At present, more than 700 million people live with caloric hunger, and more than two billion suffer from micronutrient deficiencies, known as 'hidden hunger'. From an agricultural viewpoint, three major objectives need to be worked towards simultaneously to achieve zero hunger (the United Nations Sustainable Development Goal 2): (1) enhanced yield; (2) higher vitamin and mineral density to sustain recommended daily intake (multi-biofortification); and (3) enhanced climate-change resilience. Although the Green Revolution increased global calorie production, it exacerbated hidden hunger by prioritizing high yield over nutritional quality. Stress from global climate change has been shown to reduce the densities of several micronutrients. CRISPR-Cas, which allows genome editing with extremely high precision, has emerged as a groundbreaking breeding technology that has already been adopted by many countries. Here we examine how CRISPR-Cas-based approaches could be used to achieve biofortification targets by enhancing micronutrient densities to the levels necessary to alleviate dietary vitamin and mineral deficiencies. Given the limited time frame available to achieve zero hunger, we argue that CRISPR-Cas technologies should be combined with metabolic engineering based on transformation and other technologies. We also consider untapped resources beyond metabolic pathways and current CRISPR-Cas methodologies to address one of the most important societal issues of the twenty-first century.

Geminiviruses pose a severe threat to grain and vegetable crops worldwide, often resulting in significant economic losses. In cultivated tomato (Solanum lycopersicum), Ty resistance alleles have been introduced from wild tomato relatives, providing partial to strong resistance to geminivirus infections. The Ty-6 resistance locus from Solanum chilense was previously mapped to chromosome 10. It was recently shown to contain a mutant allele of the DNA POLYMERASE DELTA 1 (POLD1) gene that provides resistance to Tomato yellow leaf curl virus (TYLCV) infections. However, the resistance mechanism remained unknown. Here, we report another POLD1 allele at the Ty-6 locus of S. chilense with an E622D mutation in the catalytic site of the POLD1 protein. POLD1E622D is maintained as a heterozygous dominant allele in S. chilense and the AVTO2225 breeding line. It provides full resistance to the severe TYLCV Thailand (TYLCTHV) strain. The E622D amino acid change does not alter the predicted structure of POLD1. Replication of the TYLCTHV genome in plants carrying the POLD1E622D allele is severely compromised by a high frequency of mutations that accumulate in viral DNA, which results in nonfunctional proteins that are essential for continuous viral replication. Ectopically expressing the POLD1E622D allele cDNA alone causes mutations in TYLCTHV genes in inoculated leaves. S. chilense and AVTO2225 plants carrying the POLD1E622D allele mount a hypersensitive response after TYLCTHV infection, indicating that the defective virus genome cannot suppress the plant defense. The dominant POLD1E622D allele is therefore an effective resistance gene that geminiviruses cannot overcome.

A core tenet of food security is ensuring people have access to sufficient nutritious food. Vitamin B1 (thiamine) is an essential micronutrient for humans, deficiency in which causes numerous diseases of the nervous and cardiovascular systems (Dhir et al., 2019). Such ailments are particularly associated with populations that have a high carbohydrate intake, and especially those that have sustenance diets comprised largely of cereals such as rice. Thiamine insufficiency is a major public health concern in Asian countries, for example, in Cambodia 27%–100% of infants and women are deficient and account for up to 45% of deaths in under 5-year-olds (Johnson et al., 2019). Rice is a staple crop for half of the global population, but seeds are low in thiamine content, and polishing (i.e. removal of the embryo and bran layers) further aggravates chronic deficiencies as up to 90% of the thiamine content is in the removed tissues (Strobbe et al., 2021). Previous biofortification attempts increased thiamine content in leaves and unpolished seeds, but the trait failed to be retained in polished grains, sometimes negatively impacting yield (Dong et al., 2016; Strobbe et al., 2021), and no field trials have been reported that are necessary to confirm achievements. Natural variation studies revealed no substantial differences in thiamine content in rice seeds and minimal correlation with the expression of known biosynthesis genes (Mangel et al., 2022), discouraging exploitation via this route. Furthermore, the molecular form of vitamin B1 is an important consideration because the biosynthesis of the coenzyme thiamine diphosphate (TDP) is tightly regulated and necessary for cellular homeostasis, whereas supplementing with thiamine suggests that this form is innocuous (Pourcel et al., 2013). Indeed, mature cereal seeds are a natural sink for thiamine (rather than TDP, Figure S1), which is associated with certain members of the large diverse class of albumin proteins (Watanabe et al., 2001). Significantly, a sequence has been reported from this class encoding a thiamine binding protein (TBP) from Sesamum indicum (Si, sesame) (Watanabe et al., 2001). Here, we address previous bottlenecks related to enhancing thiamine content in rice endosperm and field performance in a proof-of-concept study. We expressed SiTBP under the control of the rice endosperm-specific glutelin D-1 promoter in the rice japonica model variety TP309 (wild type) (Figure 1a), after confirmation of thiamine binding to a codon-optimized SiTBP expressed in Escherichia coli (Figure S2a–c). Among 45 transformants, 3 with independent single T-DNA insertion events were selected to homozygosity and were morphologically similar to wild type, but had increased thiamine content in the polished grain (L14, 20, 26). The lines were sown in an experimental field in Taiwan (GPS coordinates 24°04′41.3″N 120°42′53.8″E) initially for bulking and then monitored for their agricultural performance during season 1 in years 2022 and 2023. Each line was grown on three randomized plots of 6 × 8 plants each. The transgenic plants expressing SiTBP could not be distinguished morphologically from the wild type (Figure 1b). Various agronomic traits were recorded in each year such as plant height, tiller and panicle number, single-plant yield, 100-grain weight and fertility. No statistically significant differences were observed between the lines (Figure 1c–i; Figure S3a–f). The vitamin B1 contents of leaf and unpolished seed material indicated no differences in vitamer levels of thiamine (Figure 1j,k; Figure S3g). In contrast, thiamine levels in the polished grains of the transgenic lines were increased 3 to 4-fold in each line compared to wild-type polished grains (Figure 1l; Figure S3h). Thus, SiTBP boosts the accumulation of thiamine in the endosperm without impacting yield. A proportion of this increase appears to be derived from the bran and/or embryo (Figure 1m; Figure S3i). Expression of the transgene was stable in the field, and expression of the biosynthesis genes was not changed with the exception of a small but significant decrease in the kinase OsTPKc in some of the transgenic lines (Figure S4). This represents a key step forward in vitamin B1 biofortification by potentially achieving 35%–27% of the RDI (1.2–1.4 mg/day) for adults and lactating women with a bowl of rice (ca. 315 g), respectively. The next step in realizing the goal of vitamin B1-biofortified plants will be to pursue this approach in customer-preferred elite varieties. This could be combined with the appropriate expression of the dual thiamine/POLYAMINE UPTAKE TRANSPORTER identified in Arabidopsis (Martinis et al., 2016) to further facilitate enhanced endosperm thiamine content. Notably, exogenous thiamine application has been discussed as a strategy to improve content (Li et al., 2022), but this may switch off thiamine biosynthesis de novo through extant TDP riboswitches and moreover, affect plant microbiome communities. Thus, we consider it more advantageous to purposefully engineer endogenous thiamine increases in target plant tissues by expanding the strategy for increasing thiamine content in grain endosperm as we present here to enhance the nutritional quality of rice for human health. We thank Céline Roux (University of Geneva) for technical assistance in this project. WG thanks the Taiwan Ministry of Education (Project 110-2634-F-005-006) and Yushan Fellow Program for financial support. The authors declare no competing interests. TBF conceived the project. TBF, JBF and WG contributed to the design of experiments. TBF, ID, JBF, KW, FHC, SHC, BM and WG performed the experiments and analysed the data. TBF wrote the manuscript. WG and JBF performed edits to the manuscript. Data available on request from the authors. Figure S1 Vitamin B1 profile of leaves and seeds of field grown rice plants. Figure S2 SiTBP sequence and analysis of purified recombinant protein from Escherichia coli. Figure S3 Agronomic traits and thiamine content for field grown rice lines in 2023. Figure S4 Relative transcript levels in field grown wild type and transgenic rice lines in season 1 of year 2022. Table S1 Fertilizer components. Table S2 Primers used in this study. Forward (F), reverse (R). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

There is an urgent need to stimulate agricultural output in many tropical and subtropical countries of the world to combat hunger and malnutrition. The starchy crop cassava (Manihot esculenta), growing even under sub-optimal conditions, is a key staple food in these regions, providing millions of people with food. Cassava biotechnology is an important technique benefiting agricultural progress, but successful implementation of many biotechnological concepts depends on the availability of the right spatiotemporal expression tools. Yet, well-characterized cassava promoters are scarce in the public domain. In this study, we investigate the promoter activity and tissue specificity of 24 different promoter elements in stably transformed cassava plants. We show that many of the investigated promoters, especially from other species, have surprisingly low activity and/or tissue specificity, but feature several promoter sequences that can drive tissue-specific expression in either autotrophic-, transport- or storage tissues. We especially highlight pAtCAB1, pMePsbR, and pSlRBCS2 as strong and specific source promoters, pAtSUC2, pMeSWEET1-like, and pMeSUS1 as valuable tools for phloem and phloem parenchyma expression, and pStB33, pMeGPT, pStGBSS1, as well as pStPatatin Class I, as strong and specific promoters for heterotrophic storage tissues. We hope that the provided information and sequences prove valuable to the cassava community by contributing to the successful implementation of biotechnological concepts aimed at the improvement of cassava nutritional value and productivity.

Cytosine DNA methylation is involved in biological processes such as transposable element (TE) silencing, imprinting, and X chromosome inactivation. Plant methylation is mediated by MET1 (mammalian DNMT1), DRM2 (mammalian DNMT3), and two plant-specific DNA methyltransferases, CMT2 and CMT3 (Law and Jacobsen, 2010). De novo DNA methylation in plants is established by DRM2 via the plant specific RNA-directed DNA methylation (RdDM) pathway that depends on two DNA-dependent RNA polymerases, Pol IV and Pol V (Gallego-Bartolome et al ., 2019; Law and Jacobsen, 2010; Stroud et al ., 2013). The DNA methylome of cassava has been previously documented based on its haploid collapsed genome (Wang et al ., 2015). Since the cassava genome is highly heterozygous, DNA methylome analysis of the haplotype-collapsed genome misses many features of the methylome. With the development of long read sequencing and chromosomal conformation capture techniques, haplotype resolved genomes are available for highly heterozygous genomes (Mansfeld et al ., 2021; Qi et al ., 2022; Zhou et al ., 2020), which provides high-quality reference genomes facilitating studies of haplotype resolved DNA methylomes.

Intrinsic improvement of iron (Fe) concentration in rice grains, called rice Fe biofortification, is a promising countermeasure against widespread human Fe deficiency. In this study, two novel rice Fe biofortification approaches are reported. The first approach (Y approach) involved the expression of maize YELLOW STRIPE 1 controlled by the HEAVY METAL ATPASE 2 promoter. The Y approach increased the polished grain Fe concentrations up to 4.8-fold compared with the non-transgenic (NT) line. The second approach (T approach) involved the expression of rice TRANSPORTER OF MUGINEIC ACID 1 controlled by the FERRIC REDUCTASE DEFECTIVE LIKE 1 promoter. The T approach increased the polished grain Fe concentrations by up to 3.2-fold. No synergistic increases in the polished grain Fe concentrations were observed when Y and T approaches were combined (YT approach). However, the polished grain Fe concentrations further increased by 5.1- to 9.3-fold compared with the NT line, when YT approach was combined with the endosperm-specific expression of FERRITIN (YTF approach), or when YTF approach was combined with the constitutive expression of NICOTIANAMINE SYNTHASE (YTFN approach). Total grain weight per plant in most Y, T, YT, and YTFN lines was comparable to that in the NT line, while it was significantly decreased in most YTF lines. The novel approaches reported in this study expand the portfolio of genetic engineering strategies that can be used for Fe biofortification in rice.

BACKGROUND:Cassava (Manihot esculenta) is an important clonally propagated food crop in tropical and subtropical regions worldwide. Genetic gain by molecular breeding has been limited, partially because cassava is a highly heterozygous crop with a repetitive and difficult-to-assemble genome. FINDINGS:Here we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present 2 chromosome-scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. With consensus accuracy >QV46, contig N50 >18 Mb, BUSCO completeness of 99%, and 35k phased gene loci, it is the most accurate, continuous, complete, and haplotype-resolved cassava genome assembly so far. Ab initio gene prediction with RNA-seq data and Iso-Seq transcripts identified abundant novel gene loci, with enriched functionality related to chromatin organization, meristem development, and cell responses. During tissue development, differentially expressed transcripts of different haplotype origins were enriched for different functionality. In each tissue, 20-30% of transcripts showed allele-specific expression (ASE) differences. ASE bias was often tissue specific and inconsistent across different tissues. Direction-shifting was observed in <2% of the ASE transcripts. Despite high gene synteny, the HiFi genome assembly revealed extensive chromosome rearrangements and abundant intra-genomic and inter-genomic divergent sequences, with large structural variations mostly related to LTR retrotransposons. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding. CONCLUSIONS:The phased and annotated chromosome pairs allow a systematic view of the heterozygous diploid genome organization in cassava with improved accuracy, completeness, and haplotype resolution. They will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy, and continuity.

ABSTRACT Cassava mosaic disease suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to the cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for loss of resistance, we performed whole genome sequencing and genetic variant analysis and fine-mapped the CMD2 locus to a 190 kilobase interval. Data suggest that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 ( MePOLD1 ) located within this region. Virus-induced gene silencing of MePOLD1 in a Cassava mosaic disease-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional resistance alleles within MePOLD1. MePOLD1 resistance alleles represent important genetic resources for resistance breeding or genome editing, and elucidating mechanisms of resistance to geminiviruses.

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 ( MePOLD1 ) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1 . Genetic variation of MePOLD1 , therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.

Insufficient dietary intake of micronutrients contributes to the onset of deficiencies termed hidden hunger—a global health problem affecting approximately 2 billion people. Vitamin B1 (thiamine) and vitamin B6 (pyridoxine) are essential micronutrients because of their roles as enzymatic cofactors in all organisms. Metabolic engineering attempts to biofortify rice endosperm—a poor source of several micronutrients leading to deficiencies when consumed monotonously—have led to only minimal improvements in vitamin B1 and B6 contents. To determine if rice germplasm could be exploited for biofortification of rice endosperm, we screened 59 genetically diverse accessions under greenhouse conditions for variation in vitamin B1 and vitamin B6 contents across three tissue types (leaves, unpolished and polished grain). Accessions from low, intermediate and high vitamin categories that had similar vitamin levels in two greenhouse experiments were chosen for in-depth vitamer profiling and selected biosynthesis gene expression analyses. Vitamin B1 and B6 contents in polished seeds varied almost 4-fold. Genes encoding select vitamin B1 and B6 biosynthesis de novo enzymes (THIC for vitamin B1, PDX1.3a–c and PDX2 for vitamin B6) were differentially expressed in leaves across accessions contrasting in their respective vitamin contents. These expression levels did not correlate with leaf and unpolished seed vitamin contents, except for THIC expression in leaves that was positively correlated with total vitamin B1 contents in polished seeds. This study expands our knowledge of diversity in micronutrient traits in rice germplasm and provides insights into the expression of genes for vitamin B1 and B6 biosynthesis in rice.

Cassava is an important staple crop that provides food and income for about 700 million Africans. Cassava productivity in Africa is limited by viral diseases, mainly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Genetic barriers such as high heterozygosity, allopolyploidy, poor seed set, and irregular flowering constrain the development of virus-resistant cassava varieties via conventional breeding. Genetic transformation represents a valuable tool to circumvent several challenges associated with the development of virus resistance and other valuable agronomic traits in cassava. The implementation of genetic transformation in many local African cultivars is limited either by the difficulty to produce friable embryogenic callus (FEC), low transformation, and/or regeneration efficiencies. Here, we report the successful induction of organized embryogenic structures (OES) in 11 farmer-preferred cultivars locally grown in Ghana. The production of high quality FEC from one local cultivar, ADI 001, facilitated its genetic transformation with high shoot regeneration and selection efficiency, comparable to the model cassava cultivar 60444. We show that using flow cytometry for analysis of nuclear ploidy in FEC tissues prior to genetic transformation ensures the selection of genetically uniform FEC tissue for transformation. The high percentage of single insertion events in transgenic lines indicates the suitability of the ADI 001 cultivar for the introduction of virus resistance and other useful agronomic traits into the farmer-preferred cassava germplasm in Ghana and Africa.

Plant transformation remains the most sought-after technology for functional genomics and crop genetic improvement, especially for introducing specific new traits and to modify or recombine already existing traits. Along with many other agricultural technologies, the global production of genetically engineered crops has steadily grown since they were first introduced 25 years ago. Since the first transfer of DNA into plant cells using Agrobacterium tumefaciens, different transformation methods have enabled rapid advances in molecular breeding approaches to bring crop varieties with novel traits to the market that would be difficult or not possible to achieve with conventional breeding methods. Today, transformation to produce genetically engineered crops is the fastest and most widely adopted technology in agriculture. The rapidly increasing number of sequenced plant genomes and information from functional genomics data to understand gene function, together with novel gene cloning and tissue culture methods, is further accelerating crop improvement and trait development. These advances are welcome and needed to make crops more resilient to climate change and to secure their yield for feeding the increasing human population. Despite the success, transformation remains a bottleneck because many plant species and crop genotypes are recalcitrant to established tissue culture and regeneration conditions, or they show poor transformability. Improvements are possible using morphogenetic transcriptional regulators, but their broader applicability remains to be tested. Advances in genome editing techniques and direct, non-tissue culture-based transformation methods offer alternative approaches to enhance varietal development in other recalcitrant crops. Here, we review recent developments in plant transformation and regeneration, and discuss opportunities for new breeding technologies in agriculture.

Cassava brown streak disease (CBSD) caused by the Cassava brown streak virus(CBSV) and Ugandan cassava brown streak virus(UCBSV) is a threat to cassava production in Africa. The potential spread of CBSD into West Africa is a cause for concern, therefore screening for resistance in farmer-preferred genotypes is crucial for effective control and management. We multiplied a selection of eleven cassava cultivars grown by farmers in Ghana to test their response to a mixed infection of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) isolates using a stringent top-cleft graft inoculation method. Virus titers were quantified in the inoculated scions and cuttings propagated from the inoculated scions to assess virus accumulation and recovery. All cultivars were susceptible to the mixed infection although their response and symptom development varied. In the propagated infected scions, CBSV accumulated at higher titers in leaves of eight of the eleven cultivars. Visual scoring of storage roots from six-month-old virus-inoculated plants revealed the absence of CBSD-associated necrosis symptoms and detectable titers of CBSVs in the cultivar, IFAD. Although all eleven cultivars supported the replication of CBSV and UCBSV in their leaves, the absence of virus replication and CBSD-associated symptoms in the roots of some cultivars could be used as criteria to rapidly advance durable CBSD tolerance using breeding and genetic engineering approaches.

Cassava (Manihot esculentaCrantz) is one of the important staple foods in Sub-Saharan Africa. It produces starchy storage roots that provide food and income for several hundred million people, mainly in tropical agriculture zones. Increasing cassava storage root and starch yield is one of the major breeding targets with respect to securing the future food supply for the growing population of Sub-Saharan Africa. The Cassava Source-Sink (CASS) project aims to increase cassava storage root and starch yield by strategically integrating approaches from different disciplines. We present our perspective and progress on cassava as an applied research organism and provide insight into the CASS strategy, which can serve as a blueprint for the improvement of other root and tuber crops. Extensive profiling of different field-grown cassava genotypes generates information for leaf, phloem, and root metabolic and physiological processes that are relevant for biotechnological improvements. A multi-national pipeline for genetic engineering of cassava plants covers all steps from gene discovery, cloning, transformation, molecular and biochemical characterization, confined field trials, and phenotyping of the seasonal dynamics of shoot traits under field conditions. Together, the CASS project generates comprehensive data to facilitate conventional breeding strategies for high-yielding cassava genotypes. It also builds the foundation for genome-scale metabolic modelling aiming to predict targets and bottlenecks in metabolic pathways. This information is used to engineer cassava genotypes with improved source-sink relations and increased yield potential.

Understanding drought tolerance mechanisms of cassava is a pre-requisite to improve the performance of the crop in water-scarce regions. Several hypotheses have been formulated to suggest how cassava can withstand a prolonged period of drought. We performed field trials under drought conditions with a selection of 37 cassava genotypes to identify phenotypic and molecular patterns associated with drought tolerance. Plant morphologies varied significantly between cassava genotypes under drought conditions in Kenya, which indicates a strong genetic basis for phenotypic differences. Drought stress reduced yield by 59%, the number of edible storage roots by 43% and leaf retention by 50% on average. Over three years and in two experimental field sites, the most drought tolerant genotype bulked 7.1 ( +/- 2.1) t/ha yield while the most drought susceptible genotype yielded 3.3 ( +/- 1.4) t/ha under drought conditions. The significant positive correlation of yield under irrigated and nonirrigated conditions suggests that selection of genotypes with high yield performance under well-watered or control conditions should be prioritized to identify genotypes with superior performance under drought stress. The positive correlation between yield and leaf retention provided further evidence that leaf longevity positively contributes to yield in water-deficit conditions. Yield differences could be attributed in part to variation in stomatal conductance (g(s)) because selected drought tolerant genotypes maintained higher g, and delayed stomatal closure as compared to drought susceptible genotypes. Further analysis revealed that genetic or molecular differences for g s between drought tolerant and susceptible genotypes could be detected at early stages of water deficit. These differences likely involve both abscisic acid (ABA)-dependent and ABA-independent molecular pathways.

Ending all forms of hunger by 2030, as set forward in the UN-Sustainable Development Goal 2 (UN-SDG2), is a daunting but essential task, given the limited timeline ahead and the negative global health and socio-economic impact of hunger. Malnutrition or hidden hunger due to micronutrient deficiencies affects about one third of the world population and severely jeopardizes economic development. Staple crop biofortification through gene stacking, using a rational combination of conventional breeding and metabolic engineering strategies, should enable a leap forward within the coming decade. A number of specific actions and policy interventions are proposed to reach this goal.

ABSTRACTPlant growth depends on the diurnal regulation of cellular processes, but it is not well understood if and how transcriptional regulation controls diurnal fluctuations at the protein-level. Here we report a high-resolutionArabidopsis thaliana(Arabidopsis) leaf rosette proteome acquired over a 12 h light : 12 h dark diurnal cycle and the phosphoproteome immediately before and after the light-to-dark and dark-to-light transitions. We quantified nearly 5000 proteins and 800 phosphoproteins, of which 288 fluctuated in their abundance and 226 fluctuated in their phosphorylation status. Of the phosphoproteins, 60% were quantified for changes in protein abundance. This revealed six proteins involved in nitrogen and hormone metabolism that had concurrent changes in both protein abundance and phosphorylation status. The diurnal proteome and phosphoproteome changes involve proteins in key cellular processes, including protein translation, light perception, photosynthesis, metabolism and transport. The phosphoproteome at the light-dark transitions revealed the dynamics at phosphorylation sites in either anticipation of or response to a change in light regime. Phosphorylation site motif analyses implicate casein kinase II and calcium/calmodulin dependent kinases among the primary light-dark transition kinases. The comparative analysis of the diurnal proteome and diurnal and circadian transcriptome established how mRNA and protein accumulation intersect in leaves during the diurnal cycle of the plant.Summary StatementThe manuscript provides quantitative information of diurnal changes in the accumulation and phosphorylation of proteins inArabidopsis thalianarosettes grown in a 12 h photoperiod. The highly resolved time-scale of the datasets offer new proteome-level insights for future targeted studies.

Background Rice is an important food source for humans worldwide. Because of its nutritional and agricultural significance, a number of studies addressed various aspects of rice grain development and grain filling. Nevertheless, the molecular processes underlying grain filling and development, and in particular in different contributions of grain tissues to these process, are not understood.Main text Using RNA-sequencing, we profiled gene expression activity in grain tissues comprised of cross cells (CC), the nucellar epidermis (NE), ovular vascular trace (OVT), endosperm (EN) and the aleurone layer (AL). These tissues were dissected using laser capture microdissection (LCM) at three distinct grain development stages. The mRNA expression datasets offer comprehensive and new insights into the gene expression patterns in different rice grain tissues and their contributions to grain development. Comparative analysis of the different tissues revealed their similar and/or unique functions, as well as the spatio-temporal regulation of common and tissue-specific genes. The expression patterns of genes encoding hormones and transporters indicate an important role of the OVT tissue in metabolite transport during grain development. Gene co-expression network prediction on OVT-specific genes identified several distinct and common development-specific transcription factors. Further analysis of enriched DNA sequence motifs proximal to OVT-specific genes revealed known and novel DNA sequence motifs relevant to rice grain development.Conclusion Together, the dataset of gene expression in rice grain tissues is a novel and useful resource for further work to dissect the molecular and metabolic processes during rice grain development.

Cassava (Manihot esculenta) is one of the most important staple food crops worldwide. Its starchy tuberous roots supply over 800 million people with carbohydrates. Yet, surprisingly little is known about the processes involved in filling of those vital storage organs. A better understanding of cassava carbohydrate allocation and starch storage is key to improving storage root yield. Here, we studied cassava morphology and phloem sap flow from source to sink using transgenic pAtSUC2::GFP plants, the phloem tracers esculin and 5(6)-carboxyfluorescein diacetate, as well as several staining techniques. We show that cassava performs apoplasmic phloem loading in source leaves and symplasmic unloading into phloem parenchyma cells of tuberous roots. We demonstrate that vascular rays play an important role in radial transport from the phloem to xylem parenchyma cells in tuberous roots. Furthermore, enzymatic and proteomic measurements of storage root tissues confirmed high abundance and activity of enzymes involved in the sucrose synthase-mediated pathway and indicated that starch is stored most efficiently in the outer xylem layers of tuberous roots. Our findings form the basis for biotechnological approaches aimed at improved phloem loading and enhanced carbohydrate allocation and storage in order to increase tuberous root yield of cassava.