Ceramides mediate positional signals in Arabidopsis thaliana protoderm differentiation.

The differentiation of distinct cell types in appropriate patterns is a fundamental process in the development of multicellular organisms. In Arabidopsis thaliana, protoderm/epidermis differentiates as a single cell layer at the outermost position. However, little is known about the molecular nature of the positional signals that achieve correct epidermal cell differentiation. Here, we propose that very-long-chain fatty acid-containing ceramides (VLCFA-Cers) mediate positional signals by stimulating the function of ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), a master regulator of protoderm/epidermis differentiation, during lateral root development. We show that VLCFA-Cers, which are synthesized predominantly in the outermost cells, bind to the lipid-binding domain of ATML1. Importantly, this cell type-specific protein-lipid association alters the activity of ATML1 protein and consequently restricts its expression to the protoderm/epidermis through a transcriptional feedback loop. Furthermore, establishment of a compartment, enriched with VLCFA-containing sphingolipids, at the outer lateral membrane facing the external environment may function as a determinant of protodermal cell fate. Taken together, our results indicate that VLCFA-Cers play a pivotal role in directing protoderm/epidermis differentiation by mediating positional signals to ATML1.This article has an associated 'The people behind the papers' interview.

Low nitrogen conditions accelerate flowering by modulating the phosphorylation state of FLOWERING BHLH 4 in Arabidopsis.

Nitrogen (N) is an essential nutrient that affects multiple plant developmental processes, including flowering. As flowering requires resources to develop sink tissues for reproduction, nutrient availability is tightly linked to this process. Low N levels accelerate floral transition; however, the molecular mechanisms underlying this response are not well understood. Here, we identify the FLOWERING BHLH 4 (FBH4) transcription factor as a key regulator of N-responsive flowering in Arabidopsis Low N-induced early flowering is compromised in fbh quadruple mutants. We found that FBH4 is a highly phosphorylated protein and that FBH4 phosphorylation levels decrease under low N conditions. In addition, decreased phosphorylation promotes FBH4 nuclear localization and transcriptional activation of the direct target CONSTANS (CO) and downstream florigen FLOWERING LOCUS T (FT) genes. Moreover, we demonstrate that the evolutionarily conserved cellular fuel sensor SNF1-RELATED KINASE 1 (SnRK1), whose kinase activity is down-regulated under low N conditions, directly phosphorylates FBH4. SnRK1 negatively regulates CO and FT transcript levels under high N conditions. Together, these results reveal a mechanism by which N levels may fine-tune FBH4 nuclear localization by adjusting the phosphorylation state to modulate flowering time. In addition to its role in flowering regulation, we also showed that FBH4 was involved in low N-induced up-regulation of nutrient recycling and remobilization-related gene expression. Thus, our findings provide insight into N-responsive growth phase transitions and optimization of plant fitness under nutrient-limited conditions.

A conserved mechanism determines the activity of two pivotal transcription factors that control epidermal cell differentiation in Arabidopsis thaliana.

The surface of plants is covered by the epidermis, which protects the plant's body from the external environment and mediates inter-cell layer signaling to regulate plant development. Therefore, the manifestation of epidermal traits at a precise location is a prerequisite for their normal growth and development. In Arabidopsis thaliana, class IV homeodomain-leucine zipper transcription factors PROTODERMAL FACTOR2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) play redundant roles in epidermal cell differentiation. Nevertheless, several pieces of evidence suggest that the activity and/or function of PDF2 and ATML1 are regulated differently. The role of the steroidogenic acute regulatory protein-related lipid transfer (START) domain of ATML1 in restricting this protein's activity has been demonstrated; however, whether this lipid-dependent mechanism regulates PDF2 expression is unknown. In this study, we demonstrated that the START domains of PDF2 and ATML1, regulate protein turnover in a position-dependent manner and affect the dimeric proteins. Our results show that a conserved mechanism provides the basis for the functional redundancy of PDF2 and ATML1 in epidermal cell differentiation and that an unidentified regulatory layer specific to PDF2 or ATML1 is responsible for the difference in the activity and/or function of PDF2 and ATML1.

Transient activity of the florigen complex during the floral transition in Arabidopsis thaliana.

FLOWERING LOCUS T (FT) is an essential component of florigen in Arabidopsis thaliana Transcription of FT is induced in leaves, and the resulting FT protein is transported to the shoot apex, in which it initiates floral development. Previous analyses suggest that, together with the b-ZIP transcription factor FD, FT regulates the transcription of downstream targets such as APETALA1 (AP1) in floral anlagen. However, conclusive in vivo evidence that FT is transported to the shoot apex to form an FT-FD complex is lacking. Here, using an innovative in vivo imaging technique, we show that the FT-FD complex and AP1 colocalise in floral anlagen. In addition, the FT-FD complex disappears soon after the floral transition owing to a reduction in FD transcripts in the shoot apex. We further show that misinduction of FD activity after the transition leads to defective reproductive development. Taken together, our results indicate that the FT-FD complex functions as a transient stimulus and imply that a regulatory mechanism exists during the floral transition that reduces FT-FD complex levels via modulation of FD expression.

The lipid-binding START domain regulates the dimerization of ATML1 via modulating the ZIP motif activity in Arabidopsis thaliana.

In Arabidopsis thaliana, the epidermis is the outermost cell layer composed of many specialized types of epidermal cells, such as pavement cells, trichomes, and guard cells. The homeodomain-leucine zipper (HD-ZIP) class Ⅳ transcription factors (TFs), which are unique to the plant kingdom, have been recognized as key regulators of epidermis development. Unlike animal HD proteins, which can bind to DNA as monomers, plant HD-ZIP class Ⅳ TFs bind to DNA as dimers, although little is known about the regulation of their dimerization process. Here, we show that the homodimerization of ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) - HD-ZIP class Ⅳ TF that is required for protoderm development - is regulated by the lipid-binding steroidogenic acute regulatory protein-related lipid transfer (START) domain. We found that ATML1 forms homodimer through interaction via its ZIP motif in yeast and plant cells, although the interaction is abolished by generating a mutation into the lipid-binding START domain to disrupt the lipid-binding ability. These results suggest that lipidic ligands function as key regulators of protoderm development via modulating the dimerization of ATML1.

Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, -D, -F, -H and -J) are synergistically activated by Ca2+ -binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS-producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, -H and -J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or -J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.

SODIUM POTASSIUM ROOT DEFECTIVE1 regulates FLOWERING LOCUS T expression via the microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module in response to potassium conditions.

To determine flowering time, plants perceive multiple environmental stimuli and integrate these signals in the regulation of a florigen gene, FLOWERING LOCUS T (FT). It has been known that nutrient availability affects flowering time in both laboratories and fields. Nitrogen (N), phosphorus (P) and potassium (K) are the three major macronutrients which are important for plant growth and development. Although N and P stimuli can alter the expression of regulators of FT including microRNA156 (miR156) and miR156-targeted transcription factors of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family, how K+ conditions affect flowering is still unclear. We focused on SODIUM POTASSUIM ROOT DEFECTIVE1 (NaKR1) whose mutant plants showed Na+ and K+ overaccumulation and late flowering. It was reported that NaKR1 is involved in the phloem transport of FT protein. Here we report that NaKR1 is also required for the promotion of FT expression in long-day conditions. NaKR1 affects the accumulation of miR156 and SPL3 expression, suggesting that NaKR1 regulates FT expression in part through the miR156-SPL3 module. The late-flowering phenotype of the nakr1-1 mutant was partially suppressed under low K+ conditions, and miR156 abundance and SPL3 expression in the nakr1-1 mutant and, to a lesser extent, in wild-type plants responded to K+ conditions. Taken together, our findings suggest that the miR156-SPL3 module mediates regulation of FT expression by NaKR1 in response to K+ conditions. Finally, we propose a model in which NaKR1 plays dual roles in regulation of flowering, one in the regulation of florigen production, the other in that of florigen transport.

Re-Evaluation of Florigen Transport Kinetics with Separation of Functions by Mutations That Uncouple Flowering Initiation and Long-Distance Transport.

In many plants, timing of flowering is regulated by day length. In Arabidopsis, florigen, FLOWERING LOCUS T (FT) protein, is synthesized in leaf phloem companion cells in response to long days and is transported to the shoot apical meristem (SAM) through the phloem. The temporal aspects of florigen transportation have been studied in various plants by physiological experiments. Nevertheless, little is known about how FT protein transportation is regulated in Arabidopsis. In this study, we performed heat shock-based transient FT induction in a single leaf blade and detected the FT protein in the shoot apex by 2D-PAGE. We demonstrated that detectable amounts of FT were transported from the leaf to the shoot apex within 8 h, and subsequent FT-induced target gene expression was detected within 8-12 h. Furthermore, we identified three amino acid residues (V70, S76 and R83) where missense mutations led to reduced mobility. Interestingly, these FT variants lost only their transportation ability, but retained their flowering promotion capacity, suggesting that discrete amino acids are involved in flowering regulation and transport regulation. Since the interaction with FT-INTERACTING PROTEIN 1 (FTIP1) was not affected in these FT variants, we hypothesize that the three amino acid residues are not involved in the FTIP1-mediated pathway of uploading, but rather in the subsequent step(s) of FT transport.

FE Controls the Transcription of Downstream Flowering Regulators Through Two Distinct Mechanisms in Leaf Phloem Companion Cells.

In the facultative long-day plant Arabidopsis thaliana, FLOWERING LOCUS T (FT), encoding the mobile hormone florigen, plays an essential role in modulating the optimal timing of flowering to ensure reproductive success. Under inductive long-day conditions, the transcription of FT is activated by the CONSTANS (CO)/NUCLEAR FACTOR-Y (NF-Y) protein complex in leaf phloem companion cells. FT is transported to the shoot apical meristem through interaction with florigen transporters, such as SODIUM POTASSIUM ROOT DEFECTIVE 1 (NaKR1). Some regulators involved in photoperiod-dependent FT function have been reported previously; however, the molecular mechanism that coordinates FT protein synthesis and transport efficiently needs to be investigated. The present study examined the role of an Myb-related transcription factor, FE, in the activation of FT gene transcription and FT protein transport. Expression analysis using FE-inducible systems and chromatin immunoprecipitation assays showed that FE directly bound to the FT and NaKR1 promoters and activated the transcription of downstream target genes. FE failed to activate FT expression without CO function, whereas FE-mediated NaKR1 induction was not affected by CO function. Taken together, our data indicate that FE regulates the transcription of FT and florigen transporter genes via different mechanisms.

Ca2+-activated reactive oxygen species production by Arabidopsis RbohH and RbohJ is essential for proper pollen tube tip growth.

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca(2+) binding EF-hand motifs, possessed Ca(2+)-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca(2+)-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca(2+)-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca(2+). Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca(2+)-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

FE, a phloem-specific Myb-related protein, promotes flowering through transcriptional activation of FLOWERING LOCUS T and FLOWERING LOCUS T INTERACTING PROTEIN 1.

In many flowering plants, the transition to flowering is primarily affected by seasonal changes in day length (photoperiod). An inductive photoperiod promotes flowering via synthesis of a floral stimulus, called florigen. In Arabidopsis thaliana, the FLOWERING LOCUS T (FT) protein is an essential component of florigen, which is synthesized in leaf phloem companion cells and is transported through phloem tissue to the shoot apical meristem where floral morphogenesis is initiated. However, the molecular mechanism involved in the long-distance transport of FT protein remains elusive. In this study, we characterized the classic Arabidopsis mutant fe, which is involved in the photoperiodic induction of flowering, and showed that FE encodes a phloem-specific Myb-related protein that was previously reported as ALTERED PHLOEM DEVELOPMENT. Phenotypic analyses of the fe mutant showed that FT expression is reduced in leaf phloem companion cells. In addition, the transport of FT protein from leaves to the shoot apex is impaired in the fe mutant. Expression analyses further demonstrated that FE is also required for transcriptional activation of FLOWERING LOCUS T INTERACTING PROTEIN 1 (FTIP1), an essential regulator for selective trafficking of the FT protein from companion cells to sieve elements. These findings indicate that FE plays a dual role in the photoperiodic induction of flowering: as a transcriptional activator of FT on the one hand, and its transport machinery component, FTIP1, on the other hand. Thus, FE is likely to play a role in regulating FT by coordinating FT synthesis and FT transport in phloem companion cells.

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.

A low temperature-inducible protein AtSRC2 enhances the ROS-producing activity of NADPH oxidase AtRbohF.

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca(2+) and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca(2+)-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca(2+)-dependent AtRbohF-mediated ROS production and may play a role in cold responses.

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.

The florigen genes FT and TSF modulate lateral shoot outgrowth in Arabidopsis thaliana.

Successful sexual reproduction of a plant with prolific seed production requires appropriate timing of flowering and concomitant change of architecture (e.g. internode elongation and branching) to facilitate production of the optimal number of flowers while enabling continued resource production through photosynthesis. Florigen is the prime candidate for a signal linking the two processes. Growth analysis of lateral shoots in mutants of FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) revealed a delay in the onset of outgrowth and a reduction of the growth rate in ft plants in long-day (LD) conditions and in tsf plants in short-day (SD) conditions. Thus, as in the case of floral transition, FT and TSF play dominant roles in LD and SD conditions, respectively, in the promotion of lateral shoot development. Differential expression patterns of the two genes were in good agreement with their differential roles both in the floral transition and in lateral shoot development under contrasting photoperiod conditions. By manipulating florigen production after bolting of the primary shoot, it was shown that florigen promotes lateral shoot growth independently of its effect on the floral transition of the primary shoot. Analysis of growth and gene expression in lateral shoots of the mutants suggests that the loss of florigen leads to a reduced rate of flower formation on lateral shoots. Together, we propose that the two florigen genes are an important key to linking the floral transition and lateral shoot development to maximize the reproductive success of a plant.

CRYPTIC PRECOCIOUS/MED12 is a novel flowering regulator with multiple target steps in Arabidopsis.

The proper timing of flowering is of crucial importance for reproductive success of plants. Regulation of flowering is orchestrated by inputs from both environmental and endogenous signals such as daylength, light quality, temperature and hormones, and key flowering regulators construct several parallel and interactive genetic pathways. This integrative regulatory network has been proposed to create robustness as well as plasticity of the regulation. Although knowledge of key genes and their regulation has been accumulated, there still remains much to learn about how they are organized into an integrative regulatory network. Here, we have analyzed the CRYPTIC PRECOCIOUS (CRP) gene for the Arabidopsis counterpart of the MED12 subunit of the Mediator. A novel dominant mutant, crp-1D, which causes up-regulation of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), FRUITFULL (FUL) and APETALA1 (AP1) expression in a FLOWERING LOCUS T (FT)-dependent manner, was identified in an enhancer screen of the early-flowering phenotype of 35S::FT. Genetic and molecular analysis of both crp-1D and crp loss-of-function alleles showed that MED12/CRP is required not only for proper regulation of SOC1, FUL and AP1, but also for up-regulation of FT, TWIN SISTER OF FT (TSF) and FD, and down-regulation of FLOWERING LOCUS C (FLC). These observations suggest that MED12/CRP is a novel flowering regulator with multiple regulatory target steps both upstream and downstream of the key flowering regulators including FT florigen. Our work, taken together with recent studies of other Mediator subunit genes, supports an emerging view that the Mediator plays multiple roles in the regulation of flowering.

Regulation of reproductive development by non-coding RNA in Arabidopsis: to flower or not to flower.

Plants monitor environmental factors, such as temperature and day length, and also endogenous factors, such as their age and phytohormones, to decide when to flower. These cues are utilized to control expression levels of genes required for flowering. Thus, flowering time control is a unique model for understanding how gene activity is precisely regulated at the transcriptional level. In Arabidopsis, a remarkable number of non-coding RNA molecules have been identified by advanced sequencing technology. Recent progress in the flowering field has revealed several non-coding RNAs that play a major role in determining flowering time. Here, we introduce how two types of non-coding RNA species, microRNA (miRNA) and long noncoding RNA (lncRNA), contribute to flowering via regulation of target gene activity involved in this vital developmental transition.

Adaptation of a seedling micro-grafting technique to the study of long-distance signaling in flowering of Arabidopsis thaliana.

Long-distance signaling via phloem tissues is an important mechanism for inter-organ communication. Such communication allows plants to integrate environmental information into physiological and developmental responses. Grafting has provided persuasive evidence of long-distance signaling involved in various processes, including flowering, tuberization, nodulation, shoot branching, post-transcriptional gene silencing, and disease resistance. A micro-grafting technique to generate two-shoot grafts is available for young seedlings of Arabidopsis thaliana and was adapted for use in the study of flowering. Histological analysis using transgenic plants expressing beta-glucuronidase (GUS) in phloem tissues showed that phloem continuity between a stock and a scion was established between 7 and 10 days after grafting. Experiments using tracer dyes and enhanced green fluorescent protein (EGFP) showed that the phloem connection was functional and capable of effecting macromolecular transmission. Successful grafts can be obtained at high frequency (10-30%) and selected after 2-3 weeks of post-surgery growth. This method was applied successfully to the study of flowering, one of the important events regulated by long-distance signaling. This grafting technique will facilitate the study of the long-distance action of genes involved in various aspects of growth and development, and in transport of signal molecules.

Long-distance, graft-transmissible action of Arabidopsis FLOWERING LOCUS T protein to promote flowering.

Day length perceived by a leaf is a major environmental factor that controls the timing of flowering. It has been believed that a mobile, long-distance signal called florigen is produced in the leaf under inductive day length conditions, and is transported to the shoot apex where it triggers floral morphogenesis. Grafting experiments have shown that florigen is transmissible from a donor plant that has been subjected to inductive day length to an uninduced recipient plant. However, the nature of florigen has long remained elusive. Arabidopsis FLOWERING LOCUS T (FT) is expressed in cotyledons and leaves in response to inductive long days (LDs). FT protein, with a basic region/leucine zipper (bZIP) transcription factor FD, acts in the shoot apex to induce target meristem identity genes such as APETALA1 (AP1) and initiates floral morphogenesis. Recent studies have provided evidence that the FT protein in Arabidopsis and corresponding proteins in other species are an important part of florigen. Our work shows that the FT activity, either from overexpressing or inducible transgenes or from the endogenous gene, to promote flowering is transmissible through a graft junction, and that an FT protein with a T7 tag is transported from a donor scion to the apical region of recipient stock plants and becomes detectable within a day or two. The sequence and structure of mRNA are not of critical importance for the long-distance action of the FT gene. These observations led to the conclusion that the FT protein, but not mRNA, is the essential component of florigen.