Low nitrogen stress regulates chlorophyll fluorescence in coordination with photosynthesis and Rubisco efficiency of rice

Nitrogen (N) is the basis of plant growth and development and, is considered as one of the priming agents to elevate a range of stresses. Plants use solar radiations through photosynthesis, which amasses the assimilatory components of crop yield to meet the global demand for food. Nitrogen is the main regulator in the allocation of photosynthetic apparatus which changes of the photosynthesis (P_n) and quantum yield (F_v/F_m) of the plant. In the present study, dynamics of the photosynthetic establishment, N-dependent relation with chlorophyll fluorescence attributes and Rubisco efficacy was evaluated in low-N tolerant (cv. CR Dhan 311) and low-N sensitive (cv. Rasi) rice cultivars under low-N and optimum-N conditions. There was a decrease in the stored leaf N under low-N condition, resulting in the decreased P_n and F_v/F_m efficiency of the plants through depletion in the activity and content of Rubisco. The P_n and F_v/F_m followed the parallel trend of leaf N content during low-N condition along with depletion of intercellular CO₂ concentration and overall conductance under low-N condition. Photosynthetic saturation curve cleared abrupt decrease of effective quantum yield in the low-N sensitive rice cultivar than the low-N tolerant rice. Also, the rapid light curve highlighted the unacclimated regulation of photochemical and non-photochemical quenching in the low-N condition. The low-N sensitive rice cultivar triumphed non-photochemical quenching, whereas the low-N tolerant rice cultivar rose gradually during the light curve. Our study suggested that the quantum yield is the key limitation for photosynthesis in low-N condition. Regulation of Rubisco, photochemical and non-photochemical quenching may help plants to grow under low-N level.

     

OsMLH1 interacts with OsMLH3 to regulate synapsis and interference-sensitive crossover formation during meiosis in rice

Meiotic recombination is essential for reciprocal exchange of genetic information between homologous chromosomes and their subsequent proper segregation in sexually reproducing organisms. MLH1 and MLH3 belong to meiosis-specific members of the Mut L-homolog family, which are required for normal level of crossovers(COs) in some eukaryotes. However, their functions in plants need to be further elucidated.Here, we report the identification of Os MLH1 and reveal its functions during meiosis in rice. Using CRISPRCas9 approach, two independent mutants, Osmlh1-1 and Osmlh1-2, are generated and exhibited significantly reduced male fertility. In Osmlh1-1, the clearance of PAIR2 is delayed and partial ZEP1 proteins are not loaded into the chromosomes, which might be due to the deficient in resolution of interlocks at late zygotene. Thus, Os MLH1 is required for the assembly of synapsis complex. In Osmlh1-1, CO number is dropped by ～53% and the distribution of residual COs is consistent with predicted Poisson distribution,indicating that Os MLH1 is essential for the formation of interference-sensitive COs(class I COs). Os MLH1 interacts with Os MLH3 through their C-terminal domains. Mutation in Os MLH3 also affects the pollen fertility. Thus, our experiments reveal that the conserved heterodimer Mut Lg(Os MLH1-Os MLH3) is essential for the formation of class I COs in rice.     

A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis

Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.     

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Co-overproducing Rubisco and Rubisco activase enhances photosynthesis in the optimal temperature range in rice

Rubisco limits C₃ photosynthesis under some conditions and is therefore a potential target for improving photosynthetic efficiency. The overproduction of Rubisco is often accompanied by a decline in Rubisco activation, and the protein ratio of Rubisco activase (RCA) to Rubisco (RCA/Rubisco) greatly decreases in Rubisco-overproducing plants (RBCS-ox). Here, we produced transgenic rice (Oryza sativa) plants co-overproducing both Rubisco and RCA (RBCS-RCA-ox). Rubisco content in RBCS-RCA-ox plants increased by 23%–44%, and RCA/Rubisco levels were similar or higher than those of wild-type plants. However, although the activation state of Rubisco in RBCS-RCA-ox plants was enhanced, the rates of CO₂ assimilation at 25°C in RBCS-RCA-ox plants did not differ from that of wild-type plants. Alternatively, at a moderately high temperature (optimal range of 32°C–36°C), the rates of CO₂ assimilation in RBCS-ox and RBCS-RCA-ox plants were higher than in wild-type plants under conditions equal to or lower than current atmospheric CO₂ levels. The activation state of Rubisco in RBCS-RCA-ox remained higher than that of RBCS-ox plants, and activated Rubisco content in RCA overproducing, RBCS-ox, RBCS-RCA-ox, and wild-type plants was highly correlated with the initial slope of CO₂ assimilation against intercellular CO₂ pressures (A:Ci) at 36°C. Thus, a simultaneous increase in Rubisco and RCA contents leads to enhanced photosynthesis within the optimal temperature range.

A simultaneous increase in Rubisco and RCA contents in transgenic rice leads to an enhancement of photosynthesis at moderately high temperatures within the optimal temperature range.     

Green-revertible Chlorina 1 (grc1) is required for the biosynthesis of chlorophyll and the early development of chloroplasts in rice

The nuclear genes involved in chloroplast development and chlorophyll biosynthesis must be investigated to understand their functions in plant growth and development. In this study, we isolated and identified a unique leaf-color mutant of rice with a green-yellow phenotype before the four-leaf stage and named the mutation green-revertible chlorina 1 (grc1). The mutants had significantly lower plant height, number of tillers, and panicle length and headed significantly earlier than the wild type. The levels of chlorophylls, carotenoids, and chlorophyll precursors were also lower. The mutation in grc1 affected chloroplast ultrastructure, particularly thylakoid development. Genetic analysis indicated that the green-yellow phenotype was controlled by a single recessive gene. We mapped the grc1 gene to a 32.4-kb region on the long arm of chromosome 6. Through map-based cloning, we identified a 45-bp insertion in the genomic region of LOC_Os06g40080, which encoded a heme oxygenase. Expression of LOC_Os06g40080 was significantly down-regulated in the grc1 mutant. Subcellular localization showed that this heme oxygenase was localized in the chloroplast. In summary, we isolated and identified the gene for grc1, which plays an important role in chlorophyll biosynthesis and chloroplast development in rice.     

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.