Microtubule-associated ROP interactors affect microtubule dynamics and modulate cell wall patterning and root hair growth.

Rho of plant (ROP) proteins and the interactor of constitutively active ROP (ICR) family member ICR5/MIDD1 have been implicated to function as signaling modules that regulate metaxylem secondary cell wall patterning. Yet, loss-of-function mutants of ICR5 and its closest homologs have not been studied and, hence, the functions of these ICR family members are not fully established. Here, we studied the functions of ICR2 and its homolog ICR5. We show that ICR2 is a microtubule-associated protein that affects microtubule dynamics. Secondary cell wall pits in the metaxylem of Arabidopsis icr2 and icr5 single mutants and icr2 icr5 double mutants are smaller than those in wild-type Col-0 seedlings; however, they are remarkably denser, implying a complex function of ICRs in secondary cell wall patterning. ICR5 has a unique function in protoxylem secondary cell wall patterning, whereas icr2, but not icr5, mutants develop split root hairs, demonstrating functional diversification. Taken together, our results show that ICR2 and ICR5 have unique and cooperative functions as microtubule-associated proteins and as ROP effectors.

Brachypodium distachyon MAP20 functions in metaxylem pit development and contributes to drought recovery.

Pits are regions in the cell walls of plant tracheary elements that lack secondary walls. Each pit consists of a space within the secondary wall called a pit chamber, and a modified primary wall called the pit membrane. The pit membrane facilitates transport of solutions between vessel cells and restricts embolisms during drought. Here we analyzed the role of an angiosperm-specific TPX2-like microtubule protein MAP20 in pit formation using Brachypodium distachyon as a model system. Live cell imaging was used to analyze the interaction of MAP20 with microtubules and the impact of MAP20 on microtubule dynamics. MAP20-specific antibody was used to study expression and localization of MAP20 in different cell types during vascular bundle development. We used an artificial microRNAs (amiRNA) knockdown approach to determine the function of MAP20. MAP20 is expressed during the late stages of vascular bundle development and localizes around forming pits and under secondary cell wall thickenings in metaxylem cells. MAP20 suppresses microtubule depolymerization; however, unlike the animal TPX2 counterpart, MAP20 does not cooperate with the γ-tubulin ring complex in microtubule nucleation. Knockdown of MAP20 causes bigger pits, thinner pit membranes, perturbed vasculature development, lower reproductive potential and higher drought susceptibility. We conclude that MAP20 may contribute to drought adaptation by modulating pit size and pit membrane thickness in metaxylem.

Jasmonic Acid Methyl Ester Induces Xylogenesis and Modulates Auxin-Induced Xylary Cell Identity with NO Involvement.

In Arabidopsis basal hypocotyls of dark-grown seedlings, xylary cells may form from the pericycle as an alternative to adventitious roots. Several hormones may induce xylogenesis, as Jasmonic acid (JA), as well as indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) auxins, which also affect xylary identity. Studies with the ethylene (ET)-perception mutant ein3eil1 and the ET-precursor 1-aminocyclopropane-1-carboxylic acid (ACC), also demonstrate ET involvement in IBA-induced ectopic metaxylem. Moreover, nitric oxide (NO), produced after IBA/IAA-treatments, may affect JA signalling and interact positively/negatively with ET. To date, NO-involvement in ET/JA-mediated xylogenesis has never been investigated. To study this, and unravel JA-effects on xylary identity, xylogenesis was investigated in hypocotyls of seedlings treated with JA methyl-ester (JAMe) with/without ACC, IBA, IAA. Wild-type (wt) and ein3eil1 responses to hormonal treatments were compared, and the NO signal was quantified and its role evaluated by using NO-donors/scavengers. Ectopic-protoxylem increased in the wt only after treatment with JAMe(10 µM), whereas in ein3eil1 with any JAMe concentration. NO was detected in cells leading to either xylogenesis or adventitious rooting, and increased after treatment with JAMe(10 µM) combined or not with IBA(10 µM). Xylary identity changed when JAMe was applied with each auxin. Altogether, the results show that xylogenesis is induced by JA and NO positively regulates this process. In addition, NO also negatively interacts with ET-signalling and modulates auxin-induced xylary identity.

Tryptophan-dependent auxin biosynthesis is required for HD-ZIP III-mediated xylem patterning.

The development and growth of higher plants is highly dependent on the conduction of water and minerals throughout the plant by xylem vessels. In Arabidopsis roots the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. During vascular development, high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. Protoxylem specification is determined by both mobile, ground tissue-emanating miRNA165/6 species, which downregulate, and auxin concentrated by polar transport, which promotes HD-ZIP III expression. However, the factors promoting high HD-ZIP III expression for metaxylem identity have remained elusive. We show here that auxin biosynthesis promotes HD-ZIP III expression and metaxylem specification. Several auxin biosynthesis genes are expressed in the outer layers surrounding the vascular tissue in Arabidopsis root and downregulation of HD-ZIP III expression accompanied by specific defects in metaxylem development is seen in auxin biosynthesis mutants, such as trp2-12, wei8 tar2 or a quintuple yucca mutant, and in plants treated with L-kynurenine, a pharmacological inhibitor of auxin biosynthesis. Some of the patterning defects can be suppressed by synthetically elevated HD-ZIP III expression. Taken together, our results indicate that polar auxin transport, which was earlier shown to be required for protoxylem formation, is not sufficient to establish a proper xylem axis but that root-based auxin biosynthesis is additionally required.

The Arabidopsis polyamine oxidase/dehydrogenase 5 interferes with cytokinin and auxin signaling pathways to control xylem differentiation.

In plants, the polyamines putrescine, spermidine, spermine (Spm), and thermospermine (Therm-Spm) participate in several physiological processes. In particular, Therm-Spm is involved in the control of xylem differentiation, having an auxin antagonizing effect. Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis, five PAOs are present, among which AtPAO5 catalyzes the back-conversion of Spm, Therm-Spm, and N1-acetyl-Spm to spermidine. In the present study, it is shown that two loss-of-function atpao5 mutants and a 35S::AtPAO5 Arabidopsis transgenic line present phenotypical differences from the wild-type plants with regard to stem and root elongation, differences that are accompanied by changes in polyamine levels and the number of xylem vessels. It is additionally shown that cytokinin treatment, which up-regulates AtPAO5 expression in roots, differentially affects protoxylem differentiation in 35S::AtPAO5, atpao5, and wild-type roots. Together with these findings, Therm-Spm biosynthetic genes, as well as auxin-, xylem-, and cytokinin-related genes (such as ACL5, SAMDC4, PIN1, PIN6, VND6, VND7, ATHB8, PHB, CNA, PXY, XTH3, XCP1, and AHP6) are shown to be differentially expressed in the various genotypes. These data suggest that AtPAO5, being involved in the control of Therm-Spm homeostasis, participates in the tightly controlled interplay between auxin and cytokinins that is necessary for proper xylem differentiation.

Indole-3-Butyric Acid Induces Ectopic Formation of Metaxylem in the Hypocotyl of Arabidopsis thaliana without Conversion into Indole-3-Acetic Acid and with a Positive Interaction with Ethylene.

The role of the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) and of the auxin-interacting phytohormone ethylene, on the ectopic formation of primary xylem (xylogenesis in planta) is still little known. In particular, auxin/ethylene-target tissue(s), modality of the xylary process (trans-differentiation vs. de novo formation), and the kind of ectopic elements formed (metaxylem vs. protoxylem) are currently unknown. It is also unclear whether IBA may act on the process independently of conversion into IAA. To investigate these topics, histological analyses were carried out in the hypocotyls of Arabidopsis wild type seedlings and ech2ibr10 and ein3eil1 mutants, which are blocked in IBA-to-IAA conversion and ethylene signalling, respectively. The seedlings were grown under darkness with either IAA or IBA, combined or not with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Adventitious root formation was also investigated because this process may compete with xylogenesis. Our results show that ectopic formation of protoxylem and metaxylem occurred as an indirect process starting from the pericycle periclinal derivatives of the hypocotyl basal part. IAA favoured protoxylem formation, whereas IBA induced ectopic metaxylem with ethylene cooperation through the EIN3EIL1 network. Ectopic metaxylem differentiation occurred independently of IBA-to-IAA conversion as mediated by ECH2 and IBR10, and in the place of IBA-induced adventitious root formation.

Re-Evaluation of Vascular Histogenesis in the Root Tips of Selected Species in the Poaceae Using New Methods: Analysis of the Plerome, Vascular Initials, Pericycle and Late-Maturing Metaxylem Vessels.

Serial sectioning and 3D image reconstruction methods were applied to elucidate the structures of the apices of root vascular cylinders (VCs) in taxa of the Poaceae: Zea mays "Honey Bantam", Z. mays ssp. mexicana, Hordeum vulgare and Oryza sativa. The primary and nodal roots were investigated. Observations were performed using high-quality sectioning and 3D image-processing techniques improved and developed by the authors. We found that a quiescent uniseriate plerome was located at the most distal part of each VC. Vascular initials were located immediately basipetally to the plerome as a specific uniseriate layer that could be classified into central and peripheral initials that produced all the cells in the VC. No supplying of cells from the plerome to the vascular initials was observed. Numerical analysis revealed a "boundary point" along the root axis where the rate of increase of the vascular cell number markedly declined, and the VC diameter, number of vascular cells, and number of late-maturing metaxylem vessels (LMXs) at that point showed a similar relationship among the taxa and the types of roots examined (primary vs. nodal). The plerome and vascular initials layer can be considered independent after seed germination in these taxa. A boundary point at which procambial cell proliferation sharply declined was identified. The diameters of the VCs, number of LMXs, and number of vascular cells at the boundary point were found to be strongly related to each other.

Narrow lpa1 Metaxylems Enhance Drought Tolerance and Optimize Water Use for Grain Filling in Dwarf Rice.

Rice cultivation needs extensive amounts of water. Moreover, increased frequency of droughts and water scarcity has become a global concern for rice cultivation. Hence, optimization of water use is crucial for sustainable agriculture. Here, we characterized Loose Plant Architecture 1 (LPA1) in vasculature development, water transport, drought resistance, and grain yield. We performed genetic combination of lpa1 with semi-dwarf mutant to offer the optimum rice architecture for more efficient water use. LPA1 expressed in pre-vascular cells of leaf primordia regulates genes associated with carbohydrate metabolism and cell enlargement. Thus, it plays a role in metaxylem enlargement of the aerial organs. Narrow metaxylem of lpa1 exhibit leaves curling on sunny day and convey drought tolerance but reduce grain yield in mature plants. However, the genetic combination of lpa1 with semi-dwarf mutant (dep1-ko or d2) offer optimal water supply and drought resistance without impacting grain-filling rates. Our results show that water use, and transports can be genetically controlled by optimizing metaxylem vessel size and plant height, which may be utilized for enhancing drought tolerance and offers the potential solution to face the more frequent harsh climate condition in the future.

A Genome-Wide Association Study Dissects the Genetic Architecture of the Metaxylem Vessel Number in Maize Brace Roots.

Metaxylem vessels in maize brace roots are key tissue, and their number (MVN) affects plant water and inorganic salt transportation and lodging resistance. Dissecting the genetic basis of MVN in maize brace roots can help guide the genetic improvement of maize drought resistance and lodging resistance during late developmental stages. In this study, we used 508 inbred lines with tropical, subtropical, and temperate backgrounds to analyze the genetic architecture of MVN in maize brace roots. The phenotypic variation in MVN in brace roots was evaluated in three environments, which revealed broad natural variation and relative low levels of heritability (h 2 = 0.42). Stiff-stalk lines with a temperate background tended to have higher MVNs than plants in other genetic backgrounds. MVN was significantly positively correlated with plant height, tassel maximum axis length, ear length, and kernel number per row, which indicates that MVN may affect plant morphological development and yield. In addition, MVN was extremely significantly negatively correlated with brace root radius, but significantly positively correlated with brace root angle (BRA), diameter, and number, thus suggesting that the morphological function of some brace root traits may be essentially determined by MVN. Association analysis of MVN in brace roots combined 1,253,814 single nucleotide polymorphisms (SNPs) using FarmCPU revealed a total of nine SNPs significantly associated with MVN at P < 7.96 × 10-7. Five candidate genes for MVN that may participate in secondary wall formation (GRMZM2G168365, GRMZM2G470499, and GRMZM2G028982) and regulate flowering time (GRMZM2G381691 and GRMZM2G449165). These results provide useful information for understanding the genetic basis of MVN in brace root development. Further functional studies of identified candidate genes should help elucidate the molecular pathways that regulate MVN in maize brace roots.

Many paths to one goal: Identifying integrated rice root phenotypes for diverse drought environments.

Drought is a major source of yield loss in the production of rice (Oryza sativa L.), and cultivars that maintain yield under drought across environments and drought stress scenarios are urgently needed. Root phenotypes directly affect water interception and uptake, so plants with root systems optimized for water uptake under drought would likely exhibit reduced yield loss. Deeper nodal roots that have a low metabolic cost per length (i.e., cheaper roots) via smaller root diameter and/or more aerenchyma and that transport water efficiently through smaller diameter metaxylem vessels may be beneficial during drought. Subsets of the Rice Diversity Panel 1 and Azucena × IR64 recombinant inbred lines were grown in two greenhouse and two rainout shelter experiments under drought stress to assess their shoot, root anatomical, and root architectural phenotypes. Root traits and root trait plasticity in response to drought varied with genotype and environment. The best-performing groups in the rainout shelter experiments had less plasticity of living tissue area in nodal roots than the worst performing groups. Root traits under drought were partitioned into similar groups or clusters via the partitioning-around-medoids algorithm, and this revealed two favorable integrated root phenotypes common within and across environments. One favorable integrated phenotype exhibited many, deep nodal roots with larger root cross-sectional area and more aerenchyma, while the other favorable phenotype exhibited many, deep nodal roots with small root cross-sectional area and small metaxylem vessels. Deeper roots with high theoretical axial hydraulic conductance combined with reduced root metabolic cost contributed to greater shoot biomass under drought. These results reflect how some root anatomical and architectural phenes work in concert as integrated phenotypes to influence the performance of plant under drought stress. Multiple integrated root phenotypes are therefore recommended to be selected in breeding programs for improving rice yield across diverse environments and drought scenarios.

Effect of heavy metals in the cement dust pollution on morphological and anatomical characteristics of Cenchrus ciliaris L.

This present study illustrated the effect of heavy metals in cement dust pollution on Cenchrus ciliaris L. This wild plant species spread surrounding and within the contaminated area. The induced soil by cement dust was changed physically and chemically. There were alterations in morphological characters like chlorotic spot, stem shortening and leaf curling. Similarly, anatomical alterations appeared obviously like rupturing and thickening of cells. The data of control and induced plant species were analyzed statistically separately and combined respectively. The regression equations represented the interaction between control and induced plant species graphically. Cenchrus ciliaris L. can be regarded as a standard heavy metal tolerant plant species.

Micro-morphological response of some native dicotyledonous species to particulate pollutants emitted from stone crushing activities.

Stone crushing processes release particulates and associated noxious substances in our surroundings that are continuously destructing environmental conditions and ecosystem health. Morpho-anatomical changes in some medicinally important native species (Aerva javanica, Calotropis procera, Digera muricata, Euphorbia prostrata, Euploca strigosa, and Peganum harmala) exposed to heavy dust pollution were evaluated. These species selected on the basis of their ubiquitous distribution in the area. Two sites were selected in the Kirana Hills, Sargodha, one near stone crushers within 500-m radius (polluted) and the other 4 km away from the crushers (control) varying significantly in amount of dust particles received. A decrease in plant height of all species from dust-polluted sites was observed. Reduction in height was more prominent in species like C. procera and D. muricata. Stem sclerification increased in C. procera and E. prostrata from the polluted site that is an indication of better tolerance to dust pollution. C. procera showed increased stem and leaf epidermis, stem sclerenchyma, and stem vascular bundles, which can increase resistance to dust pollution. E. strigosa was the most sensitive species in which all morpho-anatomical factors decreased. Survival of plant species depended on specific structural modifications in dermal, mechanical, parenchymatous, and vascular tissue. Overall, dust pollution severely affected plant morphological and micro-morphological traits, but the response of selected species to dust pollution was variable. It is concluded that stem and leaf anatomical traits like size of dermal and storage tissue thickness and stomatal density are good indicators for biomonitoring of dust pollution.

Targeted mutation of transcription factor genes alters metaxylem vessel size and number in rice roots.

Root metaxylem vessels are responsible for axial water transport and contribute to hydraulic architecture. Variation in metaxylem vessel size and number can impact drought tolerance in crop plants, including rice, a crop that is particularly sensitive to drought. Identifying and validating candidate genes for metaxylem development would aid breeding efforts for improved varieties for drought tolerance. We identified three transcription factor candidate genes that potentially regulate metaxylem vessel size and number in rice based on orthologous annotations, published expression data, and available root and drought-related QTL data. Single gene knockout mutants were generated for each candidate using CRISPR-Cas9 genome editing. Root metaxylem vessel area and number were analyzed in 6-week-old knockout mutants and wild-type plants under well-watered and drought conditions in the greenhouse. Compared with wild type, LONESOME HIGHWAY (OsLHW) mutants had fewer, smaller metaxylem vessels in shallow roots and more, larger vessels in deep roots in drought conditions, indicating that OsLHW may be a repressor of drought-induced metaxylem plasticity. The AUXIN RESPONSE FACTOR 15 mutants showed fewer but larger metaxylem vessel area in well-watered conditions, but phenotypes were inconsistent under drought treatment. ORYZA SATIVA HOMEBOX 6 (OSH6) mutants had fewer, smaller metaxylem vessels in well-watered conditions with greater effects on xylem number than size. OSH6 mutants had larger shoots and more, deeper roots than the wild type in well-watered conditions, but there were no differences in performance under drought between mutants and wild type. Though these candidate gene mutants did not exhibit large phenotypic effects, the identification and investigation of candidate genes related to metaxylem traits in rice deepen our understanding of metaxylem development and are needed to facilitate incorporation of favorable alleles into breeding populations to improve drought stress tolerance.

24-epibrassinolide induces protection against waterlogging and alleviates impacts on the root structures, photosynthetic machinery and biomass in soybean.

Plants exhibit several restrictions under waterlogging conditions, including stomatal limitations, negative impacts on gas exchange, lower nutrient uptake and reduced growth. 24-epibrassinolide (EBR) is a polyhydroxylated steroid, with the advantages to be a natural and biodegradable molecule, presenting beneficial roles in metabolic and physiological processes. The aim of this research is to investigate whether EBR can protect soybean plants against damage caused by waterlogging and evaluate the responses associated with the root and leaf anatomy, photosynthetic machinery and biomass. This study used a completely randomized factorial design with two water conditions (control and waterlogging) and three concentrations of 24-epibrassinolide (0, 5 and 10 nM EBR). This steroid stimulated the activities of enzymes linked to the antioxidant system and resulted in minor damage to the chloroplast membranes. EBR maximized the efficiency of photosystem II and improved the gas exchange, which was explained by the higher density and index of the stomata in addition to the increased chlorophyll content and electron transport rate. In root structures, EBR mitigated the impact of waterlogging on vascular cylinder and metaxilem, suggesting maintenance and functions of these structures in plants stressed.

Evaluation of Metaxylem Vessel Histogenesis and the Occurrence of Vessel Collapse during Early Development in Primary Roots of Zea mays ssp. mexicana: A Result of Premature Programmed Cell Death?

Root apical meristem histological organization in Zea mays has been carefully studied previously. Classical histology describes its system as having a "closed organization" and a development of xylem that conforms to predictable rules. Among the first cell types to begin differentiation are late-maturing metaxylem (LMX) vessels. As part of a larger study comparing domestic maize root development to a wild subspecies of Z. mays (teosinte), we encountered a metaxylem development abnormality in a small percentage of our specimens that begged further study, as it interrupted normal maturation of LMX. Primary root tips of young seedlings of Zea mays ssp. mexicana were fixed, embedded in appropriate resins, and sectioned for light and transmission electron microscopy. Longitudinal and serial transverse sections were analyzed using computer imaging to determine the position and timing of key xylem developmental events. We observed a severe abnormality of LMX development among 3.5% of the 227 mexicana seedlings we screened. All LMX vessel elements in these abnormal roots collapsed and probably became non-functional shortly after differentiation began. Cytoplasm and nucleoplasm in the abnormal LMX elements became condensed and subdivided into irregularly-shaped "macrovesicles" as their cell walls collapsed inward. We propose that these seedlings possibly suffered from a mutation that affected the timing of the programmed cell death (PCD) that is required to produce functional xylem vessels, such that autolysis of the cytoplasm was prematurely executed, i.e., prior to the development and lignification of secondary walls.

Comparison of Promeristem Structure and Ontogeny of Procambium in Primary Roots of Zea mays ssp. Mexicana and Z. mays 'Honey Bantam' with Emphasis on Metaxylem Vessel Histogenesis.

Classical histology describes the histological organization in Zea mays as having a "closed organization" that differs from Arabidopsis with the development of xylem conforming to predictable rules. We speculated that root apical meristem organization in a wild subspecies of Z. mays (a teosinte) would differ from a domestic sweetcorn cultivar ('Honey Bantam'). Careful comparison could contribute to understanding how evolutionary processes and the domestication of maize have affected root development. Root tips of seedlings were prepared and sectioned for light microscopy. Most sections were treated with RNase before staining to increase contrast between the walls and cytoplasm. Longitudinal and serial transverse sections were analyzed using computer imaging to determine the position and timing of key xylem developmental events. Metaxylem development in mexicana teosinte differed from sweetcorn only in that the numbers of late-maturing metaxylem vessels in the latter are typically two-fold greater and the number of cells in the transverse section of procambium were greater in the latter, but parenchymatous cell sizes were not statistically different. Promeristems of both were nearly identical in size and organization, but did not operate quite as previously described. Mitotic activity was rare in the quiescent centers, but occasionally a synchronized pulse of mitoses was observed there. Our reinterpretation of histogen theory and procambium development should be useful for future detailed studies of regulation of development, and perhaps its evolution, in this species.

Characterization of Pearl Millet Root Architecture and Anatomy Reveals Three Types of Lateral Roots.

Pearl millet plays an important role for food security in arid regions of Africa and India. Nevertheless, it is considered an orphan crop as it lags far behind other cereals in terms of genetic improvement efforts. Breeding pearl millet varieties with improved root traits promises to deliver benefits in water and nutrient acquisition. Here, we characterize early pearl millet root system development using several different root phenotyping approaches that include rhizotrons and microCT. We report that early stage pearl millet root system development is characterized by a fast growing primary root that quickly colonizes deeper soil horizons. We also describe root anatomical studies that revealed three distinct types of lateral roots that form on both primary roots and crown roots. Finally, we detected significant variation for two root architectural traits, primary root lenght and lateral root density, in pearl millet inbred lines. This study provides the basis for subsequent genetic experiments to identify loci associated with interesting early root development traits in this important cereal.

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.