Alteration of Photosynthetic and Antioxidant Gene Expression in Sugarcane Infected by Multiple Mosaic Viruses

Abstract

Sugarcane mosaic virus (SCMV), sugarcane streak mosaic virus (SCSMV), and sorghum mosaic virus (SrMV) are the causative pathogens of mosaic disease. This study aimed to identify mosaic virus infection and its impact on photosynthetic and antioxidant gene expression in eight commercial sugarcane cultivars grown on sugarcane plantations in East Java, Indonesia. The disease incidence and severity were observed in symptomatic leave samples, and then the virus was identified. A high incidence and severity of mosaic symptoms were observed in the PS881 and NX04 cultivars compared with the other cultivars. RT-PCR analysis detected SCSMV infection in all cultivars; double infections with SCSMV and SCMV in the PS881, PS882, and Cening cultivars; and triple infections with SCSMV, SCMV, and SrMV in the PS881 cultivar. Ascorbate peroxidase (Apx) expression was upregulated in all virus-infected cultivars and significantly increased in the triple-infected PS881 cultivar. However, catalase (Cat) expression was only slightly increased in the PS881 cultivar. The chlorophyll content was reduced, and the PsaA gene was downregulated in all cultivars. The expression of PsaA, RbcS, and Sps was significantly suppressed in the triple-infected PS881 cultivar. Moreover, the downregulation of both the RbcS and Pepc genes was concomitant with that of their protein levels.

1. Introduction

Sugarcane (Saccharum spp.) is a major crop used for the production of sugar, biofuel, and other products, such as animal feed, paper, organic fertilizer, and industrial enzymes [1,2], and it is mainly propagated using lateral buds, which are easily transmitted and infected by sugarcane mosaic disease [3,4] and yellow leaf disease [5]. Mosaic virus is one of the most severe diseases in sugarcane and leads to the development of light and dark green irregular patterns, yellow patches, or streaks [6,7]. Multiple infections by two or more viruses frequently occur in plants and result in severe symptoms [8]. Mosaic diseases of sugarcane with multiple infections of sugarcane mosaic virus (SCMV), sugarcane streak mosaic virus (SCSMV), and sorghum mosaic virus (SrMV) have been reported in China [8,9]; SCMV and SrMV have been reported in the USA [10]; and SCMV and SCSMV have reported in India [11].

Reactive oxygen species (ROS) accumulate in response to biotic stress caused by viral infection. Mosaic virus infection significantly upregulates ROS-producing genes, such as NAD oxidase, malate dehydrogenase, and flavin-binding monooxygenase [12], and the production of ROS can potentially damage cellular components and disrupt programmed cell death [13,14]. However, accumulated ROS can be detoxified by non-enzymatic and enzymatic antioxidants, such as ascorbate peroxidase (Apx), catalase (Cat), superoxide dismutase, and glutathione reductase [15,16,17]. This antioxidant defense mechanism for scavenging ROS protects cells from oxygen radicals [18].

Under light illumination, SCMV infection stimulates malate synthesis by enhancing pyruvate orthophosphate dikinase (PPDK) activity, leading to ROS accumulation [19]. As a C4 plant, sugarcane exhibits high rates of photosynthesis under high light intensity. Phosphoenolpyruvate carboxylase (PEPC) captures atmospheric CO₂ and converts it to oxaloacetate and malate using phosphoenolpyruvate (PEP) as a substrate, and then malate is transported into the bundle sheath cell and decarboxylated to produce CO₂ molecules, which are re-fixed by ribulose-1,5-bisphosphate carboxylase (Rubisco) to form sucrose and starch [20]. To maintain the C4 cycle, PEP is regenerated from pyruvate using PPDK and then used by PEPC to produce malate. Light is well known to regulate the activity of several carbon-assimilating enzymes, including PPDK, PEPC, and sucrose-phosphate synthase (SPS) [21,22]. However, the effects of mosaic virus infections on photosynthetic activity remain poorly understood.

Mosaic diseases significantly reduce the chlorophyll content, photosynthetic efficiency, yield, and quality of sugarcane [6,23,24]. The expression of light-harvesting proteins and primary carbon-assimilating enzymes is high in both resistant cultivars [12] and non-infected sugarcane [24]. Moreover, NADPH, Rubisco, and photosystem I (PSI) reaction center expression, including the PsaA gene, is upregulated in mosaic-resistant sugarcane compared to susceptible sugarcane. Protein analysis confirmed that PEPC and Rubisco activity is decreased in virus-infected sugarcane seedlings [23,24]. Alterations in photosynthesis-related activities slow sugarcane growth [17] and reduce the sucrose content and sugarcane yield by up to 75% [6]. In addition, mosaic virus infection reduces the chlorophyll content in cassava [25] and poplar [26] and perturbs pigment biosynthesis in tomato [27]. These results indicate that mosaic virus infection downregulates the expression of photosynthesis-related genes [26,28,29].

Mosaic symptoms in sugarcane are frequently associated with several different viral infections. Multiple or mixed viral infections have also been reported in sweet potatoes in Uganda [29] and cotton plants in China [9]. Multiple infections from two or more viral interactions are categorized as synergistic, antagonistic, or neutral interactions [8,30] that induce a decline in plant vigor and productivity [31]. Combined infection by viruses that act synergistically exacerbates symptom severity and leads to plant death or severely reduced yields [32], as reported in maize [33].

In this study, mosaic viruses and their distribution were determined in sugarcane grown at four locations in East Java, Indonesia. Symptomatic sugarcane leaves were collected from the field, and SCSMV, SCMV, and SrMV were identified in the leaf samples using specific primer pairs for RT-PCR analysis. The effects of viral infection on the expression of antioxidant and photosynthetic genes were determined by RT-PCR and immunoblot analysis.

2. Materials and Methods

2.1. Observation Area and Leaf Sample Collection

Mosaic symptoms were observed in commercial sugarcane cultivars grown in four districts of East Java Province: Kediri, Lumajang, Jember, and Bondowoso. This province is the primary location of sugarcane production in Indonesia. Observations were conducted in plants at three to four months of age in sugarcane plantations under the authority of PT. Perkebunan Nusantara X and PT. Perkebunan Nusantara XI. Symptomatic leaf samples were collected from fields and assessed for disease incidence and severity. Disease incidence was determined as the percentage of sugarcane mosaic symptoms relative to the total sugarcane observed in the field, while disease severity was estimated using a previously described scoring system for young sugarcane leaf area in which symptoms at the first and third leaves from the green top are recorded [3]. The percentage severity was calculated using the following equation [34]:

$$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{a}\mathrm{g}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{s}\mathrm{e}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{t}\mathrm{y} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x} \left(\%\right)={\displaystyle \frac{\sum (\mathrm{c}\mathrm{l}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{f}\mathrm{r}\mathrm{e}\mathrm{q}\mathrm{u}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}\times \mathrm{s}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{c}\mathrm{l}\mathrm{a}\mathrm{s}\mathrm{s})}{(\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{s}\times (\mathrm{m}\mathrm{a}\mathrm{x}\mathrm{i}\mathrm{m}\mathrm{a}\mathrm{l} \mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x})}}\times 100$$

Disease incidence and severity were observed over approximately 10,000 m² for each sugarcane cultivar. Within the observed areas, a total of 61 sugarcane leaf samples with mosaic symptoms were selected, disinfected with 75% alcohol, and immersed in liquid nitrogen for further laboratory analysis.

2.2. RNA Extraction and Mosaic Virus Determination

For mosaic virus detection, RNA was extracted from sugarcane leaf samples using an RNAprep Pure Plant Plus Kit (Tiangen, Beijing, China) [35], and the concentration was quantified using a NanoVuePlus-UV Spectrophotometer (GE Healthcare, Chicago, IL, USA). The three major mosaic viruses, SCSMV, SCMV, and SrMV, were identified by RT-PCR using primer pairs specific for the CP gene, as shown in

Table 1. Description of primers used in this study.

Primer	Sequence Information (5′-3′)	Amplicon Length (bp)	Annealing (°C)	Reference
CP-SCSMV-F	5′-MTCTTCATCRGCCGCMTCRATAC-3′	335 bp	53	This study
CP-SCSMV-R	5′-AGAACTGAACCCACTTGTACGCC-3′
CP-SCMV-F	5′-GACATATGGATGTAGATGCTGGTACGACA-3′	735 bp	58	[35]
CP-SCMV-R	5′-ATGGATCCTAGTGGTGCTGCTGCACTCCC-3′
CP-SrMV-F	5′-GCAGATGCTGATGCGAAA-3′	480 bp	52	This study
CP-SrMV-R	5′-ACGCTTCAGCTGCATCAC-3′
PEPC-F	5′-TGGGTGGTGACCGTGATGG-3′	129 bp	60	This study
PEPC-R	5′-GCAGCGCCACATAGAGAGC-3′
PsaA-F	5′-AGGGGCTTATACCCTCAG-3′	121 bp	52	This study
PsaA-R	5′-GGATTAGGTGCCTAACGGAC-3′
RbcS-F	5′-CACTAGCTTCGCCAAAGT-3′	205 bp	55	This study
RbcS-R	5′-AAGCCTTCCTTGCTGAAC-3′
SPS-F	5′-GGGTCTCCATAGGACCATTA-3′	110 bp	55	This study
SPS-R	5′-GGGGTGTTATTGTGTGAGTA-3′
CatF	5′-GCTCAGTTCGACAGGGAACG-3′	208 bp	55	[16]
CatR	5′-CACGTGGATCCCTCAAGGTC-3′
ApxF	5′-GATTTGATTGCCGTGGCTGG-3′	134 bp	55	[36]
ApxR	5′-TCTTCAGGAAGTTTGCCAGTTG-3′
β-tubulinF	5′-CATCTTCGTGTGGAATCCT-3′	129 bp	55	This study
β-tubulinR	5′-AACTCCAGAACAGACCGTA-3′

. Total RNA (1 µg) was converted to cDNA using a ReverTra AceTM Kit (Toyobo, Osaka, Japan) and then subjected to a PCR reaction using a KOD-Plus-Neo Kit according to the manual instruction (Toyobo, Japan). The PCR analysis was performed at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s; annealing at 53 °C (SCSMV), 58 °C (SCMV), or 52 °C (SrMV) for 30 s; and extension at 72 °C for 1 min—followed by a final extension at 72 °C for 5 min using a T100TM Thermal Cycler (Bio-Rad, Hercules, CA, USA). PCR products were separated by agarose gel electrophoresis containing ethidium bromide and visualized using a GelDoc system (Major Science, Saratoga, CA, USA). The corresponding DNA bands were excised from the agarose gel, purified using a GenepHlow Gel/PCR Kit (Geneaid, Taipei, Taiwan), and used for nucleotide sequence determination.

2.3. DNA Sequencing and Phylogenetic Analysis

Nucleotide sequencing of the isolated PCR products was performed by a DNA sequencing provider (1st BASE, Singapore). The obtained nucleotide sequences were compared with the sequences in the NCBI virus database using BLAST/N (https://blast.ncbi.nlm.nih.gov (accessed on 8 January 2024)) to confirm the degree of sequence similarity. A phylogenetic tree was constructed from the SCSMV, SCMV, and SrMV nucleotide sequences using the neighbor-joining (NJ) method (1000 bootstrap replicates) and MEGA 11 software version 11.0.13 [36].

2.4. Quantitative Real-Time PCR (RT-qPCR)

RT-qPCR was performed using a CFX connectTM Real-Time System (Bio-Rad, USA) to analyze the expression levels of photosynthesis and antioxidant ROS-scavenging genes. The 25 µL reaction system contained 12.5 µL of 2× SYBR Green master mix (ThunderbirdTM SYBR qPCR Mix, Toyobo, Japan), 1 µL of 10 µmol/L of primers, and 1.5 µL of 50 ng cDNA, with nuclease-free water added to reach the reaction volume. The gene-specific primer pairs are listed in Table 1. The RT-qPCR program was 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, primer-dependent annealing temperatures (Table 1) for 15 s, elongation at 72 °C for 15 s, and final extension at 72 °C for 60 s. Three independent biological replicates were performed, each sample was analyzed in duplicate, and expression was normalized based on β-tubulin expression. The relative gene expression was calculated via Livak’s method using the formula 2^−∆∆CT [37].

2.5. Protein Extraction and Immunoblotting

One gram of frozen sugarcane leaf sample was ground in liquid nitrogen, and the proteins were extracted according to the method previously described [35]. The soluble protein concentration was determined using a Bradford reagent (Bio-Rad, USA). The levels of Rubisco and PEPC proteins were determined by immunoblotting using specific polyclonal antibodies against PEPC and Rubisco, with 15 µg and 10 µg soluble proteins for PEPC and Rubisco, respectively. The immunoblotting analysis was conducted according to the method previously described [38].

2.6. Analysis of the Total Chlorophyll of Leaves

The total chlorophyll content was determined using an optical method [39] with slight modifications. Briefly, symptomatic sugarcane leaves (0.1 g) were finely cut and incubated with 5 mL of a solution containing a mixture of acetone and ethanol (2:1, v/v) in the dark for 24 h. Three independent biological replicates were performed, and each sample was analyzed in triplicate. The absorbance of the solution was measured at 645 and 663 nm using a spectrophotometer (UV-VIS double-beam spectrophotometer (Hitachi, Tokyo, Japan). Solution without the sample was used as the blank. The total chlorophyll concentration was estimated using the following equation [40]:

mg total chlorophyll/g tissue = 20.2 × (A645) + 8.02 × (A663) × V × W

where:

• A645: absorbance at 645 nm;
• A663: absorbance at 663 nm;
• V: final volume of chlorophyll extract;
• W: fresh weight of leaf extract.

2.7. Statistical Analysis

Statistical analyses were performed in triplicates and represented as the mean ± standard errors. Statistical significance was calculated using Dunnett’s test with an ANOVA to determine significant differences. A p-value of 0.05 was considered statistically significant.

3. Results

3.1. Mosaic Symptom, Incidence, and Severity

Mosaic symptoms were observed over approximately 10,000 m² for each sugarcane cultivar, and 61 symptomatic leaf and 1 healthy non-symptomatic leaf samples were collected. Mosaic symptoms include blotchy or streaky patterns of yellow and green colors with varying lengths, sizes, and irregularities, and they were recorded to determine the incidence percentage. The incidence of symptoms observed in Kediri was higher than that in the other locations (Figure 1A). The highest incidence was observed in NX04, followed by PS881 (80 and 54%, respectively). However, the incidence of PS881 was slightly lower in Kediri compared with that reported in a previous study in the Jember area [3]. Furthermore, the percentage of severity was high at above 80% in all cultivars (Figure 1B), with the highest percentages found in PS58, PS881, and NX04.

To determine whether mosaic infection of one or more viral types occurred, RT-PCR was conducted using specific primer pairs for the CP gene to detect SCMV, SCSMV, and SrMV (Table 1), which are considered the major mosaic viruses in sugarcane. The analysis showed that CP-SCSMV DNA, with a molecular size of 335 kb, was present in all sugarcane leaf samples (Figure 2A), which indicated that SCSMV was more widespread in all cultivars and regions of the experiment compared with that observed in previous studies [4]. Furthermore, SCMV with a molecular size of 735 kb was found mainly in PS881 and PS882, with only a few instances observed in the Cening cultivar; however, the corresponding DNA band was not detected in NX04, NX01, HW Merah, PS58, and NXI-4T (Figure 2B). Interestingly, a specific primer for SrMV detection, which is a rare mosaic virus infection, was found in the PS881 cultivar but not in the other cultivars (Figure 2C). These results indicate the presence of multiple or mixed mosaic virus infections, including double and triple infections, which are reported for the first time in an Indonesian sugarcane plantation. Furthermore, the mosaic patterns were similar in all sugarcane varieties; however, the intensity of the symptoms varied (Figure 2D). The intensity of the mosaic symptoms was more severe in double and triple infections than in single infections.

3.2. Phylogenetic Analysis of the Mosaic Virus Isolates

To confirm infection by the mosaic virus, the corresponding CP-DNAs of SCSMV, SCMV, and SrMV were determined based on their nucleotide sequences. The sequences from each mosaic virus were correctly verified using NCBI BLAST, and phylogenetic trees were constructed using MEGA 11 software. The sequence similarities between the target sequences and other viral isolates available in GenBank were 92.11% (KP987832.1), 92.58% (MT725538.1), and 97.39% (EU189037.1) for SCSMV, SCMV, and SrMV, respectively. Phylogenetic analysis grouped SCSMV with isolates from Thailand (KP987832.1); SCMV with isolates from Florida, USA (MT725538.1) and Boetzingen, Germany (X98168.1) (Figure 3A,B); and SrMV with isolates from Argentina (EU189037.1), the USA (EF078962.1), and China (DQ227695.1) (Figure 3C).

3.3. Expression of Apx and Cat Genes

Environmental stresses, including biotic stresses, produce ROS that regulate cellular metabolism and inhibit cell growth and development. Plants have developed antioxidant mechanisms to overcome the detrimental effects of ROS production. In this study, Apx expression was upregulated in infected cultivars compared with that in asymptomatic healthy controls (Figure 4A). Moreover, the expression was substantially higher in PS881 and NX04 than in the other cultivars, while only a slight increase was observed in NX4T and NX01. Changes in Apx expression are associated with the ROS detoxification pathway in SCMV-resistant sugarcane genotypes [12]. However, whether Apx expression in sugarcane indicates the degree of resistance to viral infections is not well understood.

To confirm the response of antioxidant expression to mosaic virus infection, RNA was isolated from PS881 leaves exhibiting single, double, and triple mixed infections, and the expression of the Apx and Cat genes was determined. Mixed infections were detected in the leaves of the PS881 cultivar, as previously described (Figure 2). Apx expression was almost upregulated in all cultivars, except in NX-4T and NX01, which remained constant compared to those healthy non-infected leaves (control) (Figure 4A). The expression of Cat was slightly upregulated in single infections and increased by approximately 1.4-fold in mixed triple infections with the addition of SrMV compared to that in the control (Figure 4B). However, the expression of Apx significantly increased in the mixed infection group, and the highest expression (approximately 3.9-fold) was found in the triple-infection group. These results indicate that Apx expression was more affected by mosaic infection compared with Cat expression.

3.4. Expression of Photosynthesis Related-Genes in Response to Mosaic Virus Infection

Mosaic virus infection disturbs the chloroplast structure, which leads to leaf chlorosis, chloroplast malfunctions, and downregulated photosystem efficiency [23,41]. To observe how the viral infections disrupted the chloroplasts, the chlorophyll content of the infected leaves was determined. The content was significantly lower in the PS58, PS881, PS882, NX04, and HW Merah cultivars (Figure 5A) but only slightly reduced in the Cening, NXI-4T, and NX01 cultivars compared with the other varieties. These results clearly indicate that mosaic virus infection causes chloroplast disruption or damage that leads to disturbances in photosynthetic activity.

Quantitative RT-PCR was used to determine whether the expression of PsaA, located in the chloroplast genome, was affected by viral infection. The expression of PsaA was reduced in the single-infected leaves of all sugarcane cultivars compared to that in the healthy leaves, although the expression varied among the infected cultivars (Figure 5B). Consistent with the chlorophyll content, the expression of PsaA was markedly reduced in the leaves of PS58, PS881, and NX04. However, PsaA expression was slightly reduced in the other cultivars compared with that in the healthy plants.

This change in chlorophyll content suggests that viral infection has a significant impact on energy metabolism and photosynthetic efficiency and may contribute to alterations in the expression of photosynthetic and carbon-assimilating genes in infected plants. The expression of the photosynthetic genes for RbcS (Rubisco small subunit), Pepc, and Sps (sucrose-phosphate synthase) was measured along with that of the PsaA gene in sugarcane leaves with triple mixed infection (Figure 5C). The expression of PsaA gradually decreased with the degree of viral infection in single, double, and triple PS881 leaves. The expression of RbcS and Sps was gradually suppressed in single and double infections, while the expression of RbcS, Sps, and PsaA was almost depleted in the triple infection. Furthermore, the expression of Pepc was reduced by approximately 22.7–40.3% in the single-, double-, and triple-infected leaves compared with that in healthy leaves.

To ensure the consistency of gene expression under mosaic virus infection, which reduces photosynthetic activity, the protein content of Rubisco and PEPC was detected using immunoblotting. Rubisco and PEPC are key enzymes for carbon assimilation in C4 plants, such as sugarcane. Consistent with the gene expression, the PEPC, RbcL, and RbcS protein expression decreased according to the degree of single, double, and triple infections in the PS881 cultivar (Figure 5D). Because Rubisco proteins have a conservative and slow turnover [42], the inhibited proteins were still detectable and not as depleted as the level of mRNA in the triple-infected leaves.

4. Discussion

In this study, mosaic diseases were observed in four sugarcane plantations located in Lumajang, Jember, Bondowoso, and Kediri in East Java, Indonesia. Eight sugarcane cultivars were observed for mosaic symptoms and sampled for viral analysis. Mosaic diseases were found to have a higher incidence in Kediri than in the other locations, although they had a similarly high percentage of severity (above 80%). The higher incidence at this location may have been caused by differences in integrated disease management, such as heated water treatment prior to planting. Among the sugarcane cultivars, PS881 and NX04 showed the highest incidence and were categorized as susceptible cultivars [3,4]. RT-PCR analysis revealed that SCSMV was distributed in all sugarcane cultivars, while SCMV and SrMV were only observed in a few cultivars. The nucleotide sequences of the amplified CP gene and phylogenetic analyses confirmed the presence of SCSMV, SCMV, and SrMV in sugarcane. Interestingly, the PS882 and PS881 cells were infected with double SCSMV-SCMV and triple SCSMV-SCMV-SrMV infections, respectively. These results indicate the presence of a mixed mosaic virus infection in sugarcane, which has not been previously reported in Indonesia.

Mixed infections by two or more viruses occur naturally in plants [8,43]. Infection of sugarcane with SCSMV, SCMV, or SrMV resulted in mosaic-like symptoms, such as irregular light and dark green patterns on the leaves [7]. Infections with these three viruses in sugarcane present highly similar mosaic symptoms and are difficult to differentiate. Interactions between two or more plant viruses in mixed infections can be categorized as synergistic or antagonistic. A synergistic interaction refers to a situation in which two or more viral infections result in severe symptoms, and antagonistic interaction refers to only one of the viruses having a beneficiary and higher activity than the second virus [30]. PS881 with triple infection and PS882 with double infection showed higher disease severity (Figure 1B) and mosaic symptom intensity (Figure 2D) than the cultivars with single infection. These results indicate that mixed mosaic viral infection with SCSMV, SCMV, and SrMV results in synergistic interactions.

Infections caused by mosaic viruses induce ROS accumulation; however, plants have developed antioxidant scavenging and detoxification systems to overcome these problems. Apx is one of the most important genes for ROS scavenging [36], and its expression increased in all infected sugarcane cultivars, with the highest expression in the mixed infection of PS881 leaves (Figure 4A). Increased Apx expression can regulate ROS accumulation, which may be considered a characteristic of plant resistance to viral infections. However, elevated Apx expression may be required to balance oxidation, and antioxidation cascades to maintain redox homeostasis.

Viral infection has a significant effect on energy metabolism, respiration, and photosynthetic rates. Viral proliferation requires energy from the infected plant cells, which leads to ROS accumulation [19]. ROS play dual roles in plants. Low ROS concentrations act as a signaling molecule to promote growth and development, whereas excessive ROS accumulation triggers cellular damage, leading to cell death. Viral infection modified the chloroplast structure [25] and caused a reduction in chlorophyll content (Figure 5A) as well as the expression of PsaA, which is located in the chloroplast (Figure 5B). Chloroplasts play a central role in the generation of energy during photosynthesis. The disruption of chloroplasts causes a reduction in photosynthetic energy for carbon assimilation. The significant reduction in PsaA expression in PS881 plants with mixed infection was followed by the suppression of photosynthetic carbon-assimilating genes, such as RbcS, Pepc, and Sps, as well as their protein content (Figure 5C,D). These results indicate that mosaic virus infection damages the chlorophyll and subsequently reduces carbon assimilation activity. Thus, infection with mosaic viruses significantly reduces the photosynthetic efficiency, yield, and quality of sugarcane.

The development of virus-resistant sugarcane is considered the most effective approach for managing viral diseases. Pathogen-derived resistance (PDR) and RNA interference (RNAi) technologies have been previously demonstrated as powerful approaches for plant defense against sugarcane mosaic viruses [35,44]. Moreover, RNAi has been reported to effectively induce high resistance against mosaic viruses in sugarcane [45]. Mixed infections with SCSMV and SCMV, which present synergistic interactions, exacerbate sugarcane yield. A strategy to induce dual resistance to the mosaic virus can apply RNAi by assembling a hairpin element composed of CP gene sequences from SCSMV and SCMV in tandem and an intron sequence as a loop. Dual resistance to synergistically interacting viruses has been reported in transgenic orchids [46] and wheat [47]. Therefore, RNAi could be used to induce dual resistance to SCSMV and SCMV in sugarcane.

5. Conclusions

Mosaic symptoms were observed in eight commercial sugarcane varieties cultivated in four districts of East Java Province: Kediri, Lumajang, Jember, and Bondowoso. The incidence and severity of mosaic symptoms were higher in the NX04 and PS881 cultivars compared with the other cultivars. Single infection with SCSMV was found in all sugarcane cultivars, and triple infection was observed in the PS881 cultivar. Multiple infections prominently increased the expression of Apx, the most important antioxidant required to balance oxidation and antioxidation cascades. The chlorophyll content and chloroplast photosystem I gene expression were significantly reduced in the infected sugarcane leaves. The disruption of chloroplasts causes a reduction in photosynthetic energy for carbon assimilation. Therefore, the expression of the photosynthetic genes RbcS, Pepc, and Sps were significantly decreased in the PS881 sugarcane cultivar with multiple infections. Taken together, our results show that mosaic virus infection leads to oxidative stress and reduces photosynthetic activity in sugarcane, which may impair the crop’s growth and yield.