Characterization and fine mapping of a novel rice albino mutant low temperature albino 1

Albino mutants are useful genetic resource for studying chlorophyll biosynthesis and chloroplast development and cloning genes involved in these processes in plants.Here we report a novel rice mutant low temperature albino 1(lta1) that showed albino leaves before 4-leaf stage when grown under temperature lower than 20℃,but developed normal green leaves under temperature higher than 24℃or similar morphological phenotypes in dark as did the wild-type(WT).Our analysis showed that the contents of chlorophylls and chlorophyll precursors were remarkably decreased in the ltal mutant under low temperature compared to WT.Transmission electron microscope observation revealed that chloroplasts were defectively developed in the albino lta1 leaves,which lacked of well-stacked granum and contained less stroma lamellae.These results suggested that the lta1 mutation may delay the light-induced thylakoid assembly under low temperature.Genetic analysis indicated that the albino phenotype was controlled by a single recessive locus.Through map-based approach,we finally located the Lta1 gene to a region of 40.3 kb on the short arm of chromosome 11.There are 8 predicted open reading frames(ORFs) in this region and two of them were deleted in lta1 genome compared with the WT genome.The further characterization of the Ltal gene would provide a good approach to uncover the novel molecular mechanisms involved in chloroplast development under low temperature stress.     

Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant,But FtsZ-Based Plastid Division Is Not Essential for Chloroplast Partitioning or Plant Growth and Development

FtsZ1 and FtsZ2 are phylogenetically distinct families of FtsZ in plants that co-localize to mid-plastid rings and facilitate division of chloroplasts. In plants, altered levels of either FtsZ1 or FtsZ2 cause dose-dependent defects in chloroplast division; thus, studies on the functional relationship between FtsZgenes require careful manipulation of FtsZ levels in vivo. To define the functional relationship between the two FtsZ2 genes in Arabidopsis thaliana, FtsZ2-1 and FtsZ2-2, we expressed FtsZ2-1 in an ftsZ2-2 null mutant, and vice versa, and determined whether the chloroplast division defects were rescued in plants expressing different total levels of FtsZ2. Full rescue was observed when either the FtsZ2-1 or FtsZ2-2 level approximated total FtsZ2 levels in wild-type （WT）. Additionally, FtsZ2-2 interacts with ARC6, as shown previously for FtsZ2- 1. These data indicate that FtsZ2-1 and FtsZ2-2 are functionally redundant for chloroplast division in Arabidopsis. To rigorously validate the requirement of each FtsZ family for chloroplast division, we replaced FtsZ1 with FtsZ2 in vivo, and vice versa, while maintaining the FtsZ level in the transgenic plants equal to that of the total level in WT. Chloroplast division defects were not rescued, demonstrating conclusively that FtsZ1 and FtsZ2 are non-redundant for maintenance of WT chloroplast numbers. Finally, we generated ftsZtriple null mutants and show that plants completely devoid of FtsZ protein are viable and fertile. As plastids are presumably essential organelles, these findings suggest that an FtsZ-independent mode of plastid partitioning may occur in higher plants.     

NADPH Thioredoxin Reductase C Controls the Redox Status of Chloroplast 2-Cys Peroxiredoxins in Arabidopsis thaliana

Chloroplast 2-Cys peroxiredoxins （2-Cys Prxs） are efficiently reduced by NADPH Thioredoxin reductase C （NTRC）. To investigate the effect of light/darkness on NTRC function, the content of abundant plastidial enzymes, Rubisco, glutamine synthetase （GS）, and 2-Cys Prxs was analyzed during two consecutive days in Arabidopsis wild-type and ntrc mutant plants. No significant difference of the content of these proteins was observed during the day or the night in wildtype and mutant plants. NTRC deficiency caused a lower content of fully reduced 2-Cys Prxs, which was undetectable in darkness, suggesting that NTRC is the most important pathway for 2-Cys Prx reduction, probably the only one during the night. Arabidopsis contains two plastidial 2-Cys Prxs, A and B, for which T-DNA insertion lines were characterized showing the same phenotype as wild-type plants. Two-dimensional gel analysis of leaf extracts from these mutants allowed the identification of basic and acidic isoforms of 2-Cys Prx A and B. In-vitro assays and mass spectrometry analysis showed that the acidic isoform of both proteins is produced by overoxidation of the peroxidatic Cys residue to sulfinic acid. 2-Cys Prx overoxidation was lower in the NTRC mutant. These results show the important function of NTRC to maintain the redox equilibrium of chloroplast 2-Cys Prxs.     

Role of Arabidopsis UV RESISTANCE LOCUS 8 in Plant Growth Reduction under Osmotic Stress and Low Levels of UV-B

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UVRESISTANCE LOCUS 8 （UVR8） gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.     

In vivo Studies on the Roles of Tic55-Related Proteins in Chloroplast Protein Import in Arabidopsis thaliana

The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis： atTic55-11 and AtPTC52 （Protochlorophyllide-dependent Trans- Iocon Component, 52 kDa; has also been called atTic55-1V）. Our phylogenetic analysis shows that attic55-11 is an ortholog of psTic55 from pea （Pisum sativurn）, and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild- type： visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-11 mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-11 and AtPTC52 are not strictly required for functional protein import in Arabidopsis.     

The polyketide synthase OsPKS2 is essential for pollen exine and Ubisch body patterning in rice

Lipid and phenolic metabolism are important for pollen exine formation. In Arabidopsis, polyketide synthases(PKSs) are essential for both sporopollenin biosynthesis and exine formation. Here, we characterized the role of a polyketide synthase(OsPKS2) in male reproduction of rice(Oryza sativa). Recombinant OsPKS2 catalyzed the condensation of fatty acyl-CoA with malonyl-CoA to generate triketide and tetraketide α-pyrones, the main components of pollen exine. Indeed, the ospks2 mutant had defective exine patterning and was male sterile. However, the mutant showed no significant reduction in sporopollenin accumulation. Compared with the WT(wild type), ospks2 displayed unconfined and amorphous tectum and nexine layers in the exine, and less organized Ubisch bodies. Like the pksb/lap5 mutant of the Arabidopsis ortholog, ospks2 showed broad alterations in the profiles of anther-related phenolic compounds.However, unlike pksb/lap5, in which most detected phenolics were substantially decreased, ospks2 accumulated higher levels of phenolics. Based on these results and our observation that OsPKS2 is unable to fully restore the exine defects in the pksb/lap5, we propose that PKS proteins have functionally diversified during evolution.Collectively, our results suggest that PKSs represent a conserved and diversified biochemical pathway for anther and pollen development in higher plants.     

Identification of a delayed leaf greening gene from a mutation of pummelo

Delayed greening of young leaves is an unusual phenomenon of plants in nature. Citrus are mostly evergreen tree species. Here, a natural mutant of “Guanxi” pummelo(Citrus maxima), which shows yellow leaves at the young stage, was characterized to identify the genes underlying the trait of delayed leaf greening in plants. A segregating population with this mutant as the seed parent and a normal genotype as the pollen parent was generated. Two DNA pools respectively from the leaves of segregating seedlings with extreme phenotypes of normal leaf greening and delayed leaf greening were collected for sequencing. Bulked segregant analysis(BSA) and In Del marker analysis demonstrated that the delayed leaf greening trait is governed by a 0.3 Mb candidate region on chromosome 6. Gene expression analysis further identified a key candidate gene(Citrus Delayed Greening gene 1, CDG1) in the 0.3 Mb region, which showed significantly differential expression between the genotypes with delayed and normal leaf greening phenotypes. There was a 67 bp In Del region difference in the CDG1 promoter and the In Del region contains a TATA-box element. Confocal laser-scanning microscopy revealed that the CDG1-GFP fusion protein signals were co-localized with the chloroplast signals in the protoplasts. Overexpression of CDG1 in tobacco and Arabidopsis led to the phenotype of delayed leaf greening. These results suggest that the CDG1 gene is involved in controlling the delayed leaf greening phenotype with important functions in chloroplast development.     

The Quaternary Structure of NADPH Thioredoxin Reductase C Is Redox-Sensitive

NADPH thioredoxin reductase C （NTRC） is a chloroplast enzyme able to conjugate NADPH thioredoxin reductase （NTR） and thioredoxin （TRX） activities for the efficient reduction of 2-Cys peroxiredoxin （2-Cys PRX）. Because NADPH can be produced in chloroplasts during darkness, NTRC plays a key role for plant peroxide detoxification during the night. Here, it is shown that the quaternary structure of NTRC is highly dependent on its redox status. In vitro, most of the enzyme adopted an oligomeric state that disaggregated in dimers upon addition of NADPH, NADH, or DTT. Gel filtration and Western blot analysis of protein extracts from Arabidopsis chloroplast stroma showed that native NTRC forms aggregates, which are sensitive to NADPH and DTT, suggesting that the aggregation state might be a significant aspect of NTRC activity in vivo. Moreover, the enzyme is localized in clusters in Arabidopsis chloroplasts. NTRC triple and double mutants, A164G- V182E-R183F and A164G-R183F, replacing key residues of NADPH binding site, showed reduced activity but were still able to dimerize though with an increase in intermediary forms. Based on these results, we propose that the catalytically active form of NTRC is the dimer, which formation is induced by NADPH.     

A Rice Panicle Mutant Created by Transformation with an Antisense cDNA Library

A rice （Oryza sativa L.） mutant displaying defects in panicle development was identified among transformants in a transgenic mutagenlzed experiment using an antlsense cDNA library prepared from young rice panicles. In the mutant, the average splkelet number was reduced to 59.8 compared with 104.3 in wild-type plants. In addition, the seed-setting rate of the mutant was low （39.3%） owing to abnormal female development. Genetic analysis of T1 and T2 progeny showed that the traits segregated In a 3 （mutant） ： 1 （wild type） ratio and the mutation was cosegregated with the transgene. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses showed that the mutant had a single T-DNA insertion on chromosome 5, where no gene was tagged. Sequencing analysis found that the transgenic antisense cDNA was derived from a gene encoding an F-box protein in chromosome 7 with unidentified function. This and another four homologous genes encoding putative F-box proteins form a gene cluster. These results indicate that the phenotyplc mutations were most likely due to the silencing effect of the expressed transgenic antisense construct on the member（s） of the F-box gene cluster.     

GAMT2 Encodes a Methyltransferase of Gibberellic Acid That is Involved in Seed Maturation and Germination in Arabidopsis

Salicylic acid methyltransferase （SAMT）, benzoic acid methyltransferase （BAMT） and theobromine methyltransferase （TH） （henceforth, SABATH） family proteins belong to a unique class of mehtyltransferase that can methylate small molecular compounds Including indole-3-acidic acid （IAA）, salicylic acid （SA） and jasmonic acid （JA）, in plants. Here we report that the GAMT2 protein, which has 34.2% similarity with IAMT1 in the amino acid sequence, can methylate gibberellic acid （GA）. Biolnformatics analysis suggests that GAMT2 may be able to methylate one molecule larger than SA. GAMT2 is predominantly expressed in the developing seed embryo and endosperm in Arabidopsis. During seed germination, the expression of GAMT2 decreases until the cotyledons expand out of the seed coat. Overexpression of GAMT2 in Arabidopsis resulted in multiple phenotypes, including dwarfism, retarded growth, late flowering, and reduced fertility, which are similar to the phenotypes of GA-deficient mutants. Seed germination assay showed that GAMT2 overexpression in plants was hypersensitive to GA biosynthesis inhibitor （ancymidol） and abscisic acid （ABA） treatments, whereas the GAMT2 null mutant （SALK_075450） was slightly Insensitive to such treatments, suggesting that GAMT2 may methylate GA or ABA. Enzyme activity analysis indicated that GAMT2 was able to methylate GA3 into Methyi-GA3 in vitro, but could not methylate ABA. Microarray analysis on GAMT2 overexpression plants suggested that Methyl-GA may be an Inactive form of GA in Arabidopsis. These data suggest that GAMT2 Is Involved in seed maturation and germination by modulating GA activity.     

Blocking the Metabolism of Starch Breakdown Products in Arabidopsis Leaves Triggers Chloroplast Degradation

In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant （mex1）, which lacks the chloroplast envelope maltose transporter, accumulates high levels of maltose and starch in chloroplasts and develops a distinctive but previously unexplained chlorotic phenotype as leaves mature. The introduction of additional mutations that prevent starch synthesis, or that block maltose production from starch, also prevent chlorosis of mex1. In contrast, introduction of mutations in disproportionating enzyme （DPE1） results in the accumulation of maltotriose in addition to maltose, and greatly increases chlorosis. These data suggest a link between maltose accumulation and chloroplast homeostasis. Microscopic analyses show that the mesophyll cells in chlorotic mex1 leaves have fewer than half the number of chloroplasts than wild-type cells. Transmission electron microscopy reveals autophagy-like chloroplast degradation in both mex1 and the dpe1/mex1 double mutant. Microarray analyses reveal substantial reprogramming of metabolic and cellular processes, suggesting that organellar protein turnover is increased in mex1, though leaf senescence and senescence-related chlorophyll catabolism are not induced. We propose that the accumulation of maltose and malto-oligosaccharides causes chloroplast dysfunction, which may by signaled via a form of retrograde signaling and trigger chloroplast degradation.     

Creation of Transgenic Bananas Expressing Human Lysozyme Gene for Panama Wilt Resistance

Human lysozyme （HL） inhibits Fusarium oxysporum （FocR4） growth in vitro. To obtain transgenic bananas （Musa spp.） that are resistant to Panama wilt （F. oxysporum）, we introduced an HL gene that is driven by a constitutive cauliflower mosaic virus 35S promoter into the banana via Agrobacteriummediated transformation. PCR confirmed that 51 transgenic plants were obtained. The development of Panama wilt symptoms were examined after the plants had been grown in pots. The non-transgenic plants developed typical fusarium symptoms 60 d after FocR4 inoculation, whereas 24 of 51 transgenic plants remained healthy. The transgenic banana plants that showed resistance to FocR4 in the pots were then planted in a field that was heavily infected with FocR4 for further investigation. Eleven of 24 plants devel- oped symptoms before bud emergence; another 11 plants showed symptoms after bud emergence and the remaining two plants, H-67 and H-144, remained healthy and were able to fruit. Northern blotting analysis demonstrated that H-67 and H-144, bearing the strongest resistance to Panama wilt, had the highest level of HL expression and that the expression of HL was well correlated with the FocR4 resistance of transgenic plants. We conclude that Agrobacterium-mediated transformation, with the assistance of particle bombardment, is a powerful approach for banana transformation and that a transgenic HL gene can cause resistance of the crop to FocR4 in the field.