Parallel evolution of UbiA superfamily proteins into aromatic O-prenyltransferases in plants.

Plants produce ∼300 aromatic compounds enzymatically linked to prenyl side chains via C-O bonds. These O-prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although their O-prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromatic O-prenyltransferase (O-PT) has been isolated to date. This study describes the isolation of an aromatic O-PT gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This gene was shown responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin O-PT gene encoding a protein of the same family was identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O-prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary (specialized) metabolites via the UbiA superfamily.

Subtilase activity in intrusive cells mediates haustorium maturation in parasitic plants.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

BELL1-like homeobox genes regulate inflorescence architecture and meristem maintenance in rice.

Inflorescence architecture is diverse in angiosperms, and is mainly determined by the arrangement of the branches and flowers, known as phyllotaxy. In rice (Oryza sativa), the main inflorescence axis, called the rachis, generates primary branches in a spiral phyllotaxy, and flowers (spikelets) are formed on these branches. Here, we have studied a classical mutant, named verticillate rachis (ri), which produces branches in a partially whorled phyllotaxy. Gene isolation revealed that RI encodes a BELL1-type homeodomain transcription factor, similar to Arabidopsis PENNYWISE/BELLRINGER/REPLUMLESS, and is expressed in the specific regions within the inflorescence and branch meristems where their descendant meristems would soon initiate. Genetic combination of an ri homozygote and a mutant allele of RI-LIKE1 (RIL1) (designated ri ril1/+ plant), a close paralog of RI, enhanced the ri inflorescence phenotype, including the abnormalities in branch phyllotaxy and rachis internode patterning. During early inflorescence development, the timing and arrangement of primary branch meristem (pBM) initiation were disturbed in both ri and ri ril1/+ plants. These findings suggest that RI and RIL1 were involved in regulating the phyllotactic pattern of the pBMs to form normal inflorescences. In addition, both RI and RIL1 seem to be involved in meristem maintenance, because the ri ril1 double-mutant failed to establish or maintain the shoot apical meristem during embryogenesis.

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis.

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants.

Identifying the target genes of SUPPRESSOR OF GAMMA RESPONSE 1, a master transcription factor controlling DNA damage response in Arabidopsis.

In mammalian cells, the transcription factor p53 plays a crucial role in transmitting DNA damage signals to maintain genome integrity. However, in plants, orthologous genes for p53 and checkpoint proteins are absent. Instead, the plant-specific transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) controls most of the genes induced by gamma irradiation and promotes DNA repair, cell cycle arrest, and stem cell death. To date, the genes directly controlled by SOG1 remain largely unknown, limiting the understanding of DNA damage signaling in plants. Here, we conducted a microarray analysis and chromatin immunoprecipitation (ChIP)-sequencing, and identified 146 Arabidopsis genes as direct targets of SOG1. By using ChIP-sequencing data, we extracted the palindromic motif [CTT(N)7 AAG] as a consensus SOG1-binding sequence, which mediates target gene induction in response to DNA damage. Furthermore, DNA damage-triggered phosphorylation of SOG1 is required for efficient binding to the SOG1-binding sequence. Comparison between SOG1 and p53 target genes showed that both transcription factors control genes responsible for cell cycle regulation, such as CDK inhibitors, and DNA repair, whereas SOG1 preferentially targets genes involved in homologous recombination. We also found that defense-related genes were enriched in the SOG1 target genes. Consistent with this finding, SOG1 is required for resistance against the hemi-biotrophic fungus Colletotrichum higginsianum, suggesting that SOG1 has a unique function in controlling the immune response.

Constitutive BiP protein accumulation in Arabidopsis mutants defective in a gene encoding chloroplast-resident stearoyl-acyl carrier protein desaturase.

The unfolded protein response (UPR) occurs when protein folding and maturation are disturbed in the endoplasmic reticulum (ER). During the UPR, a number of genes including those encoding ER-resident molecular chaperones are induced. In Arabidopsis, BiP3 has been used as a UPR marker gene whose expression is strongly induced in response to ER stress. In this study, we mutagenized Arabidopsis plants expressing ß-glucuronidase (GUS) gene under the control of BiP3 promoter and isolated a mutant that exhibits strong GUS activity without treatment with ER stress inducers. By whole genome sequencing, we identified a causal gene in the mutant as SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY2 (SSI2), which encodes stearoyl-acyl carrier protein desaturase that converts stearic acids to oleic acids in the chloroplasts. In addition to GUS proteins, the ssi2 mutant accumulates endogenous BiP3 proteins without treatment by any stress reagents. Interestingly, although the degree of endogenous BiP3 protein accumulation in the ssi2 mutant was comparable to that in wild-type plants treated with the ER stress inducer tunicamycin, much less BiP3 transcripts were detected in the ssi2 mutant compared to tunicamycin-treated wild-type plants. Our finding suggests a genetic link between fatty acid metabolism in the chloroplasts and ER functions.

Transcription Factors VND1-VND3 Contribute to Cotyledon Xylem Vessel Formation.

Arabidopsis (Arabidopsis thaliana) VASCULAR-RELATED NAC-DOMAIN1 (VND1) to VND7 encode a group of NAC domain transcription factors that function as master regulators of xylem vessel element differentiation. These transcription factors activate the transcription of genes required for secondary cell wall formation and programmed cell death, key events in xylem vessel element differentiation. Because constitutive overexpression of VND6 and VND7 induces ectopic xylem vessel element differentiation, functional studies of VND proteins have largely focused on these two proteins. Here, we report the roles of VND1, VND2, and VND3 in xylem vessel formation in cotyledons. Using our newly established in vitro system in which excised Arabidopsis cotyledons are stimulated to undergo xylem cell differentiation by cytokinin, auxin, and brassinosteroid treatment, we found that ectopic xylem vessel element differentiation required VND1, VND2, and VND3 but not VND6 or VND7. The importance of VND1, VND2, and VND3 also was indicated in vivo; in the vnd1 vnd2 vnd3 seedlings, xylem vessel element differentiation of secondary veins in cotyledons was inhibited under dark conditions. Furthermore, the light responsiveness of VND gene expression was disturbed in the vnd1 vnd2 vnd3 mutant, and vnd1 vnd2 vnd3 failed to recover lateral root development in response to the change of light conditions. These findings suggest that VND1 to VND3 have specific molecular functions, possibly linking light conditions to xylem vessel formation, during seedling development.

A Missense Mutation in the NSF Gene Causes Abnormal Golgi Morphology in Arabidopsis thaliana.

The Golgi apparatus is a key station of glycosylation and membrane traffic. It consists of stacked cisternae in most eukaryotes. However, the mechanisms how the Golgi stacks are formed and maintained are still obscure. The model plant Arabidopsis thaliana provides a nice system to observe Golgi structures by light microscopy, because the Golgi in A. thaliana is in the form of mini-stacks that are distributed throughout the cytoplasm. To obtain a clue to understand the molecular basis of Golgi morphology, we took a forward-genetic approach to isolate A. thaliana mutants that show abnormal structures of the Golgi under a confocal microscope. In the present report, we describe characterization of one of such mutants, named #46-3. The #46-3 mutant showed pleiotropic Golgi phenotypes. The Golgi size was in majority smaller than the wild type, but varied from very small ones, sometimes without clear association of cis and trans cisternae, to abnormally large ones under a confocal microscope. At the ultrastructual level by electron microscopy, queer-shaped large Golgi stacks were occasionally observed. By positional mapping, genome sequencing, and complementation and allelism tests, we linked the mutant phenotype to the missense mutation D374N in the NSF gene, encoding the N-ethylmaleimide-sensitive factor (NSF), a key component of membrane fusion. This residue is near the ATP-binding site of NSF, which is very well conserved in eukaryotes, suggesting that the biochemical function of NSF is important for maintaining the normal morphology of the Golgi.Key words: Golgi morphology, N-ethylmaleimide-sensitive factor (NSF), Arabidopsis thaliana.

Identification of Arabidopsis genic and non-genic promoters by paired-end sequencing of TSS tags.

Information about transcription start sites (TSSs) provides baseline data for the analysis of promoter architecture. In this paper we used paired- and single-end deep sequencing to analyze Arabidopsis TSS tags from several libraries prepared from roots, shoots, flowers and etiolated seedlings. The clustering of approximately 33 million mapped TSS tags led to the identification of 324 461 promoters that covered 79.7% (21 672/27 206) of protein-coding genes in the Arabidopsis genome. In addition we identified intragenic, antisense and orphan promoters that were not associated with any gene models. Of these, intragenic promoters exhibited unique characteristics regarding dinucleotide sequences at TSSs and core promoter element composition, suggesting that these promoters use different mechanisms of transcriptional initiation. An analysis of base composition with regard to promoter position revealed a low GC content throughout the promoter region and several local strand biases that were evident for TATA-type promoters, but not for Coreless-type promoters. Most observed strand biases coincided with strand biases of single nucleotide polymorphism rate. Our analysis also revealed that transcription of a gene is supported by an average of 2.7 genic promoters, among which one specific promoter, designated as a top promoter, substantially determines the expression level of the gene.

Chloroplastic ATP synthase builds up a proton motive force preventing production of reactive oxygen species in photosystem I.

Over-reduction of the photosynthetic electron transport (PET) chain should be avoided, because the accumulation of reducing electron carriers produces reactive oxygen species (ROS) within photosystem I (PSI) in thylakoid membranes and causes oxidative damage to chloroplasts. To prevent production of ROS in thylakoid membranes the H+ gradient (ΔpH) needs to be built up across the thylakoid membranes to suppress the over-reduction state of the PET chain. In this study, we aimed to identify the critical component that stimulates ΔpH formation under illumination in higher plants. To do this, we screened ethyl methane sulfonate (EMS)-treated Arabidopsis thaliana, in which the formation of ΔpH is impaired and the PET chain caused over-reduction under illumination. Subsequently, we isolated an allelic mutant that carries a missense mutation in the γ-subunit of chloroplastic CF0 CF1 -ATP synthase, named hope2. We found that hope2 suppressed the formation of ΔpH during photosynthesis because of the high H+ efflux activity from the lumenal to stromal side of the thylakoid membranes via CF0 CF1 -ATP synthase. Furthermore, PSI was in a more reduced state in hope2 than in wild-type (WT) plants, and hope2 was more vulnerable to PSI photoinhibition than WT under illumination. These results suggested that chloroplastic CF0 CF1 -ATP synthase adjusts the redox state of the PET chain, especially for PSI, by modulating H+ efflux activity across the thylakoid membranes. Our findings suggest the importance of the buildup of ΔpH depending on CF0 CF1 -ATP synthase to adjust the redox state of the reaction center chlorophyll P700 in PSI and to suppress the production of ROS in PSI during photosynthesis.

Protein S-Nitrosylation Regulates Xylem Vessel Cell Differentiation in Arabidopsis.

Post-translational modifications of proteins have important roles in the regulation of protein activity. One such modification, S-nitrosylation, involves the covalent binding of nitric oxide (NO)-related species to a cysteine residue. Recent work showed that protein S-nitrosylation has crucial functions in plant development and environmental responses. In the present study, we investigated the importance of protein S-nitrosylation for xylem vessel cell differentiation using a forward genetics approach. We performed ethyl methanesulfonate mutagenesis of a transgenic Arabidopsis 35S::VND7-VP16-GR line in which the activity of VASCULAR-RELATED NAC-DOMAIN7 (VND7), a key transcription factor involved in xylem vessel cell differentiation, can be induced post-translationally by glucocorticoid treatment, with the goal of obtaining suppressor mutants that failed to differentiate ectopic xylem vessel cells; we named these mutants suppressor of ectopic vessel cell differentiation induced by VND7 (seiv) mutants. We found the seiv1 mutant to be a recessive mutant in which ectopic xylem cell differentiation was inhibited, especially in aboveground organs. In seiv1 mutants, a single nucleic acid substitution (G to A) leading to an amino acid substitution (E36K) was present in the gene encoding S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which regulates the turnover of the natural NO donor, S-nitrosoglutathione. An in vitro S-nitrosylation assay revealed that VND7 can be S-nitrosylated at Cys264 and Cys320 located near the transactivation activity-related domains, which were shown to be important for transactivation activity of VND7 by transient reporter assay. Our results suggest crucial roles for GSNOR1-regulated protein S-nitrosylation in xylem vessel cell differentiation, partly through the post-translational modification of VND7.

A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem.

The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis thaliana, several gain-of-function analyses have demonstrated that peptide ligands of the Clavata3 (CLV3)/embryo surrounding region-related (CLE) family are important for maintaining RM size. Here, we demonstrate that a plant U-box E3 ubiquitin ligase, PUB4, is a novel downstream component of CLV3/CLE signaling in the RM. Mutations in PUB4 reduced the inhibitory effect of exogenous CLV3/CLE peptide on root cell proliferation and columella stem cell maintenance. Moreover, pub4 mutants grown without exogenous CLV3/CLE peptide exhibited characteristic phenotypes in the RM, such as enhanced root growth, increased number of cortex/endodermis stem cells and decreased number of columella layers. Our phenotypic and gene expression analyses indicated that PUB4 promotes expression of a cell cycle regulatory gene, CYCD6;1, and regulates formative periclinal asymmetric cell divisions in endodermis and cortex/endodermis initial daughters. These data suggest that PUB4 functions as a global regulator of cell proliferation and the timing of asymmetric cell division that are important for final root architecture.

FT-like proteins induce transposon silencing in the shoot apex during floral induction in rice.

Floral induction is a crucial developmental step in higher plants. Florigen, a mobile floral activator that is synthesized in the leaf and transported to the shoot apex, was recently identified as a protein encoded by FLOWERING LOCUS T (FT) and its orthologs; the rice florigen is Heading date 3a (Hd3a) protein. The 14-3-3 proteins mediate the interaction of Hd3a with the transcription factor OsFD1 to form a ternary structure called the florigen activation complex on the promoter of OsMADS15, a rice APETALA1 ortholog. However, crucial information, including the spatiotemporal overlap among FT-like proteins and the components of florigen activation complex and downstream genes, remains unclear. Here, we confirm that Hd3a coexists, in the same regions of the rice shoot apex, with the other components of the florigen activation complex and its transcriptional targets. Unexpectedly, however, RNA-sequencing analysis of shoot apex from wild-type and RNA-interference plants depleted of florigen activity revealed that 4,379 transposable elements (TEs; 58% of all classifiable rice TEs) were expressed collectively in the vegetative and reproductive shoot apex. Furthermore, in the reproductive shoot apex, 214 TEs were silenced by florigen. Our results suggest a link between floral induction and regulation of TEs.

Genetic Enhancer Analysis Reveals that FLORAL ORGAN NUMBER2 and OsMADS3 Co-operatively Regulate Maintenance and Determinacy of the Flower Meristem in Rice.

Meristems such as the shoot apical meristem and flower meristem (FM) act as a reservoir of stem cells, which reproduce themselves and supply daughter cells for the differentiation of lateral organs. In Oryza sativa (rice), the FLORAL ORGAN NUMBER2 (FON2) gene, which is similar to Arabidopsis CLAVATA3, is involved in meristem maintenance. In fon2 mutants, the numbers of floral organs are increased due to an enlargement of the FM. To identify new factors regulating meristem maintenance in rice, we performed a genetic screening of mutants that enhanced the fon2 mutation, and found a mutant line (2B-424) in which pistil number was dramatically increased. By using a map-based approach and next-generation sequencing, we found that the line 2B-424 had a complete loss-of-function mutation (a large deletion) in OsMADS3, a class C MADS-box gene that is known to be involved in stamen specification. Disruption of OsMADS3 in the fon2 mutant by CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology caused a flower phenotype similar to that of 2B-424, confirming that the gene responsible for enhancement of fon2 was OsMADS3. Morphological analysis showed that the fon2 and osmads3 mutations synergistically affected pistil development and FM determinacy. We also found that whorl 3 was duplicated in mature flowers and the FM was enlarged at an early developmental stage in severe osmads3 single mutants. These findings suggest that OsMADS3 is involved not only in FM determinacy in late flower development but also in FM activity in early flower development.

WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

The Plasma Membrane H+-ATPase AHA1 Plays a Major Role in Stomatal Opening in Response to Blue Light.

Stomata open in response to a beam of weak blue light under strong red light illumination. A blue light signal is perceived by phototropins and transmitted to the plasma membrane H(+)-ATPase that drives stomatal opening. To identify the components in this pathway, we screened for mutants impaired in blue light-dependent stomatal opening. We analyzed one such mutant, provisionally named blus2 (blue light signaling2), and found that stomatal opening in leaves was impaired by 65%, although the magnitude of red light-induced opening was not affected. Blue light-dependent stomatal opening in the epidermis and H(+) pumping in guard cell protoplasts were inhibited by 70% in blus2 Whole-genome resequencing identified a mutation in the AHA1 gene of the mutant at Gly-602. T-DNA insertion mutants of AHA1 exhibited a similar phenotype to blus2; this phenotype was complemented by the AHA1 gene. We renamed blus2 as aha1-10 T-DNA insertion mutants of AHA2 and AHA5 did not show any impairment in stomatal response, although the transcript levels of AHA2 and AHA5 were higher than those of AHA1 in wild-type guard cells. Stomata in ost2, a constitutively active AHA1 mutant, did not respond to blue light. A decreased amount of H(+)-ATPase in aha1-10 accounted for the reduced stomatal blue light responses and the decrease was likely caused by proteolysis of misfolded AHA1. From these results, we conclude that AHA1 plays a major role in blue light-dependent stomatal opening in Arabidopsis and that the mutation made the AHA1 protein unstable in guard cells.

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements.

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell's metabolic activity for the biosynthesis of secondary wall polymers.

Localization of the CAPRICE-ENHANCER OF TRY AND CPC1 chimera protein in Arabidopsis root epidermis.

The CAPRICE (CPC) encodes an R3-type MYB transcription factor, which promotes root-hair differentiation. Previously, we showed that the CPC protein moves from the non-hair cell to the neighboring cell and induces root-hair differentiation in Arabidopsis. In addition, we proposed two cell-to-cell movement signal sequences, S1 and S2, in CPC. However, an S1:2xGFP:S2 chimera protein did not move between root epidermal cells. Here, we show that the S1 and S2 sequences do not confer cell-to-cell movement or nuclear localization ability to a GFP protein. The ENHANCER OF TRY AND CPC1 (ETC1) gene encodes the CPC homolog R3 MYB; this protein does not possess cell-to-cell movement ability or the S1 sequence. To elucidate whether the S1 sequence can induce cell-to-cell movement ability in ETC1, CPCp:S1:ETC1:2xGFP was constructed and introduced into Arabidopsis. Our results indicate that the addition of the S1 sequence was not sufficient for ETC1 to acquire cell-to-cell movement ability.

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.

The SAC51 Family Plays a Central Role in Thermospermine Responses in Arabidopsis.

The acaulis5 (acl5) mutant of Arabidopsis thaliana is defective in the biosynthesis of thermospermine and shows a dwarf phenotype associated with excess xylem differentiation. SAC51 was identified from a dominant suppressor of acl5, sac51-d, and encodes a basic helix-loop-helix protein. The sac51-d mutant has a premature termination codon in an upstream open reading frame (uORF) that is conserved among all four members of the SAC51 family, SAC51 and SACL1-SACL3 This suggests that thermospermine cancels the inhibitory effect of the uORF in main ORF translation. Another suppressor, sac57-d, has a mutation in the conserved uORF of SACL3 To define further the function of the SAC51 family in the thermospermine response, we analyzed T-DNA insertion mutants of each gene. Although sacl1-1 may not be a null allele, the quadruple mutant showed a semi-dwarf phenotype but with an increased level of thermospermine and decreased sensitivity to exogenous thermospermine that normally represses xylem differentiation. The sac51-1 sacl3-1 double mutant was also insensitive to thermospermine. These results suggest that SAC51 and SACL3 play a key role in thermospermine-dependent negative control of thermospermine biosynthesis and xylem differentiation. Using 5' leader-GUS (ß-glucuronidase) fusion constructs, however, we detected a significant enhancement of the GUS activity by thermospermine only in SAC51 and SACL1 constructs. Furthermore, while acl5-1 sac51-1 showed the acl5 dwarf phenotype, acl5-1 sacl3-1 exhibited an extremely tiny-plant phenotype. These results suggest a complex regulatory network for the thermospermine response in which SAC51 and SACL3 function in parallel pathways.