Decreasing daylength is the environmental signal utilized by many perennial plant systems to initiate growth cessation and to prepare for adverse environmental conditions associated with winter in temperate zones. In this study, V. riparia vines showed no difference in the rate of shoot growth in LD and SD during the first seven days of differential photoperiod treatments; thereafter, growth ceased in the SD treatment and the shoot apices senesced upon prolonged SD exposure. This data is in accordance with previous studies that found shoot length and node number were greater under long days [9, 11, 19–22].
Several proteins identified in this V. riparia study are in common with proteins identified in shoot or leaf proteome profiles of several V. vinifera and V. rotundifolia cultivars [23, 24]. However, those studies indicated that genotype was the most significant factor determining differences in protein abundance . Therefore, this study presents only the differentially abundant proteins in response to LD growth maintenance and SD induced growth cessation in V. riparia. In contrast to photoperiod studies in peach bark (Prunus persica) , which showed a small number (66) of proteins differentially abundant in response to SD, V. riparia had 216 proteins (≥ two-fold ratio, p-value ≤ 0.05) that showed differential abundance in response to SD. There were very few differentially abundant proteins in common between peach bark (a storage tissue) and grape shoot tip (predominately photosynthetic tissue) in response to photoperiod treatment. A comparison of the proteomes of the V. riparia shoot tissue exposed to LD and SD indicated a greater number of proteins in LD than in SD. Since an individual spot intensity is relative to the total intensity, this difference could be related to a higher abundance of a few major proteins in SD treatments, thus reducing the share of lower abundant proteins. In addition to differences in the number of abundant proteins, a comparison of the proteomes identified several molecular parameters that could play significant roles in plant adaptation to decreasing photoperiod.
Carbon fixation and carbohydrate metabolism
Major changes were observed in the abundance of proteins involved in the carbon assimilation process and carbohydrate metabolism in relation to photoperiod treatment. Several enzymes involved in the Calvin-Benson cycle were more abundant in LD shoot tips (Table 1 and 3): phosphoglycerate kinase (SSP6405), chloroplastic glyceraldehyde-3-phosphate dehydrogenase A (SSP7328), triose phosphate isomerase (SSP9108), and four transketolase proteins (SSP7601; SSP4314; SSP8707; SSP8602). In contrast, Rubisco (SSP8619), seven Rubisco activases (SSP1308 (Table 1 and 4); SSP1306; SSP1416; SSP1411; SSP3304; SSP1404), fructose-1,6-bisphosphatase (SSP7317), another transketolase (SSP7710), and sedoheptulose-1,7-bisphosphatase (SSP1304) were more abundant in SD shoot tips (Table 2 and 4; Figure 6).
In barley shoot apices it was noted that the rate of carbohydrate production was considerably slower in 8 h than in 16 h photoperiods . Similarly, in this study, enzymes involved in the reduction phases of the Calvin-Benson cycle are more abundant in LD shoot tips while enzymes involved in the carboxylation and regeneration phase are more abundant in SD shoot tips. In contrast, the greater recovery potential of ribulose-1, 5-bisphosphate exhibited in SD treatments may be related to an overall decrease in available carbon in comparison to the LD treatments.
Potentially higher carbohydrate availability in LD shoot tips may be responsible for the higher protein abundance of enzymes involved in glycolysis. These proteins include triose-phosphate isomerase (SSP9108), phosphoglycerate kinase (SSP6405), two phosphoglycerate mutases (SSP5604; SSP5611, both matching GSVIVP00033522001), pyruvate dehydrogenase (SSP2307), and pyruvate decarboxylase (SSP7608). In addition, the enzymes 2-isopropylmalate synthase (SSP7619) and malate dehydrogenase (SSP8316) (Table 1 and 3), which function in the pathway following glycolysis, were also more abundant in LD shoot tips.
Under LD conditions it appears that the carbon surplus promotes tissue growth by increasing the pyruvate pool. Roeske and Chollet  found that pyruvate accumulation was light dependent. The LD treated tissue had a greater abundance of sucrose synthase (SSP7708) enzymes. Similarly, an increase of sucrose synthase activity was observed in LD in soybean leaves , and a higher abundance of sucrose was observed in LD in tobacco leaves . In Arabidopsis, enzymes in the glycolysis pathway showed a decrease in activity in conjunction with decreasing photoperiods, while activity of photosynthesis and starch synthesis remained high .
A greater abundance of enzymes leading to the accumulation of starch has been observed in SD shoot tips. Analysis identified these storage enzymes as fructose bisphosphate aldolase (SSP7317), a second glyceraldehyde-3-phosphate dehydrogenase (SSP6412), and glucose-1-phosphate adenylyltransferase (SSP4401). Previous reports illustrated that plants grown in shorter photoperiods or lower light intensities usually synthesize proportionally more starch [29, 31, 32]. The present study reveals a clear contrast in carbon utilization through its enzymatic steps. While more carbon is probably accumulated and used for the plant growth in LD, under SD plants appear to store the carbon as starch.
Amino acid metabolism
Most minor amino acid abundance in plants has shown poor correlation with short term photoperiod changes [29, 33]. These insignificant associations suggested that the variation in minor amino acids cannot be traced to short-term changes in primary carbon and nitrogen assimilation . However, glutamate, glutamine, glycine, asparagine, alanine, threonine, and serine present daily variation in abundance in tobacco . These authors also reported that all amino acids assayed were more abundant in LD than SD unless they could not be detected.
Glutamate acts at the center of nitrogen flow by incorporating ammonia into the plant . Glutamine synthetases are especially important in the transport of nitrogen in aerial parts of the plant, and play different roles according to their cellular localization. Two glutamine synthetases have been detected as differentially abundant. One, presumably cytosolic (SSP4315), was more abundant in 7LD (Table 1), and the second, likely chloroplastic (SSP5407), was more abundant in 28SD. A third glutamine synthetase (GSVIVP00030210001) has been identified on two proximal spots (SSP3311; SSP3313); SSP3311 was more abundant in 7LD and SSP3313 was more abundant in 7SD. The differentiation between these glutamine synthetase spots is not likely caused by phosphorylation because SSP3313 has a slightly higher molecular weight (Mw) and the impact of phosphorylation on Mw is not generally noticeable in 2D gels. Over abundance of the cytosolic isoform in 7LD could be related to a greater nitrogen uptake in LD . The major role of the chloroplastic isoform of glutamine synthetase in leaves is thought to be re-assimilation of the NH3 generated in photorespiration . Glutamine synthetases are known to interact with 14-3-3 proteins . Seven 14-3-3 proteins have been identified as differentially abundant in the present study, but only three correlate strictly with glutamine synthetase abundance. One 14-3-3 protein in LD (SSP0224) (Table 1) and two in SD (SSP0227; SSP0109) (Table 2) could also be involved in glutamine synthetase regulation during photoperiod.
In addition to chloroplastic glutamine synthetase, other enzymes involved in photorespiration  have been seen as more abundant in SD shoot tips. Decarboxylating glycine dehydrogenase (SSP8713) and two phosphoglycolate phosphatases (SSP2202; SSP3111) (Table 4) were differentially abundant in the 28 day treatments. Increased photorespiration in plants has been observed in the dark [39, 40]. Photorespiration commonly produces reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) , which can be toxic to plants at certain concentrations . SD plants are known to cope better with H2O2 toxicity than LD plants . Overabundance of enzymes in SD tissue related to ascorbate metabolism, which is involved in the detoxification of reactive oxygen species , also supports the hypothesis that the grapevine leaves have a higher level of peroxides under SD treatments. Monodehydroascorbate reductase (NADH) (SSP7406), dehydroascorbate reductase (SSP5106), and L-galactose 1-phosphate phosphatase (SSP2209) (Table 4), enzymes related to ascorbate biosynthesis, were all found in greater abundance in SD shoot tips. ROS such as H2O2 often elicit various physiological processes as signal molecules. H2O2 is produced during photosynthesis and photorespiration, and interacts with thiol-containing proteins. H2O2 directly activates numerous signaling pathways and transcription factors that regulate gene expression. Most research discusses the role of hydrogen peroxide in photorespiration and stress signaling, but it was not until recently that H2O2 was linked with cell growth and other cellular processes [41, 44]. Hydroxyl radicals may have an active role in cell wall loosening . Fry and colleagues suggest that ascorbate, H2O2, and copper ions (Cu+2) could interact to form OH radicals that actively loosen cell walls [46–48].
Additional enzymes involved in the metabolism of amino acids have been identified as more abundant in LD shoot tips (Table 1 and 3), possibly linked to a greater requirement of metabolites during growth. Aspartate semialdehyde dehydrogenase (SSP4320) forms an early branch point in the metabolic pathway forming lysine, methionine, leucine, and isoleucine from aspartate . Enzymes involved downstream in the amino acids biosynthetic pathways have also been identified, including two ketol-acid reductoisomerase spots (SSP6614 and SSP6517, both matching GSVIVP00018719001) and dihydroxy-acid dehydratase (SSP7613), which are involved in the biosynthesis of isoleucine and valine. Furthermore, the 5-me-tetrahydropteroyltriglu-homocys S-Me-transferase (SSP8731;, SSP8726; SSP9702; SSP8718; SSP8723; SSP8706, all matching GSVIVP00003836001) and S-adenosylmethionine synthetase (SSP5408 matching GSVIVP00019707001; SSP6425; SSP5415 matching GSVIVP00028192001) are involved in methionine metabolism. Additionally, a cysteine synthase (SSP6307) has also been identified as more abundant in LD shoot tips.
Phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, which are important compounds for protection against UV photo-damage in plants . Effects of light treatment on phenylpropanoids have been widely studied in grape berries because of their important organoleptic properties. UV is known to increase phenolic composition in grape berries , and photoperiod has been identified as directly affecting the flavonoid composition. Flavonoid compounds decreased in SD versus LD in Xanthium, including anthocyanidin (quercitin), caffeoyl quinic acid, and bulk phenols . In this study, three enzymes involved in the flavonoid biosynthesis were more abundant in LD shoot tips (Table 1 and 3): chalcone synthase (SSP8417), chalcone isomerase (SSP2120), and leucoanthocyanidin dioxgenase (SSP6413). Polyphenols, which also play an important role in protection against oxidation, and anthocyanidin reductase (SSP7313; SSP7325) were more abundant in LD shoot tips. Cinnamyl alcohol dehydrogenase (SSP6205), an enzyme that catalyzes the final step for production of lignin monomers, was also more abundant in LD shoot tips. Both cinnamyl alcohol dehydrogenase and lignin content have been shown to be enhanced by light in Pinus radiata callus cultures  and Arabidopsis roots .
Surprisingly, a large number of proteins involved in photosystem II (PSII) (SSP2206; SSP1121; SSP1116; SSP3010), light harvest complex (LHC) subunit (SSP2101; SSP1008; SSP0008; SSP0006; SSP1002), and one involved in photosystem I (SSP1006) were more abundant in SD shoot tips (Table 2 and 4). These observations were unexpected since photoassimilate incorporation related proteins are more abundant in LD shoot tips (see previous carbon fixation section). However, several explanations are possible for these observations. Light stress-related oxidative damage causes protein degradation in PSII  and it could potentially be more dramatic under LD, leading to fewer PSII proteins. It is also noted that the leaves in the SD shoot tip are older than those in LD, since shoot growth ceases in the SD treatment and the LD shoot continues to grow and initiate new leaves. Thus the SD leaves may simply contain a greater number of photosystem complexes. Fewer photosystems does not necessarily reflect a decreased efficiency of the photosynthetic system, but rather an indication of leaf maturity and the fact that the photoassimilates are exported from the older mature leaves to the shoot tips. Mor and Halevy  and Lepistö et al.  observed a similar pattern in LHC proteins in rose (Rosa) shoots and Arabidopsis leaves respectively and showed that the photochemical efficiency of PSII was not affected by day length.
The chaperonin TCP-1 is involved in cytoskeleton organization and keeps cytoskeletal proteins folded. Six of the eight subunits of the chaperonin TCP-1 complex were more abundant in 28LD shoot tips (SSP6609; SSP5617; SSP8609; SSP6606; SSP4517; SSP8605), (Table 3). Actin and tubulin monomers both interact with TCP-1 in order to reach their native states. Brackley and Grantham  and Himmelspach et al.  observed that abundance of TCP-1 subunits is age dependent but not growth dependent. This suggests that the greater abundance of TCP-1 subunits in the LD shoot tips was more related to the fact that the tissues are younger in the actively growing LD shoot tips than in the SD shoot tips. Consistently, tubulin proteins (SSP2406; SSP2516; SSP2404; SSP1516) (Table 1 and 3) were more abundant in LD shoot tips. Also, seven proteasome subunits were more abundant in LD (SSP2221; SSP4520; SSP8103; SSP7108; SSP2008; SSP6014; SSP7520). Proteasome plays an important role in plant life cycle processes; among them, cell division, growth and light signaling, which would all be higher in the actively growing LD shoot tip .