Differential Two-Dimensional Protein Patterns as Related to Tissue Specificity and Water Conditions in Brassica napus var oleifera Root System

Differential two-dimensional protein patterns as related to tissue specificity and water conditions were investigated within Brassica napus var oleifera root system. The different parts of the root system (tap root, lateral roots, and drought-induced short roots) were analyzed under various moisture regimes (regular watering at field capacity, progressive drought stress, and rewatering). Tissue specificity was evident from 25 differences in protein patterns (qualitative and quantitative) between well-watered lateral and tap roots. Twice as many polypeptides (52) were drought-affected and the response to the water stress was shown to be similar in both root types. In addition, more than half of the polypeptides detected as organ-specific were affected by drought. Based upon the trend of variation observed under drought and rehydration, three categories of polypeptides could be defined that might be differently involved in drought susceptibility or tolerance. A highly differentiated protein pattern characterized the drought-induced short roots. This pattern appeared as far from the watered as from the water-stressed normal roots. In particular, 13 unique polypeptides were detected which could be relevant to their adaptive morphogenesis and/or their specific drought tolerance induction. Upon rehydration, their polypeptide pattern and their specific morphology returned to a normal well-watered lateral root type.     

Differential Hypocotyl and Root Development of Arabidopsis Auxin-Insensitive Mutants

Arabidopsis , aux1-7, axr1-3 and axr2-1, grown in a natural sandy soil, without sucrose supplementation. The three mutants showed impaired epidermal cell elongation in the hypocotyls of 15-day-old seedlings, with axr2-1 showing the most marked effects. In addition, the roots of axr2-1 elongated faster and presented a more extended meristematic zone than the other genotypes. Unchanged epidermal cell length in the differentiation zone of axr2-1 relative to the wild-type suggested enhancement of cell proliferation. These alterations may have affected the timing and site of emergence of the root hairs, starting later and further from the root tip than in the other genotypes. Similarly to the wild-type, no root hair growth was initiated in axr2-1 drought-induced short roots, although the epidermis was differentiated into trichoblasts and atrichoblasts. On rehydration of the short roots, hair formation occurred from trichoblasts prior to epidermal cell elongation. Therefore, auxin-insensitivity in the axr2-1 mutant did not result in alterations of the hair-forming process itself. The differential development of axr2-1 seedlings, relative to the other auxin-insensitive mutants, suggested that the AXR2 gene has a complex, regulatory function in multiple hormone signaling. Received 26 July 2000/ Accepted in revised form 28 February 2001     

A model system to study the effects of elevated CO2 on the developmental physiology of roots: the use of Arabidopsis thaliana

Three developmental changes were observed in the roots of Arabidopsis thaliana (Columbia) when shoots were exposed to elevated CO2. (I) The allometric coefficient, k, was enhanced significantly (P<0.001), (ii) primary root length and root extension rate were enhanced (P<0.001). Accelerated cortical cell expansion contributed to this effect and was associated with increased cell wall extensibility, measured as % plasticity. (iii) Lateral root formation and extension were also increased in elevated CO2 (P<0.05).These results illustrate that root growth and structure was altered following exposure to elevated CO2. The changes observed suggest that Arabidopsis provides a useful model which should, in future, be amenable to study using appropriate mutants allowing the genetic basis of the responses to be identified.Keywords: Arabidopsis thaliana, elevated CO2, cell expansion, lateral root formation, allometric coefficient.      

Azospirillum brasilense ameliorates the response of Arabidopsis thaliana to drought mainly via enhancement of ABA levels

Production of phytohormones is one of the main mechanisms to explain the beneficial effects of plant growth‐promoting rhizobacteria (PGPR) such as Azospirillum sp. The PGPRs induce plant growth and development, and reduce stress susceptibility. However, little is known regarding the stress‐related phytohormone abscisic acid (ABA) produced by bacteria. We investigated the effects of Azospirillum brasilense Sp 245 strain on Arabidopsis thaliana Col‐0 and aba2‐1 mutant plants, evaluating the morphophysiological and biochemical responses when watered and in drought. We used an in vitro‐grown system to study changes in the root volume and architecture after inoculation with Azospirillum in Arabidopsis wild‐type Col‐0 and on the mutant aba2‐1, during early growth. To examine Arabidopsis development and reproductive success as affected by the bacteria, ABA and drought, a pot experiment using Arabidopsis Col‐0 plants was also carried out. Azospirillum brasilense augmented plant biomass, altered root architecture by increasing lateral roots number, stimulated photosynthetic and photoprotective pigments and retarded water loss in correlation with incremented ABA levels. As well, inoculation improved plants seed yield, plants survival, proline levels and relative leaf water content; it also decreased stomatal conductance, malondialdehyde and relative soil water content in plants submitted to drought. Arabidopsis inoculation with A. brasilense improved plants performance, especially in drought.     

An improved procedure for transformingArabidopsis thaliana (Landsbergerecta) root explant

Agrobacterium tumefaciens-mediated transformation has been widely used in molecular characterization of genes inArabidopsis thaliana. A number of procedures have been developed for transformation ofArabidopsis explants usingAgrobacterium. This paper describes an improved protocol for transformation ofArabidopsis thaliana root explants. Most significantly, using this protocol one can achieve efficient root regeneration of transformation in Landsbergerecta, an ecotype which is widely used in genetic and molecular analyses and which has been difficult to transform in the past. Additional modifications allow easy production of roots for transformation and regeneration of large numbers of transformation t shoots.     

Growth and ultrastructure ofArabidopsis root hairs: therhd3 mutation alters vacuole enlargement and tip growth

The root hairs of plants are tubular projections of root epidermal cells and are suitable for investigating the control of cellular morphogenesis. In wild-typeArabidopsis thaliana (L.) Heynh, growing root hairs were found to exhibit cellular expansion limited to the apical end of the cell, a polarized distribution of organelles in the cytoplasm, and vesicles of several types located near the growing tip. Therhd3 mutant produces short and wavy root hairs with an average volume less than one-third of the wild-type hairs, indicating abnormal cell expansion. The mutant hairs display a striking reduction in vacuole size and a corresponding increase in the relative proportion of cytoplasm throughout hair development. Bead-labeling experiments and ultrastructural analyses indicate that the wavy-hair phenotype of the mutant is caused by asymmetric tip growth, possibly due to abnormally distributed vesicles in cortical areas flanking the hair tips. It is suggested that a major effect of therhd3 mutation is to inhibit vacuole enlargement which normally accompanies root hair cell expansion.     

Effect of Lysophosphatidylethanolamine and Brassinosteroids on Development of <Emphasis Type="Italic">Arabidopsis</Emphasis> Roots

Exogenously applied lysophosphatidylethanolamine (LPE) increased the growth of primary roots and the formation of lateral roots in Arabidopsis thaliana. In the presence of brassinolide, lateral root formation induced by LPE was enhanced, implying that both LPE and brassinosteroids (BR) interact positively in the development of Arabidopsis roots. Co-treatment with LPE and BRs increased the bending activity in the rice lamina inclination assay compared to that when BRs were applied alone, suggesting that LPE seems to exert its activity via BRs activity. RT-PCR revealed that LPE did not alter the expressions of genes involved in the biosynthesis of BRs but did activate the expressions of BR signaling genes in A. thaliana. In a BR-insensitive mutant, bri1, enhanced gravitropic response by LPE in wild-type A. thaliana was diminished. In conclusion, LPE is a positive regulator for the growth and development of Arabidopsis roots, and this process seems to be enhanced by BR signaling rather than by increase in endogenous levels of BRs in A. thaliana.     

Gram-positive rhizobacterium <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> FZB42 colonizes three types of plants in different patterns

The colonization of three types of different plants, Zea mays, Arabidopsis thaliana, and Lemna minor, by GFP-labeled Gram-positive rhizobacterium Bacillus amyloliquefaciens FZB42 was studied in gnotobiotic systems using confocal laser scanning microscopy and electron microscopy. It was demonstrated that FZB42 was able to colonize all the plants. On one hand, similar to some Gram-negative rhizobacteria like Pseudomonas, FZB42 favored the areas such as the concavities in root surfaces and the junctions where lateral roots occurred from the primary roots; on the other hand, we clearly demonstrated that root hairs were a popular habitat to the Gram-positive rhizobacterium. FZB42 exhibited a specific colonization pattern on each of the three types of plants. On Arabidopsis, tips of primary roots were favored by FZB42 but not so on maize. On Lemna, FZB42 accumulated preferably along the grooves between epidermal cells of roots and in the concave spaces on ventral sides of fronds. The results suggested L. minor to be a promising tool for investigations on plant-microbial interaction due to a series of advantages it has. Colonization of maize and Arabidopsis roots by FZB42 was also studied in the soil system. Comparatively, higher amount of FZB42 inoculum (∼10⁸ CFU/ml) was required for detectable root colonization in the soil system, where the preference of FZB42 cells to root hairs were also observed.     

Root Growth of Arabidopsis thaliana Is Regulated by Ethylene and Abscisic Acid Signaling Interaction in Response to HrpNEa, a Bacterial Protein of Harpin Group

HrpN_Ea is a harpin protein from Erwinia amylovora, a bacterial pathogen that causes fire blight in rosaceous plants. Treating plants with HrpN_Ea stimulates ethylene and abscisic acid (ABA) to induce plant growth and drought tolerance, respectively. Herein, we report that both growth hormones cooperate to mediate the role of HrpN_Ea in promoting root growth of Arabidopsis thaliana seedlings. Root growth is promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking of germinating seeds of wild-type (WT) Arabidopsis in a solution of HrpN_Ea. However, these responses are arrested by inhibiting WT roots from synthesizing ethylene as well as sensing of ABA and ethylene. The effects of HrpN_Ea on roots are also nullified in ethylene-insensitive etr1-1 and ein5-1 mutants and in the ABA-insensitive mutant abi2-1 of Arabidopsis. These results provide evidence for presence of a relationship between root growth enhancement and signaling by ABA and ethylene in response to HrpN_Ea. Nevertheless, when HrpN_Ea is applied to leaves, ethylene signaling is active in the absence of ABA signaling to promote plant growth. This suggests the presence of a different signaling mechanism in leaves from that in roots. X. Ren and F. Liu contributed equally to this study and are regarded as joint first authors     

Arabidopsis N-MYC DOWNREGULATED-LIKE1, a Positive Regulator of Auxin Transport in a G Protein–Mediated Pathway

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gβγ dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single β (AGB1) or the two γ (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.     

Microtubule reorganization as a response to implementation of NO signals in plant cells

The effects of an exogeneous NO donor, sodium nitroprusside, on the orientation and organization of cortical microtubules in Arabidopsis thaliana root cells expressing GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10–500 μM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in the differentiation zone. The influence of sodium nitroprusside revealed in changes in the orientation and organization of cortical microtubules in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of the elongation zone, where native transverse orientation of cortical microtubules turned into random, oblique, or longitudinal relative to the primary root axis. We suggest that NO, as one of the intracellular secondary messengers, triggers cell differentiation by reorientation of cortical microtubules, possibly via tubulin nitrotyrosination.     

Root hair-specific EXPANSIN B genes have been selected for graminaceae root hairs

Cell differentiation ultimately relies on the regulation of cell type-specific genes. For a root hair cell to undergo morphogenesis, diverse cellular processes including cell-wall loosening must occur in a root hair cell-specific manner. Previously, we identified and characterized root hairspecific cis-elements (RHE) from the genes encoding the cell wall-loosening protein EXPANSIN A (EXPA) which functions preferentially on dicot cell walls. This study reports two root hair-specific grass EXPB genes that contain RHEs. These genes are thought to encode proteins that function more efficiently on grass cell walls. The proximal promoter regions of two orthologous EXPB genes from rice (Oryza sativa; OsEXPB5) and barley (Hordeum vulgare; HvEXPB1) included RHE motifs. These promoters could direct root hair-specific expression of green fluorescent protein (GFP) in the roots of rice and Arabidopsis (Arabidopsis thaliana). Promoter deletion analyses demonstrated that the RHE motifs are necessary for root hairspecific expression of these EXPB promoters. Phylogenetic analysis of EXP protein sequences indicated that grass EXPBs are the only orthologs to these root hair-specific EXPBs, separating dicot EXPBs to distal branches of the tree. These results suggest that RHE-containing root hair-specific EXPB genes have evolved for grass-specific cell wall modification during root hair morphogenesis.     

Cellular Differentiation and Leaf Morphogenesis in Arabdopsis

A focused approach that exploits a single plant species, namely, Arabidopsis thaliana, as a means to understand how leaf cells differentiate and the factors that govern overall leaf morphogenesis has begun to generate a significant body of knowledge in this model plant. Although many studies have concentrated on specific cell types and factors that control their differentiation, some degree of consensus is starting to be reached. However, an understanding of specific mechanisms by which cells differentiate in relation to their position, that appears to be an overriding factor in this process, is not yet in place for cell types in the Arabidopsis leaf. It is clear that perturbations in cellular development within the leaf do not necessarily have a general effect on morphogenesis. Environmental factors, particularly light, have been known to affect leaf cell differentiation and expansion, and endogenous hormones also appear to play an important role, through mechanisms that are beginning to be uncovered. It is likely that continued identification of genes involved in leaf development and their regulation in relation to positional information or other cues will lead to a clearer understanding of the control of differentiation and morphogenesis in the Arabidopsis leaf.     

Towards the era of comparative evolutionary genomics in Brassicaceae

The vast genetic diversity, specific genome organization and sequencing of the Arabidopsis thaliana genome made crucifers an ideal group for comparative genomic studies. Arabidopsis genomic resources have greatly expedited comparative genomics within Brassicaceae and fostered the establishment of new Arabidopsis relative model systems (ARMS). The extent of genome colinearity, modes and evolutionary rates of genome alterations are being analyzed by genetic mapping with ever increasing levels of precision. Comparative cytogenetic studies in Brassicaceae are employing various chromosome landmarks and cytogenetic techniques, including localization of rDNA, variation in centromeric satellite repeats, genomic in situ hybridization (GISH), fluorescence ISH using bacterial artificial chromosomes (BAC FISH), and large-scale comparative chromosome painting. Some genome alterations may represent rare genomic changes (RGCs) and thus have the potential to resolve complex/conflicting phylogenetic relationships inferred from DNA sequencing. Comparative genomics should increasingly be integrated with molecular phylogenetics and population genetics to elucidate the processes responsible for genetic variation in Brassicaceae.     

Fibres and Fibre-sclereids in Wild-typeArabidopsis thaliana

Examination of the fibre system in roots and shoots ofArabidopsisthaliana(L.) Heynh. revealed three types of fibres, distinguishedaccording to their site of differentiation: (1) short fibre-sclereidsin the secondary phloem of roots and shoots at the rosette level;(2) long fibres in the secondary xylem of the main root; and(3) very long fibres in the xylem of the inflorescence stems.These three types are in addition to the small number of primaryphloem fibres that are formed even in smallA. thalianaindividuals.These findings provide a basis for the use ofArabidopsis thalianaasa model system to study the processes leading to fibre differentiationin dicotyledons. Arabidopsis thaliana; differentiation; fibre-sclereids; inflorescences; phloem fibres; xylem     

Abscisic Acid Elicits the Water-Stress Response in Root Hairs of Arabidopsis thaliana

Water stress has been shown to cause root hairs to become short and bulbous. Because abscisic acid (ABA) mediates a variety of water-stress responses, we investigated the response of Arabidopsis thaliana root hairs to ABA. When wild-type root hairs were treated with ABA, they exhibited the water-stress response. The Arabidopsis mutants abi1 and abi2, which are insensitive to ABA at the seedling stage, did not display the root hair response. These data suggest that ABA may mediate the response of root hairs to water stress. The drought response of root hairs resulting in an inhibition of tip growth will provide an easy screen to select mutations that are insensitive to ABA and/or involved in tip growth.     

The responsiveness of the <Emphasis Type="Italic">IAA2</Emphasis> promoter to IAA and IBA is differentially affected in <Emphasis Type="Italic">Arabidopsis</Emphasis> roots and shoots by flavonoids

The structural features of flavonoids which are involved in the modulation of auxin distribution in Arabidopsis thaliana were evaluated. An auxin-inducible promoter IAA2 fused to a reporter gene (GUS) was used to monitor the tissue responsiveness to auxins. The following aspects were investigated: 1) the influence of flavonoids (quercetin, naringenin, kaempferol, myricetin and isorhamnetin) on the distribution of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) in roots and leaves, 2) differences in flavonoid uptake into roots and shoots depending on flavonoid concentration in the medium, and 3) influence of structurally different flavonoids on the gravitropic response and growth of roots. The same flavonoids differently affected IAA and IBA distribution in leaves and roots. There were several structural requirements for the flavonoids which resulted in the changes of auxin response/distribution. Great differences between the ability of shoots and roots to take up quercetin were showed. Also, flavonoids influenced gravitropism and root growth of Arabidopsis seedlings in a structure-dependent manner.