Boron supply restores aluminum-blocked auxin transport by the modulation of PIN2 trafficking in the root apical transition zone.

The supply of boron (B) alleviates the toxic effects of aluminum (Al) on root growth; however, the mechanistic basis of this process remains elusive. This study filled this knowledge gap, demonstrating that boron modifies auxin distribution and transport in Al-exposed Arabidopsis roots. In B-deprived roots, treatment with Al induced an increase in auxin content in the root apical meristem zone (MZ) and transition zone (TZ), whereas in the elongation zone (EZ) the auxin content was decreased beyond the level required for adequate growth. These distribution patterns are explained by the fact that basipetal auxin transport from the TZ to the EZ was disrupted by Al-inhibited PIN-FORMED 2 (PIN2) endocytosis. Experiments involving the modulation of protein biosynthesis by cycloheximide (CHX) and transcriptional regulation by cordycepin (COR) demonstrated that the Al-induced increase of PIN2 membrane proteins was dependent upon the inhibition of PIN2 endocytosis, rather than on the transcriptional regulation of the PIN2 gene. Experiments reporting on the profiling of Al3+ and PIN2 proteins revealed that the inhibition of endocytosis of PIN2 proteins was the result of Al-induced limitation of the fluidity of the plasma membrane. The supply of B mediated the turnover of PIN2 endosomes conjugated with indole-3-acetic acid (IAA), and thus restored the Al-induced inhibition of IAA transport through the TZ to the EZ. Overall, the reported results demonstrate that boron supply mediates PIN2 endosome-based auxin transport to alleviate Al toxicity in plant roots.

Mapping sites of gibberellin biosynthesis in the Arabidopsis root tip.

Root elongation depends on the action of the gibberellin (GA) growth hormones, which promote cell production in the root meristem and cell expansion in the elongation zone. Sites of GA biosynthesis in the roots of 7-d-old Arabidopsis thaliana seedlings were investigated using tissue-specific GA inactivation in wild-type (Col-0) or rescue of GA-deficient dwarf mutants. Tissue-specific GA depletion was achieved by ectopic expression of the GA-inactivating enzyme AtGA2ox2, which is specific for C19 -GAs, and AtGA2ox7, which acts on C20 -GA precursors. In addition, tissue-specific rescue of ga20ox triple and ga3ox double mutants was shown. Furthermore, GUS reporter lines for major GA20ox, GA3ox and GA2ox genes were used to observe their expression domains in the root. The effects of expressing these constructs on the lengths of the root apical meristem and cortical cells in the elongation zone confirmed that roots are autonomous for GA biosynthesis, which occurs in multiple tissues, with the endodermis a major site of synthesis. The results are consistent with the early stages of GA biosynthesis within the root occurring in the meristematic region and indicate that the penultimate step of GA biosynthesis, GA 20-oxidation, is required in both the meristem and elongation zone.

Root vacuolar Na+ sequestration but not exclusion from uptake correlates with barley salt tolerance.

Soil salinity is a major constraint for the global agricultural production. For many decades, Na+ exclusion from uptake has been the key trait targeted in breeding programs; yet, no major breakthrough in creating salt-tolerant germplasm was achieved. In this work, we have combined the microelectrode ion flux estimation (MIFE) technique for non-invasive ion flux measurements with confocal fluorescence dye imaging technique to screen 45 accessions of barley to reveal the relative contribution of Na+ exclusion from the cytosol to the apoplast and its vacuolar sequestration in the root apex, for the overall salinity stress tolerance. We show that Na+ /H+ antiporter-mediated Na+ extrusion from the root plays a minor role in the overall salt tolerance in barley. At the same time, a strong and positive correlation was found between root vacuolar Na+ sequestration ability and the overall salt tolerance. The inability of salt-sensitive genotypes to sequester Na+ in root vacuoles was in contrast to significantly higher expression levels of both HvNHX1 tonoplast Na+ /H+ antiporters and HvVP1 H+ -pumps compared with tolerant genotypes. These data are interpreted as a failure of sensitive varieties to prevent Na+ back-leak into the cytosol and existence of a futile Na+ cycle at the tonoplast. Taken together, our results demonstrated that root vacuolar Na+ sequestration but not exclusion from uptake played the main role in barley salinity tolerance, and suggested that the focus of the breeding programs should be shifted from targeting genes mediating Na+ exclusion from uptake by roots to more efficient root vacuolar Na+ sequestration.

Longitudinal zonation pattern in Arabidopsis root tip defined by a multiple structural change algorithm.

Background and Aims The Arabidopsis thaliana root is a key experimental system in developmental biology. Despite its importance, we are still lacking an objective and broadly applicable approach for identification of number and position of developmental domains or zones along the longitudinal axis of the root apex or boundaries between them, which is essential for understanding the mechanisms underlying cell proliferation, elongation and differentiation dynamics during root development. Methods We used a statistics approach, the multiple structural change algorithm (MSC), for estimating the number and position of developmental transitions in the growing portion of the root apex. Once the positions of the transitions between domains and zones were determined, linear models were used to estimate the critical size of dividing cells (LcritD) and other parameters. Key Results The MSC approach enabled identification of three discrete regions in the growing parts of the root that correspond to the proliferation domain (PD), the transition domain (TD) and the elongation zone (EZ). Simultaneous application of the MSC approach and G2-to-M transition (CycB1;1DB:GFP) and endoreduplication (pCCS52A1:GUS) molecular markers confirmed the presence and position of the TD. We also found that the MADS-box gene XAANTAL1 (XAL1) is required for the wild-type (wt) PD increase in length during the first 2 weeks of growth. Contrary to wt, in the xal1 loss-of-function mutant the increase and acceleration of root growth were not detected. We also found alterations in LcritD in xal1 compared with wt, which was associated with longer cell cycle duration in the mutant. Conclusions The MSC approach is a useful, objective and versatile tool for identification of the PD, TD and EZ and boundaries between them in the root apices and can be used for the phenotyping of different genetic backgrounds, experimental treatments or developmental changes within a genotype. The tool is publicly available at www.ibiologia.com.mx/MSC_analysis.

Organellar genome copy number variation and integrity during moderate maturation of roots and leaves of maize seedlings.

Little information is available about organellar genome copy numbers and integrity in plant roots, although it was reported recently that the plastid and mitochondrial genomes were damaged under light, resulting in non-functional fragments in green seedling leaves in a maize line. In the present study, we investigated organellar genome copy numbers and integrity, after assessing the cellular ploidy, in seedling leaves and roots of two elite maize (Zea mays) cultivars using both long-fragment polymerase chain reaction (long-PCR) and real-time quantitative polymerase chain reaction (qPCR, a type of short-PCR). Since maize leaf and root cells are mainly diploid according to chromosome number counting and the literature, the DNA amount ratio between the organellar genomes and the nuclear genome could be used to estimate average organellar genome copy numbers per cell. In the present study, both long-PCR and qPCR analyses found that green leaves had dramatically more plastid DNA and less mitochondrial DNA than roots had in both cultivars. The similarity in results from long-PCR and qPCR suggests that green leaves and roots during moderate maturation have largely intact plastid and mitochondrial genomes. The high resolution of qPCR led to the detection of an increase in copies in the plastid genome and a decrease in copies in the analyzed mitochondrial sub-genomes during the moderate maturation of seedling leaves and roots. These results suggest that green seedling leaves and roots of these two maize cultivars during moderate maturation had essentially intact organellar genomes, an increased copy number of the plastid genome, and decreased copy numbers of certain mitochondrial sub-genomes.

QTL for the Kinematic Traits That Define the Arabidopsis Root Elongation Zone and Their Relationship to Gravitropism.

Cell expansion in a discrete region called the elongation zone drives root elongation. Analyzing time lapse images can quantify the expansion in kinematic terms as if it were fluid flow. We used horizontal microscopes to collect images from which custom software extracted the length of the elongation zone, the peak relative elemental growth rate (REGR) within it, the axial position of the REGR peak, and the root elongation rate. Automation enabled these kinematic traits to be measured in 1575 Arabidopsis seedlings representing 162 recombinant inbred lines (RILs) derived from a cross of Cvi and Ler ecotypes. We mapped ten quantitative trait loci (QTL), affecting the four kinematic traits. Three QTL affected two or more traits in these vertically oriented seedlings. We compared this genetic architecture with that previously determined for gravitropism using the same RIL population. The major QTL peaks for the kinematic traits did not overlap with the gravitropism QTL. Furthermore, no single kinematic trait correlated with quantitative descriptors of the gravitropism response curve across this population. In addition to mapping QTL for growth zone traits, this study showed that the size and shape of the elongation zone may vary widely without affecting the differential growth induced by gravity.

Effects of polygalacturonase overexpression on pectin distribution in the elongation zones of roots under aluminium stress.

The roots of many plant species contain large amounts of pectin and it contributes to the formation of the rhizosphere. In the present study, the relationship between the root-tip pectin content and aluminium (Al) tolerance in wild-type (WT) and demethylesterified pectin degradation enzyme gene overexpressor (OsPG2-FOX) rice lines was compared. OsPG2-FOX rice showed reduced pectin content in roots, even under control conditions; Al treatment reduced root elongation and the pectin content in the root elongation zone. Wild-type rice showed more pectin accumulation in the root elongation zone after Al treatment. Relative to WT rice, OsPG2-FOX rice showed more Al accumulation in the root elongation zone. These results indicate that the amount of pectin influences Al tolerance and that the distribution of pectin in the root elongation zone inhibits Al accumulation in rice roots. Pectin accumulation in cell walls in the root elongation zone may play a role in protecting rice plants from the Al-induced inhibition of root elongation by regulating pectin distribution.

Combined Approach of GWAS and Phylogenetic Analyses to Identify New Candidate Genes That Participate in Arabidopsis thaliana Primary Root Development Using Cellular Measurements and Primary Root Length.

Genome-wide association studies (GWAS) have allowed the identification of different loci associated with primary root (PR) growth, and Arabidopsis is an excellent model for these studies. The PR length is controlled by cell proliferation, elongation, and differentiation; however, the specific contribution of proliferation and differentiation in the control of PR growth is still poorly studied. To this end, we analyzed 124 accessions and used a GWAS approach to identify potential causal genomic regions related to four traits: PR length, growth rate, cell proliferation and cell differentiation. Twenty-three genes and five statistically significant SNPs were identified. The SNP with the highest score mapped to the fifth exon of NAC048 and this change makes a missense variant in only 33.3% of the accessions with a large PR, compared with the accessions with a short PR length. Moreover, we detected five more SNPs in this gene and in NAC3 that allow us to discover closely related accessions according to the phylogenetic tree analysis. We also found that the association between genetic variants among the 18 genes with the highest scores in our GWAS and the phenotypic classes into which we divided our accessions are not straightforward and likely follow historical patterns.

Comparative Transcriptome Analysis Reveals Common and Developmental Stage-Specific Genes That Respond to Low Nitrogen in Maize Leaves.

A growing leaf can be divided into three sections: division zone, elongation zone, and maturation zone. In previous studies, low nitrogen (LN) inhibited maize growth and development, especially leaf growth; however, the gene expression in response to LN in different regions in leaf were not clear. Here, using hydroponics and a transcriptome approach, we systematically analyzed the molecular responses of those zones and differentially expressed genes (DEG) in response to LN supply. Developmental stage-specific genes (SGs) were highly stage-specific and involved in distinct biological processes. SGs from division (SGs-DZ) and elongation zones (SGs-EZ) were more related to developmentally dependent processes, whereas SGs of the maturation zone (SGs-MZ) were more related to metabolic processes. The common genes (CGs) were overrepresented in carbon and N metabolism, suggesting that rebalancing carbon and N metabolism in maize leaves under LN condition was independent of developmental stage. Coexpression modules (CMs) were also constructed in our experiment and a total of eight CMs were detected. Most of SGs-DZ and SGs-EZ were classified into a set termed CM turquoise, which was mainly enriched in ribosome and DNA replication, whereas several genes from SGs-MZ and CGs were clustered into CM blue, which mainly focused on photosynthesis and carbon metabolism. Finally, a comprehensive coexpression network was extracted from CM blue, and several maize CONSTANS-LIKE(ZmCOL) genes seemed to participate in regulating photosynthesis in maize leaves under LN condition in a developmental stage-specific manner. With this study, we uncovered the LN-responsive CGs and SGs that are important for promoting plant growth and development under insufficient nitrogen supply.

High ammonium inhibits root growth in Arabidopsis thaliana by promoting auxin conjugation rather than inhibiting auxin biosynthesis.

Ammonium (NH4+) inhibits primary root (PR) growth in most plant species when present even at moderate concentrations. Previous studies have shown that transport of indole-3-acetic acid (IAA) is critical to maintaining root elongation under high-NH4+ stress. However, the precise regulation of IAA homeostasis under high-NH4+ stress (HAS) remains unclear. In this study, qRT-PCR, RNA-seq, free IAA and IAA conjugate and PR elongation measurements were conducted in genetic mutants to investigate the role of IAA biosynthesis and conjugation under HAS. Our data clearly show that HAS decreases free IAA in roots by increasing IAA inactivation but does not decrease IAA biosynthesis, and that the IAA-conjugating genes GH3.1, GH3.2, GH3.3, GH3.4, and GH3.6 function as the key genes in regulating high-NH4+ sensitivity in the roots. Furthermore, the analysis of promoter::GUS staining in situ and genetic mutants reveals that HAS promotes IAA conjugation in the elongation zone (EZ), which may be responsible for the PR inhibition observed under HAS. This study provides potential new insight into the role of auxin in the improvement of tolerance to NH4+.

leafkin-An R package for automated kinematic data analysis of monocot leaves.

Growth is one of the most studied plant responses. At the cellular level, plant growth is driven by cell division and cell expansion. A means to quantify these two cellular processes is through kinematic analysis, a methodology that has been developed and perfected over the past decades, with in-depth descriptions of the methodology available. Unfortunately, after performing the lab work, researchers are required to perform time-consuming, repetitive and error-prone calculations. To lower the barrier towards this final step in the analysis and to aid researchers currently applying this technique, we have created leafkin, an R-package to perform all the calculations involved in the kinematic analysis of monocot leaves using only four functions. These functions support leaf elongation rate calculations, fitting of cell length profiles, extraction of fitted cell lengths and execution of kinematic equations. With the leafkin package, kinematic analysis of monocot leaves becomes more accessible than before.

Gravity Signaling in Flowering Plant Roots.

Roots typically grow downward into the soil where they anchor the plant and take up water and nutrients necessary for plant growth and development. While the primary roots usually grow vertically downward, laterals often follow a gravity set point angle that allows them to explore the surrounding environment. These responses can be modified by developmental and environmental cues. This review discusses the molecular mechanisms that govern root gravitropism in flowering plant roots. In this system, the primary site of gravity sensing within the root cap is physically separated from the site of curvature response at the elongation zone. Gravity sensing involves the sedimentation of starch-filled plastids (statoliths) within the columella cells of the root cap (the statocytes), which triggers a relocalization of plasma membrane-associated PIN auxin efflux facilitators to the lower side of the cell. This process is associated with the recruitment of RLD regulators of vesicular trafficking to the lower membrane by LAZY proteins. PIN relocalization leads to the formation of a lateral gradient of auxin across the root cap. Upon transmission to the elongation zone, this auxin gradient triggers a downward curvature. We review the molecular mechanisms that control this process in primary roots and discuss recent insights into the regulation of oblique growth in lateral roots and its impact on root-system architecture, soil exploration and plant adaptation to stressful environments.

Dual Color Sensors for Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells.

Spatiotemporal changes in cellular calcium (Ca2+) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca2+ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca2+ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca2+ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca2+ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca2+ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca2+ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca2+ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca2+ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.

Cell Wall Heterogeneity in Root Development of Arabidopsis.

Plant cell walls provide stability and protection to plant cells. During growth and development the composition of cell walls changes, but provides enough strength to withstand the turgor of the cells. Hence, cell walls are highly flexible and diverse in nature. These characteristics are important during root growth, as plant roots consist of radial patterns of cells that have diverse functions and that are at different developmental stages along the growth axis. Young stem cell daughters undergo a series of rapid cell divisions, during which new cell walls are formed that are highly dynamic, and that support rapid anisotropic cell expansion. Once the cells have differentiated, the walls of specific cell types need to comply with and support different cell functions. For example, a newly formed root hair needs to be able to break through the surrounding soil, while endodermal cells modify their walls at distinct positions to form Casparian strips between them. Hence, the cell walls are modified and rebuilt while cells transit through different developmental stages. In addition, the cell walls of roots readjust to their environment to support growth and to maximize nutrient uptake. Many of these modifications are likely driven by different developmental and stress signaling pathways. However, our understanding of how such pathways affect cell wall modifications and what enzymes are involved remain largely unknown. In this review we aim to compile data linking cell wall content and re-modeling to developmental stages of root cells, and dissect how root cell walls respond to certain environmental changes.

Interaction of galactoglucomannan oligosaccharides with auxin involves changes in flavonoid accumulation.

Galactoglucomannan oligosaccharides (GGMOs) are signalling molecules originating from plant cell walls influencing plant growth and defence reactions. The present study focused on their interaction with exogenous IAA (indole-3-acetic acid). GGMOs acted as auxin antagonists and diminished the effect of IAA on Arabidopsis primary root growth. Their effect is associated with meristem enlargement and prolongation of the elongation zone. Reduction of the elongation zone was a consequence of the IAA action, but IAA did not affect the size of the meristem. In the absence of auxin, GGMOs stimulated root growth, meristem enlargement and elongation zone prolongation. It is assumed that the effect of GGMOs in the absence of exogenous auxin resulted from their interaction with the endogenous form. In the presence of auxin transport inhibitor GGMOs did not affect root growth. It is known that flavonoids are auxin transport modulators but this is the first study suggesting the role of flavonoids in GGMOs' signalling. The accumulation of flavonoids in the meristem and elongation zone decreased in GGMOs' treatments in comparison with the control. These oligosaccharides also diminished the effect of IAA on the flavonoids' elevation. The fact that GGMOs decreased the accumulation of flavonoids, known to be modulators of auxin transport, and the loss of GGMOs' activity in the presence of the auxin transport inhibitor indicates that the root growth stimulation caused by GGMOs could be related to changes in auxin transport, possibly mediated by flavonoids.

Modeling Root Zone Effects on Preferred Pathways for the Passive Transport of Ions and Water in Plant Roots.

We extend a model of ion and water transport through a root to describe transport along and through a root exhibiting a complexity of differentiation zones. Attention is focused on convective and diffusive transport, both radially and longitudinally, through different root tissue types (radial differentiation) and root developmental zones (longitudinal differentiation). Model transport parameters are selected to mimic the relative abilities of the different tissues and developmental zones to transport water and ions. For each transport scenario in this extensive simulations study, we quantify the optimal 3D flow path taken by water and ions, in response to internal barriers such as the Casparian strip and suberin lamellae. We present and discuss both transient and steady state results of ion concentrations as well as ion and water fluxes. We find that the peak in passive uptake of ions and water occurs at the start of the differentiation zone. In addition, our results show that the level of transpiration has a significant impact on the distribution of ions within the root as well as the rate of ion and water uptake in the differentiation zone, while not impacting on transport in the elongation zone. From our model results we infer information about the active transport of ions in the different developmental zones. In particular, our results suggest that any uptake measured in the elongation zone under steady state conditions is likely to be due to active transport.

Changes in axial hydraulic conductivity along elongating leaf blades in relation to xylem maturation in tall fescue.

Xylem maturation in elongating leaf blades of tall fescue (Festuca arundinacea) was studied using staining and microcasting. Three distinctive regions were identified in the blade: (1) a basal region, in which elongation was occurring and protoxylem (PX) vessels were functioning throughout; (2) a maturation region, in which elongation had stopped and narrow (NMX) and large (LMX) metaxylem vessels were beginning to function; (3) a distal, mature region in which most of the longitudinal water movements occurred in the LMX. The axial hydraulic conductivity (Kh ) was measured in leaf sections from all these regions and compared with the theoretical axial hydraulic conductivity (Kt ) computed from the diameter of individual inner vessels. Kt was proportional to Kh throughout the leaf, but Kt was about three times Kh . The changes in Kh and Kt along the leaf reflected the different stages of xylem maturation. In the basal 60 mm region, Kh was about 0.30±0.07 mmol s-1 mm MPa-1 . Beyond that region, Kh rapidly increased with metaxylem element maturation to a maximum value of 5.0±0.3 mmol s-1 mm MPa-1 , 105 mm from the leaf base. It then decreased to 3.5±0.2 mmol s-1 mm MPa-1 near the leaf tip. The basal expanding region was observed to restrict longitudinal water movement. There was a close relationship between the water deposition rate in the elongation zone and the sum of the perimeters of PX vessels. The implications of this longitudinal vasculature on the partitioning of water between growth and transpiration is discussed.

Inoculation Method for Studying Early Responses of Glycine max to Heterodera glycines.

An inoculation technique was developed for studying molecular responses of soybean to the soybean cyst nematode (Heterodera glycines). Effect of inoculum age (0-7 days after eggs were released from cysts) and inoculation site (meristem, elongation, or differentiation zone) on infection were tested on four soybean genotypes. Two genotypes (PI 437654 and cv. Peking) were resistant and two (cv. Essex and cv. Hutcheson) were susceptible to race 3 of H. glycines. Inoculum consisting of second-stage juveniles (J2) was prepared by gently agitating nematode eggs at 75 revolutions per minute at 28 degrees C for various intervals. Infection rates were monitored cytologically. The most consistent infection rate was obtained with 48-hour-old inoculum containing more than 80% J2. More than 100 juveniles/root were observed after inoculation with the 48-hour-old inoculum placed at the root elongation zone, in both resistant and susceptible soybeans. Horizontal orientation of roots during inoculation, the use of concentrated J2 inoculurn (500 J2 in 125 mul/root), and restriction of inoculum to the root elongation zone facilitated synchronous root infection.

The Root Apex of Arabidopsis thaliana Consists of Four Distinct Zones of Growth Activities: Meristematic Zone, Transition Zone, Fast Elongation Zone and Growth Terminating Zone.

In the growing apex of Arabidopsis thaliana primary roots, cells proceed through four distinct phases of cellular activities. These zones and their boundaries can be well defined based on their characteristic cellular activities. The meristematic zone comprises, and is limited to, all cells that undergo mitotic divisions. Detailed in vivo analysis of transgenic lines reveals that, in the Columbia-0 ecotype, the meristem stretches up to 200 microm away from the junction between root and root cap (RCJ). In the transition zone, 200 to about 520 microm away from the RCJ, cells undergo physiological changes as they prepare for their fast elongation. Upon entering the transition zone, they progressively develop a central vacuole, polarize the cytoskeleton and remodel their cell walls. Cells grow slowly during this transition: it takes ten hours to triplicate cell length from 8.5 to about 35 microm in the trichoblast cell files. In the fast elongation zone, which covers the zone from 520 to about 850 microm from the RCJ, cell length quadruplicates to about 140 microm in only two hours. This is accompanied by drastic and specific cell wall alterations. Finally, root hairs fully develop in the growth terminating zone, where root cells undergo a minor elongation to reach their mature lengths.