OsARG encodes an arginase that plays critical roles in panicle development and grain production in rice

Nitrogen is a crucial nutrient for plant growth and development. Arginine is considered to be an important amino acid for nitrogen transport and storage, playing a crucial role during plant seedling development. However, little is known about the role of arginine in nitrogen remobilization at the reproductive stage. We isolated a rice mutant nglf‐1 with reduced plant height, small panicle and grain size, and low seed‐setting rate (10% in nglf‐1 compared to 93% in wild‐type). Map‐based cloning revealed that the mutant phenotype was caused by loss of function of a gene (OsARG) encoding an arginine hydrolysis enzyme, which is consistent with arginine accumulation in the mutant. The phenotype was partially corrected supplying exogenous nitrogen, and fully corrected by expression of a wild‐type OsARG transgene. Over‐expression of OsARG in rice (cv. Kitaake) increased grain number per plant under nitrogen‐limited conditions. OsARG was ubiquitously expressed in various organs, but most strongly in developing panicles. The OsARG protein was localized in the mitochondria, consistent with other arginases. Our results suggest that the arginase encoded by OsARG, a key enzyme in Arg catabolism, plays a critical role during panicle development, especially under conditions of insufficient exogenous nitrogen. OsARG is a potential target for crop improvement.     

Morpho-physiological variations in response to NaCl stress during vegetative and reproductive development of rice

The complex nature of plant resistance to adverse environmental conditions, such as salinity and drought requires a better understanding of the stress-induced changes that may be involved in tolerance mechanisms. Here we investigate stress-related morpho-physiological effects during vegetative and reproductive growth in two Japonica rice cultivars (Bomba and Bahia) exposed to a range of NaCl concentrations from the seedling stage. The stress-related detrimental effects were observed either earlier or to a higher extent in cv. Bomba than in Bahia. Damages to the photosynthetic apparatus were related to loss of chlorophyll (Chl) and to a decrease of the maximum potential efficiency of PSII (F _v /F _m), affecting negatively net CO₂ assimilation rate (P _N). Stress-related leaf anatomical alterations were analysed during the vegetative and reproductive stages. The size of bulliform cells as well as dimensions related to the vascular system increased under mild stress but decreased in the longer term or under higher stress level. The pattern of the anatomical alterations observed at the reproductive stage under 20 mM NaCl was reflected in poor panicle development and yield loss, with effects more pronounced in cv. Bomba than in Bahia. In summary, our results show that some physiological and, particularly, leaf anatomical responses induced by NaCl stress are distinctive indicators of sensitivity to salt stress in rice cultivars.     

Comparison of Membrane Pluidity of Seedling Mitochondria of Chilling Sensitive and Chilling Tolerant Rice

The membrane fluidity of seedling mitochondria of chilling-sensitive rice and that of chilling-tolerant rice were compared by using spin labeled stearic acid: 5, 12 16-NS and fluorescent probe DPH. From the ESR spectra using 5-NS as a spin labeled probe it clearly showed that the calculated order parameter (S) of seedling mitochondria of chilling-sensitive rice Qiu Guang was obviously higher than that of chilling-resistant rice Ji Geng 44. Similar results were obtained when seedling'mitochondria of another species of chilling sensitive Zao Jin were compared with those of chilling tolerant rice Ji Geng 60. Moreover, the difference of order parameters between Ji Geng 44 and Ji Geng 60 was quite small, but both of them are obviously lower than those of chilling-sensitive rice Qiu Guang or Zao Jin. The results using spin labeled probe 12-NS, 16-NS clearly showed that the relative correlation times (τc) of seedling mitochondria of chilling-sensitive rice Qiu Guang or Zao Jin was markedly higher than that of the chilling tolerant rice Ji Geng 44 or Ji Geng 60. A comparison of membrane fluidity of seedling mitochondria of chilling-sensitive and chilling-resistant rice using fluorescent probe DPH was also carried out. Similar results were obtained and showed that the fluidity of mitochondrial membrane of chilling resistant rice seedling was obviously higher than that of the chilling-sensitive ones. Thus, it seemed that the fluidity of mitochondrial membrane might be used as a biophysical test for screening chilling tolerance of rice at seedling stage.     

Effects of anoxia on mitochondrial biogenesis in rice shoots: modification of in organello translation characteristics

Shoots of germinating rice (Oryza sativa L.) seedlings are able to grow under anoxia and to withstand long periods of anoxic treatment. Mitochondria were purified from aerobically germinated and anaerobically treated rice shoots by differential and isopycnic centrifugation and were found to consist of two subpopulations. The mitochondrial subpopulation of higher density was used for further characterization. Ultrastructural studies showed anaerobic mitochondria to be significantly different from aerobic mitochondria, with a matrix of lower density and more developed cristae. Aerobic and anaerobic mitochondria also differed in their specific activities for fumarase and succinate dehydrogenase, which were significantly lower after the anoxic treatment. In vivo labeling of seedlings with l-[³⁵S]methionine and subsequent isolation of the mitochondria indicated that anoxia induced a drastic decrease, but not a total inactivation, of the synthesis of mitochondrial proteins. In organello protein synthesis showed that anaerobic mitochondria were able to synthesize most of the polypeptides synthesized by aerobic mitochondria, although only in the presence of exogenous ATP, as would occur under anoxia. Anaerobic mitochondria, but not aerobic mitochondria, could carry out protein synthesis without a functional respiratory chain. Thus, mitochondrial protein synthesis was found to be potentially functional in the rice shoot under anoxia.     

Nucleo-cytoplasmic interactions affect RNA editing of cox2, atp6 and atp9 in alloplasmic male-sterile rice (Oryza sativa L.) lines

RNA editing plays an important role in the regulation of mitochondrial gene expression in flowering plants. In this study, we examined RNA editing of the mitochondrial genes cox2, atp6 and atp9 in five isonuclear alloplasmic male-sterile lines (IAMSLs) of rice to investigate whether different cytoplasmic types affect RNA editing. Although many editing sites were conserved among the three genes, we found that the editing efficiency of certain sites was significantly different between different IAMSLs or between IAMSLs and their corresponding cytoplasmic donor CMS lines. Furthermore, several editing sites were found to be either present or absent in certain IAMSLs and their corresponding CMS lines. These results indicate that nuclear loci, as well as unknown editing factors within the mitochondria of different cytoplasmic types, may be involved in RNA editing, and they suggest that RNA editing in plant mitochondria is affected by nucleo-cytoplasmic interactions.     

Comparative analysis of sequences of mitochondrial genomes of wild abortive male sterile (WA-CMS) and male fertile lines of rice,development of functional markers for WA-CMS trait and their use in assessment of genetic purity of seeds of WA-CMS lines

WA-CMS system based rice hybrids are widely adopted in many rice growing countries, including India. Even though it is well known that the trait is controlled by mitochondria, the genes underpinning the trait remain enigmatic. In the present study, a complete genome-wide comparative sequence analysis was performed using draft mitochondrial genomes of WA-CMS and male fertile lines in a step-wise manner, progressively covering 5–10 kb every time through BLASTN tool. The sequence polymorphisms identified in different mitochondrial regions were targeted to develop two different sets of dominant PCR-based markers, one consisting of six markers targeting WA-CMS mitochondria, the other set consisting of five markers targeting male fertile mitochondria in addition to development of a set of eight co-dominant PCR-based markers targeting both the genomes. When a set of candidate genes/ORFs reported earlier to be associated with WA-CMS trait in rice were analyzed through RT-PCR of RNA isolated from immature rice florets, it was observed that the chimeric ORF, WA352 is expressed only in WA-CMS line and hybrid (i.e. genotypes containing sterile mitochondria), indicating it’s candidacy for the WA-CMS trait. Targeting the functional nucleotide polymorphism between WA-CMS and maintainer mitochondria with respect to WA352, two dominant markers, one targeting sterile and another targeting fertile mitochondria were developed. In addition, a robust, co-dominant functional marker targeting the candidate gene was also developed and validated for its utility in identification of genetic impurities in seed lots of WA-CMS lines.     

Mitochondrial ribosome assembly in Neurospora crassa. Chloramphenicol inhibits the maturation of small ribosomal subunits.

Recent results suggest that, in Neurospora crassa, one small subunit mitochondrial ribosomal protein (S-4a, M_r 52,000) is synthesized intramitochondrially (Lambowitz et al., 1976). We now find that, when wild-type cells are treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of 30 S mitochondrial ribosomal subunits is rapidly inhibited and there is an accumulation of CAP-30 S particles which sediment slightly behind mature small subunits. Electrophoretic analysis suggests that the CAP-30 S particles are deficient in several proteins including S-4a and that they are enriched in a precursor RNA species that is slightly longer than 19 S RNA. Chloramphenicol also appears to inhibit the maturation of 50 S ribosomal subunits, but this effect is much less pronounced. Continued incubation in chloramphenicol leads to a decrease in the proportion of total mitochondrial ribonucleoprotein present as monomers, possibly reflecting the depletion of competent subunits. After long-term (17 h) growth in chloramphenicol, mitochondrial ribosome profiles from wild-type cells show decreased ratios of small to large subunits, a feature which is also characteristic of the poky (mi-1) mutant. Pulse-labeling experiments combined with electrophoretic analysis show that the synthesis of mitochondrial ribosomal RNAs is relatively unaffected by chloramphenicol and that, despite the deficiency of small subunits, 19 S and 25 S RNA are present in normal ratios in whole mitochondria. By contrast, 19 S RNA in poky mitochondria is rapidly degraded leading to a decreased ratio of 19 S to 25 S RNA. The significance of these results with respect to the etiology of the poky mutation is discussed and a model of mitochondrial ribosome assembly that incorporates all available data is presented.     

Mitochondrial development during Drosophila oogenesis: distribution,density and in situ RNA hybridizations

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15 000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.     

Wheat beta-expansin (EXPB11) genes: Identification of the expressed gene on chromosome 3BS carrying a pollen allergen domain

Background

Cytoplasmic male sterility (CMS) has often been associated with abnormal mitochondrial open reading frames. The mitochondrial gene orfH79 is a candidate gene for causing the CMS trait in CMS-Honglian (CMS-HL) rice. However, whether the orfH79 expression can actually induce CMS in rice remains unclear.

Results

Western blot analysis revealed that the ORFH79 protein is mainly present in mitochondria of CMS-HL rice and is absent in the fertile line. To investigate the function of ORFH79 protein in mitochondria, this gene was fused to a mitochondrial transit peptide sequence and used to transform wild type rice, where its expression induced the gametophytic male sterile phenotype. In addition, excessive accumulation of reactive oxygen species (ROS) in the microspore, a reduced ATP/ADP ratio, decreased mitochondrial membrane potential and a lower respiration rate in the transgenic plants were found to be similar to those in CMS-HL rice. Moreover, retarded growth of primary and lateral roots accompanied by abnormal accumulation of ROS in the root tip was observed in both transgenic rice and CMS-HL rice (YTA).

Conclusion

These results suggest that the expression of orfH79 in mitochondria impairs mitochondrial function, which affects the development of both male gametophytes and the roots of CMS-HL rice.     

ATP-dependent proteases in biogenesis and maintenance of plant mitochondria

ATP-dependent proteases from three families have been identified experimentally in Arabidopsis mitochondria: four FtsH proteases (AtFtsH3, AtFtsH4, AtFtsH10, and AtFtsH11), two Lon proteases (AtLon1 and AtLon4), and one Clp protease (AtClpP2 with regulatory subunit AtClpX). In this review we discuss their submitochondrial localization, expression profiles and proposed functions, with special emphasis on their impact on plant growth and development. The best characterized plant mitochondrial ATP-dependent proteases are AtLon1 and AtFtsH4. It has been proposed that AtLon1 is necessary for proper mitochondrial biogenesis during seedling establishment, whereas AtFtsH4 is involved in maintaining mitochondrial homeostasis late in rosette development under short-day photoperiod.     

Genetic Components of Heterosis for Seedling Traits in an Elite Rice Hybrid Analyzed Using an Immortalized F_2 Population

Utilization of heterosis has greatly contributed to rice productivity in China and many Asian countries. Superior hybrids usually show heterosis at two stages: canopy development at vegetative stage and panicle development at reproductive stage resulting in heterosis in yield. Although the genetic basis of heterosis in rice has been extensively investigated, all the previous studies focused on yield traits at maturity stage. In this study, we analyzed the genetic basis of heterosis at seedling stage making use of an "immortalized F2" population composed of 105 hybrids produced by intercrossing recombinant inbred lines(RILs) from a cross between Zhenshan 97 and Minghui 63,the parents of Shanyou 63, which is an elite hybrid widely grown in China. Eight seedling traits, seedling height, tiller number, leaf number, root number, maximum root length, root dry weight, shoot dry weight and total dry weight, were investigated using hydroponic culture. We analyzed single-locus and digenic genetic effects at the whole genome level using an ultrahigh-density SNP bin map obtained by population re-sequencing. The analysis revealed large numbers of heterotic effects for seedling traits including dominance, overdominance and digenic dominance(epistasis) in both positive and negative directions. Overdominance effects were prevalent for all the traits, and digenic dominance effects also accounted for a large portion of the genetic effects. The results suggested that cumulative small advantages of the single-locus effects and two-locus interactions, most of which could not be detected statistically, could explain the genetic basis of seedling heterosis of the F_1 hybrid.     

Aberrant vegetative and reproductive development by overexpression and lethality by silencing of OsHAP3E in rice

We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase. In the reproductive phase, dense panicle was developed, and occasionally successive generation of lateral rachises and formation of double flowers were observed. These phenotypes indicate association of OsHAP3E with determination of floral meristem identity. On the other hand, repression of OsHAP3E by RNAi or by overexpressing chimeric repressor fusion constructs brought about lethality to transformed cells, and almost no transformant was obtained. This suggests that the OsHAP3E function is essential for rice cells. Altogether, our loss-of-function and gain-of-function analyses suggest that OsHAP3E plays important pleiotropic roles in vegetative and reproductive development or basic cellular processes in rice.     

Nitrogen fertilizer increases spikelet number per panicle by enhancing cytokinin synthesis in rice

Key message

Nitrogen fertilizer enhances local cytokinin synthesis to increase flower numbers in the panicles of rice. Localized cytokinin biosynthesis is an important response to nitrogen.

Abstract

Flower number per panicle is one of the most important traits in rice productivity determination. The number of flowers is established in the early stages of panicle development. Nitrogen fertilizer application before panicle initiation is well known to increase flower number. Nitrogen increases cytokinin (CKs) biosynthesis in plants, and CKs have very similar effects as nitrogen fertilizer on panicle branching. The effects of nitrogen fertilizer on panicle branching may be mediated by CKs, in which accumulation in the inflorescence meristem can regulate panicle development, resulting in increased numbers of flowers and branches. Adenosine phosphate-isopentenyltransferase (IPT) catalyzes the rate-limiting step of CKs biosynthesis. We analyzed the effect of nitrogen fertilizer (urea) on the expression of OsIPT genes (OsIPTs). The results showed that OsIPTs were markedly increased, and CKs accumulated in panicle when nitrogen fertilizer was applied. CKs biosynthesis in the roots and leaves was not up-regulated by nitrogen. These results suggest that nitrogen fertilizer enhances local CKs synthesis to increase flower numbers in the panicles of rice. Localized CKs biosynthesis is an important response to nitrogen.