Ectopic expression of choline oxidase (codA) gene from Arthrobacter globiformis confers drought stress tolerance in transgenic sugarcane

Abstract

Drought is a serious problem that impacts sugarcane production and productivity worldwide. In this current investigation, a codon-optimized choline oxidase (codA) gene was transformed into Saccharum hybrid cultivar Co 86032 through Agrobacterium-mediated transformation. The transgenic events with the codA gene driven by the portubi882 (PD2) promoter accumulated elevated levels of glycine betaine (5 - 10µg/g) whereas untransformed control plants accumulated less than 1.5µg/g which in turn maintained the plant health by sustaining transpiration rate (4 - 5 µmol of H₂O/cm²/s) and photosynthetic efficiency (30 - 34 µmol/Co₂/s) whereas the control plants suffered from 50% reduction under water-deficit stress condition. Morpho-anatomic cross-sections of both transgenic events and control plants exhibited significant differences in the epidermal layer and sclerenchyma cells under stress conditions. The relative water content (71 - 76%) and chlorophyll fluorescence (0.60 - 0.72 Fv/Fm) were higher in transgenic events compared to control plants respectively recorded 59% and 0.50 respectively. In addition, significantly elevated activity of antioxidant enzymes viz., superoxide dismutase (95 - 102 U/g), catalase (65 - 73 umol/min/g), ascorbate peroxidase (1700 - 1900 umol/min/mg) and glutathione reductase (17 - 20 umol/min/mg) were observed in transgenic events along with reduced levels of hydrogen peroxide (14 - 16 µmol/g) and malondialdehyde (14 - 17 nmol/g) content. Transgenic events recorded significantly higher arial biomass content compared to untransformed plant after the drought stress. Overall, the increased expression levels of codA gene in sugarcane events resulted in an enhanced ability to withstand water-deficit conditions.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-024-04151-y.

Keywords: Antioxidants; Drought stress; Overexpression; Relative water content.

Fig. 1

Visual representation of the plant expression vector pCAMBIA1305.1 with portubi882 promoter and codA gene

Fig. 2

Confirmation of codA gene and portubi882 promoter through restriction digestion

Fig. 3

Response to water-shortage stress on wild type and transgenic events

Fig. 4

Anatomic cross-section of root and leaves during normal irrigation and water-deficit conditions. A Normal irrigation, B Transgenic plant (codA8) under water-deficit stress, exhibiting better root and leaf structure under stress, including thicker epidermis, increased sclerenchyma and pericycle, and protected xylem, phloem, and bulliform cells, C Control plant under water-deficit stress. (a- epidermis, b- exodermis, c- sclerenchyma, d- parenchyma, e- aerenchyma, f- endodermis, g- metaxylem, h- protoxylem, i- phloem, j- pith, k- pericycle.)

Fig. 5

Relative water content of transgenic and control plant samples during 0 day and 10th day of water-deficit conditions. Asterisk (*) and (**) indicate significant differences (P < 0.05; P < 0.01; Student's t- test) between transgenic events with respect to its control plants. The error bar signifies the standard error value

Fig. 6

Chlorophyll fluorescence of transgenic events and control plants at 0 day and 10th day of water-deficit conditions. Asterisk (*) and (**) indicate significant differences (P < 0.05; P < 0.01; Student's t- test) between transgenic events with respect to its control plants. The error bar signifies the standard error value

Fig. 7

Cell-membrane thermostability of codA transgenic events and control plants at 0 day and 10th day of water-deficit conditions. Asterisk (*) and (**) indicates significance at P values below 0.05 and 0.01 as per the Student’s t- test. The error bar represents the standard error

Fig. 8

Transpiration rate of transgenic events and control plants during normal irrigation and water-deficit conditions. Asterisk (*) and (**) indicate significant differences (P < 0.05 and P < 0.01, Student’s t- test) between transgenic events with respect to its control plants. The error bar represents the standard error

Fig. 9

Efficiency of photosynthetic processes in genetically modified events and control plants during normal irrigation and water-deficit condition s. Asterisk (*) and (**) indicate significant differences (P < 0.05; P < 0.01; Student's t- test) between transgenic events with respect to its control plants. The error bar reflects the standard error measurement

Fig. 10

Superoxide dismutase (A); Catalase (B); Ascorbate peroxidase (C) and Glutathione reductase (D) activity of both control and transgenic events during normal irrigation and water-deficit conditions. Data representation is based on the mean of three replications. Asterisks (*) highlight a significant disparity at P < 0.05 according to Duncan’s multiple range test. Standard error values are shown with the error bar

Fig. 11

Relative proline (A), hydrogen peroxide (B), and malondialdehyde (MDA) (C) content of codA overexpressing Transformed lines and wild type under 0 and 10th day of water-deficit conditions. The data is depicted as the central tendency value of three replications. An asterisk (*) denotes a substantial variation at P < 0.05 according to Duncan’s multiple range test. The error bar illustrates the standard error margin

Fig. 12

Glycine betaine levels of transgenic events and control plants on 0 and 10th day of water-deficit condition. GB extracted from control and transgenic events are averaged over three replicates. Asterisk (*) and (**) indicate significant differences (P < 0.05; P < 0.01; Student's t- test) between transgenic events with respect to its control plants. The error bar signifies the standard error value

Fig. 13

Total areal biomass content of codA transgenic sugarcane events and WT (Co 86032). A Fresh and dry biomass of transgenic events and control under irrigated conditions; B Fresh and dry biomass of transgenic events and control after 10 days of drought conditions. All transgenic events exhibited significant difference compared to WT

Fig. 14

Expression levels of codA gene in transgenic events and wild type plants under abiotic stress conditions. Data are presented as a mean of six replications and the standard error is denoted by the error bars. Significant distinctions between transgenic events and control plants are marked by asterisks (*) (P < 0.05; Student’s t- test)