Hormonal control of medial-lateral growth and vein formation in the maize leaf.

Parallel veins are characteristic of monocots, including grasses (Poaceae). Therefore, how parallel veins develop as the leaf grows in the medial-lateral (ML) dimension is a key question in grass leaf development. Using fluorescent protein reporters, we mapped auxin, cytokinin (CK), and gibberellic acid (GA) response patterns in maize (Zea mays) leaf primordia. We further defined the roles of these hormones in ML growth and vein formation through combinatorial genetic analyses and measurement of hormone concentrations. We discovered a novel pattern of auxin response in the adaxial protoderm that we hypothesize has important implications for the orderly formation of 3° veins early in leaf development. In addition, we found an auxin transport and response pattern in the margins that correlate with the transition from ML to proximal-distal growth. We present evidence that auxin efflux precedes CK response in procambial strand development. We also determined that GA plays an early role in the shoot apical meristem as well as a later role in the primordium to restrict ML growth. We propose an integrative model whereby auxin regulates ML growth and vein formation in the maize leaf through control of GA and CK.

Pleiotropic and nonredundant effects of an auxin importer in Setaria and maize.

Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in green millet (Setaria viridis) and maize (Zea mays), we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A green fluorescent protein (GFP)-tagged construct of the Setaria AUX1 protein Sparse Panicle1 (SPP1) under its native promoter showed that SPP1 localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis indicated that most gene expression modules are conserved between mutant and wild-type plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, and leaf and root development. The AUX1 importers are thus not fully redundant in S. viridis. Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins.

grasviq: an image analysis framework for automatically quantifying vein number and morphology in grass leaves.

Beyond facilitating transport and providing mechanical support to the leaf, veins have important roles in the performance and productivity of plants and the ecosystem. In recent decades, computational image analysis has accelerated the extraction and quantification of vein traits, benefiting fields of research from agriculture to climatology. However, most of the existing leaf vein image analysis programs have been developed for the reticulate venation found in dicots. Despite the agroeconomic importance of cereal grass crops, like Oryza sativa (rice) and Zea mays (maize), a dedicated image analysis program for the parallel venation found in monocots has yet to be developed. To address the need for an image-based vein phenotyping tool for model and agronomic grass species, we developed the grass vein image quantification (grasviq) framework. Designed specifically for parallel venation, this framework automatically segments and quantifies vein patterns from images of cleared leaf pieces using classical computer vision techniques. Using image data sets from maize inbred lines and auxin biosynthesis and transport mutants in maize, we demonstrate the utility of grasviq for quantifying important vein traits, including vein density, vein width and interveinal distance. Furthermore, we show that the framework can resolve quantitative differences and identify vein patterning defects, which is advantageous for genetic experiments and mutant screens. We report that grasviq can perform high-throughput vein quantification, with precision on a par with that of manual quantification. Therefore, we envision that grasviq will be adopted for vein phenomics in maize and other grass species.

Defects in meristem maintenance, cell division, and cytokinin signaling are early responses in the boron deficient maize mutant tassel-less1.

Meristems house the stem cells needed for the developmental plasticity observed in adverse environmental conditions and are crucial for determining plant architecture. Meristem development is particularly sensitive to deficiencies of the micronutrient boron, yet how boron integrates into meristem development pathways is unknown. We addressed this question using the boron-deficient maize mutant, tassel-less1 (tls1). Reduced boron uptake in tls1 leads to a progressive impairment of meristem development that manifests in vegetative and reproductive defects. We show, that the tls1 tassel phenotype (male reproductive structure) was partially suppressed by mutations in the CLAVATA1 (CLV1)-ortholog, thick tassel dwarf1 (td1), but not by other mutants in the well characterized CLV-WUSCHEL pathway, which controls meristem size. The suppression of tls1 by td1 correlates with altered signaling of the phytohormone cytokinin. In contrast, mutations in the meristem maintenance gene knotted1 (kn1) enhanced both vegetative and reproductive defects in tls1. In addition, reduced transcript levels of kn1 and cell cycle genes are early defects in tls1 tassel meristems. Our results show that specific meristem maintenance and hormone pathways are affected in tls1, and suggest that reduced boron levels induced by tls1 are the underlying cause of the observed defects. We, therefore, provide new insights into the molecular mechanisms affected by boron deficiency in maize, leading to a better understanding of how genetic and environmental factors integrate during shoot meristem development.

From element to development: the power of the essential micronutrient boron to shape morphological processes in plants.

Deficiency of the essential nutrient boron (B) in the soil is one of the most widespread micronutrient deficiencies worldwide, leading to developmental defects in root and shoot tissues of plants, and severe yield reductions in many crops. Despite this agricultural importance, the underlying mechanisms of how B shapes plant developmental and morphological processes are still not unequivocally understood in detail. This review evaluates experimental approaches that address our current understanding of how B influences plant morphological processes by focusing on developmental defects observed under B deficiency. We assess what is known about mechanisms that control B homeostasis and specifically highlight: (i) limitations in the methodology that is used to induce B deficiency; (ii) differences between mutant phenotypes and normal plants grown under B deficiency; and (iii) recent research on analyzing interactions between B and phytohormones. Our analysis highlights the need for standardized methodology to evaluate the roles of B in the cell wall versus other parts of the cell.

Assessment of a 18F-Phenylboronic Acid Radiotracer for Imaging Boron in Maize.

Boron (B) is an essential plant micronutrient. Deficiencies of B have drastic consequences on plant development leading to crop yield losses and reductions in root and shoot growth. Understanding the molecular and cellular consequences of B deficiency is challenging, partly because of the limited availability of B imaging techniques. In this report we demonstrate the efficacy of using 4-fluorophenylboronic acid (FPBA) as a B imaging agent, which is a derivative of the B deficiency mimic phenylboronic acid (PBA). We show that radioactively labelled [18F]FPBA (t½=110 m) accumulates at the root tip, the root elongation zone and at lateral root initiation sites in maize roots, and also translocates to the shoot where it accumulates along the leaf edges. Treatment of maize seedlings using FPBA and PBA causes a shortened primary root phenotype with absence of lateral roots in a dose-dependent manner. The primary root defects can be partially rescued by the addition of boric acid indicating that PBA can be used to induce B deficiency in maize and that radioactively labelled FPBA can be used to image sites of B demand on a tissue level.

Increased transpiration is correlated with reduced boron deficiency symptoms in the maize tassel-less1 mutant.

Loss-of-function mutations of the tassel-less1 (tls1) gene in maize, which is the co-ortholog of the Arabidopsis boron (B) importer NIP5;1, leads to the loss of reproductive structures (tassels and ears). The tls1 phenotypes can be rescued by B supplementation in the field and in the greenhouse. As the rescue with B supplementation is variable in the field, we investigated additional abiotic factors, potentially causing this variation in controlled greenhouse conditions. We found that the B-dependent rescue of the tls1 mutant tassel phenotype was enhanced when plants were grown with a mix of high pressure sodium (HPS) and metal halide (MH) lamps. Normal and tls1 plants had a significant increase in transpiration and increased B content in the leaves in the greenhouse with the addition of MH lamps. Our findings imply that B transport to the shoot is enhanced through increased transpiration, which suggests that the xylem transpiration stream provides a significant supply of B in maize.

Transport of boron by the tassel-less1 aquaporin is critical for vegetative and reproductive development in maize.

The element boron (B) is an essential plant micronutrient, and B deficiency results in significant crop losses worldwide. The maize (Zea mays) tassel-less1 (tls1) mutant has defects in vegetative and inflorescence development, comparable to the effects of B deficiency. Positional cloning revealed that tls1 encodes a protein in the aquaporin family co-orthologous to known B channel proteins in other species. Transport assays show that the TLS1 protein facilitates the movement of B and water into Xenopus laevis oocytes. B content is reduced in tls1 mutants, and application of B rescues the mutant phenotype, indicating that the TLS1 protein facilitates the movement of B in planta. B is required to cross-link the pectic polysaccharide rhamnogalacturonan II (RG-II) in the cell wall, and the percentage of RG-II dimers is reduced in tls1 inflorescences, indicating that the defects may result from altered cell wall properties. Plants heterozygous for both tls1 and rotten ear (rte), the proposed B efflux transporter, exhibit a dosage-dependent defect in inflorescence development under B-limited conditions, indicating that both TLS1 and RTE function in the same biological processes. Together, our data provide evidence that TLS1 is a B transport facilitator in maize, highlighting the importance of B homeostasis in meristem function.

vanishing tassel2 encodes a grass-specific tryptophan aminotransferase required for vegetative and reproductive development in maize.

Auxin plays a fundamental role in organogenesis in plants. Multiple pathways for auxin biosynthesis have been proposed, but none of the predicted pathways are completely understood. Here, we report the positional cloning and characterization of the vanishing tassel2 (vt2) gene of maize (Zea mays). Phylogenetic analyses indicate that vt2 is a co-ortholog of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), which converts Trp to indole-3-pyruvic acid in one of four hypothesized Trp-dependent auxin biosynthesis pathways. Unlike single mutations in TAA1, which cause subtle morphological phenotypes in Arabidopsis thaliana, vt2 mutants have dramatic effects on vegetative and reproductive development. vt2 mutants share many similarities with sparse inflorescence1 (spi1) mutants in maize. spi1 is proposed to encode an enzyme in the tryptamine pathway for Trp-dependent auxin biosynthesis, although this biochemical activity has recently been questioned. Surprisingly, spi1 vt2 double mutants had only a slightly more severe phenotype than vt2 single mutants. Furthermore, both spi1 and vt2 single mutants exhibited a reduction in free auxin levels, but the spi1 vt2 double mutants did not have a further reduction compared with vt2 single mutants. Therefore, both spi1 and vt2 function in auxin biosynthesis in maize, possibly in the same pathway rather than independently as previously proposed.

The main auxin biosynthesis pathway in Arabidopsis.

The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.

A maize thiamine auxotroph is defective in shoot meristem maintenance.

Plant shoots undergo organogenesis throughout their life cycle via the perpetuation of stem cell pools called shoot apical meristems (SAMs). SAM maintenance requires the coordinated equilibrium between stem cell division and differentiation and is regulated by integrated networks of gene expression, hormonal signaling, and metabolite sensing. Here, we show that the maize (Zea mays) mutant bladekiller1-R (blk1-R) is defective in leaf blade development and meristem maintenance and exhibits a progressive reduction in SAM size that results in premature shoot abortion. Molecular markers for stem cell maintenance and organ initiation reveal that both of these meristematic functions are progressively compromised in blk1-R mutants, especially in the inflorescence and floral meristems. Positional cloning of blk1-R identified a predicted missense mutation in a highly conserved amino acid encoded by thiamine biosynthesis2 (thi2). Consistent with chromosome dosage studies suggesting that blk1-R is a null mutation, biochemical analyses confirm that the wild-type THI2 enzyme copurifies with a thiazole precursor to thiamine, whereas the mutant enzyme does not. Heterologous expression studies confirm that THI2 is targeted to chloroplasts. All blk1-R mutant phenotypes are rescued by exogenous thiamine supplementation, suggesting that blk1-R is a thiamine auxotroph. These results provide insight into the role of metabolic cofactors, such as thiamine, during the proliferation of stem and initial cell populations.

Hormonal regulation of inflorescence and intercalary meristems in grasses.

Hormones played a fundamental role in improvement of yield in cereal grasses. Natural variants affecting gibberellic acid (GA) and auxin pathways were used to breed semi-dwarf varieties of rice, wheat, and sorghum, during the "Green Revolution" in the 20th century. Since then, variants with altered GA and cytokinin homeostasis have been used to breed cereals with increased grain number. These yield improvements were enabled by hormonal regulation of intercalary and inflorescence meristems. Recent advances have highlighted additional pathways, beyond the traditional CLAVATA-WUSCHEL pathway, in the regulation of auxin and cytokinin in inflorescence meristems, and have expanded our understanding of the role of GA in intercalary meristems.

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging.

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study early leaf development. Here, we describe methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging. The first method uses a wire stripper to remove the upper portions of older leaves, exposing the tip of the leaf primordium and allowing its measurement for more accurate transverse section sampling. The second method uses clear, double-sided nano tape to unroll and mount whole-leaf primordia for imaging. We show the utility of the two methods in visualizing and analyzing fluorescent protein reporters in maize. These methods provide a solution to the challenges presented by the distinctive morphology of maize leaf primordia and will be useful for visualizing and quantifying leaf anatomical and developmental traits in maize and other grass species.

Mapping Maize Mutants Using Bulked-Segregant Analysis and Next-Generation Sequencing.

Forward genetics is used to identify the genetic basis for a phenotype. The approach involves identifying a mutant organism exhibiting a phenotype of interest and then mapping the causative locus or gene. Bulked-segregant analysis (BSA) is a quick and effective approach to map mutants using pools of mutants and wild-type plants from a segregating population to identify linkage of the mutant phenotype, and this approach has been successfully used in plants. Traditional linkage mapping approaches are outdated and time intensive, and can be very difficult. With the highly evolved development and reduction in cost of high-throughput sequencing, this new approach combined with BSA has become extremely effective in multiple plant species, including Zea mays (maize). While the approach is incredibly powerful, careful experimental design, bioinformatic mapping techniques, and interpretation of results are important to obtain the desired results in an effective and timely manner. Poor design of a mapping population, limitations in bioinformatic experience, and inadequate understanding of sequence data are limitations of these approaches for the researcher. Here, we describe a straightforward protocol for mapping mutations responsible for a phenotype of interest in maize, using high-throughput sequencing and BSA. Specifically, we discuss relevant aspects of developing a mutant mapping population. This is followed by a detailed protocol for DNA preparation and analysis of short-read sequences to map and identify candidate causative mutations responsible for the mutant phenotype of interest. We provide command-line and perl scripts to complete the bioinformatic analysis of the mutant sequence data. This protocol lays out the design of the BSA, bioinformatic approaches, and interpreting the sequencing data. These methods are very adaptable to any forward genetics experiment and provide a step-by-step approach to identifying the genetic basis of a maize mutant phenotype. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Bulked-segregant analysis and high-throughput sequencing to map maize mutants.

Molecular, cellular, and developmental foundations of grass diversity.

Humans have cultivated grasses for food, feed, beverages, and construction materials for millennia. Grasses also dominate the landscape in vast parts of the world, where they have adapted morphologically and physiologically, diversifying to form ~12,000 species. Sequences of hundreds of grass genomes show that they are essentially collinear; nonetheless, not all species have the same complement of genes. Here, we focus on the molecular, cellular, and developmental bases of grain yield and dispersal-traits that are essential for domestication. Distinct genes, networks, and pathways were selected in different crop species, reflecting underlying genomic diversity. With increasing genomic resources becoming available in nondomesticated species, we anticipate advances in coming years that illuminate the ecological and economic success of the grasses.

Carbon-11 Radiotracing Reveals Physiological and Metabolic Responses of Maize Grown under Different Regimes of Boron Treatment.

In agriculture, boron is known to play a critical role in healthy plant growth. To dissect the role of boron in maize metabolism, radioactive carbon-11 (t½ 20.4 min) was used to examine the physiological and metabolic responses of 3-week-old B73 maize plants to different levels of boron spanning 0 mM, 0.05 mM, and 0.5 mM boric acid (BA) treatments. Growth behavior, of both shoots and roots, was recorded and correlated to plant physiological responses. 11CO2 fixation, leaf export of [11C]-photosynthates, and their rate of transport increased systematically with increasing BA concentrations, while the fraction of [11C]-photosynthates delivered to the roots under 0 mM and 0.5 mM BA treatments was lower than under 0.05 mM BA treatment, likely due to changes in root growth. Additionally, solid-phase extraction coupled with gamma counting, radio-fluorescence thin layer chromatography, and radio-fluorescence high-performance liquid chromatography techniques applied to tissue extracts provided insight into the effects of BA treatment on 'new' carbon (as 11C) metabolism. Most notable was the strong influence reducing boron levels had on raising 11C partitioning into glutamine, aspartic acid, and asparagine. Altogether, the growth of maize under different regimes of boron affected 11CO2 fixation, its metabolism and allocation belowground, and altered root growth. Finally, inductively coupled plasma mass spectrometry provided insight into the effects of BA treatment on plant uptake of other essential nutrients. Here, levels of boron and zinc systematically increased in foliar tissues with increasing BA concentration. However, levels of magnesium, potassium, calcium, manganese, and iron remained unaffected by treatment. The rise in foliar zinc levels with increased BA concentration may contribute to improved 11CO2 fixation under these conditions.

sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize.

The plant growth hormone auxin plays a critical role in the initiation of lateral organs and meristems. Here, we identify and characterize a mutant, sparse inflorescence1 (spi1), which has defects in the initiation of axillary meristems and lateral organs during vegetative and inflorescence development in maize. Positional cloning shows that spi1 encodes a flavin monooxygenase similar to the YUCCA (YUC) genes of Arabidopsis, which are involved in local auxin biosynthesis in various plant tissues. In Arabidopsis, loss of function of single members of the YUC family has no obvious effect, but in maize the mutation of a single yuc locus causes severe developmental defects. Phylogenetic analysis of the different members of the YUC family in moss, monocot, and eudicot species shows that there have been independent expansions of the family in monocots and eudicots. spi1 belongs to a monocot-specific clade, within which the role of individual YUC genes has diversified. These observations, together with expression and functional data, suggest that spi1 has evolved a dominant role in auxin biosynthesis that is essential for normal maize inflorescence development. Analysis of the interaction between spi1 and genes regulating auxin transport indicate that auxin transport and biosynthesis function synergistically to regulate the formation of axillary meristems and lateral organs in maize.

The Molecular Basis of Kale Domestication: Transcriptional Profiling of Developing Leaves Provides New Insights Into the Evolution of a Brassica oleracea Vegetative Morphotype.

Morphotypes of Brassica oleracea are the result of a dynamic interaction between genes that regulate the transition between vegetative and reproductive stages and those that regulate leaf morphology and plant architecture. In kales, ornate leaves, extended vegetative phase, and nutritional quality are some of the characters potentially selected by humans during domestication. We used a combination of developmental studies and transcriptomics to understand the vegetative domestication syndrome of kale. To identify candidate genes that are responsible for the evolution of domestic kale, we searched for transcriptome-wide differences among three vegetative B. oleracea morphotypes. RNA-seq experiments were used to understand the global pattern of expressed genes during a mixture of stages at one time in kale, cabbage, and the rapid cycling kale line TO1000. We identified gene expression patterns that differ among morphotypes and estimate the contribution of morphotype-specific gene expression that sets kale apart (3958 differentially expressed genes). Differentially expressed genes that regulate the vegetative to reproductive transition were abundant in all morphotypes. Genes involved in leaf morphology, plant architecture, defense, and nutrition were differentially expressed in kale. This allowed us to identify a set of candidate genes we suggest may be important in the kale domestication syndrome. Understanding candidate genes responsible for kale domestication is of importance to ultimately improve Cole crop production.

sparse inflorescence1, barren inflorescence1 and barren stalk1 promote cell elongation in maize inflorescence development.

The sparse inflorescence1 (spi1), Barren inflorescence1 (Bif1), barren inflorescence2 (bif2), and barren stalk1 (ba1) mutants produce fewer branches and spikelets in the inflorescence due to defects in auxin biosynthesis, transport, or response. We report that spi1, bif1, and ba1, but not bif2, also function in promoting cell elongation in the inflorescence.

Shoot branching.

All plant shoots can be described as a series of developmental modules termed phytomers, which are produced from shoot apical meristems. A phytomer generally consists of a leaf, a stem segment, and a secondary shoot meristem. The fate and activity adopted by these secondary, axillary shoot meristems is the major source of evolutionary and environmental diversity in shoot system architecture. Axillary meristem fate and activity are regulated by the interplay of genetic programs with the environment. Recent results show that these inputs are channeled through interacting hormonal and transcription factor regulatory networks. Comparison of the factors involved in regulating the function of diverse axillary meristem types both within and between species is gradually revealing a pattern in which a common basic program has been modified to produce a range of axillary meristem types.