SummaryThe chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially speci®es a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild-type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild-type plastid genome occurred in all transplastomic lines. These ®ndings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the ®rst higher plant chloroplast genes identi®ed as being indispensable.

Mechanisms related to biotic interactions, such as pathogen attack, herbivory and symbiosis are important challenges to higher plants and have been widely studied especially for breeding purposes. The present review focuses on a special category of defense molecules, the plant antimicrobial peptides, providing an overview of their main molecular features and structures.

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 C(4) 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.

Sugarcane (Saccharum spp.) is the most promising crop for renewable energy. Among the diverse stresses that affect plant productivity, drought stress frequently causes losses in sugarcane fields. Although several studies have addressed plant responses to drought using controlled environments, plant responses under field conditions are largely unknown. Recently, microRNA (miRNA)-mediated post-transcriptional regulation has been described as an important and decisive component in vegetal development and stress resistance modulation. The role of miRNAs in sugarcane responses to drought under field conditions is currently not known. Two sugarcane cultivars differing in drought tolerance were grown in the field with and without irrigation (rainfed) for 7 months. By using small RNA deep sequencing, we were able to identify 18 miRNA families comprising 30 mature miRNA sequences. Among these families, we found 13 mature miRNAs that were differentially expressed in drought-stressed plants. Seven miRNAs were differentially expressed in both cultivars. The target genes for many of the differentially expressed mature miRNAs were predicted, and some of them were validated by quantitative reverse transcription PCR. Among the targets, we found transcription factors, transporters, proteins associated with senescence, and proteins involved with flower development. All of these data increase our understanding of the role of miRNAs in the complex regulation of drought stress in field-grown sugarcane, providing valuable tools to develop new sugarcane cultivars tolerant to drought stress.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-012-1795-7) contains supplementary material, which is available to authorized users.

Soil salinity is a limiting factor to sugar cane crop development, although in general plants present variable mechanisms of tolerance to salinity stress. The molecular basis underlying these mechanisms can be inferred by using proteomic analysis. Thus, the objective of this work was to identify differentially expressed proteins in sugar cane plants submitted to salinity stress. For that, a greenhouse experiment was established with four sugar cane varieties and two salt conditions, 0 mM (control) and 200 mM NaCl. Physiological and proteomics analyses were performed after 2 and 72 h of stress induction by salt. Distinct physiological responses to salinity stress were observed in the varieties and linked to tolerance mechanisms. In proteomic analysis, the roots soluble protein fraction was extracted, quantified, and analyzed through bidimensional electrophoresis. Gel images analyses were done computationally, where in each contrast only one variable was considered (salinity condition or variety). Differential spots were excised, digested by trypsin, and identified via mass spectrometry. The tolerant variety RB867515 showed the highest accumulation of proteins involved in growth, development, carbohydrate and energy metabolism, reactive oxygen species metabolization, protein protection, and membrane stabilization after 2 h of stress. On the other hand, the presence of these proteins in the sensitive variety was verified only in stress treatment after 72 h. These data indicate that these stress responses pathways play a role in the tolerance to salinity in sugar cane, and their effectiveness for phenotypical tolerance depends on early stress detection and activation of the coding genes expression.