Five unaddressed questions about cytokinin biosynthesis.

Cytokinins, a class of phytohormones, play crucial roles in regulating plant growth and stress responses through finely tuned feedback loops involving metabolic and signaling cascades. Cytokinin metabolism modulates the abundance of these biologically active molecules. Over the past 25 years, studies have identified key genes involved in cytokinin biosynthesis and inactivation pathways. Nevertheless, several gaps remain in our understanding, particularly regarding the movement of intermediate metabolites between subcellular compartments and the discrepancy between the product of adenosine phosphate-isopentenyltransferase (IPT) and the substrate preferences of subsequent reactions. In addition, recent gene discoveries related to lonely guy (LOG)-independent pathways suggest a spatial extension of cytokinin biosynthesis into the apoplast. Other intriguing issues remain to be addressed, i.e., elucidating the synthetic pathway for cis-zeatin and unraveling the molecular mechanisms governing selective substrate use by the cytokinin biosynthetic enzyme tumor morphology root (Tmr) derived from the phytopathogen Agrobacterium tumefaciens during crown gall formation. Further studies are needed to reveal a fully comprehensive picture of cytokinin metabolism.

Autonomous differentiation of transgenic cells requiring no external hormone application: the endogenous gene expression and phytohormone behaviors.

The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.

Plant hormone profiling of scion and rootstock incision sites and intra- and inter-family graft junctions in Nicotiana benthamiana.

Many previous studies have suggested that various plant hormones play essential roles in the grafting process. In this study, to understand the plant hormones that accumulate in the graft junctions, whether these are supplied from the scion or rootstock, and how these hormones play a role in the grafting process, we performed a hormonome analysis that accumulated in the incision site of the upper plants from the incision as "ungrafted scion" and lower plants from the incision as "ungrafted rootstock" in Nicotiana benthamiana. The results revealed that indole-3-acetic acid (IAA) and gibberellic acid (GA), which regulate cell division; abscisic acid (ABA) and jasmonic acid (JA), which regulate xylem formation; cytokinin (CK), which regulates callus formation, show different accumulation patterns in the incision sites of the ungrafted scion and rootstock. In addition, to try discussing the differences in the degree and speed of each event during the grafting process between intra- and inter-family grafting by determining the concentration and accumulation timing of plant hormones in the graft junctions, we performed hormonome analysis of graft junctions of intra-family grafted plants with N. benthamiana as scion and Solanum lycopersicum as rootstock (Nb/Sl) and inter-family grafted plants with N. benthamiana as scion and Arabidopsis thaliana as rootstock (Nb/At), using the ability of Nicotiana species to graft with many plant species. The results revealed that ABA and CK showed different accumulation timings; IAA, JA, and salicylic acid (SA) showed similar accumulation timings, while different accumulated concentrations in the graft junctions of Nb/Sl and Nb/At. This information is important for understanding the molecular mechanisms of plant hormones in the grafting process and the differences in molecular mechanisms between intra- and inter-family grafting.

Photosynthetic-Product-Dependent Activation of Plasma Membrane H+-ATPase and Nitrate Uptake in Arabidopsis Leaves.

Plasma membrane (PM) proton-translocating adenosine triphosphatase (H+-ATPase) is a pivotal enzyme for plant growth and development that acts as a primary transporter and is activated by phosphorylation of the penultimate residue, threonine, at the C-terminus. Small Auxin-Up RNA family proteins maintain the phosphorylation level via inhibiting dephosphorylation of the residue by protein phosphatase 2C-D clade. Photosynthetically active radiation activates PM H+-ATPase via phosphorylation in mesophyll cells of Arabidopsis thaliana, and phosphorylation of PM H+-ATPase depends on photosynthesis and photosynthesis-related sugar supplementation, such as sucrose, fructose and glucose. However, the molecular mechanism and physiological role of photosynthesis-dependent PM H+-ATPase activation are still unknown. Analysis using sugar analogs, such as palatinose, turanose and 2-deoxy glucose, revealed that sucrose metabolites and products of glycolysis such as pyruvate induce phosphorylation of PM H+-ATPase. Transcriptome analysis showed that the novel isoform of the Small Auxin-Up RNA genes, SAUR30, is upregulated in a light- and sucrose-dependent manner. Time-course analyses of sucrose supplementation showed that the phosphorylation level of PM H+-ATPase increased within 10 min, but the expression level of SAUR30 increased later than 10 min. The results suggest that two temporal regulations may participate in the regulation of PM H+-ATPase. Interestingly, a 15NO3- uptake assay in leaves showed that light increases 15NO3- uptake and that increment of 15NO3- uptake depends on PM H+-ATPase activity. The results opened the possibility of the physiological role of photosynthesis-dependent PM H+-ATPase activation in the uptake of NO3-. We speculate that PM H+-ATPase may connect photosynthesis and nitrogen metabolism in leaves.

A Synthetic Minimal Beating Axoneme.

Cilia and flagella are beating rod-like organelles that enable the directional movement of microorganisms in fluids and fluid transport along the surface of biological organisms or inside organs. The molecular motor axonemal dynein drives their beating by interacting with microtubules. Constructing synthetic beating systems with axonemal dynein capable of mimicking ciliary beating still represents a major challenge. Here, the bottom-up engineering of a sustained beating synthoneme consisting of a pair of microtubules connected by a series of periodic arrays of approximately eight axonemal dyneins is reported. A model leads to the understanding of the motion through the cooperative, cyclic association-dissociation of the molecular motor from the microtubules. The synthoneme represents a bottom-up self-organized bio-molecular machine at the nanoscale with cilia-like properties.

Systemic Regulation of Iron Acquisition by Arabidopsis in Environments with Heterogeneous Iron Distributions.

Nutrient distribution within the soil is generally heterogeneous. Plants, therefore, have evolved sophisticated systemic processes enabling them to optimize their nutrient acquisition efficiency. By organ-to-organ communication in Arabidopsis thaliana, for instance, iron (Fe) starvation in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. This compensatory response through Fe-starvation-triggered organ-to-organ communication includes the upregulation of Iron-regulated transporter 1 (IRT1) gene expression on the Fe-sufficient side of the root; however, the molecular basis underlying this long-distance signaling remains unclear. Here, we analyzed gene expression by RNA-seq analysis of Fe-starved split-root cultures. Genome-wide expression analysis showed that localized Fe depletion in roots upregulated several genes involved in Fe uptake and signaling, such as IRT1, in a distant part of the root exposed to Fe-sufficient conditions. This result indicates that long-distance signaling for Fe demand alters the expression of a subset of genes responsible for Fe uptake and coumarin biosynthesis to maintain a level of Fe acquisition sufficient for the entire plant. Loss of IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) leads to the disruption of compensatory upregulation of IRT1 in the root surrounded by sufficient Fe. In addition, our split-root culture-based analysis provides evidence that the IMA3/FEP1-MYB10/72 pathway mediates long-distance signaling in Fe homeostasis through the regulation of coumarin biosynthesis. These data suggest that the signaling of IMA/FEP, a ubiquitous family of metal-binding peptides, is critical for organ-to-organ communication in response to Fe starvation under heterogeneous Fe conditions in the surrounding environment.

Differences in xylem development between Dutch and Japanese tomato (Solanum lycopersicum) correlate with cytokinin levels in hypocotyls.

BACKGROUND AND AIMS: Dutch tomato cultivars tend to have a greater yield than Japanese cultivars even if they are grown under the same conditions. Factors contributing to the increased yield of the Dutch cultivars were a greater light use efficiency and greater leaf photosynthetic rate. On the other hand, the relationship between tomato yields and anatomical traits is still unclear. The aim of this study is to identify the anatomical traits related to the difference in yield between Dutch and Japanese cultivars. METHODS: Anatomical properties were compared during different growth stages of Dutch and Japanese tomatoes. Hormone profiles and related gene expression in hypocotyls of Dutch and Japanese cultivars were compared in the hypocotyls of 3- and 4-week-old plants. KEY RESULTS: Dutch cultivars have a more developed secondary xylem than Japanese cultivars, which would allow for greater transport of water, mineral nutrients and phytohormones to the shoots. The areas and ratios of the xylem in the hypocotyls of 3- to 6-week-old plants were larger in the Dutch cultivars. In reciprocal grafts of the Japanese and Dutch cultivars, xylem development at the scion and rootstock depended on the scion cultivar, suggesting that some factors in the scion are responsible for the difference in xylem development. The cytokinin content, especially the level of N6-(Δ 2-isopentenyl) adenine (iP)-type cytokinin, was higher in the Dutch cultivars. This result was supported by the greater expression of Sl-IPT3 (a cytokinin biosynthesis gene) and Sl-RR16/17 (a cytokinin-responsive gene) in the Dutch cultivars. CONCLUSIONS: These results suggest that iP-type cytokinins, which are locally synthesized in the hypocotyl, contribute to xylem development. The greater xylem development in Dutch cultivars might contribute to the high yield of the tomato.

Columnar growth phenotype in apple results from gibberellin deficiency by ectopic expression of a dioxygenase gene.

The apple cultivar McIntosh Wijcik, which is a mutant of 'McIntosh', exhibits a columnar growth phenotype (short internodes, few lateral branches, many spurs, etc.) that is controlled by a dominant Co gene. The candidate gene (MdDOX-Co), encoding a 2-oxoglutarate-dependent dioxygenase, is located adjacent to an insertion mutation. Non-columnar apples express MdDOX-Co in the roots, whereas columnar apples express MdDOX-Co in the aerial parts as well as in the roots. However, the function of MdDOX-Co remains unknown. Here, we characterized tobacco plants overexpressing MdDOX-Co. The tobacco plants showed the typical dwarf phenotype, which was restored by application of gibberellin A3 (GA3). Moreover, the dwarf tobacco plants had low concentrations of endogenous bioactive gibberellin A1 (GA1) and gibberellin A4 (GA4). Similarly, 'McIntosh Wijcik' contained low endogenous GA4 concentration and its dwarf traits (short main shoot and internodes) were partially reversed by GA3 application. These results indicate that MdDOX-Co is associated with bioactive GA deficiency. Interestingly, GA3 application to apple trees also resulted in an increased number of lateral branches and a decrease in flower bud number, indicating that gibberellin (GA) plays important roles in regulating apple tree architecture by affecting both lateral branch formation (vegetative growth) and flower bud formation (reproductive growth). We propose that a deficiency of bioactive GA by ectopic expression of MdDOX-Co in the aerial parts of columnar apples not only induces dwarf phenotypes but also inhibits lateral branch development and promotes flower bud formation, and assembly of these multiple phenotypes constructs the columnar tree form.

Force-Generating Mechanism of Axonemal Dynein in Solo and Ensemble.

In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowledge and perspectives of the force-generating mechanism of axonemal dyneins in solo and in ensemble.

Sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated CO2.

Carbon availability is a major regulatory factor in plant growth and development. Cytokinins, plant hormones that play important roles in various aspects of growth and development, have been implicated in the carbon-dependent regulation of plant growth; however, the details of their involvement remain to be elucidated. Here, we report that sugar-induced cytokinin biosynthesis plays a role in growth enhancement under elevated CO2 in Arabidopsis thaliana. Growing Arabidopsis seedlings under elevated CO2 resulted in an accumulation of cytokinin precursors that preceded growth enhancement. In roots, elevated CO2 induced two genes involved in de novo cytokinin biosynthesis: an adenosine phosphate-isopentenyltransferase gene, AtIPT3, and a cytochrome P450 monooxygenase gene, CYP735A2. The expression of these genes was inhibited by a photosynthesis inhibitor, DCMU, under elevated CO2, and was enhanced by sugar supplements, indicating that photosynthetically generated sugars are responsible for the induction. Consistently, cytokinin precursor accumulation was enhanced by sugar supplements. Cytokinin biosynthetic mutants were impaired in growth enhancement under elevated CO2, demonstrating the involvement of de novo cytokinin biosynthesis for a robust growth response. We propose that plants employ a system to regulate growth in response to elevated CO2 in which photosynthetically generated sugars induce de novo cytokinin biosynthesis for growth regulation.

An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice.

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR-Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.

The roles of a flagellar HSP40 ensuring rhythmic beating.

HSP40s are regarded as cochaperones, perpetually shuttling client polypeptides to HSP70s for refolding. However, many HSP40s that are central for disparate processes diverge from this paradigm. To elucidate the noncanonical mechanisms, we investigated HSP40 in the radial spoke (RS) complex in flagella. Disruption of the gene by the MRC1 transposon in Chlamydomonas resulted in jerky flagella. Traditional electron microscopy, cryo-electron tomography, and sub-tomogram analysis revealed RSs of various altered morphologies that, unexpectedly, differed between the two RS species. This indicates that HSP40 locks the RS into a functionally rigid conformation, facilitating its interactions with the adjacent central pair apparatus for transducing locally varied mechanical feedback, which permits rhythmic beating. Missing HSP40, like missing RSs, could be restored in a tip-to-base direction when HSP40 mutants fused with a HSP40 donor cell. However, without concomitant de novo RS assembly, the repair was exceedingly slow, suggesting HSP40/RS-coupled intraflagellar trafficking and assembly. Biochemical analysis and modeling uncovered spoke HSP40's cochaperone traits. On the basis of our data, we propose that HSP40 accompanies its client RS precursor when traveling to the flagellar tip. Upon arrival, both refold in concert to assemble into the mature configuration. HSP40's roles in chaperoning and structural maintenance shed new light on its versatility and flagellar biology.

Ethylene-gibberellin signaling underlies adaptation of rice to periodic flooding.

Most plants do poorly when flooded. Certain rice varieties, known as deepwater rice, survive periodic flooding and consequent oxygen deficiency by activating internode growth of stems to keep above the water. Here, we identify the gibberellin biosynthesis gene, SD1 (SEMIDWARF1), whose loss-of-function allele catapulted the rice Green Revolution, as being responsible for submergence-induced internode elongation. When submerged, plants carrying the deepwater rice-specific SD1 haplotype amplify a signaling relay in which the SD1 gene is transcriptionally activated by an ethylene-responsive transcription factor, OsEIL1a. The SD1 protein directs increased synthesis of gibberellins, largely GA4, which promote internode elongation. Evolutionary analysis shows that the deepwater rice-specific haplotype was derived from standing variation in wild rice and selected for deepwater rice cultivation in Bangladesh.

Effects of overproduced ethylene on the contents of other phytohormones and expression of their key biosynthetic genes.

Ethylene is involved in regulation of various aspects of plant growth and development. Physiological and genetic analyses have indicated the existence of crosstalk between ethylene and other phytohormones, including auxin, cytokinin (CK), abscisic acid (ABA), gibberellin (GA), salicylic acid (SA), jasmonic acid (JA), brassinosteroid (BR) and strigolactone (SL) in regulation of different developmental processes. However, the effects of ethylene on the biosynthesis and contents of these hormones are not fully understood. Here, we investigated how overproduction of ethylene may affect the contents of other plant hormones using the ethylene-overproducing mutant ethylene-overproducer 1 (eto1-1). The contents of various hormones and transcript levels of the associated biosynthetic genes in the 10-day-old Arabidopsis eto1-1 mutant and wild-type (WT) plants were determined and compared. Higher levels of CK and ABA, while lower levels of auxin, SA and GA were observed in eto1-1 plants in comparison with WT, which was supported by the up- or down-regulation of their biosynthetic genes. Although we could not quantify the BR and SL contents in Arabidopsis, we observed that the transcript levels of the potential rate-limiting BR and SL biosynthetic genes were increased in the eto1-1 versus WT plants, suggesting that BR and SL levels might be enhanced by ethylene overproduction. JA level was not affected by overproduction of ethylene, which might be explained by unaltered expression level of the proposed rate-limiting JA biosynthetic gene allene oxide synthase. Taken together, our results suggest that ET affects the levels of auxin, CK, ABA, SA and GA, and potentially BR and SL, by influencing the expression of genes involved in the rate-limiting steps of their biosynthesis.

Systemic transport of trans-zeatin and its precursor have differing roles in Arabidopsis shoots.

Organ-to-organ signal transmission is essential for higher organisms to ensure coordinated biological reactions during metabolism and morphogenesis. Similar to organs in animals, plant organs communicate by various signalling molecules. Among them, cytokinins, a class of phytohormones, play a key role as root-to-shoot long-distance signals, regulating various growth and developmental processes in shoots1,2. Previous studies have proposed that trans-zeatin-riboside, a type of cytokinin precursor, is a major long-distance signalling form in xylem vessels and its action depends on metabolic conversion via the LONELY GUY enzyme in proximity to the site of action3-5. Here we report an additional long-distance signalling form of cytokinin: trans-zeatin, an active form. Grafting between various cytokinin biosynthetic and transportation mutants revealed that root-to-shoot translocation of trans-zeatin, a minor component of xylem cytokinin, controls leaf size but not meristem activity-related traits, whereas that of trans-zeatin riboside is sufficient for regulating both traits. Considering the ratio of trans-zeatin to trans-zeatin-riboside in xylem and their delivery rate change in response to environmental conditions, this dual long-distance cytokinin signalling system allows plants to fine-tune the manner of shoot growth to adapt to fluctuating environments.

Lack of Cytosolic Glutamine Synthetase1;2 Activity Reduces Nitrogen-Dependent Biosynthesis of Cytokinin Required for Axillary Bud Outgrowth in Rice Seedlings.

A mutation abolishing cytosolic glutamine synthetase1;2 (GS1;2) activity impairs assimilation of ammonium into glutamine in both roots and basal portions of shoots, and severely decreases axillary bud outgrowth (tillering) in mutant rice seedlings. Although the gs1;2 mutant phenotype is independent of strigolactone, which inhibits tillering, it also demonstrates glutamine- or related metabolite-responsive biosynthesis of cytokinin (CK), which promotes tillering. Here, we examined the connection between GS1;2 and CK biosynthesis during tillering, focusing on basal portions of the shoots as well as apical and axillary bud meristems in the gs1;2 mutant. Despite a sufficient ammonium supply, decreases in precursor CK contents and a decrease in ammonium assimilation into glutamine were observed in basal portions of mutant shoots. Reintroducing expression of OsGS1;2 cDNA driven by its own promoter restored precursor CK contents and ammonium assimilation to wild-type levels. In basal portions of the shoots, glutamine-responsive adenosine phosphate-isopentenyltransferase4 (OsIPT4), which is also predominant in rice roots, was the predominant isogene for IPT, which synthesizes CK. Cell-specific expression of OsIPT4 in phloem companion cells in nodal vascular anastomoses connected to the axillary bud vasculature also decreased in the gs1;2 mutant. Expression of CK-responsive type-A response regulator genes as local indicators of active CKs was also abolished in the axillary bud meristem of the mutant. These results suggest that the lack of GS1;2 activity decreased levels of glutamine or a related metabolite required for CK biosynthesis, causing a deficiency in active CK in the axillary bud meristem necessary for tillering.

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity.

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N(6)-(∆(2)-isopentenyl)adenine and dimethylated N(6)-(∆(2)-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N(6)-(∆(2)-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N(6)-(∆(2)-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.

Ethylene suppresses tomato (Solanum lycopersicum) fruit set through modification of gibberellin metabolism.

Fruit set in angiosperms marks the transition from flowering to fruit production and a commitment to seed dispersal. Studies with Solanum lycopersicum (tomato) fruit have shown that pollination and subsequent fertilization induce the biosynthesis of several hormones, including auxin and gibberellins (GAs), which stimulate fruit set. Circumstantial evidence suggests that the gaseous hormone ethylene may also influence fruit set, but this has yet to be substantiated with molecular or mechanistic data. Here, we examined fruit set at the biochemical and genetic levels, using hormone and inhibitor treatments, and mutants that affect auxin or ethylene signaling. The expression of system-1 ethylene biosynthetic genes and the production of ethylene decreased during pollination-dependent fruit set in wild-type tomato and during pollination-independent fruit set in the auxin hypersensitive mutant iaa9-3. Blocking ethylene perception in emasculated flowers, using either the ethylene-insensitive Sletr1-1 mutation or 1-methylcyclopropene (1-MCP), resulted in elongated parthenocarpic fruit and increased cell expansion, whereas simultaneous treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) inhibited parthenocarpy. Additionally, the application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to pollinated ovaries reduced fruit set. Furthermore, Sletr1-1 parthenocarpic fruits did not exhibit increased auxin accumulation, but rather had elevated levels of bioactive GAs, most likely reflecting an increase in transcripts encoding the GA-biosynthetic enzyme SlGA20ox3, as well as a reduction in the levels of transcripts encoding the GA-inactivating enzymes SlGA2ox4 and SlGA2ox5. Taken together, our results suggest that ethylene plays a role in tomato fruit set by suppressing GA metabolism.

Cell dedifferentiation and organogenesis in vitro require more snRNA than does seedling development in Arabidopsis thaliana.

Small nuclear RNA (snRNA) is a class of non-coding RNAs that processes pre-mRNA and rRNA. Transcription of abundant snRNA species is regulated by the snRNA activating protein complex (SNAPc), which is conserved among multicellular organisms including plants. SRD2, a putative subunit of SNAPc in Arabidopsis thaliana, is essential for development, and the point mutation srd2-1 causes severe defects in hypocotyl dedifferentiation and de novo meristem formation. Based on phenotypic analysis of srd2-1 mutant plants, we previously proposed that snRNA content is a limiting factor in dedifferentiation in plant cells. Here, we performed functional complementation analysis of srd2-1 using transgenic srd2-1 Arabidopsis plants harboring SRD2 homologs from Populus trichocarpa (poplar), Nicotiana tabacum (tobacco), Oryza sativa (rice), the moss Physcomitrella patens, and Homo sapiens (human) under the control of the Arabidopsis SRD2 promoter. Only rice SRD2 suppressed the faulty tissue culture responses of srd2-1, and restore the snRNA levels; however, interestingly, all SRD2 homologs except poplar SRD2 rescued the srd2-1 defects in seedling development. These findings demonstrated that cell dedifferentiation and organogenesis induced during tissue culture require higher snRNA levels than does seedling development.

CLUMSY VEIN, the Arabidopsis DEAH-box Prp16 ortholog, is required for auxin-mediated development.

Pre-messenger RNA (pre-mRNA) splicing is essential in eukaryotic cells. In animals and yeasts, the DEAH-box RNA-dependent ATPase Prp16 mediates conformational change of the spliceosome, thereby facilitating pre-mRNA splicing. In yeasts, Prp16 also plays an important role in splicing fidelity. Conversely, PRP16 orthologs in Chlamydomonas reinhardtii and nematode do not have an important role in general pre-mRNA splicing, but are required for gene silencing and sex determination, respectively. Functions of PRP16 orthologs in higher plants have not been described until now. Here we show that the CLUMSY VEIN (CUV) gene encoding the unique Prp16 ortholog in Arabidopsis thaliana facilitates auxin-mediated development including male-gametophyte transmission, apical-basal patterning of embryonic and gynoecium development, stamen development, phyllotactic flower positioning, and vascular development. cuv-1 mutation differentially affects splicing and expression of genes involved in auxin biosynthesis, polar auxin transport, auxin perception and auxin signaling. The cuv-1 mutation does not have an equal influence on pre-mRNA substrates. We propose that Arabidopsis PRP16/CUV differentially facilitates expression of genes, which include genes involved in auxin biosynthesis, transport, perception and signaling, thereby collectively influencing auxin-mediated development.