Seed Quality, Chlorophyll and Carotene Content in Brassica Juncea L. Leaves at Two Growth Stages in Response to Rhizospheric Bacteria

Introduction

The contemporary vegetable production systems, heavily reliant on soil fertilizers, have led to an escalation in soil health degradation. The World Food Program (WFP) aiming to improve food production efficiency is equally concerned with minimizing the adverse impacts on ecosystems and human health. In pursuit of this goal, the utilization of plant biostimulants, natural substances distinct from fertilizers and pesticides, has emerged as a promising alternative to address the challenges stemming from the industrialization of agriculture as advocated by the World Bank.

Brassica juncea L., a member of Brassicaceae family, stands out as a commercially important major oilseed crop grown widely in North America, Asia, Europe, and Northern Africa. Brassica juncea is a high-fiber, mineral-rich, and phytochemical-rich food crop grown annually with major contributors in global annual production being the European Union (30.87%), Canada (26.36%), China (20.41%), India (8.54%), Australia (4.93%), and Others (8.89%) (Source: Agricultural statistics at a Glance 2022). Plant growth and developments are significantly influenced by PGPR which modifies the chemistry of the rhizosphere providing disease resistance/and higher nutrient absorption, resulting in high biomass production. Microbial-inoculated Brassica sp. has been reported to improve photosynthesis under stress by reducing stomatal resistance, thereby enhancing biomass^{2, 3}. This increase in yield can be attributed to higher photosynthetic activities and an efficient uptake of nutrients and water.

Microbial inoculants are largely being studied for their remedial effects on stress generated oxidative bursts affecting the photosynthetic apparatus, proteins, membrane lipids and other cellular components⁴. The composition of leaf chlorophyll and carotenoid undergoes changes in trees across different phenological stages⁵ and along light canopy gradients⁶, as well as in response to various biotic^{7, 8} and abiotic stressors⁹ like heat-waves, drought^10,11, UV-B¹², and elevated CO₂ and O₃^13,14. Leaf pigments are vital for seedling growth and protection. As seedling mature, the balance and amount of leaf pigments adjust to optimize photosynthesis and protection. While PGPRs are well studied for their effects on promoting plant growth, their influence on content of various leaf pigments and their variations has not been thoroughly investigated. Therefore, the study aimed to explore the impact of various PGPRs on the growth, photosynthetic efficiency, and leaf pigments of Brassica juncea seedlings.

Materials and Methods

The study was performed at Banaras Hindu University (25.2677° N, 82.9913° E) situated in district Varanasi of India. It has a lenient, hot, and temperate climate with an average annual temperature of 26.1°C and rainfall of 1110 mm.

Seed bio-priming and growth conditions

The seeds of Indian mustard (Brassica juncea L.) procured from the Indian Agriculture Research Institute (IARI), New Delhi, India, were surface sterilized by 1% sodium hypochlorite (NaOCl) for 30 seconds, then allowed to air dry after two rounds of rinsing in sterile distilled water under laminar air-flow. Bacterial strains isolated from fields of northern India with plant growth-promoting traits tested on wheat were selected for bio-priming mustard seeds. The selected PGPRs were screened for biochemical and growth-promoting traits in our lab as already reported¹⁵. Molecular identification (16S rRNA) of bacterial strains was done earlier and Gene Bank accession number obtained were viz JRBHU1 (Burkholderia paludism-MK439528), JRBHU4 (Pseudomonas lactis-MK500865), JRBHU5 (Pseudomonas azotoformans-MK500938), JRBHU6 (Burkholderia seminalis-MK500868), JRBHU9 (Enterobacter hormaechei-MK500940), JRBHU10 (Enterobacter cloacae-MK501756), JRBHU11 (Bacillus subtilis-MN759630), and P21 (Pseudomonas gessardii RRBHU-1-OK427346). Each of the eight bacterial strains was grown in nutrient broth medium on a rotary shaker at 27^° C for 24 hours. Subsequently, the mustard seeds were immersed in 25 millilitres of nutrient media having pre-screened bacterial suspensions (10⁹ CFU/ml) and were maintained at 28 ± 2° C at 90 rpm for 10-12 hours and seeds were sown without any treatment in the control group. Seeds were further dried overnight under laminar air flow and used for greenhouse experiment. An amalgamation of soil, sand, and vermiculite (4:1:1 v/v) was autoclaved for three consecutive days. Bio-primed seeds were then sown in a greenhouse at 25/20° C, 16/8 h day/night photoperiod, 500-700 µmol m^-2 s^-1 photosynthetic photon flux density (PPFD), and 70% relative humidity. The treatments were randomly distributed with three replications each.

Seed quality and seedling parameter

The germination percentage was calculated after five days and paper towel method was used to determine the Vigour index (VI). After 15 days 3 plants from each treatment were carefully removed from pots and properly washed for analyzing the different plant growth parameters. Measurements of shoot and root length, leaf number and area, live fine fuel moisture (LFFM) fresh and dry biomasses and relative water content (RWC) were taken for all treatments along-with controls. The shoots and roots were individually oven-dried at 105°C for 24 hours to determine their dry weight. Fresh samples were used for leaf pigment analyses.

Leaf chlorophyll and carotene extraction

Chlorophylls and carotenoids are fat-soluble compounds, and acetone provides sharp absorption peaks, making it the preferred solvent for chlorophyll assays¹⁶. Therefore, approximately equal amounts of leaf tissue were collected from all three uprooted plants and bulked for each treatment. Leaf tissue (20mg) was macerated in a pestle and mortar in 5ml of 80% acetone. These extracts were adjusted up-to 10 ml of final volume. To avoid pigments photo-bleaching, extraction and concentration procedures were performed in subdued light.

UV induced chlorophyll fluorescence

Preliminary chlorophyll estimations in samples were done based on fluorescence emission on UV exposure¹⁷. The fluorescence intensities emitted were dependent on the amount of light absorbed by the chloroplast present in the mesophyll and were graded as +++ (very high), ++ (high), and + (moderate) based on visual observation. To compare the fluorescence intensities emitted by the chloroplast equal amounts of Brassica leaf extract were taken in culture tubes and UV radiations were passed through Benchmark UV lamp (UV intensity = +500 µW/cm2, and wavelength = 254 nm) in a dark room maintained at 20° C.

Spectrophotometry

The absorbance of leaf extracts was measured using Jasco V-670 UV-VIS-NIR spectrophotometer model in a range of 400–700 nm. The absorption peaks of chlorophylls can shift to longer wavelengths when the solvent’s polarity and/or water concentration increase, with the shift being more pronounced in the blue region than the red region. Chlorophyll a and chlorophyll b absorb light in narrow bands in the blue and red spectrum ranges, whereas carotenoids have broader absorption bands in the blue range¹⁸. For the identification and quantification of carotenoids, spectral absorption was used and α-carotene was measured at 444nm, and β-carotene at 452nm¹⁹. Peaks of carotenoids’ absorption can differ depending on the solvent type and water content, with shifts occurring at higher water content. This shift can be one nm or more, depending on the solvent and carotenoid type²⁰.

Data analysis

Chlorophylls, total chlorophylls, and carotenoids concentration were calculated by the below mentioned formulae. The pigments maximum absorption wavelength was determined as it shifted with the growth stages, as mentioned in equations 1 to 6.

Seedlings stage,

Chl a=12.21 A₆₆₃–2.81 A₆₄₆, Chl b=20.13 A₆₄₆–5.03 A₆₆₃                                                   (1)

Chlorophyll (a + b) = (8.05A₆₆₃ + 20.29A₆₄₆)                                                                      (2)

For β carotene (1000A₄₅₂) or for α carotene (1000A₄₄₄)-1.82chl a_c–85.02chl b_c/198   (3)

One-month plant stage,

Chl a=12.21 A₆₇₆–2.81 A₆₄₆, Chl b=20.13 A₆₄₆–5.03 A₆₇₆                                                 (4)

Chlorophyll (a + b) = (8.05A₆₇₆ + 20.29A₆₄₆)                                                                      (5)

For β carotene (1000A₅₃₇) or for α carotene (1000A₅₂₄)-1.82chl a_c–85.02chl b_c/198          (6)

Abbreviations: A = absorbance, chl = chlorophyll, c = pigment content in µg/ml of extract.

The experiments were conducted in triplicates in a randomized design, and the data were analyzed using software SPSS-version 20. The LSD test was used to compare means, and a 0.05 probability level was set for analyzing the critical difference among isolates in each treatment.

Results

Rhizobacterial effects on plant growth

It was over-all observed that rhizospheric bacteria improved plant growth and nearly 100% seed germination was recorded in treatments viz. JRBHU1, JRBHU5, JRBHU6, and P21 (figure 1). Treatment of JRBHU5 resulted in the longest root with 84% increase, followed by P21 (79%). JRBHU5 also showed the highest length of shoot with an increase of 127%, followed by P21 with 120% increase. Other strains exhibited substantially increases. The VI showed significant differences in JRBHU1, JRBHU5, JRBHU6, and P21 treatments. Additionally, the root:shoot length ratios, first and second internode distances were also obtained, as mentioned in Table 1.

Figure 1: Plant growth and vigour index of Brassica juncea L. after various PGPR   treatments at two different growth stages (A) Seedlings (B) One-month plants Click here to view Figure

Table 1: Seed germination and seedling parameters measured after different bacterial treatments. Click here to view Table

Impact of PGPR on plant biomass

The effects of rhizospheric bacteria on plant biomass was mostly insignificant for most of the isolates except for JRBHU5 which showed an increase of 1.3% in root dry biomass followed by P21 with 0.9% increase. Similarly, an increase of 5.6% in shoot dry weight was observed in JRBHU5 followed by P21 (4.8%) (Table1). Plants inoculated with PGPR isolates showed a significant increase in live fine fuel moisture content in shoots, with treatment JRBHU5 showing the highest LFFM value followed by JRBHU6 and P21 as depicted in figure 2E.

Impact of PGPR on leaf attributes

Evaluation of leaf variables viz. leaf number, petiole length, leaf area, RWC, and LDMC showed considerable effects of different bacterial treatments. The leaf counts and petiole length showed variations among which JRBHU5 followed by P21 treated plants had the highest leaf count as well as petiole length among all the treatments (Figures 2A and B). Inoculation of JRBHU5 also increased the leaf area maximally among all (Figure 2D). A slight rise in RWC was observed after different treatments for most of the isolates. However significant increase in RWC was observed in JRBHU6 (117.934%), and P21 (117.9%) inoculated plants (Figure 2C). Highest impact on LDMC with 14.5% increment was observed after JRBHU5 treatment followed by P21 (12.84%) and JRBHU6 (10.5%) (Figure 2F). The effect of bacterial isolates JRBHU1, JRBHU5, JRBHU6, and P21 on LFFM was highly significant.

Turkey’s multiple range test at p ≤ 0.05 was used to compare treatments, with results shown as means of three replicates and slanting bars suggest standard error of mean.

Figure 2: Effects of bacterial seed bio-priming on different leaf parameters (A) Leaf number (B) Petiole length (C) RWC (D) Leaf area (E) LFFM (F) LDMC Click here to view Figure

Bacterial effects on leaf pigments

To get a preliminary idea on effects of bacterial treatments on B. juncea leaf pigments, a fluorescence study was done. Chlorophyll molecules extracted from Brassica plant leaves re-emit red light as fluorescence in varying amounts under UV light exposure depending upon the chlorophyll content which was graded in the scale (+, ++, +++) based on visual observation (Table 2). Bacterial treated young seedlings and one-month-old treated plants showed a dark ruby color as compared to the control. An immense rise in red intimacy was visually observed in treatments JRBHU1, JRBHU5, JRBHU6, and P21 at both seedlings and one-month stage as depicted in Figures 3 A and B.

Figure 3: Leaf pigment fluorescence under UV light exposure at different growth stages (A) Seedlings (B) One-month plants Click here to view Figure

Table 2: Pursuance of PGPRs on UV-induced chlorophyll fluorescence

Bacterial isolates	Seedlings	One-month plants
JRBHU1	+ + +	+ + +
JRBHU4	+ +	+ +
JRBHU5	+ + +	+ + +
JRBHU6	+ + +	+ + +
JRBHU9	+ +	+ +
JRBHU10	+ +	+ +
JRBHU11	+ +	+ +
P21	+ + +	+ + +
Control	+	+
+: Visual intensity of red fluorescence

 Absorption maxima and spectra

The absorption maxima of leaf pigments being very narrow are often affected by the spectrophotometer instrument²¹. To measure the accurate effect of bacterial treatments on leaf pigment content at different growth stages the absorption maxima were first calculated for each pigment (chlorophylls, carotenoids) in the range 400–700 nm with the laboratory spectrophotometer (model Jasco V-670). The literature clearly advocates that at wavelength deviation of more than 1nm, it is advisable to measure absorbance maxima. Similarly, the wavelength regions should not exceed 2nm for the same equations to be applied to any extraction solvent. The differences in absorption maxima of different pigments in acetone extract as mentioned in literature are given in Supplementary Table 1. The absorption spectrum of Brassica leaf extract containing mixtures of chl a and b, β and α carotenes obtained over acetone solvent at two different growth stages were measured to find the absorption peak. Accordingly, the spectrophotometer readings were made as to 663nm for chl a and 646nm for chl b in seedlings, 676nm for chl a and 646nm for chl b in one-month plant. Similarly, the wavelengths for β and α carotenes were set at 452 and 444nm (seedlings) and 537 and 524nm (one-month plant) as depicted in Figure 4A and B. The solvent-specific calculation of the pigment content at different growth stages was assessed by using the relevant equations (Equations 1 to 6).

Figure 4: Absorption spectra of different pigments of B. juncea L. leaf after different bacterial treatments at different growth stages (A) Seedlings (B) One-month. Click here to view Figure

Quantification of pigments

Leaf pigments are critical indicators of plant’s photosynthetic and photo-protection status. Their concentrations vary across different growth stages and are affected by multiple factors. It was found that the chlorophyll content in one-month plants was significantly higher than carotenoids whereas the reverse was observed at seedlings stage.  Chl a content was much higher than that of Chl b at the seedlings stage (Figure 5A) but was found to degrade rapidly with plant development as at one-month stage, the content of Chl b increased (Figure 5B). The content of α carotene was slightly higher than that of β carotene at seedlings (Figure 5A), while after one-month the β carotene got increased. (Figure 5B). With plant growth, the chlorophyll contents improved while the carotenoid contents decreased. Similarly, among the chlorophyll pigments, the chlorophyll b content increased from 0.317 to 34.577 mg/mL, ranging from the seedling stage to the one-month stage (Figures 5 A and B). The trend of variation in leaf pigments with growth stages was observed to be similar in both control and bacterial treated plants. However, the different bacterial treatments showed significant effects on amount of chlorophyll and carotenoid contents. At seedlings stage, the bacterial influence was more on chl a, which was high compared to chl b. Similarly, at one month stage, the influence was more on chl b which was higher in amount than chl a. This overall reflected that the rhizospheric bacteria had strong influence on plant physiological and biochemical pathways involved in synthesis and degradation. Among all the tested bacteria higher chl b content at one month stage was observed in JRBHU5 and P21 inoculated plants that was 23 and 29 folds higher than the seedlings plants respectively. Most of the bacterial strains also influenced the carotenoid contents of Brassica. PGPR strains JRBHU5 and P21 were most influential in affecting both synthesis and degradation of carotenoid compounds.

Figure 5: Effect of bacterial seed biopriming on leaf chlorophyll and carotenoid contents at different growth stages (A) Seedlings (B) One-month Click here to view Figure

The pigment ratio in Brassica varied with different rhizobacterial treatments and growth stages. Chl a:Chl b showed 53.83% and 47.27% increases in JRBHU9 and control plants at the seedling stage, whereas 72.6% and 67.9% increases in control and JRBHU4 plants was observed after one month stage (Figure 6A). Total chlorophyll (a+b) content was higher in treatments, with JRBHU5 showing an increase of 76.71% followed by P21 with 63.75% increase at seedlings stage whereas at one-month plants this was 110% and 109% for the same treatments (Figure 6B). Total carotenoids (X+C) increased in treatments JRBHU5 (41.79%), and P21 (38.81%) at seedlings stage while control (-32.45%), and JRBHU4 (-35.88%) decreased in one-month plants (Figure 6C). Ratio of total chlorophyll to total carotenoids was higher in JRBHU5 (18.55%), and P21 (16.44%) in seedlings while P21 (-21.61%) and JRBHU1 (-21.72%) decreased in one-month plants (Figure 6D). Photosynthetic pigment concentration increased by a certain percentage compared to the control in Brassica juncea (Table 3).

Figure 6: Impact of PGPRs on the concentration of photosynthetic pigments of B. juncea L. at seedlings and one month stage (A) Chl a/b (B) Chl (a+b) (C) (X+C) (D) Chl (a+b) / (X+C) Click here to view Figure

Table 3: Percent increment in leaf pigments of B. juncea L. after different PGPR treatments

(A) Seedlings
Bacterial isolates	Chl a%	Chl b%	β car %	α car %
JRBHU1	292.2 ± 51.120a	397.2± 40.925bc	343.6 ± 3.815ab	313.4 ± 14.205bc
JRBHU4	20.0 ± 7.944c	88.0 ± 2.168d	62.0 ± 8.191c	40.4± 0.905e
JRBHU5	420.2 ± 50.658a	695.1 ± 109.480a	416.9 ± 45.929a	402.6 ± 5.875a
JRBHU6	273.5 ± 44.151ab	375.8 ± 5.414bc	312.2 ± 28.580b	284.8 ± 0.409c
JRBHU9	54.9 ± 6.974c	45.1± 0.990d	43.4 ± 10.754c	33.0 ± 4.311e
JRBHU10	92.9 ± 16.312bc	174.1 ± 17.382bcd	134.3 ± 9.153c	112.9 ± 1.531d
JRBHU11	88.4 ± 4.258bc	135.1 ± 19.672cd	104.5 ± 14.869c	92.2± 10.446de
P21	359.1 ± 70.920a	452.8 ± 73.480ab	383.2± 0.994ab	369.6 ± 32.401ab
(B) One-month plants
Bacterial isolates	Chl a%	Chl b%	β car%	α car%
JRBHU1	26.2 ± 0.982a	50.6 ± 0.569abc	41.1 ± 0.307ab	43.6± 0.021abc
JRBHU4	3.6 ± 1.522b	11.1 ± 5.049e	10.5 ± 4.842c	10.6 ± 5.015d
JRBHU5	31.9 ± 1.531a	61.9± 3.909a	53.8 ± 5.410a	55.5 ± 5.309ab
JRBHU6	30.1 ± 0.204a	56.8 ± 2.668ab	42.5 ± 8.554ab	47.5 ± 6.259ab
JRBHU9	3.3 ± 1.357b	25.2 ± 3.141de	24.4 ± 3.372bc	24.8 ± 3.447cd
JRBHU10	9.8 ± 4.521b	41.3 ± 4.401bcd	41.0 ± 4.758ab	41.4 ± 4.738abc
JRBHU11	10.0 ± 4.951b	37.5 ± 2.936cd	33.1 ± 1.074abc	34.8 ± 1.865bc
P21	30.6 ± 1.481a	61.5 ± 3.531a	55.4 ± 4.983a	56.7 ± 4.876a
Abbreviation: Chl- chlorophyll, Car- carotene The data represents the means of three replicates ± SE, with significant differences observed within the same column with different letters at p-value ≤ 0.05.

Pearson correlation coefficients computed among the photosynthetic pigments of Brassica to identify the linearity of the correlation among the pigments with respect to growth stages was found to be significant at p ≤ 0.05 at both growth stages. The correlation results showed that carotenoid content was strongly related to chlorophyll content expressed at different growth conditions. The data analysis showed that chl b was strongly and highly impacted by chl a, and carotenes at seedlings stage, while at one month stage chlorophyll a was found to be positively and highly correlated with chlorophyll b, α carotene, and β carotene. While a positive correlation was found among the different chlorophyll and carotenoid pigments at seedlings stage, after one month a negative correlation was observed among the different chlorophyll and carotenoid pigments due to steady decrease in chlorophyll a and α carotene with plant growth. Thus, increase in carotenoids at seedling stage promoted the chl a synthesis while after one month it promoted the chl b synthesis.

Discussion

The study investigated the influence of eight rhizobacterial isolates on the plant growth, development and pigment concentrations of B. juncea L. (Indian mustard). PGPRs in eukaryotic hosts can produce reactive oxygen species (ROS) and cause oxidation bursts within the endophytic cell due to severe conditions like reduced space, nutrition, and host-mediated regulation²⁸. Screening an efficient PGPR by analyzing its effect on various physiological and morphological plant parameters in a good practice³. The vigour index was chosen as the preliminary screening factor, as the growth of a plant is directly influenced by three crucial growth attributes: germination percentage, root length, and shoot length^29,30. Vigour index of plants after being inoculated by JRBHU5 and P21 increased by 105.5% and 95.25% compared to the control, indicating that JRBHU5 and P21 were the finest strains. PGPR treatments resulted in increased chlorophyll content, root-shoot diameter, root-shoot length and weight showing delayed leaf senescence compared to non-inoculated controls in cauliflower, muskmelon, and watermelon transplants^31,32, Cicer arietinum seedlings³³, maize seedlings inoculated with Burkholderia phytofirmans and Enterobacter sp.³⁴, and cabbage seedlings inoculated with Bacillus megaterium and Pseudomonas agglomerans^35,36. Inoculation of JRBHU5 exceeded the control’s shoot dry biomass, possibly due to enhanced photosynthate assimilation and biomass aggregation. Plants inoculated with JRBHU5 showed comparable shoot and roots elongation, suggesting both below and above ground portions growing in response to PGPR. Root water intake and transpiration loss significantly impact the moisture content in shoots and leaves. LFFM, or shoot water content, is an indicator of combustibility. Inoculation of bacterial isolates showed increased shoot moisture, rehydrating the plant and contributing to biomass formation. JRBHU6 (92.4%) and P21 (84.35%) displayed significant increases in LFFM, with JRBHU5 (93.65%) showing the highest value. Plants inoculated with JRBHU5 showed a significant increase in leaf area, which improved the photosynthetic and energy harvesting capabilities, thereby improving plant yield³⁷. Improvements were observed in the RWC of plants treated with JRBHU6 and P21 rhizobacterial strains, indicating lateral roots’ role in nutrient and water transport, as they increase root surface area for absorption³⁸. Bacillus52 and Pseudomonas putida51 treatment has been reported to induce soil micro-aggregation, leading to increased soil water content and water availability in the leaves of Eclipta prostrate³⁹. Plants in drought-prone areas respond better to steady tissue hydration and as rhizospheric bacterial strains accelerate imbibition it also influenced the RWC⁴⁰. PGPRs were crucial in affecting leaf mass and water retention processes along with a significant impact of LDMC^41,42.

Rhizobacterial treatments also significantly affected the Brassica’s leaf pigments content at different growth stages. PGPR inoculation responses are higher at early growth stages, possibly due to root nutrient exchanges becoming more intense during vegetative growth⁴³. Exposure to UV light to study the chlorophyll fluorescence revealed a significant increase in red intensity in treatments JRBHU1, JRBHU5, JRBHU6, and P21 at both seedling and one-month stages. UV light promotes electrons from the S₀ state to the S₂ state in chlorophyll, allowing pigments to observe only blue-violet wavelengths. When excited with ultraviolet light, in the absence of an electron transport chain, chlorophyll molecules fluoresce in red zone, indicating their return to their ground state⁴⁴. The Brassica leaf extract exhibited variations in absorption maxima at different growth stages due to redshift. The study also confirms the influence of refractive index and dielectric constant of solvents on the wavelength shifts in chlorophyll absorption⁴⁵. It is well documented that the interaction between chlorophyll molecules can cause a red shift in absorption spectra^18,46. The rhizobacterial treatments prominently increased the pigment concentrations over the control. Compared among the treatments JRBHU5, JRBHU6, JRBHU1, and P21 showed a higher pigment amount. In seedlings, Chl a was abundant compared to Chl b, but ratio got reversed in one-month old plants. The concentration of α carotene was higher than β carotene at seedlings stage whereas in one-month plants carotene ratio were found negative due to higher concentration of chl b than chl a. Carotene is an accessory light-harvesting pigment transferring its excitation energy to chlorophyll⁴⁷. Rhizospheric bacterial treatments significantly affected chlorophyll and carotenoid ratios, with JRBHU5 and P21 being most effective. Seedlings had higher chl a: chl b, (x+c), and (chl a+b: x+c), while after one-month plants had higher (chl a+b) and the chl a:chl b in leaves was approximately 3:1. The study clearly indicated that growth conditions and environmental factors have significant impact on pigments ratio. Low light and shade increases LHCPs proportion, resulting in lower chlorophyll a/b values, higher chlorophyll a/carotene values and total chlorophyll/carotenoids, indicating functional pigment equipment towards light adaptation⁴⁸. Lichtenthaler and Buschmann (1984) suggested that a decrease in Chl a: Chl b could be attributed to the expansion of PS II’s antenna system, as the pigment antenna system is the sole reservoir for Chl b⁴⁹. Bacterial strains inoculated in B. juncea L.  influenced the PS II antenna system size, as an increased chl b concentration was observed after treatments. Leaf greenness which is an indication of the higher ratio of total chlorophyll to carotenoids was increased in response to PGPR, while senescence/stress/damage triggers faster chlorophyll breakdown²¹. The content of chlorophylls to carotenes in leaves after treatments was in the following order JRBHU5>P21>JRBHU6>JRBHU1 indicating a delayed senescence. Thus, the bacteria showed significant effects on maintaining the photosynthetic efficiency of plants, delaying senescence, and regulating the aging process of plants probably by gene duplication, telomerase activity or by increasing meristematic cells⁵⁰. The acdS gene, which codes for ACC deaminase activity, has been identified in Pseudomonas sp which helps in stress alleviation, subsequently increasing the total chlorophyll content⁵¹. Studies have reported the variation in chlorophyll pigments according to season. Variations in the ratio of chl a: total chlorophyll in trees are reported to be highest in early season and minimal in mid-season, with no variability observed in chl b: total chlorophyll across seasons. Pigment derivatives and degradation products influence chlorophyll renewal especially in the mid-growing season⁵². Similarly in the present study variations in the concentrations of chlorophyll a and b was observed during various growth phases. It is crucial to note that chl a and b are reported to be inter-convertible in the chlorophyll cycle⁵³, as chl b is synthesized from chl a through chlorophyllide anoxygenase (CAO) enzyme⁵⁴. In the present study, the concentration of photosynthetic pigment got enhanced relative to the control, indicating a considerable effect of PGPRs treatments. Total chlorophyll content was also higher in Bacillus licheniformis inoculated plants compared to Pseudomonas plecoglossicida and control plants. Among all the bacterial treatments, JRBHU5 and P21 were found to be most influential in affecting the leaf pigments. Both these bacteria promoted the synthesis of chlorophyll a during the seedling stage, and after one-month, their effects were more pronounced on chlorophyll b favoring the normally observed inter-conversions in plant pigments at different growth stages. As plants grow from seedling to juvenile stage, the amounts of chlorophyll increase which affects the chlorophyll to carotenoid ratio, and the bacteria were found to promote the natural process quantitatively as well.

Conclusion

Rhizobacteria Burkholderia spp. (JRBHU1, JRBHU6), Enterobacter spp. (JRBHU9, JRBHU10), Bacillus spp. (JRBHU11), and Pseudomonas spp. (JRBHU4, JRBHU5, P21) enhanced plant growth and development, particularly by influencing photosynthetic pigments in Indian mustard (Brassica juncea L.) at various growth stages. Chlorophyll content in one-month plants was higher than carotenoids, compared to the seedling stage. Studies on chlorophyll regeneration can be facilitated by examining pigment derivatives, precursors, and degradation products of key pigments. JRBHU5 as well as P21 exhibited the most significant influence on leaf pigments ratio, promoting the synthesis of chl a during the seedling stage also facilitating inter-conversions in plant pigments at different growth stages.

Acknowledgements

The authors are highly grateful to Banaras Hindu University for the support extended under the Institute of Eminence (IoE) scheme.

Funding Sources

The grant received was from Banaras HIndu University under the IoE Scheme grant no. 6031.

Conflict of Interest

The authors declare that there is no conflict of interest.

Data Availability

This article has all the data that was generated or evaluated during this investigation, and the data can be reproduced and is provided by the authors upon request.

Author’s contribution

Surya Prakash Dube: Data curation; Formal analysis; Investigation; Methodology; Visualization; Roles/Writing – original draft. Riddha Dey:  Validation; Visualization; Writing. Seema Devi: Validation; Visualization; Writing. Richa Raghuwanshi: Conceptualization; Project administration; Resources; Supervision; Validation; Visualization; Review & editing.

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Supplementary Table 1:  Wavelength maxima (A_max) of leaf chlorophyll (a and b), and carotenes (α and β) in acetone solvent.

Amax for Chl a [nm]	Amax for Chl b [nm]	Amax for (x + c) [nm]	Amax for β car [nm]	Amax for α car [nm]	Reference
428.5blue	452.5blue				22
			452blue	444blue	19
428blue	453blue	470blue			21
663red	646red				23
663red	645red				24
663red	645red	470blue			25
663red	645red	480blue and 510blue			26
663red	645red	470blue			27
Abbreviations: Chl- Chlorophyll, Car- Carotenes, (x + c) = Total carotenoids