Exploring the role of HaTIPs genes in enhancing drought tolerance in sunflower.

BACKGROUND: Activity of plant aquaporins (AQPs) is extremely sensitive to environmental variables such as temperature, drought, atmospheric vapor pressure deficit, cell water status and also appears to be closely associated with the expression of plant tolerance to various stresses. The spatial and temporal expression patterns of genes of Tonoplast Intrinsic Proteins (TIPs) in various crops indicate the complex and diverse regulation of these proteins and are important in understanding their key role in plant growth, development and stress responses. METHODS AND RESULTS: Based on phylogenetic analysis, six distinct HaTIPs were selected for studying their spatial and temporal expression in sunflower (Helianthus annuus). In this study semi quantitative polymerase chain reaction (semi q-PCR) and real time polymerase chain reaction (q-PCR) analysis were used to study the spatial and temporal expression of HaTIPs in sunflower. The results indicated that all of HaTIPs showed differential expression specific to both the tissues and the accessions. Moreover, the expression of all HaTIPs was higher in cross compared to the parents. Results of semi q-PCR and real time PCR indicated an upregulation of expression of HaTIP-RB7 and HaTIP7 in drought tolerant entries at 12 h of 20% polyethylene glycol (PEG) treatment compared to 0 h. CONCLUSION: Hence these genes can be utilized as potential target in improving water use efficiency and for further genetic manipulation for the development of drought tolerant sunflower. This study may further contribute to our better understanding regarding the precise role of HaTIPs through their spatial and temporal expression analysis and their application in sunflower drought stress responses.

Genome-Wide Characterization and Sequence Polymorphism Analyses of Glycine max Fibrillin (FBN) Revealed Its Role in Response to Drought Condition.

The fibrillin (FBN) gene family is widely distributed in all photosynthetic organisms. Members of this gene family are involved in plant growth and development and their response to various biotic and abiotic stress factors. In this study, 16 members of FBN were identified in Glycine max and characterized by using different bioinformatics tools. Phylogenetic analysis classified FBN genes into seven groups. The presence of stress-related cis-elements in the upstream region of GmFBN highlighted their role in tolerance against abiotic stresses. To further decipher the function, physiochemical properties, conserved motifs, chromosomal localization, subcellular localization, and cis-acting regulatory elements were also analyzed. Gene expression analysis based on FPKM values revealed that GmFBNs greatly enhanced soybean drought tolerance and controlled the expression of several genes involved in drought response, except for GmFBN-4, GmFBN-5, GmFBN-6, GmFBN-7 and GmFBN-9. For high throughput genotyping, an SNP-based CAPS marker was also developed for the GmFBN-15 gene. The CAPS marker differentiated soybean genotypes based on the presence of either the GmFBN-15-G or GmFBN-15-A alleles in the CDS region. Association analysis showed that G. max accessions containing the GmFBN-15-A allele at the respective locus showed higher thousand seed weight compared to accessions containing the GmFBN-15-G allele. This research has provided the basic information to further decipher the function of FBN in soybean.

Genetic Effects of GA-Responsive Dwarfing Gene Rht13 on Plant Height, Peduncle Length, Internodal Length and Grain Yield of Wheat under Drought Stress.

Reduction in plant height is generally associated with an increase in lodging resistance, drought tolerance and grain yield of wheat worldwide. Historically, a significant increase in grain yield was observed through the introduction of semi-dwarf wheat varieties utilizing the gibberellic acid-insensitive Rht genes (Rht1 or Rht2). The gibberellic acid sensitive (GA-sensitive) reduced height (Rht) genes are available that are alternatives to gibberellic acid insensitive (GA-insensitive) Rht genes, having a neutral effect on coleoptile length seedling vigor suggesting their potential in using alone or in combination with GA-insensitive Rht genes to improve grain yield and drought tolerance in wheat. This study was conducted to evaluate parents and F1 crosses under drought stress. The crossing was done using line × tester mating design, comprising eight lines and five testers having different GA-sensitive and GA-insensitive Rht genes. Parents and F1 crosses were sown in the field under RCBD with three replications in normal and drought stress. Data were recorded for morpho-physiological traits. The mean comparison showed significant differences among parents and hybrids for most of the studies' traits. The general combining ability showed that line 1 is the good general combiner for days to heading, lodging (%), plant height, peduncle length, internodal length and days to maturity under normal conditions while L5 was the good general cobiner for chlorophyll contents and stomatal conductance both under normal and drought stress. The spcaicfic combing ability estimases showed that the cross L1 × T1 was best for days to heading, lodging (%), plant height and internodal length both under normal and drought stress. F1 hybrids showed a significant reduction in plant height (18-25%), peduncle length (20-28%) and increased grain yield (15-18%) under drought stress. Expression analysis showed upregulation of Rht13 at the middle part of the peduncle internode under drought stress. From the expression analysis, five crosses were selected, and their segregating population was raised and space-plated. Rht13 genes reduced plant height (-30 to -45%), peduncle length (-30 to -53%), peduncle internode length (-28% to -48%), increased spike length (+20% to +50%), number of grains per spike (+17 to +26%) and grain yield per plant (+29% to +50%) compared to Rht1 gene. These results suggested the possibility of using the GA-sensitive Rht13 gene for the development of high-yielding and drought-tolerant wheat varieties.

Genome-wide characterization and sequence polymorphism analyses of cysteine-rich poly comb-like protein in Glycine max.

Cysteine-rich poly comb-like protein (CPP) is a member of cysteine-rich transcription factors that regulates plant growth and development. In the present work, we characterized twelve CPP transcription factors encoding genes in soybean (Glycine max). Phylogenetic analyses classified CPP genes into six clades. Sequence logos analyses between G. max and G. soja amino acid residues exhibited high conservation. The presence of growth and stress-related cis-acting elements in the upstream regions of GmCPPs highlight their role in plant development and tolerance against abiotic stress. Ka/Ks levels showed that GmCPPs experienced limited selection pressure with limited functional divergence arising from segmental or whole genome duplication events. By using the PAN-genome of soybean, a single nucleotide polymorphism was identified in GmCPP-6. To perform high throughput genotyping, a kompetitive allele-specific PCR (KASP) marker was developed. Association analyses indicated that GmCPP-6-T allele of GmCPP-6 (in exon region) was associated with higher thousand seed weight under both water regimes (well-water and water-limited). Taken together, these results provide vital information to further decipher the biological functions of CPP genes in soybean molecular breeding.

Characterization of Histone H3 Gene Family Reveals That GmHH3-3 is Associated With Higher Seed Weight in Glycine max.

The main function of histone protein is to provide support to the structure of chromosomes. It helps in binding a long thread of DNA into a more condensed shape to fit into the nucleus. From histone variants, histone H3 (HH3) plays a crucial role in plant growth and development. Characterization of histones has not been reported in Glycine max till now. The objective of this study was to characterize the HH3 gene family for molecular breeding of G. max. In this study, 17 HH3 members in G. max were identified by performing local BLASTp using HH3 members from Arabidopsis as a query. Phylogenetic analysis classified HH3 genes in seven clades. Sequence logo analysis among Arabidopsis thaliana, Oryza sativa, and Glycine max showed a higher level of similarity in amino acids. Furthermore, conserveness of G. max HH3 genes was also confirmed by Gene Structure Display. Ten paralogous gene pairs were identified in GmHH3 genes in the Glycine max genome by conducting collinearity analysis. G. max HH3 genes have experienced strong purifying selection pressure, with limited functional divergence originating from the segmental and whole-genome duplication, as evidenced by the Ka/Ks ratio. The KASP marker was developed for GmHH3-3 gene. Genotyping was performed on 46 G. max genotypes. This differentiation was based upon the presence of either GmHH3-3-C or GmHH3-3-T allele in the CDS region. The results showed that G. max accessions containing the GmHH3-3-T allele at respective locus showed higher thousand seed weight than that of those accessions that contain the GmHH3-3-C allele. This research provides the basic information to further decipher the function of HH3 in soybean.

Leaf rolling dynamics for atmospheric moisture harvesting in wheat plant as an adaptation to arid environments.

Plant species surviving in the arid regions have developed novel leaf features to harvest atmospheric water. Before the collected water evaporates, it is absorbed and transported for storage within the tissues and move toward the root zone through the unique chemistry of leaf structures. Deep insights into such features reveal that similarities can be found in the wheat plant. Therefore, this study aimed to evaluate the leaf rolling dynamics among wheat genotypes and their relationships with moisture harvesting and its movement on the leaf surface. For this purpose, genotypes were characterized for leaf rolling at three distinct growth stages (tillering, booting, and spike emergence). The contact angle of leaf surface dynamics (adaxial and abaxial), water budget, and morphophysiological traits of genotypes were measured. The results indicate that leaf rolling varies from inward to twisting type among genotypes and positively affected the water use efficiency and soil moisture difference at all growth stages under normal and drought conditions. Results of wetting property (hydrophilic < 90°) of the leaf surface were positively associated with the atmospheric water collection (4-7 ml). The lower values of contact angle hysteresis (12-19°) also support this mechanism. Thus, genotypes with leaf rolling dynamics (inward rolled and twisted) and surface wettability is an efficient fog harvesting system in wheat for interception and utilization of fog water in drought-prone areas. These results can be exploited to develop self-irrigated and drought-tolerant crops.

Comparative Efficiency of Native Insect Pollinators in Reproductive Performance of Medicago sativa L. in Pakistan.

Lucerne (Medicago sativa L.) is a cross-pollinated crop and requires entomophilous pollination for tripping of flowers and subsequent pod and seed set. To discover the best pollinators for lucerne seed production, a two-year field trial was carried out at the research farm of MNS University of Agriculture, Multan, Pakistan. Abundance and diversity of insect pollinators along with the foraging behavior were recorded in terms of tripping efficiency, stay time, visitation rate and pollen harvest. The single-visit efficiency of abundant insect pollinators was also evaluated in terms of number of seeds and seed weight per raceme along with germination percentage. Ten most abundant floral visitors (five solitary bee species, three honeybee species and two syrphid fly species) were tested for their pollination efficiency. Honeybees were most abundant in both the years followed by the solitary bees and syrphid flies. Single-visit efficacy in terms of number of pods per raceme, number of seeds per raceme, 1000 seed weight and germination percentage revealed Megachile cephalotes as the most efficient insect pollinator followed by Megachile hera and Amegilla sp. Future studies should investigate the biology and ecology of these bee species with special emphasis on their nesting behavior and seasonality.

Characterization of Vascular plant One-Zinc finger (VOZ) in soybean (Glycine max and Glycine soja) and their expression analyses under drought condition.

Vascular plant one-zinc-finger (VOZ) transcription factors regulate plant growth and development under drought conditions. Six VOZ transcription factors encoding genes exist in soybean genome (both in Glycine max and Glycine soja). Herein, GmVOZs and GsVOZs were identified through in silico analysis and characterized with different bioinformatics tools and expression analysis. Phylogenetic analysis classified VOZ genes in four groups. Sequence logos analysis among G. max and G. soja amino acid residues revealed higher conservation. Presence of stress related cis-elements in the upstream regions of GmVOZs and GsVOZs highlights their role in tolerance against abiotic stresses. The collinearity analysis identified 14 paralogous/orthologous gene pairs within and between G. max and G. soja. The Ka/Ks values showed that soybean VOZ genes underwent selection pressure with limited functional deviation arising from whole genome and segmental duplication. The GmVOZs and GsVOZs were found to express in roots and leaves at seedling stage. The qRT-PCR revealed that GmVOZs and GsVOZs transcripts can be regulated by abiotic stresses such as polyethylene glycol (PEG). The findings of this study will provide a reference to decipher physiological and molecular functions of VOZ genes in soybean.

Total parotidectomy under local anesthesia: a novel technique.

Parotidectomy is a common procedure usually done for a parotid mass necessitating a histological diagnosis. Operation is normally performed under General anesthesia with a nerve stimulator to facilitate facial nerve stimulation. We describe a new technique with reports of three cases, making total parotidectomy under local anesthesia possible. The ascending cervical branch of cervical plexus and the auriculotemporal nerve were anesthetized by bupivacaine 0.25% (2mg/kg) and lignocaine with adrenaline 7 mg/kg. Effective onset of anesthesia was within 15-25 minutes and the operations lasted between 2-3 hours without any complications. This offers advantage in high-risk patients where general anesthesia is contraindicated. The facial nerve can be easily identified with on command movements by the patient rendering the use of nerve stimulator or injection of the dye superfluous. This technique makes total parotidectomy an outpatient procedure and facilitates an early discharge.

Do clinical manifestations of Systemic Lupus Erythematosus in Pakistan correlate with rest of Asia?

OBJECTIVE: Systemic Lupus Erythematosus (SLE) is known to be different among people with different racial, geographical and socio-economic back grounds. Asia has diverse ethnic groups broadly, Orientals in the East and Southeast Asia, Indians in South Asia and Arabs in the Middle East. These regions differ significantly from the Caucasians with reference to SLE. The purpose of this study was, therefore, to delineate the clinical pattern and disease course in Pakistani patients with SLE and compare it with Asian data. METHODS: Patients with SLE fulfilling the clinical and laboratory criteria of the American Rheumatism Association admitted at the Aga Khan University Hospital between 1986 and 2001 were studied by means of a retrospective review of their records. The results were compared with various studies in different regions of Asia. RESULTS: Demographically, it was seen that SLE is a disease predominantly of females in their third decade, which is generally consistent with Asian data. There was less cutaneous manifestations, arthritis, serositis, haematological and renal involvement compared to various regions in Asia. The neurological manifestations of SLE, however, place Pakistani patients in the middle of a spectrum between South Asians and other Asian races. CONCLUSION: This study has shown that the clinical characteristics of SLE patients in our country may be different to those of other Asian races. Although our population is similar to South Asians, but clinical manifestations of our SLE patients are considerably different, suggesting some unknown etiology. Further studies are required to confirm the above results and to find statistically sounder associations.

Renal involvement in systemic lupus erythematosus in Pakistan.

OBJECTIVE: To find the prevalence of lupus nephritis, delineate its clinical, immunological and therapeutic characteristics and compare them with the data worldwide. PATIENTS AND METHODS: Between 1985 and 2001, 198 patients with SLE fulfilling the clinical and laboratory criteria of the American Rheumatism Association (ARA) admitted to the hospital were studied by means of a retrospective review of their records. RESULTS: Renal involvement was found in 89 (45%) patients. Biopsy showed lupus nephritis in 42 patients; there were 9 male and 33 females. Mean age at initial presentation was 27 years and mean duration of follow-up was 2 .3 years. The histological types (WHO Classification) were mainly class. 4 (n = 27), class 3 (n = 7) and class 5 (n = 6). Immunoflourescence showed a predominantly granular pattern of IgG, IgA and C3. Renal manifestations included renal failure (50%), microscopic hematuria (67%), active urine sediment (22%), and proteinuria (74%). Proteinuria was nephrotic range in 45% patients. Treatment was with combinations of prednisolone and cyclophosphamide (n = 13), prednisolone and azathioprine (n = 27). 19 patients received high dose methyl prednisolone (1 gm/day for 3 days). There was no difference in mortality rate between prednisolone and cyclophosphamide and prednisolone and azathioprine treatment groups. The overall mortality rate was 17% (n = 7). Mortality was higher in WHO class 4 and 5 as compared to class 2 and 3 (p < 0.001). CONCLUSION: The prevalence of lupus nephritis in our population is an intermediate between Caucasians and other Asians. Certain clinical characteristics in our patients with lupus nephritis are different as compared to various other studies. Because of limited resources for treatment in developing countries, we believe that patients with lupus nephritis should be treated with improved ancillary medical therapies and more effective immunosuppressive regimens.