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SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.

SUGARWIN1 and 2 are defense proteins from sugarcane. Their gene expression is known to be induced in response to wound and Diatraea saccharalis damage. Although the recombinant SUGARWIN protein does not affect insect development, it promotes significant morphological and physiological changes in Fusarium verticillioides and Colletotrichum falcatum, which lead to fungal cell death via apoptosis. In this study, we deepen our understanding of the role of SUGARWINs in plant defense and the molecular mechanisms by which these proteins affect fungi by elucidating their molecular targets. Our results show that SUGARWINs play an important role in plant defense against opportunistic pathogens. We demonstrated that SUGARWINs are induced by C. falcatum, and the induction of SUGARWINs can vary among sugarcane varieties. The sugarcane variety exhibiting the highest level of SUGARWIN induction exhibited a considerable reduction in C. falcatum infection. Furthermore, SUGARWIN1 exhibited ribonuclease, chitosanase, and chitinase activity, whereas SUGARWIN2 exhibited only chitosanase activity. This variable enzymatic specificity seems to be the result of divergent amino acid composition within the substrate-binding site.

Many plant genes have their expression modulated by stress conditions. Here, we used Arabidopsis FtsH5 protease, which expression is regulated by light stress, as bait in a yeast two-hybrid screen to search for new proteins involved in the stress response. As a result, we found FIP (FtsH5 Interacting Protein), which possesses an amino proximal cleavable transit peptide, a hydrophobic membrane-anchoring region, and a carboxyl proximal C4-type zinc-finger domain. In vivo experiments using FIP fused to green fluorescent protein (GFP) showed a plastid localization. This finding was corroborated by chloroplast import assays that showed FIP inserted in the thylakoid membrane. FIP expression was down-regulated in plants exposed to high light intensity, oxidative, salt, and osmotic stresses, whereas mutant plants expressing low levels of FIP were more tolerant to these abiotic stresses. Our data shows a new thylakoid-membrane protein involved with abiotic stress response in Arabidopsis thaliana.

Many plant genes have their expression modulated by stress conditions. Here, we used Arabidopsis FtsH5 protease, which expression is regulated by light stress, as bait in a yeast two-hybrid screen to search for new proteins involved in the stress response. As a result, we found FIP (FtsH5 Interacting Protein), which possesses an amino proximal cleavable transit peptide, a hydrophobic membrane-anchoring region, and a carboxyl proximal C4-type zinc-finger domain. In vivo experiments using FIP fused to green fluorescent protein (GFP) showed a plastid localization. This finding was corroborated by chloroplast import assays that showed FIP inserted in the thylakoid membrane. FIP expression was down-regulated in plants exposed to high light intensity, oxidative, salt, and osmotic stresses, whereas mutant plants expressing low levels of FIP were more tolerant to these abiotic stresses. Our data shows a new thylakoid-membrane protein involved with abiotic stress response in Arabidopsis thaliana.

Summary The peptide RALF regulates cell expansion via an as-yet-unknown mechanism. Here, we provide evidence that AtRALF1 may be negatively regulating cell expansion by interfering with the brassinosteroid signalling pathway., Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide’s mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also showed an increase in the number of lateral roots, whereas AtRALF1 overexpression produced the opposite effect. In addition, four AtRALF1-inducible genes were identified: two genes encoding proline-rich proteins (AtPRP1 and AtPRP3), one encoding a hydroxyproline-rich glycoprotein (AtHRPG2), and one encoding a xyloglucan endotransglucosylase (TCH4). These genes were expressed in roots and involved in cell-wall rearrangement, and their induction was concentration dependent. Furthermore, AtRALF1-overexpressing plants were less sensitive to exogenous brassinolide (BL); upon BL treatment, the plants showed no increase in root length and a compromised increase in hypocotyl elongation. In addition, the treatment had no effect on the number of emerged lateral roots. AtRALF1 also induces two brassinosteroid (BR)-downregulated genes involved in the BR biosynthetic pathway: the cytochrome P450 monooxygenases CONSTITUTIVE PHOTOMORPHISM AND DWARFISM (CPD) and DWARF4 (DWF4). Simultaneous treatment with both AtRALF1 and BL caused a reduction in AtRALF1-inducible gene expression levels, suggesting that these signals may compete for components shared by both pathways. Taken together, these results indicate an opposing effect of AtRALF1 and BL, and suggest that RALF’s mechanism of action could be to interfere with the BR signalling pathway.

The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.

Rapid alkalinization factor (RALF) is a peptide signal that plays a role in plant cell expansion. We have recently proposed that AtRALF1 negatively regulates root cell elongation and lateral root formation by opposing the effects of brassinosteroid (BR). We reported 6 AtRALF1-inducible cell wall-related genes and 2 P450 monooxygenase -encoding genes involved in the BR biosynthetic pathway. The AtRALF1-inducible genes implicated in cell wall remodeling were not downregulated by brassinolide (BL) treatment alone; their induction was only compromised following simultaneous treatment with AtRALF1 and BL. We further examined the cell wall-remodeling gene EXPANSIN A5 (AtEXPA5), which is upregulated by BL and has been shown to positively affect root cell elongation. Herein, we report that AtEXPA5 expression is downregulated by AtRALF1 in a dose-dependent manner in the roots and hypocotyls of Arabidopsis plants. AtEXPA5 is also downregulated in plants that overexpress AtRALF1, and it is upregulated in plants in which the AtRALF1 gene is partially silenced. The AtRALF1 peptide is also able to repress AtEXPA5 induction following a pre-treatment with BL. A schematic diagram showing the gene regulatory network connecting the recently reported genes with the regulation of cell expansion by AtEXPA5 is presented.

The Arabidopsis thaliana THI1 protein is involved in thiamine biosynthesis and is targeted to both chloroplasts and mitochondria by N-terminal control regions. To investigate thi1 expression, a series of thi1 promoter deletions were fused to the beta-glucuronidase (GUS) reporter gene. Transgenic plants were generated and expression patterns obtained under different environmental conditions. The results show that expression derived from the thi1 promoter is detected early on during development and continues throughout the plant's life cycle. High levels of GUS expression are observed in both shoots and roots during vegetative growth although, in roots, expression is restricted to the vascular system. Deletion analysis of the thi1 promoter region identified a region that is responsive to light. The smallest fragment (designated Pthi322) encompasses 306 bp and possesses all the essential signals for tissue specificity, as well as responsiveness to stress conditions such as sugar deprivation, high salinity, and hypoxia.

In this review, plant-insect interaction is discussed as a dynamic system, subjected to continual variation and change. Plants developed different mechanisms to reduce insect attack, including specific responses that activate different metabolic pathways which considerably alter their chemical and physical aspects. On the other hand, insects developed several strategies to overcome plant defense barriers, allowing them to feed, grow and reproduce on their host plants. This review foccuses on several aspects of this complex interaction between plants and insects, including chemical-derived substances, protein-derived molecules and volatile compounds of plants whereas metabolization, sequestration or avoidance are in turn employed by the insects.

The rapid alkalinization factor (RALF) peptide negatively regulates cell expansion, and an antagonistic relationship has been demonstrated between AtRALF1, a root-specific RALF isoform in Arabidopsis, and brassinosteroids (BRs). An evaluation of the response of BR signaling mutants to AtRALF1 revealed that BRI1-associated receptor kinase1 (bak1) mutants are insensitive to AtRALF1 root growth inhibition activity. BAK1 was essential for the induction of AtRALF1-responsive genes but showed no effect on the mobilization of Ca2+ and alkalinization responses. Homozygous plants accumulating AtRALF1 and lacking the BAK1 gene did not exhibit the characteristic semi-dwarf phenotype of AtRALF1-overexpressors. Biochemical evidence indicates that AtRALF1 and BAK1 physically interact with a Kd of 4.6 μM and acridinium-labeled AtRALF1 was used to demonstrate that part of the specific binding of AtRALF1 to intact seedlings and to a microsomal fraction derived from the roots of Arabidopsis plants is BAK1-dependent. Moreover, AtRALF1 induces an increase in BAK1 phosphorylation, suggesting that the binding of AtRALF1 to BAK1 is functional. These findings show that BAK1 contains an additional AtRALF1 binding site, indicating that this protein may be part of a AtRALF1-containing complex as a co-receptor, and it is required for the negative regulation of cell expansion., Author summary The rapid alkalinization factor (RALF) is a small peptide hormone that binds to FERONIA receptor and inhibits cell expansion. The AtRALF1, a root-specific RALF isoform in Arabidopsis, opposes brassinosteroid (BR) effects on root cell elongation. To address the role of AtRALF1 and BRs in regulating cell expansion, we evaluated the response of BR signaling mutants to AtRALF1. We found that the BRI1-associated receptor kinase1 (bak1) mutants are insensitive to AtRALF1 root growth inhibition activity. Although BAK1 showed no effect on the mobilization of Ca2+ and alkalinization responses, it was required for the induction of AtRALF1-responsive genes and for the semi-dwarf phenotype of AtRALF1-overexpressing plants. Different experiments demonstrated that AtRALF1 and BAK1 interact physically in a specific manner. We also suggested that the binding of AtRALF1 to BAK1 is functional because AtRALF1 induces an increase in BAK1 phosphorylation. Our findings indicate that BAK1 plays an important role in AtRALF1 signaling.

Plants respond to pathogens and insect attacks by inducing and accumulating a large set of defense-related proteins. Two homologues of a barley wound-inducible protein (BARWIN) have been characterized in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins). Induction of SUGARWINs occurs in response to Diatraea saccharalis damage but not to pathogen infection. In addition, the protein itself does not show any effect on insect development; instead, it has antimicrobial activities toward Fusarium verticillioides, an opportunistic fungus that usually occurs after D. saccharalis borer attacks on sugarcane. In this study, we sought to evaluate the specificity of SUGARWIN2 to better understand its mechanism of action against phytopathogens and the associations between fungi and insects that affect plants. We used Colletotrichum falcatum, a fungus that causes red rot disease in sugarcane fields infested by D. saccharalis, and Ceratocystis paradoxa, which causes pineapple disease in sugarcane. We also tested whether SUGARWIN2 is able to cause cell death in Aspergillus nidulans, a fungus that does not infect sugarcane, and in the model yeast Saccharomyces cerevisiae, which is used for bioethanol production. Recombinant SUGARWIN2 altered C. falcatum morphology by increasing vacuolization, points of fractures and a leak of intracellular material, leading to germling apoptosis. In C. paradoxa, SUGARWIN2 showed increased vacuolization in hyphae but did not kill the fungi. Neither the non-pathogenic fungus A. nidulans nor the yeast S. cerevisiae was affected by recombinant SUGARWIN2, suggesting that the protein is specific to sugarcane opportunistic fungal pathogens.

thi1 has been recently isolated fromArabidopsis thaliana and is probably involved in both thiamine biosynthesis and as protection of organellar DNA from damage. Studies of thiamine biosynthesis in plants suggests a plastid location for the pathway, which is in agreement with the predicted THI1 N-terminal chloroplastic transit peptide (TP). On the other hand, thiamine is synthesized in mitochondria in yeast cells. Interestingly, A. thalianathi1 cDNA complements a yeast strain disrupted for the homologous gene. Analysis of THI1 amino acid sequence revealed the presence of a putative amphiphilic α-helix, which is typical for mitochondrial presequences, located downstream of the chloroplast transit peptide. To define the putative role of the two predicted targeting sequences in tandem, we produced two chimeric genes encompassing the chloroplastic THI1 TP and either 4 or 27 (including the putative mitochondrial presequence) N-terminal residues of the mature THI1, both linked to the reporter (gusA) gene. Analysis of GUS distribution in subcellular fractions of transgenic plants revealed that in the construct retaining only 4 residues of mature THI1, GUS was found in the chloroplastic fraction. Extension of the THI1 transit peptide to 27 residues of the mature protein allowed import and processing of GUS into both mitochondria and chloroplasts. Direct analysis by immunogold-labeling with an anti-THI1 polyclonal antibody identified THI1 in both organelles in Arabidopsis. We also provide evidence that the precursors of both organellar isoforms are encoded by a single nuclear transcript. Thus, THI1 is targeted simultaneously to mitochondria and chloroplasts by a post transcriptional mechanism.

Potato tubers were transformed with a chimeric gene made by the fusion of the soybean leghemoglobin encoding gene (lba) with the chloroplastic targeting sequence from Rubisco. This construct was placed under the control of the strong constitutive 35S promoter and the 3' nontranslated region of Rubisco from pea. Leghemoglobin expression on kanamycin-resistant plants was monitored by RT-PCR. Furthermore, immunodetection of subcellular fractions of transgenic plants revealed that leghemoglobin was imported and correctively processed inside the organelle. In addition, analysis of transgenic plants revealed reduced growth and decreased tuber production compared with the untransformed plants. It is suggested that leghemoglobin expression in potato chloroplasts interferes with aerobic metabolism, leading to physiological and morphological changes.

Several attempts have been made to alter the aerobic metabolism of plants, especially those related to the oxygenation or carboxylation of Rubisco. However, designing a more efficient Rubisco protein is rather problematic since its structural manipulation leads frequently to an enhancement of oxygenase activity, which is responsible for photorespiratory losses. In order to reduce oxygen availability inside the chloroplast, a chimeric gene consisting of a soybean leghemoglobin cDNA (lba) ligated to the chloroplast targeting signal sequence of the Rubisco small subunit gene, was introduced and expressed in Nicotiana tabacum. Lb was efficiently imported and correctly processed inside the chloroplasts of transgenic tobacco plants. Furthermore, the level of Lb expression in leaf tissue ranged from 0.01 to 0.1%. Analysis of photosynthesis, starch, sucrose and enzymes involved in aerobic metabolism, revealed that despite the high affinity of Lb for oxygen, no significant difference was observed in relation to the control plants. These results suggest that higher Lb concentrations would be required inside the chloroplasts in order to interfere on aerobic metabolism.

Summary The triose-phosphate-3-phosphoglycerate phosphate translocator (TPT) is a major protein of the chloroplast inner envelope. It is synthesized in the cytosol as a larger molecular weight precursor with an unusual transit peptide. Analysis of its primary structure reveals a similarity to mitochondrial presequences due to the ability to form a positively charged amphiphilic α-helix. In order to determine the import properties of TPT transit sequence into isolated mitochondria, it was linked to the reporter protein chloramphenicol acetyl transferase (CAT). Chimeric proteins were assayed in in vitro import experiments and the energy requirements were determined. The results obtained show that the TPT transit peptide functions in vitro as a typical mitochondrial targeting signal and the import process is similar to that used by mitochondrial precursors.

RALF is a small (5 kDa) and ubiquitous plant peptide signal. It was first isolated from tobacco leaf protein extracts owing to its capacity to alkalinize the extracellular media of cell suspensions. RALFs inhibit root growth and hypocotyl elongation, and a role for RALFs in cell expansion has also been proposed. Arabidopsis has 37 RALF isoforms (AtRALF), but only a small group of nine has high primary structure identity to the original RALF peptide isolated from tobacco. Herein, we report the heterologous production of these nine peptides in Escherichia coli and the evaluation of their activity in five biological assays. All AtRALF peptides produced showed strong alkalinizing activities, with the exception of the pollen-specific isoform AtRALF4. Although it exhibited no inhibitory activity in the root growth and hypocotyl elongation assays, AtRALF4 is a strong inhibitor of pollen germination. Our data demonstrate that the divergence in the tissue specificity and gene expression patterns of the different AtRALFs does not change the fact that their main role seems to be the regulation of cell expansion. Furthermore, different activities in the alkalinization assays upon the addition of two consecutive and saturating doses of the peptides suggest that the peptides are likely being sensed by specific receptors.

Rapid alkalinization factor (RALF) is part of a growing family of small peptides with hormone characteristics in plants. Initially isolated from leaves of tobacco plants, RALF peptides can be found throughout the plant kingdom and they are expressed ubiquitously in plants. We took advantage of the small gene family size of RALF genes in sugarcane and the ordered cellular growth of the grass sugarcane leaves to gain information about the function of RALF peptides in plants. Here we report the isolation of two RALF peptides from leaves of sugarcane plants using the alkalinization assay. SacRALF1 was the most abundant and, when added to culture media, inhibited growth of microcalli derived from cell suspension cultures at concentrations as low as 0.1 microM. Microcalli exposed to exogenous SacRALF1 for 5 days showed a reduced number of elongated cells. Only four copies of SacRALF genes were found in sugarcane plants. All four SacRALF genes are highly expressed in young and expanding leaves and show a low or undetectable level of expression in expanded leaves. In half-emerged leaf blades, SacRALF transcripts were found at high levels at the basal portion of the leaf and at low levels at the apical portion. Gene expression analyzes localize SacRALF genes in elongation zones of roots and leaves. Mature leaves, which are devoid of expanding cells, do not show considerable expression of SacRALF genes. Our findings are consistent with SacRALF genes playing a role in plant development potentially regulating tissue expansion.

We have characterized potato (Solanum tuberosum L.) plants expressing a soybean leghemoglobin that is targeted to plastids. Transgenic plants displayed a dwarf phenotype caused by short internode length, and exhibited increased tuberization in vitro. Under in vivo conditions that do not promote tuberization, plants showed smaller parenchymal cells than control plants. Analysis of gibberellin (GA) concentrations indicated that the transgenic plants have a substantial reduction (approximately 10-fold) of bioactive GA1 concentration in shoots. Application of GA3 to the shoot apex of the transformed plants completely restored the wild type phenotype suggesting that GA-biosynthesis rather than signal transduction was limiting. Since the first stage of the GA-biosynthetic pathway is located in the plastid, these results suggest that an early step in the pathway may be affected by the presence of the leghemoglobin.

Sugarcane is a highly productive crop used for centuries as the main source of sugar and recently to produce ethanol, a renewable bio-fuel energy source. There is increased interest in this crop due to the impending need to decrease fossil fuel usage. Sugarcane has a highly polyploid genome. Expressed sequence tag (EST) sequencing has significantly contributed to gene discovery and expression studies used to associate function with sugarcane genes. A significant amount of data exists on regulatory events controlling responses to herbivory, drought, and phosphate deficiency, which cause important constraints on yield and on endophytic bacteria, which are highly beneficial. The means to reduce drought, phosphate deficiency, and herbivory by the sugarcane borer have a negative impact on the environment. Improved tolerance for these constraints is being sought. Sugarcane's ability to accumulate sucrose up to 16% of its culm dry weight is a challenge for genetic manipulation. Genome-based technology such as cDNA microarray data indicates genes associated with sugar content that may be used to develop new varieties improved for sucrose content or for traits that restrict the expansion of the cultivated land. The genes can also be used as molecular markers of agronomic traits in traditional breeding programs.

The biogenesis of organelles and the maintenance of cell functions in multi-compartmentalized plant cells require a specific protein delivery mechanism to ensure efficient and effective translocation of proteins to their respective destinations. Increasing numbers of studies demonstrate that some proteins are targeted simultaneously to more than one compartment by a range of mechanisms, involving composite targeting sequences and/or transcriptional and translational controls. Recent data indicate that the final destination of a protein might respond to changes in the environment; this underlines the complexity of cell engineering that is required to localize a protein.