April 23, 2014

Review: Sugar demand, not auxin, is the initial regulator of apical dominance

PNAS April 7, 2014

(click here to download the original article) 

Michael G. Masona, John J. Rossb, Benjamin A. Babstc, Brittany N. Wienclawc, and Christine A. Beveridgea,*

aSchool of Biological Sciences, The University of Queensland, St. Lucia, QLD 4072, Australia; bSchool of Plant Science, University of Tasmania, Sandy Bay, TAS
7005, Australia
cBiosciences Department, Brookhaven National Laboratory, Upton, NY 11973-5000
* To whom correspondence should be addressed (c.beveridge@uq.edu.au)

            The role of auxin in maintaining apical dominance in plants has been a mainstay of plant physiology for 80 years. Auxin synthesized in the apical meristem moves downward, inhibiting lateral bud growth to promote upward growth at the apex (Taiz and Zeiger, 1991), a good strategy for capturing light in a competitive environment. If the meristem is removed, auxin supply is cut off and bud dormancy is broken. Undergraduate students still conduct the classic experiment in General Botany involving auxin application to cut bean stalks to restore apical dominance, confirming in theory and practice the power of indole acetic acid to maintain bud dormancy. When I conducted this same experiment as a student at Humboldt State in the 90s, we replicated its effect with naphthalene acetic acid. Due to a common structural motif of both molecules, either could simulate the effect of an intact meristem, more proof than an undergraduate ordinarily required to accept the theory put forth in textbooks. It is therefore no less than astonishing to see this theory overturned. This has been brought about by recent evidence that casts serious doubt on auxin as the primary motivator of bud release (Cline, 1996; Beveridge et al., 2000; Cline et al., 2001; Morris et al., 2005). This shift in thinking has culminated in a report last week by the group of Christine Beveridge at the University of Queensland, Australia, published in the Proceedings of the National Academy of Sciences (Mason et al., 2014).
The authors used digital time-lapse photography to observe lateral bud growth following selective removal of the apical meristem or leaves lower down the stem. In this way, they show that the emerging leaves at the apex constitute a sucrose sink whose demand for sugar helps keep lateral buds in a dormant state. The constant supply of auxin from the apex is evidently only a part of dormancy, and it can be broken while auxin remains if sucrose is suddenly made available, for example by the removal of the sugar sink at the apex. To demonstrate this, the authors removed the apex of pea plants and measured the rate at which the lateral buds grew. When plants were decapitated, the kinetics of lateral bud growth easily outpaced the downward rate of auxin depletion once its source at the apex was removed. Bud release preceded the disappearance of the hormone once thought to be the key to repressing lateral bud growth. Instead, dormancy was being broken by something else. That something else was sucrose, which could be supplied exogenously with the same effect, i.e. bud release, even while the apical meristem was still intact. Using 11C labeled sucrose, the rate of downward sugar travel to dormant buds was shown to coincide with their release much more closely than the depletion of auxin. This effect could be blocked by girdling the stem above the youngest nodes, effectively blocking sugar transport. By selectively removing certain leaves at various distances from the apex, the authors narrowed down both the source and the sink of the sugars which effected the transition to lateral bud growth. The mature leaves closest to dormant buds provide the sucrose which triggers lateral growth, a signal that is substantially more available in the absence of the emerging leaves at the apex. These developing leaves have not yet become photosynthetically self-sufficient and ordinarily monopolize these sugars during the early stages of their development at the expense of buds lower on the stem. Bud dormancy is partly maintained by the BRANCHED1 (BRC1) protein which inhibits cell cycle and meristem activity (Gonzáez-Grandío et al., 2013). The levels of transcripts encoding this protein are controlled by cytokinin and strigolactone in pea, and the authors show that both decapitation (remove of sucrose sink) and sucrose application suppress BRC1 transcript accumulation.
These findings mark a significant change in our view of one of the most basic processes in plant development, and it has come about recently through a series of papers which have re-examined long held assumptions about the role of auxin. Additional work in this area will continue to elaborate the role of auxin in maintaining bud dormancy in conjunction with other hormones such as strigolactone and cytokinin, but it is clear that sucrose availability is necessary and sufficient to break dormancy. New avenues for research have been opened by this work regarding the interaction of gene expression, hormonal action, and metabolite supply in activating lateral branch growth. Future work in this area has great potential to affect how we conduct many basic aspects of agriculture and how we view the role of metabolites in controlling central processes in plant biology.

Beveridge CA, Symons GM, Turnbull CGN (2000) Auxin inhibition of decapitation-induced branching is dependent on graft-transmissible signals regulated by genes Rms1 and Rms2. Plant Physiol 123: 689-698
Cline MG (1996) Exogenous auxin effects on lateral bud outgrowth in decapitated shoots. Ann Botany 78: 255-266
Cline MG, Chatfield SP, Leyser O (2001) NAA restores apical dominance in the axr3-1 mutant of Arabidopsis thaliana. Ann Botany 87: 61-65
Gonzáez-Grandío E, Poza-Carrión C, Sorzano COS, Cubas P (2013) BRANCHED1 promotes axillary bud dormancy in response to shade in Arabidopsis. Plant Cell 25: 834-850
Mason MG, Ross JJ, Babst BA, Wienclaw BN, Beveridge CA (2014) Sugar demand, not auxin, is the initial regulator of apical dominance. Proc Natl Acad Sci
Morris SE, Cox MCH, Ross JJ, Krisantini S, Beveridge CA (2005) Auxin dynamics after decapitation are not correlated with the initial growth of axillary buds. Plant Physiol 138: 1665-1672
Taiz L, Zeiger E (1991) Plant Physiology. Benjamin Cummings Publishing Co., Redwood City, California

April 15, 2014

Review: The Most Deeply Conserved Noncoding Sequences in Plants Serve Similar Functions to Those in Vertebrates Despite Large Differences in Evolutionary Rates

Published online before print March 2014, The Plant Cell tpc.113.121905

Diane Burgess* and Michael Freeling

Department of Plant and Microbial Biology, University of California, Berkeley, California 94720
* Corresponsing author

Several types of non-coding DNA serve regulatory functions in the genomes of plants. Burgess and Freeling1 report recently on conserved non-coding sequences (CNSs) in plants that apparently arose early in the evolution of life as they are also represented in vertebrates as long (>100 bp), highly conserved regulatory sequences that may lie megabases away from their targets, particularly in the case of mammals. Identifying such regulatory sequences in the non-coding regions of plants has been complicated by the fact that the corresponding sequences of plants are much shorter (approx. 25 bp) and have greater sequence variability. Burgess and Freeling apply robust bioinformatics methods to identify a group of 37 CNSs that are conserved among all flowering plants. Plants CNSs are also different from their vertebrate counterparts in that they are frequently no more than 11 kb away from their targets. They may occur either upstream or downstream of their targets, or in intronic regions within the target gene itself.
How CNSs function to regulate gene expression is not well understood, but these sequences are often highly enriched in transcription factor binding sites. G-boxes, which are frequent motifs in light responsive genes, are common in CNSs, as are WRKY-box motifs (related to biotic and abiotic stress responses) and MYC2 bind sites, which are closely linked to jasmonic acid responses.
Many of the intragenic CNSs identified by Burgess and Freeling form RNA secondary structures which convey their regulatory function. One example is the 3’UTR of THIC, which acts as a riboswitch, changing conformation by finding thiamine pyrophosphate which ultimately promotes alternative splicing and affects transcript stability2. CNSs may also play a role in alternative splicing, especially those located in alternative exons with premature stop codons.
Despite the highly conserved nature of these regulatory sequences across diverse plant lines, very few have been studied on a functional level. Until more functional data becomes available for these regulators, the function of most is largely the subject of speculation. However, comparisons can be drawn to vertebrate systems to gain some insight into their function, and Burgess and Freeling reference the different life strategies employed by animals and plants which may also reflect on how their respective CNSs have evolved. For instance, they note that polyploidy is a frequent occurrence in plant genomes and that this may be responsible for plant CNSs being shorter and less conserved than similar sequences in vertebrates since polyploidy may lead to a relaxation of the constraints which lead to conservation of sequence. It is also interesting to note that plants have evolved more efficient mechanisms to remove junk DNA from their genomes than animals. Animals typically rely on a slow pathway involving pseudogenization of redundant or non-functional genes whereas plant genomes remove redundant and functionless DNA through a deletion mechanism. Finally, the higher offspring number of plants means they can afford to lose larger numbers to purifying selection, which ultimately leads to shorter CNSs which evolve more quickly. The present work should provide a useful framework for contextualizing future discoveries of functional regulatory sequences, and facilitate the distinction of truly functional non-coding sequences from the significant portion of all eukaryotic genomes with no apparent function whatsoever.

1.         Burgess, D. & Freeling, M. The most deeply conserved noncoding sequences in plants serve similar functions to those in vertebrates despite large differences in evolutionary rates. Plant Cell (2014).
2.         Wachter, A. et al. Riboswitch control of gene expression in plants by splicing and alternative 3'end processing of mRNAs. Plant Cell 19, 3437-3450 (2007).

April 4, 2014

Review: Contribution of NAC transcription factors to plant adaptation to land

March 20 2014 Science 28 March 2014: Vol. 343 no. 6178 pp. 1505-1508
DOI: 10.1126/science.1248417

Bo Xu1, Misato Ohtani2, Masatoshi Yamaguchi1, Kiminori Toyooka2, Mayumi Wakazaki2, Mayuko Sato2, Minoru Kubo3, Yoshimi Nakano1, Ryosuke Sano1, Yuji Hiwatashi3, Takashi Murata3, Tetsuya Kurata1, Arata Yoneda1, Ko Kato1, Mitsuyasu Hasebe3,4, Taku Demura1,2,*
* Corresponding author

1Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
2RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa 230-0045, Japan.
3National Institute for Basic Biology, Okazaki, Aich 444-8585, Japan.
4School of Life Science, Graduate University for Advanced Studies, Okazaki, Aich 444-8585, Japan.

The colonization of land by early plants required the evolution of two specialized features which were not necessary for life in an aquatic environment: water-conducting tissues and support structures. Japanese researchers lead by Prof. Taku Demura of the RIKEN Center recently reported that the evolution of a family of transcription factors known as NO APICAL MERISTEM (NAC) may indeed have been responsible for the acquisition of both structures1. Although most current information on the evolution and function of water-conducting elements comes from studies using Arabidopsis2-4, the well characterized moss Physcomitrella patens is a valuable model system which frequently provides insights of how early plant evolution may have taken place.

It had been previously suggested from Arabidopsis studies that both fiber cells and xylem vessels share an evolutionary origin5, and in modern vascular plants these structures have functions in support and water transport, respectively. In moss, the homologous structures are known as stereids and hydroids. In the present work, Xu et al. characterize the regulation of NAC transcription factors in Physcomitrella (here referred to as PpVNS proteins, which include VDN [VASCULAR RELATED NAC-DOMAN], NST [NAC SECONDARY WALL THICKENING PROMOTING FACTOR], and SMB [SOMBRERO-related] and examine their role in the development in stereid and hydroid cell types. The evolutionary link between NAC transcription factors in bryophytes and flowering plants is not obvious since xylem vessels and fiber cells are only produced during the sporophytic generation of vascular plants, while the homologous stereids and hydroids of mosses are only present in the gametophytic generation.

Using qRTPCR on gametophyte and protonemata tissues, GUS-staining of transformed moss lines and anatomical evaluation of ppvns knock-out mutants, Xu et al. established PpVNS1, 6, and 7 as proteins with critical roles in the development of both hydroids and stereids in the gametophyte leaf, whereas PpVNS4 controls development of hydroids in the gametophyte stem (and to a lesser extent, central and transfer cells in the sporophyte foot). Mutants defective in one or more PpVNS gene displayed reduced capacity to conduct water, an enhanced sensitivity to wilting, and reduced stereid cell wall thickness. Unlike stereids of wild type plants, those of ppvns triple mutants retained their cellular contents, including plastids. Overexpression of PpVNS proteins using an inducible promoter provoked cell death, chloroplast loss, and protoplasm shrinkage in hydroids. This effect was also seen when moss was transformed with an Arabidopsis NAC protein (VDN7), which further established the evolutionary link between the homologous genes of bryophyte and flowering plants. When Arabidopsis was transformed with PpVNS proteins, ectopic secondary wall thickening was observed. 

Together, the authors present a powerful argument for the role of these NAC proteins in the functionalization of both support cells and water conducting vessels by promoting cell wall thickening (in stereids) and clearance of cellular contents (in hydroids). Because the conductivity of water is much more efficient in dead, empty cells than in living parenchyma cells with normal cellular contents, the evolution of the cell death program may well have facilitated the evolution of water conduction. Without the latter, the colonization of land by aquatic plants was all but impossible. Indeed, the evolution of this family of transcription factors may have been a key innovation that allowed early plants not only to colonize land but also to evolve large, complex body types with increased requirements for water transport. The authors are congratulated on this important advance in our understanding of the role of these proteins in support and water-conducting cells types both in bryophytes as well as in vascular plants.Additionally, this study provides new insights into how evolution may proceed with major ecological consequences, such as adaptation of an entire group of organisms to a new habitat, brought about by the simple innovation of extant transcription factors.

Questions: This work prompts many questions regarding the evolution of NAC proteins and mode of action in vascular plants. What is the phylogenetic relationship between NACs of Physcomitrella and Arabidopsis? Has this group of transcription factors acquired any new functions during the evolution of angiosperms or are they always related to the development of either fibers of xylem vessels? How precisely do NAC transcription factors induce cell death, e.g. what are their primary targets and which transcripts are the first to be activated towards this end? Are there any obvious biotechnological applications which these new insights into NAC protein function might provide?

1.         Xu, B. et al. Contribution of NAC transcription factors to plant adaptation to land. Science 343, 1505-1508 (2014).
2.         Kubo, M. et al. Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev 19, 1855-1860 (2005).
3.         Ohashi-Ito, K., Oda, Y. & Fukuda, H. Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 directly regulates the genes that govern programmed cell death and secondary wall formation during xylem differentiation. Plant Cell 22, 3461-3473 (2010).
4.         Yamaguchi, M. et al. VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system. Plant Physiol 153, 906-914 (2010).
5.         Zhong, R., Demura, T. & Ye, Z.-H. SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell 18, 3158-3170 (2006).

April 1, 2014

Review: Fast retrograde signaling in response to high light Involves metabolite export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF transcription factors in Arabidopsis

Published online at The Plant Cell before print: www.plantcell.org/cgi/doi/10.1105/tpc.113.121061

Marc Oliver Vogela, Marten Moorea, Katharina Königa, Pascal Pecherb, Khalid Alsharafa a, Justin Lee b, and Karl-Josef Dietza*
* Corresponding author
a Biochemistry and Physiology of Plants, Bielefeld University, 33501 Bielefeld, Germany
b Leibniz Institute of Plant Biochemistry, 06120 Halle, Germany
    As photosynthetic organisms, it follows naturally that plants are highly sensitive to changes in their light environment. Plants must adjust their metabolism and transcriptional programs rapidly in response to such changes. Complex regulatory cascades enable the plant to react quickly at transcript, protein, and metabolite levels that facilitate continual adjustment to environmental changes, including light levels. Because most of the protein machinery dealing with light is located in the chloroplast, while most genes encoding such proteins are part of the nuclear genome, a communication network would seem necessary by which chloroplasts can signal changes to the nucleus via direct feedback regarding the status of the cellular pool of chloroplasts. Such communication is in fact well documented and has been dubbed retrograde signaling. 

    The sensitivity to light changes provided by this light-sensitive network alternately helps the organism conserve resources when light is low and quickly ramp up photosynthetic rates when light levels are high. The responsiveness to light becomes especially important under high light intensity, i.e. light stress. The potential for irreversible photooxidative damage is significant, and dangerous imbalances to metabolite pools can occur without metabolic overflow shunts that avoid destabilizing metabolic buildups. The many responses to changes in ambient light must be implemented in a coordinated manner in the plant cell, and the implications of this phenomenon on agriculture are significant enough to make this a highly competitive and active area of original research.

    German investigators at Bielefeld University and the Leibniz Institute for Plant Biochemistry have published a report in The Plant Cell which advances our knowledge of the early events in high light signaling. Using Arabidopsis, Vogel et al.1 examined changes in transcript levels in a family of transcription factors known as APETALA2/ETHYLENE RESPONSE FACTOR (ERF). Quantitative PCR revealed that multiple ERF genes were upregulated in a little as 10 minutes following transfer of dark adapted plants to high light. However, this induction was blocked in tpt2, a mutant defective in the triose phosphate transporter which shuttles dihydroxyacetone phosphate (DHAP) between the plastid and cytosol. This implicated metabolite transport in the early signaling response to light. This process was necessary to activate MAP kinase 6 (MAPK6), which in turn activates ERFs.

    Antibodies which specifically recognize phosphorylated forms of MAPK6 were used to show that the activation of this protein occurs on the timescale of 1-2 minutes after transfer of plants to light. However, MAPK6 transcripts do not change upon light exposure; only phosphorylation of extant MAPK6 protein is required, versus translation of new protein, in effect shortening the response time of this signaling cascade. The authors pinpointed a time frame of 1-2 minutes as the point of no return for ERF transcriptional activation based on the fast activation of MAPK6. The well known biotic stress responsive gene PR1 as well as heat shock proteins and chitinases were upregulated downstream as a result of ERF activation, a result supported by microarray data using the erf6 mutant. Because of kinetic data in light induction time courses, the authors were able to rule out H2O2 as an early signal due to its late appear (>10 min) on the time scale of responses relative to MAPK6 activation and triose phosphate efflux from the plastid (seconds after light exposure). 

    The authors propose a model for early events in high light signaling which begins with efflux of triose phosphates as a direct response to the transition to high light due to increased flux in the Calvin-Benson-Bassham cycle. This is followed by activation of MAPK6 by unidentified protein kinases and transcription factor which in turn induce transcript accumulation of ERF transcription factors. Following de novo synthesis of the encoded ERF proteins, the metabolic shift in preparation for high light conditions is completed by the induction of heat shock proteins, additional cascade components, and formation of H2O2, a previously known metabolite signal belonging to the reactive oxygen species (ROS) group. The authors are congratulated for the publication of this excellent work which advances our understanding of the elements which govern the light response process in plants.

    Additional questions: While considerable evidence is presented to clearly define novel components of light signaling, the metabolic data and the specific role of metabolite efflux are less clear. How can the authors be certain it is DHAP export from the plastid which promotes MAPK6 phosphorylation? While the tpt2 mutant data is convincing, it is unclear how this specific metabolite can exert the alleged effects. Also, the authors conclude by mentioning a variety of metabolites which could feasibly contribute to operational retrograde signaling, including H2O2, singlet oxygen, and hormones. However, one molecule could be added to this list: 2-C-methylerythritol-2,4-cyclodiphosphate, an intermediate of the 2-C-methylerythritol phosphate pathway for plastid isoprenoid biosynthesis. This intermediate accumulates under high light2 and has been implicated in biotic3 and abiotic4 stress signaling by export from the plastid. Given the sensitivity of MEcPP to light and its alleged secondary signaling function outside the plastid, it seems plausible that it could also play a role in the high light response described by Vogel et al. The present work provides new perspective on light signaling and suggests several new avenues of research.

1.         Vogel, M. O. et al. Fast retrograde signaling in response to high light Involves metabolite export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF transcription factors in Arabidopsis. Plant Cell (2014).
2.         Li, Z. & Sharkey, T. D. Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illumination isoprene burst from leaves. Plant, Cell & Environment 36, 429-437 (2012).
3.         Gil, M. J., Coego, A., Mauch-Mani, B., Jorda, L. & Vera, P. The Arabidopsis csb3 mutant reveals a regulatory link between salicylic acid-mediated disease resistance and the methyl-erythritol 4-phosphate pathway. Plant J 44, 155-166 (2005).
4.         Xiao, Y. et al. Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes. Cell 149, 1525-1535 (2012).