Gerardo A. Aguado-Santacruz

Environmental factors and community dynamics at the southernmost part of the North American Graminetum – I. On the contribution of climatic factors to temporal variation in species composition

Compositional patterns of vegetation and their relationship to temporal and spatial environmental variation, with emphasis on climatic factors, were investigated in plant communities located in the southernmost portion of the North American Graminetum, in central México. Data from 353 samples, obtained in four ecologically contrasting plant communities during 11 years, were analyzed by partial canonical correspondence analysis. Eight climatic variables and eighteen covariables (seventeen edaphic and one resource management) were included in the ordination. A relationship between floristic change and weather variation, once covariables effects were fitted, was examined. Despite a strong contrast in ecological conditions among study sites, a set of four climatic variables was finally found in which each variable contributed independently and statistically (P < 0.01) to the total variance in the vegetation data. Thus, environmental variables other than climatic could not conceal the important role of weather as a mediator of floristic change through time. Summer precipitation and summer maximum temperature showed the highest correlations with the first two species axes, 0.77 and –0.39, respectively. Contribution of these two climatic variables to variance in the vegetation data explained by environmental variables was approximately 81%. Annual species were abundant during rainy years, while abundance of perennial grasses and shrubs showed no clear relationship to weather variation. This study explicitly probes the important role of rain patterns in shaping structure and composition of semiarid communities and considers how other environmental factors can affect plant communities at the southernmost part of the North American Graminetum.

Genetic variability of Bouteloua gracilis populations differing in forage production at the southernmost part of the North American Graminetum

Bouteloua gracilis (blue grama grass) native populations have been shown to be highly variable, however the genetic basis of this variability has not been well established. Determining the extent of genetic variability within and among plant populations have important repercussions for the management and conservation of species, and in particular for those subjected to intensive use such as forage plants. Using RAPD, this study was undertaken to investigate the genetic variability of four B. gracilis native populations developed in three grasslands and one shrubland at the southernmost part of the North American Graminetum in México. Significant differences in grass aboveground production were found among the study sites, while considerable genetic variation within each of the four blue grama populations evaluated was detected. The molecular analysis, based on 55 individuals, revealed a total of 108 scorable repeatable bands, with 99 of them being polymorphic (overall polymorphism= 91.7%). Within every population each individual was genetically distinct and no population-specific bands (fixed marker differences) were identified. Pair-wise ΦST comparisons indicated that the four blue grama populations examined were significantly different in their genetic constitution (P<0.001). AMOVA revealed that most of the genetic variation detected in Bouteloua gracilis was explained by intra- (88.53%), rather than by inter-population (11.47%) differences. UPGMA based on the ΦST values indicated that the blue grama population collected from the shrubland displayed the RAPD profiles that most differed among the study sites. Possible causes of these results could reside on intensive grazing reducing, and proper management conserving, the forage production and genetic diversity of blue grama native populations. Our results are consistent with previous studies analyzing population genetic variation in outcrossing grasses and, in particular, with ecological and cytological evidence for a high genetic variability in native populations of B. gracilis. The implications of our findings and prospective studies to be undertaken using molecular tools in the study of blue grama biology and ecology are discussed.

TISSUE CULTURE AND PLANT REGENERATION OF BLUE GRAMA GRASS, BOUTELOUA GRACILIS (H.B.K.) LAG. EX STEUD.

SummaryAs a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N6-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l−1 (4.52 μM) 2,4-D plus 0.5 mg l−1 (2.22 μM) BA. and 2 mg l−1 (8.88 μM) BA plus 1 mg l−1 (4.14 μM) Picloram with or without 40 mg l−1 (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l−1 (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l−1 (2.22 μM) BA, or 1 mg l−1 (4.52 μM) 2,4-D with 0.50 mg l−1 (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l−1 (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l−1 (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l−1 (8,28 μM) Picloram, 1 mg l−1 (4.44 μM) BA and 40 mg l−1 (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.

Environmental factors and community dynamics at the southernmost part of the North American Graminetum

We have delineated the spatio-temporal plant assemblages prevailing under contrasting environmental conditions at the southernmost part of the North American Graminetum in central México and discuss the relative importance of these factors in determining plant community structure and composition. 353 line-transect samples were collected during 11 years from exclosures and adjacent grazed areas within three Bouteloua-dominated grasslands and one Acacia schaffneri shrubland and analyzed using TWINSPAN. In addition, eight edaphic variables were utilized to evaluate similarities in soil properties among sites, using unweighted-pair groups method. Results from TWINSPAN were translated into mosaic patterns to show the distribution of communities as a function of environmental factors over time. Under no or moderate grazing, summer precipitation promotes an initial differentiation of vegetation into high, low or average rainfall communities. This situation is altered in grasslands degraded by intensive grazing pressure, where rainfall patterns play a subordinate role to that of livestock herbivory. Soil influences are secondary to those of precipitation and grazing in affecting grassland structure and composition. In shrubland, community stability is related to high floristic and edaphic spatial heterogeneity in the face of climatic variability.

Genetic transformation of blue grama grass with the rolA gene from Agrobacterium rhizogenes: regeneration of transgenic plants involves a "hairy embryo" stage

Until recently, information about the effects of transforming plants with the rolA gene of Agrobacterium rhizogenes has been restricted mainly to dicots in which a severely wrinkled phenotype, reduced internode distances, and abnormal reproductive development were commonly observed. In this work, we analyzed the effects associated with the expression of this gene in a new genetic context: the forage grass genome. Transgenic P35S•rolA plants of blue grama grass (Bouteloua gracilis) were obtained by a biolistic approach employing embryogenic chlorophyllic cells as the target material. Four independent transgenic lines with regeneration capacity were recovered, which showed stable integration of this transgene as demonstrated by polymerase chain reaction and Southern blot hybridization. Growth of the rolA-transformed lines under greenhouse conditions provided evidence for a new biotechnological application for the rolA gene in plants, namely, the improvement of biomass production in forage grasses. Additionally, we described here a new phenotypic marker (referred to here as the “hairy embryo” syndrome) that can be instrumental for the early identification of transformation events when transforming grasses with this gene.

Identification of miRNA from Bouteloua gracilis, a drought tolerant grass, by deep sequencing and their in silico analysis

BACKGROUND MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate signal transduction, development, metabolism, and stress responses in plants through post-transcriptional degradation and/or translational repression of target mRNAs. Several studies have addressed the role of miRNAs in model plant species, but miRNA expression and function in economically important forage crops, such as Bouteloua gracilis (Poaceae), a high-quality and drought-resistant grass distributed in semiarid regions of the United States and northern Mexico remain unknown. RESULTS We applied high-throughput sequencing technology and bioinformatics analysis and identified 31 conserved miRNA families and 53 novel putative miRNAs with different abundance of reads in chlorophyllic cell cultures derived from B. gracilis. Some conserved miRNA families were highly abundant and possessed predicted targets involved in metabolism, plant growth and development, and stress responses. We also predicted additional identified novel miRNAs with specific targets, including B. gracilis ESTs, which were detected under drought stress conditions. CONCLUSIONS Here we report 31 conserved miRNA families and 53 putative novel miRNAs in B. gracilis. Our results suggested the presence of regulatory miRNAs involved in modulating physiological and stress responses in this grass species.

Comparison of the structure and organization of the rrna operons of Bouteloua gracilis and Zea mays

Aguado-Santacruz, G. A., Betancourt-Guerra, D. A., Siquerios-Cendón, T., Arévalo-Gallegos, S., Rivera-Chavira, B. E., Nevarez-Moorillon, G. V., Moreno-Gómez, B. and Rascón-Cruz, Q. 2011. Comparison of the structure and organization of therrnaoperons of Bouteloua gracilisandZea mays. Can. J. Plant Sci. 91: 107-116. We studied the genomic structure of Bouteloua gracilis chloroplast DNA (cpDNA) and compared it with the sequenced ribosomal RNA spacer region from other cereals. This will allow us to understand chloroplast topology and the recombination ability of cpDNA. The development of potential tools for biotechnology applied to cereals can be focused through the study of cpDNA in family related grasses, such as B. gracilis. cpDNA was prepared from green B. gracilis and Zea mays plants using a modified NaCl method. A 2332 bp intergenic spacer (IGS) region (rrna16S-trnI-trnA-rrna23S) from B. gracilis was sequenced, which showed great similarity (at least 92%) to IGS region from Z. mays, Oryza sativa and Saccharum officinarum. A physical map constructed by Southern hybridization using petA, psbA, psbD, ndhA, rbcL, 16S and 23S rDNA digoxigenin-labelled probes showed low organizational resemblance to maize cpDNA. Moreover, when compared to a similar fragment of Z. mays, a 239 bp intron deletion was found in the trnI gene in the B. gracilis cpDNA. Restriction and hybridization analyses suggested that the B. gracilis cpDNA has a molecular weight of 130 Kb. We expect that the findings reported in this work can be a baseline for increasing our knowledge in chloroplast organization in grasses and for the development of molecular tools.