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Pollen exine is composed of finely-organized nexine, bacula and tectum, and is crucial for pollen viability and function. Pollen exine development involves a complicated molecular network that coordinates the interaction between pollen and tapetal cells, as well as the biosynthesis, transport and assembly of sporopollenin precursors; however, our understanding of this network is very limited. Here, we report the roles of PEM1, a member of methyl-CpG-binding domain family, in rice pollen development. PEM1 expressed constitutively and, in anthers, its expression was detectable in tapetal cells and pollen. This predicted PEM1 protein of 240 kDa had multiple epigenetic-related domains. pem1 mutants exhibited abnormal Ubisch bodies, delayed exine occurrence and, finally, defective exine, including invisible bacula, amorphous and thickened nexine and tectum layer structures, and also had the phenotype of increased anther cuticle. The mutation in PEM1 did not affect the timely degradation of tapetum. Lipidomics revealed much higher wax and cutin contents in mutant anthers than in wild-type. Accordingly, this mutation up-regulated the expression of a set of genes implicated in transcriptional repression, signaling and diverse metabolic pathways. These results indicate that PEM1 mediates Ubisch body formation and pollen exine development mainly by negatively modulating the expression of genes. Thus, the PEM1-mediated molecular network represents a route for insights into mechanisms underlying pollen development. PEM1 may be a master regulator of pollen exine development.

G-protein signaling and ubiquitin-dependent degradation are both involved in grain development in rice, but how these pathways are coordinated in regulating this process is unknown. Here, we show that Chang Li Geng 1 (CLG1), which encodes an E3 ligase, regulates grain size by targeting the Gγ protein GS3, a negative regulator of grain length, for degradation. Overexpression of CLG1 led to increased grain length, while overexpression of mutated CLG1 with changes in three conserved amino acids decreased grain length. We found that CLG1 physically interacts with and ubiquitinats GS3which is subsequently degraded through the endosome degradation pathway, leading to increased grain size. Furthermore, we identified a critical SNP in the exon 3 of CLG1 that is significantly associated with grain size variation in a core collection of cultivated rice. This SNP results in an amino acid substitution from Arg to Ser at position 163 of CLG1 that enhances the E3 ligase activity of CLG1 and thus increases rice grain size. Both the expression level of CLG1 and the SNP CLG1163S may be useful variations for manipulating grain size in rice.

Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.

Summary Coordinating stress defense and plant growth is a survival strategy for adaptation to different environments that contains a series of processes, such as, cell growth, division and differentiation. However, little is known about the coordination mechanism for protein conformation change.A cyclophilin OsCYP20‐2 with a variant interacts with SLENDER RICE1 (SLR1) and OsFSD2 in the nucleus and chloroplasts, respectively, to integrate chilling tolerance and cell elongation in rice (Oryza sativa) (FSD2, Fe-superoxide dismutase 2).Mass spectrum assay showed that OsNuCYP20‐2 localized at the nucleus (nuclear located OsCYP20‐2) was a new variant of OsCYP20‐2 that truncated 71 amino‐acid residues in N‐terminal. The loss‐of function OsCYP20‐2 mutant showed sensitivity to chilling stress with accumulation of extra reactive oxygen species (ROS). In chloroplasts, the full‐length OsCYP20‐2 promotes OsFSD2 forming homodimers which enhance its activity, eliminating the accumulation of ROS under chilling stress. However, the mutant had shorter epidermal cells in comparison with wild‐type Hwayoung (HY). In the nucleus, OsCYP20‐2 caused conformation change of SLR1 to promote its degradation for cell elongation.Our data reveal a cyclophilin with a variant with dual‐localization in chloroplasts and the nucleus, which mediate chilling tolerance and cell elongation.

The organization of microtubules into a bipolar spindle is essential for chromosome segregation. Both centrosome and chromatin-dependent spindle assembly mechanisms are well studied in mouse, Drosophila melanogaster, and Xenopus oocytes; however, the mechanism of bipolar spindle assembly in plant meiosis remains elusive. According to our observations of microtubule assembly in Oryza sativa, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum, we propose that a key step of plant bipolar spindle assembly is the correction of the multipolar spindle into a bipolar spindle at metaphase I. The multipolar spindles failed to transition into bipolar ones in OsmtopVIB with the defect in double-strand break (DSB) formation. However, bipolar spindles were normally assembled in several other mutants lacking DSB formation, such as Osspo11-1, pair2, and crc1, indicating that bipolar spindle assembly is independent of DSB formation. We further revealed that the mono-orientation of sister kinetochores was prevalent in OsmtopVIB, whereas biorientation of sister kinetochores was frequently observed in Osspo11-1, pair2, and crc1. In addition, mutations of the cohesion subunit OsREC8 resulted in biorientation of sister kinetochores as well as bipolar spindles even in the background of OsmtopVIB. Therefore, we propose that biorientation of the kinetochore is required for bipolar spindle assembly in the absence of homologous recombination.

O-GlcNAcylation and phosphorylation are two posttranslational modifications that antagonistically regulate protein function. However, the regulation of and the cross talk between these two protein modifications are poorly understood in plants. Here we investigated the role of O-GlcNAcylation during vernalization, a process whereby prolonged cold exposure promotes flowering in winter wheat (Triticum aestivum), and analyzed the dynamic profile of O-GlcNAcylated and phosphorylated proteins in response to vernalization. Altering O-GlcNAc signaling by chemical inhibitors affected the vernalization response, modifying the expression of VRN genes and subsequently affecting flowering transition. Over a vernalization time-course, O-GlcNAcylated and phosphorylated peptides were enriched from winter wheat plumules by Lectin weak affinity chromatography and iTRAQ-TiO2, respectively. Subsequent mass spectrometry and gene ontology term enrichment analysis identified 168 O-GlcNAcylated proteins that are mainly involved in responses to abiotic stimulus and hormones, metabolic processing, and gene expression; and 124 differentially expressed phosphorylated proteins that participate in translation, transcription, and metabolic processing. Of note, 31 vernalization-associated proteins were identified that carried both phosphorylation and O-GlcNAcylation modifications, of which the majority (97%) exhibited the coexisting module and the remainder exhibited the potential competitive module. Among these, TaGRP2 was decorated with dynamic O-GlcNAcylation (S87) and phosphorylation (S152) modifications, and the mutation of S87 and S152 affected the binding of TaGRP2 to the RIP3 motif of TaVRN1 in vitro. Our data suggest that a dynamic network of O-GlcNAcylation and phosphorylation at key pathway nodes regulate the vernalization response and mediate flowering in wheat.

Low temperature is one of the abiotic stressors that affect growth and productivity of rice. The plant hormone gibberellin not only regulates growth and development but is also involved in stress defense. Our rice seedling experiments demonstrated that overexpression of SLR1, a gene that encodes the rice DELLA protein, enhanced chilling tolerance. In contrast, overexpression of the active GA synthesis gene OsGA20ox1 reduced chilling tolerance, indicating that weakening GA signaling promoted plant defense against cold stress. CoIP-MS and BiFC assays showed that SLR1 physically interacted with OsGRF6. After cold treatment and recovery, the survival rates of OsGRF6-overexpression lines and an osgrf6 mutant and its complementary lines indicated that OsGRF6 is a negative regulator of chilling tolerance in rice. The yeast one-hybrid, qRT-PCR, and transactivation assays showed that both SLR1 and OsGRF6 can bind to the promoter of the active GA catabolic gene OsGA2ox1, where SLR1 promoted and OsGRF6 suppressed OsGA2ox1 expression. At normal temperature, OsGRF6 was responsible for maintaining active GA levels by inhibiting OsGA2ox1. When rice seedlings were subjected to chilling stress, the repressive effect of OsGRF6 on OsGA2ox1 was released by cold-induced SLR1, which activated OsGA2ox1 expression to decrease the active GA levels, enhancing chilling tolerance. These results suggest that OsGRF6 is an important regulator in the balance between growth and chilling tolerance in rice.

Background Grain size is one of the key agronomic traits that impact grain yield. Several regulatory pathways had been reported to participate in grain size determination via cell expansion or proliferation in rice. However, little is known about cyclophilin and spliceosome participation in grain shape regulation. Results Here, we identified OsCYP20–2, a cyclophilin that influences spliceosome assembly to determine grain length. oscyp20–2 t1, a knock out mutant of OsCYP20–2 caused by T-DNA insertion, produced shorter grains with deficient cell elongation. Through yeast two-hybrid screening and pull-down assays, OsSYF2, a pre-mRNA splicing factor, was identified as an interacting protein of OsCYP20–2. The phenotypes of transgenic lines indicated that OsSYF2 positively regulates grain length via its influence on cell expansion. Transcriptomic analysis showed that OsSYF2 controls the expression and pre-mRNA alternative splicing of genes involved in sugar metabolism. In addition, these two genes have similar effects on panicle architecture. Conclusions Taken together, OsSYF2, an interacting protein of OsCYP20–2, controls grain length and panicle architecture by regulating the alternative splicing of pre-mRNA involved in cell elongation and sugar metabolism.

Improving chilling tolerance is a major target of rice breeding. The OsMAPK3-OsbHLH002-OsTPP1 signalling pathway enhances chilling tolerance in rice: the kinase is activated by cold stress, and subsequently the transcription factor is phosphorylated by the activated kinase, triggering the expression of cold response genes. However, it is largely unknown how this pathway is suppressed in time to avoid it being in a continuously activated state. We found that a novel type 2C protein phosphatase, OsPP2C27, functions as a negative regulator of the OsMAPK3-OsbHLH002-OsTPP1 pathway. A dynamic change in OsMAPK3 activity was found during cold treatment. We show that OsPP2C27 interacts physically with and dephosphorylates OsMAPK3 in vitro and in vivo. Interestingly, OsPP2C27 can also directly dephosphorylate OsbHLH002, the target of OsMAPK3. After cold treatment, survival rates were higher in OsPP2C27-RNAi lines and a T-DNA insertion mutant, and lower in OsPP2C27-overexpression lines, compared to wild type. Moreover, expression of the OsTPP1 and OsDREBs were increased in OsPP2C27-RNAi lines and decreased in OsPP2C27-overexpression lines. These results indicate that cold-induced OsPP2C27 negatively regulates the OsMAPK3-OsbHLH002-OsTPP1 signalling pathway by directly dephosphorylating both phospho-OsMAPK3 and phospho-OsbHLH002, preventing the sustained activation of a positive pathway for cold stress and maintaining normal growth under chilling conditions.

Summary Plants modify their development to adapt to their environment, protecting themselves from detrimental conditions such as chilling stress by triggering a variety of signaling pathways; however, little is known about how plants coordinate developmental patterns and stress responses at the molecular level.Here, we demonstrate that interacting transcription factors OsMADS57 and OsTB1 directly target the defense gene OsWRKY94 and the organogenesis gene D14 to trade off the functions controlling/moderating rice tolerance to cold.Overexpression of OsMADS57 maintains rice tiller growth under chilling stress. OsMADS57 binds directly to the promoter of OsWRKY94, activating its transcription for the cold stress response, while suppressing its activity under normal temperatures. In addition, OsWRKY94 was directly targeted and suppressed by OsTB1 under both normal and chilling temperatures. However, D14 transcription was directly promoted by OsMADS57 for suppressing tillering under the chilling treatment, whereas D14 was repressed for enhancing tillering under normal condition.We demonstrated that OsMADS57 and OsTB1 conversely affect rice chilling tolerance via targeting OsWRKY94.Our findings highlight a molecular genetic mechanism coordinating organogenesis and chilling tolerance in rice, which supports and extends recent work suggesting that chilling stress environments influence organ differentiation.

Genetic improvement of plant architecture is one of the strategies for increasing the yield potential of rice (Oryza sativa). Although great progress has been made in the understanding of plant architecture regulation, the precise mechanism is still an urgent need to be revealed. Here, we report that over-expression of OsMIR396d in rice results in semidwarf and increased leaf angle, a typical phenotype of brassinosteroid (BR) enhanced mutant. OsmiR396d is involved in the interaction network of BR and gibberellin (GA) signaling. In OsMIR396d over-expression plants, BR signaling was enhanced. In contrast, both the signaling and biosynthesis of GA were impaired. BRASSINAZOLE-RESISTANT1, a core transcription activator of BR signaling, directly promoted the accumulation of OsmiR396d, which controlled BR response and GA biosynthesis by regulating the expression of different target genes respectively. GROWTH REGULATING FACTOR 6, one of OsmiR396d targets, participated in GA biosynthesis and signal transduction but was not directly involved in BR signaling. This study provides a new insight into the understanding of interaction between BR and GA from multiple levels on controlling plant architecture.

Extreme weather events can cause heat stress that decreases crop production. Recent studies have demonstrated that protein degradation and rRNA homeostasis as well as transcription factors are involved in the thermoresponse in plants. However, how RNA modifications contribute to temperature stress response in plant remains largely unknown. Herein, we identified OsNSUN2 as an RNA 5-methylcytosine (m5C) methyltransferase in rice. osnsun2 mutant displayed severe temperature- and light-dependent lesion-mimic phenotypes and heat-stress hypersensitivity. Heat stress enhanced the OsNSUN2-dependent m5C modification of mRNAs involved in photosynthesis and detoxification systems, such as β-OsLCY, OsHO2, OsPAL1, and OsGLYI4, which increased protein synthesis. Furthermore, the photosystem of osnsun2 mutant was vulnerable to high ambient temperature and failed to undergo repair under tolerable heat stress. Thus, OsNSUN2 mutation reduced photosynthesis efficiency and accumulated excessive reactive oxygen species upon heat treatment. Our findings demonstrate an important mechanism of mRNA m5C-dependent heat acclimation in rice.

Calcineurin B-like interacting protein kinases (CIPKs) play important roles via environmental stress. However, less is known how to sense the stress in molecular structure conformation level. Here, an OsCIPK7 mutant via TILLING procedure with a point mutation in the kinase domain showed increased chilling tolerance, which could be potentially used in the molecular breeding. We found that this point mutation of OsCIPK7 led to a conformational change in the activation loop of the kinase domain, subsequently with an increase of protein kinase activity, thus conferred an increased tolerance to chilling stress.

Ubiquitination is a major modifier of signaling in all eukaryotes that results in the conjugation of ubiquitin to the lysine residues of acceptor proteins. The targeted protein is then subjected to degradation by the 26S proteasome, the major protein degradation system in eukaryotes. The ubiquitin–proteasome system (UPS) greatly influences plant growth and development by modulating the activity, localization, and stability of proteins. Plants are frequently exposed to various abiotic stresses during their life cycles; they rely on proteomic plasticity achieved by the UPS to adapt to unfavorable environmental conditions. In stress signal pathways, a large number of components are modified by specific ubiquitination machinery. In this review, we highlight recent advances in understanding the roles of ubiquitination in plant responses to abiotic stresses, including salt and drought, temperature, ultraviolet (UV), and nutrient availability. The review focuses primarily on the roles of the UPS. In salt and/or drought stress signaling, a number of E3 ligases mediate the stress response in both abscisic acid (ABA)-dependent and ABA-independant pathways. The UPS-mediated regulation of several key ABA-regulated transcriptional factors, e.g. ABI3 and ABI5, has been well documented. In cold signaling, the transcription factor ICE1 is targeted by E3 ligase HOSI for proteosomal degradation. Under UV stress, CUL4-DDB1A-DDB2 E3 ligase participates in DNA excision repair, and COP1 interacts with the UVR8 mediated UV response. The UPS is also involved in the uptake, transport, and homeostasis of nutrients such as iron, phosphorus, and nitrogen. SIZ1-mediated sumoylation, a ubiquitin-like modification, is necessary for a number of processes involved in plant responses to abiotic stresses. A challenge moving forward for researchers is to define more UPS components and to characterize their functions in plant responses to stress conditions; there is particular interest in identifying the ubiquitination targets that function in specific stress signaling pathways.

Inflorescence and spikelet development determine grain yields in cereals. Although multiple genes are known to be involved in the regulation of floral organogenesis, the underlying molecular network remains unclear in cereals. Here, we report that the rice (Oryza sativa) microRNA396d (OsmiR396d) and its Os Growth Regulating Factor (OsGRF) targets, together with Os Growth Regulating Factor-Interacting Factor1 (OsGIF1), are involved in the regulation of floral organ development through the rice JMJD2 family jmjC gene 706 (OsJMJ706) and crinkly4 receptor-like kinase (OsCR4). Transgenic knockdown lines of OsGRF6, a predicted target of OsmiR396d, and overexpression lines of OsmiR396d showed similar defects in floral organ development, including open husks, long sterile lemmas, and altered floral organ morphology. These defects were almost completely rescued by overexpression of OsGRF6. OsGRF6 and its ortholog OsGRF10 were the most highly expressed OsGRF family members in young inflorescences, and the grf6/grf10 double mutant displayed abnormal florets. OsGRF6/OsGRF10 localized to the nucleus, and electrophoretic mobility shift assays revealed that both OsGRF6 and OsGRF10 bind the GA response element in the promoters of OsJMJ706 and OsCR4, which were reported to participate in the regulation of floral organ development. In addition, OsGRF6 and OsGRF10 could transactivate OsJMJ706 and OsCR4, an activity that was enhanced in the presence of OsGIF1, which can bind both OsGRF6 and OsGRF10. Together, our results suggest that OsmiR396d regulates the expression of OsGRF genes, which function with OsGIF1 in floret development through targeting of JMJ706 and OsCR4. This work thus reveals a microRNA-mediated regulation module for controlling spikelet development in rice.

Polygalacturonase (PG), one of the hydrolases responsible for cell wall pectin degradation, is involved in organ consenescence and biotic stress in plants. PG1 is composed of a catalytic subunit, PG2, and a non-catalytic PG1β subunit. OsBURP16 belongs to the PG1β-like subfamily of BURP-family genes and encodes one putative PG1β subunit precursor in rice (Oryza sativa L.). Transcription of OsBURP16 is induced by cold, salinity and drought stresses, as well as by abscisic acid (ABA) treatment. Analysis of plant survival rates, relative ion leakage rates, accumulation levels of H2O2 and water loss rates of leaves showed that overexpression of OsBURP16 enhanced sensitivity to cold, salinity and drought stresses compared with controls. Young leaves of Ubi::OsBURP16 transgenic plants showed reduced cell adhesion and increased cuticular transpiration rate. Mechanical strength measurement of Ubi::OsBURP16 plants showed that reduced force was required to break leaves as compared with wild type. Transgenic rice showed enhanced PG activity and reduced pectin content. All these results suggested that overexpression of OsBURP16 caused pectin degradation and affected cell wall integrity as well as transpiration rate, which decreased tolerance to abiotic stresses. The cell wall is a barrier against biotic and abiotic stresses. Overexpression of stress-inducible OsBURP16, the beta-subunit of polygalacturonase 1, decreases pectin contents and cell adhesion in rice. Analyses of plant survival, ion leakage, H2O2 levels, and leaf water loss showed that these effects of overexpression were accompanied by enhanced sensitivity to cold, salinity and drought compared to the wild-type. Our data therefore provide new information on links between polygalacturonase activity and abiotic stress resistance in rice.

Leaf senescence is related to the grain-filling rate and grain weight in cereals. Many components involved in senescence regulation at either the genetic or physiological level are known. However, less is known about molecular regulation mechanisms. Here, we report that OsFBK12 (an F-box protein containing a Kelch repeat motif) interacts with S-ADENOSYL-l-METHIONINE SYNTHETASE1 (SAMS1) to regulate leaf senescence and seed size as well as grain number in rice (Oryza sativa). Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays indicate that OsFBK12 interacts with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and with OsSAMS1. Biochemical and physiological data showed that OsFBK12 targets OsSAMS1 for degradation. OsFBK12-RNA interference lines and OsSAMS1 overexpression lines showed increased ethylene levels, while OsFBK12-OX lines and OsSAMS1-RNA interference plants exhibited decreased ethylene. Phenotypically, overexpression of OsFBK12 led to a delay in leaf senescence and germination and increased seed size, whereas knockdown lines of either OsFBK12 or OsSAMS1 promoted the senescence program. Our results suggest that OsFBK12 is involved in the 26S proteasome pathway by interacting with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and that it targets the substrate OsSAMS1 for degradation, triggering changes in ethylene levels for the regulation of leaf senescence and grain size. These data have potential applications in the molecular breeding of rice.

Iron (Fe) is one of the essential mineral elements for plant growth and development. Acquisition of Fe by plants is mediated by a complex network involving Fe mobilization, uptake by root cells, and transport within plants. Here, we evaluated the role of a previously clarified gene encoding a receptor-like protein from rice, OsRMC, in the regulation of Fe acquisition by comparing Fe concentration, biomass, and expression patterns of genes associated with Fe mobilization and transport in wild-type (WT) rice with those in OsRMC overexpression and RNA interference (RNAi) knockdown transgenic rice plants. Expression of OsRMC was upregulated in both shoots and roots upon exposure of WT to Fe-deficient medium. Expression levels of OsRMC were positively correlated with Fe concentration in rice plants under both Fe-sufficient and Fe-deficient conditions such that overexpression and RNAi lines had higher and lower Fe concentration in both roots and shoots than WT plants, respectively. Moreover, overexpression of OsRMC conferred greater accumulation of Fe in mature seeds under Fe-sufficient conditions. OsRMC may also play a role in regulation of Fe deficiency-induced changes in root growth, as evidenced by greater and smaller root systems of OsRMC overexpression lines and RNAi lines than WT under Fe-deficient conditions, respectively. Several Fe deficiency-responsive genes including OsDMAS1, OsNAS1, OsNAS2, OsNAAT1, OsIRT1, OsYSL15, and OsIRO2 were up- and downregulated in OsRMC-overexpressing and RNAi plants compared with WT rice plants. These novel findings highlight an important role of OsRMC played in mediation of Fe acquisition and root growth in rice, particularly under Fe-deficient conditions.

Brassinosteroids (BRs) regulate many physiological processes during plant development, including flowering. However, little is known about the components of BR signaling that mediate flowering. Here, we report that BRASSINAZOLE-RESISTANT1 (BZR1), the conformation of which is altered by a cyclophilin (CYP20-2), binds cis-elements in the FLOWERING LOCUS D (FLD) promoter to regulate flowering. Both bzr1-1D and fld-4 showed delayed flowering. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that BZR1 bound to a putative BR response cis-element and suppressed the expression of FLD. Overexpression of FLD partially rescued the late flowering of pBZR1:mBZR1(Pro234-Leu)-CFP (mx3). Yeast two-hybrid and pull-down assays demonstrated that BZR1 interacts with CYP20-2. Arabidopsis thaliana CYP20-2 had greater peptidyl-prolyl cis-trans isomerase activity than did wheat (Triticum aestivum) CYP20-2. Fourier transform infrared spectroscopy revealed conformation changes in BZR1, dependent on interaction with CYP20-2. Due to differences in activity and substrate preference between CYP20-2 proteins from wheat and Arabidopsis, At-CYP20-2-overexpressing lines showed earlier flowering, whereas Ta CYP20-2 lines flowered later. Immunoblot and chromatin immunoprecipitation assays showed that histone H3 trimethyl Lys4 and H3 acetylation levels were negatively correlated with the transcription of FLD (a putative histone demethylase) in various lines. Therefore, a conformational change of BZR1 mediated by CYP20-2 causes altered flowering through modulation of FLD expression.

The basic helix–loop–helix family of proteins, which function as transcription factors, have been intensively studied in plants and animals. However, the molecular mechanism of these factors contributing to stress tolerance is unknown. Here, we report on the overexpression of OrbHLH001 from Dongxiang wild rice (Oryza rufipogon) conferring salt tolerance in transgenic rice plants. The expression of OrbHLH001 was tissue specific, mainly in phloem tissues throughout the plant. Ion assay with the scanning ion-selective electrode technique showed that NaCl stress has a greater influence on Na+ efflux and K+ influx in OrbHLH001-overexpressed plants than the wild type. OrbHLH001 protein can induce the expression of OsAKT1 to regulate the Na+/K+ ratio in OrbHLH001-overexpressed plants by specifically binding to an E-box motif in the promoter region of OsAKT1. The mechanism may have potential use in rice molecular breeding.

Kinesin superfamily proteins are important microtubule-based motor proteins with a kinesin motor domain that is conserved among all eukaryotic organisms. They are responsible for unidirectionally transporting various cargoes, including membranous organelles, protein complexes, and mRNAs. They also play critical roles in mitosis, morphogenesis, and signal transduction. Most kinesins in plants are evolutionarily divergent from their counterparts in animals and fungi. The mitotic kinesins in the plant kinesin-5 and kinesin-14 subfamilies appear to be similar to those in fungi and animals. However, others with nonmotor sequences are unique to plants. The kinesins affect microtubule organization, organelle distribution, vesicle transport, and cellulose microfibril order. Ultimately, plant kinesins contribute directly or indirectly to cell division and cell growth in various tissues. Here, we review a novel function of kinesins with transcription activation activity in regulating gibberellin biosynthesis and cell growth. These findings will open exciting new areas of kinesin research.

Abscisic acid (ABA) regulates many aspects of plant development, including seed dormancy and germination, root growth and stomatal closure. Plant SKP1 proteins are subunits of the SCF complex E3 ligases, which regulate several phytohormone signalling pathways through protein degradation. However, little is known about SKP1 proteins participating in ABA signalling. Here, we report that the overexpression of Triticum aestivum SKP1-like 1 (TSK1) in Arabidopsis thaliana (Arabidopsis) resulted in delayed seed germination and hypersensitivity to ABA. The opening of stomatal guard cells and the transcription of several ABA-responsive genes were affected in transgenic plants. In contrast, Arabidopsis skp1-like 1 (ask1)/ask1 ASK2/ask2 seedlings exhibited reduced ABA sensitivity. Furthermore, the transcription of ASK1 and ASK2 was down-regulated in abi1-1 and abi5-1 mutants compared with that in wild type. ASK1 or ASK2 overexpression could rescue or partially rescue the ABA insensitivity of abi5-1 mutants, respectively. Our work demonstrates that SKP1 is involved in ABA signalling and that SKP1-like genes may positively regulate ABA signalling by SCF-mediated protein degradation.

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA(3)) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA(1) and real-time PCR quantitative assay analyses confirmed that the decrease of GA(1) in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice.

Plants adapt their growth and development in response to perceived salt stress. Although DELLA-dependent growth restraint is thought to be an integration of the plant's response to salt stress, little is known about how histone modification confers salt stress and, in turn, affects development. Here, we report that floral initiator Shk1 kinase binding protein1 (SKB1) and histone4 arginine3 (H4R3) symmetric dimethylation (H4R3sme2) integrate responses to plant developmental progress and salt stress. Mutation of SKB1 results in salt hypersensitivity, late flowering, and growth retardation. SKB1 associates with chromatin and thereby increases the H4R3sme2 level to suppress the transcription of FLOWERING LOCUS C (FLC) and a number of stress-responsive genes. During salt stress, the H4R3sme2 level is reduced, as a consequence of SKB1 disassociating from chromatin to induce the expression of FLC and the stress-responsive genes but increasing the methylation of small nuclear ribonucleoprotein Sm-like4 (LSM4). Splicing defects are observed in the skb1 and lsm4 mutants, which are sensitive to salt. We propose that SKB1 mediates plant development and the salt response by altering the methylation status of H4R3sme2 and LSM4 and linking transcription to pre-mRNA splicing.

With global climate change, abnormally low temperatures have affected the world's rice production. Many genes have been shown to be essential for molecular improvement of rice cold-tolerance traits. However, less is known about the molecular cellular mechanism of their response to cold stress. Here, we investigated OsRAN2 involved in regulation of cell division during cold stress in rice. Expression of OsRAN2 was increased under cold treatment, but not during salt and drought stress. The mean root mitotic index was closely related to the expression level of OsRAN2. Knockdown transgenic rice lines showed an aberrant organization of spindles during mitosis and stunted growth during development. Overexpression of OsRAN2 enhanced cold tolerance in rice. The transgenic rice overexpressing OsRAN2 showed maintained cell division, decreased proportion of cells with intranuclear tubulin and formation of a normal nuclear envelope under the cold condition. Our study suggests a mechanism for OsRAN2 in regulating cold resistance in rice by maintaining cell division through promoting the normal export of intranuclear tubulin at the end of mitosis. This insight could help improve the cold-tolerance trait in rice.

In rice (Oryza sativa), brassinosteroids (BRs) induce cell elongation at the adaxial side of the lamina joint to promote leaf bending. We identified a rice mutant (ili1-D) showing an increased lamina inclination phenotype similar to that caused by BR treatment. The ili1-D mutant overexpresses an HLH protein homologous to Arabidopsis thaliana Paclobutrazol Resistance1 (PRE1) and the human Inhibitor of DNA binding proteins. Overexpression and RNA interference suppression of ILI1 increase and reduce, respectively, rice laminar inclination, confirming a positive role of ILI1 in leaf bending. ILI1 and PRE1 interact with basic helix-loop-helix (bHLH) protein IBH1 (ILI1 binding bHLH), whose overexpression causes erect leaf in rice and dwarfism in Arabidopsis. Overexpression of ILI1 or PRE1 increases cell elongation and suppresses dwarf phenotypes caused by overexpression of IBH1 in Arabidopsis. Thus, ILI1 and PRE1 may inactivate inhibitory bHLH transcription factors through heterodimerization. BR increases the RNA levels of ILI1 and PRE1 but represses IBH1 through the transcription factor BZR1. The spatial and temporal expression patterns support roles of ILI1 in laminar joint bending and PRE1/At IBH1 in the transition from growth of young organs to growth arrest. These results demonstrate a conserved mechanism of BR regulation of plant development through a pair of antagonizing HLH/bHLH transcription factors that act downstream of BZR1 in Arabidopsis and rice.

Previously, OsRAA1, an AtFPF1 homologue gene, was found to play an important role in modulating rice root development. In the current study, OsRAA1 was overexpressed in Arabidopsis, and the transgenic plants showed early flowering and elongated hypocotyl phenotypes as compared with the wild-type under white-light conditions. The hypocotyls of transgenic lines were twice as long as those of wild-type plants under red-light conditions but were indistinguishable from those of the wild-type under blue and far-red light and darkness. In addition, the phenotypes of AtFPF1 transgenic lines were similar to those of OsRAA1 transgenic lines. These results suggested that OsRAA1/AtFPF1 protein is involved in regulating flowering time and plays an important role in the inhibition of hypocotyl elongation under continuous red light. The functions were preserved during the evolution.

Salt stress adversely affects plant growth and development. Some plants reduce the damage of high-salt stress by expressing a series of salt-responsive genes. Studies of the molecular mechanism of the salt-stress response have focused on the characterization of components involved in signal perception and transduction. In the present work, we cloned and characterized a basic helix-loop-helix (bHLH) encoding gene, OrbHLH2, from wild rice (Oryza rufipogon), which encodes a homologue protein of ICE1 in Arabidopsis. OrbHLH2 protein localized in the nucleus. Overexpression of OrbHLH2 in Arabidopsis conferred increased tolerance to salt and osmotic stress, and the stress-responsive genes DREB1A/CBF3, RD29A, COR15A and KIN1 were upregulated in transgenic plants. Abscisic acid (ABA) treatment showed a similar effect on the seed germination or transcriptional expression of stress-responsive genes in both wild type and OrbHLH2-overexpressed plants, which implies that OrbHLH2 does not depend on ABA in responding to salt stress. OrbHLH2 may function as a transcription factor and positively regulate salt-stress signals independent of ABA in Arabidopsis, which provides some useful data for improving salt tolerance in crops.

MYB transcription factors play central roles in plant responses to abiotic stresses. How stress affects development is poorly understood. Here, we show that OsMYB3R-2 functions in both stress and developmental processes in rice (Oryza sativa). Transgenic plants overexpressing OsMYB3R-2 exhibited enhanced cold tolerance. Cold treatment greatly induced the expression of OsMYB3R-2, which encodes an active transcription factor. We show that OsMYB3R-2 specifically bound to a mitosis-specific activator cis-element, (T/C)C(T/C)AACGG(T/C)(T/C)A, a conserved sequence that was found in promoters of cyclin genes such as OsCycB1;1 and OsKNOLLE2. In addition, overexpression of OsMYB3R-2 in rice led to higher transcript levels of several G2/M phase-specific genes, including OsCycB1;1, OsCycB2;1, OsCycB2;2, and OsCDC20.1, than those in OsMYB3R-2 antisense lines or wild-type plants in response to cold treatment. Flow cytometry analysis revealed an increased cell mitotic index in overexpressed transgenic lines of OsMYB3R-2 after cold treatment. Furthermore, resistance to cold stress in the transgenic plants overexpressing OsCycB1;1 was also enhanced. The level of cellular free proline was increased in the overexpressed rice lines of OsMYB3R-2 and OsCycB1;1 transgenic plants compared with wild-type plants under the cold treatment. These results suggest that OsMYB3R-2 targets OsCycB1;1 and regulates the progress of the cell cycle during chilling stress. OsCPT1, which may be involved in the dehydration-responsive element-binding factor 1A pathway, showed the same transcription pattern in response to cold as did OsCycB1;1 in transgenic rice. Therefore, a cold resistance mechanism in rice could be mediated by regulating the cell cycle, which is controlled by key genes including OsMYB3R-2.

Gibberellins (GAs) and brassinosteroids (BRs), two growth-promoting phytohormones, regulate many common physiological processes. Their interactions at the molecular level remain unclear. Here, we demonstrate that OsGSR1, a member of the GAST (GA-stimulated transcript) gene family, is induced by GA and repressed by BR. RNA interference (RNAi) transgenic rice plants with reduced OsGSR1 expression show phenotypes similar to plants deficient in BR, including short primary roots, erect leaves and reduced fertility. The OsGSR1 RNAi transgenic rice shows a reduced level of endogenous BR, and the dwarf phenotype could be rescued by the application of brassinolide. The yeast two-hybrid assay revealed that OsGSR1 interacts with DIM/DWF1, an enzyme that catalyzes the conversion from 24-methylenecholesterol to campesterol in BR biosynthesis. These results suggest that OsGSR1 activates BR synthesis by directly regulating a BR biosynthetic enzyme at the post-translational level. Furthermore, OsGSR1 RNAi plants show a reduced sensitivity to GA treatment, an increased expression of the GA biosynthetic gene OsGA20ox2, which is feedback inhibited by GA signaling, and an elevated level of endogenous GA: together, these suggest that OsGSR1 is a positive regulator of GA signaling. These results demonstrate that OsGSR1 plays important roles in both BR and GA pathways, and also mediates an interaction between the two signaling pathways.

Jasmonic acid (JA) is a well-known defence hormone, but its biological function and mechanism in rice root development are less understood. Here, we describe a JA-induced putative receptor-like protein (OsRLK, AAL87185) functioning in root development in rice. RNA in situ hybridization revealed that the gene was expressed largely in roots, and a fusion protein showed its localization on the plasma membrane. The primary roots in RNAi transgenic rice plants meandered and curled more easily than wild-type (WT) roots under JA treatment. Thus, this gene was renamed Oryza sativa root meander curling (OsRMC). The transgenic primary roots were shorter, the number of adventitious roots increased and the number of lateral roots decreased as compared to the WT. As well, the second sheath was reduced in length. Growth of both primary roots and second sheaths was sensitive to JA treatment. No significant change of JA level appeared in the roots between the transgenic rice line and WT. Expression of RSOsPR10, involved in the JA signalling pathway, was induced in transgenic rice. Western blotting revealed OsRMC induced by JA. Our results suggest that OsRMC of the DUF26 subfamily involved in JA signal transduction mediates root development and negatively regulates root curling in rice.

Lectin plays an important role in defense signaling in plants, but its function in plant growth and development is not well known. Previously, we cloned a rice (Oryza sativa L.) gene OsJAC1 encoding a mannose-binding jacalin-related lectin, and found that OsJAC1 was jasmonic acid (JA) inducible. Here we cloned the promoter of OsJAC1, and GUS activity was detected in young roots, coleoptiles, sheaths, leaves, nodes of stems, stems, rachises, pistils, stamens and lemmas of OsJAC1::GUS transgenic rice, suggesting that OsJAC1 is a constitutive expression gene in rice. Moreover, OsJAC1-overexpressed (Ubi::OsJAC1) rice showed dwarfism with shorter coleptiles resulting from the failure of cell elongation of coleoptiles. In addition, compared with coleoptiles of wild-type plants, those of OsJAC1 overexpression rice were more sensitive to JA treatment. These data revealed that, besides its roles in defense response, lectin plays an important role in rice growth and development.

Development and organogenesis in both dicot and monocot plants are highly dependent on polar auxin transport (PAT), which requires the proper asymmetric localization of both auxin influx and efflux carriers. In the model dicot plant Arabidopsis thaliana, the trafficking and localization of auxin efflux facilitators such as PIN-FORMED1 (PIN1) are mediated by GNOM, a guanine-nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, but molecular regulators of the auxin influx facilitators remain unknown. Here, we show that over-expression of OsAGAP, an ARF-GTPase-activating protein (ARF-GAP) in rice, impaired PAT and interfered with both primary and lateral root development. The lateral root phenotype could be rescued by the membrane-permeable auxin 1-naphthyl acetic acid, but not by indole 3-acetic acid (IAA) or by 2,4-dichloro-phenoxyacetic acid, which require influx facilitators to enter the cells. OsAGAP-over-expressing plants had alterations in vesicle trafficking and localization of the presumptive A. thaliana auxin-influx carrier AUX1, but not in the localization of the auxin efflux facilitators. Together, our data suggest that OsAGAP has a specific role in regulating vesicle trafficking pathways such as the auxin influx pathway, which in turn controls auxin-dependent root growth in plants.

We report here the characterization of function of a novel gene, OsSIN (Orayza sativa SHORT INTERNODES). OsSIN with no intron, encodes a 106 amino acid polypeptide and has no homology proteins in GenBank. The gene shows predominant expression in shoots and is localized in the nucleus. Constitutive expression of OsSIN resulted in dwarfism and reduced number of inferior spikelets. The functional strength was correlated with the level of transgene expression. Transgenic plants were more insensitive to endogenous gibberellic acid (GA) levels than the wild type. The rice with OsSIN overexpression was less sensitive to GA than the wild type in terms of the second leaf sheath length. Expression of OsGAI at protein level was increased in OsSIN-overexpressed rice compared with the wild type. Our results suggest that OsSIN is a new member in the GA pathway regulating rice stem development.

A predicted protein encoded by a cDNA of TaRAN1 shared homology up to 84% to the Ran family. Purified protein expressed in Escherichia coli (E. coli) retains specific GTP-binding activity. It got localized to the nucleus predominantly. The TaRAN1 transcript was expressed more abundantly in young stems and flower buds, little in old leaves in wheat. Fission yeast cells over-expressed and TaRAN1 exhibited a number of novel cell biological phenotypes reminiscent of genome integrity defects. These included elongated and multinucleated cell, G2 cell cycle delay, hypersensitivity to UV. Antisense expression of the TaRAN1 caused Schizosaccharomyces pombe (S. pombe) cells to round up, with highly condensed nucleus, and suppressed the growth rates. The number of nucleomixis and anucleate cells were increased dramatically. The results suggested that TaRAN1 protein might be involved in regulational mechanism of cell division and development and highlighted a role of the TaRAN1 in regulation of genome integrity via maintaining chromatin structures.

In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene.

Summary Improvement of chilling tolerance is a major target in rice breeding. The signaling pathways regulating chilling consist of complex networks, including key transcription factors and their targets. However, it remains largely unknown how transcription factors are activated by chilling stress. Here, we report that the transcription factor OsbHLH002/OsICE1 is phosphorylated by OsMAPK3 under chilling stress. The osbhlh002-1 knockout mutant and antisense transgenic plants showed chilling hypersensitivity, whereas OsbHLH002- overexpressing plants exhibited enhanced chilling tolerance. OsbHLH002 can directly target OsTPP1 , which encodes a key enzyme for trehalose biosynthesis. OsMAPK3 interacts with OsbHLH002 to prevent its ubiquitination by the E3 ligase OsHOS1. Under chilling stress, active OsMAPK3 phosphorylates OsbHLH002, leading to accumulation of phospho-OsbHLH002, which promotes OsTPP1 expression and increases trehalose content and resistance to chilling damage. Taken together, these results indicate that OsbHLH002 is phosphorylated by OsMAPK3, which enhances OsbHLH002 activation to its target OsTPP1 during chilling stress.

Summary Rice is sensitive to cold and can be grown only in certain climate zones. Human selection of japonica rice has extended its growth zone to regions with lower temperature, while the molecular basis of this adaptation remains unknown. Here, we identify the quantitative trait locus COLD1 that confers chilling tolerance in japonica rice. Overexpression of COLD1 jap significantly enhances chilling tolerance, whereas rice lines with deficiency or downregulation of COLD1 jap are sensitive to cold. COLD1 encodes a regulator of G-protein signaling that localizes on plasma membrane and endoplasmic reticulum (ER). It interacts with the G-protein α subunit to activate the Ca 2+ channel for sensing low temperature and to accelerate G-protein GTPase activity. We further identify that a SNP in COLD1 , SNP2, originated from Chinese Oryza rufipogon , is responsible for the ability of COLD jap/ind to confer chilling tolerance, supporting the importance of COLD1 in plant adaptation.

Vernalization, sensing of prolonged cold, is important for seasonal flowering in eudicots and monocots. While vernalization silences a repressor (FLC, MADS-box transcription factor) in eudicots, it induces an activator (TaVRN1, an AP1 clade MADS-box transcription factor) in monocots. The mechanism for TaVRN1 induction during vernalization is not well understood. Here we reveal a novel mechanism for controlling TaVRN1 mRNA accumulation in response to prolonged cold sensing in wheat. The carbohydrate-binding protein VER2, a jacalin lectin, promotes TaVRN1 upregulation by physically interacting with the RNA-binding protein TaGRP2. TaGRP2 binds to TaVRN1 pre-mRNA and inhibits TaVRN1 mRNA accumulation. The physical interaction between VER2 and TaGRP2 is controlled by TaGRP2 O-GlcNAc modification, which gradually increases during vernalization. The interaction between VER2 and O-GlcNAc-TaGRP2 reduces TaGRP2 protein accumulation in the nucleus and/or promotes TaGRP2 dissociation from TaVRN1, leading to TaVRN1 mRNA accumulation. Our data reveal a new mechanism for sensing prolonged cold in temperate cereals., In temperate cereals such as winter wheat, prolonged periods of cold induce upregulation of TaVRN1 to control the timing of seasonal flowering. Xiao et al. show TaVRN1 mRNA accumulation is regulated by O-GlcNAc modification of the RNA-binding protein TaGRP2, which increases progressively in response to cold.

Based on the enhancement of the fluorescence intensity of the complex of 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline (FCPBSQ) with Co2+ by nucleic acid in the presence of Tween 80 and in the weakly basic medium, a fluorescence method for the determination of nucleic acid was proposed. The calibration graphs for the determination of denatured calf thymus DNA (ct DNA), fish sperm DNA (fs DNA), and yeast RNA (yt RNA) were obtained in concentration ranges 0.050–4.0, 0.10–3.0, and 0.050–3.0 μg/ml with limits of detection of 0.010, 0.030, and 0.020 μg/ml, respectively. The method has been satisfactorily used for the determination of DNA in wheat cell extraction and ct DNA, fs DNA, and yt RNA in synthetic samples. Investigations on the binding mode by the Scatchard plots method suggested that both intercalation and electrostatic binding modes existed in this system.

Rice tillering is a multigenic trait that influences grain yield, but its regulation molecular module is poorly understood. Here we report that OsMADS57 interacts with OsTB1 (TEOSINTE BRANCHED1) and targets D14 (Dwarf14) to control the outgrowth of axillary buds in rice. An activation-tagged mutant osmads57-1 and OsMADS57-overexpression lines showed increased tillers, whereas OsMADS57 antisense lines had fewer tillers. OsMIR444a-overexpressing lines exhibited suppressed OsMADS57 expression and tillering. Furthermore, osmads57-1 was insensitive to strigolactone treatment to inhibit axillary bud outgrowth, and OsMADS57’s function in tillering was dependent on D14. D14 expression was downregulated in osmads57-1, but upregulated in antisense and OsMIR444a-overexpressing lines. OsMADS57 bound to the CArG motif [C(A/T)TTAAAAAG] in the promoter and directly suppressed D14 expression. Interaction of OsMADS57 with OsTB1 reduced OsMADS57 inhibition of D14 transcription. Therefore, OsMIR444a-regulated OsMADS57, together with OsTB1, target D14 to control tillering. This regulation mechanism could have important application in rice molecular breeding programs focused on high grain yield., Tillering is a multigenic complex trait that influences grain yield in cereal; however, the molecular network for its regulation remains unclear. Guo et al. show that OsMADS57, a transcription factor controlled by miR444a, interacts with OsTEOSINTE BRANCHED1 and targets DWARF14 to control tillering in rice.

Polar auxin transport, which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers, mediates various processes of plant growth and development. Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein, GNOM. However, the mediation of auxin influx carrier recycling is poorly understood. Here, we report that overexpression of OsAGAP, an ARF-GTPase-activating protein in rice, stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1. AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption. Furthermore, OsAGAP is involved in actin cytoskeletal organization, and its overexpression tends to reduce the thickness and bundling of actin filaments. Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells, and was only slightly promoted when the actin filaments were completely disrupted by Lat B. Thus, we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin -deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA3) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice.

Protein degradation by the ubiquitin-proteasome system is necessary for a normal cell cycle. As compared with knowledge of the mechanism in animals and yeast, that in plants is less known. Here we summarize research into the regulatory mechanism of protein degradation in the cell cycle in plants. Anaphase-promoting complex/cyclosome (APC), in the E3 family of enzymes, plays an important role in maintaining normal mitosis. APC activation and substrate specificity is determined by its activators, which can recognize the destruction box (D-box) in APC target proteins. Oryza sativa root architecture-associated 1 (OsRAA1) with GTP-binding activity was originally cloned from rice. Overexpression of of OsRAA1 inhibits the growth of primary roots in rice. Knockdown lines showed reduced height of seedlings because of abnormal cell division. OsRAA1 transgenic rice and fission yeast show a higher proportion of metaphase cells than that of controls, which suggests a blocked transition from metaphase to anaphase during mitosis. OsRAA1 co-localizes with spindle tubulin. It contains the D-box motif and interacts with OsRPT4 of the regulatory particle of 26S proteasome. OsRAA1 may be a cell cycle inhibitor that can be degraded by the ubiquitin-proteasome system, and its disruption is necessary for the transition from metaphase to anaphase during root growth in rice.

BACKGROUND: Although cytokinins have been known for decades to play important roles in the regulation of plant growth and development, our knowledge of the regulatory mechanism of endogenous content of specific cytokinins remains limited. METHODOLOGY/PRINCIPAL FINDINGS: Here, we characterized two SOB five-like (SOFL) genes, AtSOFL1 and AtSOFL2, in Arabidopsis (Arabidopsis thaliana) and showed that they acted redundantly in regulating specific cytokinin levels. Analysis of the translational fusion AtSOFL1:AtSOFL1-GUS and AtSOFL2:AtSOFL2-GUS indicated that AtSOFL1 and AtSOFL2 exhibited similar expression patterns. Both proteins were predominantly expressed in the vascular tissues of developing leaves, flowers and siliques, but barely detectable in roots and stems. Overexpression of either AtSOFL1 or AtSOFL2 led to increased cytokinin content and obvious corresponding mutant phenotypes for both transgenic seedlings and adult plants. In addition, overexpression and site-directed mutagenesis experiments demonstrated that the SOFL domains are necessary for AtSOFL2's overexpression phenotypes. Silencing or disrupting either AtSOFL1 or AtSOFL2 caused no obvious developmental defects. Endogenous cytokinin analysis, however, revealed that compared to the wild type control, the SOFL1-RNAi62 sofl2-1 double mutant accumulated lower levels of trans-zeatin riboside monophosphate (tZRMP) and N(6)-(Delta(2)-isopentenyl)adenosine monophosphate (iPRMP), which are biosynthetic intermediates of bioactive cytokinins. The double mutant also displayed decreased response to exogenous cytokinin in both callus-formation and inhibition-of-hypocotyl-elongation assays. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that in plants AtSOFL1 and AtSOFL2 work redundantly as positive modulators in the fine-tuning of specific cytokinin levels as well as responsiveness.

Carotenoids, a class of natural pigments found in all photosynthetic organisms, are involved in a variety of physiological processes, including coloration, photoprotection, biosynthesis of abscisic acid (ABA) and chloroplast biogenesis. Although carotenoid biosynthesis has been well studied biochemically, the genetic basis of the pathway is not well understood. Here, we report the characterization of two allelic Arabidopsis mutants, spontaneous cell death1-1 (spc1-1) and spc1-2. The weak allele spc1-1 mutant showed characteristics of bleached leaves, accumulation of superoxide and mosaic cell death. The strong mutant allele spc1-2 caused a complete arrest of plant growth and development shortly after germination, leading to a seedling-lethal phenotype. Genetic and molecular analyses indicated that SPC1 encodes a putative zeta-carotene desaturase (ZDS) in the carotenoid biosynthesis pathway. Analysis of carotenoids revealed that several major carotenoid compounds downstream of SPC1/ZDS were substantially reduced in spc1-1, suggesting that SPC1 is a functional ZDS. Consistent with the downregulated expression of CAO and PORB, the chlorophyll content was decreased in spc1-1 plants. In addition, expression of Lhcb1.1, Lhcb1.4 and RbcS was absent in spc1-2, suggesting the possible involvement of carotenoids in the plastid-to-nucleus retrograde signaling. The spc1-1 mutant also displays an ABA-deficient phenotype that can be partially rescued by the externally supplied phytohormone. These results suggest that SPC1/ZDS is essential for biosynthesis of carotenoids and plays a crucial role in plant growth and development.

We used a cDNA microarray approach to monitor the expression profile of rice (Oryza sativa) under cold stress and identified 328 cold-regulated genes. Thirteen such genes encoding MYB, homeodomain, and zinc finger proteins with unknown functions showed a significant change in expression under 72-h cold stress. Among them, OsMYB3R-2 was selected for further study. Unlike most plant R2R3 MYB transcription factors, OsMYB3R-2 has three imperfect repeats in the DNA-binding domain, the same as in animal c-MYB proteins. Expression of OsMYB3R-2 was induced by cold, drought, and salt stress. The Arabidopsis (Arabidopsis thaliana) transgenic plants overexpressing OsMYB3R-2 showed increased tolerance to cold, drought, and salt stress, and the seed germination of transgenic plants was more tolerant to abscisic acid or NaCl than that of wild type. The expression of some clod-related genes, such as dehydration-responsive element-binding protein 2A, COR15a, and RCI2A, was increased to a higher level in OsMYB3R-2-overexpressing plants than in wild type. These results suggest that OsMYB3R-2 acts as a master switch in stress tolerance.

Rice (Oryza sativa L.) plant is sensitive to chilling, particularly at early stages of seedling development. Here a novel cold-inducible gene, designated OsCOIN (Oryza sativa cold-inducible), was isolated and characterized. Results showed that OsCOIN protein, a RING finger protein, was localized in both nuclear and cytoplasm membrane. OsCOIN is expressed in all rice organs and strongly induced by low temperature, ABA, salt and drought. Over-expression of OsCOIN in transgenic rice lines significantly enhanced their tolerance to cold, salt and drought, accompanied by an up-regulation of OsP5CS expression and an increase of cellular proline level.

Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1’s function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1’s regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis.

Heterotrimeric G proteins are known to function as messengers in numerous signal transduction pathways. The null mutation of RGA (rice heterotrimeric G protein alpha subunit), which encodes the alpha subunit of heterotrimeric G protein in rice, causes severe dwarfism and reduced responsiveness to gibberellic acid in rice. However, less is known about heterotrimeric G protein in brassinosteroid (BR) signaling, one of the well-understood phytohormone pathways. In the present study, we used root elongation inhibition assay, lamina inclination assay and coleoptile elongation analysis to demonstrated reduced sensitivity of d1 mutant plants (caused by the null mutation of RGA) to 24-epibrassinolide (24-epiBL), which belongs to brassinosteroids and plays a wide variety of roles in plant growth and development. Moreover, RGA transcript level was decreased in 24-epiBL-treated seedlings in a dose-dependent manner. Our results show that RGA is involved in rice brassinosteroid response, which may be beneficial to elucidate the molecular mechanisms of G protein signaling and provide a novel perspective to understand BR signaling in higher plants.