Requirement of KNAT1/BP for the Development of Abscission Zones in Arabidopsis thaliana

The KNAT1 gene is a member of the Class I KNOXhomeobox gene family and is thought to play an important role in meristem development and leaf morphogenesis. Recent studies have demonstrated that KNAT1/BP regulates the architecture of the inflorescence by affecting pedicle development in Arabidopsis thaliana. Herein, we report the characterization of an Arabidopsis T-DNA insertion mutant that shares considerable phenotypic similarity to the previously identified mutant brevipedicle （bp）. Molecular and genetic analyses showed that the mutant is allelic to bp and that the T-DNA is located within the first helix of the KNAT1 homeodomain （HD）. Although the mutation causes a typical abnormality of short pedicles, propendent siliques, and semidwarfism, no obvious defects are observed in the vegetative stage. A study on cell morphology showed that asymmetrical division and inhibition of cell elongation contribute to the downward-pointing and shorter pedicle phenotype. Loss of KNAT/BPfunction results in the abnormal development of abscission zones. Mlcroarray analysis of gene expression profiling suggests that KNAT1/BP may regulate abscission zone development through hormone signaling and hormone metabolism in Arabidopsis.     

Interallelic complementation at the Arabidopsis CRE1 locus uncovers independent pathways for the proliferation of vascular initials and canonical cytokinin signalling

The differentiation of vascular tissue plays a central role in root architecture and its functionality. Regardless of its importance, the molecular mechanisms involved in the inception of vascular morphogenesis and their interaction with hormones are only now beginning to be understood. The characterisation of the WOODEN LEG (wol/cre1 mutant), impaired in procambial cell proliferation and the identification of WOL/CRE1 as a cytokinin receptor, provided the first genetic evidence pointing to a role of cytokinins in the formation of vascular initials. However, the striking wol phenotype in vascular differentiation is unique among all the available cre1 alleles collection. In this work, we identified a mutant with identical deficiencies in vascular differentiation as wol. Complementation analysis revealed that this mutant rescued the wol short-root phenotype. However, genetic characterisation of the mutant showed that the mutation was located at the CRE1 locus, indicating that both alleles displayed interallelic complementation. Trans-heterozygotes characterisation showed that these plants fully restored the deficiency in vascular differentiation but not the canonical cytokinin signalling. Furthermore, we show that, as measured in root growth inhibition, calli regeneration assays and northern analysis, the original wol allele is in fact more sensitive to cytokinins than the trans-heterozygous plants, or some cre1 alleles showing wild-type vascular morphogenesis. Thus, there is no strict correlation between the phenotype in vascular differentiation displayed by the cre1/wol alleles and canonical cytokinin signalling. These results indicate that at least partially independent regulatory circuits may operate in procambial cell proliferation and in cytokinin responsiveness exerted through the CRE1 receptor.     

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, micro-structure of shoot apical meristem (SAM) and histo-chemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addi-tion, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, lead-ing to alterations in the SAM and a series of phenotypes in the mutant.     

The type-A response regulator,ARR15, acts as a negative regulator in the cytokinin-mediated signal transduction in Arabidopsis thaliana

The Arabidopsis thaliana AHK4 histidine kinase (also known as CRE1 or WOL) acts as a cytokinin signal transducer, presumably, in concert with downstream components, such as histidine-containing phosphotransfer factors (AHPs) and response regulators (ARRs), through the histidine-to-aspartate (His-->Asp) phosphorelay. Among 10 members of the type-A ARR family, the cytokinin-induced expression of ARR15 in roots is selectively impaired in the cre1-1 mutant, which carries a mutation in the AHK4 gene, suggesting a link between this type-A response regulator and the AHK4-mediated cytokinin signal transduction in roots. To address this issue further, we characterized a T-DNA insertion mutant of ARR15, and also constructed transgenic lines (referred to as ARR15-ox) that overexpress the ARR15 gene in a manner independent of cytokinin. While the T-DNA insertion mutant (arr15-1) showed no apparent phenotype, the cytokinin-independent overexpression of ARR15 in ARR15-ox plants resulted in a reduced sensitivity toward exogenously applied cytokinin, not only in elongation of roots in plants, but also in green callus formation (or shoot formation) in explants. Cytokinin-induced expressions of certain type-A ARRs were also down-regulated in ARR15-ox plants. These results support the view that ARR15 acts as a repressor that mediates a negative feedback loop in the cytokinin and AHK4-mediated His-->Asp phosphorelay.     

IRREGULAR TRICHOME BRANCH 2 (ITB2) encodes a putative aminophospholipid translocase that regulates trichome branch elongation in Arabidopsis

The P4 ATPase family in Arabidopsis consists of 12 members that encode putative aminophospholipid translocases (ALA1–12). Until recently, no mutations in these genes have been shown to cause a visible phenotype, although reduced expression of ALA1 in transgenic plants expressing an antisense construct has been shown to result in reduced plant size when plants were grown under cold conditions. During a genetic screen for mutations that affect trichome shape, we isolated several alleles of the irregular trichome branch 2 ( itb2 ) mutation. Subsequent positional cloning of this locus showed that ITB2 encoded ALA3 . Phenotypic and genetic analyses of multiple itb2 alleles, including the T-DNA insertion alleles, showed that the loss of ITB2 / ALA3 function leads to aberrant trichome expansion, reduced primary root growth and longer root hairs. We also found that itb2 / ala3 mutant pollen does not grow as well as wild-type pollen, leading to severe segregation distortion. Our results suggest that aminophospholipid translocases play an important role in the polar growth of plant cells, which is consistent with the proposed role of ALA3 in membrane trafficking. Furthermore, itb2 / ala3 mutants provide a convenient visible phenotype for further genetic analysis of the ALA family in Arabidopsis.     

hoc: An Arabidopsis mutant overproducing cytokinins and expressing high in vitro organogenic capacity

A novel Arabidopsis thaliana mutant, named hoc, was found to have an high organogenic capacity for shoot regeneration. The HOC locus may be involved in cytokinin metabolism leading to cytokinin-overproduction. In vitro, hoc root explants develop many shoots in the absence of exogenous growth regulators. The mutant displays a bushy phenotype with supernumerary rosettes and with normal phyllotaxy, resulting from precocious axillary meristem development. Genetic and molecular analyses show that the high shoot regeneration and the bushy phenotype are controlled by a recessive single gene, located on chromosome I, next to the GAPB CAPS marker. The mapping data and allelism tests reveal that the hoc mutant is not allelic to other reported Arabidopsis growth-regulator mutants. In darkness the hoc mutant is de-etiolated, with a short hypocotyl, opened cotyledons and true leaves. Growth regulator assays reveal that the mutant accumulates cytokinins at about two- and sevenfold the cytokinin level of wild-type plants in its aerial parts and roots, respectively. Consequently, the elevated amounts of endogenous cytokinins in hoc plants are associated with high organogenic capacity and hence bushy phenotype. Thus hoc is the first cytokinin-overproducing Arabidopsis mutant capable of auto-regenerating shoots without exogenous growth regulators.     

The genetic locus At1g73660 encodes a putative MAPKKK and negatively regulates salt tolerance in Arabidopsis

An Arabidopsis mutant with improved salt tolerant germination was isolated from a T-DNA insertion library and designated as AT6. This mutant also exhibited improved salt tolerance phenotype in later developmental stages. But no apparent difference was observed in response to ABA, GA or ethylene during germination between the mutant and the wildtype. The T-DNA was inserted in the At1g73660 locus that coded for a putative MAPKKK. Genetic and multiple mutant allele analyses confirmed that the knockout of this gene resulted in improved salt tolerance phenotype and provided strong evidence that the genetic locus At1g73660 negatively regulated salt tolerance in Arabidopsis. The At1g73660 was down regulated in response to salt stress in the mutants, which is consistent with its role as a negative regulator. It is therefore hypothesized that the AT1g73660 may serve as one of the off-switches of stress responses that are required for unstressed conditions.     

Arabidopsis SOI33/AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transport ers in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtIPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that SOI33 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryotic cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and trans zeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hyper sensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of 3H labeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation inAtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/ AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

Arabidopsis SOI33／AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transporters in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtlPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that S0133 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryodc cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and transzeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hypersensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of^3Hlabeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation in AtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

Isolation and characterization of a novel semi-lethal Arabidopsis thaliana mutant of gene for pentatricopeptide (PPR) repeat-containing protein

A novel Arabidopsis thaliana mutant of one member of the pentatricopeptide repeat (PPR) gene family has been identified among T-DNA insertion lines. Tagging of the At1g53330 gene caused the appearance of a semi-lethal mutation with a complex phenotypic expression from embryo lethality associated with the abnormal pattern of cell division during globular to heart transition to fertile plants with just subtle phenotypic changes. The PPR protein At1g53330.1 was predicted to be targeted to mitochondria by TargetP and MitoProt programs. Complementation analysis confirmed that the phenotype is a result of a single T-DNA integration. A thorough functional analysis of this mutant aimed at finding a particular organelle target of At1g53330.1 protein will follow.     

An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.     

The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling

Arabidopsis thaliana histidine phosphotransfer proteins (AHPs) are similar to bacterial and yeast histidine phosphotransfer proteins (HPts), which act in multistep phosphorelay signaling pathways. A phosphorelay pathway is the current model for cytokinin signaling. To assess the role of AHPs in cytokinin signaling, we isolated T-DNA insertions in the five AHP genes that are predicted to encode functional HPts and constructed multiple insertion mutants, including an ahp1,2,3,4,5 quintuple mutant. Single ahp mutants were indistinguishable from wild-type seedlings in cytokinin response assays. However, various higher-order mutants displayed reduced sensitivity to cytokinin in diverse cytokinin assays, indicating both a positive role for AHPs in cytokinin signaling and functional overlap among the AHPs. In contrast with the other four AHPs, AHP4 may play a negative role in some cytokinin responses. The quintuple ahp mutant showed various abnormalities in growth and development, including reduced fertility, increased seed size, reduced vascular development, and a shortened primary root. These data indicate that most of the AHPs are redundant, positive regulators of cytokinin signaling and affect multiple aspects of plant development.     

Molecular genetic approaches to plant development.

Higher plant morphogenesis has received renewed interest over the past few years. The improvement of molecular genetic approaches to generate tagged developmental mutants, for instance by T-DNA insertion, facilitated the isolation and characterization of the altered genes. Here we present recent progress on flower and root morphogenesis in the small crucifer Arabidopsis thaliana. The current model of Arabidopsis flower development is presented. We report on FLOWER1 (Fl1), which is a T-DNA-tagged ap2 allele. Our observations indicate that this Fl1 mutant has, besides the homeotic Ap2 phenotype, an aberrant seed coat, suggesting that this gene has also a function late in flower development. Furthermore, we present a brief summary about root development and focus on the super root (Sur) mutant, which is an ethyl methanesulfonate-induced mutant that produces excess lateral roots. Root explants of the Sur mutant, that do not develop further than the 4-leaf stage, can be induced to produce normal-looking shoots and flowers by addition of only cytokinin to the medium. The phenotype of Sur and its relation to the action of phytohormones is discussed.     

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.