Identification of Sorghum (Sorghum bicolor (L.) Moench) Genotypes with Potential for Hydric and Heat Stress Tolerance in Northeastern Mexico.

Sorghum (Sorghum bicolor (L.) Moench) is cultivated in regions with frequent drought periods and high temperatures, conditions that have intensified in the last decades. One of the most important photosynthetic components, sensible to hydric stress, is maximum quantum yield for photosystem II (PSII, or Fv/Fm). The objective of the present study was to identify sorghum genotypes with tolerance to hydric and heat stress. The treatments were hydric status (hydric stress or non-hydric stress (irrigation)), the plant's developmental stages (pre or post-anthesis), and six genotypes. The response variables were Fv/Fm; photosynthetic rate (PN); stomatal conductance (gs); transpiration rate (E); relative water content (RWC); damage to cell membrane (DCM) at temperatures of 40 and 45 °C; and agronomic variables. The experiment was conducted in pots in open sky in Marín, N.L., in the dry and hot northeast Mexico. The treatment design was a split-split plot design, with three factors. Hydric stress diminished the functioning of the photosynthetic apparatus by 63%, due to damage caused to PSII. Pre-anthesis was the most vulnerable stage to hydric stress as it decreased the weight of grains per panicle (85%), number of grains per panicle (69%), and weight of 100 grains (46%). Genotypes LER 1 and LER 2 were identified as tolerant to hydric stress, as they had lower damage to PSII; LER 1 and LEB 2 for their superior RWC; and LER 1 as a thermo tolerant genotype, due to its lower DCM at 45 °C. It was concluded that LER 1 could have the potential for both hydric and heat stress tolerance in the arid northeast Mexico.

Plastid transformation: advances and challenges for its implementation in agricultural crops

Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology's high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.

Characterization of Trehalose-6-phosphate Synthase and Trehalose-6-phosphate Phosphatase Genes and Analysis of its Differential Expression in Maize (Zea mays) Seedlings under Drought Stress.

Maize is the most important crop around the world and it is highly sensitive to abiotic stress caused by drought, excessive salinity, and extreme temperature. In plants, trehalose has been widely studied for its role in plant adaptation to different abiotic stresses such as drought, high and low temperature, and osmotic stress. Thus, the aim of this work was to clone and characterize at molecular level the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) genes from maize and to evaluate its differential expression in maize seedlings under drought stress. To carry out this, resistant and susceptible maize lines were subjected to drought stress during 72 h. Two full-length cDNAs of TPS and one of TPP were cloned and sequenced. Then, TPS and TPP amino acid sequences were aligned with their homologs from different species, showing highly conserved domains and the same catalytic sites. Relative expression of both genes was evaluated by RT-qPCR at different time points. The expression pattern showed significant induction after 0.5 h in resistant lines and after two to four hours in susceptible plants, showing their participation in drought stress response.

Biofunctional properties of bioactive peptide fractions from protein isolates of moringa seed (Moringa oleifera).

Moringa oleifera (Moringaceae) is a specie of significant importance because of its multiple nutraceutical properties, that has led to increase in its consumption. The seeds contain a high percentage of protein (37.48%). However, little is known about the bioactive properties of these proteins and peptides, especially those generated by enzymatic hydrolysis. The objective of this study was to evaluate the biofunctional properties of total hydrolysates (TH) and peptide fractions from protein isolates of moringa seeds. Isoelectric protein isolates were prepared and TH were obtained by digestion with trypsin, chymotrypsin and pepsin-trypsin for 2.5 and 5 h. TH were fractioned by ultrafiltration (UF) with a 10 kDa membrane to generate the peptide fractions. In all treatments, the antioxidant capacity was significantly higher in peptide fractions > 10 kDa with 5 h of hydrolysis. The results showed that the fraction > 10 kDa of pepsin-trypsin digested for 5 h presented a better Angiotensin Converting Enzyme inhibition (ACE-I) activity with an IC50 of 0.224 µg/µl. Also, antidiabetic activity was enhanced in pepsin-trypsin treatment with 5 h of hydrolysis showing an IC50 of 0.123 µg/µl. Finally, this study showed that hydrolysates of moringa seed proteins had excellent in vitro nutraceutical potential.

Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.

Visual spectinomycin resistance (aadA(au)) gene for facile identification of transplastomic sectors in tobacco leaves.

Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar (bar(au)) gene that confers a golden leaf phenotype and a spectinomycin resistance (aadA) gene that is necessary for the introduction of the bar(au) gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadA(au) transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aadA(au) genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome.

Study of plastid genome stability in tobacco reveals that the loss of marker genes is more likely by gene conversion than by recombination between 34-bp loxP repeats.

In transformed tobacco (Nicotiana tabacum) plastids, we flank the marker genes with recombinase target sites to facilitate their posttransformation excision. The P1 phage loxP sites are identical 34-bp direct repeats, whereas the phiC31 phage attB/attP sites are 54- and 215-bp sequences with partial homology within the 54-bp region. Deletions in the plastid genome are known to occur by recombination between directly repeated sequences. Our objective was to test whether or not the marker genes may be lost by homologous recombination via the directly repeated target sites in the absence of site-specific recombinases. The sequence between the target sites was the bar(au) gene that causes a golden-yellow (aurea) leaf color, so that the loss of the bar(au) gene can be readily detected by the appearance of green sectors. We report here that transplastomes carrying the bar(au) gene marker between recombinase target sites are relatively stable because no green sectors were detected in approximately 36,000 seedlings (Nt-pSS33 lines) carrying attB/attP-flanked bar(au) gene and in approximately 38,000 seedlings (Nt-pSS42 lines) carrying loxP-flanked bar(au) gene. Exceptions were six uniformly green plants in the Nt-pSS42-7A progeny. Sequencing the region of plastid DNA that may derive from the vector indicated that the bar(au) gene in the six green plants was lost by gene conversion using wild-type plastid DNA as template rather than by deletion via directly repeated loxP sites. Thus, the recombinase target sites incorporated in the plastid genome for marker gene excisions are too short to mediate the loss of marker genes by homologous recombination at a measurable frequency.

Next generation synthetic vectors for transformation of the plastid genome of higher plants.

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites.