Chloroplast Differentiation Research Articles

Marchantia polymorpha GOLDEN2-LIKE transcriptional factor; acentral regulator of chloroplast and plant vegetative development.

The GOLDEN2-LIKE (GLK) transcription factors act as a central regulatory node involved in both developmental processes and environmental responses. Marchantia polymorpha, a basal terrestrial plant with strategic evolutionary position, contains a single GLK representative that possesses an additional domain compared to spermatophytes. We analyzed the role of MpGLK in chloroplast biogenesis and development by altering its levels, preforming transcriptomic profiling and conducting chromatin immunoprecipitation. Decreased MpGLK levels impair chloroplast differentiation and disrupt the expression of photosynthesis-associated nuclear genes, while overexpressing MpGLK leads to ectopic chloroplast biogenesis. This demonstrates the MpGLK functions as a bona fide GLK protein, likely representing an ancestral GLK architecture. Altering MpGLK levels directly regulates the expression of genes involved in Chl synthesis and degradation, similar to processes observed in eudicots, and causes various developmental defects in Marchantia, including the formation of dorsal structures such as air pores and gemma cups. MpGLK, also directly activates MpMAX2 gene expression, regulating the timing of gemma cup development. Our study shows that MpGLK functions as a master regulator, potentially coupling chloroplast development with vegetative reproduction. This illustrates the complex regulatory networks governing chloroplast function and plant development communication and highlight the evolutionary conservation of GLK-mediated regulatory processes across plant species.

Orchestration of Photosynthesis-Associated Gene Expression and Galactolipid Biosynthesis during Chloroplast Differentiation in Plants.

The chloroplast thylakoid membrane is composed of membrane lipids and photosynthetic protein complexes, and the orchestration of thylakoid lipid biosynthesis and photosynthesis-associated protein accumulation is considered important for thylakoid development. Galactolipids consist of ∼80% of the thylakoid lipids, and their biosynthesis is fundamental for chloroplast development. We previously reported that the suppression of galactolipid biosynthesis decreased the expression of photosynthesis-associated nuclear-encoded genes (PhAPGs) and photosynthesis-associated plastid-encoded genes (PhAPGs). However, the mechanism for coordinative regulation between galactolipid biosynthesis in plastids and the expression of PhANGs and PhAPGs remains largely unknown. To elucidate this mechanism, we investigated the gene expression patterns in galactolipid-deficient Arabidopsis seedlings during the de-etiolation process. We found that galactolipids are crucial for inducing both the transcript accumulation of PhANGs and PhAPGs and the accumulation of plastid-encoded photosynthesis-associated proteins in developing chloroplasts. Genetic analysis indicates the contribution of the GENOMES UNCOUPLED1 (GUN1)-mediated plastid-to-nucleus signaling pathway to PhANG regulation in response to galactolipid levels. Previous studies suggested that the accumulation of GUN1 reflects the state of protein homeostasis in plastids and alters the PhANG expression level. Thus, we propose a model that galactolipid biosynthesis determines the protein homeostasis in plastids in the initial phase of de-etiolation and optimizes GUN1-dependent signaling to regulate the PhANG expression. This mechanism might contribute to orchestrating the biosynthesis of lipids and proteins for the biogenesis of functional chloroplasts in plants.

Plastid terminal oxidase is required for chloroplast biogenesis in barley.

Chloroplast biogenesis is critical for crop biomass and economic yield. However, chloroplast development is a very complicated process coordinated by cross-communication between the nucleus and plastids, and the underlying mechanisms have not been fully revealed. To explore the regulatory machinery for chloroplast biogenesis, we conducted map-based cloning of the Grandpa 1 (Gpa1) gene regulating chloroplast development in barley. The spontaneous mutation gpa1.a caused a variegation phenotype of the leaf, dwarfed growth, reduced grain yield, and increased tiller number. Genetic mapping anchored the Gpa1 gene onto 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. One gene (HORVU.MOREX.r3.2HG0213170) in the delimited region encodes a putative plastid terminal oxidase (PTOX) in thylakoid membranes, which is homologous to IMMUTANS (IM) of Arabidopsis. The IM gene is required for chloroplast biogenesis and maintenance of functional thylakoids in Arabidopsis. Using CRISPR technology and gene transformation, we functionally validated that the PTOX-encoding gene, HORVU.MOREX.r3.2HG0213170, is the causal gene of Gpa1. Gene expression and chemical analysis revealed that the carotenoid biosynthesis pathway is suppressed by the gpa1 mutation, rendering mutants vulnerable to photobleaching. Our results showed that the overtillering associated with the gpa1 mutation was caused by the lower accumulation of carotenoid-derived strigolactones (SLs) in the mutant. The cloning of Gpa1 not only improves our understanding of the molecular mechanisms underlying chloroplast biosynthesis but also indicates that the PTOX activity is conserved between monocots and dicots for the establishment of the photosynthesis factory.

Long-distance dispersal drives the genetic variation and historical demography of Quercus magnoliifolia and Quercus resinosa (Fagaceae) in the Mexican highlands

While hybridization and introgression can have a strong adaptive importance, it can impede divergence of species. Quercus magnoliifolia and Q. resinosa are two endemic oak species distributed across the Mexican highlands. These species diverged ecological and morphologically; however, no nuclear genetic differentiation is evident. In this study, we determined the mechanisms that shape patterns of genetic variation and establish the role of migration and hybridization in the evolutionary history of these two oak species. To do this, Bayesian approaches were used for inference on migration rates and directionality and timing of divergence between species using chloroplast microsatellites. We then integrated species distribution models to infer the geographic distribution of Q. magnoliifolia and Q. resinosa during Last Interglacial, Last Glacial Maximum, and Mid-Holocene time frames. We failed in distinguishing a unique genetic composition for each species. Chloroplast differentiation was more congruent with geography than the taxonomic status of each species. Our study revealed that after the divergence (c. 10 Mya) of these two oak species, high rates of introgression took place at the end of the Pleistocene. Furthermore, past distribution models predicted that Q. magnoliifolia and Q. resinosa have likely been in sympatry presumable since Last Glacial Maximum and Q. resinosa probably expanded geographically towards its current distribution around Mid-Holocene. This expansion was supported by testing migration models, suggesting recent establishment of Q. resinosa to the north of Trans-Mexican Volcanic Belt. We hypothesized that after Q. magnoliifolia and Q. resinosa diverged, colonization events followed by hybridization between oaks and long-distance seed dispersal occurred, explaining the present-day patterns of distribution of chloroplast diversity. We propose that divergence of species remains mainly on loci under natural selection, providing evidence on the “porous” nature of species boundaries among oaks.

Plastid 2-Cys peroxiredoxins are essential for embryogenesis in Arabidopsis

The redox couple formed by NADPH-dependent thioredoxin reductase C (NTRC) and 2-Cys peroxiredoxins (Prxs) allows fine-tuning chloroplast performance in response to light intensity changes. Accordingly, the Arabidopsis 2cpab mutant lacking 2-Cys Prxs shows growth inhibition and sensitivity to light stress. However, this mutant also shows defective post-germinative growth, suggesting a relevant role of plastid redox systems in seed development, which is so far unknown. To address this issue, we first analyzed the pattern of expression of NTRC and 2-Cys Prxs in developing seeds. Transgenic lines expressing GFP fusions of these proteins showed their expression in developing embryos, which was low at the globular stage and increased at heart and torpedo stages, coincident with embryo chloroplast differentiation, and confirmed the plastid localization of these enzymes. The 2cpab mutant produced white and abortive seeds, which contained lower and altered composition of fatty acids, thus showing the relevance of 2-Cys Prxs in embryogenesis. Most embryos of white and abortive seeds of the 2cpab mutant were arrested at heart and torpedo stages of embryogenesis suggesting an essential function of 2-Cys Prxs in embryo chloroplast differentiation. This phenotype was not recovered by a mutant version of 2-Cys Prx A replacing the peroxidatic Cys by Ser. Neither the lack nor the overexpression of NTRC had any effect on seed development indicating that the function of 2-Cys Prxs at these early stages of development is independent of NTRC, in clear contrast with the operation of these regulatory redox systems in leaves chloroplasts.

Advances in albicidin

Xanthomonas albilineans (Ashby) Downson is a quarantine pest for importing plants to China that causes leaf scald bacterial disease on sugarcane. X. albilineans produces a potent phytotoxin/antibiotic called albicidin. As a pathogenic factor, albicidin causes typical white leaf stripes by inhibiting plastid DNA gyrase and disturbing chloroplast differentiation. Meanwhile, the antibacterial activity of albicidin gives X. albilineans a competitive advantage against rival bacteria during their colonization. Furthermore, albicidin has a rapid bactericidal activity against a variety of Gram-positive and Gram-negative pathogenic bacteria of human species at nanomolar concentrations, making it a potential antimicrobial drug for clinical application. This article reviews the advances of albicidin from the aspects of its molecular structure, traditional extraction methods, mechanism of action, biosynthetic genes and processes, chemical synthesis method and improvement, in order to provide insights into the prevention and treatment of the sugarcane leaf scald disease, and the development of new antibiotics.

Regulation of ppGpp Synthesis and Its Impact on Chloroplast Biogenesis during Early Leaf Development in Rice.

Guanosine tetraphosphate (ppGpp) is known as an alarmone that mediates bacterial stress responses. In plants, ppGpp is synthesized in chloroplasts from GTP and ATP and functions as a regulator of chloroplast gene expression to affect photosynthesis and plant growth. This observation indicates that ppGpp metabolism is closely related to chloroplast function, but the regulation of ppGpp and its role in chloroplast differentiation are not well understood. In rice, ppGpp directly inhibits plastidial guanylate kinase (GKpm), a key enzyme in GTP biosynthesis. GKpm is highly expressed during early leaf development in rice, and the GKpm-deficient mutant, virescent-2 (v2), develops chloroplast-deficient chlorotic leaves under low-temperature conditions. To examine the relationship between GTP synthesis and ppGpp homeostasis, we generated transgenic rice plants over-expressing RSH3, a protein known to act as a ppGpp synthase. When RSH3 was overexpressed in v2, the leaf chlorosis was more severe. Although the RSH3 overexpression in the wild type caused no visible effects, pulse amplitude modulation fluorometer measurements indicated that photosynthetic rates were reduced in this line. This finding implies that the regulation of ppGpp synthesis in rice is involved in the maintenance of the GTP pool required to regulate plastid gene expression during early chloroplast biogenesis. We further investigated changes in the expressions of RelA/SpoT Homolog (RSH) genes encoding ppGpp synthases and hydrolases during the same period. Comparing the expression of these genes with the cellular ppGpp content suggests that the basal ppGpp level is determined by the antagonistic action of multiple RSH enzymatic activities during early leaf development in rice.

Accelerated remodeling of the mesophyll-bundle sheath interface in the maize C4 cycle mutant leaves

C4 photosynthesis in the maize leaf involves the exchange of organic acids between mesophyll (M) and the bundle sheath (BS) cells. The transport is mediated by plasmodesmata embedded in the suberized cell wall. We examined the maize Kranz anatomy with a focus on the plasmodesmata and cell wall suberization with microscopy methods. In the young leaf zone where M and BS cells had indistinguishable proplastids, plasmodesmata were simple and no suberin was detected. In leaf zones where dimorphic chloroplasts were evident, the plasmodesma acquired sphincter and cytoplasmic sleeves, and suberin was discerned. These modifications were accompanied by a drop in symplastic dye mobility at the M-BS boundary. We compared the kinetics of chloroplast differentiation and the modifications in M-BS connectivity in ppdk and dct2 mutants where C4 cycle is affected. The rate of chloroplast diversification did not alter, but plasmodesma remodeling, symplastic transport inhibition, and cell wall suberization were observed from younger leaf zone in the mutants than in wild type. Our results indicate that inactivation of the C4 genes accelerated the changes in the M-BS interface, and the reduced permeability suggests that symplastic transport between M and BS could be regulated for normal operation of C4 cycle.

A Plastid-Bound Ankyrin Repeat Protein Controls Gametophyte and Early Embryo Development in Arabidopsis thaliana.

Proplastids are essential precursors for multi-fate plastid biogenesis, including chloroplast differentiation, a powerhouse for photosynthesis in plants. Arabidopsis ankyrin repeat protein (AKRP, AT5G66055) is a plastid-localized protein with a putative function in plastid differentiation and morphogenesis. Loss of function of akrp leads to embryo developmental arrest. Whether AKRP is critical pre-fertilization has remained unresolved. Here, using reverse genetics, we report a new allele, akrp-3, that exhibited a reduced frequency of mutant embryos (<13%) compared to previously reported alleles. akrp-3 affected both male and female gametophytes resulting in reduced viability, incompetence in pollen tube attraction, altered gametic cell fate, and embryo arrest that were depleted of chlorophyll. AKRP is widely expressed, and the AKRP-GFP fusion localized to plastids of both gametophytes, in isolated chloroplast and co-localized with a plastid marker in pollen and pollen tubes. Cell-type-specific complementation of akrp-3 hinted at the developmental timing at which AKRP might play an essential role. Our findings provide a plausible insight into the crucial role of AKRP in the differentiation of both gametophytes and coupling embryo development with chlorophyll synthesis.

Starch accumulation in bean fruit pericarp is mediated by the differentiation of chloroplasts into amyloplasts

The sucrose supply to bean fruits remains almost constant during seed development, and the early stages of this process are characterized by a significant amount of starch and soluble sugars (glucose, fructose and sucrose) accumulated in the pericarp. Bean fruits are photosynthetically active; however, our results indicated that starch synthesis in the pericarp was largely dependent on the photosynthetic activity of the leaves. The photosynthetic activity and the amount of the Rubisco large subunit were gradually reduced in the fruit pericarp, and a large increase in the amount of the ADP-glucose pyrophosphorylase small subunit (AGPase SS) was observed. These changes suggested differentiation of chloroplasts into amyloplasts. Pericarp chloroplasts imported glucose 1-P to support starch synthesis, and their differentiation into amyloplasts allowed the surplus sucrose to be used in the synthesis of starch, which was later degraded to meet the needs of fast-growing seeds. Starch stored in the bean fruit pericarp was not degraded in response to drought stress, but it was rapidly used under severe nutrient restriction. Together, this work indicated that starch accumulation in the pericarp of bean fruits is important to adjust the needs of developing seeds to the amount of sucrose that is provided to fruits.

The complete plastid genome of Selaginella erythropus (Selaginellaceae), a species with distinctive giant chloroplasts

The plastid genome of the deep-shade plant Selaginella erythropus, which has highly unusual chloroplasts, was characterized using Illumina pair-end sequencing. This plastome is 140,151 bp in length with a large single-copy region (LSC) of 56,133 bp, a small single-copy region (SSC) of 61,268 bp, and two direct repeats (DRs) of 11,375 bp. The overall GC content is 50.68%, while those of LSC, SSC, and DR are 48.96%, 50.3%, and 55.96%, respectively. The plastome contains 102 genes, including 76 protein-coding, 15 tRNA (12 tRNA species), and 8 rRNA genes (4 rRNA species). The phylogenetic analysis shows that S. erythropus is closely related to S. moellendorffii and S. doederleinii. This result is consistent with the previous phylogenetic relationship inferred from multiple plastid and nuclear loci. However, only S. erythropus has the two-zoned giant chloroplast, the bizonoplast. The plastome provides an excellent reference for understanding the unique chloroplast differentiation in Selaginellaceae.

Arabidopsis EGY1 Is Critical for Chloroplast Development in Leaf Epidermal Guard Cells.

In Arabidopsis thaliana, the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) gene encodes a thylakoid membrane-localized protease involved in chloroplast development in leaf mesophyll cells. Recently, EGY1 was also found to be crucial for the maintenance of grana in mesophyll chloroplasts. To further explore the function of EGY1 in leaf tissues, we examined the phenotype of chloroplasts in the leaf epidermal guard cells and pavement cells of two 40Ar17+ irradiation-derived mutants, Ar50-33-pg1 and egy1-4. Fluorescence microscopy revealed that fully expanded leaves of both egy1 mutants showed severe chlorophyll deficiency in both epidermal cell types. Guard cells in the egy1 mutant exhibited permanent defects in chloroplast formation during leaf expansion. Labeling of plastids with CaMV35S or Protodermal Factor1 (PDF1) promoter-driven stroma-targeted fluorescent proteins revealed that egy1 guard cells contained the normal number of plastids, but with moderately reduced size, compared with wild-type guard cells. Transmission electron microscopy further revealed that the development of thylakoids was impaired in the plastids of egy1 mutant guard mother cells, guard cells, and pavement cells. Collectively, these observations demonstrate that EGY1 is involved in chloroplast formation in the leaf epidermis and is particularly critical for chloroplast differentiation in guard cells.

The Role of Membranes and Lipid-Protein Interactions in the Mg-Branch of Tetrapyrrole Biosynthesis.

Chlorophyll (Chl) is essential for photosynthesis and needs to be produced throughout the whole plant life, especially under changing light intensity and stress conditions which may result in the destruction and elimination of these pigments. All steps of the Mg-branch of tetrapyrrole biosynthesis leading to Chl formation are carried out by enzymes associated with plastid membranes. Still the significance of these protein-membrane and protein-lipid interactions in Chl synthesis and chloroplast differentiation are not very well-understood. In this review, we provide an overview on Chl biosynthesis in angiosperms with emphasis on its association with membranes and lipids. Moreover, the last steps of the pathway including the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the biosynthesis of the isoprenoid phytyl moiety and the esterification of Chlide are also summarized. The unique biochemical and photophysical properties of the light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) enzyme catalyzing Pchlide photoreduction and located to peculiar tubuloreticular prolamellar body (PLB) membranes of light-deprived tissues of angiosperms and to envelope membranes, as well as to thylakoids (especially grana margins) are also reviewed. Data about the factors influencing tubuloreticular membrane formation within cells, the spectroscopic properties and the in vitro reconstitution of the native LPOR enzyme complexes are also critically discussed.

Genetic analysis of the barley variegation mutant, grandpa1.a

BackgroundProviding the photosynthesis factory for plants, chloroplasts are critical for crop biomass and economic yield. However, chloroplast development is a complicated process, coordinated by the cross-communication between the nucleus and plastids, and the underlying biogenesis mechanism has not been fully revealed. Variegation mutants have provided ideal models to identify genes or factors involved in chloroplast development. Well-developed chloroplasts are present in the green tissue areas, while the white areas contain undifferentiated plastids that are deficient in chlorophyll. Unlike albino plants, variegation mutants survive to maturity and enable investigation into the signaling pathways underlying chloroplast biogenesis. The allelic variegated mutants in barley, grandpa 1 (gpa1), have long been identified but have not been genetically characterized.ResultsWe characterized and genetically analyzed the grandpa1.a (gpa1.a) mutant. The chloroplast ultrastructure was evaluated using transmission electron microscopy (TEM), and it was confirmed that chloroplast biogenesis was disrupted in the white sections of gpa1.a. To determine the precise position of Gpa1, a high-resolution genetic map was constructed. Segregating individuals were genotyped with the barley 50 k iSelect SNP Array, and the linked SNPs were converted to PCR-based markers for genetic mapping. The Gpa1 gene was mapped to chromosome 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. In the variegated gpa1.a mutant, we identified a large deletion in this gene cluster that eliminates a putative plastid terminal oxidase (PTOX).ConclusionsHere we characterized and genetically mapped the gpa1.a mutation causing a variegation phenotype in barley. The PTOX-encoding gene in the delimited region is a promising candidate for Gpa1. Therefore, the present study provides a foundation for the cloning of Gpa1, which will elevate our understanding of the molecular mechanisms underlying chloroplast biogenesis, particularly in monocot plants.

A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis.

Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling 'Structure Establishment Phase' followed by a 'Chloroplast Proliferation Phase' during cell expansion. Moreover, these data detail thylakoid membrane expansion during de-etiolation at the seedling level and the relative contribution and differential regulation of proteins and lipids at each developmental stage. Altogether, we establish a roadmap for chloroplast differentiation, a critical process for plant photoautotrophic growth and survival.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

BackgroundMicrospore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.ResultsWe performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants.ConclusionsProplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum

Light controls several aspects of plant development through a complex signalling cascade. Several B-box domain containing proteins (BBX) were identified as regulators of Arabidopsis thaliana seedling photomorphogenesis. However, the knowledge about the role of this protein family in other physiological processes and species remains scarce. To fill this gap, here BBX protein encoding genes in tomato genome were characterised. The robust phylogeny obtained revealed how the domain diversity in this protein family evolved in Viridiplantae and allowed the precise identification of 31 tomato SlBBX proteins. The mRNA profiling in different organs revealed that SlBBX genes are regulated by light and their transcripts accumulation is directly affected by the chloroplast maturation status in both vegetative and fruit tissues. As tomato fruits develops, three SlBBXs were found to be upregulated in the early stages, controlled by the proper chloroplast differentiation and by the PHYTOCHROME (PHY)-dependent light perception. Upon ripening, other three SlBBXs were transcriptionally induced by RIPENING INHIBITOR master transcriptional factor, as well as by PHY-mediated signalling and proper plastid biogenesis. Altogether, the results obtained revealed a conserved role of SlBBX gene family in the light signalling cascade and identified putative members affecting tomato fruit development and ripening.

RNAi silenced ζ-carotene desaturase developed variegated tomato transformants with increased phytoene content

ζ-carotene desaturase (ZDS) is one of the enzymes that catalyzes the conversion of ζ-carotene to lycopene. However, the role of ZDS has been not studied extensively in tomato. In this study, to examine the role of ZDS in tomato, the expression of ZDS was downregulated by RNA interference. The relative mRNA levels of ZDS in RNAi lines were reduced by more than 75% compared to wild-type plants. The genetically modified plants exhibited altered morphological characteristics such as photobleached leaves, increased leaf length, longer petioles, larger leaf surface areas and whitish-green tomato fruits. Chromatographic determination showed a significant increase in phytoene in both leaves and fruits of the RI lines, whereas other types of carotenoids were significantly decreased compared to controls. qPCR analysis revealed significant changes in the expression of PSY, PDS and LCYB. The endogenous hormones and flavonoid levels were altered in leaves and fruits of ZDS-silenced plants. Electron microscopic studies revealed hampered chloroplast differentiation and disrupted thylakoid structure in the ZDS-suppressed lines. GC–MS analysis showed that the level of several organic acids, amino acids and fatty acids were decreased in the RI lines, whereas the sugars and sugar derivatives were slightly increased compared to controls. Thus, these results imply a potential for ZDS in carotenoid enhancement through genetic engineering of the carotenoid pathway.

Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development

Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts.

Role of Cytokinins for Interactions of Plants With Microbial Pathogens and Pest Insects.

It has been recognized that cytokinins are plant hormones that influence not only numerous aspects of plant growth, development and physiology, including cell division, chloroplast differentiation and delay of senescence but the interaction with other organisms, including pathogens. Cytokinins are not only produced by plants but are also by other prokaryotic and eukaryotic organism such as bacteria, fungi, microalgae and insects. Notably, cytokinins are produced both by pathogenic and also beneficial microbes and are known to induce resistance in plants against pathogen infections. In this review the contrasting role of cytokinin for the defence and susceptibility of plants against bacterial and fungal pathogen and pest insects is assessed. We also discuss the cross talk of cytokinins with other phytohormones and the underlying mechanism involved in enhancing plant immunity against pathogen infections and explore possible practical applications in crop plant production.