Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) plays major roles in photorespiration and primary nitrogen assimilation. However, due to no mutant or knockdown lines of OsFd-GOGAT have been reported in rice (Oryza sativa L.), the contribution of OsFd-GOGAT to rice foliar nitrogen metabolism remains little up-to-date. Here, we isolated a rice premature leaf senescence mutant named gogat1, which was reduced in 67% of the total GOGAT enzyme activity in leaves. The gogat1 mutant exhibited chlorosis under natural condition, while showed less extent premature leaf senescence under low light treatment. The gogat1 locus was mapped to a 54.1 kb region on chromosome 7, and the sequencing of OsFd-GOGAT showed one substitution (A to T) at the 3017th nucleotide of the open reading frame, leading to the amino-acid substitution of leucine changed to histidine. The gogat1 mutant showed reduced seed setting rate, while the grain protein content in gogat1 mutant was significantly higher than that in wild type. Meanwhile, during the grain-filling stage, total amino acids in the up three leaves and the upmost internode were increased dramatically. The results in this study suggested that OsFd-GOGAT might participate in nitrogen remobilization during leaf senescence, which provides a potential way to improve nitrogen use efficiency in rice.
The present study was conducted at the Biotechnology Laboratory, Biotechnology Division, Bangladesh Agricultural Research Institute (BARI), Gazipur during the period from September 2004 to June 2005 to investigate the effect of different concentrations of BAP and NAA on virus free plant regeneration, shoot multiplication and different concentrations of IBA and IAA on in vitro root formation of banana cv. BARI Banana-I. The culture meristem first turned brown in colour in 4-5 days which grew into a green globular hard coat mass after 30-35 days. From this ball like structure, adventitious plantlets were developed. Among the different concentrations, 7.5 mg/l BAP + 0.5 mg/l NAA showed highest shoot proliferation of 0.75, 2.75 and 6.25 shoots per explant at 10, 20 and 30 DAI, respectively. The longest shoot (1.03, 2.45 and 3.38 cm) at 10, 20 and 30 DAI, respectively, was produced by the treatment combination of 7.5 mg/l BAP + 0.5 mg/l NAA. The maximum number of leaves (2.50, 3.25 and 7.00 leaves/explant at 10, 20 and 30 DAI) were produced on the medium supplemented with the same treatment and it also produced the longest leaves, 0.85, 2.70 and 4.23 cm at 10, 20 and 30 DAI, respectively. For root initiation half strength MS medium supplemented with different levels of IBA (0, 0.5, 1 .0 and 1.50 mg/l) and IAA (0, 0.5 and 1.0 mg/l) was used. Root numbers varied with different concentrations of IBA and IAA. The highest number of roots were produced by 0.5 mg/l IAA + 0.5 mg/l IBA. The highest length (2.93, 4.63 and 5.88 cm) was recorded at 10, 20 and 30 DAI in the same treatment which was statistically significant. Meristem derived plantlets were transferred to poly bags containing 1:1 (ground soil : cowdung) mixture after 7 days hardening in room temperature (28-30°C) and established plantlet was ready for planting. Key Words: Banana; regeneration; micropropagation; plantlet. DOI: 10.3329/bjar.v34i4.5840Bangladesh J. Agril. Res. 34(4) : 645-659, December 2009
Salinity stress is one of the most prominent abiotic stresses that negatively affect crop production. Transcription factors (TFs) are involved in the absorption, transport, or compartmentation of sodium (Na+) or potassium (K+) to resist salt stress. The basic helix–loop–helix (bHLH) is a TF gene family critical for plant growth and stress responses, including salinity. Herein, we used the CRISPR/Cas9 strategy to generate the gene editing mutant to investigate the role of OsbHLH024 in rice under salt stress. The A nucleotide base deletion was identified in the osbhlh024 mutant (A91). Exposure of the A91 under salt stress resulted in a significant increase in the shoot weight, the total chlorophyll content, and the chlorophyll fluorescence. Moreover, high antioxidant activities coincided with less reactive oxygen species (ROS) and stabilized levels of MDA in the A91. This better control of oxidative stress was accompanied by fewer Na+ but more K+, and a balanced level of Ca2+, Zn2+, and Mg2+ in the shoot and root of the A91, allowing it to withstand salt stress. Furthermore, the A91 also presented a significantly up-regulated expression of the ion transporter genes (OsHKT1;3, OsHAK7, and OsSOS1) in the shoot when exposed to salt stress. These findings imply that the OsbHLH024 might play the role of a negative regulator of salt stress, which will help to understand better the molecular basis of rice production improvement under salt stress.
Isolating and characterizing mutants with altered senescence phenotypes is one of the ways to understand the molecular basis of leaf aging. Using ethyl methane sulfonate mutagenesis, a new rice (Oryza sativa) mutant, brown midrib leaf (bml), was isolated from the indica cultivar ‘Zhenong34’. The bml mutants had brown midribs in their leaves and initiated senescence prematurely, at the onset of heading. The mutants had abnormal cells with degraded chloroplasts and contained less chlorophyll compared to the wild type (WT). The bml mutant showed excessive accumulation of reactive oxygen species (ROS), increased activities of superoxide dismutase, catalase, and malondialdehyde, upregulation of senescence-induced STAY-GREEN genes and senescence-related transcription factors, and down regulation of photosynthesis-related genes. The levels of abscisic acid (ABA) and jasmonic acid (JA) were increased in bml with the upregulation of some ABA and JA biosynthetic genes. In pathogen response, bml demonstrated higher resistance against Xanthomonas oryzae pv. oryzae and upregulation of four pathogenesis-related genes compared to the WT. A genetic study confirmed that the bml trait was caused by a single recessive nuclear gene (BML). A map-based cloning using insertion/deletion markers confirmed that BML was located in the 57.32kb interval between the L5IS7 and L5IS11 markers on the short arm of chromosome 5. A sequence analysis of the candidate region identified a 1 bp substitution (G to A) in the 5′-UTR (+98) of bml. BML is a candidate gene associated with leaf senescence, ROS regulation, and disease response, also involved in hormone signaling in rice. Therefore, this gene might be useful in marker-assisted backcrossing/gene editing to improve rice cultivars.
A rice receptor-like kinase gene OSBBS1/OsRLCK109 was identified; this gene played vital roles in leaf senescence and the salt stress response. Early leaf senescence can cause negative effects on rice yield, but the underlying molecular regulation is not fully understood. bilateral blade senescence 1 (bbs1), an early leaf senescence mutant with a premature senescence phenotype that occurs mainly performing at the leaf margins, was isolated from a rice mutant population generated by ethylmethane sulfonate (EMS) treatment. The mutant showed premature leaf senescence beginning at the tillering stage and exhibited severe symptoms at the late grain-filling stage. bbs1 showed accelerated dark-induced leaf senescence. The OsBBS1 gene was cloned by a map-based cloning strategy, and a guanine (G) insertion was found in the first exon of LOC_Os03g24930. This gene encodes a receptor-like cytoplasmic kinase and was named OsRLCK109 in a previous study. Transgenic LOC_Os03g24930 knockout plants generated by a CRISPR/Cas9 strategy exhibited similar early leaf senescence phenotypes as did the bbs1 mutant, which confirmed that LOC_Os03g24930 was the OsBBS1 gene. OsBBS1/OsRLCK109 was expressed in all detected tissues and was predominantly expressed in the main vein region of mature leaves. The expression of OsBBS1 could be greatly induced by salt stress, and the bbs1 mutant exhibited hypersensitivity to salt stress. In conclusion, this is the first identification of OsRLCKs participating in leaf senescence and playing critical roles in the salt stress response in rice (Oryza sativa L.).
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