EUI1, encoding a putative cytochrome P450 monooxygenase, regulates internode elongation by modulating gibberellin responses in rice

Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice.     

Downregulation of OsPK1 Contributes to Oxidative Stress and the Variations in ABA/GA Balance in Rice

Abscisic acid (ABA) is a phytohormone that aids plants in coping with stress conditions. ABA and gibberellin (GA) are hormone partners that function via a complicated and antagonistic network. In ospk1, a dwarf rice mutant, the contents of ABA in the youngest leaf sheaths of 6-week-old seedlings and the uppermost internodes of heading stage plants were both increased, and the synthesis of bioactive GAs was suppressed, which may disharmonize ABA/GA balance. In ospk1, expression of three putative enzyme genes related to stress response was upregulated. A strong browning symptom was observed in the second internode (Int2, counted from the top) and part of panicles of ospk1 at the late productive phase. Furthermore, higher levels of H₂O₂ in flag leaf and Int2 were observed in ospk1 than those in wild type. These data suggest that ospk1 may undergo certain stress, especially oxidative stress. Here, we provide evidences that the downregulation of OsPK1 (a cytosolic pyruvate kinase) in ospk1 mutant results in variations in ABA/GA balance in rice and contributes to oxidative stress, which provide a new clue for understanding the connection of pyruvate kinase, ABA/GA balance, and oxidative stress in rice.     

The role of a class III gibberellin 2‐oxidase in tomato internode elongation

A network of environmental inputs and internal signaling controls plant growth, development and organ elongation. In particular, the growth‐promoting hormone gibberellin (GA) has been shown to play a significant role in organ elongation. The use of tomato as a model organism to study elongation presents an opportunity to study the genetic control of internode‐specific elongation in a eudicot species with a sympodial growth habit and substantial internodes that can and do respond to external stimuli. To investigate internode elongation, a mutant with an elongated hypocotyl and internodes but wild‐type petioles was identified through a forward genetic screen. In addition to stem‐specific elongation, this mutant, named tomato internode elongated ‐1 (tie‐1) is more sensitive to the GA biosynthetic inhibitor paclobutrazol and has altered levels of intermediate and bioactive GAs compared with wild‐type plants. The mutation responsible for the internode elongation phenotype was mapped to GA2oxidase 7, a class III GA 2‐oxidase in the GA biosynthetic pathway, through a bulked segregant analysis and bioinformatic pipeline, and confirmed by transgenic complementation. Furthermore, bacterially expressed recombinant TIE protein was shown to have bona fide GA 2‐oxidase activity. These results define a critical role for this gene in internode elongation and are significant because they further the understanding of the role of GA biosynthetic genes in organ‐specific elongation.     

Sites of gibberellin biosynthesis in pea seedlings

Potential sites of gibberellin biosynthesis in 10-day-old `Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [¹⁴C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.     

Rice DWARF AND LOW-TILLERING and the homeodomain protein OSH15 interact to regulate internode elongation via orchestrating brassinosteroid signaling and metabolism

Brassinosteroid (BR) phytohormones play crucial roles in regulating internode elongation in rice (Oryza sativa). However, the underlying mechanism remains largely unclear. The dwarf and low-tillering (dlt) mutant is a mild BR-signaling-defective mutant. Here, we identify two dlt enhancers that show more severe shortening of the lower internodes compared to the uppermost internode (IN1). Both mutants carry alleles of ORYZA SATIVA HOMEOBOX 15 (OSH15), the founding gene for dwarf6-type mutants, which have shortened lower internodes but not IN1. Consistent with the mutant phenotype, OSH15 expression is much stronger in lower internodes, particularly in IN2, than IN1. The osh15 single mutants have impaired BR sensitivity accompanied by enhanced BR synthesis in seedlings. DLT physically interacts with OSH15 to co-regulate many genes in seedlings and internodes. OSH15 targets and promotes the expression of the BR receptor gene BR INSENSITIVE1 (OsBRI1), and DLT facilitates this regulation in a dosage-dependent manner. In osh15, dlt, and osh15 dlt, BR levels are higher in seedlings and panicles, but unexpectedly lower in internodes compared with the wild-type. Taken together, our results suggest that DLT interacts with OSH15, which functions in the lower internodes, to modulate rice internode elongation via orchestrating BR signaling and metabolism.

DWARF AND LOW-TILLERING interacts with the homeodomain protein OSH15, which directly targets the brassinosteroid receptor gene OsBRI1 and is expressed in lower internodes, to regulate the internode elongation via modulating brassinosteroid signaling and metabolism.

IN A NUTSHELL Background: Rice culms consist of five to seven internodes and the length of these internodes determines plant height and resistance to wind, which is crucial for field performance. Brassinosteroid (BR) plant hormones are involved in regulating plant height because defects in BR synthesis or signaling (such as mutants in the BR receptor gene BRASSINOSTEROID INSENSITIVE 1 (OsBRI1)) usually result in dwarfism with specific shortening of the lower internodes or the second internode (IN2) compared to that of the uppermost/first internode (IN1). This pattern is known as d6 or dm-type dwarfism. Question: We wanted to know how BRs are involved in organizing the different internodes and therefore, we carried out a large-scale screen for mutants with altered internode organization pattern using the mild BR signaling-defective mutant dwarf and low-tillering (dlt). Findings: We identified two mutants showing specific shortening of the lower internodes, that is d6-type dwarfism. Both mutants have the same causal gene, namely, OSH15, which encodes a homeodomain-containing protein. OSH15 can directly interact with DLT, forming a protein complex to regulate BR contents and BR signaling. For example, DLT–OSH15 directly binds the promoter of OsBRI1 to promote gene expression. OSH15 expression is strong in the lower internodes, particularly in IN2, and DLT shows an opposite expression pattern. Therefore, the protein complex has different levels in different internodes, exerting different effects on BR levels and signaling to modulate internode organization. Next steps: Scientists aim to use BR-related genes to engineer plant height and grain size and thus produce new crops having improved grain yield and lodging resistance. The discovery of the DLT–OSH15–OsBRI1 module could help achieve this goal. Next, we will try to uncover how BRs coordinate internode elongation with panicle development.     

The biophysical basis of elongation growth in internodes of deepwater rice

Partial submergence induces rapid internodal elongation in deepwater rice (Oryza sativa L., cv Habiganj Aman II). We measured in vivo extensibility, tissue tension, hydraulic conductance and osmotic potential in the region of cell elongation in the uppermost internode. The in vivo extensibility of the internode, measured by stretching of living tissue with a custom-made constant stress extensiometer, rose rapidly following submergence of the plant. Both the elastic (E_el) and plastic (E_pl) extensibility increased when growth of the internode was induced. The submerged internode displayed tissue tension (elastic outward bending of longitudinally split internode sections); in air-grown control internodes, no such bending occurred. The hydraulic conductance, estimated from the kinetics of tissue shrinkage in 0.5 molar mannitol and subsequent swelling in distilled water, was not changed by submergence. The osmotic potential, measured with a dew-point hygrometer using frozen-thawed tissue, was only 18% less negative in the submerged internode than in the air-grown control. This indicates that osmoregulation takes place in rapidly elongating rice internodes. We suggest that the rapid expansion of the newly formed internodal cells of submerged plants is controlled by the yielding properties (E_pl) of the cell walls. Experiments with excised stem sections indicate that gibberellin is involved in increasing the E_pl of the elongating cell walls.     

DWARF50 (D50), a rice (Oryza sativa L.) gene encoding inositol polyphosphate 5‐phosphatase,is required for proper development of intercalary meristem

Rice internodes are vital for supporting high‐yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell‐producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5‐phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization.     

Inhibition of Tiller Bud Outgrowth in the tin Mutant of Wheat Is Associated with Precocious Internode Development

Tillering (branching) is a major yield component and, therefore, a target for improving the yield of crops. However, tillering is regulated by complex interactions of endogenous and environmental signals, and the knowledge required to achieve optimal tiller number through genetic and agronomic means is still lacking. Regulatory mechanisms may be revealed through physiological and molecular characterization of naturally occurring and induced tillering mutants in the major crops. Here we characterize a reduced tillering (tin, for tiller inhibition) mutant of wheat (Triticum aestivum). The reduced tillering in tin is due to early cessation of tiller bud outgrowth during the transition of the shoot apex from the vegetative to the reproductive stage. There was no observed difference in the development of the main stem shoot apex between tin and the wild type. However, tin initiated internode development earlier and, unlike the wild type, the basal internodes in tin were solid rather than hollow. We hypothesize that tin represents a novel type of reduced tillering mutant associated with precocious internode elongation that diverts sucrose (Suc) away from developing tillers. Consistent with this hypothesis, we have observed upregulation of a gene induced by Suc starvation, downregulation of a Suc-inducible gene, and a reduced Suc content in dormant tin buds. The increased expression of the wheat Dormancy-associated (DRM1-like) and Teosinte Branched1 (TB1-like) genes and the reduced expression of cell cycle genes also indicate bud dormancy in tin. These results highlight the significance of Suc in shoot branching and the possibility of optimizing tillering by manipulating the timing of internode elongation.     

Role of polyamines in gibberellin-induced internode growth in peas

To determine the requirement for polyamines in gibberellin (GA) induced internode growth polyamine content was measured in internodes of peas of various internode phenotypes (slender, tall, dwarf, nana) with and without applied gibberellin (GA₃) and polyamine synthesis inhibitors. Polyamines were assayed as dansyl derivatives which were separated by reverse phase high performance liquid chromatography and detected by fluorescence spectrophotometry. The amounts of polyamines in the different genetic lines of peas, which differed in internode lengths and extractable GA content, correlated with the extent of internode elongation. High polyamine concentrations were associated with young internodes and decreased with internode expansion. Extremely short internodes of nana plants without GA exhibited equal or higher amine concentrations relative to internodes of other lines of peas and GA-stimulated nana seedlings. The polyamine synthesis inhibitors, α-difluoromethylornithine and α-difluoromethylarginine, independently or in combination, inhibited polyamine accumulation and internode elongation of tall peas and GA-stimulated nana plants. Agmatine and putrescine restored growth and endogenous polyamine content to variable degrees. However, exogenous polyamines were not effective in promoting growth unless intracellular amines were partially depleted.

These results suggest that polyamines do not have a role in cell elongation, but may be required to support cell proliferation. Polyamines do not mediate the entire action of GA in internode growth of peas since GA induction of growth involves both cell division and cell elongation, whereas polyamines appear to affect cell division only.

     

Isolation of rice dwarf mutants with ectopic deposition of phenolic components including lignin in parenchyma cell walls of internodes

Rice internodes must have the proper shape to support high-yielding panicles. The shape of internodes is controlled by various factors involved in their formation, such as developmental patterns, cell division, cell elongation, and cell wall biosynthesis. To understand the regulation of internode development, we screened dwarf mutants to identify those with a phenotype of ectopic deposits of phenolic components in parenchyma cell walls of internodes. We named these mutants ectopic deposition of phenolic components1 (edp1). Two alleles were identified, edp1-1 and edp1-2. Furthermore, these mutants showed disordered cell files in internode parenchyma. These abnormal phenotypes were very similar to that of a previously reported dwarf50 (d50) mutant. Genetic analyses of edp1 mutants revealed that the edp1 loci are distinct from d50. Our results indicate that analyses of edp1 mutants as well as the d50 mutant will be useful for understanding the molecular mechanisms behind ectopic deposition of cell wall phenolic components in internode parenchyma cells and the regulation of internode development.     

Identification and characterization of SHORTENED UPPERMOST INTERNODE 1, a gene negatively regulating uppermost internode elongation in rice

In rice, the elongated internodes are derived from the vegetative shoot apical meristem (SAM), and the transition of the SAM from the vegetative to the reproductive stage induces internode elongation. In this study, we characterize two shortened uppermost internode mutants (sui1-1 and sui1-2). During the seedling and tillering stages, sui1 plants are morphologically similar to wild-type plants. However, at the heading stage, the sui1-1 mutant exhibits a shortened uppermost internode and a partly sheathed panicle, and the sui1-2 mutant shows an extremely shortened uppermost internode and a fully sheathed panicle. Gibberellin treatment results in elongation of every internode, but the shortened uppermost internode phenotype remains unaltered. Microscopic analysis indicates that cell length of sui1-1 uppermost internode exhibits decreased. Map-based cloning revealed that SUI1 is located on Chromosome 1, and encodes a putative phosphatidyl serine synthase (PSS) family protein. Searches for matches in protein databases showed that OsSUI1 contains the InterPro domain IPR004277, which is conserved in both animal and plant kingdoms. Introduction of a wild-type SUI1 gene fully rescued the mutant phenotype of sui1-1 and sui1-2, confirming the identity of the cloned gene. Consistent with these results, the SUI1-RNAi transgenic plants displayed decreased elongation of the uppermost internode. Our results suggest that SUI1 plays an important role in regulating uppermost internode length by decreasing longitudinal cell length in rice.     

Gibberellins in Diffusates from Shoots of Apple Trees

Excised shoot apices, leaves and internodes from shoots of apple trees (Malus×domestica) give off gibberellins by diffusion on agar. A methanol extract of the agar was prepared, the extract separated on thin layer plates, and the gibberellin activity estimated by means of Rumex and lettuce hypocotyl bioassays. The largest amounts of gibberellin are found in diffusates from the shoot apex, the two upper leaves and the two upper internodes. Several promotive fractions are found on the chromatograms as well as growth inhibitors. Removal of young leaves retards elongation of the internodes. Probably gibberellins produced in young leaves exercise some control over this process. The growth regulators Alar and CCC also retard internode elongation. Diffusates from shoots treated with these substances were also examined. Preliminary results suggest that the amount of diffusible gibberellins from treated shoots is not reduced.     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.     

SUI-family genes encode phosphatidylserine synthases and regulate stem development in rice

In vascular plants, the regulation of stem cell niche determines development of aerial shoot which consists of stems and lateral organs. Intercalary meristem (IM) controls internode elongation in rice and other grasses, however little attention has been paid to the underlying mechanism of stem cell maintenance. Here, we investigated the stem development in rice and showed that the Shortened Uppermost Internode 1 (SUI1) family of genes are pivotal for development of rice stems. We demonstrated that SUI-family genes regulate the development of IM for internode elongation and also the cell expansion of the panicle stem rachis in rice. The SUI-family genes encoded base-exchange types of phosphatidylserine synthases (PSSs), which possessed enzymatic activity in a yeast complementary assay. Overexpression of SUI1 and SUI2 caused outgrowths of internodes during vegetative development, and we showed that expression patterns of Oryza Sativa Homeobox 15 (OSH15) and Histone4 were impaired. Furthermore, genome-wide gene expression analysis revealed that overexpression and RNA knockdown of SUI-family genes affected downstream gene expression related to phospholipid metabolic pathways. Moreover, using Ultra-performance liquid chromatography–quadrupole time of flight-mass spectrometry, we analyzed PS contents in different genetic backgrounds of rice and showed that the quantity of very long chain fatty acids PS is affected by transgene of SUI-family genes. Our study reveals a new mechanism conveyed by the SUI1 pathway and provides evidence to link lipid metabolism with plant stem cell maintenance.     

Accelerated shoot overgrowth of rice mutant <Emphasis Type="Italic">ao-1</Emphasis> is epistatic to gibberellin-sensitive and -insensitive dwarf mutants

 A shoot overgrowth mutant of rice (Oryza sativa L.), accelerated internode overgrowth-1 (ao-1), is marked by accelerated longitudinal elongation of aerial parts and overgrowth of internodes at the vegetative stage. The physiological properties of ao-1 were similar to those of wild plants treated with a saturating level of exogenous gibberellins (GAs), except for the internode-overgrowth phenotype, which was not mimicked by GA-treated wild plants. The ao-1 mutant was less sensitive to a GA biosynthesis inhibitor, Uniconazole-P, than the wild type. Dwarf alleles of three loci, including two GA-sensitive and one GA-insensitive mutation, were introduced to produce double-mutants with ao-1, but the overgrowth phenotype was not suppressed in double-homozygous mutants. These results suggest that the overgrowth phenotype of ao-1 is caused by abolition of GA signaling rather than by GA overproduction. It is likely that a part of the shoot regulation system of ao-1 is saturated with the GA signal. As a possible model consistent with the results, we propose that AO-1 protein acts as a negative regulator in GA signal transduction. Received: August 14, 2001 / Accepted: February 8, 2002     

Internode length inPisum: Genelkc

A new single gene-recessive internode length mutant inPisum, lkc, is characterized. The internodes oflkc plants are 30–40% shorter than those of comparableLkc plants, and this is attributable to reductions in both cell length and the number of cells per internode. Dwarfism in the mutant is not due to modified gibberellin (GA) levels, as determined by gas chromatography-selected ion monitoring (GC-SIM) for GA₁ and GA₂₀, and bioassay (rice cv. Tan-ginbozu). Furthermore,lkc plants are not as responsive as the wild-type to applied GA₁. The diminished stature oflkc plants appears to result from a direct or indirect interference with the transduction of the GA₁ signal.