Thomas L. Slewinski

Sucrose transporter1 functions in phloem loading in maize leaves

In most plants, sucrose is exported from source leaves to carbon-importing sink tissues to sustain their growth and metabolism. Apoplastic phloem-loading species require sucrose transporters (SUTs) to transport sucrose into the phloem. In many dicot plants, genetic and biochemical evidence has established that SUT1-type proteins function in phloem loading. However, the role of SUT1 in phloem loading in monocot plants is not clear since the rice (Oryza sativa) and sugarcane (Saccharum hybrid) SUT1 orthologues do not appear to function in phloem loading of sucrose. A SUT1 gene was previously cloned from maize (Zea mays) and shown to have expression and biochemical activity consistent with a hypothesized role in phloem loading. To determine the biological function of SUT1 in maize, a sut1 mutant was isolated and characterized. sut1 mutant plants hyperaccumulate carbohydrates in mature leaves and display leaf chlorosis with premature senescence. In addition, sut1 mutants have greatly reduced stature, altered biomass partitioning, delayed flowering, and stunted tassel development. Cold-girdling wild-type leaves to block phloem transport phenocopied the sut1 mutants, supporting a role for maize SUT1 in sucrose export. Furthermore, application of 14C-sucrose to abraded sut1 mutant and wild-type leaves showed that sucrose export was greatly diminished in sut1 mutants compared with wild type. Collectively, these data demonstrate that SUT1 is crucial for efficient phloem loading of sucrose in maize leaves.

vanishing tassel2 Encodes a Grass-Specific Tryptophan Aminotransferase Required for Vegetative and Reproductive Development in Maize

This study characterizes the vanishing tassel2 (vt2) mutant of maize, which has reduced levels of auxin and severe defects in vegetative and reproductive development. It finds that vt2 encodes a co-ortholog of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1, which functions in auxin biosynthesis, and provides evidence that vt2 and spi1, a YUCCA-like gene, may act in the same auxin biosynthetic pathway. Auxin plays a fundamental role in organogenesis in plants. Multiple pathways for auxin biosynthesis have been proposed, but none of the predicted pathways are completely understood. Here, we report the positional cloning and characterization of the vanishing tassel2 (vt2) gene of maize (Zea mays). Phylogenetic analyses indicate that vt2 is a co-ortholog of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), which converts Trp to indole-3-pyruvic acid in one of four hypothesized Trp-dependent auxin biosynthesis pathways. Unlike single mutations in TAA1, which cause subtle morphological phenotypes in Arabidopsis thaliana, vt2 mutants have dramatic effects on vegetative and reproductive development. vt2 mutants share many similarities with sparse inflorescence1 (spi1) mutants in maize. spi1 is proposed to encode an enzyme in the tryptamine pathway for Trp-dependent auxin biosynthesis, although this biochemical activity has recently been questioned. Surprisingly, spi1 vt2 double mutants had only a slightly more severe phenotype than vt2 single mutants. Furthermore, both spi1 and vt2 single mutants exhibited a reduction in free auxin levels, but the spi1 vt2 double mutants did not have a further reduction compared with vt2 single mutants. Therefore, both spi1 and vt2 function in auxin biosynthesis in maize, possibly in the same pathway rather than independently as previously proposed.

Genetic Control of Carbon Partitioning in Grasses: Roles of Sucrose Transporters and Tie-dyed Loci in Phloem Loading

Plants have specialized organs for distinct functions. Leaves perform photosynthesis and fix carbon, whereas roots absorb water and minerals. To distribute resources between these organs, plants have a vasculature composed of phloem and xylem. The xylem conducts water and minerals from the roots up

Tie-dyed1 Encodes a Novel, Phloem-Expressed Transmembrane Protein That Functions in Carbohydrate Partitioning

Carbon is partitioned between export from the leaf and retention within the leaf, and this process is essential for all aspects of plant growth and development. In most plants, sucrose is loaded into the phloem of carbon-exporting leaves (sources), transported through the veins, and unloaded into carbon-importing tissues (sinks). We have taken a genetic approach to identify genes regulating carbon partitioning in maize (Zea mays). We identified a collection of mutants, called the tie-dyed (tdy) loci, that hyperaccumulate carbohydrates in regions of their leaves. To understand the molecular function of Tdy1, we cloned the gene. Tdy1 encodes a novel transmembrane protein present only in grasses, although two protein domains are conserved across angiosperms. We found that Tdy1 is expressed exclusively in phloem cells of both source and sink tissues, suggesting that Tdy1 may play a role in phloem loading and unloading processes. In addition, Tdy1 RNA accumulates in protophloem cells upon differentiation, suggesting that Tdy1 may function as soon as phloem cells become competent to transport assimilates. Monitoring the movement of a fluorescent, soluble dye showed that tdy1 leaves have retarded phloem loading. However, once the dye entered into the phloem, solute transport appeared equal in wild-type and tdy1 mutant plants, suggesting that tdy1 plants are not defective in phloem unloading. Therefore, even though Tdy1 RNA accumulates in source and sink tissues, we propose that TDY1 functions in carbon partitioning by promoting phloem loading. Possible roles for TDY1 are discussed.

Maize SUT1 functions in phloem loading

The functions of dicot sucrose transporters (SUTs) in apoplastic phloem loading of sucrose are well established; however, whether SUTs similarly function in monocots was unresolved. To address this question, we recently provided genetic evidence that ZmSUT1 from maize (Zea mays) is required for efficient phloem loading. sut1-m1 mutant plants hyperaccumulate carbohydrates in leaves, are defective in loading sucrose into the phloem, and have altered biomass partitioning. Presumably due to the hyperaccumulation of soluble sugars in leaves, mutations in ZmSUT1 lead to downregulation of chlorophyll accumulation, photosynthesis and stomatal conductance. However, because we had identified only a single mutant allele, we were not able to exclude the possibility that the mutant phenotypes were instead caused by a closely linked mutation. Based on a novel aspect of the sut1 mutant phenotype, secretion of a concentrated sugar solution from leaf hydathodes, we identified an additional mutant allele, sut1-m4. This confirms that the mutation of SUT1 is responsible for the impairment in phloem loading. In addition, the sut1-m4 mutant does not accumulate transcripts, supporting the findings reported previously that the original mutant allele is also a null mutation. Collectively, these data demonstrate that ZmSUT1 functions to phloem load sucrose in maize leaves.

The Psychedelic Genes of Maize Redundantly Promote Carbohydrate Export From Leaves

Whole-plant carbohydrate partitioning involves the assimilation of carbon in leaves and its translocation to nonphotosynthetic tissues. This process is fundamental to plant growth and development, but its regulation is poorly understood. To identify genes controlling carbohydrate partitioning, we isolated mutants that are defective in exporting fixed carbon from leaves. Here we describe psychedelic (psc), a new mutant of maize (Zea mays) that is perturbed in carbohydrate partitioning. psc mutants exhibit stable, discrete chlorotic and green regions within their leaves. psc chlorotic tissues hyperaccumulate starch and soluble sugars, while psc green tissues appear comparable to wild-type leaves. The psc chlorotic and green tissue boundaries are usually delineated by larger veins, suggesting that translocation of a mobile compound through the veins may influence the tissue phenotype. psc mutants display altered biomass partitioning, which is consistent with reduced carbohydrate export from leaves to developing tissues. We determined that the psc mutation is unlinked to previously characterized maize leaf carbohydrate hyperaccumulation mutants. Additionally, we found that the psc mutant phenotype is inherited as a recessive, duplicate-factor trait in some inbred lines. Genetic analyses with other maize mutants with variegated leaves and impaired carbohydrate partitioning suggest that Psc defines an independent pathway. Therefore, investigations into the psc mutation have uncovered two previously unknown genes that redundantly function to regulate carbohydrate partitioning in maize.

Camouflage Patterning in Maize Leaves Results from a Defect in Porphobilinogen Deaminase

Maize leaves are produced from polarized cell divisions that result in clonal cell lineages arrayed along the long axis of the leaf. We utilized this stereotypical division pattern to identify a collection of mutants that form chloroplast pigmentation sectors that violate the clonal cell lineages. Here, we describe the camouflage1 (cf1) mutant, which develops nonclonal, yellow-green sectors in its leaves. We cloned the cf1 gene by transposon tagging and determined that it encodes porphobilinogen deaminase (PBGD), an enzyme that functions early in chlorophyll and heme biosynthesis. While PBGD has been characterized biochemically, no viable mutations in this gene have been reported in plants. To investigate the in vivo function of PBGD, we characterized the cf1 mutant. Histological analyses revealed that cf1 yellow sectors display the novel phenotype of bundle sheath cell-specific death. Light-shift experiments determined that constant light suppressed cf1 sector formation, a dark/light transition is required to induce yellow sectors, and that sectors form only during a limited time of leaf development. Biochemical experiments determined that cf1 mutant leaves have decreased PBGD activity and increased levels of the enzyme substrate in both green and yellow regions. Furthermore, the cf1 yellow regions displayed a reduction in catalase activity. A threshold model is hypothesized to explain the cf1 variegation and incorporates photosynthetic cell differentiation, reactive oxygen species scavenging, and PBGD function.

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.

Maize Carbohydrate partitioning defective1 impacts carbohydrate distribution, callose accumulation, and phloem function

The maize Carbohydrate partitioning defective1 mutant causes ectopic callose formation in sieve elements, which partially inhibits carbohydrate partitioning and is associated with hyperactive defense responses under insect pest exposure.