VviPLATZ1 is a major factor that controls female flower morphology determination in grapevine.

Plant genetic sex determinants that mediate the transition to dioecy are predicted to be diverse, as this type of mating system independently evolved multiple times in angiosperms. Wild Vitis species are dioecious with individuals producing morphologically distinct female or male flowers; whereas, modern domesticated Vitis vinifera cultivars form hermaphrodite flowers capable of self-pollination. Here, we identify the VviPLATZ1 transcription factor as a key candidate female flower morphology factor that localizes to the Vitis SEX-DETERMINING REGION. The expression pattern of this gene correlates with the formation reflex stamens, a prominent morphological phenotype of female flowers. After generating CRISPR/Cas9 gene-edited alleles in a hermaphrodite genotype, phenotype analysis shows that individual homozygous lines produce flowers with reflex stamens. Taken together, our results demonstrate that loss of VviPLATZ1 function is a major factor that controls female flower morphology in Vitis.

The role of auxin and sugar signaling in dominance inhibition of inflorescence growth by fruit load.

Dominance inhibition of shoot growth by fruit load is a major factor that regulates shoot architecture and limits yield in agriculture and horticulture crops. In annual plants, the inhibition of inflorescence growth by fruit load occurs at a late stage of inflorescence development termed the end of flowering transition. Physiological studies show this transition is mediated by production and export of auxin from developing fruits in close proximity to the inflorescence apex. In the meristem, cessation of inflorescence growth is controlled in part by the age-dependent pathway, which regulates the timing of arrest. Here, we show the end of flowering transition is a two-step process in Arabidopsis (Arabidopsis thaliana). The first stage is characterized by a cessation of inflorescence growth, while immature fruit continues to develop. At this stage, dominance inhibition of inflorescence growth by fruit load is associated with a selective dampening of auxin transport in the apical region of the stem. Subsequently, an increase in auxin response in the vascular tissues of the apical stem where developing fruits are attached marks the second stage for the end of flowering transition. Similar to the vegetative and floral transition, the end of flowering transition is associated with a change in sugar signaling and metabolism in the inflorescence apex. Taken together, our results suggest that during the end of flowering transition, dominance inhibition of inflorescence shoot growth by fruit load is mediated by auxin and sugar signaling.

Genetic identification of SNP markers linked to a new grape phylloxera resistant locus in Vitis cinerea for marker-assisted selection.

BACKGROUND: Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major insect pest that negatively impacts commercial grapevine performance worldwide. Consequently, the use of phylloxera resistant rootstocks is an essential component of vineyard management. However, the majority of commercially available rootstocks used in viticulture production provide limited levels of grape phylloxera resistance, in part due to the adaptation of phylloxera biotypes to different Vitis species. Therefore, there is pressing need to develop new rootstocks better adapted to specific grape growing regions with complete resistance to grape phylloxera biotypes. RESULTS: Grapevine rootstock breeding material, including an accession of Vitis cinerea and V. aestivalis, DRX55 ([M. rotundifolia x V. vinifera] x open pollinated) and MS27-31 (M. rotundifolia specific hybrid), provided complete resistance to grape phylloxera in potted plant assays. To map the genetic factor(s) of grape phylloxera resistance, a F1 V. cinerea x V. vinifera Riesling population was screened for resistance. Heritability analysis indicates that the V. cinerea accession contained a single allele referred as RESISTANCE TO DAKTULOSPHAIRA VITIFOLIAE 2 (RDV2) that confers grape phylloxera resistance. Using genetic maps constructed with pseudo-testcross markers for V. cinerea and Riesling, a single phylloxera resistance locus was identified in V. cinerea. After validating SNPs at the RDV2 locus, interval and linkage mapping showed that grape phylloxera resistance mapped to linkage group 14 at position 16.7 cM. CONCLUSION: The mapping of RDV2 and the validation of markers linked to grape phylloxera resistance provides the basis to breed new rootstocks via marker-assisted selection that improve vineyard performance.

SNP markers tightly linked to root knot nematode resistance in grapevine (Vitis cinerea) identified by a genotyping-by-sequencing approach followed by Sequenom MassARRAY validation.

Plant parasitic nematodes, including root knot nematode Meloidogyne species, cause extensive damage to agriculture and horticultural crops. As Vitis vinifera cultivars are susceptible to root knot nematode parasitism, rootstocks resistant to these soil pests provide a sustainable approach to maintain grapevine production. Currently, most of the commercially available root knot nematode resistant rootstocks are highly vigorous and take up excess potassium, which reduces wine quality. As a result, there is a pressing need to breed new root knot nematode resistant rootstocks, which have no impact on wine quality. To develop molecular markers that predict root knot nematode resistance for marker assisted breeding, a genetic approach was employed to identify a root knot nematode resistance locus in grapevine. To this end, a Meloidogyne javanica resistant Vitis cinerea accession was crossed to a susceptible Vitis vinifera cultivar Riesling and results from screening the F1 individuals support a model that root knot nematode resistance, is conferred by a single dominant allele, referred as MELOIDOGYNE JAVANICA RESISTANCE1 (MJR1). Further, MJR1 resistance appears to be mediated by a hypersensitive response that occurs in the root apical meristem. Single nucleotide polymorphisms (SNPs) were identified using genotyping-by-sequencing and results from association and genetic mapping identified the MJR1 locus, which is located on chromosome 18 in the Vitis cinerea accession. Validation of the SNPs linked to the MJR1 locus using a Sequenom MassARRAY platform found that only 50% could be validated. The validated SNPs that flank and co-segregate with the MJR1 locus can be used for marker-assisted selection for Meloidogyne javanica resistance in grapevine.

Constraints to obtaining consistent annual yields in perennial tree crops. I: Heavy fruit load dominates over vegetative growth.

Farmers lack effective methods to achieve and maintain stable production from year to year in many commercial fruit crops. Annual fruit yield within a region often alternates between high and low fruit load and is termed alternate bearing. The underlying cause of alternate bearing is the negative impact of high fruit load on vegetative growth and next year's flowering. In this review, we emphasize common responses of diverse perennials to heavy crop load. We present botanical, ecological and horticultural perspectives on irregular bearing. The later part of this review focuses on understanding how high fruit load dominates over vegetative growth. We discuss sink strengths and putative mobile signals (hormones), perhaps seed-derived. We highlight gaps in current understanding of alternate bearing, and discuss new approaches to better understand fruit load dominance. Assuming the effect of high fruit load may be related to other mechanisms of sink partitioning, other forms of dominance are presented such as apical, first fruit and king fruit dominance. Dominance seems to be enforced, in independent cases through the establishment of a polar auxin transport system from the stronger sink. Once established this somehow perturbs the transport of auxin out of weaker sinks. Possibly, fruit derived auxin may alter the polar auxin transport system of the shoot to inhibit shoot growth.

Constraints to obtaining consistent annual yields in perennials. II: Environment and fruit load affect induction of flowering.

In many commercial fruit crop species, high fruit load inhibits vegetative growth and floral induction. As a result, trees that had a high fruit load will bear few flowers and fruit the following year, along with abundant vegetative growth. We previously discussed how high fruit load interferes with concurrent shoot growth. Here we focus on how high fruit load impacts the process of flowering. Ascertaining the precise time at which specific buds begin the floral transition in each species is challenging. The use of indirect approaches to determine time of floral induction or evocation may lead to questionable conclusions. Annual and perennial plants appear to use conserved proteins for flowering induction and initiation. The accumulation or reduction of transcripts encoding proteins similar to Arabidopsis (annual) FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1), respectively, correlates well with flower induction in several diverse species. The recent use of such markers provides a means to formulate an accurate timeframe for floral induction in different species and holds promise in providing new insight into this important developmental event. A role for hormones in modulating the inhibitory effect of fruit load on floral induction is also discussed.

Regulation of shoot meristem integrity during Arabidopsis vegetative development.

Shoot growth and development is mediated by the activity of the shoot meristem, which initiates leaves and axillary meristems. Meristem maintenance is achieved by a poorly understood process that functions to sustain the balance of stem cell perpetuation in the central zone (CZ) and organogenesis in the peripheral zone (PZ). A recent study showed that two related homeodomain transcription factors, pennywise (PNY) and pound-foolish (PNF), regulate meristem maintenance by controlling the integrity of the CZ. The non-flower producing phenotype displayed by pny pnf plants can be rescued by genetically increasing the size of the shoot meristem. In this addendum, we show that augmenting the size of the central region of pny pnf shoot meristems partially rescues the meristem termination phenotype that occurs during early stages of vegetative development. Thus, regulation of CZ integrity by PNY and PNF is crucial for vegetative and reproductive development.

Regulation of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes/microRNA156 module by the homeodomain proteins PENNYWISE and POUND-FOOLISH in Arabidopsis.

The morphology of inflorescences is regulated in part by the temporal and spatial events that regulate flower specification. In Arabidopsis, an endogenous flowering time pathway mediated by a subset of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factors, including SPL3, SPL4, and SPL5, function to specify flowers by activating floral meristem identity genes. During shoot development, SPL3, SPL4, and SPL5 are post-transcriptionally regulated by microRNA156 (miR156). The photoperiod regulated florigenic signal, FLOWERING LOCUS T (FT), promotes floral induction, in part by activating SPL3, SPL4, and SPL5. In turn, these SPLs function in parallel with FT to specify flower meristems. Two related BELL1-like homeobox genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) expressed in the shoot apical meristem are absolutely required for the specification of floral meristems. Genetic studies show that the floral specification function of FT depends upon PNY and PNF; however, the interplay between these homeodomain proteins and SPLs is not known. In this manuscript, we show that the photoperiodic floral induction of SPL3, SPL4, and SPL5 is dependent upon PNY and PNF. Further, PNY and PNF also control SPL3, SPL4, and SPL5 expression by negatively regulating miR156. Lastly, ectopic expression of SPL4 partially rescues the pny pnf non-flower-producing phenotype, while overexpression of SPL3 or SPL5 in pny pnf plants was unable to restore flower specification. These results suggest that: (1) SPL3, SPL4, and SPL5 function is dependent upon PNY and PNF, or (2) expression of multiple SPLs is required for floral specification in pny pnf plants.

The role of PENNYWISE and POUND-FOOLISH in the maintenance of the shoot apical meristem in Arabidopsis.

Growth of the aerial part of the plant is dependent upon the maintenance of the shoot apical meristem (SAM). A balance between the self-renewing stem cells in the central zone (CZ) and organogenesis in the peripheral zone (PZ) is essential for the integrity, function, and maintenance of the SAM. Understanding how the SAM maintains a balance between stem cell perpetuation and organogenesis is a central question in plant biology. Two related BELL1-like homeodomain proteins, PENNYWISE (PNY) and POUND-FOOLISH (PNF), act to specify floral meristems during reproductive development. However, genetic studies also show that PNY and PNF regulate the maintenance of the SAM. To understand the role of PNY and PNF in meristem maintenance, the expression patterns for genes that specifically localize to the peripheral and central regions of the SAM were examined in Arabidopsis (Arabidopsis thaliana). Results from these experiments indicate that the integrity of the CZ is impaired in pny pnf plants, which alters the balance of stem cell renewal and organogenesis. As a result, pools of CZ cells may be allocated into initiating leaf primordia. Consistent with these results, the integrity of the central region of pny pnf SAMs can be partially restored by increasing the size of the CZ. Interestingly, flower specification is also reestablished by augmenting the size of the SAM in pny pnf plants. Taken together, we propose that PNY and PNF act to restrict organogenesis to the PZ by maintaining a boundary between the CZ and PZ.

Specification of reproductive meristems requires the combined function of SHOOT MERISTEMLESS and floral integrators FLOWERING LOCUS T and FD during Arabidopsis inflorescence development.

In Arabidopsis floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene LEAFY. The floral specification activity of FT is dependent upon two related BELL1-like homeobox (BLH) genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) which are required for floral evocation. PNY and PNF interact with a subset of KNOTTED1-LIKE homeobox proteins including SHOOT MERISTEMLESS (STM). Genetic analyses show that these BLH proteins function with STM to specify flowers and internodes during inflorescence development. In this study, experimental evidence demonstrates that the specification of flower and coflorescence meristems requires the combined activities of FT-FD and STM. FT and FD also regulate meristem maintenance during inflorescence development. In plants with reduced STM function, ectopic FT and FD promote the formation of axillary meristems during inflorescence development. Lastly, gene expression studies indicate that STM functions with FT-FD and AGAMOUS-LIKE 24 (AGL24)-SUPPRESSOR OF OVEREXPRESSION OF CONTANS1 (SOC1) complexes to up-regulate flower meristem identity genes during inflorescence development.

gorgon, a novel missense mutation in the SHOOT MERISTEMLESS gene, impairs shoot meristem homeostasis in Arabidopsis.

The shoot meristem is a group of self-perpetuating cells that ultimately gives rise to the aerial parts of plants. The Arabidopsis thaliana SHOOT MERISTEMLESS (STM) gene, which encodes a knotted1-like homeobox transcription factor, is required for shoot meristem formation and maintenance, and loss-of-function mutations in the gene result in complete loss or premature termination of the shoot meristem. Here, we report a novel missense allele of STM, gorgon (gor), which displays striking differences in shoot meristem defects compared with known stm alleles. The gor phenotype results from substitution of the highly conserved arginine at position 53 of the homeodomain, which is important for DNA binding in other homeodomain proteins. In gor, the shoot meristem enlarges continuously during post-embryonic development and the floral meristems frequently develop additional whorls. These phenotypes, together with enlarged expression domains of meristem markers, indicate that the mutation affects shoot meristem activity in the opposite direction to other loss-of-function alleles. However, detailed genetic analyses and overexpression studies indicate that gor represents a novel type of hypomorphic alleles rather than the hypermorph that is suggested by the phenotype. Consistently, the gor allele strictly requires the functional PENNYWISE (PNY) gene, which encodes a known binding partner of the STM protein, to maintain shoot meristem activity, whereas the wild-type allele efficiently maintains the meristem even in the absence of PNY. Our results suggest a critical role for Arg53 of the homeodomain in STM function and that the gor mutation at this residue impairs shoot meristem homeostasis.

A novel role of BELL1-like homeobox genes, PENNYWISE and POUND-FOOLISH, in floral patterning.

Flowers are determinate shoots comprised of perianth and reproductive organs displayed in a whorled phyllotactic pattern. Floral organ identity genes display region-specific expression patterns in the developing flower. In Arabidopsis, floral organ identity genes are activated by LEAFY (LFY), which functions with region-specific co-regulators, UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS), to up-regulate homeotic genes in specific whorls of the flower. PENNYWISE (PNY) and POUND-FOOLISH (PNF) are redundant functioning BELL1-like homeodomain proteins that are expressed in shoot and floral meristems. During flower development, PNY functions with a co-repressor complex to down-regulate the homeotic gene, AGAMOUS (AG), in the outer whorls of the flower. However, the function of PNY as well as PNF in regulating floral organ identity in the central whorls of the flower is not known. In this report, we show that combining mutations in PNY and PNF enhance the floral patterning phenotypes of weak and strong alleles of lfy, indicating that these BELL1-like homeodomain proteins play a role in the specification of petals, stamens and carpels during flower development. Expression studies show that PNY and PNF positively regulate the homeotic genes, APETALA3 and AG, in the inner whorls of the flower. Moreover, PNY and PNF function in parallel with LFY, UFO and WUS to regulate homeotic gene expression. Since PNY and PNF interact with the KNOTTED1-like homeodomain proteins, SHOOTMERISTEMLESS (STM) and KNOTTED-LIKE from ARABIDOPSIS THALIANA2 (KNAT2) that regulate floral development, we propose that PNY/PNF-STM and PNY/PNF-KNAT2 complexes function in the inner whorls to regulate flower patterning events.

Regulatory networks that function to specify flower meristems require the function of homeobox genes PENNYWISE and POUND-FOOLISH in Arabidopsis.

Flowering is a major developmental phase change that transforms the fate of the shoot apical meristem (SAM) from a leaf-bearing vegetative meristem to that of a flower-producing inflorescence meristem. In Arabidopsis, floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene, LEAFY (LFY). Two redundant functioning homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), which are expressed in the vegetative and inflorescence SAM, regulate patterning events during reproductive development, including floral specification. To determine the role of PNY and PNF in the floral specification network, we characterized the genetic relationship of these homeobox genes with LFY and FT. Results from this study demonstrate that LFY functions downstream of PNY and PNF. Ectopic expression of LFY promotes flower formation in pny pnf plants, while the flower specification activity of ectopic FT is severely attenuated. Genetic analysis shows that when mutations in pny and pnf genes are combined with lfy, a synergistic phenotype is displayed that significantly reduces floral specification and alters inflorescence patterning events. In conclusion, results from this study support a model in which PNY and PNF promote LFY expression during reproductive development. At the same time, the flower formation activity of FT is dependent upon the function of PNY and PNF.

LATERAL ORGAN BOUNDARIES defines a new family of DNA-binding transcription factors and can interact with specific bHLH proteins.

Conserved in a variety of evolutionarily divergent plant species, LOB DOMAIN (LBD) genes define a large, plant-specific family of largely unknown function. LBD genes have been implicated in a variety of developmental processes in plants, although to date, relatively few members have been assigned functions. LBD proteins have previously been predicted to be transcription factors, however supporting evidence has only been circumstantial. To address the biochemical function of LBD proteins, we identified a 6-bp consensus motif recognized by a wide cross-section of LBD proteins, and showed that LATERAL ORGAN BOUNDARIES (LOB), the founding member of the family, is a transcriptional activator in yeast. Thus, the LBD genes encode a novel class of DNA-binding transcription factors. Post-translational regulation of transcription factors is often crucial for control of gene expression. In our study, we demonstrate that members of the basic helix-loop-helix (bHLH) family of transcription factors are capable of interacting with LOB. The expression patterns of bHLH048 and LOB overlap at lateral organ boundaries. Interestingly, the interaction of bHLH048 with LOB results in reduced affinity of LOB for the consensus DNA motif. Thus, our studies suggest that bHLH048 post-translationally regulates the function of LOB at lateral organ boundaries.

Arabidopsis inflorescence architecture requires the activities of KNOX-BELL homeodomain heterodimers.

In flowering plants, post-embryonic development is mediated by the activity of shoot and root apical meristems. Shoot architecture results from activity of the shoot apical meristem (SAM), which initiates primordia, including leaves, internodes and axillary meristems, repetitively from its flanks. Axillary meristems can develop into secondary shoots or flowers. In Arabidopsis, two paralogous BEL1-like (BELL) homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), expressed in the SAM, encode DNA-binding proteins that are essential for specifying floral primordia and establishing early internode patterning events during inflorescence development. Biochemical studies show that PNY associates with the knotted1-like homeobox (KNOX) proteins, SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). PNY-BP heterodimers are essential for establishing early internode patterning events, while PNY-STM heterodimers are critical for SAM function. In this report, we examined the role of PNY, PNF and STM during development. First, we show that PNF interacts with STM and BP indicating that PNY and PNF are redundant functioning proteins. Inflorescence development, but not vegetative development, is sensitive to the dosage levels of PNY, PNF and STM. Characterization of stm-10, a weak allele in the Columbia ecotype, indicates that STM is also involved in floral specification and internode development. Our examination of the genetic requirements for PNY, PNF and STM demonstrates that these KNOX-BELL heterodimers control floral specification, internode patterning and the maintenance of boundaries between initiating floral primordia and the inflorescence meristem.

The role of knox genes in plant development.

knox genes encode homeodomain-containing transcription factors that are required for meristem maintenance and proper patterning of organ initiation. In plants with simple leaves, knox genes are expressed exclusively in the meristem and stem, but in dissected leaves, they are also expressed in leaf primordia, suggesting that they may play a role in the diversity of leaf form. This hypothesis is supported by the intriguing phenotypes found in gain-of-function mutations where knox gene misexpression affects leaf and petal shape. Similar phenotypes are also found in recessive mutations of genes that function to negatively regulate knox genes. KNOX proteins function as heterodimers with other homeodomains in the TALE superclass. The gibberellin and lignin biosynthetic pathways are known to be negatively regulated by KNOX proteins, which results in indeterminate cell fates.

Competence to respond to floral inductive signals requires the homeobox genes PENNYWISE and POUND-FOOLISH.

The transition from vegetative to reproductive development establishes new growth patterns required for flowering. This switch is controlled by environmental and/or intrinsic developmental cues that converge at the shoot apical meristem (SAM). During this developmental transition, floral inductive signals cause the vegetative meristem to undergo morphological changes that are essential for flowering. Arabidopsis plants containing null mutations in two paralogous BEL1-like (BELL) homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), disrupt the transition from vegetative to reproductive development. These double mutants are completely unable to flower even though the SAM displays morphological and molecular changes that are consistent with having received floral inductive signals. These studies establish a link between the competence to receive floral inductive signals and restructuring of the SAM during floral evocation.

The interaction of two homeobox genes, BREVIPEDICELLUS and PENNYWISE, regulates internode patterning in the Arabidopsis inflorescence.

Plant architecture results from the activity of the shoot apical meristem, which initiates leaves, internodes, and axillary meristems. KNOTTED1-like homeobox (KNOX) genes are expressed in specific patterns in the shoot apical meristem and play important roles in plant architecture. KNOX proteins interact with BEL1-like (BELL) homeodomain proteins and together bind a target sequence with high affinity. We have obtained a mutation in one of the Arabidopsis BELL genes, PENNYWISE (PNY), that appears phenotypically similar to the KNOX mutant brevipedicellus (bp). Both bp and pny have randomly shorter internodes and display a slight increase in the number of axillary branches. The double mutant shows a synergistic phenotype of extremely short internodes interspersed with long internodes and increased branching. PNY is expressed in inflorescence and floral meristems and overlaps with BP in a discrete domain of the inflorescence meristem where we propose the internode is patterned. The physical association of the PNY and BP proteins suggests that they participate in a complex that regulates early patterning events in the inflorescence meristem.

Selective interaction of plant homeodomain proteins mediates high DNA-binding affinity.

Understanding molecular mechanisms that control cell fate in the shoot apical meristem is a fundamental question in plant development. Genetic and molecular studies demonstrate that maize KNOTTED1 (KN1) of the TALE (3-aa acid loop extension) class of homeodomain (HD) proteins is involved in shoot apical meristem function. We show that KN1 interacts with knotted interacting protein (KIP), a BEL1-like TALE HD protein. Interaction between KN1 and KIP is mediated by conserved domains in the N termini of both proteins. The KN1 DNA-binding sequence, TGACAG(G/C)T, was biochemically identified, and in vitro DNA-binding assays show that individually KN1 and the HD of KIP bind specifically to this motif with low affinity. The KN1-KIP complex, however, binds specifically to this DNA-binding motif with high affinity, indicating that the association of KN1 and KIP may function in transcriptional regulation.