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Auxin-mediated cell cycle activation during early lateral root initiation.

Lateral root formation can be divided into two major phases: pericycle activation and meristem establishment. In Arabidopsis, the first lateral root initiation event is spatially and temporally asynchronous and involves a limited number of cells in the xylem pericycle. To study the molecular regulation during pericycle activation, we developed a lateral root-inducible system. Successive treatments with an auxin transport inhibitor and exogenous auxin were used to prevent the first formative divisions and then to activate the entire pericycle. Our morphological and molecular data show that, in this inducible system, xylem pericycle activation was synchronized and enhanced to cover the entire length of the root. The results also indicate that the inducible system can be considered a novel in planta system for the study of synchronized cell cycle reactivation. In addition, the expression patterns of Kip-Related Protein2 (KRP2) in the pericycle and its ectopic expression data revealed that the cyclin-dependent kinase inhibitor plays a significant role in the regulation of lateral root initiation. KRP2 appears to regulate early lateral root initiation by blocking the G1-to-S transition and to be regulated transcriptionally by auxin.

Arabidopsis↗

The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.

Acrolein↗

Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis.

Uptake of external sulfate from the environment and use of internal vacuolar sulfate pools are two important aspects of the acquisition of sulfur for metabolism. In this study, we demonstrated that the vacuolar SULTR4-type sulfate transporter facilitates the efflux of sulfate from the vacuoles and plays critical roles in optimizing the internal distribution of sulfate in Arabidopsis thaliana. SULTR4;1-green fluorescent protein (GFP) and SULTR4;2-GFP fusion proteins were expressed under the control of their own promoters in transgenic Arabidopsis. The fusion proteins were accumulated specifically in the tonoplast membranes and were localized predominantly in the pericycle and xylem parenchyma cells of roots and hypocotyls. In roots, SULTR4;1 was constantly accumulated regardless of the changes of sulfur conditions, whereas SULTR4;2 became abundant by sulfur limitation. In shoots, both transporters were accumulated by sulfur limitation. Vacuoles isolated from callus of the sultr4;1 sultr4;2 double knockout showed excess accumulation of sulfate, which was substantially decreased by overexpression of SULTR4;1-GFP. In seedlings, the supplied [(35)S]sulfate was retained in the root tissue of the sultr4;1 sultr4;2 double knockout mutant. Comparison of the double and single knockouts suggested that SULTR4;1 plays a major role and SULTR4;2 has a supplementary function. Overexpression of SULTR4;1-GFP significantly decreased accumulation of [(35)S]sulfate in the root tissue, complementing the phenotype of the double mutant. These results suggested that SULTR4-type transporters, particularly SULTR4;1, actively mediate the efflux of sulfate from the vacuole lumen into the cytoplasm and influence the capacity for vacuolar storage of sulfate in the root tissue. The efflux function will promote rapid turnover of sulfate from the vacuoles particularly in the vasculature under conditions of low-sulfur supply, which will optimize the symplastic (cytoplasmic) flux of sulfate channeled toward the xylem vessels.

Arabidopsis↗

Constitutively high expression of the histidine biosynthetic pathway contributes to nickel tolerance in hyperaccumulator plants.

Plants that hyperaccumulate Ni exhibit an exceptional degree of Ni tolerance and the ability to translocate Ni in large amounts from root to shoot. In hyperaccumulator plants in the genus Alyssum, free His is an important Ni binding ligand that increases in the xylem proportionately to root Ni uptake. To determine the molecular basis of the His response and its contribution to Ni tolerance, transcripts representing seven of the eight enzymes involved in His biosynthesis were investigated in the hyperaccumulator species Alyssum lesbiacum by RNA gel blot analysis. None of the transcripts changed in abundance in either root or shoot tissue when plants were exposed to Ni, but transcript levels were constitutively higher in A. lesbiacum than in the congeneric nonaccumulator A. montanum, especially for the first enzyme in the biosynthetic pathway, ATP-phosphoribosyltransferase (ATP-PRT). Comparison with the weak hyperaccumulator A. serpyllifolium revealed a close correlation between Ni tolerance, root His concentration, and ATP-PRT transcript abundance. Overexpression of an A. lesbiacum ATP-PRT cDNA in transgenic Arabidopsis thaliana increased the pool of free His up to 15-fold in shoot tissue, without affecting the concentration of any other amino acid. His-overproducing lines also displayed elevated tolerance to Ni but did not exhibit increased Ni concentrations in either xylem sap or shoot tissue, suggesting that additional factors are necessary to recapitulate the complete hyperaccumulator phenotype. These results suggest that ATP-PRT expression plays a major role in regulating the pool of free His and contributes to the exceptional Ni tolerance of hyperaccumulator Alyssum species.

ATP Phosphoribosyltransferase↗

Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis.

The irregular xylem 1 (irx1) mutant of Arabidopsis has a severe deficiency in the deposition of cellulose in secondary cell walls, which results in collapsed xylem cells. This mutation has been mapped to a 140-kb region of chromosome 4. A cellulose synthase catalytic subunit was found to be located in this region, and genomic clones containing this gene complemented the irx1 mutation. IRX1 shows homology to a previously described cellulose synthase (IRX3). Analysis of the irx1 and irx3 mutant phenotypes demonstrates that both IRX1 and IRX3 are essential for the production of cellulose in the same cell. Thus, IRX1 and IRX3 define distinct classes of catalytic subunits that are both essential for cellulose synthesis in plants. This finding is supported by coprecipitation of IRX1 with IRX3, suggesting that IRX1 and IRX3 are part of the same complex.

Amino Acid Sequence↗

sas1, an Arabidopsis mutant overaccumulating sodium in the shoot, shows deficiency in the control of the root radial transport of sodium.

A recessive mutation of Arabidopsis designated sas1 (for sodium overaccumulation in shoot) that was mapped to the bottom of chromosome III resulted in a two- to sevenfold overaccumulation of Na(+) in shoots compared with wild-type plants. sas1 is a pleiotropic mutation that also caused severe growth reduction. The impact of NaCl stress on growth was similar for sas1 and wild-type plants; however, with regard to survival, sas1 plants displayed increased sensitivity to NaCl and LiCl treatments compared with wild-type plants. sas1 mutants overaccumulated Na(+) and its toxic structural analog Li(+), but not K(+), Mg(2)+, or Ca(2)+. Sodium accumulated preferentially over K(+) in a similar manner for sas1 and wild-type plants. Sodium overaccumulation occurred in all of the aerial organs of intact sas1 plants but not in roots. Sodium-treated leaf fragments or calli displayed similar Na(+) accumulation levels for sas1 and wild-type tissues. This suggested that the sas1 mutation impaired Na(+) long-distance transport from roots to shoots. The transpiration stream was similar in sas1 and wild-type plants, whereas the Na(+) concentration in the xylem sap of sas1 plants was 5.5-fold higher than that of wild-type plants. These results suggest that the sas1 mutation disrupts control of the radial transport of Na(+) from the soil solution to the xylem vessels.

Arabidopsis↗

Auxin transport promotes Arabidopsis lateral root initiation.

Lateral root development in Arabidopsis provides a model for the study of hormonal signals that regulate postembryonic organogenesis in higher plants. Lateral roots originate from pairs of pericycle cells, in several cell files positioned opposite the xylem pole, that initiate a series of asymmetric, transverse divisions. The auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) arrests lateral root development by blocking the first transverse division(s). We investigated the basis of NPA action by using a cell-specific reporter to demonstrate that xylem pole pericycle cells retain their identity in the presence of the auxin transport inhibitor. However, NPA causes indoleacetic acid (IAA) to accumulate in the root apex while reducing levels in basal tissues critical for lateral root initiation. This pattern of IAA redistribution is consistent with NPA blocking basipetal IAA movement from the root tip. Characterization of lateral root development in the shoot meristemless1 mutant demonstrates that root basipetal and leaf acropetal auxin transport activities are required during the initiation and emergence phases, respectively, of lateral root development.

Arabidopsis↗

Regulation of the Osmotin Gene Promoter.

By introducing a chimeric gene fusion of the osmotin promoter and [beta]-glucuronidase into tobacco by Agrobacterium-mediated transformation, we have demonstrated a very specific pattern of temporal and spatial regulation of the osmotin promoter during normal plant development and after adaptation to NaCl. We have found that the osmotin promoter has a very high natural level of activity in mature pollen grains during anther dehiscence and in pericarp tissue at the final, desiccating stages of fruit development. GUS activity was rapidly lost after pollen germination. The osmotin promoter thus appears to be unique among active pollen promoters described to date in that it is active only in dehydrated pollen. The osmotin promoter was also active in corolla tissue at the onset of senescence. Adaptation of plants to NaCl highly stimulated osmotin promoter activity in epidermal and cortex parenchyma cells in the root elongation zone; in epidermis and xylem parenchyma cells in stem internodes; and in epidermis, mesophyll, and xylem parenchyma cells in developed leaves. The spatial and temporal expression pattern of the osmotin gene appears consistent with both osmotic and pathogen defense functions of the gene.

Journal Article↗

Transcriptomic and proteomic signatures underlying nymphal adaptation and foam production in the forage pest Mahanarva spectabilis.

The spittlebug Mahanarva spectabilis (Distant, 1909) (Hemiptera: Cercopidae) is an important pest of forage grasses in South America, where its nymphs cause pasture damage by feeding on xylem sap and producing a characteristic foam that protects them against environmental stressors. To investigate the molecular basis of this adaptation, we integrated RNA-seq analysis of nymphs with LC-MS/MS proteomics of the Batelli gland, the primary source of foam secretion. De novo assembly of 100,666 unigenes revealed broad functional diversity, with strong representation of detoxification enzymes (CYP450s, GSTs, UGTs, carboxylesterases), transporters and ion pumps, cuticle proteins, and stress- and immunity-related genes. Nearly 16% of loci exhibited alternative splicing, particularly within detoxification, chemosensory and osmoregulatory gene families, highlighting evidence of transcriptomic variability. Signal peptide and secreted protein predictions identified 168 high-confidence candidate secreted proteins, including detoxification enzymes, proteases, structural proteins and immune-related factors, several of which are consistent with antimicrobial and surfactant-related functions. Proteomic profiling of the Batelli gland confirmed 500 proteins, enriched in chaperones, metabolic enzymes, detoxification pathways and osmoregulatory components, with the most abundant proteins corresponding to Hsp70 chaperones, ATP synthases, cuticle proteins and carbonic anhydrases. Together, these results provide an integrative transcriptomic and proteomic overview for M. spectabilis nymphs, highlighting genes and proteins associated with xylem feeding, foam production and responses potentially related to environmental stress tolerance. This comprehensive dataset not only advances the understanding of spittlebug biology but also identifies candidate molecular targets that may inform innovative strategies for controlling nymphal stages and mitigating spittlebug damage in forage systems.

Animals↗

Changes in root water flow properties of solution culture-grown trembling aspen (Populus tremuloides) seedlings under different intensities of water-deficit stress.

To study the effects of water-deficit stress on root water flow properties in trembling aspen (Populus tremuloides Michx.), seedlings were grown in solution culture and subjected to water-deficit stress by placing their roots in sealed high humidity chambers. After 17 h of stress treatment, seedlings showed mild stress (MS) symptoms with a decline in shoot water potentials. Within 20 h, shoot water potentials rapidly declined, and severe stress (SS) symptoms were present. Root hydraulic conductivity (L(pr)) increased more than two-fold and the relative concentration of apoplastic tracer dye trisodium 3-hydroxy-5, 8, 10-pyrenetrisulphonate (PTS(3)) in xylem exudate decreased by 73.6% in MS seedlings. Conversely, L(pr) decreased (55.3%) and PTS(3) increased (28.6%) in SS seedlings. Treatment of roots with 0.1 mM mercuric chloride decreased root volume flow density (J(v)) by about 29.0% in control and MS plants with no decrease measured in SS seedlings. Mercuric chloride also increased PTS(3) concentration in xylem exudate of control (59%) and MS (86%) seedlings with no change observed in SS plants. The results suggest that aquaporin-mediated transport is important in the regulation of root water flow under drought stress and that root water flow properties are strongly affected by the stress level. Regulation of root water flow may represent an important drought-stress resistance mechanism.

Journal Article↗

Ion-mediated increase in the hydraulic conductivity of Laurel stems: role of pits and consequences for the impact of cavitation on water transport.

Changes in hydraulic conductivity (K(h)) were measured in stems of Laurus nobilis L. during perfusion with KCl, NaCl or sucrose solutions. Ionic solutes induced marked increase of K(h) with respect to deionized water but sucrose had no effect. The kinetics of KCl-induced K(h) increase was measured together with changes in [K(+)] of the perfused solution. K(h) increases were paralleled by increases in the [K(+)](out)/[K(+)](in) ratio. Samples of different lengths or with increasing percentage loss of conductivity (PLC) due to xylem cavitation were tested, with the aim of increasing radial flow through intervessel pits. KCl solutions enhanced the K(h) of 12-cm-long samples with a concentration-dependent effect up to 100 mm KCl. DeltaK(h) increased from 3 to 30% in 1.5- and 12-cm-long samples, respectively and remained constant for longer samples. Increasing PLC induced an exponential increase in DeltaK(h). PLC measured with KCl solutions was significantly less than that measured with deionized water, suggesting that measurements of PLC can be affected by the composition of the perfused solution. Experiments support the hypothesis that the 'ionic effect' is mediated by physico-chemical changes of pectins of the pit membranes and raise the possibility that plants might alter the ionic composition of the xylem sap to alleviate the hydraulic impact of cavitation.

Biological Transport↗

Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle.

In angiosperms, root branching requires a continuous re-initiation of new root meristems. Through some unknown mechanism, in most eudicots pericycle cells positioned against the protoxylem change identity and initiate patterned division, leading to formation of lateral root primordia that further develop into lateral roots. This process is auxin-regulated. We have observed that three mutations in the Diageotropica (Dgt) gene in tomato prevent primordium formation. Detailed analysis of one of these mutants, dgt1-1, demonstrated that the mutation does not abolish the proliferative capacity of the xylem-adjacent pericycle in the differentiated root portion. Files of shortened pericycle cells found in dgt1-1 roots were unrelated to primordium formation. Auxin application stimulated this unusual proliferation, leading to formation of a multi-layered xylem-adjacent pericycle, but did not rescue the primordium formation. In contrast to wild type, auxin could not induce any cell divisions in the pericycle of the most distal dgt1-1 root-tip portion. In wild-type roots, the Dgt gene promoter was expressed strongly in lateral root primordia starting from their initiation, and on auxin treatment was induced in the primary root meristem. Auxin level and distribution were altered in dgt1-1 root tissues, as judged by direct auxin measurements, and the tissue-specific expression of an auxin-response reporter was altered in transgenic plants. Together, our data demonstrate that the Dgt gene product, a type-A cyclophilin, is essential for morphogenesis of lateral root primordia, and that the dgt mutations uncouple patterned cell division in lateral root initiation from proliferative cell division in the pericycle.

Arabidopsis↗

Leaf hydraulic architecture correlates with regeneration irradiance in tropical rainforest trees.

The leaf hydraulic conductance (K(leaf)) is a major determinant of plant water transport capacity. Here, we measured K(leaf), and its basis in the resistances of leaf components, for fully illuminated leaves of five tree species that regenerate in deep shade, and five that regenerate in gaps or clearings, in Panamanian lowland tropical rainforest. We also determined coordination with stomatal characters and leaf mass per area. K(leaf) varied 10-fold across species, and was 3-fold higher in sun- than in shade-establishing species. On average, 12% of leaf hydraulic resistance (= 1/K(leaf)) was located in the petiole, 25% in the major veins, 25% in the minor veins, and 39% outside the xylem. Sun-establishing species had a higher proportion of leaf resistance in the xylem. Across species, component resistances correlated linearly with total leaf resistance. K(leaf) correlated tightly with indices of stomatal pore area, indicating a coordination of liquid- and vapor-phase conductances shifted relative to that of temperate woody species. Leaf hydraulic properties are integrally linked in the complex of traits that define differences in water use and carbon economy across habitats and vegetation zones.

Photobiology↗

Silicon uptake and transport is an active process in Cucumis sativus.

Cucumis sativus is a species known to accumulate high levels of silicon (Si) in the tops, though the mechanism for its high Si uptake is little understood. In a series of hydroponic experiments, we examined uptake and xylem loading of Si in C. sativus along with Vicia faba at three levels of Si (0.085, 0.17 and 1.70 mm). Measured Si uptake in C. sativus was more than twice as high as calculated from the rate of transpiration assuming no discrimination between silicic acid and water in uptake. Measured Si uptake in V. faba, however, was significantly lower than the calculated uptake. Concentration of Si in xylem exudates was several-fold higher in C. sativus, but was significantly lower in V. faba compared with the Si concentration in external solutions, regardless of Si levels. Silicon uptake was strongly inhibited by low temperature and 2,4-dinitrophenol, a metabolic inhibitor, in C. sativus but not in V. faba. It can be concluded that Si uptake and transport in C. sativus is active and independent of external Si concentrations, in contrast to the process in V. faba.

2,4-Dinitrophenol↗

A novel form of resistance in rice to the angiosperm parasite Striga hermonthica.

The root hemiparasitic weed Striga hermonthica is a serious constraint to grain production of economically important cereals in sub-Saharan Africa. Breeding for parasite resistance in cereals is widely recognized as the most sustainable form of long-term control; however, advances have been limited owing to a lack of cereal germplasm demonstrating postattachment resistance to Striga. Here, we identify a cultivar of rice (Nipponbare) that exhibits strong postattachment resistance to S. hermonthica; the parasite penetrates the host root cortex but does not form parasite-host xylem-xylem connections. In order to identify the genomic regions contributing to this resistance, a mapping population of backcross inbred lines between the resistant (Nipponbare) and susceptible (Kasalath) parents were evaluated for resistance to S. hermonthica. Composite interval mapping located seven putative quantitative trait loci (QTL) explaining 31% of the overall phenotypic variance; a second, independent, screen confirmed four of these QTL. Relative to the parental lines, allelic substitutions at these QTL altered the phenotype by at least 0.5 of a phenotypic standard deviation. Thus, they should be regarded as major genes and are likely to be useful in breeding programmes to enhance host resistance.

Chromosome Mapping↗

Integrative vector for stable transformation and expression of a beta-1,3-glucanase gene in Clavibacter xyli subsp. cynodontis.

Clavibacter xyli subsp. cynodontis is an endophytic Gram-positive bacterium that is able to colonize the xylem tissue of various plants. We constructed an integrative vector that can carry foreign genes into a repetitive chromosomal element of C. xyli subsp. cynodontis. Using this vector, we transformed C. xyli subsp. cynodontis with a gene coding for an endoglucanase enzyme under the control of a strong promoter that was previously isolated from the genome of C. xyli subsp. cynodontis. The transformed bacteria efficiently expressed active glucanase and secreted it into the culture medium. The vector has the advantage of being stably maintained in the chromosome; the transformations were maintained without antibiotic selection both in in vitro culture and in bacteria growing in maize xylem.

Chromosomes, Bacterial↗

Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera.

Xylella fastidiosa causes Pierce's disease of grapevine as well as several other major agricultural diseases but is a benign endophyte in most host plants. X. fastidiosa colonizes the xylem vessels of host plants and is transmitted by xylem sap-feeding insect vectors. To understand better the pattern of host colonization and its relationship to disease, we engineered X. fastidiosa to express a green fluorescent protein (Gfp) constitutively and performed confocal laser-scanning microscopic analysis of colonization in a susceptible host, Vitis vinifera. In symptomatic leaves, the fraction of vessels colonized by X. fastidiosa was fivefold higher than in nearby asymptomatic leaves. The fraction of vessels completely blocked by X. fastidiosa colonies increased 40-fold in symptomatic leaves and was the feature of colonization most dramatically linked to symptoms. Therefore, the extent of vessel blockage by bacterial colonization is highly likely to be a crucial variable in symptom expression. Intriguingly, a high proportion (>80%) of colonized vessels were not blocked in infected leaves and instead had small colonies or solitary cells, suggesting that vessel blockage is not a colonization strategy employed by the pathogen but, rather, a by-product of endophytic colonization. We present evidence for X. fastidiosa movement through bordered pits to neighboring vessels and propose that vessel-to-vessel movement is a key colonization strategy whose failure results in vessel plugging and disease.

Green Fluorescent Proteins↗

Colonization of Vitis vinifera by a green fluorescence protein-labeled, gfp-marked strain of Xylophilus ampelinus, the causal agent of bacterial necrosis of grapevine.

The dynamics of Xylophilus ampelinus were studied in Vitis vinifera cv. Ugni blanc using gfp-marked bacterial strains to evaluate the relative importance of epiphytic and endophytic phases of plant colonization in disease development. Currently, bacterial necrosis of grapevine is of economic importance in vineyards in three regions in France: the Cognac, Armagnac, and Die areas. This disease is responsible for progressive destruction of vine shoots, leading to their death. We constructed gfp-marked strains of the CFBP2098 strain of X. ampelinus for histological studies. We studied the colonization of young plants of V. vinifera cv. Ugni blanc by X. ampelinus after three types of artificial contamination in a growth chamber and in a greenhouse. (i) After wounding of the stem and inoculation, the bacteria progressed down to the crown through the xylem vessels, where they organized into biofilms. (ii) When the bacteria were forced into woody cuttings, they rarely colonized the emerging plantlets. Xylem vessels could play a key role in the multiplication and conservation of the bacteria, rather than being a route for plant colonization. (iii) When bacterial suspensions were sprayed onto the plants, bacteria progressed in two directions: both in emerging organs and down to the crown, thus displaying the importance of epiphytic colonization in disease development.

Betaproteobacteria↗