Mitigation of Huanglongbing: Implications of a Biologically Enhanced Nutritional Program on Yield, Pathogen Localization, and Host Gene Expression Profiles.

Huanglongbing (HLB, citrus greening disease), the most destructive disease affecting citrus production, is primarily linked to the gram-negative, insect-vectored, phloem-inhabiting α-proteobacterium 'Candidatus Liberibacter asiaticus' (CLas). With no effective treatment available, management strategies have largely focused on the use of insecticides in addition to the destruction of infected trees, which are environmentally hazardous and cost-prohibitive for growers, respectively. A major limitation to combating HLB is the inability to isolate CLas in axenic culture, which hinders in vitro studies and creates a need for robust in situ CLas detection and visualization methods. The aim of this study was to investigate the efficacy of a nutritional program-based approach for HLB treatment, and to explore the effectiveness of an enhanced immunodetection method to detect CLas-infected tissues. To achieve this, four different biologically enhanced nutritional programs (bENPs; P1, P2, P3, and P4) were tested on CLas-infected citrus trees. Structured illumination microscopy preceded by a modified immunolabeling process and transmission electron microscopy were used to show treatment-dependent reduction of CLas cells in phloem tissues. No sieve pore plugging was seen in the leaves of P2 trees. This was accompanied by an 80% annual increase in fruit number per tree and 1,503 (611 upregulated and 892 downregulated) differentially expressed genes. These included an MLRQ subunit gene, UDP-glucose transferase, and genes associated with the alpha-amino linolenic acid metabolism pathway in P2 trees. Taken together, the results highlight a major role for bENPs as a viable, sustainable, and cost effective option for HLB management.

Comparative Clinical Evaluation of Semilunar Coronally Positioned Flap Alone and Semilunar Coronally Positioned Flap in Conjunction with Free Gingival Graft for Root Coverage.

Gingival recession accounts for apical migration of the gingival margin, resulting in exposure of the cementoenamel junction and root surface, with exposure of the root surface linked to deteriorated esthetic appearance and increased dentinal hypersensitivity. Various surgical techniques have been used to correct labial gingival recession defects. The present study evaluated and compared the results of semilunar coronally positioned flap (SCPF) alone and in conjunction with free gingival graft (FGG) for the treatment of Miller Class I and II gingival recession defects in maxillary anterior teeth. A total of 20 bilateral Miller Class I and II gingival recession sites were included and randomly allocated (n = 10 sites/group) to either the semilunar coronally positioned flap technique alone (SCPF group; control) or with FGG (SCPF+FGG group; test). Longitudinal alterations in probing depth (PD), recession width (RW), recession height (RH), width of keratinized tissue (WKT), and clinical attachment level (CAL) were measured and analyzed for both groups at 1-, 3-, 6-, and 12-month follow-ups. Both groups saw a significant decrease in RH, RW, and CAL and a significant increase in WKT. No statistically significant difference was observed in the final root coverage outcome between both groups in terms of RH, RW, and CAL, but a significant increase in WKT was seen with SCPF+FGG. Both techniques demonstrated optimal results without significant differences in the final root coverage outcomes except for WKT, which had a statistically significant increase in the SCPF+FGG group.

The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO2-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.

Mutation of the membrane-associated M1 protease APM1 results in distinct embryonic and seedling developmental defects in Arabidopsis.

Aminopeptidase M1 (APM1), a single copy gene in Arabidopsis thaliana, encodes a metallopeptidase originally identified via its affinity for, and hydrolysis of, the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Mutations in this gene result in haploinsufficiency. Loss-of-function mutants show irregular, uncoordinated cell divisions throughout embryogenesis, affecting the shape and number of cotyledons and the hypophysis, and is seedling lethal at 5 d after germination due to root growth arrest. Quiescent center and cell cycle markers show no signals in apm1-1 knockdown mutants, and the ground tissue specifiers SHORTROOT and SCARECROW are misexpressed or mislocalized. apm1 mutants have multiple, fused cotyledons and hypocotyls with enlarged epidermal cells with cell adhesion defects. apm1 alleles show defects in gravitropism and auxin transport. Gravistimulation decreases APM1 expression in auxin-accumulating root epidermal cells, and auxin treatment increases expression in the stele. On sucrose gradients, APM1 occurs in unique light membrane fractions. APM1 localizes at the margins of Golgi cisternae, plasma membrane, select multivesicular bodies, tonoplast, dense intravacuolar bodies, and maturing metaxylem cells. APM1 associates with brefeldin A-sensitive endomembrane structures and the plasma membrane in cortical and epidermal cells. The auxin-related phenotypes and mislocalization of auxin efflux proteins in apm1 are consistent with biochemical interactions between APM1 and NPA.

Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis.

Directional transport of the phytohormone auxin is established primarily at the point of cellular efflux and is required for the establishment and maintenance of plant polarity. Studies in whole plants and heterologous systems indicate that PIN-FORMED (PIN) and P-glycoprotein (PGP) transport proteins mediate the cellular efflux of natural and synthetic auxins. However, aromatic anion transport resulting from PGP and PIN expression in nonplant systems was also found to lack the high level of substrate specificity seen in planta. Furthermore, previous reports that PGP19 stabilizes PIN1 on the plasma membrane suggested that PIN-PGP interactions might regulate polar auxin efflux. Here, we show that PGP1 and PGP19 colocalized with PIN1 in the shoot apex in Arabidopsis thaliana and with PIN1 and PIN2 in root tissues. Specific PGP-PIN interactions were seen in yeast two-hybrid and coimmunoprecipitation assays. PIN-PGP interactions appeared to enhance transport activity and, to a greater extent, substrate/inhibitor specificities when coexpressed in heterologous systems. By contrast, no interactions between PGPs and the AUXIN1 influx carrier were observed. Phenotypes of pin and pgp mutants suggest discrete functional roles in auxin transport, but pin pgp mutants exhibited phenotypes that are both additive and synergistic. These results suggest that PINs and PGPs characterize coordinated, independent auxin transport mechanisms but also function interactively in a tissue-specific manner.

PGP4, an ATP binding cassette P-glycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots.

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid-reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells.

Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana.

Aglycone flavonols are thought to modulate auxin transport in Arabidopsis thaliana via an as yet undefined mechanism. Biochemical studies suggest that flavonoids interact with regulatory proteins rather than directly with the PIN auxin efflux facilitator proteins. Auxin transport is enhanced in the absence of flavonoids (transparent testa4 [tt4]) and reduced in the presence of excess flavonols (tt7 and tt3). Steady state PIN mRNA levels in roots inversely correlate with auxin movement in tt mutants. PIN gene transcription and protein localization in flavonoid-deficient mutants appear to be modulated by developmental cues and are auxin responsive. Modulation of PIN gene expression and protein distribution by localized auxin accumulations occurs in the wild type as well. Flavonoids inhibit auxin transport primarily at the shoot apex and root tip and appear to modulate vesicular cycling of PIN1 at the root tip. In some auxin-accumulating tissues, flavonoid increases and changes in flavonoid speciation are subsequent to auxin accumulation.