Plant development: Laying the foundation for high-performance photosynthesis.

A new study shows that TOO MANY LATERALS/WIP6 acts as a key regulator of vein specification and development across C3 and C4 photosynthetic grasses.

Targeted suppression of gibberellin biosynthetic genes ZmGA20ox3 and ZmGA20ox5 produces a short stature maize ideotype.

Maize is one of the world's most widely cultivated crops. As future demands for maize will continue to rise, fields will face ever more frequent and extreme weather patterns that directly affect crop productivity. Development of environmentally resilient crops with improved standability in the field, like wheat and rice, was enabled by shifting the architecture of plants to a short stature ideotype. However, such architectural change has not been implemented in maize due to the unique interactions between gibberellin (GA) and floral morphology which limited the use of the same type of mutations as in rice and wheat. Here, we report the development of a short stature maize ideotype in commercial hybrid germplasm, which was generated by targeted suppression of the biosynthetic pathway for GA. To accomplish this, we utilized a dominant, miRNA-based construct expressed in a hemizygous state to selectively reduce expression of the ZmGA20ox3 and ZmGA20ox5 genes that control GA biosynthesis primarily in vegetative tissues. Suppression of both genes resulted in the reduction of GA levels leading to inhibition of cell elongation in internodal tissues, which reduced plant height. Expression of the miRNA did not alter GA levels in reproductive tissues, and thus, the reproductive potential of the plants remained unchanged. As a result, we developed a dominant, short-stature maize ideotype that is conducive for the commercial production of hybrid maize. We expect that the new maize ideotype would enable more efficient and more sustainable maize farming for a growing world population.

Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning.

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

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.

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

Plants synthesize carbohydrates in photosynthetic tissues, with the majority of plants transporting sucrose to non-photosynthetic tissues to sustain growth and development. While the anatomical, biochemical, and physiological processes regulating sucrose long-distance transport are well characterized, little is known concerning the genes controlling whole-plant carbohydrate partitioning. To identify loci influencing carbon export from leaves, we screened mutagenized maize plants for phenotypes associated with reduced carbohydrate transport, including chlorosis and excessive starch and soluble sugars in leaves. Carbohydrate partitioning defective1 (Cpd1) was identified as a semi-dominant mutant exhibiting these phenotypes. Phloem transport experiments suggested that the hyperaccumulation of starch and soluble sugars in the Cpd1/+ mutant leaves was due to inhibited sucrose export. Interestingly, ectopic callose deposits were observed in the phloem of mutant leaves, and probably underlie the decreased transport. In addition to the carbohydrate hyperaccumulation phenotype, Cpd1/+ mutants overaccumulate benzoxazinoid defense compounds and exhibit increased tolerance when attacked by aphids. However, double mutant studies between Cpd1/+ and benzoxazinoid-less plants indicate that the ectopic callose and carbon hyperaccumulation are independent of benzoxazinoid production. Based on the formation of callose occlusions in the developing phloem, we hypothesize that the cpd1 gene functions early in phloem development, thereby impacting whole-plant carbohydrate partitioning.

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.

Symplastic phloem loading in poplar.

Sap is driven through phloem sieve tubes by an osmotically generated pressure gradient between source and sink tissues. In many plants, source pressure results from thermodynamically active loading in which energy is used to transfer sucrose (Suc) from mesophyll cells to the phloem of leaf minor veins against a concentration gradient. However, in some species, almost all trees, correlative evidence suggests that sugar migrates passively through plasmodesmata from mesophyll cells into the sieve elements. The possibility of alternate loading mechanisms has important ramifications for the regulation of phloem transport and source-sink interactions. Here, we provide experimental evidence that, in gray poplar (Populus tremula × Populus alba), Suc enters the phloem through plasmodesmata. Transgenic plants were generated with yeast invertase in the cell walls to prevent Suc loading by this route. The constructs were driven either by the constitutive 35S promoter or the minor vein-specific galactinol synthase promoter. Transgenic plants grew at the same rate as the wild type without symptoms of loading inhibition, such as accumulation of carbohydrates or leaf chlorosis. Rates of photosynthesis were normal. In contrast, alfalfa (Medicago sativa) plants, which have limited numbers of plasmodesmata between mesophyll and phloem, displayed typical symptoms of loading inhibition when transformed with the same DNA constructs. The results are consistent with passive loading of Suc through plasmodesmata in poplar. We also noted defense-related symptoms in leaves of transgenic poplar when the plants were abruptly exposed to excessively high temperatures, adding to evidence that hexose is involved in triggering the hypersensitive response.

Using evolution as a guide to engineer kranz-type c4 photosynthesis.

Kranz-type C4 photosynthesis has independently and rapidly evolved over 60 times to dramatically increase radiation use efficiency in both monocots and eudicots. Indeed, it is one of the most exceptional examples of convergent evolution in the history of life. The repeated and rapid evolution of Kranz-type C4 suggests that it may be a derivative of a conserved developmental pathway that is present in all angiosperms. Here, I argue that the Kranz-type C4 photosynthetic system is an extension of the endodermis/starch sheath, that is normally only found in the roots and stems, into photosynthetic structures such as leaves. Support for this hypothesis was recently provided by a study that showed that the same genetic pathway that gives rise to the endodermis in roots, the SCARECROW/SHORT-ROOT radial patterning system, also regulates the development of Kranz anatomy and C4 physiology in leaves. This new hypothesis for the evolution of Kranz-type C4 photosynthesis has opened new opportunities to explore the underlying genetic networks that regulate the development and physiology of C4 and provides new potential avenues for the engineering of the mechanism into C3 crops.

Structural and functional heterogeneity in phloem loading and transport.

The phloem is often regarded as a relatively straightforward transport system composed of loading (collection), long-distance (transport), and unloading (release) zones. While this simple view is necessary and useful in many contexts, it belies the reality, which is that the phloem is inherently complex. At least three types of sieve element-companion cell complexes are found in minor veins of leaves. Individual species may have more than one type, indicating that they employ multiple loading strategies, even in the same vein. Gene expression data in particular point to heterogeneity in sieve element-companion cell complexes of minor veins, perhaps in all flowering plants. Phloem heterogeneity in the transport phloem is also evident in many species based on anatomical, biochemical and gene expression data. In this regard, members of the Cucurbitaceae are especially complex and interesting. We conclude that a hidden world of specialized phloem function awaits discovery.

The tie-dyed pathway promotes symplastic trafficking in the phloem.

The tie-dyed1 (tdy1) and tdy2 mutants of maize exhibit leaf regions with starch hyperaccumulation and display unusual genetic interactions, suggesting they function in the same physiological process. Tdy2 encodes a putative callose synthase and is expressed in developing vascular tissues of immature leaves. Radiolabelling experiments and transmission electron microscopy (TEM) revealed symplastic trafficking within the phloem was perturbed at the companion cell/sieve element interface. Here, we show that as reported for tdy2 mutants, tdy1 yellow leaf regions display an excessive oil-droplet phenotype in the companion cells. Based on the proposed function of Tdy2 as a callose synthase, our previous work characterizing Tdy1 as a novel, transmembrane-localized protein, and the present findings, we speculate how TDY1 and TDY2 might interact to promote symplastic transport of both solutes and developmentally instructive macromolecules during vascular development at the companion cell/sieve element interface.

Scarecrow plays a role in establishing Kranz anatomy in maize leaves.

More than a quarter of the primary productivity on land, and a large fraction of the food that humans consume, is contributed by plants that fix atmospheric CO(2) by C(4) photosynthesis. It has been estimated that transferring the C(4) pathway to C(3) crops could boost yield by 50% and also increase water use efficiency and reduce the need for fertilizer, particularly in dry, hot environments. The high productivity of maize (Zea mays), sugarcane (Saccharum spp.) and several emerging bioenergy grasses is due largely to C(4) photosynthesis, which is enabled by the orderly arrangement, in concentric rings, of specialized bundle sheath and mesophyll cells in leaves in a pattern known as Kranz anatomy. Here we show that PIN, the auxin efflux protein, is present in the end walls of maize bundle sheath cells, as it is in the endodermis of the root. Since this marker suggests the expression of endodermal genetic programs in bundle sheath cells, we determined whether the transcription factor SCARECROW, which regulates structural differentiation of the root endodermis, also plays a role in the development of Kranz anatomy in maize. Mutations in the Scarecrow gene result in proliferation of bundle sheath cells, abnormal differentiation of bundle sheath chloroplasts, vein disorientation, loss of minor veins and reduction of vein density. Further characterization of this signal transduction pathway should facilitate the transfer of the C(4) trait into C(3) crop species, including rice.

Tie-dyed2 encodes a callose synthase that functions in vein development and affects symplastic trafficking within the phloem of maize leaves.

The tie-dyed2 (tdy2) mutant of maize (Zea mays) displays variegated green and yellow leaves. Intriguingly, the yellow leaf tissues hyperaccumulate starch and sucrose, the soluble sugar transported long distance through the phloem of veins. To determine the molecular basis for Tdy2 function, we cloned the gene and found that Tdy2 encodes a callose synthase. RNA in situ hybridizations revealed that in developing leaves, Tdy2 was most highly expressed in the vascular tissue. Comparative expression analysis with the vascular marker maize PINFORMED1a-yellow fluorescent protein confirmed that Tdy2 was expressed in developing vein tissues. To ascertain whether the defect in tdy2 leaves affected the movement of sucrose into the phloem or its long-distance transport, we performed radiolabeled and fluorescent dye tracer assays. The results showed that tdy2 yellow leaf regions were defective in phloem export but competent in long-distance transport. Furthermore, transmission electron microscopy of tdy2 yellow leaf regions showed incomplete vascular differentiation and implicated a defect in cell-to-cell solute movement between phloem companion cells and sieve elements. The disruption of sucrose movement in the phloem in tdy2 mutants provides evidence that the Tdy2 callose synthase functions in vascular maturation and that the vascular defects result in impaired symplastic trafficking into the phloem translocation stream.

Non-structural carbohydrate partitioning in grass stems: a target to increase yield stability, stress tolerance, and biofuel production.

A dramatic change in agricultural crops is needed in order to keep pace with the demands of an increasing human population, exponential need for renewable fuels, and uncertain climatic changes. Grasses make up the vast majority of agricultural commodities. How these grasses capture, transport, and store carbohydrates underpins all aspects of crop productivity. Sink-source dynamics within the plant direct how much, where, and when carbohydrates are allocated, as well as determine the harvestable tissue. Carbohydrate partitioning can limit the yield capacity of these plants, thus offering a potential target for crop improvement. Grasses have the ability to buffer this sink-source interaction by transiently storing carbohydrates in stem tissue when production from the source is greater than whole-plant demand. These reserves improve yield stability in grain crops by providing an alternative source when photosynthetic capacity is reduced during the later phases of grain filling, or during periods of environmental and biotic stresses. Domesticated grasses such as sugarcane and sweet sorghum have undergone selection for high accumulation of stem carbohydrates, which serve as the primary sources of sugars for human and animal consumption, as well as ethanol production for fuel. With the enormous expectations placed on agricultural production in the near future, research into carbohydrate partitioning in grasses is essential for maintaining and increasing yields in grass crops. This review highlights the current knowledge of non-structural carbohydrate dynamics in grass stems and discusses the impacts of stem reserves in essential agronomic grasses.

SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors.

Patterning of the floral organs is exquisitely controlled and executed by four classes of homeotic regulators. Among these, the class B and class C floral homeotic regulators are of central importance as they specify the male and female reproductive organs. Inappropriate induction of the class B gene APETALA3 (AP3) and the class C gene AGAMOUS (AG) causes reduced reproductive fitness and is prevented by polycomb repression. At the onset of flower patterning, polycomb repression needs to be overcome to allow induction of AP3 and AG and formation of the reproductive organs. We show that the SWI2/SNF2 chromatin-remodeling ATPases SPLAYED (SYD) and BRAHMA (BRM) are redundantly required for flower patterning and for the activation of AP3 and AG. The SWI2/SNF2 ATPases are recruited to the regulatory regions of AP3 and AG during flower development and physically interact with two direct transcriptional activators of class B and class C gene expression, LEAFY (LFY) and SEPALLATA3 (SEP3). SYD and LFY association with the AP3 and AG regulatory loci peaks at the same time during flower patterning, and SYD binding to these loci is compromised in lfy and lfy sep3 mutants. This suggests a mechanism for SWI2/SNF2 ATPase recruitment to these loci at the right stage and in the correct cells. SYD and BRM act as trithorax proteins, and the requirement for SYD and BRM in flower patterning can be overcome by partial loss of polycomb activity in curly leaf (clf) mutants, implicating the SWI2/SNF2 chromatin remodelers in reversal of polycomb repression.

Diverse functional roles of monosaccharide transporters and their homologs in vascular plants: a physiological perspective.

Vascular plants contain two gene families that encode monosaccharide transporter proteins. The classical monosaccharide transporter(-like) gene superfamily is large and functionally diverse, while the recently identified SWEET transporter family is smaller and, thus far, only found to transport glucose. These transporters play essential roles at many levels, ranging from organelles to the whole plant. Many family members are essential for cellular homeostasis and reproductive success. Although most transporters do not directly participate in long-distance transport, their indirect roles greatly impact carbon allocation and transport flux to the heterotrophic tissues of the plant. Functional characterization of some members from both gene families has revealed their diverse roles in carbohydrate partitioning, phloem function, resource allocation, plant defense, and sugar signaling. This review highlights the broad impacts and implications of monosaccharide transport by describing some of the functional roles of the monosaccharide transporter(-like) superfamily and the SWEET transporter family.

vanishing tassel2 encodes a grass-specific tryptophan aminotransferase required for vegetative and reproductive development in maize.

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.

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.