Lessons learned from metabolic engineering in hairy roots: Transcriptome and metabolic profile changes caused by Rhizobium-mediated plant transformation in Cucurbitaceae species.

Cucurbitaceae species are used in traditional medicine around the world. Cucurbitacins are highly oxygenated triterpenoids found in Cucurbitaceae species and exhibit potent anticancer activity alone and in combination with other existing chemotherapeutic drugs. Therefore, increasing production of these specialized metabolites is of great relevance. We recently showed that hairy roots of Cucurbita pepo can be used as a platform for metabolic engineering of cucurbitacins to modify their structure and increase their production. To study the changes in cucurbitacin accumulation upon formation of hairy roots, an empty vector (EV) control and Cucurbitacin inducing bHLH transcription factor 1 (CpCUCbH1)-overexpressing hairy roots of C. pepo were compared to untransformed (WT) roots. Whilst CpCUCbH1-overexpression increased production of cucurbitacins I and B by 5-fold, and cucurbitacin E by 3-fold when compared to EV lines, this increase was not significantly different when compared to WT roots. This indicated that Rhizobium rhizogenes transformation lowered the cucurbitacins levels in hairy roots, but that increasing expression of cucurbitacin biosynthetic genes by CpCUCbH1-overexpression restored cucurbitacin production to WT levels. Subsequent metabolomic and RNA-seq analysis indicated that the metabolic profile and transcriptome of hairy roots was significantly changed when compared to WT roots. Interestingly, it was observed that 11% of the differentially expressed genes were transcription factors. It was noteworthy that the majority of transcripts showing highest Pearson correlation coefficients to the Rhizobium rhizogenes genes rolB, rolC and ORF13a, were predicted to be transcription factors. In summary, hairy roots are an excellent platform for metabolic engineering of plant specialized metabolites, but these extensive transcriptome and metabolic profile changes should be considered in subsequent studies.

Plant Growth-Promoting Rhizobacteria Improve Growth and Fruit Quality of Cucumber under Greenhouse Conditions.

Cucumber fruit is rich in fiber, carbohydrates, protein, magnesium, iron, vitamin B, vitamin C, flavonoids, phenolic compounds, and antioxidants. Agrochemical-based production of cucumber has tripled yields; however, excessive synthetic fertilization has caused problems in the accumulation of salts in the soil and has increased production costs. The objective of this study was to evaluate the effect of three strains of plant growth-promoting rhizobacteria (PGPR) on cucumber fruit growth and quality under greenhouse conditions. The rhizobacteria Pseudomonas paralactis (KBendo6p7), Sinorhizobium meliloti (KBecto9p6), and Acinetobacter radioresistens (KBendo3p1) was adjusted to 1 × 108 CFU mL-1. The results indicated that the inoculation with PGPR improved plant height, stem diameter, root length, secondary roots, biomass, fruit size, fruit diameter, and yield, as well as nutraceutical quality and antioxidant capacity, significantly increasing the response of plants inoculated with A.radioresistens and S.meliloti in comparison to the control. In sum, our findings showed the potential functions of the use of beneficial bacteria such as PGPR for crop production to reduce costs, decrease pollution, and achieve world food safety and security.

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution.

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild-type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss-of-function mutations, or in a dominant (gain-of-function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.

The cysteine-rich receptor-like protein kinase CRK28 modulates Arabidopsis growth and development and influences abscisic acid responses.

MAIN CONCLUSION: CRK28, a cysteine-rich receptor-like kinase, plays a role in root organogenesis and overall growth of plants and antagonizes abscisic acid response in seed germination and primary root growth. Receptor-like kinases (RLK) orchestrate development and adaptation to environmental changes in plants. One of the largest RLK groups comprises cysteine-rich receptor-like kinases (CRKs), for which the function of most members remains unknown. In this report, we show that the loss of function of CRK28 led to the formation of roots that are longer and more branched than the parental (Col-0) plantlets, and this correlates with an enhanced domain of the mitotic reporter CycB1:uidA in primary root meristems, whereas CRK28 overexpressing lines had the opposite phenotype, including slow root growth and reduced lateral root formation. Epidermal cell analyses revealed that crk28 mutants had reduced root hair length and increased trichome number, whereas 35S::CRK28 lines present primary roots with longer root hairs but lesser trichomes in leaves. The overall growth in soil of crk28 mutant and CRK28 overexpressing lines was reduced or enhanced, respectively, when compared to the parental (Col-0) seedlings, while germination, root growth and expression analyses of ABI3 and ABI5 further showed that CRK28 modulates ABA responses, which may be important to fine-tune plant morphogenesis. Our study unravels the participation of RLK signaling in root growth and epidermal cell differentiation.

Ontogenetic Change in the Venom of Mexican Black-Tailed Rattlesnakes (Crotalus molossus nigrescens).

Ontogenetic changes in venom composition have important ecological implications due the relevance of venom in prey acquisition and defense. Additionally, intraspecific venom variation has direct medical consequences for the treatment of snakebite. However, ontogenetic changes are not well documented in most species. The Mexican Black-tailed Rattlesnake (Crotalus molossus nigrescens) is large-bodied and broadly distributed in Mexico. To document venom variation and test for ontogenetic changes in venom composition, we obtained venom samples from twenty-seven C. m. nigrescens with different total body lengths (TBL) from eight states in Mexico. The primary components in the venom were detected by reverse-phase HPLC, western blot, and mass spectrometry. In addition, we evaluated the biochemical (proteolytic, coagulant and fibrinogenolytic activities) and biological (LD50 and hemorrhagic activity) activities of the venoms. Finally, we tested for recognition and neutralization of Mexican antivenoms against venoms of juvenile and adult snakes. We detected clear ontogenetic venom variation in C. m. nigrescens. Venoms from younger snakes contained more crotamine-like myotoxins and snake venom serine proteinases than venoms from older snakes; however, an increase of snake venom metalloproteinases was detected in venoms of larger snakes. Venoms from juvenile snakes were, in general, more toxic and procoagulant than venoms from adults; however, adult venoms were more proteolytic. Most of the venoms analyzed were hemorrhagic. Importantly, Mexican antivenoms had difficulties recognizing low molecular mass proteins (<12 kDa) of venoms from both juvenile and adult snakes. The antivenoms did not neutralize the crotamine effect caused by the venom of juveniles. Thus, we suggest that Mexican antivenoms would have difficulty neutralizing some human envenomations and, therefore, it may be necessary improve the immunization mixture in Mexican antivenoms to account for low molecular mass proteins, like myotoxins.

Dinámica de la diversidad temporal de los hongos micorrícicos arbusculares de Larrea tridentata (Sesse & Mocino ex DC) Coville en un ecosistema semiárido / Temporal diversity dynamics of the arbuscular mycorrhizal fungi of Larrea tridentata (Sesse & Mocino ex DC) Coville in a semi-arid ecosystem

Los hongos micorrícicos arbusculares (HMA) de los ambientes áridos y semiáridos son importantes para el desarrollo de las plantas que crecen bajo estrés biótico y abiótico en áreas naturales o en agroecosistemas. Existe poca información sobre la diversidad temporal de los HMA en plantas perennes de ecosistemas áridos en el norte de México. En este estudiose evaluaron la colonización micorrícica y la diversidad temporal de los HMA en la rizosfera de Larrea tridentata, planta perenne de amplia distribución en el Desierto Chihuahuense. Se obtuvieron muestras de la rizosfera y de raíces de 15 plantas, en 3 fechas de muestreo del año 2015. Se encontró un total de 17 especies de HMA, distribuidas en 12 géneros y 7 familias dentro del phylum Glomeromycota. La especie más abundante fue Funneliformis geosporum. Esta especie pertenece a la familia Glomeraceae, la única que estuvo representada con varios géneros en L. tridentata. El mayor porcentaje de micorrización se presentó en febrero, con un 83,22%, en septiembre fue del 75,27% y en mayo del 65,27%. El muestreo realizado en febrero presentó el mayor número de especies (16), seguido por el de mayo (15) y el de septiembre (12). El análisis estadístico mostró diferencias significativas en el número de esporas entre los muestreos de febrero y los de mayo y septiembre.

Arbuscular mycorrhizal fungi (AMF) of arid and semiarid ecosystems are important for the development of plants that grow under biotic stress in wild or in agro-ecosystems. There is little information on the temporal diversity of these organisms in perennial plants from arid ecosystems in northern Mexico. On this study, the mycorrhizal colonization and the temporal diversity of AMF in the rhizosphere of Larrea tridentata, perennial plant abundant in the Chihuahuan Desert region were explored. Samples of the rhizosphere and roots of fifteen plants in each of the three sampling dates during the 2015 year were obtained. A total of 17 species of HMA belonging to 12 genera and 7 families within the phylum Glomeromycota in all three sampling dates were found. Funneliformis geosporum was the dominant species belonging to the family Glomeraceae which possess the highest genera number on L. tridentata.The highest mycorrhization percentage was in February with 83.22, followed by September and May with 75.27 and 65.27%, respectively. A maximum of 16 AM fungal species were isolated and identified from L. tridentata rhizosphere in February, 15 species in May and 12 species in September. Statistical analysis showed significant differences between sampling dates in the spores number.

[Temporal diversity dynamics of the arbuscular mycorrhizal fungi of Larrea tridentata (Sesse & Mocino ex DC) Coville in a semi-arid ecosystem]. / Dinámica de la diversidad temporal de los hongos micorrícicos arbusculares de Larrea tridentata (Sesse & Mocino ex DC) Coville en un ecosistema semiárido.

Arbuscular mycorrhizal fungi (AMF) of arid and semiarid ecosystems are important for the development of plants that grow under biotic stress in wild or in agro-ecosystems. There is little information on the temporal diversity of these organisms in perennial plants from arid ecosystems in northern Mexico. On this study, the mycorrhizal colonization and the temporal diversity of AMF in the rhizosphere of Larrea tridentata, perennial plant abundant in the Chihuahuan Desert region were explored. Samples of the rhizosphere and roots of fifteen plants in each of the three sampling dates during the 2015 year were obtained. A total of 17 species of HMA belonging to 12 genera and 7 families within the phylum Glomeromycota in all three sampling dates were found. Funneliformis geosporum was the dominant species belonging to the family Glomeraceae which possess the highest genera number on L. tridentata. The highest mycorrhization percentage was in February with 83.22, followed by September and May with 75.27 and 65.27%, respectively. A maximum of 16 AM fungal species were isolated and identified from L. tridentata rhizosphere in February, 15 species in May and 12 species in September. Statistical analysis showed significant differences between sampling dates in the spores number.

HR4 gene is induced in the Arabidopsis-Trichoderma atroviride beneficial interaction.

Plants are constantly exposed to microbes, for this reason they have evolved sophisticated strategies to perceive and identify biotic interactions. Thus, plants have large collections of so-called resistance (R) proteins that recognize specific microbe factors as signals of invasion. One of these proteins is codified by the Arabidopsis thaliana HR4 gene in the Col-0 ecotype that is homologous to RPW8 genes present in the Ms-0 ecotype. In this study, we investigated the expression patterns of the HR4 gene in Arabidopsis seedlings interacting with the beneficial fungus Trichoderma atroviride. We observed the induction of the HR4 gene mainly at 96 hpi when the fungus interaction was established. Furthermore, we found that the HR4 gene was differentially regulated in interactions with the beneficial bacterium Pseudomonas fluorescens and the pathogenic bacterium P. syringae. When hormone treatments were applied to A. thaliana (Col-0), each hormone treatment induced changes in HR4 gene expression. On the other hand, the expression of the RPW8.1 and RPW8.2 genes of Arabidopsis ecotype Ms-0 in interaction with T. atroviride was assessed. Interestingly, these genes are interaction-responsive; in particular, the RPW8.1 gene shows a very high level of expression in the later stages of interaction. These results indicate that HR4 and RPW8 genes could play a role in the establishment of Arabidopsis interactions with beneficial microbes.