NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2 interact to regulate gene networks in maize endosperm development.

NAKED ENDOSPERM1 (NKD1), NKD2, and OPAQUE2 (O2) are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these 3 factors in coregulating gene networks, we developed a set of nkd1, nkd2, and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the 8 genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 d after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2, and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early and then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins, and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2, and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.

Rearrangement with the nkd2 promoter contributed to allelic diversity of the r1 gene in maize (Zea mays).

The maize red1 (r1) locus regulates anthocyanin accumulation and is a classic model for allelic diversity; changes in regulatory regions are responsible for most of the variation in gene expression patterns. Here, an intrachromosomal rearrangement between the distal upstream region of r1 and the region of naked endosperm 2 (nkd2) upstream to the third exon generated a nkd2 null allele lacking the first three exons, and the R1-st (stippled) allele with a novel r1 5' promoter region homologous to 5' regions from nkd2-B73. R1-sc:124 (an R1-st derivative) shows increased and earlier expression than a standard R1-g allele, as well as ectopic expression in the starchy endosperm compartment. Laser capture microdissection and RNA sequencing indicated that ectopic R1-sc:124 expression impacted expression of genes associated with RNA modification. The expression of R1-sc:124 resembled nkd2-W22 expression, suggesting that nkd2 regulatory sequences may influence the expression of R1-sc:124. The r1-sc:m3 allele is derived from R1-sc:124 by an insertion of a Ds6 transposon in intron 4. This insertion blocks anthocyanin regulation by causing mis-splicing that eliminates exon 5 from the mRNA. This allele serves as an important launch site for Ac/Ds mutagenesis studies, and two Ds6 insertions believed to be associated with nkd2 mutant alleles were actually located in the r1 5' region. Among annotated genomes of teosinte and maize varieties, the nkd2 and r1 loci showed conserved overall gene structures, similar to the B73 reference genome, suggesting that the nkd2-r1 rearrangement may be a recent event.

Maize Endosperm Development: Tissues, Cells, Molecular Regulation and Grain Quality Improvement.

Maize endosperm plays important roles in human diet, animal feed and industrial applications. Knowing the mechanisms that regulate maize endosperm development could facilitate the improvement of grain quality. This review provides a detailed account of maize endosperm development at the cellular and histological levels. It features the stages of early development as well as developmental patterns of the various individual tissues and cell types. It then covers molecular genetics, gene expression networks, and current understanding of key regulators as they affect the development of each tissue. The article then briefly considers key changes that have occurred in endosperm development during maize domestication. Finally, it considers prospects for how knowledge of the regulation of endosperm development could be utilized to enhance maize grain quality to improve agronomic performance, nutrition and economic value.

Comparative transcriptomics and network analysis define gene coexpression modules that control maize aleurone development and auxin signaling.

The naked endosperm1 (nkd1), naked endosperm2 (nkd2), and thick aleurone1 (thk1) genes are important regulators of maize (Zea mays L.) endosperm development. Double mutants of nkd1 and nkd2 (nkd1,2) show multiple aleurone (AL) cell layers with disrupted AL cell differentiation, whereas mutants of thk1 cause multiple cell layers of fully differentiated AL cells. Here, we conducted a comparative analysis of nkd1,2 and thk1 mutant endosperm transcriptomes to study how these factors regulate gene networks to control AL layer specification and cell differentiation. Weighted gene coexpression network analysis was incorporated with published laser capture microdissected transcriptome datasets to identify a coexpression module associated with AL development. In this module, both Nkd1,2+ and Thk1+ appear to regulate cell cycle and division, whereas Nkd1,2+, but not Thk1+, regulate auxin signaling. Further investigation of nkd1,2 differentially expressed genes combined with published putative targets of auxin response factors (ARFs) identified 61 AL-preferential genes that may be directly activated by NKD-modulated ARFs. All 61 genes were upregulated in nkd1,2 mutant and the enriched Gene Ontology terms suggested that they are associated with hormone crosstalk, lipid metabolism, and developmental growth. Expression of a transgenic DR5-red fluorescent protein auxin reporter was significantly higher in nkd1,2 mutant endosperm than in wild type, supporting the prediction that Nkd1,2+ negatively regulate auxin signaling in developing AL. Overall, these results suggest that Nkd1,2+ and Thk1+ may normally restrict AL development to a single cell layer by limiting cell division, and that Nkd1,2+ restrict auxin signaling in the AL to maintain normal cell patterning and differentiation processes.

The thick aleurone1 Gene Encodes a NOT1 Subunit of the CCR4-NOT Complex and Regulates Cell Patterning in Endosperm.

Maize (Zea mays) thick aleurone1 (thk1-R) mutants form multiple aleurone layers in the endosperm and have arrested embryogenesis. Prior studies suggest that thk1 functions downstream of defective kernel1 (dek1) in a regulatory pathway that controls aleurone cell fate and other endosperm traits. The original thk1-R mutant contained an ∼2-Mb multigene deletion, which precluded identification of the causal gene. Here, ethyl methanesulfonate mutagenesis produced additional alleles, and RNA sequencing from developing endosperm was used to identify a candidate gene based on differential expression compared with the wild-type progenitor. Gene editing confirmed the gene identity by producing mutant alleles that failed to complement existing thk1 mutants and that produced multiple-aleurone homozygous phenotypes. Thk1 encodes a homolog of NEGATIVE ON TATA-LESS1, a protein that acts as a scaffold for the CARBON CATABOLITE REPRESSION4-NEGATIVE ON TATA-LESS complex. This complex is highly conserved and essential in all eukaryotes for regulating a wide array of gene expression and cellular activities. Maize also harbors a duplicate locus, thick aleurone-like1, which likely accounts for the ability of thk1 mutants to form viable cells. Transcriptomic analysis indicated that THK1 regulates activities involving cell division, signaling, differentiation, and metabolism. Identification of thk1 provides an important new component of the DEK1 regulatory system that patterns cell fate in endosperm.

Redundant SCARECROW genes pattern distinct cell layers in roots and leaves of maize.

The highly efficient C4 photosynthetic pathway is facilitated by 'Kranz' leaf anatomy. In Kranz leaves, closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and mesophyll (outer) cells. Here, we demonstrate that, in the C4 monocot maize, Kranz patterning is regulated by redundant function of SCARECROW 1 (ZmSCR1) and a previously uncharacterized homeologue: ZmSCR1h. ZmSCR1 and ZmSCR1h transcripts accumulate in ground meristem cells of developing leaf primordia and in Zmscr1;Zmscr1h mutant leaves, most veins are separated by one rather than two mesophyll cells; many veins have sclerenchyma above and/or below instead of mesophyll cells; and supernumerary bundle sheath cells develop. The mutant defects are unified by compromised mesophyll cell development. In addition to Kranz defects, Zmscr1;Zmscr1h mutants fail to form an organized endodermal layer in the root. Collectively, these data indicate that ZmSCR1 and ZmSCR1h redundantly regulate cell-type patterning in both the leaves and roots of maize. Leaf and root pathways are distinguished, however, by the cell layer in which they operate - mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature.

Fitness Costs and Variation in Transmission Distortion Associated with the Abnormal Chromosome 10 Meiotic Drive System in Maize.

Meiotic drive describes a process whereby selfish genetic elements are transmitted at levels greater than Mendelian expectations. Maize abnormal chromosome 10 (Ab10) encodes a meiotic drive system that exhibits strong preferential segregation through female gametes. We performed transmission assays on nine Ab10 chromosomes from landraces and teosinte lines and found a transmission advantage of 62-79% in heterozygotes. Despite this transmission advantage, Ab10 is present at low frequencies in natural populations, suggesting that it carries large negative fitness consequences. We measured pollen transmission, the percentage of live pollen, seed production, and seed size to estimate several of the possible fitness effects of Ab10. We found no evidence that Ab10 affects pollen transmission, i.e., Ab10 and N10 pollen are transmitted equally from heterozygous fathers. However, at the diploid (sporophyte) level, both heterozygous and homozygous Ab10-I-MMR individuals show decreased pollen viability, decreased seed set, and decreased seed weight. The observed fitness costs can nearly but not entirely account for the observed frequencies of Ab10. Sequence analysis shows a surprising amount of molecular variation among Ab10 haplotypes, suggesting that there may be other phenotypic variables that contribute to the low but stable equilibrium frequencies.

Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize.

Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S) of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer) mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds), Enhancer/Suppressor-mutator (En/Spm), and Mutator (Mu) transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development.

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

NKD Transcription Factors Are Central Regulators of Maize Endosperm Development.

NAKED ENDOSPERM1 (NKD1) and NKD2 are duplicate INDETERMINATE DOMAIN (IDD) transcription factors important for maize (Zea mays) endosperm development. RNA-seq analysis of the nkd1 nkd2 mutant endosperm revealed that NKD1 and NKD2 influence 6.4% of the transcriptome in developing aleurone and 6.7% in starchy endosperm. Processes regulated by NKD1 and NKD2 include gene expression, epigenetic functions, cell growth and division, hormone pathways, and resource reserve deposition. The NKD1 and NKD2 proteins bind a consensus DNA sequence of TTGTCGT with slightly different properties. This motif was enriched in the promoters of gene transcripts differentially expressed (DE) in mutant endosperm. DE genes with a NKD binding motif in the 5' promoter region were considered as likely direct targets of NKD1 and NKD2 regulation, and these putative direct target genes were notably enriched for storage proteins. Transcription assays demonstrate that NKD1 and NKD2 can directly regulate gene transcription, including activation of opaque2 and viviparous1 promoters. NKD2 functions as a negative regulator of nkd1 transcription, consistent with previously reported feedback regulation. NKD1 and NKD2 can homo- and heterodimerize through their ID domains. These analyses implicate NKD1 and NKD2 as central regulators of gene expression in developing maize endosperm.

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture.

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.

The naked endosperm genes encode duplicate INDETERMINATE domain transcription factors required for maize endosperm cell patterning and differentiation.

The aleurone is the outermost layer of cereal endosperm and functions to digest storage products accumulated in starchy endosperm cells as well as to confer important dietary health benefits. Whereas normal maize (Zea mays [Zm]) has a single aleurone layer, naked endosperm (nkd) mutants produce multiple outer cell layers of partially differentiated cells that show sporadic expression of aleurone identity markers such as a viviparous1 promoter-ß-glucuronidase transgene. The 15:1 F2 segregation ratio suggested that two recessive genes were involved, and map-based cloning identified two homologous genes in duplicated regions of the genome. The nkd1 and nkd2 genes encode the INDETERMINATE1 domain (IDD) containing transcription factors ZmIDDveg9 and ZmIDD9 on chromosomes 2 and 10, respectively. Independent mutant alleles of nkd1 and nkd2, as well as nkd2-RNA interference lines in which both nkd genes were knocked down, also showed the nkd mutant phenotype, confirming the gene identities. In wild-type kernels, the nkd transcripts were most abundant around 11 to 16 d after pollination. The NKD proteins have putative nuclear localization signals, and green fluorescent protein fusion proteins showed nuclear localization. The mutant phenotype and gene identities suggest that NKD controls a gene regulatory network involved in aleurone cell fate specification and cell differentiation.

A putative plant organelle RNA recognition protein gene is essential for maize kernel development.

Basal endosperm transfer layer (BETL) cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm, supplying the grain with compounds required for embryo development and storage reserve accumulation. Here, we analyze the maize (Zea mays L.) empty pericarp6 (emp6) mutant, which causes early arrest in grain development. The Emp6+gene function is required independently in both the embryo and endosperm. The emp6 mutant causes a notable effect on the differentiation of BETL cells; the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels. Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition (PORR) protein, 1 of 15 PORR family members in maize. The emp6 transcript is widely detected in plant tissues with highest levels in embryos and developing kernels. EMP6-green fluorescent protein (GFP) fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria. These results suggest that BETL cell differentiation might be particularly energy intensive, or alternatively, that mitochondria might confer a developmental function.

Using transposons for genetic mosaic analysis of plant development.

Genetic mosaics, or chimeras, are individual organisms composed of cells or tissues of two or more distinct genotypes. They are experimentally useful for addressing several key biological questions. These include fate mapping through analysis of marked clonal lineages, analyzing cell or tissue interactions such as the induction of developmental events, and analyzing whether a gene acts cell autonomously. Genetic mosaics can arise in many ways, including through the action of transposable elements. Naturally occurring transposons can generate genetic mosaics by somatically inserting into a gene to cause a mutant sector, somatically excising from a mutant gene to create a revertant wild-type sector, or causing chromosomal breaks or rearrangements leading to loss of a gene or genes. Transposons have also been cleverly engineered to allow the generation of marked somatic sectors, sometimes in controlled ways. Here we review ways in which transposon-induced genetic mosaics have been used experimentally, the various methods that have been used, and general considerations for designing genetic mosaic studies using transposon methods.

Endosperm imprinting: a child custody battle?

Endosperm gene imprinting has long been speculated to control nutrient allocation to seeds. For the first time, an imprinted gene directly involved in this process has been identified.

Endosperm development: dynamic processes and cellular innovations underlying sibling altruism.

The endosperm is a product of fertilization that evolved to support and nourish its genetic twin sibling embryo. Cereal endosperm accumulates starch and protein stores, which later support the germinating seedling. These nutritional stores prompted the domestication of cereals and are the focus of ongoing efforts for crop improvement and biotechnological innovations. Endosperm development entails several novel modifications to basic cellular and developmental processes. Cereals display nuclear endosperm development, which begins with a period of free nuclear division to generate a coenocyte. Cytoskeletal arrays distribute nuclei around the periphery of the cytoplasm and direct the subsequent deposition of cell wall material during cellularization. Positional cues and signaling systems function dynamically in the specification of the four major cell types: transfer cells, embryo-surrounding cells, starchy endosperm (SE), and aleurone. Genome balance, epigenetic gene regulation, and parent-of-origin effects are essential for directing these processes. Transfer cells transport solutes, including sugars and amino acids, from the maternal plant tissues into the developing grain where they are partitioned between embryo and SE cells. Cells of the embryo-surrounding region appear to coordinate development of the embryo and endosperm. As the seed matures, SE cells assimilate starch and protein stores, undergo DNA endoreduplication, and finally undergo programmed cell death. In contrast, aleurone cells follow a maturation program similar to the embryo, allowing them to survive desiccation. At germination, the aleurone cells secrete amylases and proteases that hydrolyze the storage products of the SE to nourish the germinating seedling.

Maize opaque5 encodes monogalactosyldiacylglycerol synthase and specifically affects galactolipids necessary for amyloplast and chloroplast function.

The maize (Zea mays) opaque5 (o5) locus was shown to encode the monogalactosyldiacylglycerol synthase MGD1. Null and point mutations of o5 that affect the vitreous nature of mature endosperm engendered an allelic series of lines with stepwise reductions in gene function. C(18:3)/C(18:2) galactolipid abundance in seedling leaves was reduced proportionally, without significant effects on total galactolipid content. This alteration in polar lipid composition disrupted the organization of thylakoid membranes into granal stacks. Total galactolipid abundance in endosperm was strongly reduced in o5(-) mutants, causing developmental defects and changes in starch production such that the normal simple granules were replaced with compound granules separated by amyloplast membrane. Complete loss of MGD1 function in a null mutant caused kernel lethality owing to failure in both endosperm and embryo development. The data demonstrate that low-abundance galactolipids with five double bonds serve functions in plastid membranes that are not replaced by the predominant species with six double bonds. Furthermore, the data identify a function of amyloplast membranes in the development of starch granules. Finally, the specific changes in lipid composition suggest that MGD1 can distinguish the constituency of acyl groups on its diacylglycerol substrate based upon the degree of desaturation.

The thick aleurone1 mutant defines a negative regulation of maize aleurone cell fate that functions downstream of defective kernel1.

The maize (Zea mays) aleurone layer occupies the single outermost layer of the endosperm. The defective kernel1 (dek1) gene is a central regulator required for aleurone cell fate specification. dek1 mutants have pleiotropic phenotypes including lack of aleurone cells, aborted embryos, carotenoid deficiency, and a soft, floury endosperm deficient in zeins. Here we describe the thick aleurone1 (thk1) mutant that defines a novel negative function in the regulation of aleurone differentiation. Mutants possess multiple layers of aleurone cells as well as aborted embryos. Clonal sectors of thk1 mutant tissue in otherwise normal endosperm showed localized expression of the phenotype with sharp boundaries, indicating a localized cellular function for the gene. Sectors in leaves showed expanded epidermal cell morphology but the mutant epidermis generally remained in a single cell layer. Double mutant analysis indicated that the thk1 mutant is epistatic to dek1 for several aspects of the pleiotropic dek1 phenotype. dek1 mutant endosperm that was mosaic for thk1 mutant sectors showed localized patches of multilayered aleurone. Localized sectors were surrounded by halos of carotenoid pigments and double mutant kernels had restored zein profiles. In sum, loss of thk1 function restored the ability of dek1 mutant endosperm to accumulate carotenoids and zeins and to differentiate aleurone. Therefore the thk1 mutation defines a negative regulator that functions downstream of dek1 in the signaling system that controls aleurone specification and other aspects of endosperm development. The thk1 mutation was found to be caused by a deletion of approximately 2 megabases.

Regulation of aleurone development in cereal grains.

The aleurone layer of cereal grains is important biologically as well as nutritionally and economically. Here, current knowledge on the regulation of aleurone development is reviewed. Recent reports suggest that the control of aleurone development is more complex than earlier models portrayed. Multiple levels of genetic regulation control aleurone cell fate, differentiation, and organization. The hormones auxin and cytokinin can also influence aleurone development. New technical advances promise to facilitate future progress.

From dwarves to giants? Plant height manipulation for biomass yield.

The increasing demand for lignocellulosic biomass for the production of biofuels provides value to vegetative plant tissue and leads to a paradigm shift for optimizing plant architecture in bioenergy crops. Plant height (PHT) is among the most important biomass yield components and is the focus of this review, with emphasis on the energy grasses maize (Zea mays) and sorghum (Sorghum bicolor). We discuss the scientific advances in the identification of PHT quantitative trait loci (QTLs) and the understanding of pathways and genes controlling PHT, especially gibberellins and brassinosteroids. We consider pleiotropic effects of QTLs or genes affecting PHT on other agronomically important traits and, finally, we discuss strategies for applying this knowledge to the improvement of dual-purpose or dedicated bioenergy crops.