In vitro and in vivo screening for the identification of salt-tolerant sugarcane (Saccharum officinarum L.) clones: molecular,biochemical, and physiological responses to salt stress

Sugarcane is a glycophyte whose growth and yield can be negatively affected by salt stress. As the arable lands with potential saline soils expand annually, the increase of salt-tolerance in sugarcane cultivars is highly desired. We, herein, employed in vitro and in vivo conditions in order to screen sugarcane plants for salt tolerance at the cellular and at the whole plant levels. Calli of sugarcane cv. Khon Kaen 3 (KK3) were selected after culturing in selective media containing various NaCl concentrations, and regenerated plants were then reselected after culturing in selective media containing higher NaCl concentrations. The surviving plants were finally selected after an exposure to 254 mM NaCl under greenhouse conditions. A total of 11 sugarcane plants survived the selection process. Four plants that exhibited tolerance to the four different salt concentrations applied during the aforementioned screening process were then selected for the undertaking of further molecular, biochemical, and physiological studies. The construction of a dendrogram has revealed that the most salt-tolerant plant was characterized by the lowest genetic similarity to the original cultivar. The relative expression levels of six genes (i.e., SoDREB, SoNHX1, SoSOS1, SoHKT, SoBADH, and SoMIPS) were found to be significantly higher in the salt-tolerance clones than those measured in the original plant. The measured proline levels, the glycine betaine content, the relative water content, the SPAD unit, the contents of chlorophyll a and b, as well as the K⁺/Na⁺ ratios of the salt-tolerant clones were also found to be significantly higher than those of the original plant.When the salt-tolerant clones were grown in a low saline soil, they exhibited a higher Brix percentage than that of the original cultivar.     

1-Aminocyclopropane-1-carboxylate (ACC) Deaminase-Producing Endophytic Diazotrophic <Emphasis Type="Italic">Enterobacter</Emphasis> sp. EN-21 Modulates Salt–Stress Response in Sugarcane

Soil salinity is a wide-reaching environmental problem that lowers the yield of commercial crops such as maize, rice, and sugarcane. In this study, we examined the effect of 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing endophytic diazotrophic Enterobacter sp. EN-21 on growth promotion, salt tolerance, and root colonization of sugarcane. Enterobacter sp. EN-21 inoculated and uninoculated sugarcane plants were grown in a greenhouse with and without 200 mM NaCl for 7 days. Sugarcane inoculated with Enterobacter sp. EN-21 substantially increased in total plant length, dry, and fresh weights in both non-salt and salt treatments. Under the salt–stress condition, Enterobacter sp. EN-21 significantly reduced proline, malondialdehyde, ethylene emission, and Na⁺ accumulation in sugarcane but markedly increased total chlorophyll content and K⁺ accumulation. The gfp-tagged Enterobacter sp. EN-21 was observed to colonize early at the root cap, root hairs, and lateral root junctions of sugarcane and later localized in intercellular spaces. Altogether the results of this study indicated that ACC deaminase-producing Enterobacter sp. EN-21 is a true endophyte and able to promote growth and enhance salt tolerance in sugarcane.     

Reuteran and levan as carbohydrate sinks in transgenic sugarcane

The present study reports the effect of high molecular weight bacterial fructan (levan) and glucan (reuteran) on growth and carbohydrate partitioning in transgenic sugarcane plants. These biopolymers are products of bacterial glycosyltransferases, enzymes that catalyze the polymerization of glucose or fructose residues from sucrose. Constructs, targeted to different subcellular compartments (cell wall and cytosol) and driven by the Cauliflower mosaic virus-35S: maize-ubiquitin promoter, were introduced into sugarcane by biolistic transformation. Polysaccharide accumulation severely affected growth of callus suspension cultures. Regeneration of embryonic callus tissue into plants proved problematic for cell wall-targeted lines. When targeted to the cytosol, only plants with relative low levels of biopolymer accumulation survived. In internodal stalk tissue that accumulate reuteran (max 0.03?mg/g FW), sucrose content (ca 60?mg/g FW) was not affected, while starch content (<0.4?mg/g FW) was increased up to four times. Total carbohydrate content was not significantly altered. On the other hand, starch and sucrose levels were significantly reduced in plants accumulating levan (max 0.01?mg/g FW). Heterologous expression resulted in a reduction in total carbohydrate assimilation rather than a simple diversion by competition for substrate.     

Effects of salt stress in relation to osmotic adjustment on sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) callus cultures

In vitro responses of embryogenic sugarcane (Saccharum officinarum L.; cv. CoC-671) calli stressed with different levels of NaCl (0.0, 42.8, 85.6, 128.3, 171.1, 213.9 or 256.7 mM) were studied. The results showed that a significant decrease in callus growth and cell viability occurred with ≥85.6 mM NaCl. Higher amounts of free proline and glycine betaine were accumulated in NaCl-stressed calli. Although the leached and retained Na⁺ contents increased, the retained K⁺ content decreased with increasing levels of NaCl. Such a mechanism implies that sugarcane can be considered as a Na⁺-excluder. The accumulation of salt ions and osmolytes could play an important role in osmotic adjustment in sugarcane cells under salt stress.     

Salicylic Acid Induces Salinity Tolerance in Tomato (Lycopersicon esculentum cv. Roma): Associated Changes in Gas Exchange, Water Relations and Membrane Stabilisation

The present study investigates the role of salicylic acid (SA) in inducing plant tolerance to salinity. The application of 0.1 mM SA to tomato [Lycopersicon esculentum Mill.] plants via root drenching provided protection against 150 mM or 200 mM NaCl stress. SA treated plants had greater survival and relative shoot growth rate compared to untreated plants when exposed to salt stress. At 200 mM salt, shoot growth rates were approximately 4 times higher in SA treated plants than untreated plants. Application of SA increased photosynthetic rates in salt stressed plants and may have contributed to the enhanced survival. Transpiration rates and stomatal conductance were also significantly higher in SA treated plants under saline stress conditions. SA application reduced electrolyte leakage by 44% in 150 mM NaCl and 32% in 200 mM NaCl, compared to untreated plants, indicating possible protection of integrity of the cellular membrane. Beneficial effects of SA in saline conditions include sustaining the photosynthetic/transpiration activity and consequently growth, and may have contributed to the reduction or total avoidance of necrosis. SA, when used in appropriate concentrations, alleviates salinity stress without compromising the plants ability for growth under a favourable environment.     

Effects of salt on growth,ion accumulation,photosynthesis and leaf anatomy of the mangrove,<Emphasis Type="Italic"> Bruguiera parviflora</Emphasis>

The effects of a range of salinity (0, 100, 200 and 400 mM NaCl) on growth, ion accumulation, photosynthesis and anatomical changes of leaves were studied in the mangrove, Bruguiera parviflora of the family Rhizophoraceae under hydroponically cultured conditions. The growth rates measured in terms of plant height, fresh and dry weight and leaf area were maximal in culture treated with 100 mM NaCl and decreased at higher concentrations. A significant increase of Na⁺ content of leaves from 46.01 mmol m^-2 in the absence of NaCl to 140.55 mmol m^-2 in plants treated with 400 mM NaCl was recorded. The corresponding Cl^- contents were 26.92 mmol m^-2 and 97.89 mmol m^-2. There was no significant alteration of the endogenous level of K⁺ and Fe²⁺ in leaves. A drop of Ca²⁺ and Mg²⁺ content of leaves upon salt accumulation suggests increasing membrane stability and decreased chlorophyll content respectively. Total chlorophyll content decreased from 83.44 g cm^-2 in untreated plants to 46.56 g cm^-2 in plants treated with 400 mM NaCl, suggesting that NaCl has a limiting effect on photochemistry that ultimately affects photosynthesis by inhibiting chlorophyll synthesis (ca. 50% loss in chlorophyll). Light-saturated rates of photosynthesis decreased by 22% in plants treated with 400 mM NaCl compared with untreated plants. Both mesophyll and stomatal conductance by CO₂ diffusion decreased linearly in leaves with increasing salt concentration. Stomatal and mesophyll conductance decreased by 49% and 52% respectively after 45 days in 400 mM NaCl compared with conductance in the absence of NaCl. Scanning electron microscope study revealed a decreased stomatal pore area (63%) in plants treated with 400 mM NaCl compared with untreated plants, which might be responsible for decreased stomatal conductance. Epidermal and mesophyll thickness and intercellular spaces decreased significantly in leaves after treatment with 400 mM NaCl compared with untreated leaves. These changes in mesophyll anatomy might have accounted for the decreased mesophyll conductance. We conclude that high salinity reduces photosynthesis in leaves of B. parviflora, primarily by reducing diffusion of CO₂ to the chloroplast, both by stomatal closure and by changes in mesophyll structure, which decreased the conductance to CO₂ within the leaf, as well as by affecting the photochemistry of the leaves.     

Analysis of fatty acid composition of PGPR <Emphasis Type="Italic">Klebsiella</Emphasis> sp. SBP-8 and its role in ameliorating salt stress in wheat

A halotolerant plant-growth-promoting rhizobacteria (PGPR) can ameliorate salt stress in associated plants by various mechanisms. Therefore, the present study aimed to characterize a PGPR Klebsiella sp. SBP-8 for its ability to tolerate salt stress and to study the mechanism of PGPR-mediated mitigation of salt stress in the wheat plant. The abiotic stressors result in multiple changes in the fatty acid composition of Klebsiella sp. SBP-8, helping the membrane to keep its integrity, fluidity, and function for its growth under salt (NaCl) stress conditions. The changes in fatty acid composition of test organism were analyzed by fatty acid methyl ester (FAME) analysis under varying saline conditions. The spectroscopy (GC-MS) profile of cell extract at different salt concentrations was comprised of hydrocarbons, and fatty alcohols with varying carbon chain length. Inoculation of Klebsiella sp. SBP-8 to wheat seedling showed increase in proline, total soluble sugar, and total protein content of treated plants. Bacterial inoculation also decreased the concentration of salinity-induced malondialdehyde (MDA) content. In addition, bacterial inoculation also increased the various antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) in treated plants. It is likely that bacterial inoculation alleviated the salt stress to wheat plant by co-ordination of antioxidative machinery, and improvement in osmolyte contents. Therefore, the present study suggests that bacterial-inoculated wheat plants were able to cope better with salt stress than uninoculated control, therefore it can serve as a promising bio-inoculant for enhancing the growth of wheat like cereal crops under saline stress.     

Mature‐stem expression of a silencing‐resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally‐controlled expression of a silencing‐resistant gene encoding a vacuole‐targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole‐cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high‐isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value‐added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing‐resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field‐grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale.     

An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens

This is the first successful report of the recovery of morphologically normal transgenic sugarcane plants from co-cultivation of calluses with Agrobacterium tumefaciens. Transformation frequencies (total of transgenic plants/number of cell clusters) were between 9.4 × 10–3 and 1.15 × 10–2. In our experiments, both LBA4404 (pTOK233) and EHA101 (pMTCA3IG), carrying a super-binary vector or supervirulent strain, respectively, were successful for sugarcane transformation. We found that three main factors: (1) the use of young regenerable calluses as target explants; (2) induction and/or improvement of the A. tumefaciens virulence system with sugarcane cell cultures and (3) pre-induction of organogenesis or somatic-embryogenesis-like sexual embryos, seem to be crucial in order to increase the cells competence for T-DNA transfer process. Patterns generated by Southern hybridization confirmed that T-DNAs were randomly integrated into sugarcane genome without th e persistence of A. tumefaciens in the transgenic plants     

Cell ultrastructure and fatty acid composition of lipids in vegetative organs of <Emphasis Type="Italic">Chenopodium album</Emphasis> L. under salt stress conditions

White goosefoot plants (Chenopodium album L. of the family Chenopodiaceae) grown at various NaCl concentrations (3–350 mM) in the nutrient solution were used to study the cell ultrastructure as well as the qualitative and quantitative composition of fatty acids in the lipids of vegetative organs. In addition, the biomass of Ch. album vegetative organs, the water content, and the concentrations of K⁺, Na⁺, and Cl^– were determined. The growth rates of plants raised at NaCl concentrations up to 200–250 mM were the same as for the control plants grown at 3 mM NaCl; the growth parameters remained rather high even at NaCl concentrations of 300–350 mM. The water content in Ch. album organs remained high at all NaCl concentrations tested. Analysis of the ionic status of Ch. album revealed a comparatively high K⁺ content in plant organs. At low NaCl concentrations in the nutrient solution, K⁺ ions were the dominant contributors to the osmolarity (the total concentration of osmotically active substances) and, consequently, to the lowered cell water potential in leaves and roots. As the concentration of NaCl was increased, the plant organs accumulated larger amounts of Na⁺ and Cl^–, and the contribution of these ion species to osmolarity became increasingly noticeable. At 300–350 mM NaCl the contribution of Na⁺ and Cl^– to osmolarity was comparable to that of K⁺. An electron microscopy study of Ch. album cells revealed that, apart from the usual response to salinity manifested in typical ultrastructural changes of chloroplasts, mitochondria, and the cytosol, the salinity response comprised the enhanced formation of endocytic structures and exosomes and stimulation of autophagy. It is supposed that activation of these processes is related to the removal from the cytoplasm of toxic substances and the cell structures impaired by salt stress conditions. The qualitative and quantitative composition of fatty acids in the lipids of Ch. album organs was hardly affected by NaCl level. These findings are consistent with the high salt tolerance of Ch. album, manifested specifically in retention of growth functions under wide-range variations of NaCl concentration in the nutrient solution and in maintenance of K⁺, Na⁺, and Cl^– content in organs at a constant level characteristic of untreated plants.     

<i>Azospirillum brasilense</i> and <i>Saccharomyces cerevisiae</i> as Alternative for Decrease the Effect of Salinity Stress in Tomato (<i>Lycopersicon esculentum</i>) Growth

The salinity stress is one of the most relevant abiotic stresses that affects the agricultural production. The present study was performed to study the improvement of the salt tolerance of tomato plants which is known for their susceptibility to salt stress. The present study aimed to assess to what extent strain Azospirillum brasilense (N040) and Saccharomyces cerevisiae improve the salt tolerance to tomato plants treated with different salt concentration. The inoculant strain A. brasilense (N040) was previously adapted to survive up to 7% NaCl in the basal media. A greenhouse experiment was conducted to evaluate the effect of this inoculation on growth parameter such as: plant height, root length, fresh and dry weight, fruits fresh weight, chlorophyll content, proline and total soluble sugar in tomato plants under salt stress condition. The results revealed that co-inoculation of Azospirillum brasilense (N040) and Saccharomyces cerevisiae significantly increased the level of proline (8.63 mg/g FW) and total soluble sugar (120 mg/g FW) of leaves under salinity condition comparing to non-inoculated plants (2.3 mg/g FW and 70 mg/g FW, respectively). Plants co-inoculated with adapted strain of A. brasilense and S. cerevisiae showed the highest significant (p < 0.01) increase in fruit yield (1166.6 g/plant), plant high (115 cm) and roots length (52.6) compared whit un-inoculated control plants (42 g/pant, 43.3 cm and 29.6 cm, respectively). In contrast, Na⁺ ion content was significantly decreased in the leaves of salt stressed plants treated with the A. brasilense (N040) and S. cerevisiae. Finally, the results showed that dual benefits provided by both A. brasilense (N040) and S. cerevisiae can provide a major way to improve tomato yields in saline soils.     

Sugarcane white leaf phytoplasma in tissue culture: long-term maintenance,transmission, and oxytetracycline remission

Sugarcane white leaf (SCWL)-diseased sugarcane plants collected from Udornthani Province, in north-eastern Thailand, were the source for tissue culture experiments. Explants from axillary buds, meristem tips, and leaves grew optimally in Murashige-Skoog medium containing 0.5 mg/l -naphthaleneacetic acid, 0.5 mg/l 6-benzylaminopurine, and 15% coconut water. Callus development and shoot/root proliferation were more rapid in cultures from diseased than from healthy plants. Disease symptoms continued for 6 years after culture initiation, and SCWL phytoplasma persisted, as confirmed by polymerase chain reaction using both 16S rDNA and 16S-23S rDNA primers. Phytoplasmas in the cultured plantlets were transmissible by grafting to sugarcane and periwinkle, and by feeding of the leafhopper vector Matsumuratettix hiroglyphicus to sugarcane. Although 50% of the plantlets were killed by oxytetracycline at 500 mg/ml, 70–100% of plantlets grown with 200–500 mg/ml oxytetracycline showed symptom remission through 5–8 subcultures. Typical phytoplasma-like bodies, visible by electron microscopy in sieve tubes of untreated diseased plantlets, were absent in antibiotic-treated plantlets. Thus, tissue culture provides a convenient and reliable in vivo system for investigation of SCWL phytoplasma. A preliminary report of this study was presented at the Eighth International Congress of Plant Pathology, Christchurch, New Zealand, 2–7 February 2003     

Kinetin improves photosynthetic and antioxidant responses of <Emphasis Type="Italic">Nigella sativa</Emphasis> to counteract salt stress

The present study involves analysis of growth, photosynthesis, oxidant (H₂O₂) accumulation, and antioxidant enzyme activities in Nigella sativa L. as affected by foliar kinetin (KIN) application during salt stress. The test plants were treated with 75 or 150 mM NaCl since germination and sprayed with either water or 10 μM KIN in 25 days after emergence. Salt stress, especially at the higher NaCl concentration, was found to induce a substantial decrease in leaf relative water content and subsequently in leaf area and stomatal conductance; chlorophyll content and δ-aminolevulinic acid dehydratase (ALA-D) activity were also affected, resulting in the lower net photosynthetic rate and dry matter production. Moreover, H₂O₂ content increased in the salt-treated plants, concomitant with an increase in superoxide dismutase and peroxidase activities; however, the activity of catalase declined. Meanwhile KIN was found to reduce appreciably the adverse effects of salinity, besides favorably modulating antioxidant enzyme activities and alleviating oxidative stress in the test plants, to result in a higher yield as compared to the untreated stressed plants. Overall, the results indicate an optimization of antioxidant defense mechanisms and physiological processes by KIN and a significant role of exogenous phytohormones in conferring salt tolerance.     

Changes in water relations,photosynthetic activity and proline accumulation in one-year-old olive trees (<Emphasis Type="Italic">Olea europaea</Emphasis> L. cv. Chemlali) in response to NaCl salinity

The comparative responses of young olive trees (Olea europaea L. cv “Chemlali”) to different NaCl salinity levels were investigated over 11 months. One-year-old own rooted plants were grown in 10-L pots containing sand and perlite mixture (1:3 v/v). Trees were subjected to three irrigation treatments: CP (control plants that were irrigated with fresh water); SS1 (salt stressed plants irrigated with water containing 100 mM NaCl) and SS2 plants (salt stressed plants irrigated with water containing 200 mM NaCl). Shoot elongation rate, relative water content, leaf water potential and net carbon dioxide exchange rates decreased significantly with increased NaCl salinity level. Under stressed conditions, the increase of Na⁺ and Cl⁻ ions in both leaves and roots was accompanied with that of proline and soluble sugars. The above results show that the accumulation of proline and sugars under stressed conditions could play a role in salt tolerance. The absence of toxicity symptoms under both stress treatments and the superior photosynthetic activity recorded in SS1-treated plants suggest that cv Chemlali is better able to acclimatize to 100 mM NaCl than at 200 mM NaCl. Our findings indicate that saline water containing 100 mM NaCl, the most available water in arid region in Tunisia, can be recommended for the irrigation of cv Chemlali in the arid south of Tunisia.     

Pseudomonas spp. Mediate defense response in sugarcane through differential exudation of root phenolics

Pseudomonas spp., a ubiquitous biocontrol agent, protects the plants from phytopathogens by suppressing them directly by reinforcing the plant’s intrinsic defense mechanism. Root exudated phenolics play an important role in establishing the rhizobacteria population and cross the host boundaries in beneficial plant–microbe interaction. In this study, Pseudomonas spp. HU-8 & HU-9 antagonized the sugarcane red rot pathogen (C. falcatum) and showed a positive chemotactic response against different concentrations (10–30 µM) of synthetic phenolic acids like p-coumaric, vanillic, and 3,4 di-hydroxybenzoic acid. In a pot experiment, they effectively colonized the sugarcane rhizosphere and mediated defense response in sugarcane plants challenged with red rot pathogen C. falcatum by regulating the exudation of root phenolics under hydroponic conditions. They significantly induced the activity of the antioxidant enzymes CAT (1.24–1.64 fold), PO (0.78–1.61 fold), PAL (0.77–0.97 fold), and PPO (3.67–3.73 fold) over untreated plants in sugarcane. They also induced the total phenolic contents (TPC) in sugarcane in the presence (6.56–10.29 mg/g GAE) and absence (2.89–4.16 mg/g GAE) of the pathogen quantified through the Folin-Ciocalteu (FC) method. However, their effect was lower than that of the pathogen (4.34–8 mg/g GAE). The Pseudomonas spp. significantly colonized the sugarcane rhizosphere by maintaining a cell population of (1.0E + 07–1.3E + 08 CFU/mL). A significant positive Pearson’s correlation was observed between the root exudated total phenolic contents, antioxidant enzymatic activities, and rhizospheric population of inoculated bacteria. The 16S rRNA and rpoD gene analysis showed sequence conservation (C: 0.707), average number of nucleotide differences (k: 199.816), nucleotide diversity, (Pi): 0.09819), average number of informative nucleotide sites per site (Psi: 0.01275), GC content (0.57), and polymorphic sites (n = 656). These diverse Pseudomonas spp. could be an ideal bio-inoculants for a broad range of hosts especially graminaceous crops.     

Stress-induced Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene confers tolerance to salt stress in transgenic sugarcane

High salinity interferes in sugarcane growth and development, affecting not only crop yield but also reducing sucrose concentration in culms. Sugarcane plants submitted to salt stress can accumulate compatible solutes, such as proline, which may counteract the effects of salt accumulation in the vacuole and scavenge reactive oxygen species. The objective of this study was to evaluate the response to salt stress of sugarcane plants transformed with the Vigna aconitifolia P5CS gene, which encodes ?1-pyrroline-5-carboxylate synthetase, under the control of a stress-induced promoter AIPC (ABA-inducible promoter complex). For this, 4-month-old clonally multiplied sugarcane plants from two transformation events were irrigated every 2 days with 1/10 Hoagland’s solution supplemented with 100, 150 and 200 NaCl, progressively, during 28 days. Transgenic lines showed increased transgene expression in 3.75-fold when compared with the control plants after 9 days of irrigation with saline water, which can explain the higher proline concentration found in these plants. At the end of the experiment (day 28), the transgenic lines accumulated up to 25 % higher amounts of proline when compared with non-transformed control plants. Stress response in transgenic plants was also accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation in leaves, lower Na⁺ accumulation in leaves and maintenance of photochemical efficiency of PSII. Thus, proline contributed to the protection of the photosynthetic apparatus and the prevention of oxidative damage in transgenic sugarcane under salt stress.     

Systems biology and metabolic modelling unveils limitations to polyhydroxybutyrate accumulation in sugarcane leaves; lessons for C4 engineering

In planta production of the bioplastic polyhydroxybutyrate (PHB) is one important way in which plant biotechnology can address environmental problems and emerging issues related to peak oil. However, high biomass C₄ plants such as maize, switch grass and sugarcane develop adverse phenotypes including stunting, chlorosis and reduced biomass as PHB levels in leaves increase. In this study, we explore limitations to PHB accumulation in sugarcane chloroplasts using a systems biology approach, coupled with a metabolic model of C₄ photosynthesis. Decreased assimilation was evident in high PHB‐producing sugarcane plants, which also showed a dramatic decrease in sucrose and starch content of leaves. A subtle decrease in the C/N ratio was found which was not associated with a decrease in total protein content. An increase in amino acids used for nitrogen recapture was also observed. Based on the accumulation of substrates of ATP‐dependent reactions, we hypothesized ATP starvation in bundle sheath chloroplasts. This was supported by mRNA differential expression patterns. The disruption in ATP supply in bundle sheath cells appears to be linked to the physical presence of the PHB polymer which may disrupt photosynthesis by scattering photosynthetically active radiation and/or physically disrupting thylakoid membranes.