Characterization of SgALMT genes reveals the function of SgALMT2 in conferring aluminum tolerance in Stylosanthes guianensis through the mediation of malate exudation.

Aluminum (Al) toxicity is the major constraint on plant growth and productivity in acidic soils. An adaptive mechanism to enhance Al tolerance in plants is mediated malate exudation from roots through the involvement of ALMT (Al-activated malate transporter) channels. The underlying Al tolerance mechanisms of stylo (Stylosanthes guianensis), an important tropical legume that exhibits superior Al tolerance, remain largely unknown, and knowledge of the potential contribution of ALMT genes to Al detoxification in stylo is limited. In this study, stylo root growth was inhibited by Al toxicity, accompanied by increases in malate and citrate exudation from roots. A total of 11 ALMT genes were subsequently identified in the stylo genome and named SgALMT1 to SgALMT11. Diverse responses to metal stresses were observed for these SgALMT genes in stylo roots. Among them, the expressions of 6 out of the 11 SgALMTs were upregulated by Al toxicity. SgALMT2, a root-specific and Al-activated gene, was selected for functional characterization. Subcellular localization analysis revealed that the SgALMT2 protein is localized to the plasma membrane. The function of SgALMT2 in mediating malate release was confirmed by analysis of the malate exudation rate from transgenic composite stylo plants overexpressing SgALMT2. Furthermore, overexpression of SgALMT2 led to increased root growth in transgenic stylo plants treated with Al through decreased Al accumulation in roots. Taken together, the results of this study suggest that malate secretion mediated by SgALMT2 contributes to the ability of stylo to cope with Al toxicity.

Multi-omics analysis reveals the roles of purple acid phosphatases in organic phosphorus utilization by the tropical legume Stylosanthes guianensis.

Stylo (Stylosanthes guianensis) is a tropical legume known for its exceptional tolerance to low phosphate (Pi), a trait believed to be linked to its high acid phosphatase (APase) activity. Previous studies have observed genotypic variations in APase activity in stylo; however, the gene encoding the crucial APase responsible for this variation remains unidentified. In this study, transcriptomic and proteomic analyses were employed to identify eight Pi starvation-inducible (PSI) APases belonging to the purple APase (PAP) family in the roots of stylo and seven in the leaves. Among these PSI-PAPs, SgPAP7 exhibited a significantly positive correlation in its expression levels with the activities of both internal APase and root-associated APase across 20 stylo genotypes under low-Pi conditions. Furthermore, the recombinant SgPAP7 displayed high catalytic activity toward adenosine 5'-diphosphate (ADP) and phosphoenolpyruvate (PEP) in vitro. Overexpression (OE) of SgPAP7 in Arabidopsis facilitated exogenous organic phosphorus utilization. Moreover, SgPAP7 OE lines showed lower shoot ADP and PEP levels than the wild type, implying that SgPAP7 is involved in the catabolism and recycling of endogenous ADP and PEP, which could be beneficial for plant growth in low-Pi soils. In conclusion, SgPAP7 is a key gene with a major role in stylo adaptation to low-Pi conditions by facilitating the utilization of both exogenous and endogenous organic phosphorus sources. It may also function as a PEP phosphatase involved in a glycolytic bypass pathway that minimizes the need for adenylates and Pi. Thus, SgPAP7 could be a promising target for improving tolerance of crops to low-Pi availability.

Development of transgenic composite Stylosanthes plants to study root growth regulated by a ß-expansin gene, SgEXPB1, under phosphorus deficiency.

KEY MESSAGE: A highly efficient transformation procedure to generate transgenic Stylosanthes roots was established. SgEXPB1 is involved in Stylosanthes root growth under phosphorus deficiency. Stylo (Stylosanthes spp.) is an important forage legume widely applied in agricultural systems in the tropics. Due to the recalcitrance of stylo genetic transformation, functional characterization of candidate genes involved in stylo root growth is limited. This study established an efficient procedure for Agrobacterium rhizogenes-mediated transformation for generating transgenic composite plants of S. guianensis cultivar 'Reyan No. 5'. Results showed that composite stylo plants with transgenic hairy roots were efficiently generated by A. rhizogenes strains K599 and Arqual, infecting the residual hypocotyl at 1.0 cm of length below the cotyledon leaves of 9-d-old seedlings, leading to a high transformation efficiency of > 95% based on histochemical ß-glucuronidase (GUS) staining. Notably, 100% of GUS staining-positive hairy roots can be achieved per composite stylo plant. Subsequently, SgEXPB1, a ß-expansin gene up-regulated by phosphorus (P) deficiency in stylo roots, was successfully overexpressed in hairy roots. Analysis of hairy roots showed that root growth and P concentration in the transgenic composite plants were increased by SgEXPB1 overexpression under low-P treatment. Taken together, a highly efficient A. rhizogenes-mediated transformation procedure for generating composite stylo plants was established to study the function of SgEXPB1, revealing that this gene is involved in stylo root growth during P deficiency.

Dissection of Crop Metabolome Responses to Nitrogen, Phosphorus, Potassium, and Other Nutrient Deficiencies.

Crop growth and yield often face sophisticated environmental stresses, especially the low availability of mineral nutrients in soils, such as deficiencies of nitrogen, phosphorus, potassium, and others. Thus, it is of great importance to understand the mechanisms of crop response to mineral nutrient deficiencies, as a basis to contribute to genetic improvement and breeding of crop varieties with high nutrient efficiency for sustainable agriculture. With the advent of large-scale omics approaches, the metabolome based on mass spectrometry has been employed as a powerful and useful technique to dissect the biochemical, molecular, and genetic bases of metabolisms in many crops. Numerous metabolites have been demonstrated to play essential roles in plant growth and cellular stress response to nutrient limitations. Therefore, the purpose of this review was to summarize the recent advances in the dissection of crop metabolism responses to deficiencies of mineral nutrients, as well as the underlying adaptive mechanisms. This review is intended to provide insights into and perspectives on developing crop varieties with high nutrient efficiency through metabolite-based crop improvement.

Differential expressions and enzymatic properties of malate dehydrogenases in response to nutrient and metal stresses in Stylosanthes guianensis.

Malate dehydrogenase (MDH, EC 1.1.1.37) is a key enzyme that catalyzes a reversible NAD-dependent dehydrogenase reaction from oxaloacetate (OAA) to malate. Although MDH has been documented to participate in cellular metabolism and redox homeostasis in plants, the roles of MDH members in the tropical legume Stylosanthes guianensis (stylo) remain less definitive. In this study, except SgMDH1 that had been previously characterized, six novel MDH genes were isolated from stylo and were then designated as SgMDH2 to SgMDH7. All of the SgMDH proteins possessed the common features of NAD binding, dimerization interface and substrate binding sites. Expression analysis showed that three SgMDHs exhibited preferential expressions in leaves, and one SgMDH was mainly expressed in roots. Furthermore, SgMDHs were regulated by nutrient deficiencies in stylo roots, especially for phosphorus (-P) and potassium (-K) deficiencies. Differential responses of SgMDHs to trace metal stress and heavy metal toxicity were observed in stylo roots, suggesting the involvement of SgMDHs in the response of stylo to metal stresses. The six novel SgMDHs were subsequently expressed and purified from Escherichia coli to analyze their biochemical properties. Although SgMDHs exhibited variations in subcellular localizations, each SgMDH protein displayed a high level of catalytic efficiency towards OAA and NADH but a low level of catalytic efficiency towards malate and NAD+. In addition, the activities of recombinant SgMDH proteins were pH-dependent and temperature-sensitive, and exhibited differential regulations by various metal ions. These results together suggest the potential roles of SgMDHs in stylo coping with nutrient and metal stresses.

Physiological responses and transcriptomic changes reveal the mechanisms underlying adaptation of Stylosanthes guianensis to phosphorus deficiency.

BACKGROUND: Phosphorus (P) is an essential macronutrient for plant growth that participates in a series of biological processes. Thus, P deficiency limits crop growth and yield. Although Stylosanthes guianensis (stylo) is an important tropical legume that displays adaptation to low phosphate (Pi) availability, its adaptive mechanisms remain largely unknown. RESULTS: In this study, differences in low-P stress tolerance were investigated using two stylo cultivars ('RY2' and 'RY5') that were grown in hydroponics. Results showed that cultivar RY2 was better adapted to Pi starvation than RY5, as reflected by lower values of relative decrease rates of growth parameters than RY5 at low-P stress, especially for the reduction of shoot and root dry weight. Furthermore, RY2 exhibited higher P acquisition efficiency than RY5 under the same P treatment, although P utilization efficiency was similar between the two cultivars. In addition, better root growth performance and higher leaf and root APase activities were observed with RY2 compared to RY5. Subsequent RNA-seq analysis revealed 8,348 genes that were differentially expressed under P deficient and sufficient conditions in RY2 roots, with many Pi starvation regulated genes associated with P metabolic process, protein modification process, transport and other metabolic processes. A group of differentially expressed genes (DEGs) involved in Pi uptake and Pi homeostasis were identified, such as genes encoding Pi transporter (PT), purple acid phosphatase (PAP), and multidrug and toxin extrusion (MATE). Furthermore, a variety of genes related to transcription factors and regulators involved in Pi signaling, including genes belonging to the PHOSPHATE STARVATION RESPONSE 1-like (PHR1), WRKY and the SYG1/PHO81/XPR1 (SPX) domain, were also regulated by P deficiency in stylo roots. CONCLUSIONS: This study reveals the possible mechanisms underlying the adaptation of stylo to P deficiency. The low-P tolerance in stylo is probably manifested through regulation of root growth, Pi acquisition and cellular Pi homeostasis as well as Pi signaling pathway. The identified genes involved in low-P tolerance can be potentially used to design the breeding strategy for developing P-efficient stylo cultivars to grow on acid soils in the tropics.

Effect of feed supplements on dry season milk yield and profitability of crossbred cows in Honduras.

The contribution of dry season silage feeding on daily milk yield (MY) and dairying profitability in terms of income over feed cost (IOFC) was evaluated in dual-purpose cattle production systems in Honduras. MY records of 34 farms from two milk collection centres were collected over a 2-year period. Farms were surveyed to obtain information on the type, quantity and cost of supplemented feed, breed type and number of lactating cows in each month. Farms were classified in silage farms (SF, with a short silage supplementation period), non-silage farms (NSF) and prototype farms (PF, with an extended silage supplementation period). Data were analysed using descriptive statistics and a linear mixed model approach. PF had significantly higher MY than SF and NSF but, due to higher expenses for both concentrate and silage, similar IOFC compared to NSF. SF had similar MY but lower IOFC compared to NSF, due to higher feed expenses. The effect of silage feeding, particularly maize silage, on MY was significant and superior to that of other forage supplements. Silage supplementation contributed to the highest MY and IOFC on farms with crossbred cows of >62.5 % Bos taurus and to the second highest profitability on farms with >87.5 % Bos indicus share. It is concluded that silage can play an important role in drought-constrained areas of the tropics and can contribute to profitable dairying, irrespective of breed.

Land degradation on barren hills: a case study in northeast Vietnam.

The term "barren hills" has been a keyword for land degradation in the uplands of Vietnam for over a decade. Nevertheless, the "barren" land is still not adequately ecologically characterized. In this work, we analyze land use-induced changes in vegetation and soil properties along a sequence of barren hills types formed on one physiotope. The study is undertaken in the Bac Kan province, one of the poorest upland regions where livestock plays an important role. A transition from an old-growth laurel forest to a sparse manmade grassland is characterized by a total of 177 species, rapid species turnover, and discrete dominants, and an overwhelming effect of disturbance history on both soil and vegetation patterning. Land degradation is most apparent in land use-induced maintenance of arrested successions, and the regeneration course is shifted towards drier formations. We hypothesize a conceptual model as an aid to understanding the process of early fallow differentiation in response to the patterned, fine-scale disturbances. The larger-scale implications of the observed trends in regeneration potentials deviation, and, in particular, the effect of water buffaloes in halting fallow successions, are discussed.

Extent and structure of genetic diversity in a collection of the tropical multipurpose shrub legume Cratylia argentea (Desv.) O. Kuntze as revealed by RAPD markers

The tropical multipurpose shrub legume Cratylia argentea is well adapted to acid soils of low to medium fertility and has excellent drought-tolerance. Due to its high nutritive value it is particularly suited as forage for dry-season supplementation. A collection of 47 C. argentea accessions in a collection, derived from seed replicating of original accessions with differing geographic origin and morphological and agronomic characteristics was investigated using molecular markers (RAPD (random amplified polymorphic DNA)). Genetic diversity (H T = 0.145) in the collection was low, with 30% of differentiation among groups and high genetic similarity among accessions (GS = 0.805). Within-accession variability was high. One taxonomic mismatch and five possible duplicate accessions were identified. Our results suggest that the genetic diversity in the C. argentea accessions studied is relatively homogeneously distributed, indicating the likelihood of extensive outcrossing. The genetic diversity of original accessions should be assessed to determine if outcrossing has occurred during or before ex situ storage. This might also support any decision on whether accessions should be bulked rather than maintaining them individually.

Effect of the tropical grass Brachiaria brizantha (Hochst. ex A. Rich.) Stapf on microbial population and activity in petroleum-contaminated soil.

The effect of the tropical pasture grass Brachiaria brizantha on numbers of bacteria, fungi and degraders of alkanes, aromatics, cycloalkanes and crude oil in petroleum hydrocarbon contaminated and uncontaminated savannah soil was evaluated. Substrate induced soil respiration and soil pH were compared between planted and unplanted soil. B. brizantha had a mostly increasing effect on microbial numbers. As an exception, growth of bacteria was not or negatively affected. Microbial respiration and pH were always lower in planted than in unplanted soil. Low pH may result from enhanced oil degradation in planted soil leading to an accumulation of organic acids. A comparable stimulation of crude oil degraders and fungi in planted soil points to the importance of fungi. Since they tolerate lower pH values than bacteria, they are considered to play a central role in oil degradation. Given that the enhancement of crude oil degradation under the influence of B. brizantha could not clearly be correlated to microbial numbers and activity, other factors like oxygen availability, plant enzymes and synergistic degradation by microbial consortia have to be considered.

Potential of vetiver (vetiveria zizanioides (L.) Nash) for phytoremediation of petroleum hydrocarbon-contaminated soils in Venezuela.

Venezuela is one of the largest oil producers in the world. For the rehabilitation of oil-contaminated sites, phytoremediation represents a promising technology whereby plants are used to enhance biodegradation processes in soil. A greenhouse study was conducted to determine the tolerance of vetiver (Vetiveria zizanioides (L.) Nash) to a Venezuelan heavy crude oil in soil. Additionally, the plant's potential for stimulating the biodegradation processes of petroleum hydrocarbons was tested under the application of two fertilizer levels. In the presence of contaminants, biomass and plant height were significantly reduced. As for fertilization, the lower fertilizer level led to higher biomass production. The specific root surface area was reduced under the effects of petroleum. However, vetiver was found to tolerate crude-oil contamination in a concentration of 5% (w/w). Concerning total oil and grease content in soil, no significant decrease under the influence of vetiver was detected when compared to the unplanted control. Thus, there was no evidence of vetiver enhancing the biodegradation of crude oil in soil under the conditions of this trial. However, uses of vetiver grass in relation to petroleum-contaminated soils are promising for amelioration of slightly polluted sites, to allow other species to get established and for erosion control.

Influence of fertilizer levels on phytoremediation of crude oil-contaminated soils with the tropical pasture grass Brachiaria brizantha (hochst. ex a. rich.) stapf.

Determination of fertilizer levels in phytoremediation of petroleum hydrocarbons is a complex issue, since nutrient demands of the plant and of degrading microorganisms in the rhizosphere have to be considered In the present work, three fertilizer levels were tested in a greenhouse experiment with the aim of optimizing growth of the tropical pasture grass Brachiaria brizantha and enhance microbial degradation of heavy crude oil in soil Fertilizer was applied twice in a concentration of 200, 300, and 400 mg each of N, P, and K per kg soil before and after the first sampling (14 wk). The medium fertilizer concentration resulted in best root growth and highest absolute oil dissipation (18.4%) after 22 wk The highest concentration produced best shoot growth and highest relative oil dissipation after 14 wk (10.5% less than unplanted control). In general, degradation of total oil and grease was higher in planted than in unplanted soil, but differences diminished toward the end of the experiment. Next to fertiizer quantity, its composition is an important factor to be further studied, including the form of available nitrogen (N-NO3- vs. N-NH4+). Field trials are considered indispensable for further phytoremediation studies, since greenhouse experiments produce particular water and nutrient conditions.

Phytoremediation in the tropics--influence of heavy crude oil on root morphological characteristics of graminoids.

When studying species for phytoremediation of petroleum-contaminated soils, one of the main traits is the root zone where enhanced petroleum degradation takes place. Root morphological characteristics of three tropical graminoids were studied. Specific root length (SRL), surface area, volume and average root diameter (ARD) of plants grown in crude oil-contaminated and uncontaminated soil were compared. Brachiaria brizantha and Cyperus aggregatus showed coarser roots in polluted soil compared to the control as expressed in an increased ARD. B. brizantha had a significantly larger specific root surface area in contaminated soil. Additionally, a shift of SRL and surface area per diameter class towards higher diameters was found. Oil contamination also caused a significantly smaller SRL and surface area in the finest diameter class of C. aggregatus. The root structure of Eleusine indica was not significantly affected by crude oil. Higher specific root surface area was related to higher degradation of petroleum hydrocarbons found in previous studies.