Transgenic sugarcane with higher levels of BRK1 showed improved drought tolerance.

KEY MESSAGE: Transgenic sugarcane overexpressing BRK1 showed improved tolerance to drought stress through modulation of actin polymerization and formation of interlocking marginal lobes in epidermal leaf cells, a typical feature associated with BRK1 expression under drought stress. BRICK1 (BRK1) genes promote leaf epidermal cell morphogenesis and division in plants that involves local actin polymerization. Although the changes in actin filament organization during drought have been reported, the role of BRK in stress tolerance remains unknown. In our previous work, the drought-tolerant Erianthus arundinaceus exhibited high levels of the BRK gene expression under drought stress. Therefore, in the present study, the drought-responsive gene, BRK1 from Saccharum spontaneum, was transformed into sugarcane to test if it conferred drought tolerance in the commercial sugarcane cultivar Co 86032. The transgenic lines were subjected to drought stress, and analyzed using physiological parameters for drought stress. The drought-induced BRK1-overexpressing lines of sugarcane exhibited significantly higher transgene expression compared with the wild-type control and also showed improved physiological parameters. In addition, the formation of interlocking marginal lobes in the epidermal leaf cells, a typical feature associated with BRK1 expression, was observed in all transgenic BRK1 lines during drought stress. This is the first report to suggest that BRK1 plays a role in sugarcane acclimation to drought stress and may prove to be a potential candidate in genetic engineering of plants for enhanced biomass production under drought stress conditions.

Gene Co-Expression Analysis Reveals Transcriptome Divergence between Wild and Cultivated Sugarcane under Drought Stress.

Drought is the main abiotic stress that constrains sugarcane growth and production. To understand the molecular mechanisms that govern drought stress, we performed a comprehensive comparative analysis of physiological changes and transcriptome dynamics related to drought stress of highly drought-resistant (ROC22, cultivated genotype) and weakly drought-resistant (Badila, wild genotype) sugarcane, in a time-course experiment (0 h, 4 h, 8 h, 16 h and 32 h). Physiological examination reviewed that ROC22, which shows superior drought tolerance relative to Badila, has high performance photosynthesis and better anti-oxidation defenses under drought conditions. The time series dataset enabled the identification of important hubs and connections of gene expression networks. We identified 36,956 differentially expressed genes (DEGs) in response to drought stress. Of these, 15,871 DEGs were shared by the two genotypes, and 16,662 and 4423 DEGs were unique to ROC22 and Badila, respectively. Abscisic acid (ABA)-activated signaling pathway, response to water deprivation, response to salt stress and photosynthesis-related processes showed significant enrichment in the two genotypes under drought stress. At 4 h of drought stress, ROC22 had earlier stress signal transduction and specific up-regulation of the processes response to ABA, L-proline biosynthesis and MAPK signaling pathway-plant than Badila. WGCNA analysis used to compile a gene regulatory network for ROC22 and Badila leaves exposed to drought stress revealed important candidate genes, including several classical transcription factors: NAC87, JAMYB, bHLH84, NAC21/22, HOX24 and MYB102, which are related to some antioxidants and trehalose, and other genes. These results provide new insights and resources for future research and cultivation of drought-tolerant sugarcane varieties.

A PIP-mediated osmotic stress signaling cascade plays a positive role in the salt tolerance of sugarcane.

BACKGROUND: Plasma membrane intrinsic proteins (PIPs) are plant channel proteins involved in water deficit and salinity tolerance. PIPs play a major role in plant cell water balance and responses to salt stress. Although sugarcane is prone to high salt stress, there is no report on PIPs in sugarcane. RESULTS: In the present study, eight PIP family genes, termed ScPIP1-1, ScPIP1-2, ScPIP1-3, ScPIP1-4, ScPIP2-1, ScPIP2-2, ScPIP2-4 and ScPIP2-5, were obtained based on the sugarcane transcriptome database. Then, ScPIP2-1 in sugarcane was cloned and characterized. Confocal microscopy observation indicated that ScPIP2-1 was located in the plasma membrane and cytoplasm. A yeast two-hybridization experiment revealed that ScPIP2-1 does not have transcriptional activity. Real time quantitative PCR (RT-qPCR) analysis showed that ScPIP2-1 was mainly expressed in the leaf, root and bud, and its expression levels in both below- and aboveground tissues of ROC22 were up-regulated by abscisic acid (ABA), polyethylene glycol (PEG) 6000 and sodium chloride (NaCl) stresses. The chlorophyll content and ion leakage measurement suggested that ScPIP2-1 played a significant role in salt stress resistance in Nicotiana benthamiana through the transient expression test. Overexpression of ScPIP2-1 in Arabidopsis thaliana proved that this gene enhanced the salt tolerance of transgenic plants at the phenotypic (healthier state, more stable relative water content and longer root length), physiologic (more stable ion leakage, lower malondialdehyde content, higher proline content and superoxide dismutase activity) and molecular levels (higher expression levels of AtKIN2, AtP5CS1, AtP5CS2, AtDREB2, AtRD29A, AtNHX1, AtSOS1 and AtHKT1 genes and a lower expression level of the AtTRX5 gene). CONCLUSIONS: This study revealed that the ScPIP2-1-mediated osmotic stress signaling cascade played a positive role in plant response to salt stress.

Proposal of an index of stability for evaluating plant drought memory: A case study in sugarcane.

Stability is a key trait for plant growth and development in a changing environment, involving homeostasis and resilience. While homeostasis refers to the maintenance of the internal structural and functional plant integrity, resilience is associated with the plant ability in returning to the initial conditions after a given disturbance. Such concepts are especially relevant for perennial and semi-perennial plants facing seasonal and frequent stress conditions. Although plant memory is closely associated with plant performance under recurrent stresses, to date, there is no study evaluating how stress memory is linked to stability under varying water conditions. Herein, we evaluated the association between drought stability and memory in sugarcane plants and proposed a new stability index to evaluate plant memory. Two datasets were analyzed, the first deals with leaf gas exchange and photochemistry of sugarcane plants grown in nutrient solution and exposed to one, two or three water deficit cycles. The second takes into account the physiological performance of sugarcane propagules obtained by vegetative propagation from plants that faced drought. To quantify sugarcane stability, we estimated the drought impact, the disturbance rate (DR), drought perturbation, and recovery rate (RR) for plants from both datasets. Drought memory - given by improved performance after previous stress events or when origin material faced drought - was detected in both datasets, changing either DR or RR. Based on these indices, we proposed the overall stability (OSt), defined as the ratio between RR and DR. While DR is associated to plant homeostasis, RR is a measure of plant resilience. Sugarcane plants exposed to three cycles of water deficit or those propagules originated from stressed plants presented the highest OSt values, showing higher RR and/or lower DR when compared to well-watered plants or to propagules from well-watered plants. Regarding the physiological traits evaluated, leaf CO2 assimilation and stomatal conductance were the most consistent variables in revealing drought stability and memory. Concluding, OSt revealed consistently patterns of response associated with plant memory, besides quantifying plant stability under stressful conditions.

Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.).

Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane.

Assessing single effects of sugarcane pesticides fipronil and 2,4-D on plants and soil organisms.

The continuous growth in global population since the beginning of the 20th century result in the necessity of food and energy provision favoring the intensive use of agricultural products such as pesticides. Although pesticides are important to prevent losses in the conventional chemically based agriculture, they frequently present side effects, which goes against agricultural production. The use of pesticides cause direct and indirect effects to soil organisms unbalancing essential soil processes (e.g. primary production, organic matter decomposition, nutrient cycling). Under tropical conditions, very little is known regarding the effects of pesticides to terrestrial organisms. Hence, the aim of the present study was to assess the ecotoxicological effects of the herbicide DMA® 806 BR (active ingredient: 2,4-D) and the insecticide Regent® 800 WG (active ingredient: fipronil), on terrestrial plant species (the dicot Raphanus sativus var. acanthioformis and the monocot Allium cepa), and soil invertebrates (the collembolan Folsomia candida and the enchytraeid Enchytraeus crypticus), using natural (NS) and artificial soils (TAS). For both pesticides, negative effects on non-target species were observed at concentrations lower than the doses recommended to prevent pests in sugarcane fields. For both soils, the dicot species was the most affected by the herbicide (R. sativus > A. cepa > F. candida > E. crypticus) and the collembolan species was the most affected by the insecticide (F. candida > E. crypticus = R. sativus = A. cepa). Although the order of the organisms' sensitivity for both pesticides was the same in both soils, results showed that the extent of the effects was soil dependent. Considering the ecologically relevant concentrations tested, and their severe effects to non-target organisms, it may be concluded that the use of fipronil and 2,4-D under recommended conditions may pose a risk to the terrestrial environment.

hRNAi-mediated knock-down of Sphenophorus levis V-ATPase E in transgenic sugarcane (Saccharum spp interspecific hybrid) affects the insect growth and survival.

KEY MESSAGE: Transgenic sugarcane expressing V-ATPase subunit E dsRNA affects growth and survival of Sphenophorus levis. Plants being sessile organisms are constantly confronted with several biotic and abiotic stresses. Sugarcane (Saccharum spp) is a major tropical crop widely cultivated for its sugar and other by-products. In Brazil, sugarcane plantations account for significant production losses due to Sphenophorus levis (sugarcane weevil) infestations. With the existing control measures being less effective, there arises a necessity for advanced strategies. Our bioassay injection experiments with V-ATPase E dsRNA in S. levis larvae showed significant mortality and reduction in transcription levels. Furthermore, we down-regulated the V-ATPase E gene of S. levis in transgenic sugarcane using an RNAi approach. The resultant RNAi transgenic lines exhibited reduction in larval growth and survival, without compromising plant performance under controlled environment. Our results illustrate that RNAi-mediated down-regulation of key genes is a promising approach in imparting resistance to sugarcane weevil.

Effect of sugar feedback regulation on major genes and proteins of photosynthesis in sugarcane leaves.

Productivity of sugarcane (Saccharum spp.) relies upon sucrose production in leaves and movement to sinks. The feedback regulatory effect of sugar upon photosynthesis balances this process involving Phosphoenolpyruvate carboxylase (PEPCase) and Rubisco where greater understanding in this area may allow manipulation to achieve higher yields. Accumulation of sucrose in leaves and decreased photosynthesis are early symptoms of the condition called yellow canopy syndrome (YCS) in sugarcane, which presents as a system in which to study sucrose feedback regulation. This work investigates changes in gene expression and protein abundance which coincide with the sugar accumulation in the leaves of YCS symptomatic sugarcane. During the early-stage of sugar accumulation, the levels of the Photosystem II core protein D1, and PsbQ of the oxygen-evolving complex decreased significantly. Transcript levels of these proteins also decreased, suggesting both nuclear and chloroplast gene expression were affected early in sugar build-up of YCS development. Transcript level of primary carbon fixation reactions enzyme NADP malate dehydrogenase was especially downregulated. However, PEPCase, decarboxylation and re-fixation (Rubisco) enzymes were not negatively regulated at the transcript or protein abundance level. Phosphoenolpyruvate carboxykinase was upregulated in both gene expression and protein abundance. The Calvin cycle in the bundle sheath was sensitive through the CP12 protein. Two isoforms of CP12 were found, one of which showed downregulation which coincided with a decrease in CP12 protein. This suggests transcript and protein decrease of PEPCase and Rubisco may be secondary regulation points of the sugar feedback regulation process upon photosynthesis in sugarcane leaves.

Amino Acid and Carbohydrate Metabolism Are Coordinated to Maintain Energetic Balance during Drought in Sugarcane.

The ability to expand crop plantations without irrigation is a major goal to increase agriculture sustainability. To achieve this end, we need to understand the mechanisms that govern plant growth responses under drought conditions. In this study, we combined physiological, transcriptomic, and genomic data to provide a comprehensive picture of drought and recovery responses in the leaves and roots of sugarcane. Transcriptomic profiling using oligoarrays and RNA-seq identified 2898 (out of 21,902) and 46,062 (out of 373,869) transcripts as differentially expressed, respectively. Co-expression analysis revealed modules enriched in photosynthesis, small molecule metabolism, alpha-amino acid metabolism, trehalose biosynthesis, serine family amino acid metabolism, and carbohydrate transport. Together, our findings reveal that carbohydrate metabolism is coordinated with the degradation of amino acids to provide carbon skeletons to the tricarboxylic acid cycle. This coordination may help to maintain energetic balance during drought stress adaptation, facilitating recovery after the stress is alleviated. Our results shed light on candidate regulatory elements and pave the way to biotechnology strategies towards the development of drought-tolerant sugarcane plants.

Gene expression profiling of reactive oxygen species (ROS) and antioxidant defense system following Sugarcane mosaic virus (SCMV) infection.

BACKGROUND: Viruses are infectious pathogens, and plant virus epidemics can have devastating consequences to crop yield and quality. Sugarcane mosaic virus (SCMV, belonging to family Potyviridae) is one of the leading pathogens that affect the sugarcane crop every year. To combat the pathogens' attack, plants generate reactive oxygen species (ROS) as the first line of defense whose sophisticated balance is achieved through well-organized antioxidant scavenging pathways. RESULTS: In this study, we investigated the changes occurring at the transcriptomic level of ROS associated and ROS detoxification pathways of SCMV resistant (B-48) and susceptible (Badila) sugarcane genotypes, using Saccharum spontaneum L. genome assembly as a reference genome. Transcriptomic data highlighted the significant upregulation of ROS producing genes such as NADH oxidase, malate dehydrogenase and flavin-binding monooxygenase, in Badila genotype after SCMV pathogenicity. To scavenge the ROS, the Badila genotype illustrated a substantial enhancement of antioxidants i.e. glutathione s-transferase (GST), as compared to its resistant counterpart. GST is supposed to be a key indicator of pathogen attacks on the plant. A remarkably lower GST expression in B-48, as compared to Badila, indicated the development of resistance in this genotype. Additionally, we characterized the critical transcription factors (TFs) involved in endowing resistance to B-48. Among these, WRKY, AP2, NAC, bZIP, and bHLH showed enhanced expression in the B-48 genotype. Our results also confirmed the linkage of transcriptomic data with the enzymatic and qPCR data. The estimation of enzymatic activities for superoxide dismutase, catalase, ascorbate peroxidase, and phenylalanine ammonia-lyase supported the transcriptomic data and evinced higher resistance in B-48 genotype. CONCLUSION: The current study supported the efficiency of the B-48 genotype under SCMV infection. Moreover, comparative transcriptomic data has been presented to highlight the role of significant transcription factors conferring resistance to this genotype. This study provides an in-depth knowledge of the expression profiling of defense mechanisms in sugarcane.

ScAOC1, an allene oxide cyclase gene, confers defense response to biotic and abiotic stresses in sugarcane.

KEY MESSAGE: An allene oxide cyclase gene which is involved in defense against biotic and abiotic stresses was cloned and characterized in sugarcane. Allene oxide cyclase (AOC), a key enzyme in jasmonate acid (JA) biosynthesis, affects the stereoisomerism and biological activity of JA molecules, and plays an important role in plant stress resistance. In this study, four SsAOC alleles (SsAOC1-SsAOC4), which shared similar gene structure and were located on Chr1A, Chr1B, Chr1C, and Chr1D, respectively, were mined from sugarcane wild species Saccharum spontaneum, and a homologous gene ScAOC1 (GenBank Accession Number: MK674849) was cloned from sugarcane hybrid variety Yacheng05-179 inoculated with Sporisorium scitamineum for 48 h. ScAOC1 and SsAOC1-SsAOC4 were alkaline, unstable, hydrophilic, and non-secretory proteins, which possess the same set of conserved motifs and were clustered into one group in the phylogenetic analysis. ScAOC1 was expressed in all sugarcane tissues, but with different levels. After infection by S. scitamineum, the transcripts of ScAOC1 were increased significantly both in the smut-susceptible (ROC22) and resistant (Yacheng05-179) varieties, but its transcripts were more accumulated and lasted for a longer period in the smut-resistant variety than in the smut-susceptible one. ScAOC1 was down-regulated under MeJA and NaCl treatments, but up-regulated under SA, ABA, PEG, and cold stresses. Transiently overexpressing ScAOC1 gene into Nicotiana benthamiana leaves regulated the responses of N. benthamiana to two pathogens Ralstonia solanacearum and Fusarium solani var. coeruleum. Furthermore, prokaryotic expression analysis showed overexpression of ScAOC1 in Escherichia coli BL21 could enhance its tolerance to NaCl, mannitol, and cold stimuli. These results indicated that ScAOC1 may play an active role in response to biotic and abiotic stresses in sugarcane.

Ectopic expression of DJ-1/PfpI domain containing Erianthus arundinaceus Glyoxalase III (EaGly III) enhances drought tolerance in sugarcane.

KEY MESSAGE: Sugarcane transgenic overexpressing EaGly III from Erianthus arundinaceus showed enhanced water deficit stress tolerance. Methylglyoxal (MG), an α-ketoaldehyde formed from either glycolysis or TCA cycle, is capable of causing total cellular damage via the generation of reactive oxygen species (ROS), advanced glycation end products (AGEs) and nucleic acid degradation. Glyoxalase pathway is a ubiquitous pathway known for detoxification of MG, involving key enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Recently, a novel and an additional enzyme in glyoxalase pathway, viz., glyoxalase III (Gly III), has been discovered which possesses DJ-1/PfpI domain recognized for detoxifying MG in a single step process without requirement of any coenzyme. In the present study, a Gly III gene isolated from Erianthus arundinaceus, a wild relative of sugarcane, overexpressed in commercially cultivated sugarcane hybrid Co 86032 was assessed for drought tolerance. Morphometric observations revealed that transgenic sugarcane overexpressing EaGly III acquired drought tolerance trait. Oxidative damage caused by triggering generation of ROS has been determined to be low in transgenic plants as compared to wild type (WT). Transgenics resulted in higher relative water content, chlorophyll content, gas exchange parameters, photosynthetic efficiency, proline content and soluble sugars upon water deficit stress. In addition, higher and stable level of superoxide dismutase and peroxidase activities were observed along with minimal lipid peroxidation during drought stress signifying the tolerance mechanism exhibited by transgenic events. There was no significant structural change observed in the root anatomy of transgenic plants. Altogether, EaGly III gene could be considered as a potential candidate for conferring water deficit stress tolerance for sugarcane and other agricultural crops.

Seeds attached to refrigerated shipping containers represent a substantial risk of nonnative plant species introduction and establishment.

The initial processes for successful biological invasions are transport, introduction, and establishment. These can be directly influenced or completely avoided through activities that reduce the number and frequency of entering nonnative propagules. Economic and environmental benefits through preventative monitoring programs at early stages of invasion far outweigh the long-term costs associated with mitigating ecological and economic impacts once nonnative species establish and spread. In this study, we identified 30 taxa of hitchhiking plant propagules on the air-intake grilles of refrigerated shipping containers arriving into a United States seaport from a port on the Pacific coast of South America. The four monocotyledonous taxa with the highest number of seeds collected were analyzed; we estimated propagule pressure, germination, and survivorship of these taxa, and we used the estimates to determine likelihood of establishment. At the levels of propagule pressure estimated here, non-zero germination and survival rates resulted in high establishment probabilities even when escape rates from shipping containers were modelled to be exceedingly low. Our results suggest high invasion risk for nonnative taxa including Saccharum spontaneum L., a listed Federal Noxious Weed. Currently, not all shipping containers arriving at USA ports are thoroughly inspected due to limited personnel and funding for biological invasion prevention. Our results indicate that there is a significant risk from only a few propagules escaping into the environment from this source, and we propose possible solutions for reducing this risk.

Drought-induced alterations in photosynthetic, ultrastructural and biochemical traits of contrasting sugarcane genotypes.

Drought is an important factor which limits growth of sugarcane. To elucidate the physiological and biochemical mechanisms of tolerance, a pot experiment was conducted at Sugarcane Research Institute, Kaiyuan, China. Two genotypes (Yuetang 93-159-sensitive and Yunzhe 05-51-tolerant), were subjected to three treatments; 70±5% (control), 50±5% (moderate drought) and 30±5% (severe drought) of soil field capacity. The results demonstrated that drought induced considerable decline in morpho-physiological, biochemical and anatomical parameters of both genotypes, with more pronounced detrimental effects on Yuetang 93-159 than on Yunzhe 05-51. Yunzhe 05-51 exhibited more tolerance by showing higher dry biomass, photosynthesis and antioxidant enzyme activities. Compared with Yuetang 93-159, Yunzhe 05-51 exhibited higher soluble sugar, soluble protein and proline contents under stress. Yunzhe 05-51 illustrated comparatively well-composed chloroplast structure under drought stress. It is concluded that the tolerance of Yunzhe 05-51 was attributed to improved antioxidant activities, osmolyte accumulation and enhanced photosynthesis. These findings may provide valuable information for future studies on molecular mechanism of tolerance.

Physiological and molecular mechanisms governing the effect of virus-free chewing cane seedlings on yield and quality.

The effects of increasing yield and quality of virus-free chewing cane seedlings and their physiological and molecular basis were studied in this study. Results showed that compared with infected seedlings (the control), the yield of chewing cane stems grown from virus-free seedlings increased by 21.81-29.93%, stem length increased by 28.66-34.49 cm, internode length increased by 2.16-2.68 cm, the single stem weight increased by 20.10-27.68%, the reducing sugar increased by 0.91-1.15% (absolute value), and sucrose increased by - 0.06-1.33% (absolute value). The decrease in sucrose content did not reach significant level, but all other parameters were reached significant level. The chlorophyll content, photosynthetic parameters such as stomatal conductance (Gs), net photosynthetic rate (Pn) and transpiration rate (Tr), the activity of photosynthetic key enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC), and gene (pepc, rbcS, and rbcL) expression levels were all greater in virus-free seedlings than infected seedlings. The content of superoxide anion (O2-) and malondialdehyde (MDA) in virus-free seedlings was lower than infected seedlings at the main growth stage. With increased development, the activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were gradually higher in virus-free seedlings than infected seedlings. Our results indicate that virus-free seedlings may improve photosynthesis efficiency and promote photosynthesis by increasing chlorophyll content, photosynthetic key enzyme activity, and the gene expression levels in leaves. By increasing the activity of antioxidant enzymes, reducing the degree of membrane lipid peroxidation, and improving the stress resistance of chewing cane, the virus-free chewing cane seedlings increased yield and quality. Our findings provide a scientific and theoretical basis for the promotion and application of virus-free chewing cane seedlings.

Allele specific expression of Dof genes responding to hormones and abiotic stresses in sugarcane.

Dof transcription factors plant-specific and associates with growth and development in plants. We conducted comprehensive and systematic analyses of Dof transcription factors in sugarcane, and identified 29 SsDof transcription factors in sugarcane genome. Those SsDof genes were divided into five groups, with similar gene structures and conserved motifs within the same groups. Segmental duplications are predominant in the evolution of Dof in sugarcane. Cis-element analysis suggested that the functions of SsDofs were involved in growth and development, hormones and abiotic stresses responses in sugarcane. Expression patterns indicated that SsDof7, SsDof23 and SsDof24 had a comparatively high expression in all detected tissues, indicating these genes are crucial in sugarcane growth and development. Moreover, we examined the transcription levels of SsDofs under four plant hormone treatments, SsDof7-3 and SsDof7-4 were down-regulated after ABA treatment, while SsDof7-1 and SsDof7-2 were induced after the same treatment, indicating different alleles may play different roles in response to plant hormones. We also analyzed SsDofs' expression profiling under four abiotic stresses, SsDof5 and SsDof28 significantly responded to these four stresses, indicating they are associate with abiotic stresses responses. Collectively, our results yielded allele specific expression of Dof genes responding to hormones and abiotic stresses in sugarcane, and their cis-elements could be crucial for sugarcane improvement.

Genomic and metabolic differences between Pseudomonas putida populations inhabiting sugarcane rhizosphere or bulk soil.

Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant growth promotion and bioremediation. Here we compared a set of 19 P. putida isolates obtained from sugarcane rhizosphere or bulk soil using a population genomics approach aiming to assess genomic and metabolic differences between populations from these habitats. Phylogenomics placed rhizosphere versus bulk soil strains in separate clades clustering with different type strains of the P. putida group. Multivariate analyses indicated that the rhizosphere and bulk soil isolates form distinct populations. Comparative genomics identified several genetic functions (GO-terms) significantly different between populations, including some exclusively present in the rhizosphere or bulk soil strains, such as D-galactonic acid catabolism and cellulose biosynthesis, respectively. The metabolic profiles of rhizosphere and bulk soil populations analyzed by Biolog Ecoplates also differ significantly, most notably by the higher oxidation of D-galactonic/D-galacturonic acid by the rhizosphere population. Accordingly, D-galactonate catabolism operon (dgo) was present in all rhizosphere isolates and absent in the bulk soil population. This study showed that sugarcane rhizosphere and bulk soil harbor different populations of P. putida and identified genes and functions potentially associated with their soil niches.

Foliar application of gamma radiation processed chitosan triggered distinctive biological responses in sugarcane under water deficit stress conditions.

Chitosan, being one of the most promising biological macromolecules, has an immense scope in agriculture to boost crop growth and defense responses. In this study, chitosan was exposed to gamma rays in order to obtain a low molecular weight derivative. Viscometric characterization showed a sharp decrease in molecular weight and FTIR based analysis confirmed retention of structural integrity of the polymer upon gamma irradiation. Assessments of various physiological and biochemical attributes were carried out on sugarcane plantlets that were subjected to progressive water deficit stress. The irradiated chitosan was found to differentially ameliorate water deficit stress tolerance against that of normal chitosan through positive modulation of various gas exchange parameters alongside significant improvement in relative tissue water content, SOD activity, soluble sugars and adenine energetics. Furthermore, application of irradiated chitosan significantly reduced cell membrane damage, lipid peroxidation, H2O2 and free-proline accumulations. This is the first report on the use of gamma irradiated chitosan to alleviate water deficit stress tolerance in sugarcane. Overall comparative assessments showed that differential plant responses were triggered upon foliar application of normal and gamma irradiated chitosan in sugarcane plants grown under water deficit stress conditions.

Ethylene-mediated improvement in sucrose accumulation in ripening sugarcane involves increased sink strength.

BACKGROUND: Sugarcane is a major crop producing about 80% of sugar globally. Increasing sugar content is a top priority for sugarcane breeding programs worldwide, however, the progress is extremely slow. Owing to its commercial significance, the physiology of sucrose accumulation has been studied extensively but it did not lead to any significant practical outcomes. Recent molecular studies are beginning to recognize genes and gene networks associated with this phenomenon. To further advance our molecular understanding of sucrose accumulation, we altered sucrose content of sugarcane genotypes with inherently large variation for sucrose accumulation using a sugarcane ripener, ethylene, and studied their transcriptomes to identify genes associated with the phenomenon. RESULTS: Sucrose content variation in the experimental genotypes was substantial, with the top-performing clone producing almost 60% more sucrose than the poorest performer. Ethylene treatment increased stem sucrose content but that occurred only in low-sugar genotype. Transcriptomic analyses have identified about 160,000 unigenes of which 86,000 annotated genes were classified into functional groups associated with carbohydrate metabolism, signaling, localization, transport, hydrolysis, growth, catalytic activity, membrane and storage, suggesting the structural and functional specification, including sucrose accumulation, occurring in maturing internodes. About 25,000 genes were differentially expressed between all genotypes and treatments combined. Genotype had a dominant effect on differential gene expression than ethylene treatment. Sucrose and starch metabolism genes were more responsive to ethylene treatment in low-sugar genotype. Ethylene caused differential gene expression of many stress-related transcription factors, carbohydrate metabolism, hormone metabolism and epigenetic modification. Ethylene-induced expression of ethylene-responsive transcription factors, cytosolic acid- and cell wall-bound invertases, and ATPase was more pronounced in low- than in high-sugar genotype, suggesting an ethylene-stimulated sink activity and consequent increased sucrose accumulation in low-sugar genotype. CONCLUSION: Ethylene-induced sucrose accumulation is more pronounced in low-sugar sugarcane genotype, and this is possibly achieved by the preferential activation of genes such as invertases that increase sink strength in the stem. The relatively high enrichment of differentially expressed genes associated with hormone metabolism and signaling and stress suggests a strong hormonal regulation of source-sink activity, growth and sucrose accumulation in sugarcane.

Diel oscillations in cell wall components and soluble sugars as a response to short-day in sugarcane (Saccharum sp.).

BACKGROUND: Sugarcane is a tropical crop that can accumulate high concentration of sucrose in the stem as a storage carbohydrate. For that reason, sugarcane accounts for approximately 75% of all the sugar produced in the world and has become the main sugar source to produce first-generation bioethanol in Brazil. Daily rhythms cause plants to adapt and coordinate their metabolism to achieve maximum photosynthesis and carbohydrate production throughout the day. Circadian rhythms arise from the interaction of an internal oscillator and external stimuli, whereas diel rhythms occur in response to a light-dark cycle. Diel signalling contributes to synchronizing circadian rhythms to photoperiods, and levels of carbohydrates oscillate in a diel fashion. Under regular photoperiods, they are synthesized during the daytime and consumed throughout the night as an energy reserve. However, short days can induce higher rates of synthesis during daytime and lower rates of consumption in the dark. Cell wall carbohydrates are also diurnally regulated, and it has been shown that celluloses, hemicelluloses and pectin are deposited/degraded at different times of the day. To assess the diel carbohydrate profile in young sugarcane plants, we measured soluble sugars and cell wall components along a time course in plants subjected either to a regular day or short day. RESULTS: Short-day influenced sucrose synthesis and cell wall components. In short-day a 44% increase in sucrose concentration was detected in the dark, but was stable during the day. Cellulose, hemicellulose and pectin also fluctuate within a 24 h interval when subjected to a short day. A 38% increase in leaf sheath cellulose was observed from the middle of the day to the first hour of the night. Leaf sheath pectin and hemicellulose also increased from the day to the night, while it decreased in leaves. CONCLUSIONS: The presented data show diurnal patterns of soluble sugar metabolism together with temporal regulation of cell wall metabolism for a short day, suggesting that diel signalling has a role in how sugarcane manages sugar accumulation and partitioning. Understanding cell wall synthesis/degradation dynamics may help to improve the yield of sugarcane.