The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse biological processes. Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photoreceptor’s signaling network, we have undertaken a comprehensive computational analysis of the Arabidopsis genome sequence databases to define the scope and features of the bHLH family. Using a set of criteria derived from a previously defined consensus motif, we identified 147 bHLH protein‐encoding genes, making this one of the largest transcription factor families in Arabidopsis. Phylogenetic analysis of the bHLH domain sequences permits classification of these genes into 21 subfamilies. The evolutionary and potential functional relationships implied by this analysis are supported by other criteria, including the chromosomal distribution of these genes relative to duplicated genome segments, the conservation of variant exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Considerable diversity in DNA binding site specificity among family members is predicted, and marked divergence in protein sequence outside of the conserved bHLH domain is observed. Together with the established propensity of bHLH factors to engage in varying degrees of homodimerization and heterodimerization, these observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs. We provide evidence from yeast two-hybrid and in vitro binding assays that two related phytochrome-interacting members in the Arabidopsis family, PIF3 and PIF4, can form both homodimers and heterodimers and that all three dimeric configurations can bind specifically to the G-box DNA sequence motif CACGTG. These data are consistent, in principle, with the operation of this combinatorial mechanism in Arabidopsis.

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The Arabidopsis basic/helix-loop-helix transcription factor family.

A major component in the regulatory network controlling fruit ripening is likely to be the gene at the tomato Colorless non-ripening (Cnr) locus. The Cnr mutation results in colorless fruits with a substantial loss of cell-to-cell adhesion. The nature of the mutation and the identity of the Cnr gene were previously unknown. Using positional cloning and virus-induced gene silencing, here we demonstrate that an SBP-box (SQUAMOSA promoter binding protein-like) gene resides at the Cnr locus. Furthermore, the Cnr phenotype results from a spontaneous epigenetic change in the SBP-box promoter. The discovery that Cnr is an epimutation was unexpected, as very few spontaneous epimutations have been described in plants. This study demonstrates that an SBP-box gene is critical for normal ripening and highlights the likely importance of epialleles in plant development and the generation of natural variation.

A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening.

Increasing crop yield is a major challenge for modern agriculture. The development of new plant types, which is known as ideal plant architecture (IPA), has been proposed as a means to enhance rice yield potential over that of existing high-yield varieties. Here, we report the cloning and characterization of a semidominant quantitative trait locus, IPA1 (Ideal Plant Architecture 1), which profoundly changes rice plant architecture and substantially enhances rice grain yield. The IPA1 quantitative trait locus encodes OsSPL14 (SOUAMOSA PROMOTER BINDING PROTEIN-LIKE 14) and is regulated by microRNA (miRNA) OsmiR156 in vivo. We demonstrate that a point mutation in OsSPL14 perturbs OsmiR156-directed regulation of OsSPL14, generating an 'ideal' rice plant with a reduced tiller number, increased lodging resistance and enhanced grain yield. Our study suggests that OsSPL14 may help improve rice grain yield by facilitating the breeding of new elite rice varieties.

Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice

SPL3, SPL4 and SPL5 ( SPL3/4/5 ) are closely related members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE family of transcription factors in Arabidopsis , and have a target site for the microRNA miR156 in their 3 ′ UTR. The phenotype of Arabidopsis plants constitutively expressing miR156 -sensitive and miR156 -insensitive forms of SPL3/4/5 revealed that all three genes promote vegetative phase change and flowering, and are strongly repressed by miR156 . Constitutive expression of miR156a prolonged the expression of juvenile vegetative traits and delayed flowering. This phenotype was largely corrected by constitutive expression of a miR156 -insensitive form of SPL3 . The juvenile-to-adult transition is accompanied by a decrease in the level of miR156 and an increase in the abundance of SPL3 mRNA. The complementary effect of hasty on the miR156 and SPL3 transcripts, as well as the miR156 -dependent temporal expression pattern of a 35S :: GUS-SPL3 transgene, suggest that the decrease in miR156 is responsible for the increase in SPL3 expression during this transition. SPL3 mRNA is elevated by mutations in ZIPPY/AGO7, RNA DEPENDENT RNA POLYMERASE 6 ( RDR6 ) and SUPPRESSOR OF GENE SILENCING 3 ( SGS3 ), indicating that it is directly or indirectly regulated by RNAi. However, our results indicate that RNAi does not contribute to the temporal expression pattern of this gene. We conclude that vegetative phase change in Arabidopsis is regulated by an increase in the expression of SPL3 and probably also SPL4 and SPL5, and that this increase is a consequence of a decrease in the level of miR156 .

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Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3.

Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nat Genet

Visual screening of a T-DNA mutagenised population of Arabidopsis thaliana for an absence of silique elongation lead to the isolation of the aborted microspores (ams) mutant that shows a sporophytic recessive male sterile phenotype. Homozygous mutant plants are completely devoid of mature pollen. Pollen degeneration occurs shortly after release of the microspores from the tetrad, prior to pollen mitosis I. Premature tapetum and microspore degeneration are the primary defects caused by this lesion, while a secondary effect is visualised in the stamen filaments, which are reduced in length and lie beneath the receptive stigma at flower opening. The disrupted gene was isolated and revealed a T-DNA element to be inserted into the eighth exon of a basic helix-loop-helix (bHLH) gene located on chromosome II. This protein sequence contains a basic DNA binding domain and two alpha helices separated by a loop, typical of a transcription factor belonging to the MYC sub family of bHLH genes. Therefore, AMS plays a crucial role in tapetal cell development and the post-meiotic transcriptional regulation of microspore development within the developing anther.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.2003.01644.x

The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor

SQUAMOSA PROMOTER BINDING PROTEIN-box genes (SBP-box genes) encode plant-specific proteins that share a highly conserved DNA binding domain, the SBP domain. Although likely to represent transcription factors, little is known about their role in development. In Arabidopsis, SBP-box genes constitute a structurally heterogeneous family of 16 members known as SPL genes. For one of these genes, SPL8, we isolated three independent transposon-tagged mutants, all of which exhibited a strong reduction in fertility. Microscopic analysis revealed that this reduced fertility is attributable primarily to abnormally developed microsporangia, which exhibit premeiotic abortion of the sporocytes. In addition to its role in microsporogenesis, the SPL8 knockout also seems to affect megasporogenesis, trichome formation on sepals, and stamen filament elongation. The SPL8 mutants described help to uncover the roles of SBP-box genes in plant development.

/pdf/spl8-an-sbp-box-gene-that-affects-pollen-sac-development-in-344ynn0s69.pdf

SPL8, an SBP-box gene that affects pollen sac development in Arabidopsis.

A male sterile mutant with a defect in anther dehiscence was identified in an Arabidopsis thaliana population mutagenized with the Zea mays transposon En-1/Spm. Mutants produce viable pollen that can fertilize when released mechanically from the anthers. Mutant stamens are of normal size and shape, but lack cell wall fortifications in the endothecial cell layer of the anther, which are required for the dehiscence process. The mutant phenotype was shown to be caused by a transposon insertion in AtMYB26, disrupting the putative DNA-binding domain of this R2R3-type MYB transcription factor. RT-PCR revealed that expression of AtMYB26 is restricted to inflorescences. Sterility was shown to be stable under several environmental conditions. The high stability of the sterile phenotype, together with the fact that pollen is functional, makes AtMYB26 and its orthologs a valuable tool for manipulating male fertility in higher plants.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.2003.01745.x

Disruption of Arabidopsis thaliana MYB26 results in male sterility due to non-dehiscent anthers.

Plastids of nongreen tissues can import carbon in the form of glucose 6-phosphate via the glucose 6-phosphate/phosphate translocator (GPT). The Arabidopsis thaliana genome contains two homologous GPT genes, AtGPT1 and AtGPT2. Both proteins show glucose 6-phosphate translocator activity after reconstitution in liposomes, and each of them can rescue the low-starch leaf phenotype of the pgi1 mutant (which lacks plastid phosphoglucoisomerase), indicating that the two proteins are also functional in planta. AtGPT1 transcripts are ubiquitously expressed during plant development, with highest expression in stamens, whereas AtGPT2 expression is restricted to a few tissues, including senescing leaves. Disruption of GPT2 has no obvious effect on growth and development under greenhouse conditions, whereas the mutations gpt1-1 and gpt1-2 are lethal. In both gpt1 lines, distorted segregation ratios, reduced efficiency of transmission in males and females, and inability to complete pollen and ovule development were observed, indicating profound defects in gametogenesis. Embryo sac development is arrested in the gpt1 mutants at a stage before the fusion of the polar nuclei. Mutant pollen development is associated with reduced formation of lipid bodies and small vesicles and the disappearance of dispersed vacuoles, which results in disintegration of the pollen structure. Taken together, our results indicate that GPT1-mediated import of glucose 6-phosphate into nongreen plastids is crucial for gametophyte development. We suggest that loss of GPT1 function results in disruption of the oxidative pentose phosphate cycle, which in turn affects fatty acid biosynthesis.

Ulrike S. Unte

Papers

The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor

SPL8, an SBP-box gene that affects pollen sac development in Arabidopsis.

Disruption of Arabidopsis thaliana MYB26 results in male sterility due to non-dehiscent anthers.

The Arabidopsis Plastidic Glucose 6-Phosphate/Phosphate Translocator GPT1 Is Essential for Pollen Maturation and Embryo Sac Development