Traveling with purpose: cell-to-cell transport of plant mRNAs.

Messenger RNAs (mRNAs) in multicellular organisms can act as signals transported cell-to-cell and over long distances. In plants, mRNAs traffic cell-to-cell via plasmodesmata (PDs) and over long distances via the phloem vascular system to control diverse biological processes - such as cell fate and tissue patterning - in destination organs. Research on long-distance transport of mRNAs in plants has made remarkable progress, including the cataloguing of many mobile mRNAs, characterization of mRNA features important for transport, identification of mRNA-binding proteins involved in their transport, and understanding of the physiological roles of mRNA transport. However, information on short-range mRNA cell-to-cell transport is still limited. This review discusses the regulatory mechanisms and physiological functions of mRNA transport at the cellular and whole plant levels.

Sugar loading is not required for phloem sap flow in maize plants.

Phloem transport of photoassimilates from leaves to non-photosynthetic organs, such as the root and shoot apices and reproductive organs, is crucial to plant growth and yield. For nearly 90 years, evidence has been generally consistent with the theory of a pressure-flow mechanism of phloem transport. Central to this hypothesis is the loading of osmolytes, principally sugars, into the phloem to generate the osmotic pressure that propels bulk flow. Here we used genetic and light manipulations to test whether sugar import into the phloem is required as the driving force for phloem sap flow. Using carbon-11 radiotracer, we show that a maize sucrose transporter1 (sut1) loss-of-function mutant has severely reduced export of carbon from photosynthetic leaves (only ~4% of the wild type level). Yet, the mutant remarkably maintains phloem pressure at ~100% and sap flow speeds at ~50-75% of those of wild type. Potassium (K+) abundance in the phloem was elevated in sut1 mutant leaves. Fluid dynamic modelling supports the conclusion that increased K+ loading compensated for decreased sucrose loading to maintain phloem pressure, and thereby maintained phloem transport via the pressure-flow mechanism. Furthermore, these results suggest that sap flow and transport of other phloem-mobile nutrients and signalling molecules could be regulated independently of sugar loading into the phloem, potentially influencing carbon-nutrient homoeostasis and the distribution of signalling molecules in plants encountering different environmental conditions.

An Optimized Whole-Mount Immunofluorescence Method for Shoot Apices.

The localization of a protein provides important information about its biological functions. The visualization of proteins by immunofluorescence has become an essential approach in cell biology. Here, we describe an easy-to-follow immunofluorescence protocol to localize proteins in whole-mount tissues of maize (Zea mays) and Arabidopsis. We present the whole-mount immunofluorescence procedure using maize ear primordia and Arabidopsis inflorescence apices as examples, followed by tips and suggestions for each step. In addition, we provide a supporting protocol to describe the use of an ImageJ plug-in to analyze colocalization. This protocol has been optimized to observe proteins in 2-5 mm maize ear primordia or in developing Arabidopsis inflorescence apices; however, it can be used as a reference to perform whole-mount immunofluorescence in other plant tissues and species. © 2021 Wiley Periodicals LLC. Basic Protocol: Whole-mount immunofluorescence for maize and Arabidopsis shoot apices Support Protocol: Measure colocalization by JACoP plugin in ImageJ.

The Kernel Size-Related Quantitative Trait Locus qKW9 Encodes a Pentatricopeptide Repeat Protein That Aaffects Photosynthesis and Grain Filling.

In maize (Zea mays), kernel weight is an important component of yield that has been selected during domestication. Many genes associated with kernel weight have been identified through mutant analysis. Most are involved in the biogenesis and functional maintenance of organelles or other fundamental cellular activities. However, few quantitative trait loci (QTLs) underlying quantitative variation in kernel weight have been cloned. Here, we characterize a QTL, qKW9, associated with maize kernel weight. This QTL encodes a DYW motif pentatricopeptide repeat protein involved in C-to-U editing of ndhB, a subunit of the chloroplast NADH dehydrogenase-like complex. In a null qkw9 background, C-to-U editing of ndhB was abolished, and photosynthesis was reduced, resulting in less maternal photosynthate available for grain filling. Characterization of qKW9 highlights the importance of optimizing photosynthesis for maize grain yield production.

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.

Sugar Transporters in Plants: New Insights and Discoveries.

Carbohydrate partitioning is the process of carbon assimilation and distribution from source tissues, such as leaves, to sink tissues, such as stems, roots and seeds. Sucrose, the primary carbohydrate transported long distance in many plant species, is loaded into the phloem and unloaded into distal sink tissues. However, many factors, both genetic and environmental, influence sucrose metabolism and transport. Therefore, understanding the function and regulation of sugar transporters and sucrose metabolic enzymes is key to improving agriculture. In this review, we highlight recent findings that (i) address the path of phloem loading of sucrose in rice and maize leaves; (ii) discuss the phloem unloading pathways in stems and roots and the sugar transporters putatively involved; (iii) describe how heat and drought stress impact carbohydrate partitioning and phloem transport; (iv) shed light on how plant pathogens hijack sugar transporters to obtain carbohydrates for pathogen survival, and how the plant employs sugar transporters to defend against pathogens; and (v) discuss novel roles for sugar transporters in plant biology. These exciting discoveries and insights provide valuable knowledge that will ultimately help mitigate the impending societal challenges due to global climate change and a growing population by improving crop yield and enhancing renewable energy production.

In vivo transport of three radioactive [18F]-fluorinated deoxysucrose analogs by the maize sucrose transporter ZmSUT1.

Sucrose transporter (SUT) proteins translocate sucrose across cell membranes; however, mechanistic aspects of sucrose binding by SUTs are not well resolved. Specific hydroxyl groups in sucrose participate in hydrogen bonding with SUT proteins. We previously reported that substituting a radioactive fluorine-18 [18F] at the C-6' position within the fructosyl moiety of sucrose did not affect sucrose transport by the maize (Zea mays) ZmSUT1 protein. To determine how 18F substitution of hydroxyl groups at two other positions within sucrose, the C-1' in the fructosyl moiety or the C-6 in the glucosyl moiety, impact sucrose transport, we synthesized 1'-[F18]fluoro-1'-deoxysucrose and 6-[F18]fluoro-6-deoxysucrose ([18F]FDS) analogs. Each [18F]FDS derivative was independently introduced into wild-type or sut1 mutant plants, which are defective in sucrose phloem loading. All three (1'-, 6'-, and 6-) [18F]FDS derivatives were efficiently and equally translocated, similarly to carbon-14 [14C]-labeled sucrose. Hence, individually replacing the hydroxyl groups at these positions within sucrose does not interfere with substrate recognition, binding, or membrane transport processes, and hydroxyl groups at these three positions are not essential for hydrogen bonding between sucrose and ZmSUT1. [18F]FDS imaging afforded several advantages compared to [14C]-sucrose detection. We calculated that 1'-[18F]FDS was transported at approximately a rate of 0.90 ± 0.15 m.h-1 in wild-type leaves, and at 0.68 ± 0.25 m.h-1 in sut1 mutant leaves. Collectively, our data indicated that [18F]FDS analogs are valuable tools to probe sucrose-SUT interactions and to monitor sucrose transport in plants.

Sucrose transporter2 contributes to maize growth, development, and crop yield.

During daylight, plants produce excess photosynthates, including sucrose, which is temporarily stored in the vacuole. At night, plants remobilize sucrose to sustain metabolism and growth. Based on homology to other sucrose transporter (SUT) proteins, we hypothesized the maize (Zea mays) SUCROSE TRANSPORTER2 (ZmSUT2) protein functions as a sucrose/H+ symporter on the vacuolar membrane to export transiently stored sucrose. To understand the biological role of ZmSut2, we examined its spatial and temporal gene expression, determined the protein subcellular localization, and characterized loss-of-function mutations. ZmSut2 mRNA was ubiquitously expressed and exhibited diurnal cycling in transcript abundance. Expressing a translational fusion of ZmSUT2 fused to a red fluorescent protein in maize mesophyll cell protoplasts revealed that the protein localized to the tonoplast. Under field conditions, zmsut2 mutant plants grew slower, possessed smaller tassels and ears, and produced fewer kernels when compared to wild-type siblings. zmsut2 mutants also accumulated two-fold more sucrose, glucose, and fructose as well as starch in source leaves compared to wild type. These findings suggest (i) ZmSUT2 functions to remobilize sucrose out of the vacuole for subsequent use in growing tissues; and (ii) its function provides an important contribution to maize development and agronomic yield.

An Inexpensive, Easy-to-Use, and Highly Customizable Growth Chamber Optimized for Growing Large Plants.

The ability to grow plants in highly controlled and reproducible environments is a critical factor for successful plant biology experiments. This protocol describes a simple and inexpensive method for constructing a fully automatic controlled growth chamber that can be easily adapted in plant biology laboratories as well as classrooms. All the materials described in this protocol can be found in garden and home improvement stores or through websites, making the procurement and setup for growing plants in a controlled environment less expensive and convenient. Furthermore, the system is highly customizable and can be used to study plant responses to numerous abiotic and biotic stress conditions. The growth chamber is designed to enable growth and characterization of large plants, such as maize and soybean. © 2017 by John Wiley & Sons, Inc.

Lenalidomide in myelodysplastic syndrome and multiple myeloma.

The use of thalidomide is limited by adverse effects of sedation, constipation, neuropathy and thromboembolism. In order to discover more potent and less toxic immunomodulators than thalidomide, its chemical structure was modified and lenalidomide was formed. Lenalidomide is approved by the US FDA for the treatment of patients with low-risk myelodysplastic syndrome (MDS) with deletion 5q cytogenetic abnormality. Two studies and a case report have evaluated lenalidomide in these MDS patients and showed significantly higher cytogenetic responses and durable red blood cell transfusion independence. Lenalidomide should be the drug of choice for patients with low and intermediate-1 risk MDS (based on the International Prognostic Scoring System) with chromosome 5q31 deletion with or without other karyotype abnormalities. Lenalidomide, in combination with dexamethasone, has been compared with dexamethasone alone in patients with relapsed or refractory multiple myeloma (MM) in two studies (MM-009 in North America and MM-010 in Europe, Israel and Australia). In these two phase III trials, lenalidomide demonstrated impressive (58-59%) response rates with dexamethasone. Lenalidomide has also been shown to overcome thalidomide resistance in MM patients. Therefore, the lenalidomide plus dexamethasone regimen provides another treatment option, in addition to first line MM treatment regimens of bortezomib, thalidomide or high-dose dexamethasone, for the treatment of relapsed or refractory MM. Lenalidomide does not produce significant sedation, constipation or neuropathy, but does lead to significant myelosuppression, unlike thalidomide. The prescribing information has a black box warning for risk of myelosuppression, deep vein thrombosis/pulmonary embolism and teratogenicity. Administration of lenalidomide is recommended at a starting dose of 10 mg/day orally for deletion 5q in MDS patients. Significant risk of myelosuppression may lead to dose reduction in the majority of these patients. Clinical trials of relapsed and refractory MM have shown that lenalidomide is clinically efficacious at a dosage of 25 mg/day when administered in combination with dexamethasone. Lenalidomide should be continued until disease progression in both MDS and MM patients.