Natural alleles of the abscisic acid catabolism gene ZmAbh4 modulate water use efficiency and carbon isotope discrimination in maize.

Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ13C) during photosynthesis in the C4 crop maize. Changes in Δ13C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ13C through a single gene with potential applications in maize breeding.

A genomic panel for studying C3-C4 intermediate photosynthesis in the Brassiceae tribe.

Research on C4 and C3-C4 photosynthesis has attracted significant attention because the understanding of the genetic underpinnings of these traits will support the introduction of its characteristics into commercially relevant crop species. We used a panel of 19 taxa of 18 Brassiceae species with different photosynthesis characteristics (C3 and C3-C4) with the following objectives: (i) create draft genome assemblies and annotations, (ii) quantify orthology levels using synteny maps between all pairs of taxa, (iii) ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠describe the phylogenetic relatedness across all the species, and (iv) track the evolution of C3-C4 intermediate photosynthesis in the Brassiceae tribe. Our results indicate that the draft de novo genome assemblies are of high quality and cover at least 90% of the gene space. Therewith we more than doubled the sampling depth of genomes of the Brassiceae tribe that comprises commercially important as well as biologically interesting species. The gene annotation generated high-quality gene models, and for most genes extensive upstream sequences are available for all taxa, yielding potential to explore variants in regulatory sequences. The genome-based phylogenetic tree of the Brassiceae contained two main clades and indicated that the C3-C4 intermediate photosynthesis has evolved five times independently. Furthermore, our study provides the first genomic support of the hypothesis that Diplotaxis muralis is a natural hybrid of D. tenuifolia and D. viminea. Altogether, the de novo genome assemblies and the annotations reported in this study are a valuable resource for research on the evolution of C3-C4 intermediate photosynthesis.

Brassicaceae display variation in efficiency of photorespiratory carbon-recapturing mechanisms.

Carbon-concentrating mechanisms enhance the carboxylase efficiency of Rubisco by providing supra-atmospheric concentrations of CO2 in its surroundings. Beside the C4 photosynthesis pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 ppm and 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed 'C3-C4 intermediates'. In the present study, we perform a physiological, biochemical, and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4-classified taxa, indicating a crucial role for anatomical features in CO2-concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of phosphoenolpyruvate carboxylase activities suggested that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct photosynthesis type that is beneficial in some environments.

Flowering Time-Regulated Genes in Maize Include the Transcription Factor ZmMADS1.

Flowering time (FTi) control is well examined in the long-day plant Arabidopsis (Arabidopsis thaliana), and increasing knowledge is available for the short-day plant rice (Oryza sativa). In contrast, little is known in the day-neutral and agronomically important crop plant maize (Zea mays). To learn more about FTi and to identify novel regulators in this species, we first compared the time points of floral transition of almost 30 maize inbred lines and show that tropical lines exhibit a delay in flowering transition of more than 3 weeks under long-day conditions compared with European flint lines adapted to temperate climate zones. We further analyzed the leaf transcriptomes of four lines that exhibit strong differences in flowering transition to identify new key players of the flowering control network in maize. We found strong differences among regulated genes between these lines and thus assume that the regulation of FTi is very complex in maize. Especially genes encoding MADS box transcriptional regulators are up-regulated in leaves during the meristem transition. ZmMADS1 was selected for functional studies. We demonstrate that it represents a functional ortholog of the central FTi integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) of Arabidopsis. RNA interference-mediated down-regulation of ZmMADS1 resulted in a delay of FTi in maize, while strong overexpression caused an early-flowering phenotype, indicating its role as a flowering activator. Taken together, we report that ZmMADS1 represents a positive FTi regulator that shares an evolutionarily conserved function with SOC1 and may now serve as an ideal stating point to study the integration and variation of FTi pathways also in maize.

Photosynthesis in C3-C4 intermediate Moricandia species.

Evolution of C4 photosynthesis is not distributed evenly in the plant kingdom. Particularly interesting is the situation in the Brassicaceae, because the family contains no C4 species, but several C3-C4 intermediates, mainly in the genus Moricandia Investigation of leaf anatomy, gas exchange parameters, the metabolome, and the transcriptome of two C3-C4 intermediate Moricandia species, M. arvensis and M. suffruticosa, and their close C3 relative M. moricandioides enabled us to unravel the specific C3-C4 characteristics in these Moricandia lines. Reduced CO2 compensation points in these lines were accompanied by anatomical adjustments, such as centripetal concentration of organelles in the bundle sheath, and metabolic adjustments, such as the balancing of C and N metabolism between mesophyll and bundle sheath cells by multiple pathways. Evolution from C3 to C3-C4 intermediacy was probably facilitated first by loss of one copy of the glycine decarboxylase P-protein, followed by dominant activity of a bundle sheath-specific element in its promoter. In contrast to recent models, installation of the C3-C4 pathway was not accompanied by enhanced activity of the C4 cycle. Our results indicate that metabolic limitations connected to N metabolism or anatomical limitations connected to vein density could have constrained evolution of C4 in Moricandia.

The Road to C4 Photosynthesis: Evolution of a Complex Trait via Intermediary States.

C4 photosynthesis enables high photosynthetic energy conversion efficiency as well as high nitrogen and water use efficiencies. Given the multitude of biochemical, structural and molecular changes in comparison with C3 photosynthesis, it appears unlikely that such a complex trait would evolve in a single step. C4 photosynthesis is therefore believed to have evolved from the ancestral C3 state via intermediary stages. Consequently, the identification and detailed characterization of plant species representing transitory states between C3 and C4 is important for the reconstruction of the sequence of evolutionary events, especially since C4 evolution occurred in very different phylogenetic backgrounds. There is also significant interest in engineering of C4 or at least C4-like elements into C3 crop plants. A detailed and mechanistic understanding of C3-C4 intermediates is likely to provide guidance for the experimental design of such approaches. Here we provide an overview on the most relevant results obtained on C3-C4 intermediates to date. Recent knowledge gains in this field will be described in more detail. We thereby concentrate especially on biochemical and physiological work. Finally, we will provide a perspective and outlook on the continued importance of research on C3-C4 intermediates.

An integrated functional approach to dissect systemic responses in maize to arbuscular mycorrhizal symbiosis.

Most terrestrial plants benefit from the symbiosis with arbuscular mycorrhizal fungi (AMF) mainly under nutrient-limited conditions. Here the crop plant Zea mays was grown with and without AMF in a bi-compartmented system separating plant and phosphate (Pi) source by a hyphae-permeable membrane. Thus, Pi was preferentially taken up via the mycorrhizal Pi uptake pathway while other nutrients were ubiquitously available. To study systemic effects of mycorrhizal Pi uptake on leaf status, leaves of these plants that display an increased biomass in the presence of AMF were subjected to simultaneous ionomic, transcriptomic and metabolomic analyses. We observed robust changes of the leaf elemental composition, that is, increase of P, S and Zn and decrease of Mn, Co and Li concentration in mycorrhizal plants. Although changes in anthocyanin and lipid metabolism point to an improved P status, a global increase in C versus N metabolism highlights the redistribution of metabolic pools including carbohydrates and amino acids. Strikingly, an induction of systemic defence gene expression and concomitant accumulation of secondary metabolites such as the terpenoids alpha- and beta-amyrin suggest priming of mycorrhizal maize leaves as a mycorrhiza-specific response. This work emphasizes the importance of AM symbiosis for the physiological status of plant leaves and could lead to strategies for optimized breeding of crop species with high growth potential.

Systems analysis of a maize leaf developmental gradient redefines the current C4 model and provides candidates for regulation.

We systematically analyzed a developmental gradient of the third maize (Zea mays) leaf from the point of emergence into the light to the tip in 10 continuous leaf slices to study organ development and physiological and biochemical functions. Transcriptome analysis, oxygen sensitivity of photosynthesis, and photosynthetic rate measurements showed that the maize leaf undergoes a sink-to-source transition without an intermediate phase of C(3) photosynthesis or operation of a photorespiratory carbon pump. Metabolome and transcriptome analysis, chlorophyll and protein measurements, as well as dry weight determination, showed continuous gradients for all analyzed items. The absence of binary on-off switches and regulons pointed to a morphogradient along the leaf as the determining factor of developmental stage. Analysis of transcription factors for differential expression along the leaf gradient defined a list of putative regulators orchestrating the sink-to-source transition and establishment of C(4) photosynthesis. Finally, transcriptome and metabolome analysis, as well as enzyme activity measurements, and absolute quantification of selected metabolites revised the current model of maize C(4) photosynthesis. All data sets are included within the publication to serve as a resource for maize leaf systems biology.

Predmoter-cross-species prediction of plant promoter and enhancer regions.

Motivation: Identifying cis-regulatory elements (CREs) is crucial for analyzing gene regulatory networks. Next generation sequencing methods were developed to identify CREs but represent a considerable expenditure for targeted analysis of few genomic loci. Thus, predicting the outputs of these methods would significantly cut costs and time investment. Results: We present Predmoter, a deep neural network that predicts base-wise Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) and histone Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) read coverage for plant genomes. Predmoter uses only the DNA sequence as input. We trained our final model on 21 species for 13 of which ATAC-seq data and for 17 of which ChIP-seq data was publicly available. We evaluated our models on Arabidopsis thaliana and Oryza sativa. Our best models showed accurate predictions in peak position and pattern for ATAC- and histone ChIP-seq. Annotating putatively accessible chromatin regions provides valuable input for the identification of CREs. In conjunction with other in silico data, this can significantly reduce the search space for experimentally verifiable DNA-protein interaction pairs. Availability and implementation: The source code for Predmoter is available at: https://github.com/weberlab-hhu/Predmoter. Predmoter takes a fasta file as input and outputs h5, and optionally bigWig and bedGraph files.

Adaptation of maize source leaf metabolism to stress related disturbances in carbon, nitrogen and phosphorus balance.

BACKGROUND: Abiotic stress causes disturbances in the cellular homeostasis. Re-adjustment of balance in carbon, nitrogen and phosphorus metabolism therefore plays a central role in stress adaptation. However, it is currently unknown which parts of the primary cell metabolism follow common patterns under different stress conditions and which represent specific responses. RESULTS: To address these questions, changes in transcriptome, metabolome and ionome were analyzed in maize source leaves from plants suffering low temperature, low nitrogen (N) and low phosphorus (P) stress. The selection of maize as study object provided data directly from an important crop species and the so far underexplored C4 metabolism. Growth retardation was comparable under all tested stress conditions. The only primary metabolic pathway responding similar to all stresses was nitrate assimilation, which was down-regulated. The largest group of commonly regulated transcripts followed the expression pattern: down under low temperature and low N, but up under low P. Several members of this transcript cluster could be connected to P metabolism and correlated negatively to different phosphate concentration in the leaf tissue. Accumulation of starch under low temperature and low N stress, but decrease in starch levels under low P conditions indicated that only low P treated leaves suffered carbon starvation. CONCLUSIONS: Maize employs very different strategies to manage N and P metabolism under stress. While nitrate assimilation was regulated depending on demand by growth processes, phosphate concentrations changed depending on availability, thus building up reserves under excess conditions. Carbon and energy metabolism of the C4 maize leaves were particularly sensitive to P starvation.

Maize source leaf adaptation to nitrogen deficiency affects not only nitrogen and carbon metabolism but also control of phosphate homeostasis.

Crop plant development is strongly dependent on the availability of nitrogen (N) in the soil and the efficiency of N utilization for biomass production and yield. However, knowledge about molecular responses to N deprivation derives mainly from the study of model species. In this article, the metabolic adaptation of source leaves to low N was analyzed in maize (Zea mays) seedlings by parallel measurements of transcriptome and metabolome profiling. Inbred lines A188 and B73 were cultivated under sufficient (15 mM) or limiting (0.15 mM) nitrate supply for up to 30 d. Limited availability of N caused strong shifts in the metabolite profile of leaves. The transcriptome was less affected by the N stress but showed strong genotype- and age-dependent patterns. N starvation initiated the selective down-regulation of processes involved in nitrate reduction and amino acid assimilation; ammonium assimilation-related transcripts, on the other hand, were not influenced. Carbon assimilation-related transcripts were characterized by high transcriptional coordination and general down-regulation under low-N conditions. N deprivation caused a slight accumulation of starch but also directed increased amounts of carbohydrates into the cell wall and secondary metabolites. The decrease in N availability also resulted in accumulation of phosphate and strong down-regulation of genes usually involved in phosphate starvation response, underlining the great importance of phosphate homeostasis control under stress conditions.

Photorespiration is the solution, not the problem.

The entry of carbon dioxide from the atmosphere into the biosphere is mediated by the enzyme Rubisco, which catalyzes the carboxylation of ribulose 1,5-bisphosphate (RuBP) as the entry reaction of the Calvin Benson Bassham cycle (CBBC), leading to the formation of 2 molecules of 3-phosphoglyceric acid (3PGA) per CO2 fixed. 3PGA is reduced to triose phosphates at the expense of NADPH + H+ and ATP that are provided by the photosynthetic light reactions. Triose phosphates are the principal products of the CBBC and the precursors for almost any compound in the biosphere.

Improving photosynthetic efficiency toward food security: Strategies, advances, and perspectives.

Photosynthesis in crops and natural vegetation allows light energy to be converted into chemical energy and thus forms the foundation for almost all terrestrial trophic networks on Earth. The efficiency of photosynthetic energy conversion plays a crucial role in determining the portion of incident solar radiation that can be used to generate plant biomass throughout a growth season. Consequently, alongside the factors such as resource availability, crop management, crop selection, maintenance costs, and intrinsic yield potential, photosynthetic energy use efficiency significantly influences crop yield. Photosynthetic efficiency is relevant to sustainability and food security because it affects water use efficiency, nutrient use efficiency, and land use efficiency. This review focuses specifically on the potential for improvements in photosynthetic efficiency to drive a sustainable increase in crop yields. We discuss bypassing photorespiration, enhancing light use efficiency, harnessing natural variation in photosynthetic parameters for breeding purposes, and adopting new-to-nature approaches that show promise for achieving unprecedented gains in photosynthetic efficiency.

OPTIMAS-DW: a comprehensive transcriptomics, metabolomics, ionomics, proteomics and phenomics data resource for maize.

BACKGROUND: Maize is a major crop plant, grown for human and animal nutrition, as well as a renewable resource for bioenergy. When looking at the problems of limited fossil fuels, the growth of the world's population or the world's climate change, it is important to find ways to increase the yield and biomass of maize and to study how it reacts to specific abiotic and biotic stress situations. Within the OPTIMAS systems biology project maize plants were grown under a large set of controlled stress conditions, phenotypically characterised and plant material was harvested to analyse the effect of specific environmental conditions or developmental stages. Transcriptomic, metabolomic, ionomic and proteomic parameters were measured from the same plant material allowing the comparison of results across different omics domains. A data warehouse was developed to store experimental data as well as analysis results of the performed experiments. DESCRIPTION: The OPTIMAS Data Warehouse (OPTIMAS-DW) is a comprehensive data collection for maize and integrates data from different data domains such as transcriptomics, metabolomics, ionomics, proteomics and phenomics. Within the OPTIMAS project, a 44K oligo chip was designed and annotated to describe the functions of the selected unigenes. Several treatment- and plant growth stage experiments were performed and measured data were filled into data templates and imported into the data warehouse by a Java based import tool. A web interface allows users to browse through all stored experiment data in OPTIMAS-DW including all data domains. Furthermore, the user can filter the data to extract information of particular interest. All data can be exported into different file formats for further data analysis and visualisation. The data analysis integrates data from different data domains and enables the user to find answers to different systems biology questions. Finally, maize specific pathway information is provided. CONCLUSIONS: With OPTIMAS-DW a data warehouse for maize was established, which is able to handle different data domains, comprises several analysis results that will support researchers within their work and supports systems biological research in particular. The system is available at http://www.optimas-bioenergy.org/optimas_dw.

Transport Proteins Enabling Plant Photorespiratory Metabolism.

Photorespiration (PR) is a metabolic repair pathway that acts in oxygenic photosynthetic organisms to degrade a toxic product of oxygen fixation generated by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase. Within the metabolic pathway, energy is consumed and carbon dioxide released. Consequently, PR is seen as a wasteful process making it a promising target for engineering to enhance plant productivity. Transport and channel proteins connect the organelles accomplishing the PR pathway-chloroplast, peroxisome, and mitochondrion-and thus enable efficient flux of PR metabolites. Although the pathway and the enzymes catalyzing the biochemical reactions have been the focus of research for the last several decades, the knowledge about transport proteins involved in PR is still limited. This review presents a timely state of knowledge with regard to metabolite channeling in PR and the participating proteins. The significance of transporters for implementation of synthetic bypasses to PR is highlighted. As an excursion, the physiological contribution of transport proteins that are involved in C4 metabolism is discussed.

Whole genome sequencing and assembly data of Moricandia moricandioides and M. arvensis.

Moricandia is a genus belonging to the family Brassicaceae. C3 and C3-C4 photosynthesis Moricandia species exist in a close phylogenetic proximity, as well as to Brassica crops. Here, we performed PacBio genome sequencing on M. moricandioides and M. arvensis. The genomes were assembled using Flye assembler, then polished with Illumina reads and reduced duplication with Purge Haplotigs. The total length of genome assemblies of M. moricandioides and M. arvensis was 498 Mbp and 759 Mbp, respectively. These data will be useful for studies of the genetic control of C3-C4 characteristics, therefore gaining new insights into the early evolutionary steps of C4 photosynthesis. Further, it can be integrated into Brassica crop breeding. The data can be accessed at ENA under the project number PRJEB39764.

New Insights Into the Evolution of C4 Photosynthesis Offered by the Tarenaya Cluster of Cleomaceae.

Cleomaceae is closely related to Brassicaceae and includes C3, C3-C4, and C4 species. Thus, this family represents an interesting system for studying the evolution of the carbon concentrating mechanism. However, inadequate genetic information on Cleomaceae limits their research applications. Here, we characterized 22 Cleomaceae accessions [3 genera (Cleoserrata, Gynandropsis, and Tarenaya) and 11 species] in terms of genome size; molecular phylogeny; as well as anatomical, biochemical, and photosynthetic traits. We clustered the species into seven groups based on genome size. Interestingly, despite clear differences in genome size (2C, ranging from 0.55 to 1.3 pg) in Tarenaya spp., this variation was not consistent with phylogenetic grouping based on the internal transcribed spacer (ITS) marker, suggesting the occurrence of multiple polyploidy events within this genus. Moreover, only G. gynandra, which possesses a large nuclear genome, exhibited the C4 metabolism. Among the C3-like species, we observed intra- and interspecific variation in nuclear genome size as well as in biochemical, physiological, and anatomical traits. Furthermore, the C3-like species had increased venation density and bundle sheath cell size, compared to C4 species, which likely predisposed the former lineages to C4 photosynthesis. Accordingly, our findings demonstrate the potential of Cleomaceae, mainly members of Tarenaya, in offering novel insights into the evolution of C4 photosynthesis.

Recombinant protease inhibitors for herbivore pest control: a multitrophic perspective.

Protease inhibitors are a promising complement to Bt toxins for the development of insect-resistant transgenic crops, but their limited specificity against proteolytic enzymes and the ubiquity of protease-dependent processes in living organisms raise questions about their eventual non-target effects in agroecosystems. After a brief overview of the main factors driving the impacts of insect-resistant transgenic crops on non-target organisms, the possible effects of protease inhibitors are discussed from a multitrophic perspective, taking into account not only the target herbivore proteases but also the proteases of other organisms found along the trophic chain, including the plant itself. Major progress has been achieved in recent years towards the design of highly potent broad-spectrum inhibitors and the field deployment of protease inhibitor-expressing transgenic plants resistant to major herbivore pests. A thorough assessment of the current literature suggests that, whereas the non-specific inhibitory effects of recombinant protease inhibitors in plant food webs could often be negligible and their 'unintended' pleiotropic effects in planta of potential agronomic value, the innocuity of these proteins might always remain an issue to be assessed empirically, on a case-by-case basis.

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus.

The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of digestive cysteine protease activities. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet assay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg-MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with the broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatin-infiltrated banana stem disks. These promising results, which illustrate the susceptibility of C. sordidus to plant cystatins, are discussed in the light of recent hypotheses suggesting a key role for cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.