RESUMEN
Sugarcane root systems are poorly studied and understood due to the perennial nature, tall stature, and the long cropping cycle. Whilst some field studies gave insights into sugarcane root traits, there is no detailed description of root and root system traits available. The objectives of our work were to establish a baseline of sugarcane root trait values that will serve for future studies, and to characterize the degree of root system resilience when restricting tiller number. We first conducted an initial screening for root trait diversity on a collection of twenty cultivars representative of sugarcane breeding from 1930 to now. Then we investigated the effect of reduced tillering, via manual de-tillering, on the plant root and root system traits of five varieties grown under optimal conditions in a glasshouse for 1700°Cd. In addition to establishing baseline means and variation for sugarcane root trait values that could serve as a reference for crop models, we demonstrated that the sugarcane root mass fraction was extremely resilient to drastic reduction in tiller number. Restricted plants were effectively maintaining their root system configuration (opening angle) by dramatically increasing the number of nodal roots produced per tiller as well as maximizing total root length by increasing the specific root length. Using this knowledge of sugarcane root traits in combination with the specific agronomic constraints for sugarcane will now underpin the development of a root system ideotype for sugarcane to enable targeted root trait selection for improving crop productivity.
RESUMEN
How sucrose transporters (SUTs) regulate phloem unloading in monocot stems is poorly understood and particularly so for species storing high Suc concentrations. To this end, Sorghum bicolor SUTs SbSUT1 and SbSUT5 were characterized by determining their transport properties heterologously expressed in yeast or Xenopus laevis oocytes, and their in planta cellular and subcellular localization. The plasma membrane-localized SbSUT1 and SbSUT5 exhibited a strong selectivity for Suc and high Suc affinities in X. laevis oocytes at pH 5-SbSUT1, 6.3 ± 0.7 mm, and SbSUT5, 2.4 ± 0.5 mm Suc. The Suc affinity of SbSUT1 was dependent on membrane potential and pH. In contrast, SbSUT5 Suc affinity was independent of membrane potential and pH but supported high transport rates at neutral pH. Suc transport by the tonoplast localized SbSUT4 could not be detected using yeast or X. laevis oocytes. Across internode development, SUTs, other than SbSUT4, were immunolocalized to sieve elements, while for elongating and recently elongated internodes, SUTs also were detected in storage parenchyma cells. We conclude that apoplasmic Suc unloading from de-energized protophloem sieve elements in meristematic zones may be mediated by reversal of SbSUT1 and/or by uniporting SWEETs. Storage parenchyma localized SbSUT1 and SbSUT5 may accumulate Suc from the stem apoplasms of elongating and recently elongated internodes, whereas SbSUT4 may function to release Suc from vacuoles. Transiting from an apoplasmic to symplasmic unloading pathway as the stem matures, SbSUT1 and SbSUT5 increasingly function in Suc retrieval into metaphloem sieve elements to maintain a high turgor to drive symplasmic unloading by bulk flow.
Asunto(s)
Floema/metabolismo , Proteínas de Plantas/metabolismo , Tallos de la Planta/crecimiento & desarrollo , Sorghum/metabolismo , Animales , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Oocitos/metabolismo , Proteínas de Plantas/genética , Tallos de la Planta/metabolismo , Sacarosa/metabolismo , Xenopus laevis/metabolismoRESUMEN
Sugarcane (Saccharum spp. hybrids) accumulates high concentrations of sucrose in its mature stalk and a considerable portion of carbohydrate metabolism is also devoted to cell wall synthesis and fibre production. We examined tissue-specific expression patterns to explore the spatial deployment of pathways responsible for sucrose accumulation and fibre synthesis within the stalk. We performed expression profiling of storage parenchyma, vascular bundles and rind dissected from a maturing stalk internode of sugarcane, identifying ten cellulose synthase subunit genes and examining significant differences in the expression of their corresponding transcripts and those of several sugar transporters. These were correlated with differential expression patterns for transcripts of genes encoding COBRA-like proteins and other cell wall metabolism-related proteins. The sugar transporters genes ShPST2a, ShPST2b and ShSUT4 were significantly up-regulated in storage parenchyma while ShSUT1 was up-regulated in vascular bundles. Two co-ordinately expressed groups of cell wall related transcripts were also identified. One group, associated with primary cell wall synthesis (ShCesA1, ShCesA7, ShCesA9 and Shbk2l3), was up-regulated in parenchyma. The other group, associated with secondary cell wall synthesis (ShCesA10, ShCesA11, ShCesA12 and Shbk-2), was up-regulated in rind. In transformed sugarcane plants, the ShCesA7 promoter conferred stable expression of green fluorescent protein preferentially in the storage parenchyma of the maturing stalk internode. Our results indicate that there is spatial separation for elevated expression of these important targets in both sucrose accumulation and cell wall synthesis, allowing for increased clarity in our understanding of sucrose transport and fibre synthesis in sugarcane.
Asunto(s)
Genes de Plantas , Glucosiltransferasas/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Plantas/genética , Saccharum/genética , Saccharum/metabolismo , Pared Celular/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , Filogenia , Haz Vascular de Plantas/genética , Haz Vascular de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Distribución TisularRESUMEN
Accurate and timely detection of transgene copy number in sugarcane is currently hampered by the requirement to use Southern blotting, needing relatively large amounts of genomic DNA and, therefore, the continued growth and maintenance of bulky plants in containment glasshouses. In addition, the sugarcane genome is both polyploid and aneuploid, complicating the identification of appropriate genes for use as references in the development of a high-throughput method. Using bioinformatic techniques followed by in vitro testing, two genes that appear to occur once per base genome of sugarcane were identified. Using these genes as reference genes, a high-throughput assay employing RT-qPCR was developed and tested using a group of sugarcane plants that contained unknown numbers of copies of the nptII gene encoding kanamycin resistance. Using this assay, transgene copy numbers from 3 to more than 50 were identified. In comparison, Southern blotting accurately identified the number of transgene copies for one line and by inference for another, but was not able to provide an accurate estimation for transgenic lines containing numerous copies of the nptII gene. Using the reference genes identified in this study, a high-throughput assay for the determination of transgene copy number was developed and tested for sugarcane. This method requires much less input DNA, can be performed much earlier in the production of transgenic sugarcane plants and allows much more efficient assessment of numerous potentially transgenic lines than Southern blotting.
Asunto(s)
Dosificación de Gen , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Saccharum/genética , Transgenes , Southern Blotting , Cartilla de ADN , Ensayos Analíticos de Alto Rendimiento , Resistencia a la Kanamicina/genética , Plantas Modificadas Genéticamente/genética , Sorghum/genéticaRESUMEN
Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (ß-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form.
Asunto(s)
Proteínas de Plantas/metabolismo , Saccharum/enzimología , Vacuolas/enzimología , beta-Fructofuranosidasa/metabolismo , Secuencia de Aminoácidos , Núcleo Celular , Citoplasma , Hexosas/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Transporte de Proteínas , Análisis de Secuencia de Proteína , Sacarosa/metabolismoRESUMEN
AtSUC9 (At5g06170), a sucrose (Suc) transporter from Arabidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ultrahigh affinity for Suc (K(0.5) = 0.066 +/- 0.025 mm). AtSUC9 showed low substrate specificity, similar to AtSUC2 (At1g22710), and transported a wide range of glucosides, including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and alpha-methyl-d-glucose. The ability of AtSUC9 to transport 10 glucosides was compared directly with that of AtSUC2, HvSUT1 (from barley [Hordeum vulgare]), and ShSUT1 (from sugarcane [Saccharum hybrid]), and results indicate that type I and type II Suc transporters have different substrate specificities. AtSUC9 protein was localized to the plasma membrane by transient expression in onion (Allium cepa) epidermis. Using a whole-gene translational fusion to beta-glucuronidase, AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression in Arabidopsis was dependent on intragenic sequence, and this was found to also be true for AtSUC1 (At1g71880) but not AtSUC2. Plants containing mutations in Suc transporter gene AtSUC9 were found to have an early flowering phenotype under short-day conditions. The transport properties of AtSUC9 indicate that it is uniquely suited to provide cellular uptake of Suc at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Proteínas de Transporte de Membrana/fisiología , Proteínas de Plantas/fisiología , Sacarosa/metabolismo , Animales , Arabidopsis/anatomía & histología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arbutina/metabolismo , Alcoholes Bencílicos/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucósidos/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Elementos Reguladores de la Transcripción , Especificidad por Sustrato , XenopusRESUMEN
Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K(0.5) = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K(i)) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.
Asunto(s)
Proteínas de Transporte de Monosacáridos/metabolismo , Saccharum/metabolismo , Sacarosa/análogos & derivados , Sacarosa/metabolismo , Animales , Alcoholes Bencílicos/metabolismo , Transporte Biológico , Clonación Molecular , Glucósidos/metabolismo , Maltosa/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Sacarosa/antagonistas & inhibidores , Sacarosa/farmacología , Xenopus laevisRESUMEN
A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K(m) for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes.