Search

Brassinosteroids (BRs) play vital roles in the plant life cycle and synthetic BRs are widely used to increase crop yield and plant stress tolerance. Among them are 24R-methyl-epibrassinolide (24-EBL) and 24S-ethyl-28-homobrassinolide (28-HBL), which differ from brassinolide (BL, the most active BR) at the C-24 position. Although it is well known that 24-EBL is 10% active as BL, there is no consensus on the bioactivity of 28-HBL. A recent outpouring of research interest in 28-HBL on major crops accompanied with a surge of industrial-scale synthesis that produces mixtures of active (22R,23R)-28-HBL and inactive (22S,23S)-28HBL, demands a standardized assay system capable of analyzing different synthetic “28-HBL” products. In this study, the relative bioactivity of 28-HBL to BL and 24-EBL, including its capacity to induce the well-established BR responses at molecular, biochemical, and physiological levels, was systematically analyzed using the whole seedlings of the wild-type and BR-deficient mutant of Arabidopsis thaliana. These multi-level bioassays consistently showed that 28-HBL exhibits a much stronger bioactivity than 24-EBL and is almost as active as BL in rescuing the short hypocotyl phenotype of the dark-grown det2 mutant. These results are consistent with the previously established structure–activity relationship of BRs, proving that this multi-level whole seedling bioassay system could be used to analyze different batches of industrially produced 28-HBL or other BL analogs to ensure the full potential of BRs in modern agriculture.

Unfolded protein response (UPR) is a conserved signaling pathway that is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER) and stimulates production of ER chaperones to restore ER proteostasis. However, little is known how UPR-induced proteins return to their pre-stress levels upon removal of ER stress. TUNICAMYCIN-INDUCED1 (TIN1) is an Arabidopsis protein that is normally expressed in pollen but is rapidly induced by ER stresses in vegetative tissues. Here we show that the ER stress-induced TIN1 is rapidly degraded in the UPR recovery phase. We found that TIN1 degradation depends on its asparagine-linked glycans and requires both EMS-mutagenized bri1 suppressor 5 (EBS5) and EBS6 for its recruitment to the ER-associated degradation (ERAD) complex. Loss-of-function mutations in Arabidopsis ERAD components greatly stabilize TIN1. Interestingly, two other UPR-induced proteins that are coexpressed with TIN1 remained stable upon removal of ER stress, suggesting that rapid degradation during the stress-recovery phase likely applies to a subset of UPR-induced proteins. Further investigation should uncover the mechanisms by which the ERAD machinery differentially recognizes UPR-induced ER proteins.

Protein-targeting technologies represent essential approaches in biological research. Protein knockdown tools developed recently in mammalian cells by exploiting natural degradation mechanisms allow for precise determination of protein function and discovery of degrader-type drugs. However, no method to directly target endogenous proteins for degradation is currently available in plants. Here, we describe a novel method for targeted protein clearance by engineering an autophagy receptor with a binder to provide target specificity and an ATG8-binding motif (AIM) to link the targets to nascent autophagosomes, thus harnessing the autophagy machinery for degradation. We demonstrate its specificity and broad potentials by degrading various fluorescence-tagged proteins, including cytosolic mCherry, the nucleus-localized bZIP transcription factor TGA5, and the plasma membrane-anchored brassinosteroid receptor BRI1, as well as fluorescence-coated peroxisomes, using a tobacco-based transient expression system. Stable expression of AIM-based autophagy receptors in Arabidopsis further confirms the feasibility of this approach in selective autophagy of endogenous proteins. With its wide substrate scope and its specificity, our concept of engineered AIM-based selective autophagy could provide a convenient and robust research tool for manipulating endogenous proteins in plants and may open an avenue toward degradation of cytoplasmic components other than proteins in plant research.

Endoplasmic reticulum-associated degradation (ERAD) is a unique mechanism to degrade misfolded proteins via complexes containing several highly-conserved ER-anchored ubiquitin ligases such as HMG-CoA reductase degradation1 (Hrd1). Arabidopsis has a similar Hrd1-containing ERAD machinery; however, our knowledge of this complex is limited. Here we report two closely-related Arabidopsis proteins, Protein Associated With Hrd1-1 (PAWH1) and PAWH2, which share a conserved domain with yeast Altered Inheritance of Mitochondria24. PAWH1 and PAWH2 localize to the ER membrane and associate with Hrd1 via EMS-mutagenized Bri1 Suppressor7 (EBS7), a plant-specific component of the Hrd1 complex. Simultaneously elimination of two PAWHs constitutively activates the unfolded protein response and compromises stress tolerance. Importantly, the pawh1 pawh2 double mutation reduces the protein abundance of EBS7 and Hrd1 and inhibits degradation of several ERAD substrates. Our study not only discovers additional plant-specific components of the Arabidopsis Hrd1 complex but also reveals a distinct mechanism for regulating the Hrd1 stability., Endoplasmic reticulum (ER)-associated degradation (ERAD) removes misfolded proteins from the secretory pathway. Here the authors identify two plant-specific proteins in Arabidopsis, PAWH1 and PAWH2, that bind to and stabilise the ER-anchored ubiquitin ligase Hrd1.

BRASSINOSTEROID-INSENSITIVE1 (BRI1) is a leucine-rich-repeat receptor-like kinase that functions as the cell surface receptor for brassinosteroids (BRs). Previous studies showed that BRI1 requires its kinase activity to transduce the extracellular BR signal into the nucleus. Among the many reported mutant bri1 alleles, bri1-301 is unique, as its glycine-989-to-isoleucine mutation completely inhibits its kinase activity in vitro but only gives rise to a weak dwarf phenotype compared with strong or null bri1 alleles, raising the question of whether kinase activity is essential for the biological function of BRI1. Here, we show that the Arabidopsis (Arabidopsis thaliana) bri1-301 mutant receptor exhibits weak BR-triggered phosphorylation in vivo and absolutely requires its kinase activity for the limited growth that occurs in the bri1-301 mutant. We also show that bri1-301 is a temperature-sensitive misfolded protein that is rapidly degraded in the endoplasmic reticulum and at the plasma membrane by yet unknown mechanisms. A temperature increase from 22°C to 29°C reduced the protein stability and biochemical activity of bri1-301, likely due to temperature-enhanced protein misfolding. The bri1-301 protein could be used as a model to study the degradation machinery for misfolded membrane proteins with cytosolic structural lesions and the plasma membrane-associated protein quality-control mechanism.

To adapt to constantly changing environmental conditions, plants have evolved sophisticated tolerance mechanisms to integrate various stress signals and to coordinate plant growth and development. It is well known that inter-organellar communications play important roles in maintaining cellular homeostasis in response to environmental stresses. The endoplasmic reticulum (ER), extending throughout the cytoplasm of eukaryotic cells, is a central organelle involved in lipid metabolism, Ca2+ homeostasis, and synthesis and folding of secretory and transmembrane proteins crucial to perceive and transduce environmental signals. The ER communicates with the nucleus via the highly conserved unfolded protein response pathway to mitigate ER stress. Importantly, recent studies have revealed that the dynamic ER network physically interacts with other intracellular organelles and endomembrane compartments, such as the Golgi complex, mitochondria, chloroplast, peroxisome, vacuole, and the plasma membrane, through multiple membrane contact sites between closely apposed organelles. In this review, we will discuss the signaling and metabolite exchanges between the ER and other organelles during abiotic stress responses in plants as well as the ER-organelle membrane contact sites and their associated tethering complexes.

Grain size is an important agronomic trait affecting grain yield, but the underlying molecular mechanisms remain to be elucidated. Here, we isolated a dominant mutant, big grain3 (bg3-D), which exhibits a remarkable increase of grain size caused by activation of the PURINE PERMEASE gene, OsPUP4. BG3/OsPUP4 is predominantly expressed in vascular tissues and is specifically suppressed by exogenous cytokinin application. Hormone profiling revealed that the distribution of different cytokinin forms, in roots and shoots of the bg3-D mutant, is altered. Quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that expression of rice cytokinin type-A RESPONSE REGULATOR (OsRR) genes is enhanced in the roots of the bg3-D mutant. These results suggest that OsPUP4 might contribute to the long-distance transport of cytokinin, by reinforcing cytokinin loading into vascular bundle cells. Furthermore, plants overexpressing OsPUP7, the closest homolog of OsPUP4, also exhibited a similar phenotype to the bg3-D mutant. Interestingly, subcellular localization demonstrated that OsPUP4 was localized on the plasma membrane, whereas OsPUP7 was localized to the endoplasmic reticulum. Based on these findings, we propose that OsPUP4 and OsPUP7 function in a linear pathway to direct cytokinin cell-to-cell transport, affecting both its long-distance movement and local allocation.

To ensure high crop yields in a sustainable manner, a comprehensive understanding of the control of nutrient acquisition is required. In particular, the signalling networks controlling the coordinated utilization of the two most highly demanded mineral nutrients, nitrogen and phosphorus, are of utmost importance. Here, we reveal a mechanism by which nitrate activates both phosphate and nitrate utilization in rice (Oryza sativa L.). We show that the nitrate sensor NRT1.1B interacts with a phosphate signalling repressor SPX4. Nitrate perception strengthens the NRT1.1B-SPX4 interaction and promotes the ubiquitination and degradation of SPX4 by recruiting NRT1.1B interacting protein 1 (NBIP1), an E3 ubiquitin ligase. This in turn allows the key transcription factor of phosphate signalling, PHR2, to translocate to the nucleus and initiate the transcription of phosphorus utilization genes. Interestingly, the central transcription factor of nitrate signalling, NLP3, is also under the control of SPX4. Thus, nitrate-triggered degradation of SPX4 activates both phosphate- and nitrate-responsive genes, implementing the coordinated utilization of nitrogen and phosphorus.

Due to an error in combining the figure, an incorrect version of Fig. 9e was presented in the original publication.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The bZIP transcription factor (TF) family plays an important role in the abscisic acid (ABA) signaling pathway of abiotic stress in plants. We here report the cloning and characterization of OsbZIP71, which encodes a rice bZIP TF. Functional analysis showed that OsbZIP71 is a nuclear-localized protein that specifically binds to the G-box motif, but has no transcriptional activity both in yeast and rice protoplasts. In yeast two-hybrid assays, OsbZIP71 can form both homodimers and heterodimers with Group C members of the bZIP gene family. Expression of OsbZIP71 was strongly induced by drought, polyethylene glycol (PEG), and ABA treatments, but repressed by salt treatment. OsbZIP71 overexpressing (p35S::OsbZIP71) rice significantly improved tolerance to drought, salt and PEG osmotic stresses. In contrast, RNAi knockdown transgenic lines were much more sensitive to salt, PEG osmotic stresses, and also ABA treatment. Inducible expression (RD29A::OsbZIP71) lines were significantly improved their tolerance to PEG osmotic stresses, but hypersensitivity to salt, and insensitivity to ABA. Real-time PCR analysis revealed that the abiotic stress-related genes, OsVHA-B, OsNHX1, COR413-TM1, and OsMyb4, were up-regulated in overexpressing lines, while these same genes were down-regulated in RNAi lines. Chromatin immunoprecipitation analysis confirmed that OsbZIP71 directly binds the promoters of OsNHX1 and COR413-TM1 in vivo. These results suggest that OsbZIP71 may play an important role in ABA-mediated drought and salt tolerance in rice.

In this study, we characterized the semi-dominant mutant nls1-1D (necrotic leaf sheath 1) of rice, which displays spontaneous lesions, specifically on leaf sheaths, with a developmental pattern. nls1-1D plants also exhibited constitutively activated defense responses, including extensive cell death, excess hydrogen peroxide and salicylic acid (SA) accumulation, up-regulated expressions of pathogenesis-related genes, and enhanced resistance to bacterial pathogens. Map-based cloning revealed that NLS1 encodes a typical CC-NB-LRR-type protein in rice. The nls1-1D mutation causes a S367N substitution in the non-conserved region close to the GLPL motif of the NB domain. An adjacent S366T substitution was found in another semi-dominant mutant, nls1-2D, which exhibited the same phenotypes as nls1-1D. Combined analyses of wild-type plants transformed with the mutant NLS1 gene (nls1-1D), NLS1 RNAi and over-expression transgenic lines showed that nls1-2D is allelic to nls1-1D, and both mutations may cause constitutive auto-activation of the NLS1 R protein. Further real-time PCR analysis revealed that NLS1 is expressed constitutively in an age-dependent manner. In addition, because the morphology and constitutive defense responses of nls1-1D were not suppressed by blocking SA or NPR1 transcript accumulation, we suggest that NLS1 mediates both SA and NPR1-independent defense signaling pathways in rice.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is an essential part of an ER-localized protein quality-control system for eliminating terminally misfolded proteins. Recent studies have demonstrated that the ERAD machinery is conserved among yeast, animals, and plants; however, it remains unknown if the plant ERAD system involves plant-specific components. Here we report that the Arabidopsis ethyl methanesulfonate-mutagenized brassinosteroid-insensitive 1 suppressor 7 (EBS7) gene encodes an ER membrane-localized ERAD component that is highly conserved in land plants. Loss-of-function ebs7 mutations prevent ERAD of brassinosteroid insensitive 1-9 (bri1-9) and bri1-5, two ER-retained mutant variants of the cell-surface receptor for brassinosteroids (BRs). As a result, the two mutant receptors accumulate in the ER and consequently leak to the plasma membrane, resulting in the restoration of BR sensitivity and phenotypic suppression of the bri1-9 and bri1-5 mutants. EBS7 accumulates under ER stress, and its mutations lead to hypersensitivity to ER and salt stresses. EBS7 interacts with the ER membrane-anchored ubiquitin ligase Arabidopsis thaliana HMG-CoA reductase degradation 1a (AtHrd1a), one of the central components of the Arabidopsis ERAD machinery, and an ebs7 mutation destabilizes AtHrd1a to reduce polyubiquitination of bri1-9. Taken together, our results uncover a plant-specific component of a plant ERAD pathway and also suggest its likely biochemical function.

Grain size is one of the key factors determining grain yield. However, it remains largely unknown how grain size is regulated by developmental signals. Here, we report the identification and characterization of a dominant mutant big grain1 (Bg1-D) that shows an extra-large grain phenotype from our rice T-DNA insertion population. Overexpression of BG1 leads to significantly increased grain size, and the severe lines exhibit obviously perturbed gravitropism. In addition, the mutant has increased sensitivities to both auxin and N-1-naphthylphthalamic acid, an auxin transport inhibitor, whereas knockdown of BG1 results in decreased sensitivities and smaller grains. Moreover, BG1 is specifically induced by auxin treatment, preferentially expresses in the vascular tissue of culms and young panicles, and encodes a novel membrane-localized protein, strongly suggesting its role in regulating auxin transport. Consistent with this finding, the mutant has increased auxin basipetal transport and altered auxin distribution, whereas the knockdown plants have decreased auxin transport. Manipulation of BG1 in both rice and Arabidopsis can enhance plant biomass, seed weight, and yield. Taking these data together, we identify a novel positive regulator of auxin response and transport in a crop plant and demonstrate its role in regulating grain size, thus illuminating a new strategy to improve plant productivity.

Given the continuously growing population and decreasing arable land, food shortage is becoming one of the most serious global problems in this century1. Grain size is one of the determining factors for grain yield and thus is a prime target for genetic breeding2,3. Although a number of quantitative trait loci (QTLs) associated with rice grain size have been identified in the past decade, mechanisms underlying their functions remain largely unknown4,5. Here we show that a grain-length-associated QTL, GL2, has the potential to improve grain weight and grain yield up to 27.1% and 16.6%, respectively. We also show that GL2 is allelic to OsGRF4 and that it contains mutations in the miR396 targeting sequence. Because of the mutation, GL2 has a moderately increased expression level, which consequently activates brassinosteroid responses by upregulating a large number of brassinosteroid-induced genes to promote grain development. Furthermore, we found that GSK2, the central negative regulator of rice brassinosteroid signalling, directly interacts with OsGRF4 and inhibits its transcription activation activity to mediate the specific regulation of grain length by the hormone. Thus, this work demonstrates the feasibility of modulating specific brassinosteroid responses to improve plant productivity. To understand the mechanisms of grain size control, researchers experimentally reveal that the molecular module miR396/GRF4 regulates rice grain size by activating brassinosteroid signalling. Modulating miR396/GRF4 or brassinosteroid responses can thus be used to improve crop yield.

Asian cultivated rice (Oryza sativa L.) consists of two main subspecies, indica and japonica. Indica has higher nitrate-absorption activity than japonica, but the molecular mechanisms underlying that activity remain elusive. Here we show that variation in a nitrate-transporter gene, NRT1.1B (OsNPF6.5), may contribute to this divergence in nitrate use. Phylogenetic analysis revealed that NRT1.1B diverges between indica and japonica. NRT1.1B-indica variation was associated with enhanced nitrate uptake and root-to-shoot transport and upregulated expression of nitrate-responsive genes. The selection signature of NRT1.1B-indica suggests that nitrate-use divergence occurred during rice domestication. Notably, field tests with near-isogenic and transgenic lines confirmed that the japonica variety carrying the NRT1.1B-indica allele had significantly improved grain yield and nitrogen-use efficiency (NUE) compared to the variety without that allele. Our results show that variation in NRT1.1B largely explains nitrate-use divergence between indica and japonica and that NRT1.1B-indica can potentially improve the NUE of japonica.

Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA1 levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana.

It has long been established that premature leaf senescence negatively impacts the yield stability of rice, but the underlying molecular mechanism driving this relationship remains largely unknown. Here, we identified a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). PS1 encodes a plant-specific NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2) transcriptional activator, Oryza sativa NAC-like, activated by apetala3/pistillata (OsNAP). Overexpression of OsNAP significantly promoted senescence, whereas knockdown of OsNAP produced a marked delay of senescence, confirming the role of this gene in the development of rice senescence. OsNAP expression was tightly linked with the onset of leaf senescence in an age-dependent manner. Similarly, ChIP-PCR and yeast one-hybrid assays demonstrated that OsNAP positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and nutrient transport and other genes associated with senescence, suggesting that OsNAP is an ideal marker of senescence onset in rice. Further analysis determined that OsNAP is induced specifically by abscisic acid (ABA), whereas its expression is repressed in both aba1 and aba2, two ABA biosynthetic mutants. Moreover, ABA content is reduced significantly in ps1-D mutants, indicating a feedback repression of OsNAP on ABA biosynthesis. Our data suggest that OsNAP serves as an important link between ABA and leaf senescence. Additionally, reduced OsNAP expression leads to delayed leaf senescence and an extended grain-filling period, resulting in a 6.3% and 10.3% increase in the grain yield of two independent representative RNAi lines, respectively. Thus, fine-tuning OsNAP expression should be a useful strategy for improving rice yield in the future.

Chromatin modifications affect flowering time in the long-day plant Arabidopsis thaliana, but the role of histone methylation in flowering time regulation of rice (Oryza sativa), a short-day plant, remains to be elucidated. We identified a late-flowering long vegetative phase1 (lvp1) mutant in rice and used map-based cloning to reveal that lvp1 affects the SET domain group protein 724 (SDG724). SDG724 functions as a histone methyltransferase in vitro and contributes to a major fraction of global histone H3 lysine 36 (H3K36) methylation in vivo. Expression analyses of flowering time genes in wild-type and lvp1 mutants revealed that Early heading date1, but not Heading date1, are misregulated in lvp1 mutants. In addition, the double mutant of lvp1 with photoperiod sensitivity5 (se5) flowered later than the se5 single mutant, indicating that lvp1 delays flowering time irrespective of photoperiod. Chromatin immunoprecipitation assays showed that lvp1 had reduced levels of H3K36me2/3 at MADS50 and RFT1. This suggests that the divergent functions of paralogs RFT1 and Hd3a, and of MADS50 and MADS51, are in part due to differential H3K36me2/3 deposition, which also correlates with higher expression levels of MADS50 and RFT1 in flowering promotion in rice.

In Arabidopsis thaliana, the GSK3/SHAGGY-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2) plays a critical role in the brassinosteroid (BR) signaling pathway by negatively regulating the activities of bri1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 family transcription factors that regulate the expression of downstream BR-responsive genes. In this study, we analyzed the function of a rice (Oryza sativa) GSK3/SHAGGY-like kinase (GSK2), which is one of the orthologs of BIN2. Overexpression of GSK2 (Go) led to plants with typical BR loss-of-function phenotypes, and suppression of GSK2 resulted in enhanced BR signaling phenotypes. DWARF AND LOW-TILLERING (DLT) is a positive regulator that mediates several BR responses in rice. Suppression of DLT can enhance the phenotypes of BR receptor mutant d61-1, and overexpression of DLT obviously suppressed the BR loss-of-function phenotypes of both d61-1 and Go, suggesting that DLT functions downstream of GSK2 to modulate BR responses. Indeed, GSK2 can interact with DLT and phosphorylate DLT. Moreover, brassinolide treatment can induce the dephosphorylation of DLT, leading to the accumulation of dephosphorylated DLT protein. In GSK2 transgenic plants, the DLT phosphorylation level is dictated by the GSK2 level. These results demonstrate that DLT is a GSK2 substrate, further reinforcing that the BIN2/GSK2 kinase has multiple substrates that carry out various BR responses.

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H2O2) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H2O2-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H2O2-induced leaf cell death in rice.

Photosynthesis is the final determinator for crop yield. To gain insight into genes controlling photosynthetic capacity, we selected from our large T-DNA mutant population a rice stunted growth mutant with decreased carbon assimilate and yield production named photoassimilate defective1 (phd1). Molecular and biochemical analyses revealed that PHD1 encodes a novel chloroplast-localized UDP-glucose epimerase (UGE), which is conserved in the plant kingdom. The chloroplast localization of PHD1 was confirmed by immunoblots, immunocytochemistry, and UGE activity in isolated chloroplasts, which was approximately 50% lower in the phd1-1 mutant than in the wild type. In addition, the amounts of UDP-glucose and UDP-galactose substrates in chloroplasts were significantly higher and lower, respectively, indicating that PHD1 was responsible for a major part of UGE activity in plastids. The relative amount of monogalactosyldiacylglycerol (MGDG), a major chloroplast membrane galactolipid, was decreased in the mutant, while the digalactosyldiacylglycerol (DGDG) amount was not significantly altered, suggesting that PHD1 participates mainly in UDP-galactose supply for MGDG biosynthesis in chloroplasts. The phd1 mutant showed decreased chlorophyll content, photosynthetic activity, and altered chloroplast ultrastructure, suggesting that a correct amount of galactoglycerolipids and the ratio of glycolipids versus phospholipids are necessary for proper chloroplast function. Downregulated expression of starch biosynthesis genes and upregulated expression of sucrose cleavage genes might be a result of reduced photosynthetic activity and account for the decreased starch and sucrose levels seen in phd1 leaves. PHD1 overexpression increased photosynthetic efficiency, biomass, and grain production, suggesting that PHD1 plays an important role in supplying sufficient galactolipids to thylakoid membranes for proper chloroplast biogenesis and photosynthetic activity. These findings will be useful for improving crop yields and for bioenergy crop engineering., Author Summary Photosynthesis is carried out in chloroplast, a plant-specific organelle. Photosynthetic membranes in chloroplasts contain high levels of glycolipids, and UDP-galactose is a dominating donor for glycolipid biosynthesis. Although glycolipid assembly of photosynthetic membranes has been characterized at the genetic and enzymatic level, the mechanism of substrate supply of UDP-galactose for the glycolipid biosynthetic pathway remains obscure. By genetic screening of rice mutants that are impaired in photosynthetic capacity and carbon assimilation, we identified PHD1 as a novel nucleotide sugar epimerase involved in a process of glycolipid biosynthesis and participating in photosynthetic membrane biogenesis. PHD1 was preferentially expressed in green and meristem tissues, and the PHD1 protein was targeted to chloroplasts. We revealed that UDP-galactose for glycolipid biosynthesis catalyzed by the new enzyme was generated inside chloroplasts, and the reduced amounts of glycolipids in the mutant led to decreased chlorophyll content and photosynthetic activity. Overexpression of this gene lead to growth acceleration, enhanced photosynthetic efficiency, and finally improved biomass and grain yield in rice. These results suggest that PHD1 has significant economic implications in both traditional crop improvement and bioenergy crop production.

In this study, we characterized the semi-dominant mutant nls1-1D (necrotic leaf sheath 1) of rice, which displays spontaneous lesions, specifically on leaf sheaths, with a developmental pattern. nls1-1D plants also exhibited constitutively activated defense responses, including extensive cell death, excess hydrogen peroxide and salicylic acid (SA) accumulation, up-regulated expressions of pathogenesis-related genes, and enhanced resistance to bacterial pathogens. Map-based cloning revealed that NLS1 encodes a typical CC-NB-LRR-type protein in rice. The nls1-1D mutation causes a S367N substitution in the non-conserved region close to the GLPL motif of the NB domain. An adjacent S366T substitution was found in another semi-dominant mutant, nls1-2D, which exhibited the same phenotypes as nls1-1D. Combined analyses of wild-type plants transformed with the mutant NLS1 gene (nls1-1D), NLS1 RNAi and over-expression transgenic lines showed that nls1-2D is allelic to nls1-1D, and both mutations may cause constitutive auto-activation of the NLS1 R protein. Further real-time PCR analysis revealed that NLS1 is expressed constitutively in an age-dependent manner. In addition, because the morphology and constitutive defense responses of nls1-1D were not suppressed by blocking SA or NPR1 transcript accumulation, we suggest that NLS1 mediates both SA and NPR1-independent defense signaling pathways in rice.

Although phosphate (Pi) starvation signaling is well studied in Arabidopsis (Arabidopsis thaliana), it is still largely unknown in rice (Oryza sativa). In this work, a rice leaf tip necrosis1 (ltn1) mutant was identified and characterized. Map-based cloning identified LTN1 as LOC_Os05g48390, the putative ortholog of Arabidopsis PHO2, which plays important roles in Pi starvation signaling. Analysis of transgenic plants harboring a LTN1 promoter::β-glucuronidase construct revealed that LTN1 was preferentially expressed in vascular tissues. The ltn1 mutant exhibited increased Pi uptake and translocation, which led to Pi overaccumulation in shoots. In association with enhanced Pi uptake and transport, some Pi transporters were up-regulated in the ltn1 mutant in the presence of sufficient Pi. Furthermore, the elongation of primary and adventitious roots was enhanced in the ltn1 mutant under Pi starvation, suggesting that LTN1 is involved in Pi-dependent root architecture alteration. Under Pi-sufficient conditions, typical Pi starvation responses such as stimulation of phosphatase and RNase activities, lipid composition alteration, nitrogen assimilation repression, and increased metal uptake were also activated in ltn1. Moreover, analysis of OsmiR399-overexpressing plants showed that LTN1 was down-regulated by OsmiR399. Our results strongly indicate that LTN1 is a crucial Pi starvation signaling component downstream of miR399 involved in the regulation of multiple Pi starvation responses in rice.

Lesion mimic is necrotic lesions on plant leaf or stem in the absence of pathogenic infection, and its exact biological mechanism is varied. By a large-scale screening of our T-DNA mutant population, we identified a mutant rice lesion initiation 1 (rlin1), which was controlled by a single nuclear recessive gene. Map-based cloning revealed that RLIN1 encoded a putative coproporphyrinogen III oxidase in tetrapyrrole biosynthesis pathway. Sequencing results showed that a G to T substitution occurred in the second exon of RLIN1 and led to a missense mutation from Asp to Tyr. Ectopic expression of RLIN1 could rescue rlin1 lesion mimic phenotype. Histochemical analysis demonstrated that lesion formation in rlin1 was light-dependent accompanied by reactive oxygen species accumulated. These results suggest that tetrapyrrole participates in lesion formation in rice.