Sugarcane (
spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression ...analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR.
In this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and RefFinder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples,
and
genes were more suitable as reference genes, whereas
was the best reference one in shoot roots.
Reference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions.
Drought is the most significant environmental stress for agricultural production worldwide, and tremendous efforts have been made to improve crop yield under the increasing water scarcity. ...Transcription factors are major players in the regulation of water stress-related genes in plants. Recently, different MYB transcription factors were characterized for their involvement in drought response. A sugarcane R2R3-MYB gene (ScMYBAS1) and its four alternative forms of transcript (ScMYAS1-2, ScMYBAS1-3, ScMYBAS1-4 and ScMYBAS1-5) were identified in this study. The subcellular localization, in Nicotiniana benthamiana, of the TFs fused in frame with GFP revealed that ScMYBAS1-2-GFP and ScMYBAS1-3-GFP were observed in the nucleus. The overexpression of ScMYBAS1-2 and ScMYBAS1-3 spliced transcripts in rice promoted change in plant growth under both well-watered and drought conditions. The ScMYBAS1-2 and ScMYBAS1-3 transgenic lines revealed a higher relative water content (RWC) compared to the wild type before maximum stress under drought conditions. The ScMYBAS1-2 transgenic lines showed a reduction in biomass (total dry weight). Conversely, ScMYBAS1-3 showed an increased biomass (total dry weight) relative to the wild-type. The overexpression of ScMYBAS1-3 in rice transgenic lines showed involvement with drought tolerance and biomass and, for this reason, was considered a good target for plant transformation, particularly for use in developing genotypes with drought tolerance and biomass accumulation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it ...has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on
tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars.
The selection of reference genes in sugarcane under Sugarcane mosaic virus (SCMV) infection has not been reported and is indispensable to get reliable reverse transcription quantitative PCR (RT-qPCR) ...results for validation of transcriptome analysis. In this regard, seven potential reference genes were tested by RT-qPCR and ranked according to their stability using BestKeeper, NormFinder and GeNorm algorithms, and RefFinder WEB-based software in an experiment performed with samples from two sugarcane cultivars contrasting for SCMV resistance, when mechanically inoculated with a severe SCMV strain and using mock inoculated plant controls.
The genes Uridylate kinase (UK) and Ubiquitin-conjugating enzyme 18 (UBC18) were the most stable according to GeNorm algorithm and the Pearson correlation coefficients with the BestKeeper index. On the other hand, ribosomal protein L35-4 (RPL1), Actin (ACT) and Ubiquitin1 (UBQ1) were the least stable genes for all algorithms tested.
The agricultural sugarcane residues, bagasse and straws, can be used for second-generation ethanol (2GE) production by the cellulose conversion into glucose (saccharification). However, the lignin ...content negatively impacts the saccharification process. This polymer is mainly composed of guaiacyl (G), hydroxyphenyl (H), and syringyl (S) units, the latter formed in the ferulate 5-hydroxylase (F5H) branch of the lignin biosynthesis pathway. We have generated transgenic lines overexpressing
under the control of the
(cinnamate 4-hydroxylase) rice promoter, which led to a significant increase of up to 160% in the S/G ratio and 63% in the saccharification efficiency in leaves. Nevertheless, the content of lignin was unchanged in this organ. In culms, neither the S/G ratio nor sucrose accumulation was altered, suggesting that
overexpression would not affect first-generation ethanol production. Interestingly, the bagasse showed a significantly higher fiber content. Our results indicate that the tissue-specific manipulation of the biosynthetic branch leading to S unit formation is industrially advantageous and has established a foundation for further studies aiming at refining lignin modifications. Thus, the
overexpression in sugarcane emerges as an efficient strategy to improve 2GE production from straw.
Dirigent (DIR) proteins, encoded by
DIR
genes, are referred to as “dirigent” because they direct the outcome of the coupling of the monolignol coniferyl alcohol into (+) or (−) pinoresinol, the first ...intermediates in the enantiocomplementary pathways for lignan biosynthesis. DIR domain-containing or DIR-like proteins are, thus, termed for not having a clear characterization. A transcriptome- and genome-wide survey of DIR domain-containing proteins in sugarcane was carried out, in addition to phylogenetic, physicochemical and transcriptional analyses. A total of 120 non-redundant sequences containing the DIR domain were identified and classified into 64 groups according to phylogenetic and sequence alignment analyses. In silico analysis of transcript abundance showed that these sequences are expressed at low levels in leaves and genes in the same phylogenetic clade have similar expression patterns. Expression analysis of
ShDIR1
-
like
transcripts in the culm internodes of sugarcane demonstrates their abundance in mature internodes, their induction by nitrogen fertilization and their predominant expression in cells that have a lignified secondary cell wall, such as vascular bundles of young internodes and parenchymal cells of the pith of mature internodes. Due to the lack of information about the functional role of DIR in plants, a possible relationship is discussed between the
ShDIR1
-
like
transcriptional profile and cell wall development in parenchyma cells of sugarcane culm, which typically accumulates large amounts of sucrose. The number of genes encoding the DIR domain-containing proteins in sugarcane is intriguing and is an indication per se that these proteins may have an important metabolic role and thus deserve to be better studied.
Sugarcane yellow leaf disease (YLD) caused by sugarcane yellow leaf virus (ScYLV) is a major threat for the sugarcane industry worldwide, and the aphid Melanaphis sacchari is its main vector. ...Breeding programs in Brazil have provided cultivars with intermediate resistance to ScYLV, whereas the incidence of ScYLV has been underestimated partly due to the complexity of YLD symptom expression and identification. Here, we evaluated YLD symptoms in a field assay using eight sugarcane genotypes comprising six well-established commercial high-sucrose cultivars, one biomass yield cultivar, and a susceptible reference under greenhouse conditions, along with estimation of virus titer through RT-qPCR from leaf samples. Additionally, a free-choice bioassay was used to determine the number of aphids feeding on the SCYLV-infected cultivars. Most of the cultivars showed some degree of resistance to YLD, while also revealing positive RT-qPCR results for ScYLV and virus titers with non-significant correlation with YLD severity. The cultivars IACSP01-5503 and IACBIO-266 were similar in terms of aphid preference and ScYLV resistance traits, whereas the least preferred cultivar by M. sacchari, IACSP96-7569, showed intermediate symptoms but similar virus titer to the susceptible reference, SP71-6163. We conclude that current genetic resistance incorporated into sugarcane commercial cultivars does not effectively prevent the spread of ScYLV by its main aphid vector.
A sugarcane gene encoding a
,
, was induced under drought stress. To elucidate its biological function, we integrated a
-overexpression construction into the rice Nipponbare genome via
-mediated ...transformation. Two transgenic lines with a single copy gene in T
were selected and evaluated in both the T
and T
generations. Transgenic lines had drastically improved survival rate under water deficit conditions, at rates close to 100%, while WT did not survive. Besides, transgenic lines had improved biomass production and higher tillering under water deficit conditions compared with WT plants. Reduced pectin and hemicellulose contents were observed in transgenic lines compared with wild-type plants under both well-watered and water deficit conditions, whereas cellulose content was unchanged in line #17 and reduced in line #29 under conditions of low water availability. Changes in lignin content under water deficit were only observed in line #17. However, improvements in saccharification were found in both transgenic lines along with changes in the expression of
and
secondary cell wall biosynthesis genes.
-overexpression up-regulated the expression of the
,
,
, and
genes in rice stems under well-watered conditions. Taken together, our data suggest that
has the potential for improving drought tolerance, plant biomass accumulation, and saccharification efficiency.
One of the major challenges involved in biofuel production from plant biomass is increasing the saccharification efficiency impaired by cell wall recalcitrance. The sugarcane transcription factor ...ShSHN1 was overexpressed in rice to evaluate its impacts on cell wall composition and potential for the development of refined lignocellulosic feedstocks for biofuel manufacturing. Plant phenotyping included biomass and cell wall component determination. Additionally, gene expression of biosynthetic cell wall polymers and wax/cutin-related genes were evaluated. ShSHN1 ectopic overexpression promoted an increase in biomass (91–340%), pectin (26–209%), cellulose content (10–22%) and saccharification efficiency (5–53%) in rice transgenic plants, as well as a reduction in the lignin content (17–35%) and an altered lignin composition. Such an increase in pectin is a new finding and opens up new research pathways for cell wall manipulation of energy crops. Furthermore, ShSHN1 overexpression led to changes in the expression patterns of all genes analyzed, suggesting a possible relation between ShSHN1 and the regulatory mechanisms involved in secondary plant cell wall formation. Saccharification improvement and biomass increase made the perfect combination for second generation ethanol production. This synergistic gain is discussed based on structural polysaccharides and lignin modification. Phenotypes and gene expression analysis provide insights into possible SHINE mechanisms of action and biological role in cell wall component biosynthesis. These results demonstrate the great potential of ShSHN1 to improve grass feedstocks as a source of energy.
•Overexpression of ShSHN1 coordinates the biosynthesis of cell wall components in rice.•Rice ShSHN1 transgenic lines exhibited increase in pectin and decrease in lignin.•Genetic manipulation promoted biomass gain and improved saccharification efficiency.
ABSTRACT Purpose To evaluate the biomechanical properties of a novel total hip replacement femoral stem. Methods Eight pairs of femurs from dog cadavers were used. The femurs were separated into ...different groups. A novel femoral stem with a convex proximal portion (Stem B) was biomechanically evaluated and compared to awell-known veterinary collared stem (Stem A). Femoral stems were inserted into the contralateral femurs from the same dog, forming 16 constructs. A flexo-compression load was applied on the axial axis of each sample. Maximum strength, deflection, stiffness, and energy absorption were analysed. Results Group B constructs showed significantly higher values (p ? 0.05) for the variables, except stiffness. The mean maximum strength was 1,347 ± 357 N for Group A and 1,805 ± 123 N for Group B (p ? 0.0069). The mean deflection was5.54 ± 2.63 mm for Group A and 10.03 ± 3.99 mm for Group B (p ? 0.0056). For the energy variable, the force was 6,203 ± 3,488 N/mm for Group A and 12,885 ± 5,056 N/mm for Group B (p ? 0.0054). Stem B had greater maximum strength, deflection, and energy. Conclusions The new stem was effective in neutralizing the impact of axial flexion-compression stresses during biomechanical tests in cadaveric models.
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