Synchronized flowering in pomegranate, following pruning, is associated with expression of the FLOWERING LOCUS T homolog, PgFT1.

Flowering in angiosperms is a crucial event that marks the transition from the vegetative to the reproductive phase. In many perennials, pruning is an important horticultural practice that induces synchronized and profuse flowering. In pomegranate, vegetative growth immediately after pruning is associated with activation of PgCENa, a flowering suppressor of the phosphatidyl ethanolamine binding protein (PEBP) family, while a reduction is associated with synchronous flowering. We show that flowering in pomegranate is activated by expression of another PEBP family member, PgFT1, a homolog of the FLOWERING LOCUS T (FT) gene that promotes flowering. PgFT1 shows a rapid reduction in expression during the extensive vegetative growth immediately after pruning but shows robust expression during synchronous flowering post-pruning, in flower-bearing shoots but not in branches that do not bear flowers. A continuous low-level flowering in the absence of pruning is associated with continuous but reduced expression of PgFT1. Flowering by heterologous expression of PgFT1 in Arabidopsis is affected by a single amino acid change in the C-terminal region of PgFT1, which upon correction, promotes flowering in Arabidopsis. Our study provides insights into the molecular mechanisms by which pruning affects flowering pathways in tropical perennial fruit plants such as pomegranate.

Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV).

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl ß-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.

Studies on the expression patterns of the circadian rhythm regulated genes in mango.

Mango, an important fruit crop of the tropical and subtropical regions shows alternate bearing in most varieties causing a financial loss to the farmer. Genetic reasons for this undesirable trait have not been studied so far. In our attempts to investigate the genetic reasons for alternate bearing we have initiated studies on genes associated with the induction, repression and regulation of flowering in mango. We have previously identified and characterized FLOWERING LOCUS T (FT) genes that induce flowering and two TERMINAL FLOWER1 (TFL1) genes that repress flowering. In this communication, we have explored the association of GI-FKF1-CDF1-CO module with the regulation of flowering in mango. The role of this module in regulating flowering has been well documented in photoperiod sensitive plants. We have characterized these genes and their expressions during flowering in Ratna variety as also their diurnal fluctuations and tissue specific expressions. The data taken together suggest that GI-FKF1-CDF1-CO module may also be employed by mango in regulating its flowering. Further, we suggest that the temperature dependent flowering in mango is probably associated with the presence of temperature sensitive elements present in the promoter region of one of the GIGANTEA genes that have been shown to be closely associated with floral induction. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01053-8.

Independent and combined abiotic stresses affect the physiology and expression patterns of DREB genes differently in stress-susceptible and resistant genotypes of banana.

In tropics, combined stresses of drought and heat often reduce crop productivity in plants like Musa acuminata L. We compared responses of two contrasting banana genotypes, namely the drought-sensitive Grand Nain (GN; AAA genome) and drought tolerant Hill banana (HB; AAB genome) to individual drought, heat and their combination under controlled and field conditions. Drought and combined drought and heat treatments caused greater reduction in leaf relative water content and greater increase in ion leakage and H2 O2 content in GN plants, especially in early stages, while the responses were more pronounced in HB at later stages. A combination of drought and heat increased the severity of responses. Real-time expression patterns of the A-1 and A-2 group DEHYDRATION-RESPONSIVE ELEMENT BINDING (DREB) genes revealed greater changes in expression in leaves of HB plants for both the individual stresses under controlled conditions compared to GN plants. A combination of heat and drought, however, activated most DREB genes in GN but surprisingly suppressed their expression in HB in controlled and field conditions. Its response seems correlated to a better stomatal control over transpiration in HB and a DREB-independent pathway for the more severe combined stresses unlike in GN. Most of the DREB genes had abscisic acid (ABA)-responsive elements in their promoters and were also activated by ABA suggesting at least partial dependence on ABA. This study provides valuable information on physiological and molecular responses of the two genotypes to individual and combined drought and heat stresses.

Molecular characterization of CONSTANS-Like (COL) genes in banana (Musa acuminata L. AAA Group, cv. Grand Nain).

The CONSTANS (CO) family is an important regulator of flowering in photoperiod sensitive plants. But information regarding their role in day neutral plants is limited. We report identification of nine Group I type CONSTANS-like (COL) genes of banana and their characterization for their age dependent, diurnal and tissue-specific expression. Our studies show that the Group I genes are conserved in structure to members in other plants. Expression of these genes shows a distinct circadian regulation with a peak during light period. Developmental stage specific expression reveals high level transcript accumulation of two genes, MaCOL3a and MaCOL3b, well before flowering and until the initiation of flowering. A decrease in their transcript levels after initiation of flowering is followed by an increase in transcription of other members that coincides with the continued development of the inflorescence and fruiting. CO binding cis-elements are observed in at least three FT -like genes in banana suggesting possible CO-FT interactions that might regulate flowering. Distinct tissue specific expression patterns are observed for different family members in mature leaves, apical inflorescence, bracts, fruit skin and fruit pulp suggesting possible roles other than flowering. This is the first exhaustive study of the COL genes belonging to Group I of banana.

Improved abiotic stress tolerance in Arabidopsis by constitutive active form of a banana DREB2 type transcription factor, MaDREB20.CA, than its native form, MaDREB20.

Banana is grown as one of the important fruit crops in tropical and subtropical regions of the world. In this study, we report induced expression of a dehydration responsive element binding 2 (DREB2) gene (MaDREB20) under individual heat, drought, and combined drought and heat stress in root of two banana genotypes Grand Nain (GN) and Hill Banana (HB). Motif analysis of MaDREB20 protein demonstrated the presence of a negative regulatory domain (NRD) or PEST motif between 150 and 184 amino acids. Transgenic Arabidopsis overexpressing MaDREB20 gene showed more survival rate, above-ground biomass, seed yield, leaf relative water content, and proline content but less ion leakage and malonaldehyde content, revealing improved tolerance against heat and drought as well as their combination than the wild-type. Overexpression of MaDREB20.CA (constitutive active form of MaDREB20 after removal of PEST region) showed better abiotic stress tolerance in Arabidopsis than its native form (MaDREB20). Transgenic Arabidopsis overexpressing MaDREB20 and MaDREB20.CA genes appeared to be associated with reduced stomatal densities under normal condition, better regulation of stomatal aperture under drought than in wild-type plants, and differential regulation of downstream target (AtTCH4 and AtIAA1) genes under heat, drought, and combined stress. Taken together, our findings revealed important functions of MaDREB20 in abiotic stress responses in transgenic Arabidopsis and could form a basis for CRISPR/Cas9-mediated removal of its NRD to enhance stress tolerance in banana.

RAPD based genetic variability among cultivated varieties of Aonla (Indian Gooseberry, Phyllanthus emblica L.).

Aonla, the Indian Gooseberry (Phyllanthus emblica) is widely grown in India due to its neutraceutical properties. Investigations on the use of RAPD markers enabled us to estimate genetic variability among commercially cultivated varieties. This study also enabled us to distinguish these varieties using a set of four decamer primers, which was otherwise difficult by using morphological markers. Cluster analysis revealed three different groups of varieties directly associated to their place of origin. RAPD markers were also able to differentiate varieties of same origin or even selection from same parents. This information can be used for identification of varieties and further crop improvement programme.

Indeterminate growth of the umbel inflorescence and bulb is associated with increased expression of the TFL1 homologue, AcTFL1, in onion.

TERMINAL FLOWER1 (TFL1) is a key gene for maintenance of vegetative and inflorescence indeterminacy and architecture. In onion, flowering and bulbing are two distinct developmental phases, each under complex environmental regulatory control. We have identified two CEN/TFL1-like genes from onion designated as AcTFL1 and AcCEN1. AcTFL1 is expressed during bulbing and inflorescence development with expression increasing with indeterminate growth of the umbel and the bulb suggesting possible conservation of function. Increase in AcTFL1 expression during umbel growth is associated with a simultaneous reduction in expression of AcLFY. Expression of AcTFL1 within the bulb is lowest in the outermost layers and highest in the innermost (youngest) layers. Bulb storage at room temperature or in cold leads to a gradual reduction in AcTFL1 levels in the meristem-containing tissues, the decrease being faster in the variety not requiring vernalization. Constitutive expression of AcTFL1, but not AcCEN1 complements the Arabidopsis tfl1-14 mutant and delays flowering in wild type suggesting conservation of the AcTFL1 function even in the distantly related Arabidopsis. Taken together, AcTFL1 appears to be the functional counterpart of TFL1 and regulates indeterminate growth of the umbel inflorescence as well as bulb development in onion.

Differential regulation of the banana stress NAC family by individual and combined stresses of drought and heat in susceptible and resistant genotypes.

Banana, an important tropical fruit crop, often faces drought, heat and its combination during its growth, leading to decreased yields. The combined stresses caused 100% yield loss in Grand Nain (GN) as compared to only 46% in Hill Banana (HB). To understand the response of combined stresses, we studied the stress-responsive NAC gene sub-family under individual and combined drought/heat stresses under controlled and field conditions in the stress-sensitive GN (AAA genotype) and stress-tolerant HB (AAB genotype). Under drought, expression of most stress-NACs increased with progression of drought in either one or the other genotype with little overlap. Heat stress caused a continuous decline in expression of most genes in HB unlike in GN where many NACs were up-regulated although to a lesser scale than for drought. Combination of the two stresses elicited a very different response compared with individual stresses. GN responded strongly to the combined stress with up-regulation of most genes unlike that seen in drought. Surprisingly, NAC genes in HB did not respond much to the more severe combination of the stresses despite being up-regulated strongly by drought. The response of the NACs to combined field stress was similar to that under controlled conditions. Most of the stress-NACs were strongly up-regulated upon treatment with exogenous ABA within 30-60 min, the increase being more prominent in GN. The studies suggest that the B genome in the stress-tolerant HB may counter more drastic combined stresses without taking recourse to the expression of stress NACs.

Characterization of two TERMINAL FLOWER1 homologs PgTFL1 and PgCENa from pomegranate (Punica granatum L.).

FLOWERING LOCUS T (FT) and TERMINAL FLOWER1/CENTRORADIALIS (TFL1/CEN) are the key regulators of flowering time in plants with FT promoting flowering and TFL1 repressing flowering. TFL1 also controls floral meristem identity and its maintenance. In this study we have characterized two pomegranate (Punica granatum L.) TFL1/CEN-like genes designated as PgTFL1 and PgCENa. The expression of PgTFL1 and PgCENa fluctuated through alternate pruning and flowering cycles, being highly expressed during the vegetative phase (immediately after pruning) and decreasing gradually in the months thereafter such that their lowest levels, especially for PgCENa coincided with the flowering phase. Both the genes are able to functionally suppress the Arabidopsis tfl1-14 mutant flowering defect. Their expression in Arabidopsis resulted in delayed flowering time, increased plant height and leaf number, branches and shoot buds as compared with wild type, suggesting that PgTFL1 and PgCENa are bonafide homologs of TFL1. However, both the genes show distinct expression patterns, being expressed differentially in vegetative shoot apex and floral bud samples. While PgTFL1 expression was low in vegetative shoot apex and high in flower bud, PgCENa expression showed the opposite trend. These results suggest that the two TFL1s in pomegranate may be utilized to control distinct developmental processes, namely repression of flowering by PgCENa and development and growth of the reproductive tissues by PgTFL1 via distinct temporal and developmental regulation of their expression.

Analysis of synonymous codon usage bias and phylogeny of coat protein gene in banana bract mosaic virus isolates.

The banana is one of the world's most important livelihood crops. Banana plants are principally infected by four virus species, Banana bunchy top virus (genus Babuvirus), Cucumber mosaic virus (genus Cucumovirus), Banana streak virus (genus Badnavirus) and Banana bract mosaic virus (genus Potyvirus). The objective of this study is to understand the codon usage pattern and phylogeny of coat protein gene in different banana bract mosaic virus (BBrMV) isolates. The BBrMV Coat Protein (CP) gene was amplified from BBrMV infected banana plant samples collected from different districts of Tamil Nadu and Karnataka, India. Six new BBrMV isolates were submitted to National Center for Biotechnology Information. Phylogenetic analysis and codon usage indices were studied along with other isolates of BBrMV. Phylogenetic analysis of CP genes shows that most of BBrMV isolates are closely related to each other except KF385484.1 and KF385478.1. Relative codon usage patterns among different BBrMV isolates were calculated by software CodonW version 1.4.2. In BBrMV, codons with A-ended or U ended are the most preferential except the Leu and Gln whose optimized codons are CAG and UUG ending by G. The codon usage patterns of BBrMV isolates are principally influenced by mutational bias; however, compositional constraints along with mutational bias also play a major role.

Flowering time in banana (Musa spp.), a day neutral plant, is controlled by at least three FLOWERING LOCUS T homologues.

Banana is an important day neutral food crop with a long flowering/fruiting cycle that is affected by hot summers or cold winters in different places. Manipulating its life cycle requires an understanding of its flowering time machinery to bypass these stresses. Twelve FLOWERING LOCUS T (FT) and two TWIN SISTER OF FT (TSF) members were isolated from banana and their organization and expression pattern studied during development in two varieties that differ in flowering time namely Grand Nain (AAA genotype) and Hill banana (AAB genotype). The expression of at least 3 genes namely MaFT1, MaFT2 and MaFT5 (and to some extent MaFT7) increases just prior to initiation of flowering. These four genes and five others (MaFT3, MaFT4, MaFT8, MaFT12 and MaTSF1 could suppress the delayed flowering defect in the Arabidopsis ft-10 mutant and induce early flowering upon over-expression in the Col-0 ecotype. Most genes are diurnally regulated and differentially expressed during development and in various vegetative and reproductive tissues suggesting roles besides flowering. Subtle amino acid changes in these FT/TSF-like proteins provide interesting insights into the structure/function relationships of banana FTs vis-à-vis Arabidopsis. The studies provide a means for manipulation of flowering in banana for better management of resources and to reduce losses through abiotic stresses.

Association of interleukin-10 promoter polymorphism (-1082 g/a) and gastric cancer in andhra pradesh population of South India.

BACKGROUND: Gastric Cancer (GC) is one of the most commonly diagnosed malignancies. Genetic variation in genes encoding cytokines and their receptors, determine the intensity of the inflammatory response, which may contribute to individual differences in the outcome and severity of the disease. Interleukin-10 (IL-10) is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions. Polymorphisms in the IL-10 gene promoter genetically determine inter-individual differences in IL-10 production. In the present study, we investigated the association between the IL-10 -1082 G/A polymorphism and the susceptibility to gastric cancer in a South Indian population from Andhra Pradesh. METHODS: We genotyped 100 patients diagnosed with gastric cancer and 132 healthy control subjects for -1082G/A single nucleotide polymorphism by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) method followed by agarose gel electrophoresis. RESULTS: The distribution of IL-10 genotypes at -1082 G/A were GG 18 %, GA 35% and AA 47 % in gastric cancer patients and GG 31.82 %, GA 37.88 % and AA 30.3% in control subjects. The allelic frequencies of G and A were 0.355 and 0.645 in GC patients and 0.508 and 0.492 in control subjects respectively. The IL-10 -1082 A allele was associated with risk of gastric cancer (OR=1.873, 95%CI-1.285-2.73and P= 0.001048**). CONCLUSION: Our study indicates that allele A of IL-10-1082 G/A polymorphism may be considered as one of the important risk factor in the etiology of gastric cancer.

Protection of the hemopoietic system by Podophyllum hexandrum against gamma radiation-induced damage.

A semi-purified extract of low-altitude Podophyllum hexandrum (REC-2001) containing a relatively low content of podophyllotoxin (3.25 %) exhibited potent antioxidant ability in lipid media (at 1000 microg/mLagainst 0.25 kGy) and significant (p < 0.05) hydroxyl ion scavenging potential (78.83 % at 500 microg/mL). In vitro investigations revealed the ability of REC-2001 to significantly (p < 0.05) reduce radiation-induced hemolysis (2 microg/mL; 46.184 %) and nitric oxide scavenging levels (IC (50): 792 +/- 1.25 microg/mL). Protection of the hemopoietic system of Strain 'A' mice administered 20 mg/kg BW REC-2001 30 min prior to lethal irradiation (10 Gy) was recorded and was mediated by free radical scavenging and lowering of lipid oxidation. Further studies investigating the effects of REC-2001 on stem cell modulation are warranted.