Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Evaluation of Morpho-Physiological and Yield-Associated Traits of Rice (Oryza sativa L.) Landraces Combined with Marker-Assisted Selection under High-Temperature Stress and Elevated Atmospheric CO2 Levels.

Rice (Oryza sativa L.) is an important cereal crop worldwide due to its long domestication history. North-Eastern India (NEI) is one of the origins of indica rice and contains various native landraces that can withstand climatic changes. The present study compared NEI rice landraces to a check variety for phenological, morpho-physiological, and yield-associated traits under high temperatures (HTs) and elevated CO2 (eCO2) levels using molecular markers. The first experiment tested 75 rice landraces for HT tolerance. Seven better-performing landraces and the check variety (N22) were evaluated for the above traits in bioreactors for two years (2019 and 2020) under control (T1) and two stress treatments [mild stress or T2 (eCO2 550 ppm + 4 °C more than ambient temperature) and severe stress or T3 (eCO2 750 ppm + 6 °C more than ambient temperature)]. The findings showed that moderate stress (T2) improved plant height (PH), leaf number (LN), leaf area (LA), spikelets panicle-1 (S/P), thousand-grain weight (TGW), harvest index (HI), and grain production. HT and eCO2 in T3 significantly decreased all genotypes' metrics, including grain yield (GY). Pollen traits are strongly and positively associated with spikelet fertility at maturity and GY under stress conditions. Shoot biomass positively affected yield-associated traits including S/P, TGW, HI, and GY. This study recorded an average reduction of 8.09% GY across two seasons in response to the conditions simulated in T3. Overall, two landraces-Kohima special and Lisem-were found to be more responsive compared to other the landraces as well as N22 under stress conditions, with a higher yield and biomass increment. SCoT-marker-assisted genotyping amplified 77 alleles, 55 of which were polymorphic, with polymorphism information content (PIC) values from 0.22 to 0.67. The study reveals genetic variation among the rice lines and supports Kohima Special and Lisem's close relationship. These two better-performing rice landraces are useful pre-breeding resources for future rice-breeding programs to increase stress tolerance, especially to HT and high eCO2 levels under changing climatic situations.

Combinatorial impacts of elevated CO2 and temperature affect growth, development, and fruit yield in Capsicum chinense Jacq.

Hot chilli ('Bhut Jolokia') (Capsicum chinense Jacq.) is the hottest chilli widely grown in the North-Eastern region of India for its high pungency. However, little information is available on its physiology, growth and developmental parameters including yield. Therefore, the present research was undertaken to study the physiological responses of Bhut Jolokia under elevated CO2 (eCO2) and temperature. Two germplasms from two different agro-climatic zones (Assam and Manipur) within the North-East region of India were collected based on the pungency. The present study explored the interactive effect of eCO2 [at 380, 550, 750 ppm (parts per million)] and temperature (at ambient, > 2 °C above ambient, and > 4 °C above ambient) on various physiological processes, and expression of some photosynthesis and capsaicin related genes in both the germplasms. Results revealed an increase (> 1-2 fold) in the net photosynthetic rate (Pn), carbohydrate content, and C: N ratio in 'Bhut Jolokia' under eCO2 and elevated temperature regimes compared to ambient conditions within the germplasms. Gene expression studies revealed an up-regulation of photosynthesis-related genes such as Cs RuBPC2 (Ribulose biphosphate carboxylase 2) and Cs SPS (Sucrose phosphate synthase) which, explained the higher Pn under eCO2 and temperature conditions. Both the germplasm showed better performance under CTGT-II (Carbon dioxide Temperature Gradient Tunnel having 550 ppm CO2 and temperature of 2 °C above ambient) in terms of various physiological parameters and up-regulation of key photosynthesis-related genes. An up-regulation of the Cs  capsaicin synthase gene was also evident in the study, which could be due to the metabolite readjustment in 'Bhut Jolokia'. In addition, the cultivar from Manipur (cv. 1) had less fruit drop compared to the cultivar from Assam (cv. 2) in CTGT II. The data indicated that 550 ppm of eCO2 and temperature elevation of > 2 °C above the ambient with CTGT-II favored the growth and development of 'Bhut Jolokia'. Thus, results suggest that Bhut Jolokia grown under the elevation of CO2 up to 550 ppm and temperature above 2 °C than ambient may support the growth, development, and yield. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01294-9.

Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina.

BACKGROUND: Pumilio RNA-binding proteins are evolutionarily conserved throughout eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Although a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. The species of the genus Chara have been known to be most closely related to land plants, as they share several characteristics with modern Embryophyta. RESULTS: In this study, we identified two putative nucleolar Pumilio protein genes, namely, ChPUM2 and ChPUM3, from the transcriptome of Chara corallina. Of the two ChPUM proteins, ChPUM2 was most similar in amino acid sequence (27% identity and 45% homology) and predicted protein structure to Arabidopsis APUM23, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of 35S:APUM23-GFP and 35S:APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23, but 35S:ChPUM3/apum24+/- plants did not rescue that of apum24. Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GGAAUUGACGG) for APUM23 was conserved in Arabidopsis and C. corallina, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in C. corallina than in A. thaliana. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain, which had a longer random coil in ChPUM3 than in APUM24. CONCLUSIONS: ChPUM2 of C. corallina was functional in Arabidopsis, similar to APUM23, but ChPUM3 did not substitute for APUM24 in Arabidopsis. Protein homology modeling showed high coverage between APUM23 and ChPUM2, but displayed structural differences between APUM24 and ChPUM3. Together with the protein structure of ChPUM3 itself, a short ITS2 of Arabidopsis pre-rRNA may interrupt the binding of ChPUM3 to 3'-extended 5.8S pre-rRNA.

Growth, development and nitrogen uptake efficiency of some sali rice genotypes under delayed dates of sowing.

Sali rice which is the major rice crop of Assam faces recurrent floods co-inciding with different phonological stages, especially the seedling stage. Owing to the damage caused to the seedlings, the transplanting also gets delayed. Delayed transplanting results in poor grain yield due to poor biomass accumulation as influenced by prevailing photoperiodic and thermal regimes during that period of the year. From this angle, selection of suitable genotypes appears to be the viable option that can have better early vegetative growth by utilizing available resources and should possess considerable degree of thermo- and photo- insensitivity. Keeping in view the above points, a study was conducted during the sali seasons at the experimental plots of Instructional cum Research (ICR) farm, Assam Agricultural University, Jorhat with already shortlisted seven sali rice genotypes namely, Satya, Luit, Monoharsali, Jaya, Bordhan, Basundhara and Srimanta under delayed dates of sowing using thirty days old seedlings for transplanting laid out in split plot design. Results revealed that as compared to timely sowing, delayed sowing resulted in progressively lower values of various physiological parameters. While comparison was made between timely sowing and the deferred dates of sowing lowest reduction in the values of grain yield were recorded in genotypes of Manoharsali and Srimanta (35.66% and 35.03% with a delay of transplanting by 35 days compared to the recommended date of sowing i.e., 15th June). These two genotypes recorded better performance in terms of parameters like leaf area index, nitrogen accumulation in biomass and plant biomass etc. The better performing genotypes namely Srimanta and Monoharsali recorded higher values of nitrogen uptake efficiency.

Differential proteomics of Picrorhiza kurrooa Royle ex Benth. in response to dark stress.

Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) an important medicinal herb of western Himalayan region has been used to treat various diseases and disorders. Over-harvesting and lack of cultivation has led to its entry in Red Data Book as an endangered species. Further, its very restrictive habitat and lesser biomass production are major limitations for bringing it under commercial cultivation. All these issues necessitate deeper insights into mechanisms governing its growth and interaction with the environmental cues. Light may be one of the important factors to be studied for its role in regulating growth and adaptation of Picrorhiza as in natural habitat it prefers shady niches. Keeping this in view, proteome of Picrorhiza kept under light vis-à-vis under dark was analysed and compared. Leaf as well as root proteome of Picrorhiza was studied. Denaturing two dimensional gel electrophoresis and mass spectrometry techniques were used to detect and identify differentially expressed proteins, respectively. Twenty two proteins from leaf and 25 proteins from root showed differential expression levels under dark and light conditions. Among the differentially expressed proteins, majority were those involved in metabolism, protein synthesis, and stress and defense response. Other differentially expressed proteins were those involved in photosynthetic process, photorespiration and few proteins were with unknown function indicating that many different processes work together to establish a new cellular homeostasis in response to dark and light conditions. Proteins found to be differentially expressed under light vis-à-vis dark conditions suggested a range of biochemical pathways and processes being associated with response of plant to dark conditions. The identified proteins may be utilized for developing strategies for improving the biomass production/performance of Picrorhiza under varied light/dark habitats.