Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea

Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.     

Flavonoids: from cell cycle regulation to biotechnology

Flavonoids have been proposed to play diverse roles in plant growth and development, including defense, symbiosis, pollen development and male fertility, polar auxin transport, and protection against ultraviolet radiation. Recently, a new role in cell cycle regulation has emerged. Genetic alteration of glucuronide metabolism by altered expression of a Pisum sativum UDP-glucuronosyltransferase (PsUGT1) results in an altered cell cycle in pea, alfalfa, and Arabidopsis. In alfalfa, altered expression of PsUGT1 results in accumulation of a flavonoid-like compound that suppresses growth of cultured cells. The results are consistent with the hypothesis that PsUGT1 functions by controlling cellular levels of a factor controlling cell cycle (FCC).     

Nucleoside diphosphate kinase required for coleoptile elongation in rice

Although several nucleoside diphosphate (NDP) kinase genes have been cloned in plants, little is known about the functional significance of this enzyme during plant growth and development. We introduced a chimeric gene encoding an antisense RNA of NDP kinase under the control of the Arabidopsis heat shock protein HSP81-1 promoter into rice (Oryza sativa L.) plants using the Agrobacterium tumefaciens transformation system. The expression of antisense RNA down-regulated the accumulation of mRNA, resulting in reduced enzyme activity even under the standard growth temperature (25 degrees C) in transgenic plants. Following heat shock treatment (37 degrees C), NDP kinase activities in some transgenic rice plants were more reduced than those grown under 25 degrees C. The comparison of the coleoptile growth under submersion showed that cell elongation process was inhibited in antisense NDP kinase transgenic plants, suggesting that an altered guanine nucleotide level may be responsible for the processes.     

Expression of MsLEC1- and MsLEC2-antisense genes in alfalfa plant lines causes severe embryogenic, developmental and reproductive abnormalities

Although it has been proposed that plant lectins play a number of roles, the function of these proteins in normal plant growth and development has been unclear. To analyze the functions of putative alfalfa lectin genes, lines of transgenic alfalfa plants expressing approximately half of the open reading frame of MsLEC1 or MsLEC2, in the antisense or sense orientation, were established and analyzed. The antisense plants displayed severe abnormalities in embryogenesis, and both vegetative and reproductive development were perturbed. Some differences were observed between MsLEC1- and MsLEC2-antisense plants, and abnormalities were especially severe during the early stages of development in both the primary and secondary transgenic generations. In contrast, vector-control and sense-transgene plants exhibited normal growth and development. MsLEC1 and MsLEC2 mRNA accumulation levels were reduced in cognate antisense plants, especially during the later stages of embryogenesis, but also tended to be low in MsLEC1 sense-transgene plants. However, correlated with the phenotypic abnormalities observed in the MsLEC1-antisense plants was the specific reduction in the accumulation of a candidate MsLEC1 protein. Our results suggest that the MsLEC1 and MsLEC2 gene products, in addition to being important for embryogenesis, are required throughout alfalfa development.     

Identification and characterization of a rhizosphere β-galactosidase from Pisum sativum L.

Plant enzyme activities in the rhizosphere potentially are a resource for improved plant nutrition, soil fertility, bioremediation, and disease resistance. Here we report that a border cell specific β-galactosidase is secreted into the acidic extracellular environment surrounding root tips of pea, as well as bean, alfalfa, barrel medic, sorghum, and maize. No enzyme activity was detected in radish and Arabidopsis, species that do not produce viable border cells. The secreted enzyme activity was inhibited by galactose and 2-phenylethyl 1-thio-β-d-galactopyranoside (PETG) at concentrations that altered root growth without causing cell death. A tomato galactanase encoding gene was used as a probe to isolate a full length pea cDNA clone (BRDgal1) from a root cap-border cell cDNA library. Southern blot analysis using full length BRDgal1 as a probe revealed 1–2 related sequences within the pea genome. BRDgal1 mRNA expression was analysed by whole mount in situ hybridization (WISH) and found to occur in the outermost peripheral layer of the cap and in suspensions of detached border cells. No expression was detected within the body of the root cap. Repeated efforts to develop viable hairy root clones expressing BRDgal1 antisense mRNA under the control of the CaMV35S promoter, whose expression in the root cap is limited to cells at the root cap periphery only during root emergence, were unsuccessful. These data suggest that altered expression of this enzyme is deleterious to early root development. The first two authors contributed equally to the completion of this project.     

Lethality of inducible,meristem-localized ectopic β-glucuronidase expression in plants

GUSA fromEscherichia coli, encoded by theuidA gene, has been successfully used as a plant reporter system for more than a decade with no reported deleterious effects. However, when expressed in coordination with a UDP-glucuronosyltransferase isolated from the root cap meristem ofPisum sativum (PsUGT1) at the onset of mitosis, GUSA expression was lethal in pea, alfalfa, andArabidopsis thaliana. These unexpected results indicate that, under some circumstances, using GUSA in plants is incompatible with life and suggest that the cell-specific lethal phenotype might be useful in selecting for genes specifically involved in regulating the G2-M phase of the cell cycle.     

Inducible expression of <Emphasis Type="Italic">Pisum sativum</Emphasis> xyloglucan fucosyltransferase in the pea root cap meristem,and effects of antisense mRNA expression on root cap cell wall structural integrity

Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.     

Altered susceptibility to infection by <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Nectria haematococca</Emphasis> in alfalfa roots with altered cell cycle

Most infections of plant roots are initiated in the region of elongation; the mechanism for this tissue-specific localization pattern is unknown. In alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter, the cell cycle in roots is completed in 48 h instead of 24 h, and border cell number is decreased by more than 99%. These plants were found to exhibit increased root-tip infection by a fungal pathogen and reduced nodule formation by a bacterial symbiont. Thus, the frequency of infection in the region of elongation by Nectria haematocca was unaffected, but infection of the root tip was increased by more than 90%; early stages of Sinorhizobium meliloti infection and nodule morphology were normal, but the frequency of nodulation was fourfold lower than in wild-type roots.     

Effect of pectin methylesterase gene expression on pea root development.

Expression of an inducible gene with sequences common to genes encoding pectin methylesterase (PME) was found to be tightly correlated, both spatially and temporally, with border cell separation in pea root caps. Partial inhibition of the gene's expression by antisense mRNA in transgenic pea hairy roots prevented the normal separation of root border cells from the root tip into the external environment. This phenotype was correlated with an increase in extracellular pH, reduced root elongation, and altered cellular morphology. The translation product of the gene exhibited PME activity in vitro. These results are consistent with the long-standing hypothesis that the demethylation of pectin by PME plays a key role in cell wall metabolism.     

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) stress genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.Abbreviations YEM yeast extract-mannitol - GUS glucuronidase - PAL phenylalanine ammonium lyase - CHS chalcone syntase     

The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development

Copper plays a dual role in aerobic organisms, as both an essential and a potentially toxic element. To ensure copper availability while avoiding its toxic effects, organisms have developed complex homeostatic networks to control copper uptake, distribution, and utilization. In eukaryotes, including yeasts and mammals, high affinity copper uptake is mediated by the Ctr family of copper transporters. This work is the first report on the physiological function of copper transport in Arabidopsis thaliana. We have studied the expression pattern of COPT1 in transgenic plants expressing a reporter gene under the control of the COPT1 promoter. The reporter gene is highly expressed in embryos, trichomes, stomata, pollen, and root tips. The involvement of COPT1 in copper acquisition was investigated in CaMV35S::COPT1 antisense transgenic plants. Consistent with a decrease in COPT1 expression and the associated copper deprivation, these plants exhibit increased mRNA levels of genes that are down-regulated by copper, decreased rates of (64)Cu uptake by seedlings and reduced steady state levels of copper as measured by atomic absorption spectroscopy in mature leaves. Interestingly, COPT1 antisense plants also display dramatically increased root length, which is completely and specifically reversed by copper addition, and an increased sensitivity to growth inhibition by the copper-specific chelator bathocuproine disulfonic acid. Furthermore, COPT1 antisense plants exhibit pollen development defects that are specifically reversed by copper. Taken together, these studies reveal striking plant growth and development roles for copper acquisition by high affinity copper transporters.     

Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa

Plant root development is an essential determinant of plant growth and crop yield that could be enhanced by induced changes in the expression of root-specific regulatory factors. We reported previously that Alfin1 binds DNA in a sequence-specific manner and that Alfin1 overexpression in transgenic alfalfa (Medicago sativa L.) enhances expression of the salt-inducible MsPRP2 gene in roots, suggesting that Alfin1 functions to regulate gene expression in roots. Here we show that Alfin1 is an essential gene for root growth and that its overexpression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions. Alfin1-binding sites occur in promoters of genes expressed in roots of a wide variety of plant species and we propose that it is a general root growth regulator. Even though Alfin1 overexpression was under the control of the CaMV 35S promoter, plant shoot growth was not adversely affected. We show further that introduction of the Alfin1 transgene in plants confers a dominant characteristic that significantly increases plant growth and salt tolerance.     

Modifying expression of closely related UDP-glycosyltransferases from pea and Arabidopsis results in altered root development and function

Glycosyltransferases (GTs) play diverse roles in cellular metabolism by modifying the activities of structural and regulatory metabolites. Previous studies indicated that a Pisum sativum UDP-glycosyltransferase (PsUGT1) is essential for plant development, and suggested a role for this enzyme in the regulation of the cell division cycle. Here we report that recombinant PsUGT1 expressed in vitro exhibits activity on diverse flavonoids including kaempferol. In Arabidopsis expressing PsUGT1, gravity sensing is impaired, and this loss of function is corrected by exogenous addition of kaempferol. HPLC of tissue extracts of Arabidopsis expressing PsUGT1 revealed the accumulation of glycosides of kaempferol, but not of other related flavonoids. A search of the NCBI gene bank ( http://www.ncbi.nlm.nih.gov/ ) using PsUGT1 revealed that six genes from the Arabidopsis AtUGT85A subfamily show similarities both in DNA and protein sequences ( Woo et al. 2007 ). In the current study, we examined the hypothesis that one or more members of this family, like PsUGT1, is required for Arabidopsis development. Altered expression of AtUGT85A7, but not other AtUGT85A subfamily members, resulted in changes in life cycle, leaf morphology, auxin response, and root development, including loss of gravity sensing. The phenotypes of plants where AtUGT85A7 gene expression was suppressed, by RNAi mutagenesis, were very similar to those occurring in plants with altered expression of PsUGT1.     

Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant

A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the CaMV—35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes . Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing β-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.