A Plate Growth Assay to Quantify Embryonic Root Development of Zea mays.

Murashige-Skoog medium solutions have been used in a variety of plant plate growth assays, yet most research uses Arabidopsis thaliana as the study organism. For larger seeds such as maize (Zea mays), most protocols employ a paper towel roll method for experiments, which often involves wrapping maize seedlings in wet, sterile germination paper. What the paper towel roll method lacks, however, is the ability to image the roots over time without risk of contamination. Here, we describe a sterile plate growth assay that contains Murashige-Skoog medium to grow seedlings starting two days after germination. This protocol uses a section of a paper towel roll method to achieve uniform germination of maize seedlings, which are sterilely transferred onto large acrylic plates for the duration of the experiment. The media can undergo modification to include an assortment of plant hormones, exogenous sugars, and other chemicals. The acrylic plates allow researchers to freely image the plate without disturbing the seedlings and control the environment in which the seedlings are grown, such as modifications in temperature and light. Additionally, the protocol is widely adaptable for use with other cereal crops. Key features • Builds upon plate growth methods routinely used for Arabidopsis seedlings but that are inadequate for maize. • Real-time photographic analysis of seedlings up to two weeks following germination. • Allows for testing of various growth conditions involving an assortment of additives and/or modification of environmental conditions. • Samples are able to be collected for genotype screening.

A Functionalizable Analog of the Yariv Reagent for AGP Imaging using Fluorescence Microscopy.

Small molecule fluorescent probes that bind selectively to plant cell wall polysaccharides have been instrumental in elucidating the localization and function of these glycans. Arabinogalactan proteins (AGPs) are cell wall proteoglycans implicated in essential functions such as cell signaling, plant growth, and programmed cell death. There is currently no small molecule probe capable of fluorescently labeling AGPs. The Yariv reagents are the only small molecules that bind AGPs, and have been used to study AGP function and isolate AGPs via precipitation of an AGP-Yariv complex. However, the Yariv reagents are not fluorescent, rendering them ineffective for localization studies using fluorescence microscopy. A fluorescent version of a Yariv reagent that is capable of both binding as well as imaging AGPs would provide a powerful tool for studying AGPs in planta. Herein, we describe the synthesis of an azido analog of the Yariv reagent that can be further functionalized with a fluorophore to provide a glycoconjugate that binds AGPs and is fluorescent. We show that the modified reagent binds gum arabic in in vitro binding assays when used in conjunction with the ßGlcYariv reagent. Fluorescent imaging of AGPs in fixed maize leaf tissue enables localization of AGPs to cell walls in the leaf. Significantly, imaging can also be carried out using fresh tissue. This represents the first small molecule probe that can be used to visualize AGPs using fluorescence microscopy.

De novo transcriptome assembly from the nodal root growth zone of hydrated and water-deficit stressed maize inbred line FR697.

Certain cultivars of maize show increased tolerance to water deficit conditions by maintenance of root growth. To better understand the molecular mechanisms related to this adaptation, nodal root growth zone samples were collected from the reference inbred line B73 and inbred line FR697, which exhibits a relatively greater ability to maintain root elongation under water deficits. Plants were grown under various water stress levels in both field and controlled environment settings. FR697-specific RNA-Seq datasets were generated and used for a de novo transcriptome assembly to characterize any genotype-specific genetic features. The assembly was aided by an Iso-Seq library of transcripts generated from various FR697 plant tissue samples. The Necklace pipeline was used to combine a Trinity de novo assembly along with a reference guided assembly and the Viridiplantae proteome to generate an annotated consensus "SuperTranscriptome" assembly of 47,915 transcripts with a N50 of 3152 bp in length. The results were compared by Blastn to maize reference genes, a Benchmarking Universal Single-Copy Orthologs (BUSCO) genome completeness report and compared with three maize reference genomes. The resultant 'SuperTranscriptome' was demonstrated to be of high-quality and will serve as an important reference for analysis of the maize nodal root transcriptomic response to environmental perturbations.

Bioinformatics for plant and agricultural discoveries in the age of multiomics: A review and case study of maize nodal root growth under water deficit.

Advances in next-generation sequencing and other high-throughput technologies have facilitated multiomics research, such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, and phenomics. The resultant emerging multiomics data have brought new challenges as well as opportunities, as seen in the plant and agriculture science domains. We reviewed several bioinformatic and computational methods, models, and platforms, and we have highlighted some of our in-house developed efforts aimed at multiomics data analysis, integration, and management issues faced by the research community. A case study using multiomics datasets generated from our studies of maize nodal root growth under water deficit stress demonstrates the power of these datasets and some other publicly available tools. This analysis also sheds light on the landscape of such applied bioinformatic tools currently available for plant and crop science studies and introduces emerging trends and how they may affect the future.

Phloem anatomy and function as shaped by the cell wall.

The partitioning of assimilated carbon is a complex process that involves the loading, long-distance transport, and subsequent unloading of carbohydrates from source to sink tissues. The network of plumbing that facilitates this coordinated process is the phloem tissue. Our understanding of the physiology of phloem transport has grown tremendously since the modern theory of mass flow was first put forward, aided by the concomitant progress of technology and experimental methodologies. Recent findings have put a renewed emphasis on the underlying anatomy of the phloem, and in particular the important role that cell walls play in enabling the high-pressure flow of photoassimilates through the sieve element. This review will briefly summarize the foundational work in phloem anatomy and highlight recent work exploring the physiology of phloem cell wall structure and mechanics.

Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning.

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

Maize Carbohydrate Partitioning Defective33 Encodes an MCTP Protein and Functions in Sucrose Export from Leaves.

To sustain plant growth, development, and crop yield, sucrose must be transported from leaves to distant parts of the plant, such as seeds and roots. To identify genes that regulate sucrose accumulation and transport in maize (Zea mays), we isolated carbohydrate partitioning defective33 (cpd33), a recessive mutant that accumulated excess starch and soluble sugars in mature leaves. The cpd33 mutants also exhibited chlorosis in the leaf blades, greatly diminished plant growth, and reduced fertility. Cpd33 encodes a protein containing multiple C2 domains and transmembrane regions. Subcellular localization experiments showed the CPD33 protein localized to plasmodesmata (PD), the plasma membrane, and the endoplasmic reticulum. We also found that a loss-of-function mutant of the CPD33 homolog in Arabidopsis, QUIRKY, had a similar carbohydrate hyperaccumulation phenotype. Radioactively labeled sucrose transport assays showed that sucrose export was significantly lower in cpd33 mutant leaves relative to wild-type leaves. However, PD transport in the adaxial-abaxial direction was unaffected in cpd33 mutant leaves. Intriguingly, transmission electron microscopy revealed fewer PD at the companion cell-sieve element interface in mutant phloem tissue, providing a possible explanation for the reduced sucrose export in mutant leaves. Collectively, our results suggest that CPD33 functions to promote symplastic transport into sieve elements.

The barren stalk2 Gene Is Required for Axillary Meristem Development in Maize.

The diversity of plant architecture is determined by axillary meristems (AMs). AMs are produced from small groups of stem cells in the axils of leaf primordia and generate vegetative branches and reproductive inflorescences. Previous studies identified genes critical for AM development that function in auxin biosynthesis, transport, and signaling. barren stalk1 (ba1), a basic helix-loop-helix transcription factor, acts downstream of auxin to control AM formation. Here, we report the cloning and characterization of barren stalk2 (ba2), a mutant that fails to produce ears and has fewer branches and spikelets in the tassel, indicating that ba2 functions in reproductive AM development. Furthermore, the ba2 mutation suppresses tiller growth in the teosinte branched1 mutant, indicating that ba2 also plays an essential role in vegetative AM development. The ba2 gene encodes a protein that co-localizes and heterodimerizes with BA1 in the nucleus. Characterization of the genetic interaction between ba2 and ba1 demonstrates that ba1 shows a gene dosage effect in ba2 mutants, providing further evidence that BA1 and BA2 act together in the same pathway. Characterization of the molecular and genetic interaction between ba2 and additional genes required for the regulation of ba1 further supports this finding. The ba1 and ba2 genes are orthologs of rice genes, LAX PANICLE1 (LAX1) and LAX2, respectively, hence providing insights into pathways controlling AMs development in grasses.

Vacuolar Na⁺/H⁺ NHX-Type Antiporters Are Required for Cellular K⁺ Homeostasis, Microtubule Organization and Directional Root Growth.

Na⁺/H⁺ antiporters (NHXs) are integral membrane transporters that catalyze the electroneutral exchange of K⁺ or Na⁺ for H⁺ and are implicated in cell expansion, development, pH and ion homeostasis and salt tolerance. Arabidopsis contains four vacuolar NHX isoforms (NHX1-NHX4), but only the functional roles for NHX1 and NHX2 have been assessed thus far. Colocalization studies indicated that NHX3 and NHX4 colocalize to the tonoplast. To investigate the role of all vacuolar NHX isoforms, a quadruple knockout nhx1nhx2nhx3nhx4, lacking all vacuolar NHXs, was generated. Seedlings of nhx1nhx2nhx3nhx4 displayed significantly reduced growth, with markedly shorter hypocotyls. Under high K⁺, but not Na⁺, pronounced root skewing occurred in nhx1nhx2nhx3nhx4, suggesting that the organization of the cytoskeleton might be perturbed. Whole mount immunolabeling of cortical microtubules indicated that high K⁺ caused significant microtubule reorganization in nhx1nhx2nhx3nhx4 root cells of the elongation zone. Using microtubule stabilizing (Taxol) and destabilizing (propyzamide) drugs, we found that the effect of K⁺ on nhx1nhx2nhx3nhx4 root growth was antagonistic to that of Taxol, whereas elevated K⁺ exacerbated the endogenous effect of propyzamide on root skewing. Collectively, our results suggest that altered K⁺ homeostasis leads to an increase in the dynamics of cortical microtubule reorganization in nhx1nhx2nhx3nhx4 root epidermal cells of the elongation zone.