By Chan Yul Yoo, Paul M. Hasegawa, and Michael V. Mickelbart
Regulation of Stomatal Density factor for improved water use efficiency.
Introduction
A decline in global water availability and increased agricultural drought have resulted in significant reductions in crop production, which in turn has intensified research into more efficient water use in plants. Opening stomata to admit CO2 for carbon assimilation necessary for growth, development, and yield/production causes simultaneous water loss via transpiration. Stomatal pore size or number (density) influences transpiration, CO2 uptake, and water use efficiency (WUE)1,2,3. Methods to induce stomatal closure reduce stomatal pore size, thereby increasing drought tolerance and, potentially, WUE4. However, this drought tolerance strategy usually results in a reduction in biomass and/or yield, due to reduced CO2 uptake for carbon assimilation, often referred to as yield penalty.
Analysis of natural genetic variation indicates that high WUE is correlated with low transpiration rather than high CO2 assimilation4, implying that plants have evolved to improve WUE by regulating transpiration. Quantitative
trait loci (QTL) analysis for natural variation of WUE in Arabidopsis ecotypes indicates the ERECTA locus affects WUE through various mechanisms, including stomatal development5. Several genes, including HDG11, GPA1, and CA1/4, have been also implicated in the regulation of stomatal development for WUE6,7,8. Recently, we determined that the Arabidopsis GTL1 transcription factor negatively regulates WUE through stomatal density control by which AtGTL1 transrepresses SDD1, a negative regulator of stomatal density9,10.
Molecular, anatomical, and physiological functions of GTL1
GTL1 expression was down-regulated by water-deficit stress, and loss of GTL1 function increased WUE and survival in response to water-deficit stress. gtl1 plants had lower daytime transpiration rates, but biomass accumulation was not different in mutant and wild-type plants, which resulted in higher integrated WUE in gtl1 plants. Lower daytime transpiration in gtl1 plants led to higher leaf water status and greater water-deficit tolerance. Leaf gas exchange analysis indicated that transpiration and stomatal conductance was lower, while net CO2 assimilation in gtl1 leaves was not different from wild-type leaves. Therefore, consistent with integrated WUE, leaf instantaneous WUE was also higher in gtl1 leaves. Stomatal pore size and ABA response in gtl1 plants was similar to wild-type plants. However, abaxial stomatal density was approximately 25% lower in gtl1 plants compared with the wild type, which was correlated with an approximately 25% lower transpiration rate in gtl1 plants. Consequently, a ~25% reduction of stomatal density in abaxial leaves caused ~25% reduction of transpiration, but did not inhibit CO2 uptake under experimental conditions. This resulted in higher WUE without any adverse effect on biomass accumulation and drought tolerance.
Gene expression analysis indicated that SDD1, a negative regulator of stomatal density, was highly expressed in gtl1 leaves, which is consistent with reduced stomatal density. Expression of MITOGEN-ACTIVATED KINASE 3 (MPK3) and MPK6, downstream components of SDD1, were also highly expressed in gtl1 leaves, indicating that the negative regulatory pathway (SDD1 8 MPK3/6) of stomatal development was activated in gtl1 leaves compared to wild type. Consistently, stomatal precursor cells, such as meristemoids and guard mother cells, were still observed in fully expanded gtl1 leaves, indicating that stomatal development may have been inhibited or delayed.
GTL1 promoter fused with ß-glucuronidase (GUS) and GTL1 promoter fused with GTL1 cDNA and green fluorescence protein (GFP) reporter systems indicated that GTL1 was expressed and localized in the nucleus of the abaxial epidermis. These data suggest that GTL1 protein represses SDD1 expression to regulate stomatal density in the abaxial epidermis. gtl1 plants also had larger pavement cells, and therefore, a lower pavement cell density. Recently, Breuer et al.11 reported that GTL1 negatively regulates ploidy-dependent trichome cell growth by repressing endoreduplication. It is possible that larger pavement cells in gtl1 plants were due to increased ploidy level. Together, delayed stomatal development due to higher SDD1 expression and larger pavement cells due to unrepressed endoreduplication contributed to reduced stomatal density.
Analysis of DNA sequences in the SDD1 promoter revealed that it contains a potential GTL1-binding element: a GT3-box (GGTAAA)12. An in vitro DNA binding electrophoretic mobility shift assay (EMSA) indicated that the N-terminal DNA-binding domain of GTL1 binds directly to the fragment of the SDD1 promoter that includes the GT3-box. Site-directed mutagenesis showed that the GT3-box is specifically responsible for the interaction with GTL1. Chromatin-immunoprecipitation in vivo assays demonstrated that GTL1 protein is associated with the region around the GT3-box in the SDD1 promoter.
GTL1 acts as a transcriptional repressor of SDD1 to modulate stomatal density in response to environmental changes such as water availability. Down-regulation of GTL1 in response to water-deficit stress activates SDD1 expression to reduce stomatal density and transpiration, which improves WUE and drought tolerance. It is postulated that GTL1 receives an environmental signal and translates that signal to transcriptional regulation for an adaptive response that prevents excess water loss.
Potential for crop improvement
GTL1 is a member of the GT2 transcription factor family containing two DNA-binding domains12,13. Using the full-length amino acid sequences in the genome databases of several crop species, the Basic Local Alignment Search Tool (BLAST) was used for phylogenetic analysis to identify putative GTL1 orthologs in other plant species, including
the monocots Oryza sativa (rice), Sorghum bicolor (sorghum), and Zea mays (maize), and the dicots Glycine
max (soybean), Populus trichocarpa (Poplar), and Solanum lycopersicum (tomato). In future work, we hope to characterize GTL1 orthologs in crop species to determine if regulation of these genes can lead to improved WUE in these economically important plants.
References
1. Kim TH, Böhmer M, Hu H, Nishimura N, Schroeder JI. Guard cell signal transduction network: advances in understanding abscisic acid, CO2, and Ca2+ signaling. Annu Rev Plant Biol 61, 561-591 (2010).
2. Bergmann DC, Sack FD. Stomatal development. Annu Rev Plant Biol 58, 163-181 (2007).
3. Casson SA, Hetherington AM. Environmental regulation of stomatal development. Curr Opin Plant Biol 13, 90-95 (2010).
4. Yoo CY, Pence HE, Hasegawa PM, Mickelbart MV. Regulation of transpiration to improve crop water use. Crit Reviews Plant Sci 28, 410-431 (2009).
5. Masle J, Gilmore SR, Farquhar GD. The ERECTA gene regulates plant transpiration efficiency in Arabidopsis. Nature; 436, 866-870 (2005).
6. Yu H, Chen X, Hong YY, Wang Y, Xu P, Ke SD, Liu HY, Zhu JK, Oliver DJ, Xiang CB. Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density. Plant Cell; 20, 1134-1151 (2008).
7. Nilson SE, Assmann SM. The -subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency. Plant Physiol; 152, 2067-2077 (2010).
8. Hu H, Boisson-Dernier A, Israelsson-Nordström M, Böhmer M, Xue S, Ries A, Godoski J, Kuhn JM, Schroeder JI. Carbonic anhydrases are upstream regulators of CO2-controlled stomatal movements in guard cells. Nat Cell Biol 12, 87-93 (2010).
9. Yoo CY, Pence HE, Jin JB, Miura K, Gosney MJ, Hasegawa PM, Mickelbart MV. The Arabidopsis GTL1 transcription factor regulates water use efficiency and drought tolerance by modulating stomatal density via transrepression of SDD1. Plant Cell; 22, 4128-4141 (2010).
10. von Groll U, Berger D, Altmann T. The subtilisin-like serine protease SDD1 mediates cell-to-cell signaling during Arabidopsis stomatal development. Plant Cell; 19, 63-83 (2002).
11. Breuer C, Kawamura A, Ichikawa T, Tominaga-Wada R, Wada T, Knodou Y, Muto S, Matsui M, Sugimoto K. The trihelix transcription factor GTL1 regulates ploidy-dependent cell growth in the Arabidopsis trichome. Plant Cell 21, 2307-2322 (2009).
12. Zhu DX. Regulatory mechanism of plant gene transcription by GT-elements and GT-factors. Trends Plant Sci 4, 210-214, (1999).
13. Ni M, Dehesh K, Tepperman JM, Quail PH. GT-2: In vivo transcriptional activation activity and definition of novel twin DNA binding domains with reciprocal target sequence selectivity. Plant Cell 8,1041-1059 (1996).
Michael V Mickelbart
Assistant Professor Horticulture
Department: Horticulture and Landscape Architecture
mmickelb@purdue.edu
