Physiological Traits for Shortening Crop Duration and Improving Productivity of Greengram (Vigna radiata L. Wilczek) Under High Temperature

Abstract

Greengram is an important protein-rich food legume crop. During the reproductive stage, high temperatures cause flower drop, induce male sterility, impair anthesis, and shortens the grain-filling period. Initially, 116 genotypes were evaluated for 3 years in two locations, and based on flowering, biomass, and yield attributes, they were grouped into four major clusters. A panel of 17 contrasting genotypes was selected for their heat tolerance in high-temperature greenhouses. The seedlings of the selected genotypes were exposed to heat shock in the range 37°C-52°C and their recovery after heat shock was assessed at 30°C. The seedlings of EC 398889 turned completely green and rejuvenated, while those of LGG 460 failed to recover, therefore, EC 398889 and LGG 460 were identified as heat-tolerant and heat-sensitive genotypes, respectively. Except for EC 398889, the remaining genotypes could not survive after heat shock. Fresh seeds of EC 398889 and LGG 460 were planted in field and pollen fertility and sucrose-synthase (SuSy) activity in grains were assessed at high temperatures. The pollen germination and SuSy activity were normal even at temperatures beyond 40°C in EC 398889 and high SuSy activity enabled faster grain filling than in LGG 460. The precise phenotyping demonstrated significant differences in the light-temperature response of photosynthesis, chlorophyll fluorescence imaging of quantum yield (Fv/Fm), and electron transport rate (ETR) between heat-tolerant (EC 398889) and heat-sensitive (LGG 460) genotypes. Molecular profiling of selected accessions showed polymorphism with 11 SSR markers and the markers CEDG147, CEDG247, and CEDG044 distinguished tolerant and sensitive groups of accessions.

Keywords: acquired thermotolerance; chlorophyll fluorescence; photosynthesis; sucrose synthase; thermo-tolerance.

Figure 1

Temperature regime during crop duration of greengram at two experimental sites.

Figure 2

Performance of a heat tolerant (EC 398889) and heat sensitive (LGG 460) genotypes at high temperature (45/25⁰C max/min) and 14 h day length (Heat tolerant genotype showed pod formation while sensitive genotype without pods at high temperature).

Figure 3

Seedling viability and regeneration after heat shock at 52⁰C in heat tolerant (EC 398889) (A–C) and heat sensitive (LGG 460) genotype (D–F). (A and D) showing results of TTC test; (B and E) showing amount of chlorophyll accumulation and (C and F) showing rejuvenation or failure of normal growth of plants after heat treatment.

Figure 4

Molecular profiling of amplified DNA of leaf samples extracted from selected heat sensitive and heat tolerant greengram genotypes. Marker CEDG 147, L-100 bp ladder; 1. (HUM 12); 2. (Ganga 8); 3. (EC 398889); 4. (IPM-02-03); 5.(IPM-02-14); 6. (LGG 460); 7. (Kopergaon); 8. (NSB 007).

Figure 5

SDS-PAGE of protein profile (leaf) of heat sensitive (LGG 460) and tolerant (EC 398889) genotypes preadapted to normal (25/18 ⁰C) and high temperature (43/35⁰ C) conditions. Lanes of SDS-PAGE represented Protein markers (1); while (2 and 3) for LGG 460 adapted to low (25/18⁰C) and high (43/35⁰C) temperature (3), respectively; The lanes (4 and 5) for EC 398889 adapted to low (25/18 ⁰C) and high (43/35⁰C) temperature, respectively. Additional protein band between 91–137 kDa appeared in EC 398889 adapted to high temperature regime (as shown in circle).

Figure 6

Response of net photosynthetic rate (A), stomatal conductance (B), and transpiration rate (C) to increasing temperatures (20⁰C, 30⁰C, and 40⁰C) in preadapted plants to low temperature (LT) or high temperature (HT) conditions in heat tolerant (EC 398889) and heat sensitive (LGG 460) genotypes. (D) represents light response of photosynthetic electron transport rate, ETR 30⁰C and 40⁰C in heat tolerant (EC 398889) and heat sensitive (LGG 460) genotypes. (A–C) Each value represents mean of three replications with standard error of mean (SEm) shown by error bar. Analysis of variance test using two factors factorial design (Genotype,G and Temperature, T) showed significant interaction effects (GxT) at P ≤ 0.01 on photosynthesis, stomatal conductance and transpiration with CD values 0.76**, 0.32**, and 0.15**, respectively, for treatment mean comparison. While (D), three factors such as Genotype,G (2), temperature,T (2), and irradiance levels, L (13) were taken into account to test the significance level of interaction among these factors (GxTxL) which was shown by CD value 2.1** (P ≤ 0.01) for treatment mean comparison.

Figure 7

Relationship among specific leaf area (SLA), SPAD chlorophyll meter reading (SCMR), and carbon discrimination (D¹³C) in selected greengram genotypes. The heat tolerant genotype (EC 398889) showed lower, SLA (A) and D¹³C (B) values indicating higher photosynthate partitioning and water-use efficiency as compared to heat sensitive genotype (LGG 460) (A, B). Each value represents mean of five replications.

Figure 8

Changes in chlorophyll fluorescence images (F₀, minimal; F_m, maximal, and F_v/F_m, ratio of variable to maximal fluorescence or quantum yield) at two temperatures (30⁰C normal and 43⁰C high temperature) in heat tolerant (EC 398889) and heat sensitive (LGG 460) genotypes. (A–C; top view): Fluorescence images at 30⁰C in dark-adapted leaves (LGG 460). (D–F; top view): Fluorescence images at 30⁰C in light-adapted leaves (LGG 460). (A–C; bottom view): Fluorescence images at 43⁰C in dark-adapted leaves (LGG 460). (D–F; bottom view): Fluorescence images 43⁰C in light-adapted leaves (LGG 460). (G–I; top view): Fluorescence images at 30⁰C in dark-adapted leaves (EC 398889). (J–L; top view): Fluorescence images at 30⁰C in light-adapted leaves (EC 398889). (G–I; bottom view): Fluorescence images at 43⁰C in dark-adapted leaves (EC 398889). (J–L; bottom view): Fluorescence images at 43⁰C in light-adapted leaves (EC 398889).

Figure 9

Sucrose synthase (SuSy) activity in developing grains of field-grown greengram (heat tolerant EC 398889 and sensitive LGG 460) genotypes at different pod development stages. Each value represents mean of three replications.Treatment means comparison (Genotype, G and days after anthesis, D) and significance levels of difference (CD) between genotype and days after anthesis activating SuSy was performed based upon the CD value 751.1** of interaction effects of Genotype x days (GxD) significant at P ≤ 0.01.

Figure 10

Pollen germination of heat tolerant (EC 398889) and sensitive (LGG 460) greengram genotypes at different temperatures. (A,B,C,D,E,F,G,H) represents pollen tube growth of heat tolerant (EC 398889) and (I,J,K,L,M,N,O,P) for heat sensitive (LGG 460) genotypes with progressive increase in the temperature from 29⁰C to 43⁰C.Abnormalities in pollen tube growth in heat sensitive genotype LGG 460 was noticed at much lower temperature starting from 39⁰C onwards than heat tolerant ones EC 398889 that had occurred at higher temperature 43⁰C.