Non-green Tissues Research Articles

JrPPO1/2 play distinct roles in regulating walnut fruit browning by different spatiotemporal expression and enzymatic characteristics

Polyphenol oxidase (PPO) activity drives walnut fruit browning, but the roles of its only two-family genes, JrPPO1 and JrPPO2, remain unclear. This study explores the spatiotemporal expression and enzymatic characteristics of JrPPO1 and JrPPO2 in walnut. Treatment with the PPO activator CuSO4 and H2O2 accelerated fruit browning and up-regulated JrPPO1/2 expression, whereas treatment with the PPO inhibitor ascorbic acid delayed browning, down-regulating JrPPO1 and up-regulating JrPPO2 expression. Compared to mJrPPO1, mJrPPO2 can exhibited better enzyme activity at higher temperatures (47 °C) and in more acidic environments (pH 4.25). mJrPPO2 exhibited a higher substrate specificity over mJrPPO1, and the preferred substrates are catechol, chlorogenic acid, and epicatechin. Additionally, mJrPPO2 adapted better to low concentration of oxygen (as low as 1.0% O2) and slightly elevated CO2 levels compared to mJrPPO1. Subcellular localization and spatiotemporal expression patterns showed that JrPPO1 is only expressed in green tissues and located in chloroplasts, while JrPPO2 is also located in chloroplasts, partly associated with membranes, and is expressed in both green and non-green tissues. Silencing JrPPO1/2 with virus-induced gene silencing (VIGS) reduced fruit browning, maintained higher total phenols, and decreased MDA production. Notably, silencing JrPPO1 had a greater impact on browning than JrPPO2, indicating JrPPO1's greater contribution to PPO activity and fruit browning in walnut fruits. Consequently, JrPPO1 can be effectively regulated both at the molecular level and by manipulating environmental conditions, to achieve the objective of controlling fruit browning.

Onion cryptochrome 1 (AcCRY1) regulates photomorphogenesis and photoperiod flowering in Arabidopsis and exploration of its functional mechanisms under blue light

Cryptochromes (CRYs), as blue-light photoreceptors, play a crucial role in regulating flowering time and hypocotyl and cotyledon development. Their physiological functions have been extensively studied in various plant species. However, research on onions remains limited. In this study, we identified AcCRY1 and conducted preliminary investigations into its function. Our results demonstrate that AcCRY1 possesses a conserved domain typical of cryptochromes with high homology to those found in monocots. Furthermore, we examined the expression level of AcCRY1 in onion. The green tissues is significantly higher compared to non-green tissues, and it exhibits a significant response to blue-light induction. AcCRY1 demonstrates cytoplasmic localization under blue-light conditions, while it localizes in the nucleus during darkness, indicating a strong dependence on blue-light for its subcellular distribution. In comparison to cry1, overexpression of AcCRY1 leads to a significant shorten in seedling hypocotyl length, notable expansion of cotyledons, and acceleration of flowering time. The yeast two-hybrid experiment demonstrated the in vitro interaction between AcCRY1, AcCOP1, and AcSPA1. Additionally, BIFC analysis confirmed their interaction in Onion epidermis. Notably, under blue-light conditions, a significantly enhanced binding activity was observed compared to dark conditions. These findings establish a functional foundation for the regulatory role of AcCRY1 in important physiological processes of onion and provide initial insights into the underlying molecular mechanisms.

Engineered Expression of Vip3A in Green Tissues as a Feasible Approach for the Control of Insect Pests in Maize.

Genetic engineering technology offers opportunities to improve many important agronomic traits in crops, including insect-resistance. However, genetically modified (GM) exogenous proteins in edible tissues of transgenic crops has become an issue of intense public concern. To advance the application of GM techniques in maize, a Cre/loxP-based strategy was developed for manipulating the transgenes in green tissues while locking them in non-green tissues. In the strategy, the site-specific excision can be used to switch on or off the expression of transgenes at specific tissues. In this work, two basic transgenic maize, named KEY, carrying the Cre gene, and LOCK, containing the Vip3A gene with a blocked element, were obtained based on their separate fusion gene cassettes. The expression level and concentration of Vip3A were observed with a high specific accumulation in the green tissues (leaf and stem), and only a small amount was observed in the root and kernel tissues in the KEY × LOCK hybrids. The insect resistance of transgenic maize against two common lepidopteran pests, Ostrinia furnacalis and Spodoptera frugiperda, was assessed in the laboratory and field. The results indicate that the hybrids possessed high resistance levels against the two pests, with mortality rates above 73.6% and damage scales below 2.4 compared with the control group. Our results suggest that the Cre/loxP-mediated genetic engineering approach has a competitive advantage in GM maize. Overall, the findings from this study are significant for providing a feasible strategy for transgenes avoiding expression in edible parts and exploring novel techniques toward the biosafety of GM plants.

Green tissue-targeted expression of the Cry1Ab/c gene in transgenic maize using the Cre/loxP system as an alternative strategy against lepidopteran pests.

Genetically modified (GM) proteins in edible tissues of transgenic maize are of intense public concern. We provided a Cre/loxP-based strategy for manipulating the expression of transgenes in green tissues while locking them in nongreen tissues. First, the Cre gene was driven by the green tissue-specific promoter Zm1rbcS to generate transgenic maize KEY. Meanwhile, a gene cassette containing a Nos terminator (NosT) in front of the Cry1Ab/c gene was driven by the strong promoter ZmUbi to generate another transgenic maize LOCK. By crossing KEY and LOCK plants, the expressed Cre recombinase under the control of the Zm1rbcS promoter from KEY maize accurately removed the NosT of LOCK maize. Consequently, the expression of blocked Cry1Ab/c was enabled in specific green tissues in their hybrids. The expression level and concentration of Cry1Ab/c were observed using a strategy with high specific accumulation in green tissues (leaf and stem). Still, only a small or absent amount was observed in root and kernel tissues. Furthermore, we assessed the bioactivity of transgenic maize against 2 common lepidopteran pests, Ostrinia furnacalis and Spodoptera frugiperda, in the laboratory and field. The transgenic plants showed high plant resistance levels against the 2 pests, with mortality rates above 97.2% and damage scales below 2.2 compared with the control group. These findings are significant for exploring novel genetic engineering techniques in GM maize and providing a feasible strategy for transgenes avoiding expression in edible parts. In addition, implementing the Cre/loxP-mediated system could relieve public sentiment toward the biosafety of GM plants.

Activity of Arabidopsis Rubisco small subunit promoter in various tissues of chickpea.

The activity of a green tissue-specific promoter of the Rubisco small subunit gene fromArabidopsis(AraSSU) was studied using transgenic chickpea lines. We generated transgenic chickpea lines expressing anAraSSUpromoter-drivencry2Aagene throughthe Agrobacterium-mediatedtransformation method. Lines withAraSSUexpressed the gene in all green tissues at high levels (> 90ng/mg of fresh weight tissue) compared to lines generated usingCaMV35S(< 10ng/mg FW). We used vertical cross sections of various tissues of homozygous progeny using microtome for immunolocalization. The immunolocalization showed the expression of thecry2Aagene in the green mesophyll cells of the leaves of bothAraSSUand CaMV35 chickpea lines. Moreover, the accumulation ofAraSSU-regulatedCry2Aa protein was also observed in vascular tissues, including enucleate sieve elements and their companion cells. However, no expression was observed in the roots ofAraSSUlines. In the case ofCaMV35lines, the transgene expression was observed in all the tissues. Since our data indicated that theAraSSUpromoter is active in non-green tissues such as vascular bundles. Therefore, we validated this by RT-PCR. We found Cry2Aa RNA transcripts in leaves, stems without epidermis (for vascular tissues), and roots with and without epidermis. Thus,the AraSSUpromoter is active in all above-ground tissues of the chickpea plant.

Cytosolic Nudix Hydrolase 1 Is Involved in Geranyl β-Primeveroside Production in Tea.

Geraniol is a potent tea odorant and exists mainly as geranyl glycoside in Camellia sinensis. Understanding the mechanisms of geraniol biosynthesis at molecular levels in tea plants is of great importance for practical improvement of tea aroma. In this study, geraniol and its glycosides from tea plants were examined using liquid chromatography coupled with mass spectrometry. Two candidate geraniol synthase (GES) genes (CsTPS) and two Nudix hydrolase genes (CsNUDX1-cyto and CsNUDX1-chlo) from the tea genome were functionally investigated through gene transcription manipulation and gene chemical product analyses. Our data showed that in tea leaves, levels of geranyl β-primeveroside were dramatically higher than those of geranyl β-glucoside, while free geraniol was undetectable in this study. A tempo-spatial variation of geranyl β-primeveroside abundance in tea plants existed, with high levels in young and green tissues and low levels in mature or non-green tissues. Cytosolic CsNUDX1-cyto showed higher hydrolysis activity of geranyl-pyrophosphate to geranyl-monophosphate (GP) in vitro than did chloroplastidial CsNUDX1-chlo. A transgenic study revealed that expression of CsNUDX1-cyto resulted in significantly more geranyl β-primeveroside in transgenic Nicotiana benthamiana compared with non-transgenic wild-type, whereas expression of CsNUDX1-chlo had no effect. An antisense oligo-deoxynucleotide study confirmed that suppression of CsNUDX1-cyto transcription in tea shoots led to a significant decrease in geranyl β-primeveroside abundance. Additionally, CsNUDX1-cyto transcript levels and geranyl β-primeveroside abundances shared the same tempo-spatial patterns in different organs in the tea cultivar “Shucha Zao,” indicating that CsNUDX1-cyto is important for geranyl β-primeveroside formation in tea plants. Results also suggested that neither of the two candidate GES genes in tea plants did not function as GES in transgenic N. benthamiana. All our data indicated that CsNUDX1-cyto is involved in geranyl β-primeveroside production in tea plants. Our speculation about possible conversion from the chemical product of CsNUDX1-cyto to geranyl β-primeveroside in plants was also discussed.

Transgene-free genome editing supports CCD4 role as a negative regulator of β-carotene in banana.

This study aims to understand the regulatory mechanism of the β-carotene homeostasis by establishing transgene-free genome editing in banana. Carotenoid cleavage dioxygenases (CCDs) belong to a miniature gene family having an imperative role in the intricated carotenoid metabolism in plants. Here, the expression pattern of multiple CCDs was correlated with the levels of carotenoid accumulation in two contrasting cultivars, viz., Nendran (high β-carotene) and Rasthali (low β-carotene). The higher expression of the RAS-CCD4 inversely correlated with β-carotene accumulation in fruit-pulp of the Rasthali. The docking analysis followed enzyme assay of purified RAS-CCD4 suggested β-carotene and 10-apo-β-carotenal as its preferred substrates. Bacterial complementation assay affirmed RAS-CCD4 role in β-carotene degradation and then overexpression of the RAS-CCD4 in the Arabidopsis thaliana further validated results in-vivo by the significant reduction in β-carotene. Subsequently, CRISPR/Cas9 mediated editing of CCD4 was demonstrated in the protoplasts and embryogenic cell lines of Rasthali. The carotenoid profiling in stable mutant lines revealed higher fold β-carotene accumulation in non-green tissue (roots) than in green tissue (leaf) compared with the unedited control plants. The differential expression of carotenoid pathway genes was correlated with the metabolites in the edited lines. The study suggests that carotenoid catabolism is regulated by the CCD4 in tissue and cultivar specific manners, and also demonstrated the use of the genome editing tool in developing transgene-free biofortified banana.

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism.

Sustainable and economically viable support for an ever-increasing global population requires a paradigm shift in agricultural productivity, including the application of biotechnology to generate future crop plants. Current genetic engineering approaches aimed at enhancing the photosynthetic efficiency or composition of the harvested tissues involve relatively simple manipulations of endogenous metabolism. However, radical rewiring of central metabolism using new-to-nature pathways, so-called “synthetic metabolism”, may be needed to really bring about significant step changes. In many cases, this will require re-programming the metabolism of the chloroplast, or other plastids in non-green tissues, through a combination of chloroplast and nuclear engineering. However, current technologies for sophisticated chloroplast engineering (“transplastomics”) of plants are limited to just a handful of species. Moreover, the testing of metabolic rewiring in the chloroplast of plant models is often impractical given their obligate phototrophy, the extended time needed to create stable non-chimeric transplastomic lines, and the technical challenges associated with regeneration of whole plants. In contrast, the unicellular green alga, Chlamydomonas reinhardtii is a facultative heterotroph that allows for extensive modification of chloroplast function, including non-photosynthetic designs. Moreover, chloroplast engineering in C. reinhardtii is facile, with the ability to generate novel lines in a matter of weeks, and a well-defined molecular toolbox allows for rapid iterations of the “Design-Build-Test-Learn” (DBTL) cycle of modern synthetic biology approaches. The recent development of combinatorial DNA assembly pipelines for designing and building transgene clusters, simple methods for marker-free delivery of these clusters into the chloroplast genome, and the pre-existing wealth of knowledge regarding chloroplast gene expression and regulation in C. reinhardtii further adds to the versatility of transplastomics using this organism. Herein, we review the inherent advantages of the algal chloroplast as a simple and tractable testbed for metabolic engineering designs, which could then be implemented in higher plants.

Plastid transformation: Advances and challenges for its implementation in agricultural crops

Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology’s high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots. How to cite: Quintín Rascón-Cruz Q, González-Barriga CD, Iglesias-Figueroa BF, et al. Plastid transformation: Advances and challenges for its implementation in agricultural crops. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.005

The genome of Ricinus communis encodes a single glycolate oxidase with different functions in photosynthetic and heterotrophic organs

Main conclusionThe biochemical characterization of glycolate oxidase in Ricinus communis hints to different physiological functions of the enzyme depending on the organ in which it is active.Enzymatic activities of the photorespiratory pathway are not restricted to green tissues but are present also in heterotrophic organs. High glycolate oxidase (GOX) activity was detected in the endosperm of Ricinus communis. Phylogenetic analysis of the Ricinus l-2-hydroxy acid oxidase (Rc(l)-2-HAOX) family indicated that Rc(l)-2-HAOX1 to Rc(l)-2-HAOX3 cluster with the group containing streptophyte long-chain 2-hydroxy acid oxidases, whereas Rc(l)-2-HAOX4 clusters with the group containing streptophyte GOX. Rc(l)-2-HAOX4 is the closest relative to the photorespiratory GOX genes of Arabidopsis. We obtained Rc(l)-2-HAOX4 as a recombinant protein and analyze its kinetic properties in comparison to the Arabidopsis photorespiratory GOX. We also analyzed the expression of all Rc(l)-2-HAOXs and conducted metabolite profiling of different Ricinus organs. Phylogenetic analysis indicates that Rc(l)-2-HAOX4 is the only GOX encoded in the Ricinus genome (RcGOX). RcGOX has properties resembling those of the photorespiratory GOX of Arabidopsis. We found that glycolate, the substrate of GOX, is highly abundant in non-green tissues, such as roots, embryo of germinating seeds and dry seeds. We propose that RcGOX fulfills different physiological functions depending on the organ in which it is active. In autotrophic organs it oxidizes glycolate into glyoxylate as part of the photorespiratory pathway. In fast growing heterotrophic organs, it is most probably involved in the production of serine to feed the folate pathway for special demands of those tissues.

Quantification of Carotenoid Pathway Flux in Green and Nongreen Systems.

Metabolite accumulation in plant tissues represents the transient net result of their constant biosynthesis and degradation. For carotenoids, degradation might occur enzymatically by carotenoid cleavage producing plant hormones and volatiles or by nonenzymatic oxidation, both depending on environmental and developmental conditions. Carotenoid biosynthesis is therefore constantly regulated at various levels to attain sufficient carotenoid accumulation, mainly for photosynthesis and photoprotection. Due to the plenitude of carotenoids and their degradation products, it is not feasible to investigate overall carotenoid biosynthetic activity and its regulation by the quantification of all carotenoids including their derivatives. This is an issue encountered in investigations on many other highly branched pathways. We therefore present protocols to determine carotenoid biosynthesis flux in a given plant tissue by HPLC quantification of phytoene, the first pathway-specific intermediate and precursor of all carotenoids synthesized by phytoene synthase (PSY). For this purpose, enzymatic metabolization of phytoene in the tissue under investigation is prevented by treatment with the bleaching herbicide norflurazon, blocking the carotenogenic pathway downstream of PSY. As phytoene is more resistant to oxidation than desaturated carotenoids, the rate of phytoene biosynthesis serves as a good measure for total carotenogenic flux in a given tissue. The method is described for Arabidopsis for two photosynthetically active sample types, namely, seedlings and leaves, as well as for seed-derived callus as nongreen tissue. It should be realizable using only a relatively simple experimental setup and is applicable to other plant tissues as well as to different plant species. Additionally, similar experimental setups could be a useful tool to investigate total flux and turnover rates in other biosynthetic pathways.

Albino Leaf 2 is involved in the splicing of chloroplast group I and II introns in rice.

Chloroplasts play an essential role in plant growth and development through manipulating photosynthesis and the production of hormones and metabolites. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, identification of novel components is still lacking. We isolated a rice (Oryza sativa) mutant, termed albino leaf 2 (al2), using genetic screening. Phenotypic analysis revealed that the al2 mutation caused obvious albino leaves at the early developmental stage, eventually leading to al2 seedling death. Electron microscopy investigations indicated that the chloroplast structure was disrupted in the al2 mutants at an early developmental stage and subsequently resulted in the breakdown of the entire chloroplast. Molecular cloning illustrated that AL2 encodes a chloroplast group IIA intron splicing facilitator (CRS1) in rice, which was confirmed by a genetic complementation experiment. Moreover, our results demonstrated that AL2 was constitutively expressed in various tissues, including green and non-green tissues. Interestingly, we found that the expression levels of a subset of chloroplast genes that contain group IIA and IIB introns were significantly reduced in the al2 mutant compared to that in the wild type, suggesting that AL2 is a functional CRS1 in rice. Differing from the orthologous CRS1 in maize and Arabidopsis that only regulates splicing of the chloroplast group II intron, our results demonstrated that the AL2 gene is also likely to be involved in the splicing of the chloroplast group I intron. They also showed that disruption of AL2 results in the altered expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded polymerases and nuclear-encoded chloroplast genes. Taken together, these findings shed new light on the function of nuclear-encoded chloroplast group I and II intron splicing factors in rice.

Suppression of nighttime sap flux with lower stem photosynthesis in Eucalyptus trees

It is widely accepted that substantial nighttime sap flux (J s,n) or transpiration (E) occurs in most plants, but the physiological implications are poorly known. It has been hypothesized that J s,n or E serves to enhance nitrogen uptake or deliver oxygen; however, no clear evidence is currently available. In this study, sap flux (J s) in Eucalyptus grandis × urophylla with apparent stem photosynthesis was measured, including control trees which were covered by aluminum foil (approximately 1/3 of tree height) to block stem photosynthesis. We hypothesized that the nighttime water flux would be suppressed in trees with lower stem photosynthesis. The results showed that the green tissue degraded after 3 months, demonstrating a decrease in stem photosynthesis. The daytime J s decreased by 21.47%, while J s,n decreased by 12.03% in covered trees as compared to that of control, and the difference was statistically significant (P < 0.01). The linear quantile regression model showed that J s,n decreased for a given daytime transpiration water loss, indicating that J s,n was suppressed by lower stem photosynthesis in covered trees. Predawn (ψ pd) of covered trees was marginally higher than that of control while lower at predawn stomatal conductance (g s), indicating a suppressed water flux in covered trees. There was no difference in leaf carbon content and δ(13)C between the two groups, while leaf nitrogen content and δ(15)N were significantly higher in covered trees than that of the control (P < 0.05), indicating that J s,n was not used for nitrogen uptake. These results suggest that J s,n may act as an oxygen pathway since green tissue has a higher respiration or oxygen demand than non-green tissue. Thus, this study demonstrated the physiological implications of J s,n and the possible benefits of nighttime water use or E by the tree.

NADH-dependent glutamate synthase participated in ammonium assimilation in Arabidopsis root

Higher plants have 2 GOGAT species, Fd-GOGAT and NADH-GOGAT. While Fd-GOGAT mainly assimilates ammonium in leaves, which is derived from photorespiration, the function of NADH-GOGAT, which is highly expressed in roots,1 needs to be elucidated. The aim of this study was to clarify the role of NADH-GOGAT in Arabidopsis roots. The supply of ammonium to the roots caused an accumulation of NADH-GOGAT, while Fd-GOGAT 1 and Fd-GOGAT 2 showed no response. A promoter–GUS fusion analysis and immunohistochemistry showed that NADH-GOGAT was located in non-green tissues like vascular bundles, shoot apical meristem, pollen, stigma, and roots. The localization of NADH-GOGAT and Fd-GOGAT was not overlapped. NADH-GOGAT T-DNA insertion lines showed a reduction of glutamate and biomass under normal CO2 conditions. These data emphasizes the importance of NADH-GOGAT in the ammonium assimilation of Arabidopsis roots.

NADH-dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2-oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin-dependent glutamate synthase (Fd-GOGAT) and nicotinamide adenine dinucleotide (NADH)-GOGAT. While Fd-GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH-GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH-GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real-time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH-GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH-GOGAT and Fd-GOGAT did not fully overlap. Promoter-β-glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH-GOGAT was highly accumulated in non-green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH-GOGAT transfer DNA (T-DNA) insertion lines under normal CO2 condition. The data emphasize the importance of NADH-GOGAT in the ammonium assimilation in Arabidopsis roots.

Or mutation leads to photo-oxidative stress responses in cauliflower (Brassica oleracea) seedlings during de-etiolation

The Orange (Or) gene is a gene mutation that can increase carotenoid content in plant tissues normally devoid of pigments. It affects plastid division and is involved in the differentiation of proplastids or non-colored plastids into chromoplasts. In this study, the de-etiolation process of the wild type (WT) cauliflower (Brassica oleracea L. var. botrytis) and Or mutant seedlings was investigated. We analyzed pigment content, plastid development, transcript abundance and protein levels of genes involved in the de-etiolation process. The results showed that Or can increase the carotenoid content in green tissues, although not as effectively as in non-green tissues, and this effect might be caused by the changes in biosynthetic pathway genes at both transcriptional and post-transcriptional levels. There was no significant difference in the plastid development process between the two lines. However, the increased content of antheraxanthin and anthocyanin, and higher expression levels of violaxanthin de-epoxidase gene (VDE) suggested a stress situation leading to photoinhibition and enhanced photoprotection in the Or mutant. The up-regulated expression levels of the reactive oxygen species (ROS)-induced genes, ZAT10 for salt tolerance zinc finger protein and ASCORBATE PEROXIDASE2 (APX2), suggested the existence of photo-oxidative stress in the Or mutant. In summary, abovementioned findings provide additional insight into the functions of the Or gene in different tissues and at different developmental stages.

Differential regulation of Arabidopsis plastid gene expression and RNA editing in non-photosynthetic tissues

RNA editing is one of the post-transcriptional processes that commonly occur in plant plastids and mitochondria. In Arabidopsis, 34 C-to-U RNA editing events, affecting transcripts of 18 plastid genes, have been identified. Here, we examined the editing and expression of these transcripts in different organs, and in green and non-green seedlings (etiolated, cia5-2, ispF and ispG albino mutants, lincomycin-, and norflurazon-treated). The editing efficiency of Arabidopsis plastid transcripts varies from site to site, and may be specifically regulated in different tissues. Steady state levels of plastid transcripts are low or undetectable in etiolated seedlings, but most editing sites are edited with efficiencies similar to those observed in green seedlings. By contrast, the editing of some sites is completely lost or significantly reduced in other non-green tissues; for instance, the editing of ndhB-149, ndhB-1255, and ndhD-2 is completely lost in roots and in lincomycin-treated seedlings. The editing of ndhD-2 is also completely lost in albino mutants and norflurazon-treated seedlings. However, matK-640 is completely edited, and accD-794, atpF-92, psbE-214, psbF-77, psbZ-50, and rps14-50 are completely or highly edited in both green and non-green tissues. In addition, the expression of nucleus-encoded RNA polymerase dependent transcripts is specifically induced by lincomycin, and the splicing of ndhB transcripts is significantly reduced in the albino mutants and inhibitor-treated seedlings. Our results indicate that plastid gene expression, and the splicing and editing of plastid transcripts are specifically and differentially regulated in various types of non-green tissues.

Tissue-Specific and Developmental Expression Pattern of Abscisic Acid Biosynthetic Genes in Peach Fruit: Possible Role of the Hormone in the Coordinated Growth of Seed and Mesocarp

Peach fruit tissues start differentiating into mesocarp, endocarp, and seed from the first phases of development. A seed is necessary for fruit set, and it may strongly regulate the growth of the whole fruit through metabolic or hormonal signals. Although the importance of sugar and hormone signaling in growing fruit, such as peach, has been analyzed extensively, no conclusions as to the cross-talk between these signal molecules and their role in seed and flesh development have previously been made. The present study provides insight into the regulatory steps of the carotenoid/ABA biosynthetic pathway to establish possible relationships between growth and accumulation of pigments, sugar, and hormone in the different tissues of peach fruit (Prunus persica L. Batsch, cv Redhaven). In the flesh, the transcriptional pattern of most of the genes involved in the ABA synthesis exhibits a good association with both color changes and hormone accumulation. In contrast, in the seed, along with other nongreen tissues, this association is not conceivable. The behavior of zeaxanthin epoxidase genes (Ppzep) well represents the presence of distinct regulatory mechanisms at various steps of the pathway and in a tissue-specific manner. Moreover, the key role of 9-cis-epoxycarotenoid dioxygenase (NCED) enzyme in regulating ABA synthesis appears to be substantiated by observed Ppnced expression profiles, both in the flesh and in the seed. Based on the results obtained in this study, a crucial connection between ABA biosynthesis, sugar content, and sucrose cleavage enzymes (sucrose synthase), at different stages of fruit development, is proposed.

Design of chimeric expression elements that confer high‐level gene activity in chromoplasts

Non-green plastids, such as chromoplasts, generally have much lower activity of gene expression than chloroplasts in photosynthetically active tissues. Suppression of plastid genes in non-green tissues occurs through a complex interplay of transcriptional and translational control, with the contribution of regulation of transcript abundance versus translational activity being highly variable between genes. Here, we have investigated whether the low expression of the plastid genome in chromoplasts results from inherent limitations in gene expression capacity, or can be overcome by designing appropriate combinations of promoters and translation initiation signals in the 5' untranslated region (5'-UTR). We constructed chimeric expression elements that combine promoters and 5'-UTRs from plastid genes, which are suppressed during chloroplast-to-chromoplast conversion in Solanum lycopersicum (tomato) fruit ripening, either just at the translational level or just at the level of mRNA accumulation. These chimeric expression elements were introduced into the tomato plastid genome by stable chloroplast transformation. We report the identification of promoter-UTR combinations that confer high-level gene expression in chromoplasts of ripe tomato fruits, resulting in the accumulation of reporter protein GFP to up to 1% of total cellular protein. Our work demonstrates that non-green plastids are capable of expressing genes to high levels. Moreover, the chimeric cis-elements for chromoplasts developed here are widely applicable in basic and applied research using transplastomic methods.

Identification of cis‐elements conferring high levels of gene expression in non‐green plastids

Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research.