The small genome of Arabidopsis contains at least six expressed alpha-tubulin genes.

The goal of our investigations is to define the genetic control of microtubule-based processes in a higher plant. The available evidence suggests that we have achieved our first objective: the characterization of the complete alpha-tubulin and beta-tubulin gene families of Arabidopsis. Four additional alpha-tubulin genes (TUA2, TUA4, TUA5, and TUA6) of Arabidopsis have been cloned and sequenced to complete the analysis of the gene structure for all six alpha-tubulin genes detectable on DNA gel blots of Arabidopsis genomic DNA hybridized with alpha-tubulin coding sequences. TUA1 and TUA3 were characterized earlier in our laboratory. Noncoding gene-specific hybridization probes have been constructed for all six alpha-tubulin genes and used in RNA gel blot analyses to demonstrate that all six genes are transcribed. The six genes encode four different alpha-tubulin isoforms; TUA2 and TUA4 encode a single isoform, as do TUA3 and TUA5. Two-dimensional protein gel immunoblot analyses have resolved at least four alpha-tubulin isoforms from plant tissues, suggesting that all of the predicted TUA gene products are synthesized in vivo.     

A novel function for a redox-related LEA protein (SAG21/AtLEA5) in root development and biotic stress responses

SAG21/AtLEA5 belongs to the late embryogenesis-associated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, we characterized root and shoot development and response to biotic stress in SAG21/AtLEA5 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass. Primary root length was reduced in AS lines, as was the number of laterals relative to the primary root. Root hair number was unchanged but root hair length was proportional to SAG21/AtLEA5 expression level, with longer root hairs in OEX lines and shorter root hairs in AS, relative to wild type. Growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21/AtLEA5 expression; however, growth of an avirulent P.syringae strain was unaffected. A SAG21/AtLEA5-YFP fusion was localized to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling, which in turn, impacts on root development and pathogen responses.     

Increase in expression level of alpha-tubulin gene in Arabidopsis seedlings under hypergravity conditions.

Under hypergravity conditions, elongation growth of plant shoots is suppressed. The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method showed that a gene encoding alpha-tubulin, which is a component of microtubules, was up-regulated by hypergravity. In Arabidopsis six genes encoding alpha-tubulin (TUA1-TUA6) have been identified. In the present study, we examined the dose-response and the time course relations of the changes in the expression of all six alpha-tubulin genes in Arabidopsis hypocotyls grown under hypergravity conditions. The expression levels of all six alpha-tubulin genes, TUA1-TUA6, were increased by increasing gravity, although the extent was variable among genes. The increase in expression of all alpha-tubulin genes was detected within a few hours, when the seedlings grown at 1 g were transferred to 300 g condition. These results suggest that Arabidopsis hypocotyls regulate the expression level of six alpha-tubulin genes promptly in response to gravity stimuli. The increase in the amount of microtubules due to the activation of tubulin gene expression may be involved in the regulation by gravity signal of shoot growth.     

A Single Amino-Acid Substitution at Lysine 40 of an Arabidopsis thaliana α-tubulin Causes Extensive Cell Proliferation and Expansion Defects

Microtubules are highly dynamic cytoskeletal polymers of α/β‐tubulin heterodimers that undergo multiple post‐translational modifications essential for various cellular functions in eukaryotes. The lysine 40 (K40) is largely conserved in α‐tubulins in many eukaryote species, and the post‐translational modification by acetylation at K40 is critical for neuronal development in vertebrates. However, the biological function of K40 of α‐tubulins in plants remains unexplored. In this study, we show in Arabidopsis thaliana that constitutive expression of mutated forms of α‐tubulin6 (TUA6) at K40 (TUA6^K40A or TUA6^K40Q), in which K40 is replaced by alanine or glutamine, result in severely reduced plant size. Phenotypic characterization of the 35S:TUA6^K40A transgenic plants revealed that both cell proliferation and cell expansion were affected. Cytological and biochemical analyses showed that the accumulation of α‐ and β‐tubulin proteins was significantly reduced in the transgenic plants, and the cortical microtubule arrays were severely disrupted, indicating that K40 of the plant α‐tubulin is critical in maintaining microtubule stability. We also constructed 35S:TUA6^K40R transgenic plants in which K40 of the engineered TUA6 protein is replaced by an arginine, and found that the 35S:TUA6^K40R plants were phenotypically indistinguishable from the wild‐type. Since lysine and arginine are similar in biochemical nature but arginine cannot be acetylated, these results suggest a structural importance for K40 of α‐tubulins in cell division and expansion.     

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

Manifestation of heterosis during early maize (Zea mays L.) root development

Heterosis is typically detected in adult hybrid plants as increased yield or vigor compared to their parental inbred lines. Only little is known about the manifestation of heterosis during early postembryonic development. Objective of this study was to identify heterotic traits during early maize root development. Four German inbred lines of the flint (UH002 and UH005) and dent (UH250 and UH301) pool and the 12 reciprocal hybrids generated from these inbred lines were subjected to a morphological and histological analysis during early root development. Primary root length and width were measured daily in a time course between 3 and 7 days after germination (DAG) and displayed average midparent heterosis (MPH) of 17–25% and 1–7%, respectively. Longitudinal size of cortical cells in primary roots was determined 5 DAG and displayed on average 24% MPH thus demonstrating that enlarged primary roots of hybrids can mainly be attributed to elongated cortical cells. The number of seminal roots determined 14 DAG showed on average 18% MPH. Lateral root density of all tested hybrids was determined 5 DAG. This root trait showed the highest degree of heterosis with an average MPH value of 51%. This study demonstrated that heterosis is already manifesting during the very early stages of root development a few days after germination. The young root system is therefore a suitable model for subsequent molecular studies of the early stages of heterosis manifestation during seedling development.     

Is microtubule disassembly a trigger for cold acclimation?

Cold acclimation was followed in three cultivars of winter wheat (Triticum aestivum L.) that differ in freezing tolerance, using root growth as the indicator. During acclimation (followed through 7 d at 4 degrees C), growth rate progressively recovered. The recovery was fast in the tolerant, slow in the sensitive cultivars. The development of freezing tolerance was followed by a challenging cold shock administered after various time intervals of acclimation. Acclimation proceeded faster in the tolerant cultivars. Microtubules were monitored during the acclimation period. A rapid, but transient partial disassembly in the tolerant cultivars preceded the formation of cold-stable microtubules and the recovery of growth rate. In contrast, this transient disassembly was absent in the sensitive cultivar. When a transient disassembly was artificially generated by a pulse-treatment with the antimicrotubular herbicide pronamide, this could induce freezing tolerance. The appearance of cold-stable microtubules was accompanied by a reduced abundance of type TUA1/2 alpha-tubulin isotypes. These findings are discussed with respect to a role of microtubule disassembly in the sensing of low-temperature stress.     

Morphological,genetic and molecular characteristics of barley root hair mutants

Root hairs are tubular outgrowths of specialized epidermal cells called trichoblasts. They affect anchoring plants in soil, the uptake of water and nutrients and are the sites of the interaction between plants and microorganisms. Nineteen root hair mutants of barley representing different stages of root hair development were subjected to detailed morphological and genetic analyses. Each mutant was monogenic and recessive. An allelism test revealed that nine loci were responsible for the mutated root hair phenotypes in the collection and 1–4 mutated allelic forms were identified at each locus. Genetic relationships between the genes responsible for different stages of root hair formation were established. The linkage groups of four loci rhl1, rhp1, rhi1 and rhs1, which had previously been mapped on chromosomes 7H, 1H, 6H and 5H, respectively, were enriched with new markers that flank the genes at a distance of 0.16 cM to 4.6 cM. The chromosomal position of three new genes – two that are responsible for the development of short root hairs (rhs2 and rhs3) and the gene that controls an irregular root hair pattern (rhi2) – were mapped on chromosomes 6H, 2H and 1H, respectively. A comparative analysis of the agrobotanical parameters between some mutants and their respective parental lines showed that mutations in genes responsible for root hair development had no effect on the agrobotanical performance of plants that were grown under controlled conditions. The presented mutant collection is a valuable tool for further identification of genes controlling root hair development in barley.     

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri T_L-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.     

MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development

Previous data have suggested an involvement of MDR/PGP-like ABC transporters in transport of the plant hormone auxin and, recently, AtPGP1 has been demonstrated to catalyze the primary active export of auxin. Here we show that related isoform AtPGP4 is expressed predominantly during early root development. AtPGP4 loss-of-function plants reveal enhanced lateral root initiation and root hair lengths both known to be under the control of auxin. Further, atpgp4 plants show altered sensitivities toward auxin and the auxin transport inhibitor, NPA. Finally, mutant roots reveal elevated free auxin levels and reduced auxin transport capacities. These results together with yeast growth assays suggest a direct involvement of AtPGP4 in auxin transport processes controlling lateral root and root hair development.     

A zinc finger protein,interacted with cyclophilin,affects root development via IAA pathway in rice

The plant hormone auxin plays a crucial role in lateral root development. To better understand the molecular mechanisms underlying lateral root formation,an auxin-responsive gene OsCYP2(Os02g0121300) was characterized from rice. Compared to the wild type,OsCYP2-RNAi(RNA interference) lines exhibited distinctive defects in lateral root development. Yeast two-hybrid and glutathione S-transferase pull-down results confirmed that OsCYP2 interacted with a C2HC-type zinc finger protein(OsZFP, Os01g0252900) which is located in the rice nucleus. T_2 OsZFP-RNAi lines had significantly fewer lateral roots than did wild-type plants, which suggests a role for OsCYP2 and OsZFP in regulating lateral root development.Quantitative real-time polymerase chain reaction showed that the expression of certain Aux/IAA(auxin/indole-3-acetic acid) genes was altered in OsCYP2-and Os ZFP-RNAi lines in response to IAA. These findings imply that OsCYP2 and OsZFP participate in IAA signal pathways controlling lateral root development. More importantly, OsIAA11 showed functional redundancy not only in OsCYP2-RNAi lines but also in Os ZFP-RNAi lines, which provides important clues for the elucidation of mechanisms controlling lateral root development in response to auxin.     

NtGNL1 is involved in embryonic cell division patterning, root elongation, and pollen tube growth in tobacco

The function of the ARF-GEF family has drawn great attention recently, especially GNOM and GNL1, owing to their important role in plant development. A homolog of GBF was identified in Nicotiana tabacum, named NtGNL1, which is ubiquitously expressed throughout the tobacco life cycle. In NtGNL1 RNAi plants, irregular orientation of cell division and asynchronous cell development during early embryogenesis disrupted the symmetry of the developing embryo. In addition, root growth in transgenic lines was significantly slower than that in wild-type plants, although the structure of the root tip was largely intact. Pollen germination and pollen tube growth were also inhibited in the transgenic lines, and the tip of the pollen tube presented various aberrant morphologies in one of the transgenic lines. The phenotypes of different NtGNL1 RNAi transgenic lines suggest that the NtGNL1 is likely to be involved not only in embryogenesis and postembryonic development, but also in sexual reproduction; thus, NtGNL1 may play multiple and critical roles in plant development.     

Combining enhanced root and shoot growth reveals cross talk between pathways that control plant organ size in Arabidopsis

Functionally distinct Arabidopsis (Arabidopsis thaliana) genes that positively affect root or shoot growth when ectopically expressed were combined to explore the feasibility of enhanced biomass production. Enhanced root growth resulting from cytokinin deficiency was obtained by overexpressing CYTOKININ OXIDASE/DEHYDROGENASE3 (CKX3) under the control of the root-specific PYK10 promoter. Plants harboring the PYK10-CKX3 construct were crossed with four different transgenic lines showing enhanced leaf growth. For all combinations, the phenotypic traits of the individual lines could be combined, resulting in an overall growth increase. Unexpectedly, three out of four combinations had more than additive effects. Both leaf and root growth were synergistically enhanced in plants ectopically expressing CKX3 and BRASSINOSTEROID INSENSITIVE1, indicating cross talk between cytokinins and brassinosteroids. In agreement, treatment of PYK10-CKX3 plants with brassinolide resulted in a dramatic increase in lateral root growth that could not be observed in wild-type plants. Coexpression of CKX3 and the GROWTH-REGULATING FACTOR5 (GRF5) antagonized the effects of GRF5 overexpression, revealing an interplay between cytokinins and GRF5 during leaf cell proliferation. The combined overexpression of CKX3 and GIBBERELLIN 20-OXIDASE1 led to a synergistic increase in leaf growth, suggesting an antagonistic growth control by cytokinins and gibberellins. Only additive effects on root and shoot growth were visible in plants ectopically expressing both CKX3 and ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1, hinting at an independent action mode. Our results show new interactions and contribute to the molecular and physiological understanding of biomass production at the whole plant level.