iTRAQ-Based Quantitative Proteomics Analysis Reveals the Mechanism of Golden-Yellow Leaf Mutant in Hybrid Paper Mulberry

Plant growth and development relies on the conversion of light energy into chemical energy, which takes place in the leaves. Chlorophyll mutant variations are important for studying certain physiological processes, including chlorophyll metabolism, chloroplast biogenesis, and photosynthesis. To uncover the mechanisms of the golden-yellow phenotype of the hybrid paper mulberry plant, this study used physiological, cytological, and iTRAQ-based proteomic analyses to compare the green and golden-yellow leaves of hybrid paper mulberry. Physiological results showed that the mutants of hybrid paper mulberry showed golden-yellow leaves, reduced chlorophyll, and carotenoid content, and increased flavonoid content compared with wild-type plants. Cytological observations revealed defective chloroplasts in the mesophyll cells of the mutants. Results demonstrated that 4766 proteins were identified from the hybrid paper mulberry leaves, of which 168 proteins displayed differential accumulations between the green and mutant leaves. The differentially accumulated proteins were primarily involved in chlorophyll synthesis, carotenoid metabolism, and photosynthesis. In addition, differentially accumulated proteins are associated with ribosome pathways and could enable plants to adapt to environmental conditions by regulating the proteome to reduce the impact of chlorophyll reduction on growth and survival. Altogether, this study provides a better understanding of the formation mechanism of the golden-yellow leaf phenotype by combining proteomic approaches.

A Single Nucleotide Substitution of GSAM Gene Causes Massive Accumulation of Glutamate 1-Semialdehyde and Yellow Leaf Phenotype in Rice

Background Tetrapyrroles play indispensable roles in various biological processes. In higher plants, glutamate 1-semialdehyde 2,1-aminomutase (GSAM) converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA), which is the rate-limiting step of tetrapyrrole biosynthesis. Up to now, GSAM genes have been successively identified from many species. Besides, it was found that GSAM could form a dimeric protein with itself by x-ray crystallography. However, no mutant of GSAM has been identified in monocotyledonous plants, and no experiment on interaction of GSAM protein with itself has been reported so far. Result We isolated a yellow leaf mutant, ys53, in rice (Oryza sativa). The mutant showed decreased photosynthetic pigment contents, suppressed chloroplast development, and reduced photosynthetic capacity. In consequence, its major agronomic traits were significantly affected. Map-based cloning revealed that the candidate gene was LOC_Os08g41990 encoding GSAM protein. In ys53 mutant, a single nucleotide substitution in this gene caused an amino acid change in the encoded protein, so its ALA-synthesis ability was significantly reduced and GSA was massively accumulated. Complementation assays suggested the mutant phenotype of ys53 could be rescued by introducing wild-type OsGSAM gene, confirming that the point mutation in OsGSAM is the cause of the mutant phenotype. OsGSAM is mainly expressed in green tissues, and its encoded protein is localized to chloroplast. qRT-PCR analysis indicated that the mutation of OsGSAM not only affected the expressions of tetrapyrrole biosynthetic genes, but also influenced those of photosynthetic genes in rice. In addition, the yeast two-hybrid experiment showed that OsGSAM protein could interact with itself, which could largely depend on the two specific regions containing the 81th–160th and the 321th–400th amino acid residues at its N- and C-terminals, respectively. Conclusions We successfully characterized rice GSAM gene by a yellow leaf mutant and map-based cloning approach. Meanwhile, we verified that OsGSAM protein could interact with itself mainly by means of the two specific regions of amino acid residues at its N- and C-terminals, respectively.

Analysis of Classic Tomato Mutants Reveals Influence of Leaf Vein Density on Fruit BRIX

Tomato bipinnate (bip) is a classic leaf mutant, with highly increased leaf complexity resulting from the loss of function of a BEL-LIKE HOMEODAMAIN (BELL) gene. Here, we analyzed several bip mutants and their isogenic wildtype backgrounds for a suite of leaf morphology traits, ranging from leaf complexity, leaflet shape and size, to leaf vascular density to investigate how changes in leaf morphology influence fruit traits. Our analyses showed an unexpected relationship between leaf vein density and fruit sugar levels, where leaf vein density was negatively correlated with fruit BRIX. RNA-Seq analysis suggested variation in Glucose-6-phosphate translocator2 (GPT2) gene expression caused correlated changes in leaf vein density and BRIX when bip mutant and wildtype were compared, suggesting that the correlation between leaf vein density and fruit sugar may result from the genes regulating leaf vein development that are also involved in regulating leaf sugar biosynthesis. Our results provide a resource for further exploration of the genetic basis for the complex relationship between fruit quality and leaf traits in natural populations.

Comparative transcriptome analysis of differentially expressed genes related to the physiological changes of yellow-green leaf mutant of maize

Chlorophylls, green pigments in chloroplasts, are essential for photosynthesis. Reduction in chlorophyll content may result in retarded growth, dwarfism, and sterility. In this study, a yellow-green leaf mutant of maize, indicative of abnormity in chlorophyll content, was identified. The physiological parameters of this mutant were measured. Next, global gene expression of this mutant was determined using transcriptome analysis and compared to that of wild-type maize plants. The yellow-green leaf mutant of maize was found to contain lower contents of chlorophyll a, chlorophyll b and carotenoid compounds. It contained fewer active PSII centers and displayed lower values of original chlorophyll fluorescence parameters than the wild-type plants. The real-time fluorescence yield, the electron transport rate, and the net photosynthetic rate of the mutant plants showed reduction as well. In contrast, the maximum photochemical quantum yield of PSII of the mutant plants was similar to that of the wild-type plants. Comparative transcriptome analysis of the mutant plants and wild-type plants led to the identification of differentially expressed 1,122 genes, of which 536 genes were up-regulated and 586 genes down-regulated in the mutant. Five genes in the chlorophyll metabolism pathway, nine genes in the tricarboxylic acid cycle and seven genes related to the conversion of sucrose to starch displayed down-regulated expression. In contrast, genes encoding a photosystem II reaction center PsbP family protein and the PGR5-like protein 1A (PGRL1A) exhibited increased transcript abundance.

Curled Flag Leaf 2, Encoding a Cytochrome P450 Protein, Regulated by the Transcription Factor Roc5, Influences Flag Leaf Development in Rice

Moderate curling generally causes upright leaf blades, which favors the establishment of ideal plant architecture and increases the photosynthetic efficiency of the population, both of which are desirable traits for super hybrid rice (Oryza sativa L.). In this study, we identified a novel curled-leaf mutant, curled flag leaf 2 (cfl2), which shows specific curling at the base of the flag leaf owing to abnormal epidermal development, caused by enlarged bulliform cells and increased number of papillae with the disordered distribution. Map-based cloning reveals that CFL2 encodes a cytochrome P450 protein and corresponds to the previously reported OsCYP96B4. CFL2 was expressed in all analyzed tissues with differential abundance and was downregulated in the clf1 mutant [a mutant harbors a mutation in the homeodomain leucine zipper IV (HD-ZIP IV) transcription factor Roc5]. Yeast one-hybrid and transient expression assays confirm that Roc5 could directly bind to the cis-element L1 box in the promoter of CFL2 before activating CFL2 expression. RNA sequencing reveals that genes associated with cellulose biosynthesis and cell wall-related processes were significantly upregulated in the cfl2 mutant. The components of cell wall, such as lignin, cellulose, and some kinds of monosaccharide, were altered dramatically in the cfl2 mutant when compared with wild-type “Jinhui10” (WT). Taken together, CFL2, as a target gene of Roc5, plays an important role in the regulation of flag leaf shape by influencing epidermis and cell wall development.

520YS , Encoding an Ankyrin Repeat Protein, is Involved in Chloroplast Development in Rice

BackgroundThe ankyrin repeat (ANK) proteins are widely distributed in organisms ranging from viruses to plants, which play key roles in plastid differentiation, embryogenesis, chloroplast biogenesis and so on. However, only a few ANK genes have been identified in rice.ResultsIn this study, we isolated a yellow-green leaf mutant, 520ys, from japonica rice cultivar Nipponbare through ethyl methane sulfonate mutagenesis. The mutant exhibited a yellow-green leaf phenotype throughout the life cycle, arrested development of chloroplasts, reduced levels of photosynthetic pigments, and accumulated reactive oxide species. Map-based cloning suggested that the candidate gene was LOC_Os07g33660, which encodes an expressed protein containing one ankyrin repeat and showing sequence similarity with the Arabidopsis LTD/GDC1 (At1g50900). Transgenic complementation experiment confirmed that LOC_Os07g33660 is the causal gene for the mutant type of 520ys. 520YS (LOC_Os07g33660) is mainly expressed in green tissues and its encoded protein is targeted to the chloroplast. In 520ys mutant, expression levels of four light-harvesting chlorophyll a/b-binding protein translocation-related genes and eight photosynthesis-related genes were significantly down-regulated.ConclusionWe characterized a novel ANK gene, 520YS, which plays a key role in chloroplast development in rice.