David J. Hannapel

Dynamics of a Mobile RNA of Potato Involved in a Long-Distance Signaling Pathway

BEL1-like transcription factors interact with Knotted1 types to regulate numerous developmental processes. In potato (Solanum tuberosum), the BEL1 transcription factor St BEL5 and its protein partner POTH1 regulate tuber formation by mediating hormone levels in the stolon tip. The accumulation of St BEL5 RNA increases in response to short-day photoperiods, inductive for tuber formation. RNA detection methods and heterografting experiments demonstrate that BEL5 transcripts are present in phloem cells and move across a graft union to localize in stolon tips, the site of tuber induction. This movement of RNA to stolon tips is correlated with enhanced tuber production. Overexpression of BEL5 transcripts that include the untranslated sequences of the BEL5 transcript endows transgenic lines with the capacity to overcome the inhibitory effects of long days on tuber formation. Addition of the untranslated regions leads to preferential accumulation of the BEL5 RNA in stolon tips under short-day conditions. Using a leaf-specific promoter, the movement of BEL5 RNA to stolon tips was facilitated by a short-day photoperiod, and this movement was correlated with enhanced tuber production. These results implicate the transcripts of St BEL5 in a long-distance signaling pathway that are delivered to the target organ via the phloem stream.

Lipoxygenase Is Involved in the Control of Potato Tuber Development

Plant lipoxygenases (LOXs) are a functionally diverse class of dioxygenases implicated in physiological processes such as growth, senescence, and stress-related responses. LOXs incorporate oxygen into their fatty acid substrates and produce hydroperoxide fatty acids that are precursors of jasmonic acid and related compounds. Here, we report the involvement of the tuber-associated LOXs, designated the Lox1 class, in the control of tuber growth. RNA hybridization analysis showed that the accumulation of Lox1 class transcripts was restricted to developing tubers, stolons, and roots and that mRNA accumulation correlated positively with tuber initiation and growth. In situ hybridization showed that Lox1 class transcripts accumulated in the apical and subapical regions of the newly formed tuber, specifically in the vascular tissue of the perimedullary region, the site of the most active cell growth during tuber enlargement. Suppression mutants produced by expressing antisense coding sequence of a specific tuber LOX, designated POTLX-1, exhibited a significant reduction in LOX activity in stolons and tubers. The suppression of LOX activity correlated with reduced tuber yield, decreased average tuber size, and a disruption of tuber formation. Our results indicate that the pathway initiated by the expression of the Lox1 class genes of potato is involved in the regulation of tuber enlargement.

Overexpression of a knotted-like homeobox gene of potato alters vegetative development by decreasing gibberellin accumulation.

Potato (Solanum tuberosum) homeobox 1 (POTH1) is a class I homeobox gene isolated from an early-stage tuber cDNA library. The RNA expression pattern ofPOTH1, unlike that of most other class Iknotted-like homeobox genes, is widespread in the cells of both indeterminate and differentiated tissues. Using in situ hybridization, POTH1 transcripts were detected in meristematic cells, leaf primordia, and the vascular procambium of the young stem. Overexpression of POTH1 produced dwarf plants with altered leaf morphology. Leaves were reduced in size and displayed a “mouse-ear” phenotype. The mid-vein was less prominent, resulting in a palmate venation pattern. The overall plant height of overexpression lines was reduced due to a decrease in internode length. Levels of intermediates in the gibberellin (GA) biosynthetic pathway were altered, and the bioactive GA, GA1, was reduced by one-half in sense mutants. Accumulation of mRNA for GA 20-oxidase1, a key biosynthetic enzyme, decreased in overexpression lines. In vitro tuberization was enhanced under both short- and long-day photoperiods in several POTH1 overexpression lines. Sense lines produced more tubers at a faster rate than controls. These results imply that POTH1 mediates the development of potato by acting as a negative regulator of GA biosynthesis.

Interacting Transcription Factors from the Three-Amino Acid Loop Extension Superclass Regulate Tuber Formation

Using the yeast (Saccharomyces cerevisiae) two-hybrid system and a potato (Solanum tuberosum) KNOX protein, designated POTH1, as bait, we have identified seven distinct interacting proteins from a stolon library of potato. All seven cDNAs are members of the BEL1-like family of transcription factors. Among these proteins, there are at least four regions of high sequence conservation including the homeodomain, the proline-tyrosine-proline three-amino acid loop extension, the SKY box, and a 120-amino acid region upstream from the homeodomain. Through deletion analysis, we identified a protein-binding domain present in the carboxy end of the KNOX domain of POTH1. The protein-binding domain in the BEL1 protein is located in the amino-terminal one-half of the 120-residue conserved region of the BELs. RNA-blot analysis showed differential patterns of RNA accumulation for the BELs in various potato organs. The level of StBEL5 mRNA increased in response to a short-day photoperiod in both leaves and stolons. Similar to sense mutants of POTH1, transgenic lines that overexpressed StBEL5 exhibited enhanced tuber formation even under noninductive conditions. Unlike POTH1 sense lines, however, these BEL lines did not exhibit the extreme leaf and stem morphology characteristic of KNOX overexpressers and displayed a more rapid rate of growth than control plants. Both StBEL5 and POTH1 sense lines exhibited an increase in cytokinin levels in shoot tips. StBEL5 lines also exhibited a decrease in the levels of GA 20-oxidase1 mRNA in stolon tips from long-day plants. Our results demonstrate an interaction between KNOX and BEL1-like transcription factors of potato that may potentially regulate processes of development.

Untranslated Regions of a Mobile Transcript Mediate RNA Metabolism

BEL1-like transcription factors are ubiquitous in plants and interact with KNOTTED1 types to regulate numerous developmental processes. In potato (Solanum tuberosum subsp. andigena), the BEL1-like transcription factor StBEL5 and its Knox protein partner regulate tuber formation by targeting genes that control growth. RNA detection methods and heterografting experiments demonstrated that StBEL5 transcripts are present in phloem cells and move across a graft union to localize in stolon tips, the site of tuber induction. This movement of RNA originates in leaf veins and petioles and is induced by a short-day photoperiod, regulated by the untranslated regions, and correlated with enhanced tuber production. Assays for RNA mobility suggest that both 5′ and 3′ untranslated regions contribute to the preferential accumulation of the StBEL5 RNA but that the 3′ untranslated region may contribute more to transport from the leaf to the stem and into the stolons. Addition of the StBEL5 untranslated regions to another BEL1-like mRNA resulted in its preferential transport to stolon tips and enhanced tuber production. Transcript stability assays showed that the untranslated regions and a long-day photoperiod enhanced StBEL5 RNA stability in shoot tips. Upon fusion of the untranslated regions of StBEL5 to a β-glucuronidase marker, translation in tobacco (Nicotiana tabacum) protoplasts was repressed by those constructs containing the 3′ untranslated sequence. These results demonstrate that the untranslated regions of the mRNA of StBEL5 are involved in mediating its long-distance transport, in maintaining transcript stability, and in controlling translation.

Suppression of a Vegetative MADS Box Gene of Potato Activates Axillary Meristem Development

Potato MADS box 1 (POTM1) is a member of the SQUAMOSA-like family of plant MADS box genes isolated from an early stage tuber cDNA library. The RNA ofPOTM1 is most abundant in vegetative meristems of potato (Solanum tuberosum), accumulating specifically in the tunica and corpus layers of the meristem, the procambium, the lamina of new leaves, and newly formed axillary meristems. Transgenic lines with reduced levels of POTM1 mRNA exhibited decreased apical dominance accompanied by a compact growth habit and a reduction in leaf size. Suppression lines produced truncated shoot clusters from stem buds and, in a model system, exhibited enhanced axillary bud growth instead of producing a tuber. This enhanced axillary bud growth was not the result of increased axillary bud formation. Tuber yields were reduced and rooting of cuttings was strongly inhibited inPOTM1 suppression lines. Both starch accumulation and the activation of cell division occurred in specific regions of the vegetative meristems of the POTM1 transgenic lines. Cytokinin levels in axillary buds of a transgenic suppression line increased 2- to 3-fold. These results imply that POTM1mediates the control of axillary bud development by regulating cell growth in vegetative meristems.

The mRNA of a Knotted1-like transcription factor of potato is phloem mobile.

Potato Homeobox1 (POTH1) is a Knotted1-like transcription factor from the Three Amino Acid Loop Extension (TALE) superfamily that is involved in numerous aspects of development in potato (Solanum tuberosum L). POTH1 interacts with its protein partner, StBEL5, to facilitate binding to specific target genes to modulate hormone levels, mediate leaf architecture, and enhance tuber formation. In this study, promoter analyses show that the upstream sequence of POTH1 drives β-glucuronidase activity in response to light and in association with phloem cells in both petioles and stems. Because POTH1 transcripts have previously been detected in phloem cells, long-distance movement of its mRNA was tested. Using RT-PCR and transgenic potato lines over-expressing POTH1, in vitro micrografts demonstrated unilateral movement of POTH1 RNA in a rootward direction. Movement across a graft union into leaves from newly arising axillary shoots and roots of wild type stocks was verified using soil-grown tobacco heterografts. Leaves from the wild type stock containing the mobile POTH1 RNA exhibited a reduction in leaf size relative to leaves from wild type grafts. Both untranslated regions of POTH1 when fused to an expression marker β-glucuronidase, repressed its translation in tobacco protoplasts. RNA/protein binding assays demonstrated that the UTRs of POTH1 bind to two RNA-binding proteins, a polypyrimidine tract-binding protein and an alba-domain type. Conserved glycerol-responsive elements (GRE), specific to alba-domain interaction, are duplicated in both the 5′ and 3′ untranslated regions of POTH1. These results suggest that POTH1 functions as a mobile signal in regulating development.

Proteinase-inhibitor activity and wound-inducible gene expression of the 22-kDa potato-tuber proteins.

Using a proteinase-inhibition assay, we have demonstrated that the 22-kilodalton (kDa) potato (Solanum tuberosum L.) tuber proteins are strong inhibitors of serine proteinases. Two out of three purified proteins from the 22-kDa family of potato-tuber proteins were effective inhibitors of both trypsin and chymotrypsin, while the third, with a molecular mass (Mr) of approx. 24 kDa, inhibited only trypsin activity. Comparison of the amino-acid sequence of the putative reactive sites of several proteinase inhibitors with the deduced sequence of the 22-kDa protein showed that the 22-kDa protein contained sequences potentially possessing “doubleheaded” sites of inhibition, one against trypsin and another against chymotrypsin. The genes coding for the 22-kDa proteins were developmentally regulated in tubers and environmentally regulated in leaves. Wound induction of the genes coding for the 22-kDa potatotuber proteins was detected at the RNA level. In leaves, transcripts of the 22-kDa protein family were detected 6 h after wounding and were highest after 12 h in locally wounded leaves. The strongest induction occurred systemically in response to mechanical wounding in non-wounded leaves. Cross-hybridization of a cDNA, p34021, which codes for the 22-kDa tuber protein, with both proteinase-inhibitor I and II cDNAs and with a second family of 20-kDa potato-tuber cDNAs showed no cross-homology. Members of this second group of 20-kDa potato-tuber proteins also exhibited wound-induction in leaves at the RNA level.

The Impact of the Long-Distance Transport of a BEL1-Like Messenger RNA on Development

Summary: Through regulation by light, a mobile mRNA of potato is involved in controlling processes of growth and in mediating the activation of its own gene. BEL1- and KNOTTED1-type proteins are transcription factors from the three-amino-loop-extension superclass that interact in a tandem complex to regulate the expression of target genes. In potato (Solanum tuberosum), StBEL5 and its Knox protein partner regulate tuberization by targeting genes that control growth. RNA movement assays demonstrated that StBEL5 transcripts move through the phloem to stolon tips, the site of tuber induction. StBEL5 messenger RNA originates in the leaf, and its movement to stolons is induced by a short-day photoperiod. Here, we report the movement of StBEL5 RNA to roots correlated with increased growth, changes in morphology, and accumulation of GA2-oxidase1, YUCCA1a, and ISOPENTENYL TRANSFERASE transcripts. Transcription of StBEL5 in leaves is induced by light but insensitive to photoperiod, whereas in stolon tips growing in the dark, promoter activity is enhanced by short days. The heterodimer of StBEL5 and POTH1, a KNOTTED1-type transcription factor, binds to a tandem TTGAC-TTGAC motif that is essential for regulating transcription. The discovery of an inverted tandem motif in the StBEL5 promoter with TTGAC motifs on opposite strands may explain the induction of StBEL5 promoter activity in stolon tips under short days. Using transgenic potato lines, deletion of one of the TTGAC motifs from the StBEL5 promoter results in the reduction of GUS activity in new tubers and roots. Gel-shift assays demonstrate BEL5/POTH1 binding specificity to the motifs present in the StBEL5 promoter and a double tandem motif present in the StGA2-oxidase1 promoter. These results suggest that, in addition to tuberization, the movement of StBEL5 messenger RNA regulates other aspects of vegetative development.

Tissue integrity and RNA quality of laser microdissected phloem of potato

The phloem is an important conduit for the transport of signaling molecules including RNA. Phloem sap has served as a source of RNA to profile uncontaminated phloem transcriptomes but its collection is difficult in many species. Laser capture microdissection techniques offer a valuable alternative for isolating RNA from specific vascular cells. In potato (Solanum tuberosum L.), there are seven BEL1-like transcription factors expressed throughout the plant with diverse functions. The RNA of one of these, StBEL5, moves through the phloem from the leaf to stolon tips to regulate tuber formation. In this study, the presence of several BEL RNAs and one Knotted1-like RNA was determined in phloem cells collected by laser microdissection coupled to laser pressure catapulting (LMPC). Three fixatives were compared for their effect on cell morphology and RNA quality in transverse sections of stems of potato. For optimum tissue integrity and quality of RNA from potato stem sections, the best results were achieved using ethanol acetate as the fixative. In addition, the RT-PCR results demonstrated the presence of six out of seven of the StBEL RNAs and a potato Knox RNA in phloem cells.