Environmental Regulation of Lateral Root Initiation in Arabidopsis

Plant morphology is dramatically influenced by environmental signals. The growth and development of the root system is an excellent example of this developmental plasticity. Both the number and placement of lateral roots are highly responsive to nutritional cues. This indicates that there must be a signal transduction pathway that interprets complex environmental conditions and makes the "decision" to form a lateral root at a particular time and place. Lateral roots originate from differentiated cells in adult tissues. These cells must reenter the cell cycle, proliferate, and redifferentiate to produce all of the cell types that make up a new organ. Almost nothing is known about how lateral root initiation is regulated or coordinated with growth conditions. Here, we report a novel growth assay that allows this regulatory mechanism to be dissected in Arabidopsis. When Arabidopsis seedlings are grown on nutrient media with a high sucrose to nitrogen ratio, lateral root initiation is dramatically repressed. Auxin localization appears to be a key factor in this nutrient-mediated repression of lateral root initiation. We have isolated a mutant, lateral root initiation 1 (lin1), that overcomes the repressive conditions. This mutant produces a highly branched root system on media with high sucrose to nitrogen ratios. The lin1 phenotype is specific to these growth conditions, suggesting that the lin1 gene is involved in coordinating lateral root initiation with nutritional cues. Therefore, these studies provide novel insights into the mechanisms that regulate the earliest steps in lateral root initiation and that coordinate plant development with the environment.     

The role of sucrose and nitrogen in adventitious root formation on cultured rose shoots

Cultured shoots ofRosa ‘Improved Blaze’ were used to determine the effects of sucrose and inorganic nitrogen on adventitious root formation. Shoots grown in media containing high sucrose concentrations (146.07–262.93 mM) produced more and longer roots than those grown in media containing 0–87.64 mM sucrose. This response to sucrose was related to the metabolism of sucrose rather than its osmotic properties since the use of mannitol and 3-O-methyl-α-D-glucopyranoside as osmotic substitutes did not reproduce the effect on rooting. The number and length of roots increased when the shoots were grown in media with the nitrogen concentration of the Murashige-Skoog (MS) salt formulation reduced from 60 to 7.5 mM. Neither nitrate (NO ₃ ⁻ ) nor ammonium (NH ₄ ⁺ ) alone at any of the concentrations tested had the effect on rooting that both had together in the ratio of the MS salt formulation. When the sucrose and nitrogen concentrations were both varied, the greatest rate of root initiation occurred on shoots grown in media with a high sucrose to nitrogen concentration ratio.     

PP2A mediates lateral root development under NaCl-induced osmotic stress throughout auxin redistribution in Arabidopsis thaliana

Aim

Auxin plays an important role in modulating root system architecture. The effect of salinity on root development has been extensively studied; however, evidence on how salinity affects lateral root development and its underlying molecular mechanism is scarce. Here, we analyzed the role of protein phosphatase PP2A activity in auxin redistribution during Arabidopsis root system adaptation under NaCl-induced osmotic stress.

Method

Arabidopsis Col-0 and DR5::UidA seedlings were grown in MS media containing NaCl alone or in combination with the auxin transport inhibitor naphthylphthalamic acid, the synthetic auxin α-Naphthaleneacetic acid or the phosphatase inhibitor Okadaic acid. After 8 days, primary root length and lateral root number in seedlings were quantified and the auxin distribution was analyzed.

Results

Promotion of primary root shortening and lateral root development induced by osmotic stress correlated with an increase in active auxin content and a >50 % reduction in protein phosphatase type 2A (PP2A) activity. Moreover, the observed effects on seedlings under osmotic stress are more pronounced with the PP2A inhibitor Okadaic acid.

Conclusion

Our data suggest PP2A is a positive regulator of osmotic stress-induced root system architecture modulation, involving auxin redistribution in Arabidopsis thaliana.     

The role of miR156/SPLs modules in Arabidopsis lateral root development

miR156 is an evolutionarily highly conserved miRNA in plants that defines an age‐dependent flowering pathway. The investigations thus far have largely, if not exclusively, confined to plant aerial organs. Root branching architecture is a major determinant of water and nutrients uptake for plants. We show here that MIR156 genes are differentially expressed in specific cells/tissues of lateral roots. Plants overexpressing miR156 produce more lateral roots whereas reducing miR156 levels leads to fewer lateral roots. We demonstrate that at least one representative from the three groups of miR156 targets SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE (SPL) genes: SPL3, SPL9 and SPL10 are involved in the repression of lateral root growth, with SPL10 playing a dominant role. In addition, both MIR156 and SPLs are responsive to auxin signaling suggesting that miR156/SPL modules might be involved in the proper timing of the lateral root developmental progression. Collectively, these results unravel a role for miR156/SPLs modules in lateral root development in Arabidopsis.     

Characterization of CYCLOPHILLIN38 shows that a photosynthesis-derived systemic signal controls lateral root emergence

Photosynthesis in leaves generates fixed-carbon resources and essential metabolites that support sink tissues, such as roots. Two of these metabolites, sucrose and auxin, promote growth in root systems, but the explicit connection between photosynthetic activity and control of root architecture has not been explored. Through a mutant screen to identify pathways regulating root system architecture, we identified a mutation in the Arabidopsis thaliana CYCLOPHILIN 38 (CYP38) gene, which causes accumulation of pre-emergent stage lateral roots. CYP38 was previously reported to stabilize photosystem II (PSII) in chloroplasts. CYP38 expression is enriched in shoots, and grafting experiments show that the gene acts non-cell-autonomously to promote lateral root emergence. Growth of wild-type plants under low-light conditions phenocopies the cyp38 lateral root emergence defect, as does the inhibition of PSII-dependent electron transport or Nicotinamide adenine dinucleotide phosphate (NADPH) production. Importantly, these perturbations to photosynthetic activity rapidly suppress lateral root emergence, which is separate from their effects on shoot size. Supplementary exogenous sucrose largely rescued primary root (PR) growth in cyp38, but not lateral root growth. Auxin (indole-3-acetic acid (IAA)) biosynthesis from tryptophan is dependent on reductant generated during photosynthesis. Consistently, we found that wild-type seedlings grown under low light and cyp38 mutants have highly diminished levels of IAA in root tissues. IAA treatment rescued the cyp38 lateral root defect, revealing that photosynthesis promotes lateral root emergence partly through IAA biosynthesis. These data directly confirm the importance of CYP38-dependent photosynthetic activity in supporting root growth, and define the specific contributions of two metabolites in refining root architecture under light-limited conditions.

Lateral root emergence is regulated via systemic signaling that incorporates photosynthesis-dependent redox control and auxin biosynthesis.     

Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

INTRODUCTIONAuxin plays an important role in regu1ating celldivision, e1ongation and differentiatiou, vascular tis-sue fOrmation[1], pollen deve1opment[2] and 1eafyhead fOrmation[3]. Adrin polar transport is be-1ieved to invohe in a variety of important growthand developmenial processes, including the patternfOrmation of eInbryO, leaf morphogenesis and theroot gravity response[4--8]. Auxin po1ar transportinhibitor has been proved essential illterference ofataln transport leading to patte…     

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT

Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.     

Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of light and auxin

Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-¹⁴C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH₃ content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-¹⁴C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of ¹⁴CO₂ followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.     

Cytokinins act directly on lateral root founder cells to inhibit root initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.     

D‐Root: a system for cultivating plants with the roots in darkness or under different light conditions

In nature roots grow in the dark and away from light (negative phototropism). However, most current research in root biology has been carried out with the root system grown in the presence of light. Here, we have engineered a device, called Dark‐Root (D‐Root), to grow plants in vitro with the aerial part exposed to the normal light/dark photoperiod while the roots are in the dark or exposed to specific wavelengths or light intensities. D‐Root provides an efficient system for cultivating a large number of seedlings and easily characterizing root architecture in the dark. At the morphological level, root illumination shortens root length and promotes early emergence of lateral roots, therefore inducing expansion of the root system. Surprisingly, root illumination also affects shoot development, including flowering time. Our analyses also show that root illumination alters the proper response to hormones or abiotic stress (e.g. salt or osmotic stress) and nutrient starvation, enhancing inhibition of root growth. In conclusion, D‐Root provides a growing system closer to the natural one for assaying Arabidopsis plants, and therefore its use will contribute to a better understanding of the mechanisms involved in root development, hormonal signaling and stress responses.     

Differential effects of sucrose and auxin on localized phosphate deficiency-induced modulation of different traits of root system architecture in Arabidopsis

Phosphorus, one of the essential elements for plants, is often a limiting nutrient in soils. Low phosphate (Pi) availability induces sugar-dependent systemic expression of genes and modulates the root system architecture (RSA). Here, we present the differential effects of sucrose (Suc) and auxin on the Pi deficiency responses of the primary and lateral roots of Arabidopsis (Arabidopsis thaliana). Inhibition of primary root growth and loss of meristematic activity were evident in seedlings grown under Pi deficiency with or without Suc. Although auxin supplementation also inhibited primary root growth, loss of meristematic activity was observed specifically under Pi deficiency with or without Suc. The results suggested that Suc and auxin do not influence the mechanism involved in localized Pi sensing that regulates growth of the primary root and therefore delineates it from sugar-dependent systemic Pi starvation responses. However, the interaction between Pi and Suc was evident on the development of the lateral roots and root hairs in the seedlings grown under varying levels of Pi and Suc. Although the Pi+ Suc- condition suppressed lateral root development, induction of few laterals under the Pi- Suc- condition point to increased sensitivity of the roots to auxin during Pi deprivation. This was supported by expression analyses of DR5uidA, root basipetal transport assay of auxin, and RSA of the pgp19 mutant exhibiting reduced auxin transport. A significant increase in the number of lateral roots under the Pi- Suc- condition in the chalcone synthase mutant (tt4-2) indicated a potential role for flavonoids in auxin-mediated Pi deficiency-induced modulation of RSA. The study thus demonstrated differential roles of Suc and auxin in the developmental responses of ontogenetically distinct root traits during Pi deprivation. In addition, lack of cross talk between local and systemic Pi sensing as revealed by the seedlings grown under either the Pi- Suc- condition or in the heterogeneous Pi environment highlighted the coexistence of Suc-independent and Suc-dependent regulatory mechanisms that constitute Pi starvation responses.     

Stimulation of in vitro root and shoot growth of potato by increasing sucrose concentration in the presence of fluridone,an inhibitor of abscisic acid synthesis

Fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, strongly stimulated rooting of nodal stem segments of potato (Solanum tuberosum L.) cultivar Arran Banner cultured in darkness on tuberisation medium. Inclusion of 10^-6 M ABA in the culture medium prevented this rooting response, indicating that root proliferation in the presence of fluridone could be due to inhibition of ABA synthesis. The rooting response to fluridone (increased total root number and root fresh weight) was obtained only at high sucrose concentrations (0.175 and 0.234 M) and was demonstrated with two potato cultivars and two culture media; one which favoured tuberisation and one which did not. Shoot numbers were also increased, but to a lesser extent than root numbers, and total fresh weight of plant material per culture was greatly increased by inclusion of both fluridone (10^-6 or 10^-5 M) and 0.234 M sucrose in the culture medium. The role of sucrose was not simply osmotic because when the osmolarity of fluridone medium was increased using mixtures of mannitol and sucrose, no root proliferation occurred unless sucrose predominated in the mixture.     

Review: role of carbon sources for in vitro plant growth and development

In vitro plant cells, tissues and organ cultures are not fully autotrophic establishing a need for carbohydrates in culture media to maintain the osmotic potential, as well as to serve as energy and carbon sources for developmental processes including shoot proliferation, root induction as well as emission, embryogenesis and organogenesis, which are highly energy demanding developmental processes in plant biology. A variety of carbon sources (both reducing and non-reducing) are used in culture media depending upon genotypes and specific stages of growth. However, sucrose is most widely used as a major transport-sugar in the phloem sap of many plants. In micropropagation systems, morphogenetic potential of plant tissues can greatly be manipulated by varying type and concentration of carbon sources. The present article reviews the past and current findings on carbon sources and their sustainable utilization for in vitro plant tissue culture to achieve better growth rate and development.     

AtCYT-INV1 in Arabidopsis Sugar Signaling

Sugar acts as a signal molecule and plays a pivotal role in plant development and stress response. Neutral/alkaline invertases found only in photosynthetic bacteria and plants is sucrose-specific enzymes cleave sucrose into glucose and fructose. We have identified a gene for neutral/alkaline invertase in Arabidopsis designated as AtCYT-INV1 which is involved in sugar/ABA signaling and plays multiple roles in plant development and osmotic stress-induced inhibition on lateral root growth.Key Words: Arabidopsis thaliana, AtCTY-INV1, sugar signaling     

Repression of early lateral root initiation events by transient water deficit in barley and maize

The formation of lateral roots (LRs) is a key driver of root system architecture and developmental plasticity. The first stage of LR formation, which leads to the acquisition of founder cell identity in the pericycle, is the primary determinant of root branching patterns. The fact that initiation events occur asynchronously in a very small number of cells inside the parent root has been a major difficulty in the study of the molecular regulation of branching patterns. Inducible systems that trigger synchronous lateral formation at predictable sites have proven extremely valuable in Arabidopsis to decipher the first steps of LR formation. Here, we present a LR repression system for cereals that relies on a transient water-deficit treatment, which blocks LR initiation before the first formative divisions. Using a time-lapse approach, we analysed the dynamics of this repression along growing roots and were able to show that it targets a very narrow developmental window of the initiation process. Interestingly, the repression can be exploited to obtain negative control root samples where LR initiation is absent. This system could be instrumental in the analysis of the molecular basis of drought-responsive as well as intrinsic pathways of LR formation in cereals.     

Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system

The postembryonic developmental program of the plant root system is plastic and allows changes in root architecture to adapt to environmental conditions such as water and nutrient availability. Among essential nutrients, phosphorus (P) often limits plant productivity because of its low mobility in soil. Therefore, the architecture of the root system may determine the capacity of the plant to acquire this nutrient. We studied the effect of P availability on the development of the root system in Arabidopsis. We found that at P-limiting conditions (<50 microM), the Arabidopsis root system undergoes major architectural changes in terms of lateral root number, lateral root density, and primary root length. Treatment with auxins and auxin antagonists indicate that these changes are related to an increase in auxin sensitivity in the roots of P-deprived Arabidopsis seedlings. It was also found that the axr1-3, axr2-1, and axr4-1 Arabidopsis mutants have normal responses to low P availability conditions, whereas the iaa28-1 mutant shows resistance to the stimulatory effects of low P on root hair and lateral root formation. Analysis of ethylene signaling mutants and treatments with 1-aminocyclopropane-1-carboxylic acid showed that ethylene does not promote lateral root formation under P deprivation. These results suggest that in Arabidopsis, auxin sensitivity may play a fundamental role in the modifications of root architecture by P availability.     

High root biomass production in anchored Arabidopsis plants grown in axenic sucrose supplemented liquid culture

There are many benefits to growing Arabidopsis in solution-based media, especially when large amounts of root tissue are required for molecular and biochemical studies. Roots grown in soil are brittle and tend to break easily when removed from their substrate. We have developed an axenic liquid culture system that simplifies growing large amounts of roots from intact plants. This technique consists of germinating 15 seeds on 2.5 cm2 stainless steel screens placed on half-strength semisolid Murashige and Skoog medium containing 1% or 2% sucrose. The screens anchor and support the plantlets in an upright position while keeping the roots and shoots separate. The seedlings are transferred with forceps to 125-mL wide-mouth Erlenmeyer flasks containing 10 mL of half-strength Murashige and Skoog liquid medium and 1% sucrose. The flasks are placed onto a floor rotary shaker under fluorescent lights. After 3 days, the sucrose is increased to 3% and the volume to 15 mL for 7 days. During any further experimental manipulations, sucrose is not supplied. The media is changed every 3-4 days to replenish the nutrients. The presence of sucrose in the media dramatically increases the biomass, and large amounts of root tissue can easily be harvested.