NOA1 Functions in a Temperature-Dependent Manner to Regulate Chlorophyll Biosynthesis and Rubisco Formation in Rice

NITRIC OXIDE-ASSOCIATED1 (NOA1) encodes a circularly permuted GTPase (cGTPase) known to be essential for ribosome assembly in plants. While the reduced chlorophyll and Rubisco phenotypes were formerly noticed in both NOA1-supressed rice and Arabidopsis, a detailed insight is still necessary. In this study, by using RNAi transgenic rice, we further demonstrate that NOA1 functions in a temperature-dependent manner to regulate chlorophyll and Rubisco levels. When plants were grown at 30°C, the chlorophyll and Rubisco levels in OsNOA1-silenced plants were only slightly lower than those in WT. However, at 22°C, the silenced plants accumulated far less chlorophyll and Rubisco than WT. It was further revealed that the regulation of chlorophyll and Rubisco occurs at the anabolic level. Etiolated WT seedlings restored chlorophyll and Rubisco accumulations readily once returned to light, at either 30°C or 15°C. Etiolated OsNOA1-silenced plants accumulated chlorophyll and Rubisco to normal levels only at 30°C, and lost this ability at low temperature. On the other hand, de-etiolated OsNOA1-silenced seedlings maintained similar levels of chlorophyll and Rubisco as WT, even after being shifted to 15°C for various times. Further expression analyses identified several candidate genes, including OsPorA (NADPH: protochlorophyllide oxidoreductase A), OsrbcL (Rubisco large subunit), OsRALyase (Ribosomal RNA apurinic site specific lyase) and OsPuf4 (RNA-binding protein of the Puf family), which may be involved in OsNOA1-regulated chlorophyll biosynthesis and Rubisco formation. Overall, our results suggest OsNOA1 functions in a temperature-dependent manner to regulate chlorophyll biosynthesis, Rubisco formation and plastid development in rice

Glycolate Oxidase Isozymes Are Coordinately Controlled by GLO1 and GLO4 in Rice

Glycolate oxidase (GLO) is a key enzyme in photorespiratory metabolism. Four putative GLO genes were identified in the rice genome, but how each gene member contributes to GLO activities, particularly to its isozyme profile, is not well understood. In this study, we analyzed how each gene plays a role in isozyme formation and enzymatic activities in both yeast cells and rice tissues. Five GLO isozymes were detected in rice leaves. GLO1 and GLO4 are predominately expressed in rice leaves, while GLO3 and GLO5 are mainly expressed in the root. Enzymatic assays showed that all yeast-expressed GLO members except GLO5 have enzymatic activities. Further analyses suggested that GLO1, GLO3 and GLO4 interacted with each other, but no interactions were observed for GLO5. GLO1/GLO4 co-expressed in yeast exhibited the same isozyme pattern as that from rice leaves. When either GLO1 or GLO4 was silenced, expressions of both genes were simultaneously suppressed and most of the GLO activities were lost, and consistent with this observation, little GLO isozyme protein was detected in the silenced plants. In contrast, no observable effect was detected when GLO3 was suppressed. Comparative analyses between the GLO isoforms expressed in yeast and the isozymes from rice leaves indicated that two of the five isozymes are homo-oligomers composed of either GLO1 or GLO4, and the other three are hetero-oligomers composed of both GLO1 and GLO4. Our current data suggest that GLO isozymes are coordinately controlled by GLO1 and GLO4 in rice, and the existence of GLO isozymes and GLO molecular and compositional complexities implicate potential novel roles for GLO in plants

Experimental and numerical studies on spray characteristics of an internal oscillating nozzle

© 2019 by Begell House, Inc. An open atomization test bench based on high-speed Schlieren technology and a Malvern particle size analyzer was developed to investigate the effects of different injection pressures (0.12 MPa to 0.24 MPa) on spray characteristics of an internal oscillating nozzle, including the spatial distribution of flow rate, oscillation frequency, spray cone angle, spatial distribution of spray particle size, and velocity. A numerical investigation that simultaneously considered the internal flow field and the external spray within the same computational field was performed to reveal the oscillation mechanism. The experimental results indicated that the spray of the internal oscillating nozzle shows a fan shape distribution with small flow in the middle and large distribution on both sides. The flow rate gradually increases with the rising of injection pressure and reaches its maximum at 0.24 MPa when the distance from the nozzle is constant. The oscillating frequency keeps an upward tendency with a maximum growth rate when the injection pressure ascends from 0.15 MPa to 0.18 MPa. The spray cone angle does not change significantly with the increase of the injection pressure, fluctuating at approximately 41.8 degrees. Moreover, a critical injection pressure is obtained, below which the droplet size increases with the rise of the injection pressure and above which the droplet size declines moderately. The numerical investigation revealed that the oscillation phenomenon was generated due to the periodic establishing and vanishing of the pressure gradient within the feedback channels, and the Coanda effect occurred in the main flow passage

Catalytic activities of the GLO isoforms expressed in yeast.

<p>Y-GLO1S2, Y-GLO3S2, Y-GLO4S2, Y-GLO5S2 represent the crude enzyme extracted from yeast cells expressing pYES2-<i>GLO1</i>, pYES2-<i>GLO3</i>, pYES2-<i>GLO4</i>, pYES2-<i>GLO5</i>, respectively. Y-GLO1S3, Y-GLO3S3, Y-GLO4S3, represent the crude enzyme extracted from yeast cells expressing pYES3-<i>GLO1</i>, pYES3-<i>GLO3</i>, pYES3-<i>GLO4</i>, respectively. Y-GLO1S3+3S2, Y-GLO1S3+4S2, Y-GLO1S3+5S2, Y-GLO4S3+3S2, Y-GLO4S3+5S2, Y-GLO3S3+5S2, represent the crude enzyme extracted from yeast cells co-expressing pYES3-<i>GLO1</i> and pYES2-<i>GLO3</i>, pYES3-<i>GLO1</i> and pYES2-<i>GLO4</i>, pYES3-<i>GLO1</i> and pYES2-<i>GLO5</i>, pYES3-<i>GLO4</i> and pYES2-<i>GLO3</i>, pYES3-<i>GLO4</i> and pYES2-<i>GLO5</i>, pYES3-<i>GLO3</i> and pYES2-<i>GLO5</i>, respectively. R-GLO represents the crude enzyme extracted from leaves of rice. The data represent means ±SD of 3 independent experiments.</p

GLO isozyme patterns and the expression of various <i>GLO</i> members in rice.

<p>(<b>A</b>) Enzymatic activity staining showing patterns of GLO isozymes. GLO enzyme was extracted from rice leaves and separated by 6% CN-PAGE at a running pH of 10.2. The number above each lane indicates activity units (µmol H₂O₂ min⁻¹ mg⁻¹ protein) loaded. Arrows point to each isozyme band. This result is representative of five independent experiments. (<b>B</b>) mRNA transcript abundance of the four <i>GLO</i> genes (<i>GLO1, GLO3, GLO4,</i> and <i>GLO5</i>) in rice leaves and roots was determined by real-time quantitative RT-PCR. The second leaf from the top and 5 cm of roots were detached from plants for RNA isolation. Relative mRNA levels were graphed based on the mRNA level of Leaf-<i>GLO1</i> as 1. The data represent means ±SD of 3 independent experiments.</p

BiFC detection of interactions among the proteins encoded by the four <i>GLO</i> genes.

<p>Every two <i>GLO</i> genes were co-expressed in rice protoplasts and BiFC visualization assays were carried out to test the interaction as indicated: (A) NYFP-tagged GLO1 and CYFP-tagged GLO3; (B) NYFP-tagged GLO1 and CYFP-tagged GLO4; (C) NYFP-tagged GLO1 and CYFP-tagged GLO5; (D) NYFP-tagged GLO3 and CYFP-tagged GLO4; (E) NYFP-tagged GLO3 and CYFP-tagged GLO5; (F) NYFP-tagged GLO4 and CYFP-tagged GLO5; (G) NYFP and CYFP. This result is representative of three independent experiments.</p

Subcellular localization of GLO isoforms.

<p>The YFP-tagged GLOs and CFP-tagged PTS1 fusion constructs were used in protoplast transient expression system to determine the subcellular localization of GLOs, the CFP-tagged PTS1 was used as the peroxisome marker. A1–A4: transfection of rice protoplasts; B1–B4: transfection of <i>Arabidopsis</i> protoplasts. This result is representative of three independent experiments.</p

Comparison between the GLO isoforms expressed in yeast and those extracted from rice leaves.

<p>GLO isozymes were separated in 6% uniform CN-PAGE at a running pH of 10.2. Y-GLO1, Y-GLO3, Y-GLO4, represent the enzyme extracted from yeast cells expressing pYES3-<i>GLO1</i>, pYES3-<i>GLO3</i>, pYES3-<i>GLO4</i>, respectively. Y-GLO3+4, Y-GLO3+1, Y-GLO1+4, represent the enzyme extracted from yeast cells co-expressing pYES3-<i>GLO3</i> and pYES2-<i>GLO4</i>, pYES3-<i>GLO3</i> and pYES2-<i>GLO1</i>, pYES3-<i>GLO1</i> and pYES2-<i>GLO4</i>, respectively. R-GLO represents the enzyme extracted from leaves of rice, and R-OxGLO4 represents the enzyme extracted from leaves of <i>GLO4</i>-overexpressed transgenic rice. This result is representative of five independent experiments.</p

Interactions of the GLOs determined by His-tag pull down assay.

<p>GLO-his means the 6 amino acids on the C-terminus of GLO was mutated to a 6×his-tag. The interactions between every two GLOs are evaluated by calculating the activity recovery rate. The data are means of 3 independent experiments.</p