has been identified as an important molecule for the circulation and delivery of metals since it participates in the loading of copper (Cu) and nickel in xylem and prevents Fe precipitation in leaves. Nicotianamine is a precursor of phytosiderophores, which are high-affinity Fe ligands exclusively synthesized by Poaceae species and excreted by roots for the chelation and acquisition of Fe. Maize YS1 is the founding member of a family of membrane transporters called YS1-like (YSL), which functions in root Fe-phytosiderophore uptake from the soil. Next to this well-known Fe acquisition role, most of the other YSL family members are likely to function in plant-wide distribution of metals since (a) they are produced in vascular tissues throughout the plant and (b) they are found in non-Poaceae species that do not synthesize phytosiderophores. The hypothesized activity as Fe-nicotianamine transporters of several YSL members has been demonstrated experimentally by heterologous expression in yeast or by electrophysiology in Xenopus oocytes but, despite numerous attempts, proof of the arabidopsis YSL substrate specificity is still lacking. Reverse genetics, however, has revealed a role for AtYSL members in the remobilization of Cu and zinc from senescing leaves, in the formation of pollen and in the Fe, zinc and Cu loading of seeds. Conclusions Preliminary data on the YSL family of transporters clearly argues in favour of its role in the long-distance transport of metals through and between vascular tissues to eventually support gametogenesis and embryo development.

Involvement of the xyloglucan endotransglycosylase/hydrolases encoded by celery XTH1 and Arabidopsis XTH33 in the phloem response to aphids.

During infestation, phloem-feeding insects induce transcriptional reprogramming in plants that may lead to protection. Transcripts of the celery XTH1 gene, encoding a xyloglucan endotransglycosylase/hydrolase (XTH), were previously found to accumulate systemically in celery (Apium graveolens) phloem, following infestation with the generalist aphid Myzus persicae. XTH1 induction was specific to the phloem but was not correlated with an increase in xyloglucan endotransglycosylase (XET) activity in the phloem. XTH1 is homologous to the Arabidopsis thaliana XTH33 gene. XTH33 expression was investigated following M. persicae infestation. The pattern of XTH33 expression is tightly controlled during development and indicates a possible role in cell expansion. An xth33 mutant was assayed for preference assay with M. persicae. Aphids settled preferentially on the mutant rather than on the wild type. This suggests that XTH33 is involved in protecting plants against aphids; therefore, that cell wall modification can alter the preference of aphids for a particular plant. Nevertheless, the ectopic expression of XTH33 in phloem tissue was not sufficient to confer protection, demonstrating that modifying the expression of this single gene does not readily alter plant-aphid interactions.

Protophloem differentiation in early Arabidopsis thaliana development.

During Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell divisions, giving rise to vascular precursor cells (protophloem and protoxylem precursors). After germination, these cells terminally differentiate into specialized conducting cells, referred to as protophloem and protoxylem cells. Few readily identifiable markers of the onset of specification and differentiation are available, hampering the molecular genetic analysis of protophloem development. Confocal microscopy was used to investigate the patterning and differentiation of phloem cells during early plant development. Longitudinal divisions of phloem initials allowed the identification of protophloem precursor cells and adjacent metaphloem initials along the length of the plant. During germination, protophloem differentiation was observed at two independent locations, in the cotyledons and the hypocotyl. In both locations, differentiation was concomitant with cell elongation. We identified five gene-trap lines (PD1-PD5) with marker gene expression in immature protophloem elements. The spatio-temporal marker expression pattern of the lines divides them into two groups. The early specification markers PD4 and PD5 were expressed in developing organs before procambium formation and then became restricted to phloem initial cells. The protophloem precursor markers PD1-PD3 were expressed in differentiating protophloem cells at different stages of their development. All markers were expressed transiently and iteratively during the differentiation of protophloem in newly formed organs. Flanking genes were identified for four out of five gene-trap insertion lines. The possible function of these genes with respect to phloem differentiation is discussed.

Systemic response to aphid infestation by Myzus persicae in the phloem of Apium graveolens.

Little is known about the molecular processes involved in the phloem response to aphid feeding. We investigated molecular responses to aphid feeding on celery (Apium graveolenscv. Dulce) plants infested with the aphid Myzus persicae, as a means of identifying changes in phloem function. We used celery as our model species as it is easy to separate the phloem from the surrounding tissues in the petioles of mature leaves of this species. We generated a total of 1187 expressed sequence tags (ESTs), corresponding to 891 non-redundant genes. We analysed these ESTs in silico after cDNA macroarray hybridisation. Aphid feeding led to significant increase in RNA accumulation for 126 different genes. Different patterns of deregulation were observed, including transitory or stable induction 3 or 7 days after infestation. The genes affected belonged to various functional categories and were induced systemically in the phloem after infestation. In particular, genes involved in cell wall modification, water transport, vitamin biosynthesis, photosynthesis, carbon assimilation and nitrogen and carbon mobilisation were up-regulated in the phloem. Further analysis of the response in the phloem or xylem suggested that a component of the response was developed more specifically in the phloem. However, this component was different from the stress responses in the phloem driven by pathogen infection. Our results indicate that the phloem is actively involved in multiple adjustments, recruiting metabolic pathways and in structural changes far from aphid feeding sites. However, they also suggest that the phloem displays specific mechanisms that may not be induced in other tissues.