Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development

A highly efficient and synchronousin vitro tuberization system is described. One-node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for bothin vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz-and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud.These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development.     

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 of POTM1 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 in POTM1 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 POTM1 mediates the control of axillary bud development by regulating cell growth in vegetative meristems.     

Induction and accumulation of major tuber proteins of potato in stems and petioles

A family of immunologically identical glycoproteins with apparent molecular weights of approximately 40,000 are among the major tuber proteins of potato (Solanum tuberosum L.). These proteins, as purified by ion-exchange and affinity chromatography, have been given the trivial name `patatin.' To determine if patatin can be used as a biochemical marker to study the process of tuberization, its amount was measured in a variety of tissues by rocket immunoelectrophoresis and by enzyme-linked immunosorbent assay (ELISA).

Patatin comprises 40 to 45% of the soluble protein in tubers regardless of whether they are formed on underground stolons or from axillary buds of stem cuttings. Under normal conditions, patatin is present in only trace amounts, if at all, in leaves, stems, or roots of plants which are either actively forming tubers or which have been grown under long days to prevent tuberization. However, if tubers and axillary buds are removed, patatin can accumulate in stems and petioles. This accumulation occurred without any obvious tuber-like swelling and would occur even under long days. In all tissues containing large amounts of patatin, the other tuber proteins were also found as well as large amounts of starch.

     

Comparison of axillary bud growth and patatin accumulation in potato leaf cuttings as assays for tuber induction

Single-node leaf cuttings from potatoes (Solanum tuberosum L.) cvs. Norland, Superior, Norchip, and Kennebec, were used to assess tuber induction in plants grown under 12, 16, and 20 h daily irradiation (400 micromol s-1 m-2 PPF). Leaf cuttings were taken from plants at four, six and 15 weeks after planting and cultured for 14 d in sand trays in humid environments. Tuber induction was determined by visually rating the type of growth at the attached axillary bud, and by measuring the accumulation of the major tuber protein, patatin, in the base of the petioles. Axillary buds from leaf cuttings of plants grown under the 12 h photoperiod consistently formed round, sessile tubers at the axils for all four cultivars at all harvests. Buds from cuttings of plants grown under the 16 and 20 h photoperiods exhibited mixed tuber, stolon, and leafy shoot growth. Patatin accumulation was highest in petioles of cuttings taken from 12 h plants for all cultivars at all harvests, with levels in 16 and 20 h cuttings approx. one-half that of the 12 h cuttings. Trends, both in visual ratings of axillary buds and in petiole patatin accumulation, followed the harvest index (ratio of tuber to total plant dry matter), suggesting that either method is an acceptable assay for tuber induction in the potato.     

Symplastic connection is required for bud outgrowth following dormancy in potato (Solanum tuberosum L.) tubers

To gain greater insight into the mechanism of dormancy release in the potato tuber, an investigation into physiological and biochemical changes in tuber and bud tissues during the transition from bud dormancy (immediately after harvest) to active bud growth was undertaken. Within the tuber, a rapid shift from storage metabolism (starch synthesis) to reserve mobilization within days of detachment from the mother plant suggested transition from sink to source. Over the same period, a shift in the pattern of [U-(14)C]sucrose uptake by tuber discs from diffuse to punctate accumulation was consistent with a transition from phloem unloading to phloem loading within the tuber parenchyma. There were no gross differences in metabolic capacity between resting and actively growing tuber buds as determined by [U-(14)C]glucose labelling. However, marked differences in metabolite pools were observed with large increases in starch and sucrose, and the accumulation of several organic acids in growing buds. Carboxyfluorescein labelling of tubers clearly demonstrated strong symplastic connection in actively growing buds and symplastic isolation in resting buds. It is proposed that potato tubers rapidly undergo metabolic transitions consistent with bud outgrowth; however, growth is initially prevented by substrate limitation mediated via symplastic isolation.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Molecular cloning and analysis of four potato tuber mRNAs

Tuberization in potato is a complex developmental process involving the expression of a specific set of genes leading to the synthesis of tuber proteins. We here report the cloning and analysis of mRNAs encoding tuber proteins. From a potato tuber cDNA library four different recombinants were isolated which hybridized predominantly with tuber mRNAs. Northern blot hybridization experiments showed that three of them, pPATB2, p303 and p340, can be regarded as tuber-specific while the fourth, p322, hybridizes to tuber and stem mRNA. Hybrid-selected in vitro translation and nucleotide sequence analysis indicate that pPATB2 and p303 represent patatin and the proteinase inhibitor II mRNA respectively. Recombinant p322 represents an mRNA encoding a polypeptide having homology with the soybean Bowman-Birk proteinase inhibitor while p340 represents an mRNA encoding a polypeptide showing homology with the winged bean Kunitz trypsin inhibitor. In total, these four polypeptides constitute approximately 50% of the soluble tuber protein. Using Southern blot analysis of potato DNA we estimate that these mRNAs are encoded by small multigene families.     

Expression of a Patatin-like Protein in the Anthers of Potato and Sweet Pepper Flowers

Patatin, the major glycoprotein in potato tubers, is encoded by a multigene family. RNA and protein analyses reveal that a homologous mRNA and an immunologically cross-reacting protein can be found in potato flowers, which is similar to patatin in that it displays a lipid acyl hydrolase activity. The patatin-like protein found in flowers has a higher molecular weight than the authentic tuber patatin. Deglycosylation experiments show that this is not due to differences in the glycosylation pattern. Immunocytochemical analysis shows the patatin-like protein to be present only in the epidermal cell layer of the anther, the exothecium, and in petals of potato flowers. Furthermore, the fact that a patatin-like protein can be detected in a similar tissue in sweet pepper, another solanaceous plant, could give a clue concerning the evolutionary origin of patatin.     

Gene expression during tuber development in potato plants

Potato tubers are modified stems that have differentiated into storage organs. Factors such as day-length, nitrogen supply, and levels of the phytohormones cytokinin and gibberellic acid, are known to control tuberization. Morphological changes during tuber initiation are accompanied by the accumulation of a characteristic set of proteins, thought to be involved in N-storage (i.e. patatin) or defense against microbial or insect attack (i.e. proteinase inhibitor II). Additionally, deposition of large amounts of starch occurs during tuber formation, which is paralleled by an increase in sucrose synthase and other enzymes involved in starch biosynthesis (i.e. ADP-glucose pyrophosphorylase, starch synthases, and branching enzyme). Potential controlling mechanisms for genes expressed during tuberization are discussed.     

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.     

Analysis of Cycles of Dormancy and Growth in Pea Axillary Buds Based on mRNA Accumulation Patterns of Cell Cycle-Related Genes

Axillary buds of pea (Pisum sativum L. cv. Alaska) do not growon intact plants. Dormant axillary buds can be stimulated togrow rapidly after decapitation. Here, we isolated cDNAs ofPCNA, cyclinB, cyclinD, and cdc2 from pea. The mRNA expressionlevels of these genes were very low in dormant axillary buds,whereas they remarkably increased after decapitation. Basedon the mRNA accumulation patterns of these genes, we found thatmost cells in dormant axillary buds are arrested at the G₁ phasein the cell cycle. There are four buds at the second node onpea seedlings. After decapitation, mRNAs became abundant inthe large and small buds and were kept during the following3 d. After 4 d, mRNAs were still present in the large bud, butnot in the small bud. However, after removal of the large bud,the mRNA levels started to increase again in the small bud.These mRNA accumulation patterns were the same as those afterthe first decapitation. These results suggested that most cellsin axillary buds at the second node are arrested at the G₁]phase again and have the capacity to undergo multiple cyclesof dormancy and growth. Moreover, in situ hybridization analysesdemonstrated that PCNA mRNA increased in all parts of the axillarybuds after decapitation. (Received October 31, 1997; Accepted December 11, 1997)     

Cytokinin-cytokinin-Induced tuber formation Tuber Formation on Stolons of Solanum tuberosum

Kinetin-induced tuberization of isolated stolons was investigated with regard to accumulation of labelled kinetin, starch, protein and nucleic acid synthesis. Using kinetin-8-¹⁴C, it was found that more labelled meterial appeared at the locus of tyber formation that in other parts of the stolon. Substantial accumulation was evident before visible signs of tuber formation. The basal portion of the stolon also accumulated substantial amounts of labelled material. Kinetin-treated stolons showed extensive starch accumulation which was not evident in gibberellic acid-treated or untreated stolons. Starch accumulation occurred before any visible sign of tuber formation. There was no marked differences in the ability of the apical 0.5 cm of kinetin-treated and untrated stolons to incorporate ¹⁴ C uridine into RNA and ¹⁴C leucine into TCA precipitable protein. From these results it was concluded that kinetin-induced tuber formation may not involve the synthesis of new proteins. It is suggested that kinetin may be regulating tuber formation by suppressing starch hydrolase activity and stimulating starch synthetase activity whereas gibberellic acid inhibits tuber formation by promoting starch hydrolase activity or by suppressing starch synthesizing enzymes.     

Sucrose Mobilisation in Sugarcane Stalk Induced by Heterotrophic Axillary Bud Growth

A theoretical high-yield sugarcane biofactory can be idealised as containing culm tissue that functions as a secondary source tissue rather than a sink. To investigate this potential process, heterotrophic axillary bud outgrowth from sugarcane (Saccharum spp. hybrids) setts was used as a model system to demonstrate that sucrose is a mobilisable carbon source. The outgrowth and subsequent biomass accumulation of axillary buds from two-eye setts of mature sugarcane stalks grown in the dark was used to measure carbon mobilisation from sett internode pith tissue. After 42 days growth 99.0 ± 0.72% of sett internode pith sucrose was depleted and 2.66 ± 0.16 g of new tissue accumulated. Comparison with a control treatment in which axillary buds were excised at day zero demonstrated that carbon mobilisation was driven by the accumulation of new biomass. Profiling of soluble carbohydrates (viz. sucrose, glucose and fructose), starch, total soluble protein, total amino nitrogen, free amino acids and total insoluble material showed that the sucrose stored in the sett internode pith was the only available carbon source of sufficient size at day zero for the observed biomass accumulation. Other metabolites mobilised were glucose, fructose and some amino acids, notably isoleucine and leucine that were depleted in shoot treatment setts at day 42.     

Effect of Sucrose on Starch Accumulation in and Adventitious Bud Formation on Embryos of Picea abies

Adventitious buds were initiated on embryos of Picea abies (L.)Karst. after a pulse treatment with cytokinin. The initial stagesof bud formation could take place on culture medium lackingsucrose, but sucrose was required for further development ofmeristematic centres into bud primordia and buds. Sucrose atone per cent was optimal for adventitious bud formation. Embryoscultured on media containing sucrose started to accumulate starchduring the first day. Starch accumulation occurred especiallyin the cortex cells where starch grains were frequently presentin the chloroplasts. The starch accumulation increased withhigher sucrose concentrations in the culture medium. Embryoscultured on medium lacking sucrose did not accumulate starchbefore the formation of meristematic centres. Starch accumulationwas never observed in meristematic cells from which adventitiousbud primordia developed. Picea abies (L.) Karst., Norway spruce, adventitious bud, starch accumulation, sucrose concentration     

Histological and immunohistochemical studies on flower induction in the olive tree (Olea europaea L.)

The aim of this research was to study flower bud differentiation processes in two oil olive cultivars from Tuscan germplasm (Leccino and Puntino). The effect of fruit-set was studied using 'ON' (with fruits) and 'OFF' (without fruits) shoots. Axillary buds were periodically collected at different phenological stages, from endocarp sclerification (July) until budbreak in the following spring. Thin sections were analysed using histology (apex size), histochemistry (RNA, starch and soluble carbohydrates) and cytokinin immunocytochemistry (zeatin localisation). The micromorphological observations and histochemical procedures did not allow us to distinguish axillary buds sampled from 'ON' and 'OFF' shoots. Cytokinin immunocytochemistry revealed early different localisation patterns between 'ON' and 'OFF' samples. Zeatin accumulated only in 'OFF' axillary bud meristems, particularly in July, when endocarp sclerification of fruits from the previous flowering is taking place. At this time, a strong RNA signal was also observed. Both these signals were correlated with floral evocation, and their coincidence with a phenological stage of development provided a useful tool to determine the time when axillary buds switch from the vegetative to the reproductive phase.     

Acropetal disappearance of PsAD1 protein in pea axillary buds after the release of apical dominance

We recently isolated PsAD1 cDNA from pea (Pisum sativum L. cv. Alaska) seedlings, whose mRNA abundantly accumulated in dormant axillary buds and disappeared after decapitation [Madoka and Mori (2000) Plant Cell Physiol. 41: 274]. To further elucidate the function of PsAD1, we investigated the temporal and spatial distribution patterns of PsAD1 protein using Western blot and immunocytochemical analyses. Western blot analyses showed that accumulation patterns of PsAD1 protein in axillary buds after decapitation and in response to IAA and 6-benzyladenine were the same as those of PsAD1 mRNA. Immunocytochemical analyses showed that (1) PsAD1 proteins were localized in the procambia, leaf primordia, apical meristem, and secondary axillary buds in the dormant axillary bud, and this distribution was the same as that of PsAD1 mRNA, (2) PsAD1 proteins acropetally disappeared after decapitation, and (3) the growth of axillary buds occurred in the same manner. These acropetal changes occur in a manner similar to the way in which the procambium differentiates into vascular tissue. These results suggest that PsAD1 plays some role in the inhibition of growth and differentiation, or in the maintenance of the dormant state in axillary buds.