Source-to-sink partitioning is altered by changes in the expression of the transcription factor AtHB5 in Arabidopsis.

Carbohydrates are transported from source to sink tissues. The efficiency of this transport determines plant growth and development. The process is finely regulated and transcription factors are crucial in its modulation. AtHB5 is a homeodomain-leucine zipper I transcription factor that is repressed during stem maturation. However, its function in this developmental event is unknown. Here, we investigated the expression pattern and role of AtHB5. AtHB5 was expressed in roots, hypocotyls, stems, petioles, pedicels, and central leaf veins. athb5 mutant plants exhibited wider and more lignified stems than controls, whereas AtHB5 overexpressors showed the opposite phenotype. Cross sections of athb5 mutant stems showed enlarged vascular bundle, xylem, phloem, and petiole areas, whereas AtHB5 overexpressors had callose deposits. Several genes involved in starch biosynthesis and degradation had altered transcript levels in athb5 mutants and AtHB5 overexpressors. Rosette and stem biomass was enhanced in athb5 mutants, positively impacting seed yield, protein, and lipid content. Moreover, these effects were more evident in debranched plants. Finally, transport to roots was significantly slowed in AtHB5 overexpressors. Altogether, the results indicated that AtHB5 is a negative modulator of carbon partitioning and sucrose transport from source to sink tissues, and its overexpression diminished plant biomass and seed yield.

The Arabidopsis transcription factors AtPHL1 and AtHB23 act together promoting carbohydrate transport from pedicel-silique nodes to seeds.

Carbohydrates are produced in green tissues through photosynthesis and then transported to sink tissues. Carbon partitioning is a strategic process, fine regulated, involving specific sucrose transporters in each connecting tissue. Here we report that a screening of an Arabidopsis transcription factor (TF) library using the homeodomain-leucine zipper I member AtHB23 as bait, allowed identifying the TF AtPHL1 interacting with the former. An independent Y2H assay, and in planta by BiFC, confirmed such interaction. AtHB23 and AtPHL1 coexpressed in the pedicel-silique nodes and the funiculus. Mutant plants (phl1, and amiR23) showed a marked reduction of lipid content in seeds, although lipid composition did not change compared to the wild type. While protein and carbohydrate contents were not significantly different between mutants and control mature seeds, we observed a reduced carbohydrate content in mutant plants young siliques (7 days after pollination). Moreover, using a CFDA probe, we revealed an impaired transport to the seeds, and the gene encoding the carbohydrate transporters SWEET10 and SWEET11, usually expressed in connecting tissues, was repressed in the amiR23 and phl1 mutant plants. Altogether, the results indicated that AtHB23 and AtPHL1 act together, promoting sucrose transport, and the lack of any of them provoked a reduction in seeds lipid content.

Successful field performance in warm and dry environments of soybean expressing the sunflower transcription factor HB4.

Soybean yield is limited primarily by abiotic constraints. No transgenic soybean with improved abiotic stress tolerance is commercially available. We transformed soybean plants with genetic constructs able to express the sunflower transcription factor HaHB4, which confers drought tolerance to Arabidopsis and wheat. One line (b10H) carrying the sunflower promoter was chosen among three independent lines because it exhibited the best performance in seed yield, and was evaluated in the greenhouse and in 27 field trials in different environments in Argentina. In greenhouse experiments, transgenic plants showed increased seed yield under stress conditions together with greater epicotyl diameter, larger xylem area, and increased water use efficiency compared with controls. They also exhibited enhanced seed yield in warm and dry field conditions. This response was accompanied by an increase in seed number that was not compensated by a decrease in individual seed weight. Transcriptome analysis of plants from a field trial with maximum difference in seed yield between genotypes indicated the induction of genes encoding redox and heat shock proteins in b10H. Collectively, our results indicate that soybeans transformed with HaHB4 are expected to have a reduced seed yield penalty when cultivated in warm and dry conditions, which constitute the best target environments for this technology.

The AtHB1 Transcription Factor Controls the miR164-CUC2 Regulatory Node to Modulate Leaf Development.

The presence of small tooth-like indentations, or serrations, characterizes leaf margins of Arabidopsis thaliana plants. The NAC family member CUP-SHAPED COTYLEDON 2 (CUC2), which undergoes post-transcriptional gene silencing by three micro-RNA genes (MIR164A, B and C), controls the extension of leaf serration. Here, we analyzed the role of AtHB1, a transcription factor (TF) belonging to the homeodomain-leucine zipper subfamily I, in shaping leaf margins. Using mutants with an impaired silencing pathway as background, we obtained transgenic plants expressing AtHB1 over 100 times compared to controls. These plants presented an atypical developmental phenotype characterized by leaves with deep serration. Transcript measurements revealed that CUC2 expression was induced in plants overexpressing AtHB1 and repressed in athb1 mutants, indicating a positive regulation exerted by this TF. Moreover, molecular analyses of AtHB1 overexpressing and mutant plants revealed that AtHB1 represses MIR164 transcription. We found that overexpression of MIR164B was able to reverse the serration phenotype of plants overexpressing AtHB1. Finally, chromatin immunoprecipitation assays revealed that AtHB1 was able to bind in vivo the promoter regions of all three MIR164 encoding loci. Altogether, our results indicate that AtHB1 directly represses MIR164 expression to enhance leaf serration by increasing CUC2 levels.

Maize expressing the sunflower transcription factor HaHB11 has improved productivity in controlled and field conditions.

HaHB11 is a sunflower transcription factor from the homeodomain-leucine zipper I family. Transgenic Arabidopsis plants expressing HaHB11 had larger rosettes and improved seed yield. In this work maize plants from hybrid HiII were transformed with 35S:HaHB11, ZmUBI:HaHB11 and ProHaHB11:HaHB11 and then backcrossed to B73 to obtain a more homozygous inbred phenotype. Transgene expression levels were stable at least during three generations. Greenhouse-grown HaHB11 transgenic lines had larger leaf area and delayed senescence than controls, together with increased total biomass (up to 25%) and seed yield (up to 28%). Field trials conducted with T2 and T4 generations indicated that enhanced leaf area (up to 18%), stem diameter (up to 28%) and total biomass (up to 40%) as well as delayed leaf senescence were maintained among transgenic individuals when upscaling from pots in the greenhouse to communal plants in the field. The T4 field-grown transgenic generation had increased light interception and radiation use efficiency as well as seed yield (43-47% for events driven by the 35S promoter). Results suggest that HaHB11 is a promising tool for crop improvement because differential traits observed in the Arabidopsis model plant were preserved in a crop like maize independently of growth conditions and backcross level.

AtHB23 participates in the gene regulatory network controlling root branching, and reveals differences between secondary and tertiary roots.

In Arabidopsis, lateral root (LR) development is mainly controlled by several known auxin-regulated transcription factors (TFs). Here, we show that AtHB23 (a homeodomain-leucine zipper I TF) participates in this intricate network. Our study of the expression pattern of AtHB23 revealed that it is transcriptionally activated in the early stages of secondary LR primordium (LRP). We found that AtHB23 directly limits the expression of LBD16, a key factor in LR initiation, and also directly induces the auxin transporter gene LAX3. We propose that this HD-Zip I mediates the regulation of LAX3 by ARF7/19. Furthermore, AtHB23 plays distinct roles during the formation of secondary and tertiary roots, exhibiting differential expression patterns. ATHB23 is expressed throughout the tertiary root primordium, whereas it is restricted to early stages in secondary primordia, likely later repressing LBD16 in tertiary LR development and further inhibiting root emergence. Our results suggest that different genetic programs govern the formation of LRP from the main or secondary roots, thereby shaping the global dynamic architecture of the root system.

Arabidopsis and sunflower plants with increased xylem area show enhanced seed yield.

Plant architecture plasticity determines the efficiency at harvesting and plays a major role defining biomass and seed yield. We observed that several previously described transgenic genotypes exhibiting increased seed yield also show wider stems and more vascular bundles than wild-type plants. Here, the relationship between these characteristics and seed yield was investigated. Hanging weight on the main stem of Arabidopsis plants provoked significant stem widening. Such widening was accompanied by an increase in the number of vascular bundles and about 100% of yield increase. In parallel, lignin deposition diminished. Vascular bundle formation started in the upper internode and continued downstream. AUX/LAX carriers were essential for this response. The increase of vascular bundles was reverted 3 weeks after the treatment leading to an enlarged xylem area. Aux1, lax1, and lax3 mutant plants were also able to enlarge their stems after the treatment, whereas lax2 plants did not. However, none of these mutants exhibited more vascular bundles or seed yield compared with untreated plants. Weight-induced xylem area enhancement and increased seed yield were also observed in sunflower plants. Altogether these results showed a strong correlation between the number of vascular bundles and enhanced seed yield under a long-day photoperiod. Furthermore, changes in the levels of auxin carriers affected both these processes in the same manner, suggesting that there may be an underlying causality.

The antagonistic basic helix-loop-helix partners BEE and IBH1 contribute to control plant tolerance to abiotic stress.

The bHLH family is composed by canonical and non-canonical transcription factors (TFs) that differ in the presence or absence of their DNA-binding domain, respectively. Since both types of bHLH proteins are able to dimerize, their relative abundance impacts their biological activity. Among this TF family BEE and IBH are canonical and non-canonical bHLHs, respectively and previous reports indicated that BEE2 and IBH1 dimerize. Wondering whether BEE TFs participate in the abiotic stress response and how the dimerization with IBH1 could regulate their role in Arabidopsis, double bee1/bee2 and triple bee1/bee2/bee3 mutants were tested under salinity and drought stresses. The bee1/bee2/bee3 mutant showed an enhanced tolerance whereas the double mutant behaved similar to wild type plants. These results indicated that BEE genes play a role in the stress response and also put in evidence the redundancy within the BEE family. Moreover, ectopic expression of IBH1 on different mutant backgrounds improved plant tolerance to abiotic stress, independently of the background. However, the yield of these transgenic plants was penalized with abortive seeds. Our results suggest that BEE genes are negative regulators of physiological responses to abiotic stress whereas IBH1 is a positive modulator via different pathways, one of them involving BEE TFs.

Arabidopsis thaliana homeodomain-leucine zipper type I transcription factors contribute to control leaf venation patterning.

Venation patterning is a taxonomic attribute for classification of plants and it also plays a role in the interaction of plants with the environment. Despite its importance, the molecular physiology controlling this aspect of plant development is still poorly understood. Auxin plays a central role modulating the final vein network and patterning. This addendum discusses recent findings on the role of homeodomain-leucine zipper (HD-Zip) transcription factors on the regulation of leaf venation patterning. Moreno-Piovano et al. reported that ectopic expression of a sunflower HD-Zip I gene, HaHB4, increased the asymmetry of leaf venation. Even more, this work showed that auxin transport in the leaf through LAX carriers controls venation patterning. Here, we provide evidence indicating that some Arabidopsis thaliana HD-Zip I genes play a role in the determination of the final leaf venation patterning. We propose that these genes contribute to regulate vein patterning, likely controlling auxin homeostasis.

A role for LAX2 in regulating xylem development and lateral-vein symmetry in the leaf.

Background and Aims: The symmetry of venation patterning in leaves is highly conserved within a plant species. Auxins are involved in this process and also in xylem vasculature development. Studying transgenic Arabidopsis plants ectopically expressing the sunflower transcription factor HaHB4, it was observed that there was a significant lateral-vein asymmetry in leaves and in xylem formation compared to wild type plants. To unravel the molecular mechanisms behind this phenotype, genes differentially expressed in these plants and related to auxin influx were investigated. Methods: Candidate genes responsible for the observed phenotypes were selected using a co-expression analysis. Single and multiple mutants in auxin influx carriers were characterized by morphological, physiological and molecular techniques. The analysis was further complemented by restoring the wild type (WT) phenotype by mutant complementation studies and using transgenic soybean plants ectopically expressing HaHB4 . Key Results: LAX2 , down-regulated in HaHB4 transgenic plants, was bioinformatically chosen as a candidate gene. The quadruple mutant aux1 lax1 lax2 lax3 and the single mutants, except lax1, presented an enhanced asymmetry in venation patterning. Additionally, the xylem vasculature of the lax2 mutant and the HaHB4 -expressing plants differed from the WT vasculature, including increased xylem length and number of xylem cell rows. Complementation of the lax2 mutant with the LAX2 gene restored both lateral-vein symmetry and xylem/stem area ratio in the stem, showing that auxin homeostasis is required to achieve normal vascular development. Interestingly, soybean plants ectopically expressing HaHB4 also showed an increased asymmetry in the venation patterning, accompanied by the repression of several GmLAX genes. Conclusions: Auxin influx carriers have a significant role in leaf venation pattering in leaves and, in particular, LAX2 is required for normal xylem development, probablt controlling auxin homeostasis.

A uORF Represses the Transcription Factor AtHB1 in Aerial Tissues to Avoid a Deleterious Phenotype.

AtHB1 is an Arabidopsis (Arabidopsis thaliana) homeodomain-leucine zipper transcription factor that participates in hypocotyl elongation under short-day conditions. Here, we show that its expression is posttranscriptionally regulated by an upstream open reading frame (uORF) located in its 5' untranslated region. This uORF encodes a highly conserved peptide (CPuORF) that is present in varied monocot and dicot species. The Arabidopsis uORF and its maize (Zea mays) homolog repressed the translation of the main open reading frame in cis, independent of the sequence of the latter. Published ribosome footprinting results and the analysis of a frame-shifted uORF, in which the repression capability was lost, indicated that the uORF causes ribosome stalling. The regulation exerted by the CPuORF was tissue specific and did not act in the absence of light. Moreover, a photosynthetic signal is needed for the CPuORF action, since plants with uncoupled chloroplasts did not show uORF-dependent repression. Plants transformed with the native AtHB1 promoter driving AtHB1 expression did not show differential phenotypes, whereas those transformed with a construct in which the uORF was mutated exhibited serrated leaves, compact rosettes, and, most significantly, short nondehiscent anthers and siliques containing fewer or no seeds. Thus, we propose that the uncontrolled expression of AtHB1 is deleterious for the plant and, hence, finely repressed by a translational mechanism.

JUNGBRUNNEN1 Confers Drought Tolerance Downstream of the HD-Zip I Transcription Factor AtHB13.

Low water availability is the major environmental factor limiting growth and productivity of plants and crops and is therefore considered of high importance for agriculture affected by climate change. Identifying regulatory components controlling the response and tolerance to drought stress is thus of major importance. The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) from Arabidopsis thaliana extends leaf longevity under non-stress growth conditions, lowers cellular hydrogen peroxide (H2O2) level, and enhances tolerance against heat stress and salinity. Here, we additionally find that JUB1 strongly increases tolerance to drought stress in Arabidopsis when expressed from both, a constitutive (CaMV 35S) and an abiotic stress-induced (RD29A) promoter. Employing a yeast one-hybrid screen we identified HD-Zip class I TF AtHB13 as an upstream regulator of JUB1. AtHB13 has previously been reported to act as a positive regulator of drought tolerance. AtHB13 and JUB1 thereby establish a joint drought stress control module.

The sunflower transcription factor HaHB11 confers tolerance to water deficit and salinity to transgenic Arabidopsis and alfalfa plants.

Homeodomain-leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom; members of subfamily I are known to be involved in abiotic stress responses. HaHB11 belongs to this subfamily and it was previously shown that it is able to confer improved yield and tolerance to flooding via a quiescent strategy. Here we show that HaHB11 expression is induced by ABA, NaCl and water deficit in sunflower seedlings and leaves. Arabidopsis transgenic plants expressing HaHB11, controlled either by its own promoter or by the constitutive 35S CaMV, presented rolled leaves and longer roots than WT when grown under standard conditions. In addition, these plants showed wider stems and more vascular bundles. To deal with drought, HaHB11 transgenic plants closed their stomata faster and lost less water than controls, triggering an enhanced tolerance to such stress condition and also to salinity stress. Concomitantly, ABA-synthesis and sensing related genes were differentially regulated in HaHB11 transgenic plants. Either under long-term salinity stress or mild drought stress, HaHB11 transgenic plants did not exhibit yield penalties. Moreover, alfalfa transgenic plants were generated which also showed enhanced drought tolerance. Altogether, the results indicated that HaHB11 was able to confer drought and salinity tolerance via a complex mechanism which involves morphological, physiological and molecular changes.

A matter of quantity: Common features in the drought response of transgenic plants overexpressing HD-Zip I transcription factors.

Plant responses to water deficit involve complex molecular mechanisms in which transcription factors have key roles. Previous reports ectopically overexpressed a few members of the homeodomain-leucine zipper I (HD-Zip I) family of transcription factors from different species, and the obtained transgenic plants exhibited drought tolerance which extent depended on the level of overexpression, triggering diverse molecular and physiological pathways. Here we show that most HD-Zip I genes are regulated by drought in the vegetative and/or reproductive stages. Moreover, uncharacterized members of this family were expressed as transgenes both in Col-0 and rdr6-12 backgrounds and were able to enhance drought tolerance in host plants. The extent of such tolerance depended on the expression level of the transgene and was significantly higher in transgenic rdr6-12 than in Col-0. Comparative transcriptome analyses of Arabidopsis thaliana plants overexpressing HD-Zip I proteins indicated that many members have common targets. Moreover, the water deficit tolerance exhibited by these plants is likely due to the induction and repression of certain of these common HD-Zip I-regulated genes. However, each HD-Zip I member regulates other pathways, which, in some cases, generate differential and potentially undesirable traits in addition to drought tolerance. In conclusion, only a few members of this family could become valuable tools to improve drought-tolerance.

A sunflower WRKY transcription factor stimulates the mobilization of seed-stored reserves during germination and post-germination growth.

KEY MESSAGE: The sunflower transcription factor HaWRKY10 stimulates reserves mobilization in Arabidopsis. Gene expression and enzymes activity assays indicated that lipolysis and gluconeogenesis were increased. Microarray results suggested a parallelism in sunflower. Germinating oilseeds converts stored lipids into sugars, and thereafter in metabolic energy that is used in seedling growth and establishment. During germination, the induced lipolysis linked to the glyoxylate pathway and gluconeogenesis produces sucrose, which is then transported to the embryo and driven through catabolic routes. Herein, we report that the sunflower transcription factor HaWRKY10 regulates carbon partitioning by reducing carbohydrate catabolism and increasing lipolysis and gluconeogenesis. HaWRKY10 was regulated by abscisic acid and gibberellins in the embryo leaves 48 h after seed imbibition and highly expressed during sunflower seed germination and seedling growth, concomitantly with lipid mobilization. Sunflower leaf disks overexpressing HaWRKY10 showed repressed expression of genes related to sucrose cleavage and glycolysis compared with controls. Moreover, HaWRKY10 constitutive expression in Arabidopsis seeds produced higher decrease in lipid reserves, whereas starch and sucrose were more preserved compared with wild type. Gene transcripts abundance and enzyme activities involved in stored lipid mobilization and gluconeogenesis increased more in transgenic than in wild type seeds 36 h after imbibition, whereas the negative regulator of lipid mobilization, ABI4, was repressed. Altogether, the results point out a functional parallelism between tissues and plant species, and reveal HaWRKY10 as a positive regulator of storage reserve mobilization in sunflower.

The sunflower transcription factor HaHB11 improves yield, biomass and tolerance to flooding in transgenic Arabidopsis plants.

HaHB11 is a member of the sunflower homeodomain-leucine zipper I subfamily of transcription factors. The analysis of a sunflower microarray hybridized with RNA from HaHB11-transformed leaf-disks indicated the regulation of many genes encoding enzymes from glycolisis and fermentative pathways. A 1300bp promoter sequence, fused to the GUS reporter gene, was used to transform Arabidopsis plants showing an induction of expression after flooding treatments, concurrently with HaHB11 regulation by submergence in sunflower. Arabidopsis transgenic plants expressing HaHB11 under the control of the CaMV 35S promoter and its own promoter were obtained and these plants exhibited significant increases in rosette and stem biomass. All the lines produced more seeds than controls and particularly, those of high expression level doubled seeds yield. Transgenic plants also showed tolerance to flooding stress, both to submergence and waterlogging. Carbohydrates contents were higher in the transgenics compared to wild type and decreased less after submergence treatments. Finally, transcript levels of selected genes involved in glycolisis and fermentative pathways as well as the corresponding enzymatic activities were assessed both, in sunflower and transgenic Arabidopsis plants, before and after submergence. Altogether, the present work leads us to propose HaHB11 as a biotechnological tool to improve crops yield, biomass and flooding tolerance.

A Predictive Coexpression Network Identifies Novel Genes Controlling the Seed-to-Seedling Phase Transition in Arabidopsis thaliana.

The transition from a quiescent dry seed to an actively growing photoautotrophic seedling is a complex and crucial trait for plant propagation. This study provides a detailed description of global gene expression in seven successive developmental stages of seedling establishment in Arabidopsis (Arabidopsis thaliana). Using the transcriptome signature from these developmental stages, we obtained a coexpression gene network that highlights interactions between known regulators of the seed-to-seedling transition and predicts the functions of uncharacterized genes in seedling establishment. The coexpressed gene data sets together with the transcriptional module indicate biological functions related to seedling establishment. Characterization of the homeodomain leucine zipper I transcription factor AtHB13, which is expressed during the seed-to-seedling transition, demonstrated that this gene regulates some of the network nodes and affects late seedling establishment. Knockout mutants for athb13 showed increased primary root length as compared with wild-type (Columbia-0) seedlings, suggesting that this transcription factor is a negative regulator of early root growth, possibly repressing cell division and/or cell elongation or the length of time that cells elongate. The signal transduction pathways present during the early phases of the seed-to-seedling transition anticipate the control of important events for a vigorous seedling, such as root growth. This study demonstrates that a gene coexpression network together with transcriptional modules can provide insights that are not derived from comparative transcript profiling alone.

The sunflower transcription factor HaWRKY76 confers drought and flood tolerance to Arabidopsis thaliana plants without yield penalty.

KEY MESSAGE: Arabidopsis transgenic plants expressing the sunflower transcription factor HaWRKY76 exhibit increased yield and tolerance to drought and flood stresses. The genetic construct containing HaWRKY76 is proposed as a potential biotechnological tool to improve crops. Water deficit and water excess are abiotic stress factors that seriously affect crops worldwide. To increase the tolerance to such stresses without causing yield penalty constitutes a major goal for biotechnologists. In this survey, we report that HaWRKY76, a divergent sunflower WRKY transcription factor, is able to confer both dehydration and submergence tolerance to Arabidopsis transgenic plants without yield penalty. The expression pattern of HaWRKY76 was analyzed in plants grown in standard conditions and under different watering regimes indicating a regulation by water availability. The corresponding cDNA was isolated and cloned under the control of a constitutive promoter and Arabidopsis plants were transformed with this construct. These transgenic plants presented higher biomass, seed production and sucrose content than controls in standard growth conditions. Moreover, they exhibited tolerance to mild drought or flood (complete submergence/waterlogging) stresses as well as the same or increased yield, depending on the stress severity and plant developmental stage, compared with controls. Drought tolerance occurred via an ABA-independent mechanism and induction of stomatal closure. Submergence tolerance can be explained by the carbohydrate (sucrose and starch) preservation achieved through the repression of fermentation pathways. Higher cell membrane stability and chlorenchyma maintenance could be the nexus between tolerance responses in front of both stresses. Altogether, the obtained results indicated that HaWRKY76 can be a potential biotechnological tool to improve crops yield as well as drought and flood tolerances.

Functional characterization of the homeodomain leucine zipper I transcription factor AtHB13 reveals a crucial role in Arabidopsis development.

AtHB13 is a homeodomain leucine zipper I transcription factor whose function in development is largely unknown. AtHB13 and AtHB23 mutant and silenced lines were characterized by expression studies, reciprocal crosses, complementation, molecular analyses, and developmental phenotypes. The athb13-1 and athb13-2 mutants, athb23 silenced, and athb13/athb23 double-silenced plants exhibited faster elongation rates of their inflorescence stems, whereas only athb13-1 and the double-knockdown athb13/athb23 exhibited shorter siliques, fewer seeds, and unfertilized ovules compared with the wild type (WT). The cell sizes of mutant and WT plants were similar, indicating that these transcription factors probably affect cell division. Reciprocal crosses between athb13-1 and the WT genotype indicated that the silique defect was male specific. Pollen hydration assays indicated that the pollen grains of the athb13-1 mutant were unable to germinate on stigmas. AtHB23-silenced plants exhibited normal siliques, whereas double-knockdown athb13/athb23 plants were similar to athb13-1 plants. Both AtHB13 and AtHB23 were able to rescue the abnormal silique phenotype. AtHB23 was upregulated in athb13-2 plants, whereas its transcript levels in athb13-1 mutants were not significantly increased. Transcriptome analysis comparing athb13-1 and WT inflorescences revealed that a large number of genes, including several involved in pollen coat formation, are regulated by AtHB13. Finally, athb13-1 complementation with mutated versions of AtHB13 confirmed that two different tryptophans in its C terminus are essential. We conclude that AtHB13 and AtHB23 play independent, negative developmental roles in stem elongation, whereas only AtHB13 is crucial for pollen germination. Furthermore, AtHB23, which does not normally exert a functional role in pollen, can act as a substitute for AtHB13.