Cankui Zhang

Structural and Metabolic Transitions of C4 Leaf Development and Differentiation Defined by Microscopy and Quantitative Proteomics in Maize

This study presents a systems analysis of maize C4 leaf development and cell-specific differentiation as well as the leaf sink-source transition and associated changes. Five phases (transitions) of development and differentiation were recognized, and several regulatory and signaling proteins involved with some of these phases identified. C4 grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C4 photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C4 differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C4 specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database.

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.

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.

Allocation, stress tolerance and carbon transport in plants: how does phloem physiology affect plant ecology?

Despite the crucial role of carbon transport in whole plant physiology and its impact on plant-environment interactions and ecosystem function, relatively little research has tried to examine how phloem physiology impacts plant ecology. In this review, we highlight several areas of active research where inquiry into phloem physiology has increased our understanding of whole plant function and ecological processes. We consider how xylem-phloem interactions impact plant drought tolerance and reproduction, how phloem transport influences carbon allocation in trees and carbon cycling in ecosystems and how phloem function mediates plant relations with insects, pests, microbes and symbiotes. We argue that in spite of challenges that exist in studying phloem physiology, it is critical that we consider the role of this dynamic vascular system when examining the relationship between plants and their biotic and abiotic environment.

The Origin and Composition of Cucurbit "Phloem" Exudate

Cucurbits exude profusely when stems or petioles are cut. We conducted studies on pumpkin (Cucurbita maxima) and cucumber (Cucumis sativus) to determine the origin and composition of the exudate. Morphometric analysis indicated that the exudate is too voluminous to derive exclusively from the phloem. Cold, which inhibits phloem transport, did not interfere with exudation. However, ice water applied to the roots, which reduces root pressure, rapidly diminished exudation rate. Sap was seen by microscopic examination to flow primarily from the fascicular phloem in cucumber, and several other cucurbit species, but primarily from the extrafascicular phloem in pumpkin. Following exposure of leaves to 14CO2, radiolabeled stachyose and other sugars were detected in the exudate in proportions expected of authentic phloem sap. Most of this radiolabel was released during the first 20 s. Sugars in exudate were dilute. The sugar composition of exudate from extrafascicular phloem near the edge of the stem differed from that of other sources in that it was high in hexose and low in stachyose. We conclude that sap is released from cucurbit phloem upon wounding but contributes negligibly to total exudate volume. The sap is diluted by water from cut cells, the apoplast, and the xylem. Small amounts of dilute, mobile sap from sieve elements can be obtained, although there is evidence that it is contaminated by the contents of other cell types. The function of P-proteins may be to prevent water loss from the xylem as well as nutrient loss from the phloem.

Downregulating the sucrose transporter VpSUT1 in Verbascum phoeniceum does not inhibit phloem loading

Sucrose is loaded into the phloem in the minor veins of leaves before export. Two active, species-specific loading mechanisms have been proposed. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the putative second mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the phloem, not by transporter activity. Several sucrose-transporting plants have been shown to be apoplastic loaders by downregulating sucrose transporter 1 (SUT1), leading to accumulation of sugars and leaf chlorosis. In this study we compared the effect of downregulating SUT1 in Nicotiana tabacum, a sucrose transporter, and Verbascum phoeniceum, a species that transports raffinose and stachyose. To test the effectiveness of RNAi downregulation, we measured SUT1 mRNA levels and sucrose-H+ symport in leaf discs. Mild NtSUT1 downregulation in N. tabacum resulted in the pronounced phenotype associated with loading inhibition. In contrast, no such phenotype developed when VpSUT1 was downregulated in V. phoeniceum, leaving minimal sucrose transport activity. Only those plants with the most severe VpSUT1 downregulation accumulated more carbohydrate than usual and these plants were normal by other criteria: growth rate, photosynthesis, and ability to clear starch during the night. The results provide direct evidence that the mechanism of phloem loading in V. phoeniceum does not require active sucrose uptake from the apoplast and strongly supports the conclusion that the loading pathway is symplastic in this species.

Symplastic Phloem Loading in Poplar

Sucrose enters the phloem in poplar leaves through plasmodesmata. 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.

StMYB44 negatively regulates phosphate transport by suppressing expression of PHOSPHATE1 in potato

&NA; Phosphorus is an important macronutrient for plant growth, but often deficient in soil. To understand the molecular basis of the complex responses of potato (Solanum tuberosum L.) to phosphate (Pi) deficiency stress, the RNA‐Seq approach was taken to identify genes responding to Pi starvation in potato roots. A total of 359 differentially expressed genes were identified, among which the Solanum tuberosum transcription factor gene MYB44 (StMYB44) was found to be down‐regulated by Pi starvation. StMYB44 was ubiquitously expressed in potato tissues and organs, and StMYB44 protein was exclusively localized in the nucleus. Overexpression of StMYB44 in potato resulted in lower accumulation of Pi in shoots. Transcriptomic analysis indicated that the abundance of S. tuberosum PHOSPHATE1 (StPHO1), a Pi transport‐related gene, was reduced in StMYB44 overexpression lines. In contrast, knock‐out of StMYB44 by a CRISPR/Cas9 system failed to increase transcription of StPHO1. Moreover, StMYB44 was found to interact in the nucleus with AtWRKY6, a known Arabidopsis transcription factor directly regulating PHO1 expression, and StWRKY6, indicating that StMYB44 could be a member of the regulatory complex controlling transcription of StPHO1. Taken together, our study demonstrates that StMYB44 negatively regulates Pi transport in potato by suppressing StPHO1 expression.

The complex character of photosynthesis in cucumber fruit

Highlight Photosynthesis by cucumber fruits, through direct fixation of atmospheric CO2 and recapture of respired CO2, makes an important contribution to fruit growth.

Elucidation of the Mechanisms of Long-Distance mRNA Movement in a Nicotiana benthamiana/Tomato Heterograft System

Messenger RNA is degraded as it moves through the tomato phloem, and the mobility of a transcript cannot be reliably predicted based on its abundance in the Nicotiana benthamiana leaf or on whether it harbors a tRNA-like structural motif. Recent heterograft analyses showed that large-scale messenger RNA (mRNA) movement takes place in the phloem, but the number of mobile transcripts reported varies widely. However, our knowledge of the mechanisms underlying large-scale mRNA movement remains limited. In this study, using a Nicotiana benthamiana/tomato (Solanum lycopersicum) heterograft system and a transgenic approach involving potato (Solanum tuberosum), we found that: (1) the overall mRNA abundance in the leaf is not a good indicator of transcript mobility to the root; (2) increasing the expression levels of nonmobile mRNAs in the companion cells does not promote their mobility; (3) mobile mRNAs undergo degradation during their movement; and (4) some mRNAs arriving in roots move back to shoots. These results indicate that mRNA movement has both regulated and unregulated components. The cellular origins of mobile mRNAs may differ between herbaceous and woody species. Taken together, these findings suggest that the long-distance movement of mRNAs is a complex process and that elucidating the physiological roles associated with this movement is challenging but remains an important task for future research.