Juliana de Maria Felix

Sugarcane genes associated with sucrose content

Background -Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcanes sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants.Results -We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways.Conclusion -Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.

Fly‐pollinated Pleurothallis (Orchidaceae) species have high genetic variability: evidence from isozyme markers

We conducted an isozyme study in 22 populations of five Pleurothallis (Orchidaceae) species (12 loci in nine enzymatic systems). The genetic variability in all populations is surprisingly high (P = 58-83%, A = 2.1-3.8, H(e) = 0.25-0.43) in spite of the fact that the five species are pollinated by small flies whose behavior enables self-pollination. We suggest that self-incompatibility, inbreeding depression, and mechanical barriers that prevent self-pollination in these species are responsible for the maintainance of the high genetic variability. These traits are uncommon in Orchidaceae, but have been observed in these and some other species pollinated by flies or other pollinators with behavior that facilitates self-pollination. The genetic similarity among conspecific populations is also high for species with very short-range flying pollinators. Only one population of P. teres presented values of genetic similarity lower than usually observed in allopatric conspecific populations. Morphology, however, does not support its segregation as a new taxon. All species can be recognized by their enzymatic patterns, and the results agree with recently proposed taxonomic realignments. Conversely, the supposed affinities among these species based on floral morphology are not supported, and we hypothesize that it may be due to convergence in species with similar pollinators.

Identification of new ABA- and MEJA-activated sugarcane bZIP genes by data mining in the SUCEST database.

Sugarcane is generally propagated by cuttings of the stalk containing one or more lateral buds, which will develop into a new plant. The transition from the dormant into the active stage constitutes a complex phenomenon characterized by changes in accumulation of phytohormones and several other physiological aspects. Abscisic acid (ABA) and methyl-jasmonate (MeJA) are major signaling molecules, which influence plant development and stress responses. These plant regulators modulate gene expression with the participation of many transcriptional factors. Basic leucine zipper proteins (bZIPs) form a large family of transcriptional factors involved in a variety of plant physiological processes, such as development and responses to stress. Query sequences consisting of full-length protein sequence of each of the Arabidopsis bZIP families were utilized to screen the sugarcane EST database (SUCEST) and 86 sugarcane assembled sequences (SAS) coding for bZIPs were identified. cDNA arrays and RNA-gel blots were used to study the expression of these sugarcane bZIP genes during early plantlet development and in response to ABA and MeJA. Six bZIP genes were found to be differentially expressed during development. ABA and MeJA modulated the expression of eight sugarcane bZIP genes. Our findings provide novel insights into the expression of this large protein family of transcriptional factors in sugarcane.

Expression Profile of Signal Transduction Components in a Sugarcane Population Segregating for Sugar Content

Sucrose is the major product of photosynthesis in many higher plants. It is transported from the source tissue through the phloem to various sink tissues to support plant growth, development and reproduction. Knowledge on the signal transduction pathways involved in sucrose synthesis in mature leaves is limited. Using a microarray approach, we analyzed the expression profiles of 1920 sugarcane genes encoding signal transduction elements, transcription factors and stress-related proteins. We used individuals from a population segregating for sugar content and gene expression profiles were obtained from seven individuals with highest and seven with lowest sugar content. Surprisingly, from the 24 differentially expressed genes, 19 were more expressed in plants containing low-sugar content. Three of these genes encoded 14-3-3 like proteins, which have been found to reduce sucrose phosphate synthase (SPS) activity. Another encoded an SNF1-related protein similar to a protein kinase that phosphorylates SPS in vitro making it a target for the interaction with 14-3-3 proteins. The up-regulation of eight stress related genes in the lower sugar content plants supports a view that sugar levels modulate a complex signal transduction network that seems to involve responses that are related to stress. Evidence that hormone signaling is related to the sucrose content was also found. These data reinforced the usefulness of genomic approaches to uncover how sucrose metabolism can be regulated in sugarcane.

Prospecting sugarcane genes involved in aluminum tolerance

Aluminum is one of the major factors that affect plant development in acid soils, causing a substantial reduction in yield in many crops. In South America, about 66% of the land surface is made up of acid soils where high aluminum saturation is one of the main limiting factors for agriculture. The biochemical and molecular basis of aluminum tolerance in plants is far from being completely understood despite a growing number of studies, and in the specific case of sugarcane there are virtually no reports on the effects of gene regulation on aluminum stress. The objective of the work presented in this paper was to prospect the sugarcane expressed sequence tag (SUCEST) data bank for sugarcane genes related to several biochemical pathways known to be involved in the responses to aluminum toxicity in other plant species and yeast. Sugarcane genes similar to most of these genes were found, including those coding for enzymes that alleviate oxidative stress or combat infection by pathogens and those which code for proteins responsible for the release of organic acids and signal transducers. The role of these genes in aluminum tolerance mechanisms is reviewed. Due to the high level of genomic conservation in related grasses such as maize, barley, sorghum and sugarcane, these genes may be valuable tools which will help us to better understand and to manipulate aluminum tolerance in these species.

Characterization of a sugarcane (Saccharum spp.) gene homolog to the brassinosteroid insensitive1-associated receptor kinase 1 that is associated to sugar content.

The present article reports on the characterization of ScBAK1, a leucine-rich repeat receptor-like kinase from sugarcane (Saccharum spp.), expressed predominantly in bundle-sheath cells of the mature leaf and potentially involved in cellular signaling cascades mediated by high levels of sugar in this organ. In this report, it was shown that the ScBAK1 sequence was similar to the brassinosteroid insensitive1-associated receptor kinase1 (BAK1). The putative cytoplasmatic domain of ScBAK1 contains all the amino acids characteristic of protein kinases, and the extracellular domain contains five leucine-rich repeats and a putative leucine zipper. Transcripts of ScBAK1 were almost undetectable in sugarcane roots or in any other sink tissue, but accumulated abundantly in the mature leaves. The ScBAK1 expression was higher in the higher sugar content individuals from a population segregating for sugar content throughout the growing season. In situ hybridization in sugarcane leaves showed that the ScBAK1 mRNA accumulated at much higher levels in bundle-sheath cells than in mesophyll cells. In addition, using biolistic bombardment of onion epidermal cells, it was shown that ScBAK1-GFP fusions were localized in the plasma membrane as predicted for a receptor kinase. All together, the present data indicate that ScBAK1 might be a receptor involved in the regulation of specific processes in bundle-sheath cells and in sucrose synthesis in mature sugarcane leaves.

Evaluation of the performance of different plastics used to seal nylon cDNA arrays

Os arranjos de cDNA sao uma poderosa ferramenta para o estudo de padroes de expressao genica. Os arranjos em membranas de nailon apresentam ainda a vantagem de poderem ser reutilizados diversas vezes. Porem, um ponto bastante delicado em estudos de expressao genica em larga escala e a sensibilidade. No caso de arranjos em membranas de nailon, a deteccao dos sinais pode ser afetada pelo envoltorio plastico utilizado para manter as membranas umidas. Nesse estudo, nos avaliamos os efeitos de cinco tipos de plasticos na transmissao radioativa detectada, no numero de genes com sinal acima da emissao de fundo e na variabilidade dos dados. O plastico produzido com polietileno com 69 μm de espessura apresentou uma forte interferencia na emissao radioativa, bloqueando 68.7% do sinal detectado. Este bloqueio na transmitância diminuiu o numero de genes detectados e aumentou a variabilidade dos dados. Outros plasticos mais finos tiveram resultados melhores. Apesar de plasticos feitos de cloreto de polivinilideno e cloreto de polivinila (ambos com 13 μm de espessura) e polietileno (29 e 7 μm de espessura) terem diferentes niveis de transmitância, todos apresentaram performances semelhantes nos testes realizados. Cloreto de polivinilideno e polietileno com 29 μm de espessura foram os plasticos escolhidos devido a facilidade de manuseio. Para outros tipos de plasticos, e recomendavel realizar um teste de suas performances antes de utiliza-los para envolver membranas de nailon, de forma a obter o maximo de informacao dos experimentos com arranjos de cDNA.

Using macroarrays containing sugarcane ESTs to identify aluminium-induced genes in maize.

Aluminium (A1) toxicity affects the productivity of several crops in acid soils, which comprise up to 40% of arable land in the world. The first and most dramatic symptom of A1 toxicity is the inhibition of root growth. Our group has been involved in the Sugarcane Expressed Sequence Tag (SUCEST) project which has generated around 50,000 genes from several tissues. Based on the high level of nucleotide identity found between sugarcane and maize genes, radiolabeled cDNA probes from maize would be expected to hybridize with the sugarcane ESTs on the arrays. In this work, we have confirmed this hypothesis by showing that sugarcane macroarrays can be used to identify A1-induced genes in maize.