Green Tissue Research Articles

Achieving High Expression of Cry in Green Tissues and Negligible Expression in Endosperm Simultaneously via rbcS Gene Fusion Strategy in Rice.

To allay excessive public concern about the safety of transgenic foods, and to optimize insect-resistant genes expression to delay the evolution of resistance in pests, we developed a promising strategy to fuse the GOI (gene of interest) with OsrbcS (rice small subunit of ribulose bisphosphate carboxylase/oxygenase) in transgenic rice, which acted as a carrier, driven by the OsrbcS native promoter to sequester its expression in green tissues. Using eYFP as a trial, we reported a high-level accumulation of eYFP in green tissue and almost none in the seed and root of the fused construct compared to the non-fused construct. After applying this fusion strategy in insect-resistant rice breeding, recombinant OsrbcS-Cry1Ab/Cry1Ac expressed rice plants conferred high resistance to leaffolders and striped stem borers, among which two single-copy lines possessed normal agronomic performance in the field. Specifically, Cry1Ab/Cry1Ac protein levels in single-copy construct transgenic lines ranged from 1.8 to 11.5 µg g-1 in the leaf, higher than the Actin I promoter-driven control, T51-1, about 1.78 µg g-1 in the leaf, but negligible (only 0.00012-0.00117 µg g-1) in endosperm by ELISA analysis. Our study provided a novel approach to creating Cry1Ab/Cry1Ac-free endosperm rice with a high level of insect-resistant protein in green tissues through the simultaneous usage of the OsrbcS promoter and OsrbcS as a fusion partner.

Installing the neurospora carotenoid pathway in plants enables cytosolic formation of provitamin A and its sequestration in lipid droplets

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs, including β-carotene. This strategy led to the accumulation of significant amounts of phytoene and γ- and β-carotene, in addition to fungal, health-promoting carotenes with 13 conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs), which represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, β-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidial β-carotene. Moreover, engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of β-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Collectively, our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and thus paves the way for further optimization of carotenoid biofortification in crops.

Cambrian 'sap-sucking' molluscan radulae among small carbonaceous fossils (SCFs).

Molluscs have produced an extensive fossil record, owing to the prevalence of robust biomineralized shells among this clade. By contrast, most other components of molluscan anatomy are seldom preserved. Importantly, little is known of the evolutionary history of the unique molluscan feeding apparatus-the radula. A scarcity of fossil radulae has hampered our understanding of the ancestral condition, and of the dietary ecology of early molluscs. The handful of known fossil radulae all point to early molluscs as simple deposit feeders that obtained food via rasping or scraping. This study reports microscopic radulae preserved as 'small carbonaceous fossils' (SCFs) from Cambrian (Stage 4-Wuliuan, approximately 514-504.5 Ma) strata of Sweden. These rare fossil radulae offer novel insights into the feeding anatomy and ecology of early molluscs. Each radula comprises a uniseriate arc of (≤10) blade-shaped teeth, fringed by a slicing keel. This distinctive morphology is strikingly convergent with the radulae of extant sacoglossan heterobranch gastropods-such radulae are specially adapted for piercing the cell walls of green algal tissues to enable suctorial feeding on the cytoplasm contents. Discovery of analogous Cambrian radulae demonstrates this specialized form of herbivory had already evolved among molluscs more than half a billion years ago.

Melon yellow-green plant (Cmygp) encodes a Golden2-like transcription factor regulating chlorophyll synthesis and chloroplast development.

A SNP mutation in CmYGP gene encoding Golden2-like transcription factor is responsible for melon yellow-green plant trait. Chlorophylls are essential and beneficial substances for both plant and human health. Identifying the regulatory network of chlorophyll is necessary to improve the nutritional quality of fruits. At least six etiolation genes have been identified in different melon varieties, but none of them have been cloned, and the molecular mechanisms underlying chlorophyll synthesis and chloroplast development in melon remain unclear. Here, the NSL73046, a yellow-green plant (Cmygp) mutant, enabled the map-based cloning of the first etiolation gene in melon. CmYGP encodes a Golden2-like transcription factor. Spatiotemporal expression analyses confirmed the high CmYGP expression in all green tissues, particularly in young leaves and fruit peels. Virus-induced gene silencing and the development of near-isogenic line by marker-assisted selection further confirmed that downregulation of CmYGP can reduce chloroplast number and chlorophyll content, thereby resulting in yellow-green leaves and fruits in melon, and overexpression of CmYGP in tomatoes also led to dark-green leaves and fruits. RNA-seq analysis revealed that CmYGP greatly affected the expression of key genes associated with chloroplast development. Taken together, these findings demonstrated that CmYGP regulate chlorophyll synthesis and chloroplast development thus affect fruit development in melon. This study also offers a new strategy to enhance fruit quality in melon.

Novel Insights into the Contribution of Cyclic Electron Flow to Cotton Bracts in Response to High Light.

Cyclic electron flow around photosystem I (CEF-PSI) is shown to be an important protective mechanism to photosynthesis in cotton leaves. However, it is still unclear how CEF-PSI is regulated in non-foliar green photosynthetic tissues such as bracts. In order to learn more about the regulatory function of photoprotection in bracts, we investigated the CEF-PSI attributes in Yunnan 1 cotton genotypes (Gossypium bar-badense L.) between leaves and bracts. Our findings demonstrated that cotton bracts possessed PROTON GRADIENT REGULATION5 (PGR5)-mediated and the choroplastic NAD(P)H dehydrogenase (NDH)-mediated CEF-PSI by the same mechanism as leaves, albeit at a lower rate than in leaves. The ATP synthase activity of bracts was also lower, while the proton gradient across thylakoid membrane (ΔpH), rate of synthesis of zeaxanthin, and heat dissipation were higher than those of the leaves. These results imply that cotton leaves under high light conditions primarily depend on CEF to activate ATP synthase and optimize ATP/NADPH. In contrast, bracts mainly protect photosynthesis by establishing a ΔpH through CEF to stimulate the heat dissipation process.

Silencing of Pepper CaFtsH1 or CaFtsH8 Genes Alters Normal Leaf Development.

Filamentation temperature-sensitive H (FtsH) is a proteolytic enzyme that plays an important role in plant photomorphogenesis and stress resistance. However, information regarding the FtsH family genes in pepper is limited. In our research, through genome-wide identification, 18 members of the pepper FtsH family (including five FtsHi members) were identified and renamed based on phylogenetic analysis. CaFtsH1 and CaFtsH8 were found to be essential for pepper chloroplast development and photosynthesis because FtsH5 and FtsH2 were lost in Solanaceae diploids. We found that the CaFtsH1 and CaFtsH8 proteins were located in the chloroplasts and specifically expressed in pepper green tissues. Meanwhile, CaFtsH1 and CaFtsH8-silenced plants created by virus-induced gene silencing exhibited albino leaf phenotypes. In addition, CaFtsH1-silenced plants were observed to contain very few dysplastic chloroplasts and lost the capacity for photoautotrophic growth. Transcriptome analysis revealed that the expression of chloroplast-related genes such as those coding the photosynthesis-antenna protein and structural proteins was downregulated in CaFtsH1-silenced plants, resulting in the inability to form normal chloroplasts. This study improves our understanding of pepper chloroplast formation and photosynthesis through the identification and functional study of CaFtsH genes.

Two isoforms of Arabidopsis protoporphyrinogen oxidase localize in different plastidal membranes.

All land plants encode 2 isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis (Arabidopsis thaliana), the effects of PPO2 deficiency have not been investigated in detail. We identified 2 ppo2 T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, substantial changes in PPO activity were measured in etiolated and root tissues. However, ppo1 ppo2 double mutants were embryo-lethal. To shed light on possible functional differences between the 2 isoforms, PPO2 was overexpressed in the ppo1 background. Although the ppo1 phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid membranes. Mitochondrial localization of PPO2 in Arabidopsis was ruled out. Since Arabidopsis PPO2 does not encode a cleavable transit peptide, integration of the protein into the chloroplast envelope must make use of a noncanonical import route. However, when a chloroplast transit peptide was fused to the N-terminus of PPO2, the enzyme was detected predominantly in thylakoid membranes and was able to fully complement ppo1. Thus, the 2 PPO isoforms in Arabidopsis are functionally equivalent but spatially separated. Their distinctive localizations within plastids thus enable the synthesis of discrete subpools of the PPO product protoporphyrin IX, which may serve different cellular needs.

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.

Bioaccessibility of glucosinolates, isothiocyanates and inorganic micronutrients in cruciferous vegetables through INFOGEST static in vitro digestion model

Green tissues and seeds from cruciferous vegetables growing in conventional and ecological conditions (Brassica carinata; Brassica rapa; Eruca vesicaria and Sinapis alba) were analyzed to determine their contents of glucosinolates, isotihiocyanates (ITCs) and inorganic micronutrients (Ca, Cr, Cu, Fe, Mn, Ni, Se and Zn), and the bioaccessibility of these compounds. Regarding total contents and bioaccessibility values of these compounds, no clear difference was found between the organic and conventional systems. Glucosinolates bioaccessibility present in green tissues were high, with values around 60–78%. In additon, it was quantified in bioaccessible fraction ITCs concentrations such as Allyl – ITC; 3 – Buten – 1 – yl – ITC and 4 – Penten – 1 – yl – ITC. Trace elements bioaccessibility in green tissues was also high for Ca (2.26–7.66 mg/g), Cu (0.60–2.78 µg/g), Se (9.93–74.71 µg/Kg) and Zn (12.98–20.15 µg/g). By contrast, the bioaccessibility of glucosinolates and trace elements in cruciferous seeds was extremely low. With the exception of Cu, these bioaccessibility percentages did not exceed 1% in most cases.

Comparison between the stem and leaf photosynthetic productivity in Eucalyptus urophylla plantations with different age.

We developed a more realistic modeling framework by integrating stem photosynthesis into the canopy carbon assimilation model to compare the photosynthetic productivity between the stem and leaf of Eucalyptus urophylla plantations. Stems of Eucalyptus species with smooth outer bark have photosynthetic green tissue that can recycle internal stem CO2. However, the potential contribution of stem photosynthesis to forest productivity has not previously been adequately quantified, and we also do not know how it compares to leaf photosynthetic productivity. To assist in addressing this knowledge gap, we conducted field surveys in Eucalyptus urophylla plantations of different ages and developed a more realistic modeling framework by integrating stem photosynthesis into the existing canopy carbon assimilation model. We calculated the proportion of tree stems shaded by neighboring tree trunks based on Poisson spatial point process. Under the stand density of 2000 trees per hectare, the light absorption area of tree trunks of 2-year-old and 7-year-old E. urophylla plantations were 0.11 (± 0.15) and 0.35 (± 0.12) m2 stem m-2 land, the stem photosynthetic productivity (GPPstem) was 0.72 (± 0.45) and 1.81 (± 1.12) mol C m-2month-1, and the ratios of GPPstem to leaf photosynthetic productivity (GPPleaf) were 5.10 and 8.17% for 2- and 7-year-old plantations, respectively. Overall, this study presents the feasibility of incorporating stem photosynthesis into the productivity prediction of E. urophylla plantations by developing the stem light absorption model.

Genome-wide analysis of the polyphenol oxidase gene family reveals that MaPPO1 and MaPPO6 are the main contributors to fruit browning in Musa acuminate.

Polyphenol oxidases (PPOs), which are widely present in plants, play an important role in the growth, development, and stress responses. They can catalyze the oxidization of polyphenols and result in the browning of damaged or cut fruit, which seriously affects fruit quality and compromises the sale of fruit. In banana (Musa acuminata, AAA group), 10 PPO genes were determined based on the availability of a high-quality genome sequence, but the role of PPO genes in fruit browning remains unclear. In this study, we analyzed the physicochemical properties, gene structure, conserved structural domains, and evolutionary relationship of the PPO gene family of banana. The expression patterns were analyzed based on omics data and verified by qRT-PCR analysis. Transient expression assay in tobacco leaves was used to identify the subcellular localization of selected MaPPOs, and we analyzed the polyphenol oxidase activity using recombinant MaPPOs and transient expression assay. We found that more than two-thirds of the MaPPO genes had one intron, and all contained three conserved structural domains of PPO, except MaPPO4. Phylogenetic tree analysis revealed that MaPPO genes were categorized into five groups. MaPPOs did not cluster with Rosaceae and Solanaceae, indicating distant affinities, and MaPPO6/7/8/9/10 clustered into an individual group. Transcriptome, proteome, and expression analyses showed that MaPPO1 exhibits preferential expression in fruit tissue and is highly expressed at respiratory climacteric during fruit ripening. Other examined MaPPO genes were detectable in at least five different tissues. In mature green fruit tissue, MaPPO1 and MaPPO6 were the most abundant. Furthermore, MaPPO1 and MaPPO7 localized in chloroplasts, and MaPPO6 was a chloroplast- and Endoplasmic Reticulum (ER)-localized protein, whereas MaPPO10 only localized in the ER. In addition, the enzyme activity in vivo and in vitro of the selected MaPPO protein showed that MaPPO1 had the highest PPO activity, followed by MaPPO6. These results imply that MaPPO1 and MaPPO6 are the main contributors to banana fruit browning and lay the foundation for the development of banana varieties with low fruit browning.

Terahertz Vibrational Modes of Sodium Magnesium Chlorophyllin and Chlorophyll in Plant Leaves

The low-frequency (terahertz) vibrational spectroscopy has been investigated experimentally for two chlorophyll species, Chl-a and one of its magnesium derivatives (Chl-Mg-Na). The combination of terahertz time-domain spectroscopy and Fourier transform infrared spectroscopy has enabled a broad frequency range to be covered (0.2 to 18 THz). For Chl-Mg-Na, the terahertz spectra show clear and well-marked features at 1.44, 1.64, and 1.83 THz dominated by intermolecular interactions. The frequency dependent refractive index and absorption coefficient of Chl-Mg-Na were both determined using the Fit@TDS software. Below 1.0 THz, a refractive index of 2.09 was measured. In order to acquire further understanding of the observed vibrational modes, a detailed study of the temperature dependence of the line positions of the lowest modes in Chl-Mg-Na was performed. As the temperature was increased from 88 K to 298 K, the feature at 1.83 THz experienced a notable red shift of frequency and line shape broadening, whereas the feature at 1.44 THz showed little change. These results suggest that the 1.83 THz feature is dominated by intermolecular motions occurring over the crystalline unit cell of the Chl-Mg-Na molecular crystal. Finally, terahertz time-domain spectroscopy was used to acquire the spectra of an ornamental plant bearing yellow-green variegated leaves (ivy, Aureomarginata variety), the yellow sectors having lower chlorophyll content compared to the green sectors. In dehydrated green tissue, the chlorophyll molecules showed well-marked intermolecular vibrational modes at 1.86 THz, indicating that chlorophyll molecules are prone to packing with an ordered molecular arrangement. These results demonstrate the potential application of THz spectroscopy in the field of agronomy.

Carbon and Nitrogen Allocation between the Sink and Source Leaf Tissue in Response to the Excess Excitation Energy Conditions.

Plants are inevitably exposed to extreme climatic conditions that lead to a disturbed balance between the amount of absorbed energy and their ability to process it. Variegated leaves with photosynthetically active green leaf tissue (GL) and photosynthetically inactive white leaf tissue (WL) are an excellent model system to study source-sink interactions within the same leaf under the same microenvironmental conditions. We demonstrated that under excess excitation energy (EEE) conditions (high irradiance and lower temperature), regulated metabolic reprogramming in both leaf tissues allowed an increased consumption of reducing equivalents, as evidenced by preserved maximum efficiency of photosystem II (ФPSII) at the end of the experiment. GL of the EEE-treated plants employed two strategies: (i) the accumulation of flavonoid glycosides, especially cyanidin glycosides, as an alternative electron sink, and (ii) cell wall stiffening by cellulose, pectin, and lignin accumulation. On the other hand, WL increased the amount of free amino acids, mainly arginine, asparagine, branched-chain and aromatic amino acids, as well as kaempferol and quercetin glycosides. Thus, WL acts as an important energy escape valve that is required in order to maintain the successful performance of the GL sectors under EEE conditions. Finally, this role could be an adaptive value of variegation, as no consistent conclusions about its ecological benefits have been proposed so far.

Carotenoid metabolism: New insights and synthetic approaches.

Carotenoids are well-known isoprenoid pigments naturally produced by plants, algae, photosynthetic bacteria as well as by several heterotrophic microorganisms. In plants, they are synthesized in plastids where they play essential roles in light-harvesting and in protecting the photosynthetic apparatus from reactive oxygen species (ROS). Carotenoids are also precursors of bioactive metabolites called apocarotenoids, including vitamin A and the phytohormones abscisic acid (ABA) and strigolactones (SLs). Genetic engineering of carotenogenesis made possible the enhancement of the nutritional value of many crops. New metabolic engineering approaches have recently been developed to modulate carotenoid content, including the employment of CRISPR technologies for single-base editing and the integration of exogenous genes into specific "safe harbors" in the genome. In addition, recent studies revealed the option of synthetic conversion of leaf chloroplasts into chromoplasts, thus increasing carotenoid storage capacity and boosting the nutritional value of green plant tissues. Moreover, transient gene expression through viral vectors allowed the accumulation of carotenoids outside the plastid. Furthermore, the utilization of engineered microorganisms allowed efficient mass production of carotenoids, making it convenient for industrial practices. Interestingly, manipulation of carotenoid biosynthesis can also influence plant architecture, and positively impact growth and yield, making it an important target for crop improvements beyond biofortification. Here, we briefly describe carotenoid biosynthesis and highlight the latest advances and discoveries related to synthetic carotenoid metabolism in plants and microorganisms.

Biosynthesis of Chlorophyll and Other Isoprenoids in the Plastid of Red Grape Berry Skins.

Despite current knowledge showing that fruits like tomato and grape berries accumulate different components of the light reactions and Calvin cycle, the role of green tissues in fruits is not yet fully understood. In mature tomato fruits, chlorophylls are degraded and replaced by carotenoids through the conversion of chloroplasts in chromoplasts, while in red grape berries, chloroplasts persist at maturity and chlorophylls are masked by anthocyanins. To study isoprenoid and lipid metabolism in grape skin chloroplasts, metabolites of enriched organelle fractions were analyzed by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) and the expression of key genes was evaluated by real-time polymerase chain reaction (PCR) in berry skins and leaves. Overall, the results indicated that chloroplasts of the grape berry skins, as with leaf chloroplasts, share conserved mechanisms of synthesis (and degradation) of important components of the photosynthetic machinery. Some of these components, such as chlorophylls and their precursors, and catabolites, carotenoids, quinones, and lipids have important roles in grape and wine sensory characteristics.

Photosynthesis - beyond the leaf.

Although leaves are considered the main site for photosynthesis, other green nonfoliar tissues can carry out considerable amounts of photosynthetic carbon assimilation. With photosynthesis, a potential target for improving crop productivity, physiology and contribution of nonfoliar tissues to overall plant carbon acquisition is gaining increasing attention. This review will provide an overview of nonfoliar photosynthesis, the role of stomata in these tissues and methodologies for quantification and the contribution to overall carbon gain.

Stomatal conductance in rice leaves and panicles responds differently to abscisic acid and soil drought.

Improvement of photosynthesis in non-foliar green tissues is beneficial for enhancing crop yield. Recently, we have demonstrated that panicle stomatal conductance is a major limiting factor for photosynthesis. However, mechanisms underlying the responses of panicle stomatal conductance (gs,panicle) and photosynthesis (Apanicle) to environmental stimuli remain unknown. In the present study, the responses of gs,panicle and leaf stomatal conductance (gs,leaf) to exogenous application of abscisic acid and step-changes in vapor pressure deficit were investigated at the anthesis stage in pot-grown rice plants. Furthermore, the effects of drought on Apanicle and leaf photosynthesis (Aleaf) were examined. Smearing and xylem feeding of abscisic acid significantly decreased gs,leaf. In contrast, while smearing of abscisic acid substantially increased gs,panicle, its xylem feeding dramatically decreased gs,panicle. In addition, both gs,leaf and gs,panicle effectively responded to step changes in vapor pressure deficit. Furthermore, both Aleaf and Apanicle were sensitive to plant dehydration; however, given the lower sensitivity of panicle water potential than leaf water potential to drought, Apanicle was less sensitive to soil drought than Aleaf. These findings indicate that gs,panicle is hydropassively regulated, while panicle photosynthesis is less sensitive to drought.

Fungal Metabarcoding Data for Two Grapevine Varieties (Regent and Vitis vinifera ‘Cabernet-Sauvignon’) Inoculated with Powdery Mildew (Erysiphe necator) Under Drought Conditions

Fungal Metabarcoding Data for Two Grapevine Varieties (Regent and <i>Vitis vinifera</i> ‘Cabernet-Sauvignon’) Inoculated with Powdery Mildew (<i>Erysiphe necator</i>) Under Drought Conditions

First Report of Bacterial Leaf Scald of Plum Caused by Xylella fastidiosa in Texas.

Xylella fastidiosa is the etiological agent of Plum Leaf Scald (Greco et al. 2021). The disease was first reported in Argentina (Fernandez-Valiela et al. 1954) and then Brazil and Paraguay (French et al. 1978). In the USA, Plum Leaf Scald has been reported in the Southeastern United States (Wells et al. 1981a) and California (Hernandez-Martinez et al. 2009). In August 2021, during the Stone Fruit Survey of FY2020, plum trees (Mexican variety, Prunus mexicana) with symptoms of leaf scald, were observed in a Central Texas orchard with approximately 7% of trees exhibiting symptoms. Leaf margins were asymmetrically scorched, with necrotic areas that transitioned into chlorotic and healthy green tissues. To detect the presence of the pathogen, leaf sample petioles were tested using a double-antibody sandwich (DAS) ELISA® with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) according to manufacturer's guidelines. X. fastidiosa was detected in 20 of the 35 symptomatic samples. To confirm ELISA results, total DNA was extracted from the plant samples using the Plant DNeasy® kit (Qiagen Co. Hilden, Germany) following the manufacturer's protocol. All 20 ELISA-positive samples tested positive in a X. fastidiosa-specific real time PCR assay, using the primers XF1F and XF1R and probe XF1p (Schaad et al. 2002). Moreover, the ELISA-negative samples were also negative for PCR assay. Symptomatic samples were used to isolate the pathogen. Samples were debarked, surface-sterilized and xylem fluid collected. The fluid was gently imprinted on buffered charcoal yeast extract (BCYE) (Wells et al. 1981b) or periwinkle wilt modified (PWM) agar plates (Summer et al. 2010). After 10 days of incubation, individual colonies were observed. The colonies were slightly convex, white, opalescent, mucoid, circular with entire margins and with smooth surfaces on both media plates. Isolated colonies were triple-streak single colony purified and archived. Genomic DNA was extracted from four purified isolates using the DNeasy Blood and Tissue Qiagen® Kit, to conduct conventional PCR using HL5/HL6 (Francis et al. 2006), which identified the isolates as X. fastidiosa. Using the 16S rRNA primer pair U3/U4 (James 2010), amplicons were sequenced and compared against the NCBI database using the BLASTn algorithm. Comparative sequence analysis of amplicons from the four isolates were identical and indicated that the isolates were 100% identical to X. fastidiosa subsp. multiplex RIV5 (CP064326.1) from cherry plum, and IVIA5901 (CP047134.1) from almond. The sequences of all four isolates were deposited into NCBI GenBank, with the accession numbers OM617940 (467), OM617941 (470), OM617942 (471) and OM617943 (468). To our knowledge, this is the first report of X. fastidiosa associated with plum leaf scald in Texas, extending the geographical range of this important bacterial disease, in the Southern United States. This study highlights the importance of routine scouting of agricultural settings with a view to assessing and detecting early threats from either pests or disease and implementing relevant management strategies.

High-throughput discovery of plastid genes causing albino phenotypes in ornamental chimeric plants.

Chimeric plants composed of green and albino tissues have great ornamental value. To unveil the functional genes responsible for albino phenotypes in chimeric plants, we inspected the complete plastid genomes (plastomes) in green and albino leaf tissues from 23 ornamental chimeric plants belonging to 20 species, including monocots, dicots, and gymnosperms. In nine chimeric plants, plastomes were identical between green and albino tissues. Meanwhile, another 14 chimeric plants were heteroplasmic, showing a mutation between green and albino tissues. We identified 14 different point mutations in eight functional plastid genes related to plastid-encoded RNA polymerase (rpo) or photosystems which caused albinism in the chimeric plants. Among them, 12 were deleterious mutations in the target genes, in which early termination appeared due to small deletion-mediated frameshift or single nucleotide substitution. Another was single nucleotide substitution in an intron of the ycf3 and the other was a missense mutation in coding region of the rpoC2 gene. We inspected chlorophyll structure, protein functional model of the rpoC2, and expression levels of the related genes in green and albino tissues of Reynoutria japonica. A single amino acid change, histidine-to-proline substitution, in the rpoC2 protein may destabilize the peripheral helix of plastid-encoded RNA polymerase, impairing the biosynthesis of the photosynthesis system in the albino tissue of R. japonica chimera plant.