Composition and Flowering Quality of Cacahuacintle Maize Populations from the High Valleys of Mexico.

Cacahuacintle is one of the maize types with great demand for pozole preparation; however, little is known about the variation in chemical composition and flowered grain quality among populations. Physicochemical characteristics, flowered grain quality, pasting properties, and starch microstructure were evaluated in 33 populations of Cacahuacintle maize collected in Valles Altos, Mexico. The seeds samples of corn were obtained in 2017 from local farmers in the states of Mexico, Puebla, and Tlaxcala. Results were analyzed under a completely randomized design, and the ANOVA, Tukey test, and principal components were obtained. The ANOVA showed significance (p ≤ 0.05) in 18 of the 22 variables evaluated. The TE-6, AM-7, and CA-6 populations were outstanding for the good quality of their protein, pasting viscosity, and flowered grain quality. Nine populations collected in Calimaya, estate of Mexico, and Serdan Valley, state of Puebla, presented excellent physical, pasting, and flowery grain characteristics, with reduced protein content and lysine and tryptophan values typical of maize with normal endosperm. The softness of the endosperm grain, starch microstructural, and pasting characteristics of Cacahuacintle maize populations have a fundamental role in reducing the time and increasing the flowered grain volume, properties that were different from those observed in the Chalqueño, included as dent maize check. Variations in grain quality among Cacahuacintle maize populations is an important genetic resource for the improvement of the nutritional and flowering quality of Cacahuacintle maize.

A B73×Palomero Toluqueño mapping population reveals local adaptation in Mexican highland maize.

Generations of farmer selection in the central Mexican highlands have produced unique maize varieties adapted to the challenges of the local environment. In addition to possessing great agronomic and cultural value, Mexican highland maize represents a good system for the study of local adaptation and acquisition of adaptive phenotypes under cultivation. In this study, we characterize a recombinant inbred line population derived from the B73 reference line and the Mexican highland maize variety Palomero Toluqueño. B73 and Palomero Toluqueño showed classic rank-changing differences in performance between lowland and highland field sites, indicative of local adaptation. Quantitative trait mapping identified genomic regions linked to effects on yield components that were conditionally expressed depending on the environment. For the principal genomic regions associated with ear weight and total kernel number, the Palomero Toluqueño allele conferred an advantage specifically in the highland site, consistent with local adaptation. We identified Palomero Toluqueño alleles associated with expression of characteristic highland traits, including reduced tassel branching, increased sheath pigmentation and the presence of sheath macrohairs. The oligogenic architecture of these three morphological traits supports their role in adaptation, suggesting they have arisen from consistent directional selection acting at distinct points across the genome. We discuss these results in the context of the origin of phenotypic novelty during selection, commenting on the role of de novo mutation and the acquisition of adaptive variation by gene flow from endemic wild relatives.

Phylogeny, Diversification Rate, and Divergence Time of Agave sensu lato (Asparagaceae), a Group of Recent Origin in the Process of Diversification.

Agave sensu lato is one of the most diverse and complex genera of Asparagaceae, with more than 250 species. The morphological, ecological, and evolutionary diversity of the group has complicated its taxonomical study. We conducted phylogenetic analyses of DNA sequence data to reconstruct the phylogenetic relationships of the Agave genus. We included 107 species of the Asparagaceae family from which 83 correspond to the Agave sensu lato clade (Agave sensu stricto + Polianthes + Manfreda and Prochnyanthes, which together represent 30% of the genus) and as outgroups the genera Dasylirion, Hesperoyucca, Chlorogalum, Camassia, Hesperaloe, Yucca, Beschorneria, and Furcraea, in order to estimate the age and propose the history of their diversification. Previous studies postulated the relevance of the Miocene in the speciation rates of the agaves, as well as the relevance of the type of inflorescence in its diversification. However, these assertions have not been well supported. The analysis of chloroplast regions resulted in low resolution, which could be the consequence of the few variable sites. On the other hand, the internal transcribed spacer (ITS) implemented in our analysis ensued in higher resolution and better support values. Our phylogenetic analyses recovered five groups; one is the Striatae group, which is the sister group to Agave sensu stricto clade. Within this clade, we found three main groups with high support; these groups are not related with previous morphological proposals. We also analyzed the dates of origin and diversification rates. A Bayesian analysis of macroevolutionary mixtures indicated two significant shifts; the first was identified at 6.18 Ma, where the speciation rate increased to 4.10 species/Mya, this shift occurred during the late Miocene period, characterized by the emergence of arid biomes in North America. The second was identified at a stem age of 2.68 Ma where the speciation rate increased to 6.04 species/Mya. Concerning the ancestral reconstruction state of the inflorescence type in the Agave sensu stricto clade, the spike inflorescence character was predominant in the early-diverging groups, whereas the late-diverging groups present panicle inflorescences as the predominant character and higher speciation rates.

Mayahuelin, a Type I Ribosome Inactivating Protein: Characterization, Evolution, and Utilization in Phylogenetic Analyses of Agave.

Agaves resist extreme heat and drought. In A. tequilana var. azul, the central spike of the rosette -containing the shoot apical meristem and folded leaves in early stages of development- is remarkably heat tolerant. We found that the most abundant protein in this organ is a 27 kDa protein. This protein was named mayahuelin to honor Mayáhuel, the agave goddess in the Aztec pantheon. LC-MS/MS analyses identified mayahuelin as a type I RIP (Ribosome Inactivating Protein). In addition to the spike, mayahuelin was expressed in the peduncle and in seeds, whereas in mature leaves, anthers, filaments, pistils, and tepals was absent. Anti-mayahuelin antibody raised against the A. tequilana var. azul protein revealed strong signals in spike leaves of A. angustifolia, A. bracteosa, A. rhodacantha, and A. vilmoriniana, and moderate signals in A. isthmensis, A. kerchovei, A. striata ssp. falcata, and A. titanota, indicating conservation at the protein level throughout the Agave genus. As in charybdin, a type I RIP characterized in Drimia maritima, mayahuelin from A. tequilana var. azul contains a natural aa substitution (Y76D) in one out of four aa comprising the active site. The RIP gene family in A. tequilana var. azul consists of at least 12 genes and Mayahuelin is the only member encoding active site substitutions. Unlike canonical plant RIPs, expression of Mayahuelin gene in S. cerevisiae did not compromise growth. The inhibitory activity of the purified protein on a wheat germ in vitro translation system was moderate. Mayahuelin orthologs from other Agave species displayed one of six alleles at Y76: (Y/Y, D/D, S/S, Y/D, Y/S, D/S) and proved to be useful markers for phylogenetic analysis. Homozygous alleles were more frequent in wild accessions whereas heterozygous alleles were more frequent in cultivars. Mayahuelin sequences from different wild populations of A. angustifolia and A. rhodacantha allowed the identification of accessions closely related to azul, manso, sigüín, mano larga, and bermejo varieties of A. tequilana and var. espadín of A. angustifolia. Four A. rhodacantha accessions and A. angustifolia var. espadín were closer relatives of A. tequilana var. azul than A. angustifolia wild accessions or other A. tequilana varieties.

Molecular Markers Associated With Chemical Analysis: A Powerful Tool for Quality Control Assessment of Copalchi Medicinal Plant Complex.

The copalchi complex, Hintonia latiflora, H. standleyana, and Exostema caribaeum, is widely used in Mexico for treating diabetes and gastrointestinal disorders. The first therapeutic use for H. latiflora bark was registered in the "Florentine Codex" in the sixteenth century. The latest pharmacological and phytochemical studies revealed that the infusion of the leaves have hypoglycemic, antihyperglycemic and gastroprotective activities. For these reasons the monograph of the main copalchi species, H. latiflora, was recently added to the Mexican Herbal Pharmacopoeia. Nevertheless, quality control parameters are focused to the bark but not to the leaves. Moreover, information about other Rubiaceae species is needed. The main goal of this study was to generate molecular and chemical markers for quality control of the copalchi complex raw material. In addition, the resolution of the taxonomical ambiguity between H. latiflora and H. standleyana, as well as the testing of the molecular and chemical markers in different geographical batches, were aims of this study. The molecular markers and chemical profiles of the leaf infusions were generated considering three different populations for H. latiflora and separate individuals of the three species (HL, n = 10; HS, n = 3; EC, n = 4). The molecular markers matK, rbcL, trnH-psbA, rpl32-trnL, and ITS2 were tested for their discriminating capabilities. Chemical profiles of the leaf infusions were obtained by means of HPLC analyses using chlorogenic acid and 4-phenylcoumarins as chemical markers. The concatenated sequence of the molecular markers trnH-psbA, rpl32-trnL, and ITS2 clearly distinguished the three taxa, clarifying the taxonomical ambiguity of the Hintonia genus. Additionally, the chemical profiles allowed the unequivocal identification of each species supporting the molecular results; the geographical origin of the samples did not modify neither the chemical profiles nor the concatenated sequence of H. latiflora, suggesting that it is a robust identity test. The complementary use of molecular and chemical markers will assure the quality of plant material used in traditional medicine for therapeutic purposes, and should be valuable new information for the National Health authorities as a part of the Mexican Herbal Pharmacopoeia.

Synergy between root hydrotropic response and root biomass in maize (Zea mays L.) enhances drought avoidance.

Roots of higher plants change their growth direction in response to moisture, avoiding drought and gaining maximum advantage for development. This response is termed hydrotropism. There have been few studies of root hydrotropism in grasses, particularly in maize. Our goal was to test whether an enhanced hydrotropic response of maize roots correlates with a better adaptation to drought and partial/lateral irrigation in field studies. We developed a laboratory bioassay for testing hydrotropic response in primary roots of 47 maize elite DTMA (Drought Tolerant Maize for Africa) hybrids. After phenotyping these hybrids in the laboratory, selected lines were tested in the field. Three robust and three weak hybrids were evaluated employing three irrigation procedures: normal irrigation, partial lateral irrigation and drought. Hybrids with a robust hydrotropic response showed growth and developmental patterns, under drought and partial lateral irrigation, that differed from weak hydrotropic responders. A correlation between root crown biomass and grain yield in hybrids with robust hydrotropic response was detected. Hybrids with robust hydrotropic response showed earlier female flowering whereas several root system traits, such as projected root area, median width, maximum width, skeleton width, skeleton nodes, average tip diameter, rooting depth skeleton, thinner aboveground crown roots, as well as stem diameter, were considerably higher than in weak hydrotropic responders in the three irrigation procedures utilized. These results demonstrate the benefit of intensive phenotyping of hydrotropism in primary roots since maize plants that display a robust hydrotropic response grew better under drought and partial lateral irrigation, indicating that a selection for robust hydrotropism might be a promising breeding strategy to improve drought avoidance in maize.

Erratum to: A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis.

The original version of this article unfortunately contains a mistake. The authors have inadvertently incorrectly listed the concentration of TCA in the acetone/TCA/ß-ME solution in the materials and methods section of this paper. The TCA concentration in Sects. 2.3.2 and 2.3.5 should be 10% TCA, making the solution acetone/10% TCA/0.07% ß-ME. It is now corrected with this erratum.

A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis.

Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-ß-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.

Suppression of the tert-butylhydroquinone toxicity by its grafting onto chitosan and further cross-linking to agavin toward a novel antioxidant and prebiotic material.

The enzyme-mediated grafting of tert-butylhydroquinone (TBHQ) onto chitosan and further crosslinking to agave inulin (agavin) has been successfully achieved in a mild and non-toxic two-step route. The resulting products were characterized by nuclear magnetic resonance (NMR) and Infra-red spectroscopies to assess the molecular structure. The study of acute oral toxicity in mice revealed no adverse short-term effects of consumption in the synthesized materials with non-toxicity evidence until 2000 mg/kg through an oral acute administration. Importantly, this study proves that the compound maintains the radical scavenging capacity of the phenolic antioxidant upon ferric-reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays with a measured half-maximal inhibitory concentration (IC50) for the best case of 1.54 g/L based on inhibition of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid diammonium salt (ABTS). Additionally, the novel compound presented high prebiotic activities as ascertained in the presence of lactic acid bacteria (LAB).

Complex regulation of Hsf1-Skn7 activities by the catalytic subunits of PKA in Saccharomyces cerevisiae: experimental and computational evidences.

BACKGROUND: The cAMP-dependent protein kinase regulatory network (PKA-RN) regulates metabolism, memory, learning, development, and response to stress. Previous models of this network considered the catalytic subunits (CS) as a single entity, overlooking their functional individualities. Furthermore, PKA-RN dynamics are often measured through cAMP levels in nutrient-depleted cells shortly after being fed with glucose, dismissing downstream physiological processes. RESULTS: Here we show that temperature stress, along with deletion of PKA-RN genes, significantly affected HSE-dependent gene expression and the dynamics of the PKA-RN in cells growing in exponential phase. Our genetic analysis revealed complex regulatory interactions between the CS that influenced the inhibition of Hsf1/Skn7 transcription factors. Accordingly, we found new roles in growth control and stress response for Hsf1/Skn7 when PKA activity was low (cdc25Δ cells). Experimental results were used to propose an interaction scheme for the PKA-RN and to build an extension of a classic synchronous discrete modeling framework. Our computational model reproduced the experimental data and predicted complex interactions between the CS and the existence of a repressor of Hsf1/Skn7 that is activated by the CS. Additional genetic analysis identified Ssa1 and Ssa2 chaperones as such repressors. Further modeling of the new data foresaw a third repressor of Hsf1/Skn7, active only in the absence of Tpk2. By averaging the network state over all its attractors, a good quantitative agreement between computational and experimental results was obtained, as the averages reflected more accurately the population measurements. CONCLUSIONS: The assumption of PKA being one molecular entity has hindered the study of a wide range of behaviors. Additionally, the dynamics of HSE-dependent gene expression cannot be simulated accurately by considering the activity of single PKA-RN components (i.e., cAMP, individual CS, Bcy1, etc.). We show that the differential roles of the CS are essential to understand the dynamics of the PKA-RN and its targets. Our systems level approach, which combined experimental results with theoretical modeling, unveils the relevance of the interaction scheme for the CS and offers quantitative predictions for several scenarios (WT vs. mutants in PKA-RN genes and growth at optimal temperature vs. heat shock).

The absence of heat shock protein HSP101 affects the proteome of mature and germinating maize embryos.

Maize heat shock protein HSP101 accumulates during embryo maturation and desiccation and persists at high levels during the first 24 h following kernel imbibition in the absence of heat stress. This protein has a known function in disaggregation of high molecular weight complexes and has been proposed to be a translational regulator of specific mRNAs. Here, a global proteomic approach was used to identify changes in the maize proteome due to the absence of HSP101 in embryos from mature-dry or 24 h-imbibed kernels. A total of 26 protein spots from the mature dry embryo exhibited statistically significant expression changes in the L10 inbred hsp101 mutant (hsp101-m5::Mu1/hsp101-m5::Mu1) line as compared to the corresponding wild type (Hsp101/Hsp101). Additional six spots reproducibly showed qualitative changes between the mutant and wild-type mature and germinating embryos. Several chaperones, translation-related proteins, actin, and enzymes participating in cytokinin metabolism were identified in these spots by tandem mass-spectrometry (MS). The proteomic changes partially explain the altered root growth and architecture observed in young hsp101 mutant seedlings. In addition, specific protein de novo synthesis was altered in the 24 h-imbibed mutant embryos indicating that maize HSP101 functions as both chaperone and translational regulator during germination. Supporting this, HSP101 was found as part of Cap-binding and translation initiation complexes during early kernel imbibition. Overall, these findings expose the relevance of maize HSP101 for protein synthesis and balance mechanisms during germination.

Role of HSP101 in the stimulation of nodal root development from the coleoptilar node by light and temperature in maize (Zea mays L.) seedlings.

Nodal roots (NRs) constitute the prevalent root system of adult maize plants. NRs emerge from stem nodes located below or above ground, and little is known about their inducing factors. Here, it is shown that precocious development of NRs at the coleoptilar node (NRCNs) occurred in maize seedlings when: (i) dark grown and stimulated by the concurrent action of a single light shock of low intensity white light (2 µmol m(-2) s(-1)) and a single heat shock; (ii) grown under a photoperiod of low intensity light (0.1 µmol m(-2) s(-1)); or (iii) grown in the dark under a thermoperiod (28 °C/34 °C). The light shock effects were synergistic with heat shock and with the photoperiod, whereas the thermoperiodical and photoperiodical effects were additive. Dissection of the primary root or the root cap, to mimic the fatal consequences of severe heat shock, caused negligible effects on NRCN formation, indicating that the shoot is directly involved in perception of the heat shock-inducible signal that triggered NRCN formation. A comparison between hsp101-m5::Mu1/hsp101-m5::Mu1 and Hsp101/Hsp101 seedlings indicated that the heat shock protein 101 (HSP101) chaperone inhibited NRCN formation in the light and in the dark. Stimulation of precocious NRCN formation by light and heat shocks was affected by genetic background and by the stage of seedling development. HSP101 protein levels increased in the coleoptilar node of induced wild-type plants, particularly in the procambial region, where NRCN formation originated. The adaptive relevance of development of NRCNs in response to these environmental cues and hypothetical mechanisms of regulation by HSP101 are discussed.

Small heat-shock proteins and leaf cooling capacity account for the unusual heat tolerance of the central spike leaves in Agave tequilana var. Weber.

Agaves are perennial crassulacean acid metabolism (CAM) plants distributed in tropical and subtropical arid environments, features that are attractive for studying the heat-shock response. In agaves, the stress response can be analysed easily during leaf development, as they form a spirally shaped rosette, having the meristem surrounded by folded leaves in the centre (spike) and the unfolded and more mature leaves in the periphery. Here, we report that the spike of Agave tequilana is the most thermotolerant part of the rosette withstanding shocks of up to 55 degrees C. This finding was inconsistent with the patterns of heat-shock protein (Hsp) gene expression, as maximal accumulation of Hsp transcripts was at 44 degrees C in all sectors (spike, inner, middle and outer). However, levels of small HSP (sHSP)-CI and sHSP-CII proteins were conspicuously higher in spike leaves at all temperatures correlating with their thermotolerance. In addition, spike leaves showed a higher stomatal density and abated more efficiently their temperature several degrees below that of air. We propose that the greater capacity for leaf cooling during the day in response to heat stress, and the elevated levels of sHSPs, constitute part of a set of strategies that protect the SAM and folded leaves of A. tequilana from high temperatures.

Cap-independent translation of maize Hsp101.

Maize embryonic axes contain stored mRNAs, some of which are able to undergo cap-independent translation initiation during germination. The Hsp101 mRNA, encoding a heat shock protein, is essential for thermo-tolerance induction and is present among the stored transcripts. This research aimed to investigate whether the Hsp101 transcript is IRES-driven regulated upon heat stress. Hsp101 transcribed either in vitro or in vivo was efficiently translated via a cap-independent mechanism. This was observed either in an animal in vitro translation system containing proteolytically cleaved eukaryotic initiation factor eIF4G or in a plant system lacking both eIF4E and eIFiso4E initiation factors. Deletion of the 5' untranslated region (UTR) from the Hsp101 mRNA abolished its cap-independent translation indicating that this nucleotide sequence is required to confer cap-independent initiation. Bicistronic constructs containing the Hsp101 mRNA 5'UTR in sense and anti-sense directions between two reporter genes were translated in both cap-independent systems. A similar bicistronic construct containing a viral internal ribosome entry site (IRES) element between the reporter genes was used as control. Internal translation of the second reporter gene was observed when the Hsp101 5'UTR was in the sense but not in the anti-sense orientation in the bicistronic construct. Taken together, these data suggest that the 5'UTR of maize Hsp101, a plant cellular mRNA, functions as an IRES-like element accounting for its cap-independent translation during heat stress.

New roles for CDC25 in growth control, galactose regulation and cellular differentiation in Saccharomyces cerevisiae.

Living organisms display large differences in stress resistance throughout their life cycles. To study the coordinated regulation of development and stress responses in exponentially growing yeast, mutants that displayed elevated heat-shock resistance at this stage were screened for. Here, two new mutant alleles of CDC25 in Saccharomyces cerevisiae, cdc25-21 and cdc25-22, are described. During exponential growth in glucose at 25 degrees C, these mutants are resistant to heat, oxidative, osmotic and ionic shock, accumulate stress-protein transcripts, show slow growth rates, thick cell walls and glycogen hyperaccumulation and lack cAMP signalling in response to glucose. Genetic and cellular analyses revealed that the stationary-phase phenotypes of cdc25-21 and cdc25-22 mutants are not due to entrance to a G(0) state during exponential growth, but are the result of a prolonged G(1) phase. It was found that, in the W303 background, CDC25 is dispensable for growth in glucose media. However, CDC25 is essential for growth in galactose, in non-fermentable carbon sources and under continuous incubation at 38 degrees C. In conclusion, the function of the catalytic, C-terminal domain of Cdc25p is not only important for fermentative growth, but also for growth in non-fermentable carbon sources and to trigger galactose derepression.

Maize HSP101 plays important roles in both induced and basal thermotolerance and primary root growth.

HSP101 belongs to the ClpB protein subfamily whose members promote the renaturation of protein aggregates and are essential for the induction of thermotolerance. We found that maize HSP101 accumulated in mature kernels in the absence of heat stress. At optimal temperatures, HSP101 disappeared within the first 3 days after imbibition, although its levels increased in response to heat shock. In embryonic cells, HSP101 concentrated in the nucleus and in some nucleoli. Hsp101 maps near the umc132 and npi280 markers on chromosome 6. Five maize hsp101-m-::Mu1 alleles were isolated. Mutants were null for HSP101 and defective in both induced and basal thermotolerance. Moreover, during the first 3 days after imbibition, primary roots grew faster in the mutants at optimal temperature. Thus, HSP101 is a nucleus-localized protein that, in addition to its role in thermotolerance, negatively influences the growth rate of the primary root. HSP101 is dispensable for proper embryo and whole plant development in the absence of heat stress.