# Overexpression of sirohydrochlorin ferrochelatase boosts nitrogen and carbon assimilation and overcomes nutrient deficiency in Arabidopsis thaliana

Naveen C Joshi, Laurent Dufossé, Baishnab C Tripathy

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Naveen C Joshi, Laurent Dufossé, Baishnab C Tripathy. Overexpression of sirohydrochlorin ferrochelatase boosts nitrogen and carbon assimilation and overcomes nutrient deficiency in Arabidopsis thaliana. Plant stress, 2026, 19, pp.101143. 10.1016/j.stress.2025.101143. hal-05509442

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# Overexpression of sirohydrochlorin ferrochelatase boosts nitrogen and carbon assimilation and overcomes nutrient deficiency in *Arabidopsis thaliana*

Naveen C. Joshi<sup>a</sup>, Laurent Dufossé<sup>b,* ID</sup>, Baishnab C. Tripathy<sup>c,*</sup>

## ARTICLE INFO

### Keywords:

Nitrite reductase

Nitrogen assimilation

Photosynthesis

Siroheme

Sirohydrochlorin ferrochelatase

Sulfite reductase

## ABSTRACT

Nitrogen (N) deficiency in soil results in the reduction of plant productivity and emission of greenhouse nitrogenous gases from the applied nitrogenous fertilizers and formation of sodic soil. To overcome soil N-deficiency the rate of N-assimilation was augmented by overexpression of *AtSirB* coding for sirohydrochlorin ferrochelatase, responsible for Fe insertion to the tetrapyrrole moiety of sirohydrochlorin in *Arabidopsis thaliana* under the control of 35S promoter for increased synthesis of siroheme. The siroheme is a cofactor for the plasticid enzymes nitrite reductase (NiR) and sulfite reductase (SiR), which reduce nitrite (NO₂) and sulfite (SO₃²⁻) to ammonium (NH₄⁺) and sulfide (S²⁻) respectively. Increased gene and protein expression of *AtSirB* resulted in heightened *NiR* and *SiR* gene expression and protein abundance, boosting NiR and nitrate reductases (NR) activities that resulted in increased protein and chlorophyll (Chl) content in *Arabidopsis thaliana*. Conversely, antisense plants showed decreased protein levels due to reduced NR and NiR activities. *AtSirB* overexpressors due to their higher protein and Chl contents had increased photosynthesis, and biomass. Under N and sulfur (S) limitations, these overexpressors outperformed wild type (WT) and antisense plants. These results demonstrate the crucial role of *AtSirB* and siroheme in N and carbon (C) assimilation. Furthermore, they indicate the potential of *SirB* to enhance crop productivity, alleviate N and S deficiency across different cropping regimes.

### Abbreviations

AtSirB *Arabidopsis thaliana* Sirohydrochlorin ferrochelatase

NiR Nitrite reductase

SiR Sulfite reductase

NR Nitrate reductase

Chl Chlorophyll

NUE Nitrogen Use Efficiency

NADPH Nicotinamide adenine dinucleotide phosphate (reduced form)

UPM1 Uroporphyrinogen III methyltransferase 1

GS Glutamine synthetase

GOGAT Glutamate synthase

Fd-GOGAT Ferredoxin-dependent glutamate synthase

WT Wild type

AAT Aspartate aminotransferase

PCR Polymerase chain reaction

cDNA Complementary DNA

FP Forward primer

RP Reverse primer

SD Standard deviation

DW Dry weight

PAM Pulse amplitude modulation

Fv/Fm Maximum quantum efficiency of PSII

φPSII Effective quantum yield of PSII

LRE Light responsive element

BCIP 5-bromo-4-chloro-3-indolyl phosphate

NBT Nitro blue tetrazolium

HATS High-affinity nitrate transporter system

PSI Photosystem I

SUE -Sulfur Use Efficiency

<footnote><sup>a</sup> Amity institute of microbial technology, Amity University, Uttar Pradesh 201303, India</footnote>

<footnote><sup>b</sup> Laboratoire CHEMBIOPRO (Chimie et Biotechnologie des Produits Naturels), ESIROI Agroalimentaire, Université de la Réunion, 15 Avenue René Cassin—CS 92003, Saint-Denis Cedex 09, La Réunion 97744, France</footnote>

<footnote><sup>c</sup> Department of Biotechnology, Sharada University, Uttar Pradesh 201310, India</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: laurent.dufosse@univ-reunion.fr (L. Dufossé), baishnabtripathy@yahoo.com (B.C. Tripathy).</footnote>
# 1. Introduction

Plants require an external source of nitrogen (N), which they obtain from the soil either as nitrate (NO₃) or ammonia (NH₃). The application of nitrogenous fertilizers in the soil leads to the emission of the greenhouse gas nitrous oxide, which is responsible for global warming. NH₃ leached from soil causes eutrophication in water bodies (Stevens, 2019; Anas et al., 2020). Conversely, N deficiency negatively impacts crop growth and yield. Therefore, improving nitrogen use efficiency (NUE) can enhance the agronomic values of fertilizers by boosting crop yield, saving energy, and reducing negative environmental impacts (Congreves et al., 2021).

The metabolisms of carbon (C), N, and sulfur (S) are closely interconnected. N and S metabolism are predominantly regulated by the uptake of NO₃ and sulfate (SO₄²⁻) through the root system, followed by their enzymatic reduction to ammonium (NH₄⁺) or sulfide (S²⁻). In contrast, C metabolism is primarily driven by photosynthetic carbon fixation and respiration. After being absorbed from the soil, nitrate is transformed into nitrite (NO₂) by nitrate reductase (NR) using NADPH as a reductant (Campbell, 1999; Costa-Broseta et al., 2021). Nitrite migrates from the cytosol to chloroplasts, where it undergoes six electron reduction mediated by plastidic nitrite reductase (NiR), often using ferredoxin as a reductant to form ammonium (Campbell, 1999; Yoneyama et al., 2020). Similarly, a six-electron reduction of sulfite (SO₃²⁻), to sulfide (S²⁻) is accomplished in the plastids of the photosynthetic organisms, mediated by sulfite reductase (SiR) (Crane et al., 1996; Khan et al., 2010).

Siroheme is the prosthetic group for both NiR and SiR (Stroupe et al.; 2009). It is a closed tetrapyrrole that contains Fe, and its biosynthesis begins from uroporphyrinogen III, an intermediate of chlorophyll (Chl) and heme biosynthesis pathways (Murphy and Siegel, 1973; Murphy et al., 1974). Siroheme biosynthesis involves three enzymatic steps: (i) two methylations reactions, (ii) oxidation, and (iii) ferrochelatation (Wu et al., 1991; Tripathy et al., 2010). Precorrin-1 and precorrin-2 are produced via methylation by uroporphyrinogen III methyltransferase (UPM1) (Warren et al., 1990). Precorrin-2 is then dehydrogenated to form sirohydrochlorin. Sirohydrochlorin ferrochelatase (SirB), which contains a 2Fe-2S center, is the terminal enzyme in the siroheme biosynthesis pathway. It inserts Fe into the center of sirohydrochlorin to produce siroheme (Raux-Deery et al., 2005).

Recently, research has focused on the effectiveness of N consumption, including plant N absorption, acquisition, and utilization (Zhang et al., 2023; Zayed et al. 2023). Nitrate transporters are essential components of N homeostasis in plants (Alduko et al. 2023). In the low-affinity nitrate transporter mutant *chl1*, overexpression of *CHL1* led to the restoration of low and high-affinity nitrate uptake in *Arabidopsis* (Liu et al., 1999). In rice, overexpression of aspartate aminotransferase (AAT), a crucial enzyme in the production of amino acids, led to a significantly higher leaf AAT activity and increased seed levels of both amino acids and proteins (Zhou et al., 2009).

However, manipulating NR has yielded mixed results regarding NUE, protein content, and photosynthetic efficiency. Constitutive expression of NR demonstrated a temporary delay in the drought induced loss in NR activity, decreased nitrate content, and increased glutamine accumulation, which resulted in an increased amino acid pool in tobacco (Ferrario-me et al., 1998) and potato (Djennane et al., 2002). Nevertheless, the total N content and protein contents remains unaltered. Similar efforts to overexpress the *NiR* gene in tobacco and *Arabidopsis* led to higher levels of *NiR* transcripts but lower enzyme activity levels due to post-translational modifications (Crété et al., 1997). Although *NiR* enrichment enhances NO₂ assimilation in *Arabidopsis*, *NiR* is post-transcriptionally regulated by NO₃ or NR activity (Takahashi et al., 2001).

The glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway is primarily responsible for ammonium assimilation in plants (Fortunato et al., 2023). Tobacco plants overexpressing

NADH-glutamate synthase showed higher C and N assimilation (Chichkova et al., 2001). Conversely, plants with underexpressed GS2 were exhibited reduced photorespiration, and were severely photo-inhibited under high light conditions (Migge and Becker, 2000). Over-expression of chloroplastic GS2 in rice enhanced photorespiration and salt tolerance in transgenic plants (Hoshida et al., 2000). GS2 over-expression in tobacco was linked to both the drop in the leaf ammonium pool and the buildup of several free amino acids, like glutamate and glutamine (Migge et al., 2000). Reduced expression of Fd-GOGAT in a barley mutant showed a decrease in leaf protein and NO₃ content (Häusler et al., 1994). In tobacco, overexpression of cytosolic pea GS1 improved plant growth and protein content (Fuentes et al., 2001). However, there are contradictory reports of the overexpression of GS1 and its impact on NUE. In poplar, GS1 overexpression increased NUE (Man et al. (2005), whereas overexpression of the soybean GS1 in pea plants did not increase the NUE (Fei et al., 2006). In addition to GS/GOGAT, arginine metabolism contributes significantly to NUE. Arginine functions as both a storage and transport form of organics N and serves as a precursor for protein, polyamines and nitric oxide (NO). In *Malus hupehensis*, exogenous arginine improved nutrient uptake, photosynthesis, and chlorophyll content under N deficiency, while also enhancing amino acid metabolism and antioxidant synthesis. Overall, arginine contributes to stronger photosynthesis, nutrient adsorption, and stress tolerance under low N conditions (Chen et al., 2022).

Although all of these genes involved in secondary N assimilation seem to have the potential to increase NUE, the extent of their efficacy needs to be carefully investigated across various crops and cropping regimes. The effectiveness of genetically altering NR/NiR for significant increase in NUE met with only partial success and varies among plant species.

SO₄²⁻ assimilation by plants is necessary for life on Earth, as it is an essential source for synthesizing S-containing amino acids cysteine and methionine (Tripathy et al., 2010; Garai and Tripathy, 2018). After being absorbed from the soil, SO₄²⁻ is activated to become adenosine 5'-phosphosulfate (APS). The primary assimilation process involves the reduction of APS to SO₃²⁻, followed by its further reduction to S²⁻ (Saito, 2004). Plastid localized SiR converts SO₃²⁻ to S²⁻ via a six-electron reduction process mediated by the prosthetic group siroheme. Besides providing S²⁻ for the synthesis of cysteine, methionine, Fe-S centers, glutathione, and several other metabolic intermediates, SiR also protects plants from SO₃²⁻ toxicity. The expression of genes coding for NiR and SiR, responsible for N and S assimilation, is controlled by phytochrome (Sharma and Sopory, 1984; Faure et al., 1991; Sherameti et al., 2002). In addition, the expression of SirB, which is responsible for synthesizing siroheme (the cofactor for both NiR and SiR), is also light-regulated. The SirB promoter contains light responsive elements (LREs) that modulate SirB expression in response to light (Garai et al., 2016). Thus, the expression of both the apoproteins of NiR and SiR and their shared cofactor (siroheme) is light-regulated. In *A. thaliana*, the *AtSirB* [AT1G50170] codes for SirB, a nuclear-encoded enzyme with a 46 amino acid long transit peptide (Raux-Deery et al., 2005). SirB is essential for plants, as its knockout in *Arabidopsis* results in embryo lethality (Raux-Deery et al., 2005). In this study, we demonstrate that the overexpression of *AtSirB* in *A. thaliana* results in increased NiR and SiR expression, augmented NiR activity, higher protein content, and improved photosynthetic efficiency. Under N and S limiting conditions, the over-expressers outgrow the wild type (WT) and exhibited superior photosynthetic efficiency. Conversely, the *AtSirB* antisense plants either perished or showed reduced NUE, decreased protein content, lower photosynthetic efficiency, and were susceptible to N or S starvation.

# 2. Experimental procedure

## 2.1. Plant materials and growth conditions

The *Arabidopsis thaliana* columbia-0 (Wild-type: WT) and *AtSirB*-
overexpressing lines (*AtSirBx*, S1& S2) and silencing lines antisense (A1&A2) were cultivated in a growth chamber (Conviron, Canada) (100 µmoles photons m⁻²s⁻¹), and a 14 h light/10 h dark photoperiod at 22 °C (*Pattnayak and Tripathy, 2002; Pattanayak et al., 2005*).

## 2.2. Cloning and transformation of *AtSirB* in *Arabidopsis*

*AtSirB* was amplified from *Arabidopsis* cDNA from mature leaf, with the help of gene-specific forward (FP) and reverse (RP) primers designed based on the to *AtSirB* cDNA sequence (**Supplementary Table S 1**). For the amplification, a PCR program was designed having 94 °C for 5 min followed by 30 cycles of 94 °C (denaturation) for 30 s, 56 °C (annealing) for 30 s, 72 °C (extension) for 45 s using a standard PCR reaction set up. The resulting amplification product was gel purified and ligated to a pGEMT-easy (Promega, USA) vector. The recombinant plasmid (pGEMT-easy-*AtSirB*) was transformed into competent *E.coli* (DH5α) cells. Plasmid DNA was prepared, and the nucleotide sequence of the *AtSirB* was confirmed by sequencing using a standard procedure (Sambrook et al., 1989). The modified pCAMBIA1304 binary vector was used to clone the full-length *AtSirB* cDNA from the recombinant pGEMT-Easy plasmid after it had been EcoRI digested and the product had been oriented in both sense and antisense. *Arabidopsis thaliana* (Col-0) was transformed using the vacuum infiltration method after the pCAMBIA1304: *AtSirB* construct was transformed to the *Agrobacterium tumefaciens* strain GV3101 (Clough and Bent, 1998; Das and Joshi, 2011). Primary transformants were cultivated until T3 generation after being screened in half-MS containing 50 mg/L kanamycin to establish homozygous lines for further study. The sense orientation of *AtSirBx* plants was done by gDNA PCR using 35S Int- FP and *AtSirB* RP, whereas the antisense orientation of antisense transgenic plants was tested using 35S Int- FP and *AtSirB* FP.

## 2.3. qRT-PCR analysis to check *AtSirB* mRNA expression in *AtSirB* transgenic plants

TRIzol reagent (Invitrogen, USA) was used to isolate total RNA from 15-day-old *Arabidopsis* seedlings. To ensure no DNA was present, the RNA was processed with RNase-free DNase I (Promega, USA). In a 50 µl reaction volume, 2 µg of total RNA, an oligo (dT) primer, and AMV reverse transcriptase (Promega, USA) were used to produce cDNA for reverse transcription (RT) PCR (*Pattanayak and Tripathy, 2011*). The cDNA was diluted 1:10 and utilized as a template for qRT-PCR reaction with 2µl of the diluted cDNA. The gene-specific primers mentioned in **Supplementary Table 1** were used in PCR reactions per the manufacturer's instructions using an applied biosystems (Life Technologies, Carlsbad, CA) real-time PCR system. Using the comparative CT approach (*Schmittgen and Livak, 2008*), expression was normalized to actin. The resulting data are shown as the standard deviation and relative means of three biological replicates.

## 2.4. Antisense mRNA expression in antisense *AtSirB* plants

Gene-specific primers were employed to prime first-strand cDNA synthesis rather than oligo (dT) or random hexamers. Reverse transcription from RNA obtained from *AtSirB* transgenic strains was done using the forward primer to look for antisense mRNA. Only the antisense mRNA is bound by the forward primer. As a result, if there is no anti-sense mRNA, no cDNA synthesis will occur from the forward primer (*Song and Wang, 2009*). The result demonstrates that the *AtSirB* gene product was amplified when PCR was performed using the cDNA prepared from gene-specific forward primer only. cDNA prepared using total RNA from WT plants was used as a template for another set of PCR that served as negative control.

## 2.5. Total protein quantification from *AtSirB* transgenic seedlings

To calculate the total protein content, seedlings were homogenized with liquid N. The homogenized tissues were mixed with 1 ml of an isolation buffer of 56 mM Na₂CO₃, 5 mM DTT, 10 mM isoascorbate, 12 % sucrose, 2 mM EDTA, and 2 % SDS. The samples were adequately vortexed after being transferred from the homogenate to the eppendorf tube. Samples were heated at 80 °C for 20 min, followed by centrifugation at 13,000 rpm for 5 min. A new eppendorf tube was used to collect the supernatant. The protein content in various extracts was estimated using Bradford's method (Bradford, 1976). A polyclonal antibody (1/1000 dilution) produced against the *AtSirB* protein was used to blot approximately 20 µg of the total protein supernatant separated onto SDS PAGE (Laemmli, 1970). Antibody against NiR (1/20, 000 dilution) was prepared commercially, and SiR (1/20,000 dilution) antibody (*Zea mays*) was kindly donated by Toshiharu Hase, Institute for Protein Research, Osaka University, Japan.

Crude protein extracts were prepared from *Arabidopsis* plants for immunoblot analysis, as described by *Pattanayak and Tripathy (2016)*. Total protein (20 µg) was resolved in 12.5 % SDS-PAGE and was then transferred onto nitrocellulose membranes. Blots were stained for alkaline phosphatase using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) (*Pattnayak and Tripathy, 2002*).

## 2.6. Total N quantification in *AtSirB* transgenic plants

N quantification was done on a Euro- E.A. elemental analyzer or CHNS analyser. Approximately 5–10 mg of dried leaves (at 105 °C for 24 hr) was weighed and used for the analysis. The standard used for the analysis is L-cysteine. N present in the cysteine was calculated in %. So the amount of N present in plant samples was in percentage (%) of g dry weight (gDW).

## 2.7. Determination of nitrogen use efficiency (NUE)

NUE was calculated as the protocol developed by *Perchlik and Tegeder (2017)*. NUE = g Biomass DW per plant/ g Nitrogen supplied to the growth medium

## 2.8. Determination of sulfur use efficiency (SUE)

SUE was calculated as the protocol developed by *Chowdhury et al. (2020)* and *Perchlik and Tegeder (2017)*. SUE = g Biomass DW per plant/ g Sulfur supplied to the growth medium

## 2.10. 2.9. Chl estimation in *AtSirB* transgenics

Chl was extracted from plant tissues in a safe green light. Leaf samples (as well as seedlings) were homogenized in 10 ml of 90 % cold ammonical acetone using a previously chilled mortar and pestle. For each batch, more than ten replicates were collected. Homogenate was centrifuged at 4 °C for 10 min at 10,000 rpm. The absorbance of the supernatant was measured at 663 nm, and 645 nm to calculate total Chl. Chl was estimated according to (Porra et al., 1989).

## 2.11. 2.10 Pulse amplitude modulation (PAM) measurements in *AtSirB* transgenics

All quantifications of Chl fluorescence were made using a PAM-2100 fluorometer (Walz, Effelteich, Germany). The leaf was dark-adapted using leaf clips from the walz company for 20 min before each measurement. The optimum quantum efficiency of PSII in the dark-adapted leaves was calculated as Fv/Fm = (Fm-F0)/Fm, where F0 is the initial fluorescence and Fm is the maximum fluorescence (Harbinson et al., 1989). The effective PSII quantum yield (ΦPSII) was calculated
according to Genty et al. (1989) and Harbinson et al. (1989) by the formula $\phi PSII = (Fm' - Ft) / Fm'$, where Fm' is defined as the maximal fluorescence yield in a pulse of saturating light when the sample is pre-illuminated. Ft represents the measured fluorescence yield at any given time (t) (Kandoi et al., 2022).

## 2.12. 2.11 Measurement of morphological parameters in AtSirB transgenics

WT and *AtSirB* transgenic plants were raised for three weeks in MS medium. The fresh weight of the plants was measured after they were removed from petri plates and placed on tissue paper to absorb moisture. Whole plants were wrapped in aluminium foil and baked at 70 °C for 48 h to determine their dry weight. Primary root length was measured with image J software, and the numbers of lateral roots were counted individually under a light microscope.

## 2.13. 2.12 Quantification of NIR and NR activity in AtSirB transgenics

### 2.13.1. 2.12.1 Standard curve for NO₂

The standard curve for NO₂ was made by different concentrations of NaNO₂ (10 µg/ml stock) in water with sulphanilamide and N-1-naphthyl-ethylene-diamine dihydrochloride (NED). A series of concentrations of NaNO₂ was prepared by adding 0 µg, 0.2 µg, 0.4 µg, 0.6 µg, 0.8 µg, 1.0 µg, and 1.2 µg of solution from a 10µg/ml stock solution of NaNO₂ and adjusted to a final volume of 1 ml with deionized water. To the above NO₂ solution, 0.5 ml of 1 % (w/v) sulfanilamide dissolved in 3 N HCl and 0.5 ml of 0.02 % (w/v) NED were added to each tube. The reaction was incubated for 10 min and the colour developed. The absorbance was measured at 540 nm to generate the standard curve.

### 2.13.2. 2.12.2 NiR assay

Approximately 100 mg of leaves of 3-4-weeks-old plants of *AtSirB* transgenic lines and WT were taken and ground in powder form. It was followed by mixing with 0.3 ml of extraction solution (per 100 mg of tissue) that contained 50 mM potassium phosphate buffer (pH 7.5), 1 mM EDTA, 10 mM mercaptoethanol, and 100 µM PMSF. The supernatant from the centrifugation of the homogenate at 4 °C and 13,000 rpm for 15 min in a chilled microfuge was utilized for the NiR assay. The amount of protein was calculated (Bradford, 1976). The decline of NO₂ ion in the assay mixture was assessed to monitor NiR activity with a minor modification (Takahashi et al. 2001). A 1.5 ml centrifuge tube was filled with 200 µl of the crude enzyme extract, and 195 µl of the assay solution, which included 50 mM potassium phosphate buffer (pH 7.5), 1 mM NaNO₂, and 1 mM methyl viologen, was then added. The reaction was initiated by adding 105 µl of 57.4 mM Na₂S₂O₄ in 290 mM NaHCO₃ at 30 °C for 15 min. After vigorously mixing a 20µl sample in a fresh tube with 980 µl of water to halt the reaction, 500 µl of 1 % (w/v) sulfanilamide in 3 N HCl and 500 µl of 0.02 % (w/v) NED were added. NiR activity was assessed regarding moles of NO₂ decreased (mg protein)⁻¹min⁻¹ by taking absorbance at 540 nm.

### 2.13.3. 2.12.3 NR assay

NR activity was measured as described by Kaiser and Lewis (1984), with a minor change to determine NO₂ ion formation in the reaction volume. Approximately 100 mg of leaves of 3-4-weeks grown plants of transgenic line or WT were grounded in a mortar and pestle with 0.5 ml of grinding buffer containing 0.1 M tris-HCl (pH 7.5), 5 mM cysteine, and 1 mM EDTA for 5 min. The homogenate was centrifuged at 4 °C at 13,000 rpm for 20 min in a refrigerated microfuge, and the supernatant was taken for enzyme assay. The protein was quantified according to Bradford (1976). The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.5), 20 mM KNO₃, 0.1 mM NADH and 0.2 ml of crude enzyme extract in 1 ml of reaction volume. At 30 °C, the mixture was incubated for 15 min. After the incubation period, the reaction was stopped by adding 0.5 ml of 0.02 % (w/v) N-1-naphthyl

ethylenediamine dihydrochloride (NED) and 0.5 ml of 1 % (w/v) sulphanilamide in 3 N HCl. The mixture was centrifuged at 13,000 rpm for 15 min. The supernatant was used for measuring NO₂ content by taking the absorbance at 540 nm. NR activity was measured in terms of an increase in NO₂ in the reaction mixture. The enzyme activity was measured as µmoles of NO₂ produced (mg protein)⁻¹min⁻¹.

## 2.14. 2.13 AtSirB transgenic plants in N -limiting medium

Plants were grown photo-periodically (16 h L/8 h D) at 22 °C in 100 µmoles photon m⁻² s⁻¹ light intensity for 15 days in normal MS medium. After 15 days, plants of equal size were taken and transferred to optimal (1 N) or N-limiting (0.1 N) hoagland media in an agar plate (Supplementary Table S 2). After 7 days of growth in N-limiting medium, WT and *AtSirB* sense and antisense plants were analyzed. Chl, Chl a fluorescence (PAM), protein, and NiR activities were measured.

### 2.14.1. 2.13.1 Accumulation of starch in *AtSirB* transgenic plants in N limiting medium

Plants were grown photo-periodically (16 h L/8 h D) at 22 °C in 100 µmoles photon m⁻² s⁻¹ light intensity for 15 days in normal MS medium. After 15 days, plants of equal height were transferred to optimal or N-limiting media prepared in an agar plate with a hoagland solution as in Supplementary Table S 2. After 7 days of growth in N-limiting medium, WT, *AtSirB* sense, and antisense plants were kept in methanol for 24 h. Subsequently, plants were stained with 0.2 % w/v of iodine solution that developed a blue color due to starch accumulation in the leaves in N-deficient plants (Hermans et al., 2016).

## 2.15. 2.14 AtSirB transgenic plants in S -limiting medium

WT and *AtSirB* transgenic plants were grown photo-periodically (16 h L/8 h D) at 22 °C in 100 µmoles photon m⁻² s⁻¹ light intensity for 15 days in normal MS medium. After 15 days, plants of equal size were taken and transferred to optimal (1S) or S limiting (0.1S) hoagland media in an agar plate (Supplementary Table S 2). After 7 days of growth in S-limiting medium, WT, and *AtSirB* and antisense plants were analyzed. Chl, carotenoids, Chl a fluorescence (PAM), and protein contents were measured.

## 2.16. 215 Statistical data analysis

Data are presented as means ± standard deviation (SD) for a minimum of five samples (n ≥ 5), depending on the experiment. Error bars in the graphs represent the standard deviation. Statistical significance was assessed using single-factor analysis of variance (ANOVA) in Microsoft Excel (Supplementary Table S 3). Significant differences are indicated with asterisks: *P* < 0.05, **P** < 0.01, ***P** < 0.001, and ****P < 0.0001.

# 3. Results

## 3.1. Generation and identification of AtSirB transgenic in Arabidopsis thaliana

A transgenic method was employed to characterize the function of Sirohydrochlorin ferrochelatase (AtSirB). Two constructs containing *AtSirB* cDNA in a sense (Overexpression) and antisense (silencing) orientation with CaMV 35S promoter were transformed into *Arabidopsis* (Col-0) by *Agrobacterium*-mediated transformation (Fig. 1A). Over more than ten transgenic lines were generated. Using kanamycin-specific primers, PCR was used to detect transgenics. PCR findings showed that around 90 % of the plants had incorporated the construct. All transgenic lines were segregated in a 3:1 ratio in a kanamycin plate, demonstrating a single integration of the transgene. Those who survived were grown for the next generation (T2). The resulting seeds were
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Transformation of *Arabidopsis* by using *AtSirB* cDNA: *AtSirB* cloned in pCAMBIA1304 modified plant transformation vector in sense (overexpression) and antisense orientation were used to transform *Arabidopsis* plants. (A) Vector constructs, (B) confirmation of T3 generation of *AtSirB* transgenic line by PCR by using gDNA as template and 35S forward primer and gene specific forward and reverse primers for antisense and sense plants, (C) Expression of *AtSirB* cDNA in transgenic over-expresser (S1 & S2) and antisense (A1&A2) by qRT-PCR. (D) Antisense transgenic plants were further confirmed by antisense RNA analysis by strand-specific RT-PCR, (E) *AtSirB* Protein expression in transgenic plants was checked by immunoblot against *AtSirB* antibody; standard deviation is shown by the error bar. WT, S1, S2 and A1, A2 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P* < 0.01, ***P* < 0.001, ****P* < 0.0001).</figcaption></figure>

grown in kanamycin plates and were selected for kanamycin resistance. By employing 35S internal forward primer, gene-specific reverse primers (over-expresser), and gene-specific forward primers (antisense), gDNA PCR was used to verify T3 generations of homozygous transgenic plants (Fig. 1B).

Once the transgene integration was confirmed by gDNA PCR, gene expression of *AtSirB* in WT and transgenic lines was checked by qRT-PCR analysis. The qRT-PCR reveals that, compared to WT, the *AtSirB* over-expresser lines (S1 and S2) had a 5-7-fold increase in *AtSirB* gene expression. In antisense lines (A1 and A2), *AtSirB* expression decreased by ~70–80 % (Fig. 1C). To probe further, the antisense *AtSirB* message abundance was studied. Since the forward primer only binds to antisense mRNA, it was possible to identify antisense mRNA using reverse transcription of RNA isolated from *AtSirB* antisense transgenic plants. The forward and reverse primers for *AtSirB* were used to create PCR targets from the first-strand reactions. The *AtSirB* was amplified by PCR using the cDNA prepared from the forward primer. The antisense A1 and A2 lines show a band corresponding to the *AtSirB* size of 678 bp, which confirms the antisense expression (Fig. 1D).

Immunoblot analysis was conducted to study if the overexpression of *AtSirB* in transgenic plants resulted in an expected change in *AtSirB* protein expression. A polyclonal antibody preparation developed against the *Arabidopsis* *AtSirB* protein in our laboratory was used to detect the *AtSirB* expression in *AtSirB* transgenic plants. Immunoblot analysis showed higher *AtSirB* protein levels in S1 and S2 lines whereas expression was lower in A1 and A2 transgenic lines. It proved that *AtSirB* was highly expressed in over-expresser plants and minimally in antisense plants (Fig. 1E).

## 3.2. *AtSirB* modification changes plant morphology in *Arabidopsis*

The morphology of the *AtSirB* transgenic plants changed in a typical manner. The S1 and S2 plants were bigger and greener than the WT plants. In contrast, the antisense lines A1 and A2 survived and were

smaller than WT (Fig. 2A). Most of our antisense lines bleached and perished (Fig. 2B). *AtSirB* transgenic root morphology changed, especially in S1 and S2 plants (Supplementary Figure S 1). Compared to WT, the number of lateral roots were higher in S1 and S2 plants. The number of lateral roots of antisense plants were the same as that of WT (Fig. 2C). In contrast to antisense plants, rosette diameter, fresh weight, and dry weight of S1 and S2 increased by 60–70 % (Figs. 2D, 2E, 2F).

## 3.3. Modulation of *N* assimilation by *AtSirB*

*SirB* is the terminal enzyme responsible for the synthesis of siroheme, the cofactor of NiR. To probe if the increased expression of cofactor siroheme synthesizing gene *AtSirB* could modulate the expression of NiR apoprotein and NiR activity, its expression and enzymatic reaction were monitored.

### 3.3.1. Gene expression and enzymatic activity of *NiR* in *AtSirB* transgenic plants

The qRT-PCR of *NiR* (*At2g15620*) revealed that message abundance of *NiR* increased as a result of *AtSirB* overexpression. The qRT-PCR indicated that the *NiR* gene expression in the overexpression lines, S1 and S2, was 90 % and 80 % higher than WT. However, in antisense lines (A1 & A2), there were no significant changes in the gene expression of *NiR* (Fig. 3A). Due to increased message abundance for NiR, the NiR apoprotein increased in S1 and S2 over-expresser lines. The western blot further revealed that in A1 and A2 antisense lines, there was no significant change in NiR protein level (Fig. 3B, 3C). The NiR enzymatic activity was measured to examine the consequences of the altered expression of *AtSirB* in transgenic plants. Compared to WT, NiR activity increased by approximately 25 % in S1 and 20 % in S2 lines due to the increased availability of the cofactor siroheme. However, in antisense plants, its enzymatic activity declined by 43 % and 37 % for A1 and A2 lines, respectively (Fig. 3D). In S1 and S2 overexpresser plants, higher activity of NiR efficiently consumes its substrate NO₂ whose over-
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

**Fig. 2.** Phenotype and Morphological parameters *AtSirB* transgenic plants. (A) Phenotypes of *AtSirB* sense (S 1 & S 2) and antisense (A 1 & A 2) lines grown for 3 weeks, (B) antisense lines which could not survive due to excessive bleaching, (C) lateral roots per plant, (D) rosette diameter, (E) fresh weight and (F) Dry weight. An average of 10 replicates makes up each data point. Standard deviation is shown by the error bar. WT, S1, and A1 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P** < 0.01, ***P** < 0.001, ****P < 0.0001).

<figure><img src="image_8.png" /></figure>

Most of the Antisense lines Could not survive
due to excessive bleaching

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

accumulation could be toxic to the plant.

### 3.3.2. Gene expression and enzymatic activity of NR in transgenic

The cytoplasmic NR, which converts NO₃ to NO₂, supplies the substrate for NiR. Consequently, their stoichiometry needs to be regulated. To probe further, transcript abundance and NR activity in WT and transgenic lines were examined. In S1 and S2 over-expresser lines, the gene *Nia1*, which codes for NR, showed increases in expression by 60 % and 50 %, respectively (Fig. 3E). To ascertain if NiR could modulate NR, its activity was estimated in WT and *AtSirB* transgenic plants (S1, S2 and A1, A2). NR activity increased in S1 plants by ~26 % and in S2 plants by 21 % over WT. Conversely, antisense plants' activity declined by ~12 % and 11 % in A1 and A2 plants, respectively (Fig. 3F).

## 3.4. *AtSirB* overexpression increased total protein and N levels

### 3.4.1. Total protein content

The *AtSirB* plants had higher levels of expression and activity of enzymes involved in N assimilation. As a result, the total protein content of transgenics increased. Compared to WT, S1 and S2 had a 27 % and 26 % higher protein content, respectively. In antisense plants, enzymatic activities of NiR and NR were substantially reduced, likely due to siroheme limitation. It resulted in lower protein content (15 %–18 %) of antisense plants (Fig. 4A).

### 3.4.2. N content

The significant increase in the enzymatic activity of two crucial enzymes involved in N metabolism and total protein contents led us to quantify total N content in WT and *AtSirB* transgenic plants. N content

increased by ~35 % and 33 % in S1 and S2 overexpressors and declined by ~16 % and 15 % in A1 and A2 antisense plants compared to WT (Fig. 4B). It is in agreement with that of the total protein contents.

### 3.4.3. Nitrogen use efficiency (NUE) in WT and transgenic plants

The NUE of WT and *AtSirB* transgenic plants. The NUE was significantly increased by 37 % and 35 % in S1 and S2 overexpressors and declined by 33 % and 25 % in A1 and A2 antisense plants in comparison to WT (Fig. 4C). It is in the agreement with the increase of total protein and N contents of transgenic plants.

### 3.4.4. *AtSirB* manipulation modulates chlorophyll content and photosynthetic efficiency

#### 3.4.4.1. Chlorophyll content. *AtSirB* overexpressor S1 and S2 had higher amounts of total Chl (~32 %) than WT. Compared to WT plants, overall Chl content decreased (~30 %) in *AtSirB* antisense A1 and A2 plants (Fig. 5A).

#### 3.4.4.2. Photosynthetic efficiency. Chl fluorescence is a non-invasive marker of photosynthetic ability in plants (Padhi et al., 2021). To ascertain whether plants with higher Chl and protein concentrations had higher photosynthetic ability, three Chl a fluorescence metrics, Fo, Fv/Fm, and the quantum yield of PSII (ΦPSII), were measured. The Fo, the minimal fluorescence increased in S1 and S2, likely due to an increase in Chl content. In the A1 and A2, the Fo declined, likely due to a reduction in Chl content (Fig. 5B). The maximum photochemical efficiency of the dark-adapted leaves, measured as Fv/Fm ratio, significantly increased in S1 plants compared to WT plants. In contrast, it
<figure><img src="image_11.png" /></figure>

**Fig. 3.** NiR and NR gene expression and activities in *AtSirB* transgenic plants. WT and *AtSirB* sense lines (S1, S2) and antisense lines (A1, A2) plants raised at 22 °C under 14 h L/10 h D photoperiod in a light intensity of 100 µmoles photons m⁻² s⁻¹ for 3 weeks in MS medium and their vital parameters were analysed. (A) qRT-PCR of gene expression of nitrite reductase (NiR), (B) NiR protein abundance using immunoblot, (C) densitometric analysis of NiR immunoblot, (D) nitrite reductase (NiR) activity estimated in the leaves of 3-week-old WT and transgenic plants, (E) Nitrate reductase (*Nia1*) gene expression was quantified by qRT-PCR, (F) nitrate reductase activity was measured in leaf of 3-week-old WT and transgenic plants. An average of 5 replicates make up each data point. The standard deviation is shown as the error bar. WT, S1, S2, A1 and A2 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P** < 0.01, ***P** < 0.001, ****P < 0.0001). “ns” indicates for no significant change in the expression.

<figure><img src="image_12.png" /></figure>

**Fig. 4.** Total protein, N and NUE in *AtSirB* transgenic plants. WT and *AtSirB* sense lines (S1, S2) and antisense lines (A1, A2) plants raised at 22 °C under 14 h L/10 h D photoperiod in a light intensity of 100 µmoles photons m⁻² s⁻¹ for 3 weeks in MS medium and their vital parameters were analysed. (A) total protein content of *Arabidopsis* seedling, (B) N content was quantified by CHNS analyzer using 1- Cysteine as standard, (C) NUE quantification. An average of 5–10 replicates make up each data point. The standard deviation is shown as the error bar. WT, S1, S2, A1 and A2 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P* < 0.01, ***P* < 0.001, ****P* < 0.0001). “ns” indicates for no significant change in the expression.

decreased by almost 20 % in A1 plants (Fig. 5C). As expected, the φPSII, the quantum yield of PSII in light-adapted plants, reduced in response to rising light intensities in WT and transgenic plants. In saturating light intensities, the φPSII of S1 and S2 lines were 40 % and 32 % higher than WT, respectively. Conversely, in the antisense A1 and A2 lines, the φPSII was substantially lower than WT at saturating light intensities (Fig. 5D).

### 3.5. Overexpression of *AtSirB* alleviates N-deficiency

To ascertain if SirB overexpression could mitigate N deficiency, the NO₃⁻ content in the growth media was reduced, and the WT and transgenic plants were cultivated under N-limiting conditions. After 7 days in the N-limiting medium, there was a visible difference in *AtSirB* transgenic plants compared to WT. The phenotype of WT plants under 0.1 N condition appears pale due to N deprivation. The S1 plants appeared greener than WT when grown in identical growth conditions. However, in A1 plants, the N starvation caused severe phenotypic changes,

including bleaching (Fig. 6). Total Chl contents were higher in S1 plants than WT and lower in A1 plants at all N levels. The Chl contents of S1 were 87 % higher than WT at 0.1 N levels. The Chl content decreased by 30 % compared to WT at 0.1 N level in A1 plants (Fig. 7A). Total protein content was higher in S1 and lower in A1 plants when compared to WT. Under normal (1 N) N level, protein contents of S1 were 35 % higher, and in A1, the same was lower by 11 % than that of WT. The protein contents of S1 plants were 95 % higher than WT's in 0.1 N levels. In A1 plants, protein contents declined by 32 % compared to WT (Fig. 7B). To comprehend the mechanism underlying the tolerance of *AtSirB* transgenic plants to N deprivation, the activities of N assimilating enzymes were investigated in WT and transgenic plants grown in optimum and N-limiting medium, compared to WT, NiR activity at optimum N level (1 N) increased by 30 % in S1 plants. In A1 plants, the same declined by 45 %. The degree of NiR activity augmentation in S1 rose in N-limiting conditions compared to optimal N levels, whereas it reduced in A1 plants. S1 plants had NiR activities that were 72 % greater in 0.1 N levels
<figure><img src="image_13.png" /><figcaption>**Fig. 5.** Photosynthetic efficiency and chlorophyll content of WT and *AtSirB* transgenic plants. WT and *AtSirB* transgenic sense (S1, S2) and antisense (A1, A2) plants were grown at 22 °C under 14 h L/10 h D photoperiod in a light intensity of 100 µmoles photons m⁻² s⁻¹ for three weeks in MS medium and their (A) Chl, (B) F0, (C) Fv/Fm, and (D) φPSII were measured. An average of 5–10 replicates makes up each data point. Standard deviation is shown as the error bar. WT, S1, S2 and A1, A2 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P** < 0.01, ***P** < 0.001, ****P < 0.0001).</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Fig. 6.** Phenotypes of WT and *AtSirB* transgenic plants in N-limiting condition. Plants were grown at 22 °C under 14 h L/10 h D photoperiod in (100 µmoles photons m⁻² s⁻¹). WT and *AtSirB* transgenic plants (S1 and A1) raised under normal (1.0 N) MS for 15 days and subsequently transferred for 7 days to Nitrogen limitation medium (0.1 N). Nitrogen concentration for 1.0 N was 5 mM.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Fig. 7.** Physiological responses of WT and transgenic plants to N-limitation. WT and *AtSirB* transgenic plants (S1 and A1) were grown at 22 °C under 14 h L/10 h D photoperiod in a light intensity of 100 µmoles photons m⁻² s⁻¹ in MS for 15 days and subsequently sifted for 7 days into N-limitation condition (0.1 N) and their physiological parameters were monitored. (A) Total Chl, (B) total protein, (C) NiR activity, and (D) NUE, were measured. An average of 5–10 replicates makes up each data point. The standard deviation is shown by the error bar. WT, S1, and A1 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P** < 0.01, ***P** < 0.001, ****P** < 0.0001).</figcaption></figure>

than WT plants. In the A1 plants, NiR activity dropped by 54 % compared to WT (Fig. 7C). As NiR activity was significantly enhanced in S1 plants under both optimum and limiting nitrogen (N) conditions, a similar trend was observed in NUE. In S1 plants, NUE increased by 30 % under optimum N and by 100 % under limiting N conditions. In contrast, A1 plants showed a marked reduction in NUE, decreasing by 25 % under optimum N and by 53 % under limiting N conditions (Fig. 7D).

### 3.5.1. Photosynthetic responses of WT and transgenic plants grown in optimal and N-limiting conditions

Fo, Fv/Fm, and φPSII were examined after 7 days of growth on N-deficient media. Fo declined in N-limiting conditions in WT, S1, and A1 plants, although S1 plants had high Fo in comparison to WT, whereas A1 had lower Fo than WT at all N levels (Fig. 8A). In comparison to WT plants grown at all N levels, S1 plants had a higher Fv/Fm ratio.

<figure><img src="image_16.png" /><figcaption>**Fig. 8.** Photosynthetic responses of WT and transgenic plants to N-limitation. WT and *AtSirB* transgenic plants (S1 and A1) were grown at 22 °C under 14 h L/10 h D photoperiod in a light intensity of 100 µmoles photons m⁻² s⁻¹ in MS for 15 days and subsequently sifted for 7 days into nitrogen limitation condition (0.1 N) and their photosynthetic parameters were monitored. (A) Fo, (B) Fv/Fm, (C), φPSII measured by pulse amplitude modulated (PAM) fluorometer. An average of 10 replicates makes up each data point. The standard deviation is shown by the error bar. WT, S1, and A1 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P** < 0.01, ***P** < 0.001, ****P** < 0.0001).</figcaption></figure>
Conversely, in A1 plants, the same declined (Fig. 8B). Both in N-sufficient and N-limiting conditions, the φPSII increased in response to increasing light intensities. In *AtSirBx* plants, the φPSII was higher in S1 in limiting and saturating light intensities. The higher light-saturated rate implies an increase in the antenna size and the components of the electron transport chain in *AtSirBx* plants, and the increased slope of φPSII at limiting light intensities supports enhanced energy collection by the light-harvesting complex. In N-deficient media, the φPSII was higher in overexpressers and lower in antisense plants (Fig. 8C).

### 3.5.2. Alteration of carbohydrate metabolism in WT and *AtSirB* transgenic plants

Sugars and starch build up in leaves due to an N deficit as the carbon skeletons cannot be utilized for amino acid and protein synthesis. As revealed from starch iodide test the starch did not accumulate in WT, S1, and A1 plants in optimal N concentration. However, under N-limitation (0.1 N), carbohydrates accumulated in the leaves of WT and A1 plants. However, the S1 plants did not accumulate carbohydrates under identical conditions, suggesting they could tolerate extreme N-starvation (Supplementary Figure S 2).

### 3.6. *AtSirB* overexpression overcomes S deficiency

To investigate the role of *AtSirB* in S metabolism, WT, and *AtSirB* plants were grown in S-limiting media. Compared to WT plants, *AtSirB* transgenic plants showed a noticeable change in the phenotype after 7 days of growth on S-deficient media. The phenotype of WT plants looked pale-green after 7 days of growth in S starvation (0.1S) media. The S1 plants were greener than WT under the same growth conditions. However, the S-deprivation of antisense plants resulted in severe bleaching of leaves (Fig. 9A). As the siroheme is a prosthetic group of SiR (At5g04590), its expression was studied in optimally grown (1S) WT, *AtSirBx* (S1, S2) and *AtSirB* antisense (A1, A2) plants. Compared to WT, SiR expression increased by 55 % and 50 % in S1 and S2 plants, respectively. In A1 and A2 plants, SiR message abundance was almost similar to the WT (Fig. 9B). The protein abundance of SiR was examined

by Western blot to see whether gene expression and protein abundance are correlated. In consonance with gene expression, the S1 and S2 lines displayed an increase in SiR protein abundance over the WT. However, their abundance in the A1 and A2 lines was similar to WT (Fig. 9C). To probe further, WT and *AtSirB* transgenic plants were grown in optimal (1S) and minimal (0.1S) S growth media and their protein contents were measured. The S1 plants had a 28 % higher total protein content than WT plants in optimal growth conditions (1S). Conversely, protein content was reduced (25 %) in antisense A1 plants. In the S-deficient (0.1S) growth condition the protein content of S1 was 46 % higher than that of WT. Conversely, the protein content was 51 % lower than WT in A1 plants grown in an S-limiting media (Fig. 10A).

In S1 plants grown in optimal S medium (1S), total Chl contents were higher (32 %) than WT plants. However, in A1 plants, Chl content declined by 27 %. In the S-deficient (0.1S) growth condition the Chl content of S1 was 51 % higher than that of WT. Conversely, the Chl content was 71 % lower than WT in A1 plants grown in an S-starvation media (Fig. 10B).

### 3.6.1. Photosynthetic response of WT and transgenic plants grown in S-limitation conditions

The Fo, Fv/Fm, and φPSII were measured in the S-limitating medium. Fo declined in S-limiting condition in WT, S1, and A1 plants, although S1 plants had higher Fo than WT, whereas A1 had lower Fo than WT at all S levels (Supplementary Figure S 3A). Similarly, in comparison to WT, S1 plants had a higher Fv/Fm ratio, whereas in antisense plants, the Fv/Fm ratio decreased (Supplementary Figure S 3B). As expected, the φPSII decreased in response to photo actice radiation (PAR). However, the φPSII of S1 plants was higher than WT in limiting and saturating light intensities. In the S-starvation media, in A1 plants, the φPSII was lower than WT (Fig. 10C). As the *AtSirB* over-expressors were growing better in the S-limiting condition, SUE of the WT and *AtSirB* transgenic plants were measured. In the sense S1 plants, SUE increased by 35 % under optimum S and by 85 % under limiting S conditions. In contrast, the antisense, A1 plants showed a marked reduction in SUE, decreasing by 35 % under optimum S and by 47 %

<figure><img src="image_17.png" /><figcaption>**Fig. 9.** Phenotypes of *WT* and *AtSirB* transgenic plants in S-limiting condition and *SiR* gene and protein expression. WT and *AtSirB* (S1 and A1) plants were grown at 22 °C under 14 h L / 10 h D photoperiod in a light intensity of 100 µmoles photons m⁻² s⁻¹ for 15 days in optimal sulfur media (1S) and subsequently transferred into minimal sulfur (0.1S) media for 7 days. Concentration of S in normal media (1S) was 2 mM. (A) Phenotype of WT and *AtSirB* transgenic plants in S-limitation condition, (B) the *SiR* gene expression measured by qRT-PCR, (C) protein abundance measured by Western blot using antibodies against sulfite reductase (Zea mays). An average of 3 replicates makes up each data point. The standard deviation is shown as the error bar. WT, S1, S2, A1, and A2 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P* < 0.01, ***P* < 0.001, ****P* < 0.0001). “ns” indicates for no significant change in the expression.</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>**Fig. 10.** Physiological responses of *WT* and *AtSirB* transgenic plants to S- limitation. WT and *AtSirB* (S1 and A1) plants were grown at 22 °C under 14 h L / 10 h D photoperiod in a light intensity of 100 µmoles photons m⁻² s⁻¹ for 15 days in optimal sulfur media (1S) and subsequently transferred into minimal sulfur (0.1S) media for 7 days. Concentration of Sulfur in normal media (1S) was 2 mM. (A) Total protein and, (B) Total Chl, (C) φPSII, and (D) SUE were measured. An average of 10 replicates makes up each data point. The standard deviation is shown as the error bar. WT, S1, S2, A1, and A2 significantly differ from each other as indicated by the (*) asterisk (*P* < 0.05, **P** < 0.01, ***P** < 0.001, ****P < 0.0001). “ns” indicates for no significant change in the expression.</figcaption></figure>

under limiting S conditions (Fig. 10D).

**4. Discussion**

Although C assimilation is a well-established light-dependent pro-
cess, the assimilation of N and S is also influenced by light, albeit indi-
rectly (Neuenschwander et al., 1991; Becker et al., 1992; Pilgrim et al.,
1993; Liester, D. 2023; Kumari et al., 2024). Transcript levels of *NR* and
*NiR* are known to be co-induced by both *NO*₃ and light (Faure et al.,
1991). The reducing side of photosystem I (PSI) serves as a control point
for the mutual regulation of the *NO*₂ and *CO*₂ assimilation pathways,
mediated through the photosynthetic electron transport chain
(Commichau et al., 2006; Ivanov et al., 2022). Furthermore,
ferredoxin-NADP reductase and NiR compete for the reduced ferredoxin,
highlighting a key point of interaction in the regulation of nitrogen
metabolism (Hirasawa et al., 2009). *NO*₂ reduction facilitates the syn-
thesis of ATP for alternate purposes, including the synthesis of proteins
and carbohydrates in chloroplasts. The gene expression of NR is regu-
lated by light via phytochromes (Sharma and Sopory, 1984; Sherameti
et al., 2002). The *AtSirB* is responsible for N and S metabolism in plants,
and these processes are dependent on light for proper utilization of N
and S compounds taken up from the soil (LaBrie and Crawford, 1994;
Bork et al., 1998). We have shown that the *AtSirB* is a light-regulated
gene that is present in a minimal amount in etiolated tissues and

highly upregulated after light exposure due to light-responsive elements
in its promoter (Garai et al., 2016). It may be inferred that similar to the
light-regulated expression of apoenzyme, the gene expression of the
enzyme required to synthesize the cofactor is also photo-regulated
(Rajasekhar and Mohr, 1986; Becker, Foyer, and Caboche, 1992; Garai
et al., 2016).

Our study on the overexpression of AtSirB working in the branched
pathway of plant tetrapyrrole biosynthesis and is responsible for the
synthesis of siroheme that governs N and S metabolism, demonstrates the
co-modulation of NiR and SiR, which are essential for amino acid and
protein synthesis. The qRT-PCR results showed that AtSirB expression
increased in *AtSirBx* plants and declined in antisense plants. Similarly,
protein expression increased in *AtSirBx* lines and reduced in antisense
lines. In comparison to WT, *AtSirBx* plants were larger and greener in
color.

In plants, protein synthesis and Chl synthesis are highly interde-
pendent (Biswal et al., 2012). Importantly, precorrin II synthesized by
UPM1 is usually not converted to protoporphyrin IX but serves as a
committed precursor of siroheme biosynthesis. The expression of all Chl
biosynthetic enzymes is tightly regulated, and even an increased de-
mand at the later stages of Chl synthesis for example, the conversion of
protochlorophyllide to chlorophyllide via overexpression of the penul-
timate enzyme protochlorophyllide oxidoreductase (POR) induces
expression of all genes in the Chl biosynthetic pathway (Pattnayak and
Tripathy, 2011). Similarly, overexpression of chlorophyllide a oxygenase (CAO), which converts chlorophyllide a to chlorophyllide b, activates the gene and protein expression of all enzymes in the pathway, leading to enhanced tetrapyrrole synthesis. Similarly, diversion of uroporphyrinogen III from the Chl biosynthetic pathway through SirB overexpression is expected to augment both protein and gene expression of Chl biosynthesis. The increased Chl content observed in our *AtSirB* overexpressors suggests a fine-tuned coordination between siroheme and Chl biosynthetic pathways.

The most of *AtSirB* antisense plants exhibited chlorosis and were non-viable, likely due to substantial silencing of *AtSirB* expression. This suppression is presumed to disrupt the activities of nitrite reductase (NiR) and sulfite reductase (SiR), leading to the intracellular accumulation of cytotoxic intermediates such as NO₂ and SO₃²⁻ causing plant mortality (Bork et al., 1998; Takahashi et al., 2001). Futher, the antisense plants that survived had reduced total protein and Chl content due to the downregulation of nitrite reductase activity resulting in reduced plant growth and biomass. In contrast to antisense and WT plants, FW and DW of *AtSirBx* were higher. It could be because of overexpressers assimilating N and C at higher rates. Compared to WT, the number of lateral roots per plant was higher in *AtSirBx* plants. However, the antisense plants had the same number of lateral roots as the WT plants. The present study reveals that the reduction of NiR activity in *AtSirB* antisense plants down-regulates NR reaction to avoid excess accumulation of toxic nitrite. The downregulated NR function could reduce nitrate uptake from the growth medium by suppressing the root-localized high-affinity nitrate transport system (HATS). Therefore, plants would stifle the development of nitrate-absorbing lateral roots. It is further supported by Little et al. (2005), who showed that a defect in HATS stimulates lateral root initiation in *Arabidopsis*.

Similarly, the total protein of antisense plants declined by 18 %, primarily due to reduced NiR activity resulting from decreased siroheme biosynthesis. Although NiR apoprotein content remained largely unchanged, the limited availability of siroheme led to a 60 % reduction in NiR activity. NR activity also declined, likely as a protective response to avoid toxic NO₂ accumulation. Althoguh siroheme is not directly required for NR activity, NO₃⁻ reduction still dropped by 23 %. This suggests a coordinated regulation of NR and NiR activities in both overexpressing and antisense plants.

Despite a 60 % decline in NiR activity in antisense plants, the total protein content was reduced by only 18 %. This suggests that when grown in an optimal NO₃⁻ containing growth medium, plants may partially compensate for reduced NiR activity, which is crucial for protein synthesis. Conversely, *AtSirBx* plants with higher NiR activity exhibited enhanced NR function to supply NO₂ as a substrate for the augmented NiR reaction in the overexpressor. To stimulate NR activity, the NO₃⁻ needs to be absorbed in large amounts; therefore, the sense plants showed increased lateral root growth to promote nitrate uptake (Little et al., 2005).

In *AtSirBx* lines, NiR gene (*Nii*) and protein expression increased, which resulted in increased NiR activity. The increase in NiR activity was due to the enhanced availability of the apoprotein and the prosthetic group siroheme in *AtSirBx* plants. Due to augmented NiR and NR activities, the protein content of the overexpressers was higher than that of the WT. Our results demonstrate a co-regulation of *NiR* and *NR* gene expression and their activity, which play a pivotal role in N assimilation in non-leguminous plants. Chl synthesis is one of the most significant cellular processes for plants' photosynthesis. Chl content increased significantly in *AtSirBx* lines and decreased in antisense lines. It is consistent with increased or decreased protein content in the transgenics.

Although numerous previous attempts have been made to enhance NUE and protein content of plants by overexpressing NiR, NR, and high affinity nitrate transporters, these strategies have largely failed to improve the protein content in transgenic lines (Vincentz and Caboche, 1991; Quilleré et al., 1994; Crétet et al., 1997; Fraisier et al., 2000;

Takahashi et al., 2001; Djennane et al., 2002;). In our study, NiR assembly and activity appear to be significantly enhanced by the increased synthesis of the siroheme in *AtSirBx* plants. As siroheme is limiting factor, its augmented synthesis in *AtSirBx* plants bind to NiR apoproteins to enhance NiR activity, modulating NR reaction and ultimately increasing protein content. The resulting rise in protein content, combined with enhanced photosynthetic activity and improved NUE leads to a larger plant phenotype and higher biomass accumulation in *AtSirBx* plants. These finding demonstrate that boosting the synthesis of the siroheme cofactor is more effective for increasing, NUE, protein content and biomass than merely increasing levels of NiR or SiR apoproteins (Fig. 11). Consistently, our earlier study on the siroheme biosynthetic gene *AtUPM1* showed that its overexpression in *Arabidopsis thaliana* also increased protein accumulation in transgenic plants (Garai and Tripathy, 2018). Similarly, Castello et al. (2021) reported that overexpression of the cyanobacterial NOS gene enhanced NUE, improved yield, and increased tolerance to N-limiting conditions. Likewise, overexpression of the AMINO ACID PERMEASE 1 (AAP1) gene in pea plants significantly improved NUE under both low- and high-N conditions (Perchlik and Tegeder, 2017). In agreement with our results, the NUE was consistently higher under N-limiting conditions as compared to optimal N supply. These further support our observations that NUE under low N was greater than under high N in both WT and S1 plants. Moreover, S1 plants had higher NUE in both N-sufficient and N-limiting conditions, whereas the antisense A1 plants showed lower NUE under both conditions. Similarly, Xie et al. (2025) demonstrated that overexpression of G-proteins in *Brassica napus* L. enhanced NUE and promoted growth under N-limiting conditions. Collectively, these studies support our conclusion that overexpression of *AtSirB* improves NUE, total protein content, and nitrogen assimilation, thereby enhancing plant tolerance to N-limiting environment.

## 4.1. Overexpression of *AtSirB* protects plants from N deficiency

The *AtSirBx* plants were greener and had more Chl than WT under N limitation (0.1 N). Conversely, *AtSirB* antisense plants displayed pronounced phenotypic abnormalities under N-deficient conditions, including severe bleaching at 0.1 N, which corelated with a significant decline in both protein and Chl contents. *Synechocystis* and *Chlorella* grown in N-deficient media exhibited reduced photosynthetic activity, smaller antennas, slower electron transport rates, decreased C-dependent oxygen evolution and lower biomass production, aligning with our finding (Salomon et al., 2013; Kumari et al., 2021). The *AtSirBx* plants had a substantially higher protein content (95 %) than WT plants under N-limitation. Conversely, the antisense plants showed a substantial reduction in protein content (32 %) under identical conditions. This decline in protein content of plants grown in nitrate-deficient media was due to decreased NiR activity and impaired N assimilation.

Under optimal N conditions, transgenic plants exhibited higher $\phi$PSII values than WT plants at both limiting and saturating light intensities. This suggests improved light utilization, likely due to an increased abundance of the light-harvesting complex and more efficient use of absorbed energy, resulting from a robust PSII. Conversely, the $\phi$PSII were lower in antisense plants than in the WT under both low and high light intensities, indicating reduced light absorption and utilization likely due to lower abundance of light-harvesting complex and inefficient use of absorbed energy by a frail PSII.

The Fv/Fm ratio decreased in WT, sense, and antisense plants in N-limiting condition. However, the *AtSirBx* plants consistently showed higher Fv/Fm ratios than WT at all N levels, while the *AtSirB* antisense plants always had smaller Fv/Fm ratios. The increased protein content of *AtSirBx* plants contributed to the better development of the thylakoid membranes even under N-limiting condition. In normal and N-limiting condition, the $\phi$PSII was higher in *AtSirBx* and lower in antisense plants in all measured light intensities. In normal growth media, the Fv/Fm value is essentially constant but declined with nutrient constraints,
<figure><img src="image_19.png" /><figcaption>Fig. 11. A cartoon of *SirB* expression in *Arabidopsis*. Overexpression of *SirB* (Sirohydrochlorin ferrochelatase) increases the abundance of Siroheme in over-expresser plants, further inducing expression/activity of nitrite reductase and sulfite reductase in over-expressers. Induction of NiR and SiR enhances the nutrient use of overexpresser plants. Enhancement of nutrient use increases total protein content which ultimately increases the photosynthetic efficiency (PE) of *AtSirB* over-expressing *Arabidopsis* plants.</figcaption></figure>

which can be used to identify nutritional limitations (White et al., 2011; Zhang et al., 2013). In the WT and *AtSirBx* plants, the φPSII increased in response to a rise in PAR in N-sufficient and N-deficient media, albeit to a larger extent in the overexpressers. In *AtSirBx* plants, the greater light-saturated rate denotes an increase in the components of the electron transport chain. In contrast, the increased slope of φPSII at limiting light intensities shows improved energy collection by the larger light-harvesting complex. The φPSII decreased in antisense plants at low and high light intensities in all N levels, indicating a decline in energy capture and usage due to decreased light-harvesting antennae and reduced abundance of the electron transport components.

N deficiency is known to suppress plant growth and reduce grain yield compared with N sufficient conditions, and it promotes starch accumulation in leaves due to an increase in both the number and size of starch granules in bundle sheath cells (Ning et al., 2018). Our findings align with these observations: in *AtSirB*-overexpressing lines, which exhibit improved N assimilation efficiency, no detectable starch accumulation was observed under N-limited conditions. In contrast, WT and antisense lines showed significant starch accumulation under N-limiting condition. This suggests that the improved NUE in *AtSirB* overexpressors prevents the C-N imbalance typically caused by N deficiency and they do not accumulate starch even at N-limiting condition.

## 4.2. Overexpression of *AtSirB* protects plants from S deficiency

As siroheme also serves as the prosthetic group of SiR, our study demonstrates that *AtSirBx* plants are better equipped to counter S deficiency. Our results show that in a S-limiting medium (0.1S), the WT plants had very low protein and Chl, and appeared pale green. However, the *AtSirBx* plants, due to better utilization of available low amounts of S, had higher Chl and were greener than WT. Under S-limiting condition, the *AtSirB* antisense plants exhibited severe phenotypical changes; plants became severely blanched due to reduced Chl contents. *AtSirBx*

plants showed increased SiR protein abundance over that of WT. In agreement with protein expression, SiR mRNA expression substantially higher in *AtSirBx* plants compared to WT. In antisense plants, SiR mRNA abundance was slightly reduced, and there was no significant change in the abundance of SiR apoprotein.

Compared to WT, *AtSirBx* plants having more Chl and protein levels and showed higher photosynthetic rates under both optimal (1S) and limiting (0.1S) sulfur conditions. Conversely, the photosynthetic capacity of antisense plants was reduced due of their lower protein and Chl content. The Fv/Fm, a measure of the plant's quantum efficiency in the dark-adapted plants, declined when WT, *AtSirBx*, and antisense plants were grown on the S-limiting medium. The antisense plants had a lower Fv/Fm than the WT, whereas the *AtSirBx* plants consistently had a higher Fv/Fm ratio under both S-optimal and S-limiting growth conditions. This was attributed to the increased protein contents of *AtSirBx* plants, which contributed to the better development of the thylakoid membranes in S-limiting growth conditions. The φPSII in *AtSirBx* plants was higher under normal and S-deficient conditions than in antisense plants. The φPSII increased in response to PAR; the *AtSirBx* plants maintained higher φPSII in both low and high light intensities, indicating an improved light-harvesting complex and more efficient electron transport components of PSII in the overexpressers. Under S-deficiency, the φPSII declined in WT and, to a larger extent, in antisense plants, suggesting loss of their PSII antennae and electron transport components. In S-deficient *AtSirBx* plants, the φPSII was much higher than in S-starved WT plants, suggesting preservation of photosynthetic apparatus and improved tolerance to S-deficiency. The *AtSirBx* displayed higher SUE under both S-limiting and S-optimum conditions. While SUE increased significantly in both the wild-type (WT) and *AtSirB* transgenic lines under S-limitation, the overexpressor consistently maintained higher SUE across both conditions. In contrast, the antisense plants had lower SUE than the WT in both conditions, following a trend similar to that observed for NUE.
## 5. Conclusion

The present study demonstrates that overexpression of AtSIRb substantially enhances N and C assimilation, resulting in increased protein and Chl content, larger biomass, and improved photosynthetic efficiency under both optimal and limiting N and S conditions. By boosting siroheme synthesis, a key cofactor for nitrite and sulfite reductase, *AtSIRb* plants showed coordinated up-regulation of NR, NiR, and SiR gene and protein expression and increased NiR and NR activities, allowing for effective utilization of available nutrients, better maintenance of photosynthetic apparatus, and improved tolerance to N and S deficiencies. These findings highlight that stimulating cofactor synthesis could be more effective than solely increasing apoenzyme abundance for enhancing NUE, SUE and biomass accumulation in plants. Potentially, our results could be exploited to genetically transformed important crop plants for better NUE, SUE and reduced application of fertilizers in the soil.

### CRediT authorship contribution statement

**Naveen C. Joshi:** Writing – original draft, Methodology, Investigation. **Laurent Dufossé:** Writing – review & editing, Visualization, Validation. **Baishnab C. Tripathy:** Writing – review & editing, Supervision, Methodology, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgment

The authors thank the Department of Biotechnology, Govt. of India for financial assistance. This work is supported by DST/SERB-SRG (#SRG/2020/002237) awarded to Naveen Chandra Joshi and the Department of Biotechnology grant (#BT/PR11896/BPA/118/3/2014) to Baishnab C. Tripathy. Laurent Dufossé deeply thanks the Conseil Régional de La Réunion and the Conseil Régional de Bretagne for the continuous support of research activities dedicated to biotechnology.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.stress.2025.101143.

### Data availability

Data will be made available on request.

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