Mix-method toolbox for monitoring greenhouse gas production and microbiome responses to soil amendments.

In this study, we adopt an interdisciplinary approach, integrating agronomic field experiments with soil chemistry, molecular biology techniques, and statistics to investigate the impact of organic residue amendments, such as vinasse (a by-product of sugarcane ethanol production), on soil microbiome and greenhouse gas (GHG) production. The research investigates the effects of distinct disturbances, including organic residue application alone or combined with inorganic N fertilizer on the environment. The methods assess soil microbiome dynamics (composition and function), GHG emissions, and plant productivity. Detailed steps for field experimental setup, soil sampling, soil chemical analyses, determination of bacterial and fungal community diversity, quantification of genes related to nitrification and denitrification pathways, measurement and analysis of gas fluxes (N2O, CH4, and CO2), and determination of plant productivity are provided. The outcomes of the methods are detailed in our publications (Lourenço et al., 2018a; Lourenço et al., 2018b; Lourenço et al., 2019; Lourenço et al., 2020). Additionally, the statistical methods and scripts used for analyzing large datasets are outlined. The aim is to assist researchers by addressing common challenges in large-scale field experiments, offering practical recommendations to avoid common pitfalls, and proposing potential analyses, thereby encouraging collaboration among diverse research groups.•Interdisciplinary methods and scientific questions allow for exploring broader interconnected environmental problems.•The proposed method can serve as a model and protocol for evaluating the impact of soil amendments on soil microbiome, GHG emissions, and plant productivity, promoting more sustainable management practices.•Time-series data can offer detailed insights into specific ecosystems, particularly concerning soil microbiota (taxonomy and functions).

Carbon and Nutrients from Organic Residues Modulate the Dynamics of Prokaryotic and Fungal Communities.

Inputs of carbon (C) and nutrients from organic residues may select specific microbes and shape the soil microbial community. However, little is known about the abiotic filtering of the same residues with different nutrient concentrations applied to the soil. In our study, we explored how applying organic residue, vinasse, as fertilizer in its natural state (V) versus its concentrated form (CV) impacts soil microbiota. We conducted two field experiments, evaluating soil prokaryotic and fungal communities over 24 and 45 days with vinasse (V or CV) plus N fertilizer. We used 16S rRNA gene and ITS amplicon sequencing. Inorganic N had no significant impact on bacterial and fungal diversity compared to the control. However, the varying concentrations of organic C and nutrients in vinasse significantly influenced the soil microbiome structure, with smaller effects observed for V compared to CV. Prokaryotic and fungal communities were not correlated (co-inertia: RV coefficient = 0.1517, p = 0.9708). Vinasse did not change the total bacterial but increased the total fungal abundance. A higher C input enhanced the prokaryotic but reduced the fungal diversity. Our findings highlight vinasse's role as an abiotic filter shaping soil microbial communities, with distinct effects on prokaryotic and fungal communities. Vinasse primarily selects fast-growing microorganisms, shedding light on the intricate dynamics between organic residues, nutrient concentrations, and soil microbes.

Microbiome resilience of Amazonian forests: Agroforest divergence to bacteria and secondary forest succession convergence to fungi.

An alarming and increasing deforestation rate threatens Amazon tropical ecosystems and subsequent degradation due to frequent fires. Agroforestry systems (AFS) may offer a sustainable alternative, reportedly mimicking the plant-soil interactions of the natural mature forest (MF). However, the role of microbial community in tropical AFS remains largely unknown. This knowledge is crucial for evaluating the sustainability of AFS and practices given the key role of microbes in the aboveground-belowground interactions. The current study, by comparing different AFS and successions of secondary and MFs, showed that AFS fostered distinct groups of bacterial community, diverging from the MFs, likely a result of management practices while secondary forests converged to the same soil microbiome found in the MF, by favoring the same groups of fungi. Model simulations reveal that AFS would require profound changes in aboveground biomass and in soil factors to reach the same microbiome found in MFs. In summary, AFS practices did not result in ecosystems mimicking natural forest plant-soil interactions but rather reshaped the ecosystem to a completely different relation between aboveground biomass, soil abiotic properties, and the soil microbiome.

Rhizosphere microbiome response to host genetic variability: a trade-off between bacterial and fungal community assembly.

Rhizosphere microbial community composition is strongly influenced by plant species and cultivar. However, our understanding of the impact of plant cultivar genetic variability on microbial assembly composition remains limited. Here, we took advantage of vegetatively propagated chrysanthemum (Chrysanthemum indicum L.) as a plant model and induced roots in five commercial cultivars: Barolo, Chic, Chic 45, Chic Cream and Haydar. We observed strong rhizosphere selection for the bacterial community but weaker selection for the fungal community. The genetic distance between cultivars explained 42.83% of the total dissimilarity between the bacteria selected by the different cultivars. By contrast, rhizosphere fungal selection was not significantly linked to plant genetic dissimilarity. Each chrysanthemum cultivar selected unique bacterial and fungal genera in the rhizosphere. We also observed a trade-off in the rhizosphere selection of bacteria and fungi in which the cultivar with the strongest selection of fungal communities showed the weakest bacterial selection. Finally, bacterial and fungal family taxonomic groups consistently selected by all cultivars were identified (bacteria Chitinophagaceae, Beijerinckiaceae and Acidobacteriaceae, and fungi Pseudeurotiaceae and Chrysozymaceae). Taken together, our findings suggest that chrysanthemum cultivars select distinct rhizosphere microbiomes and share a common core of microbes partially explained by the genetic dissimilarity between cultivars.

Plant-Growth Endophytic Bacteria Improve Nutrient Use Efficiency and Modulate Foliar N-Metabolites in Sugarcane Seedling.

Beneficial plant-microbe interactions lead to physiological and biochemical changes that may result in plant-growth promotion. This study evaluated the effect of the interaction between sugarcane and endophytic bacterial strains on plant physiological and biochemical responses under two levels of nitrogen (N) fertilization. Six strains of endophytic bacteria, previously selected as plant growth-promoting bacteria (PGPB), were used to inoculate sugarcane mini stalks, with and without N fertilization. After 45 days, biomass production; shoot nutrient concentrations; foliar polyamine and free amino acid profiles; activities of nitrate reductase and glutamine synthase; and the relative transcript levels of the GS1, GS2, and SHR5 genes in sugarcane leaves were determined. All six endophytic strains promoted sugarcane growth, increasing shoot and root biomass, plant nutritional status, and the use efficiency of most nutrients. The inoculation-induced changes at the biochemical level altered the foliar free amino acid and polyamine profiles, mainly regarding the relative concentrations of citrulline, putrescine, glycine, alanine, glutamate, glutamine, proline, and aspartate. The transcription of GS1, GS2, and SHR5 was higher in the N fertilized seedlings, and almost not altered by endophytic bacterial strains. The endophytic strains promoted sugarcane seedlings growth mainly by improving nutrient efficiency. This improvement could not be explained by their ability to induce the production of amino acid and polyamine composts, or GS1, GS2, and SHR5, showing that complex interactions may be associated with enhancement of the sugarcane seedlings' performance by endophytic bacteria. The strains demonstrated biotechnological potential for sugarcane seedling production.

Methods to Identify Soil Microbial Bioindicators of Sustainable Management of Bioenergy Crops.

Here we describe a suite of methods to identify potential taxonomic and functional soil microbial indicators of soil quality and plant health in biofuel crops in various areas and land types. This approach draws on tools to assess microbial diversity, greenhouse gas fluxes, and soil physicochemical properties in bioenergy cropping systems. Integrative statistical models are then used to identify potential microbial indicators for sustainable management of bioenergy crops.

Unraveling the xylanolytic potential of Acidobacteria bacterium AB60 from Cerrado soils.

The presence of genes for glycosyl hydrolases in many Acidobacteria genomes indicates an important role in the degradation of plant cell wall material. Acidobacteria bacterium AB60 was obtained from Cerrado oligotrophic soil in Brazil, where this phylum is abundant. The 16S rRNA gene analyses showed that AB60 was closely related to the genera Occallatibacter and Telmatobacter. However, AB60 grew on xylan as carbon source, which was not observed in Occallatibacter species; but growth was not detected on medium containing carboxymethyl cellulose, as observed in Telmatobacter. Nevertheless, the genome analysis of AB60 revealed genes for the enzymes involved in cellulose as well as xylan degradation. In addition to enzymes involved in xylan degradation, α-l-rhamnosidase was detected in the cultures of AB60. Functional screening of a small-insert genomic library did not identify any clones capable of carboxymethyl cellulose degradation, but open reading frames coding α-l-arabinofuranosidase and α-l-rhamnosidase were present in clones showing xylan degradation halos. Both enzymes act on the lateral chains of heteropolymers such as pectin and some hemicelluloses. These results indicate that the hydrolysis of α-linked sugars may offer a metabolic niche for slow-growing Acidobacteria, allowing them to co-exist with other plant-degrading microbes that hydrolyze ß-linked sugars from cellulose or hemicellulose backbones.

Dynamics and resilience of soil mycobiome under multiple organic and inorganic pulse disturbances.

Disturbances in soil can cause short-term soil changes, consequently changes in microbial community what may result in long-lasting ecological effects. Here, we evaluate how multiple pulse disturbances effect the dynamics and resilience of fungal community, and the co-occurrence of fungal and bacterial communities in a 389 days field experiment. We used soil under sugarcane cultivation as soil ecosystem model, and organic residue (vinasse - by-product of sugarcane ethanol production) combined or not with inorganic (organic residue applied 30 days before or together with mineral N fertilizer) amendments as disturbances. Application of organic residue alone as a single disturbance or 30 days prior to a second disturbance with mineral N resulted in similar changes in the fungal community. The simultaneous application of organic and mineral N as a single pulse disturbance had the greatest impact on the fungal community. Organic amendment increased the abundance of saprotrophs, fungal species capable of denitrification, and fungi described to have copiotrophic and oligotrophic lifestyles. Furthermore, the changes in the fungal community were not correlated with the changes in the bacterial community. The fungal community was neither resistant nor resilient to organic and inorganic disturbances over the one-year sampling period. Our findings provide insights on the immediate and delayed responses of the fungal community over one year to disturbance by organic and inorganic amendments.

Microbial N-cycling gene abundance is affected by cover crop specie and development stage in an integrated cropping system.

Grasses of the Urochloa genus have been widely used in crop-livestock integration systems or as cover crops in no-till systems such as in rotation with maize. Some species of Urochloa have mechanisms to reduce nitrification. However, the responses of microbial functions in crop-rotation systems with grasses and its consequence on soil N dynamics are not well-understood. In this study, the soil nitrification potential and the abundance of ammonifying microorganisms, total bacteria and total archaea (16S rRNA gene), nitrogen-fixing bacteria (NFB, nifH), ammonia-oxidizing bacteria (AOB, amoA) and archaea (AOA, amoA) were assessed in soil cultivated with ruzigrass (Urochloa ruziziensis), palisade grass (Urochloa brizantha) and Guinea grass (Panicum maximum). The abundance of ammonifying microorganisms was not affected by ruzigrass. Ruzigrass increased the soil nitrification potential compared with palisade and Guinea grass. Ruzigrass increased the abundance of N-fixing microorganisms at the middle and late growth stages. The abundances of nitrifying microorganisms and N-fixers in soil were positively correlated with the soil N-NH4+ content. Thus, biological nitrogen fixation might be an important input of N in systems of rotational production of maize with forage grasses. The abundance of microorganisms related to ammonification, nitrification and nitrogen fixing and ammonia-oxidizing archea was related to the development stage of the forage grass.

Assessing nickel tolerance of bacteria isolated from serpentine soils.

Serpentine soils present unique characteristics such as a low Ca/Mg ratio, low concentration of nutrients, and a high concentration of heavy metals, especially nickel. Soil bacterial isolates from an ultramafic complex located in the tropical savanna known as the Brazilian Cerrado were studied. Nickel-tolerant bacteria were obtained, and their ability to remove nickel from a culture medium was assessed. Bacterial isolates presented higher tolerance to nickel salts than previously reported for bacteria obtained from serpentine environments in other regions of the world. In addition, the quantification of nickel in cell pellets indicated that at least four isolates may adsorb soluble forms of nickel. It is expected that information gathered in this study will support future efforts to exploit serpentine soil bacteria for biotechnological processes involving nickel decontamination from environmental samples.

Strategies to mitigate the nitrous oxide emissions from nitrogen fertilizer applied with organic fertilizers in sugarcane.

Vinasse is a major byproduct of the sugarcane biofuel industry, recycled in the fields. However, there is evidence that the application of vinasse with mineral nitrogen (N) fertilizers in sugarcane enhances the emission of greenhouse gases (GHGs). Therefore, strategies are needed to decrease the environmental impacts caused by both inputs. We carried out three sugarcane field experiments by applying N fertilizer (ammonium nitrate) with types of vinasses (concentrated-CV and standard-V) in different combinations (vinasses with N fertilizer and vinasses one month before or after mineral N fertilization). The gases nitrous oxide (N2O), carbon dioxide (CO2), and methane (CH4) were measured in one experiment fertilized in the beginning (fall/winter = dry season) and two experiments fertilized in the end (spring = rainy season) of the harvest season. Sugarcane fields were sinks rather than sources of CH4, while total carbon emitted as CO2 was similar between seasons and treatments. The effect of mineral fertilization and vinasses (CV and V) on N2O emissions was highly dependent on soil moisture (rain events). The N2O-N fertilizer emission factor (EF) varied from 0.07% to 0.51%, whereas the average EF of V and CV were 0.66% and 0.34%, respectively. On average across the three experiments, the combination of vinasse (CV or V) with N fertilizer increased the N2O emissions 2.9-fold compared to that of N fertilizer alone. For CV + N, the EF was 0.94% of the applied N and 0.23% of the ammonium nitrate-N, and for V + N (EF = 0.47%), increased emissions were observed in two out of three experiments. The strategy of anticipating or postponing vinasse application by one month with respect to mineral N reduced the N2O emissions by 51% for CV, but not for V. Therefore, to avoid boosting N2O emissions, we suggest applying vinasses (CV and V) before or after mineral N fertilization.

Exploitation of new endophytic bacteria and their ability to promote sugarcane growth and nitrogen nutrition.

Few studies have evaluated endophytic bacteria in relation to plant growth promotion, nitrogen uptake and biological control. The aim of this study was to molecularly and physiologically characterize thirteen endophytic bacteria strains, evaluate their biological control properties and their ability to promote plant growth and plant N nutrition. All the strains produced indole acetic acid and promoted increase of plant biomass, N accumulative amount and N-use efficiency index. None of the strains carries the nifH gene. Four strains stimulated plant nitrate reductase activity, four solubilized phosphate, nine produced siderophores and none produced HCN. Seven strains inhibited Bipolaris sacchari growth and one was antagonistic to Ceratocystis paradoxa. The pathogens were inhibited by the production of diffusible and volatile metabolites by the bacterial strains. Moreover, this is the first study to demonstrate the effect of Delftia acidovorans on sugarcane plant growth, nitrogen metabolism improvement and antagonism to B. sacchari. The most efficient strains in promoting plant growth and exhibiting antagonistic activities towards fungal pathogens were Herbaspirillum frinsingense (IAC-BECa-152) and three Pantoea dispersa strains (IAC-BECa-128, IAC-BECa-129, and IAC-BECa-132). These bacteria show potential to be used as inoculants for sustainable agricultural management, mainly at the seedling production phase.

Recycling organic residues in agriculture impacts soil-borne microbial community structure, function and N2O emissions.

Recycling residues is a sustainable alternative to improve soil structure and increase the stock of nutrients. However, information about the magnitude and duration of disturbances caused by crop and industrial wastes on soil microbial community structure and function is still scarce. The objective of this study was to investigate how added residues from industry and crops together with nitrogen (N) fertiliser affect the microbial community structure and function, and nitrous oxide (N2O) emissions. The experimental sugarcane field had the following treatments: (I) control with nitrogen, phosphorus, and potassium (NPK), (II) sugarcane straw with NPK, (III) vinasse (by-product of ethanol industry) with NP, and (IV) vinasse plus sugarcane straw with NP. Soil samples were collected on days 1, 3, 6, 11, 24 and 46 of the experiment for DNA extraction and metagenome sequencing. N2O emissions were also measured. Treatments with straw and vinasse residues induced changes in soil microbial composition and potential functions. The change in the microbial community was highest in the treatments with straw addition with functions related to decomposition of different ranges of C-compounds overrepresented while in vinasse treatment, the functions related to spore-producing microorganisms were overrepresented. Furthermore, all additional residues increased microorganisms related to the nitrogen metabolism and vinasse with straw had a synergetic effect on the highest N2O emissions. The results highlight the importance of residues and fertiliser management in sustainable agriculture.

Optimized medium culture for Acidobacteria subdivision 1 strains.

Members of subdivision 1 of the phylum Acidobacteria were grown at different pH values in a new medium formulation named PSYL 5, which includes sucrose as a carbon source and other compounds (such as KH2PO4 and MgSO4.7H2O). Growth rate was nearly constant at pH 5.0 and declined at pH 3-4 and 6-7. However, it was found that effects involving good carbon/nitrogen ratios and pH on the growth of the members of Acidobacteria subdivision 1 were significant, and the strongest effect of these conditions was at pH 5.0. In addition, incubation time of 48, 72, 96 and 120 h was shorter than that described previously for members of Acidobacteria subdivision 1 on solid laboratory media.

Fungal Community Assembly in the Amazonian Dark Earth.

Here, we compare the fungal community composition and diversity in Amazonian Dark Earth (ADE) and the respective non-anthropogenic origin adjacent (ADJ) soils from four different sites in Brazilian Central Amazon using pyrosequencing of 18S ribosomal RNA (rRNA) gene. Fungal community composition in ADE soils were more similar to each other than their ADJ soils, except for only one site. Phosphorus and aluminum saturation were the main soil chemical factors contributing to ADE and ADJ fungal community dissimilarities. Differences in fungal richness were not observed between ADE and ADJ soil pairs regarding to the most sites. In general, the most dominant subphyla present in the soils were Pezizomycotina, Agaricomycotina, and Mortierellomycotina. The most abundant operational taxonomic units (OTUs) in ADE showed similarities with the entomopathogenic fungus Cordyceps confragosa and the saprobes Fomitopsis pinicola, Acremonium vitellinum, and Mortierellaceae sp., whereas OTUs similar to Aspergillus niger, Lithothelium septemseptatum, Heliocephala gracillis, and Pestalosphaeria sp. were more abundant in ADJ soils. Differences in fungal community composition were associated to soil chemical factors in ADE (P, Ca, Zn, Mg, organic matter, sum of bases, and base saturation) and ADJ (Al, potential acidity, Al saturation, B, and Fe) soils. These results contribute to a deeper view of the fungi communities in ADE and open new perspectives for entomopathogenic fungi studies.

Verrucomicrobial community structure and abundance as indicators for changes in chemical factors linked to soil fertility.

Here we show that verrucomicrobial community structure and abundance are extremely sensitive to changes in chemical factors linked to soil fertility. Terminal restriction fragment length polymorphism fingerprint and real-time quantitative PCR assay were used to analyze changes in verrucomicrobial communities associated with contrasting soil nutrient conditions in tropical regions. In case study Model I ("Slash-and-burn deforestation") the verrucomicrobial community structures revealed disparate patterns in nutrient-enriched soils after slash-and-burn deforestation and natural nutrient-poor soils under an adjacent primary forest in the Amazonia (R = 0.819, P = 0.002). The relative proportion of Verrucomicrobia declined in response to increased soil fertility after slash-and-burn deforestation, accounting on average, for 4 and 2 % of the total bacterial signal, in natural nutrient-poor forest soils and nutrient-enriched deforested soils, respectively. In case study Model II ("Management practices for sugarcane") disparate patterns were revealed in sugarcane rhizosphere sampled on optimal and deficient soil fertility for sugarcane (R = 0.786, P = 0.002). Verrucomicrobial community abundance in sugarcane rhizosphere was negatively correlated with soil fertility, accounting for 2 and 5 % of the total bacterial signal, under optimal and deficient soil fertility conditions for sugarcane, respectively. In nutrient-enriched soils, verrucomicrobial community structures were related to soil factors linked to soil fertility, such as total nitrogen, phosphorus, potassium and sum of bases, i.e., the sum of calcium, magnesium and potassium contents. We conclude that community structure and abundance represent important ecological aspects in soil verrucomicrobial communities for tracking the changes in chemical factors linked to soil fertility under tropical environmental conditions.

Amazonian dark Earth and plant species from the Amazon region contribute to shape rhizosphere bacterial communities.

Amazonian Dark Earths (ADE) or Terra Preta de Índio formed in the past by pre-Columbian populations are highly sustained fertile soils supported by microbial communities that differ from those extant in adjacent soils. These soils are found in the Amazon region and are considered as a model soil when compared to the surrounding and background soils. The aim of this study was to assess the effects of ADE and its surrounding soil on the rhizosphere bacterial communities of two leguminous plant species that frequently occur in the Amazon region in forest sites (Mimosa debilis) and open areas (Senna alata). Bacterial community structure was evaluated using terminal restriction fragment length polymorphism (T-RFLP) and bacterial community composition by V4 16S rRNA gene region pyrosequencing. T-RFLP analysis showed effect of soil types and plant species on rhizosphere bacterial community structure. Differential abundance of bacterial phyla, such as Acidobacteria, Actinobacteria, Verrucomicrobia, and Firmicutes, revealed that soil type contributes to shape the bacterial communities. Furthermore, bacterial phyla such as Firmicutes and Nitrospira were mostly influenced by plant species. Plant roots influenced several soil chemical properties, especially when plants were grown in ADE. These results showed that differences observed in rhizosphere bacterial community structure and composition can be influenced by plant species and soil fertility due to variation in soil attributes.

Sulphur-oxidizing and sulphate-reducing communities in Brazilian mangrove sediments.

Mangrove soils are anaerobic environments rich in sulphate and organic matter. Although the sulphur cycle is one of the major actors in this ecosystem, little is known regarding the sulphur bacteria communities in mangrove soils. We investigated the abundance, composition and diversity of sulphur-oxidizing (SOB) and sulphate-reducing (SRB) bacteria in sediments from three Brazilian mangrove communities: two contaminated, one with oil (OilMgv) and one with urban waste and sludge (AntMgv), and one pristine (PrsMgv). The community structures were assessed using quantitative real-time polymerase chain reaction (qPCR), polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries, using genes for the enzymes adenosine-5'-phosphosulphate reductase (aprA) and sulphite reductase (Dsr) (dsrB). The abundance for qPCR showed the ratio dsrB/aprA to be variable among mangroves and higher according to the gradient observed for oil contamination in the OilMgv. The PCR-DGGE patterns analysed by Nonmetric Multidimensional Scaling revealed differences among the structures of the three mangrove communities. The clone libraries showed that Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria were the most abundant groups associated with sulphur cycling in mangrove sediments. We conclude that the microbial SOB and SRB communities in mangrove soils are different in each mangrove forest and that such microbial communities could possibly be used as a proxy for contamination in mangrove forests.

Soil-borne bacterial structure and diversity does not reflect community activity in Pampa biome.

The Pampa biome is considered one of the main hotspots of the world's biodiversity and it is estimated that half of its original vegetation was removed and converted to agricultural land and tree plantations. Although an increasing amount of knowledge is being assembled regarding the response of soil bacterial communities to land use change, to the associated plant community and to soil properties, our understanding about how these interactions affect the microbial community from the Brazilian Pampa is still poor and incomplete. In this study, we hypothesized that the same soil type from the same geographic region but under distinct land use present dissimilar soil bacterial communities. To test this hypothesis, we assessed the soil bacterial communities from four land-uses within the same soil type by 454-pyrosequencing of 16S rRNA gene and by soil microbial activity analyzes. We found that the same soil type under different land uses harbor similar (but not equal) bacterial communities and the differences were controlled by many microbial taxa. No differences regarding diversity and richness between natural areas and areas under anthropogenic disturbance were detected. However, the measures of microbial activity did not converge with the 16S rRNA data supporting the idea that the coupling between functioning and composition of bacterial communities is not necessarily correlated.

Identification of 14-3-3-like protein in sugarcane (Saccharum officinarum)

Seqüências completas de três cDNAs que codificam proteínas 14-3-3 de cana-de-açúcar (Saccharum officinarum) foram encontradas no projeto genoma EST de cana-de-açúcar. As proteínas codificadas por essas seqüências foram identificadas baseando-se no agrupamento de ESTs de cana-de-açúcar e, três "clusters" (SCCCLR1022 DO5.g, SCCCRZl001D02.g e SCBFLR1026E02.g) foram similares a proteínas 14-3-3 de outras monocotiledôneas. O cluster SCCCLR1022DO5.g apresentou similaridade de 99 por cento com a proteína 14-3-3 de milho (gi/1345587) e, os clusters SCCCRZ1001D02.g e SCBFLR1026E02.g foram similares a proteína 14-3-3 de arroz (gi/7435022), 96 por cento e 94 por cento, respectivamente. Embora proteínas 14-3-3 têm sido relatadas como sendo específicas a organismos, tecidos e órgäos, todos os "clusters" de cana-de-açúcar correspondentes a 14-3-3 foram provenientes de cDNAs advindos de diferentes tecidos.