Shuping Xing

miR156-Targeted and Nontargeted SBP-Box Transcription Factors Act in Concert to Secure Male Fertility in Arabidopsis

This work uses genetic and histologic analyses to show that a subgroup of plant-specific and evolutionary conserved SBP-box genes is absolutely required for male fertility in Arabidopsis. Either together with or independently of SPOROCYTELESS/NOZZLE, another early anther gene, these SBP-box transcription factors promote sporogenous cell and parietal cell formation in early anthers. The SBP-box transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE8 (SPL8) is required for proper development of sporogenic tissues in Arabidopsis thaliana. Here, we show that the semisterile phenotype of SPL8 loss-of-function mutants is due to partial functional redundancy with several other members of the Arabidopsis SPL gene family. In contrast with SPL8, the transcripts of these latter SPL genes are all targeted by miR156/7. Whereas the introduction of single miR156/7-resistant SPL transgenes could only partially restore spl8 mutant fertility, constitutive overexpression of miR156 in an spl8 mutant background resulted in fully sterile plants. Histological analysis of the anthers of such sterile plants revealed an almost complete absence of sporogenous and anther wall tissue differentiation, a phenotype similar to that reported for sporocyteless/nozzle (spl/nzz) mutant anthers. Expression studies indicated a functional requirement for miR156/7-targeted SPL genes limited to early anther development. Accordingly, several miR156/7-encoding loci were found expressed in anther tissues at later stages of development. We conclude that fully fertile Arabidopsis flowers require the action of multiple miR156/7-targeted SPL genes in concert with SPL8. Either together with SPL/NZZ or independently, these SPL genes act to regulate genes mediating cell division, differentiation, and specification early in anther development. Furthermore, SPL8 in particular may be required to secure fertility of the very first flowers when floral transition-related miR156/7 levels might not have sufficiently declined.

ROXY1, a member of the plant glutaredoxin family, is required for petal development in Arabidopsis thaliana

We isolated three alleles of an Arabidopsis thaliana gene named ROXY1, which initiates a reduced number of petal primordia and exhibits abnormalities during further petal development. The defects are restricted to the second whorl of the flower and independent of organ identity. ROXY1 belongs to a subgroup of glutaredoxins that are specific for higher plants and we present data on the first characterization of a mutant from this large Arabidopsis gene family for which information is scarce. ROXY1 is predominantly expressed in tissues that give rise to new flower primordia, including petal precursor cells and petal primordia. Occasionally, filamentous organs with stigmatic structures are formed in the second whorl of the roxy1 mutant, indicative for an ectopic function of the class C gene AGAMOUS (AG). The function of ROXY1 in the negative regulation of AG is corroborated by premature and ectopic AG expression in roxy1-3 ap1-10 double mutants, as well as by enhanced first whorl carpeloidy in double mutants of roxy1 with repressors of AG, such as ap2 or lug. Glutaredoxins are oxidoreductases that oxidize or reduce conserved cysteine-containing motifs. Mutagenesis of conserved cysteines within the ROXY1 protein demonstrates the importance of cysteine 49 for its function. Our data demonstrate that, unexpectedly, a plant glutaredoxin is involved in flower development, probably by mediating post-translational modifications of target proteins required for normal petal organ initiation and morphogenesis.

Genomic organization, phylogenetic comparison and differential expression of the SBP-box family of transcription factors in tomato

SBP-box genes represent transcription factors ubiquitously found in the plant kingdom and recognized as important regulators of many different aspects of plant development. In this study, 15 SBP-box gene family members were identified in tomato and analyzed with respect to their genomic organization and other structural features. Phylogenetic reconstruction based on the DNA-binding SBP-domain, allowed the classification of the SlySBP proteins into eight groups representing clear orthologous relationships to family members of other flowering plants and the moss Physcomitrella. In order to have a better understanding of their possible function in the development of a fleshy-fruit species like tomato, the mRNA expression levels of all SlySBP genes were quantified in vegetative and reproductive organs of plants, at different stages of growth. As transcripts of ten SlySBP genes were found to carry putative miR156- and miR157-response elements, the expression levels of the corresponding microRNAs were determined as well, revealing different patterns of expression. In addition, eight putative miR156 and four miR157 encoding loci could be identified in the tomato genome, four of them forming a polycistronic cluster. Whereas miR156 and miR157 levels were highest in seedlings, leaves and anthers of young flowers, most miR156-targeted SlySBP genes were found to be expressed in young inflorescences and during fruit development and ripening, suggesting a particularly important role during tomato reproductive growth. The data presented provide a basis for future clarification of the various functions that SBP-box gene family members play in tomato growth and development.

SPL8 and miR156-targeted SPL genes redundantly regulate Arabidopsis gynoecium differential patterning

SPL8 and miR156-targeted SPL genes are known to play an essential role in Arabidopsis anther development. Here we show that these SPL genes are also expressed within the developing gynoecium, where they redundantly control development of the female reproductive tract. Whereas the gynoecium morphology in the spl8 single mutant is largely normal, additional down-regulation of miR156-targeted SPL genes results in a shortened style and an apically swollen ovary narrowing onto an elongated gynophore. In particular, the septum does not form properly and lacks a transmitting tract. Loss of SPL8 function enhances the mutant phenotypes of ett, crc and spt, indicating a functional overlap between SPL8 and these genes in regulating gynoecium development. Furthermore, gynoecium development of 35S:MIR156b spl8-1 double mutants shows enhanced sensitivity to a polar auxin transport inhibitor, and the expression pattern of the auxin biosynthesis gene YUCCA4 is altered compared to wild-type. Our observations imply that SPL8 and miR156-targeted SPL genes control gynoecium patterning through interference with auxin homeostasis and signalling.

Conserved Functions of Arabidopsis and Rice CC-Type Glutaredoxins in Flower Development and Pathogen Response

Glutaredoxins (GRXs) are ubiquitous oxidoreductases that play a crucial role in response to oxidative stress by reducing disulfides in various organisms. In planta, three different GRX classes have been identified according to their active site motifs. CPYC and CGFS classes are found in all organisms, whereas the CC-type class is specific for higher land plants. Recently, two Arabidopsis CC-type GRXs, ROXY1 and ROXY2, were shown to exert crucial functions in petal and anther initiation and differentiation. To analyze the function of CC-type GRXs in the distantly related monocots, we isolated and characterized OsROXY1 and OsROXY2-two rice homologs of ROXY1. Both genes are expressed in vegetative and reproductive stages. Although rice flower morphology is distinct from eudicots, OsROXY1/2 floral expression patterns are similar to their Arabidopsis counterparts ROXY1/2. Complementation experiments demonstrate that OsROXY1 and OsROXY2 can fully rescue the roxy1 floral mutant phenotype. Overexpression of OsROXY1, OsROXY2, and ROXY1 in Arabidopsis causes similar vegetative and reproductive plant developmental defects. ROXY1 and its rice homologs thus exert a conserved function during eudicot and monocot flower development. Strikingly, overexpression of these CC-type GRXs also leads to an increased accumulation of hydrogen peroxide levels and hyper-susceptibility to infection from the necrotrophic pathogen Botrytis cinerea, revealing the importance of balanced redox processes in flower organ development and pathogen defence.

Pollen Lethality: A Phenomenon in Arabidopsis RNA Interference Plants

To study gene function, generation of loss-of-function mutants by RNA interference (RNAi) approaches and T-DNA insertional mutagenesis are widely used in plant science and have proven to be very successful in determining gene functions. AGAMOUS-LIKE18 ( AGL18 ) is a MADS-box gene that is expressed

Functional characterisation of Arabidopsis SPL7 conserved protein domains suggests novel regulatory mechanisms in the Cu deficiency response

BackgroundThe Arabidopsis SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor SPL7 reprograms cellular gene expression to adapt plant growth and cellular metabolism to copper (Cu) limited culture conditions. Plant cells require Cu to maintain essential processes, such as photosynthesis, scavenging reactive oxygen species, cell wall lignification and hormone sensing. More specifically, SPL7 activity promotes a high-affinity Cu-uptake system and optimizes Cu (re-)distribution to essential Cu-proteins by means of specific miRNAs targeting mRNA transcripts for those dispensable. However, the functional mechanism underlying SPL7 activation is still to be elucidated. As SPL7 transcript levels are largely non-responsive to Cu availability, post-translational modification seems an obvious possibility. Previously, it was reported that the SPL7 SBP domain does not bind to DNA in vitro in the presence of Cu ions and that SPL7 interacts with a kin17 domain protein to raise SPL7-target gene expression upon Cu deprivation. Here we report how additional conserved SPL7 protein domains may contribute to the Cu deficiency response in Arabidopsis.ResultsCytological and biochemical approaches confirmed an operative transmembrane domain (TMD) and uncovered a dual localisation of SPL7 between the nucleus and an endomembrane system, most likely the endoplasmic reticulum (ER). This new perspective unveiled a possible link between Cu deficit and ER stress, a metabolic dysfunction found capable of inducing SPL7 targets in an SPL7-dependent manner. Moreover, in vivo protein-protein interaction assays revealed that SPL7 is able to homodimerize, probably mediated by the IRPGC domain. These observations, in combination with the constitutive activation of SPL7 targets, when ectopically expressing the N-terminal part of SPL7 including the SBP domain, shed some light on the mechanisms governing SPL7 function.ConclusionsHere, we propose a revised model of SPL7 activation and regulation. According to our results, SPL7 would be initially located to endomembranes and activated during ER stress as a result of Cu deficiency. Furthermore, we added the SPL7 dimerization in the presence of Cu ions as an additional regulatory mechanism to modulate the Cu deficiency response.

New players unveiled in early anther development

Anther development is an important process for the successful sexual reproduction in plants. Whereas the regulation of the late stages of anther development is quite well described in A. thaliana, little is known about the regulation of the early stages of this process. Two novel groups of factors involved in these early stages have recently been described, namely ROXYs, members of the Glutaredoxin (GRX) family of small and ubiquitous oxidoreductases involved in various cellular and stress-related responses, and SBP-box Genes. ROXYs belong to the CC-type of GRXs with a CCXC active motif and are specific for higher plants. SBP-box genes encode for SQUAMOSA PROMOTER BINDING PROTEIN transcription factors, many of which are targeted by miR156 and miR157. Strikingly, both the enzymes and the transcription factors represent evolutionary conserved gene families and loss-of-function of these genes exhibits similar anther phenotypes, e.g. arresting sporogenous cell formation and missing pollen sacs. This mini-review gives an overview of how these factors affect early anther development and discusses a possible relationship between these factors and other known early anther genes.

A Conserved KIN17 Curved DNA-Binding Domain Protein Assembles with SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 to Adapt Arabidopsis Growth and Development to Limiting Copper Availability

An Arabidopsis homolog of KIN17 physically interacts with the transcription factor SPL7 to prevent impaired plant growth during periods of limited copper availability. Proper copper (Cu) homeostasis is required by living organisms to maintain essential cellular functions. In the model plant Arabidopsis (Arabidopsis thaliana), the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7) transcription factor participates in reprogramming global gene expression during Cu insufficiency in order to improve the metal uptake and prioritize its distribution to Cu proteins of major importance. As a consequence, spl7 null mutants show morphological and physiological disorders during Cu-limited growth, resulting in lower fresh weight, reduced root elongation, and chlorosis. On the other hand, the Arabidopsis KIN17 homolog belongs to a well-conserved family of essential eukaryotic nuclear proteins known to be stress activated and involved in DNA and possibly RNA metabolism in mammals. In the study presented here, we uncovered that Arabidopsis KIN17 participates in promoting the Cu deficiency response by means of a direct interaction with SPL7. Moreover, the double mutant kin17-1 spl7-2 displays an enhanced Cu-dependent phenotype involving growth arrest, oxidative stress, floral bud abortion, and pollen inviability. Taken together, the data presented here provide evidence for SPL7 and KIN17 protein interaction as a point of convergence in response to both Cu deficiency and oxidative stress.

SPL8 Acts Together with the Brassinosteroid-Signaling Component BIM1 in Controlling Arabidopsis thaliana Male Fertility.

The non-miR156 targeted SBP-box gene SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8), plays an important role in Arabidopsis anther development, where its loss-of-function results in a semi-sterile phenotype. Fully male-sterile plants are obtained when a spl8 loss-of-function mutation is introduced into a 35S:MIR156 genetic background, thereby revealing functional redundancy between SPL8 and miR156-targeted SBP-box genes. Here, we show that BIM1, a gene encoding a bHLH protein involved in brassinosteroid signaling and embryonic patterning, functions redundantly with SPL8 in its requirement for male fertility. Although bim1 single mutants displayed a mild fertility problem due to shortened filaments in some flowers, mutation of BIM1 significantly enhanced the semi-sterile phenotype of the spl8 mutant. Expression of both SPL8 and BIM1 was detected in overlapping expression domains during early anther developmental stages. Our data suggest that in regulating anther development, SPL8 and BIM1 function cooperatively in a common complex or in synergistic pathways. Phylogenetic analysis supports the idea of an evolutionary conserved function for both genes in angiosperm anther development.