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The stay-green mutant of Arabidopsis thaliana, ore10 forms stable light-harvesting complex II (LHCII) aggregates during dark-induced senescence, which showed a single base deletion (G1351) in the coding region of the pheophytinase (PPH) gene. PPH specifically dephytylates the Mg-free chlorophyll (Chl) pigment pheophytin, yielding pheophorbide. In both ore10 and pph-1 mutants, pheophytin a accumulated due to the deficiency of PPH gene, but the amount was relatively smaller than that of degraded Chl, and most of the pheophytin a was bound to the stable LHCII forming aggregates with some other Chl-protein (CP) complexes. Comparison of Chl a/b ratios in thylakoids, aggregates, and LHCII indicated that the suppression of Chl b to Chl a conversion was stronger when Chl b reductase was missing and weak when PPH is missing in the large Chl catabolic complex, which allowed the partial degradation of Chl b. These results suggest that the PPH-dependent pathway is not specific for LHCII, but common for all CP complexes, including LHCII. In PPH-deficient mutants, the degradation of LHCII was suppressed by the formation of aggregates, and some of the remaining CP complexes and pheophytin a were included in the aggregates. Non-included CP complexes were degraded via an unknown mechanism.

The previously published version of this Article contained errors in Figure 5. In panel c, the second and fourth blot images were incorrectly labeled ‘α-Myc’ and should have been labelled ‘α-HA’. These errors have been corrected in both the PDF and HTML versions of the Article.

Background Rice zebra mutants are leaf variegation mutants that exhibit transverse sectors of green/yellow or green/white in developing or mature leaves. In most cases, leaf variegation is caused by defects in chloroplast biogenesis pathways, leading to an accumulation of reactive oxygen species in a transverse pattern in the leaves. Here, we examine a new type of leaf variegation mutant in rice, zebra3 (z3), which exhibits transverse dark-green/green sectors in mature leaves and lacks the typical yellow or white sectors. Results Map-based cloning revealed that the Z3 locus encodes a putative citrate transporter that belongs to the citrate-metal hydrogen symport (CitMHS) family. CitMHS family members have been extensively studied in bacteria and function as secondary transporters that can transport metal-citrate complexes, but whether CitMHS family transporters exist in eukaryotes remains unknown. To investigate whether Z3 acts as a citrate transporter in rice, we measured citrate levels in wild-type leaves and in the dark-green and green sectors of the leaves of z3 mutants. The results showed that citrates accumulated to high levels in the dark-green sectors of z3 mutant leaves, but not in the green sectors as compared with the wild-type leaves. Conclusions These results suggest that leaf variegation in the z3 mutant is caused by an unbalanced accumulation of citrate in a transverse pattern in the leaves. Taking these results together, we propose that Z3 plays an important role in citrate transport and distribution during leaf development and is a possible candidate for a CitMHS family member in plants. Electronic supplementary material The online version of this article (10.1186/s12284-017-0196-8) contains supplementary material, which is available to authorized users.

In Arabidopsis thaliana, CONSTANS (CO) plays an essential role in the regulation of photoperiodic flowering under long-day conditions. CO protein is stable only in the afternoon of long days, when it induces the expression of FLOWERING LOCUS T (FT), which promotes flowering. The blue-light photoreceptor FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1) interacts with CO and stabilizes it by an unknown mechanism. Here, we provide genetic and biochemical evidence that FKF1 inhibits CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1)-dependent CO degradation. Light-activated FKF1 has no apparent effect on COP1 stability but can interact with and negatively regulate COP1. We show that FKF1 can inhibit COP1 homo-dimerization. Mutation of the coiled-coil domain in COP1, which prevents dimer formation, impairs COP1 function in coordinating flowering time. Based on these results, we propose a model whereby the light- and day length-dependent interaction between FKF1 and COP1 controls CO stability to regulate flowering time., CONSTANS promotes flowering under long-day conditions in Arabidopsis but is rapidly degraded in short-day conditions. Here the authors show that the blue-light photoreceptor FKF1 can interact with the E3 ligase COP1 in a light-dependent manner and prevent degradation of CO in long-day conditions.

To improve our understanding of the molecular–genetic mechanisms governing leaf and root development in rice (Oryza sativa), we investigated narrow leaf1 (nal1), a pleiotropic mutant with short and narrow leaves, semi-dwarf stature, and fewer adventitious (or crown) roots. The narrow leaf5 (nal5) and nal1 mutants display similar defects in leaf and root development. Sequence analysis and complementation tests showed that nal5 is allelic to nal1. NAL1 encodes a putative trypsin-like serine/cysteine protease; the coding region of nal5 contains a missense mutation and nal1 harbors a 30-bp deletion. Quantitative real-time PCR revealed that nal1 mutants have altered expression levels of many OSHB, YABBY, and PIN-FORMED genes associated with leaf development and auxin transport. In addition, expression levels of CROWN ROOTLESS genes are markedly down-regulated in nal1. These results indicate that NAL1 functions in the regulation of both leaf and adventitious root development at the transcriptional level. Notably, exogenous auxin treatment rescued the reduced number of adventitious roots in nal1. Based on our results, we propose that NAL1 plays important roles in adventitious root development in rice.

In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. It was recently reported that ZEBRA2 encodes carotenoid isomerase (CRTISO) and that low levels of lutein, an essential carotenoid for non-photochemical quenching, cause leaf variegation in z2 mutants. However, we found that the z2 mutant phenotype was completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants under CL. In addition, three types of reactive oxygen species (ROS; superoxide [O₂⁻], hydrogen peroxide [H₂O₂], and singlet oxygen [¹O₂]) accumulated to high levels in z2 mutants grown under short-day conditions (SD; alternate 10-h light/14-h dark; restrictive), but do not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than normal in both permissive CL and restrictive SD growth conditions, indicating that deficiency of these two carotenoids is not responsible for the leaf variegation phenotype. We found that the CRTISO substrate tetra-Cis-lycopene accumulated during the dark periods under SD, but not under CL conditions. Its accumulation was also positively correlated with ¹O₂ levels generated during the light period, which consequently altered the expression of ¹O₂-responsive and cell death-related genes in the variegated z2 leaves. Taking these results together, we propose that the z2 leaf variegation can be largely attributed to photoperiodic accumulation of tetra-cis-lycopene and generation of excessive ¹O₂ under natural day-night conditions.

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most serious diseases in wheat (Triticum aestivum) and barley (Hordeum vulgare). Dahongmil is an elite Korean wheat cultivar with relatively high resistance to FHB. To identify differentially expressed genes in the resistant cultivar Dahongmil and the susceptible cultivar Urimil after inoculation of F. graminearum, we used the Affymetrix GeneChip® Wheat Genome Array to identify 328 ESTs that were differentially expressed in inoculated seedling tissues of the two cultivars. From these, we selected 16 induced genes and found that they have defense functions, such as genes encoding pathogen resistance proteins, oxidative stress-related proteins, metabolism, and proteins involved in defense mechanisms. To verify the DNA microarray results, we tested seven of these genes by semiquantitative RT-PCR and confirmed that these defense- and stress-related genes were expressed at much higher levels in the resistant Dahongmil cultivar. We next developed a hypothetical functional gene network and identified 89 interaction pairs mediated by four of the differentially expressed genes in the hypothetical network. We further refined the network by identifying nine genes showing significant up- or down-regulation after FHB challenge in the resistant cultivar and two genes having multiple interactions with queried proteins. We hope that the set of induced genes identified in this study can be used for development of new wheat and barley cultivars with improved resistance to FHB.

Tillering in rice (Oryza sativa L.) is an important agronomic trait that enhances grain production. A tiller is a specialized grain-bearing branch that is formed on a non-elongated basal internode that grows independently of the mother stem. Transgenic rice over-expressing the transcription factorOsTB1, a homologue of maizeTB1 (Teosinte Branched 1), exhibits markedly reduced lateral branching without the propagation of axillary buds being affected. However, the tillering mechanism remains unknown. Therefore, to further understand that mechanism, we applied proteomics methodology to isolate the proteins involved. Using two-dimensional gel electrophoresis and mass spectrometry, our analysis of the basal nodes from two rice cultivars that differ in their numbers of tillers showed that a rice serine proteinase inhibitor, OsSerpin, accumulates in great amounts in high-tillering ‘Hwachung’ rice. Northern blot analysis revealed that much moreOsSerpin transcript is found in ‘Hwachung’ than in relatively low-tillering ‘Hanmaeum’, likely because of high levels of transcription. Therefore, our data suggest that OsSerpin content determines the extent of lateral branching.

Arabidopsis flowers early under long days (LD) and late under short days (SD).The repressor of photomorphogenesis DE-ETIOLATED1 (DET1) delaysflowering; det1-1 mutants flower early, especially under SD, but themolecular mechanism of DET1 regulation remains unknown. Here we examine theregulatory function of DET1 in repression of flowering. Under SD, the det1-1mutation causes daytime expression of FKF1 and CO; however, theiraltered expression has only a small effect on early flowering in det1-1mutants. Notably, DET1 interacts with GI and binding of GI to the FT promoterincreases in det1-1 mutants, suggesting that DET1 mainly restricts GIfunction, directly promoting FT expression independent of COexpression. Moreover, DET1 interacts with MSI4/FVE, which epigenetically inhibitsFLC expression, indicating that the lack of FLC expression indet1-1 mutants likely involves altered histone modifications at theFLC locus. These data demonstrate that DET1 acts in both photoperiod andautonomous pathways to inhibit expression of FT and SOC1. Consistentwith this, the early flowering of det1-1 mutants disappears completely in theft-1 soc1-2 double mutant background. Thus, we propose that DET1 is astrong repressor of flowering and has a pivotal role in maintaining photoperiodsensitivity in the regulation of flowering time.

The identification of molecular markers and marker-aided selection are essential to the efficient breeding of drought-tolerant plants. However, because that characteristic is controlled by many quantitative trait loci, such markers that can screen and trace desirable barley genotypes in a segregating population or germplasm have not yet been determined. Relative water content has been used to estimate drought tolerance in plants because it is highly correlated with the drought index of yield. To develop reliable gene-specific markers for identifying tolerant versus susceptible genotypes, we performed suppression subtractive hybridization to identify candidate genes. We used two domestic barley cultivars, one having the highest RWC (drought-tolerant ‘Chalbori’) and the other having the lowest (drought-susceptible ‘Daebaekbori’). In response to dehydration at the early seedling stage, rapid upregulation ofDehydrin3 (Dhn3) andDhn4 occurred in the drought-tolerant genotypes, but not in the susceptible ones. Similar results were obtained with mature plants growing under frequent drought stress in the greenhouse. In addition,Dhn3 andDhn4 conferred higher drought tolerance when they were over-expressed in transgenicArabidopsis. Thus, in addition to using assessments of RWC, we propose thatDhn3 andDhn4 expressions can serve as drought-induced gene-specific markers to determine drought-tolerant barley genotypes at the seedling stage.

Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg(2+) into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.

Leaf color turns yellow during senescence due to the degradation of chlorophylls and photosynthetic proteins. A stay green mutant was isolated from the glutinous japonica rice Hwacheong- wx through N-methyl-N-nitrosourea mutagenesis. Leaves of the mutant remained green, while turning yellow in those of the wild-type rice during senescence. The stay green phenotype was controlled by a single recessive nuclear gene, tentatively symbolized as sgr(t). All the phenotypic characteristics of the mutant were the same as those of the wild-type lines except for the stay green trait. The leaf chlorophyll concentration of the mutant was similar to that of the wild-type before heading, but decreased steeply in the wild-type during grain filling, while very slowly in the mutant. However, no difference in photosynthetic activity was observed between the stay green mutant and the yellowing wild-type leaves, indicating that senescence is proceeding normally in the mutant leaves and that the mutation affects the rate of chlorophyll degradation during the leaf senescence. Using phenotypic and molecular markers, we mapped the sgr(t) locus to the long arm of chromosome 9 between RFLP markers RG662 and C985 at 1.8- and 2.1-cM intervals, respectively.