ATML1 and PDF2 Play a Redundant and Essential Role in Arabidopsis Embryo Development.

The epidermis of shoot organs in plants develops from the outermost layer (L1) of the shoot apical meristem. In Arabidopsis, a pair of homeobox genes, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) and PROTODERMAL FACTOR2 (PDF2), play a role in regulating the expression of L1-specific genes. atml1-1 pdf2-1 double mutants show striking defects in the differentiation of shoot epidermal cells. However, because atml1-1 and pdf2-1 have a T-DNA inserted downstream of the respective homeobox sequences, these alleles may not represent null mutations. Here we characterized additional mutant alleles that have a T-DNA insertion at different positions of each gene. Double mutants of a strong atml1-3 allele with each pdf2 allele were found to cause embryonic arrest at the globular stage. Although with low frequency, all double mutant combinations of a weak atml1-1 allele with each pdf2 allele germinated and showed phenotypes defective in shoot epidermal cell differentiation. We further confirmed that transgenic induction of PDF2 fused to the Drosophila Engrailed repressor domain temporarily interferes with epidermal cell differentiation in the wild-type background. These results indicate that ATML1 and PDF2 act redundantly as a positive regulator of shoot epidermal cell differentiation and at least one copy of these genes is essential for embryo development.

Control of embryonic meristem initiation in Arabidopsis by PHD-finger protein complexes.

Plant growth is directed by the activity of stem cells within meristems. The first meristems are established during early embryogenesis, and this process involves the specification of both stem cells and their organizer cells. One of the earliest events in root meristem initiation is marked by re-specification of the uppermost suspensor cell as hypophysis, the precursor of the organizer. The transcription factor MONOPTEROS (MP) is a key regulator of hypophysis specification, and does so in part by promoting the transport of the plant hormone auxin and by activating the expression of TARGET OF MP (TMO) transcription factors, both of which are required for hypophysis specification. The mechanisms leading to the activation of these genes by MP in a chromatin context are not understood. Here, we show that the PHD-finger proteins OBERON (OBE) and TITANIA (TTA) are essential for MP-dependent embryonic root meristem initiation. TTA1 and TTA2 are functionally redundant and function in the same pathway as OBE1 and OBE2. These PHD-finger proteins interact with each other, and genetic analysis shows that OBE-TTA heterotypic protein complexes promote embryonic root meristem initiation. Furthermore, while MP expression is unaffected by mutations in OBE/TTA genes, expression of MP targets TMO5 and TMO7 is locally lost in obe1 obe2 embryos. PHD-finger proteins have been shown to act in initiation of transcription by interacting with nucleosomes. Indeed, we found that OBE1 binds to chromatin at the TMO7 locus, suggesting a role in its MP-dependent activation. Our data indicate that PHD-finger protein complexes are crucial for the activation of MP-dependent gene expression during embryonic root meristem initiation, and provide a starting point for studying the mechanisms of developmental gene activation within a chromatin context in plants.

Mutations in epidermis-specific HD-ZIP IV genes affect floral organ identity in Arabidopsis thaliana.

Development of the epidermis involves members of the class-IV homeodomain-leucine zipper (HD-ZIP IV) transcription factors. The Arabidopsis HD-ZIP IV family consists of 16 members, among which PROTODERMAL FACTOR 2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) play an indispensable role in the differentiation of shoot epidermal cells; however, the functions of other HD-ZIP IV genes that are also expressed specifically in the shoot epidermis remain to be fully elucidated. We constructed double mutant combinations of these HD-ZIP IV mutant alleles and found that the double mutants of pdf2-1 with homeodomain glabrous1-1 (hdg1-1), hdg2-3, hdg5-1 and hdg12-2 produced abnormal flowers with sepaloid petals and carpelloid stamens in association with the reduced expression of the petal and stamen identity gene APETALA 3 (AP3). Expression of another petal and stamen identity gene PISTILATA (PI) was less affected in these mutants. We confirmed that AP3 expression in pdf2-1 hdg2-3 was normally induced at the initial stages of flower development, but was attenuated both in the epidermis and internal cell layers of developing flowers. As the expression of PDF2 and these HD-ZIP IV genes during floral organ formation is exclusively limited to the epidermal cell layer, these double mutations may have non-cell-autonomous effects on AP3 expression in the internal cell layers. Our results suggest that cooperative functions of PDF2 and other members of the HD-ZIP IV family in the epidermis are crucial for normal development of floral organs in Arabidopsis.

LEAFY controls Arabidopsis pedicel length and orientation by affecting adaxial-abaxial cell fate.

Pedicel length and orientation (angle) contribute to the diversity of inflorescence architecture, and are important for optimal positioning of the flowers. However, relatively little is known about pedicel development. We previously described the Arabidopsis CORYMBOSA1 (CRM1)/BIG gene, which affects inflorescence architecture by controlling pedicel elongation and orientation. Here, we performed a suppressor screen using the partial loss-of-function allele crm1-13 to identify genes and pathways that affect pedicel development. We identified a hypomorph allele of the meristem identity regulator LEAFY (LFY) as the suppressor. Consistent with this, crm1 pedicels had elevated LFY levels and conditional gain of LFY function produced downward-bending pedicels. Steroid activation of 35S:LFY-GR plants caused a reduction in the cortical cell length in the abaxial domain and additional defects associated with adaxialization. Further analyses of loss of LFY function revealed that LFY is required for reduced cortical cell elongation at the adaxial side of the pedicel base. Defects in conditional LFY gain-of-function pedicels were correlated with decreased BREVIPEDICELLUS (BP) expression, while ASYMMETRIC LEAVES2 (AS2), a transcriptional repressor of BP, and REVOLUTA, a promoter of adaxial cell fate, were highly and ectopically expressed in LFY gain-of-function pedicels. LFY bound to cis-regulatory regions upstream of AS2, and as2 mutations partially suppressed the pedicel length and orientation defects caused by increased LFY activity. These data suggest that LFY activity promotes adaxial cell fate and hence the proper orientation and length of the pedicel partly by directly activating AS2 expression, which suppresses BP expression.

Characterization of EMU, the Arabidopsis homolog of the yeast THO complex member HPR1.

Diverse and precise control is essential for eukaryotic gene expression. This is accomplished through the recruitment of a myriad of proteins to a nascent messenger RNA (mRNA) to mediate modifications, such as capping, splicing, 3'-end processing, and export. Despite being important for every cell, however, the mechanism by which the formation of diverse messenger ribonucleoprotein (mRNP) particles contributes to maintaining intricate systems in the multicellular organism remains incompletely defined. We identified and characterized a mutant gene named erecta mRNA under-expressed (emu) that leads to the defective mRNA accumulation of ERECTA, a developmental regulator in the model plant Arabidopsis thaliana. EMU encodes a protein homologous to a component of the THO complex that is required for the generation of functional mRNPs. Further analysis suggested that EMU is genetically associated with SERRATE, HYPONASTIC LEAVES1, and ARGONAUTE1, which are required for proper RNA maturation or action. Furthermore, mutations in another THO-related gene led to embryonic lethality. These findings support the presence and importance of the THO-related complex in plants as well as yeast and vertebrates.

Genetic regulation of time to flower in Arabidopsis thaliana.

In Arabidopsis thaliana, the initiation of flowering is carried out by four genetic pathways: gibberellin, autonomous, vernalization, and light-dependent pathways. These processes are integrated by the function of the genes FD, FE, FWA, PDF2, SOC1, and FT at the integration pathway. The integrated signal of the floral induction is transmitted to the floral meristem identity genes LFY and AP1, and floral morphogenesis is performed.

A novel mutation in KNOPF uncovers the role of alpha-glucosidase I during post-embryonic development in Arabidopsis thaliana.

N-glycosylation is a common protein modification. Joining of polypeptide and carbohydrate elements into hybrid molecules provides an opportunity to fine-tune protein properties. However, the role of N-glycosylation on the development of multicellular organisms remains elusive. Here we report a hypomorphic allele of KNOPF/GLUCOSIDASE 1, which allows us to describe the effects of impaired alpha-glucosidase I on post-embryonic development of plants for the first time. This knf-101 mutation alters cell shape but does not affect cell arrangements, except for the patterning of specialized epidermal cells, delineating the significance of N-glycan processing during epidermal development in Arabidopsis.

An inversion identified in acl1-1 mutant functions as an enhancer of the acl1-1 phenotype.

The Arabidopsis acaulis1-1 (acl1-1) mutant exhibits severe growth defects when grown at 22 degrees C. The leaves are tiny and curled and the inflorescence stems are short. We identified an inversion mutation in the original acl1-1 plants. The acl1-1 plants were crossed with Columbia wild-type, and the acl1-1 phenotype and the inversion were segregated in the F2 generation. Compared to the original acl1-1 plants with the inversion, the genuine acl1-1 plants without the inversion grew larger and their inflorescence stems grew longer at 22 degrees C. When the plants were grown at 24 degrees C, the differences in growth became more apparent. We investigated the expression of genes located in the inversion. Two genes that were located at each end of the inversion were disrupted, and full-length transcripts were not expressed. Expressions of some genes within and adjacent to the inversion were also altered. Our results indicate that the expression of multiple genes may be involved in the enhancement of the acl1-1 phenotype.

Metabolic changes in Arabidopsis thaliana expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D.

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 microM L-Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3 M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3 M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.

Role of the ACL2 locus in flower stalk elongation in Arabidopsis thaliana.

The acaulis2 (acl2) mutant of Arabidopsis thaliana shows a defect in flower stalk elongation. We identified the mutation point of acl2 by map-based cloning. The ACL2 locus is located within an approximately 320-kb region at around 100 map units on chromosome 1. One nucleotide substitution was detected in this region in the acl2 mutant, but no significant open reading frames were found around this mutation point. When wild-type DNA fragments containing the mutation point were introduced into acl2 mutant plants, some transgenic plants partially or almost completely recovered from the defect in flower stalk elongation. 3'-RACE experiments showed that bidirectional transcripts containing the acl2 mutation point were expressed, and the Plant MPSS database revealed that several small RNAs were produced from this region. Microarray analysis showed that transcription of many genes is activated in flower stalks of acl2 mutant plants. Overexpression of some of these genes caused a dwarf phenotype in wild-type plants. These results suggest the following novel mechanism for control of the elongation of flower stalks. Bidirectional non-coding RNAs are transcribed from the ACL2 locus, and small RNAs are generated from them in flower stalks. These small RNAs repress the transcription of a set of genes whose expression represses flower stalk elongation, and flower stalks are therefore fully elongated.

The plant exon finder: a tool for precise detection of exons using a T-DNA-based tagging approach.

We have developed a new tool, named the plant exon finder (PEF), for identifying exons in plant genome sequences as an applied technique of T-DNA insertional mutagenesis. The T-DNA constructs contain a heat-shock gene promoter or the cauliflower mosaic virus (CaMV) 35S promoter, followed by the first exon of an Arabidopsis gene with its start codon and the intron donor sequence facing the T-DNA left border (LB) in order to trap exons in the genome. The constructs were used to make transgenic Arabidopsis plants. We generated 280 transgenic lines and identified 156 T-DNA-tagged readthrough transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) using an oligo(dT)-linker primer and a T-DNA-specific primer. Sequence analysis of the RT-PCR products revealed that 18 of them carried cDNAs processed by the use of an intron acceptor sequence adjacent to T-DNA insertion sites and 11 of them were in-frame fusions. In one case, the readthrough transcript trapped an exon located 1.6 kb downstream of the site of the insertion.

Characterization of the spermidine synthase-related gene family in Arabidopsis thaliana.

The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase-deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells.

Overexpression of the TIR-X gene results in a dwarf phenotype and activation of defense-related gene expression in Arabidopsis thaliana.

The Arabidopsis genome encodes various proteins with a Toll/interleukin-1 receptor (TIR) domain. Many of these proteins also contain nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains and function as resistance (R) proteins. However, the protein encoded by At2g32140 (a TIR-X gene) contains a TIR domain but lacks NBS and LRR domains. We found that transgenic plants overexpressing At2g32140 displayed a dwarf phenotype and showed increased expression of defense-related genes. In general, the growth defect caused by activation of defense responses is suppressed under high-temperature conditions. However, transgenic plants overexpressing At2g32140 displayed a much stronger dwarf phenotype at 28°C than at 22°C. This dwarf phenotype was suppressed under the combination with known salicylic-acid pathway mutants. These findings suggest that At2g32140 encodes a protein involved in the plant defense response.

The role of CORYMBOSA1/BIG and auxin in the growth of Arabidopsis pedicel and internode.

The developmental regulation of pedicel and internode ensures that plants of the same species have similar inflorescence architecture. In Arabidopsis, the elongation of pedicels and internodes must be temporally and spatially regulated. We previously isolated the corymbosa1 (crm1) mutant, which has a corymb-like inflorescence because of shortened pedicels and internodes. CRM1/BIG encodes a membrane-associated protein and is required for auxin transport. Although CRM1/BIG and auxin have important roles in the development of pedicels and internodes, the developmental changes in cell growth in pedicels and internodes have not been examined. Here, we investigated the role of auxin in the cell growth of pedicels and internodes. Wild-type plants had rapid pedicel and internodal elongation that resulted from the temporal control of cell division and elongation. The crm1 mutants had defects in cell division and elongation. Auxin signaling and cell cycle gene expression in crm1 inflorescences were lower than those in the wild type. Moreover, wild-type plants treated with an auxin transport inhibitor and mutants defective in auxin signaling had shorter pedicels and internodes, with fewer and smaller cells. Our results suggest that auxin transport and signaling have important roles in controlling the proliferation and elongation of pedicel and internodal cells.

Allele-specific effects of PDF2 on floral morphology in Arabidopsis thaliana.

The class IV Homeodomain-leucine zipper (HD-ZIP IV) gene family includes several genes that are functionally significant in epidermal development. Our recent study revealed that double mutants of the epidermis-expressed HD-ZIP IV members, PROTODERMAL FACTOR2 (PDF2) in combination with some HOMEODOMAIN GLABROUS (HDG, pronounced "hedge") genes, affect stamen development and specification of petal and stamen identity, possibly in a non cell-autonomous manner. However, the effect of the pdf2 mutations on the floral development was largely different depending on T-DNA insertion locations: pdf2-1 hdg flowers exhibited homeotic conversion of petals and stamens, while pdf2-2 hdg flowers had only a reduced number of stamens. Here, we used 2 additional pdf2 alleles to make double mutants and found that their floral phenotypes were rather similar to those of pdf2-2 hdg. The allele-specific effect caused by pdf2-1, which carries a T-DNA in a steroidogenic acute regulatory protein-related lipid transfer (START) domain-encoding region, suggests the importance of the START domain in proper function of HD-ZIP IV proteins.

Overexpression of the NAC transcription factor family gene ANAC036 results in a dwarf phenotype in Arabidopsis thaliana.

NAC proteins comprise one of the largest families of transcription factors in the plant genome. They are known to be involved in various aspects of plant development, but the functions of most of them have not yet been determined. ANAC036, a member of the Arabidopsis NAC transcription factor family, contains unique sequences that are conserved among various NAC proteins found in other plant species. Expression analysis of the ANAC036 gene indicated that this gene was strongly expressed in leaves. Transgenic plants overexpressing the ANAC036 gene showed a semidwarf phenotype. The lengths of leaf blades, petioles and stems of these plants were smaller than those in wild-type plants. Microscopy revealed that cell sizes in leaves and stems of these plants were smaller than those in wild-type plants. These findings suggested that ANAC036 and its orthologues are involved in the growth of leaf cells.

The Arabidopsis OBERON1 and OBERON2 genes encode plant homeodomain finger proteins and are required for apical meristem maintenance.

Maintenance of the stem cell population located at the apical meristems is essential for repetitive organ initiation during the development of higher plants. Here, we have characterized the roles of OBERON1 (OBE1) and its paralog OBERON2 (OBE2), which encode plant homeodomain finger proteins, in the maintenance and/or establishment of the meristems in Arabidopsis. Although the obe1 and obe2 single mutants were indistinguishable from wild-type plants, the obe1 obe2 double mutant displayed premature termination of the shoot meristem, suggesting that OBE1 and OBE2 function redundantly. Further analyses revealed that OBE1 and OBE2 allow the plant cells to acquire meristematic activity via the WUSCHEL-CLAVATA pathway, which is required for the maintenance of the stem cell population, and they function parallel to the SHOOT MERISTEMLESS gene, which is required for preventing cell differentiation in the shoot meristem. In addition, obe1 obe2 mutants failed to establish the root apical meristem, lacking both the initial cells and the quiescent center. In situ hybridization revealed that expression of PLETHORA and SCARECROW, which are required for stem cell specification and maintenance in the root meristem, was lost from obe1 obe2 mutant embryos. Taken together, these data suggest that the OBE1 and OBE2 genes are functionally redundant and crucial for the maintenance and/or establishment of both the shoot and root meristems.

CRM1/BIG-mediated auxin action regulates Arabidopsis inflorescence development.

The shape of the inflorescence in Arabidopsis thaliana ecotype Columbia is a raceme with individual flowers developing acropetally. The ecotype Landsberg harboring the erecta (er) mutation shows a corymb-like inflorescence, namely a compact inflorescence with a flattened arrangement of flower buds at the tip. To gain insight into inflorescence development, we previously isolated corymb-like inflorescence mutants, named corymbosa1 (crm1), and found that the corymb-like inflorescence in crm1-1 was due to reduced cell elongation of pedicels and stem internodes. Double mutants of crm1 with er and crm2, and crm1-1 crm2-1 er-105 triple mutants show an additive phenotype. crm1-1 is caused by a mutation in BIG, which is required for polar auxin transport. CRM1/BIG is expressed in inflorescence meristems, floral meristems and vascular tissues. We analyzed a collection of 12 reduced lateral root formation (rlr) mutants, which are allelic to crm1-1, and categorized the mutants into three classes, depending on the plant developmental defects. Although all 12 alleles had new stop codons, the phenotype of heterozygous crm1-1/doc1-1 and Northern blotting suggest that new crm1/big mutant alleles are hypomorphic. Auxin-responsive DR5rev::GFP expression was decreased in crm1-1 vasculature of pedicels and stem internodes. PINFORMED1 (PIN1) and CRM1/BIG are expressed in vasculature of pedicels and stem internodes. The severity of corymb-like inflorescence in crm1/big mutants correlated with increased levels of PIN1. Our results suggest that CRM1/BIG controls the elongation of the pedicels and stem internodes through auxin action.

The dwarf phenotype of the Arabidopsis acl5 mutant is suppressed by a mutation in an upstream ORF of a bHLH gene.

Loss-of-function mutants of the Arabidopsis thaliana ACAULIS 5 (ACL5) gene, which encodes spermine synthase, exhibit a severe dwarf phenotype. To elucidate the ACL5-mediated regulatory pathways of stem internode elongation, we isolated four suppressor of acaulis (sac) mutants that reverse the acl5 dwarf phenotype. Because these mutants do not rescue the dwarfism of known phytohormone-related mutants, the SAC genes appear to act specifically on the ACL5 pathways. We identify the gene responsible for the dominant sac51-d mutant, which almost completely suppresses the acl5 phenotype. sac51-d disrupts a short upstream open reading frame (uORF) of SAC51, which encodes a bHLH-type transcription factor. Our results indicate that premature termination of the uORF in sac51-d results in an increase in its own transcript level, probably as a result of an increased translation of the main ORF. We suggest a model in which ACL5 plays a role in the translational activation of SAC51, which may lead to the expression of a subset of genes required for stem elongation.

Characterization of the class IV homeodomain-Leucine Zipper gene family in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) genome contains 16 genes belonging to the class IV homeodomain-Leucine zipper gene family. These include GLABRA2, ANTHOCYANINLESS2, FWA, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), and PROTODERMAL FACTOR2 (PDF2). Our previous study revealed that atml1 pdf2 double mutants have severe defects in the shoot epidermal cell differentiation. Here, we have characterized additional members of this gene family, which we designated HOMEODOMAIN GLABROUS1 (HDG1) through HDG12. Analyses of transgenic Arabidopsis plants carrying the gene-specific promoter fused to the bacterial beta-glucuronidase reporter gene revealed that some of the promoters have high activities in the epidermal layer of the shoot apical meristem and developing shoot organs, while others are temporarily active during reproductive organ development. Expression profiles of highly conserved paralogous gene pairs within the family were found to be not necessarily overlapping. Analyses of T-DNA insertion mutants of these HDG genes revealed that all mutants except hdg11 alleles exhibit no abnormal phenotypes. hdg11 mutants show excess branching of the trichome. This phenotype is enhanced in hdg11 hdg12 double mutants. Double mutants were constructed for other paralogous gene pairs and genes within the same subfamily. However, novel phenotypes were observed only for hdg3 atml1 and hdg3 pdf2 mutants that both exhibited defects in cotyledon development. These observations suggest that some of the class IV homeodomain-Leucine zipper members act redundantly with other members of the family during various aspects of cell differentiation. DNA-binding sites were determined for two of the family members using polymerase chain reaction-assisted DNA selection from random oligonucleotides with their recombinant proteins. The binding sites were found to be similar to those previously identified for ATML1 and PDF2, which correspond to the pseudopalindromic sequence 5'-GCATTAAATGC-3' as the preferential binding site.