Roots are crucial for nutrient and water acquisition and can be targeted to enhance plant productivity under a broad range of growing conditions. A current challenge for plant breeding is the limited ability to phenotype and select for desirable root characteristics due to their underground location. Plant breeding efforts aimed at modifying root traits can result in novel, more stress-tolerant crops and increased yield by enhancing the capacity of the plant for soil exploration and, thus, water and nutrient acquisition. Available approaches for root phenotyping in laboratory, greenhouse and field encompass simple agar plates to labor-intensive root digging (i.e., shovelomics) and soil boring methods, the construction of underground root observation stations and sophisticated computer-assisted root imaging. Here, we summarize root architectural traits relevant to crop productivity, survey root phenotyping strategies and describe their advantages, limitations and practical value for crop and forage breeding programs.

Summary• The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic, biochemical and bioinformatics studies.• Heterologous expression in yeast, and gene expression and localization in plants were used to characterize all six members of an Arabidopsis thaliana membrane transporter family designated here as PHT4. PHT4 proteins share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride.• All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid envelope, and the sixth resides in the Golgi apparatus.• PHT4 genes are expressed in both roots and leaves, although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. In plants, NAEs are generally elevated in desiccated seeds, suggesting that they may play a role in seed physiology. NAE and abscisic acid (ABA) levels were depleted during seed germination, and both metabolites inhibited the growth of Arabidopsis thaliana seedlings within a similar developmental window. Combined application of low levels of ABA and NAE produced a more dramatic reduction in germination and growth than either compound alone. Transcript profiling and gene expression studies in NAE-treated seedlings revealed elevated transcripts for a number of ABA-responsive genes and genes typically enriched in desiccated seeds. The levels of ABI3 transcripts were inversely associated with NAE-modulated growth. Overexpression of the Arabidopsis NAE degrading enzyme fatty acid amide hydrolase resulted in seedlings that were hypersensitive to ABA, whereas the ABAinsensitive mutants, abi1-1, abi2-1, and abi3-1, exhibited reduced sensitivity to NAE. Collectively, our data indicate that an intact ABA signaling pathway is required for NAE action and that NAE may intersect the ABA pathway downstream from ABA. We propose that NAE metabolism interacts with ABA in the negative regulation of seedling development and that normal seedling establishment depends on the reduction of the endogenous levels of both metabolites.

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using 86Rb+ as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a K  m of 136 and 196 μ  m, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment.

Plant development is regulated by numerous chemicals derived from a multitude of metabolic pathways. However, we know very little about the biological effects and functions of many of these metabolites in the cell. N-Acylethanolamines (NAEs) are a group of lipid mediators that play important roles in mammalian physiology. Despite the intriguing similarities between animals and plants in NAE metabolism and perception, not much is known about the precise function of these metabolites in plant physiology. In plants, NAEs have been shown to inhibit phospholipase Dalpha (PLDalpha) activity, interfere with abscisic acid-induced stomatal closure, and retard Arabidopsis seedling development. 1-Butanol, an antagonist of PLD-dependent phosphatidic acid production, was reported to induce defects in Arabidopsis seedling development that were somewhat similar to effects induced by elevated levels of NAE. This raised the possibility that the impact of NAE on seedling growth could be mediated in part via its influence on PLD activity. To begin to address this possibility, we conducted a detailed, comparative analysis of the effects of 1-butanol and N-lauroylethanolamine (NAE 12:0) on Arabidopsis root cell division, in vivo cytoskeletal organization, seed germination, and seedling growth. Although both NAE 12:0 and 1-butanol induced profound cytoskeletal and morphological alterations in seedlings, there were distinct differences in their overall effects. 1-Butanol induced more pronounced modifications in cytoskeletal organization, seedling growth, and cell division at concentrations severalfold higher than NAE 12:0. We propose that these compounds mediate their differential effects on cellular organization and seedling growth, in part through the differential modulation of specific PLD isoforms.

Potato virus X (PVX) encodes three proteins named TGBp1, TGBp2, and TGBp3 which are required for virus cell-to-cell movement. To determine whether PVX TGB proteins interact during virus cell-cell movement, GFP was fused to each TGB coding sequence within the viral genome. Confocal microscopy was used to study subcellular accumulation of each protein in virus-infected plants and protoplasts. GFP:TGBp2 and TGBp3:GFP were both seen in the ER, ER-associated granular vesicles, and perinuclear X-bodies suggesting that these proteins interact in the same subdomains of the endomembrane network. When plasmids expressing CFP:TGBp2 and TGBp3:GFP were co-delivered to tobacco leaf epidermal cells, the fluorescent signals overlapped in ER-associated granular vesicles indicating that these proteins colocalize in this subcellular compartment. GFP:TGBp1 was seen in the nucleus, cytoplasm, rod-like inclusion bodies, and in punctate sites embedded in the cell wall. The puncta were reminiscent of previous reports showing viral proteins in plasmodesmata. Experiments using CFP:TGBp1 and YFP:TGBp2 or TGBp3:GFP showed CFP:TGBp1 remained in the cytoplasm surrounding the endomembrane network. There was no evidence that the granular vesicles contained TGBp1. Yeast two hybrid experiments showed TGBp1 self associates but failed to detect interactions between TGBp1 and TGBp2 or TGBp3. These experiments indicate that the PVX TGB proteins have complex subcellular accumulation patterns and likely cooperate across subcellular compartments to promote virus infection.

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs.