Gary S. Ditta

SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis

The fruit, which mediates the maturation and dispersal of seeds, is a complex structure unique to flowering plants. Seed dispersal in plants such as Arabidopsis occurs by a process called fruit dehiscence, or pod shatter. Few studies have focused on identifying genes that regulate this process, in spite of the agronomic value of controlling seed dispersal in crop plants such as canola. Here we show that the closely related SHATTERPROOF (SHP1) and SHATTERPROOF2 (SHP2 ) MADS-box genes are required for fruit dehiscence in Arabidopsis. Moreover, SHP1 and SHP2 are functionally redundant, as neither single mutant displays a novel phenotype. Our studies of shp1 shp2 fruit, and of plants constitutively expressing SHP1 and SHP2, show that these two genes control dehiscence zone differentiation and promote the lignification of adjacent cells. Our results indicate that further analysis of the molecular events underlying fruit dehiscence may allow genetic manipulation of pod shatter in crop plants.

Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression

Derivatives of plasmid pRK290 that are useful for cloning and for analyzing gene expression in a wide variety of Gram-negative bacteria are described. A smaller broad host range plasmid derived from RK2, with properties similar to that of pRK290, is also described.

The SEP4 Gene of Arabidopsis thaliana Functions in Floral Organ and Meristem Identity

The ABC model of flower organ identity is widely recognized as providing a framework for understanding the specification of flower organs in diverse plant species. Recent studies in Arabidopsis thaliana have shown that three closely related MADS-box genes, SEPALLATA1 (SEP1), SEP2 and SEP3, are required to specify petals, stamens, and carpels because these organs are converted into sepals in sep1 sep2 sep3 triple mutants. Additional studies indicate that the SEP proteins form multimeric complexes with the products of the B and C organ identity genes. Here, we characterize the SEP4 gene, which shares extensive sequence similarity to and an overlapping expression pattern with the other SEP genes. Although sep4 single mutants display a phenotype similar to that of wild-type plants, we find that floral organs are converted into leaf-like organs in sep1 sep2 sep3 sep4 quadruple mutants, indicating the involvement of all four SEP genes in the development of sepals. We also find that SEP4 contributes to the development of petals, stamens, and carpels in addition to sepals and that it plays an important role in meristem identity. These and other data demonstrate that the SEP genes play central roles in flower meristem identity and organ identity.

Assessing the redundancy of MADS-box genes during carpel and ovule development.

Carpels are essential for sexual plant reproduction because they house the ovules and subsequently develop into fruits that protect, nourish and ultimately disperse the seeds. The AGAMOUS (AG) gene is necessary for plant sexual reproduction because stamens and carpels are absent from ag mutant flowers. However, the fact that sepals are converted into carpelloid organs in certain mutant backgrounds even in the absence of AG activity indicates that an AG-independent carpel-development pathway exists. AG is a member of a monophyletic clade of MADS-box genes that includes SHATTERPROOF1 (SHP1), SHP2 and SEEDSTICK (STK), indicating that these four genes might share partly redundant activities. Here we show that the SHP genes are responsible for AG-independent carpel development. We also show that the STK gene is required for normal development of the funiculus, an umbilical-cord-like structure that connects the developing seed to the fruit, and for dispersal of the seeds when the fruit matures. We further show that all four members of the AG clade are required for specifying the identity of ovules, the landmark invention during the course of vascular plant evolution that enabled seed plants to become the most successful group of land plants.

Diverse Roles for MADS Box Genes in Arabidopsis Development

Members of the MADS box gene family play important roles in flower development from the early step of determining the identity of floral meristems to specifying the identity of floral organ primordia later in flower development. We describe here the isolation and characterization of six additional members of this family, increasing the number of reported Arabidopsis MADS box genes to 17. All 11 members reported prior to this study are expressed in flowers, and the majority of them are floral specific. RNA expression analyses of the six genes reported here indicate that two genes, AGL11 and AGL13 (AGL for AGAMOUS-like), are preferentially expressed in ovules, but each has a distinct expression pattern. AGL15 is preferentially expressed in embryos, with its onset at or before the octant stage early in embryo development. AGL12, AGL14, and AGL17 are all preferentially expressed in root tissues and therefore represent the only characterized MADS box genes expressed in roots. Phylogenetic analyses showed that the two genes expressed in ovules are closely related to previously isolated MADS box genes, whereas the four genes showing nonfloral expression are more distantly related. Data from this and previous studies indicate that in addition to their proven role in flower development, MADS box genes are likely to play roles in many other aspects of plant development.

Interactions among APETALA1 , LEAFY , and TERMINAL FLOWER1 Specify Meristem Fate

Upon floral induction, the primary shoot meristem of an Arabidopsis plant begins to produce flower meristems rather than leaf primordia on its flanks. Assignment of floral fate to lateral meristems is primarily due to the cooperative activity of the flower meristem identity genes LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER. We present evidence here that AP1 expression in lateral meristems is activated by at least two independent pathways, one of which is regulated by LFY. In lfy mutants, the onset of AP1 expression is delayed, indicating that LFY is formally a positive regulator of AP1. We have found that AP1, in turn, can positively regulate LFY, because LFY is expressed prematurely in the converted floral meristems of plants constitutively expressing AP1. Shoot meristems maintain an identity distinct from that of flower meristems, in part through the action of genes such as TERMINAL FLOWER1 (TFL1), which bars AP1 and LFY expression from the inflorescence shoot meristem. We show here that this negative regulation can be mutual because TFL1 expression is downregulated in plants constitutively expressing AP1. Therefore, the normally sharp phase transition between the production of leaves with associated shoots and formation of the flowers, which occurs upon floral induction, is promoted by positive feedback interactions between LFY and AP1, together with negative interactions of these two genes with TFL1.

MADS-Box Protein Complexes Control Carpel and Ovule Development in Arabidopsis

The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.

The HECATE genes regulate female reproductive tract development in Arabidopsis thaliana.

Successful fertilization in plants requires the properly coordinated development of female reproductive tissues, including stigma, style, septum and transmitting tract. We have identified three closely related genes, HECATE1 (HEC1), HECATE2 (HEC2) and HECATE3 (HEC3), the expression domains of which encompass these regions of the Arabidopsis gynoecium. The HEC genes encode putative basic helix-loop-helix (bHLH) transcription factors with overlapping functionality. Depending on the amount of HEC function missing, plants exhibit varying degrees of infertility, defects in septum, transmitting tract and stigma development and impaired pollen tube growth. The observed phenotypes are similar to those reported for mutations in the SPATULA (SPT) gene, which also encodes a bHLH transcription factor required for development of the same female tissues. We show that the HEC proteins can dimerize with SPT in a yeast two-hybrid system, indicating that the HEC genes work in concert with SPT to coordinately regulate development of the female reproductive tract. Furthermore, when the HEC genes are ectopically expressed from the CaMV 35S promoter, some of the resulting transgenic plants show pin-shaped inflorescences, suggesting that the HEC genes are probably involved in auxin-mediated control of gynoecium patterning.

Structural relationships among rhizobium meliloti symbiotic promoters

Symbiotic nitrogen fixation by Rhizobium meliloti requires the developmentally specific expression of certain bacterial genes. One set of these genes encodes the subunits of nitrogenase, the enzyme responsible for the reduction of atmospheric dinitrogen to ammonia, and another set consists of closely linked genes also essential for nitrogen fixation. Examination of promoter and probable regulatory regions for these gene sets has revealed extensive DNA sequence conservation for more than 160 bp upstream of the respective transcription start points. Three such promoter regions have been identified in the nitrogen fixation (nif) gene cluster of R. meliloti strain 102F34. Using one of these promoter regions as a hybridization probe, three additional sequences were found in the genome of this strain. The DNA of other R. meliloti strains and Rhizobium species were also examined for homology to the symbiotically regulated promoters of R. meliloti 102F34. DNA sequences homologous to these R. meliloti promoters were found among diverse rhizobia, and in at least some cases were associated with nif genes.

Modular structure of Fix J: homology of the transcriptional activator domain with the -35 binding domain of sigma factors

The FixL/Fix J two‐component system is a global regulator of nitrogen‐fixation genes in Rhizobium meliloti. The transcriptional activator FixJ contains two modules: its N‐terminal module is homologous with other two‐component regulators; its C‐terminal module shows homology with various transcriptional activators, and with the C‐terminal region of σ factors, which is involved in the discrimination of the ‐35 region of bacterial promoters. We show that C‐terminal module of Fix J contains the entire transcription activation function, and that the N‐terminal module regulates this activity negatively. Oligonucleotide directed mutagenesis of the transcriptional activator module demonstrated the importance of a potential helix‐turn‐helix structure.