Short grain1 decreases organ elongation and brassinosteroid response in rice

We identified a short-grain mutant (Short grain1 (Sg1) Dominant) via phenotypic screening of 13,000 rice (Oryza sativa) activation-tagged lines. The causative gene, SG1, encodes a protein with unknown function that is preferentially expressed in roots and developing panicles. Overexpression of SG1 in rice produced a phenotype with short grains and dwarfing reminiscent of brassinosteroid (BR)-deficient mutants, with wide, dark-green, and erect leaves. However, the endogenous BR level in the SG1 overexpressor (SG1:OX) plants was comparable to the wild type. SG1:OX plants were insensitive to brassinolide in the lamina inclination assay. Therefore, SG1 appears to decrease responses to BRs. Despite shorter organs in the SG1:OX plants, their cell size was not decreased in the SG1:OX plants. Therefore, SG1 decreases organ elongation by decreasing cell proliferation. In contrast to the SG1:OX plants, RNA interference knockdown plants that down-regulated SG1 and a related gene, SG1-LIKE PROTEIN1, had longer grains and internodes in rachis branches than in the wild type. Taken together, these results suggest that SG1 decreases responses to BRs and elongation of organs such as seeds and the internodes of rachis branches through decreased cellular proliferation.     

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.     

Vacuolar proton pumps and aquaporins involved in rapid internode elongation of deepwater rice

Rapid growth of the submerged shoots of deepwater rice is essential for survival during the rainy season. We investigated changes in the expression of vacuolar H(+)-ATPase (V-ATPase), H(+)-pyrophosphatase (V-PPase), and aquaporins under submerged conditions. The amounts of vacuolar proton pumps, which support the active transport of ions into the vacuoles, were maintained on a membrane protein basis in the developing vacuoles. Among the six isogenes of V-PPase, OsVHP1;3 was markedly enhanced by submersion. The gene expression of efficient water channels, OsTIP1;1, OsTIP2;2, OsPIP1;1, OsPIP2;1, and OsPIP2;2, was markedly enhanced by submersion. The increase in aquaporin expression might support quick elongation of internodes. The mRNA levels of OsNIP2;2 and OsNIP3;1, which transport silicic and boric acids respectively, clearly decreased. The present study indicates that internodes of deepwater rice upregulate vacuolar proton pumps and water channel aquaporins and downregulate aquaporins that allow permeation of the substrates that suppress internode growth.     

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.     

Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization

Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes.     

Temperature-dependent internode elongation in vegetative plants of Arabidopsis thaliana lacking phytochrome B and cryptochrome 1

 Vegetative plants of Arabidopsis thaliana (L.) Heynh. form a compact rosette of leaves in which internode growth is virtually arrested. Rapid extension of the internodes occurs after flower buds are present in the reproductive apex. Under natural radiation, continuous light from fluorescent lamps, or short photoperiods of light from fluorescent lamps, plants of the phyB cry1 double mutant (lacking both phytochrome B and cryptochrome 1) did not form normal rosettes because all the internodes showed some degree of elongation. Internode elongation was weak in the phyB single mutant and absent in the cry1 mutant, indicating redundancy between phytochrome B and cryptochrome 1. The absence of phytochrome A caused no effects. The failure to form normal rosettes was conditional because internode elongation was arrested at low temperatures in all the mutant combinations. In contrast, the temperature dependence of phytochrome B and cryptochrome 1 effects on hypocotyl growth was weak. The elongation of the internodes in phyB cry1 was not accompanied by early flowering as showed by the lack of effects on the final number of leaves. Apex dissection indicated that in phyB cry1 double mutants internode elongation anticipated the transition from the vegetative to the reproductive stage. Thus, stem growth in Arabidopsis thaliana is not fully dependent on the program of reproductive development. Received: 2 June 1999 / Accepted: 13 August 1999     

Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P - 3, P - 4, and P + 5 (relative to the phosphorylated Ser at P = 0).     

Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of noncoding RNAs involved in developmental regulation, genome maintenance, and defense in eukaryotes. The activity of Dicer or Dicer-like (DCL) proteins is required for the maturation of miRNAs and siRNAs. In this study, we cloned and sequenced 66 candidate rice (Oryza sativa) miRNAs out of 1,650 small RNA sequences (19 to approximately 25 nt), and they could be further grouped into 21 families, 12 of which are newly identified and three of which, OsmiR528, OsmiR529, and OsmiR530, have been confirmed by northern blot. To study the function of rice DCL proteins (OsDCLs) in the biogenesis of miRNAs and siRNAs, we searched genome databases and identified four OsDCLs. An RNA interference approach was applied to knock down two OsDCLs, OsDCL1 and OsDCL4, respectively. Strong loss of function of OsDCL1IR transformants that expressed inverted repeats of OsDCL1 resulted in developmental arrest at the seedling stage, and weak loss of function of OsDCL1IR transformants caused pleiotropic developmental defects. Moreover, all miRNAs tested were greatly reduced in OsDCL1IR but not OsDCL4IR transformants, indicating that OsDCL1 plays a critical role in miRNA processing in rice. In contrast, the production of siRNA from transgenic inverted repeats and endogenous CentO regions were not affected in either OsDCL1IR or OsDCL4IR transformants, suggesting that the production of miRNAs and siRNAs is via distinct OsDCLs.     

A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

The microtubules (MTs) of higher plant cells are organized into arrays with essential functions in plant cell growth and differentiation; however, molecular mechanisms underlying the organization and regulation of these arrays remain largely unknown. We have approached this problem using tubulin affinity chromatography to isolate carrot proteins that interact with MTs. From these proteins, a 50-kD polypeptide was selectively purified by exploiting its Ca(2+)-dependent binding to calmodulin (CaM). This polypeptide was identified as a homolog of elongation factor-1 alpha (EF-1 alpha)--a highly conserved and ubiquitous protein translation factor. The carrot EF-1 alpha homolog bundles MTs in vitro, and moreover, this bundling is modulated by the addition of Ca2+ and CaM together (Ca2+/CaM). A direct binding between the EF-1 alpha homolog and MTs was demonstrated, providing novel evidence for such an interaction. Based on these findings, and others discussed herein, we propose that an EF-1 alpha homolog mediates the lateral association of MTs in plant cells by a Ca2+/CaM-sensitive mechanism.     

Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis

We have isolated a new recessive dwarf mutant of rice (Oryza sativa L. cv Nipponbare). Under normal growth conditions, the mutant has very short leaf sheaths; has short, curled, and frizzled leaf blades; has few tillers; and is sterile. Longitudinal sections of the leaf sheaths revealed that the cell length along the longitudinal axis is reduced, which explains the short leaf sheaths. Transverse sections of the leaf blades revealed enlargement of the motor cells along the dorsal-ventral axis, which explains the curled and frizzled leaf blades. In addition, the number of crown roots was smaller and the growth of branch roots was weaker than those in the wild-type plant. Because exogenously supplied brassinolide considerably restored the normal phenotypes, we designated the mutant brassinosteroid-dependent 1 (brd1). Further, under darkness, brd1 showed constitutive photomorphogenesis. Quantitative analyses of endogenous sterols and brassinosteroids (BRs) indicated that BR-6-oxidase, a BR biosynthesis enzyme, would be defective. In fact, a 0.2-kb deletion was detected in the genomic region of OsBR6ox (a rice BR-6-oxidase gene) in the brd1 mutant. These results indicate that BRs are involved in many morphological and physiological processes in rice, including the elongation and unrolling of leaves, development of tillers, skotomorphogenesis, root differentiation, and reproductive growth, and that the defect of BR-6-oxidase caused the brd1 phenotype.     

Barley morphology, genetics and hormonal regulation of internode elongation modelled by a relational growth grammar

A multiscaled ecophysiological model of barley (Hordeum vulgare) development is presented here. The model is based on the new formalism of relational growth grammars (RGG), an extension of L-systems, and implemented using the new modelling language XL. It is executable in the interactive modelling platform GroIMP. The model consists of a set of morphogenetic rules, combined with a metabolic regulatory network, which simulates the biosynthesis of gibberellic acid (GA1). GA1 and two of its metabolic precursors are transported along the developing simulated structure. Local concentrations of GA1 determine internode elongation. Furthermore, virtual barley individuals are chosen interactively from a population, based on genotype, and (sexual or asexual) reproduction is simulated. Genotype and phenotype of the population are visualized. Seven Mendelian genes have been implemented in the model so far; some of these directly influence the GA-regulation network. The model exemplifies and validates the new formalism and modelling language. RGG have the capability to represent genetic, metabolic and morphological aspects of plant development and reproduction, all within the same framework.     

Anatomical analysis of growth and developmental patterns in the internode of deepwater rice

Submergence of the stem induces rapid internodal elongation in deepwater rice (Oryza sativa L. cv. Habiganj Aman II). A comparative anatomical study of internodes isolated from airgrown and partially submerged rice plants was undertaken to localize and characterize regions of growth and differentiation in rice stems. Longitudinal sections were examined by light and scanning-electron microscopy. Based on cell-size analysis, three zones of internodal development were recognized: a zone of cell division and elongation at the base of the internode, designated the intercalary meristem (IM); a zone of cell elongation without concomitant cell division; and a zone of cell differentiation where neither cell division nor elongation occur. The primary effects of submergence on internodal development were a threefold increase in the number of cells per cell file resulting from a decrease in the cell-cycle time from 24 to 7 h within the IM; an expansion of the cell-elongation zone from 5 to 15 mm leading to a threefold greater final cell length; and a suppression of tissue differentiation as indicated by reduced chlorophyll content and a lack of secondary wall formation in xylem and cortical sclerenchyma. These data indicate that growth of deepwater-rice internoes involves a balance between elongation and differentiation of the stem. Submergence shifts this balance in favor of growth.Abbreviations GA gibberellin - IM intercalary meristem     

The Rice brassinosteroid-deficient dwarf2 mutant, defective in the rice homolog of Arabidopsis DIMINUTO/DWARF1, is rescued by the endogenously accumulated alternative bioactive brassinosteroid, dolichosterone

We have identified a rice (Oryza sativa) brassinosteroid (BR)-deficient mutant, BR-deficient dwarf2 (brd2). The brd2 locus contains a single base deletion in the coding region of Dim/dwf1, a homolog of Arabidopsis thaliana DIMINUTO/DWARF1 (DIM/DWF1). Introduction of the wild-type Dim/dwf1 gene into brd2 restored the normal phenotype. Overproduction and repression of Dim/dwf1 resulted in contrasting phenotypes, with repressors mimicking the brd2 phenotype and overproducers having large stature with increased numbers of flowers and seeds. Although brd2 contains low levels of common 6-oxo-type BRs, the severity of the brd2 phenotype is much milder than brd1 mutants and most similar to d2 and d11, which show a semidwarf phenotype at the young seedling stage. Quantitative analysis suggested that in brd2, the 24-methylene BR biosynthesis pathway is activated and the uncommon BR, dolichosterone (DS), is produced. DS enhances the rice lamina joint bending angle, rescues the brd1 dwarf phenotype, and inhibits root elongation, indicating that DS is a bioactive BR in rice. Based on these observations, we discuss an alternative BR biosynthetic pathway that produces DS when Dim/dwf1 is defective.     

Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.     

Morphological alteration caused by brassinosteroid insensitivity increases the biomass and grain production of rice

The rice (Oryza sativa) dwarf mutant d61 phenotype is caused by loss of function of a rice BRASSINOSTEROID INSENSITIVE1 ortholog, OsBRI1. We have identified nine d61 alleles, the weakest of which, d61-7, confers agronomically important traits such as semidwarf stature and erect leaves. Because erect-leaf habit is considered to increase light capture for photosynthesis, we compared the biomass and grain production of wild-type and d61-7 rice. The biomass of wild type was 38% higher than that of d61-7 at harvest under conventional planting density because of the dwarfism of d61-7. However, the biomass of d61-7 was 35% higher than that of wild type at high planting density. The grain yield of wild type reached a maximum at middensity, but the yield of d61-7 continued to increase with planting density. These results indicate that d61-7 produces biomass more effectively than wild type, and consequently more effectively assimilates the biomass in reproductive organ development at high planting density. However, the small grain size of d61-7 counters any increase in grain yield, leading to the same grain yield as that of wild type even at high density. We therefore produced transgenic rice with partial suppression of endogenous OsBRI1 expression to obtain the erect-leaf phenotype without grain changes. The estimated grain yield of these transformants was about 30% higher than that of wild type at high density. These results demonstrate the feasibility of generating erect-leaf plants by modifying the expression of the brassinosteroid receptor gene in transgenic rice plants.     

Expression and function of the equine herpesvirus 1 virion-associated host shutoff homolog.

The ability of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of the UL41 gene product, the virion-associated host shutoff (vhs) protein. To date, UL41 homologs have been identified in the genomes of three other alphaherpesviruses: equine herpesvirus 1 (EHV-1), varicella-zoster virus, and pseudorabies virus, but very little is known about the putative products of these homologous genes. Our earlier observations that no rapid early host protein shutoff occurred in EHV-1-infected cells led us to test EHV-1 vhs activity more thoroughly and to examine the expression and function of the EHV-1 UL41 homolog, ORF19. In the present study, the effects of EHV-1 and HSV-1 infections on cellular protein synthesis and mRNA degradation were compared at various multiplicities of infection in several cell types under an actinomycin D block. No virion-associated inhibition of cellular protein synthesis or vhs-induced cellular mRNA degradation was detected in cells infected with any of three EHV-1 strains (Ab4, KyA, and KyD) at multiplicities of infection at which HSV-1 strain F exhibited maximal vhs activity. However, further analyses revealed that (i) the EHV-1 vhs homolog gene, ORF19, was transcribed and translated into a 58-kDa protein in infected cells; (ii) the ORF19 protein was packaged into viral particles in amounts detectable in Western blots (immunoblots) with monoclonal antibodies; (iii) in cotransfection vhs activity assays, transiently-expressed ORF19 protein had intrinsic vhs activity comparable to that of wild-type HSV-1 vhs; and (iv) this intrinsic vhs activity was ablated by in vitro site-directed mutations in which either the functionally inactive HSV-1 vhs1 UL41 mutation (Thr at position 214 replaced by Ile [Thr-214-->Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 192-->Phe). From these results we conclude that EHV-1's low vhs activity in infected cells is not a reflection of the ORF19 protein's intrinsic vhs activity but may be due instead to the amount of ORF19 protein associated with viral particles or to modulation of ORF19 protein's intrinsic activity by another viral component(s).