A flow cytometry-based analysis to establish a cell cycle synchronization protocol for Saccharum spp.

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 µM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 µM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.

Biochemical and kinetic characterization of the recombinant GH28 Stereum purpureum endopolygalacturonase and its biotechnological application.

Endopolygalacturonase (EndoPG) from Stereum purpureum was expressed as a soluble protein in Pichia pastoris GS115, where after 3 days methanol induction the enzyme activity in the culture supernatant was 40 U mL-1. After purification by IMAC, SDS-PAGE analysis showed that the molecular weight of EndoPG was approximately 60.0 kDa. The carbohydrate content of the recombinant enzyme was estimated to be 67.0% (w/w). The optimum temperature and pH of catalysis were 60-70 °C and pH of 4.5, respectively. The enzyme was highly stable over the pH range 6.0-8.0 and retained approximately 60% of its initial activity after incubation at 70 °C for 30 min. The enzyme showed a specific activity of 5040.0 ±â€¯217 U mg-1 and hydrolyzed citrus pectin with Vmax and a KM of 4947.10 ±â€¯393.63 U mg-1 and 2.45 ±â€¯0.23 mg mL-1, respectively, and showed a catalytic efficiency of 2052.90 ±â€¯193.54 mL mg-1 s-1. EndoPG alone reduced the viscosity of papaya juice by 20% after 30 min, and increased its transmittance about 50% with a concomitant reduction of the color by about 55% after 5 h of enzymatic treatment. For apple juice, the relative reduction of viscosity was 30% after 5 h, and the reduction of the color was 30% with a 12% increase in transmittance. Supplementation of a commercial enzymatic cocktail for lignocellulose saccharification with EndoPG increased total reducing sugar release by 8.6 ±â€¯2.1% against sugar cane bagasse, indicating improved access of the cellulolytic enzymes to the biomass polysaccharides.

Cell Wall Proteome of Sugarcane Young and Mature Leaves and Stems.

By characterizing the cell wall proteomes of different sugarcane organs (leaves and stems) at two developmental stages (young vs mature/apical vs basal), it is possible to address unique characteristics in each of them. Four-month-old leaves show a higher proportion of oxido-reductases and proteins related to lipid metabolism (LM), besides a lower proportion of proteins acting on polysaccharides, in comparison to 4-month-old internodes. It is possible to note that sugarcane leaves and young stems have the highest LM rate than all species, which is assumed to be linked to cuticle formation. The data generated enrich the number of cell wall proteins (CWPs) identified in sugarcane, reaching 277. To our knowledge, sugarcane has now the second higher coverage of monocot CWP in plants.

Cell wall metabolism and hexose allocation contribute to biomass accumulation in high yielding extreme segregants of a Saccharum interspecific F2 population.

BACKGROUND: Sugarcane is an emerging dual-purpose biofuel crop for energy and sugar production, owing to its rapid growth rate, high sucrose storage in the stems, and high lignocellulosic yield. It has the highest biomass production reaching 1.9 billion tonnes in 2014 worldwide. RESULTS: To improve sugarcane biomass accumulation, we developed an interspecific cross between Saccharum officinarum 'LA Purple' and Saccharum robustum 'MOL5829'. Selected F1 individuals were self-pollinated to generate a transgressive F2 population with a wide range of biomass yield. Leaf and stem internodes of fourteen high biomass and eight low biomass F2 extreme segregants were used for RNA-seq to decipher the molecular mechanism of rapid plant growth and dry weight accumulation. Gene Ontology terms involved in cell wall metabolism and carbohydrate catabolism were enriched among 3274 differentially expressed genes between high and low biomass groups. Up-regulation of cellulose metabolism, pectin degradation and lignin biosynthesis genes were observed in the high biomass group, in conjunction with higher transcript levels of callose metabolic genes and the cell wall loosening enzyme expansin. Furthermore, UDP-glucose biosynthesis and sucrose conversion genes were differentially expressed between the two groups. A positive correlation between stem glucose, but not sucrose, levels and dry weight was detected. CONCLUSIONS: We thus postulated that the high biomass sugarcane plants rapidly convert sucrose to UDP-glucose, which is the building block of cell wall polymers and callose, in order to maintain the rapid plant growth. The gene interaction of cell wall metabolism, hexose allocation and cell division contributes to biomass yield.

Influence of ozonolysis time during sugarcane pretreatment: Effects on the fiber and enzymatic saccharification.

Modifications in sugarcane bagasse (SCB) from ozonolysis (O) NaOH (B) and ultrasound (U) (OBU) treatment for cellulosic ethanol production by enzymatic hydrolysis, were evaluated when increasing the exposure time of SCB to ozone. The lignin, cellulose, and hemicellulose after treatment were quantified: lignin removal and a consequent increase in cellulose content were shown using an infrared spectroscopic technique (ATR-FTIR) and chemical characterization. X-ray diffraction analysis (XRD) proved that OBU treatment does not affect the crystalline cellulose portion and electron microscopy techniques established that the fiber region most affected by the OBU treatment was the secondary cell wall, where the greatest lignin content is located. For OBU-60 treatment the lignin content was reduced and consequently there was a significant increase in cellulose content. After enzymatic hydrolysis, this pretreated SCB released 418mgglucose/g, corresponding to six times more than untreated SCB and a yield of 93% of the cellulose available.

Molecular Cloning, Characterization, and Expression Analysis of Lignin Genes from Sugarcane Genotypes Varying in Lignin Content.

Sugarcane (Saccharum spp.) is one of the highest biomass-producing plant and the best lignocellulosic feedstock for ethanol production. To achieve more efficient conversion of biomass to ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Therefore, with this objective, here, we report a systematic study on lignin content, deposition, identification, and cloning of genes involved in lignin biosynthesis and their differential expression in five sugarcane clones, EC11003, EC11010, IK 76-91, IK 76-99, and Co 86032. Lignin content among the clones varied from 26.87 to 23.19 % with the highest in the clone EC11010 and the lowest in high sugar Co86032. Lignin deposition studied through phloroglucinol staining of the cell walls implied that the sclerenchyma cells of the energy canes (EC11010 and EC11003) have more lignin deposition followed by the Erianthus (IK 76-91 and IK 76-99) clones whereas Co86032 has the minimum amount of lignin deposition. We cloned partial coding regions of important genes of lignification COMT (650 bp), CCR (332 bp), and PAL (650 bp) from Erianthus, wild relative of sugarcane followed by the expression analysis through real-time PCR. Differential expression analysis showed high level of expression for the three genes in the energy cane EC11010.

Effect of pretreatment and enzymatic hydrolysis on the physical-chemical composition and morphologic structure of sugarcane bagasse and sugarcane straw.

The present work aimed to study the effect of the pretreatment of sugarcane bagasse and straw with microwave irradiation in aqueous and acid glycerol solutions on their chemical composition, fiber structure and the efficiency of subsequent enzymatic hydrolysis. Thermogravimetric analysis showed that the pretreatment acted mainly on the lignin and hemicellulose fractions of the bagasse, whereas, in the straw, lesser structural and chemical changes were observed. The images from transmission electron microscopy (TEM) revealed that treating bagasse and straw with acid glycerol solution loosened the cell walls and there was a breakdown in the pit membrane. The treated material was submitted to hydrolysis for 72h and higher yields of reducing sugars were observed compared to the untreated material (250.9mg/g from straw and 197.4mg/g from bagasse). TEM images after hydrolysis confirmed the possible points of access of the enzymes to the secondary cell wall region of the pretreated biomass.

Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production.

The acetyl bromide method is faster, simpler and presents best recovery of lignin in different herbaceous tissues than Klason and thioglycolic acid methods.

We compared the amount of lignin as determined by the three most traditional methods for lignin measurement in three tissues (sugarcane bagasse, soybean roots and soybean seed coat) contrasting for lignin amount and composition. Although all methods presented high reproducibility, major inconsistencies among them were found. The amount of lignin determined by thioglycolic acid method was severely lower than that provided by the other methods (up to 95%) in all tissues analyzed. Klason method was quite similar to acetyl bromide in tissues containing higher amounts of lignin, but presented lower recovery of lignin in the less lignified tissue. To investigate the causes of the inconsistencies observed, we determined the monomer composition of all plant materials, but found no correlation. We found that the low recovery of lignin presented by the thioglycolic acid method were due losses of lignin in the residues disposed throughout the procedures. The production of furfurals by acetyl bromide method does not explain the differences observed. The acetyl bromide method is the simplest and fastest among the methods evaluated presenting similar or best recovery of lignin in all the tissues assessed.

Cell wall proteomics of sugarcane cell suspension cultures.

The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl2 and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second-generation ethanol production.

GISH: resolving interspecific and intergeneric hybrids.

Genomic in situ hybridization (GISH) is an invaluable cytogenetic technique which enables the visualization of whole genomes in hybrids and polyploidy taxa. Total genomic DNA from one or two different species/genome is used as a probe, labeled with a fluorochrome and directly detected on mitotic chromosomes from root-tip meristems. In sugarcane we were able to characterize interspecific hybrids of two closely related species as well as intergeneric hybrids of two closely related genera.

Non-invasive monitoring of sucrose mobilization from culm storage parenchyma by magnetic resonance spectroscopy.

Because sucrose stored in mature stalks (in excess of 40% of stalk dry weight) can be wholly mobilized to supply carbon for the growth of heterotrophic tissues, we propose that sucrose mobilization requires a net sink-to-source transition that acts in toto within sett internode storage parenchyma. Based on our data we propose that mobilization of sucrose from culm storage parenchyma requires minimal investment of metabolic resources, and that the mechanism of sucrose mobilization is metabolically neutral. By magnetic resonance spectroscopy and phloem-specific tracer dyes, strong evidence was found that sucrose is mobilized from sett storage parenchyma via phloem to the growing shoot tissue. An analysis of the enzyme activities involved in sucrose metabolism and glycolysis suggested that sucrose synthase activity is downregulated due to the effects of sucrose mobilization. Overall, metabolism in storage parenchyma shifts from futile cycling to a more quiescent state during sucrose mobilization.

Suspension cell culture as a tool for the characterization of class III peroxidases in sugarcane.

Secreted class III peroxidases (EC 1.11.1.7) are implicated in a broad range of physiological processes throughout the plant life cycle. However, the unambiguous determination of the precise biological role of an individual class III peroxidase isoenzyme is still a difficult task due to genetic redundancy and broad substrate specificity in vitro. In addition, many difficulties are encountered during extraction and analysis of cell wall proteins. Since class III peroxidases are also secreted into the apoplast, the use of suspension cell cultures can facilitate isolation and functional characterization of individual isoforms. Here, we report on the characterization of class III peroxidases secreted in the spent medium of sugarcane suspension cell cultures. After treatment with specific inducers of cell wall lignification, peroxidases were isolated and activities assayed with guaiacol, syringaldazine and coniferyl alcohol. Enzymatic activity was not significantly different after treatments, regardless of the substrate, with the exception of methyl-jasmonate treatment, which led to a decreased guaiacol peroxidase activity. Remarkably, peroxidases isolated from the medium were capable of oxidizing syringaldazine, an analog to sinapyl alcohol, suggesting that sugarcane cultures can produce peroxidases putatively correlated to lignification. A proteomic approach using activity staining of 2-DE gels revealed a complex isoperoxidase profile, composed predominantly of cationic isoforms. Individual spots were excised and analyzed by LC-ESI-Q-TOF and homology-based search against the Sugarcane EST Database resulted in the identification of several proteins. Spatio-temporal expression pattern of selected genes was determined for validation of identified class III peroxidases that were preferentially expressed during sugarcane stem development.

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.

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns.

BACKGROUND: Sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs) are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out. RESULTS: Sixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. FISH analysis resulted in the expected pattern of Gypsy/heterochromatin, Copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. Analysis of related ESTs and RT-PCR showed transcriptional variation between tissues and families. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements. CONCLUSIONS: Individual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNA's that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome.

Unprecedented enhancement of transient gene expression from minimal cassettes using a double terminator.

The potential of using vector-free minimal gene cassettes (MGCs) with a double terminator for the enhancement and stabilization of transgene expression was tested in sugarcane biolistic transformation. The MGC system used consisted of the enhanced yellow fluorescent protein (EYFP) reporter gene driven by the maize ubiquitin-1 (Ubi) promoter and a single or double terminator from nopaline synthase (Tnos) or/and Cauliflower mosaic virus 35S (35ST). Transient EYFP expression from Tnos or 35ST single terminator MGC was very low and unstable, typically peaking early (8-16 h) and diminishing rapidly (48-72 h) after bombardment. Addition of a ~260 bp vector sequence (VS) to the single MGC downstream of Tnos (Tnos + VS) or 35ST (35ST + VS) enhanced EYFP expression by 1.25- to 25-fold. However, a much more significant increase in EYFP expression was achieved when the VS in 35ST + VS was replaced by Tnos to generate a 35ST-Tnos double terminator MGC, reaching its maximum at 24 h post-bombardment. The enhanced EYFP expression from the double terminator MGC was maintained for a long period of time (168 h), resulting in an overall increase of 5- to 65-fold and 10- to 160-fold as compared to the 35ST and Tnos single terminator MGCs, respectively. The efficiency of the double terminator MGC in enhancing EYFP expression was also demonstrated in sorghum and tobacco, suggesting that the underlying mechanism is highly conserved among monocots and dicots. Our results also suggest the involvement of posttranscriptional gene silencing in the reduced and unstable transgene expression from single terminator MGCs in plants.

In-situ monitoring of chromium cytotoxicity in sugarcane.

The potential of in-situ monitoring of cytotoxic effects of chromium through root-tip assay was studied in a sugarcane cultivar CoLk 8102 (Saccharum spp. hybrid). Sugarcane setts supplied with graded concentrations of chromium (VI), exhibited a reduction of 85.92 and 95.10 % in mean root length at 40 and 80 ppm Cr dosages along with 61.25 and 82.50% reduction in mean root number/node respectively. Mitotic index of root tip cells of treated setts declined and the frequency of aberrant mitotic phases increased pari passu to the increasing chromium concentration. To compare and quantify the effect of graded chromium dosages on frequency of chromosome aberrations vis-à-vis inhibition of mitotic activity, a 'Decretion factor' (D.F.) has been used for the first time. The value of DF increased with the increase in the chromium dosages. The increase in chromosome aberration frequency was low at low chromium dosages (1 or 2 ppm), but the high Cr dosages (40 and 80 ppm), induced sharp reduction in mitotic efficiency of root system along with anomalies in the process of cell division and induced chromosome aberrations in sugarcane root meristem, which in turn affected the over all plant growth.

Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes.

BACKGROUND: Sugarcane (Saccharum spp.) has become an increasingly important crop for its leading role in biofuel production. The high sugar content species S. officinarum is an octoploid without known diploid or tetraploid progenitors. Commercial sugarcane cultivars are hybrids between S. officinarum and wild species S. spontaneum with ploidy at approximately 12x. The complex autopolyploid sugarcane genome has not been characterized at the DNA sequence level. RESULTS: The microsynteny between sugarcane and sorghum was assessed by comparing 454 pyrosequences of 20 sugarcane bacterial artificial chromosomes (BACs) with sorghum sequences. These 20 BACs were selected by hybridization of 1961 single copy sorghum overgo probes to the sugarcane BAC library with one sugarcane BAC corresponding to each of the 20 sorghum chromosome arms. The genic regions of the sugarcane BACs shared an average of 95.2% sequence identity with sorghum, and the sorghum genome was used as a template to order sequence contigs covering 78.2% of the 20 BAC sequences. About 53.1% of the sugarcane BAC sequences are aligned with sorghum sequence. The unaligned regions contain non-coding and repetitive sequences. Within the aligned sequences, 209 genes were annotated in sugarcane and 202 in sorghum. Seventeen genes appeared to be sugarcane-specific and all validated by sugarcane ESTs, while 12 appeared sorghum-specific but only one validated by sorghum ESTs. Twelve of the 17 sugarcane-specific genes have no match in the non-redundant protein database in GenBank, perhaps encoding proteins for sugarcane-specific processes. The sorghum orthologous regions appeared to have expanded relative to sugarcane, mostly by the increase of retrotransposons. CONCLUSIONS: The sugarcane and sorghum genomes are mostly collinear in the genic regions, and the sorghum genome can be used as a template for assembling much of the genic DNA of the autopolyploid sugarcane genome. The comparable gene density between sugarcane BACs and corresponding sorghum sequences defied the notion that polyploidy species might have faster pace of gene loss due to the redundancy of multiple alleles at each locus.

SacRALF1, a peptide signal from the grass sugarcane (Saccharum spp.), is potentially involved in the regulation of tissue expansion.

Rapid alkalinization factor (RALF) is part of a growing family of small peptides with hormone characteristics in plants. Initially isolated from leaves of tobacco plants, RALF peptides can be found throughout the plant kingdom and they are expressed ubiquitously in plants. We took advantage of the small gene family size of RALF genes in sugarcane and the ordered cellular growth of the grass sugarcane leaves to gain information about the function of RALF peptides in plants. Here we report the isolation of two RALF peptides from leaves of sugarcane plants using the alkalinization assay. SacRALF1 was the most abundant and, when added to culture media, inhibited growth of microcalli derived from cell suspension cultures at concentrations as low as 0.1 microM. Microcalli exposed to exogenous SacRALF1 for 5 days showed a reduced number of elongated cells. Only four copies of SacRALF genes were found in sugarcane plants. All four SacRALF genes are highly expressed in young and expanding leaves and show a low or undetectable level of expression in expanded leaves. In half-emerged leaf blades, SacRALF transcripts were found at high levels at the basal portion of the leaf and at low levels at the apical portion. Gene expression analyzes localize SacRALF genes in elongation zones of roots and leaves. Mature leaves, which are devoid of expanding cells, do not show considerable expression of SacRALF genes. Our findings are consistent with SacRALF genes playing a role in plant development potentially regulating tissue expansion.

Efficient targeting of polyhydroxybutyrate biosynthetic enzymes to plant peroxisomes requires more than three amino acids in the carboxyl-terminal signal.

Metabolic engineering of plant peroxisomes for biotechnological purposes typically requires efficient peroxisomal targeting of heterologous proteins. Type I peroxisomal targeting signals (PTS1) consist of three uncleaved amino acids (SKL or a conserved variant) at the carboxyl terminus and direct nuclear-encoded proteins into the peroxisomes of eukaryotic cells. PTS1 fusion with a heterologous protein results in peroxisomal targeting of that protein, but the minimal length of PTS1 required for efficient targeting in plants is vague. Here, we determine short effective PTS1 sequences derived from plant peroxisomal proteins to target four heterologous proteins, namely the green fluorescent protein (GFP) and the three enzymes required for polyhydroxybutyrate (PHB) production, PhaA, PhaB and PhaC, each fused to the C-terminus of GFP. Transient expression analysis in leaf cells of Saccharum sp. (sugarcane interspecific hybrids) indicated that a three amino acid (ARL) PTS1 effectively targeted only GFP and PhaB to peroxisomes. The same signal was not sufficient to target PhaA and only inefficiently targeted PhaC. An alternative, prototypic three amino acid (SKL) PTS1 was also insufficient to target PhaA and inefficient in targeting PhaC, whilst a six amino acid (RAVARL) PTS1 efficiently targeted both of these enzymes. This study highlights the need for more than a three amino acid PTS1 to target some heterologous proteins to plant peroxisomes.