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Serial analysis of gene expression in sugarcane (Saccharum spp.) leaves revealed alternative C4 metabolism and putative antisense transcripts

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Abstract

Sugarcane (Saccharum spp.) is a highly efficient biomass and sugar producing crop. Leaf reactions have been considered as potential rate-limiting step for sucrose accumulation in sugarcane stalks. To characterize the sugarcane leaf transcriptome, field-grown mature leaves from cultivar “SP80-3280” were analyzed using Serial Analysis of Gene Expression (SAGE). From 480 sequenced clones, 9,482 valid tags were extracted, with 5,227 unique sequences, from which 3,659 (70%) matched at least a sugarcane assembled sequence (SAS) with putative function; while 872 tags (16.7%) matched SAS with unknown function; 523 (10%) matched SAS without a putative annotation; and only 173 (3.3%) did not match any sugarcane ESTs. Based on gene ontology (GO), photosystem (PS) I reaction center was identified as the most frequent gene product location, followed by the remaining sites of PS I, PS II and thylakoid complexes. For metabolic processes, photosynthesis light harvesting complexes; carbon fixation; and chlorophyll biosynthesis were the most enriched GO-terms. Considering the alternative photosynthetic C4 cycles, tag frequencies related to phosphoenolpyruvate carboxykinase (PEPCK) and aspartate aminotransferase compared to those for NADP+-malic enzyme (NADP-ME) and NADP-malate dehydrogenase, suggested that PEPCK-type decarboxylation appeared to predominate over NADP-ME in mature leaves, although both may occur, opposite to currently assumed in sugarcane. From the unique tag set, 894 tags (17.1%) were assigned as potentially derived from antisense transcripts, while 73 tags (1.4%) were assigned to more than one SAS, suggesting the occurrence of alternative processing. The occurrence of antisense was validated by quantitative reverse transcription amplification. Sugarcane leaf transcriptome provided new insights for functional studies associated with sucrose synthesis and accumulation.

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Abbreviations

ABA:

Abscisic acid

bp:

Base pairs

CAB:

Chlorophyll a/b-binding protein

EST:

Expressed sequence tag

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GLGI:

Generation of longer cDNA fragments for gene identification

GO:

Gene ontology

GRBP:

Glycine-rich RNA-binding proteins

MPSS:

Massive parallel signature sequencing

MTN:

Metallothionein

NAD:ME:

NAD+-malic enzyme

NADP-ME:

NADP+-malic enzyme

NMDH:

NADP-malate dehydrogenase

PEPCK:

Phosphoenolpyruvate carboxykinase

PAGE:

Polyacrylamide gel electrophoresis

PS:

Photosystem

PCR:

Polymerase chain reaction

RT-qPCR:

Quantitative reverse transcription polymerase chain reaction

SAGE:

Serial analysis of gene expression

SAS:

Sugarcane assembled sequences

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Acknowledgements

Authors wish to thank Dr Eugenio Cesar Ulian, from “Centro de Tecnologia Canavieira”, for providing plant material; Renato Vicentini and Prof Marcelo Menossi (CBMEG, UNICAMP) for support in bioinformatics; FAPESP for financial support (02/04024-7; 02/04025-7) and CNPq. Technical assistance by Angela Artero, Danielle Scotton and Jeanne Machado was greatly appreciated. We wish to thank anonymous reviewers whose comments greatly improved the manuscript.

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  1. Laboratório de Melhoramento de Plantas, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 303, CP 96, Piracicaba, SP, 13400-970, Brazil

    Tercilio Calsa Jr & Antonio Figueira

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  1. Tercilio Calsa Jr
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Correspondence to Antonio Figueira.

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11103_2006_9121_MOESM1_ESM.pdf

Genes chosen to be validated by quantitative reverse transcription amplification, with respective tentative consensus identification as found in TIGR (http://www.tigr.org); annotation; primer sequences; and expected product size (PDF 103 kb)

ESM2 (XLS 23 kb)

ESM3 (XLS 20 kb)

Fig4

Alternative C4-cycle and carbon fixation metabolic pathways, adapted from Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg). Enzymes are named according to EC number: EC 4.1.1.31: phosphoenolpyruvate carboxylase; EC 1.1.1.82: NADP+-malate dehydrogenase; EC 1.1.1.40: malate dehydrogenase; EC 2.7.9.1 pyruvate, phosphate dikinase; EC 2.6.1.2: alanine transaminase; EC 2.6.1.1: aspartate transaminase; EC 4.1.1.49: phosphoenolpyruvate carboxykinase; EC 2.7.1.40: phosphoenolpyruvate kinase; EC 2.6.1.1: aspartate transaminase; EC 1.1.1.37: malate dehydrogenase; EC 1.1.1.39: NAD-malic enzyme; EC 4.1.1.39: ribulose-bisphosphate carboxylase; EC 2.7.2.3: phosphoglycerate kinase; EC 1.2.1.13: NADP-glyceraldehyde-3-phosphate dehydrogenase; EC 4.1.2.13: fructose-bisphosphate aldolase; EC 5.3.1.1: triose phosphoisomerase ; EC 3.1.3.11: fructose 1,6-bisphosphate 1-phosphatase; EC 2.2.1.1: transketolase; EC 4.1.2.9: phosphoketolase; EC 4.1.2.13: fructose 1,6-diphosphate aldolase; EC 3.1.3.37: sedoheptulose 1,7-diphospate phosphatase; EC 2.7.1.14: sedoheptulokinase; EC 2.2.1.1: transketolase; EC 5.3.1.6: ribose-5-phosphate isomerase; EC 5.1.3.1: ribulose-phosphate 3-epimerase; EC 4.1.2.9: phosphoketolase; EC 2.7.1.19: ribulose-5-phosphate kinase (GIF 143 kb)

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Calsa, T., Figueira, A. Serial analysis of gene expression in sugarcane (Saccharum spp.) leaves revealed alternative C4 metabolism and putative antisense transcripts. Plant Mol Biol 63, 745–762 (2007). https://doi.org/10.1007/s11103-006-9121-z

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